# Patent

## Title
Diagnostic method using PALB2

## Abstract

The present invention provides a method for detecting mutations in the PALB2 gene in pancreatic cancer patients and in individuals having a family history of pancreatic cancer. Methods are also provided for diagnosing a predisposition to pancreatic cancer, for predicting a patient's response to pancreatic cancer therapies, and for treating pancreatic cancer, based on presence of a PALB2 mutation or abberant PALB2 gene expression in a patient.

## Background
This application incorporates expressly by reference the content of U.S. patent applications Ser. No. 61/157,700 filed Mar. 5, 2009 and Ser. No. 61/264,019 filed Nov. 24, 2009.


STATEMENT GOVERNMENT INTEREST
Funding for the work described herein was provided, in part, by the federal government, which may have certain rights in the invention according to the terms of National Institute of Health grants CA62924, CA123483, and RO1CA97075.


FIELD OF THE INVENTION
The invention generally relates to personalized medicine, and particularly to method of diagnosis in pancreatic cancer.
BACKGROUND OF THE INVENTION
Pancreatic cancer is one of the most deadly cancers, in part because it is often undetected until an advanced stage. If a patient is diagnosed at a late stage, the survival rate at five years after diagnosis is only about 5 percent. Early diagnosis at an early stage however can lead to significantly improved survival outcome. It is estimated that as many as 10% of pancreatic cancers are familial or hereditary. For familial pancreatic cancer, if a predisposition in a patient having a family history can be detected before cancer develops, then early start of surveillance and screening could increase the chance of early diagnosis and improved prognosis. Five different genetic syndromes have been associated with predisposition to pancreatic cancer. These include BRCA2 mutations, familial atypical multiple mole melanoma (FAMMM), Peutz-Jeghers Syndrome, hereditary pancreatitis, and the hereditary non-polyposis colorectal cancer (HNPCC) syndrome. See e.g., Tascilar et al., Anal. Cell Pathol., 19(3-4):105-10 (1999). However, many familial pancreatic cancer cases can not be explained by any of these five genetic syndromes. Clearly there is a need to identify additional genetic predisposition markers for pancreatic cancer.
Another difficulty in pancreatic cancer is that there is no effective treatment for advanced pancreatic cancer. Gemcitabine, the current standard of care, results in an average progression free survival of 3-4 months and median survival of 5-6 months. See O'Reilly, Gastrointest. Cancer Res., 3(2 Suppl):S11-5 (2009. Second line treatment after gemcitabine failure is even less effective with median survival of approximately 3 months. See, Moore et al., J. Clin. Oncol., 25(15):1960-6 (2007); Philip, Gastrointest. Cancer Res., 2(4 Suppl):S16-9 (2008). One strategy actively sought to improve outcome is to personalize patient treatment. In recent years, with the ability to interrogate the entire human cancer genome, it is becoming apparent that some cancers can be effectively treated by targeting specific somatic genetic alterations present in the cancers. This is perhaps best exemplified by the observation that patients with lung cancer harboring mutations in the epidermal growth factor (EGFR) gene respond rather dramatically to agents that target this receptor. Lynch et al., N. Engl. J. Med., 350(21):2129-39 (2004); Paez et al., Science, 304(5676):1497-500 (2004). Clearly, there is also a need for biomarkers predictive of therapy outcome and useful in personalized pancreatic cancer treatment.
BRIEF SUMMARY OF THE INVENTION
The inventors have now surprisingly discovered that PALB2 mutations are associated with a predisposition to pancreatic cancer. PALB2 appears to be the second most commonly mutated gene for hereditary pancreatic cancer, after BRCA2. The inventors have also surprisingly discovered that some pancreatic cancer patients harbor somatic PALB2 mutations in their pancreatic tumors. In addition, it has also been surprisingly discovered that pancreatic cancer patients having PALB2 mutations are especially responsive to treatment with a DNA damaging agent such as mitomycin C and cisplatin.
