WO2013190089A1 - Biomarqueurs moléculaires permettant de prédire l'issue dans le cancer du poumon - Google Patents

Biomarqueurs moléculaires permettant de prédire l'issue dans le cancer du poumon Download PDF

Info

Publication number
WO2013190089A1
WO2013190089A1 PCT/EP2013/062992 EP2013062992W WO2013190089A1 WO 2013190089 A1 WO2013190089 A1 WO 2013190089A1 EP 2013062992 W EP2013062992 W EP 2013062992W WO 2013190089 A1 WO2013190089 A1 WO 2013190089A1
Authority
WO
WIPO (PCT)
Prior art keywords
egfr
mutation
inhibitor
chemotherapy
lung cancer
Prior art date
Application number
PCT/EP2013/062992
Other languages
English (en)
Inventor
Rafael Rosell Costa
Miguel Tarón Roca
Original Assignee
Pangaea Biotech, S.L.
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 Pangaea Biotech, S.L. filed Critical Pangaea Biotech, S.L.
Publication of WO2013190089A1 publication Critical patent/WO2013190089A1/fr

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
    • 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
    • C12Q2600/118—Prognosis of disease development
    • 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
    • C12Q2600/158—Expression markers

Definitions

  • the invention relates to the field of pharmacogenomics and, more in particular, to methods for predicting the survival of a lung cancer patient to a treatment with conventional chemotherapy or with an EGFR tyrosine kinase inhibitor-based chemotherapy, based on the detection of one sensitivity mutation in the EGFR gene towards an inhibitor of EGFR tyrosine kinase activity in a bio fluid of the patient.
  • Non-small-cell lung cancer accounts for approximately 80% of all lung cancers, with 1.2 million new cases worldwide each year. NSCLC resulted in more than one million deaths worldwide in 2001 and is the leading cause of cancer-related mortality in both men and women (31% and 25%, respectively). The prognosis of advanced NSCLC is dismal. A recent Eastern Cooperative Oncology Group trial of 1155 patients showed no differences among the chemotherapies used: cisplatin/paclitaxel, cisplatin/gemcitabine, cisplatin/docetaxel and carboplatin/paclitaxel. Overall median time to progression was 3.6 months, and median survival was 7.9 months.
  • patients with NSCLC can be divided into three groups that reflect both the extent of the disease and the treatment approach:
  • the first group of patients has tumors that are surgically resectable (generally stage I, stage II, and selected stage III tumors). This group has the best prognosis.
  • the second group includes patients with either locally (T3-T4) and/or regionally (N2-N3) advanced lung cancer. Patients with unresectable or N2-N3 disease are treated with radiation therapy in combination with chemotherapy.
  • Selected patients with T3 or N2 disease can be treated effectively with surgical resection and either preoperative or postoperative chemotherapy or chemoradiation therapy.
  • the final group includes patients with distant metastases (Ml). This group can be treated with palliative radiation therapy or chemotherapy.
  • First-line treatment of NSCLC is carried out by platinum-based chemotherapy.
  • NSCLC non-small cell lung carcinoma
  • Erlotinib has been shown to improve progression-free survival compared with chemotherapy when given as first-line treatment for Asian patients with non-small-cell lung cancer (NSCLC) with activating EGFR mutations (Mitsudomi et al, Lancet Oncol 2010; 11 : 121-28). These results have been recently confirmed by Rosell et al. (Lancet Oncology, DOI: 10.1016/S1470-2045(11)70393-X) in non-Asian patients with advanced NSCLC by a prospective head-to-head phase 3 study comparing efficacy and safety of first-line erlotinib with platinum-based chemotherapy in patients.
  • the invention relates to a method for predicting the outcome of a patient suffering lung cancer after treatment with chemotherapy, with an EGFR inhibitor or a combination thereof wherein said lung cancer carries at least an activating EGFR mutation, said method comprising determining in a bio fluid of said patient the levels of the cell-free EGFR gene carrying said at least one activating EGFR mutation wherein the detection of cell-free EGFR gene containing said mutation or the detection of an increased level thereof with respect to a reference value is indicative of a poor outcome of the patient or wherein an absence of detection of the cell- free EGFR gene containing said mutation or the detection of decreased levels thereof with respect to a reference value is indicative of a good outcome of the patient.
  • the invention in a second aspect, relates to a method of prognosing or classifying a subject with lung cancer positive for an activating EGFR mutation and treated with chemotherapy, with an EGFR inhibitor or a combination thereof said method comprising determining in a biofluid of said patient the level of cell-free EGFR gene carrying said EGFR mutation wherein the detection of cell-free EGFR gene containing said mutation or the detection of an increased level thereof with respect to a reference value is is used to prognose or classify the subject with lung cancer into a poor outcome group or wherein an absence of detection of the cell-free EGFR gene containing said mutation or the detection of decreased levels thereof with respect to a reference value is is used to prognose or classify the subject with lung cancer into a good outcome group.
  • the invention relates to a computer system that is provided with means for implementing the methods according to the invention, to a computer program comprising a programming code to execute the steps of the methods according to invention if carried out in a computer and to a computer-readable data medium comprising a computer program according to the invention in the form of a computer- readable programming code.
  • the invention relates to a method for the further treatment of lung cancer in a subject and to a kit for for determining the outcome of a subject suffering from lung cancer.
  • Figure 1 Progression free survival in patients treated with chemotherapy according to EGFR mutations in cfDNA.
  • Figure 2 Progression free survival in patients treated with erlotinib according to EGFR mutations in cfDNA.
  • Figure 3 Overall survival according to EGFR mutations in cfDNA.
  • the authors of the present invention have observed that, surprisingly, the level of cell-free EGFR carrying activating EGFR mutations in the serum of patients with non- small-cell lung cancer (NSCLC) carrying activating EGFR mutations can be used as a marker for outcome of the patients in response to conventional therapy or to therapy based on the use of inhibitors of the EGFR tyrosine kinase.
  • NSCLC non- small-cell lung cancer
  • the invention relates to a method (first method of the invention) for predicting the outcome of a patient suffering lung cancer after treatment with chemotherapy, with an EGFR inhibitor or a combination thereof wherein said lung cancer carries at least an activating EGFR mutation, said method comprising determining in a biofluid of said patient the levels of the cell-free EGFR gene carrying said at least one activating EGFR mutation wherein the detection of cell-free EGFR gene containing said mutation or the detection of an increased level thereof with respect to a reference value is indicative of a poor outcome of the patient or wherein an absence of detection of the cell-free EGFR gene containing said mutation or the detection of decreased levels thereof with respect to a reference value is indicative of a good outcome of the patient.
  • predicting refers to the determination of the likelihood that the patient will respond either favorably or unfavorably to a given therapy.
  • prediction relates to an individual assessment of any parameter that can be useful in determining the evolution of a patient.
  • the prediction of the clinical response to the treatment with a biological drug although preferred to be, need not be correct for 100% of the subjects to be diagnosed or evaluated. The term, however, requires that a statistically significant portion of subjects can be identified as having an increased probability of having a positive response.
  • Whether a subject is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test, etc. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983.
  • Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90% at least 95%.
  • the p-values are, preferably, 0.05, 0.02 or 0.01.
  • outcome refers to any clinical observation or measurement relating to a patient's reaction to a therapy.
  • clinical outcomes include tumor response (TR), overall survival (OS), progression free survival (PFS), disease free survival, time to tumor recurrence (TTR), time to tumor progression (TTP), relative risk (RR), toxicity or side effect.
  • TR tumor response
  • OS overall survival
  • PFS progression free survival
  • TTR time to tumor recurrence
  • TTP time to tumor progression
  • RR relative risk
  • toxicity or side effect toxicity or side effect.
  • “Overall Survival” (OS) intends a prolongation in life expectancy as compared to naive or untreated individuals or patients.
  • progression free survival PFS
  • TTP Time to Tumor Progression
  • Progression-free survival includes the amount of time patients have experienced a complete response or a partial response, as well as the amount of time patients have experienced stable disease.
  • Tumor Recurrence as used herein and as defined by the National Cancer Institute is cancer that has recurred (come back), usually after a period of time during which the cancer could not be detected. The cancer may come back to the same place as the original (primary) tumor or to another place in the body. It is also called recurrent cancer.
  • TTR Time to Tumor Recurrence
  • TTR is defined as the time from the date of diagnosis of the cancer to the date of first recurrence, death, or until last contact if the patient was free of any tumor recurrence at the time of last contact.
  • Relative Risk in statistics and mathematical epidemiology, refers to the risk of an event (or of developing a disease) relative to exposure. Relative risk is a ratio of the probability of the event occurring in the exposed group versus a non- exposed group.
  • the outcome of the patient is determined as progression- free survival or as survival.
  • patient refers to all animals classified as mammals and includes, but is not restricted to, domestic and farm animals, primates and humans, e.g., human beings, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents.
  • the patient is a male or female human of any age or race.
  • the patient that has had lung cancer still is considered to have lung cancer.
  • lung cancer is meant to refer to any cancer of the lung and includes non-small cell lung carcinomas and small cell lung carcinomas.
  • the methods of the invention are applicable to a subject suffering from NSCLC and/or that has suffered NSCLC.
  • the NSCLC is selected from squamous cell carcinoma of the lung, large cell carcinoma of the lung, and adenocarcinoma of the lung.
  • the present method can also be applicable to a subject that has suffered or is suffering from any stage of NSCLC (stages 0, IA, IB, Ila, lib, Ilia, Illb o IV).
  • the patient has had advanced lung cancer.
  • lung cancer with an activating EGFR mutation refers to cancer wherein the EGFR gene carries one or more mutations in the tyrosine kinase domain which results in that the tyrosine kinase activity of EGFR is increased with respect to the wild type EGFR.
  • These mutants are also characterised in that they show increased sensitivity to EGFR tyrosine kinase specific inhibitor such as erlotinib.
  • Cancers carrying such a mutation can be identified by determining in a sample of the tumor the presence of the EGFR mutation. Suitable samples include, e.g., tumor biopsies which are excised from the tissue using any suitable method in the art.
  • samples of a particular cell type may be micro- dissected using laser-capture micro-dissection ("LCM") techniques, as described in U.S. Pat. Nos. 5,843,657, 6,251,516 Bl, and 6,969,614 Bl, each of which is hereby incorporated by reference in its entirety.
  • LCM laser-capture micro-dissection
  • the cells of interest can be identified, e.g., by morphology, in situ immunohistochemistry, or fluorescent microscopy.
  • Methods for determining whether a given mutant is an activating EGFR mutation or confers sensitivity to a tyrosine kinase activity have been described in detail in the prior art and include, among others, a method as described in WO2006091889 based on the detection of the autophosphorylation capacity of EGFR as measured in cells over- expressing the variant EGFR in response to the treatment with a gefmtib (IressaTM) or panitumumab.
  • kinase or phosphorylation activity can be increased (e.g., by at least 5 percent, 10 percent, 15 percent, 20 percent, 30 percent, 40 percent, 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, or even 100 percent), as compared to wild type EGFR.
  • Activating EGFR mutations include one or more deletions, substitutions, or additions in the amino acid or nucleotide sequences of EGFR protein, or EGFR coding sequence.
  • the activating EGFR mutant can also include one or more deletions, substitutions, or additions, or a fragment thereof, as long as the mutant retains or increases tyrosine kinase activity, compared to wild type EGFR.
  • Particular EGFR mutants are described herein, where mutations are provided relative to the position of an amino acid in human EGFR, as described in the sequence provided in NCBI GenBank Reference Sequence: NP 005219.2.
  • Exemplary EGFR mutations include, for example, mutations in exon 18, 19, 20 or 21.
  • EGFR mutants showing an increased sensitivity to tyrosine kinase inhibitors include, without limitation, mutations at positions L858 in exon 21 such as L858R, L858P, L861Q or L861 point mutations in the activation loop (exon 21), in-frame deletion/insertion mutations in the ELREA sequence (exon 19) such as the E746-R748 deletion, the E746-A750 deletion, the E746-R748 deletion together with E749Q and A750P substitutions, del L747-E749 deletion combined with the A750P substitution, the L747S substitution in combination with the R748-P753 deletion, the L747-S752 deletion in combination with the E746V substitution, the L747- T751 deletion combined with an serine insertion, the AI insertion at positions M766- A767, the SVA insertion at positions S768-V769, or substitutions in at position 719 in the nucleotide binding loop (
