WO2010076887A1 - Biomarqueurs prédictifs utiles pour une thérapie anticancéreuse à médiation par un inhibiteur de wee1 - Google Patents

Biomarqueurs prédictifs utiles pour une thérapie anticancéreuse à médiation par un inhibiteur de wee1 Download PDF

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WO2010076887A1
WO2010076887A1 PCT/JP2009/071849 JP2009071849W WO2010076887A1 WO 2010076887 A1 WO2010076887 A1 WO 2010076887A1 JP 2009071849 W JP2009071849 W JP 2009071849W WO 2010076887 A1 WO2010076887 A1 WO 2010076887A1
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inhibitor
wee
expression
patient
gene
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PCT/JP2009/071849
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English (en)
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Shinji Mizuarai
Hiraku Itadani
Kazunori Yamanaka
Toshihide Nishibata
Tsuyoshi Arai
Hiroshi Hirai
Hidehito Kotani
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Banyu Pharmaceutical Co.,Ltd.
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Publication of WO2010076887A1 publication Critical patent/WO2010076887A1/fr

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    • 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
    • C12Q2600/112—Disease subtyping, staging or classification

Definitions

  • the present invention relates generally to the identification of potential responder biomarker gene set(s) whose expression levels are useful for predicting a patient's response to treatment with an antiproliferative agent, particularly one that is responsive to a Weel inhibitor. More, the invention provides a clinician with the tools necessary to predict a patient's potential response to treatment an antiproliferative agent such as a Weel inhibitor. In certain embodiments, the invention provides a skilled artisan with the means to identify whether a patient presenting with a cancerous condition, in particular, a condition mediated by a dysfunctional or aberrant p53, is likely to respond to treatment with a Weel inhibitor, prior to dosing with the Weel inhibitor.
  • the ability to screen a potential patient's sensitivity to treatment with a Weel inhibitor pre-dose via utilizing methods of the invention, e.g., by quantifying biomarker expression prior to administering the Weel inhibitor, is an advantage over current treatment standards because it not only allows for early intervention but it also prevents subjecting a patient to the unnecessary side effects of treatment with an anti-cancer agent. As well, it provides the clinician with important information about a patient's potential response at an earlier stage of the treatment protocol and in those rare cases where the data read out relative to the biomarker signature detailed herein suggests that the patient is unlikely to respond, it prevents the subject from incurring any additional discomfort and / or side effects related to treatment with the particular anti-cancer agent.
  • the present invention relates to the identification of biomarker gene sets whose expression levels are useful for evaluating and classifying biological samples to determine exposure to a biological active dose of a Wee 1 inhibitor, early prediction of response to a cancer therapy treatment, and pre-dose exposure prediction of sensitivity or resistance of a sample to Wee 1 inhibitor, among other uses.
  • the present invention also relates generally to a method for predicting a response to a cancer therapy treatment with a Wee 1 inhibitor by determining expression level(s) of the biomarker gene sets.
  • the availability of the end point biomarkers makes them useful in analyzing, at a molecular level, the disease-related genes of an individual, which ultimately will allow for a more personalized approach in treating individual patients or sub-population of patients sharing similar traits, with fewer complications and side-effects.
  • Weel is a serine/threonine kinase regulating G2/M cell cycle checkpoint.
  • DNA damaging agent causes cell cycle arrest at G2/M phase in cancer cells.
  • Weel inhibition specifically causes cell death in p53 negative tumor.
  • the concept for Weel inhibitor is to selectively sensitize p53-deficient tumors to different chemotherapies without increasing the chemotherapy-related toxicity to normal tissues.
  • Biomarkers used to measure a response to an intervention are called surrogate endpoint biomarkers or SEBs (Kelloff et al., 1996, Cancer Epidemiology, Biomarkers and Prev., 5:355-360.
  • biomarkers include genetic markers (e.g., nuclear aberrations [such as micronuclei], gene amplification, and mutation), cellular markers (e.g., differentiation markers and measures of proliferation, such as thymidine labeling index), histologic markers (e.g., premalignant lesions, such as leukoplakia and colonic polyps), and biochemical and pharmacologic markers (e.g., ornithine decarboxylase activity, and radiology imaging reagents).
  • genetic markers e.g., nuclear aberrations [such as micronuclei], gene amplification, and mutation
  • cellular markers e.g., differentiation markers and measures of proliferation, such as thymidine labeling index
  • histologic markers e
  • biomarkers may be carried out by analyzing changes in specific polypeptides, metabolites or mRNAs, as predicted by the known biology associated with the molecule targeted by the agent of interest.
  • biomarkers can be identified by analyzing global changes in polypeptides or mRNA in cells or tissues exposed to efficacious doses of the agent.
  • the pre-dose expression levels of at least one or more of the biomarkers detailed herein will also find use in predicting a patient's response to a Weel inhibitor, as disclosed herein, and based upon the readout, followed by treatment with a Weel inhibitor.
  • these biomarkers can be used to tailor a patient's clinical protocol, such as, by example, being able to predict a patient's response to a particular treatment protocol with a particular therapeutic moiety, or determining that a patient target tissue has received a biologically effective dose of a therapeutic agent.
  • the present invention relates generally to the identification of biomarker gene set(s) whose expression level(s) are useful for evaluating and classifying biological samples to determine exposure to a biological active dose of a Wee 1 inhibitor, early prediction of response to a cancer therapy treatment, and pre-dose exposure prediction of sensitivity or resistance to an anti-cancer agent, such as, for example, a Wee 1 inhibitor.
  • the present invention also relates generally to a method for predicting a patient's response to treatment with a Wee 1 inhibitor by determining, for example, expression level(s) of one or more of the biomarker gene or gene set(s) disclosed herein.
  • the invention further provides the above method(s), applied to classifying patients as having good prognosis or poor prognosis relative to treatment with a Weel inhibitor.
  • the invention provides that the method may be used wherein a plurality of genes is at least 5, or 10 or 15 of the Weel markers listed in Table 1 or 2.
  • the optimum 15 markers listed in Table 1 or the 12 markers listed in Table 2 are used.
  • at least one or more of the biomarker gene markers listed in one of Table 1 or Table 2 are used.
  • the invention provides that at least 1, 5, 7, 10 or 15 gene markers listed in Table 1 or 2 may be used.
  • the invention describes the link between particular biomarker genes detailed herein and a Wee 1 inhibitor.
  • the specification demonstrates that expression of at least one or more of the biomarker genes in cancer cells are altered following exposure to a Wee 1 inhibitor. This, in turn, demonstrates their utility as potential diagnostic and prognostic biomarkers relative to the use of a Weel inhibitor.
  • the biomarkers comprise one or more genes whose expression levels were altered following exposure to an anti-cancer agent, e.g., Weel inhibitor.
  • the biomarkers comprises one or more genes whose expression levels were altered between high-sensitivity cells and low-sensitivity cells to an anti-cancer agent, e.g., Weel inhibitor.
  • the biomarkers are at least one biomarker gene or its gene product selected from the group consisting of Clspn, Fancd2, Ccnel, Mem 10, Stmnl, Fbxo5, Ccna2, Spbc25, Brrnl, Atf5, Vim, Cnne2, Myb, Egrl and Histlh2bd.
  • the biomarkers comprise at least one biomarker gene or its gene product selected from the group consisting of SPRY2, CCNI, JUNB, SMAD2, SHCl, MADlLl, GADD45GLP1, CKAP5, TUBB4, BCATl, MCM8 and TLK2. See Table 2.
  • Table 2 lists one variant of each biomarker genes as a representative example thereof, but any variant of each biomarker gene can be equally used for the purpose of the present invention.
  • FIG 1 shows hierarchical clustering of up-/down-regulated genes in skin sample of nude rat treated with Gemcitabine/Compound A.
  • FIG 2 shows hierarchical clustering of up-/down-regulated genes in both
  • FIG 3 shows another hierarchical clustering of up-/down-regulated genes in both TOV21G-derived p53 positive and negative matched pair cell lines treated with Gemcitabine/Compound A.
  • FIG 4 shows quantitative real-time RT-PCR analysis of expression pharmacodynamic biomarker genes in xenograft tumor, Widr cells.
  • FIG 5 shows sensitivity of 22 NSCLC cell lines with p53 deficient to Cisplatin/
  • FIG 6 shows hierarchical clustering analysis of up-/down-regulated genes in p53 deficient 22 NSCLC cell lines treated with Cisplatin/Compound A.
  • FIG 7 shows the result of leave-one-out-cross-validation test of the 134 genes which show high prediction accuracy for the sensitivity of Cisplatin/Compound A combination treatment.
  • FIG 8 shows the result of leave-one-out-cross-validation test of the 134 genes which shows high prediction accuracy for the sensitivity of Carboplatin/Compound A and Gemcitabine/Compound A.
  • FIG 9 shows expression ratio of MADl and SMAD in hyper-responder and normal-responder p53 deficient NSCLC cell lines treated with Gemcitabine/Compound A, Carboplatin/Compound A and Cisplatin/Compound A.
  • the present invention relies on the surprising discovery of identifying pharmacodynamic and responder biomarker(s) for a Weel inhibitor, which will have utility in predicting efficacy of Weel inhibitor as well as in predicting a patient's response to a treatment protocol comprising a Weel inhibitor.
  • the discovery is based, in part, on the observation that since Weel inhibition causes G2 checkpoint abrogation and induces premature mitosis in the presence of DNA damaging agents, the same should be observed via a change in the expression of mRNA levels of certain genes responsive to or are modulated in response to Weel inhibition
  • mRNA expression of at least one or more genes preferably a set of genes (Weel pharmacodynamic gene signature or a Wee responder gene signature) as disclosed herein was specifically altered, e.g., increased or decreased in response to treatment by administration of a Weel inhibitor in a biological sample. The altered expression or change in expression was observed in both skin and cancers.
