Title:
Methods for Treating Cancer with a WEE1 Inhibitor
Kind Code:
A1


Abstract:
The present invention relates generally to the use of gene mutations, whose presence or absence are useful for predicting a patient's response to treatment with an anti-proliferative agent, in particular a WEE1 inhibitor. The presence or absence of a mutation to the TP53 gene, can be used to predict response to treatment with a WEE1 inhibitor in a patient presenting with a cancerous condition.



Inventors:
Marton, Matthew J. (Whitehouse Station, NJ, US)
Benita, Yair (Tel Aviv, IL)
Application Number:
15/314650
Publication Date:
04/06/2017
Filing Date:
05/26/2015
Assignee:
Merck Sharp & Dohme Corp. (Rahway, NJ, US)
Primary Class:
International Classes:
A61K31/519; A61K31/337; A61K31/555; C12Q1/68
View Patent Images:
Related US Applications:



Foreign References:
WO2011027800A12011-03-10
Primary Examiner:
PODGORSKI, DANIEL MICHAEL
Attorney, Agent or Firm:
MERCK (P O BOX 2000 RAHWAY NJ 07065-0907)
Claims:
1. A method of treating cancer in a patient comprising the steps of: 1) selecting a patient diagnosed with cancer that has one or more TP53 gene mutations according to Table 3, or at least one basepair insertion or deletion in the TP53 gene that causes a frameshift in encoding the p53 protein resulting in loss of function, or a combination thereof in the cancer cell; 2) administering a therapeutically effective amount of a WEE1 inhibitor and optionally one or more additional anti-cancer agents to the patient.

2. A method of treating cancer in a patient, in which the patient is diagnosed with cancer and has one or more TP53 gene mutations according to Table 3, or at least one basepair insertion or deletion in the TP53 gene that causes a frameshift in encoding the p53 protein resulting in loss of function, or a combination thereof in the cancer cell; comprising the step of administering a therapeutically effective amount of a WEE1 inhibitor and optionally one or more additional anti-cancer agents to the patient.

3. A method of treating cancer in a patient, comprising the step of administering a therapeutically effective amount of a WEE1 inhibitor and optionally one or more additional anti-cancer agents to the patient, wherein the patient is diagnosed with cancer and has one or more TP53 gene mutations according to Table 3, or at least one basepair insertion or deletion in the TP53 gene that causes a frameshift in encoding the p53 protein resulting in loss of function, or a combination thereof in the cancer cell.

4. The method of claim 1, wherein the TP53 gene mutation has an evidence score that is equal or greater than 2.5 according to Table 3.

5. The method of claim 1, wherein the TP53 gene mutation has an evidence score that is equal or greater than 3 according to Table 3.

6. The method of claim 1, wherein the TP53 gene mutation has an evidence score that is equal or greater than 3.5 according to Table 3.

7. The method of claim 1, wherein the TP53 gene mutation has an evidence score that is equal or greater than 4.0 according to Table 3.

8. The method of claim 1, wherein the TP53 gene mutation has an evidence score that is equal or greater than 4.5 according to Table 3.

9. The method of claim 1, wherein the TP53 gene mutation has an evidence score that is equal to 5.0 according to Table 3.

10. The method of claim 1, wherein the patient has at least one of the TP53 gene mutations resulting in the amino acid change selected from the group consisting of C238F, R248W and R273L according to Table 3, a stop codon at the codon encoding E298 in the p53 protein, or a deletion of a basepair in the codon encoding V157 in the p53 protein.

11. The method of claim 1, wherein the WEE1 inhibitor is embedded image or a pharmaceutically acceptable salt thereof.

12. The method of claim 1, wherein the WEE1 inhibitor is embedded image or a pharmaceutically acceptable salt thereof.

13. The method of claim 1, wherein the cancer is selected from the group consisting of ovarian cancer, melanoma, lung cancer, colorectal cancer, colon cancer, rectum cancer, prostate cancer, and breast cancer.

14. The method of claim 1, wherein the cancer is ovarian cancer.

15. The method of claim 1, wherein the cancer is lung cancer.

16. The method of claim 1, wherein the anti-cancer agent is selected from the group consisting of: 5-FU, carboplatin, doxorubicin, etoposide, gemcitabine, irinotecan, mitomycin, temozolomide and topotecan.

17. The method of claim 1, wherein the anti-cancer agent is carboplatin and paclitaxel.

Description:

FIELD OF THE INVENTION

The present invention relates generally to the use of gene mutations, whose presence or absence are useful for predicting a patient's response to treatment with an anti-proliferative agent, in particular a WEE1 inhibitor. The presence or absence of a mutation to the TP53 gene, can be used to predict response to treatment with a WEE1 inhibitor in a patient presenting with a cancerous condition.

BACKGROUND OF THE INVENTION

Many commonly used anti-cancer drugs indiscriminately target DNA in dividing cells and ultimately cause DNA damage. This, in turn, triggers activation of cell cycle checkpoints which arrest progression of the cell cycle (at the G1, S, or G2/M phases) with the purpose of allowing time for the DNA to be repaired before the cell undergoes DNA replication or division. From a therapeutic standpoint, inhibition of checkpoint kinases that mediate cell cycle arrest could force tumor cells to continue cell division before chemically-induced DNA damage is repaired, eventually causing apoptosis or mitotic catastrophe (Medema, R. H. and Macurek, L., Oncogene, 2012, 31(21):2601-2613). Cell line studies support this hypothesis and show chemosensitization and radiosensitization by pharmacologic or genetic disruption of checkpoint kinase activity including CHK1, WEE1, ATR, and ATM. Inhibitors against these kinases are at various stages of preclinical and clinical development for their ability to sensitize tumor cells to therapeutic DNA damage.

The checkpoint kinase WEE1 catalyzes an inhibitory phosphorylation of both CDK1 (CDC2) and CDK2 on tyrosine 15 (Parker, L. L. and Piwnica-Worms, H., Science, 1992, 257(5078):1955-1957; Watanabe, N., et al., Embo J., 1995, 14(9):1878-1891). WEE1-dependent inhibition of CDK1 and CDK2 arrests the cell cycle in response to extrinsically induced DNA damage (Hamer, P. C. D., et al., Clin. Cancer Res., 2011, 17(13):4200-4207). WEE1 activity is also essential for the unperturbed cell cycle (Mcgowan, C. H. and Russell, P., Embo J., 1993, 12(1):75-85; Tominaga, Y., et al., Intl. J. Biol. Sci., 2006, 2(4):161-170). Cell synchronization studies in normal human fibroblasts revealed that similar amounts of WEE1 protein were detected in both S and G2/M phases, but that its greatest activity was in S phase of the cell cycle (Watanabe, N., 1995). Further, upon conditional WEE1 knockout in mouse embryonic fibroblasts (MEFs), cells show evidence of genomic instability, malfunctioning checkpoints, and premature mitosis (Tominaga, et al., 2006). This phenotype was explained in part by recent findings that demonstrate a critical role for WEE1 in DNA synthesis. Knockdown of WEE1, in the absence of DNA damaging agents, led to rapid and robust detection of DNA double strand breaks specifically in S-phase cells undergoing DNA replication (Beck, H., et al., J. Cell Biol., 2010, 188(5):629-638; Dominguez-Kelly, R., et al., J. Cell Biol., 2011, 194(4):567-579). Data support a model of WEE1-dependent genomic stability in which WEE1 knockdown or inhibition leads to aberrantly high activity of CDK 1 and 2, resulting in inappropriately timed firing of excessive DNA replication origins that quickly depletes nucleotide pools and leads to stalled replication forks which, in the absence of WEE1 activity, are substrates for DNA exonucleases and resolve into DNA doubles strand breaks (Beck, H., et al., 2012).

Deregulated WEE1 expression or activity is believed to be a hallmark of pathology in several types of cancer. WEE1 is often overexpressed in glioblastomas and its activity protects this tumor type from mitotic catastrophe such that high WEE1 levels are associated with poor prognosis (Mir, S. E., et al., Cancer Cell, 2010, 18(3):244-257). High expression of WEE1 was found in malignant melanoma and correlated with poor disease-free survival in this population (Magnussen, G. I., et al., Plos One, 2012, 7(6)). Aberrant WEE1 expression has been implicated in additional tumor types such as hepatocellular carcinoma (Masaki, T., et al., Hepatology, 2003, 37(3):534-543), breast cancer (Iorns, E., et al., Plos One, 2009, 4(4)), colon carcinoma (Backert, S., et al., Intl., J. Cancer, 1999, 82(6):868-874)), lung carcinoma (Yoshida, T., et al., Annals of Oncology, 2004, 15(2):252-256) and head and neck squamous cell carcinoma (Wu, Z. X., et al., Mol. &Cell. Proteomics, 2011, 10(12)). Advanced tumors with an increased level of genomic instability may require functional checkpoints to allow for repair of such lethal DNA damage. As such, WEE1 represents an attractive target in advanced tumors where its inhibition is believed to result in irreparable DNA damage (Sorensen, C. S. and Syljuasen, R. G., Nuc. Acids Res., 2012, 40(2):477-486).

The TP53 gene, which encodes the p53 protein, is an important regulator of the cell-cycle as a key regulator of the G1 checkpoints and is one of the most frequently mutated genes in cancer (Molinari, M., Cell. Prolif., 2000, 33:261-174). Cells lacking the G1 checkpoint are predicted to be more dependent on the WEE1-mediated S or G2 checkpoint. Thus, p53-deficient tumors treated with G2 checkpoint abrogators may be particularly susceptible to DNA damage (Kawabe, T., Mol. Cancer Ther., 2004, 3:513-519; Bucher, N., and Britten, C. D., Br. J. Cancer, 2008, 98:523-528).

There is a need for biomarkers that can be used to predict which patients are amenable to treatment with specific therapies, particularly for patients who are non-responsive or who are likely to become refractive to first line therapies. It is, therefore, an object of this invention to provide biomarkers to select patients likely to respond to treatment with a WEE1 inhibitor.

SUMMARY OF THE INVENTION

The instant invention relates generally to the identification of TP53 gene mutations whose presence or absence are useful for evaluating and classifying patients for treatment with a WEE1 inhibitor. In one embodiment of the invention, the TP53 gene mutations resulting in loss of function are used to identify patients likely to respond to treatment with a WEE1 inhibitor. In another embodiment, the invention is a method for treating a patient diagnosed with a WEE1 associated cancer with a WEE1 inhibitor, wherein the cancer cells of said patient are characterized by the presence of a mutation in TP53 which renders p53 non-functional. In still another embodiment, the invention is a method for treating a cancer patient who is responsive or predicted to be responsive to treatment with a WEE1 inhibitor, wherein the cancer cells of said patient are characterized by the presence of a mutation in TP53 which renders p53 non-functional.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Effect of TP53 mutation status on the degree of synergy observed between WEE1-1 and various DNA-damaging agents.

DETAILED DESCRIPTION OF THE INVENTION

Many anti-cancer treatments act by damaging DNA, which subsequently initiates the DNA damage response (DDR) and activates checkpoint kinases to arrest division while the DNA is repaired. WEE1, a tyrosine kinase, is activated by the DDR to phosphorylate and inhibit cyclin dependent kinases (CDKs) 1 and 2 and, as such, arrest cell division. Inhibiting WEE1 potentiates DNA damaging treatments by abrogating cell cycle arrest and proper DNA repair.