  • Further activating mutations include, without limitation, mutations in the kinase domain, G719A, L858R, E746K, L747S, E749Q, A750P, A755V, V765M, S7681, L858P, E746-R748 del, R748-P753 del, M766-A767 Al ins, S768- V769 SVA ins, P772-H773 NS ins, 2402OC, 24820A, 2486T>C, 2491 G>C, 24940C, 251 0OT, 25390A, 25490T, 25630T, 2819T>C, 2482-2490 del, 2486-2503 del, 2544-2545 ins GCCATA, 2554-2555 ins CCAGCGTGG, or 2562-2563 ins AACTCC.
  • Other examples of EGFR activating mutations are known in the art (see e.g., US Patent Publication No. 2005
  • the patient shows at least a mutation conferring sensitivity to tyrosine kinase inhibitors.
  • the patient shows a first mutation selected from the group of the L858R substitution and the ELREA deletion in exon 19.
  • chemotherapy refers to any treatment that reduces, prevents, mitigates, limits, and/or delays the growth of metastases or neoplasms, or kills neoplastic cells directly by necrosis or apoptosis of neoplasms or any other mechanism, or that can be otherwise used to reduce, prevent, mitigate, limit, and/or delay the growth of metastases or neoplasms in a subject with neoplastic disease.
  • Suitable chemotherapeutic treatments include, without limitation, plant alkaloids, such as vincristine, vinblastine and etoposide; anthracycline antibiotics including doxorubicin, epirubicin, daunorubicin; fluorouracil; antibiotics including bleomycin, mitomycin, plicamycin, dactinomycin; topoisomerase inhibitors, such as camptothecin and its analogues; and platinum compounds, including cisplatin and its analogues, such as carboplatin.
  • plant alkaloids such as vincristine, vinblastine and etoposide
  • anthracycline antibiotics including doxorubicin, epirubicin, daunorubicin
  • fluorouracil antibiotics including bleomycin, mitomycin, plicamycin, dactinomycin
  • topoisomerase inhibitors such as camptothecin and its analogues
  • platinum compounds including cisplatin and its
  • chemotherapeutic agents suitable for use include, asparaginase, busulfan, chlorambucil, cyclophosphamide, cytarabine, dacarbazine, estramustine phosphate sodium, floxuridine, fluorouracil (5-FU), hydroxyurea (hydroxycarbamide), ifosfamide, lornustine (CCNU), mechlorethamine HC1 (nitrogen mustard), melphalan, mercaptopurine, methotrexate (MTX), mitomycin, mitotane, mitsxantrone,, procarbazine, streptozocin,, thioguanine, thiotepa, amsacrine (m-AMSA), azacitidine,, hexamethylmeiamine (HMM),, mitoguazone (methyl-GAG; methyl giyoxal bis- guanyihydrazone; MG
  • the chemotherapy is a therapy with a platinum-based compound.
  • platinum-based compound refers to a compound comprising a heavy metal complex containing a central atom of platinum surrounded by organic and/or inorganic functionalities.
  • platinum-based drugs include cisplatin, carboplatin, oxilaplatin, nedaplatin, spiroplatin, iproplatin, satraplatin, pharmaceutically acceptable salts thereof, stereoisomers thereof, derivatives thereof, analogs thereof, and combinations thereof.
  • an "EGFR inhibitor” is any agent that inhibits (blocks, reduces, antagonizes, decreases, reverses) the expression and/or biological activity of an epidermal growth factor receptor (EGFR), including any EGFR. Therefore, an inhibitor can include, but is not limited to, a product of drug/compound/peptide design or selection, an antibody or antigen binding fragment thereof, a protein, a peptide, a nucleic acid (including ribozymes, antisense, RNAi and aptamers), or any other agent that inhibits the expression and/or biological activity of an EGFR.
  • EGFR EGFR
  • ErbBl epidermal growth factor receptor
  • HER2/neu ErbB2
  • HER3 ErbB3
  • ErbB4 ErbB4
  • variants thereof e.g. a deletion mutant EGFR as in Humphrey et al. (Proc. Natl. Acad. Sci. USA, 1990, 87:4207-4211).
  • the EGFR is human.
  • the EGFR inhibitor is an EGFR tyrosine kinase inhibitor.
  • the type of EGFR tyrosine kinase inhibitor therapy for use according to the method of the present invention is not particularly limiting and may include any of the inhibitors mentioned above.
  • the EGFR tyrosine kinase inhibitor is a dual EGFR inhibitor, a dual EGFR tyrosine kinase inhibitor or a EGFR tyrosine kinase inhibitor specific for EGFR carrying a resistance mutation.
  • dual EGFR inhibitor refers to a composition which is capable of simultaneously inhibiting the tyrosine kinase activity of the intracellular domain of EGFR as well as its activation by the binding of the ligand to the extracellular domain.
  • Illustrative and non- limitative example of such an inhibitor is, e.g. the composition comprising cetuximab (C225) as inhibitor of the extracellular domain and erlotinib (E) as inhibitor of the tyrosine kinase activity of the intracellular domain.
  • EGFR tyrosine kinase inhibitor refers to a compound which is capable of simultaneously inhibiting EGFR and HER2 activity.
  • examples of such compounds include the EGFR and HER2 inhibitor CI- 1033 (formerly known as PD 183805; Pfizer); the EGFR and HER2 inhibitor GW-2016 (also known as GW-572016 or lapatinib ditosylate; GSK); the EGFR and JAK 2/3 inhibitor AG490 (a tyrphostin); the EGFR and HER2 inhibitor ARRY-334543 (Array BioPharma); BIBW- 2992, an irreversible dual EGFR HER2 kinase inhibitor (Boehringer Ingelheim Corp.)
  • the EGFR inhibitor-based chemotherapy is an inhibitor of the EGFR tyrosine kinase.
  • EGFR tyrosine kinase inhibitor relates to a chemical substance inhibiting "tyrosine kinase” which transfers a ⁇ -phosphate group of ATP to a hydroxy group of a specific tyrosine in protein catalyzed by the tyrosine kinase domain of the receptor for epidermal growth factor (EGFR). Tyrosine kinase activity is measured by detecting phosphorylation of a protein.
  • EGFR tyrosine kinase inhibitors are known in the art. For example, a tyrosine kinase inhibitor is identified by detecting a decrease the tyrosine mediated transfer phosphate from ATP to protein tyrosine residues.
  • the tyrosine kinase inhibitor is for example an erbB tyrosine kinase inhibitor.
  • the tyrosine kinase inhibitor is an EGFR tyrosine kinase inhibitor.
  • the tyrosine kinase inhibitor is a reversible tyrosine kinase inhibitor.
  • the tyrosine kinase inhibitor is an irreversible tyrosine kinase inhibitor.
  • Reversible tyrosine kinase inhibitors include for example, HKI-272, BIBW2992, EKB-569 or CL-387,785 or mimetics or derivatives thereof.
  • Other tyrosine kinase inhibitors include those described in U.S. Pat. Nos. 6,384,051, 6,288,082 and US Application No. 20050059678, each of which is hereby incorporated by reference in their entirety.
  • EGFR tyrosine kinase inhibitors include, for example quinazoline EGFR kinase inhibitors, pyrido- pyrimidine EGFR kinase inhibitors, pyrimido-pyrimidine EGFR kinase inhibitors, pyrrolo- pyrimidine EGFR kinase inhibitors, pyrazolo -pyrimidine EGFR kinase inhibitors, phenylamino- pyrimidine EGFR kinase inhibitors, oxindole EGFR kinase inhibitors, indolocarbazole EGFR kinase inhibitors, phthalazine EGFR kinase inhibitors, isoflavone EGFR kinase inhibitors, quinalone EGFR kinase inhibitors, and tyrphostin EGFR kinase inhibitors, such as those described in the following patent publications, and all pharmaceutically acceptable salts
  • Additional non-limiting examples of low molecular weight EGFR kinase inhibitors include any of the EGFR tyrosine kinase inhibitors described in Traxler, P., 1998, Exp. Opin. Ther. Patents 8(12): 1599-1625.
  • low molecular weight EGFR tyrosine kinase inhibitors that can be used according to the present invention include [6,7-bis(2- methoxyethoxy)-4-quinazolin-4-yl]- (3-ethynylphenyl)amine (also known as OSI-774, erlotinib, or TARCEVA (erlotinib HC1); OSI Pharmaceuticals/Genentech/Roche) (U.S. Pat. No. 5,747,498; International Patent Publication No. WO 01/34574, and Moyer, J. D. et al. (1997) Cancer Res.
  • CI- 1033 (formerly known as PD183805; Pfizer) (Sherwood et al, 1999, Proc. Am. Assoc. Cancer Res. 40:723); PD-158780 (Pfizer); AG-1478 (University of California); CGP-59326 (Novartis); PKI-166 (Novartis); EKB-569 (Wyeth); GW-2016 (also known as GW-572016 or lapatinib ditosylate; GSK); and gefitinib (also known as ZD 1839 or IRESSA.TM.; Astrazeneca) (Woodburn et al, 1997, Proc. Am. Assoc. Cancer Res.
  • a particularly preferred low molecular weight EGFR kinase inhibitor that can be used according to the present invention is [6,7-bis(2-methoxyethoxy)-4-quinazolin-4-yl]-(3-ethynylphenyl) amine (i.e. erlotinib), its hydrochloride salt (i.e. erlotinib HC1, TARCEVA), or other salt forms (e.g. erlotinib mesylate).
  • EGFR tyrosine kinase inhibitors also include, for example multi-kinase inhibitors that have activity on EGFR kinase, i.e. inhibitors that inhibit EGFR kinase and one or more additional kinases.
  • Examples of such compounds include the EGFR and HER2 inhibitor CI- 1033 (formerly known as PD 183805; Pfizer); the EGFR and HER2 inhibitor GW-2016 (also known as GW- 572016 or lapatinib ditosylate; GSK); the EGFR and JAK 2/3 inhibitor AG490 (a tyrphostin); the EGFR and HER2 inhibitor ARRY-334543 (Array BioPharma); BIBW-2992, an irreversible dual EGFR/HER2 kinase inhibitor (Boehringer Ingelheim Corp.); the EGFR and HER2 inhibitor EKB-569 (Wyeth); the VEGF-R2 and EGFR inhibitor ZD6474 (also known as ZACTIMA ;AstraZeneca Pharmaceuticals), and the EGFR and HER2 inhibitor BMS-599626 (Bristol-Myers Squibb).
  • an antisense strategy may be used to interfere with the kinase activity of a variant EGFR.
  • This approach may, for instance, utilize antisense nucleic acids or ribozymes that block translation of a specific mRNA, either by masking that mRNA with an antisense nucleic acid or cleaving it with a ribozyme.
  • antisense technology see, e.g., Antisense DNA and RNA, (Cold Spring Harbor Laboratory, D. Melton, ed., 1988).
  • RNA interference (RNAi) technology prevents the expression of gene by using small RNA molecules such as small interfering RNAs (siRNAs). This technology in turn takes advantage of the fact that RNAi is a natural biological mechanism for silencing genes in most cells of many living organisms, from plants to insects to mammals (McManus et al., Nature Reviews Genetics, 2002, 3(10) p. 737). RNAi prevents a gene from producing a functional protein by ensuring that the molecule intermediate, the messenger RNA copy of the gene is destroyed. siRNAs can be used in a naked form and incorporated in a vector, as described below.
  • aptamers useful in the present invention may be identified using the SELEX process.
  • the methods of SELEX have been described in, for example, U. S. Patent Nos. 5,707,796, 5,763,177, 6,011,577, 5,580,737, 5,567,588, and 5,660,985.
  • an “antisense nucleic acid” or “antisense oligonucleotide” is a single stranded nucleic acid molecule, which, on hybridizing under cytoplasmic conditions with complementary bases in a RNA or DNA molecule, inhibits the latter's role. If the RNA is a messenger RNA transcript, the antisense nucleic acid is a counter-transcript or mRNA-interfering complementary nucleic acid. As presently used, “antisense” broadly includes RNA-RNA interactions, RNA- DNA interactions, ribozymes, RNAi, aptamers and Rnase-H mediated arrest.
  • Ribozymes are RNA molecules possessing the ability to specifically cleave other single stranded RNA molecules in a manner somewhat analogous to DNA restriction endonucleases. Ribozymes were discovered from the observation that certain mRNAs have the ability to excise their own introns. By modifying the nucleotide sequence of these ribozymes, researchers have been able to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, 1989, Science 245(4915) p. 276). Because they are sequence-specific, only mRNAs with particular sequences are inactivated.
  • Antisense nucleic acid molecules can be encoded by a recombinant gene for expression in a cell (e.g., U.S. patent No 5,814,500; U.S. 5,811,234), or alternatively they can be prepared synthetically (e.g., u.s. patent No 5,780,607).
  • siRNAs have been described in Brummelkamp et al, Science 296; 550-
  • siRNA directed specifically against the mutated EGFR should not inhibit the wild-type EGFR. This is significant because it is generally believed that the "side effects" of gefitinib treatment, which include diarrhea and dermatitis, are a consequence of inhibition of EGFR in normal tissues that require its function.
  • the compounds are antisense molecules specific for human sequences coding for an EGFR having at least one variance in its kinase domain.
  • the administered therapeutic agent may be an antisense oligonucleotides, particularly synthetic oligonucleotides; having chemical modifications from native nucleic acids, or nucleic acid constructs that express such anti-sense molecules as RNA.
  • the antisense sequence is complementary to the mRNA of the targeted EGFR genes, and inhibits expression of the targeted gene products (see e.g. Nyce et al. (1997) Nature 385:720).
  • Antisense molecules inhibit gene expression by reducing the amount of mRNA available for translation, through activation of RNAse H or steric hindrance.
  • One or a combination of antisense molecules may be administered, where a combination may comprise multiple different sequences from a single targeted gene, or sequences that complement several different genes.
  • a preferred target gene is an EGFR with at least one nucleic acid variance in its kinase domain.
  • the antisense sequence will have the same species of origin as the animal host.
  • Antisense molecules may be produced by expression of all or a part of the target gene sequence in an appropriate vector, where the vector is introduced and expressed in the targeted cells.
  • the transcriptional initiation will be oriented such that the antisense strand is produced as an RNA molecule.
  • the anti-sense RNA hybridizes with the endogenous sense strand mRNA, thereby blocking expression of the targeted gene.
  • the native transcriptional initiation region, or an exogenous transcriptional initiation region may be employed.
  • the promoter may be introduced by recombinant methods in vitro, or as the result of homologous integration of the sequence into a chromosome.
