WO2010020619A2 - Susceptibility to dasatinib - Google Patents

Susceptibility to dasatinib Download PDF

Info

Publication number
WO2010020619A2
WO2010020619A2 PCT/EP2009/060641 EP2009060641W WO2010020619A2 WO 2010020619 A2 WO2010020619 A2 WO 2010020619A2 EP 2009060641 W EP2009060641 W EP 2009060641W WO 2010020619 A2 WO2010020619 A2 WO 2010020619A2
Authority
WO
WIPO (PCT)
Prior art keywords
cell
src
epha3
frk
treatment
Prior art date
Application number
PCT/EP2009/060641
Other languages
French (fr)
Other versions
WO2010020619A3 (en
Inventor
Roman Thomas
Martin Sos
Jonathan Weiss
Thomas Zander
Peter Frommolt
Original Assignee
MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Universität Zu Köln
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V., Universität Zu Köln filed Critical MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Publication of WO2010020619A2 publication Critical patent/WO2010020619A2/en
Publication of WO2010020619A3 publication Critical patent/WO2010020619A3/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
    • C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
    • C12Q2537/157—A reaction step characterised by the number of molecules incorporated or released
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds

Definitions

  • the present invention relates to a method of selecting (a) cell(s). (a) tissue(s) or (a) cell culture(s) with susceptibility to dasatinib. Also a method for determining the responsiveness of a mammalian tumor cell or cancer cell to treatment with dasatinib is described herein.
  • an in vitro method for the identification of a responder for or a patient sensitive to an dasatinib uses of an oligo- or polynucleotide capable of detecting (an) the amplification status of at least one gene selected from the group consisting of SRC, EPHA3, FRX. EPHA5. EPHA8. YES, ABL2, LCK, and BLK gene are provided.
  • the present invention also relates to a method of diagnosing non-small cell lung cancer and a method of monitoring the efficacy of a treatment of said cancer.
  • a method of predicting the efficacy of a cancer treatment is described, in particular a non-small cell lung cancer.
  • a (transgenic) non-human animal or a (transgenic) cell having at least one amplified marker gene as defined herein for screening and/or validation of a medicament for the treatment of said cancer is described and a kit useful for carrying out the methods described herein is provided.
  • the ERBB2/Her2-targeted antibody trastuzumab shrinks tumors in women with £ ⁇ J3J?2-amplified breast cancer (Slamon et al., 2001);
  • the ABL/KIT/ PDGFR inhibitor imatinib induces responses in patients with chronic myeloid leukemia carrying the BCRJABL translocation (Druker et a!.. 2001 a; Druker et al., 2001b) as well as in gastrointestinal stromal tumors and melanomas bearing (see Hodi et al..
  • Non-small cell lung cancer is one of the two main types of lung carcinoma, non- small cell (80.4%) and small-cell (16.8%) lung carcinoma, the classification being based on histological criteria.
  • the non-small cell lung carcinomas have a similar prognosis and similar management and comprise three sub-types: squamous cell lung carcinoma, adenocarcinoma and large cell lung carcinoma.
  • Squamous ceil lung carcinoma (31.1% of lung cancers) often starts near a central bronchus and commonly shows cavitation and necrosis within the center of the cancer.
  • Adenocarcinoma (29,4% of lung cancers) mostly originates in peripheral lung tissue and is usually associated with smoking.
  • Large cell lung carcinoma (10.7% of lung cancers) is a fast- growing form that develops near the surface of the lung.
  • Common treatments of NSCLC include surgery, chemotherapy, and radiation therapy, fn particular, NSCLC is treated with adjuvant chemotherapy (i.e. chemotherapy after surgery).
  • Targeted therapies for NSCLC have also been developed.
  • gefitinib which targets the tyrosine kinase domain of EGFR (epidermal growth factor) is used in the treatment of NSCLC.
  • Erlotinib another tyrosine kinase inhibitor, has been shown to increase survival in lung cancer patients.
  • the angiogenesis inhibitor bevacizumab (in combination with paclitaxel and carboplatin) is known to improve the survival of patients with advanced non-small cell lung carcinoma.
  • Further drugs under evaluation in the treatment of NSCLC are cyclo-oxygenase-2 inhibitors, proteasome inhibitors and bexarotene.
  • dasatinib a BCR/ABL and Src family tyrosine kinases inhibitor
  • BMS-354825 a drug produced by Bristol-Myers Squibb and sold under the trade name Sprycel.
  • Dasatinib is used in the treatment of patients with chronic myelogenous leukemia (CML) after imatinib treatment and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph 4 - ALL). It is also being assessed for use in metastatic melanoma.
  • CML chronic myelogenous leukemia
  • Ph 4 - ALL Philadelphia chromosome-positive acute lymphoblastic leukemia
  • NSCLC tumor-specific tumor entity
  • a specific tumor entity e.g "NSCLC " '
  • NSCLC tumor-specific tumor entity
  • a specific tumor entity e.g "NSCLC " '
  • NSCLC tumor-specific tumor entity
  • NSCLC tumor-specific tumor entity
  • many patients with tumors belonging to the same tumor entity would profit from an identification of markers/predictors for susceptibility of the specific tumor type to (a) certain drug(s)/compound(s).
  • the identification prior to initiation/completion of clinical trials is desirable.
  • Cancer cell lines may be used in corresponding in vitro experiments for identification of drugs to which NSCLC tumors/tumor cell are susceptible; yet. the validity and clinical interpretability of these widely used models have been questioned. In addition, cell lines are frequently thought to be genomically disarrayed and unstable and therefore likely poorly representative of primary tumors. Furthermore, the genetic diversity of histopathologically defined classes of tumors is often substantial: e.g., the clinical tumor entity non-small cell lung cancer (NSCLC) comprises EGFR- and KRAS-irmtant lung adenocarcinomas as well as KRA S-mutant squamous-cell lung cancers. Thus, any representative pre-clinical model would need to capture the nature of lesions of primary tumors as well as their distribution in the histopathologically defined cohort.
  • NSCLC clinical tumor entity non-small cell lung cancer
  • somatic genetic alterations (lesions) in cancer have been causally linked with response to targeted therapeutics as they frequently expose a specific dependence on activated oncogenic signaling pathways.
  • an identification of compounds/drugs to which tumors are susceptible is often time-consuming and cost-intensive since these compounds/drugs may only be identified after completion of clinical trials.
  • the technical problem underlying the present invention is the provision of means and methods for the evaluation of cells, in particular tumor cells, for their susceptibility or responsiveness to anti-cancer treatment,
  • the present invention relates to a method of selecting (a) cell(s). (a) tissue(s) or (a) cell cult ⁇ jre(s) with susceptibility to dasatinib, comprising the steps'
  • the method may additionally comprise the steps (i) contacting said cell(s). tissue(s) or cell culture(s) with dasatinib; and (ii) evaluating viability of said cell(s), tissue(s) or cell culture(s) contacted with dasatinib. It is of note that steps (I) and (ii) may be performed prior to step (a) but also after step (a) or, optionally after step (b). Said steps (i) and (ii) may in particular serve as further experimental proof that the selected cell, tissue or cell culture that comprises (a) gene amplification is susceptible in its viability to dasatinib.
  • cell, tissue and cell culture is not only limited to isolated cells, tissues and ceil cultures but also comprises the use of samples, i.e. biological, medical or pathological samples that consist of fluids that comprise such cells, tissues or cell cultures.
  • a fluid may be a body fluid or also excrements and may also be a culture sample, like the culture medium from cultured cells or cultured tissues.
  • the body fluids may comprise, but are not limited to blood, serum, plasma, urine, saliva, synovial fluid, spinal fluid, cerebrospinal fluid, tears, stool and the like.
  • the gist of the present invention lies in the fact that a method is provided that allows for the determination of the susceptibility of a given cell, tissue or cells in a tissue, (or a cell culture or individual cells in such a cell culture, or as will be explained below, (a) cell(s) in a biological/medical/palhological sample) for the anti-cancer or antiproliferative treatment with dasatinib.
  • a method is provided that allows for the determination of the susceptibility of a given cell, tissue or cells in a tissue, (or a cell culture or individual cells in such a cell culture, or as will be explained below, (a) cell(s) in a biological/medical/palhological sample) for the anti-cancer or antiproliferative treatment with dasatinib.
  • a biological/medical/palhological sample for the anti-cancer or antiproliferative treatment with dasatinib.
  • the present invention does not only provide for a method for selecting cells/tissues/cell cultures which are susceptible to dasatinib. but also for an in vitro method for assessing an individual, i.e. a human or animal patient, for its potential responsiveness to an anti-cancer or anti-proliferate treatment with dasatinib.
  • the present invention provides not only for the possibility to select cells, tissues and cell cultures that are susceptible for dasatinib treatment (i.e. the selection of e.g.
  • dasatinib drugs with a structural similarity to dasatinib may be tested or which are useful in screening methods for compounds that are suspected to function like dasatinib) but also for a method to evaluate whether a given patient, preferably a human patient, in need of treatment but also prevention of a proliferative disease, is a responder for dasatinib treatment. Most preferably, the responsiveness of a given patient to dasatinib is tested. Dasatinib is described herein below in more detail.
  • the selection method of a dasatinib responding cell or a responding patient comprises a step, wherein (a) cell(s), (a) tissue(s) or (a) cell culture(s) with (a) gene amplification(s) above normal of at least one of SRC, EPH ⁇ 3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK is selected.
  • Said cell/tissue may also be derived from a human sample or from a body fluid that comprises such a cell, for example a cancer cell.
  • EPHA8, YES, ABL2, LCK and'or BLK is indicative for susceptibility to dasatinib.
  • the present invention relates in particular to a method for determining the responsiveness of a mammalian tumor cell or cancer cell to treatment with dasatinib, said method comprising determining the amplification status of at least one gene selected from SRC, EPHA3, FRK, EPHA5. EPHA8. YES, ABL2, LCK and/or BLK in said tumor cell, wherein said amplification status is indicative of whether the cell is likely to respond or is responsive to the treatment.
  • a determination may take place on an individual, isolated tumor cell.
  • Such an evaluation may also be carried out on biological/medical/pathological samples, like body fluids, isolated body tissue samples and the like, wherein said samples preferably comprise cells or cell debris to be analyzed.
  • markers which can predict the outcome of an anti-cancer therapy with dasatinib prior to and during treatment.
  • Subject of the present invention is a method for diagnosing an individual who is to be subjected to or is being subjected to an anti -cancer treatment or an anti-pro liferative treatment to asses the responsiveness to dasatinib prior, during and/or after dasatinib treatment which comprises the steps of (a) detection of the gene amplification status of at least one gene selected from the group consisting of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK, and BLK in a biological/medical/pathological sample wherein the gene amplification above normal of at least one of said genes is indicative for the responsiveness to dasatinib treatment prior, during and after treatment with dasatinib; and (b) sorting the individual into responder or Non-responder based on detection of said gene amplification of at least one of said genes.
  • the invention provides for markers which can predict the outcome of an anti- cancer/anti-pro liferative treatment with dasatinib prior to treatment in addition to during and/or after treatment.
  • the present invention solves the above identified technical problem since, as documented herein below and in the appended examples, it was surprisingly found that the presence of (a) gene amplification above normal of at least one of SRC, EPHA3, FRK. EPHA5, EPHA8, YES. AB L2, LCK, and/or BLK in (a) cell(s), (a) tissue(s) or (a) cell culture (or in a biological sample comprising cells or cell debris) is highly predictive for susceptibility of said cell(s), tissue(s) or cell culture(s) (or the individual who provided said biological sample) to dasatinib.
  • the presence of (a) gene amplif ⁇ cation(s) above normal of at least one of SRC is highly predictive for susceptibility of said cell(s), tissue(s) or cell culture(s) (or the individual who provided said biological sample) to dasatinib.
  • EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK was surprisingly identified as a marker /predictor for responsiveness to treatment with dasatinib or for susceptibility to dasatinib.
  • the terms "marker for responsiveness to treatment with dasatinib”7'"marker for susceptibility to dasatinib " and "predictor for responsiveness to treatment with an dasatinib'V'predictor for susceptibility to dasatinib” can be used interchangeably and refer to (a) gene amplification(s) of said genes, whereby the amplification status is indicative for susceptibility to dasatinib.
  • a gene amplification is defined herein as amplification of the gene above normal.
  • “'Normal amplification * 7"Normal amplification status” used herein refers to the presence of two copies of a gene in the genome. "Amplification above normal' * refers accordingly to the presence of at least three copies of a gene in a genome. The presence of (a) gene amplification(s) of the genes defined herein above correlates significantly (p ⁇ 0.05) with a responsiveness to treatment with dasatinib or susceptibility to dasatinib.
  • LCK and/or BLK as markers for susceptibility of tumor cell(s) to an dasatinib allows for the first time a reliable identification of subjects/patients which can be specifically and efficiently treated with dasatinib.
  • the likelihood for susceptibility of patients having no gene amplification above normal of at least one of said genes to treatment with dasatinib is below 10 %.
  • the probability for susceptibility of patients having (a) gene amplification(s) above normal of one of said genes to treatment with dasatinib rises 5-fold to about 50 %.
  • the probability for susceptibility of patients having (a) gene amplification(s) above normal of two of said genes to treatment with dasatinib rises 8-fold to about 80 %.
  • the gene-expression signatures described by Huang was in no way predictive of responsiveness to dasatinib.
  • the chromosomal gene copy number signature i.e. the gene amplification above normal of at least one of the following genes SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK ) described herein represents an important and unexpected contribution to the art and a significant improvement.
  • the present invention is illustrated by the experiments described in the appended Example.
  • the gene amplification status above normal of at least one of the following genes SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK was surprisingly identified as predictor/marker for susceptibility to dasatinib.
  • the gene amplification status of said gene(s) was identified as predictor/marker for susceptibility to dasatinib using the representative NSCLC (non small cell lung cancer) cell line collection as demonstrated in the appended example.
  • One advantage of the present method is the fact that it allows for an in vitro selection of (a) cell(s), (a) tissue(s) or (a) cell culture with susceptibility to dasatinib/responsive to treatment with dasatinib.
  • genomically annotated NSCLC cell lines are used that are representative of the genetic diversity, the transcriptional profile and the phenotypic properties of primary NSCLC (non-small cell lung cancer) tumors. It is shown herein by integrated genomic profiling on a global scale that the genomes of non-small cell lung cancer (NSCLC) ceil lines are highly representative of several primary NSCLC tumors isolated by surgery from patients in gene copy number, oncogene mutation and gene expression space.
  • using said NSCLC cell line collection in context of the present invention may avoid animal tests or voluntary tests with, cancer patients; at the same time use of said cell line collection allows, in contrast to methods known in the art.
  • EPHA5, EPHA8. YES, ABL2, LCK and/orBLK as markers for susceptibility to dasatinib.
  • NSCLC non-small cell lung cancer
  • EGFR mutations were confirmed to predict sensitivity to EGFR inhibitors (erlotinib. PD168393, vandetanib) (Arao et al., 2004; Lynch et al, 2004; Paez et al., 2004; Pao et ah, 2004; Sos et al., 2008) which is in accordance with prior ail observations.
  • EGFR inhibitors in EGFR-mutant NSCLC cell lines has been described in the prior art (McDermott et al. 2007; Paez et ah, 2004; Tracy et al.. 2004).
  • EGFff-routations were identified as markers/predictors of susceptibility to EGFR mhibition (p ⁇ 0.0001) using the systematic cell-based compound screening followed by computational prediction of sensitivity based on lesion profiles, EGFR mutations were also identified herein as predictors/markers for susceptibility to the SRC/ ABL inhibitor dasatinib. These findings suggest that mutant EGFR might be a target of dasatinib (Song et al, 2006). However, not only EGFR-mutations were identified and confirmed as predictors/markers for susceptibility to EGFR inhibitors but also and unexpectedly the amplification status of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2. LCK and/or BLK as predictors/markers for susceptibility to dasatinib.
  • SRC is the relevant target in cells with SRC amplification by showing growth inhibition and cell death induced by sliRNA- mediated knockdown of SRC.
  • the finding that SRC is indeed the relevant target of dasatinib has been further confirmed in a chemical genetics approach that can be considered as the most stringent assay for target validation (Du et al., 2009).
  • This assay exploits the fact that dasatinib is an ATP-competitive hinge region-binding compound that is highly sensitive to modifications of amino acids regulating the entrance to the ATP binding pocket.
  • a change of the threonin to a stericall '"bulky" methionin at the amino acid position 341 lead to a physical barrier that acts as a "gatekeeper” and prevents the binding of dasatinib to the hinge region.
  • the gatekeeper region is a unique residue in the entrance of the ATP-binding pocket that is occupied by competitive inhibitors and thus a common position for the occurance of resistance mutations that abrogate the activity of such compounds.
  • SRC has been ectopically expressed with and without the above described gatekeeper mutation at the amino acid position 341 (i.e., threonin to methionin [T341M]) and it was found herein that the cells stably expressing the gatekeeper mutation were fully resistant to dasatinib treatment, This demonstrates that SRC is indeed the relevant target of dasatinib in SRC-amplified cells.
  • susceptibility to dasatinib(s)/ responsiveness to treatment with dasatinib are well known in the art.
  • susceptibility to dasatinib/responsiveness to treatment with dasatinib may be determined by contacting (a) cell(s) 5 (a) tissue(s), or (a) cell culture(s) with dasatinib and determining the viability of said (a) cell(s), (a) tissue(s), or (a) cell culture(s) after contacting.
  • These above-mentioned methods for determining the susceptibility to dasatinib(s)/ responsiveness to treatment with dasatinib may. for example, comprise an evaluation/determination step, which may.
  • tissue(s) or cell culture(s) contacted with/exposed to an dasatinib or fa) mammalian cellfs) or cancer cell treated with an dasatinib For example, (a) cell(s), (a) tissue(s) or (a) cell culture(s) described herein above may show decreased viability upon contacting/exposing/treating with a dasatinib.
  • the ce ⁇ l(s). tissue(s) or cell culture(s) may show an at least 10 %, 20 %, 30 % 5 40 %.
  • control cell(s), tissue(s) or cell culture(s) not contacted/exposed/treated with an dasatinib Preferably, the control cell(s).
  • tissue(s) or cell culture(s) will be identical to the cell(s).
  • ce ⁇ l(s), (a) tissue(s) or (a) cell culture(s) contacled/exposed/treated with dasatinib and showing, for example, a decreased viability as described herein above, can be considered as being susceptible to dasatinib.
  • tissue(s) or cell culture(s) contacled/exposed/treated with dasatinib and showing, for example, a decreased viability as described herein above can be considered as being susceptible to dasatinib.
  • mammalian tumor cellfs) or (a) cancer cell(s) treated with dasatinib showing such a decreased viability can be considered as responsive to treatment with dasatinib.
  • a reduction in viability may. for example, be reflected in a decreased proliferation, such as 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %. 80 %, and most preferably, 90 % reduction in proliferation compared to control cell(s), tissue(s) or cell culture(s) not contacted/exposed/treated with dasatinib.
  • the decreased proliferation may be quantitated, for example, by measuring the total cell volume, tissue volume or cell culture volume using standard techniques.
  • the difference in proliferation between contacted/exposed/treated cell(s), tissue(s) or cell culture(s) and corresponding controls as defined herein may, for example, be evaluated/determined by measuring the volume of the cell(s), tissue(s) or cell culture(s) taking advantage of standard techniques. Said evaluation/determination may be performed in various points in time, for example, 15 minutes, 30 minutes. 60 minutes. 2 hours, 5 hours, 18 hours. 24 hours. 2 days, 3 days, 4 days, 5 days, six days and/or seven days after contacting/treating said cell(s), tissue(s) or cell culture(s) with an dasatinib or exposing said cell(s), tissue(s) or cell culture(s) to an dasatinib.
  • said evaluation/determination may be performed repeatedly, for example, at 15 minutes, 30 minutes and 60 minutes after said contacting/exposing/treating.
  • said cell(s). tissue(s) or cell culture(s) may be contacted /treated not oniy once with said dasatinib or exposed to said dasatinib but several times (e.g. 2 times, 3 times. 5 times, 10 times or 20 times) under various conditions (e.g. same concentration of inhibitor, different concentration of inhibitor, inhibitor comprised in a composition with different stabilizers, diluents, and/or carriers and the like).
  • said optionally repeated evaluation/determination may be performed after the final contacting/treating with or exposing to said dasatinib or in between said above-mentioned various contacting/exposing/treating steps.
  • These tests may include but are not limited to, measurements of Annexin-V exposure on the outer membrane, cell cycle analyses, propidium iodide staining, TUNEL assay, DNA fragmentation assays, nuclear condensation assays, KI-67 staining, resazurm staining, protein cleavage assays (e.g., PARP or Caspase-3) and others.
  • dasatinib is a known tyrosine kinase inhibitor.
  • the respective formula is given herein below:
  • HTS high throughput screening
  • tissue(s) and/or cell culture(s) for responsiveness/sensitivity to dasatinib.
  • Suitable (HTS) approaches are known in the art.
  • An exemplary protocol for such a screening method is also provided in the appended examples; a person skilled in the art is readily in the position to adapt this protocol or known HTS approaches to the performance of the methods of the present invention.
  • Screening-assays are usually performed in liquid phase, wherein for each cell/tissue/cell culture to be tested at least one reaction batch is made.
  • Typical containers to be used are micro titer plates having for example, 384, 1536. or 3456 wells (i.e. multiples of the "original" 96 reaction vessels).
  • Robotics, data processing and control software and sensitive detectors are further commonly used components of a HTS device.
  • robot system which transport micro titer plates from station to station for addition and mixing of sample(s) and reagent(s), incubating the reagents, and final readout (detection).
  • ITTS can be used in the simultaneous preparation, incubation, and analysis of many plates.
  • the assay can be performed in a singly reaction (which is usually preferred), may, however, also comprise washing and/or transfer steps. Detection can be performed taking advantage of radioactivity, luminescence or fluorescence, like fiuorescence-resonance-energytransfer (FRET) and fluorescence polarisation (FP) and the like.
  • FRET fiuorescence-resonance-energytransfer
  • FP fluorescence polarisation
  • the biological samples described herein can also be used in such a context.
  • cellular assays and in vivo assays can be employed in HTS.
  • Cellular assays may also comprise cellular extracts, i.e. extracts from cells, tissues and the like.
  • cell(s) or tissue(s) as biological sample (in particular a sample obtained from a patient/subject suffering or being prone to suffer from cancer), whereas in vivo assays (wherein suitable animal models are employed, e.g. the herein described mouse models) are particularly useful in the validation/monitoring of the treatment with dasatinib.
  • in vivo assays wherein suitable animal models are employed, e.g. the herein described mouse models
  • follow up assays can be performed by re-running the experiment to collect further data on a narrowed set (e.g. samples found "positive" in the first assay), confirming and refining observations.
  • a suitable readout in animal (in vivo) models is tumor growth (or respectively the complete or partial inhibition of tumor growth and/or its remission).
  • the herein described HTS methods for the detection of copy number changes include but are not limited to techniques such as single nucleotide polymorphism (SNP)-array and Comparative Genomic Hybridization (CGH)-arrays.
  • SNP-array technology allows parallel interrogation of up to two million genomic locations in high-throughput. After DNA labelling and hybridization, fluorescence intensities are measured for each allele of each SNP and genomic copy number changes can be inferred.
  • the CGH technique allows the detection of "tumor” and "normal” tissue extracts, differently fluorescence labeled, on the same glass slide and the relative copy number changes (amplifications and deletions) can thus be inferred.
  • cell(s) refers to a single cell or a plurality of cells.
  • plural of cells means in the context of the present invention a group of cells comprising more than a single cell. Thereby, the cells out of said group of cells may have a similar function. Said cells may be connected cells and/or separate cells.
  • tissue in the context of the present invention particularly means a group of cells that perform a similar function.
  • culture(s) means in context of the present invention cells as defined herein above which are grown/cultured under controlled conditions.
  • Cell culture(s) comprise in particular cells (derived/obtained) from multicellular eukaryotes, preferably animals as defined elsewhere herein. It is to be understood that the term "cell culture(s)” as used herein refers also "tissue culture(s)” and/or “organ culture(s)", an “organ” being a group of tissues which perform the same function.
  • the cell(s), tissue(s) or cell culture(s) to be selected comprise/are derived from or are (a) tumor cell(s).
  • the tumor cells may, for example, be obtained from a biopsy, in particular a biopsy/biopsies from a patient/subject suffering from or being prone to suffering from non-small cell lung cancer. It is preferred herein that said subject is a human.
  • the term "mammalian tumor cell(s)" used herein refers to (a) tumor cell(s) which is derived from or is a tumor cell from a mammal, the term mammal being derived herein below.
  • tumor cells may be obtained from a biopsy, in particular a biopsy/biopsies from a patient/ subject suffering from non-small cell lung cancer or a patient/subject being prone to suffer from said disorders.
  • tissue cell also relates to "cancer cells”
  • said tumor cell or cancer cell may be obtained from any biological source/organism, particularly any biological source/organism, suffering from or being prone to suffer from the above-mentioned non-small cell lung cancer.
  • the (tumor) cell(s) or (cancer) cell to be contacted is (are) obtained/derived from an animal. More preferably, said (turnor)/cancer cell(s) is (are) derived from a mammal
  • animal or “mammal”
  • Non-limiting examples for mammals are even-toed ungulates such as sheep, cattle and pig, odd-toed angulates such as horses as well as carnivores such as cats and dogs.
  • DNA samples are derived from organisms that are economically, agronomically or scientifically important.
  • Scientifically or experimentally important organisms include, but are not limited to. mice, rats, rabbits, guinea pigs and pigs.
  • the tumor cell(s) may also be obtained from primates which comprise lemurs, monkeys and apes.
  • the meaning of the terms “primate”, “lemur”, “monkey * ' and “ape” is known and may, for example, be deduced by an artisan from Wehner und Gehring (1995, Thieme Veriag).
  • the tumor or cancer cell(s) is (are) most preferably derived from a human being suffering from the above-mentioned non-small cell lung cancer, pancreatic cancer, colorectal cancer, breast cancer, leukemias.
  • particular useful cells, in particular- tumor or cancer cells are, accordingly, human cells. These cells can be obtained from e.g. biopsies or from biological samples but the term "cell” also relates to in vitro cultured cells.
  • the present invention relates to an in vitro method for the identification of a responder for or a patient sensitive to dasatinib, said method comprising the following steps: (a) obtaining a sample from a patient suspected to suffer from or being prone to suffer from non- small cell lung cancer; and
  • Said sample may, for example, be obtained by (a) biopsy (biopsies).
  • said sample is obtained from a patient suspected to suffer from or being prone to suffer from non-small cell lung cancer. It is preferred herein that said sample is obtained from (a) tumor(s) and, accordingly, is (a) tumor cell(s) or (a) tumor tissue(s).
  • tumor, sample(s) may be obtained from subjects/patients suffering from non-small cell lung cancer.
  • SRC single as marker in context of the present invention
  • EPHA3 or FRK alone as marker.
  • the amplification status of at least two genes selected from the group of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and BLK is assessed or evaluated.
  • the amplification status of SRC in combination with at least one further of the above 8 remaining genes is assessed or evaluated.
  • Exemplary combinations which are preferred in this context are: SRC and EPHA3; SRC and FRK; SRC. EPHA3 and FRK.
  • SRQ EPHA3 and EPHA5 SRC, EPHA3 and EPHA8: SRC, EPHA3 and YES; SRC 5 EPHA3 and ABL2; SRC, EPHA3 and LCK; SRC. EPHA3 and BLK; SRC. FRK and EPHA5: SRC. FRK and EPHA8; SRC, FRK and YES; SRC 5 FRK and ABL2;
  • SRC EPHA5 and EPHA8; SRC, EPHA5 and YES; SRC. EPHA5 and ABL2; SRC, EPHA5 and LCK; SRC, EPHA5 and BLK;
  • SRC EPHA8 and YES; SRC. EPHA8 and ABL2; SRC, EPHA8 and LCK; SRC, EPHA8 and BLK;
  • SRC SRC, EPHA3, and two further markers
  • SRC SRC.
  • SRC EPHA3. EPHA5 and EPHA8; SRC, EPHA3, EPHA5 and YES; SRC, EPHA3, EPHA5 and ABL2; SRC, EPHA3, EPHA5 and LCK; SRC, EPHA3, EPHA5 and BLK; SRC, EPHA3, EPHA8 and YES; SRC, EPHA3, EPHA8 and ABL2; SRC, EPHA3, EPHA8 and LCK; SRC, EPHA3, EPHA8 and BLK;
  • SRC 5 EPHA3, YES and ABL2; SRC, EPHA3, YES and LCK; SRC, EPHA3, YES and BLK: SRC, EPHA3, ABL2 and LCK; SRC, EPHA3, ABL2 and BLK; or SRC, EPHA3, LCK and BLK; wherein combinations which comprise SRC, EPHA3 and FRK are particularly preferred.
  • Preferred combinations of 4 genes are SRC, FRK, EPHA5 and EPHA8; SRC, FRK, EPHA5 and YES; SRC, FRIC, EPHA5 and
  • ABL2 ABL2; SRC, FRK, EPHA5 and LCK; SRC, FRK, EPHA5 and BLK;
  • SRC SRC, FRK, EPHA8 and YES; SRC. FRK, EPHA8 and ABL2; SRC, FRK, EPHA8 and LCK;
  • SRC SRC with any of the remaining markers EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK, and BLK are easily deduclble by a skilled person, wherein SRC can be combined with at least one, two, three, four, five, six, seven, or eightfurther markers is envisaged. Again, combinations comprising SRC and EPHA3; SRC and FRK; or SRC, EPFIA3 and FRK are preferred.
  • EPHA3 or FRK alone can be used as marker in context of the present invention.
  • the amplification status of EPHA3 and/or FRK in combination with at least one further of the above remaining genes is assessed or evaluated.