WEE1-1, also known as 2-allyl-1-[6-(1-hydroxy-1-methylethyl)pyridin-2-yl]-6-{[4-(4-methylpiperazin-1-yl)phenyl]amino}-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-on e, is a potent (IC50=5.2 nM) and selective ATP-competitive small molecule inhibitor of WEE1 (Hirai, H., et al., Mol. Cancer Ther., 2009, 8(11):2992-3000) that is currently under clinical development as an anti-tumor agent in combination with standard of care (SOC) chemotherapeutics (Stathis, A. and Oza A., Drug News &Perspectives, 2010, 23(7):425-429; Schellens, J. H. M., et al., J. Clin. Oncol., 2011, 29:2011 (suppl; abstr 3068); Mizuarai, S., et al., Mol. Cancer, 2009, 8:34). Previous studies on WEE1-1 have demonstrated its potential as an adjunct or sensitizer to currently used standard of care (SOC) chemotherapeutics by its ability to force unscheduled mitosis that ultimately results in apoptosis or mitotic catastrophe (Hirai, H., et al., Cancer Biol. &Ther., 2010, 9(7):514-522; Aarts, M., et al., Cancer Discovery, 2012, 2(6):524-539; Indovina, P. and Giordano A., Cancer Biol. &Ther., 2010, 9(7); 523-525; Wang, Y. L., et al., Cancer Biol. &Ther., 2004, 3(3):305-313). However, the potential therapeutic effect of WEE1 inhibition in the absence of SOC chemotherapy is less defined. RNAi knockdown of WEE1 inhibited proliferation of cancer cell lines (Iorns, E., et al., Cancer Targets, 2009, Plos One, 4(4); Murrow, L. M., et al., Breast Cancer Research and Treatment, 2010, 122(2):347-357) and recently it was demonstrated that WEE1-1 alone can induce apoptosis in sarcoma cell lines treated in vitro (Kreahling, J. M., et al., Mol. Cancer Ther., 2012, 11(1):174-182).

The p53 protein is encoded by the TP53 gene. Pre-clinical studies have suggested that WEE1-1 may be selectively effective in patients having p53-defective tumors, that is, in patients whose tumors harbor mutations in TP53 which render p53 non-functional. While over 25,000 mutations in the TP53 gene have been reported, not every mutation is expected to lead to a loss of function. Moreover, even as to mutations associated with loss of function, all are not equal in their ability to extinguish the functionality of p53.

Applicants herein have developed a method to identify a subset of loss of function mutations, referred to as a “p53 filter”, by assigning to each type of mutation (see Table 1) a point value related to the likelihood that the mutation results in loss of function of p53, and in one embodiment, sensitivity to treatment with a WEE1 inhibitor, such as WEE1-1. As shown in Table 3, the evidence score of each gene mutation was calculated based on adding together the point values for the mutation: stop codon mutation, splice site mutation, dominant negative mutation, p53 signature p value <0.05, mutation reported >10× times in somatic tissue at amino acid level, mutation reported >10× in somatic tissue at nucleotide level. The TP53 mutation score of a patient is calculated by identifying the various gene mutations in Table 3 in the cancer cell of the patient, and adding together the evidence score of each identified gene mutation.

Thus, in one embodiment of the invention, patients whose TP53 mutation score is 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 or greater according to Table 3 are identified as those patients most likely to respond to treatment with a WEE1 inhibitor and are selected for treatment with a WEE1 inhibitor. In another embodiment of the invention, patients treated with a WEE1 inhibitor whose TP53 mutation score is 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 or greater according to Table 3 are identified as patients most likely to continue to respond to treatment and are selected to continue treatment with a WEE1 inhibitor.

The GENBANK accession number of the p53 protein is NM000546. The GENBANK accession number of the p53 gene is X54156.1. The TP53 gene sequence is also available from the IARC database (http://p53.iarc.fr). While different databases may use different nucleotide or amino acid numbering systems, based on the nucleotide and amino acid information in Table 3, one skilled in the art can readily identify the gene mutation and its location in the p53 gene.

The present invention provides a method of treating cancer or modulating WEE1 activity in a patient comprising the step of:

    • 1) selecting a patient diagnosed with cancer that has one or more TP53 gene mutations according to Table 3, at least one basepair insertion or deletion in the TP53 gene that causes a frameshift in encoding the p53 protein resulting in loss of function, or a combination thereof in the cancer cell;
    • 2) administering a therapeutically effective amount of a WEE1 inhibitor and optionally one or more additional anti-cancer agents to the patient.

The present invention also provides a method of treating cancer or modulating WEE1 activity in a patient, in which the patient is diagnosed with cancer and has one or more TP53 gene mutations according to Table 3, at least one basepair insertion or deletion in the TP53 gene that causes a frameshift in encoding the p53 protein resulting in loss of function, or a combination thereof in the cancer cell; comprising the step of administering a therapeutically effective amount of a WEE1 inhibitor and optionally one or more additional anti-cancer agents to the patient.

The present invention further provides a method of treating cancer or modulating WEE1 activity in a patient, comprising the step of administering a therapeutically effective amount of a WEE1 inhibitor and optionally one or more additional anti-cancer agents to the patient, wherein the patient is diagnosed with cancer and has one or more TP53 gene mutations according to Table 3, at least one basepair insertion or deletion in the TP53 gene that causes a frameshift in encoding the p53 protein resulting in loss of function, or a combination thereof in the cancer cell. In one embodiment, the invention provides a WEE1 inhibitor for use in the treatment of cancer or modulating WEE1 activity in a patient, wherein the patient is diagnosed with cancer and has one or more TP53 gene mutations according to Table 3, or at least one basepair insertion or deletion in the TP53 gene that causes a frameshift in encoding the p53 protein resulting in loss of function, or a combination thereof in the cancer cell, and the treatment optionally comprises one or more additional anti-cancer agents.

In one embodiment, the at least one basepair insertion or deletion in the TP53 gene that causes a frameshift in encoding the p53 protein completely eliminates the DNA binding domain of p53.

In one embodiment, the TP53 gene mutation has an evidence score that is equal or greater than 2.5 according to Table 3. In another embodiment, the TP53 gene mutation has an evidence score that is equal or greater than 3 according to Table 3. In another embodiment, the TP53 gene mutation has an evidence score that is equal or greater than 3.5 according to Table 3. In a further embodiment, the TP53 gene mutation has an evidence score that is equal or greater than 4.0 according to Table 3. In yet a further embodiment, the TP53 gene mutation has an evidence score that is equal or greater than 4.5 according to Table 3. In yet another embodiment, the TP53 gene mutation has an evidence score that is equal to 5.0 according to Table 3. In yet another embodiment, the patient has at least one of the TP53 gene mutations resulting in the amino acid change selected from the group consisting of C238F, R248W and R273L according to Table 3, a stop codon at the codon encoding E298 in the p53 protein, or a deletion of a basepair in the codon encoding V157 in the p53 protein.

In another embodiment of the above method, step 1) is selecting a patient diagnosed with cancer that has two or more TP53 gene mutations according to Table 3, or three or more TP53 gene mutations according to Table 3 in the cancer cell.

In one embodiment of the invention, the AmpliChip p53 Assay (Roche Molecular Systems, Inc., Pleasanton, Calif.) is used to identify the TP53 mutation present in the samples from patients diagnosed with a TP53 associated cancer. The AmpliChip p53 test is a microarray-based resequencing test. The test is designed to detect single nucleotide substitutions and lbp deletions in the entire coding region and the flanking splice sites of exons 2-11 of the TP53 gene in either formalin-fixed paraffin-embedded tissue (FFPE) or freshly frozen tissue. The AmpliChip p53 test queries for the presence of sequence alterations through comparative analysis of the hybridization pattern of a series of probes to sample DNA and wild-type reference DNA. The highly redundant probe tiling approach is able to detect a significantly lower abundance of TP53 mutations in samples which contain mixtures of normal and tumor tissue without the need for microdissection. See Li et al. Current Genomics, 2008, 9, 466-474.

Those skilled in the art would recognize and appreciate that other methods could be employed to identify the gene mutation present, such as Sanger sequencing, massively parallel sequencing (Next-Generation Sequencing), mass spectrometry, or PCR techniques. The inventive method herein is not tied to the method used to identify the specific p53 loss-of-function mutation or the absence of any such mutation.

WEE1 Inhibitors

In an embodiment of the invention, the WEE1 inhibitor for use in the methods of the instant invention is WEE1-1, the structure of which is as shown below.

embedded image

WEE1-1 is a WEE1 inhibitor which is useful for the treatment of cancer. WEE1-1 is also known as 2-allyl-1-[6-(1-hydroxy-1-methylethyl)pyridin-2-yl]-6-{[4-(4-methylpiperazin-1-yl)phenyl]amino}-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-on e. WEE1-1 has been described in U.S. Pat. No. 7,834,019, and in PCT International Publication WO2007/126122, WO 2007/126128 and WO2008/153207, which are incorporated by reference herein in their entirety. Crystalline forms of WEE1-1 are described in US Publication US2010-0124544 and PCT International Publication WO2011/034743, which are incorporated by reference herein in their entirety.

In an embodiment of the invention, the WEE1 inhibitor for use in the instant invention is WEE1-2, the structure of which is as shown below.

embedded image

WEE1-2 is a WEE1 inhibitor which is useful for the treatment of cancer. WEE1-2 is also known as 3-(2,6-dichlorophenyl)-4-imino-7-[(2′-methyl-2′,3′-dihydro-1′H-spiro[cyclopropane-1,4′-isoquinolin]-7′-yl)amino]-3,4-dihydropyrimido[4,5-d]p yrimidin-2(1H)-one. WEE1-2 has been described in PCT International Publication WO2008/153207 and US Publication US2011-0135601, which are incorporated by reference herein in their entirety. Crystalline forms of WEE1-2 are described in International Publication WO2009/151997 and US Publication US2011-0092520.

In one embodiment, the WEE1 inhibitor is

embedded image

or a pharmaceutically acceptable salt thereof.
In another embodiment, the WEE1 inhibitor is

embedded image

or a pharmaceutically acceptable salt thereof.

The compounds used in the methods of the present invention may have asymmetric centers, chiral axes, and chiral planes (as described in: E. L. Eliel and S. H. Wilen, Stereochemistry of Carbon Compounds, John Wiley & Sons, New York, 1994, pages 1119-1190), and occur as racemates, racemic mixtures, and as individual diastereomers, with all possible isomers and mixtures thereof, including optical isomers, all such stereoisomers being included in the present invention. In addition, the compounds disclosed herein may exist as tautomers and both tautomeric forms are intended to be encompassed by the scope of the invention, even though only one tautomeric structure is depicted.

In the compounds used in the methods of the present invention, the atoms may exhibit their natural isotopic abundances, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature. The present invention is meant to include all suitable isotopic variations of the compounds disclosed herein. For example, different isotopic forms of hydrogen (H) include protium (1H) and deuterium (2H). Protium is the predominant hydrogen isotope found in nature. Enriching for deuterium may afford certain therapeutic advantages, such as increasing in vivo half-life or reducing dosage requirements, or may provide a compound useful as a standard for characterization of biological samples. Isotopically-enriched compounds disclosed herein can be prepared without undue experimentation by conventional techniques well known to those skilled in the art.

The WEE1 inhibitors used in the methods of the instant invention may also exist as various crystals, amorphous substances, pharmaceutically acceptable salts, hydrates and solvates. Further, the WEE1 inhibitors of the instant invention may be provided as prodrugs. In general, such prodrugs are functional derivatives of the WEE1 inhibitors of the instant invention that can be readily converted into compounds that are needed by living bodies. Accordingly, in the method of treatment of various cancers in the invention, the term “administration” includes not only the administration of a specific compound but also the administration of a compound which, after administered to patients, can be converted into the specific compound in the living bodies. Conventional methods for selection and production of suitable prodrug derivatives are described, for example, in “Design of Prodrugs”, ed. H. Bundgaard, Elsevier, 1985, which is referred to herein and is entirely incorporated herein as a part of the present description. Metabolites of the compound may include active compounds that are produced by putting the compound in a biological environment, and are within the scope of the compound in the invention.

In one embodiment of the invention, the WEE1 inhibitor is administered in a dose between 100 mg per day and 250 mg per day. In another embodiment of the invention, the WEE1 inhibitors may be dosed twice a day (BID) over the course of two and a half days (for a total of 5 doses) or once a day (QD) over the course of two days (for a total of 2 doses).

In another embodiment of the invention, the WEE1 inhibitor is administered in a dose between 200 mg per day and 400 mg per day, and preferably 250-350 mg per day. In an embodiment of the invention, the WEE1 inhibitors may be dosed once a day (QD) over the course of five days.

Method of Treating Cancer

Cancers that may be treated by the WEE1 inhibitors include, but are not limited to: Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma) colorectal; Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus (endometrial carcinoma), cervix (cervical carcinoma, pre tumor cervical dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes (carcinoma), breast; Hematologic: blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma]; Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis; and Adrenal glands: neuroblastoma.