  • the altered expression or change in expression was observed in lung cancers.
  • the gene expression or pharmacodynamic marker(s) of the present invention are quantifiable versus previously known pharmacodynamic marker(s) which measures the phosphorylation level of a direct substrate of Wee 1, specifically a tyrosine residue at position 15 (Tyrl5 residue) of CDC2 or previously known responder marker9s) which measure the status of p53
  • the identification and development of gene expression pharmacodynamic marker(s) disclosed herein is advantageous over the previously identified biomarker, supra, in that it is less time consuming and thus cheaper and also requires small amount of biopsied tissue.
  • Wee 1 inhibitor means any compound or agent that inhibits the activity of one or more Wee 1 proteins. The compound or agent may inhibit Wee 1 activity by direct or indirect interaction with Wee 1 protein or it may act to prevent expression of one or more Wee 1 gene.
  • the gene EST derived from that gene, the expression or level of which changes between certain conditions.
  • the gene is a marker for that condition.
  • Marker-derived polynucleotides means the RNA transcribed from a marker gene, any cDNA or cRNA produced therefrom, and any nucleic acid derived therefrom, such as synthetic nucleic acid having a sequence derived from the gene corresponding to the marker gene.
  • a “similarity value” is a number that represents the degree of similarity between two things being compared.
  • a similarity value may be a number that indicates the overall similarity between a patient's expression profile using specific phenotype-related markers and a control specific to that phenotype (for instance, the similarity to a "good prognosis" template, where the phenotype is a good prognosis).
  • the similarity value may be expressed as a similarity metric, such as a correlation coefficient, or may simply be expressed as the expression level difference, or the aggregate of the expression level differences, between a patient sample and a template.
  • the terms “measuring expression levels,” “obtaining an expression level” and the like includes methods that quantify target gene expression level exemplified by a transcript of a gene, including microRNA (miRNA) or a protein encoded by a gene, as well as methods that determine whether a gene of interest is expressed at all.
  • miRNA microRNA
  • an assay which provides a “yes” or “no” result without necessarily providing quantification of an amount of expression is an assay that "measures expression” as that term is used herein.
  • the term may include quantifying expression level of the target gene expressed in a quantitative value, for example, a fold-change in expression, up or down, relative to a control gene or relative to the same gene in another sample, or a log ratio of expression, or any visual representation thereof, such as, for example, a "heatmap" where a color intensity is representative of the amount of gene expression detected.
  • Exemplary methods for detecting the level of expression of a gene include, but are not limited to, Northern blotting, dot or slot blots, reporter gene matrix (see, for example, US 5,569,588), nuclease protection, RT-PCR, microarray profiling, differential display, SAGE (Velculescu et al., 1995, Science 270:484-87), Digital Gene Expression System (see WO2007076128; WO2007076129), multiplex mRNA assay (Tian et al., 2004 Nucleic Acids Res.
  • subject refers to an organism or to a cell sample, tissue sample or organ sample derived therefrom, including, for example, cultured cell lines, biopsy, blood sample or fluid sample containing a cell.
  • the subject or sample derived therefrom comprises a plurality of cell types.
  • the sample includes, for example, a mixture of tumor cells and normal cells.
  • the sample comprises at least 10%, 15%, 20%, et seq., 90%, or 95% tumor cells.
  • the organism is a mammal, such as a human, canine, murine, feline, bovine, ovine, swine or caprine. In a particular embodiment, the organism is a human patient.
  • Patient refers to the recipient in need of medical intervention or treatment. Mammalian and non-mammalian patients are included. In one embodiment, the patient is a mammal, such as a human, canine, murine, feline, bovine, ovine, swine or caprine. In a particular embodiment, the patient is a human.
  • the term "treating" in its various grammatical forms in relation to the present invention refers to preventing (i.e.
  • treatment may involve alleviating a symptom (i.e., not necessary all symptoms) of a disease or attenuating the progression of a disease.
  • Treatment of cancer refers to partially or totally inhibiting, delaying or preventing the progression of cancer including cancer metastasis; inhibiting, delaying or preventing the recurrence of cancer including cancer metastasis; or preventing the onset or development of cancer (chemoprevention) in a mammal, for example a human.
  • the methods of the present invention may be practiced for the treatment of chemoprevention of human patients with cancer. However, it is also likely that the methods would also be effective in the treatment of cancer in other mammals.
  • the term "therapeutically effective amount" is intended to qualify the amount of the treatment in a therapeutic regimen necessary to treat cancer.
  • the desired biological response is partial or total inhibition, delay or prevention of the progression of cancer including cancer metastasis; inhibition, delay or prevention of the recurrence of cancer including cancer metastasis; or the prevention of the onset or development of cancer (chemoprevention) in a mammal, for example a human.
  • the individual is treated with a first therapeutic agent, preferably a DNA damaging agent and/or a Wee 1 inhibitor as described herein.
  • the second therapeutic agent may be another Wee 1 inhibitor or may be any clinically established anti-cancer agent as defined herein.
  • a combinatorial treatment may include a third or even further therapeutic agent.
  • Status means a state of gene expression of a set of genetic markers whose expression is strongly correlated with a particular phenotype.
  • ER status means a state of gene expression of a set of genetic markers whose expression is strongly correlated with that of ESRl (estrogen receptor gene), wherein the pattern of these genes' expression differs detectably between tumors expressing the receptor and tumors not expressing the receptor.
  • Good prognosis means that a patient is expected to have no distant metastases of a breast tumor within five years of initial diagnosis of breast cancer.
  • “Poor prognosis” means that a patient is expected to have distant metastases of a breast tumor within five years of initial diagnosis of breast cancer.
  • Abroad aspect of the invention concerns the identification of at least one or more biomarker genes whose expression is modulated by a Weel inhibitor.
  • Table 1 and Table 2 each lists one such gene marker set (signature) whose expression level(s) are correlated with exposure to an anti-cancer agent such as Weel inhibitor.
  • the invention provides a gene set whose expression co-relates to a response to an anti-cancer agent such as a Weel inhibitor.
  • the expression levels in response to the inhibitor may change relative to the entire gene set or just one gene from the gene set or a combination of different genes selected from one of Table 1 and Table 2.
  • Methods of using some or all of the marker genes detailed in one of Table 1 or 2 to predict a subject's sensitivity or resistance to a Wee 1 inhibitor are also provided as methods for determining whether a subject has been exposed to a Wee 1 inhibitor, or predict whether treatment with a cancer therapeutic agent, particularly Wee 1 inhibitor will be effective.
  • the present invention provides gene biomarkers whose expression co-relates with exposure or treatment with a Weel inhibitor.
  • the marker sets were identified as detailed in the Examples set forth below by determining which of the numerous genes had expression patters that correlated with the conditions or indications.
  • target polynucleotide molecules are extracted from a sample taken from an individual afflicted with breast cancer.
  • the sample may be collected in any clinically acceptable manner, but must be collected such that marker-derived polynucleotides (i.e., RNA) are preserved.
  • marker-derived polynucleotides i.e., RNA
  • mRNA or nucleic acids derived therefrom i.e., cDNA or amplified DNA
  • cDNA or amplified DNA are preferably labeled distinguishably from standard or control polynucleotide molecules, and both are simultaneously or independently hybridized to a microarray comprising some or all of the markers or marker sets or subsets described above.
  • mRNA or nucleic acids derived therefrom may be labeled with the same label as the standard or control polynucleotide molecules, wherein the intensity of hybridization of each at a particular probe is compared.
  • a sample may comprise any clinically relevant tissue sample, such as a tumor biopsy or fine needle aspirate, or a sample of bodily fluid, such as blood, plasma, serum, lymph, ascitic fluid, cystic fluid, urine or nipple exudate.
  • the sample may be taken from a human, or, in a veterinary context, from non-human animals such as ruminants, horses, swine or sheep, or from domestic companion animals such as felines and canines.
  • RNA may be isolated from eukaryotic cells by procedures that involve lysis of the cells and denaturation of the proteins contained therein.
  • Cells of interest include wild-type cells (i.e., non-cancerous), drug-exposed wild-type cells, tumor- or tumor-derived cells, modified cells, normal or tumor cell line cells, and drug-exposed modified cells.
  • RNA is extracted from cells of the various types of interest using guanidinium thiocyanate lysis followed by CsCl centrifugation to separate the RNA from DNA (Chirgwin et al, Biochemistry 18:5294 5299 (1979)).
  • Poly(A)+RNA is selected by selection with oligo-dT cellulose (see Sambrook et al., MOLECULAR CLOMNG-A LABORATORY MANUAL (2ND ED.), VoIs. 1 3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. (1989).
  • separation of RNA from DNA can be accomplished by organic extraction, for example, with hot phenol or phenol/chloroform/isoamyl alcohol.
  • RNAse inhibitors may be added to the lysis buffer.
  • mRNAs include transfer RNA (tRNA) and ribosomal RNA (rRNA).
  • Most mRNAs contain a poly(A) tail at their 3' end. This allows them to be enriched by affinity chromatography, for example, using oligo(dT) or poly(U) coupled to a solid support, such as cellulose or SEPHADEX.RTM. medium (see Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, vol. 2, Current Protocols Publishing, New York (1994).
  • poly(A)+mRNA is eluted from the affinity column using 2 mM EDTA/0.1% SDS.
  • the sample of RNA can comprise a plurality of different mRNA molecules, each different mRNA molecule having a different nucleotide sequence.
  • the mRNA molecules in the RNA sample comprise at least 100 different nucleotide sequences. More preferably, the mRNA molecules of the RNA sample comprise mRNA molecules corresponding to each of the marker genes.
  • the RNA sample is a mammalian RNA sample.
  • total RNA or mRNA from cells are used in the methods of the invention.