  • Many strong promoters that are active in muscle cells are known in the art, including the ⁇ -actin promoter, SV40 early and late promoters, human cytomegalovirus promoter, retroviral LTRs, etc.
  • Transcription vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences.
  • Transcription cassettes maybe prepared comprising a transcription initiation region, the target gene or fragment thereof, and a transcriptional termination region.
  • the transcription cassettes may be introduced into a variety of vectors, e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus; and the like, where the vectors are able to transiently or stably be maintained in cells, usually for a period of at least about one day, more usually for a period of at least about several days
  • Aptamers are also useful. Aptamers are a promising new class of therapeutic oligonucleotides or peptides and are selected in vitro to specifically bind to a given target with high affinity, such as for example ligand receptors. Their binding characteristics are likely a reflection of the ability of oligonucleotides to form three dimensional structures held together by intramolecular nucleobase pairing. Aptamers are synthetic DNA, RNA or peptide sequences which may be normal and modified (e.g. peptide nucleic acid (PNA), thiophophorylated DNA, etc) that interact with a target protein, ligand (lipid, carbohydrate, metabolite, etc). In a further embodiment, RNA aptamers specific for a variant EGFR can be introduced into or expressed in a cell as a therapeutic.
  • PNA peptide nucleic acid
  • PNAs Peptide nucleic acids
  • PNA Peptide nucleic acids
  • the deoxyribose backbone of oligonucleotides has been replaced by a pseudo-peptide backbone (Nielsen et al. 1991 Science 254, 1457-1500).
  • Each subunit, or monomer has a naturally occurring or non-naturally occurring nucleobase attached to this backbone.
  • One such backbone is constructed of repeating units of N(2-amino ethyl) glycine linked through amide bonds.
  • PNA hybridises with complementary nucleic acids through Watson and Crick base pairing and helix fold.
  • the Pseudo-peptide backbone provides superior hybridization properties (Egholm et al. Nature (1993) 365, 566-568), resistance to enzymatic degradation (Demidov et al. Biochem. Pharmacol. (1994) 48, 1310-1313) and access to a variety of chemical modifications (Nielsen and Haaima Chemical Society Reviews (1997) 73-78).
  • PNAs specific for a variant EGFR can be introduced into or expressed in a cell as a therapeutic. PNAs have been described, for example, in U.S. Application No. 20040063906.
  • the EGFR inhibitor is a specific inhibitor of the tyrosine kinase activity of EGFR.
  • the inhibitor is erlotinib or gefitinib.
  • the method according to the invention allows predicting the survival of a patient after treatment with a combination of chemotherapy and treatment with an EGFR inhibitor.
  • the combination of a chemotherapy and therapy with a therapy with an EGFR inhibitor comprises the sequential administration of chemotherapy and an EGFR inhibitor or of an EGFR inhibitor and chemotherapy.
  • the method allows predicting the survival of a patient which is treated first with chemotherapy and then with an EGFR inhibitor.
  • the method allows predicting the survival in a patient which is treated first with an EGFR inhibitor and then with chemotherapy.
  • the method for predicting the outcome of a patient according to the invention comprises the determination in a biofluid of said patient of the levels of cell- free EGFR gene which contain said activating EGFR mutation.
  • cell-free DNA and "circulating DNA”, are used herein interchangeably to refer to free genomic DNA molecules that are not contained within any intact cells and can be obtained from any biofluid and, in particular, serum or plasma. It will be understood that the method of the present invention do not require the detection of the complete EGFR gene as cell-free DNA. Instead, the cell-free DNA is formed by a population of DNA molecules which are fragments of the EGFR gene which have a variable length and which contain the region of the EGFR gene wherein the activating mutation is found.
  • the cell- free DNA refers to fragments of genomic DNA having at least 10 bp, 20 bp, 30 bp, 40, bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 200 bp, 300 bp, 400, bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp, 2000 bp, 3000 bp, 4000 bp, 5000 bp, 6000 bp, 7000 bp, 8000 bp, 9000 bp, 10000 bp, 10000 bp, 10000 bp, 20000 bp, 30000 bp, 40000 bp, 50000 bp, 60000 bp, 70000 bp, 80000 bp, 90000 bp, 100000 bp or more.
  • biofluid as used herein, relates to any fluid sample which can be obtained from the subject.
  • Samples may be collected from a variety of sources from a mammal (e.g., a human), including a body fluid sample, blood, serum, plasma, sputum including saliva, plasma, nipple aspirants, synovial fluids, cerebrospinal fluids, sweat, urine, fecal matter, pancreatic fluid, trabecular fluid, cerebrospinal fluid, tears, bronchial lavage, swabbings, bronchial aspirants, semen, prostatic fluid, precervicular fluid, vaginal fluids, pre-ejaculate, etc.
  • the biofluid is blood or serum.
  • biofluid such as blood is drawn by standard methods into a collection tube.
  • said tube preferably comprises siliconized glass, either without anticoagulant for preparation of serum or with EDTA, heparin, or similar anticoagulants, most preferably EDTA, for preparation of plasma.
  • Plasma may optionally be subsequently converted to serum by incubation of the anticoagulated plasma with an equal volume of calcium chloride at 37°C for a brief period, most preferably for 1-3 minutes, until clotting takes place.
  • the clot may then be pelleted by a brief centrifugation and the deproteinized plasma removed to another tube. Alternatively, the centrifugation may be omitted.
  • Serum can also be obtained using clot activator tubes.
  • level refers to a numeric value that measures the amount or concentration of polynucleotides derived from the EGFR gene (fragments thereof) present in the sample. Since the cell- free DNA comprises a population of genomic fragments of variable length, the term “level” is to be understood as the concentration of genome fragments which comprise at least the mutation which is being studied.
  • the level of cell-free EGFR can be determined by any method suitable for determining the concentration of a specific gene in a sample.
  • cell-free EGFR levels can be determined by measuring the levels of the corresponding mRNA carrying the activating EGFR mutation or by measuring the levels of the corresponding protein carrying the activating EGFR mutation. It can be desirable to confirm mutations in genomic DNA by analysis of transcripts and/or polypeptides, in order to ensure that the detected mutation is indeed expressed in the subject.
  • nucleic acid refers to a multimeric compound comprising nucleosides or nucleoside analogues which have nitrogenous heterocyclic bases, or base analogues, which are linked by phosphodiester bonds to form a polynucleotide such as DNA.
  • DNA refers to deoxyribonucleic acid.
  • a DNA sequence is a deoxyribonucleic sequence.
  • DNA is a long polymer of nucleotides and encodes the sequence of the amino acid residues in proteins using the genetic code. Mutations in genomic nucleic acid are advantageously detected by techniques based on mobility shift in amplified nucleic acid fragments. For instance, Chen et al. (Anal. Biochem., 1996, 239:61-9), describe the detection of single-base mutations by a competitive mobility shift assay. Moreover, assays based on the technique of Marcelino et al, BioTechniques 26: 1134-1148 (June 1999) are available commercially. In a preferred example, capillary heteroduplex analysis may be used to detect the presence of mutations based on mobility shift of duplex nucleic acids in capillary systems as a result of the presence of mismatches.
  • nucleic acids for analysis from samples generally requires nucleic acid amplification.
  • Many amplification methods rely on an enzymatic chain reaction (such as a polymerase chain reaction, a ligase chain reaction, or a self-sustained sequence replication) or from the replication of all or part of the vector into which it has been cloned.
  • the amplification according to the invention is an exponential amplification, as exhibited by for example the polymerase chain reaction.
  • amplification methods have been described in the literature, for example, general reviews of these methods in Landegren, U., et al, Science, 1988, 242:229- 237 and Lewis, R., Genetic Engineering News 10: 1, 54-55 (1990).
  • amplification methods can be used in the methods of our invention, and include polymerase chain reaction (PCR), PCR in situ, ligase amplification reaction (LAR), ligase hybridisation, Qbeta bacteriophage replicase, transcription-based amplification system (TAS), genomic amplification with transcript sequencing (GAWTS), nucleic acid sequence-based amplification (NASBA) and in situ hybridisation.
  • Primers suitable for use in various amplification techniques can be prepared according to methods known in the art.
  • SCCP detection is based on the aberrant migration of single stranded mutated DNA compared to reference DNA during electrophoresis. Mutation produces conformational change in single stranded DNA, resulting in mobility shift. Fluorescent SCCP uses fluorescent-labelled primers to aid detection. Reference and mutant DNA are thus amplified using fluorescent labelled primers. The amplified DNA is denatured and snap-cooled to produce single stranded DNA molecules, which are examined by non-denaturing gel electrophoresis.
  • SSCP detection is based on the aberrant migration of single stranded mutated DNA compared to reference DNA during electrophoresis. Mutation produces conformational change in single stranded DNA, resulting in mobility shift.
  • Fluorescent SCCP uses fluorescent-labelled primers to aid detection. Reference and mutant DNA are thus amplified using fluorescent labelled primers. The amplified DNA is denatured and snap-cooled to produce single stranded DNA molecules, which are examined by non-denaturing gel electrophoresis.
  • Chemical mismatch cleavage is based on the recognition and cleavage of DNA mismatched base pairs by a combination of hydroxylamine, osmium tetroxide and piperidine.
  • CMC Chemical mismatch cleavage
  • both reference DNA and mutant DNA are amplified with fluorescent labelled primers.
  • the amplicons are hybridised and then subjected to cleavage using Osmium tetroxide, which binds to an mismatched T base, or Hydroxylamine, which binds to mismatched C base, followed by Piperidine which cleaves at the site of a modified base. Cleaved fragments are then detected by electrophoresis.
  • RFLPs restriction fragment polymorphisms
  • SNPs single nucleotide polymorphisms
  • PIRA-PCR primer-induced restriction analysis PCR
  • Primers for PIRA-PCR which introduce suitable restriction sites can be designed by computational analysis, for example as described in Xiaiyi et al. (2001) Bio informatics 17:838-839.
  • Real-time PCR also known as Quantitative PCR, Real-time Quantitative PCR, or RTQ- PCR
  • Quantitative PCR is a method of simultaneous DNA quantification and amplification (Expert Rev. Mol. Diagn. 2005(2):209-19).
  • DNA is specifically amplified by polymerase chain reaction. After each round of amplification, the DNA is quantified.
  • Common methods of quantification include the use of fluorescent dyes that intercalate with double-strand DNA and modified DNA oligonucleotides (called probes) that fluoresce when hybridised with a complementary DNA.
  • the detecting step of the method of the invention is carried out by means of nucleic acid sequencing.
  • nucleic acid sequencing methods are cycle sequencing (Sarkar et al, 1995, Nucleic Acids Res. 23: 1269-70) or direct dideoxynucleotide sequencing, in which some or the entire DNA of interest that has been harvested from the sample is used as a template for sequencing reactions.
  • An oligonucleotide primer or set of primers specific to the gene or DNA of interest is used in standard sequencing reactions.
  • DNA sequencing such as sequencing by hybridization, sequencing using a "chip" containing many oligonucleotides for hybridization (as, for example, those produced by Affymetrix Corp.; Ramsay et al, 1998, Nature Biotechnology 16: 40-44; Marshall et al, 1998, Nature Biotechnology 16: 27-31), sequencing by HPLC (DeDionisio et al, 1996, J Chromatogr A 735: 191-208), and modifications of DNA sequencing strategies such as multiplex allele-specific diagnostic assay (MASDA; Shuber et al, 1997, Hum. Molec. Genet.
  • MASDA multiplex allele-specific diagnostic assay
  • amplification can be carried out using primers that are appropriately labelled, and the amplified primer extension products can be detected using procedures and equipment for detection of the label.
  • probes of this invention are labeled with at least one detectable moiety, wherein the detectable moiety or moieties are selected from the group consisting of: a conjugate, a branched detection system, a chromophore, a fluorophore, a spin label, a radioisotope, an enzyme, a hapten, an acridinium ester and a luminescent compound.
  • the primers used can labelled with a fluorophore.
  • the reverse primer of the method of the present invention is labelled with the 6-FAM fluorophore at its 5 ' end.
  • This fluorophore emits fluorescence with a peak wavelength of 522 nm.
  • the PCR can be carried out using one of the primers labelled with, for example, either FAM, HEX, VIC or NED dyes.
  • the posterior detection and analysis of the DNA amplified with the method of the invention is carried out by the GeneScan technique as it is illustrated in EP2046985.
  • the GeneScan technique as it is illustrated in EP2046985.
  • an aliquot of the PCR reaction typically 1 ⁇
  • the sample is placed in the ABI 3130 Genetic Analyzer and capillary electrophoresis is carried out.
  • the raw data is analysed using GeneScan software. This analysis is very important since the PCR products will be sized by extrapolation to an in-sample size standard. Using this technique inventors are able to detect in a very precise and accurate manner the mutation of interest.