  • the amplification status of EPFIA3 can also be assessed/evaluated in combination with FRK, EPHA5, EPHA8, YES, ABL2, LCK, and/or BLK, wherein combinations of EPHA3 and FRK with at least one further of the remaining markers are preferred.
  • the amplification status of FRK can also be assessed/evaluated in combination with EPHA3, EPHA5, EPHA8, YES, ABL2, LCK, and/or BLK; wherein combinations of FRK and EPFIA3 with at least one further of the remaining markers are preferred.
  • EPHA 3 The following combinations of EPHA 3 are preferred: EPHA3 and SRC; EPHA3 and FRK; EPHA3 and EPHA5; EPHA3 and EPHA8; EPHA3 and YES; EPHA3 and ABL2; EPHA3 and LCK; EPHA3 and BLK. Also preferred herein are combinations of FRK and SRC; FRK and EPHA3; FRK and EPHA5; FRK and EPFIA8; FRK and YES; FRK and ABL2; FRK and LCK: FRK and BLK.
  • said at least two amplified genes are selected from the group consisting of SRC and EPHA3.
  • EPHA3 and FRK, and EPHA3 and ABL2 and, as mentioned above, it is preferred herein that said at least one gene/said at least two genes is/are present in at least 3 copies.
  • the gene amplification status may, for example, be detected, assessed or evaluated by an in situ hybridization method, comparative genomic hybridisation and smgie-nucleotide polymorphism arrays.
  • exemplary in situ hybridisations are, inter alia, fluorescent in situ hybridisation (FISH), chromogenic in situ hybridisation (CISH) and silver in situ hybridisation (SISH).
  • the present invention relates to the use of an oligo- or polynucleotide capable of detecting the amplification status of at least one gene selected from the group consisting of SRC. EPHA3. FRK. EPHA5, EPHA8. YES, ABL2, LCK 5 and BLK for diagnosing sensitivity to dasatinib.
  • said oligonucleotide is about 15 to 50 nucleotides in length.
  • a person skilled in the art is, based on his general knowledge and the teaching provided herein, easily in the position to identify and/or prepare (a) an oligo- or polynucleotide capable of detecting the amplification status of at least one gene selected from the group consisting of SRC, EPHA3, FRK. EPHA5, EPHA8, YES, ABL2, LCK and BLK for diagnosing sensitivity to dasatinib.
  • these oligo- or polynucleotides may be used as probe(s) in the detection methods described herein.
  • a skilled person will know, for example, computer programs which may be useful for the identification of corresponding probes to be used herein.
  • the SRC EPHA3. FRK, EPHA5, EPHA8.
  • LCK and/or BLK nucleic acid sequences may be used in this context for identifying specific probes for detecting the amplification status.
  • Exemplary, non- limiting SRC, EPHA3, FRK, EPHA5, EPHA8, YES 5 ABL2, LCK and/or BLK nucleic acid sequences are also available on corresponding databases, such as the NCBI database (www.ncbi.nlm. nih.gov/sites/entrez).
  • the present invention relates to a method of diagnosing (non-small cell lung cancer) in a subject/patient suspected of suffering from non-small cell lung cancer or suspected of being prone to suffering from non-small ceil lung cancer comprising the steps of a) determining in a cell or tissue sample obtained from said subject/patient the activity of at least one marker gene selected from the group consisting of SRC, EPHA3, FRK, EPHA5, EPHA8, YES 5 ABL2, LCK and BLK ; and b) comparing the activity of said at least one marker gene determined in a) with a reference activity of said at least one marker gene determined in (a sample from) a control subject/patient (healthy subject), wherein said non-small cell lung cancer is diagnosed when said activity determined in a) differs from said reference activity.
  • the present invention also relates to a method of monitoring the efficacy of a treatment of a non-small cell lung cancer in a subject/patient suffering from said disorder or being prone to suffering from said disorder comprising the steps of a) determining in a cell or tissue sample obtained from said subject/patient the activity of at least one marker gene selected from the group consisting of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and BLK ; and b) comparing the activity of said at least one marker gene determined in a) with a reference activity of said at least one marker gene, optionally determined in (a sample from) a control subject/patient (responder and/or non-responder), wherein the extent of the difference between said activity determined in a) and said reference activity is indicative for said efficacy of a treatment of a non-small cell lung cancer.
  • the term "activity" as used herein refers to the activity of a protein as described elsewhere herein.
  • the method of monitoring the efficacy of a treatment of a cancer may comprise a step of determining in a cell or tissue sample obtained from a subject/patient suffering from Non- small cell lung cancer (e.g. a biopsy) the gene amplification status of at least one gene selected from the group consisting of SRC, EPHA3, FRK. EPHA5, EPHA8. YES, ABL2, LCK and BLK .
  • a gene amplification above normal of at least one gene selected from the group consisting of SRC, EPHA3. FRK, EPHA5. EPHA8, YES, ABL2. LCK and BLK may be present in a sample before start of the treatment of a cancer. During or after treatment of the cancer, the tumor cells having said gene amplification above normal are erased or otherwise depleted. Thus, the absence of a detectable gene amplification above normal of at least one of said genes in a sample (cell samples/biopsy samples and the like) obtained from a subject/patient during or after treatment of a cancer is indicative of the efficacy of the treatment.
  • the present invention relates to a method of predicting the efficacy of a treatment of a non-small cell lung cancer for a subject/patient suffering from said disorder or being prone to suffering from said disorder comprising the steps of a) determining in a cell or tissue sample obtained from said subject/patient the activity of at least one marker gene selected from the group consisting of SRC, EPHA3, FRK. EPHA5, EPHA8. YES. ABL2.
  • the treatment of non-small cell lung cancer may comprise the administration of dasatinib.
  • the non-small cell lung cancer may, inter alia, be a squamous cell lung carcinoma, an adenocarcinoma, a large cell lung carcinoma, or an anaplastic carcinoma.
  • the subject/patient suffering from said non-small cell lung cancer or being prone to suffering from said non-small cell lung cancer may also exhibit resistance (primary or secondary) against any platinum- based, taxane-based chemotherapy, vinorelbine, gemcitinibe, erlotinib, sunitinib and/or vandetanib.
  • EPHA5, EPHA8, YES, ABL2, LCK and/or BLK as disclosed herein act as markers/predictors for susceptibility to dasatinib.
  • a responder for or a patient sensitive to dasatinib may be identified in accordance with the present method.
  • the present invention provides the possibility to recognize (aberrant) changes of SRC, EPHA3. FRK, EPPIA5, EPHA8. YES, ABL2. LCK and/or BLK activity immediately once the ⁇ ' occur, for example, by determining the activity of said marker gene(s).
  • the assessment/evaluation/detection of the amplification status of any of the above marker genes is sufficient for determining whether a patient is likely to respond to or is sensitive to dasatinib. whether a (mammalian tumor or cancer) cell is likely to respond or is responsive to treatment with dasatinib.
  • the assessment/evaluation/detection of the amplification status of any of the above marker genes (and their combinations) is also sufficient for diagnosing sensitivity to dasatinib.
  • the amplification status alone of any of the above marker genes is indicative for a sensitivity/responsiveness to dasatinib and the expression level/activity of the gene products of the above marker genes need not be determined in addition to the amplification status.
  • LCK BLK may not only be determined by measuring the expression level but also, be determined, for example, by measuring substrate turnover of SRC, EPHA3. FRK. EPHA5, EPHA8, YES. ABL2, LCK. This may be particularly useful in methods described herein for diagnosing non- small cell lung cancer, monitoring non-small cell lung cancer or predicting the efficacy of a treatment of cell lung cancer. Means and methods for determining the activity of said proteins are well known in the art and may. for example, be deduced from Lottspeich (Spektrum Akademischer Verlag, 1998).
  • determining the activity may comprise determining the expression level
  • the expression status i.e. expression of gene products such as mRNA and/or proteins
  • SRC expression status
  • LCK and/or BLK can be determined by standard techniques. Amplification of SRC, EPHA3, FRK, EPH A5, EPHAS 5 YES, ABL2. LCK and/or BLK gene does not necessarily correlate with a change in the expression level of these genes. In context of the present invention it is therefore preferred that the activity of (amplified) SRC. EPHA3, FRK, EPHA5, EPHA8, YES.
  • ABL2, LCK and/or BLK is not determined by measuring the expression status of these genes but by alternative methods which reflect, for example, the enzymatic activity of the corresponding proteins. It is of note that, for example, the enzymatic activity of proteins encoded by a (amplified) SRC, EPHA3. FRK, EPHA5, EPHA8, YES, ⁇ BL2, LCK and/or BLK may differ from that of the respective proteins encoded by wild type genes (i.e. reference activity) without a change in the expression level. Preferably, (amplified) SRC. EPHA3, FRK.
  • EPHA5, EPFIA8, YES, ABL2, LCK and/or BLK show an increased activity compared to non-amplified (amplified) SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK (controls).
  • the enzymatic activity and/or the expression level described herein above are increased.
  • Exemplary ranges of changes in the activity compared to ''normal" activity i.e. activity of (non-amplified) SRC, EPHA3.
  • FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK are described herein below.
  • 'non-amplified refers to the ''normal" amplification status, i.e. two gene copies, whereas "amplified” refers to an amplification status above normal, i.e. at least three copies of the respective gene.
  • the present invention provides the particular advantages that, by determining the amplification status of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK in accordance with this invention, (aberrant) changes of (amplified) SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK activity/expression level can be recognized early, i.e. that the efficacy of a treatment NSCLC can be monitored early and that the efficacy of a treatment of said cancer can be predicted early. Hence, also a possible resistance to the treatment can be recognized early by using the means and method of this invention.
  • the present invention relates to corresponding means, methods and uses which are based on the early recognition of (aberrant) changes of (amplified) SRC, EPHA3. FRK. EPHA5, EPHA8, YES, ABL2, LCK and/or BLK activity/expression level of the respective genes.
  • the possibility of recognizing (aberrant) changes of (amplified) SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK activity/expression level early provides several advantages, like a higher lifespan/likelihood of survival of the subject/patient (for example due to the notice of possible treatment failures and a corresponding change of the treatment regimen) and the possibility of a more efficient therapy (for example due to the possibility to avoid/recognize treatment failures early and. hence, to correspondingly change the treatment regimen early in therapy, i.e. to timely switch to a more suited inhibitor, to discontinue an expensive, ineffective treatment early after diagnosis and to opt for alternative therapy) .
  • '"early particularly means prior to (the onset of) a (complete or partial) cytogenetic or haematological response or a response measured by any type of imaging technique and/or prior to the outbreak of NSCLC (or susceptibility th ereto ) .
  • "'early" monitoring the efficacy of a therapy /treatment of said cancer may be at least 1. at least 2. at least 3, at least 4, at least 5. at least 6. at least 7, at least 10, or at least 14 days prior to (the onset of) a (partial) cytogenetic or haematological response or a response measured by any type of imaging technique to said therapy/treatment and/or at least 1. at least 2. at least 3. at least 4. at least 5 5 at least 6, at least 7, at least 10, at least 12, at least 15, or at least 18 month prior a complete cytogenetic or haematological response or a response measured by any type of imaging technique to said therapy/treatment (of the patient or control patient (responder)), wherein the longer periods are preferred.
  • ''early' ' monitoring the efficacy of a therapy/treatment of said cancer may also be at most 1 , at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 10. or at most 14 days after (onset of) the therapy /treatment of said cancer, wherein the shorter periods are preferred.
  • EPHA5, EPHA8, YES, ABL2, LCK and/or BLK activity /expression level changes upon said therapy/treatment.
  • the reference activity/expression level may be taken at the day the therapy/treatment is initiated, from the subject/patient to be treated and/or from a corresponding control subject/patient (responder/non-responder); see below.
  • '"early 1" predicting the efficacy of a therapy/treatment of the cancer defined herein may be at least 1, at least 2. at least 3, at least 4. at least 5, at least 6, at least 7, at least 10, or at least 14 days prior to (the onset of) a (partial) cytogenetic or haematological response to said therapy/treatment and/or at least 1, at least 2. at least 3. at least 4, at least 5, at least 6. at least 7, at least 10, at least 12, at least 15, or at least 18 month prior a complete cytogenetic or haematological response or a response measured by any type of imaging technique to said therapy/treatment, wherein the longer periods are preferred.
  • "early " ' predicting the efficacy of a therapy/treatment of the cancer defined herein may also be at most 1, at most 2. at most 3, at most 4. at most 5, at most 6, at most 7, at most 10, or at most 14 days after (onset of) the therapy/treatment of the cancer defined herein, wherein the shorter periods are preferred. Most preferably, it is envisaged to already monitor the efficacy of a therapy/treatment of said cancer at the day the therapy/treatment was initiated, i.e. once the (amplified) SRC 5 EPHA3, FRK. EPHA5, EPHA8. YES, ABL2, LCK, and/or BLK activity/expression level changes upon said therapy/treatment.
  • "early" predicting the efficacy of a therapy /treatment of the cancer defined herein may also be at most L at most 2, at most 3, at most 4, at most 5, at most 6, at most 7. at most 10. or at most 14 days after diagnosis of the cancer, wherein the shorter periods are preferred. Most preferably, it is envisaged to already predict the efficacy of a therapy/treatment of said cancer at the day of diagnosis.
  • the present invention is particularly useful for monitoring the efficacy of a therapy/treatment of the cancer as defined herein.
  • Corresponding means, uses and methods are provided herein.
  • monitoring the efficacy of a certain kind of therapy/treatment is regularly applied in clinical routine.
  • the skilled person is aware of the meaning of monitoring the efficacy of a certain kind of therapy/treatment.
  • the meaning of the term “monitoring " ' encompasses the meaning of terms like "tracking", '"discovering " ' etc..
  • monitoring the efficacy of a therapy/treatment of NSCLC refers to monitoring whether a subject/patient suffering from said disorder (or being prone to suffering from said cancer) responds at all to a therapy/treatment of said disorder and/or how the course of said respond is (e.g. how fast/slow the respond is and/or to what extent the respond is).
  • the present invention is further useful for predicting the efficacy of a therapy/treatment of the cancer as defined herein.
  • predicting the efficacy of a certain kind of therapy/treatment is highly desired in clinical routine, since it allows for preventing the disorder and/or increasing the efficiency of a therapy/treatment and hence, leads to savings in cost and time and to a higher lifespan/likelihood of survival or of 'Genesung' of the affected patient.
  • the definitions given with respect to the term "efficacy of a therapy/treatment of NSCLC * provided herein apply here, mutatis mutandis.
  • the term "predicting the efficacy of a therapy/treatment of NSCLC for a subject/patient” is used in basically the same sense like determining whether, and/or to what extent, a subject/patient exhibits susceptibility to such therapy/treatment, i.e. whether said subject/patient will or would respond at all to a therapy/treatment of said disorder and/or how the course of said respond will or would be (e.g. how fast/slow the respond is and/or to what extent the respond is).
  • a subject/patient exhibits susceptibility to said cancer in accordance with this invention, when its (amplified) SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK activity/expression level is aberrant.
  • said "amplification” is already “aberrant” as defined herein.
  • the '"predicting the efficacy of a therapy/treatment of the cancer defined herein" in accordance with this invention may be performed after initiation of the therapy/treatment, i.e. during the already ongoing therapy/treatment.
  • said "predicting" may be performed during the herein described monitoring the efficacy of a therapy/treatment of said cancer, preferably early after the beginning of said monitoring.
  • the predicting may be based on results from said monitoring obtained at a certain point in time of the ongoing therapy/treatment.
  • said point in time is an early point in time, like, for example that point in time, when a first result from said monitoring has been obtained.
  • the "predicting the efficacy of a therapy/treatment of the cancer defined herein" is performed during an already ongoing therapy/treatment, it refers to the following/subsequent efficacy of said therapy/treatment.
  • the "predicting the efficacy of a therapy/treatment of the cancer defined herein" in accordance with this invention may be performed (immediately) after diagnosis but, however, prior to initiation of the therapy/treatment.
  • "predicting the efficacy of a therapy/treatment of said cancer” refers to the efficacy of a therapy/treatment which has not yet been initiated (or has been initiated substantially at the same point in time when the "predicting" w r as performed.
  • one non-limiting example of a healthy control subject/patient is one having (a) non-amplified SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK gene(s). This is in contrast to an amplification leading to an aberrant activity/expression of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK, and/or BLK.
  • the amplification of said marker genes as provided herein is considered aberrant.
  • the "reference activity" of SRC is one having (a) non-amplified SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK gene(s).
  • EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK, and/or BLK or the "reference expression level" of said marker gene(s), with respect to the means, methods and uses of monitoring the efficacy of a treatment of a cancer defined herein, is that "reference activity/reference expression level" determined in (a sample of) the corresponding healthy control subject, i.e. is the "normal" activity/expression level.
  • control subject/patient is, in one embodiment, envisaged to be a subject/patient suffering from said cancer or being prone to suffering from said cancer, i.e.
  • a subject/patient having, for example, an aberrant activity/expression level of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK and, hence, not a "normal” activity or "normal expression level" of SRC, EPHA3, FRK, EPHA5, EPPIA8, YES, ABL2, LCK, and/or BLK as described in accordance with this invention.
  • “different” means and comprises “higher” or “lower”, depending on whether the cancer defined and described herein comes along with an up- or down-regulated activity of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK.
  • “different”, “higher” or “lower” means different, higher or lower than the normal (range of) activity of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or
  • “higher” or “lower” means different, higher or lower than the normal (range of) expression level of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK.
  • different, higher or lower means at least 1,5 fold, at least 2 fold, at least 2,5 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 7 fold, at least 10 fold, at least 15 fold, at least 25 fold, at least 50 fold, at least 100 fold, at least 200 fold different, higher or lower, wherein the higher values are preferred. Whether, in which direction (i.e.
  • LCK and/or BLK differs from Its corresponding reference activity/expression level of SRC, EPHA3, FRK, EPHA5. EPHA8. YES.
  • ABL2, LCK and/or BLK can easily be deduced by the skilled person based on the teaching provided herein and the common general knowledge.
  • control subject/patient is subjected to the same treatment of the cancer subject/patient described and defined herein.
  • Said control subject/patient may be a responder (positive control) or non-responder (negative control) to this treatment.
  • a subject/patient is a "'responder * ' or "non-responder " ' with respect to a certain kind of cancer treatment/therapy can be evaluated by the skilled person on the basis of his common general knowledge and/or the teaching provided herein.
  • a ''responder may be a subject/patient whose cytological/haemato logical parameters and/or (aberrant) SRC, EPHA3, FRK, EPHA5.
  • EPHA5, EPHA8, YES. ABL2, LCK and/or BLK change towards the their "normal" activity/(expression) level(s) (in a sufficient manner) upon the cancer treatment/therapy
  • a "'responder” may be a subject/ patient not suffering from one of the herein defined resistances.
  • a "'non-responder " ' may be a subject/patient whose cytological/haematological parameters and/or (aberrant) activity/expression level of SRC. EPHA3.
  • a '"non-responder may be a subject/patient suffering from one of the herein defined resistances.
  • the patient responds to cancer treatment/therapy, if the activity/expression level of SRC. EPHA3, FRK. EPHA5, EPHA8. YES, ABL2, LCK and/or BLK is reduced upon said treatment/therapy.
  • the expression/ activity of SRC. EPHA3. FRK, EPFIA5, EPHA8. YES, ABL2, LCK and/or BLK is reduced to control expression/activity (e.g. determined in a sample obtained from a person not suffering from said cancer).
  • a reduction in expression/ activity of SRC, EPHA3, FIOC, EPHA5, EPHA8. YES, ABL2. LCK and/or BLK is indicative for a successful treatment/therapy.
  • a skilled person is readily in the position to determine whether a patient responds to cancer treatment/therapy by evaluation of the expression/ activity of SRC.
  • a person skilled in the art may also determine cytological/haematological parameters characteristic for a specific cancer in order to assess whether a patient responds to cancer treatment/therapy.
  • a patient who does not respond to cancer treatment/therapy does not show a reduced expression/ activity of SRC, EPHA3.
  • one non-limiting example of a diseased control subject/patient (responder and/or non-responder) suffering from a cancer defined herein or being prone to suffering from a susceptibility thereto is one having an amplified SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK leading to an aberrant SRC.
  • EPHA5, EPHA8. YES, ABL2. LCK and/or BLK are examples of a diseased control subject/patient (responder and/or non-responder) suffering from a cancer defined herein or being prone to suffering from a susceptibility thereto.
  • the skilled person is aware of how a typical/desired response to a known therapy/treatment of NSCLC should proceed or is intended to proceed. Moreover, the skilled person can consider how a typical/desired response to a (unknown) therapy/treatment of a NSCLC proceeds or is intended to proceed. Based on this knowledge, the means, methods and uses of this invention referring to the efficacy of a therapy/treatment of such a cancer can. for example, also be carried out without employing (a sample of) a particular control subject/patient, i.e. without comparing the activity or expression level of SRC. EPHA3, FRK, EPHA5, EPHA8.
  • the subject/patient is a " 'responder". If the response of a subject/patient is slower than the "typical/desired response", the subject/patient is a "non- responder" (when no substantial response can be seen) or "weak-responder".
  • the efficacy of a cancer treatment/therapy can be determined taking account of the change in the activity/expression level of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK during the treatment/therapy.
  • a skilled person is able to assess the efficacy of a treatment by evaluating the activity/expression level of the above marker genes at various points in time during the treatment (e.g. prior to the treatment, after start of the treatment, and subseqently in intervals during the treatment). In this particular case, it may not be necessary to compare the activity/expression level with reference values (control values) as indicated above in order to assess the efficacy of the treatment. Instead it may suffice to detect the change in the activity/expression level of the marker genes in samples obtained from a treated patient after start of the treatment.
  • a (desired) efficacy of a treatment of a cancer described herein or susceptibility thereto is indicated/predicted, when the aberrant (Le, enhanced or decreased) activity or expression level of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK is shifted back towards the "normal level” of a (healthy)control subject/patient or to "normal level” of a defined responder ("positive control") due to/in consequence of said treatment of the cancer or susceptibility thereto.
  • the efficacy of a treatment of the cancer defined herein is high, when the subject/patient (to be) treated responds as fast (or even faster) and as complete as a "responder", i.e. exhibits a 'typical/desired response".
  • the subject/patient reaches the "normal" level of the relevant cytological/haematological parameters and/or SRC, EPHA3, FRK, EPHA5, EPHA8, YES 5 ABL2, LCK and/or BLK activity (and hence of the coiTcsponding marker gene expression level(s)) of a healthy subject/patient as fast as a "responder", i.e. in the same manner as in a "typical/desired response".
  • the efficacy of a treatment of the cancer is high, if the patient treated shows a "typical/desired response".
  • the efficacy is high, when the activity/expression level of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK in said patient reach a "normal” acivity/level as rapidly as in a "typical/desired response".
  • the efficacy of a treatment of the cancer defined herein is moderate/low, when the subject/patient (to be) treated responds not as fast and/or not as complete as a "responder" ' , i.e. does not exhibit a "typical/desired response " .
  • said subject/patient does not reach the ''normal" level of the relevant cytological/haematological parameters and/or activity of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2 5 LCK and/or BLK (and hence of the corresponding marker gene expression level(s)) of a healthy subject/patient as complete and/or as fast as a "responder", i.e. not in the same manner as in a "typical/desired response".
  • the efficacy of a treatment of the cancer is low, if the patient treated does not show a ''typical/desired response".
  • the efficacy is low, when the activity/expression level of SRC, EPHA3. FRK, EPHA5, EPHA8, YES. ABL2. LCK and/or BLK in said patient reaches a "norma!' " activity/level slower than in a "typical/desired response".
  • EPHA8, YES 5 ABL2, LCK and/or BLK of a "control subject/patient'” can be replaced by a '"own" reference activity or expression level sample of SRC.
  • Such an "own' " reference sample may be obtained prior to (or at the beginning of) the treatment/therapy.
  • the '"control subject/patient would be the subject/patient to be treated itself.
  • the efficacy of the cancer treatment would then be assessed on the basis of how the activity or expression level of SRC, EPHA3, FRIC 5 EPHA5.
  • the efficacy of a treatment of the cancer is assessed in accordance with specific embodiments of this invention, on the basis that the activity/expression level of SRC.
  • FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK is different from a certain or given '"reference activity/reference expression level" of SRC, EPHA3.
  • EPHA8, YES, ABL2, LCK and/or BLK EPHA8, YES, ABL2, LCK and/or BLK.
  • the efficacy of a treatment of the cancer is assessed based on the comparison of the activity/ expression level of SRC, EPHA3, FRK 5 EPHA5, EPHA8, YES, ABL2, LCK and/or BLK in a sample obtained from a patient with a reference (control) activity/expression level.
  • '"different", ''higher” or “lower” means different, higher or lower than the normal (range of) activity/expression level of SRC.
  • LCK and/or BLK means at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 7 fold, at least 10 fold, at least 15 fold, at least 25 fold, at least 50 fold, at least 100 fold or at least 200 fold different, higher or lower, wherein the higher values are preferred.
  • a certain type of cancer can be associated with increased activity/expression level of any one of the above marker genes or with a decreased increased activity/expression level of any one of the above marker genes. Since a skilled person will be aware of reference acitivity/expression levels of the marker genes (e.g. in a healthy person), he will be readily in the position to determine whether the activity/expression level of any one of the above marker genes is increased or decreased when compared to the reference acitivity/expression level.
  • control subject/patient is a responder.
  • activity/expression level of SRC, EPHA3, FRK, EPHA5, EPHAS. YES, ABL2, LCK and/or BLK is evaluated on the basis of a 'typical/desired response " , a low difference (at a certain point in time) indicates a high efficacy.
  • a responder shows expression/ activity of SRC, EPHA3.
  • EPHA5, EPHA8, YES, ABL2, LCK and/or BLK similar to a typical/desired response, wherein a typical/desired response Is indicative for a successful cancer treatment/therapy.
  • a responder may show reduced or increased expression/ activity of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2. LCK and/or BLK 5 depending on the type of cancer.
  • the cancer is, for example, characterised by a high expression/activity of at least one of the marker genes and if expression/ activity of SRC.
  • FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK, is reduced in a responder to a similar extent as in a typical/desired response, the cancer treatment/therapy can be considered successful.
  • the cancer treatment/therapy can be considered successful if the cancer is characterised by a low expression/activity of at least one of the marker genes and if expression/ activity of SRC, EPHA3, FRK 5 EPHA5, EPHA8, YES, ABL2, LCK and/or BLK, is increased in a responder to a similar extent as in a typical/desired response, the cancer treatment/therapy can be considered successful.
  • control subject/patient is a non-responder
  • activity/expression level of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK is evaluated on the basis of a reference activity/reference expression level of SRC, EPHA3, FRK, EPFIA5, EPHA8, YES, ⁇ BL2, LCK, and/or BLK obtained form the subject to be treated prior to/at the beginning of a therapy/treatment of NSCLC, a high difference (at a certain point in time) indicates a high efficacy.
  • a control sample can be obtained from a non-responder or can be obtained prior to/at the beginning of a therapy /treatment of a cancer. Accordingly, if the difference between expression/ activity of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK in a responder (or similarly in a typical/desired response) compared to said control is high, such a high difference indicates a successful treatment/therap ⁇ * (i.e.
  • a responder shows a reduced expression/ activity of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK compared to high expression/ activity of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK in a control, when the cancer is associated with such a high expression/activity.
  • the difference between the expression/ activity of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK in a responder and the control should be high.
  • EPHA8, YES 5 ABL2, LCK and/or BLK may be taken at the day of diagnosis, once the therapy/treatment is initialed, in between and/or during therapy/treatment, either from the subject/patient to be treated itself or from a corresponding control subject/patient (healthy/re sponder/non ⁇ responder).
  • EPHA5, EPHAS 5 YES 5 ABL2, LCK and/or BLK may be determined at the same or at a different point in time than the activity/expression level of
  • SRC 5 EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK, for example with respect to the course of the therapy/treatment.
  • ABL2, LCK and/or BLK can be determined in a control sample obtained from a patient (healthy tissue) or healthy person at the same time or at a different time when the cancer sample is obtained from said patient.