  • the source of the RNA can be cells of a plant or animal, human, mammal, primate, non-human animal, dog, cat, mouse, rat, bird, yeast, eukaryote, prokaryote, etc.
  • the method of the invention is used with a sample containing total mRNA or total RNA from 1.times.l ⁇ .sup.6 cells or less.
  • proteins can be isolated from the foregoing sources, by methods known in the art, for use in expression analysis at the protein level.
  • Probes to the homologs of the marker sequences disclosed herein can be employed preferably wherein non-human nucleic acid is being assayed. (i) Prediction of Sensitivity and/or Resistance of a Cell Sample to Weel inhibitor Treatment
  • the invention provides a set of 15 genetic markers whose expression is correlated with a subject's exposure to or treatment with a Weel inhibitor.
  • a set of these markers identified as useful for diagnosis or prognosis is listed in Table 1 - SEQ ID Nos. 1-15.
  • the invention also provides a method of using these markers to distinguish tumor types in diagnosis or prognosis.
  • any of the sets of markers provided above may be used alone specifically or in combination with markers outside the set. Any of the marker sets provided above may also be used in combination with other markers for Weel mediated disorders such as cancer, or for any other clinical or physiological condition.
  • the present invention provides a set of gene markers (Table 1) that can be used to classify a cell sample as having been exposed to a biologically active dose of a Wee 1 inhibitor.
  • Table 1 it is of value to determine if a particular cell population has received or been exposed to a therapeutic dose of a Wee 1 inhibitor.
  • a whole organism might be dosed with a Wee 1 inhibitor yet there is uncertainty whether target cells located in a solid tumor have been exposed to the Wee 1 inhibitor. In this situation it is desirable to have a biomarker that indicates that such tumor cells have been exposed to a therapeutic dose of the Wee 1 inhibitor.
  • the markers are listed in Table 1.
  • the invention provides a set of 15 genetic markers that can classify breast cancer patients by estrogen receptor (ER) status; i.e., distinguish between ER(+) and ER(-) patients or tumors derived from these patients.
  • ER status is an important indicator of the likelihood of a patient's response to some chemotherapies (i.e., tamoxifen). These markers are listed in Table 1.
  • the invention also provides subsets of at least 1, 2, 3, 4, or 5 markers derived from the set listed in Table 1.
  • the invention further provides a set of markers derived from Table 1 that are optimal for predicting a patient's response to a treatment protocol comprising a Weel inhibitor.
  • the selected markers are selected from Tables 1 to include at least 1, 2 or 3 up-regulated genetic markers and at least 1, 2, 3, 4, or 5 down-regulated genetic markers. In other embodiments, the selected markers are all up-regulated markers or all down-regulated markers. In certain embodiments, the selected markers are detailed in Table 1- all 15 of the markers disclosed in Table 1.
  • the markers are selected from Table 1 based upon pre-determined threshold that is based upon marker gene expression measurements in Wee 1 inhibitor treated control samples.
  • the pre-determined threshold may be a fold change, up or down, of 1.2-fold change or greater, 1.3-fold or greater or 1.4-fold or greater, or 1.5-fold or greater, 1.6-fold or greater, 1.7-fold or greater, 1.8-fold or greater, 1.9-fold or greater, 2.0-fold or greater, or 3.0-fold or greater.
  • the 2-fold means 2-fold up-regulated or l/2-fold down-regulated of the markers in Wee 1 inhibitor treated samples compared with non-treated control samples.
  • the invention also provides a method of using these sets of Wee 1 inhibitor pharmacodymanic biomarkers to classify a cell sample as having been exposed to a biologically active dose of a Weel inhibitor.
  • the method comprises: (a) calculating a measure of similarity between a first expression profile and a Wee 1 inhibitor exposure template, or calculating a first measure of similarity between the first expression profile and the Weel inhibitor exposure template and a second measure of similarity between the first expression profile and a Weel inhibitor-free exposure template, wherein the first expression profile comprises a cell sample obtained from a patient and measuring expression level of at least one or more genes as set forth in Table 1 in said cell sample derived from a patient presenting with or at risk of developing cancer (patient sample) , wherein the Wee 1 inhibitor exposure template comprises measuring the average expression level of the same gene as that in the patient sample in a plurality of treated cell samples that were exposed to a therapeutically effective dose of the Wee 1 inhibitor agent (treated sample), and where
  • the measurement step includes measuring the average expression of each of the genes set forth in Table 1, while in other embodiment, the measurement includes measuring the expression level of at least one or more or a combination of the genes set forth in Table 1.
  • the control cells may include measuring the expression level of each of the genes set forth in Table 1, which may serve as a control against which the expression levels of one or more genes as set forth in Table 1 are measured. It is understood that the expression levels in each of the patient and treated sample may comprises measuring the average level of expression of a single gene or the average expression level of a plurality of genes selected from the group detailed in Table. The same holds true for the control sample.
  • gene markers and methods are provided to predict, based upon gene expression levels and prior to any change in a subject's cancer status, whether the subject will respond to a treatment. It is understood that in numerous instances it would be valuable to determine as early as possible whether a subject, who has been treated with a cancer therapeutic is likely to respond favorably to the treatment regiment.
  • Patients who are predicted as not responding to therapy based upon the biomarker readout may then be shifted to different therapeutic agent or dosing regiment.
  • 15 gene markers are used to make a drug efficacy prediction. These markers are listed in Table 1.
  • subsets of these markers are provided comprising at least 1, 2, 3, 4, or 5 biomarkers drawn from the set of 15 markers listed in Table 1.
  • a method of using these sets of cancer therapeutic response biomarkers to predict whether a subject with cancer will respond to treatment with Wee 1 inhibitor is also provided.
  • the method of predicting a patient's response to treatment with a Weel inhibitor comprises:
  • the first expression profile comprises a clinical cell sample suspected of containing cancer cells and includes measuring nucleic acid level of expression of at least one or more genes as set forth in Table 1 in said cell sample derived from a patient presenting with or at risk of developing cancer (patient sample) and is obtained from said subject after treatment with a Weel inhibitor (patient sample), wherein the cellular proliferation therapeutic response template includes measuring the average expression of at least one or more of the genes listed in Table 1 individually, or measuring the average collective expression levels of a collection of genes - two or more genes, listed in Table 1 in a plurality of cell samples that responded to treatment with a therapeutically effective dose of said Wee 1 inhibitor (treated sample), and wherein the cellular proliferation non
  • the step of calculating a measure of similarity or similarity value is correlation coefficient.
  • gene markers and methods are provided that are useful in predicting sensitivity and/or resistance of a subject to treatment with a Wee 1 inhibitor.
  • the gene markers or subset thereof are used to make a drug response prediction based upon gene expression levels measured in a cell sample comprising tumor cells prior to Wee 1 inhibitor treatment.
  • the Wee 1 response prediction markers are listed in Table 1.
  • Table 1 lists gene markers, Clspn, Fancd2, Ccnel, McmlO, Stmnl, Fbxo5, Ccna2, Spbc25, Brrnl, Atf5, Vim, Cnne2, Myb, Egrl and Histlh2bd whose expression levels are correlated with sensitivity of cells to Wee 1 inhibitor treatment.
  • Wee 1 inhibitor sensitivity or resistance is evaluated in a subject using 2 or more gene markers selected from Table 1. In another embodiment, resistance or sensitivity are evaluated in a subject using at least 1, 2, 3, 4 or 5 markers selected from Table 1.
  • One aspect of the present invention provides a method of using these sets of Wee 1 inhibitor biomarkers to predict whether a subject with cancer will respond to treatment with a Wee 1 inhibitor.
  • the method comprising:
  • the invention provides a set of 12 genetic markers whose expression is correlated with a subject's response to a treatment with a Weel inhibitor.
  • a set of these markers identified as useful for diagnosis or pre-dose prediction is listed in Table 2 - SEQ ID Nos. 16-27.
  • the invention also provides a method of using these markers to distinguish tumor types in diagnosis or pre-dose prediction. Any of the sets of markers provided above may be used alone specifically or in combination with markers outside the set. Any of the marker sets provided above may also be used in combination with other markers for Weel mediated disorders such as cancer, or for any other clinical or physiological condition.
  • the present invention provides a set of gene markers (Table 2) that can be used to predict a cell sample as having sensitivity to a biologically active dose of a Weel inhibitor. In some instances it is of value to determine if a particular cell population has sensitivity or resistance to a therapeutic dose of a Weel inhibitor.
  • the markers are listed in Table 2.
  • the invention also provides subsets of at least 1, 2, 3, 4, or 5 markers derived from the set listed in Table 2.
  • the invention further provides a set of markers derived from Table 2 that are optimal for predicting a patient's response to a treatment protocol comprising a Weel inhibitor.
  • the selected markers are selected from Tables 2 to include at least 1, 2 or 3 up-regulated genetic markers and at least 1, 2 or 3 down-regulated genetic markers. In other embodiments, the selected markers are all up-regulated markers or all down-regulated markers relative to a reference expression level.
  • the markers are selected from Table 2 based upon a pre-determined threshold, wherein the pre-determined threshold is based upon marker gene expression measurements taken in control samples exposed to a Weel inhibitor.
  • the pre-determined threshold may be expressed in several way, including, but not limited to, a fold change, up or down, of 1.2-fold change or greater, 1.3-fold or greater or 1.4-fold or greater, or 1.5-fold or greater, 1.6-fold or greater, 1.7-fold or greater, 1.8-fold or greater, 1.9-fold or greater, 2.0-fold or greater, or 3.0-fold or greater.
  • the 2-fold means 2-fold up-regulated or l/2-fold down-regulated of the markers in Weel inhibitor treated samples compared with non-treated control samples.