  • the biofluid preferably serum or plasma
  • the biofluid may be utilized directly for identification and quantification of the mutant DNA.
  • nucleic acid is extracted from the biofluid as an initial step of the invention. In such cases, the total DNA extracted from said samples would represent the working material suitable for subsequent amplification.
  • amplification of nucleic acid is carried out.
  • the amplification of the DNA is carried out by means of PCR.
  • the general principles and conditions for amplification and detection of nucleic acids, such as using PCR, are well known for the skilled person in the art.
  • the Polymerase Chain Reaction (PCR) carried out by the method of the present invention uses appropriate and specific oligonucleotide primers or amplification oligonucleotides to specifically amplify the EGFR target sequences.
  • oligonucleotide primers or “amplification oligonucleotides” are herein used indistinguishably and refer to a polymeric nucleic acid having generally less than 1,000 residues, including those in a size range having a lower limit of about 2 to 5 residues and an upper limit of about 500 to 900 residues.
  • oligonucleotide primers are in a size range having a lower limit of about 5 to about 15 residues and an upper limit of about 100 to 200 residues. More preferably, oligonucleotide primers of the present invention are in a size range having a lower limit of about 10 to about 15 residues and an upper limit of about 17 to 100 residues.
  • oligonucleotide primers may be purified from naturally occurring nucleic acids, they are generally synthesized using any of a variety of well known enzymatic or chemical methods. In a particular embodiment of the invention, such oligonucleotide primers enable the specific amplification of the DNA fragments corresponding to the deletion of specific nucleotides in the exon 19 at the EGFR gene.
  • the method of the invention can be used for the detection of ELREA deletions at the exon 19.
  • the present invention refers to a method for the detection of 9, 12, 15, 18, or 24 nucleotides deletions in the exon 19 at the EGFR gene.
  • the method of the invention can be used for the detection of the L858R mutation at the exon 21 of the EGFR gene.
  • amplification oligonucleotide refers to an oligonucleotide that hybridizes to a target nucleic acid, or its complement, and participates in a nucleic acid amplification reaction.
  • Amplification oligonucleotides include primers and promoter- primers in which the 3' end of the oligonucleotide is extended enzymatically using another nucleic acid strand as the template.
  • an amplification oligonucleotide contains at least about 10 contiguous bases, and more preferably about 12 contiguous bases, that are complementary to a region of the target sequence (or its complementary strand).
  • Target-binding bases are preferably at least about 80%, and more preferably about 90% to 100% complementary to the sequence to which it binds.
  • An amplification oligonucleotide is preferably about 10 to about 60 bases long and may include modified nucleotides or base analogues.
  • amplify refers to a procedure to produce multiple copies of a target nucleic acid sequence or its complement or fragments thereof (i.e., the amplified product may contain less than the complete target sequence).
  • fragments may be produced by amplifying a portion of the target nucleic acid by using an amplification oligonucleotide which hybridizes to, and initiates polymerization from, an internal position of the target nucleic acid.
  • amplification methods include, for example, polymerase chain reaction (PCR) amplification, replicase-mediated amplification, ligase chain reaction (LCR) amplification, strand-displacement amplification (SDA) and transcription-associated or transcription-mediated amplification (TMA).
  • PCR amplification uses DNA polymerase, primers for opposite strands and thermal cycling to synthesize multiple copies of DNA or cDNA.
  • Replicase- mediated amplification uses QB-replicase to amplify RNA sequences.
  • LCR amplification uses at least four different oligonucleotides to amplify complementary strands of a target by using cycles of hybridization, ligation, and denaturation.
  • SDA uses a primer that contains a recognition site for a restriction endonuclease and an endonuclease that nicks one strand of a hemimodified DNA duplex that includes the target sequence, followed by a series of primer extension and strand displacement steps.
  • Transcription-associated or transcription-mediated amplification uses a primer that includes a promoter sequence and an R A polymerase specific for the promoter to produce multiple transcripts from a target sequence, thus amplifying the target sequence.
  • Preferred embodiments of the present invention amplify the EGFR target sequences using the present amplification oligonucleotides in a polymerase chain reaction (PCR) amplification.
  • PCR polymerase chain reaction
  • amplification oligonucleotides can readily be used in other methods of nucleic acid amplification that uses polymerase-mediated primer extension.
  • the mutations in EGFR are determined in serum samples as described in WO07039705 based on the use of specific Scorpion probes in combination with the Amplification Refractory Mutation System (ARMS) (Nucleic Acids Res., 1989, 17:2503-2516 and Nature Biotechnology, 1999, 17:804-807).
  • ARMS Amplification Refractory Mutation System
  • the levels of the EGFR gene carrying the activating mutation is measured by a method comprising the steps of
  • the Protein-Nucleic Acid probe which is capable of specifically recognising and hybridising with the EGFR wild type sequence thereby inhibiting its amplification has a sequence selected from the group consisting of SEQ ID NO:3 (for detecting ELREA deletions in exon 19) and SEQ ID NO: 10 (for detecting the L858R mutation in exon 21) such as it is described in WO08009740.
  • the amplifying step the nucleic acid sequence corresponding to a specific region of the EGFR gene is amplified by means of PCR using a Protein-Nucleic Acid (PNA) probe.
  • PNA Protein-Nucleic Acid
  • PNA probes are nucleic acid analogs in which the sugar phosphate backbone of a natural nucleic acid has been replaced by a synthetic peptide backbone, usually formed from N-(2-aminoethyl)-glycine units, resulting in an achiral and uncharged mimic.
  • This new molecule is chemically stable and resistant to hydro lytic (enzymatic) cleavage and thus not expected to be degraded inside a living cell.
  • PNA is still capable of sequence-specific binding to DNA as well as RNA obeying the Watson-Crick hydrogen bonding rules. Its hybrid complexes exhibit extraordinary thermal stability and display unique ionic strength properties.
  • PNA probes are preferred to nucleic acid probes because, unlike nucleic acid/nucleic acid duplexes which are destabilized under conditions of low salt, PNA/nucleic acid duplexes are formed and remain stable under conditions of very low salt.
  • factors commonly used to impose or control stringency of hybridization include formamide concentration (or other chemical denaturant reagent), salt concentration (i.e., ionic strength), hybridization temperature, detergent concentration, pH and the presence or absence of chaotropes.
  • Optimal stringency for a probe/target sequence combination is often found by the well known technique of fixing several of the aforementioned stringency factors and then determining the effect of varying a single stringency factor.
  • the same stringency factors can be modulated to thereby control the stringency of hybridization of a PNA to a nucleic acid, except that the hybridization of a PNA is fairly independent of ionic strength.
  • Optimal stringency for an assay may be experimentally determined by examination of each stringency factor until the desired degree of discrimination is achieved.
  • PNA oligomers can be prepared following standard solid-phase synthesis protocols for peptides (Merrifield, B. 1986. Solid-phase synthesis. Science 232, 341- 347) using, for example, a (methyl-benzhydryl)amine polystyrene resin as the solid support.
  • PNAs may contain a chimeric architecture, such as a PNA/DNA chimera, where a PNA oligomer is fused to a DNA oligomer.
  • the PNA probe utilized by the inventors is capable of specifically recognize and hybridize with the wild-type EGFR sequence.
  • the PNA probe to be used for carrying out the method of the present invention comprises the PNA probe described as the SEQ ID NO: 3 or SEQ ID NO: 10 in the Example accompanying the present invention.
  • Such probe is added to the PCR reaction mix thus inhibiting amplification of the wild-type allele and favouring amplification of the mutant allele present in the sample, i.e. EGFR mutant, facilitating its posterior detection.
  • a suitable PNA probe do not need to have exactly these probing nucleic acid sequences to be operative but often modified according to the particular assay conditions.
  • shorter PNA probes can be prepared by truncation of the nucleic acid sequence if the stability of the hybrid needs to be modified to thereby lower the Tm and/or adjust for stringency.
  • nucleic acid sequence may be truncated at one end and extended at the other end as long as the discriminating nucleic acid sequence remains within the sequence of the PNA probe.
  • the first method of the invention involves correlating the detection/absence of detection or the levels of the EGFR gene carrying the activating mutation with a prediction of outcome.
  • the correlation may indicate a poor outcome when cell- free EGFR gene containing said mutation is detected or wherein the levels of the EGFR gene carrying the activating mutation are increased levels with respect to a reference value.
  • the correlation may indicate a good survival when cell- free EGFR gene containing said mutation is not detected or wherein the levels of the EGFR gene carrying the activating mutation are decreased levels with respect to a reference value.
  • detection or "absence of detection” as used herein refers to the ability or inability to detect cell-free EGFR polynucleotides carrying the activating mutations using the methods provided herein.
  • reference value refers to a laboratory value used as a reference for values/data obtained by laboratory examination of patients or samples collected from patients.
  • the reference value or reference level can be an absolute value; a relative value; a value that has an upper and/or lower limit; a range of values; an average value; a median value, a mean value, or a value as compared to a particular control or baseline value.
  • the reference value can be based on a large number of samples, such as from population of subjects of the chronological age matched group, or based on a pool of samples including or excluding the sample to be tested.
  • the reference value is an absolute value.
  • the reference value is the level of mutated EGFR DNA in 2 pg/ ⁇ of diploid heterozygotic mutant genomic DNA or the level of concentration of mutated EGFR DNA in 1 pg/ ⁇ of haploid mutant genomic DNA.
  • the levels of the EGFR gene carrying the activating mutation in a bio fluid of patient can be considered as being increased if the signal corresponding to the mutated EGFR DNA detected in a given volume of biofluid of the patient is higher than the signal detected in a sample containing 2 pg/ ⁇ of genomic DNA wherein one of the copies of the EGFR gene contains the mutation under study when both the patient sample and the reference sample are analysed in parallel using the same technique.
  • the levels of the EGFR gene carrying the activating mutation in a biofluid of patient can be considered as being decreased if the levels of mutated EGFR DNA detected in a given volume of biofluid of the patient are lower than the levels detected in a sample containing 2 pg/ ⁇ of genomic DNA wherein one of the copies of the EGFR gene contains the mutation under study when both the patient sample and the reference sample are analysed in parallel using the same technique.
  • the genomic DNA used to generate the reference value is typically genomic DNA isolated from a tumor tissue carrying the mutation or genomic DNA from a cell line derived from lung cancer carrying said mutation such as, for example, cell lines carrying a deletion in exon 198 (e.g.
  • the PC9 cell line as described by Koizumi et al, (Int. J. Cancer, 2005, 116: 36-44) or the HCC2279, the HCC827 or the H4006 cell lines) as well as cells carrying the L858R mutation (e.g. the H3255) or cells carrying the L858R and the T790M mutation (the H1975 cell line).
  • the reference value is the level of mutated EGFR in a pool of samples from patients suffering from lung cancer with activating EGFR mutations.
  • mutated EGFR levels are determined in a population of subjects suffering lung cancer with activating EGFR mutations and the reference value is determined by the average or mean of the mutated EGFR levels in the population.
  • the reference value corresponds to the detection limit of the assay used for detecting mutated EGFR in the bio fluid.
  • an increase in EGFR levels of at least 1.1 -fold, 1.5- fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90- fold, 100-fold or even more compared with the reference value is considered as "increased” expression.
  • a decrease in EGFR levels below the reference value of at least 0.9-fold, 0.75-fold, 0.2-fold, 0.1-fold, 0.05-fold, 0.025- fold, 0.02-fold, 0.01 -fold, 0.005-fold or even less compared with the reference value is considered as "decreased" expression.
  • the first method of the invention does not comprise the determination of any other marker, i.e. the level of EGFR gene carrying the activating mutation can be used on its own for predicting the outcome.
  • the term "marker”, as used herein, refers to any parameter which provides a correlation with a The term “marker” is considered to include clinical markers (e.g. ECOG performance status) as well as molecular markers.
  • molecular marker refers generally to a molecule, including a gene, protein, carbohydrate structure, or glycolipid, the expression of which in or on a mammalian tissue or cell can be detected by standard methods and which is associated quantitatively or qualitatively with the presence of a biological phenomenon (e.g. outcome of a patient suffering lung cancer or lung cancer carrying an activating EGFR mutation).