  • the reference activity/reference expression level of SRC. EPHA3, FRK, EPHA5. EPHA8, YES, ABL2, LCK and/or BLK is obtained from a control subject/patient different from the subject/patient to be treated, it is preferred that the reference activity/reference expression level of SRC. EPHA3, FRIC. EPHA5. EPHA8. YES, ABL2. LCK and/or BLK is determined at the same point in time during therapy/treatment.
  • the reference activity/reference expression level of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK is obtained from the subject/patient to be treated itself, the reference activity/reference expression level of SRC, EPHA3, FRK, EPHA5. EPHAS 5 YES. ABL2. LCK and/or BLK should be determined at a different point in time during therapy/treatment to allow comparison, for example, at the beginning of (or prior to) the therapy/treatment.
  • activity/expression level of SRC 5 EPHA3. FRK. EPHA5, EPHA8, YES. ABL2. LCK and/or BLK as described herein can be determined once or. preferably, several times. For example, activities/expression levels of SRC. EPHA3, FRK, EPHA5. EPHAS 5 YES, ABL2, LCK. and/or BLK can be determined on a daily, weekly, monthly or yearly basis during therapy/treatment. Commonly, the requirements of corresponding studies would be met, if the frequency of determining activity/expression level of SRC 5 EPHA3, FRK, EPHA5, EPHA8, YES. ABL2, LCK and/or BLK decreases during process of therapy/treatment.
  • Non- limiting examples of schemes of determining activities/expression levels of SRC, EPHA3, FRK, EPFIA5, EPHA8, YES, ABL2, LCK and/or BLK in accordance with this invention are provided herein.
  • the present invention relates to the use of a (transgenic) cell or a (transgenic) non-human animal having at least one amplified marker gene as defined herein for screening and/or validation of a medicament for the treatment of non-small cell lung cancer.
  • the term "cell" as used in this context may also comprise a plurality of cells as well as cells comprised in a tissue.
  • a cell to be used may, for example, be a primary tumor cell.
  • the tumor cell or cell to be used in the screening or validation method may be obtained from samples from a (transgenic) non-human animal suffering from non-small cell lung cancer.
  • the tumor cell or cell may also be obtained from patient samples (e.g.
  • the tumor cell or cell may be a human tumor cell or cell.
  • such a cell to be used in the present screening or validation methods may be comprised in a tissue or tissue sample, like in a sample biopsy.
  • the used non-human animal or cell may be transgenic or non transgenic.
  • Transgenic in this context particularly means that at least one of the marker genes as described or defined herein is over- or under-expressed or has a higher or lower activity. For example, if dasatmib is to be screened and/or validated, it is preferred that such marker genes as SRC. EPHA3, FRK. EPHA5, EPHA8, YES, ABL2, LCK and/or BLK are over-expressed or have a higher activity.
  • Transgenic in this context may also mean that SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK is over- or under-expressed, and/or that the SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK-activity in the transgenic non-human animal or a transgenic cell is enhanced or decreased. It is also envisaged in this context that SRC 5 EPHA3, FRK. EPHA5. EPHA8, YES, ABL2.
  • LCK and/or BLK is under-expressed, and/or that the SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK-activity in the transgenic non-human animal or a transgenic cell is decreased.
  • a preferred (transgenic) non-human animal or (transgenic) cell in context of the invention suffers from NSCLC for the treatment of which the medicament is to be screened and/or validated.
  • the (transgenic) non-human animal or (transgenic) cell is particularly intended to suffer from non-small lung cancer, i.e. to have, for example, an increased SRC, EPHA3, FRK. EPHA5.
  • EPHA8. YES, ABL2, LCK and/or BLK activity and/or increased expression level of, for example, SRC. EPHA3. FRK, EPHA5. EPHA8. YES 5 ABL2, LCK and/or BLK.
  • transgenic non-human animal or “transgenic cell” as used herein refers to a non- human animal or cell, not being a human, that comprises genetic material different from the genetic material of a corresponding wild-type animal/cell
  • ""Genetic material " ' in this context may be any kind of a nucleic acid molecule, or analogues thereof, for example a nucleic acid molecule, or analogues thereof as defined herein.
  • "Different' ' in this context means additional or fewer genetic material with respect Io the genome of the wild-type animal/cell and/or rearranged genetic material, i.e. genetic material present at a different locus of the genome with respect to the genome of the wild-type animal/cell.
  • the (transgenic) non-human animal or (transgenic) cell is or is derived from a mammal.
  • Non-limiting examples of the (transgenic) non-human animal or derived (transgenic) cell are selected from the group consisting of a mouse, a rat, a rabbit, a guinea pig and a Drosophila.
  • the (transgenic) cell in accordance with this invention may be an animal cell, for example, a non-human animal cell.
  • human cells are envisaged to be employed as cells in context of the present invention.
  • such cell may be an embryonic stem cell (ES cell), particularly a non-human animal ES, like, for example, a mouse or rat ES cell.
  • the (transgenic) cell as described herein may also be used for generating the (transgenic) non-human animal as described herein.
  • the ES cell technology for generating transgenic animals is well known in the art and for example is described in Pirity et.al.( Methods Cell Biol, 1998, 57:279).
  • the (transgenic) cell may be a prokaryotic or eukaryotic cell.
  • the (transgenic) cell may be a bacterial, yeast, fungus, plant or animal cell.
  • the transformation or genetically engineering of a cell with a nucleic acid construct or vector can be carried out by standard methods, as for instance described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor. NY 5 USA; Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 1990.
  • the (transgenic) non-human animal or (transgenic) cell as described or defined in context of this invention is particularly useful in methods for screening and/or validation of a medicament for the treatment of cancers as defined and described herein.
  • These screening methods may, in particular, performed in vivo using, for example, (transgenic) animals as described herein (e.g. rats, mice and the like) and/or animals comprising (a) NSCLC cell(s), (a) tissue(s) or (a) ceil culture(s).
  • Said (a) cell(s), (a) tissue(s) or (a) cell culture(s) may, for example, be obtained/derived from (a) NSCLC tumor cell(s)/tumor(s).
  • said (a) cell(s), (a) tissue(s) or (a) cell culture(s) may be obtained from a subject/patient suffering from a NSCLC.
  • These in vivo screening methods may in particular comprise measuring and determining differences in tumor volume, for example, in the (transgenic) animals described herein above.
  • the present invention also relates to a method for screening and/or validation of a medicament for the treatment of a cancer.
  • Said method may comprise the steps of a) administering to a (transgenic) non-human animal or (transgenic) cell as defined herein said medicament to be screened/validated; b) determining in (a sample from) said animal or cell the activity or expression level of SRC, EPHA3, FRK, EPHA5. EPHA8, YES.
  • LCK and/or BLK gene in accordance with this invention c) comparing the activity or expression level of said at least one marker gene determined in b) with a reference activity or reference expression level of SRC, EPHA3, FRK, EPHA5, EPHA8, YES.
  • ABL2, LCK and/or BLK said activity or said expression level optionally determined in (a sample from) a control (transgenic) non-human animal or (transgenic) cell as defined herein to which said medicament to be screened has not been administered; and d) selecting said medicament when said activity/expression level of SRC.
  • LCK and/or BLK determined in b) differs from said reference activity/expression level determined in c).
  • “'screening and/or validation of medicaments” means, on the one hand, whether a given set of compounds comprises one or more compound(s) that can function as (a) medicament(s). and/or, on the other hand, whether (a) given compound(s) can function as (a) medicament(s). It is particularly intended that the medicaments to be screened and/or ⁇ alidated in context of this invention are medicaments for the treatment, prevention and/or amelioration of a cancer as defined herein.
  • the compound(s)/medicament(s) to be screened and/or validated may be administered to the non-human (transgenic) animal or cell described herein, and. afterwards (for example after a certain period of time sufficient to allow a compound to effect on a cancer as described herein), it is analyzed whether the cancer, or a symptom thereof, of said animal/cell is ameliorated.
  • the present invention also relates to a kit useful for carrying out the method or used of this invention.
  • said kit comprises oligonucleotides or polynucleotides capable of detecting the amplification status of at least one gene selected from the group of SRC, EPHA3, FRK 3 FPHA5 EPH A8. YES. ABL2. LCK and BLK.
  • said kit may comprise (a) compound(s) required for specifically determining the amplification status of at least one gene of SRC. EPHA3, FRK. EPHA5. EPHA8, YES, ABL2. LCK and/or BLK.
  • the kit (to be prepared in context) of this invention is a diagnostic kit.
  • the kit (to be prepared in context) of this invention or the methods and uses of the invention may further comprise or be provided with (an) instruction manual(s).
  • said instruction manual(s) may guide the skilled person (how) to determine amplification status of at least one gene of SRC, EPHA3, FRK. EPHA5, EPHA8, YES. ABL2, LCK and/or BLK., i.e. (how) to diagnose the susceptibility to dasati ⁇ ib.
  • said instruction manual(s) may comprise guidance to use or apply the herein provided methods or uses.
  • the kit (to be prepared in context) of this invention may further comprise substances/chemicals and/or equipment suitable/required for carrying out the methods and uses of this invention.
  • substances/chemicals and/or equipment are solvents, diluents and/or buffers for stabilizing and/or storing (a) compound(s) required for specifically determining the amplification status of at least one gene of SRC, EPHA3.
  • FRK. EPHA5, EPHA8. YES. ABL2, LCK and/or BLK are solvents, diluents and/or buffers for stabilizing and/or storing (a) compound(s) required for specifically determining the amplification status of at least one gene of SRC, EPHA3.
  • FRK. EPHA5, EPHA8. YES.
  • ABL2, LCK and/or BLK are solvents, diluents and/or buffers for stabilizing and/or storing (a) compound(s) required for specifically determining the amplification status of at least one gene
  • the present invention also relates to a combination of cell lines selected from the group consisting of A427, A549, CaIu-I, Calu-3. Calu-6. H1299, H1355, H1395. H1437, H1563. H1568, H1648, H1650, H1666. H1734. H1755. H1770, H1781, H1792. H1819, H1838, H1915. Hl 944. H1975. Hl 993. H2009, H2030, H2052.
  • combinations of cell lines should comprise at least 3, 4, 5, 6. 7. 8 5 9, 10, 11, 12, 13. 14, 15, 20, 25. 30, 35, 40, 45. 50. 55 or 60 of the cell lines as provided herein above.
  • combination of eel! lines should comprise at least 60 cell lines as provided herein above.
  • These cell lines, and in particular their combination are particular useful as model systems for the assessment of any potential drug susceptibility. This usefulness of these specifically selected cell lines (in combination) for the assessment of drug susceptibility is demonstrated in the appended example. All of the mentioned cell lines are available to the person skilled in the ait and the public from cell depositary institutions, in particular ATCC or DMSZ as illustrated in Figure 19 where also corresponding accession numbers for these cells are provided.
  • the combination of cell lines as defined herein above for predicting susceptibility to a drug, in particular, dasatinib is disclosed herein.
  • the combination of cell lines may be useful for predicting the susceptibility to a drug, in particular to dasatinib or responsiveness of a (mammalian) tumor cell or cancer cell to treatment with a drug, in particular dasatinib. It may also be useful in predicting whether a patient is likely to respond to or is sensitive to a drug, in particular dasatinib.
  • Corresponding means and methods for predicting susceptibility/responsiveness and the like are well known in the art and also described herein above. Accordingly, a skilled person will know how to use such a combination of cell lines in this context.
  • the present invention is further described by reference to the following non-limiting figures and examples.
  • FIG. 4 Profiles of aberrations in glioma, melanoma and lung cancer
  • A Chromosomal copy number changes of KSCLC cell lines are plotted against those of primary gliomas. Two separate figures are given for deletions (left panel, NSCLC cell lines in black, gliomas in grey) and amplifications (right panel, NSCLC cell lines in light black, gliomas in dark grey).
  • B Chromosomal copy number changes of NSCLC cell lines are plotted against those of primary melanomas (NSCLC cell lines in red respectively blue as above, melanoma short term cultures in purple).
  • Genomic similarity was analyzed by computing correlations of GlSTIC q-values for each SNP between NSCLC cell lines and the indicated cancer entity primary lung cancer, ovarian cancer, glioma, melanoma cell culture samples, normal tissues and a randomly split subset of NSCLC cell lines.
  • A Hierarchical clustering of primary lung adenocarcinomas was performed using genes identified as being differentially expressed in erlotinib-sensitive versus erlotinib-resistant NSCLC cell lines. Whitebars represent EGFR-mxAsnt tumors.
  • B The EGFR mutation signature published by Choi et al. PLoS ONE 2: el 226 (2007) was used to perform hierarchical clustering of primary lung adenocarcinomas. Red bars represent £GFi?-mutant tumors.
  • Multi-Jesion predictors of sensitivity tested with the KNN method Fisher's exact test and t- test are displayed. Only significant predictors are displayed for two different GLAD thresholds.
  • the half-maximal inhibitory concentrations (y-axis; IC 50 values) for 1 1 compounds are shown for the entire collection of NSCLC cell lines (individual cell lines, x-axis). Due to the fact that rapamycin typically fails to completely abrogate cellular proliferation (O'Reilly et al, 2006) v the 25%-inhibitory concentration is shown for this compound. Bars represent IC 50 respectively IC 25 values (y-axis) throughout the cell line collection (x-axis) ranked according to sensitivity. The maximum concentration is assigned to the IC 50 resp.
  • IC 25 value (10 ⁇ M for 17- AAG, erlotinib, vandetanib, sunitinib and PDl 68393, 30 ⁇ M for SU-11274 and dasatinib. 60 ⁇ M for VX-680, 90 ⁇ M for purvaianol and UO 126) for resistant cell lines.
  • EGFR inhibitors form a distinct subcluster, where EGFR-mutaXed samples show the highest degree of sensitivity.
  • Dasatinib represented as white to dark grey ballandsticks is modeled into the ATP binding site of EGFR.
  • lower panel The T790M mutation at the gatekeeper position of the ATP pocket, associated with secondary EGFR- inhibitor resistance in patients displaces both erlotinib and dasatinib from the ATP binding pocket of the kinase domain.
  • (D) Upper panel: Ba/F3 cells expressing mutant (del Ex 19 or Exl9/T790M) EGFR were treated for 12h with the indicated concentrations of either dasatinib or erlotinib and whole-ceil Iy sales were inimunoblotted for phospho-EGFR and EGFR.
  • Lower panel Dose-dependent growth inhibition after 96h treatment with either dasatinib or erlotinib was assessed measuring cellular ATP content.
  • FIG. 12 Mutated EGFR as a target for vandeta ⁇ ib activity.
  • Vandetanib represented as ballandsticks is modeled into the ATP binding site of EGFR based on its crystallographically determined binding mode with the RET kinase
  • right panel The T790M mutation at the gatekeeper position of the ATP pocket, associated with secondary EGFR-inhibitor resistance in patients displaces the drug from the ATP- binding pocket.
  • the crystographically determined binding mode of erlotinib asballandsticks is shown in both panels as an overlay for reference.
  • B Ba/F3 cells expressing mutant (del ExI 9 or Exl9/T790M) EGFR were treated for 12h with the indicated concentrations of either vandetanib or erlotinib and whole-ceil lysates were inimunoblotted for phospho-EGFR and EGFR.
  • C Dose-dependent growth inhibition after 96h treatment with either dasatinib or erlotinib was assessed measuring cellular ATP content.
  • FIG. 13 Lesion-based prediction for activity of 17- AAG, UO126 and dasatinib.
  • A Distribution of KRAS mutations (black columns) across the 17-AAG-sensitivity profile (IC 50 values) in the NSCLC cell line collection and the NCI-60 cell line panel. Incidence of KRAS mutation and sensitivity towards 17- A AG is represented by a Fisher's exact test for both datasets.
  • B Lysates of a KRAS wildtype (wt) and a KRAS mutated (Gl 2C) cell line treated with 17AAG at different concentrations were imraunoblotted for c-RAF, KRAS, cyclinDl and Akt.
  • C Distribution of copy number gain at 1 q21.3 (black columns) across the UO126-sensitivity profile (IC JO values) in the NSCLC cell line collection and the hypothemycin- sensitivity profile in the NCI-60 cell line panel. Incidence of amplification of Iq21.3 mutation and sensitivity towards 17-AAG is represented by a Fisher's exact test for both datasets.
  • D Cell lines were sorted according to their sensitivity to dasatinib (IC50 ⁇ l ⁇ M; light grey ). Strikingly, most dasatinib-sensitive NSCLC ceil lines are found among those with highest copy number for members of the SRC and Ephrin receptor kinase families.
  • Figure 14 Validation of the target-enriched sensitivity prediction method that yielded genomic predictors of dasatin ⁇ b sensitivity.
  • AU cell lines were sorted according to their sensitivity to erlotinib (IC 5 o ⁇ l ⁇ M; greybars). Cell lines enriched for sensitivity to erlotinib are found among those with highest copy numbers for EGFR.
  • the contingency table for EGFR (dark grey bars) amplification and erlotinib sensitivity including the p-vaiue determined using fisher ' s exact test are displayed in the right panel.
  • FIG. 16 Prostate/breast cancer -gene signature associated with dasatinib sensitivity
  • the expression levels of the respective genes were analyzed by hierarchical clustering with the dasatinib sensitivity denoted using the annotation by 0 and 1.
  • the samples with a similar expression profile across these genes are found in the same subcluster.
  • Bright spots represent genes that are repressed, dark spots represent genes that are overexpressed when compared to average mRNA expression levels.
  • Figure 17 Ail genes associated with dasatinib sensitivity in prostate cancer
  • Figure 18 shows a list of cell lines in the initially tested NSCLC cell line panel, their respective KRAS mutations and the corresponding half-maximal inhibitory concentrations (IC50).
  • Figure 19 shows a set of cell lines which are to be used in accordance with the screening methods provided herein for the identification of dings, which can be used in and -cancer treatment/anti-proliferative treatment.
  • mice expressing a Gl 2D mutation of KRAS specifically in the lung were generated by intranasal application of adenoviral Cre recombinase to Lox-Stop-Lox KRASG12D mice (upper panels from left to right) as described in the literature (Jackson. E. L. et al, Genes Dev 15:3243-3248 (2001 ): Johnson L. et al. Nature. 410(6832):! 1 11-6 (2001) and Ji H. et al.. Nature. 448 (7155):807-10 (2007)). These mice develop lethal lung adenocarcinomas at high penetrance (lower panels from left to right). Note that the images were taken from the ealier publications for illustration purposes only.
  • FIG. 21 Treatment of Los-Stop-Lox KRASG12D mice with HSP90 inhibitor Transgenic mice (as described in Figure 20) with KRAS-mutant lung tumors were treated with the HSP90 inhibitor 17-DMAG for seven days. Tumor volumes were determined by magnetic resonance imaging and shown as transthoracic images (left panels), quantified and changes in tumor volume relative to the pre-therapy images are given in percent.
  • the Example illustrates the invention.
  • Cells were obtained from ATCC (www.atcc.org), DSMZ (www.dsmz.de), from own or from other cell culture collections. Details on all cell lines are listed in Figure 1. This also contains information on providers and on culture conditions. Cells were routinely controlled for infection with my ooplasm by MycoAleit (www.cambrex.com) and were treated with antibiotics according to a previously published protocol (Uphoff and Drexler, 2005) in case of infection.
  • Genomic DNA was extracted from cell lines using the PureGene kit (www.gentra.com) and hybridized to high-density oligonucleotide arrays (Affymetrix. Santa Clara, CA) interrogating 238,000 SNP loci on all chromosomes except Y, with a median intermarker distance of 5.2 kb (mean 12.2 kb; www.affymetrix.com ).
  • Array experiments were performed according to manufacturer's instructions, SNPs were genotyped by the Affymetrix Genotyping Tools Version 2.0 software.
  • SNP array data of primary samples were obtained from the Tumor Sequencing Project (http://www.genome.gov/cancereequencing/).
  • GlSTiC Genomic Identification of Significant Targets in Cancer
  • the analysis was performed computing ratios of observed vs. expected co-occurrence frequency of individual lesions.
  • Hierarchical clustering of mutation data combined to a dichotomized version of quantitative copy number changes was performed using the reciprocal co-occurrence ratio as distance measure with average linkage method.
  • the adequate threshold for occurrence of copy number lesions depends on the overall level, of copy number alteration for that specific lesion, the sum of these ratios for three distinct thresholds was used.
  • genes EGFR, BRAF 1 ERBB2, PIK3CA, KRAS, TP53, STKIl, PTEN and CDKN2A were bidirectionally sequenced following PCR-amplificatioii of all coding exons.
  • Mutation detection for choice of appropriate therapy depending on the respective mutation has been further developed to compensate for the methodological issues connected with sequencing of tumor samples with high admixture of non-tumoral cells, in our laboratory we have therefore developed the following algorithm: if the tumor content of the tumor specimens is higher or equal than 70% estimated by conventional histomorphology, we have found Sanger dideoxy-chain-termination sequencing to be optimal in terms of cost-efficiency and sensitivity. However, when the tumor content is between 70% and 20% we have found conventional pyrosequencing as, for example, implemented in the Biotage instrument, to deliver higher sensitivity and specificity at acceptable costs.
  • next-generation sequencing as for example implemented in the Roche-454 sequencing system (Thomas et al, Nature Medicine JuI; 12(7): 852-5 2006), to be the most sensitive and accurate method in this setting. Together, this algorithm provides high sensitivity in all settings combined with maximum cost- efficiency.
  • Expression arrays Expression data were obtained using Affymetrix Ul 33A arrays from 54 of the cell lines. RNA extraction, hybridization and scanning of arrays were performed using standard procedures (Bhattacharjee et al, 2001 ). CEL files from U133A arrays were preprocessed using the dChip software. We compared the cell lines to primary lung cancer, renal cell carcinomas and lymphoma specimens as well as to the respective cell lines by hierarchical clustering. For comparison with expression profiles from further entities, we used lung cancer (Lu el a!. , 2006).
  • Erlotinib, vandetanib and sunitinib were purchased from commercial suppliers, dissolved in DMSO and stored according to manufacturer's instructions. Cells were plated into sterile microtiter plates using a Multidrop instrument (www.thermo.com) and cultured overnight. Compounds were then added in serial dilutions. Cellular viability was determined after 9 ⁇ h by measuring cellular ATP content using the CellTiter-Glo assay (www.promega.com). Plates were measured on a Mithras LB940 plate reader (www.bertholdtech.com). Half-maximal inhibitory concentrations were determined from the respective preimage under the kill curve, where the latter was smoothed according to the logistic function with the parameters appropriately chosen.
  • multi-lesion predictors of sensitivity were calculated using a KNN algorithm with a leave-one-out strategy (Golub el ai , 1999). where the same choice of samples was used as above for Fisher's exact test: For all but one sample, genetic lesions strongly discriminating between sensitive and resistant cell lines were selected and the KNN algorithm was based on these. The prediction was validated by the remaining left-out sample. The collection of features where this validation had best performance was taken as the best combined predictor to the respective compound.
  • EGFR cDNA was subcloned into pBabe-hygro vectors.
  • the most prevalent NSCLC-derived mutants http://www.sanger.ac.uk/genetics/CGP/cosmic/
  • site-directed mutagenesis Quiick-Change Mutagenesis XL kit; Stratagene, La Jolla, CA, USA
  • virus was packed and produced as previously described (Greulich et al, 2005).
  • Murine Ba/F3 cells were stably transduced with the retroviruses and after IL- 3 withdrawal, independently growing cells were chosen for further experiments.
  • the piperazine moiety of the inhibitor points out of the ATP site into the solvent while the 2-amino-thiazole forms two hydrogen bonds with the hinge region of the kinase (N 3 of the thiazole ring with the amide nitrogen of Met793 , Met318 in AbI) and the 2-amino hydrogen of dasatinib with O of Met793 (Met318 in AbI).
  • An additional hydrogen bond can form between the side chain hydroxy! of the gatekeeper Thr790 (Thr315 in AbI) and the amide nitrogen of the inhibitor.
  • the chloro-melhyl-phenyl ring of dasatinib binds into a hydrophobic pocket near the gatekeeper Thr790 and helix C and would clearly clash with the Met side chain of drug resistant EGFR- T790M.
  • I vandetanib, Nl of the quinazoline scaffold forms one key hydrogen bond to the backbone of the hinge region (Met793 in EGFR, Ala807 in RET kinase).
  • the brorao-fluoro-phenylamine moiety of vandetanib adopts a conformation similar to the ethynyl-phenylamine of erlotinib being close to the side chain of Thr790 in EGFR and Val804 in RET kinase.
  • Figures of the structures were prepared using PyMoI.
  • NP40 lysis buffer 50 mmol/L Tris-HCI (pH 7.4), 150 mmol/L NaCl. 1% NP40
  • Protein concentrations were determined using the Bicinchoninic Acid Protein Assay kit (www.piercenet.com) and equivalent amounts (40-60 ⁇ g) were subjected to SDS- PAGE on 12% gels, except where indicated. Western blotting was done as described previously (Shimamura et al, 2006).
  • Anti-EGFR, anti-phospho-EGFR (Tyr 1068 ) and anti-pAkt antibodies were purchased from Cell Signaling Technology (Beverly. MA).
  • Anti c-raf and anti-cyclin Dl antibody s were purchased from Santa Cruz.
  • Anti KRAS antibody was purchased from Merck. Results
  • NSCLC cell lines were collected from various sources ( Figure 1) and formed the basis for all subsequent experiments.
  • Cell lines were derived from tumors representing all major subtypes of NSCLC tumors, including adenocarcinoma, squamous-cell carcinoma and large- cell carcinoma.
  • Genomic Identification of Significant Targets in Cancer (GlSTIC) to statistically distinguish biologically relevant lesions from background noise (Beroukhim el al., 2007). This method assigns a statistical score to each chromosomal marker reflecting both the mean amplitude and frequency of alterations at a given locus within a data set.
  • HD homozygous deletions
  • LOH loss of heterozygosity
  • EGFR mutations define phenotypic properties of lung tumors in vitro aad in vivo
  • Activated oncogenes typically cause a transcriptional signature that can be used to identify tumors carrying such oncogenes (BiId et al, 2006; Lamb el al . 2003).
  • a transcriptional signature that can be used to identify tumors carrying such oncogenes.
  • EGFR inhibitors such as erlotimb
  • mutations in the EGFR gene Lynch el al, 2004; Paez et al, 2004; Pao et al. 2004.
  • To systematically identify genetic lesions associated with sensitivity to erlotinib we determined erlotinib sensitivity in all cell lines. Then, we analyzed the distribution of genetic lesions in sensitive compared to insensitive cell lines ( Figure 7) and further compared the mean sensitivity of cell lines with and without the respective genetic lesion. In both analyses, EGFR mutations were the best single-lesion predictor of erlotinib sensitivity ( Figure 5D, p ⁇ 0.0001).
  • UO 126 is a MEK inhibitor that also showed enhanced activity in a subset of the lung cancer cell line collection.
  • hypothemycin was used as a MEK inhibitor (Solit et al, 2006).
  • Example 2 Animal data show that mice with KRAS-driven lung adenocarcinomas are susceptible to treatment with an HSP90 inhibitor
  • mice genetically engineered to develop KRAS-driven lung adenocarcinomas are susceptible to treatment with an HSP90 inhibitor.
  • mice earn,' a Lox-Stop-Lox- KRAS_G12D gene.
  • adenoviral Cre Upon administration of adenoviral Cre by nasal inhalation, these mice develop lung cancers with high penetrance, leading to rapid death from the disease.
  • This mouse model therefore represents the most stringent and optimal model of KRAS-mutant human lung cancer and was therefore chosen for in vivo experiments; see Figure 20.
  • Mice received 20 mg/kg/d of 17-DMAG, a geldanamycin HSP90 inhibitor with almost identical structure as 17- AAG.
  • 17-DMAG a geldanamycin HSP90 inhibitor with almost identical structure as 17- AAG.
  • In vitro data confirmed that the biological effects seen with 17-AAG in the cell lines were identical to those seen with 17- DMAG (data not shown).
  • mice After only one week of treatment 2 of 3 mice showed dramatic regression of tumors as measured by MRI imaging; see Figure 21.
  • the third mice showed a slight but insignificant reduction of tumor burden, comparable to stable disease.
  • untreated mice invariably show rapid tumor progression and die quickly from disease.
  • the present invention refers to the following nucleotide sequences:
  • the present invention also provides techniques and methods wherein homologous sequences, and also genetic allelic variants and the like of the concise sequences provided herein are used. Preferably, such "variants' " are genetic variants.
  • SEQ lD No. 1 Nucleotide sequence of Homo sapiens v-abl Abelson murine leukemia viral oncogene homolog 2 (arg, Abelson-related gene) (ABL2), transcript variant c, mRKA (>gijl53266777MNM_001100108.11).
  • SEQ ID No. 2 Nucleotide sequence of Homo sapiens B lymphoid tyrosine kinase (BLK) 5 mRNA (>gi
  • EPH receptor A5 EPH receptor A5
  • transcript variant 1 mRNA (>gi
  • Nucleotide sequence of Homo sapiens v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) (SRC), transcript variant 1, mRNA (>g ⁇ 38202215
  • SEQ ID No. 9 Nucleotide sequence of Homo sapiens v-yes-1 Yamaguchi sarcoma viral oncogene homolog 1 (YESl), mRNA (>gi
  • SEQ ID No. 10 Nucleotide sequence of Homo sapiens v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), transcript variant b, mRNA (>gi
  • KRAS Kirsten rat sarcoma viral oncogene homolog
  • transcript variant b mRNA (>gi
  • Basso AD et aLAkt forms an intracellular complex with heat shock protein 90 (Hsp90) and Cdc37 and is destabilized by inhibitors of Hsp90 function. J Biol C hem 277: 39858-66 (2002).
  • Cappuzzo F et al. Epidermal growth factor receptor gene and protein and gefitinib sensitivity in non-small-cell lung cancer. J Nail Cancer Inst 97: 643-55 (2005). Chen CF et al, Molecular genetic evidence supporting a novel human hepatocellular carcinoma tumor suppressor locus at 13ql2.11. Genes Chromosomes Cancer 44: 320-8 (2005).
  • Greshock J et al. Cancer cell lines as genetic models of their parent histology: analyses based on array comparative genomic hybridization. Cancer Res 67: 3594-600 (2007).
  • Greulich H et al. Oncogenic Transformation by Inhibitor-Sensitive and -Resistant EGFR Mutants.
  • McDermott U et al. Identification of genotype -correlated sensitivity to selective kinase inhibitors by using high-throughput tumor cell line profiling. Proc Natl Acad Sci U S A 104: 19936-41 (2007).
  • Neve RM et al. A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell 10: 515-27 (2006). Nevins JR et al.. Mining gene expression profiles: expression signatures as cancer phenotypes. Nat Rev Genet 8: 601-9 (2007).
  • EGF receptor gene mutations are common in lung cancers from "never smokers” and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci US A U)U 13306-1 1 (2004).
  • Non-small-cell lung cancer and Ba/F3 transformed cells harboring the ERBB2 G77 ⁇ insV_G/C mutation are sensitive to the dual-specific epidermal growth factor receptor and ERBB2 inhibitor HKI-272. Cancer Res 66: 6487-91 (2006).