Accordingly, in a first aspect of the present invention, a method of detecting mutation is provided comprising identifying a patient diagnosed of pancreatic cancer, or having an increased risk, or a family history of, pancreatic cancer, and determining in a sample obtained from the identified patient the presence or absence of a mutation in the PALB2 gene or a reduced level of PALB2 gene expression. Optionally, the method includes a step of identifying or diagnosing a patient as having pancreatic cancer or having a family history of pancreatic cancer.
In another aspect, the present invention also provides a method for diagnosing a predisposition to, or increased risk of developing, pancreatic cancer in a patient, comprising detecting in a non-tumor sample or tumor sample obtained from an individual, a mutation in the PALB2 gene or a reduced level of PALB2 gene expression, wherein the presence of the mutation or reduced level of gene expression would indicate a predisposition to pancreatic cancer. The method for diagnosing a predisposition to pancreatic cancer according to the present invention may optionally further include a step of placing the diagnosed/identified individual under a “preventive regimen” such as increased surveillance for pancreatic cancer using diagnostic markers such as CA19-9, or frequent image scan (e.g., CT and ultrasound), or administration of preventive pharmaceuticals or nutraceuticals (e.g., vitamin D, and B vitamins such as B12, B6, and folate).
In yet another aspect of the present invention, a method is provided for predicting an individual's response to therapy. The method comprises detecting in a sample obtained from a pancreatic cancer patient, a mutation in the PALB2 gene or a reduced level of PALB2 gene expression, wherein the presence of the mutation or reduced level of gene expression would indicate that the individual has an increased likelihood of responding to a therapy that induces DNA damage or interferes with DNA damage repairs in tumor cells. Again, the sample used can be a non-tumor sample or a tumor sample.
The present invention further provides a method of treating pancreatic cancer in a patient, comprising administering to a patient identified as having a germline or somatic mutation in a PALB2 gene or a reduced level of PALB2 gene expression, a therapy that induces DNA damage or interferes with DNA damage repairs in tumor cells.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic diagram showing the location of identified PALB2 mutations. Exons are represented as boxes and introns as black lines (not to scale). Mutations previously identified in patients with familial breast cancer or Fanconi Anemia are shown in black or purple, respectively. Germline mutations identified in patients with familial pancreatic cancer are shown above the gene in red.
FIG. 2 are images of clinical outcome in the patient who was found to harbor a PALB2 mutation and was responsive to mitomycin C (MMC) and cisplatin. FIG. 2A is a chest CT image obtained in the first postoperative visit showing an enlarged left supracalvicular node (arrow). A biopsy of this node showed metastatic adenocarcinoma; FIG. 2B is an image of FDG-PET scan demonstrating increase glucose update in the above mentioned node (arrow); FIG. 2C demonstrates extensive locoregional recurrent disease after for cycles of gemcitabine; FIG. 2D is a plot showing the time-course of CA 19-9 indicating disease progression while on gemcitabine and complete normalization with MMC; FIG. 2E demonstrates late pulmonary progression with a left upper lesion after 22 months of follow up; FIG. 2F indicates decrease in pulmonary lesion size after two additional courses of MMC.
FIG. 3 includes tumor growth curves from in vivo xenograft studies indicating resistance to gemcitabine and remarkable response to MMC and cisplatin in the patient's own xenograft. Panc 185 tumor with wild-type PALB2 is presented as a control.
FIG. 4A is a schematic diagram showing the somatic mutation found in the PALB2 gene of the patient treated;
FIG. 4B is gel image of showing the co-immunoprecipitation with a monoclonal antibody against BRCA1 of the BRCA1-BRCA2 complex. No complex is identified in the PALB2 mutant tumor JH033 as compared to the wild type Panc185 tumor used as a control; and
FIG. 4C is a Western Blot image for FANCD2 ubiquitination. The upper band represents the ubiquitinaed or long form (P-FANCD2 Lys561) and the lower band represents the short, non ubiquitinated form. JH033 has competent proximal FA complex similar to the MMC resistant Pancl85 control.