  • the level of mutant EGFR in a bio fluid of samples can be used as a marker for predicting survival in patients suffering EGFR-positive lung cancer provides a method for typing or classifying a patient into a poor survival or a good survival group.
  • the invention relates to a method (second method of the invention) A method of prognosing or classifying a subject with lung cancer positive for an activating EGFR mutation and treated with chemotherapy, with an EGFR inhibitor or a combination thereof said method comprising determining in a bio fluid of said patient the level of cell-free EGFR gene carrying said EGFR mutation wherein the detection of cell-free EGFR gene containing said mutation or the detection of an increased level thereof with respect to a reference value is is used to prognose or classify the subject with lung cancer into a poor outcome group or wherein an absence of detection of the cell-free EGFR gene containing said mutation or the detection of decreased levels thereof with respect to a reference value is is used to prognose or classify the subject with lung cancer into a good outcome group.
  • prognosing means categorizing a subject into a clinical outcome group, such as a poor survival group or a good survival group.
  • a subject is classified or prognosed according to whether the subject's risk score is above or below a control value.
  • prognosing or classifying comprises a method or process of determining whether an individual with NSCLC has a good or poor outcome, or grouping an individual with NSCLC into a good outcome group or a poor outcome group, based on whether the individual's calculated risk score is above or below the control value.
  • the reference value is an absolute value.
  • the reference value is the level or concentration of mutated EGFR DNA in 2 pg/ ⁇ of diploid heterozygotic mutant genomic DNA or the level of concentration of mutated EGFR DNA in 1 pg/ ⁇ of haploid mutant genomic DNA.
  • good survival refers to an increased chance of survival as compared to patients in the "poor survival” group.
  • the bio markers of the application can prognose or classify patients into a "good survival group". These patients are at a lower risk of death after surgery.
  • the patient is classified in a good survival group, and the patient does not receive chemotherapy.
  • pool survival refers to an increased risk of death as compared to patients in the "good survival” group.
  • gene signatures of the application can prognose or classify patients into a "poor survival group". These patients are at greater risk of death after surgery.
  • the patient is classified in a poor survival group, and the patient receives a chemotherapeutic regimen.
  • the patient has suffered advanced lung cancer.
  • the lung cancer is Non Small Cell Lung Cancer.
  • the second method of the invention is carried out using serum or plasma as a biofluid.
  • the biofluid sample is obtained prior to the treatment with chemotherapy.
  • the second method of the invention is carried out in order to type or prognose a patient which has been treated with a combination of a chemotherapy and a therapy with an EGFR inhibitor.
  • the combined treatment comprises the sequential administration of chemotherapy and an EGFR inhibitor or of an EGFR inhibitor and a chemotherapy.
  • Suitable chemotherapy compounds have been defined in the first method of the invention.
  • the chemotherapy is a therapy with a platinum- based compound.
  • Suitable platinum-based compounds have been mentioned in the context of the first method of the invention.
  • the platinum- based compound is cisplatin or carboplatin.
  • the EGFR tyrosine kinase inhibitor is a dual EGFR inhibitor, a dual EGFR tyrosine kinase inhibitor or a EGFR tyrosine kinase inhibitor specific for EGFR carrying a resistance mutation.
  • the EGFR inhibitor is a EGFR tyrosine kinase inhibitor.
  • EGFR tyrosine kinase inhibitor is gefitinob or erlotinib.
  • the combination of a chemotherapy with a therapy with an EGFR inhibitor comprises the sequential administration of chemotherapy and an EGFR inhibitor or of an EGFR inhibitor and a chemotherapy.
  • the activating mutation in the EGFR gene is selected from L858R mutation and an (E)LREA deletion in exon 19 or a combination thereof.
  • the levels of EGFR carrying the activating mutation in the biofluid is measured by (i) amplifying the nucleic acid sequence corresponding to a region of the EGFR gene comprising the mutation of the EGFR gene by means of PCR using a Protein-Nucleic Acid probe, wherein said Protein-Nucleic Acid probe is capable of specifically recognising and hybridising with the EGFR wild type sequence thereby inhibiting its amplification and
  • the first method of the invention does not comprise the determination of any other marker, i.e. the level of EGFR gene carrying the activating mutation can be used on its own for predicting the outcome of the patients.
  • the invention relates to a computer system that is provided with means for implementing the first or second method according to the invention.
  • the computer system can include:
  • At least one memory containing at least one computer program adapted to control the operation of the computer system to implement a method that includes: (i) receiving DNA methylation data e.g., the methylation profile of a CUP and the methylation profile of one or more primary tumors, (ii) determining the degree of identity between the methylation profile of the CUP and the methylation profile of the primary tumors and
  • Another aspect of the present invention relates to a computer program for controlling a computer system to execute the steps according to the first, second or third method of the invention.
  • the present invention can be implemented on a stand-alone computer or as part of a networked computer system.
  • all the software and data can reside on local memory devices, for example an optical disk or flash memory device can be used to store the computer software for implementing the invention as well as the data.
  • the software or the data or both can be accessed through a network connection to remote devices.
  • the invention use a client -server environment over a public network, such as the internet or a private network to connect to data and resources stored in remote and/or centrally located locations.
  • a server including a web server can provide access, either open access, pay as you go or subscription based access to the information provided according to the invention.
  • a client computer executing a client software or program, such as a web browser, connects to the server over a network.
  • the client software or web browser provides a user interface for a user of the invention to input data and information and receive access to data and information.
  • the client software can be viewed on a local computer display or other output device and can allow the user to input information, such as by using a computer keyboard, mouse or other input device.
  • the server executes one or more computer programs that enable the client software to input data, process data according to the invention and output data to the user, as well as provide access to local and remote computer resources.
  • the user interface can include a graphical user interface comprising an access element, such as a text box, that permits entry of data from the assay, e.g., the DNA methylation data levels or DNA gene expression levels of target genes of a reference pluripotent stem cell population and/or pluripotent stem cell population of interest, as well as a display element that can provide a graphical read out of the results of a comparison with a score card, or data sets transmitted to or made available by a processor following execution of the instructions encoded on a computer- readable medium.
  • an access element such as a text box
  • a method for the further treatment of lung cancer in another aspect, relates to A method for the further treatment of lung cancer in a subject in need thereof after treatment with chemotherapy, with an EGFR inhibitor, or a combination thereof, said method comprising;
  • the reference value is 2 pg of cell-free mutated EGFR gene/ ⁇ of bio fluid.
  • the patient has advanced lung cancer.
  • the lung cancer is Non Small Cell Lung Cancer.
  • the bio fluid is serum or plasma.
  • the combination of a chemotherapy with a therapy with an EGFR inhibitor comprises the sequential administration of chemotherapy and an EGFR inhibitor or of an EGFR inhibitor and a chemotherapy.
  • the chemotherapy is a therapy with a platinum-based compound.
  • the platinum-based compound is cisplatin or carboplatin.
  • the EGFR inhibitor is a EGFR tyrosine kinase inhibitor.
  • the EGFR tyrosine kinase inhibitor is a dual EGFR inhibitor, a dual EGFR tyrosine kinase inhibitor or a EGFR tyrosine kinase inhibitor specific for EGFR carrying a resistance mutation.
  • the tyrosine- kinase inhibitor is erlotinib or gefitinib.
  • the bio fluid sample is obtained prior to the treatment of the patient with chemotherapy, with an EGFR inhibitor or the combination thereof.
  • the mutation in EGFR conferring sensitivity to an EGFR tyrosine kinase inhibitor is a L858R mutation, an (E)LREA deletion in exon 19 or a combination thereof.
  • the levels of cell-free EGFR gene in the biofluid of the patient is measured by
  • the invention in another aspect, relates to a kit for determining the outcome of a subject suffering from lung cancer after treatment with chemotherapy, with an EGFR inhibitor, or a combination thereof comprising: (a) means for quantifying in a biofluid sample of said subject the level of cell-free EGFR gene carrying at least one activating EGFR mutation; and (b) means for comparing the level quantified in (a) with a reference level.
  • the kit further comprises means for determining a therapy for further treating said lung cancer based on the comparison of the quantified expression level to the reference level.
  • the invention in another aspect, relates to a kit comprising: a) a reagent for quantifying in a biofluid sample of a subject suffering from lung cancer after treatment with chemotherapy, with an EGFR inhibitor, or a combination thereof, the level of cell- free EGFR gene carrying at least one activating EGFR mutation; and (b) one or more indices that have been predetermined to correlate levels of cell-free EGFR gene carrying at least one activating EGFR mutation in a biofluid sample to the outcome of the lung cancer treatment.
  • the patient has advanced lung cancer.
  • the lung cancer is Non Small Cell Lung Cancer.
  • the biofluid is serum or plasma.
  • the combination of a chemotherapy with a therapy with an EGFR inhibitor comprises the sequential administration of chemotherapy and an EGFR inhibitor or of an EGFR inhibitor and a chemotherapy.
  • the chemotherapy is a therapy with a platinum-based compound.
  • the platinum-based compound is cisplatin or carboplatin.
  • the EGFR inhibitor is a EGFR tyrosine kinase inhibitor.
  • the tyrosine kinase inhibitor is a dual EGFR inhibitor, a dual EGFR tyrosine kinase inhibitor or a EGFR tyrosine kinase inhibitor specific for EGFR carrying a resistance mutation.
  • the kit according to claim 57 wherein EGFR tyrosine-kinase inhibitor is erlotinib or gefitinib.
  • the mutation in EGFR conferring sensitivity to an EGFR tyrosine kinase inhibitor is a L858R mutation, an (E)LREA deletion in exon 19 or a combination thereof.
  • primers were as follows: forward 5'- GTGCATCGCTGGTAACATCC-3 ' (SEQ ID NO: 1) and reverse 5'- TGTGGAGATGAGCAGGGTCT- 3' (SEQ ID NO: 2).
  • PNA Peptide Nucleic Acid
  • the first PCR was performed in 25- ⁇ volumes adding 2 ⁇ of sample, 0.125 ⁇ of Ecotaq Polymerase (Ecogen, Barcelona, Spain), 2,5 ⁇ of PCR buffer xlO, 0,625 ⁇ dNTPs (10 mM), 0,75 ⁇ MgC12 (50 mM), 1.25 pmol of each primer (10 ⁇ ) and 12,5 ⁇ ⁇ of 10 ⁇ PNA probe.
  • Amplification was as follows: 25 cycles of 30 seconds at 94°C, 30 seconds at 64°C, and 1 minute at 72°C (exons 19 and 21), or 35 cycles of 30 seconds at 94°C, 30 seconds at 58°C, and 1 minute at 72°C (exon 20).
  • amplification was performed with the following primers: forward 5 '-ACTCTGGATCCCAGAAGGTGAG-3 ' (SEQ ID NO:4) and reverse 5 '- FAM-CC ACAC AGCAAAGCAGAAACTC-3 ' (SEQ ID NO: 5).
  • Amplification (35 cycles) was done for 30 seconds at 94°C, 30 seconds at 58°C, and 1 minute at 72°C in 25- ⁇ 1 volumes adding 2 ⁇ of sample, 0.1 ⁇ of Ecotaq Polymerase (Ecogen, Barcelona, Spain), 2,5 ⁇ of PCR buffer xlO, 0,625 ⁇ dNTPs (10 mM), 1 ⁇ MgC12 (50 mM), 1.25 pmol of each primer (10 ⁇ ) and 7,5 ⁇ ⁇ of 10 mM PNA probe.
  • One microliter of a 1/200 dilution of each PCR product was mixed with 0.5 ⁇ of size standard (Applied Biosystems) and denatured in 9 ⁇ formamide at 90°C for 5 minutes.
  • Amplification was performed in 12.5- ⁇ 1 volumes using 1 of sample, 6.25 ⁇ of Ampli Taq Gold PCR Master Mix (Applied Biosystems), 0.75 ⁇ of each primer (10 ⁇ ), 0.25 ⁇ , of probes (10 ⁇ ) and 0,625 ⁇ of PNA (10 ⁇ ). Samples were submitted to 50 cycles of 15 seconds at 94°C and 1 minute at 60°C in an Applied Biosystems 7000 real-time cycler.
  • a sample is considered positive (mutation detected) if a peak of mutated allele appears at least in one of the aliquots analyzed.
  • the number of aliquots showing a mutated peak is recorded.
  • another indicator is calculated as follows: area of the mutated peaks (in the four aliquots) / total area of the wt + mutated peaks (also in the four aliquots)
  • a sample is considered positive (mutation detected) if at least in one of the aliquots analyzed is positive.
  • EGFR mutations in cfDNA from serum were examined in the EURTAC (European Tarceva ® vs Chemotherapy) trial in European patients with advanced EGFR mutation-positive non-small-cell lung cancer.
  • Median progression- free survival (PFS) in the erlotinib group was 9.7 months, in comparison with 5.2 months in the chemotherapy group (hazard ratio [HR] 0-37, 95% CI 0-25-0-54; p ⁇ 0-0001).
  • HR for patients with EGFR mutations detected in serum was 0.25 in favour of erlotinib.
  • Table 2 Multivariate analysis of survival. No interaction between treatment arm and cfDNA EGFR mutation.