  • gene markers and methods are provided that are useful in predicting sensitivity and/or resistance of a subject to treatment with a Weel inhibitor.
  • the gene markers or subset thereof are used to make a drug response prediction based upon gene expression levels measured in a cell sample comprising tumor cells before Weel inhibitor treatment.
  • the Weel response prediction markers are listed in Table 2. Table 2 lists gene markers, SPRY2, CCNI, JUNB, SMAD2, SHCl, MADlLl, GADD45GIP1, CKAP5, TUBB4, BCATl, MCM8 and TLK2, whose expression levels are correlated with sensitivity of cells to Weel inhibitor treatment.
  • Weel inhibitor sensitivity or resistance is predicted in a subject using 2 or more gene markers selected from Table 2. In another embodiment, sensitivity or resistance are predicted in a subject using at least 1, 2, 3, 4 or 5 markers selected from Table 2.
  • One aspect of the present invention provides a method of using these sets of Weel inhibitor biomarkers to predict whether a subject with cancer will respond to treatment with a Wee 1 inhibitor.
  • the invention comprises using data obtained from the biomarker predictor set as a means of determining whether a patient should continue treatment with a WeI inhibitor or be treated with a Weel inhibitor in the first place.
  • patients exhibiting a favorable data set e.g., a sensitivity signature, may continue treatment with the Weel inhibitor or start treatment with a Weel inhibitor.
  • the methods of the invention may also be used to stratify patient population into a treatment group, e.g., those that can be treated with a Weel inhibitor and thus may be enrolled into a therapeutic regiment employing a Weel inhibitor or a non-treatment group, e.g., those that are not amenable to treatment with a Weel inhibitor.
  • the methods of the invention may also be used to identify patients who may need to be pulled out of therapeutic protocol comprising a Weel inhibitor where the biomarker signature is not positive, e.g., non-sensitive signature.
  • the method comprising:
  • the method further proposes treating the patient with a Weel inhibitor based upon the prediction, e.g., treating patients demonstrating a sensitive profile and pulling out patients from a treatment protocol comprising a Weel inhibitor if their signature profile is that of a non-responder, e.g., non-responsive to Weel inhibitor.
  • the degree of similarity between a gene expression profile obtained from a cellular sample and a template profile can be determined using any method known in the art.
  • Dai et al. describe a number of different ways of calculating gene expression templates and corresponding gene marker gene sets useful in classifying breast cancer patients (US 7,171,311; WO2002103320; WO2005086891; WO2006015312; WO2006084272).
  • Linsley et al., (US 20030104426) and Radish et al., (US 20070154931) disclose gene markers genesets and methods of calculating gene expression template useful in classifying chronic myologenous leukemia patients.
  • the method for identifying marker sets is as follows. After extraction and labeling of target polynucleotides, the expression of all markers (genes) in a sample X is compared to the expression of all markers in a standard or control.
  • the standard or control comprises target polynucleotide molecules derived from a sample from a normal individual (i.e., an individual not afflicted with cancer) or a cell sample not exposed to the Weel inhibitor.
  • the standard or control is a pool of target polynucleotide molecules. The pool may be derived from collected samples from a number of normal individuals. In certain embodiments, the pool comprises samples taken from a number of individuals having cancers responsive to a Weel inhibitor.
  • the pool comprises an artificially- generated population of nucleic acids designed to approximate the level of nucleic acid derived from each marker found in a pool of marker-derived nucleic acids derived from tumor samples.
  • the pool is derived from normal or cancer cell lines or cell line samples.
  • the comparison may be accomplished by any means known in the art. For example, expression levels of various markers may be assessed by separation of target polynucleotide molecules (e.g., RNA or cDNA) derived from the markers in agarose or polyacrylamide gels, followed by hybridization with marker-specific oligonucleotide probes. Alternatively, the comparison may be accomplished by the labeling of target polynucleotide molecules followed by separation on a sequencing gel.
  • target polynucleotide molecules e.g., RNA or cDNA
  • Polynucleotide samples are placed on the gel such that patient and control or standard polynucleotides are in adjacent lanes. Comparison of expression levels is accomplished visually or by means of densitometer. In one embodiment, the expression of all markers is assessed simultaneously by hybridization to a microarray. In each approach, markers meeting certain criteria are identified as associated with a cancer responsive to a Weel inhibitor.
  • Selection of a marker is based preferably on the difference in the expression level of at least one marker in a test sample compared to a standard or control sample. Selection may be made based upon a statistically significant up- or down regulation of the marker in the patient sample after treatment of Weel inhibitor. Selection may also be made by calculation of the statistical significance (i.e., the p-value) of the correlation between the expression of the marker and the condition or indication. Indeed, the larger the difference in expression of at least one biomarker detailed herein between the patient sample and the control or standard, the more informative the prediction. In certain embodiments, both selection criteria may be used. Thus, in one embodiment of the present invention, markers associated with a cancer responsive to a
  • Weel inhibitor are selected where the markers show both more than two-fold change (increase or decrease) in expression as compared to a standard, and the p-value for the correlation between the existence of cancerous condition and the change in marker expression is no more than 0.01 (i.e., is statistically significant).
  • the expression of the identified Weel responsive cancer markers is then used to identify markers that can differentiate tumors into clinical types.
  • c represents the clinical parameters or categories and r represents the linear, logarithmic or any transform of the ratio of expression between sample and control.
  • the significance of the correlation is calculated.
  • This significance may be calculated by any statistical means by which such significance is calculated.
  • a set of correlation data is generated using a Monte-Carlo technique to randomize the association between the expression difference of a particular marker and the clinical category.
  • the frequency distribution of markers satisfying the criteria through calculation of correlation coefficients is compared to the number of markers satisfying the criteria in the data generated through the Monte-Carlo technique.
  • the frequency distribution of markers satisfying the criteria in the Monte-Carlo runs is used to determine whether the number of markers selected by correlation with clinical data is significant. See, for example, Example 4.
  • the markers may be rank-ordered in order of significance of discrimination.
  • rank ordering is by the amplitude of correlation between the change in gene expression of the marker and the specific condition being discriminated.
  • Another, preferred, means is to use a statistical metric.
  • the metric is a Fisher-like statistic: Equation (3)
  • ⁇ x x > is the error- weighted average of the log ratio of transcript expression measurements within a first diagnostic group (e.g., ER(-), ⁇ x 2 > is the error-weighted average of log ratio within a second, related diagnostic group (e.g., ER(+)), O 1 is the variance of the log ratio within the ER(-) group and n x is the number of samples for which valid measurements of log ratios are available.
  • ⁇ 2 is the variance of log ratio within the second diagnostic group (e.g., ER(+))
  • n 2 is the number of samples for which valid measurements of log ratios are available.
  • the t-value represents the variance-compensated difference between two means.
  • the rank-ordered marker set may be used to optimize the number of markers in the set used for discrimination. This is accomplished generally in a "leave one out” method as follows. In a first run, a subset, for example 5, of the markers from the top of the ranked list is used to generate a template, where out of X samples, X-I are used to generate the template, and the status of the remaining sample is predicted. This process is repeated for every sample until every one of the X samples is predicted once. In a second run, additional markers, for example 5, are added, so that a template is now generated from 10 markers, and the outcome of the remaining sample is predicted. This process is repeated until the entire set of markers is used to generate the template.
  • type 1 error false negative
  • type 2 errors false positive
  • the optimal number of markers is that number where the type 1 error rate, or type 2 error rate, or preferably the total of type 1 and type 2 error rate is lowest.
  • validation of the marker set may be accomplished by an additional statistic, a survival model. This statistic generates the probability of cancer as measured by, for example, tumor burden as a function of time since initial diagnosis.
  • a number of models may be used, including Weibull, normal, log-normal, log logistic, log-exponential, or log-Rayleigh (Chapter 12 "Life Testing", S-PLUS 2000 GUIDE TO STATISTICS, Vol. 2, p. 368 (2000)).
  • the above methods are not limited to the identification of markers associated with a Weel inhibitor or Weel mediated cancer, but may be used to identify set of marker genes associated with any phenotype.
  • the phenotype can be the presence or absence of a disease such as cancer, or the presence or absence of any identifying clinical condition associated with that cancer.
  • the phenotype may be a prognosis such as a survival time, probability of distant metastases of a disease condition, or likelihood of a particular response to a therapeutic or prophylactic regimen.
  • the phenotype need not be cancer, or a disease; the phenotype may be a nominal characteristic associated with a healthy individual.
  • the similarity is represented by a correlation coefficient between the sample profile and the template.
  • a correlation coefficient above a correlation threshold indicates high similarity, whereas a correlation coefficient below the threshold indicates low similarity.
  • the correlation threshold is set as 0.3, 0.4, 0.5 or 0.6.
  • similarity between a sample profile and a template is represented by a distance between the sample profile and the template. In one embodiment, a distance below a given value indicates high similarity, whereas a distance equal to or greater than the given value indicates low similarity.
  • the expression levels of the marker genes in a sample may be determined by any means known in the art.
  • the expression level may be determined by isolating and determining the level (i.e., amount) of nucleic acid transcribed from each marker gene.
  • the level of specific proteins encoded by a marker gene may be determined.
  • the level of expression of specific marker genes can be accomplished by determining the amount of mRNA, or polynucleotides derived therefrom, present in a sample. Any method for determining RNA levels can be used. For example, RNA is isolated from a sample and separated on an agarose gel. The separated RNA is then transferred to a solid support, such as a filter.
  • Nucleic acid probes representing one or more markers are then hybridized to the filter by northern hybridization, and the amount of marker-derived RNA is determined. Such determination can be visual, or machine-aided, for example, by use of a densitometer. Another method of determining RNA levels is by use of a dot-blot or a slot-blot. In this method, RNA, or nucleic acid derived therefrom, from a sample is labeled. The RNA or nucleic acid derived therefrom is then hybridized to a filter containing oligonucleotides derived from one or more marker genes, wherein the oligonucleotides are placed upon the filter at discrete, easily-identifiable locations.