Abstract

The present invention relates to a method of selecting (a) cell(s), (a) tissue(s) or (a) cell culture(s) with susceptibility to dasatinib. Also a method for determining the responsiveness of a mammalian tumor cell or cancer cell to treatment with dasatinib is described herein. Furthermore, an in vitro method for the identification of a responder for or a patient sensitive to an dasatinib is disclosed and uses of an oligo- or polynucleotide capable of detecting (an) the amplification status of at least one gene selected from the group consisting of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and BLK gene are provided. The present invention also relates to a method of diagnosing non-small cell lung cancer and a method of monitoring the efficacy of a treatment of said cancer. In addition, a method of predicting the efficacy of a cancer treatment is described, in particular a non-small cell lung cancer. Also the use of a (transgenic) non-human animal or a (transgenic) cell having at least one amplified marker gene as defined herein for screening and/or validation of a medicament for the treatment of said cancer is described and a kit useful for carrying out the methods described herein is provided.

Description

Susceptibility to Dasatinib
The present invention relates to a method of selecting (a) cell(s). (a) tissue(s) or (a) cell culture(s) with susceptibility to dasatinib. Also a method for determining the responsiveness of a mammalian tumor cell or cancer cell to treatment with dasatinib is described herein.
Furthermore, an in vitro method for the identification of a responder for or a patient sensitive to an dasatinib is disclosed and uses of an oligo- or polynucleotide capable of detecting (an) the amplification status of at least one gene selected from the group consisting of SRC, EPHA3, FRX. EPHA5. EPHA8. YES, ABL2, LCK, and BLK gene are provided. The present invention also relates to a method of diagnosing non-small cell lung cancer and a method of monitoring the efficacy of a treatment of said cancer. In addition, a method of predicting the efficacy of a cancer treatment is described, in particular a non-small cell lung cancer. Also the use of a (transgenic) non-human animal or a (transgenic) cell having at least one amplified marker gene as defined herein for screening and/or validation of a medicament for the treatment of said cancer is described and a kit useful for carrying out the methods described herein is provided.
The dynamics of ongoing efforts to fully annotate the genomes of all major cancer types is reminiscent of that of the Human Genome Project. The analysis of somatic gene copy number alterations and gene mutations associated with cancer (both are here referred to as lesions) will thus provide the genetic landscape of human cancer in the near future. The medical implications of these endeavors are exemplified by the success of molecularly targeted cancer therapeutics in genetically defined tumors: the ERBB2/Her2-targeted antibody trastuzumab shrinks tumors in women with £ϋJ3J?2-amplified breast cancer (Slamon et al., 2001); the ABL/KIT/ PDGFR inhibitor imatinib induces responses in patients with chronic myeloid leukemia carrying the BCRJABL translocation (Druker et a!.. 2001 a; Druker et al., 2001b) as well as in gastrointestinal stromal tumors and melanomas bearing (see Hodi et al.. NEJM 2008) mutations in KIT or PDGFRA (Hemrich et al., 2003); finally, EGFR-mx\X<mi lung tumors are highly sensitive to the EGFR inhibitors gefitinib and erlotinib (Lynch et al., 2004; Paez et al., 2004; Pao et al, 2004). In most cases, such discoveries were made after the completion of clinical trials; and as yet no robust mechanism currently exists that permits systematic identification of lesions causing therapeutically relevant oncogene dependency prior to initiation of clinical trials involving patients.
Non-small cell lung cancer (NSCLC) is one of the two main types of lung carcinoma, non- small cell (80.4%) and small-cell (16.8%) lung carcinoma, the classification being based on histological criteria. The non-small cell lung carcinomas have a similar prognosis and similar management and comprise three sub-types: squamous cell lung carcinoma, adenocarcinoma and large cell lung carcinoma.
Squamous ceil lung carcinoma (31.1% of lung cancers) often starts near a central bronchus and commonly shows cavitation and necrosis within the center of the cancer. Adenocarcinoma (29,4% of lung cancers) mostly originates in peripheral lung tissue and is usually associated with smoking. Large cell lung carcinoma (10.7% of lung cancers) is a fast- growing form that develops near the surface of the lung. Common treatments of NSCLC include surgery, chemotherapy, and radiation therapy, fn particular, NSCLC is treated with adjuvant chemotherapy (i.e. chemotherapy after surgery).
Targeted therapies for NSCLC have also been developed. For example, gefitinib which targets the tyrosine kinase domain of EGFR (epidermal growth factor) is used in the treatment of NSCLC. Also Erlotinib. another tyrosine kinase inhibitor, has been shown to increase survival in lung cancer patients. The angiogenesis inhibitor bevacizumab (in combination with paclitaxel and carboplatin) is known to improve the survival of patients with advanced non-small cell lung carcinoma. Further drugs under evaluation in the treatment of NSCLC are cyclo-oxygenase-2 inhibitors, proteasome inhibitors and bexarotene. Also treatment of lung cancer with dasatinib (a BCR/ABL and Src family tyrosine kinases inhibitor) has been proposed: see Song et al.. (2006) Cancer Res 66 (1 1), 5542-5548. Dasatinib is also known as BMS-354825, a drug produced by Bristol-Myers Squibb and sold under the trade name Sprycel. Dasatinib is used in the treatment of patients with chronic myelogenous leukemia (CML) after imatinib treatment and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph4- ALL). It is also being assessed for use in metastatic melanoma. However, not all patients suffering from NSCLC may be effectively treated with the above- mentioned drugs, since a specific tumor entity (e.g "NSCLC"') may comprise various tumor types characterized by different genomic and/or transcriptional profiles. Therefore, even "same" tumors (tumors belonging to the same tumor entity, such as NSCLC) may not be amenable to treatment with the same drug/compound. Therefore, many patients with tumors belonging to the same tumor entity would profit from an identification of markers/predictors for susceptibility of the specific tumor type to (a) certain drug(s)/compound(s). For effective treatment of patients suffering from NSCLC, the identification prior to initiation/completion of clinical trials is desirable.
Cancer cell lines may be used in corresponding in vitro experiments for identification of drugs to which NSCLC tumors/tumor cell are susceptible; yet. the validity and clinical interpretability of these widely used models have been questioned. In addition, cell lines are frequently thought to be genomically disarrayed and unstable and therefore likely poorly representative of primary tumors. Furthermore, the genetic diversity of histopathologically defined classes of tumors is often substantial: e.g., the clinical tumor entity non-small cell lung cancer (NSCLC) comprises EGFR- and KRAS-irmtant lung adenocarcinomas as well as KRA S-mutant squamous-cell lung cancers. Thus, any representative pre-clinical model would need to capture the nature of lesions of primary tumors as well as their distribution in the histopathologically defined cohort.
Recent reports have credentialed the use of cancer cell lines in preclinical drug target validation experiments (Lin et al., 2008; McDermott et aL 2007; Neve et al.. 2006; Solit et al., 2006). However, these studies either employed cell line collections of mixed cancer lineage (Solit et al., 2006). were focused on large-scale genomic analysis of cancer cell line collections (Lin et al., 2008) or established high-throughput cell line profiling to define cell lines with exquisite inhibitor sensitivity without genomic analyses (McDermott et aL 2007). Thus, somatic genetic alterations (lesions) in cancer have been causally linked with response to targeted therapeutics as they frequently expose a specific dependence on activated oncogenic signaling pathways. However, no tools currently exist to systematically link such lesions to therapeutic vulnerability. Accordingly, an identification of compounds/drugs to which tumors are susceptible is often time-consuming and cost-intensive since these compounds/drugs may only be identified after completion of clinical trials. Thus, the technical problem underlying the present invention is the provision of means and methods for the evaluation of cells, in particular tumor cells, for their susceptibility or responsiveness to anti-cancer treatment,
The technical problem is solved by provision of the embodiments characterized in the claims.
Accordingly, the present invention relates to a method of selecting (a) cell(s). (a) tissue(s) or (a) cell cultτjre(s) with susceptibility to dasatinib, comprising the steps'
(a) evaluating the gene amplification status of at least one gene selected from the group consisting of SRC. EPHA3, FRK, EPHA5, EPHA8, YES. ABL2, LCK, and BLK in said cell, tissue or cell culture; and
(b) selecting (a) cell(s). (a) tissue(s) or (a) cell culture(s) with (a) gene amplification above normal of at least one of said genes.
The method may additionally comprise the steps (i) contacting said cell(s). tissue(s) or cell culture(s) with dasatinib; and (ii) evaluating viability of said cell(s), tissue(s) or cell culture(s) contacted with dasatinib. It is of note that steps (I) and (ii) may be performed prior to step (a) but also after step (a) or, optionally after step (b). Said steps (i) and (ii) may in particular serve as further experimental proof that the selected cell, tissue or cell culture that comprises (a) gene amplification is susceptible in its viability to dasatinib.
As used herein, the term "cell, tissue and cell culture" is not only limited to isolated cells, tissues and ceil cultures but also comprises the use of samples, i.e. biological, medical or pathological samples that consist of fluids that comprise such cells, tissues or cell cultures. Such a fluid may be a body fluid or also excrements and may also be a culture sample, like the culture medium from cultured cells or cultured tissues. The body fluids may comprise, but are not limited to blood, serum, plasma, urine, saliva, synovial fluid, spinal fluid, cerebrospinal fluid, tears, stool and the like.
Accordingly, the gist of the present invention lies in the fact that a method is provided that allows for the determination of the susceptibility of a given cell, tissue or cells in a tissue, (or a cell culture or individual cells in such a cell culture, or as will be explained below, (a) cell(s) in a biological/medical/palhological sample) for the anti-cancer or antiproliferative treatment with dasatinib. As detailed in the appended examples, it was surprisingly found that cells that comprise a gene amplification above norma] of at least one of SRC, EPHA3, FRK, EPHA5, EPHA8, YES. ABL2, LCK and/or BLK are in particular susceptible to the treatment with dasatinib.
Therefore, the present invention does not only provide for a method for selecting cells/tissues/cell cultures which are susceptible to dasatinib. but also for an in vitro method for assessing an individual, i.e. a human or animal patient, for its potential responsiveness to an anti-cancer or anti-proliferate treatment with dasatinib. The present invention provides not only for the possibility to select cells, tissues and cell cultures that are susceptible for dasatinib treatment (i.e. the selection of e.g. research tools wherein drugs with a structural similarity to dasatinib may be tested or which are useful in screening methods for compounds that are suspected to function like dasatinib) but also for a method to evaluate whether a given patient, preferably a human patient, in need of treatment but also prevention of a proliferative disease, is a responder for dasatinib treatment. Most preferably, the responsiveness of a given patient to dasatinib is tested. Dasatinib is described herein below in more detail.
The selection method of a dasatinib responding cell or a responding patient comprises a step, wherein (a) cell(s), (a) tissue(s) or (a) cell culture(s) with (a) gene amplification(s) above normal of at least one of SRC, EPHΛ3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK is selected. Said cell/tissue may also be derived from a human sample or from a body fluid that comprises such a cell, for example a cancer cell. Said gene amplification(s) above normal of at least one of SRC, EPHA3, FRK, EPHA5. EPHA8, YES, ABL2, LCK and'or BLK is indicative for susceptibility to dasatinib.
As pointed out above, and in an alternative embodiment, the present invention relates in particular to a method for determining the responsiveness of a mammalian tumor cell or cancer cell to treatment with dasatinib, said method comprising determining the amplification status of at least one gene selected from SRC, EPHA3, FRK, EPHA5. EPHA8. YES, ABL2, LCK and/or BLK in said tumor cell, wherein said amplification status is indicative of whether the cell is likely to respond or is responsive to the treatment. Such a determination may take place on an individual, isolated tumor cell. Such an evaluation may also be carried out on biological/medical/pathological samples, like body fluids, isolated body tissue samples and the like, wherein said samples preferably comprise cells or cell debris to be analyzed.
As pointed out in the technical problem above, there is a need in the art for markers, which can predict the outcome of an anti-cancer therapy with dasatinib prior to and during treatment. There is a need for stratification of patients who are to be subjected to or are being subjected to an anti -cancer therapy with dasatinib and distinguishing between dasatinib "responder" and "non-responder" patients.
Subject of the present invention is a method for diagnosing an individual who is to be subjected to or is being subjected to an anti -cancer treatment or an anti-pro liferative treatment to asses the responsiveness to dasatinib prior, during and/or after dasatinib treatment which comprises the steps of (a) detection of the gene amplification status of at least one gene selected from the group consisting of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK, and BLK in a biological/medical/pathological sample wherein the gene amplification above normal of at least one of said genes is indicative for the responsiveness to dasatinib treatment prior, during and after treatment with dasatinib; and (b) sorting the individual into responder or Non-responder based on detection of said gene amplification of at least one of said genes.
Thus, the invention provides for markers which can predict the outcome of an anti- cancer/anti-pro liferative treatment with dasatinib prior to treatment in addition to during and/or after treatment.
The present invention solves the above identified technical problem since, as documented herein below and in the appended examples, it was surprisingly found that the presence of (a) gene amplification above normal of at least one of SRC, EPHA3, FRK. EPHA5, EPHA8, YES. AB L2, LCK, and/or BLK in (a) cell(s), (a) tissue(s) or (a) cell culture (or in a biological sample comprising cells or cell debris) is highly predictive for susceptibility of said cell(s), tissue(s) or cell culture(s) (or the individual who provided said biological sample) to dasatinib. In the present invention, the presence of (a) gene amplifϊcation(s) above normal of at least one of SRC. EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK was surprisingly identified as a marker /predictor for responsiveness to treatment with dasatinib or for susceptibility to dasatinib. The terms "marker for responsiveness to treatment with dasatinib"7'"marker for susceptibility to dasatinib" and "predictor for responsiveness to treatment with an dasatinib'V'predictor for susceptibility to dasatinib" can be used interchangeably and refer to (a) gene amplification(s) of said genes, whereby the amplification status is indicative for susceptibility to dasatinib. A gene amplification is defined herein as amplification of the gene above normal. "'Normal amplification*7"Normal amplification status" used herein refers to the presence of two copies of a gene in the genome. "Amplification above normal'* refers accordingly to the presence of at least three copies of a gene in a genome. The presence of (a) gene amplification(s) of the genes defined herein above correlates significantly (p<0.05) with a responsiveness to treatment with dasatinib or susceptibility to dasatinib.
The identification of (a) gene amplification(s) above normal of at least one of SRC. EPHA3, FRK, EPHA5, EPHA8, YES, ABL2. LCK and/or BLK as markers for susceptibility of tumor cell(s) to an dasatinib allows for the first time a reliable identification of subjects/patients which can be specifically and efficiently treated with dasatinib. The likelihood for susceptibility of patients having no gene amplification above normal of at least one of said genes to treatment with dasatinib is below 10 %. In contrast thereto, it was surprisingly found in the present invention that the probability for susceptibility of patients having (a) gene amplification(s) above normal of one of said genes to treatment with dasatinib rises 5-fold to about 50 %. Unexpectedly, the probability for susceptibility of patients having (a) gene amplification(s) above normal of two of said genes to treatment with dasatinib rises 8-fold to about 80 %.
Gene amplifϊcation(s) of the above-described genes have neither been described nor proposed in the art as markers for susceptibility of (a) tumor ccli(s)/(a)tumor(s) to dasatinib. Huang (2007), Cancer Res 67, 2226-2238 describes a set of 161 genes which are to serve as predictors for susceptibility to dasatinib. However, the genes described in Huang (2007), Cancer Res 67, 2226-2238 were identified according to their respective expression pattern in cell lines sensitive to or resistant to dasatinib. Further, the cell lines used in Huang are not representative for primary NSCLC tumors (Figure. 17). In contrast thereto, in the present invention a highly representative cell line collection was used for the identification of SRC, EPHA3, FRK, EPHA5, EPHA8, YES5 ABL2, LCK and/or BLKas markers for susceptibility of tumor cell(s) to dasatinib. Unexpectedly, it was found that the amplification status of said genes is indicative for a susceptibility to dasatinib as described herein below and demonstrated in the appended example. The genes SRC, EPHA3, FRK, EPHA5, EPHA8, YES5 ABL2, LCK BLK identified herein as markers/predictors are not even mentioned in Huang (loc.cit) as markers/predictors. Furthermore ; in the representative cell line panel described herein and in the appended example, the gene-expression signatures described by Huang (loc.cit) was in no way predictive of responsiveness to dasatinib. Thus, the chromosomal gene copy number signature (i.e. the gene amplification above normal of at least one of the following genes SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK ) described herein represents an important and unexpected contribution to the art and a significant improvement.
The present invention is illustrated by the experiments described in the appended Example. As mentioned, the gene amplification status above normal of at least one of the following genes SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK was surprisingly identified as predictor/marker for susceptibility to dasatinib. The gene amplification status of said gene(s) was identified as predictor/marker for susceptibility to dasatinib using the representative NSCLC (non small cell lung cancer) cell line collection as demonstrated in the appended example.
One advantage of the present method is the fact that it allows for an in vitro selection of (a) cell(s), (a) tissue(s) or (a) cell culture with susceptibility to dasatinib/responsive to treatment with dasatinib. As documented herein, genomically annotated NSCLC cell lines are used that are representative of the genetic diversity, the transcriptional profile and the phenotypic properties of primary NSCLC (non-small cell lung cancer) tumors. It is shown herein by integrated genomic profiling on a global scale that the genomes of non-small cell lung cancer (NSCLC) ceil lines are highly representative of several primary NSCLC tumors isolated by surgery from patients in gene copy number, oncogene mutation and gene expression space. Thus, using said NSCLC cell line collection in context of the present invention may avoid animal tests or voluntary tests with, cancer patients; at the same time use of said cell line collection allows, in contrast to methods known in the art. the reliable identification of fa) amplification of at least SRC, EPHA3, FRK. EPHA5, EPHA8. YES, ABL2, LCK and/orBLK as markers for susceptibility to dasatinib.
It is envisaged that other cell line collection may be used herein as long as they are highly representative of primary NSCLC tumors, in particular in respect of gene copy number, oncogene mutation and gene expression space. Also computational approaches described herein and used in the appended example, may be used in context of the present invention for the evaluation of experimental data and/or identification of predictors/markers of (a) (tumor) cell(s), (a) (tumor) tissue(s), or (a) (tumor) cell culture for susceptibility to a dasatinib. Such computational approaches are known in the art and comprise, inter alia, K-nearest neighbour and statistical tests, such as Fisher's exact test. A person skilled in the art knows how to use these computational approaches in context of the present invention. A skilled person will also be aware of further computational approaches to be used in accordance with the present invention and may adapt said approaches based on his general knowledge. The description herein below and the appended examples provide for a specific selection of cell lines or combination of cell lines which can be used as experimental set in the assessment of markers for drug susceptibility.
A systematical annotation of the genomes of a large panel of NSCLC cell lines was performed in order to determine whether such a collection reflects the genetic diversity of primary NSCLC tumors. As described in the appended example, the phenotypic validity of this collection was demonstrated, thus confirming that the genomes of non-small cell lung cancer (NSCLC) cell lines are highly representative of primary NSCLC tumors. Said cell lines may, accordingly, be used in analysis of drug activity as a function of genomic lesions in a systematic fashion as described herein.
Using the systematic similarity profiling described in the appended example, EGFR mutations were confirmed to predict sensitivity to EGFR inhibitors (erlotinib. PD168393, vandetanib) (Arao et al., 2004; Lynch et al, 2004; Paez et al., 2004; Pao et ah, 2004; Sos et al., 2008) which is in accordance with prior ail observations. A high activity of EGFR inhibitors in EGFR-mutant NSCLC cell lines has been described in the prior art (McDermott et al. 2007; Paez et ah, 2004; Tracy et al.. 2004). As mentioned, these findings of the prior art have been confirmed herein using an unbiased computational approach employing systematic global measurements of genetic lesions. These findings described in the prior art in respect of the identification of EGFR mutations as predictors/markers for susceptibility to EGFR inhibitors have been confirmed in the present invention, thus demonstrating the overall functional biological validity of the computational approach used in the appended example for the identification of the amplification status of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK as predictors/markers for susceptibility to dasatinib. For example, EGFff-routations were identified as markers/predictors of susceptibility to EGFR mhibition (p< 0.0001) using the systematic cell-based compound screening followed by computational prediction of sensitivity based on lesion profiles, EGFR mutations were also identified herein as predictors/markers for susceptibility to the SRC/ ABL inhibitor dasatinib. These findings suggest that mutant EGFR might be a target of dasatinib (Song et al, 2006). However, not only EGFR-mutations were identified and confirmed as predictors/markers for susceptibility to EGFR inhibitors but also and unexpectedly the amplification status of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2. LCK and/or BLK as predictors/markers for susceptibility to dasatinib.
These surprising findings were confirmed in a xenograft mouse model. As demonstrated in the appended examples, an amplification above normal of the SRC gene (i.e. a copy number gain) is indicative to susceptibility to dasatinib. While a cell line found to be dasatinib- resistant in the screening experiment (talcing advantage of the cell line collection) also grew aggressively in a mouse model, the dasatinib-sensitlve tumors showed robust regressions upon treatment with dasatinib in vivo. It is also shown that tumors with copy number gain not only in SRC but also in EPHA3 shrink upon treatment with dasatinib. This shows that the findings based on the large-scale cell-based screening experiments involving genomically annotated cell line panels are confirmed in vivo.
Furthermore, it is demonstrated in the appended examples that SRC is the relevant target in cells with SRC amplification by showing growth inhibition and cell death induced by sliRNA- mediated knockdown of SRC. The finding that SRC is indeed the relevant target of dasatinib has been further confirmed in a chemical genetics approach that can be considered as the most stringent assay for target validation (Du et al., 2009). This assay exploits the fact that dasatinib is an ATP-competitive hinge region-binding compound that is highly sensitive to modifications of amino acids regulating the entrance to the ATP binding pocket. A change of the threonin to a stericall '"bulky" methionin at the amino acid position 341 lead to a physical barrier that acts as a "gatekeeper" and prevents the binding of dasatinib to the hinge region. The gatekeeper region is a unique residue in the entrance of the ATP-binding pocket that is occupied by competitive inhibitors and thus a common position for the occurance of resistance mutations that abrogate the activity of such compounds. Therefore, SRC has been ectopically expressed with and without the above described gatekeeper mutation at the amino acid position 341 (i.e., threonin to methionin [T341M]) and it was found herein that the cells stably expressing the gatekeeper mutation were fully resistant to dasatinib treatment, This demonstrates that SRC is indeed the relevant target of dasatinib in SRC-amplified cells.
It is to be understood that in-depth preclinical analysis of activity of cancer therapeutics in tumor cells requires both thorough genomic analysis of a large cell line collection of a single tumor entity and high-throughput cell line profiling, followed by genomic prediction of compound activity as described and demonstrated herein.
The terms .susceptibility to dasatinib" and "responsiveness to treatment with dasatinib" are used interchangeably in context of the present invention. Any explanations given herein in respect to "susceptibility to dasatinib'' also apply to "responsiveness to treatment with dasatinib''. mutatis mutandis, and vice versa.
Methods for determining the susceptibility to dasatinib(s)/ responsiveness to treatment with dasatinib are well known in the art. For example, susceptibility to dasatinib/responsiveness to treatment with dasatinib may be determined by contacting (a) cell(s)5 (a) tissue(s), or (a) cell culture(s) with dasatinib and determining the viability of said (a) cell(s), (a) tissue(s), or (a) cell culture(s) after contacting. These above-mentioned methods for determining the susceptibility to dasatinib(s)/ responsiveness to treatment with dasatinib may. for example, comprise an evaluation/determination step, which may. for example, include determining the viability of the cell(s). tissue(s) or cell culture(s) contacted with/exposed to an dasatinib or fa) mammalian cellfs) or cancer cell treated with an dasatinib. For example, (a) cell(s), (a) tissue(s) or (a) cell culture(s) described herein above may show decreased viability upon contacting/exposing/treating with a dasatinib. Preferably, the ceϊl(s). tissue(s) or cell culture(s) may show an at least 10 %, 20 %, 30 %5 40 %. 50 %, 60 %, 70 %, 80 %, and most preferably, 90 % reduction in viability compared to control cell(s), tissue(s) or cell culture(s) not contacted/exposed/treated with an dasatinib. Preferably, the control cell(s). (a) tissue(s) or (a) cell culture(s) will be identical to the cell(s). (a) tissue(s) or (a) cell culture(s) to be tested as described herein with the only exception that the control (s), (a) tissue(s) or (a) cell culture(s) are not contacted with/exposed to the dasatinib,
Thus (a) ceϊl(s), (a) tissue(s) or (a) cell culture(s) contacled/exposed/treated with dasatinib and showing, for example, a decreased viability as described herein above, can be considered as being susceptible to dasatinib. Correspondingly, (a) mammalian tumor cellfs) or (a) cancer cell(s) treated with dasatinib showing such a decreased viability can be considered as responsive to treatment with dasatinib.
A reduction in viability may. for example, be reflected in a decreased proliferation, such as 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %. 80 %, and most preferably, 90 % reduction in proliferation compared to control cell(s), tissue(s) or cell culture(s) not contacted/exposed/treated with dasatinib. The decreased proliferation may be quantitated, for example, by measuring the total cell volume, tissue volume or cell culture volume using standard techniques.
The difference in proliferation between contacted/exposed/treated cell(s), tissue(s) or cell culture(s) and corresponding controls as defined herein may, for example, be evaluated/determined by measuring the volume of the cell(s), tissue(s) or cell culture(s) taking advantage of standard techniques. Said evaluation/determination may be performed in various points in time, for example, 15 minutes, 30 minutes. 60 minutes. 2 hours, 5 hours, 18 hours. 24 hours. 2 days, 3 days, 4 days, 5 days, six days and/or seven days after contacting/treating said cell(s), tissue(s) or cell culture(s) with an dasatinib or exposing said cell(s), tissue(s) or cell culture(s) to an dasatinib. It is envisaged herein that said evaluation/determination may be performed repeatedly, for example, at 15 minutes, 30 minutes and 60 minutes after said contacting/exposing/treating. It is of note that said cell(s). tissue(s) or cell culture(s) may be contacted /treated not oniy once with said dasatinib or exposed to said dasatinib but several times (e.g. 2 times, 3 times. 5 times, 10 times or 20 times) under various conditions (e.g. same concentration of inhibitor, different concentration of inhibitor, inhibitor comprised in a composition with different stabilizers, diluents, and/or carriers and the like). Accordingly, said optionally repeated evaluation/determination may be performed after the final contacting/treating with or exposing to said dasatinib or in between said above-mentioned various contacting/exposing/treating steps.
The explanations given herein above in respect of the exemplary determination/evaluation step, comprising determining the proliferation of the cell(s), tissue(s) or cell culturefs) contacted with/exposed to dasatinib apply to other determination/evaluation steps described herein and further determinati on/evaluation steps a person skilled in the art will be aware of, such as measuring cellular proliferation or cell death by determining cellular ATP content or incorporation of BrDU or any other marker of cellular proliferation, by determining ceil membrane integrity or phenotypic properties of apoplotic cells or of any other phenotypic properties typical of dying cells. These tests may include but are not limited to, measurements of Annexin-V exposure on the outer membrane, cell cycle analyses, propidium iodide staining, TUNEL assay, DNA fragmentation assays, nuclear condensation assays, KI-67 staining, resazurm staining, protein cleavage assays (e.g., PARP or Caspase-3) and others.
As mentioned above, dasatinib is a known tyrosine kinase inhibitor. The respective formula is given herein below:
Figure imgf000014_0001
Also the use of high throughput screening (HTS) is envisaged in context of the present invention, in particular the screening methods of cell(s). tissue(s) and/or cell culture(s) for responsiveness/sensitivity to dasatinib. Suitable (HTS) approaches are known in the art. An exemplary protocol for such a screening method is also provided in the appended examples; a person skilled in the art is readily in the position to adapt this protocol or known HTS approaches to the performance of the methods of the present invention. Screening-assays are usually performed in liquid phase, wherein for each cell/tissue/cell culture to be tested at least one reaction batch is made. Typical containers to be used are micro titer plates having for example, 384, 1536. or 3456 wells (i.e. multiples of the "original" 96 reaction vessels).
Robotics, data processing and control software and sensitive detectors are further commonly used components of a HTS device. Often robot system are used which transport micro titer plates from station to station for addition and mixing of sample(s) and reagent(s), incubating the reagents, and final readout (detection). Usually, ITTS can be used in the simultaneous preparation, incubation, and analysis of many plates.
The assay can be performed in a singly reaction (which is usually preferred), may, however, also comprise washing and/or transfer steps. Detection can be performed taking advantage of radioactivity, luminescence or fluorescence, like fiuorescence-resonance-energytransfer (FRET) and fluorescence polarisation (FP) and the like. The biological samples described herein can also be used in such a context. In particular cellular assays and in vivo assays can be employed in HTS. Cellular assays may also comprise cellular extracts, i.e. extracts from cells, tissues and the like. However, preferred herein is the use of cell(s) or tissue(s) as biological sample (in particular a sample obtained from a patient/subject suffering or being prone to suffer from cancer), whereas in vivo assays (wherein suitable animal models are employed, e.g. the herein described mouse models) are particularly useful in the validation/monitoring of the treatment with dasatinib. Depending on the results of a first assay, follow up assays can be performed by re-running the experiment to collect further data on a narrowed set (e.g. samples found "positive" in the first assay), confirming and refining observations. A suitable readout in animal (in vivo) models is tumor growth (or respectively the complete or partial inhibition of tumor growth and/or its remission).
In a preferred embodiment the herein described HTS methods for the detection of copy number changes include but are not limited to techniques such as single nucleotide polymorphism (SNP)-array and Comparative Genomic Hybridization (CGH)-arrays. The SNP-array technology allows parallel interrogation of up to two million genomic locations in high-throughput. After DNA labelling and hybridization, fluorescence intensities are measured for each allele of each SNP and genomic copy number changes can be inferred. The CGH technique allows the detection of "tumor" and "normal" tissue extracts, differently fluorescence labeled, on the same glass slide and the relative copy number changes (amplifications and deletions) can thus be inferred.
The meaning of the terms "cell(s)", "tissue(s)" and "cell culture(s)" is well known in the art and may. for example, be deduced from 'The Cell" (Garland Publishing, Inc.). Generally, the term "cell(s)" used herein refers to a single cell or a plurality of cells. The term "plurality of cells" means in the context of the present invention a group of cells comprising more than a single cell. Thereby, the cells out of said group of cells may have a similar function. Said cells may be connected cells and/or separate cells. The term "tissue" in the context of the present invention particularly means a group of cells that perform a similar function. The term "eel! culture(s)" means in context of the present invention cells as defined herein above which are grown/cultured under controlled conditions. Cell culture(s) comprise in particular cells (derived/obtained) from multicellular eukaryotes, preferably animals as defined elsewhere herein. It is to be understood that the term "cell culture(s)" as used herein refers also "tissue culture(s)" and/or "organ culture(s)", an "organ" being a group of tissues which perform the same function.
Preferably, the cell(s), tissue(s) or cell culture(s) to be selected comprise/are derived from or are (a) tumor cell(s). The tumor cells may, for example, be obtained from a biopsy, in particular a biopsy/biopsies from a patient/subject suffering from or being prone to suffering from non-small cell lung cancer. It is preferred herein that said subject is a human. The term "mammalian tumor cell(s)" used herein refers to (a) tumor cell(s) which is derived from or is a tumor cell from a mammal, the term mammal being derived herein below. As described herein above in respect of "cell(s)", "tissue(s)" and "cell culture(s)" the "mammalian tumor cells" or cancer cells may be obtained from a biopsy, in particular a biopsy/biopsies from a patient/ subject suffering from non-small cell lung cancer or a patient/subject being prone to suffer from said disorders. The term "tumor cell" also relates to "cancer cells"
Generally, said tumor cell or cancer cell may be obtained from any biological source/organism, particularly any biological source/organism, suffering from or being prone to suffer from the above-mentioned non-small cell lung cancer.
Preferably, the (tumor) cell(s) or (cancer) cell to be contacted is (are) obtained/derived from an animal. More preferably, said (turnor)/cancer cell(s) is (are) derived from a mammal The meaning of the terms "animal" or "mammal" is well known in the art and can, for example, be deduced from Wehner und Gehring (1995; Thieme Veriag). Non-limiting examples for mammals are even-toed ungulates such as sheep, cattle and pig, odd-toed angulates such as horses as well as carnivores such as cats and dogs. In the context of this invention, it is particularly envisaged that DNA samples are derived from organisms that are economically, agronomically or scientifically important. Scientifically or experimentally important organisms include, but are not limited to. mice, rats, rabbits, guinea pigs and pigs.
The tumor cell(s) may also be obtained from primates which comprise lemurs, monkeys and apes. The meaning of the terms "primate", "lemur", "monkey*' and "ape" is known and may, for example, be deduced by an artisan from Wehner und Gehring (1995, Thieme Veriag). Λs mentioned above, the tumor or cancer cell(s) is (are) most preferably derived from a human being suffering from the above-mentioned non-small cell lung cancer, pancreatic cancer, colorectal cancer, breast cancer, leukemias. In context of this invention particular useful cells, in particular- tumor or cancer cells, are, accordingly, human cells. These cells can be obtained from e.g. biopsies or from biological samples but the term "cell" also relates to in vitro cultured cells.
Exemplary cell(s) or cell culture(s) which may be used in context of this invention are shown in appended Figure 1.
In one embodiment, the present invention relates to an in vitro method for the identification of a responder for or a patient sensitive to dasatinib, said method comprising the following steps: (a) obtaining a sample from a patient suspected to suffer from or being prone to suffer from non- small cell lung cancer; and
(b) evaluating the gene amplification status of at least one gene selected from the group of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and BLK ; whereby a gene amplification of at least one of said genes is indicative for a responding patient or is indicative for a sensitivity of said patient to dasatinib.