The term “WEE1 kinase associated cancer” as referred to in this description means a cancer associated with the activity or inhibition of WEE1 kinases including, but not limited to, brain cancer, cervicocerebral cancer, esophageal cancer, thyroid cancer, small cell cancer, non-small cell cancer, breast cancer, lung cancer, stomach cancer, gallbladder/bile duct cancer, liver cancer, pancreatic cancer, colon cancer, rectal cancer, ovarian cancer, choriocarcinoma, uterus body cancer, uterocervical cancer, renal pelvis/ureter cancer, bladder cancer, prostate cancer, penis cancer, testicles cancer, fetal cancer, Wilms' cancer, skin cancer, malignant melanoma, neuroblastoma, osteosarcoma, Ewing's tumor, soft part sarcoma, acute leukemia, chronic lymphatic leukemia, chronic myelocytic leukemia, Hodgkin's lymphoma, or as sensitizers for chemo therapy or radiation therapy of those cancers. In particular, the WEE1 inhibitor of the invention are useful as remedies, for example, for breast cancer, lung cancer, pancreatic cancer, colon cancer, ovarian cancer, acute leukemia, chronic lymphatic leukemia, chronic myelocytic leukemia, Hodgkin's lymphoma, or as sensitizers for chemotherapy or radiation therapy of those cancers.

In another embodiment, the cancer is selected from the group consisting of ovarian cancer, melanoma, lung cancer, colorectal cancer, colon cancer, rectum cancer, prostate cancer, and breast cancer.

In a further embodiment, the cancer is selected from the group consisting of ovarian carcinoma, ovary clear cell carcinoma, ovary adenocarcinoma, ovary teratocarcinoma, skin malignant melanoma, malignant melanoma, lung carcinoma, large cell lung cancer, lung adenocarcinoma, non-small cell lung cancer, lung squamous cell carcinoma, colorectal carcinoma, colorectal adenocarcinoma, colon carcinoma, rectum adenocarcinoma, prostate carcinoma, breast ductal carcinoma and breast cancer.

In yet another embodiment, the cancer is ovarian cancer. In a further embodiment, the cancer is lung cancer.

In another embodiment of the invention, a method of inhibiting or modulating WEE1 activity in a patient is provided.

The term “treatment of cancer” as referred to in this description means that an anti-cancer agent is administered to a cancer patient so as to inhibit the growth of the cancer cells in the patient.

The term “patient” or “subject” as referred to in this description means the recipient in need of medical intervention or treatment. Mammalian and non-mammalian patients or subjects are included.

Combination Therapy

Combinations of the WEE1 inhibitors with therapeutic, chemotherapeutic and anti-cancer agents in the methods of the invention are within the scope of the invention. The WEE1 inhibitors may also be administered in combination with one or more additional anti-cancer agents, wherein the amounts of the WEE1 inhibitor and the anti-cancer agent result in a therapeutic effect. Examples of such agents can be found in Cancer Principles and Practice of Oncology by V. T. Devita and S. Hellman (editors), 6th edition (Feb. 15, 2001), Lippincott Williams & Wilkins Publishers. A person of ordinary skill in the art would be able to discern which combinations of agents would be useful based on the particular characteristics of the drugs and the cancer involved. Such agents include the following: estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxic/cytostatic agents, antiproliferative agents, prenyl-protein transferase inhibitors, HMG-CoA reductase inhibitors and other angiogenesis inhibitors, HIV protease inhibitors, reverse transcriptase inhibitors, inhibitors of cell proliferation and survival signaling, bisphosphonates, aromatase inhibitors, siRNA therapeutics, γ-secretase inhibitors, agents that interfere with receptor tyrosine kinases (RTKs) and agents that interfere with cell cycle checkpoints. The WEE1 inhibitors may also be useful when co-administered with radiation therapy. The WEE1 inhibitors can be present in the same dosage unit as the anticancer agent or in separate dosage units.

Non-limiting examples of suitable anti-cancer agents include cytostatic agents, cytotoxic agents, targeted therapeutic agents (small molecules, biologics, siRNA and microRNA) against cancer and neoplastic diseases as follows:

    • 1) anti-metabolites (such as methoxtrexate, 5-fluorouracil, gemcitabine, fludarabine, capecitabine);
    • 2) alkylating agents, such as temozolomide and cyclophosphamide,
    • 3) DNA interactive and DNA damaging agents, such as cisplatin, oxaliplatin and doxorubicin,
    • 4) Ionizing irradiation, such as radiation therapy,
    • 5) topoisomerase II inhibitors, such as etoposide and doxorubicin,
    • 6) topoisomerase I inhibitors, such as irinotecan and topotecan,
    • 7) tubulin interacting agents, such as paclitaxel, docetaxel, abraxane and epothilones,
    • 8) kinesin spindle protein inhibitors,
    • 9) spindle checkpoint inhibitors,
    • 10) poly(ADP-ribose) polymerase (PARP) inhibitors, such as olaparib, MK-4827 and veliparib,
    • 11) matrix metalloprotease (MMP) inhibitors,
    • 12) protease inhibitors, such as cathepsin D and cathepsin K inhibitors,
    • 13) proteosome or ubiquitination inhibitors, such as bortezomib,
    • 14) activators of mutant p53 to restore its wild-type p53 activity,
    • 15) adenoviral-p53,
    • 16) Bcl-2 inhibitors, such as ABT-263,
    • 17) heat shock protein (HSP) modulators, such as geldanamycin and 17-AAG,
    • 18) histone deacetylase (HDAC) inhibitors, such as vorinostat (SAHA),
    • 19) sex hormone modulating agents,
      • a. anti-estrogens, such as tamoxifen and fulvestrant,
      • b. selective estrogen receptor modulators (SERM), such as raloxifene,
      • c. anti-androgens, such as bicalutamide and flutamide,
      • d. LHRH agonists, such as leuprolide,
      • e. 5α-reductase inhibitors, such as finasteride,
      • f. cytochrome P450 C17 lyase (CYP450c17, also called 17α-hydroxylase/17,20 lysase) inhibitors, such as abiraterone acetate, VN/124-1 and TAK-700,
      • g. aromatase inhibitors, such as letrozole, anastrozole and exemestane,
    • 20) EGFR kinase inhibitors, such as geftinib, erlotinib and laptinib,
    • 21) dual erbB1 and erbB2 inhibitors, such as lapatinib,
    • 22) multi-targeted kinases (serine/threonine and/or tyrosine kinase) inhibitors,
    • a. ABL kinase inhibitors, imatinib, nilotinib and dasatinib,
      • b. VEGFR-1, VEGFR-2, PDGFR, KDR, FLT, c-Kit, Tie2, Raf, MEK and ERK inhibitors, such as sunitinib, sorafenib, vandetanib, pazopanib, PLX-4032, axitinib, PTK787 and GSK-1120212,
      • c. polo-like kinase inhibitors,
      • d. aurora kinase inhibitors,
      • e. JAK inhibitors,
      • f. c-MET kinase inhibitors,
      • g. cyclin-dependent kinase inhibitors, such as CDK1 and CDK2 inhibitor SCH 727965,
      • h. PI3K and mTOR inhibitors, such as GDC-0941, BEZ-235, BKM-120 and AZD-8055,
      • i. rapamycin and its analogs, such as temsirolimus, everolimus, and deforolimus
    • 23) and other anti-cancer (also know as anti-neoplastic) agents include but are not limited to ara-C, adriamycin, cytoxan, carboplatin, uracil mustard, clormethine, ifosfsmide, melphalan, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatine, vinblastine, vincristine, vindesine, vinorelbine, navelbine, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, teniposide, cytarabine, pemetrexed, idarubicin, mithramycin, deoxycoformycin, mitomycin-C, 1-asparaginase, teniposide, ethinylestradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, dromostanolone propionate, testolactone, megestrolacetate, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, flutamide medroxyprogesteroneacetate, toremifene, goserelin, carboplatin, hydroxyurea, amsacrine, procarbazine, mitotane, mitoxantrone, levamisole, drolloxafine, hexamethylmelamine, bexxar, zevalin, trisenox, profimer, thiotepa, altretamine, doxil, ontak, depocyt, aranesp, neupogen, neulasta and kepivance,
    • 24) farnesyl protein transferase inhibitors, such as, SARASAR™ (4-[2-[4-[(11R)-3,10-dibromo-8-chloro-6, 11-dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl-]-1-piperidinyl]-2-oxoethyl]-piperidinecarboxamide and tipifarnib,
    • 25) interferons, such as Intron A and Peg-Intron,
    • 26) anti-erbB1 antibodies, such as cetuximab and panitumumab,
    • 27) anti-erbB2 antibodies, such as trastuzumab,
    • 28) anti-CD52 antibodies, such as alemtuzumab,
    • 29) anti-CD20 antibodies, such as rituximab,
    • 30) anti-CD33 antibodies, such as gemtuzumab ozogamicin,
    • 31) anti-VEGF antibodies, such as avastin,
    • 32) TRIAL ligands, such as lexatumumab, mapatumumab, and AMG-655,
    • 33) anti-CTLA-4 antibodies, such as ipilimumab,
    • 34) antibodies against CTA1, CEA, CD5, CD19, CD22, CD30, CD44, CD44V6, CD55, CD56, EpCAM, FAP, MHCII, HGF, IL-6, MUC1, PSMA, TALE, TAG-72, TRAILR, VEGFR, IGF-2 and FGF, and
    • 35) anti-IGF-1R antibodies, such as dalotuzumab (MK-0646) and robatumumab (SCH 717454).

If formulated as a fixed dose such combination products employ the WEE1 inhibitor administered in the invention within the dosage range described herein and the other pharmaceutically active agent or treatment within its dosage range. The WEE1 inhibitor may also be administered sequentially with known anticancer or cytotoxic agents when a combination formulation is inappropriate. The invention is not limited in the sequence of administration; the WEE1 inhibitor may be administered either concurrent with, prior to or after administration of the known anticancer or cytotoxic agent. Such techniques are within the skills of the persons skilled in the art as well as attending physicians.

Accordingly, in an aspect, this invention includes combinations for use in the invention comprising an amount of a WEE1 inhibitor, or a pharmaceutically acceptable salt thereof, and an amount of one or more anti-cancer treatments and anti-cancer agents listed above or below wherein the amounts of the compounds/treatments result in potential therapeutic effect. In one embodiment, the anti-cancer agent is selected from the group consisting of: 5-FU, carboplatin, doxorubicin, etoposide, gemcitabine, irinotecan, mitomycin, temozolmide and topotecan. In another embodiment, the anti-cancer agent is carboplatin. In another embodiment, the anti-cancer agents are carboplatin and paclitaxel.

“Estrogen receptor modulators” refers to compounds that interfere with or inhibit the binding of estrogen to the receptor, regardless of mechanism. Examples of estrogen receptor modulators include, but are not limited to, tamoxifen, raloxifene, idoxifene, LY353381, LY117081, toremifene, fulvestrant, 4-[7-(2,2-dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate, 4,4′-dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone, and SH646.

“Androgen receptor modulators” refers to compounds which interfere or inhibit the binding of androgens to the receptor, regardless of mechanism. Examples of androgen receptor modulators include finasteride and other 5α-reductase inhibitors, nilutamide, flutamide, bicalutamide, liarozole, and abiraterone acetate.

“Retinoid receptor modulators” refers to compounds which interfere or inhibit the binding of retinoids to the receptor, regardless of mechanism. Examples of such retinoid receptor modulators include bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid, α-difluoromethylornithine, ILX23-7553, trans-N-(4′-hydroxyphenyl) retinamide, and N-4-carboxyphenyl retinamide.

“Cytotoxic/cytostatic agents” refer to compounds which cause cell death or inhibit cell proliferation primarily by interfering directly with the cell's functioning or inhibit or interfere with cell mitosis, including alkylating agents, tumor necrosis factors, intercalators, hypoxia activatable compounds, microtubule inhibitors/microtubule-stabilizing agents, inhibitors of mitotic kinesins, histone deacetylase inhibitors, inhibitors of kinases involved in mitotic progression, inhibitors of kinases involved in growth factor and cytokine signal transduction pathways, antimetabolites, biological response modifiers, hormonal/anti-hormonal therapeutic agents, haematopoietic growth factors, monoclonal antibody targeted therapeutic agents, topoisomerase inhibitors, proteosome inhibitors, ubiquitin ligase inhibitors, and aurora kinase inhibitors.