  • Hybridization, or lack thereof, of the labeled RNA to the filter-bound oligonucleotides is determined visually or by densitometer.
  • Polynucleotides can be labeled using a radiolabel or a fluorescent (i.e., visible) label.
  • tissue array Kononen et al, Nat. Med 4(7):844-7 (1998).
  • tissue array multiple tissue samples are assessed on the same microarray. The arrays allow in situ detection of RNA and protein levels; consecutive sections allow the analysis of multiple samples simultaneously.
  • polynucleotide microarrays are used to measure expression so that the expression status of each of the markers in one or more of the inventive genesets described herein, is assessed simultaneously.
  • the microarrays of the invention preferably comprise at least 2, 3, 4, 5 or more of markers, or all of the markers, or any combination of markers, identified as classification-informative within a subject subset.
  • the actual number of informative markers the microarray comprises will vary depending upon the particular condition of interest, the number of markers identified, and, optionally, the number of informative markers found to result in the least Type I error, Type II error, or Type I and Type II error in determination of an endpoint phenotype.
  • Type I error means a false positive and “Type II error” means a false negative; in the example of prediction of therapeutic response to exposure to a Wee 1 inhibitor, Type I error is the mis-characterization of an individual with a therapeutic response to a Wee 1 inhibitor as having being a non-responder to Wee 1 inhibitor treatment, and Type II error is the mis-characterization of an individual with no response to Wee 1 inhibitor treatment as having a therapeutic response.
  • the invention provides polynucleotide arrays in which the markers identified for a particular subject subset comprise at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 98% of the probes on said array.
  • the microarray comprises a plurality of probes, wherein said plurality of probes comprise probes complementary and hybridizable to at least 75% of the Wee 1 inhibitor exposure/prediction-informative markers identified for a particular patient subset.
  • Microarrays of the invention may comprise probes complementary and hybridizable to Wee 1 inhibitor exposure/prediction-informative markers for a plurality of the subject subsets, or for each subject subset, identified for a particular condition.
  • the microarray of the invention comprises a plurality of probes complementary and hybridizable to at least 75% of the Wee 1 inhibitor exposure/prediction-informative markers identified for each subject subset identified for the condition of interest, and wherein said probes, in total, are at least 50% of the probes on said microarray.
  • the microarray is a commercially-available cDNA microarray that comprises at least two markers identified by the methods described herein.
  • a commercially-available cDNA microarray comprises all of the markers identified by the methods described herein as being informative for a patient subset for a particular condition.
  • such a microarray may comprise at least 1, 2, 3, 4 or 5 of such markers, up to the maximum number of markers identified.
  • the array comprises a plurality of probes derived from markers listed in any of Tables 1 in combination with a plurality of other probes, derived from markers not listed in any of Tables 1, that are identified as informative for the evaluation of exposure to a Wee 1 inhibitor, prediction of therapeutic response, etc. The same holds true for the sequences listed in Table 2.
  • Polynucleotides capable of specifically or selectively binding to the mRNA transcripts encoding the polypeptide biomarkers of the invention are also contemplated.
  • oligonucleotides, cDNA, DNA, RNA, PCR products, synthetic DNA, synthetic RNA, or other combinations of naturally occurring or modified nucleotides which specifically and/or selectively hybridize to one or more of the RNA products of the biomarker of the invention are useful in accordance with the invention.
  • the oligonucleotides, cDNA, DNA, RNA, PCR products, synthetic DNA, synthetic RNA, or other combinations of naturally occurring or modified nucleotides oligonucleotides which both specifically and selectively hybridize to one or more of the RNA products of the biomarker of the invention are used.
  • any method known in the art may be utilized.
  • expression based on detection of RNA which hybridizes to the genes identified and disclosed herein is used. This is readily performed by any RNA detection or amplification methods known or recognized as equivalent in the art such as, but not limited to, reverse transcription-PCR, and methods to detect the presence, or absence, of RNA stabilizing or destabilizing sequences.
  • expression based on detection of DNA status may be used. Detection of the DNA of an identified gene as may be used for genes that have increased expression in correlation with a particular outcome. This may be readily performed by PCR based methods known in the art, including, but not limited to, Q-PCR.
  • detection of the DNA of an identified gene as amplified may be used for genes that have increased expression in correlation with a particular treatment outcome. This may be readily performed by PCR based, fluorescent in situ hybridization (FISH) and chromosome in situ hybridization (CISH) methods known in the art.
  • FISH fluorescent in situ hybridization
  • CISH chromosome in situ hybridization
  • a gene expression-based expression assay based on a small number of genes i.e., about 1 to 3000 genes can be performed with relatively little effort using existing quantitative real-time PCR technology familiar to clinical laboratories.
  • Quantitative real-time PCR measures PCR product accumulation through a dual-labeled fluorigenic probe.
  • a variety of normalization methods may be used, such as an internal competitor for each target sequence, a normalization gene contained within the sample, or a housekeeping gene.
  • Sufficient RNA for real time PCR can be isolated from low milligram quantities from a subject. Quantitative thermal cyclers may now be used with microfluidics cards preloaded with reagents making routine clinical use of multigene expression-based assays a realistic goal.
  • the gene markers of the various inventive genesets or a subset of genes selected from the inventive genesets, which are assayed according to the present invention are typically in the form of total RNA or mRNA or reverse transcribed total RNA or mRNA.
  • General methods for total and mRNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology, including Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997).
  • RNA isolation can also be performed using purification kit, buffer set and protease from commercial manufacturers, such as Qiagen (Valencia, CA) and Ambion (Austin, TX), according to the manufacturer's instructions.
  • TAQman quantitative real-time PCR can be performed using commercially available PCR reagents (Applied Biosystems, Foster City, CA) and equipment, such as ABI Prism 7900HT Sequence Detection System (Applied Biosystems) according the manufacturer's instructions.
  • the system consists of a thermocycler, laser, charge-coupled device (CCD), camera, and computer.
  • the system amplifies samples in a 96-well or 384- well format on a thermocycler.
  • laser-induced fluorescent signal is collected in real-time through fiber-optics cables for all 96 wells, and detected at the CCD.
  • the system includes software for running the instrument and for analyzing the data.
  • a real-time PCR TAQman assay can be used to make gene expression measurements and perform the classification methods described herein.
  • oligonucleotide primers and probes that are complementary to or hybridize to the markers of the invention may be selected based upon the marker transcript sequences set forth in the Sequence Listing.
  • the polynucleotide used to measure the RNA products of the invention can be used as nucleic acid members stably associated with a support to comprise an array according to one aspect of the invention.
  • the length of a nucleic acid member can range from 8 to 1000 nucleotides in length and are chosen so as to be specific for the RNA products of the biomarkers of the invention, hi one embodiment, these members are selective for the RNA products of the invention.
  • the nucleic acid members may be single or double stranded, and/or may be oligonucleotides or PCR fragments amplified from cDNA. Preferably oligonucleotides are approximately 20-30 nucleotides in length.
  • ESTs are preferably 100 to 600 nucleotides in length. It will be understood to a person skilled in the art that one can utilize portions of the expressed regions of the biomarkers of the invention as a probe on the array. More particularly oligonucleotides complementary to the genes of the invention and cDNA or ESTs derived from the genes of the invention are useful. For oligonucleotide based arrays, the selection of oligonucleotides corresponding to the gene of interest which are useful as probes is well understood in the art. More particularly it is important to choose regions which will permit hybridization to the target nucleic acids. Factors such as the Tm of the oligonucleotide, the percent GC content, the degree of secondary structure and the length of nucleic acid are important factors. See for example U.S. Pat. No. 6,551,784.
  • an array of nucleic acid members stably associated with the surface of a substantially support is contacted with a sample comprising target nucleic acids under hybridization conditions sufficient to produce a hybridization pattern of complementary nucleic acid members/target complexes in which one or more complementary nucleic acid members at unique positions on the array specifically hybridize to target nucleic acids.
  • the identity of target nucleic acids which hybridize can be determined with reference to location of nucleic acid members on the array.
  • the nucleic acid members may be produced using established techniques such as polymerase chain reaction (PCR) and reverse transcription (RT). These methods are similar to those currently known in the art (see e.g., PCR Strategies, Michael A. Innis (Editor), et al. (1995) and PCR: Introduction to Biotechniques Series, C. R. Newton, A. Graham (1997)). Amplified nucleic acids are purified by methods well known in the art (e.g., column purification or alcohol precipitation). A nucleic acid is considered pure when it has been isolated so as to be substantially free of primers and incomplete products produced during the synthesis of the desired nucleic acid.
  • PCR polymerase chain reaction
  • RT reverse transcription
  • a purified nucleic acid will also be substantially free of contaminants which may hinder or otherwise mask the specific binding activity of the molecule.
  • An array comprises a plurality of nucleic acids attached to one surface of a support at a density exceeding 20 different nucleic acids/cm 2 , wherein each of the nucleic acids is attached to the surface of the support in a non-identical pre-selected region (e.g. a microarray).
  • Each associated sample on the array comprises a nucleic acid composition, of known identity, usually of known sequence, as described in greater detail below. Any conceivable substrate may be employed in the invention.
  • the nucleic acid attached to the surface of the support is DNA. In one embodiment, the nucleic acid attached to the surface of the support is cDNA or RNA. In another embodiment, the nucleic acid attached to the surface of the support is cDNA synthesized by polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • a nucleic acid member in the array is at least 10, 25, 50, 60 nucleotides in length. In one embodiment, a nucleic acid member is at least 150 nucleotides in length. Preferably, a nucleic acid member is less than 1000 nucleotides in length. More preferably, a nucleic acid member is less than 500 nucleotides in length.