Abstract

Cette invention concerne des méthodes permettant de déterminer la survie des patients atteints d'un cancer du poumon positif à la mutation de l'EGFR en fonction de la détermination des niveaux d'EFGR acellulaire portant ladite mutation. Cette invention concerne également des méthodes permettant de procéder au typage et à la classification des patients en fonction des niveaux d'EGFR circulant libre portant ladite mutation. Des programmes d'ordinateur ainsi que des systèmes et des kits pour la mise en œuvre des méthodes précitées sont également décrits.
PCT/EP2013/062992 2012-06-21 2013-06-21 Biomarqueurs moléculaires permettant de prédire l'issue dans le cancer du poumon WO2013190089A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP12382245 2012-06-21
EP12382245.4 2012-06-21

Publications (1)

Publication Number Publication Date
WO2013190089A1 true WO2013190089A1 (fr) 2013-12-27

Family

ID=48692495

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2013/062992 WO2013190089A1 (fr) 2012-06-21 2013-06-21 Biomarqueurs moléculaires permettant de prédire l'issue dans le cancer du poumon

Country Status (1)

Country Link
WO (1) WO2013190089A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109097469A (zh) * 2018-08-17 2018-12-28 上海迈景纳米科技有限公司 一种基于固相杂交技术的egfr突变基因检测方法
WO2019075251A3 (fr) * 2017-10-12 2019-07-25 Nantomics, Llc Score de cancer pour l'évaluation et la prévision de réponse à partir de fluides biologiques

Citations (68)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992020642A1 (fr) 1991-05-10 1992-11-26 Rhone-Poulenc Rorer International (Holdings) Inc. Composes aryle et heteroaryle bis monocycliques et/ou bicycliques qui inhibent la tyrosine kinase d'un recepteur du egf et/ou du pdgf
EP0520722A1 (fr) 1991-06-28 1992-12-30 Zeneca Limited Préparations thérapeutiques contenant des dérivés de quinazoline
EP0566226A1 (fr) 1992-01-20 1993-10-20 Zeneca Limited Dérivés de quinazoline
WO1995009847A1 (fr) 1993-10-01 1995-04-13 Ciba-Geigy Ag Derives pyrimidineamine et leurs procedes de preparation
WO1995019970A1 (fr) 1994-01-25 1995-07-27 Warner-Lambert Company Composes tricycliques pouvant inhiber les tyrosines kinases de la famille des recepteurs du facteur de croissance epidermique
WO1995019774A1 (fr) 1994-01-25 1995-07-27 Warner-Lambert Company Composes bicycliques permettant d'inhiber les tyrosine-kinases de la famille du recepteur du facteur de croissance de l'epiderme
EP0682027A1 (fr) 1994-05-03 1995-11-15 Ciba-Geigy Ag Dérivés de la pyrrolopyrimidine avec une activité anti-proliférative
WO1996030347A1 (fr) 1995-03-30 1996-10-03 Pfizer Inc. Derives de quinazoline
WO1996031510A1 (fr) 1995-04-03 1996-10-10 Novartis Ag Derives de pyrazole et leurs procedes de preparation
US5567588A (en) 1990-06-11 1996-10-22 University Research Corporation Systematic evolution of ligands by exponential enrichment: Solution SELEX
WO1996033980A1 (fr) 1995-04-27 1996-10-31 Zeneca Limited Derives de quinazoline
US5580737A (en) 1990-06-11 1996-12-03 Nexstar Pharmaceuticals, Inc. High-affinity nucleic acid ligands that discriminate between theophylline and caffeine
WO1997002266A1 (fr) 1995-07-06 1997-01-23 Novartis Ag Pyrrolopyrimidines et leurs procedes de preparation
WO1997003288A1 (fr) 1995-07-07 1997-01-30 Bonus Energy A/S Cadre de base pour bati de moulin a vent et moulin a vent pourvu de ce cadre de base
WO1997013771A1 (fr) 1995-10-11 1997-04-17 Glaxo Group Limited Composes hetero-aromatiques bicycliques utilises comme inhibiteurs de proteine tyrosine kinase
WO1997019065A1 (fr) 1995-11-20 1997-05-29 Celltech Therapeutics Limited 2-anilinopyrimidines substituees utiles en tant qu'inhibiteurs de proteine kinase
US5650415A (en) 1995-06-07 1997-07-22 Sugen, Inc. Quinoline compounds
WO1997027199A1 (fr) 1996-01-23 1997-07-31 Novartis Ag Pyrrolopyrimidines et leurs procedes de preparation
EP0787772A2 (fr) 1996-01-30 1997-08-06 Dow Corning Toray Silicone Company Ltd. Compositions d'élastomère de silicone
US5656643A (en) 1993-11-08 1997-08-12 Rhone-Poulenc Rorer Pharmaceuticals Inc. Bis mono-and bicyclic aryl and heteroaryl compounds which inhibit EGF and/or PDGF receptor tyrosine kinase
WO1997030034A1 (fr) 1996-02-14 1997-08-21 Zeneca Limited Derives de la quinazoline servant d'agents antitumoraux
WO1997030044A1 (fr) 1996-02-14 1997-08-21 Zeneca Limited Composes de quinazoline
US5660985A (en) 1990-06-11 1997-08-26 Nexstar Pharmaceuticals, Inc. High affinity nucleic acid ligands containing modified nucleotides
WO1997032881A1 (fr) 1996-03-06 1997-09-12 Dr. Karl Thomae Gmbh Derives de 4-amino-pyrimidine, medicaments contenant ces composes, leur utilisation et leur procede de production
WO1997032880A1 (fr) 1996-03-06 1997-09-12 Dr. Karl Thomae Gmbh PYRIMIDO[5,4-d]PYRIMIDINES, MEDICAMENTS CONTENANT CES COMPOSES, LEUR UTILISATION ET PROCEDE DE FABRICATION ASSOCIE
WO1997034895A1 (fr) 1996-03-15 1997-09-25 Novartis Ag NOUVELLES N-7-HETEROCYCLYL-PYRROLO[2,3-d]PYRIMIDINES ET LEUR UTILISATION
WO1997038994A1 (fr) 1996-04-13 1997-10-23 Zeneca Limited Derives de quinazoline
WO1997038983A1 (fr) 1996-04-12 1997-10-23 Warner-Lambert Company Inhibiteurs irreversibles de tyrosine kinases
WO1997049688A1 (fr) 1996-06-24 1997-12-31 Pfizer Inc. Derives tricycliques substitues par phenylamino, destines au traitement des maladies hyperproliferatives
US5707796A (en) 1990-06-11 1998-01-13 Nexstar Pharmaceuticals, Inc. Method for selecting nucleic acids on the basis of structure
WO1998002438A1 (fr) 1996-07-13 1998-01-22 Glaxo Group Limited Composes heteroaromatiques bicycliques en tant qu'inhibiteurs de la proteine tyrosine kinase
WO1998002437A1 (fr) 1996-07-13 1998-01-22 Glaxo Group Limited Composes heteroaromatiques bicycliques en tant qu'inhibiteurs de la proteine tyrosine kinase
WO1998002434A1 (fr) 1996-07-13 1998-01-22 Glaxo Group Limited Composes heterocycliques condenses en tant qu'inhibiteurs de la proteine tyrosine kinase
WO1998007726A1 (fr) 1996-08-23 1998-02-26 Novartis Ag Pyrrolopyrimidines substituees et procede pour leur preparation
WO1998014450A1 (fr) 1996-10-02 1998-04-09 Novartis Ag Derives de pyrimidine et procedes de preparation de ces derniers
WO1998014449A1 (fr) 1996-10-02 1998-04-09 Novartis Ag Derives de pyrazole condenses et procedes pour leur preparation
WO1998014451A1 (fr) 1996-10-02 1998-04-09 Novartis Ag Derive de pyrazole condense et procede pour sa preparation
EP0837063A1 (fr) 1996-10-17 1998-04-22 Pfizer Inc. Dérivés de 4-aminoquinazoline
WO1998017662A1 (fr) 1996-10-18 1998-04-30 Novartis Ag Derives d'heterocyclyle bicyclique a substitution phenyle et utilisation de ces derives
US5747498A (en) 1996-05-28 1998-05-05 Pfizer Inc. Alkynyl and azido-substituted 4-anilinoquinazolines
US5763177A (en) 1990-06-11 1998-06-09 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: photoselection of nucleic acid ligands and solution selex
US5780607A (en) 1995-10-13 1998-07-14 Hoffmann-La Roche Inc. Antisense oligomers
US5789427A (en) 1994-03-07 1998-08-04 Sugen, Inc. Methods and compositions for inhibiting cell proliferative disorders
WO1998033798A2 (fr) 1997-02-05 1998-08-06 Warner Lambert Company Pyrido[2,3d]pyrimidines et 4-aminopyrimidines en tant qu'inhibiteurs de la proliferation cellulaire
US5811234A (en) 1990-10-19 1998-09-22 Board Of Trustees Of University Of Illinois Methods and applications for efficient genetic suppressor elements
US5814500A (en) 1996-10-31 1998-09-29 The Johns Hopkins University School Of Medicine Delivery construct for antisense nucleic acids and methods of use
US5843657A (en) 1994-03-01 1998-12-01 The United States Of America As Represented By The Department Of Health And Human Services Isolation of cellular material under microscopic visualization
WO1999007701A1 (fr) 1997-08-05 1999-02-18 Sugen, Inc. Derives de quinoxaline tricyclique utiles en tant qu'inhibiteurs de proteine tyrosine kinase
WO1999035132A1 (fr) 1998-01-12 1999-07-15 Glaxo Group Limited Composes heterocycliques
WO1999035146A1 (fr) 1998-01-12 1999-07-15 Glaxo Group Limited Composes heteroaromatiques bicycliques agissant comme inhibiteurs de la tyrosine kinase
US6011577A (en) 1997-06-30 2000-01-04 Polaroid Corporation Modular optical print head assembly
WO2001034574A1 (fr) 1999-11-11 2001-05-17 Osi Pharmaceuticals, Inc. Polymorphe stable de chlorhydrate de n-(3-ethynylphenylamino)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine, et methodes de production et utilisations pharmaceutiques dudit polymorphe
US6251516B1 (en) 1994-03-01 2001-06-26 The United States Of America As Represented By The Department Of Health And Human Services Isolation of cellular material under microscopic visualization
US6288082B1 (en) 1998-09-29 2001-09-11 American Cyanamid Company Substituted 3-cyanoquinolines
US6384051B1 (en) 2000-03-13 2002-05-07 American Cyanamid Company Method of treating or inhibiting colonic polyps
WO2003028531A2 (fr) * 2001-09-28 2003-04-10 Oncomedx, Inc. Detection d'arn tumoral dans le plasma et le serum
US20030198627A1 (en) 2001-09-01 2003-10-23 Gert-Jan Arts siRNA knockout assay method and constructs
US6699843B2 (en) 1995-06-07 2004-03-02 Gilead Sciences, Inc. Method for treatment of tumors using nucleic acid ligands to PDGF
US20040063906A1 (en) 2000-11-24 2004-04-01 Nielsen Peter E. Pna analogues
US20050059678A1 (en) 2003-09-15 2005-03-17 Wyeth Protein tyrosine kinase enzyme inhibitors
US6969614B1 (en) 1999-02-16 2005-11-29 The United States Of America As Represented By The Department Of Health And Human Services Methods for the isolation and analysis of cellular protein content
US20050272083A1 (en) 2004-06-04 2005-12-08 Somasekar Seshagiri EGFR mutations
WO2006091889A2 (fr) 2005-02-25 2006-08-31 Michael Horn Bande transporteuse modulaire pour vehicule
WO2007039705A1 (fr) 2005-10-05 2007-04-12 Astrazeneca Uk Limited Méthode pour prédire ou surveiller la réponse d'un patient à un médicament de récepteur erbb
WO2008009740A1 (fr) 2006-07-20 2008-01-24 Pangaea Biotech, S.A. Procédé de détection de mutations egfr dans des échantillons sanguins
WO2008106453A2 (fr) * 2007-02-26 2008-09-04 John Wayne Cancer Institute Utilité de la mutation d'adn de b-raf dans le diagnostic et le traitement d'un cancer
WO2009021149A1 (fr) * 2007-08-07 2009-02-12 The Penn State Research Foundation Détection d'acides nucléiques extracellulaires associés à une tumeur dans le plasma ou le sérum sanguin
EP2468883A1 (fr) * 2010-12-22 2012-06-27 Pangaea Biotech S.L. Biomarqueurs moléculaires pour la prédiction de la réponse aux inhibiteurs de la tyrosine kinase dans le cancer du poumon