Examples of cytotoxic/cytostatic agents include, but are not limited to, platinum coordinator compounds, sertenef, cachectin, ifosfamide, tasonermin, lonidamine, carboplatin, altretamine, prednimustine, dibromodulcitol, ranimustine, fotemustine, nedaplatin, oxaliplatin, temozolomide, heptaplatin, estramustine, improsulfan tosilate, trofosfamide, nimustine, dibrospidium chloride, pumitepa, lobaplatin, satraplatin, profiromycin, cisplatin, irofulven, dexifosfamide, cis-aminedichloro(2-methyl-pyridine)platinum, benzylguanine, glufosfamide, GPX100, (trans, trans, trans)-bis-mu-(hexane-1,6-diamine)-mu-[diamine-platinum(II)]bis[diamine(chloro)platinum (II)]-tetrachloride, diarizidinylspermine, arsenic trioxide, 1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, zorubicin, idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin, pinafide, valrubicin, amrubicin, antineoplaston, 3′-deamino-3′-morpholino-13-deoxo-10-hydroxycarminomycin, annamycin, galarubicin, elinafide, MEN10755, 4-demethoxy-3-deamino-3-aziridinyl-4-methylsulphonyl-daunorubicin (see WO 00/50032).

Examples of proteosome inhibitors include but are not limited to lactacystin and MLN-341 (Velcade).

Examples of microtubule inhibitors/microtubule-stabilising agents include taxanes in general. Specific compounds include paclitaxel (Taxol®), vindesine sulfate, 3′,4′-didehydro-4′-deoxy-8′-norvincaleukoblastine, docetaxol (Taxotere®), rhizoxin, dolastatin, mivobulin isethionate, auristatin, cemadotin, RPR109881, BMS184476, vinflunine, cryptophycin, 2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl) benzene sulfonamide, anhydrovinblastine, N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-proline-t-butylamide, TDX258, the epothilones (see for example U.S. Pat. Nos. 6,284,781 and 6,288,237) and BMS188797.

Some examples of topoisomerase inhibitors are topotecan, hycaptamine, irinotecan, rubitecan, 6-ethoxypropionyl-3′,4′-O-exo-benzylidene-chartreusin, 9-methoxy-N,N-dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-(6H) propanamine, 1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:b,7]-indolizino[1,2b]quinoline-10,13(9H,15H)dione, lurtotecan, 7-[2-(N-isopropylamino)ethyl]-(20S)camptothecin, BNP1350, BNPI1100, BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxane, 2′-dimethylamino-2′-deoxy-etoposide, GL331, N-[2-(dimethylamino)ethyl]-9-hydroxy-5,6-dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxamide, asulacrine, (5a, 5aB, 8aa,9b)-9-[2-[N-[2-(dimethylamino)ethyl]-N-methylamino]ethyl]-5-[4-hydro0xy-3,5-dimethoxyphenyl]-5,5a,6,8,8a,9-hexohydrofuro(3′,4′:6,7)napht ho(2,3-d)-1,3-dioxol-6-one, 2,3-(methylenedioxy)-5-methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridinium, 6,9-bis[(2-aminoethyl)amino]benzo[g]isoguinoline-5,10-dione, 5-(3-aminopropylamino)-7,10-dihydroxy-2-(2-hydroxyethylaminomethyl)-6H-pyrazolo[4,5,1-de]acridin-6-one, N-[1-[2(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthen-4-ylmethyl]formamide, N-(2-(dimethylamino)ethyl)acridine-4-carboxamide, 6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c] quinolin-7-one, and dimesna.

Examples of inhibitors of mitotic kinesins, and in particular the human mitotic kinesin KSP, are described in Publications WO03/039460, WO03/050064, WO03/050122, WO03/049527, WO03/049679, WO03/049678, WO04/039774, WO03/079973, WO03/099211, WO03/105855, WO03/106417, WO04/037171, WO04/058148, WO04/058700, WO04/126699, WO05/018638, WO05/019206, WO05/019205, WO05/018547, WO05/017190, US2005/0176776. In an embodiment inhibitors of mitotic kinesins include, but are not limited to inhibitors of KSP, inhibitors of MKLP1, inhibitors of CENP-E, inhibitors of MCAK and inhibitors of Rab6-KIFL.

Examples of “histone deacetylase inhibitors” include, but are not limited to, SAHA, TSA, oxamflatin, PXD101, MG98 and scriptaid. Further reference to other histone deacetylase inhibitors may be found in the following manuscript; Miller, T. A. et al. J. Med. Chem. 46(24):5097-5116 (2003).

“Inhibitors of kinases involved in mitotic progression” include, but are not limited to, inhibitors of aurora kinase, inhibitors of Polo-like kinases (PLK; in particular inhibitors of PLK-1), inhibitors of bub-1 and inhibitors of bub-R1. An example of an “aurora kinase inhibitor” is VX-680.

“Antiproliferative agents” includes antisense RNA and DNA oligonucleotides such as G3139, ODN698, RVASKRAS, GEM231, and INX3001, and antimetabolites such as enocitabine, carmofur, tegafur, pentostatin, doxifluridine, trimetrexate, fludarabine, capecitabine, galocitabine, cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed, paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed, pemetrexed, nelzarabine, 2′-deoxy-2′-methylidenecytidine, 2′-fluoromethylene-2′-deoxycytidine, N-[5-(2,3-dihydro-benzofuryl)sulfonyl]-N′-(3,4-dichlorophenyl)urea, N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycero-B-L-manno-heptopyranosyl]adenine, aplidine, ecteinascidin, troxacitabine, 4-[2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b][1,4]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamic acid, aminopterin, 5-flurouracil, alanosine, 11-acetyl-8-(carbamoyloxymethyl)-4-formyl-6-methoxy-14-oxa-1,11-diazatetracyclo(7.4.1.0.0)-tetradeca-2,4,6-trien-9-yl acetic acid ester, swainsonine, lometrexol, dexrazoxane, methioninase, 2′-cyano-2′-deoxy-N4-palmitoyl-1-B-D-arabin furanosyl cytosine, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone and trastuzumab.

Examples of monoclonal antibody targeted therapeutic agents include those therapeutic agents which have cytotoxic agents or radioisotopes attached to a cancer cell specific or target cell specific monoclonal antibody. Examples include Bexxar.

“HMG-CoA reductase inhibitors” refers to inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase. Examples of HMG-CoA reductase inhibitors that may be used include but are not limited to lovastatin (MEVACOR®; see U.S. Pat. Nos. 4,231,938, 4,294,926 and 4,319,039), simvastatin (ZOCOR®; see U.S. Pat. Nos. 4,444,784, 4,820,850 and 4,916,239), pravastatin (PRAVACHOL®; see U.S. Pat. Nos. 4,346,227, 4,537,859, 4,410,629, 5,030,447 and 5,180,589), fluvastatin (LESCOL®; see U.S. Pat. Nos. 5,354,772, 4,911,165, 4,929,437, 5,189,164, 5,118,853, 5,290,946 and 5,356,896), atorvastatin (LIPITOR®; see U.S. Pat. Nos. 5,273,995, 4,681,893, 5,489,691 and 5,342,952) and cerivastatin (also known as rivastatin and BAYCHOL®; see U.S. Pat. No. 5,177,080). The structural formulas of these and additional HMG-CoA reductase inhibitors that may be used in the instant methods are described at page 87 of M. Yalpani, “Cholesterol Lowering Drugs”, Chemistry & Industry, pp. 85-89 (5 Feb. 1996) and U.S. Pat. Nos. 4,782,084 and 4,885,314. The term HMG-CoA reductase inhibitor as used herein includes all pharmaceutically acceptable lactone and open-acid forms (i.e., where the lactone ring is opened to form the free acid) as well as salt and ester forms of compounds which have HMG-CoA reductase inhibitory activity, and therefor the use of such salts, esters, open-acid and lactone forms is included within the scope of this invention.

“Prenyl-protein transferase inhibitor” refers to a compound which inhibits any one or any combination of the prenyl-protein transferase enzymes, including farnesyl-protein transferase (FPTase), geranylgeranyl-protein transferase type I (GGPTase-I), and geranylgeranyl-protein transferase type-II (GGPTase-II, also called Rab GGPTase).

Examples of prenyl-protein transferase inhibitors can be found in the following publications and patents: WO 96/30343, WO 97/18813, WO 97/21701, WO 97/23478, WO 97/38665, WO 98/28980, WO 98/29119, WO 95/32987, U.S. Pat. No. 5,420,245, U.S. Pat. No. 5,523,430, U.S. Pat. No. 5,532,359, U.S. Pat. No. 5,510,510, U.S. Pat. No. 5,589,485, U.S. Pat. No. 5,602,098, European Patent Publ. 0 618 221, European Patent Publ. 0 675 112, European Patent Publ. 0 604 181, European Patent Publ. 0 696 593, WO 94/19357, WO 95/08542, WO 95/11917, WO 95/12612, WO 95/12572, WO 95/10514, U.S. Pat. No. 5,661,152, WO 95/10515, WO 95/10516, WO 95/24612, WO 95/34535, WO 95/25086, WO 96/05529, WO 96/06138, WO 96/06193, WO 96/16443, WO 96/21701, WO 96/21456, WO 96/22278, WO 96/24611, WO 96/24612, WO 96/05168, WO 96/05169, WO 96/00736, U.S. Pat. No. 5,571,792, WO 96/17861, WO 96/33159, WO 96/34850, WO 96/34851, WO 96/30017, WO 96/30018, WO 96/30362, WO 96/30363, WO 96/31111, WO 96/31477, WO 96/31478, WO 96/31501, WO 97/00252, WO 97/03047, WO 97/03050, WO 97/04785, WO 97/02920, WO 97/17070, WO 97/23478, WO 97/26246, WO 97/30053, WO 97/44350, WO 98/02436, and U.S. Pat. No. 5,532,359. For an example of the role of a prenyl-protein transferase inhibitor on angiogenesis see European J. of Cancer, Vol. 35, No. 9, pp. 1394-1401 (1999).

“Angiogenesis inhibitors” refers to compounds that inhibit the formation of new blood vessels, regardless of mechanism. Examples of angiogenesis inhibitors include, but are not limited to, tyrosine kinase inhibitors, such as inhibitors of the tyrosine kinase receptors Flt-1 (VEGFR1) and Flk-1/KDR (VEGFR2), inhibitors of epidermal-derived, fibroblast-derived, or platelet derived growth factors, MMP (matrix metalloprotease) inhibitors, integrin blockers, interferon-α, interleukin-12, pentosan polysulfate, cyclooxygenase inhibitors, including nonsteroidal anti-inflammatories (NSAIDs) like aspirin and ibuprofen as well as selective cyclooxy-genase-2 inhibitors like celecoxib and rofecoxib (PNAS, Vol. 89, p. 7384 (1992); JNCI, Vol. 69, p. 475 (1982); Arch. Opthalmol., Vol. 108, p. 573 (1990); Anat. Rec., Vol. 238, p. 68 (1994); FEBS Letters, Vol. 372, p. 83 (1995); Clin, Orthop. Vol. 313, p. 76 (1995); J. Mol. Endocrinol., Vol. 16, p. 107 (1996); Jpn. J. Pharmacol., Vol. 75, p. 105 (1997); Cancer Res., Vol. 57, p. 1625 (1997); Cell, Vol. 93, p. 705 (1998); Intl. J. Mol. Med., Vol. 2, p. 715 (1998); J. Biol. Chem., Vol. 274, p. 9116 (1999)), steroidal anti-inflammatories (such as corticosteroids, mineralocorticoids, dexamethasone, prednisone, prednisolone, methylpred, betamethasone), carboxyamidotriazole, combretastatin A-4, squalamine, 6-O-chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin, troponin-1, angiotensin II antagonists (see Fernandez et al., J. Lab. Clin. Med. 105:141-145 (1985)), and antibodies to VEGF (see, Nature Biotechnology, Vol. 17, pp. 963-968 (October 1999); Kim et al., Nature, 362, 841-844 (1993); WO 00/44777; and WO 00/61186).