  • the nucleic acid compositions are stably associated with the surface of a support, where the support may be a flexible or rigid support.
  • stably associated is meant that each nucleic acid member maintains a unique position relative to the support under hybridization and washing conditions.
  • the samples are non-covalently or covalently stably associated with the support surface. Examples of non-covalent association include non-specific adsorption, binding based on electrostatic interactions (e.g., ion pair interactions), hydrophobic interactions, hydrogen bonding interactions, specific binding through a specific binding pair member covalently attached to the support surface, and the like.
  • covalent binding examples include covalent bonds formed between the nucleic acids and a functional group present on the surface of the rigid support (e.g., --OH), where the functional group may be naturally occurring or present as a member of an introduced linking group, as described in greater detail below.
  • a functional group present on the surface of the rigid support e.g., --OH
  • the functional group may be naturally occurring or present as a member of an introduced linking group, as described in greater detail below.
  • each composition will be sufficient to provide for adequate hybridization and detection of target nucleic acid sequences during the assay in which the array is employed.
  • the amount of each nucleic acid member stably associated with the support of the array is at least about 0.001 ng, preferably at least about 0.02 ng and more preferably at least about 0.05 ng, where the amount may be as high as 1000 ng or higher, but will usually not exceed about 20 ng.
  • the diameter of the "spot” will generally range from about 10 to 5,000 ⁇ m, usually from about 20 to 2,000 ⁇ m and more usually from about 100 to 200 ⁇ m.
  • Control nucleic acid members may be present on the array including nucleic acid members comprising oligonucleotides or nucleic acids corresponding to genomic DNA, housekeeping genes, vector sequences, plant nucleic acid sequence, negative and positive control genes, and the like. Control nucleic acid members are calibrating or control genes whose function is not to tell whether a particular "key" gene of interest is expressed, but rather to provide other useful information, such as background or basal level of expression.
  • control nucleic acids are spotted on the array and used as target expression control nucleic acids and mismatch control nucleotides to monitor non-specific binding or cross- hybridization to a nucleic acid in the sample other than the target to which the probe is directed.
  • Mismatch probes thus indicate whether a hybridization is specific or not. For example, if the target is present, the perfectly matched probes should be consistently brighter than the mismatched probes. In addition, if all control mismatches are present, the mismatch probes are used to detect a mutation.
  • Numerous methods may be used for attachment of the nucleic acid members of the invention to the substrate (a process referred to as "spotting"). For example, nucleic acids are attached using the techniques of, for example U.S. Pat. No.
  • RNA product of the invention can be done by using those polynucleotides which are specific and/or selective for the RNA products of the invention to quantitate the expression of the RNA product.
  • the polynucleotides which are specific and/or selective for the RNA products are probes or primers.
  • these polynucleotides are in the form of nucleic acid probes which can be spotted onto an array to measure RNA from the sample of an individual to be measured.
  • the polynucleotides which are specific and/or selective for the RNA products of the invention are used in the form of probes and primers in techniques such as quantitative real-time RT PCR, using for example SYBR®Green, or using TaqMan® or Molecular Beacon techniques, where the polynucleotides used are used in the form of a forward primer, a reverse primer, a TaqMan labelled probe or a Molecular Beacon labelled probe.
  • the nucleic acid derived from the sample cell(s) may be preferentially amplified by use of appropriate primers such that only the genes to be analyzed are amplified to reduce background signals from other genes expressed in the breast cell.
  • the nucleic acid from the sample may be globally amplified before hybridization to the immobilized polynucleotides.
  • RNA, or the cDNA counterpart thereof may be directly labeled and used, without amplification, by methods known in the art.
  • a "microarray” is a linear or two- dimensional array of preferably discrete regions, each having a defined area, formed on the surface of a solid support such as, but not limited to, glass, plastic, or synthetic membrane.
  • the density of the discrete regions on a microarray is determined by the total numbers of immobilized polynucleotides to be detected on the surface of a single solid phase support, preferably at least about 50/cm 2 , more preferably at least about 100/cm 2 , even more preferably at least about 500/cm 2 , but preferably below about 1,000/cm 2 .
  • the arrays contain less than about 500, about 1000, about 1500, about 2000, about 2500, or about 3000 immobilized polynucleotides in total.
  • a DNA microarray is an array of oligonucleotides or polynucleotides placed on a chip or other surfaces used to hybridize to amplified or cloned polynucleotides from a sample. Since the position of each particular group of primers in the array is known, the identities of a sample polynucleotides can be determined based on their binding to a particular position in the microarray.
  • Determining gene expression levels may be accomplished utilizing microarrays. Generally, the following steps may be involved: (a) obtaining an mRNA sample from a subject and preparing labeled nucleic acids therefrom (the "target nucleic acids” or “targets”); (b) contacting the target nucleic acids with an array under conditions sufficient for the target nucleic acids to bind to the corresponding probes on the array, for example, by hybridization or specific binding; (c) optional removal of unbound targets from the array; (d) detecting the bound targets, and (e) analyzing the results, for example, using computer based analysis methods.
  • “nucleic acid probes” or “probes” are nucleic acids attached to the array
  • target nucleic acids are nucleic acids that are hybridized to the array.
  • Nucleic acid specimens may be obtained from a subject to be tested using either "invasive” or “non-invasive” sampling means.
  • a sampling means is said to be “invasive” if it involves the collection of nucleic acids from within the skin or organs of an animal (including murine, human, ovine, equine, bovine, porcine, canine, or feline animal).
  • invasive methods include, for example, blood collection, semen collection, needle biopsy, pleural aspiration, umbilical cord biopsy. Examples of such methods are discussed by Kim, et al., (J. Virol. 66:3879-3882, 1992); Biswas, et al., (Ann. NY Acad. Sci. 590:582-583, 1990); and Biswas, et al. , (J. Clin. Microbiol. 29:2228-2233, 1991).
  • a “non-invasive” sampling means is one in which the nucleic acid molecules are recovered from an internal or external surface of the animal.
  • Examples of such "non-invasive” sampling means include, for example, “swabbing,” collection of tears, saliva, urine, fecal material etc.
  • one or more cells from the subject to be tested are obtained and RNA is isolated from the cells.
  • a sample of cells is obtained from the subject. It is also possible to obtain a cell sample from a subject, and then to enrich the sample for a desired cell type. For example, cells may be isolated from other cells using a variety of techniques, such as isolation with an antibody binding to an epitope on the cell surface of the desired cell type.
  • the desired cells are in a solid tissue
  • particular cells may be dissected, for example, by microdissection or by laser capture microdissection (LCM) (see, e.g., Bonner, et al., Science 278:1481, 1997; Emmert-Buck, et al., Science 274:998, 1996; Fend, et al., Am. J. Path. 154:61, 1999; and Murakami, et al., Kidney hit. 58:1346, 2000).
  • LCM laser capture microdissection
  • RNA may be extracted from tissue or cell samples by a variety of methods, for example, guanidium thiocyanate lysis followed by CsCl centrifugation (Chirgwin, et al.,
  • RNA from single cells may be obtained as described in methods for preparing cDNA libraries from single cells (see, e.g., Dulac, Curr. Top. Dev. Biol. 36:245, 1998; Jena, et al., J. Immunol. Methods 190:199, 1996).
  • RNA sample can be further enriched for a particular species.
  • poly(A)+RNA may be isolated from an RNA sample.
  • the RNA population may be enriched for sequences of interest by primer-specific cDNA synthesis, or multiple rounds of linear amplification based on cDNA synthesis and template- directed in vitro transcription (see, e.g., Wang, et al., Proc. Natl. Acad. Sci. USA 86:9717, 1989; Dulac, et al., supra; Jena, et al., supra).
  • RNA may be further amplified by a variety of amplification methods including, for example, PCR; ligase chain reaction (LCR) (see, e. g., Wu and Wallace, Genomics 4:560, 1989; Landegren, et al., Science 241:1077, 1988); self- sustained sequence replication (SSR) (see, e.g., Guatelli, et al., Proc. Natl. Acad. Sci. USA 87:1874, 1990); nucleic acid based sequence amplification (NASBA) and transcription amplification (see, e.g., Kwoh, et al., Proc. Natl. Acad.
  • LCR ligase chain reaction
  • SSR self- sustained sequence replication
  • NASBA nucleic acid based sequence amplification
  • transcription amplification see, e.g., Kwoh, et al., Proc. Natl. Acad.
  • PCR Technology Principles and Applications for DNA Amplification (ed. H. A. Erlich, Freeman Press, N. Y., N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (eds. Innis, et al., Academic Press, San Diego, Calif, 1990); Mattila, et al., Nucleic Acids Res. 19:4967, 1991 ; Eckert, et al., PCR Methods and Applications 1:17, 1991; PCR (eds. McPherson et al., IRL Press, Oxford); and U.S. Pat. No. 4,683,202).
  • RNA amplification and cDNA synthesis may also be conducted in cells in situ (see, e.g., Eberwine, et al. Proc. Natl. Acad. Sci. USA 89:3010, 1992).
  • all or part of a disclosed marker sequence may be amplified and detected by methods such as the polymerase chain reaction (PCR) and variations thereof, such as, but not limited to, quantitative PCR (Q-PCR), reverse transcription PCR (RT- PCR), and real-time PCR, optionally real-time RT-PCR.
  • PCR polymerase chain reaction
  • Q-PCR quantitative PCR
  • RT- PCR reverse transcription PCR
  • real-time PCR optionally real-time RT-PCR.
  • Such methods would utilize one or two primers that are complementary to portions of a disclosed sequence, where the primers are used to prime nucleic acid synthesis.
  • the newly synthesized nucleic acids are optionally labeled and may be detected directly or by hybridization to a polynucleotide of the invention.