Patent Citations (70)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5580737A (en) 1990-06-11 1996-12-03 Nexstar Pharmaceuticals, Inc. High-affinity nucleic acid ligands that discriminate between theophylline and caffeine
US5763177A (en) 1990-06-11 1998-06-09 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: photoselection of nucleic acid ligands and solution selex
US5660985A (en) 1990-06-11 1997-08-26 Nexstar Pharmaceuticals, Inc. High affinity nucleic acid ligands containing modified nucleotides
US5707796A (en) 1990-06-11 1998-01-13 Nexstar Pharmaceuticals, Inc. Method for selecting nucleic acids on the basis of structure
US5567588A (en) 1990-06-11 1996-10-22 University Research Corporation Systematic evolution of ligands by exponential enrichment: Solution SELEX
US5811234A (en) 1990-10-19 1998-09-22 Board Of Trustees Of University Of Illinois Methods and applications for efficient genetic suppressor elements
WO1992020642A1 (fr) 1991-05-10 1992-11-26 Rhone-Poulenc Rorer International (Holdings) Inc. Composes aryle et heteroaryle bis monocycliques et/ou bicycliques qui inhibent la tyrosine kinase d'un recepteur du egf et/ou du pdgf
EP0520722A1 (fr) 1991-06-28 1992-12-30 Zeneca Limited Préparations thérapeutiques contenant des dérivés de quinazoline
EP0566226A1 (fr) 1992-01-20 1993-10-20 Zeneca Limited Dérivés de quinazoline
WO1995009847A1 (fr) 1993-10-01 1995-04-13 Ciba-Geigy Ag Derives pyrimidineamine et leurs procedes de preparation
US5656643A (en) 1993-11-08 1997-08-12 Rhone-Poulenc Rorer Pharmaceuticals Inc. Bis mono-and bicyclic aryl and heteroaryl compounds which inhibit EGF and/or PDGF receptor tyrosine kinase
WO1995019970A1 (fr) 1994-01-25 1995-07-27 Warner-Lambert Company Composes tricycliques pouvant inhiber les tyrosines kinases de la famille des recepteurs du facteur de croissance epidermique
WO1995019774A1 (fr) 1994-01-25 1995-07-27 Warner-Lambert Company Composes bicycliques permettant d'inhiber les tyrosine-kinases de la famille du recepteur du facteur de croissance de l'epiderme
US5843657A (en) 1994-03-01 1998-12-01 The United States Of America As Represented By The Department Of Health And Human Services Isolation of cellular material under microscopic visualization
US6251516B1 (en) 1994-03-01 2001-06-26 The United States Of America As Represented By The Department Of Health And Human Services Isolation of cellular material under microscopic visualization
US5789427A (en) 1994-03-07 1998-08-04 Sugen, Inc. Methods and compositions for inhibiting cell proliferative disorders
EP0682027A1 (fr) 1994-05-03 1995-11-15 Ciba-Geigy Ag Dérivés de la pyrrolopyrimidine avec une activité anti-proliférative
WO1996030347A1 (fr) 1995-03-30 1996-10-03 Pfizer Inc. Derives de quinazoline
WO1996031510A1 (fr) 1995-04-03 1996-10-10 Novartis Ag Derives de pyrazole et leurs procedes de preparation
WO1996033980A1 (fr) 1995-04-27 1996-10-31 Zeneca Limited Derives de quinazoline
US5650415A (en) 1995-06-07 1997-07-22 Sugen, Inc. Quinoline compounds
US6699843B2 (en) 1995-06-07 2004-03-02 Gilead Sciences, Inc. Method for treatment of tumors using nucleic acid ligands to PDGF
WO1997002266A1 (fr) 1995-07-06 1997-01-23 Novartis Ag Pyrrolopyrimidines et leurs procedes de preparation
WO1997003288A1 (fr) 1995-07-07 1997-01-30 Bonus Energy A/S Cadre de base pour bati de moulin a vent et moulin a vent pourvu de ce cadre de base
WO1997013771A1 (fr) 1995-10-11 1997-04-17 Glaxo Group Limited Composes hetero-aromatiques bicycliques utilises comme inhibiteurs de proteine tyrosine kinase
US5780607A (en) 1995-10-13 1998-07-14 Hoffmann-La Roche Inc. Antisense oligomers
WO1997019065A1 (fr) 1995-11-20 1997-05-29 Celltech Therapeutics Limited 2-anilinopyrimidines substituees utiles en tant qu'inhibiteurs de proteine kinase
WO1997027199A1 (fr) 1996-01-23 1997-07-31 Novartis Ag Pyrrolopyrimidines et leurs procedes de preparation
EP0787772A2 (fr) 1996-01-30 1997-08-06 Dow Corning Toray Silicone Company Ltd. Compositions d'élastomère de silicone
WO1997030034A1 (fr) 1996-02-14 1997-08-21 Zeneca Limited Derives de la quinazoline servant d'agents antitumoraux
WO1997030044A1 (fr) 1996-02-14 1997-08-21 Zeneca Limited Composes de quinazoline
DE19629652A1 (de) 1996-03-06 1998-01-29 Thomae Gmbh Dr K 4-Amino-pyrimidin-Derivate, diese Verbindungen enthaltende Arzneimittel, deren Verwendung und Verfahren zu ihrer Herstellung
WO1997032880A1 (fr) 1996-03-06 1997-09-12 Dr. Karl Thomae Gmbh PYRIMIDO[5,4-d]PYRIMIDINES, MEDICAMENTS CONTENANT CES COMPOSES, LEUR UTILISATION ET PROCEDE DE FABRICATION ASSOCIE
WO1997032881A1 (fr) 1996-03-06 1997-09-12 Dr. Karl Thomae Gmbh Derives de 4-amino-pyrimidine, medicaments contenant ces composes, leur utilisation et leur procede de production
WO1997034895A1 (fr) 1996-03-15 1997-09-25 Novartis Ag NOUVELLES N-7-HETEROCYCLYL-PYRROLO[2,3-d]PYRIMIDINES ET LEUR UTILISATION
WO1997038983A1 (fr) 1996-04-12 1997-10-23 Warner-Lambert Company Inhibiteurs irreversibles de tyrosine kinases
WO1997038994A1 (fr) 1996-04-13 1997-10-23 Zeneca Limited Derives de quinazoline
US5747498A (en) 1996-05-28 1998-05-05 Pfizer Inc. Alkynyl and azido-substituted 4-anilinoquinazolines
WO1997049688A1 (fr) 1996-06-24 1997-12-31 Pfizer Inc. Derives tricycliques substitues par phenylamino, destines au traitement des maladies hyperproliferatives
WO1998002438A1 (fr) 1996-07-13 1998-01-22 Glaxo Group Limited Composes heteroaromatiques bicycliques en tant qu'inhibiteurs de la proteine tyrosine kinase
WO1998002437A1 (fr) 1996-07-13 1998-01-22 Glaxo Group Limited Composes heteroaromatiques bicycliques en tant qu'inhibiteurs de la proteine tyrosine kinase
WO1998002434A1 (fr) 1996-07-13 1998-01-22 Glaxo Group Limited Composes heterocycliques condenses en tant qu'inhibiteurs de la proteine tyrosine kinase
WO1998007726A1 (fr) 1996-08-23 1998-02-26 Novartis Ag Pyrrolopyrimidines substituees et procede pour leur preparation
WO1998014450A1 (fr) 1996-10-02 1998-04-09 Novartis Ag Derives de pyrimidine et procedes de preparation de ces derniers
WO1998014451A1 (fr) 1996-10-02 1998-04-09 Novartis Ag Derive de pyrazole condense et procede pour sa preparation
WO1998014449A1 (fr) 1996-10-02 1998-04-09 Novartis Ag Derives de pyrazole condenses et procedes pour leur preparation
EP0837063A1 (fr) 1996-10-17 1998-04-22 Pfizer Inc. Dérivés de 4-aminoquinazoline
WO1998017662A1 (fr) 1996-10-18 1998-04-30 Novartis Ag Derives d'heterocyclyle bicyclique a substitution phenyle et utilisation de ces derives
US5814500A (en) 1996-10-31 1998-09-29 The Johns Hopkins University School Of Medicine Delivery construct for antisense nucleic acids and methods of use
WO1998033798A2 (fr) 1997-02-05 1998-08-06 Warner Lambert Company Pyrido[2,3d]pyrimidines et 4-aminopyrimidines en tant qu'inhibiteurs de la proliferation cellulaire
US6011577A (en) 1997-06-30 2000-01-04 Polaroid Corporation Modular optical print head assembly
WO1999007701A1 (fr) 1997-08-05 1999-02-18 Sugen, Inc. Derives de quinoxaline tricyclique utiles en tant qu'inhibiteurs de proteine tyrosine kinase
WO1999035132A1 (fr) 1998-01-12 1999-07-15 Glaxo Group Limited Composes heterocycliques
WO1999035146A1 (fr) 1998-01-12 1999-07-15 Glaxo Group Limited Composes heteroaromatiques bicycliques agissant comme inhibiteurs de la tyrosine kinase
US6288082B1 (en) 1998-09-29 2001-09-11 American Cyanamid Company Substituted 3-cyanoquinolines
US6969614B1 (en) 1999-02-16 2005-11-29 The United States Of America As Represented By The Department Of Health And Human Services Methods for the isolation and analysis of cellular protein content
WO2001034574A1 (fr) 1999-11-11 2001-05-17 Osi Pharmaceuticals, Inc. Polymorphe stable de chlorhydrate de n-(3-ethynylphenylamino)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine, et methodes de production et utilisations pharmaceutiques dudit polymorphe
US6384051B1 (en) 2000-03-13 2002-05-07 American Cyanamid Company Method of treating or inhibiting colonic polyps
US20040063906A1 (en) 2000-11-24 2004-04-01 Nielsen Peter E. Pna analogues
US20030198627A1 (en) 2001-09-01 2003-10-23 Gert-Jan Arts siRNA knockout assay method and constructs
WO2003028531A2 (fr) * 2001-09-28 2003-04-10 Oncomedx, Inc. Detection d'arn tumoral dans le plasma et le serum
US20050059678A1 (en) 2003-09-15 2005-03-17 Wyeth Protein tyrosine kinase enzyme inhibitors
US20050272083A1 (en) 2004-06-04 2005-12-08 Somasekar Seshagiri EGFR mutations
WO2006091889A2 (fr) 2005-02-25 2006-08-31 Michael Horn Bande transporteuse modulaire pour vehicule
WO2007039705A1 (fr) 2005-10-05 2007-04-12 Astrazeneca Uk Limited Méthode pour prédire ou surveiller la réponse d'un patient à un médicament de récepteur erbb
WO2008009740A1 (fr) 2006-07-20 2008-01-24 Pangaea Biotech, S.A. Procédé de détection de mutations egfr dans des échantillons sanguins
EP2046985A1 (fr) 2006-07-20 2009-04-15 Pangaea Biotech, S.A. Procédé de détection de mutations egfr dans des échantillons sanguins
WO2008106453A2 (fr) * 2007-02-26 2008-09-04 John Wayne Cancer Institute Utilité de la mutation d'adn de b-raf dans le diagnostic et le traitement d'un cancer
WO2009021149A1 (fr) * 2007-08-07 2009-02-12 The Penn State Research Foundation Détection d'acides nucléiques extracellulaires associés à une tumeur dans le plasma ou le sérum sanguin
EP2468883A1 (fr) * 2010-12-22 2012-06-27 Pangaea Biotech S.L. Biomarqueurs moléculaires pour la prédiction de la réponse aux inhibiteurs de la tyrosine kinase dans le cancer du poumon