Other examples of angiogenesis inhibitors include, but are not limited to, endostatin, ukrain, ranpirnase, IM862, 5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-1-oxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate, acetyldinanaline, 5-amino-1-[[3,5-dichloro-4-(4-chlorobenzoyl)phenyl]methyl]-1H-1,2,3-triazole-4-carboxamide, CM101, squalamine, combretastatin, RPI4610, NX31838, sulfated mannopentaose phosphate, 7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolocarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]-bis-(1,3-naphthalene disulfonate), and 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone (SU5416).

Other therapeutic agents that modulate or inhibit angiogenesis and may also be used in combination with WEE1 inhibitors include agents that modulate or inhibit the coagulation and fibrinolysis systems (see review in Clin. Chem. La. Med. 38:679-692 (2000)). Examples of such agents that modulate or inhibit the coagulation and fibrinolysis pathways include, but are not limited to, heparin (see Thromb. Haemost. 80:10-23 (1998)), low molecular weight heparins and carboxypeptidase U inhibitors (also known as inhibitors of active thrombin activatable fibrinolysis inhibitor [TAFIa]) (see Thrombosis Res. 101:329-354 (2001)). TAFIa inhibitors have been described in U.S. Ser. Nos. 60/310,927 (filed Aug. 8, 2001) and 60/349,925 (filed Jan. 18, 2002).

“Agents that interfere with cell cycle checkpoints” refer to compounds that inhibit protein kinases that transduce cell cycle checkpoint signals, thereby sensitizing the cancer cell to DNA damaging agents. Such agents include inhibitors of ATR, ATM, the CHK11 and CHK12 kinases and cdk and cdc kinase inhibitors and are specifically exemplified by 7-hydroxystaurosporin, flavopiridol, CYC202 (Cyclacel) and BMS-387032.

“Agents that interfere with receptor tyrosine kinases (RTKs)” refer to compounds that inhibit RTKs and therefore mechanisms involved in oncogenesis and tumor progression. Such agents include inhibitors of c-Kit, Eph, PDGF, Flt3 and c-Met. Further agents include inhibitors of RTKs as described by Bume-Jensen and Hunter, Nature, 411:355-365, 2001.

“Inhibitors of cell proliferation and survival signalling pathway” refer to compounds that inhibit signal transduction cascades downstream of cell surface receptors. Such agents include inhibitors of serine/threonine kinases (including but not limited to inhibitors of Akt such as described in WO 02/083064, WO 02/083139, WO 02/083140, US 2004-0116432, WO 02/083138, US 2004-0102360, WO 03/086404, WO 03/086279, WO 03/086394, WO 03/084473, WO 03/086403, WO 2004/041162, WO 2004/096131, WO 2004/096129, WO 2004/096135, WO 2004/096130, WO 2005/100356, WO 2005/100344, US 2005/029941, US 2005/44294, US 2005/43361, 60/734,188, 60/652,737, 60/670,469), inhibitors of Raf kinase (for example PLX-4032), inhibitors of MEK (for example Arry-162, RO-4987655 and GSK-1120212), inhibitors of mTOR (for example AZD-8055, BEZ-235 and everolimus), and inhibitors of PI3K (for example GDC-0941, BKM-120).

As used above, “integrin blockers” refers to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to the αvβ3 integrin, to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to the αvβ5 integrin, to compounds which antagonize, inhibit or counteract binding of a physiological ligand to both the αvβ3 integrin and the αvβ5 integrin, and to compounds which antagonize, inhibit or counteract the activity of the particular integrin(s) expressed on capillary endothelial cells. The term also refers to antagonists of the αvβ6, αvβ8, α1β1, α2β1, α5β1, α6β1 and α6β4 integrins.

Some specific examples of tyrosine kinase inhibitors include N-(trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide, 3-[(2,4-dimethylpyrrol-5-yl)methylidenyl)indolin-2-one, 17-(allylamino)-17-demethoxygeldanamycin, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-[3-(4-morpholinyl)propoxyl]quinazoline, N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine, BIBX1382, 2,3,9,10,11,12-hexahydro-10-(hydroxymethyl)-10-hydroxy-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocin -1-one, SH268, genistein, STI571, CEP2563, 4-(3-chlorophenylamino)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidinemethane sulfonate, 4-(3-bromo-4-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, 4-(4′-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, SU6668, STI571A, N-4-chlorophenyl-4-(4-pyridylmethyl)-1-phthalazinamine, and EMD121974.

Combinations of the WEE1 inhibitor with PPAR-γ (i.e., PPAR-gamma) agonists and PPAR-δ (i.e., PPAR-delta) agonists may be useful in the treatment of certain malignancies. PPAR-γ and PPAR-δ are the nuclear peroxisome proliferator-activated receptors γ and δ. The expression of PPAR-γ on endothelial cells and its involvement in angiogenesis has been reported in the literature (see J. Cardiovasc. Pharmacol. 1998; 31:909-913; J. Biol. Chem. 1999; 274:9116-9121; Invest. Ophthalmol Vis. Sci. 2000; 41:2309-2317). More recently, PPAR-γ agonists have been shown to inhibit the angiogenic response to VEGF in vitro; both troglitazone and rosiglitazone maleate inhibit the development of retinal neovascularization in mice. (Arch. Ophthamol. 2001; 119:709-717). Examples of PPAR-γ agonists and PPAR-γ/α agonists include, but are not limited to, thiazolidinediones (such as DRF2725, CS-011, troglitazone, rosiglitazone, and pioglitazone), fenofibrate, gemfibrozil, clofibrate, GW2570, SB219994, AR-H039242, JTT-501, MCC-555, GW2331, GW409544, NN2344, KRP297, NP0110, DRF4158, NN622, GI262570, PNU182716, DRF552926, 2-[(5,7-dipropyl-3-trifluoromethyl-1,2-benzisoxazol-6-yl)oxy]-2-methylpropionic acid, and 2(R)-7-(3-(2-chloro-4-(4-fluorophenoxy) phenoxy)propoxy)-2-ethylchromane-2-carboxylic acid.

Another embodiment of the instant invention is the use of WEE1 inhibitors in combination with gene therapy for the potential treatment of cancer. For an overview of genetic strategies to treating cancer see Hall et al (Am. J. Hum. Genet. 61:785-789, 1997) and Kufe et al (Cancer Medicine, 5th Ed, pp 876-889, BC Decker, Hamilton 2000). Gene therapy can be used to deliver any tumor suppressing gene. Examples of such genes include, but are not limited to, p53, which can be delivered via recombinant virus-mediated gene transfer (see U.S. Pat. No. 6,069,134, for example), a uPA/uPAR antagonist (“Adenovirus-Mediated Delivery of a uPA/uPAR Antagonist Suppresses Angiogenesis-Dependent Tumor Growth and Dissemination in Mice,” Gene Therapy, August 1998; 5(8):1105-13), and interferon gamma (J. Immunol. 2000; 164:217-222).

The invention disclosed herein is exemplified by the following examples which should not be construed to limit the scope of the disclosure.

EXAMPLES

Example 1

Thirty-one cell lines were treated with various DNA-damaging agents with or without WEE1-1 in a cell growth assay. Immediately after cells were plated, each drug was added at four concentrations for a total of 16 treatment conditions per combination (4×4 dose grid). Ninety-six hours following treatment, cell growth was evaluated with CellTiter-Glo (Promega) in treated samples relative to vehicle (DMSO) treated samples. Synergy was quantitated as the predicted additive growth inhibition of the two drugs (using the Bliss additivity model) subtracted from the observed growth inhibition of the two drugs. Therefore, a larger positive net difference indicates greater synergy, a negative difference indicates antagonism, and values at or close to 0 indicate additivity predicted by the Bliss model. The scale is displayed in the upper left corner of FIG. 1; darkest grey denotes strong synergy, light grey denotes moderate synergy, and darker grey denotes values that are considered additive, not synergistic. Each column represents a different cell line; each row represents a pairing between WEE1-1 and different DNA-damaging agents. The cell line name is shown above the column and the tissue type from which the cell line was derived is at the bottom of the column. The 12 leftmost columns represent cell lines that are reported to be TP53 wildtype; the 19 rightmost columns represent cell lines that are reported to have mutations in TP53. Data are summarized in the table to the right. The p53+/+ column summarizes the percentage of p53+/+ cell lines that exhibited synergy in the assay (i.e., the number of darkest grey and light grey squares in a row divided by 12). The p53−/− column summarizes the percentage of p53−/− cell lines that exhibited synergy in the assay (i.e., the number of darkest grey and light grey squares in a row divided by 19). Overall, the percentage of p53 wildtype cell lines that exhibited synergy was 15/(12×9)=14%; the percentage of P53 mutant cell lines that exhibited synergy was 50/(19×9)=29%. Thus, in combination with DNA-damaging agents, WEE1-1 is more likely to lead to synergistic growth inhibition in cell lines defective in TP53.

Example 2

Identification of Loss of Function Mutations

A multiple-step approach to develop a list of mutations which would be most likely to result in a non-functional p53 protein was implemented. First, different types of mutations predicted to result in p53 loss-of-function were assigned a relative point value, as shown in the Table 1 below. For example, three points were assigned for any mutation that results in a truncation, frameshift or a splice site defect that would completely eliminate the DNA binding domain of p53, i.e., before amino acid 306. (A frameshift mutation is one that causes the ribosome to use a different reading frame on the mRNA. For example, frameshifts can occur from one or two basepair insertions or deletions). Second, published data in the International Agency for Research on Cancer (IARC) database (A. Petijean et al. Hum Mutat. 2007, 28(6):622-9) was used to assess the impact of each possible mutation on p53 function (dominant negative protein or transactivation function). Mutations shown to display dominant negative activity in a model system were assigned a point value of 2.0 points, while mutations reducing or eliminating the protein's transactivation function were assigned a value of 1 point. Third, data from an internal gene expression database and the Cell line Biomarker Discovery (CBD) expression database of 650 cancer cell lines was used to identify mutations in TP53 that resulted in gene expression patterns indicative of p53 loss-of-function. These mutations were assigned 1 point. Finally, mutational analysis of tumor samples reported to the IARC database was used to identify mutations that were observed in more than 10 somatic (i.e., tumor) samples, which were assigned a point value of 0.5 points.