Said sample may, for example, be obtained by (a) biopsy (biopsies). Preferably, said sample is obtained from a patient suspected to suffer from or being prone to suffer from non-small cell lung cancer. It is preferred herein that said sample is obtained from (a) tumor(s) and, accordingly, is (a) tumor cell(s) or (a) tumor tissue(s). Preferably, (a) tumor, sample(s) may be obtained from subjects/patients suffering from non-small cell lung cancer.
Particularly preferred is the use of SRC alone as marker in context of the present invention (i.e. the amplification status of only SRC Is assessed or evaluated). Also preferred is the use of EPHA3 or FRK alone as marker. In a preferred embodiment, in the present method the amplification status of at least two genes selected from the group of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and BLK is assessed or evaluated.
Preferably, the amplification status of SRC in combination with at least one further of the above 8 remaining genes (in particular with EPHA3 and/or FRK) is assessed or evaluated. Exemplary combinations which are preferred in this context are: SRC and EPHA3; SRC and FRK; SRC. EPHA3 and FRK. Also preferred are SRC and EPHA5; SRC and EPHA8; SRC and YES: SRC and ABL2: SRC and LCK; SRC and BLK.
Preferred combinations of 3 genes are
SRQ EPHA3 and EPHA5: SRC, EPHA3 and EPHA8: SRC, EPHA3 and YES; SRC5 EPHA3 and ABL2; SRC, EPHA3 and LCK; SRC. EPHA3 and BLK; SRC. FRK and EPHA5: SRC. FRK and EPHA8; SRC, FRK and YES; SRC5 FRK and ABL2;
SRC, FRK and LCK; SRC, FRK and BLK:
SRC. EPHA5 and EPHA8; SRC, EPHA5 and YES; SRC. EPHA5 and ABL2; SRC, EPHA5 and LCK; SRC, EPHA5 and BLK;
SRC, EPHA8 and YES; SRC. EPHA8 and ABL2; SRC, EPHA8 and LCK; SRC, EPHA8 and BLK;
SRC, YES and ABL2; SRC. YES and LCK; SRC, YES and BLK; or SRC, LCK and BLK; wherein combinations which comprise SRC and EPHA3; or SRC and FRK are particularly preferred.
Preferred combinations of 4 genes (SRC, EPHA3, and two further markers) are SRC. EPHA3, FRK and EPHA5; SRC, EPHA3. FRK and EPHA8; SRC, EPHA3, FRK and
YES; SRC. EPHA3, FRK and ABL2; SRC. EPHA3, FRK and LCK; SRC, EPHA3. FRK and
BLK;
SRC. EPHA3. EPHA5 and EPHA8; SRC, EPHA3, EPHA5 and YES; SRC, EPHA3, EPHA5 and ABL2; SRC, EPHA3, EPHA5 and LCK; SRC, EPHA3, EPHA5 and BLK; SRC, EPHA3, EPHA8 and YES; SRC, EPHA3, EPHA8 and ABL2; SRC, EPHA3, EPHA8 and LCK; SRC, EPHA3, EPHA8 and BLK;
SRC5 EPHA3, YES and ABL2; SRC, EPHA3, YES and LCK; SRC, EPHA3, YES and BLK: SRC, EPHA3, ABL2 and LCK; SRC, EPHA3, ABL2 and BLK; or SRC, EPHA3, LCK and BLK; wherein combinations which comprise SRC, EPHA3 and FRK are particularly preferred.
Preferred combinations of 4 genes (SRC. FRK5 and two further markers) are SRC, FRK, EPHA5 and EPHA8; SRC, FRK, EPHA5 and YES; SRC, FRIC, EPHA5 and
ABL2; SRC, FRK, EPHA5 and LCK; SRC, FRK, EPHA5 and BLK;
SRC, FRK, EPHA8 and YES; SRC. FRK, EPHA8 and ABL2; SRC, FRK, EPHA8 and LCK;
SRC, FRK, EPHA8 and BLK;
SRC, FRK, YES and ABL2; SRC, FRK. YES and LCK; SRC5 FRiC, YES and BLK; SRC, FRK, ABL2 and LCK; SRC, FRK, ABL2 and BLK; or
SRC, FRK, LCK and BLK; wherein combinations which comprise SRC, FRK and EPHA3 are particularly preferred.
These and further combinations of SRC with any of the remaining markers EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK, and BLK are easily deduclble by a skilled person, wherein SRC can be combined with at least one, two, three, four, five, six, seven, or eightfurther markers is envisaged. Again, combinations comprising SRC and EPHA3; SRC and FRK; or SRC, EPFIA3 and FRK are preferred.
As mentioned above, EPHA3 or FRK alone can be used as marker in context of the present invention. However, it is also preferred herein is that the amplification status of EPHA3 and/or FRK in combination with at least one further of the above remaining genes (in particular with SRC), is assessed or evaluated. The amplification status of EPFIA3 can also be assessed/evaluated in combination with FRK, EPHA5, EPHA8, YES, ABL2, LCK, and/or BLK, wherein combinations of EPHA3 and FRK with at least one further of the remaining markers are preferred. The amplification status of FRK can also be assessed/evaluated in combination with EPHA3, EPHA5, EPHA8, YES, ABL2, LCK, and/or BLK; wherein combinations of FRK and EPFIA3 with at least one further of the remaining markers are preferred.
The following combinations of EPHA 3 are preferred: EPHA3 and SRC; EPHA3 and FRK; EPHA3 and EPHA5; EPHA3 and EPHA8; EPHA3 and YES; EPHA3 and ABL2; EPHA3 and LCK; EPHA3 and BLK. Also preferred herein are combinations of FRK and SRC; FRK and EPHA3; FRK and EPHA5; FRK and EPFIA8; FRK and YES; FRK and ABL2; FRK and LCK: FRK and BLK.
The various combinations of the herein preferred markers SRC, EPHA3 and FRIC alone with at least one further of the remaining markers can easily be deduced by a skilled person and applied in accordance with the present invention. Also the use of combinations of at least one of EPHA5, EPHA8, YES, ABL2, LCK, and BLK with at least one further of the remaining markers is envisaged in context of the present invention. All these combinations are readily derivable by a skilled person in the art. The explanations given herein above regarding SRC, EPHA3 and FRK alone or in various combinations with each other apply here, mutatis mutandis, to any of the remaining markers (or various combinations thereof).
Preferably, said at least two amplified genes are selected from the group consisting of SRC and EPHA3. EPHA3 and FRK, and EPHA3 and ABL2 and, as mentioned above, it is preferred herein that said at least one gene/said at least two genes is/are present in at least 3 copies.
The gene amplification status may, for example, be detected, assessed or evaluated by an in situ hybridization method, comparative genomic hybridisation and smgie-nucleotide polymorphism arrays. Exemplary in situ hybridisations are, inter alia, fluorescent in situ hybridisation (FISH), chromogenic in situ hybridisation (CISH) and silver in situ hybridisation (SISH).
In one embodiment, the present invention relates to the use of an oligo- or polynucleotide capable of detecting the amplification status of at least one gene selected from the group consisting of SRC. EPHA3. FRK. EPHA5, EPHA8. YES, ABL2, LCK5 and BLK for diagnosing sensitivity to dasatinib. Preferably, said oligonucleotide is about 15 to 50 nucleotides in length. A person skilled in the art is, based on his general knowledge and the teaching provided herein, easily in the position to identify and/or prepare (a) an oligo- or polynucleotide capable of detecting the amplification status of at least one gene selected from the group consisting of SRC, EPHA3, FRK. EPHA5, EPHA8, YES, ABL2, LCK and BLK for diagnosing sensitivity to dasatinib. In particular these oligo- or polynucleotides may be used as probe(s) in the detection methods described herein. A skilled person will know, for example, computer programs which may be useful for the identification of corresponding probes to be used herein. For example, the SRC. EPHA3. FRK, EPHA5, EPHA8. YES. ABL2. LCK and/or BLK nucleic acid sequences (shown in appended SEQ ID NOs: 1-10) may be used in this context for identifying specific probes for detecting the amplification status. Exemplary, non- limiting SRC, EPHA3, FRK, EPHA5, EPHA8, YES5 ABL2, LCK and/or BLK nucleic acid sequences are also available on corresponding databases, such as the NCBI database (www.ncbi.nlm. nih.gov/sites/entrez).
In one embodiment, the present invention relates to a method of diagnosing (non-small cell lung cancer) in a subject/patient suspected of suffering from non-small cell lung cancer or suspected of being prone to suffering from non-small ceil lung cancer comprising the steps of a) determining in a cell or tissue sample obtained from said subject/patient the activity of at least one marker gene selected from the group consisting of SRC, EPHA3, FRK, EPHA5, EPHA8, YES5 ABL2, LCK and BLK ; and b) comparing the activity of said at least one marker gene determined in a) with a reference activity of said at least one marker gene determined in (a sample from) a control subject/patient (healthy subject), wherein said non-small cell lung cancer is diagnosed when said activity determined in a) differs from said reference activity.
The present invention also relates to a method of monitoring the efficacy of a treatment of a non-small cell lung cancer in a subject/patient suffering from said disorder or being prone to suffering from said disorder comprising the steps of a) determining in a cell or tissue sample obtained from said subject/patient the activity of at least one marker gene selected from the group consisting of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and BLK ; and b) comparing the activity of said at least one marker gene determined in a) with a reference activity of said at least one marker gene, optionally determined in (a sample from) a control subject/patient (responder and/or non-responder), wherein the extent of the difference between said activity determined in a) and said reference activity is indicative for said efficacy of a treatment of a non-small cell lung cancer.
The term "activity" as used herein refers to the activity of a protein as described elsewhere herein. The method of monitoring the efficacy of a treatment of a cancer may comprise a step of determining in a cell or tissue sample obtained from a subject/patient suffering from Non- small cell lung cancer (e.g. a biopsy) the gene amplification status of at least one gene selected from the group consisting of SRC, EPHA3, FRK. EPHA5, EPHA8. YES, ABL2, LCK and BLK .
For example, a gene amplification above normal of at least one gene selected from the group consisting of SRC, EPHA3. FRK, EPHA5. EPHA8, YES, ABL2. LCK and BLK may be present in a sample before start of the treatment of a cancer. During or after treatment of the cancer, the tumor cells having said gene amplification above normal are erased or otherwise depleted. Thus, the absence of a detectable gene amplification above normal of at least one of said genes in a sample (cell samples/biopsy samples and the like) obtained from a subject/patient during or after treatment of a cancer is indicative of the efficacy of the treatment.
In an further embodiment the present invention relates to a method of predicting the efficacy of a treatment of a non-small cell lung cancer for a subject/patient suffering from said disorder or being prone to suffering from said disorder comprising the steps of a) determining in a cell or tissue sample obtained from said subject/patient the activity of at least one marker gene selected from the group consisting of SRC, EPHA3, FRK. EPHA5, EPHA8. YES. ABL2. LCK and BLK ; and b) comparing the activity of said at least one marker gene determined in a) with a reference activity of said at least one marker gene, optionally determined in (a sample from) a control subject/patient (responder and/or non-re sponder), wherein the extent of the difference between said activity determined in a) and said reference activity is indicative for the predicted efficacy of a treatment of a non-small cell lung cancer.
The treatment of non-small cell lung cancer may comprise the administration of dasatinib. The non-small cell lung cancer may, inter alia, be a squamous cell lung carcinoma, an adenocarcinoma, a large cell lung carcinoma, or an anaplastic carcinoma. The subject/patient suffering from said non-small cell lung cancer or being prone to suffering from said non-small cell lung cancer may also exhibit resistance (primary or secondary) against any platinum- based, taxane-based chemotherapy, vinorelbine, gemcitinibe, erlotinib, sunitinib and/or vandetanib.
It has been described in context of this invention that gene amplification above normal of at least one gene of SRC, EPHA3, FRIC. EPHA5, EPHA8, YES, ABL2, LCK and/or BLK as disclosed herein act as markers/predictors for susceptibility to dasatinib. In particular a responder for or a patient sensitive to dasatinib may be identified in accordance with the present method. Accordingly, the present invention provides the possibility to recognize (aberrant) changes of SRC, EPHA3. FRK, EPPIA5, EPHA8. YES, ABL2. LCK and/or BLK activity immediately once the}' occur, for example, by determining the activity of said marker gene(s).
It is of note that the assessment/evaluation/detection of the amplification status of any of the above marker genes (and their various combinations described herein) is sufficient for determining whether a patient is likely to respond to or is sensitive to dasatinib. whether a (mammalian tumor or cancer) cell is likely to respond or is responsive to treatment with dasatinib. The assessment/evaluation/detection of the amplification status of any of the above marker genes (and their combinations) is also sufficient for diagnosing sensitivity to dasatinib. In other words, in particular in these methods described and provided in the present invention, the amplification status alone of any of the above marker genes is indicative for a sensitivity/responsiveness to dasatinib and the expression level/activity of the gene products of the above marker genes need not be determined in addition to the amplification status.
The activity of (amplified) SRC. EPHA3, FRK, EPHA5, EPHA8. YES, ABL2. LCK BLK may not only be determined by measuring the expression level but also, be determined, for example, by measuring substrate turnover of SRC, EPHA3. FRK. EPHA5, EPHA8, YES. ABL2, LCK. This may be particularly useful in methods described herein for diagnosing non- small cell lung cancer, monitoring non-small cell lung cancer or predicting the efficacy of a treatment of cell lung cancer. Means and methods for determining the activity of said proteins are well known in the art and may. for example, be deduced from Lottspeich (Spektrum Akademischer Verlag, 1998). As mentioned herein above, determining the activity may comprise determining the expression level Accordingly, in an alternative, the expression status (i.e. expression of gene products such as mRNA and/or proteins) of (amplified) SRC. EPHA3, FRK, EPHA5, EPHA8, YES, ABL2. LCK and/or BLK can be determined by standard techniques. Amplification of SRC, EPHA3, FRK, EPH A5, EPHAS5 YES, ABL2. LCK and/or BLK gene does not necessarily correlate with a change in the expression level of these genes. In context of the present invention it is therefore preferred that the activity of (amplified) SRC. EPHA3, FRK, EPHA5, EPHA8, YES. ABL2, LCK and/or BLK is not determined by measuring the expression status of these genes but by alternative methods which reflect, for example, the enzymatic activity of the corresponding proteins. It is of note that, for example, the enzymatic activity of proteins encoded by a (amplified) SRC, EPHA3. FRK, EPHA5, EPHA8, YES, ΛBL2, LCK and/or BLK may differ from that of the respective proteins encoded by wild type genes (i.e. reference activity) without a change in the expression level. Preferably, (amplified) SRC. EPHA3, FRK. EPHA5, EPFIA8, YES, ABL2, LCK and/or BLK show an increased activity compared to non-amplified (amplified) SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK (controls). In particular the enzymatic activity and/or the expression level described herein above are increased. Exemplary ranges of changes in the activity compared to ''normal" activity (i.e. activity of (non-amplified) SRC, EPHA3. FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK) are described herein below. The term •'non-amplified" used herein refers to the ''normal" amplification status, i.e. two gene copies, whereas "amplified" refers to an amplification status above normal, i.e. at least three copies of the respective gene.
Based on these findings, the present invention provides the particular advantages that, by determining the amplification status of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK in accordance with this invention, (aberrant) changes of (amplified) SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK activity/expression level can be recognized early, i.e. that the efficacy of a treatment NSCLC can be monitored early and that the efficacy of a treatment of said cancer can be predicted early. Hence, also a possible resistance to the treatment can be recognized early by using the means and method of this invention.
In a particular embodiment, the present invention relates to corresponding means, methods and uses which are based on the early recognition of (aberrant) changes of (amplified) SRC, EPHA3. FRK. EPHA5, EPHA8, YES, ABL2, LCK and/or BLK activity/expression level of the respective genes. The possibility of recognizing (aberrant) changes of (amplified) SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK activity/expression level early, provides several advantages, like a higher lifespan/likelihood of survival of the subject/patient (for example due to the notice of possible treatment failures and a corresponding change of the treatment regimen) and the possibility of a more efficient therapy (for example due to the possibility to avoid/recognize treatment failures early and. hence, to correspondingly change the treatment regimen early in therapy, i.e. to timely switch to a more suited inhibitor, to discontinue an expensive, ineffective treatment early after diagnosis and to opt for alternative therapy) .
In context of the above embodiments of this invention, '"early" particularly means prior to (the onset of) a (complete or partial) cytogenetic or haematological response or a response measured by any type of imaging technique and/or prior to the outbreak of NSCLC (or susceptibility th ereto ) .
For example, "'early" monitoring the efficacy of a therapy /treatment of said cancer may be at least 1. at least 2. at least 3, at least 4, at least 5. at least 6. at least 7, at least 10, or at least 14 days prior to (the onset of) a (partial) cytogenetic or haematological response or a response measured by any type of imaging technique to said therapy/treatment and/or at least 1. at least 2. at least 3. at least 4. at least 55 at least 6, at least 7, at least 10, at least 12, at least 15, or at least 18 month prior a complete cytogenetic or haematological response or a response measured by any type of imaging technique to said therapy/treatment (of the patient or control patient (responder)), wherein the longer periods are preferred.
Alternatively, ''early'' monitoring the efficacy of a therapy/treatment of said cancer may also be at most 1 , at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 10. or at most 14 days after (onset of) the therapy /treatment of said cancer, wherein the shorter periods are preferred. Most preferably, it is envisaged to already monitor the efficacy of a therapy/treatment of said cancer at the day the therapy/treatment was initiated, i.e. once the (amplified) SRC, EPHA3, FRK. EPHA5, EPHA8, YES, ABL2, LCK and/or BLK activity /expression level changes upon said therapy/treatment. In the following, an example of a scheme for (early) monitoring the efficacy of a therapy/treatment of the cancer defined herein in accordance with this invention is provided; « When monitoring the therapy/treatment of said cancer (for example therapy /treatment based on dasatinib as described herein, activity of (amplified) SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK may be determined daily during the first week after initiation of the therapy/treatment, weekly during the first month of the therapy/treatment and, afterwards, monthly.
• The reference activity/expression level may be taken at the day the therapy/treatment is initiated, from the subject/patient to be treated and/or from a corresponding control subject/patient (responder/non-responder); see below.
* If a rise in marker levels is observed in two consecutive samples, or if decrease in marker level is not fast enough (for example not as fast as that of responder or not sufficiently faster than that of a non-re sponder). change of treatment regimen may be considered.
It is of note that this example is in no way limiting. The skilled person is readily in the position to adapt this scheme to the particular requirements relevant for each individual case, based on the teaching provided herein an on his common general knowledge.
For example, '"early1" predicting the efficacy of a therapy/treatment of the cancer defined herein may be at least 1, at least 2. at least 3, at least 4. at least 5, at least 6, at least 7, at least 10, or at least 14 days prior to (the onset of) a (partial) cytogenetic or haematological response to said therapy/treatment and/or at least 1, at least 2. at least 3. at least 4, at least 5, at least 6. at least 7, at least 10, at least 12, at least 15, or at least 18 month prior a complete cytogenetic or haematological response or a response measured by any type of imaging technique to said therapy/treatment, wherein the longer periods are preferred.
Alternatively, "early"' predicting the efficacy of a therapy/treatment of the cancer defined herein may also be at most 1, at most 2. at most 3, at most 4. at most 5, at most 6, at most 7, at most 10, or at most 14 days after (onset of) the therapy/treatment of the cancer defined herein, wherein the shorter periods are preferred. Most preferably, it is envisaged to already monitor the efficacy of a therapy/treatment of said cancer at the day the therapy/treatment was initiated, i.e. once the (amplified) SRC5 EPHA3, FRK. EPHA5, EPHA8. YES, ABL2, LCK, and/or BLK activity/expression level changes upon said therapy/treatment.
Furthermore, "early" predicting the efficacy of a therapy /treatment of the cancer defined herein may also be at most L at most 2, at most 3, at most 4, at most 5, at most 6, at most 7. at most 10. or at most 14 days after diagnosis of the cancer, wherein the shorter periods are preferred. Most preferably, it is envisaged to already predict the efficacy of a therapy/treatment of said cancer at the day of diagnosis.
As mentioned, the present invention is particularly useful for monitoring the efficacy of a therapy/treatment of the cancer as defined herein. Corresponding means, uses and methods are provided herein. In general, monitoring the efficacy of a certain kind of therapy/treatment is regularly applied in clinical routine. Hence, the skilled person is aware of the meaning of monitoring the efficacy of a certain kind of therapy/treatment. In context of this invention, the meaning of the term "monitoring"' encompasses the meaning of terms like "tracking", '"discovering"' etc.. In particular, the term "monitoring the efficacy of a therapy/treatment of NSCLC as used herein refers to monitoring whether a subject/patient suffering from said disorder (or being prone to suffering from said cancer) responds at all to a therapy/treatment of said disorder and/or how the course of said respond is (e.g. how fast/slow the respond is and/or to what extent the respond is).
The present invention is further useful for predicting the efficacy of a therapy/treatment of the cancer as defined herein. Corresponding means, uses and methods ai-e also provided herein. In general, predicting the efficacy of a certain kind of therapy/treatment is highly desired in clinical routine, since it allows for preventing the disorder and/or increasing the efficiency of a therapy/treatment and hence, leads to savings in cost and time and to a higher lifespan/likelihood of survival or of 'Genesung' of the affected patient. The definitions given with respect to the term "efficacy of a therapy/treatment of NSCLC* provided herein apply here, mutatis mutandis. In context of this invention, the term "predicting the efficacy of a therapy/treatment of NSCLC for a subject/patient" is used in basically the same sense like determining whether, and/or to what extent, a subject/patient exhibits susceptibility to such therapy/treatment, i.e. whether said subject/patient will or would respond at all to a therapy/treatment of said disorder and/or how the course of said respond will or would be (e.g. how fast/slow the respond is and/or to what extent the respond is). In particular, a subject/patient exhibits susceptibility to said cancer in accordance with this invention, when its (amplified) SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK activity/expression level is aberrant. In this context, said "amplification" is already "aberrant" as defined herein.
In one embodiment, the '"predicting the efficacy of a therapy/treatment of the cancer defined herein" in accordance with this invention may be performed after initiation of the therapy/treatment, i.e. during the already ongoing therapy/treatment. In particular, said "predicting" may be performed during the herein described monitoring the efficacy of a therapy/treatment of said cancer, preferably early after the beginning of said monitoring. Thereby, the predicting may be based on results from said monitoring obtained at a certain point in time of the ongoing therapy/treatment. Preferably, said point in time is an early point in time, like, for example that point in time, when a first result from said monitoring has been obtained. In cases where the "predicting the efficacy of a therapy/treatment of the cancer defined herein" is performed during an already ongoing therapy/treatment, it refers to the following/subsequent efficacy of said therapy/treatment.
In another embodiment, the "predicting the efficacy of a therapy/treatment of the cancer defined herein" in accordance with this invention may be performed (immediately) after diagnosis but, however, prior to initiation of the therapy/treatment. In such cases, "predicting the efficacy of a therapy/treatment of said cancer" refers to the efficacy of a therapy/treatment which has not yet been initiated (or has been initiated substantially at the same point in time when the "predicting" wras performed.
In context of this embodiment of the invention, one non-limiting example of a healthy control subject/patient is one having (a) non-amplified SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK gene(s). This is in contrast to an amplification leading to an aberrant activity/expression of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK, and/or BLK. The amplification of said marker genes as provided herein is considered aberrant. In accordance with the above, the "reference activity" of SRC. EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK, and/or BLK or the "reference expression level" of said marker gene(s), with respect to the means, methods and uses of monitoring the efficacy of a treatment of a cancer defined herein, is that "reference activity/reference expression level" determined in (a sample of) the corresponding healthy control subject, i.e. is the "normal" activity/expression level.
It is to be understood that the activity of amplified SRC5 EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK described herein is different from the above described "reference activity/ reference expression level" of "normal" SRC5 EPHA3, FRK. EPHA5, EPHA8, YES, ABL2, LCK, and/or BLK. In particular with respect to the herein disclosed means, methods and uses of monitoring/predicting the efficacy of a treatment of the cancer as defined herein, the control subject/patient is, in one embodiment, envisaged to be a subject/patient suffering from said cancer or being prone to suffering from said cancer, i.e. a subject/patient having, for example, an aberrant activity/expression level of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK and, hence, not a "normal" activity or "normal expression level" of SRC, EPHA3, FRK, EPHA5, EPPIA8, YES, ABL2, LCK, and/or BLK as described in accordance with this invention.
Thereby, "different" means and comprises "higher" or "lower", depending on whether the cancer defined and described herein comes along with an up- or down-regulated activity of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK.
In this context, "different", "higher" or "lower" means different, higher or lower than the normal (range of) activity of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or
BLK activity. As far as the "expression level" of said marker genes is concerned, "different",
"higher" or "lower" means different, higher or lower than the normal (range of) expression level of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK. For example, different, higher or lower means at least 1,5 fold, at least 2 fold, at least 2,5 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 7 fold, at least 10 fold, at least 15 fold, at least 25 fold, at least 50 fold, at least 100 fold, at least 200 fold different, higher or lower, wherein the higher values are preferred. Whether, in which direction (i.e. higher or lower) and/or to which extent the activity and/or the expression level of SRC, EPHA3, FRK, EPHA5, EPHA8, YES. ABL2. LCK and/or BLK differs from Its corresponding reference activity/expression level of SRC, EPHA3, FRK, EPHA5. EPHA8. YES. ABL2, LCK and/or BLK , can easily be deduced by the skilled person based on the teaching provided herein and the common general knowledge.
It is preferred and envisaged in this context that the control subject/patient is subjected to the same treatment of the cancer subject/patient described and defined herein. Said control subject/patient may be a responder (positive control) or non-responder (negative control) to this treatment. Whether a subject/patient is a "'responder*' or "non-responder"' with respect to a certain kind of cancer treatment/therapy can be evaluated by the skilled person on the basis of his common general knowledge and/or the teaching provided herein. In particular, a ''responder" may be a subject/patient whose cytological/haemato logical parameters and/or (aberrant) SRC, EPHA3, FRK, EPHA5. EPHA8, YES, ABL2, LCK and/or BLK activity/expression level (and hence the corresponding marker gene expression level(s))/ activity of SRC, EPHA3. FRK. EPHA5, EPHA8, YES. ABL2, LCK and/or BLK change towards the their "normal" activity/(expression) level(s) (in a sufficient manner) upon the cancer treatment/therapy In one specific embodiment, a "'responder" may be a subject/ patient not suffering from one of the herein defined resistances. In particular, a "'non-responder"' may be a subject/patient whose cytological/haematological parameters and/or (aberrant) activity/expression level of SRC. EPHA3. FRK. EPHA5, EPHA8, YES, ABL2. LCK and/or BLK (and hence the corresponding marker gene expression level(s)) do not change towards the their '"normal" (expression) level(s) (in a sufficient manner) upon the cancer treatment/therapy. Jn one specific embodiment, a '"non-responder" may be a subject/patient suffering from one of the herein defined resistances.
Accordingly , the patient responds to cancer treatment/therapy, if the activity/expression level of SRC. EPHA3, FRK. EPHA5, EPHA8. YES, ABL2, LCK and/or BLK is reduced upon said treatment/therapy. Preferably, the expression/ activity of SRC. EPHA3. FRK, EPFIA5, EPHA8. YES, ABL2, LCK and/or BLK is reduced to control expression/activity (e.g. determined in a sample obtained from a person not suffering from said cancer). In other words, a reduction in expression/ activity of SRC, EPHA3, FIOC, EPHA5, EPHA8. YES, ABL2. LCK and/or BLK is indicative for a successful treatment/therapy. A skilled person is readily in the position to determine whether a patient responds to cancer treatment/therapy by evaluation of the expression/ activity of SRC. EPHA3, FRK, EPHA5. EPHA8, YES, ABL2, LCK and/or BLK. In addition to the evaluation of said expression/activity, a person skilled in the art may also determine cytological/haematological parameters characteristic for a specific cancer in order to assess whether a patient responds to cancer treatment/therapy.
In contrast, a patient who does not respond to cancer treatment/therapy does not show a reduced expression/ activity of SRC, EPHA3. FRK, EPHA5, EPHA8, YES- ABL23 LCK and/or BLK upon said treatment/therapy as defined herein above in context of responders/responding patients.
In context of this embodiment of the invention, one non-limiting example of a diseased control subject/patient (responder and/or non-responder) suffering from a cancer defined herein or being prone to suffering from a susceptibility thereto is one having an amplified SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK leading to an aberrant SRC. EPHA3, FRK. EPHA5, EPHA8, YES. ABL2, LCK and/or BLK activity/expression of SRC. EPF1A3, FRK. EPHA5, EPHA8. YES, ABL2. LCK and/or BLK .
The skilled person is aware of how a typical/desired response to a known therapy/treatment of NSCLC should proceed or is intended to proceed. Moreover, the skilled person can consider how a typical/desired response to a (unknown) therapy/treatment of a NSCLC proceeds or is intended to proceed. Based on this knowledge, the means, methods and uses of this invention referring to the efficacy of a therapy/treatment of such a cancer can. for example, also be carried out without employing (a sample of) a particular control subject/patient, i.e. without comparing the activity or expression level of SRC. EPHA3, FRK, EPHA5, EPHA8. YES, ABL2, LCK and/or BLK with a "reference activity" or "reference expression level" of SRC. EPHA3, FRK, EPHA5. EPHA8, YES, ABL2, LCK and/or BLK. for example in (a sample from) a control subject/patient. Simply by comparing the course of the determined "activity'' ore expression level of SRC, EPHA3, FRK, EPHAS9 EPHA8. YES, ABL2. LCK and/or BLK during the therapy/treatment of a cancer with the above-mentioned known "typical/desired response", the skilled person is able to consider about the efficacy of the therapy/treatment monitored/predicted. If the response of a subject/patient is as fast (or even faster) than the "typical/desired response", the subject/patient is a "'responder". If the response of a subject/patient is slower than the "typical/desired response", the subject/patient is a "non- responder" (when no substantial response can be seen) or "weak-responder".
Accordingly, the efficacy of a cancer treatment/therapy can be determined taking account of the change in the activity/expression level of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK during the treatment/therapy. Thus, a skilled person is able to assess the efficacy of a treatment by evaluating the activity/expression level of the above marker genes at various points in time during the treatment (e.g. prior to the treatment, after start of the treatment, and subseqently in intervals during the treatment). In this particular case, it may not be necessary to compare the activity/expression level with reference values (control values) as indicated above in order to assess the efficacy of the treatment. Instead it may suffice to detect the change in the activity/expression level of the marker genes in samples obtained from a treated patient after start of the treatment.
In general, a (desired) efficacy of a treatment of a cancer described herein or susceptibility thereto is indicated/predicted, when the aberrant (Le, enhanced or decreased) activity or expression level of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK is shifted back towards the "normal level" of a (healthy)control subject/patient or to "normal level" of a defined responder ("positive control") due to/in consequence of said treatment of the cancer or susceptibility thereto.
In context of this invention, the efficacy of a treatment of the cancer defined herein is high, when the subject/patient (to be) treated responds as fast (or even faster) and as complete as a "responder", i.e. exhibits a 'typical/desired response". This means that said subject/patient reaches the "normal" level of the relevant cytological/haematological parameters and/or SRC, EPHA3, FRK, EPHA5, EPHA8, YES5 ABL2, LCK and/or BLK activity (and hence of the coiTcsponding marker gene expression level(s)) of a healthy subject/patient as fast as a "responder", i.e. in the same manner as in a "typical/desired response".
Accordingly, the efficacy of a treatment of the cancer is high, if the patient treated shows a "typical/desired response". In other words, the efficacy is high, when the activity/expression level of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK in said patient reach a "normal" acivity/level as rapidly as in a "typical/desired response". In context of this invention, the efficacy of a treatment of the cancer defined herein is moderate/low, when the subject/patient (to be) treated responds not as fast and/or not as complete as a "responder"', i.e. does not exhibit a "typical/desired response". This means that said subject/patient does not reach the ''normal" level of the relevant cytological/haematological parameters and/or activity of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL25 LCK and/or BLK (and hence of the corresponding marker gene expression level(s)) of a healthy subject/patient as complete and/or as fast as a "responder", i.e. not in the same manner as in a "typical/desired response".
Accordingly, the efficacy of a treatment of the cancer is low, if the patient treated does not show a ''typical/desired response". In other words, the efficacy is low, when the activity/expression level of SRC, EPHA3. FRK, EPHA5, EPHA8, YES. ABL2. LCK and/or BLK in said patient reaches a "norma!'" activity/level slower than in a "typical/desired response".
In context of this invention, there is no efficacy of a treatment of the cancer at all, when the siibjectφatient (to be) treated does not respond at all.
Particularly, when the efficacy of a treatment of the cancer as monitored/predicted in context of this invention is moderate/low or when there is no such efficacy, a change of the (planned) therapy/treatment might be and should be considered.
In an alternative embodiment, of the herein disclosed means, methods and uses of monitoring/predicting, the reference activity of SRC, EPHA3. FRK1 EPHA5. EPHA8, YES5 ABL2, LCK and/or BLK of a "control subject/patient'" can be replaced by a '"own" reference activity or expression level sample of SRC. EPHA3. FRK. EPHA5, EPHA8, YES, ABL2. LCK and/or BLK from the subject/patient to be treated itself. Such an "own'" reference sample may be obtained prior to (or at the beginning of) the treatment/therapy. In this specific case, the '"control subject/patient" would be the subject/patient to be treated itself. The efficacy of the cancer treatment would then be assessed on the basis of how the activity or expression level of SRC, EPHA3, FRIC5 EPHA5. EPHA 8, YES, ABL2, LCK and/or BLK changes during treatment/therapy compared with said particular "reference activity/ expression level" of SRC, EPHA3, FRK, EPHA5, EPHA8, YES5 ABL2, LCK and/or BLK. The more significant and/or faster said change is, the more efficacious is the treatment/therapy .
As mentioned above, the efficacy of a treatment of the cancer is assessed in accordance with specific embodiments of this invention, on the basis that the activity/expression level of SRC.
EPHA3. FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK is different from a certain or given '"reference activity/reference expression level" of SRC, EPHA3. FRK, EPHA5,
EPHA8, YES, ABL2, LCK and/or BLK. Thereby, it is clear that "different'" means higher or lower, depending on whether the cancer comes along with an up- or down-regulated activity/expression level SRC, EPHA3, FRK5 EPHA5, EPHA8, YES, ABL2, LCK and/or
BLK.
In accordance with the above, the efficacy of a treatment of the cancer is assessed based on the comparison of the activity/ expression level of SRC, EPHA3, FRK5 EPHA5, EPHA8, YES, ABL2, LCK and/or BLK in a sample obtained from a patient with a reference (control) activity/expression level.
In this context, '"different", ''higher" or "lower" means different, higher or lower than the normal (range of) activity/expression level of SRC. EPHA3, FRK. EPHA5, EPHA8, YES, ABL2. LCK and/or BLK. For example, different, higher or lower means at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 7 fold, at least 10 fold, at least 15 fold, at least 25 fold, at least 50 fold, at least 100 fold or at least 200 fold different, higher or lower, wherein the higher values are preferred.
Whether, in which direction (i.e. higher or lower) and/or to which extend the activity/expression level of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK described herein differs from its corresponding "reference activity/reference expression level", can easily be deduced by the skilled person based on the teaching provided herein and the common general knowledge. Accordingly, it is possible to assess for each marker gene particularly described herein, whether a given difference between the reference activity/reference expression level of SRC5 EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK and the activity/expression level of SRC, EPHA3. FRK. EPHA5, EPHA8, YES. ABL2, LCK and/or BLK in a subject/patient to be assessed in accordance with this invention exists. This diagnostic assessment may be. in accordance with this invention the diagnostis for a NSCLC.