Non-Patent Citations (55)

* Cited by examiner, † Cited by third party
Title
"ASCO Annual Meeting Proceedings", JOURNAL OF CLINICAL ONCOLOGY, vol. 24, 2006
BRUMMELKAMP ET AL., SCIENCE, vol. 296, 2002, pages 550 - 553
CAICUN ZHOU ET AL: "Erlotinib versus chemotherapy as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer (OPTIMAL, CTONG-0802): a multicentre, open-label, randomised, phase 3 study", THE LANCET ONCOLOGY, vol. 12, no. 8, 1 August 2011 (2011-08-01), pages 735 - 742, XP055073569, ISSN: 1470-2045, DOI: 10.1016/S1470-2045(11)70184-X *
CECH, SCIENCE, vol. 245, no. 4915, 1989, pages 276
CHEN ET AL., ANAL. BIOCHEM., vol. 239, 1996, pages 61 - 9
D. MELTON,: "Antisense DNA and RNA", 1988, COLD SPRING HARBOR LABORATORY
DEDIONISIO ET AL., J CHROMATOGR A, vol. 735, 1996, pages 191 - 208
DEMIDOV ET AL., BIOCHEM. PHARMACOL., vol. 48, 1994, pages 1310 - 1313
DOWDY; WEARDEN: "Statistics for Research", 1983, JOHN WILEY & SONS
E. A. PUNNOOSE ET AL: "Evaluation of Circulating Tumor Cells and Circulating Tumor DNA in Non-Small Cell Lung Cancer: Association with Clinical Endpoints in a Phase II Clinical Trial of Pertuzumab and Erlotinib", CLINICAL CANCER RESEARCH, vol. 18, no. 8, 15 April 2012 (2012-04-15), pages 2391 - 2401, XP055073510, ISSN: 1078-0432, DOI: 10.1158/1078-0432.CCR-11-3148 *
EGHOLM ET AL., NATURE, vol. 365, 1993, pages 566 - 568
ELBASHIR S. M. ET AL., NATURE, vol. 411, 2001, pages 494 - 498
EXPERT REV. MOI. DIAGN., no. 2, 2005, pages 209 - 19
GENET TEST., vol. 1, no. 3, 1997, pages 201 - 6
GUO JIAN ET AL: "Prediction of epidermal growth factor receptor mutations in the plasma/pleural effusion to efficacy of gefitinib treatment in advanced non-small cell lung cancer", JOURNAL OF CANCER RESEARCH AND CLINICAL ONCOLOGY, SPRINGER, BERLIN, DE, vol. 136, no. 9, 14 February 2010 (2010-02-14), pages 1341 - 1347, XP019849084, ISSN: 1432-1335 *
HEID ET AL., GENOME RES., vol. 6, 1996, pages 986 - 94
HUMPHREY ET AL., PROC. NATL. ACAD. SCI. USA, vol. 87, 1990, pages 4207 - 4211
JAQUE ET AL., NATURE, vol. 418, 2002, pages 435 - 438
KIMURA HIDEHARU ET AL: "Detection of epidermal growth factor receptor mutations in serum as a predictor of the response to gefitinib in patients with non-small-cell lung cancer", CLINICAL CANCER RESEARCH, THE AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 12, no. 13, 1 July 2006 (2006-07-01), pages 3915 - 3921, XP002413945, ISSN: 1078-0432, DOI: 10.1158/1078-0432.CCR-05-2324 *
KOIZUMI ET AL., INT. J. CANCER, vol. 116, 2005, pages 36 - 44
LANDEGREN, U ET AL., SCIENCE, vol. 242, 1988, pages 229 - 237
LEWIS, R., GENETIC ENGINEERING NEWS, vol. 10, no. 1, 1990, pages 54 - 55
MARCELINO ET AL., BIOTECHNIQUES, vol. 26, June 1999 (1999-06-01), pages 1134 - 1148
MARIE BREVET ET AL: "Detection of mutations in plasma DNA from lung cancer patients by mass spectrometry genotyping is predictive of tumorstatus and response to EGFR inhibitors", LUNG CANCER, ELSEVIER, AMSTERDAM, NL, vol. 73, no. 1, 24 October 2010 (2010-10-24), pages 96 - 102, XP028225315, ISSN: 0169-5002, [retrieved on 20101110], DOI: 10.1016/J.LUNGCAN.2010.10.014 *
MARSHALL ET AL., NATURE BIOTECHNOLOGY, vol. 16, 1998, pages 27 - 31
MARTINCIC ET AL., ONCOGENE, vol. 13, 1996, pages 2039 - 44
MCCAFFREY ET AL., NATURE, vol. 418, 2002, pages 38 - 39
MCMANUS, NATURE REVIEWS GENETICS, vol. 3, no. 10, 2002, pages 737
MERRIFIELD, B.: "Solid-phase synthesis", SCIENCE, vol. 232, 1986, pages 341 - 347
METHODS MOL. MED., vol. 108, 2004, pages 173 - 88
MITSUDOMI ET AL., LANCET ONCOL, vol. 11, 2010, pages 121 - 28
MOYER, J. D. ET AL., CANCER RES., vol. 57, 1997, pages 4838 - 4848
NATURE BIOTECHNOLOGY, vol. 17, 1999, pages 804 - 807
NIELSEN ET AL., SCIENCE, vol. 254, 1991, pages 1457 - 1500
NIELSEN; HAAIMA, CHEMICAL SOCIETY REVIEWS, 1997, pages 73 - 78
NOVINA ET AL., NAT. MED., vol. 8, 2002, pages 681 - 686
NUCLEIC ACIDS RES., vol. 17, 1989, pages 2503 - 2516
NYCE ET AL., NATURE, vol. 385, 1997, pages 720
PHILIP J. JOHNSON ET AL: "Plasma Nucleic Acids in the Diagnosis and Management of Malignant Disease", CLINICAL CHEMISTRY, vol. 48, no. 8, 1 August 2002 (2002-08-01), pages 1186 - 1193, XP055042293, ISSN: 0009-9147 *
RAMSAY ET AL., NATURE BIOTECHNOLOGY, vol. 16, 1998, pages 40 - 44
ROSELL ET AL., LANCET ONCOLOGY
SARKAR ET AL., GENOMICS, vol. 13, 1992, pages 441 - 3
SARKAR ET AL., NUCLEIC ACIDS RES., vol. 23, 1995, pages 1269 - 70
SHERWOOD ET AL., PROC. AM. ASSOC. CANCER RES., vol. 40, 1999, pages 723
SHUBER ET AL., HUM. MOLEC. GENET., vol. 6, 1997, pages 337 - 47
SORENSEN BOE S ET AL: "Circulating HER2 DNA after Trastuzumab Treatment Predicts Survival and Response in Breast Cancer", ANTICANCER RESEARCH, vol. 30, no. 6, June 2010 (2010-06-01), pages 2463 - 2468, XP009164134 *
T. S.K. MOK ET AL: "Randomized, Placebo-Controlled, Phase II Study of Sequential Erlotinib and Chemotherapy As First-Line Treatment for Advanced Non-Small-Cell Lung Cancer", JOURNAL OF CLINICAL ONCOLOGY, vol. 27, no. 30, 8 September 2009 (2009-09-08), pages 5080 - 5087, XP055073589, ISSN: 0732-183X, DOI: 10.1200/JCO.2008.21.5541 *
TAKUYA ARAKI: "Clinical screening assay for EGFR exon 19 mutations using PNA-clamp smart amplification process version 2 in lung adenocarcinoma", ONCOLOGY REPORTS, 15 July 2011 (2011-07-15), XP055073548, ISSN: 1021-335X, DOI: 10.3892/or.2011.1391 *
TONY K F YUNG ET AL: "Single-molecule detection of epidermal growth factor receptor mutations in plasma by microfluidics digital PCR in non-small cell lung cancer patients", CLINICAL CANCER RESEARCH, THE AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 15, no. 6, 15 March 2009 (2009-03-15), pages 2076 - 2084, XP002628813, ISSN: 1078-0432, [retrieved on 20090310], DOI: 10.1158/1078-0432.CCR-08-2622 *
TONY S. MOK ET AL: "Gefitinib or Carboplatin-Paclitaxel in Pulmonary Adenocarcinoma", NEW ENGLAND JOURNAL OF MEDICINE, vol. 361, no. 10, 3 September 2009 (2009-09-03), pages 947 - 957, XP055073586, ISSN: 0028-4793, DOI: 10.1056/NEJMoa0810699 *
TRAXLER, P., EXP. OPIN. THER. PATENTS, vol. 8, no. 12, 1998, pages 1599 - 1625
WOODBURN ET AL., PROC. AM. ASSOC. CANCER RES., vol. 38, 1997, pages 633
XIA H. ET AL., NAT. BIOTECH., vol. 20, 2002, pages 1006 - 1010
XIAIYI ET AL., BIOINFORMATICS, vol. 17, 2001, pages 838 - 839
YI LIU ET AL: "A comparison of ARMS and direct sequencing for EGFR mutation analysis and Tyrosine Kinase Inhibitors treatment prediction in body fluid samples of Non-Small-Cell Lung Cancer patients", JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH, BIOMED CENTRAL LTD, LONDON UK, vol. 30, no. 1, 6 December 2011 (2011-12-06), pages 111, XP021132019, ISSN: 1756-9966, DOI: 10.1186/1756-9966-30-111 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019075251A3 (fr) * 2017-10-12 2019-07-25 Nantomics, Llc Score de cancer pour l'évaluation et la prévision de réponse à partir de fluides biologiques
US11810672B2 (en) 2017-10-12 2023-11-07 Nantomics, Llc Cancer score for assessment and response prediction from biological fluids
CN109097469A (zh) * 2018-08-17 2018-12-28 上海迈景纳米科技有限公司 一种基于固相杂交技术的egfr突变基因检测方法

Similar Documents

Publication Publication Date Title
JP6032616B2 (ja) Ｋｉｆ５ｂ遺伝子とｒｅｔ遺伝子との融合遺伝子、並びに該融合遺伝子を標的としたがん治療の有効性を判定する方法
AU2007275140B2 (en) Method for the detection of EGFR mutations in blood samples
US20200385817A1 (en) Compositions and methods for screening solid tumors
US20080286785A1 (en) Method to predict or monitor the response of a patient to an erbb receptor drug
US20150038520A1 (en) Molecular Biomarkers for Predicting Response to Tyrosine Kinase Inhibitors in Lung Cancer
US11275088B2 (en) BCR-ABL variants
WO2013190089A1 (fr) Biomarqueurs moléculaires permettant de prédire l'issue dans le cancer du poumon
JP2017169580A (ja) 上皮増殖因子受容体キナーゼ・ドメイン内の新規な複合突然変異
US11261482B2 (en) Composition for detecting epidermal cell growth factor receptor gene mutation, and kit comprising same
US20220073993A1 (en) Methods and kits for detecting egfr mutations
US20120316187A1 (en) Molecular biomarkers for predicting response to tyrosine kinase inhibitors in lung cancer
EP2492688A1 (fr) Biomarqueurs moléculaires pour la prédiction de la réponse à un traitement antitumoral dans le cancer du poumon
KR101169247B1 (ko) Ｅｇｆｒ 저해제 처리 마커
WO2012065705A1 (fr) Nouvelle mutation complexe dans le domaine kinase du récepteur du facteur de croissance épidermique
Vlastos et al. Mapping EGFR1 mutations in patients with lung adenocarcinoma
AU2011265464B2 (en) Methods for prediction of clinical outcome to epidermal growth factor receptor inhibitors by cancer patients
KR20100037641A (ko) Ｅｇｆｒ 저해제 처리에 대한 예측성 마커

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: 13731124

Country of ref document: EP

Kind code of ref document: A1

DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13731124

Country of ref document: EP

Kind code of ref document: A1