TABLE 1
Type of MutationPoint Value
Stop codon mutation (truncated protein) prior to codon 3063
Splice site mutation between codons 97and 3753
Frameshift mutation3
Dominant negative mutation2
P53 signature score p-value < 0.05 vs WT1
Transactivation loss-of-function mutation1
Mutation reported >10 times in somatic tissue at0.5
nucleotide level
Mutation reported >10 times in somatic tissue at0.5
amino acid level

TABLE 2
Mutation Lookup Table
E2*K120*V143MA159DC176FR196PC229*C242SR249SC275GR282G
E3*C124*V143AA161DC176YV197GY234HC242*P250LC275YR282W
Q5*Y126*Q144*Y163NC176SE198*Y234CC242WI255FC275FR282P
S6*S127PL145QY163HC176*G199*Y234*G244SL257QC275SR282Q
E11*S127TL145PY163DC176WG199EY236DG244CL257PC275*R283H
Q16*S127FW146*Y163CP177SL201*Y236HG244RE258*C275WR283P
E17*S127YD148*Y163SP177HE204*Y236NG244DE258KA276PE285*
S20*S127CP151TY163*P177RY205DY236CG244VD259YC277YE285K
W23*K132*P151SK164*H179NY205SY236SG244AD259VC277FE285V
K24*K132QP151AK164EH179YY205CY236*G245SN263*C277*E286*
E28*K132EP151RQ165*H179RY205*M237IG245RL265PP278TE286K
L35*K132RP151HS166*H179LL206*C238RG245CG266*P278SE286Q
Q38*K132MP152SQ167*H179QR209*C238YG245DG266RP278AE286G
L43*K132TP152LH168YH179*R213*C238FG245VG266EP278HE286V
E51*K132NG154VH168RE180*R213QC238*G245AG266VP278LE286A
Q52*M133KT155PH168PC182*R213LN239DM246VR267WP278RE286D
W53*C135RT155NH168LS183*H214RN239SM246LR267PG279EE287*
E56*C135SR156PE171*S183*S215IN239*M246RF270CR280*N288D
E62*C135GV157FV173MQ192*V216MS240GN247IE271*R280GK291*
R65*C135YV157LV173LH193YV216LS241TN247TV272MR280TK292*
E68*C135FV157DV173GH193DV216ES241AR248WR273CR280KE294*
W91*C135SV157GV173EH193NV216GS241PR248GR273SR280IE298*
S94*C135*V157AR175GH193LY220NS241FR248QR273GR280SK305*
Q100*C135WR158GR175CH193PY220HS241CR248LR273HD281NR306*
K101*Q136*R158SR175HH193RY220DS241YR248PR273LD281YR337C
Y103*A138PR158PR175LL194FY220CC242SR249WR273PD281H
Q104*A138GR158HR175PL194RY220SC242RR249GV274FD281G
Y107*K139*R158LC176RI195FY220*C242GR249MV274AD281V
R110LC141YA159PC176SI195TE221*C242YR249KC275RD281A
L114*C141*A159VC176GR196*E224*C242FR249TC275SD281E
PLUS ANY MUTATIONS LABELED “FRAMESHIFT” OR “SPLICE DEFECT”
*Denotes stop codon; The GENBANK accession number of the p53 protein is NM000546. The GENBANK accession number of the TP53 gene is X54156.1

The TP53 mutations in the look-up table (Table 2) include those whose evidence score is 2.0 or greater. The expanded table of TP53 mutations (Table 3) shows the location and type of mutation, as well as the point value assigned to the mutation.