As explained above, a certain type of cancer can be associated with increased activity/expression level of any one of the above marker genes or with a decreased increased activity/expression level of any one of the above marker genes. Since a skilled person will be aware of reference acitivity/expression levels of the marker genes (e.g. in a healthy person), he will be readily in the position to determine whether the activity/expression level of any one of the above marker genes is increased or decreased when compared to the reference acitivity/expression level.
As mentioned, in context of the means, methods and uses of monitoring/predicting as disclosed herein, the extent of the difference between the activity/expression level of SRC, EPHA3, FRK. EPHA5. EPHA8, YES. ABL2, LCK and/or BLK and corresponding reference activity/reference expression level of SRC, EPHA3, FRK, EPHA5. EPHA8. YES, ABL2. LCK and/or BLK is indicative for the (predicted) efficacy of the therapy/treatment of a the cancer (to be) performed.
For example, if the control subject/patient is a responder. or the activity/expression level of SRC, EPHA3, FRK, EPHA5, EPHAS. YES, ABL2, LCK and/or BLK is evaluated on the basis of a 'typical/desired response", a low difference (at a certain point in time) indicates a high efficacy.
Accordingly, a responder shows expression/ activity of SRC, EPHA3. FRK. EPHA5, EPHA8, YES, ABL2, LCK and/or BLK similar to a typical/desired response, wherein a typical/desired response Is indicative for a successful cancer treatment/therapy.
As explained herein above, a responder may show reduced or increased expression/ activity of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2. LCK and/or BLK5 depending on the type of cancer.
If the cancer is, for example, characterised by a high expression/activity of at least one of the marker genes and if expression/ activity of SRC. EPHA3. FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK, is reduced in a responder to a similar extent as in a typical/desired response, the cancer treatment/therapy can be considered successful. Vice versa, if the cancer is characterised by a low expression/activity of at least one of the marker genes and if expression/ activity of SRC, EPHA3, FRK5 EPHA5, EPHA8, YES, ABL2, LCK and/or BLK, is increased in a responder to a similar extent as in a typical/desired response, the cancer treatment/therapy can be considered successful.
In other words, if the difference between expression/ activity of SRC5 EPHA3, FRJC5 EPHA5, EPHA8, YES, ABL2, LCK and/or BLK in a responder and in a typical/desired response is low, such a low difference indicates a successful treatment/therapy (i.e. a high efficacy in the treatment/therapy) .
For example, if the control subject/patient is a non-responder, or the activity/expression level of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK is evaluated on the basis of a reference activity/reference expression level of SRC, EPHA3, FRK, EPFIA5, EPHA8, YES, ΛBL2, LCK, and/or BLK obtained form the subject to be treated prior to/at the beginning of a therapy/treatment of NSCLC, a high difference (at a certain point in time) indicates a high efficacy.
As explained above, a control sample can be obtained from a non-responder or can be obtained prior to/at the beginning of a therapy /treatment of a cancer. Accordingly, if the difference between expression/ activity of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK in a responder (or similarly in a typical/desired response) compared to said control is high, such a high difference indicates a successful treatment/therap}* (i.e. a high efficacy in the treatment/therapy), in other words, a responder shows a reduced expression/ activity of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK compared to high expression/ activity of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK in a control, when the cancer is associated with such a high expression/activity. In this context, the difference between the expression/ activity of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK in a responder and the control should be high. The same explanations apply mutatis mutandis in context of the treatment of cancer associated with a low expression/ activity of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK, wherein the expression/ activity of SRC, EPHA3, FRK. EPHA5. EPHA8, YES. ABL2, LCK and/or BLK is increased in a responder compared to a control.
As can be deduced from the above, the reference activity /reference expression level of SRC, EPHA3, FRK. EPHA5. EPHA8, YES5 ABL2, LCK and/or BLK may be taken at the day of diagnosis, once the therapy/treatment is initialed, in between and/or during therapy/treatment, either from the subject/patient to be treated itself or from a corresponding control subject/patient (healthy/re sponder/non~responder). The reference activity/reference expression level of SRC. EPHA3, FRK. EPHA5, EPHAS5 YES5 ABL2, LCK and/or BLK may be determined at the same or at a different point in time than the activity/expression level of
SRC5 EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK, for example with respect to the course of the therapy/treatment.
Accordingly, the reference activity/expression level of SRC, EPHA3, FRK5 EPHA5, EPHA8, YES. ABL2, LCK and/or BLK can be determined in a control sample obtained from a patient (healthy tissue) or healthy person at the same time or at a different time when the cancer sample is obtained from said patient.
If the reference activity/reference expression level of SRC. EPHA3, FRK, EPHA5. EPHA8, YES, ABL2, LCK and/or BLK is obtained from a control subject/patient different from the subject/patient to be treated, it is preferred that the reference activity/reference expression level of SRC. EPHA3, FRIC. EPHA5. EPHA8. YES, ABL2. LCK and/or BLK is determined at the same point in time during therapy/treatment. In particular, if the reference activity/reference expression level of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK is obtained from the subject/patient to be treated itself, the reference activity/reference expression level of SRC, EPHA3, FRK, EPHA5. EPHAS5 YES. ABL2. LCK and/or BLK should be determined at a different point in time during therapy/treatment to allow comparison, for example, at the beginning of (or prior to) the therapy/treatment.
In general, activity/expression level of SRC5 EPHA3. FRK. EPHA5, EPHA8, YES. ABL2. LCK and/or BLK as described herein can be determined once or. preferably, several times. For example, activities/expression levels of SRC. EPHA3, FRK, EPHA5. EPHAS5 YES, ABL2, LCK. and/or BLK can be determined on a daily, weekly, monthly or yearly basis during therapy/treatment. Commonly, the requirements of corresponding studies would be met, if the frequency of determining activity/expression level of SRC5 EPHA3, FRK, EPHA5, EPHA8, YES. ABL2, LCK and/or BLK decreases during process of therapy/treatment. Non- limiting examples of schemes of determining activities/expression levels of SRC, EPHA3, FRK, EPFIA5, EPHA8, YES, ABL2, LCK and/or BLK in accordance with this invention are provided herein.
It is of note that the skilled person is readily in the position to elect (a) suitable control patient(s)/subject(s) and the point(s) in time when the (reference) activity/(reference) expression levels of SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK are determined for each individual setup of the means, methods and uses provided.
In one embodiment, the present invention relates to the use of a (transgenic) cell or a (transgenic) non-human animal having at least one amplified marker gene as defined herein for screening and/or validation of a medicament for the treatment of non-small cell lung cancer. The term "cell" as used in this context may also comprise a plurality of cells as well as cells comprised in a tissue. A cell to be used may, for example, be a primary tumor cell. The tumor cell or cell to be used in the screening or validation method may be obtained from samples from a (transgenic) non-human animal suffering from non-small cell lung cancer. The tumor cell or cell may also be obtained from patient samples (e.g. biopsies), m particular a biopsy/biopsies from a patient/subject suffering from non-small cell lung cancer. Accordingly, the tumor cell or cell may be a human tumor cell or cell. Again, such a cell to be used in the present screening or validation methods may be comprised in a tissue or tissue sample, like in a sample biopsy.
The used non-human animal or cell may be transgenic or non transgenic. "Transgenic"' in this context particularly means that at least one of the marker genes as described or defined herein is over- or under-expressed or has a higher or lower activity. For example, if dasatmib is to be screened and/or validated, it is preferred that such marker genes as SRC. EPHA3, FRK. EPHA5, EPHA8, YES, ABL2, LCK and/or BLK are over-expressed or have a higher activity.
'Transgenic" in this context may also mean that SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK is over- or under-expressed, and/or that the SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK-activity in the transgenic non-human animal or a transgenic cell is enhanced or decreased. It is also envisaged in this context that SRC5 EPHA3, FRK. EPHA5. EPHA8, YES, ABL2. LCK and/or BLK is under-expressed, and/or that the SRC, EPHA3, FRK, EPHA5, EPHA8, YES, ABL2, LCK and/or BLK-activity in the transgenic non-human animal or a transgenic cell is decreased.
A preferred (transgenic) non-human animal or (transgenic) cell in context of the invention suffers from NSCLC for the treatment of which the medicament is to be screened and/or validated. For example, if a medicament for non-small lung cancer is to be screened and/or validated, the (transgenic) non-human animal or (transgenic) cell is particularly intended to suffer from non-small lung cancer, i.e. to have, for example, an increased SRC, EPHA3, FRK. EPHA5. EPHA8. YES, ABL2, LCK and/or BLK activity and/or increased expression level of, for example, SRC. EPHA3. FRK, EPHA5. EPHA8. YES5 ABL2, LCK and/or BLK.
The term '"transgenic non-human animal" or "transgenic cell" as used herein refers to a non- human animal or cell, not being a human, that comprises genetic material different from the genetic material of a corresponding wild-type animal/cell, ""Genetic material"' in this context may be any kind of a nucleic acid molecule, or analogues thereof, for example a nucleic acid molecule, or analogues thereof as defined herein. "Different'' in this context means additional or fewer genetic material with respect Io the genome of the wild-type animal/cell and/or rearranged genetic material, i.e. genetic material present at a different locus of the genome with respect to the genome of the wild-type animal/cell. An overview of examples of different expression systems to be used for generating transgenic cell/animal is, for instance, contained in Methods in Enzymology 153 (1987). 385-516. in Bitter et al. (Methods in Enzymology 153 (1987). 516-544) and in Sawers et al. (Applied Microbiology and Biotechnology 46 (1996). 1- 9), Billman-Jacobe (Current Opinion in Biotechnology 7 (1996), 500-4), Hockney (Trends in Biotechnology 12 (1994), 456-463), Griffiths et al.. (Methods in Molecular Biology 75 (1997), 427-440).
In a preferred embodiment, the (transgenic) non-human animal or (transgenic) cell is or is derived from a mammal. Non-limiting examples of the (transgenic) non-human animal or derived (transgenic) cell are selected from the group consisting of a mouse, a rat, a rabbit, a guinea pig and a Drosophila. Preferably, the (transgenic) cell in accordance with this invention may be an animal cell, for example, a non-human animal cell. However, also human cells are envisaged to be employed as cells in context of the present invention. In a non limiting example, such cell may be an embryonic stem cell (ES cell), particularly a non-human animal ES, like, for example, a mouse or rat ES cell. The (transgenic) cell as described herein, particularly the ES cell, may also be used for generating the (transgenic) non-human animal as described herein. The ES cell technology for generating transgenic animals is well known in the art and for example is described in Pirity et.al.( Methods Cell Biol, 1998, 57:279).
Generally, the (transgenic) cell may be a prokaryotic or eukaryotic cell. For example, the (transgenic) cell, may be a bacterial, yeast, fungus, plant or animal cell. In general, the transformation or genetically engineering of a cell with a nucleic acid construct or vector can be carried out by standard methods, as for instance described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor. NY5 USA; Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 1990.
The (transgenic) non-human animal or (transgenic) cell as described or defined in context of this invention is particularly useful in methods for screening and/or validation of a medicament for the treatment of cancers as defined and described herein.
These screening methods may, in particular, performed in vivo using, for example, (transgenic) animals as described herein (e.g. rats, mice and the like) and/or animals comprising (a) NSCLC cell(s), (a) tissue(s) or (a) ceil culture(s). Said (a) cell(s), (a) tissue(s) or (a) cell culture(s) may, for example, be obtained/derived from (a) NSCLC tumor cell(s)/tumor(s). In particular, said (a) cell(s), (a) tissue(s) or (a) cell culture(s) may be obtained from a subject/patient suffering from a NSCLC. These in vivo screening methods may in particular comprise measuring and determining differences in tumor volume, for example, in the (transgenic) animals described herein above.
Accordingly, the present invention also relates to a method for screening and/or validation of a medicament for the treatment of a cancer. Said method may comprise the steps of a) administering to a (transgenic) non-human animal or (transgenic) cell as defined herein said medicament to be screened/validated; b) determining in (a sample from) said animal or cell the activity or expression level of SRC, EPHA3, FRK, EPHA5. EPHA8, YES. ABL2. LCK and/or BLK gene in accordance with this invention: c) comparing the activity or expression level of said at least one marker gene determined in b) with a reference activity or reference expression level of SRC, EPHA3, FRK, EPHA5, EPHA8, YES. ABL2, LCK and/or BLK, said activity or said expression level optionally determined in (a sample from) a control (transgenic) non-human animal or (transgenic) cell as defined herein to which said medicament to be screened has not been administered; and d) selecting said medicament when said activity/expression level of SRC. EPHA3, FRK. EPHA5. EPHA8, YES, ABL2. LCK and/or BLK determined in b) differs from said reference activity/expression level determined in c).
The corresponding definitions and descriptions provided above, for example with respect to ''marker gene'\ '"therapy/treatment", "efficacy'". "NSCLC" or "susceptibility'" thereto, "(control) subjectφatient'", '"(transgenic) non-human animal" or "(transgenic) cell"', "expression level'", "'reference expression level" etc., apply here, mutatis mutandis. Particularly the relevant definitions and descriptions provided above with respect to "control subject/patient" also apply to the "control (transgenic) non-human animal" or "(transgenic) cell", mutatis mutandis.
In context of this invention, "'screening and/or validation of medicaments"1 means, on the one hand, whether a given set of compounds comprises one or more compound(s) that can function as (a) medicament(s). and/or, on the other hand, whether (a) given compound(s) can function as (a) medicament(s). It is particularly intended that the medicaments to be screened and/or \alidated in context of this invention are medicaments for the treatment, prevention and/or amelioration of a cancer as defined herein.
The skilled person is readily in the position to put this embodiment of the present invention into practice For example, by doing so, the compound(s)/medicament(s) to be screened and/or validated may be administered to the non-human (transgenic) animal or cell described herein, and. afterwards (for example after a certain period of time sufficient to allow a compound to effect on a cancer as described herein), it is analyzed whether the cancer, or a symptom thereof, of said animal/cell is ameliorated.
The present invention also relates to a kit useful for carrying out the method or used of this invention. In a preferred embodiment, said kit comprises oligonucleotides or polynucleotides capable of detecting the amplification status of at least one gene selected from the group of SRC, EPHA3, FRK3 FPHA5 EPH A8. YES. ABL2. LCK and BLK.
For example, said kit may comprise (a) compound(s) required for specifically determining the amplification status of at least one gene of SRC. EPHA3, FRK. EPHA5. EPHA8, YES, ABL2. LCK and/or BLK. In a preferred embodiment, the kit (to be prepared in context) of this invention is a diagnostic kit.
In a particularly preferred embodiment of the present invention, the kit (to be prepared in context) of this invention or the methods and uses of the invention may further comprise or be provided with (an) instruction manual(s). For example, said instruction manual(s) may guide the skilled person (how) to determine amplification status of at least one gene of SRC, EPHA3, FRK. EPHA5, EPHA8, YES. ABL2, LCK and/or BLK., i.e. (how) to diagnose the susceptibility to dasatiπib. Particularly, said instruction manual(s) may comprise guidance to use or apply the herein provided methods or uses.
The kit (to be prepared in context) of this invention may further comprise substances/chemicals and/or equipment suitable/required for carrying out the methods and uses of this invention. For example, such substances/chemicals and/or equipment are solvents, diluents and/or buffers for stabilizing and/or storing (a) compound(s) required for specifically determining the amplification status of at least one gene of SRC, EPHA3. FRK. EPHA5, EPHA8. YES. ABL2, LCK and/or BLK.
The present invention also relates to a combination of cell lines selected from the group consisting of A427, A549, CaIu-I, Calu-3. Calu-6. H1299, H1355, H1395. H1437, H1563. H1568, H1648, H1650, H1666. H1734. H1755. H1770, H1781, H1792. H1819, H1838, H1915. Hl 944. H1975. Hl 993. H2009, H2030, H2052. H2087, H2110, H2122, H2126, H2172, H2228, H23, H2347, H2444, H28, H358, H441, H460, H520, H522, H596, H647, H661, H820, HCC2935, HCC4006, HCC827, SK-LU-I5 EKVX5 H322M, HOP-62, HOP-92, Colo6995 DV-90, HCC15, HCC366, HCC44, HCC78. LCLC103H, LCLC97TM1 and LouNH91.
These combinations of cell lines should comprise at least 3, 4, 5, 6. 7. 85 9, 10, 11, 12, 13. 14, 15, 20, 25. 30, 35, 40, 45. 50. 55 or 60 of the cell lines as provided herein above. In particular the combination of eel! lines should comprise at least 60 cell lines as provided herein above These cell lines, and in particular their combination are particular useful as model systems for the assessment of any potential drug susceptibility. This usefulness of these specifically selected cell lines (in combination) for the assessment of drug susceptibility is demonstrated in the appended example. All of the mentioned cell lines are available to the person skilled in the ait and the public from cell depositary institutions, in particular ATCC or DMSZ as illustrated in Figure 19 where also corresponding accession numbers for these cells are provided.
These cell lines are well known in the art. Detailed information on these markers is also given in the Table shown in Figure 19,
Also the use of the combination of cell lines as defined herein above for predicting susceptibility to a drug, in particular, dasatinib, is disclosed herein. The combination of cell lines may be useful for predicting the susceptibility to a drug, in particular to dasatinib or responsiveness of a (mammalian) tumor cell or cancer cell to treatment with a drug, in particular dasatinib. It may also be useful in predicting whether a patient is likely to respond to or is sensitive to a drug, in particular dasatinib. Corresponding means and methods for predicting susceptibility/responsiveness and the like are well known in the art and also described herein above. Accordingly, a skilled person will know how to use such a combination of cell lines in this context. The present invention is further described by reference to the following non-limiting figures and examples.
The Figures show:
Figure 1. The NSCLC cell line collection
Overview over the NSCLC cell line collection used in the study including providers and ni orph opatho Io gical d etail s.
Figure 2. Significant lesions in lung cancer
Summary of regions with significant copy number alterations as defined by GISTIC in the cell line panel and a primary lung cancer panel.
Figure 3. Genomic validation of 84 NSCLC cell lines
(A) Chromosomal copy number changes of NSCLC cell lines are plotted against those of 371 primary NSCLC tumors. The q- values of the false discovery rates for each alteration (x-axis) are plotted at each genome position (y-axis). Figure 3Al , chromosomal losses (cell lines, grey; primary tumors, black); Figure 3A2right panel, chromosomal gains (cell lines, greycurves; primary tumors, blackcurves). Genomic positions corresponding to even- numbered chromosomes are shaded; dotted lines indicate centromeres; light grey line, q- value cutoff (0.25) for significance. Genes marked in bold represent known targets of mutation in lung adenocarcinoma. Putative targets near peaks are given in parentheses. Genes identified by GlSTlC using stringent filtering criteria for peak border detection are marked by asterisks. (B) Oncogene mutations present in NSCLC cell lines (black bars) are plotted according to their relative frequencies in comparison to primary lung tumors (empty bars) (Aviel-Ronen et al. 2006; Bamford et al, 2004: Sharma et al . 2007; Thomas et al. 2007).. (C) Hierarchical clustering of significant lesions defined by GISTIC and oncogene mutations using the reciprocal co-occurance ratio as distance measure and average linkage of clusters. The distance of the clusters is reflected in the length of the branches. Note that mutations in EGFR and KRAS occur in a mutually exclusive fashion, while EGFR mutation and amplification are found in the same cluster. (D) Transcriptional profiles from renal primary tissue (grey) and cell lines (black); lung cancer primary tissue (dark grey) and cell lines (light black); lymphoma primary tissue (light black) and lymphoma cell lines (light grey) were analyzed by hierarchical clustering. To reduce noise, probesets were filtered prior to clustering (Coefficient of variation from 1.0-10.0, present call rate 20%; absolute expression > 100 in >20% of samples).
Figure 4. Profiles of aberrations in glioma, melanoma and lung cancer (A) Chromosomal copy number changes of KSCLC cell lines are plotted against those of primary gliomas. Two separate figures are given for deletions (left panel, NSCLC cell lines in black, gliomas in grey) and amplifications (right panel, NSCLC cell lines in light black, gliomas in dark grey). (B) Chromosomal copy number changes of NSCLC cell lines are plotted against those of primary melanomas (NSCLC cell lines in red respectively blue as above, melanoma short term cultures in purple). (C) Genomic similarity was analyzed by computing correlations of GlSTIC q-values for each SNP between NSCLC cell lines and the indicated cancer entity primary lung cancer, ovarian cancer, glioma, melanoma cell culture samples, normal tissues and a randomly split subset of NSCLC cell lines.
Figure 5, Robustness of pfaenotypic properties of mutated EGFR lung cancer ceils in vivo (A) EGFR mutations induce substantial changes in gene expression as measured by principal component analysis. The first two principal components clearly distinguish cell lines with mutated (mt) EGFR (white squares) and wildtype EGFR (wt) (black dots) (PC; total 54; cumulative variance of first 2 principal components = 24.1 : %: Iogio]eigenvector| is given). (B) The signature of FGFR-mutatod cell lines (Fold change >2; absolute difference >FC2; 100, p<0.01) was used for hierarchical clustering of 123 primary adenocarcinomas (Bhattacharjee ei ah, 2001 ) annotated for the presence (EGFR mt) or absence (EGFR wt) of EGFR mutations. All EGFR-muiant samples were grouped within one cluster. (C) All 123 primary adenocarcinomas with known EGFR mutation status were grouped using RNA transcripts identified in the cell line panel to be differentially expressed between EGFR mutated (mt) and EGFR wildtype (wt) cell lines. EGFR mutant tumors were excluded from survival analyses. (D) The association between presence (amplification, green: mutation, red; deletion, yellow) of genetic lesions to erlotinib sensitivity was analyzed by a /-test and Fisher's exact test. Only EGFR mutations and amplifications can be considered statistically significant if common methods of p-value adjustment are applied (data not shown). Figure 6. Phenotypic properties of primary tumors are conserved in eriotiEib-sensitive lung cancer cells.
(A) Hierarchical clustering of primary lung adenocarcinomas was performed using genes identified as being differentially expressed in erlotinib-sensitive versus erlotinib-resistant NSCLC cell lines. Whitebars represent EGFR-mxAsnt tumors. (B) The EGFR mutation signature published by Choi et al. PLoS ONE 2: el 226 (2007) was used to perform hierarchical clustering of primary lung adenocarcinomas. Red bars represent £GFi?-mutant tumors.
Figure 7. Raw sensitivity data for screened compoimds
Overview over the half maximal inhibitory concentrations (ICso values; [μM]) derived form high-throughput cell line based screening and analysis of the respective preimage under the kill curve for each cell line and each compound.
Figure 8. Multi-lesion predictors of sensitivity
Multi-Jesion predictors of sensitivity tested with the KNN method, Fisher's exact test and t- test are displayed. Only significant predictors are displayed for two different GLAD thresholds.
Figure 9. Characterization of compounds used in the screen
Displayed are the chemical structures, the development phase and the main known targets for all compounds used in the screening experiments
Figure 10. Sensitivity profiles of compounds determined by high-througliput cell line screening
The half-maximal inhibitory concentrations (y-axis; IC50 values) for 1 1 compounds are shown for the entire collection of NSCLC cell lines (individual cell lines, x-axis). Due to the fact that rapamycin typically fails to completely abrogate cellular proliferation (O'Reilly et al, 2006)v the 25%-inhibitory concentration is shown for this compound. Bars represent IC50 respectively IC25 values (y-axis) throughout the cell line collection (x-axis) ranked according to sensitivity. The maximum concentration is assigned to the IC50 resp. IC25 value (10 μM for 17- AAG, erlotinib, vandetanib, sunitinib and PDl 68393, 30 μM for SU-11274 and dasatinib. 60 μM for VX-680, 90 μM for purvaianol and UO 126) for resistant cell lines. Figure 11. Hierarchical clustering of compound activity uncovers mutated EGFR as a target for dasatiϊiib activity.
(A) Displayed are the IC50 values (red - high compound activity; white - low compound activity) after logarithmic transformation and normalization with the mean of the respective profile for all compounds (x-axis) and all cell lines (y-axis). The presence (black dot) of absence (grey dot) of relevant lesions is annotated in the right panel. EGFR inhibitors form a distinct subcluster, where EGFR-mutaXed samples show the highest degree of sensitivity. (B) Correlation coefficients (r2; red - high correlation; white - low correlation) of the IC50 values after logarithmic transformation versus chromosomal gain (amp) and loss (del) as well as oncogene mutations (mut); copy numbers changes in regions defined by GISTIC were dichotomized and merged together with the binary mutation data. Putative target genes inside (#) and bordering (*) the region defined by GISTIC are annotated. Again, EGFR inhibitors are grouped in a separate cluster. (C) upper panel: Cry stalio graphically determined binding mode of erlotinib (grey sticks) to wild-type EGFR. Dasatinib represented as white to dark grey ballandsticks is modeled into the ATP binding site of EGFR. lower panel: The T790M mutation at the gatekeeper position of the ATP pocket, associated with secondary EGFR- inhibitor resistance in patients displaces both erlotinib and dasatinib from the ATP binding pocket of the kinase domain. (D) Upper panel: Ba/F3 cells expressing mutant (del Ex 19 or Exl9/T790M) EGFR were treated for 12h with the indicated concentrations of either dasatinib or erlotinib and whole-ceil Iy sales were inimunoblotted for phospho-EGFR and EGFR. Lower panel: Dose-dependent growth inhibition after 96h treatment with either dasatinib or erlotinib was assessed measuring cellular ATP content.
Figure 12. Mutated EGFR as a target for vandeta^ib activity. (A) left panel: Vandetanib represented as ballandsticks is modeled into the ATP binding site of EGFR based on its crystallographically determined binding mode with the RET kinase, right panel: The T790M mutation at the gatekeeper position of the ATP pocket, associated with secondary EGFR-inhibitor resistance in patients displaces the drug from the ATP- binding pocket. The crystographically determined binding mode of erlotinib asballandsticks is shown in both panels as an overlay for reference. (B) Ba/F3 cells expressing mutant (del ExI 9 or Exl9/T790M) EGFR were treated for 12h with the indicated concentrations of either vandetanib or erlotinib and whole-ceil lysates were inimunoblotted for phospho-EGFR and EGFR. (C) Dose-dependent growth inhibition after 96h treatment with either dasatinib or erlotinib was assessed measuring cellular ATP content.
Figure 13. Lesion-based prediction for activity of 17- AAG, UO126 and dasatinib. (A) Distribution of KRAS mutations (black columns) across the 17-AAG-sensitivity profile (IC50 values) in the NSCLC cell line collection and the NCI-60 cell line panel. Incidence of KRAS mutation and sensitivity towards 17- A AG is represented by a Fisher's exact test for both datasets. (B) Lysates of a KRAS wildtype (wt) and a KRAS mutated (Gl 2C) cell line treated with 17AAG at different concentrations were imraunoblotted for c-RAF, KRAS, cyclinDl and Akt. (C) Distribution of copy number gain at 1 q21.3 (black columns) across the UO126-sensitivity profile (ICJO values) in the NSCLC cell line collection and the hypothemycin- sensitivity profile in the NCI-60 cell line panel. Incidence of amplification of Iq21.3 mutation and sensitivity towards 17-AAG is represented by a Fisher's exact test for both datasets. (D) Cell lines were sorted according to their sensitivity to dasatinib (IC50<l μM; light grey ). Strikingly, most dasatinib-sensitive NSCLC ceil lines are found among those with highest copy number for members of the SRC and Ephrin receptor kinase families.
Figure 14. Validation of the target-enriched sensitivity prediction method that yielded genomic predictors of dasatinϊb sensitivity.
AU cell lines were sorted according to their sensitivity to erlotinib (IC5o<l μM; greybars). Cell lines enriched for sensitivity to erlotinib are found among those with highest copy numbers for EGFR. The contingency table for EGFR (dark grey bars) amplification and erlotinib sensitivity including the p-vaiue determined using fisher's exact test are displayed in the right panel.
Figure 15. Cluster image of the NSCLC cell lines against dasatinib using the 6 gene signature published by Huang et aL, (2007)
To evaluate the predictive value of the 6 gene signature proposed by Huang (2007). Cancer Res 67, 2226-2238, the expression levels of the respective genes were analyzed by hierarchical clustering with the dasatinib sensitivity denoted using the annotation by 0 and 1.
The samples with a similar expression profile across these genes are found in the same subcluster. Bright spots represent genes that are repressed, dark spots represent genes that are overexpressed when compared to average mRNA expression levels.
Figure 16. Prostate/breast cancer -gene signature associated with dasatinib sensitivity To evaluate the predictive value of a gene signature proposed by Huang (2007), Cancer Res 67, 2226-2238, the expression levels of the respective genes were analyzed by hierarchical clustering with the dasatinib sensitivity denoted using the annotation by 0 and 1. The samples with a similar expression profile across these genes are found in the same subcluster. Bright spots represent genes that are repressed, dark spots represent genes that are overexpressed when compared to average mRNA expression levels.
Figure 17. Ail genes associated with dasatinib sensitivity in prostate cancer
To evaluate the whole gene set signature proposed by Huang (2007), Cancer Res 67, 2226- 2238. the expression levels of the respective genes were analyzed by hierarchical clustering with the dasatinib sensitivity denoted using the annotation by 0 and L The samples with a similar expression profile across these genes are found in the same subcluster. Bright spots represent genes that are repressed, dark spots represent genes that are overexpressed when compared to average mRNA expression levels.
Figure 18. NSCLC cell ϊme panel
Figure 18 shows a list of cell lines in the initially tested NSCLC cell line panel, their respective KRAS mutations and the corresponding half-maximal inhibitory concentrations (IC50)."
Figure 19. Cell lines
Figure 19 shows a set of cell lines which are to be used in accordance with the screening methods provided herein for the identification of dings, which can be used in and -cancer treatment/anti-proliferative treatment.
Figure 20, A transgenic mouse model of KRAS-mutasit iung cancer
Transgenic mice expressing a Gl 2D mutation of KRAS specifically in the lung were generated by intranasal application of adenoviral Cre recombinase to Lox-Stop-LoxKRASG12D mice (upper panels from left to right) as described in the literature (Jackson. E. L. et al, Genes Dev 15:3243-3248 (2001 ): Johnson L. et al. Nature. 410(6832):! 1 11-6 (2001) and Ji H. et al.. Nature. 448 (7155):807-10 (2007)). These mice develop lethal lung adenocarcinomas at high penetrance (lower panels from left to right). Note that the images were taken from the ealier publications for illustration purposes only.
Figure 21. Treatment of Los-Stop-LoxKRASG12D mice with HSP90 inhibitor Transgenic mice (as described in Figure 20) with KRAS-mutant lung tumors were treated with the HSP90 inhibitor 17-DMAG for seven days. Tumor volumes were determined by magnetic resonance imaging and shown as transthoracic images (left panels), quantified and changes in tumor volume relative to the pre-therapy images are given in percent.
The Example illustrates the invention.
Example 1: Methods and Results
Cells
Cells were obtained from ATCC (www.atcc.org), DSMZ (www.dsmz.de), from own or from other cell culture collections. Details on all cell lines are listed in Figure 1. This also contains information on providers and on culture conditions. Cells were routinely controlled for infection with my ooplasm by MycoAleit (www.cambrex.com) and were treated with antibiotics according to a previously published protocol (Uphoff and Drexler, 2005) in case of infection.
SNP Arrays
Genomic DNA was extracted from cell lines using the PureGene kit (www.gentra.com) and hybridized to high-density oligonucleotide arrays (Affymetrix. Santa Clara, CA) interrogating 238,000 SNP loci on all chromosomes except Y, with a median intermarker distance of 5.2 kb (mean 12.2 kb; www.affymetrix.com ). Array experiments were performed according to manufacturer's instructions, SNPs were genotyped by the Affymetrix Genotyping Tools Version 2.0 software. SNP array data of primary samples were obtained from the Tumor Sequencing Project (http://www.genome.gov/cancereequencing/). We appjied a novel and general method for Genomic Identification of Significant Targets in Cancer (GlSTiC) (Beroukhim et al.. 2007) to analyze the dataset. Each genomic marker is scored according to an integrated measure of the prevalence and amplitude of copy-number changes (and only prevalence in the case of LOH), and the statistical significance of each score is assessed by comparison to the results expected from the background aberration rate alone. The GlSTIC algorithm was run using two different copy number thresholds to assign genomic segments across the SNP data using the Gain and Loss Analysis of DNA (GLAD) segmentation algorithm (Hupe et al. 2004) with copy number 4 (GLAD threshold 1.0) and copy number 2.14 (GLAD threshold 0.1).
Detection of homozygous deletions
For the identification of homozygous deletions, SNP data were filtered for five coherent SNPs exhibiting a copy number loss of < 0.5. The analysis was focused on focal losses, excluding entire chromosomal arms. Information about genes located in a region of homozygous deletion was based on hgl7 build of the human genome sequence from the University of California, Santa Cruz (http://genome.ucsc.edu).
Analysis of co-occurring lesions
The analysis was performed computing ratios of observed vs. expected co-occurrence frequency of individual lesions. Hierarchical clustering of mutation data combined to a dichotomized version of quantitative copy number changes was performed using the reciprocal co-occurrence ratio as distance measure with average linkage method. As the adequate threshold for occurrence of copy number lesions depends on the overall level, of copy number alteration for that specific lesion, the sum of these ratios for three distinct thresholds was used.
Mutation detection Mutation status of known oncogene mutations in the genes EGFR, BRAF, ERBB2, PIK3CA, NRAS, KRAS, ABLl, AKT2, CDK4, FGFRl1 FGFR3, FLT3, HRAS JAK2, KIT, PDGFRA and RET was determined by mass-spectrometric genotyping (Thomas el αl., 2007).. The mutation status of these genes for all cell lines was published previously (Thomas et αl. , 2007),. In addition, the genes EGFR, BRAF1 ERBB2, PIK3CA, KRAS, TP53, STKIl, PTEN and CDKN2A were bidirectionally sequenced following PCR-amplificatioii of all coding exons.
Mutation detection for choice of appropriate therapy depending on the respective mutation has been further developed to compensate for the methodological issues connected with sequencing of tumor samples with high admixture of non-tumoral cells, in our laboratory we have therefore developed the following algorithm: if the tumor content of the tumor specimens is higher or equal than 70% estimated by conventional histomorphology, we have found Sanger dideoxy-chain-termination sequencing to be optimal in terms of cost-efficiency and sensitivity. However, when the tumor content is between 70% and 20% we have found conventional pyrosequencing as, for example, implemented in the Biotage instrument, to deliver higher sensitivity and specificity at acceptable costs. If the tumor content is lower than 20%, we have found massively parallel next-generation sequencing, as for example implemented in the Roche-454 sequencing system (Thomas et al, Nature Medicine JuI; 12(7): 852-5 2006), to be the most sensitive and accurate method in this setting. Together, this algorithm provides high sensitivity in all settings combined with maximum cost- efficiency.