  • the nucleic acid molecules may be labeled to permit detection of hybridization of the nucleic acid molecules to a microarray. That is, the probe may comprise a member of a signal producing system and thus, is detectable, either directly or through combined action with one or more additional members of a signal producing system.
  • the nucleic acids may be labeled with a fluorescently labeled dNTP (see, e.g., Kricka, 1992, Nonisotopic DNA Probe Techniques, Academic Press San Diego, Calif.) , biotinylated dNTPs or rNTP followed by addition of labeled streptavidin, chemiluminescent labels, or isotopes.
  • labels include "molecular beacons" as described in Tyagi and Kramer (Nature Biotech. 14:303, 1996).
  • the newly synthesized nucleic acids may be contacted with polynucleotides (containing sequences) of the invention under conditions which allow for their hybridization.
  • Hybridization may be also determined, for example, by plasmon resonance (see, e.g., Thiel, et al. Anal. Chem. 69:4948, 1997).
  • a plurality e.g., 2 sets of target nucleic acids are labeled and used in one hybridization reaction ("multiplex" analysis).
  • one set of nucleic acids may correspond to RNA from one cell and another set of nucleic acids may correspond to RNA from another cell.
  • the plurality of sets of nucleic acids may be labeled with different labels, for example, different fluorescent labels (e.g., fluorescein and rhodamine) which have distinct emission spectra so that they can be distinguished.
  • the sets may then be mixed and hybridized simultaneously to one microarray (see, e.g., Shena, et al., Science 270:467-470, 1995).
  • Patents describing methods of using arrays in various applications include: U.S. Pat. Nos. 5,143,854; 5,288, 644; 5,324,633; 5, 432,049; 5,470,710; 5,492,806; 5,503,980; 5,510,270; 5, 525,464; 5,547,839; 5,580,732; 5,661,028; 5,848,659; and 5,874,219; the disclosures of which are herein incorporated by reference.
  • an array of oligonucleotides may be synthesized on a solid support.
  • solid supports include glass, plastics, polymers, metals, metalloids, ceramics, organics, etc.
  • chip masking technologies and photoprotective chemistry it is possible to generate ordered arrays of nucleic acid probes.
  • These arrays which are known, for example, as "DNA chips” or very large scale immobilized polymer arrays (“VLSIPS®” arrays), may include millions of defined probe regions on a substrate having an area of about 1 cm 2 to several cm 2 , thereby incorporating from a few to millions of probes (see, e.g., U.S. Pat. No. 5,631,734).
  • labeled nucleic acids may be contacted with the array under conditions sufficient for binding between the target nucleic acid and the probe on the array.
  • the hybridization conditions may be selected to provide for the desired level of hybridization specificity; that is, conditions sufficient for hybridization to occur between the labeled nucleic acids and probes on the microarray.
  • Hybridization may be carried out in conditions permitting essentially specific hybridization.
  • the length and GC content of the nucleic acid will determine the thermal melting point and thus, the hybridization conditions necessary for obtaining specific hybridization of the probe to the target nucleic acid. These factors are well known to a person of skill in the art, and may also be tested in assays.
  • An extensive guide to nucleic acid hybridization may be found in Tijssen, et al. (Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 24: Hybridization With Nucleic Acid Probes, P. Tijssen, ed. Elsevier, N. Y., (1993)).
  • the methods described above will result in the production of hybridization patterns of labeled target nucleic acids on the array surface.
  • the resultant hybridization patterns of labeled nucleic acids may be visualized or detected in a variety of ways, with the particular manner of detection selected based on the particular label of the target nucleic acid.
  • Representative detection means include scintillation counting, autoradiography, fluorescence measurement, calorimetric measurement, light emission measurement, light scattering, and the like.
  • One such method of detection utilizes an array scanner that is commercially available (Affymetrix, Santa Clara, Calif.), for example, the 417® Arrayer, the 418® Array Scanner, or the Agilent GeneArray® Scanner.
  • This scanner is controlled from a system computer with an interface and easy-to-use software tools. The output may be directly imported into or directly read by a variety of software applications. Exemplary scanning devices are described in, for example, U. S. Pat. Nos. 5,143,854 and 5,424,186.
  • Cancers amenable to treatment with a Weel inhibitor include but are not limited to acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia, Kaposi's sarcoma; breast cancers; bone cancers, brain cancers, cancers of the head and neck, gallbladder and bile duct cancers, cancers of the retina, cancers of the esophagus, gastric cancers, multiple myeloma, ovarian cancer, uterine cancer, thyroid cancer, testicular cancer, endometrial cancer, melanoma, colorectal cancer, bladder cancer, prostate cancer, lung cancer, pancreatic cancer, sarcomas, Wilms' tumor, cervical cancer, skin cancers, nasopharyngeal carcinoma, liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladder adeno carcinoma, paroti
  • the Wee 1 inhibitor can be administered by any known administration method known to a person skilled in the art.
  • routes of administration include but are not limited to oral, parenteral, intraperitoneal, intravenous, intraarterial, transdermal, sublingual, intramuscular, rectal, transbuccal, intranasal, liposomal, via inhalation, vaginal, intraoccular, via local delivery by catheter or stent, subcutaneous, intraadiposal, intraarticular, intrathecal, or in a slow release dosage form.
  • the Wee 1 inhibitors or a pharmaceutically acceptable salt or hydrate thereof can be administered in accordance with any dose and dosing schedule that, achieves a dose effective to treat cancer.
  • Weel inhibitors can be administered in a total daily dose of up to 1000 mg, preferably orally, once, twice or three times daily, continuously (every day) or intermittently (e.g., 3-5 days a week).
  • a Wee 1 inhibitor may also be administered in combination with an anti-cancer agent, wherein the amount of Wee 1 and the amount of the anti-cancer agent together comprise a therapeutically effective amount.
  • the combination therapy can provide a therapeutic advantage in view of the differential toxicity associated with the two treatment modalities. For example, treatment with Wee 1 inhibitors can lead to a particular toxicity that is not seen with the anti-cancer agent, and vice versa.
  • this differential toxicity can permit each treatment to be administered at a dose at which said toxicities do not exist or are minimal, such that together the combination therapy provides a therapeutic dose while avoiding the toxicities of each of the constituents of the combination agents.
  • the therapeutic effects achieved as a result of the combination treatment are enhanced or synergistic, for example, significantly better than additive therapeutic effects, the doses of each of the agents can be reduced even further, thus lowering the associated toxicities to an even greater extent.
  • Wee 1 inhibitor can be combined with chemotherapy and radiotherapy.
  • Wee 1 inhibitor is also combined with an anti-cancer agent, but is preferably combined with a DNA damaging agents.
  • anti-cancer agent used in a combination treatment with Wee 1 inhibitors are for example, but not limited to, gemcitabine, cisplatin, carboplatin, 5-fluorouracil, pemetrexed, doxorubicin, camptothecin and mitomycin.
  • a Wee 1 inhibitor is administered in a pharmaceutical composition, preferably suitable for oral administration.
  • Wee 1 is administered orally in a gelating capsule, which can comprise excipients such as microcrystalline cellulose, croscarmellose sodium and magnesium stearate.
  • the Wee 1 inhibitors can be administered in a total daily dose that may vary from patient to patient, and may be administered at varying dosage schedules. Suitable dosages are total daily dosage of between about 25-4000 mg/m 2 administered orally once-daily, twice-daily or three times-daily, continuous (every day) or intermittently (e.g. 3-5 days a week).
  • compositions may be administered in cycles, with rest periods in between the cycles (e.g. treatment for two to eight weeks with a rest period of up to a week between treatments).
  • rest periods e.g. treatment for two to eight weeks with a rest period of up to a week between treatments.
  • Other treatment combinations and dosing regiments are set forth in WO
  • any one or more of the specific dosages and dosage schedules of the Wee 1 inhibitors is also applicable to any one or more of the anti-cancer agents to be used in the combination treatment.
  • the specific dosage and dosage schedule of the anti-cancer agent can further vary, and the optimal dose, dosing schedule and route of administration will be determined based upon the specific anti-cancer agent that is being used.
  • WO2007/126128 was stored at -20. Purity was 99.3%.
  • Compound A was dissolved in dimethyl sulphoxide (DMSO) (SIGMA, #D2650).
  • Gemcitabine (Gem): Gemzar ® Injection (Eli Lilly Japan K.K.) was dissolved in Phosphate Buffered Saline, pH 7.4 (PBS) (Invitrogen, #10010-049) and stored at -2O 0 C.
  • PBS Phosphate Buffered Saline, pH 7.4
  • WiDr human colorectal adenocarcinoma cell line, obtained from ATCC, was cultured in DMEM; Dulbecco's Modified Eagle Medium low-glucose (Invitrogen, #11885-092) containing 10% FBS and 100 U/mL penicillin / 100 ⁇ g/mL streptomycin.
  • TOV21G matched pair cell lines human ovarian adenocarcinoma cell line, obtained from ROSETTA INPHARMATICS, were cultured in DMEM (Dulbecco's Modified Eagle Medium) high-glucose (Invitrogen, #11965-092) containing 10% FBS and 100 U/mL penicillin / 100 ⁇ g/mL streptomycin.
  • the p53 matched pair cell lines are p53 wild type TOV21G (TOV21G-vec) and p53-silenced TOV21G (TOV21G-shp53) cell lines by small hairpin RNA.
  • WiDr human colon cancer cells (right flank: 2x10 7 cells/rat) was inoculated into the hind flank of F344/N Jcl-rnu nude rats (U- 19672, female, 7wks). Two weeks later,
  • Gemcitabine was administered by IV infusion (50mg/kg). Twenty four hr later, Compound A (0.5, 1.0, 3.0 mg/kg/hr) was IV infused for 8 hr at the rate of 0.25 mL/hr. Thereafter, rats were euthanized at 8 hr and rat skin samples and tumor xenograft was excised for microarray analysis. Briefly, microarray hybridizations were performed as follows.