TABLE 3
Supporting evidence for mutations in the Mutation Lookup Table
P53
signa-Reported >10xReported >10x
Trans-P53turein somaticin somatic
WTPro-StopSpliceDominantactiva-Signa-p value <@ AA@ BaseEvi-
Co-Co-MutantteinCodonDefectNegativetionture0.05levelleveldence
NtNumdondonCodonChange(3 pts)(3 pts)(2 pts)(1 pt)P-Value(1 pt)(0.5 pt)(0.5 pt)Score
110092GAGTAGE2*Yes03
110123GAGTAGE3*Yes03
110185CAGTAGQ5*Yes13
110226TCATAAS6*Yes03
110226TCATGAS6*Yes03
1103611GAGTAGE11*Yes03
1105116CAGTAGQ16*Yes03
1105417GAATAAE17*Yes03
1106420TCATAAS20*Yes03
1106420TCATGAS20*Yes03
1107323TGGTAGW23*Yes03
1107423TGGTGAW23*Yes03
1107524AAATAAK24*Yes03
1120428GAATAAE28*Yes03
1133535TTGTAGL35*Yes03
1134338CAATAAQ38*Yes33
1135943TTGTAGL43*Yes33
1138251GAATAAE51*Yes63
1138552CAATAAQ52*Yes83
1138953TGGTAGW53*Yes73
1139053TGGTGAW53*Yes103
1139756GAATAAE56*Yes63
1141562GAATAAE62*Yes93
1142465AGATGAR65*Yes33
1143368GAGTAGE68*Yes63
1150391TGGTAGW91*Yes133.5
1150491TGGTGAW91*Yes123.5
1151294TCATAAS94*Yes23
1151294TCATGAS94*Yes23
11529100CAGTAGQ100*Yes163.5
11532101AAATAAK101*Yes13
11540103TACTAAY103*Yes33
11540103TACTAGY103*Yes13
11541104CAGTAGQ104*Yes163.5
11552107TACTAAY107*Yes43
11552107TACTAGY107*Yes53
11560110CGTCTTR110LNon1.00E−0118302
functional
11572114TTGTAGL114*Yes33
11589120AAGTAGK120*Yes23
11603124TGCTGAC124*Yes13
12366126TACTAAY126*Yes123.5
12366126TACTAGY126*Yes133.5
12367127TCCCCCS127PYesNon1.00E−02Yes364
functional
12367127TCCACCS127TNon1.00E−02Yes32
functional
12368127TCCTTCS127FNon1.00E−02Yes2232.5
functional
12368127TCCTACS127YNon1.00E−02Yes592
functional
12368127TCCTGCS127CNon1.00E−02Yes122
functional
12382132AAGTAGK132*Yes7.00E−04Yes24
12382132AAGCAGK132QNon7.00E−04Yes12173
functional
12382132AAGGAGK132ENon7.00E−04Yes16243
functional
12383132AAGAGGK132RYesNon7.00E−04Yes30575
functional
12383132AAGATGK132MNon7.00E−04Yes7122.5
functional
12383132AAGACGK132TNon7.00E−04Yes342
functional
12384132AAGAATK132NYesNon7.00E−04Yes22265
functional
12384132AAGAACK132NYesNon7.00E−04Yes22345
functional
12386133ATGAAGM133KNon2.00E−0111202
functional
12391135TGCCGCC135RNon2.00E−02Yes5162.5
functional
12391135TGCAGCC135SNon2.00E−02Yes482
functional
12391135TGCGGCC135GNon2.00E−02Yes692
functional
12392135TGCTACC135YYesNon2.00E−02Yes46805
functional
12392135TGCTTCC135FNon2.00E−02Yes34563
functional
12392135TGCTCCC135SNon2.00E−02Yes482
functional
12393135TGCTGAC135*Yes2.00E−02Yes84
12393135TGCTGGC135W2.00E−02Yes17262
12394136CAATAAQ136*Yes393.5
12400138GCCCCCA138PNon4.00E−02Yes12303
functional
12401138GCCGGCA138GNon4.00E−02Yes02
functional
12403139AAGTAGK139*Yes53
12410141TGCTACC141YNon5.00E−0161972
functional
12411141TGCTGAC141*Yes5.00E−01193.5
12415143GTGATGV143MNon1.00E−0115302
functional
12416143GTGGCGV143ANon1.00E−0113202
functional
12418144CAGTAGQ144*Yes443.5
12422145CTGCAGL145QNon14192
functional
12422145CTGCCGL145PNon15212
functional
12425146TGGTAGW146*Yes503.5
12426146TGGTGAW146*Yes513.5
12432148GATTGAD148*YesNon6.00E−03Yes4
functional
12439151CCCACCP151TNon6.00E−03Yes13213
functional
12439151CCCTCCP151SNon6.00E−03Yes58953
functional
12439151CCCGCCP151ANon6.00E−03Yes7182.5
functional
12440151CCCCGCP151RYesNon6.00E−03Yes6204.5
functional
12440151CCCCACP151HNon6.00E−03Yes1362.5
functional
12442152CCGTCGP152SNon6.00E−0121302
functional
12442152CCGCTGP152LNon6.00E−03Yes84103
functional
12449154GGCGTCG154VNon4.00E−0134642
functional
12451155ACCCCCT155PNon1.00E−0113212
functional
12452155ACCAACT155NNon1.00E−0119322
functional
12455156CGCCCCR156PNon8.00E−0123442
functional
12457157GTCTTCV157FNon1.00E−02Yes1331863
functional
12457157GTCCTCV157LNon1.00E−02Yes692
functional
12458157GTCGACV157DNon1.00E−02Yes8142.5
functional
12458157GTCGGCV157GNon1.00E−02Yes7122.5
functional
12458157GTCGCCV157ANon1.00E−02Yes122
functional
12460158CGCGGCR158GNon4.00E−03Yes10222.5
functional
12460158CGCAGCR158SNon4.00E−03Yes42
functional
12461158CGCCCCR158PYesNon4.00E−03Yes9194.5
functional
12461158CGCCACR158HNon4.00E−03Yes551063
functional
12461158CGCCTCR158LNon4.00E−03Yes1962.5
functional
12463159GCCCCCA159PNon3.00E−03Yes13303
functional
12464159GCCGTCA159VNon3.00E−03Yes28463
functional
12464159GCCGACA159DNon3.00E−03Yes6112.5
functional
12470161GCCGACA161DNon4.00E−02Yes7212.5
functional
12475163TACAACY163NNon2.00E−02Yes16233
functional
12475163TACCACY163HNon2.00E−02Yes16253
functional
12475163TACGACY163DNon2.00E−02Yes242
functional
12476163TACTGCY163CYesNon2.00E−02Yes901475
functional
12476163TACTCCY163SNon2.00E−02Yes452
functional
12477163TACTAGY163*Yes2.00E−02Yes114.5
12477163TACTAAY163*Yes2.00E−02Yes74
12478164AAGTAGK164*Yes173.5
12478164AAGGAGK164ENon11242
functional
12481165CAGTAGQ165*Yes453.5
12485166TCATAAS166*Yes153.5
12485166TCATGAS166*Yes153.5
12487167CAGTAGQ167*Yes383.5
12490168CACTACH168YYes1.00E−02Yes9143.5
12491168CACCGCH168RNon1.00E−02Yes12223
functional
12491168CACCCCH168PNon1.00E−02Yes9132.5
functional
12491168CACCTCH168LNon1.00E−02Yes582
functional
12499171GAGTAGE171*Yes213.5
12505173GTGATGV173MYesNon1.00E−02Yes35715
functional
12505173GTGTTGV173LYesNon1.00E−02Yes9654.5
functional
12505173GTGCTGV173LYesNon1.00E−02Yes9214.5
functional
12506173GTGGGGV173GNon1.00E−02Yes4162.5
functional
12506173GTGGAGV173ENon1.00E−02Yes132
functional
12511175CGCGGCR175GNon9.00E−13Yes11243
functional
12511175CGCTGCR175C9.00E−13Yes12272
12512175CGCCACR175HYesNon9.00E−13Yes69111585
functional
12512175CGCCTCR175L9.00E−13Yes18272
12512175CGCCCCR175PNon9.00E−13Yes582
functional
12514176TGCCGCC176RYesNon2.00E−03Yes8144.5
functional
12514176TGCAGCC176SNon2.00E−03Yes1202.5
functional
12514176TGCGGCC176GNon2.00E−03Yes372
functional
12515176TGCTTCC176FYes2.00E−03Yes11563.5
12515176TGCTACC176YNon2.00E−03Yes44893
functional
12515176TGCTCCC176SNon2.00E−03Yes1112.5
functional
12516176TGCTGAC176*Yes2.00E−03Yes94
12516176TGCTGGC176WNon2.00E−03Yes11203
functional
12517177CCCTCCP177SYes7.00E−018162.5
12518177CCCCACP177HYes7.00E−01252
12518177CCCCGCP177RNon7.00E−0113172
functional
12523179CATAATH179NYes2.00E−02Yes13244
12523179CATTATH179YYes2.00E−02Yes81073.5
12524179CATCGTH179RYesNon2.00E−02Yes901515
functional
12524179CATCTTH179LYes2.00E−02Yes31424
12525179CATCAGH179QNon2.00E−02Yes7162.5
functional
12525179CATCAAH179QNon2.00E−02Yes792
functional
12525179CATTAGH179*YesNon7.00E−0114
functional
12526180GAGTAGE180*Yes2.00E−01193.5
12534182TGCTGAC182*Yes73
12536183TCATGAS183*Yes263.5
12536183TCATAAS183*Yes33
12643192CAGTAGQ192*Yes963.5
12646193CATTATH193YNon5.00E−03Yes25393
functional
12646193CATGATH193DNon5.00E−03Yes7152.5
functional
12646193CATAATH193NNon5.00E−03Yes332
functional
12647193CATCTTH193LNon5.00E−03Yes28603
functional
12647193CATCCTH193PNon5.00E−03Yes12183
functional
12647193CATCGTH193RNon5.00E−03Yes58913
functional
12649194CTTTTTL194FNon2.00E−0115272
functional
12650194CTTCGTL194RNon2.00E−0126602
functional
12652195ATCTTCI195FNon3.00E−0114282
functional
12653195ATCACCI195TNon3.00E−0151892
functional
12655196CGATGAR196*Yes2.00E−012233.5
12656196CGACCAR196PNon2.00E−0112192
functional
12659197GTGGGGV197GNon2.00E−0114222
functional
12661198GAATAAE198*Yes243.5
12664199GGATGAG199*Yes5.00E−0143
12665199GGAGAAG199ENon5.00E−0111152
functional
12671201TTGTAGL201*Yes73
12679204GAGTAGE204*Yes453.5
12682205TATGATY205DNon7.00E−0213182
functional
12683205TATTCTY205SNon7.00E−0211202
functional
12683205TATTGTY205CNon7.00E−02481102
functional
12684205TATTAAY205*Yes7.00E−0243
12684205TATTAGY205*Yes7.00E−0243
12686206TTGTAGL206*Yes113.5
12694209AGATGAR209*Yes133.5
12706213CGATGAR213*Yes6.00E−0153
12707213CGACAAR213QNon6.00E−0121362
functional
12707213CGACTAR213LNon6.00E−0124382
functional
12710214CATCGTH214RNon2.00E−0141782
functional
12713215AGTATTS215INon9.00E−0214252
functional
12715216GTGATGV216MNon7.00E−04Yes43763
functional
12715216GTGTTGV216LNon7.00E−04Yes7132.5
functional
12715216GTGCTGV216LNon7.00E−04Yes712
functional
12716216GTGGAGV216ENon7.00E−04Yes472
functional
12716216GTGGGGV216GNon7.00E−04Yes352
functional
12727220TATAATY220NNon4.00E−06Yes12173
functional
12727220TATCATY220HNon4.00E−06Yes7162.5
functional
12727220TATGATY220DNon4.00E−06Yes232
functional
12728220TATTGTY220CNon4.00E−06Yes1863603
functional
12728220TATTCTY220SNon4.00E−06Yes9132.5
functional
12729220TATTAAY220*Yes4.00E−06Yes24
12729220TATTAGY220*Yes4.00E−06Yes34
12730221GAGTAGE221*Yes113.5
12739224GAGTAGE224*Yes103
13324229TGTTGAC229*Yes73
13337234TACCACY234HNon2.00E−0112272
functional
13338234TACTGCY234CNon2.00E−01651362
functional
13339234TACTAAY234*Yes2.00E−01113.5
13339234TACTAGY234*Yes2.00E−0113
13343236TACGACY236DYesNon1.00E−02Yes584
functional
13343236TACCACY236HNon1.00E−02Yes7142.5
functional
13343236TACAACY236N1.00E−02Yes11192
13344236TACTGCY236CNon1.00E−02Yes41803
functional
13344236TACTCCY236SNon1.00E−02Yes332
functional
13345236TACTAAY236*Yes1.00E−02Yes144.5
13345236TACTAGY236*Yes1.00E−02Yes84
13348237ATGATAM237IYesNon1.00E−0111213.5
functional
13348237ATGATTM237IYesNon1.00E−011503.5
functional
13348237ATGATCM237IYesNon1.00E−011133.5
functional
13349238TGTCGTC238RNon6.00E−0114232
functional
13350238TGTTATC238YYesNon6.00E−0142854
functional
13350238TGTTTTC238FYesNon6.00E−0126404
functional
13351238TGTTGAC238*Yes6.00E−0173
13352239AACGACN239DNon25482
functional
13353239AACAGCN239SYesNon1313.5
functional
13355240AGTGGTS240GNon13182
functional
13358241TCCACCS241TYesNon3.00E−06Yes594
functional
13358241TCCGCCS241ANon3.00E−06Yes6122.5
functional
13358241TCCCCCS241PNon3.00E−06Yes3102
functional
13359241TCCTTCS241FYesNon3.00E−06Yes51004.5
functional
13359241TCCTGCS241CNon3.00E−06Yes23353
functional
13359241TCCTACS241YNon3.00E−06Yes6192.5
functional
13361242TGCAGCC242SNon6.00E−03Yes10142.5
functional
13361242TGCCGCC242RNon6.00E−03Yes10132.5
functional
13361242TGCGGCC242GNon6.00E−03Yes272
functional
13362242TGCTACC242YYesNon6.00E−03Yes35545
functional
13362242TGCTTCC242FYesNon6.00E−03Yes1884.5
functional
13362242TGCTCCC242SNon6.00E−03Yes10192.5
functional
13363242TGCTGAC242*Yes6.00E−03Yes44
13363242TGCTGGC242WNon6.00E−03Yes7132.5
functional
13367244GGCAGCG244SYesNon4.00E−02Yes34705
functional
13367244GGCTGCG244CNon4.00E−02Yes30473
functional
13367244GGCCGCG244RNon4.00E−02Yes352
functional
13368244GGCGACG244DYesNon4.00E−02Yes31635
functional
13368244GGCGTCG244VNon4.00E−02Yes13243
functional
13368244GGCGCCG244ANon4.00E−02Yes9112.5
functional
13370245GGCAGCG245SYesNon6.00E−06Yes2584365
functional
13370245GGCCGCG245RYesNon6.00E−06Yes9184.5
functional
13370245GGCTGCG245CNon6.00E−06Yes43833
functional
13371245GGCGACG245DYesNon6.00E−06Yes831535
functional
13371245GGCGTCG245VNon6.00E−06Yes1732.5
functional
13371245GGCGCCG245ANon6.00E−06Yes7132.5
functional
13373246ATGGTGM246VYesNon5.00E−0225554
functional
13373246ATGTTGM246LYesNon5.00E−02273
functional
13373246ATGCTGM246LYesNon5.00E−02213
functional
13374246ATGAGGM246RYesNon5.00E−0210143.5
functional
13377247AACATCN247INon1.00E−02Yes172
functional
13377247AACACCN247TNon1.00E−02Yes442
functional
13379248CGGTGGR248WYesNon7.00E−17Yes4027075
functional
13379248CGGGGGR248GNon7.00E−17Yes10232.5
functional
13380248CGGCAGR248QYesNon7.00E−17Yes58654.5
functional
13380248CGGCTGR248LYesNon7.00E−17Yes21144.5
functional
13380248CGGCCGR248PNon7.00E−17Yes11183
functional
13382249AGGTGGR249WYesNon2.00E−03Yes23405
functional
13382249AGGGGGR249GNon2.00E−03Yes23473
functional
13383249AGGATGR249MYesNon2.00E−03Yes25665
functional
13383249AGGAAGR249KNon2.00E−03Yes14273
functional
13383249AGGACGR249TNon2.00E−03Yes15303
functional
13384249AGGAGTR249SYesNon2.00E−03Yes2823895
functional
13384249AGGAGCR249SYesNon2.00E−03Yes282405
functional
13387250CCCCTCP250LNon2.00E−03Yes51103
functional
13400255ATCTTCI255FNon15392
functional
13407257CTGCAGL257QYesNon6163.5
functional
13407257CTGCCGL257PYesNon7143.5
functional
13409258GAATAAE258*Yes8.00E−02Yes253.5
13409258GAAAAAE258KNon2.00E−03Yes67143
functional
13412259GACTACD259YYesNon7.00E−02Yes14324
functional
13413259GACGTCD259VNon7.00E−02Yes12192
functional
13774265CTGCCGL265PYesNon1203.5
functional
13776266GGATGAG266*Yes2.00E−02Yes294.5
13776266GGAAGAG266RNon2.00E−02Yes14513
functional
13776266GGACGAG266RNon2.00E−02Yes14183
functional
13777266GGAGAAG266ENon2.00E−02Yes41793
functional
13777266GGAGTAG266VNon2.00E−02Yes22553
functional
13779267CGGTGGR267WNon19312
functional
13780267CGGCCGR267PNon12172
functional
13789270TTTTGTF270CNon3.00E−0115272
functional
13791271GAGTAGE271*Yes5.00E−02173.5
13794272GTGATGV272MNon1.00E−01601032
functional
13797273CGTTGTR273CYesNon4.00E−18Yes3636645
functional
13797273CGTAGTR273SNon4.00E−18Yes1192.5
functional
13797273CGTGGTR273GNon4.00E−18Yes8172.5
functional
13798273CGTCATR273HYesNon4.00E−18Yes4348125
functional
13798273CGTCTTR273LYesNon4.00E−18Yes791425
functional
13798273CGTCCTR273PNon4.00E−18Yes22373
functional
13800274GTTTTTV274FYesNon7.00E−0114334
functional
13801274GTTGCTV274ANon7.00E−0111182
functional
13803275TGTCGTC275RNon1.00E−02Yes6152.5
functional
13803275TGTAGTC275SNon1.00E−02Yes202
functional
13803275TGTGGTC275GNon1.00E−02Yes782
functional
13804275TGTTATC275YNon1.00E−02Yes40753
functional
13804275TGTTTTC275FNon1.00E−02Yes30493
functional
13804275TGTTCTC275SNon1.00E−02Yes222
functional
13805275TGTTGAC275*Yes1.00E−02Yes34
13805275TGTTGGC275WNon1.00E−02Yes7102
functional
13806276GCCCCCA276PYesNon7.00E−0211184
functional
13810277TGTTATC277YNon1.00E−0114272
functional
13810277TGTTTTC277FNon1.00E−0119512
functional
13811277TGTTGAC277*Yes1.00E−0183
13812278CCTACTP278TNon1.00E−05Yes21323
functional
13812278CCTTCTP278SNon1.00E−05Yes46823
functional
13812278CCTGCTP278ANon1.00E−05Yes16283
functional
13813278CCTCATP278HYesNon1.00E−05Yes10144.5
functional
13813278CCTCTTP278LNon1.00E−05Yes51813
functional
13813278CCTCGTP278RNon1.00E−05Yes24423
functional
13816279GGGGAGG279EYesNon1453.5
functional
13818280AGATGAR280*Yes2.00E−0193
13818280AGAGGAR280GNon2.00E−0115412
functional
13819280AGAACAR280TYesNon2.00E−0146914
functional
13819280AGAAAAR280KNon2.00E−0137702
functional
13819280AGAATAR280INon2.00E−0111242
functional
13820280AGAAGTR280SYesNon2.00E−014193.5
functional
13820280AGAAGCR280SYesNon2.00E−01453
functional
13821281GACAACD281NYesNon4.00E−03Yes18335
functional
13821281GACTACD281YYesNon4.00E−03Yes5114.5
functional
13821281GACCACD281HNon4.00E−03Yes18383
functional
13822281GACGGCD281GYesNon4.00E−03Yes9144.5
functional
13822281GACGTCD281VNon4.00E−03Yes242
functional
13822281GACGCCD281ANon4.00E−03Yes252
functional
13823281GACGAAD281EYesNon4.00E−03Yes9204.5
functional
13823281GACGAGD281EYesNon4.00E−03Yes9244.5
functional
13824282CGGGGGR282GNon2.00E−07Yes25463
functional
13824282CGGTGGR282WNon2.00E−07Yes105542.5
functional
13825282CGGCCGR282PNon2.00E−07Yes13223
functional
13825282CGGCAGR282Q2.00E−07Yes19262
13828283CGCCACR283HNon5.00E−0111172
functional
13828283CGCCCCR283PNon5.00E−0121352
functional
13833285GAGTAGE285*Yes1.00E−01213.5
13833285GAGAAGE285KNon1.00E−01871692
functional
13834285GAGGTGE285VNon1.00E−0113192
functional
13836286GAATAAE286*Yes1.00E−03Yes214.5
13836286GAAAAAE286KNon1.00E−03Yes1832.5
functional
13836286GAACAAE286QNon1.00E−03Yes5152.5
functional
13837286GAAGGAE286GNon1.00E−03Yes14193
functional
13837286GAAGTAE286VNon1.00E−03Yes662
functional
13837286GAAGCAE286ANon1.00E−03Yes122
functional
13838286GAAGATE286DNon1.00E−03Yes132
functional
13838286GAAGACE286DNon1.00E−03Yes112
functional
13839287GAGTAGE287*Yes143.5
13842288AATGATN288DYes112
13851291AAGTAGK291*Yes73
13854292AAATAAK292*Yes23
13860294GAGTAGE294*Yes573.5
13872298GAGTAGE298*Yes643.5
13893305AAGTAGK305*Yes153.5
13896306CGATGAR306*YesNon9.00E−021644.5
functional
16900337CGCTGCR337CNon9.00E−0211182
functional
P53Reported >10xReported >10x
signaturein somaticin somatic
StopSpliceDominantTrans-P53p value <@ AA@ BaseEvi-
ProteinCodonDefectNegativeactivationSignature0.05levelleveldence
NtNumCodonChangeChange(3 pts)(3 pts)(2 pts)(1 pt)P-Value(1 pt)(0.5 pt)(0.5 pt)Score
110802-intronG > AN/AYes3
110802-intronG > CN/AYes3
110802-intronG > TN/AYes3
110812-introndel167N/AYes3
110812-intronT > AN/AYes3
110812-intronT > CN/AYes3
110812-intronT > GN/AYes3
111952-intronA > CN/AYes3
111952-intronA > GN/AYes3
111952-intronA > TN/AYes3
111962-intronG > AN/AYes3
111962-intronG > CN/AYes3
111962-intronG > TN/AYes3
112193-intronG > AN/AYes3
112193-intronG > CN/AYes3
112193-intronG > TN/AYes3
112203-intronT > AN/AYes3
112203-intronT > CN/AYes3
112203-intronT > GN/AYes3
113263-intronA > CN/AYes3
113263-intronA > GN/AYes3
113263-intronA > TN/AYes3
113273-introndel19N/AYes3
113273-intronG > AN/AYes3
113273-intronG > CN/AYes3
113273-intronG > TN/AYes3
116074-intronG > AN/AYes3
116074-intronG > CN/AYes3
116074-intronG > TN/AYes3
116084-intronins1N/AYes3
116084-intronT > AN/AYes3
116084-intronT > CN/AYes3
116084-intronT > GN/AYes3
123624-intronA > CN/AYes3
123624-intronA > GN/AYes3
123624-intronA > TN/AYes3
123624-introndel1N/AYes3
123624-intronins1N/AYes3
123634-introndel1N/AYes3
123634-intronG > AN/AYes3
123634-intronG > CN/AYes3
123634-intronG > TN/AYes3
125485-intronG > AN/AYes3
125485-intronG > CN/AYes3
125485-intronG > TN/AYes3
125495-intronT > AN/AYes3
125495-intronT > CN/AYes3
125495-intronT > GN/AYes3
126275-intronA > CN/AYes3
126275-intronA > GN/AYes3
126275-intronA > TN/AYes3
126275-introndel10N/AYes3
126285-introndel1N/AYes3
126285-intronG > AN/AYes3
126285-intronG > CN/AYes3
126285-intronG > TN/AYes3
126285-intronGG > AAN/AYes3
127426-introndel1N/AYes3
127426-intronG > AN/AYes3
127426-intronG > CN/AYes3
127426-intronG > TN/AYes3
127436-intronT > AN/AYes3
127436-intronT > CN/AYes3
127436-intronT > GN/AYes3
133086-intronA > CN/AYes3
133086-intronA > GN/AYes3
133086-intronA > TN/AYes3
133086-introndel11N/AYes3
133096-intronG > AN/AYes3
133096-intronG > CN/AYes3
133096-intronG > TN/AYes3
134207-introndel137N/AYes3
134207-introndel342(delN/AYes3
intron7)
134207-intronG > AN/AYes3
134207-intronG > CN/AYes3
134207-intronG > TN/AYes3
134217-introndel10N/AYes3
134217-intronT > AN/AYes3
134217-intronT > CN/AYes3
134217-intronT > GN/AYes3
137597-intronG > AN/AYes3
137617-intronA > CN/AYes3
137617-intronA > GN/AYes3
137617-intronA > TN/AYes3
137627-intronG > AN/AYes3
137627-intronG > CN/AYes3
137627-intronG > TN/AYes3
137627-intronins3N/AYes3
“del” denotes deletion of the number of basepairs; “ins” denotes insertion of the number of baspairs.