Expression arrays Expression data were obtained using Affymetrix Ul 33A arrays from 54 of the cell lines. RNA extraction, hybridization and scanning of arrays were performed using standard procedures (Bhattacharjee et al, 2001 ). CEL files from U133A arrays were preprocessed using the dChip software. We compared the cell lines to primary lung cancer, renal cell carcinomas and lymphoma specimens as well as to the respective cell lines by hierarchical clustering. For comparison with expression profiles from further entities, we used lung cancer (Lu el a!. , 2006). renal cell carcinoma (Lenburg et al , 2003) (Shankavaram et al, 2007) and lymphoma (Hummel et al, 2006; Rinaldi et al, 2006) specimen datasets as published in GEO array (http://www.ncbi.nlm.nih.gov/geo/). Data were processed by standard procedures, normalization was performed in dChip (http://biosunl.harvard.edu/comp]ab/dchip/). For comparison of NSCLC cell lines (U133A) and primary tumors, we used data on adenocarcinomas from Bhattacharjee and colleagues generated on U95Av2 arrays. Genes differentially expressed between cell lines with mutated EGFR and wild type EGFR (fold change between groups >2 [90% CI], absolute difference > 100 and p<0.005) respectively between erlotinib sensitive and resistant cell lines (erlotinib sensitive (IC50 < 0.5μM) vs. erlotinib resistant (IC50 > 2μM), fold change > 1.5 [90% CI], absolute difference > 100, p<0.005) were selected. For principal component analysis, the R language for statistical computing was used. Variable transcripts were identified using the following filtering criteria: coefficient of variation in f l.9;10], 40% present call rate. The first principal component described 14.5% of the overall variance, the second 9.6% and the third 8.2%. Using a cut-off of 1400, samples were grouped according to the first principal component.
Ceϊl-based screening
Erlotinib, vandetanib and sunitinib were purchased from commercial suppliers, dissolved in DMSO and stored according to manufacturer's instructions. Cells were plated into sterile microtiter plates using a Multidrop instrument (www.thermo.com) and cultured overnight. Compounds were then added in serial dilutions. Cellular viability was determined after 9όh by measuring cellular ATP content using the CellTiter-Glo assay (www.promega.com). Plates were measured on a Mithras LB940 plate reader (www.bertholdtech.com). Half-maximal inhibitory concentrations were determined from the respective preimage under the kill curve, where the latter was smoothed according to the logistic function with the parameters appropriately chosen.
Lesion-based prediction of compound sensitivity
For lesion-based prediction of sensitivity, three different approaches were applied. First, the most sensitive and most resistant samples were chosen according to their sensitivity profile. Where the sensitivity profile of the corresponding compound did not allow a clear distinction between resistant and sensitive cell lines, groups were defined by the 25th and 75th percentiles. We used Fisher's exact test to evaluate the association between the activity of the compound and the presence of significant lesions as defined by GISTiC. For this, the ceil line panel was divided according to the presence of each lesion. The logarithmically transformed IC50 values pertinent to each group were now compared by a two-sample t-test. In order to avoid an artificially low variance the t-tests were based on & fixed variance determined as the mean of the variances that were clearly distinct from zero (>0.1).
In a next step, multi-lesion predictors of sensitivity were calculated using a KNN algorithm with a leave-one-out strategy (Golub el ai , 1999). where the same choice of samples was used as above for Fisher's exact test: For all but one sample, genetic lesions strongly discriminating between sensitive and resistant cell lines were selected and the KNN algorithm was based on these. The prediction was validated by the remaining left-out sample. The collection of features where this validation had best performance was taken as the best combined predictor to the respective compound.
For identification of the best erlotinib single gene predictor, we dichotomized (threshold=0.7) our lesion data. Cell lines with an IC50<0.07 μM were defined as sensitive. For the predictor, the same cutoff values were used. Best performance in the leave-one-cross validation was obtained using 15 features. k=3 neighbours and the cosine-based metric. Due to the problem of multiple hypotheses testing, the significance of the above t-tests as well as Fisher's exact tests should be understood in an explorative rather than confirmative sense.
In order to validate the finding that single lesions may be associated with sensitivity to a specific inhibitor, we made use of the NCI-60 cancer cell line panel (http://dtp.nci.nih. go v/mtargets/mt Jmdex.html). Since the MEK inhibitor UO 126 and the Hsp90 inhibitor 17-AAG were not covered by the collection of pharmacological data, we analyzed the association of the respective lesions to hypothemycin (MEK inhibitor) and to geldananiycin (17-AAG is a geldanamycin derivate) instead. In order to compare the association between lesions and sensitivity, the number of cell lines classified as sensitive or resistant in the lung cancer cell line panel was adapted for the NCI-60 cell line collection for the respective compounds. Significance of association was analyzed by a Fisher's exact test. Due to discordant IC^o values the cell lines HOP62 and A549 were excluded from the analysis with respect to the Hsp90-inhibitors. The thresholds for Iq21.3 amplification were set according to the overall distribution of copy number changes in the respective dataset (2.7 corresponding to 33% of the NSCLC cell lines; 2.4 corresponding to 33% of the NCI-60 collection).
To evaluate the 6 gene signature proposed by Huang et al. (2007), the expression levels of the respective genes were analyzed by hierarchical clustering (Fig. 17) with the dasatinib sensitivity denoted using the annotation by 0 and 1. The samples with a similar expression profile across these genes are found in the same subcluster. However, the dasatinib-sensitive samples did not form a specific subcluster but were distributed randomly in the columns, suggesting that the signature under consideration is not suitable for prediction of dasatinib vulnerability.
Generation of EGFR-mutant Ba/F3 cells
EGFR cDNA was subcloned into pBabe-hygro vectors. The most prevalent NSCLC-derived mutants (http://www.sanger.ac.uk/genetics/CGP/cosmic/) were introduced into the retroviral construct using site-directed mutagenesis (Quick-Change Mutagenesis XL kit; Stratagene, La Jolla, CA, USA) and virus was packed and produced as previously described (Greulich et al, 2005). Murine Ba/F3 cells were stably transduced with the retroviruses and after IL- 3 withdrawal, independently growing cells were chosen for further experiments.
Structural modeling of compound binding The crystal structure of dasatinib and vandetanib bound to the RET kinase (pdb code 2IVU (Knowles et al, 2006)) was aligned to the kinase domain of EGFR bound to erlotϊnib (pdb code IMl 7 (Stamos et al, 2002)) using PyMoI (http://www.pymol.org). Based on the structural alignment of AbI with EGFR, the binding mode for dasatinib in EGFR is identical to the dasatinib-Abl complex. The piperazine moiety of the inhibitor points out of the ATP site into the solvent while the 2-amino-thiazole forms two hydrogen bonds with the hinge region of the kinase (N 3 of the thiazole ring with the amide nitrogen of Met793 , Met318 in AbI) and the 2-amino hydrogen of dasatinib with O of Met793 (Met318 in AbI). An additional hydrogen bond can form between the side chain hydroxy! of the gatekeeper Thr790 (Thr315 in AbI) and the amide nitrogen of the inhibitor. The chloro-melhyl-phenyl ring of dasatinib binds into a hydrophobic pocket near the gatekeeper Thr790 and helix C and would clearly clash with the Met side chain of drug resistant EGFR- T790M. I vandetanib, Nl of the quinazoline scaffold forms one key hydrogen bond to the backbone of the hinge region (Met793 in EGFR, Ala807 in RET kinase). The brorao-fluoro-phenylamine moiety of vandetanib adopts a conformation similar to the ethynyl-phenylamine of erlotinib being close to the side chain of Thr790 in EGFR and Val804 in RET kinase. Figures of the structures were prepared using PyMoI.
Western blot analysis
Whole-cell lysafes were prepared in NP40 lysis buffer (50 mmol/L Tris-HCI (pH 7.4), 150 mmol/L NaCl. 1% NP40) supplemented with protease and phosphatase inhibitor I and II cocktails (www.merckbiosciences.co.uk/g.asp?f=CBC/home.html) and clarified by centrifugation. Protein concentrations were determined using the Bicinchoninic Acid Protein Assay kit (www.piercenet.com) and equivalent amounts (40-60 μg) were subjected to SDS- PAGE on 12% gels, except where indicated. Western blotting was done as described previously (Shimamura et al, 2006). Anti-EGFR, anti-phospho-EGFR (Tyr1068) and anti-pAkt antibodies were purchased from Cell Signaling Technology (Beverly. MA). Anti c-raf and anti-cyclin Dl antibody s were purchased from Santa Cruz. Anti KRAS antibody was purchased from Merck. Results
A genomicaily validated collection of NSCLC cell lines
84 NSCLC cell lines were collected from various sources (Figure 1) and formed the basis for all subsequent experiments. Cell lines were derived from tumors representing all major subtypes of NSCLC tumors, including adenocarcinoma, squamous-cell carcinoma and large- cell carcinoma.
The genomic landscape of these cell lines were characterized by analyzing gene copy number alterations using high-resolution single-nucleotide polymorphism (SNP-) arrays (250K Sty 1 ; www.affymetrix.com) We used a novel analytical algorithm called Genomic Identification of Significant Targets in Cancer (GlSTIC) to statistically distinguish biologically relevant lesions from background noise (Beroukhim el al., 2007). This method assigns a statistical score to each chromosomal marker reflecting both the mean amplitude and frequency of alterations at a given locus within a data set. The application of GISTIC revealed 16 regions of recurrent, high-level copy number gain (inferred copy number > 2.14) and 20 regions of recurrent copy number loss (inferred copy number < 1.86) (Figure 2). Overall, we identified focal peaks with a median width of 1.45 Mb (median 13.5 genes/region) for amplifications and 0.45 Mb for deletions (median 1 gene/region). These regions contain lesions known to occur in NSCLC (e.g., deletion of LRPlB (2q), FHIT (3p). CDKN2A (9ρ); amplification of MYC (Sq), EGFR (7p) and ERBB2 (17q); (Figure 3A and Figure 2). Furthermore, within broad regions of copy number gain we also identified amplification of the TITFl (14q) and TERT (5p) (Figure 3A and Figure 2), recently identified by large-scale genomic profiling of primary lung adenocarcinomas (Kendall et al, 2007: Lockwood et al., 2008; Weir et al , 2007).
Analysis of homozygous deletions (HD) as well as loss of heterozygosity (LOH) is typically hampered by admixture of non-tumoral cells in primary tumors. The purity of cell line DNA permitted identification of previously unknown HD and regions of LOH, including LOH events resulting from uniparental disomy (e.g. copy-neutral events). Regions targeted by LOH encompassed genes shown to be affected by LOH in other tumor types as well as previously unrecognized LOH targets such as TSP AN 5 (4q). LRDD (l ip), SIRT3 (l ip), NLRP6 (l ip), BCL2L14 (12p), CDK.8 (13q), BCL2L12 (18q), DAPK3 (19p), or UHRFl (19p). . In this analysis known genes such as MTAP (9ρ) and LATS2 (13q) were altered by HD (Chen el al , 2005; Schmid et al. 1998) and we found novel HD of genes such as TUBA.2 or FRK. Of note, most of these regions could also be identified in primary NSCLC tumors as deleted (Weir et al , 2007); however, inferred copy numbers only inconstantly showed LOH or HD, indicating admixture of normal diploid DNA. Thus, while a recent large-scale cancer profiling study (Weir et al.., 2007) has enabled insight into the genomic landscape of lung adenocarcinoma, the use of pure populations of tumor cells further afforded discovery of previously unrecognized regions of HD and LOH.
We next compared the profile of significant amplifications and deletions in this cell line collection with that of a set of 371 primary lung adenocarcinomas (Weir et al, 2007). This comparison revealed a striking similarity between the two data sets (Figure 3A) but not between NSCLC cell lines and gliomas or melanomas (Figure 4A and 4B). A quantitative analysis of similarity by computing correlations of tj-values confirmed the similarity of primary lung cancer and lung cancer cell line (r2= 0.77) and the lack of similarity of lung cancer cell lines and primary gliomas (Beroukhim et al, 2007) (r2= 0.44) or melanoma cell lines (Lin et al , 2008) (r2== 0.44; Figure 4C). Repeated random splitting of the lung cell line data and computation of internal similarity indicated correlations between 0.82 and 0.86 whereas we found no correlation with normal tissue (r2 = 0.0195; Figure 4C). These results demonstrate that the genomic copy number landscape of NSCLC cell lines reflects that of primary NSCLC tumors, while tumors or cell lines of other lineages show a much lower degree of similarity (Greshock et al., 2007; Jong et al, 2007).. Furthermore, the distribution of oncogene mutations in the cell lines was similar to those in primary NSCLC tumors with a high prevalence of mutations in the KRAS and EGFR genes (Aviel-Ronen et al., 2006; Baraford et al, 2004; Sharma et al, 2007; Thomas et al . 2007) and rare occurrence of P1K3CA and BRAF mutations (Figure 3B). These results further validate our cell line collection on a genetic level.
The availability of two dimensions of genetic information (chromosomal copy number alterations and mutations) from the NSCLC cell lines enabled us to analyze the interactions of both types of lesions (Figure 3C). Hierarchical clustering of lesions robustly grouped both mutations and amplification of EGFR in one subcluster (ratio Q of observed vs. expected cooccurrence: C>=4.38, p= 0.001) while KRAS mutations consistently grouped in a distinct cluster. These findings corroborate prior observations in vivo where mutations in KRAS and EGFR are mutually exclusive while EGFR mutation and EGFR amplification frequently co- occur (Kaye, 2005; Pao et al, 2005b; Sharma et al, 2007).. Moreover, these results strongly suggest that each of these mutations conditions the larger sets of genomic alterations in a specific manner.
Finally, in cluster analyses of gene expression data primary lung cancer specimens (Lu et al, 2006) and lung cancer cell lines shared one cluster (Figure 3D) while renal cell carcinomas (Lenburg et al , 2003) and lymphomas (Hummel et al, 2006) clustered in a separate group. In summary, in-depth comparative analysis of orthogonal genomic data sets of a large panel of NSCLC cell lines and primary tumors demonstrates that these cell lines reflect the genetic and transcriptional landscape of primary NSCLC tumors.
EGFR mutations define phenotypic properties of lung tumors in vitro aad in vivo Activated oncogenes typically cause a transcriptional signature that can be used to identify tumors carrying such oncogenes (BiId et al, 2006; Lamb el al . 2003). However, we consistently failed to identify a gene expression signature specific for EGFR-mutmit tumors using a annotated gene expression data set of 123 primary lung adenocarcinomas (Bhattacharjee et al, 2001) (data not shown). We therefore reasoned that the cellular purity of our cell lines might enable extracting such a signature and its translation to primary tumors. We applied principal component analyses on the variable genes and found a remarkable grouping of all EGFR mutated cell lines with a significant dissociation already in the first principal component (p= 0.0005) contributing 14.5% to the overall variance (Figure 5A), Similar results were obtained by hierarchical clustering (data not shown). Using genes differentially expressed in EGFR -mutant cell lines as a surrogate feature, all of the EGFR- mutant primary tumors were grouped in a distinct cluster when performing hierarchical clustering (Figure 5B), This result was also recapitulated when selecting genes differentially expressed in erlotinib-sensitive cell lines (Figure 6A). Furthermore, patients with a tumor expressing the signature of EGFR mutated cell lines had a better overall survival than those whose tumors did not, even when excluding £GFi?-mutant tumors (Figure 5C) as seen in vivo (Eberhard et al, 2005b). These data also point to the fact that expression signatures extracted in vitro can be used to identify biologically diverse tumors in vivo, a concept recently developed and validated (Nevins and Potti, 2007). Others have recently characterized a transcriptional signature of EGFR mutant NSCLC using a small set of cell lines (Choi et at. , 2007). However, when analyzing primary lung adenocarcinomas with the signature described by Choi et al. EGFR-mutant samples were randomly distributed across the data set (Figure 6B). This finding further highlights the importance of using large cell line collections in order to represent the overall genomic diversity of primary tumors.
In patients, tumor regression or "response'" to EGFR inhibitors such as erlotimb is correlated with mutations in the EGFR gene (Lynch el al, 2004; Paez et al, 2004; Pao et al. 2004).. To systematically identify genetic lesions associated with sensitivity to erlotinib we determined erlotinib sensitivity in all cell lines. Then, we analyzed the distribution of genetic lesions in sensitive compared to insensitive cell lines (Figure 7) and further compared the mean sensitivity of cell lines with and without the respective genetic lesion. In both analyses, EGFR mutations were the best single-lesion predictor of erlotinib sensitivity (Figure 5D, p< 0.0001). Furthermore, we found a less stringent association with amplification of EGFR; however, only EGFR mutations were significant predictors of erlotinib sensitivity when adjusting for multiple hypothesis testing using either Bonferroni or FDR-based methods (data not shown).
We next used signal-to-noise based feature selection combined with the KNN algorithm (Golub et al., 1999; Reich et al.. 2006) to build a multi-lesion predictor of erlotinib sensitivity. The best performing multi-lesion predictor comprised EGFR mutations, amplification of EGFR and lack of KRAS mutations (Figure 8) which have all been implicated in determining responsiveness of NSCLC patients to EGFR inhibitors (Cappuzzo et al . 2005;
Hirsch et al. 2005: Lynch et al , 2004: Paez et al.. 2004: Pao et al, 2004: Pao et al, 2005b; Tsao et al. 2005). These results underscore the role of EGFR mutations in conferring sensitivity to erlotinib in a systematic computational analysis involving global genetic lesion profiles. Furthermore, these findings imply that essential transcriptional and cell biology phenotypes of the original tumors are preserved in the cell lines, a necessary requirement for application of such collections as proxies in preclinical drug target validation efforts.
Differential activity of compounds in clinical development in NSCLC cell lines
Having validated the cell line collection by demonstrating their genomic and phenotypic similarity to primary NSCLC tumors, we reasoned that adding complex phenotypic data might elicit additional insights into how cancer genotypes impact cell biology phenotypes. We therefore established a high-throughput cell-line screening platform that enables systematic chemical perturbations across the entire cell line panel in a short time. In our initial pilot screening experiment we profiled all cell lines against 10 inhibitors that are either under clinical evaluation or showed high activity in preclinical models; these compounds target a wide spectrum of relevant proteins in cancer (Figure 9). We treated all cell lines with these compounds and determined IC50 values (Figure 7). The resulting sensitivity patterns (Figure 10) revealed that while some of the compounds exhibited a pronounced cytotoxic activity in a small subset of cell lines (e.g. eriotinib, vandetanib, VX-680), others were active in most of the cell lines with only a minority being resistant (e.g. 17-AA G). Only two cell lines (<2%) were resistant to all of the compounds (Figure 7) suggesting that most NSCLC tumors might be amenable to targeted treatment. Overall, these observations are highly reminiscent of patient responses in clinical trials where limited subsets of patients experience partial or complete response while the majority of patients exhibit stable disease, no change or progression. Thus, high-throughput cell-line screening may help estimate the fraction of tumors that are sensitive to a novel compound in development.
Identification of relevant compound targets by similarity profiling
As an initial approach to identify shared targets of inhibitors, we performed hierarchical clustering based on similarity of sensitivity profiles (Figure 11 A) and based on the correlation between sensitivity and genomic lesion profiles (Figure 1 IB). Eriotinib and vandetanib exhibited the highest degree of similarity pointing to mutant EGFR as the critical target of vandetanib in NSCLC tumor cells (Figure 11 A and 1 1 B). The high degree of correlation (r2=
0.91; p< 0,001;) of cell line IC50 values for both compounds as well as structural modelling of vandetanib binding in the EGFR kinase domain which revealed a binding mode identical to that of eriotinib further corroborate this notion (Figure 12A). Notably, this model predicted that binding of both compounds would be prevented by the T790M resistance mutations of
EGFR: accordingly. Ba/F3 cells expressing erlotinib-sensitizing mutations of EGFR together with T790M were completely resistant to eriotinib and vandetanib (Figure 12A and 1 ID).
In addition to vandetanib, PD 168393, a known irreversible EGFR inhibitor (Sos et al , 2008), and the SRC/ABL inhibitor dasatinib (Shah et al. 2004) shared a cluster with eriotinib (Figure HA and B). Molecular modeling of dasatinib binding to EGFR predicted a binding mode similar to that of erlotinib (Figure 11 C) with a steric clash of erlotinib and dasatinib with the erlotinib resistance mutation T790M (Kobayashi et al, 2005: Pao et al, 2005a; Yun et al. 2007) (Figure 1 1C). We formally validated EGFR as a relevant tumor cell target of dasatinib by showing cytotoxicity as well as EGFR dephosphorylation (Song et al, 2006) elicited by this compound in Ba/F3 cells ectopically expressing mutant EGFR but not in those co-expressing the T790M resistance allele (Figure 1 ID). Thus, our approach identified a relevant tumor-cell target of an FDA-approved drug using a systematic unbiased approach. Note that a trial of dasatinib in patients with acquired erlotinib resistance is currently ongoing (trial-Id: NCT00570401, http://clinicaltrials.gov/ct2/home); we predict limited activity of dasatinib in those patients in which acquired resistance is due to the T790M mutation.
Supervised learning to identify predictors for inhibitor responsiveness
As an alternative method for predicting inhibitor-responsiveness from global lesion data in a systematic fashion we applied supervised learning methods (Golub et al , 1999) as we had applied for erlotinib (see above). Applying this method we identified robust, genetic lesion- based predictors for activity of erlotinib (see above), vandetanib, PD 168393. dasatinib. VX- 680, 17-AA G and UO 126 (Figure 8) Supporting the results from our cluster analyses followed by structural modeling, mutations in EGFR were the best predictor for activity of erlotinib. PDl 68393, vandetanib and dasatinib (Figure 8).
In our initial cluster analysis we found that KRAS mutations con-elated with sensitivity to the Hsp90 inhibitor 17- AAG, a geldanamycin derivative (Figure 13A). Using our KNN-based prediction approach. KRAS mutations were also predictive of 17-AAG sensitivity (ρ~ 0.0307. Figures 13 A. Validating this observation in an independent data set, we found the distribution o^ geldanamycin sensitivity and KRAS mutation in the NCI-60 cell line panel to be strikingly similar to the one observed in our panel (p= 0.049; Figure 13A). In sensitive cell lines. 17- AAG treatment reduced protein levels of c-RAF and Akt but not KRAS (Figure 13B). Since both c-RAF and Akt are known Hsp90 clients (Basso et al, 2002; Grbovic et al , 2006), KRAS mutations may lead to dependency on activation of the Akt and RAF-MEK-ERK signaling pathways thus rendering the cells sensitive to Hsp90 inhibitors.
UO 126 is a MEK inhibitor that also showed enhanced activity in a subset of the lung cancer cell line collection. Here, the KNN-prediction approach identified chromosomal gains of Iq21.3 affecting the genes ARNT and RAB 13 were robustly associated with UOl 26 sensitivity (Fisher's exact test, p= 0.0442; Figure 13C and 14A). In order to validate this finding in an independent data set, again we made use of the NCI-60 cancer cell line panel (Shoemaker, 2006) where hypothemycin was used as a MEK inhibitor (Solit et al, 2006). This cross- platform validation revealed that Iq21.3 gain predicts sensitivity to MEK inhibition in both datasets (Fisher's exact test, p-0.042 NSCLC cell lines: p=Q.035 NCI-60 collection). Thus, cell line profiling coupled to systematic, lesion-based prediction of drug sensitivity led to predictors that could be validated in an independent data set.
Compound target gene enrichment to predict sensitivity
Amplification of target genes has been demonstrated to predict vulnerability to target-specific compounds in ERBB2 amplified breast cancer and EGFR amplified lung cancer. We therefore speculated that chromosomal copy number alterations of biochemically defined drug targets could be used for prediction of sensitivity to other tyrosine kinase inhibitors. To this end we used relevant tyrosine kinase inhibitor targets defined by the quantitative dissociation constant as determined in quantitative kinase assays (Karaman et al, 2008). As a proof of principal we tested whether copy number gain in EGFR is associated with sensitivity to erlotinib. In our systematic approach erlotinib sensitive cell lines were highly enriched for amplification (en > 3) of EGFR (p= 0.000082 ) (Figure 14). We next performed a limited screen (30 cell lines) of lapatinib. a specific inhibitor of EGFR and ERBB2, Again, we observed lapatinib sensitive cell lines to be significantly enriched in cell lines with amplified EGFR or ERBB2 genes (p=0.0265: data not shown).
Encouraged by these findings we set out to test our approach for compounds with a broad range of inhibited kinases such as dasatinib (Karaman et al , 2008). Wτe ranked cell lines according to chromosomal copy number gain at either one of the biochemically most sensitive dasatinib targets (K<?< InM). Those cell lines were significantly enriched for sensitivity to this compound (p= 0.003, Figure 13D). In particular, this predictor comprised gain at gene family members of Ephrin receptor and Src kinases suggesting that NSCLC cells harboring such lesions might be exquisitely sensitive to therapeutic inhibition of the encoded proteins. By contrast, copy number gain involving loci encoding biochemically less sensitive dasatinib targets failed to show enrichment of sensitive cell lines (data not shown). We therefore conclude that in NSCLC, copy number gain of Ephrin receptor or SRC family member genes renders the cells dependent on these kinases exposing a vulnerability to therapeutic inhibition with dasatinib.
Example 2. Animal data show that mice with KRAS-driven lung adenocarcinomas are susceptible to treatment with an HSP90 inhibitor
This in vivo experiment confirms that mice genetically engineered to develop KRAS-driven lung adenocarcinomas are susceptible to treatment with an HSP90 inhibitor.
These mice earn,' a Lox-Stop-Lox- KRAS_G12D gene. Upon administration of adenoviral Cre by nasal inhalation, these mice develop lung cancers with high penetrance, leading to rapid death from the disease. This mouse model therefore represents the most stringent and optimal model of KRAS-mutant human lung cancer and was therefore chosen for in vivo experiments; see Figure 20. Mice received 20 mg/kg/d of 17-DMAG, a geldanamycin HSP90 inhibitor with almost identical structure as 17- AAG. In vitro data confirmed that the biological effects seen with 17-AAG in the cell lines were identical to those seen with 17- DMAG (data not shown).
After only one week of treatment 2 of 3 mice showed dramatic regression of tumors as measured by MRI imaging; see Figure 21. The third mice showed a slight but insignificant reduction of tumor burden, comparable to stable disease. By contrast, untreated mice invariably show rapid tumor progression and die quickly from disease.
The present invention refers to the following nucleotide sequences:
The sequences provided herein are available in the NCBI database and can be retrieved from www.ncbi.nlm.nih.gov/sites/entrez?db=gene; Theses sequences also relate to annotated and modified sequences. The present invention also provides techniques and methods wherein homologous sequences, and also genetic allelic variants and the like of the concise sequences provided herein are used. Preferably, such "variants'" are genetic variants.
SEQ lD No. 1 : Nucleotide sequence of Homo sapiens v-abl Abelson murine leukemia viral oncogene homolog 2 (arg, Abelson-related gene) (ABL2), transcript variant c, mRKA (>gijl53266777MNM_001100108.11).
SEQ ID No. 2: Nucleotide sequence of Homo sapiens B lymphoid tyrosine kinase (BLK)5 mRNA (>gi|33469981 |refjNM_001715.2|).
SEQ ID No. 3:
Nucleotide sequence of Homo sapiens EPH receptor A3 (EPHA3). transcript variant I5 mRNA (>gi!156547090|ref]NM_005233.5|).
SEQ ID No. 4:
Nucleotide sequence of Homo sapiens EPH receptor A5 (EPHA5), transcript variant 1. mRNA (>gi|5όl 19208|reflNM_004439.4|).
SEQ ID No. 5:
Nucleotide sequence of Homo sapiens EPH receptor Λ8 (EPHA8), transcript variant 2, mRNA (>gi|5577G891 |refNMJ)0l006943.1 |)
SEQ ID No. 6:
Nucleotide sequence of Homo sapiens fyn-related kinase (FRK), mRNA (>gi|31657133 |ref]NM_002031.2|) SEQ ID No. 7:
Nucleotide sequence of Homo sapiens lymphocyte-specific protein tyrosine kinase (LCK), transcript variant 1 , mRNA (>gi j 1 12789547|refjNM_001042771.11)
SEQ ID No. 8:
Nucleotide sequence of Homo sapiens v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) (SRC), transcript variant 1, mRNA (>gϋ38202215|rei]NM_005417.3|)
SEQ ID No. 9: Nucleotide sequence of Homo sapiens v-yes-1 Yamaguchi sarcoma viral oncogene homolog 1 (YESl), mRNA (>gi|51702529|ref|NM_005433.3|)
SEQ ID No. 10: Nucleotide sequence of Homo sapiens v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), transcript variant b, mRNA (>gi|34485723jref|NM_004985.3|) The sequences are available in the NCBI database and can be retrieved from w\vw.ncbi.nlm.nih.gov/sites/entrez?db=gene
References cited:
Arao T et al.. Small in-frame deletion in the epidermal growth factor receptor as a target for ZD6474. Cancer Res 64: 9101-4. (2004)
Aviel-Ronen S et al.K-ras mutations in non-small-cell lung carcinoma: a review. Clin Lung Cancer 8: 30-8 (2006).
Bamford S et al.The COSMIC (Catalogue of Somatic Mutations in Cancer) database and website. Br J Cancer 91: 355-8 (2004).
Banerji U et al.BRAF and NRAS mutations in melanoma: potential relationships to clinical response to HSP90 inhibitors. MoI Cancer Ther 7: 737-9. (2008)
Basso AD et aLAkt forms an intracellular complex with heat shock protein 90 (Hsp90) and Cdc37 and is destabilized by inhibitors of Hsp90 function. J Biol C hem 277: 39858-66 (2002).
Beroukhim R et al.Assessing the significance of chromosomal aberrations in cancer: methodology and application to glioma.. Proc Natl Acad Sd U SA 104: 20007-12 (2007).
Bhattacharjee A et al., Classification of human lung carcinomas by mRNA expression profiling reveals distinct adenocarcinoma subclasses. Proc Natl Acad Sci U S A 98: 13790-5 (2001).
BiId AH et al., Oncogenic pathway signatures in human cancers as a guide to targeted therapies. Nature 439: 353-7 (2006).
Cappuzzo F et al., Epidermal growth factor receptor gene and protein and gefitinib sensitivity in non-small-cell lung cancer. J Nail Cancer Inst 97: 643-55 (2005). Chen CF et al, Molecular genetic evidence supporting a novel human hepatocellular carcinoma tumor suppressor locus at 13ql2.11. Genes Chromosomes Cancer 44: 320-8 (2005).
Choi K et al., Tz'anscriptional profiling of non-small cell lung cancer cells with activating EGFR somatic mutations. PLoS ONE 2: el 226 (2007).
Druker BJ et al., Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome. N Engl J Med 344: 1038-42 (2001a).
Druker BJ et al., Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med 344: 1031-7 (2001b).
Eberhard DA et al., Mutations in the Epidermal Growth Factor Receptor and in KRAS Are Predicth'e and Prognostic Indicators in Patients With Non-Small-Cell Lung Cancer Treated With Chemotherapy Alone and in Combination With Erlotinib. J Clin Oncol. (2005a)
Eberhard DA et al., Mutations in the epidermal growth factor receptor and in KRAS are predictive and prognostic indicators in patients with non-small-cell lung cancer treated with chemotherapy alone and in combination with erlotinib. J Clin Oncol 23: 5900-9 (2005b).
Garraway LA et al., Integrative genomic analyses identify MITF as a lineage survival oncogene amplified in malignant melanoma. Nature 436: 117-22 (2005).
Golub TR et al.,, Molecular classification of cancer: class discovery and class prediction by gene expression monitoring. Science 286: 531-7 (1999).
Grbovic OM et al., Raf requires the Hsp90 chaperone for stability and is degraded in response to Hsp90 inhibitors. Proc Natl Acad Sci USA l x 57-62 (2006).
Greshock J et al., Cancer cell lines as genetic models of their parent histology: analyses based on array comparative genomic hybridization. Cancer Res 67: 3594-600 (2007). Greulich H et al., Oncogenic Transformation by Inhibitor-Sensitive and -Resistant EGFR Mutants. PLoS Med 2: e313 (2005).
Heinrich MC et al.. Kinase mutations and imatinib response in patients with metastatic gastrointestinal stromal tumor. J Clin Oncol 21: 4342-9 (2003).
Hirsch FR et al., Increased epidermal growth factor receptor gene copy number detected by fluorescence in situ hybridization associates with increased sensitivity to gefitinib in patients with bronchioloalveolar carcinoma subtypes: a Southwest Oncology Group Study. J Clin Oncol 23: 6838-45 (2005).
Hostein I et al., Inhibition of signal transduction by the Hsp90 inhibitor 17-allylamino-17- demethoxygeldanamycin results in cytostasis and apoptosis. Cancer Res 61: 4003-9 (2001).
Hummel M et al., A biologic definition of Burkitt's lymphoma from transcriptional and genomic profiling. N Engl, J Med 354: 2419-30 (2006).
Hupe P et al., Analysis of array CGH data: from signal ratio to gain and loss of DNA regions. Bioinformatics 20: 3413-22 (2004).
Jong K et al., Cross-platform array comparative genomic hybridization meta-analysis separates hematopoietic and mesenchymal from epithelial tumors. Oncogene 26: 1499-506 (2007).
Karaman MW et al., A quantitative analysis of kinase inhibitor selectivity. Nat Biotechnol 26: 127-32 (2008).
Kaye FJ et al., A curious link between epidermal growth factor receptor amplification and survival: effect of "allele dilution" on gefitinib sensitivity? J Natl Cancer Inst 97: 621-3 (2005). Kendall J et al.. Oncogenic cooperation and co amplification of developmental transcription factor genes in lung cancer. Proc Natl Acad Sci USA 104: 16663-8 (2007).
Knowles PP et al,, Structure and chemical inhibition of the RET tyrosine kinase domain. J Biol Chem 281: 33577-87 (2006).
Kobayashi S et al., EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med 352: 786-92 (2005).
Lamb J et al., Cell 114: 323-34 (2003).
Lenburg ME et al., Previously unidentified changes in renal cell carcinoma gene expression identified by parametric analysis of microarray data. BMC Cancer 3: 31 (2003).
Lin WM et al., Modeling genomic diversity and tumor dependency in malignant melanoma. Cancer Res 68: 664-73 (2008).
Lockwood WW et al.. DNA amplification is a ubiquitous mechanism of oncogene activation in lung and other cancers. Oncogene (2008).
Lu Y et al., A gene expression signature predicts survival of patients with stage I non-small cell lung cancer. PLoS Med 3: e467 (2006).
Lynch TJ et al., Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 350: 2129-39 (2004).
McDermott U et al.. Identification of genotype -correlated sensitivity to selective kinase inhibitors by using high-throughput tumor cell line profiling. Proc Natl Acad Sci U S A 104: 19936-41 (2007).
Neve RM et al., A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell 10: 515-27 (2006). Nevins JR et al.. Mining gene expression profiles: expression signatures as cancer phenotypes. Nat Rev Genet 8: 601-9 (2007).
O'Reilly KE et al., mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt. Cancer Res 66: 1500-8 (2006).
Paez .TG et al., EGFR mutations in lung cancer: con-elation with clinical response to gefitinib therapy. Science 304: 1497-500 (2004).
Pao W et al., EGF receptor gene mutations are common in lung cancers from "never smokers" and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci US A U)U 13306-1 1 (2004).
Pao W et al., Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med 2: e73 (2005a).
Pao W et al.. KRAS mutations and primary resistance of lung adenocarcinomas to gefitinib or erlotinib. PLoS Med 2: el7 (2005b).
Papadopoulos N et al.,The role of companion diagnostics in the development and use of mutation -targeted cancer therapies. Nat Biotechnol 24: 985-95 (2006).
Reich M et al., GenePattern 2.0. Nat Genet 38: 500-1 (2006).
Rinaldi A et al., Genomic and expression profiling identifies the B-celi associated tyrosine kinase Syk as a possible therapeutic target in mantle cell lymphoma. Br J Haematol 132: 303- 16 (2006).
Schmid M et al., Homozygous deletions of methylthioadenosine phosphorylase (MTAP) are more frequent than pl6INK4A (CDKN2) homozygous deletions in primary non-small cell lung cancers (NSCLC). Oncogene 17: 2669-75 (1998). Shah NP et al., Overriding imatinib resistance with a novel ABL kinase inhibitor. Science 305: 399-401 (2004).
Shankavaram UT et al.. Transcript and protein expression profiles of the NCI-60 cancer cell panel: an integromic microarray study. MoI Cancer Ther 6: 820-32 (2007).
Sharma SV et al., Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer 7: 169-81 (2007).
Sharp S et al., Inhibitors of the HSP90 molecular chaperone: current status. Adv Cancer Res 95: 323-48 (2006).
Shendure J et al., Advanced sequencing technologies: methods and goals. Nat Rev Gene! 5: 335-44 (2004).
Shimamura T et al.. Non-small-cell lung cancer and Ba/F3 transformed cells harboring the ERBB2 G77όinsV_G/C mutation are sensitive to the dual-specific epidermal growth factor receptor and ERBB2 inhibitor HKI-272. Cancer Res 66: 6487-91 (2006).
Shoemaker RH.The NCI60 human tumour cell line anticancer drug screen. Nat Rev Cancer 6: 813-23 (2006).
Slamon DJ et al.Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N EnglJ Med 344: 783-92 (2001).
Solit DB et al.BRAF mutation predicts sensitivity to MEK inhibition. Nature 439: 358-62 (2006).
Solit DB and Rosen NHsp90: a novel target for cancer therapy. , Curr Top Med C hem 6: 1205-14 (2006). Song L et al.Dasatinib (BMS-354825) selectively induces apoptosis in lung cancer cells dependent on epidermal growth factor receptor signaling for survival. Cancer Res 66: 5542-8 (2006).
Sos ML et al. Expression of signaling mediators downstream of EGF-receptor predict sensitivity to small molecule inhibitors directed against the EGF-receptor pathway. J Thorac Oncol 3: 170-3 (2008).
Stamos J et al. Structure of the epidermal growth factor receptor kinase domain alone and in complex with a 4-anilinoquinazoIine inhibitor. J Biol Chem 277: 46265-72 (2002).
Thomas RK et al. High-throughput oncogene mutation profiling in human cancer. Nat Genet. (2007).
Tracy S et aI.,Gefitinih induces apoptosis in the EGFRL858R non-small-cell lung cancer cell line H3255. Cancer Res 64; 7241-4 (2004).
Tsao MS et al,Erlofmib \n \ung cancer - molecular and clinical predictors of outcome. N Engl J Med 353: 133-44 (2005).
Uphoff CC and Drexler HGEradication of mycoplasma contaminations.. Methods MoI Bio! 290: 25-34 (2005).
Weir BA et al.. Characterizing the cancer genome in lung adenocarcinoma. Nature 450: 893-8 (2007).
Yim CH et al., Structures of lung cancer-derived EGFR mutants and inhibitor complexes: mechanism of activation and insights into differential inhibitor sensitivity. Cancer Cell 11: 217-27 (2007).
Huang F et al., Identification of candidate molecular markers predicting sensitivity in solid tumors to dasatinib: rationale for patients selection. E.Cancer Res. Mar l ;67(5):2226-38 (2007) Jackson, E. L. el al., Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev 15:3243-3248 (2001)
Johnson L et al., Somatic activation of the K-ras oncogene causes early onset lung cancer in mice. Nature. 410(6832):! 11 1-6 (2001).
Ji H. ei al., LKBl modulates lung cancer differentiation and metastasis. Nature. 448(7155):807-10. (2007)
Thomas RK et al., Sensitive mutation detection in heterogeneous cancer specimens by massively parallel picoliter reactor sequencing. Nat Med. Jul;12(7):852-5. Epub 2006 Jun 25. Erratum in: Nat Med.2006 Oct; 12(10): 1220.