  • microarray design for in vitro expression profiling using TOV21G-derived p53 matched pair cell lines was as follows.
  • RNA from cultured cells was isolated by using the RNeasy Mini kit (Qiagen, #74104) with DNase I (Qiagen, #79254).
  • Total RNA from tissues in rat xenograft model was isolated by Trizol reagent (Invitrogen, #15596-018), and the isolated RNA was repurified with RNeasy Mini Kit.
  • the purified RNA from each sample was converted to cDNA and hybridized to appropriate reference standards, rat skin microarray: three vehicle control samples; human cell line microarray: pooled TOV21G with control vector samples.
  • microarray analysis was performed with Rosetta/Merck microarray, Human 44k 1.1 and Rat 44k 1.1. Expression profile was analyzed by a microarray software, Resolver (Rosetta Inpharmatics) to identify the classifier genes for responder.
  • cDNA was synthesized from 1 ⁇ g of total RNA by using TaqMan Reverse Transcription Reagents (PE Applied Biosystems, #N8080234). Quantitative real-time PCR assays for human CLSPN, CCNE 1/2, MCMlO, FBXO5 and GAPDH were performed in triplicate for cDNA samples in 96-well optical plates. Data were collected and analyzed using an ABI PRISM 7700 sequence detector system (PE Applied Biosystems). Primer and probe sequences for the RT-PCR were described in Table 3. Real-time PCR for CCNE2, MCM 10 and FBXOlO was performed using SYBR Green method that does not require probe.
  • PCR was performed on the following condition: 50°C for 2 min, 95°C for 10 min, 40
  • RNA samples extracted from each rat were purified and reverse-transcribed to cDNA. They were applied to microarray analysis using Rat 44k 1.1.
  • R Correlation coefficient between phosphor-CDC2 level and expression of each gene.
  • the candidate biomarker genes show similar expression profile in both surrogate and tumor samples.
  • expression profile of TOV21G-derived p53 matched pair cell lines treated with Gemcitabine/Compound A in vitro which are shown in Figures 2 and 3 were analyzed. Genes that were commonly changed in both p53-positive and negative cell lines, because surrogate tissues possess wild-type p53 were selected.
  • the selection criteria to determine up- and down-regulated genes were as described in the experimental methods section. As a result, 56 genes were extracted as candidate genes for expression biomarker with tumor cancer cell lines.
  • the candidate genes identified in rat skin analysis and the TOV21 G cell line analysis were compared. From both experiments, Clspn, Mem 10, Fbxo5 and Ccnel and Ccne2 were selected as each was expressed in both tumor and surrogate tissues.
  • Microarray analysis in rat skin sample and TOV21 cell lines implicated Clspn, Ccnel, McmlO, Ccne2 and Fbxo5 as potential expression biomarkers.
  • changes in expression pattern were analyzed in xenograft tumors (transplanted Widr cells) by quantitative real-time PCR analysis.
  • Figure 4 details cells exhibiting increased expression levels of these genes after treatment with Gemcitabine.
  • the expression levels were down-regulated with Gemcitabine/Compound A combination treatment in dose dependent manner and significantly changed in both 1.0 and 3.0 mpk treated samples (p ⁇ 0.01), which is consistent with the data obtained from the microarray experiment.
  • Microarray analysis was performed to identify expression pharmacodynamic biomarkers, the expression of which was changed in both in vivo and in vitro samples treated with a Weel inhibitor Compound A in combination with Gemcitabine.
  • Five genes, Clspn, Ccnel, Ccne2, McmlO and Fbxo5 exhibited a significant change in their expression pattern in both surrogate and tumor samples, e.g., higher or lower levels of expression thus implicating them as surrogate Wee 1 biomarkers.
  • WO2007/126128 was stored at -20. Purity was 99.3%.
  • Compound A was dissolved in dimethyl sulphoxide (DMSO) (SIGMA, #D2650).
  • Gemcitabine (Gem): Gemzar ® Injection (Eli Lilly Japan K.K.) was dissolved in Phosphate Buffered Saline, pH 7.4 (PBS) (Invitrogen, #10010-049) and stored at -2O 0 C. 3) Carboplatin : Carboplatin (SIGMA, #C2538) was dissolved in PBS and stored at
  • Cisplatin (SIGMA, #P4394) was dissolved in PBS and stored at -20 0 C.
  • Each cell line was cultured for 24 hours, and then DNA damaging agent (Gemcitabine at 1 ⁇ 300 nM, Carboplatin at 1 ⁇ 300 uM or Cisplatin at 0.1 ⁇ 30 uM) were added and continued to be cultured for another 24 hours.
  • Weel inhibitor (Compund A) was added to the cultured cells at 30, 100 and 300 nM, and they were incubated for additional 24 hours. Then, cell viability was measured with a WST-8 kit (KISHIDA CHEMICAL CO., LTD) using a SpectraMax Plus384 plate reader (Molecular Devices Corporation). The sensitivity to the combination therapy was shown as Bliss additivism index .
  • the 22 NSCLC cell lines with deficient p53 status were analyzed for the cytotoxicity assay.
  • top 10 higher-sensitive cell lines and bottom 10 lower-sensitive cell lines were classified as hyper-responders and normal-responders respectively.
  • the P value of hypergeometric test represents the chance of seeing the observed number of overlap genes (or more) between the input set and the comparison set if input genes are randomly selected.
  • Leave-one-out cross-validation involves using a single Bliss index of one cell line from the original sample containing the 20 cell lines as the validation data, and the remaining 19 data as the training data. This is repeated such that each observation in the sample is used once as the validation data.
  • the viability of the cell lines was measured by a cytotoxic assay, and sensitivity of the cells to the combination therapy was shown as bliss additivism index.
  • the result of the treatment with Compound A/Cisplatin is shown in Figure 5.
  • the synergistic effect (excess over Bliss additivism) was variable, ranging from 4 to 32 among the cell lines treated with the Compound A /Cisplatin combination.
  • top 10 high-sensitivity cell lines and bottom 10 low-sensitivity cell lines were classified as hyper-responders and normal-responders respectively.
  • Genes differentially expressed between 10 hyper and 10 normal responder lung cancer cell lines to Compound A/Cisplatin combination treatment were selected. Genes whose expression patterns were highly correlated with the synergistic index (see materials and methods section supra) were reviewed with the objective of narrowing the gene set(s) for further study.
  • the selected signature genes could correctly distinguish the two groups by hierarchical clustering analysis as shown in Figure 6.
  • the 117 genes are shown in Table 5 and
  • the inventors further narrowed the signature gene set(s) and identified two cell cycle related genes, annotated in the hypergeometric test. Theses two genes are designated herein as MADlLl (MADl) and SMAD2.
  • the inventors next examined whether the selected two genes could be used to identify a hyper responder and predict its sensitivity to a Weel inhibitor. As shown in Figure 9, the expression ratio of MADl to SMAD2 was useful in being able to predict Weel sensitivity with an 85% of prediction accuracy. The data support the hypothesis that, given the frequent deregulation and high prediction accuracy by the two genes, the expression pattern/ratio of
  • MADl to SMAD2 will find use in predicting a patient's potential response, e.g., sensitivity to a combination therapy comprising a Weel inhibitor and a DNA damaging agent.

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Abstract

La présente invention porte sur l'identification d'ensembles de gènes biomarqueurs dont les niveaux d'expression sont utiles pour l'évaluation et la classification d'échantillons biologiques pour déterminer l'exposition à une dose biologique active d'un inhibiteur de Wee1, la prédiction précoce d'une réponse à un traitement thérapeutique anticancéreux et la prédiction avant exposition à une dose de la sensibilité ou de la résistance d'un échantillon à un inhibiteur de Wee1, entre autres utilisations. La présente invention porte également sur un procédé pour la prédiction d'une réponse à un traitement thérapeutique anticancéreux avec un inhibiteur de Wee1 par la détermination des niveaux d'expression de l'ensemble de gènes biomarqueurs.
PCT/JP2009/071849 2009-01-05 2009-12-22 Biomarqueurs prédictifs utiles pour une thérapie anticancéreuse à médiation par un inhibiteur de wee1 WO2010076887A1 (fr)

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CN114032339A (zh) * 2021-12-22 2022-02-11 湖南工程学院 一种检测鼻咽癌的超支化杂交链式反应信号放大体系、试剂盒和检测方法
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WO2022271731A1 (fr) * 2021-06-23 2022-12-29 Recurium Ip Holdings, Llc Inhibiteurs de wee1 et méthodes de traitement du cancer

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111315747A (zh) * 2018-01-05 2020-06-19 四川科伦博泰生物医药股份有限公司 二氢吡唑酮并嘧啶类化合物及其制备方法和用途
CN111315747B (zh) * 2018-01-05 2023-05-02 四川科伦博泰生物医药股份有限公司 二氢吡唑酮并嘧啶类化合物及其制备方法和用途
WO2022136916A1 (fr) * 2020-12-22 2022-06-30 Recurium Ip Holdings, Llc Inhibiteurs de wee1 et méthodes de traitement du cancer
WO2022271731A1 (fr) * 2021-06-23 2022-12-29 Recurium Ip Holdings, Llc Inhibiteurs de wee1 et méthodes de traitement du cancer
CN114032339A (zh) * 2021-12-22 2022-02-11 湖南工程学院 一种检测鼻咽癌的超支化杂交链式反应信号放大体系、试剂盒和检测方法
CN114032339B (zh) * 2021-12-22 2023-09-01 湖南工程学院 一种检测鼻咽癌的超支化杂交链式反应信号放大体系、试剂盒和检测方法

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