Example 2

A Phase II study was conducted with WEE1-1 Inhibitor combined with carboplatin in patients with p53 mutated epithelial ovarian cancer that show early relapse (<3 months) or progression during standard first line treatment with carboplatin—paclitaxel combination therapy. Patients were enrolled based on the tumor's TP53 gene sequence as determined in the AmpliChip p53 assay.

AmpliChip p53 test reagents are used to amplify products encompassing the coding regions of the p53 gene in two reactions (A and B) for all samples including a reference wildtype DNA. Exons 2, 5, 8, and 10; exon 4 upstream sequence; and internal control are in the Primer Mix A. Primer Mix B is designed and contained primers for exons 3, 6, 7, 9, and 11; exon 4 downstream sequence; and internal control. After thermal cycling, the products from Primer Mixes A and B are combined. The products generated from the A and B reactions are cleaved by a mix containing DNase I. Fragmentation is performed by recombinant DNase I to generate small DNA fragments of an average size of 50-100 nucleotides. The alkaline phosphatase in the Working Fragmentation Mix destroys the residual dNTPs from the amplification reactions. The fragmented DNA amplicons are subsequently labeled with biotin at their 3′ termini by the action of terminal transferase, using AmpliChip TdT Labeling Reagent as substrate. The biotin-labeled p53 target DNA fragments are added to the hybridization buffer containing the AmpliChip Oligonucleotide Solution which functions as a hybridization control. The mixture is hybridized to the oligonucleotides located on the AmpliChip p53 Microarray using the Affymetrix GeneChip Fluidics Station 450Dx and an AmpliChip p53 specific protocol. The hybridized AmpliChip p53 Microarray is washed and stained with a streptavidin-conjugated fluorescent dye.

Design of the AmpliChip p53 Microarray

The microarray consists of a square grid of 228,484 probes, with sides that are 11 micron each. Each probe contains multiple copies of a specific oligonucleotide sequence. A single probe set for an interrogating base position includes five probes, one probe to hybridize to the wild type, three probes to detect three possible single base pair mutations, and one probe to detect single deletion. There are at least 24 probe sets for each nucleotide position, including both sense and antisense probe sequences. A total of 1300 nucleotide positions of coding regions of exons 2-11 are tiled on AmpliChip p53. AmpliChip p53 Microarrays are manufactured using technology that combines photolithographic methods and combinatorial chemistry. Over 220,000 different oligonucleotide probes are synthesized on a glass surface to analyze both sense and antisense strands of an amplified target DNA specimen. Within the 11×11 μm2 probe microarray, each probe type is located in a specific area called a probe cell, which contains approximately 106 copies of a given probe. Probe microarrays are manufactured by light-directed combinatorial chemistry in a series of cycles. The glass substrates are coated with linkers containing photolabile protecting groups. A mask is then applied that exposes selected portions of the probe microarray. Illumination removes the photolabile protecting groups enabling selective nucleoside phosphoramidite addition only at the previously exposed sites. Next, a different mask is applied and the cycle of illumination and chemical coupling is performed again. By repeating this cycle, a specific set of oligonucleotide probes is synthesized, with each probe type in a known location. The completed probe microarrays are packaged into cartridges compatible with the GeneChip Fluidics Station 450Dx. After staining, the AmpliChip p53 Microarray is scanned by an Affymetrix GeneChip Scanner 3000Dx using a laser that excites the fluorescent label bound to the hybridized p53 target DNA fragments. The amount of emitted light is proportional to bound target DNA at each location on the probe microarray.

Data Analysis of Microarray Signals.

The p53 mutation status is determined by a p53 mutation detection algorithm, which is designed to detect single base pair substitutions and single base pair deletions of a sample in a background of wild type p53 DNA probe intensities. The algorithm first reads the probe intensities generated by the GeneChip Operating Software, Version 1.1 provided by Affymetrix. Based on the raw data, the algorithm performs an initial exon quality test to detect distinct problems in each PCR product. If an exon fails the initial quality test, the exon failure is reported and no further analysis is made. If an exon passes the test, the probe intensities are normalized by using quantile normalization in order to correct array-to-array variability.

The quality of each probe set is then examined to eliminate unreliable probe set data for further computation. Using probes sets that passed the quality tests, the algorithm makes a tentative call for each base position. Possible base calls for each nucleotide position are wild type, single base substitution, single base deletion, or no call (unable to make a call). In the case of a single base substitution, the algorithm identifies the mutated base (e.g., G®A). After the tentative calls are made, the reliability of each base call is reexamined by the algorithm to fine tune the calls using various parameters calculated from the neighboring base positions. Each exon quality is also reexamined based on the final base calls. If there are too many no calls and/or mutation calls in one exon, the data is considered as “noisy,” and the exon fails the quality test. If an exon fails, the exon failure is reported, and no calls are reported for that exon.

Tumor Response Evaluation

Patients were evaluable for response to study treatment if at least one follow-up examination was performed at the end of the second treatment cycle (at least a six-week period). Tumor response was assessed either in measurable or evaluable tumor lesions according to the RECIST 1.0 criteria (Appendix IV). In the case of stable disease, a confirmation was necessary within 6 to 8 weeks of initial assessment. In case of stable disease at the end of treatment or in case of discontinuation for unacceptable adverse experiences evaluation took place every 2 months and CA-125 (cancer antigen) was determined. In case of a CA-125 increase a CT scan was performed. In patients for whom CA-125 is not a good marker, a CT-scan was performed every 2 months, until disease progression. Patients without radiologically measurable disease had elevated CA-125 prior to start and were evaluated based on CA-125 levels. CT-scans were also performed in these patients. According to the Gynecologic Cancer Intergroup (GCIG) CA-125 response criteria, progressive disease after a complete response to primary therapy is defined as follows: the date of first elevation of CA-125 to two-fold the upper limit of normal (ULN) (documented on two occasions at least a week apart). For those with persistently elevated CA-125 levels, progression of disease is defined as the first date of CA-125≧2× the nadir value documented on two occasions no less than a week apart. The ULN for CA-125 at the NKI is 35 U/mL. However, in this study for patients with CA-125 nadir ≦10 U/mL, a confirmed value of ≧20 U/mL served as an early signal of CA-125 progression, and for patients with nadir more than 10 U/mL, a value ≧2× nadir. The definition of disease progression included both the standard RECIST criterion and the CA-125 criterion as defined above, whichever occurs first. Response regarding CA-125 is defined standards as 50% reduction during treatment of CA-125.

The following table summarizes the p53 amino acid mutation information for responders in the above clinical trial.

TABLE 4
Amino
Acid/GeneMutation listed
Patient NumberPCRmutationin Table 3Responder
1003Exon 7C238FYesYes
1008Exon 7R248WYesYes
1010Exon 8E298 stop codonYesYes
1011Exon 8R273LYesYes
1015Exon 5V157 frameshiftYesYes

Example 4

In a randomized, Phase II study evaluating WEE1-1 in combination with paclitaxel and carboplatin versus paclitaxel and carboplatin alone in adult patients with platinum sensitive p53 mutant ovarian cancer, patient enrollment was based on the presence of one or more mutations in Table 3. If the specimen has no mutations in the TP53 gene, or if it contains a mutation not listed on the Mutation Lookup Table 3, the patient will not be eligible for the study. If the specimen had at least one mutation that is listed in the Mutation Lookup Table 3, the patient was eligible for enrollment in the study.

Analysis of patient tumor tissue samples using the AmpliChip p53 assay was performed by Caris Life Sciences (Phoenix, Ariz.). The AmpliChip p53 assay characterized each specimen as ‘Mutation Not Detected,’ Mutation Detected′ or ‘Test Invalid’. A base change to a synonymous codon is treated as a Mutation Not Detected call, since it does not alter the amino acid. There are seven single-nucleotide polymorphisms reported in the IARC database R15 (2010 release) within the tiled nucleotide positions of AmpliChip 53. While three do not result in amino acid changes (codons 34, 36 and 213), four others result in the following amino acid changes: P47S, P72R, V217M and G360A. These are all treated as Mutation Not Detected, and are not reported. If at least one mutation is detected, the sample is called “Mutation Detected” and the nucleotide change detected is reported, along with the corresponding amino acid change in the p53 protein. If no base changes are detected and the test is valid, the sample is called “Mutation Not Detected.”

F.