Claims

1. A method of selecting (a) cell(s), (a) tissue(s) or (a) cell culture(s) with susceptibility to dasatinib, comprising the steps:
(a) evaluating the gene amplification status of at least one gene selected from the group consisting SRC5 EPHA3, FRK. EPHA5, EPHA8, YES, ABL2, LCK and BLK in said cell, cell tissue or cell culture; and (b) selecting (a) cell(s), (a) tissue(s) or (a) cell culture(s) with (a) gene amplification above normal of at least one of said genes,
2. The method of claim 1, further comprising the steps
(i) contacting said cell(s), tissue(s) or cell culture(s) with dasatinib: and (ii) evaluating viability of said cell(s), tissue(s) or cell culture(s) contacted with dasatinib.
3, A method for determining the responsiveness of a mammalian tumor cell or cancer cell to treatment with dasatinib. said method comprising determining the amplification status of at least one gene selected from SRC, EPHA3, FRK5 EPHA5, EPHA8. YES,
ABL2, LCK and BLK in said tumor cell or cancer cell, wherein said amplification status is indicative of whether the cell is likely to respond or is responsive to the treatment.
4. In vitro method for the identification of a responder for or a patient sensitive to dasatinib. said method comprising the following steps: (a) obtaining a sample from a patient suspected to suffer from or being prone to suffer from non-small cell lung cancer; and (b) evaluating the gene amplification status of at least one gene selected from the group of SRC. EPHA3, FRK. EPHA5, EPHA8, YES, ABL2, LCK and BLK; whereby a gene amplification of at least one of said genes is indicative for a responding patient or is indicative for a sensitivity of said patient to dasatinib.
5. The method of any one of claims 1 to 4, wherein the amplification status of at least two genes selected from the group of SRC5 EPHA3, FRK, EPHA5, EPHA8, YES, ABL2. LCK and BLK is assessed or evaluated.
6. The method of claim 5, wherein said at least two amplified genes are selected from the group consisting of EPHA3 and ABL2, EPHA3 and SRC, and EPHA3 and FRK.
7. The method of any one of claims 1 to 6. wherein said at least one gene is present in at least 3 copies.
8. The method of any one of claims 1 to 7, wherein said gene amplification status is detected, assessed or evaluated by an in situ hybridization method, comparative genomic hybridisation and single-nucleotide polymorphism arrays.
9. The method of claim 8. wherein said in situ hybridisation is selected from the group consisting of fluorescent in situ hybridisation (FISH), chromogenic in situ hybridisation (CISH) and silver in situ hybridisation (SISH).
10. The method of any one of claims 3 to 9, wherein said sample is obtained from a patient suspected to suffer from or being prone to suffer from non-small cell lung cancer.
11. Use of an oligo- or polynucleotide capable of detecting the amplification status of at least one gene selected from the group consisting of SRC, EPHA3. FRK. EPHA5,
EPHA8, YES. ABL2, LCK and BLK for diagnosing sensitivity to dasatinib.
12. The use of claim 11 , wherein said oligonucleotide is about 15 to 50 nucleotides in length,
13. A method of diagnosing in a subject/patient suspected of suffering from non-small cell lung cancer or suspected of being prone to suffering from non-small cell lung cancer comprising the steps of a) determining in a cell or tissue sample obtained from said subject/patient the activity of at least one marker gene selected from the group consisting of SRC, EPHA3, FRK, EPHA5. EPHA8. YES5 ABL2, LCK and BLK; and b) comparing the activity of said at least one marker gene determined in a) with a reference activity of said at least one marker gene determined in (a sample from) a control subject/patient (healthy subject), wherein said non-small cell lung cancer is diagnosed when said activity determined in a) differs from said reference activity.
14. A method of monitoring the efficacy of a treatment of a non-small cell lung cancer in a subject/patient suffering from said disorder or being prone to suffering from said disorder comprising the steps of a) determining in a cell or tissue sample obtained from said subject/patient the activity of at least one marker gene selected from the group consisting of SRC, EPHA3, FRK5 EPHA5, EPHA8, YES, ABL2, LCK and BLK; and b) comparing the activity of said at least one marker gene determined in a) with a reference activity of said at least one marker gene, optionally determined in (a sample from) a control subjectφatient (responder and/or non-responder), wherein the extent of the difference between said activity determined in a) and said reference activity is indicative for said efficacy of a treatment of a non-small cell lung cancer.
15. A method of predicting the efficacy of a treatment of a non-small cell lung cancer for a subject/patient suffering from said disorder or being prone to suffering from said disorder comprising the steps of a) determining in a cell or tissue sample obtained from said subject/patient the activity of at least one marker gene selected from the group consisting of SRC, EPHA3- FRK. EPHA5, EPHA8, YES, ABL2, LCK and BLK; and b) comparing the activity of said at least one marker gene determined in a) with a reference activity of said at least one marker gene, optionally determined in (a sample from) a control subject/patient (responder and/or non-responder). wherein the extent of the difference between said activity determined in a) and said reference activity is indicative for the predicted efficacy of a treatment of a non-small cell lung cancer.
16. The method of any one of claims 13 to 15, wherein said treatment of non-small cell lung cancer comprises the administration of dasatinib.
17. The method of any one of claims 10 and 13 to 16, wherein said non-small cell lung cancer is asquamous cell lung carcinoma, an adenocarcinoma, a large cell lung carcinoma, or an anaplastic carcinoma .
18. The method of any one of claims 10 and 13 to 17, wherein said subject/patient suffering from said non-small cell lung cancer or being prone to suffering from said non-small cell lung cancer exhibits resistance (primary or secondary) against any platinum -based, taxane-based chemotherapy, vinorelbine, gemcitinibe, erlotinib, sunitinib and/or vandetanib..
19. Use of a (transgenic) cell or a (transgenic) non-human animal having at least one amplified marker gene as defined in any one of claims 1 to 10 for screening and/or validation of a medicament for the treatment of non-small cell lung cancer.
20. A kit useful for carrying out the method of any one of claims 1 to 10 and 13 to 19 comprising oligonucleotides or polynucleotides capable of detecting the amplification status of at least one gene selected from the group of SRC, EPHA3, FRK, EPHA5, EPHA8, YES5 ABL2, LCK and BLK.
21. A combination of eel] lines selected from the group consisting of A427, A549, CaIu-I5 Calu-3, Calu-6, H1299, H1355, Hl 395, H1437. Hl 563, H1568, H1648, Hl 650, H1666. H1734. H1755, H1770, H1781, H1792, H1819, H1838, H1915, H1944, Hl 975, H1993, H2009, H2030, H2052. H2087, H2110. H2122, H2126, H2172,
H2228, H23, H2347, H2444, H28. H358, H441, H460, H520, H522, H596, H647, H661, H820, HCC2935, HCC4006, HCC827, SK-LU-I, EKVX, H322M, HOP-62, HOP-92, Colo699; DV-90, HCCl 5, HCC366, HCC44, HCC78, LCLCl 03H, LCLC97TM1 and LouNH91.
22. Use of the combination of cell lines as defined in claim 21 for predicting susceptibility to a drug.
23. The use of claim 22, wherein said drug is dasatinib.
PCT/EP2009/060641 2008-08-18 2009-08-17 Susceptibility to dasatinib WO2010020619A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP08014655 2008-08-18
EP08014655.8 2008-08-18

Publications (2)

Publication Number Publication Date
WO2010020619A2 true WO2010020619A2 (en) 2010-02-25
WO2010020619A3 WO2010020619A3 (en) 2010-06-24

Family

ID=41284106

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2009/060641 WO2010020619A2 (en) 2008-08-18 2009-08-17 Susceptibility to dasatinib

Country Status (1)

Country Link
WO (1) WO2010020619A2 (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2562265A1 (en) 2011-08-22 2013-02-27 Lead Discovery Center GmbH Susceptibility to selective CDK9 inhibitors
WO2013026874A1 (en) 2011-08-22 2013-02-28 Lead Discovery Center Gmbh Cdk9 inhibitors in the treatment of midline carcinoma
EP2600150A3 (en) * 2011-12-01 2014-03-05 Sysmex Corporation Method for determining sensitivity of tumor cells to dasatinib and computer program
WO2014043628A1 (en) * 2012-09-14 2014-03-20 Memorial Sloan-Kettering Cancer Center Genes associated with dasatinib sensitivity
WO2021043953A1 (en) * 2019-09-05 2021-03-11 Pamgene Bv Kinase activity signatures for predicting the response of non-small-cell lung carcinoma patients to a pd-1 or pd-l1 immune checkpoint inhibitor

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006005035A2 (en) * 2004-06-30 2006-01-12 Bristol-Myers Squibb Company Identification of polynucleotides for predicting activity of compounds that interact with and/or modulate protein tyrosine kinases and/or protein tyrosine pathways in lung cancer cells
US20070161019A1 (en) * 2005-11-04 2007-07-12 Johji Inazawa Method for detecting cancer and a method for suppressing cancer
US20070254295A1 (en) * 2006-03-17 2007-11-01 Prometheus Laboratories Inc. Methods of predicting and monitoring tyrosine kinase inhibitor therapy

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006005035A2 (en) * 2004-06-30 2006-01-12 Bristol-Myers Squibb Company Identification of polynucleotides for predicting activity of compounds that interact with and/or modulate protein tyrosine kinases and/or protein tyrosine pathways in lung cancer cells
US20070161019A1 (en) * 2005-11-04 2007-07-12 Johji Inazawa Method for detecting cancer and a method for suppressing cancer
US20070254295A1 (en) * 2006-03-17 2007-11-01 Prometheus Laboratories Inc. Methods of predicting and monitoring tyrosine kinase inhibitor therapy

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
AMACHIKA TOMOKO ET AL: "Diagnostic relevance of overexpressed mRNA of novel oncogene with kinase-domain (NOK) in lung cancers." LUNG CANCER (AMSTERDAM, NETHERLANDS) JUN 2007, vol. 56, no. 3, June 2007 (2007-06), pages 337-340, XP002575640 ISSN: 0169-5002 *
BLUME-JENSEN P ET AL: "ONCOGENIC KINASE SIGNALLING" NATURE, NATURE PUBLISHING GROUP, LONDON, GB, vol. 411, no. 6835, 17 May 2001 (2001-05-17), pages 355-365, XP001156920 ISSN: 0028-0836 *
DU JINYAN ET AL: "Bead-based profiling of tyrosine kinase phosphorylation identifies SRC as a potential target for glioblastoma therapy" NATURE BIOTECHNOLOGY, vol. 27, no. 1, January 2009 (2009-01), pages 77-83, XP002558123 ISSN: 1087-0156 *
GIACCONE G ET AL: "Src as a potential therapeutic target in non-small-cell lung cancer." ANNALS OF ONCOLOGY : OFFICIAL JOURNAL OF THE EUROPEAN SOCIETY FOR MEDICAL ONCOLOGY / ESMO JUL 2008, vol. 19, no. 7, July 2008 (2008-07), pages 1219-1223, XP002558117 ISSN: 1569-8041 *
HAFNER CHRISTIAN ET AL: "Differential gene expression of Eph receptors and ephrins in benign human tissues and cancers" CLINICAL CHEMISTRY, AMERICAN ASSOCIATION FOR CLINICAL CHEMISTRY, WASHINGTON, DC, vol. 50, no. 3, 1 March 2004 (2004-03-01), pages 490-499, XP002498092 ISSN: 0009-9147 *
HUANG FEI ET AL: "Identification of candidate molecular markers predicting sensitivity in solid tumors to dasatinib: Rationale for patient selection" CANCER RESEARCH, vol. 67, no. 5, March 2007 (2007-03), pages 2226-2238, XP002558115 ISSN: 0008-5472 cited in the application *
QUINTAS-CARDAMA A ET AL: "Activity of tyrosine kinase inhibitors against human NUP214-ABL1-positive T cell malignancies" LEUKEMIA (BASINGSTOKE), vol. 22, no. 6, June 2008 (2008-06), pages 1117-1124, XP002558118 ISSN: 0887-6924 *
SONG LANXI ET AL: "Dasatinib (BMS-354825) selectively induces apoptosis in lung cancer cells dependent on epidermal growth factor receptor signaling for survival" CANCER RESEARCH, vol. 66, no. 11, June 2006 (2006-06), pages 5542-5548, XP002558116 ISSN: 0008-5472 *
SOS ET AL: "Predicting drug susceptibility of non-small cell lung cancers based on genetic lesions" THE JOURNAL OF CLINICAL INVESTIGATION,, vol. 119, no. 6, 18 May 2009 (2009-05-18), pages 1727-1740, XP002555300 *
SURAWSKA H ET AL: "The role of ephrins and Eph receptors in cancer" CYTOKINE AND GROWTH FACTOR REVIEWS, OXFORD, GB, vol. 15, no. 6, 1 December 2004 (2004-12-01), pages 419-433, XP004652022 ISSN: 1359-6101 *
WICKS I P ET AL: "MOLECULAR CLONING OF HEK, THE GENE ENCODING A RECEPTOR TYROSINE KINASE EXPRESSED BY HUMAN LYMPHOID TUMOR CELL LINES" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA. (PNAS), NATIONAL ACADEMY OF SCIENCE, WASHINGTON, DC, US, vol. 89, 1 March 1992 (1992-03-01), pages 1611-1615, XP000615502 ISSN: 0027-8424 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2562265A1 (en) 2011-08-22 2013-02-27 Lead Discovery Center GmbH Susceptibility to selective CDK9 inhibitors
WO2013026890A1 (en) 2011-08-22 2013-02-28 Lead Discovery Center Gmbh Susceptibility to selective cdk9 inhibitors
WO2013026874A1 (en) 2011-08-22 2013-02-28 Lead Discovery Center Gmbh Cdk9 inhibitors in the treatment of midline carcinoma
EP2600150A3 (en) * 2011-12-01 2014-03-05 Sysmex Corporation Method for determining sensitivity of tumor cells to dasatinib and computer program
WO2014043628A1 (en) * 2012-09-14 2014-03-20 Memorial Sloan-Kettering Cancer Center Genes associated with dasatinib sensitivity
US10113200B2 (en) 2012-09-14 2018-10-30 Memorial Sloan-Kettering Cancer Center Genes associated with dasatinib sensitivity
WO2021043953A1 (en) * 2019-09-05 2021-03-11 Pamgene Bv Kinase activity signatures for predicting the response of non-small-cell lung carcinoma patients to a pd-1 or pd-l1 immune checkpoint inhibitor

Also Published As

Publication number Publication date
WO2010020619A3 (en) 2010-06-24

Similar Documents

Publication Publication Date Title
Ennishi et al. Genetic profiling of MYC and BCL2 in diffuse large B-cell lymphoma determines cell-of-origin–specific clinical impact
US20130023420A1 (en) Susceptibility to hsp90-inhibitors
US20200190602A1 (en) Methods and materials for assessing loss of heterozygosity
JP7186700B2 (en) Methods to Distinguish Tumor Suppressor FOXO Activity from Oxidative Stress
Sos et al. Predicting drug susceptibility of non–small cell lung cancers based on genetic lesions
Schwalbe et al. Rapid diagnosis of medulloblastoma molecular subgroups
CN102016579B (en) VEGF polymorphisms and anti-angiogenesis therapy
EP2825669A1 (en) Methods and compositions for the diagnosis, prognosis and treatment of acute myeloid leukemia
WO2012158780A2 (en) Lung cancer signature
WO2009150255A2 (en) Markers for predicting response and survival in anti-egfr treated patients
Ocak et al. High-throughput molecular analysis in lung cancer: insights into biology and potential clinical applications
Xu et al. Detection of epidermal growth factor receptor mutation in lung cancer by droplet digital polymerase chain reaction
Ong et al. A gene signature associated with PTEN activation defines good prognosis intermediate risk prostate cancer cases
WO2010020619A2 (en) Susceptibility to dasatinib
Yokogami et al. Impact of PCR-based molecular analysis in daily diagnosis for the patient with gliomas
Noushmehr et al. Detection of glioma and prognostic subtypes by non-invasive circulating cell-free DNA methylation markers
Zhang et al. Comprehensive profiling and quantitation of oncogenic mutations in non small-cell lung carcinoma using single molecule amplification and re-sequencing technology
Fettke et al. Independent prognostic impact of plasma NCOA2 alterations in metastatic castration‐resistant prostate cancer
Chung et al. Clinical applications of genomics in head and neck cancer
KR20210144863A (en) Classification of B-cell Non-Hodgkin&#39;s Lymphoma
Paquet et al. A 12‐gene signature to distinguish colon cancer patients with better clinical outcome following treatment with 5‐fluorouracil or FOLFIRI
US11214838B2 (en) Method of predicting effect of medicinal therapy on cancer
WO2018078142A1 (en) Means and methods for determining efficacy of fluorouracil (5-fu) in colorectal cancer (crc) therapy
WO2006109086A2 (en) Method to predict the sensitivity of tumors to eg5 inhibitors
Raja et al. Integrating genomics into drug discovery and development: challenges and aspirations

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09781926

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 09781926

Country of ref document: EP

Kind code of ref document: A2