DETAILED DESCRIPTION OF THE INVENTION
In accordance with a first aspect of the present invention, a method of detecting mutation is provided comprising identifying a patient diagnosed of pancreatic cancer, or having an increased risk of developing, or having a family history of, pancreatic cancer, and determining in a sample obtained from the identified patient the presence or absence of a mutation in the PALB2 gene or a reduced level of PALB2 gene expression. Optionally, the method includes a step of identifying or diagnosing a patient as having pancreatic cancer or having a family history of pancreatic cancer.
The present invention also provide a method for diagnosing a predisposition to, or an increased risk of developing, pancreatic cancer in a patient, comprising detecting in a non-tumor sample or tumor sample obtained from an individual, a mutation in the PALB2 gene or a reduced level of PALB2 gene expression, wherein the presence of the mutation or reduced level of gene expression would indicate a predisposition to pancreatic cancer. In various embodiments, the individual tested can be healthy and free of family history, or diagnosed of pancreatic cancer, or is suspected of having pancreatic cancer, or has a family history of pancreatic cancer. Preferably, a non-tumor sample, i.e., normal tissue or bodily fluid sample is used in the method. In non-tumor samples, a mutation detected would be a germline mutation.
Once an individual is identified as having a PALB2 mutation or a reduced level of PALB2 gene expression, and thus having a predisposition to, or an increased likelihood of having, pancreatic cancer, the individual can be placed under a “preventive regimen.” Thus, the method for diagnosing a predisposition to pancreatic cancer according to the present invention may optionally further include a step of placing the diagnosed/identified individual under a “preventive regimen.” As used herein, the term “preventive regimen” means any preventive measures suitable for early detection of pancreatic cancer, or preventing or delaying the onset of PALB2-associated pancreatic cancer. For example, increased surveillance for pancreatic cancer using diagnostic markers such as CA19-9, or image scan (e.g., CT and ultrasound) can be used for this purpose. Alternatively or concurrently, pharmaceuticals or nutraceuticals (e.g., vitamin D, and B vitamins such as B12, B6, and folate) can be administered to the individual to prevent or delay the onset of pancreatic cancer.
In yet another aspect of the present invention, a method is provided for predicting an individual's response to therapy. Generally, the method comprises detecting in a sample obtained from a pancreatic cancer patient, a mutation in the PALB2 gene or a reduced level of PALB2 gene expression, wherein the presence of the mutation or reduced level of gene expression would indicate that the individual has an increased likelihood of responding to a therapy that induces DNA damage or interferes with DNA damage repairs in tumor cells. Again, the sample used can be a non-tumor sample or a tumor sample.
Accordingly, the present invention also provides a method of treating pancreatic cancer in a patient, comprising administering to a patient identified as having a germline or somatic mutation in a PALB2 gene or a reduced level of PALB2 gene expression, a therapy that induces DNA damage or interferes with DNA damage repairs in tumor cells. More specifically, a method is provided which includes generally the steps of identifying a patient diagnosed of pancreatic cancer, determining in said patient the presence or absence a defect in a PALB2 gene or protein, and if the defect is detected, treating said patient with a therapeutic regimen that induces DNA damage or interferes with DNA damage repairs in tumor cells.
In the various methods of the present invention described above, PALB2 mutations refer to alterations to the PALB2 gene including deletions, insertions, duplications, substitutions, etc. As will be apparent to skilled artisans, mutations resulting in significant reduction or loss of PALB2 protein or PALB2 function are most relevant in the methods of the present invention, particularly the PALB2 function in homologous-recombination-based DNA double-strand break repair (DSBR). See e.g., Xia et al., Mol. Cell, 22:719-729 (2006). In this regard, large genomic rearrangements (typically resulting in deletion or duplication of one or more exons), stop codon mutations, and frameshift mutations are clearly deleterious, as they typically lead to truncation of PALB2 protein and loss of PALB2 function. Biochemical and cellular assays may also be used to determine whether a particular mutation is deleterious or not. Such assays are known in the art and would be apparent to skilled artisans. Examples of PALB2 mutations include those in FIG. 1. PALB2 mutations such as 172-5de1TTGT, IVS5-1 G>T, 3116delA, 3256C>T, and IVS10+2C>T are newly discovered by the inventors. Thus, in some specific embodiments of the methods of the present invention described above, the presence or absence of one or more such mutations are determined. The locations and identities of such mutations should be apparent to skilled artisans especially in view of the diagram in FIG. 1 and the publicly known reference sequences under RefSeq Nos. NG—007406.1 (genomic) and NM—024675.3 (cDNA/mRNA) in NCBI Reference Sequence Database. In this regard, the present invention also provides isolated nucleic acids (e.g., DNA) comprising a portion of the PALB2 gene sequence spanning and containing one of the mutations 172-5delTTGT, IVS5-1 G>T, 3116delA, 3256C>T, and IVS10+2C>T. Preferably, the isolated nucleic acids have a consecutive 17, 18, 19, 20, 21, 25 or 30 to about 100, 200, 300, 400 or 500 nucleotides of the PALB2 genomic DNA or cDNA, spanning and containing one of the mutations 172-5de1TTGT, IVS5-1 G>T, 3116delA, 3256C>T, and IVS10+2C>T. In one embodiment, the isolated nucleic acid has from 18, 19, 20 or 21 to about 100, 200, 300 400 or 500 nucleotides comprising at least 18, 19, 20 or 21 consecutive nucleotides of SEQ ID NO:1 spanning the nucleotides T and C at positions 24 and 25, respectively of SEQ ID NO:1. In another embodiment, the isolated nucleic acid has from 18, 19, 20 or 21 to about 100, 200, 300 400 or 500 nucleotides comprising at least 18, 19, 20 or 21 consecutive nucleotides of SEQ ID NO:2 spanning the nucleotides A and T at positions 24 and 25, respectively of SEQ ID NO:2. In another embodiment, the isolated nucleic acid has from 18, 19, 20 or 21 to about 100, 200, 300 400 or 500 nucleotides comprising at least 18, 19, 20 or 21 consecutive nucleotides of SEQ ID NO:3 spanning the nucleotide T at position 25 of SEQ ID NO:3. In yet another embodiment, the isolated nucleic acid has from 18, 19, 20 or 21 to about 100, 200, 300 400 or 500 nucleotides comprising at least 18, 19, 20 or 21 consecutive nucleotides of SEQ ID NO:4 spanning the nucleotide T at position 25 of SEQ ID NO:4. In yet another embodiment, the isolated nucleic acid has from 18, 19, 20 or 21 to about 100, 200, 300 400 or 500 nucleotides comprising at least 18, 19, 20 or 21 consecutive nucleotides of SEQ ID NO:5 spanning the nucleotide T at position 25 of SEQ ID NO:5.
For purposes of the various methods of the present invention, the diagnosis of pancreatic cancer can be done by conventional diagnostic methods known in the art. For example, CA19-9 (carbohydrate antigen 19.9) is a tumor marker often used for the diagnosis of pancreatic cancer. Imaging studies, such as computed tomography (CT scan) is useful in the identification of the location of the cancer. Endoscopic ultrasound (EUS) can also help in visualizing the location and can guide a percutaneous needle biopsy. Biopsy samples can be used in pathological analysis for definitive diagnosis.
The identification of individuals with an increased risk of family history of pancreatic cancer should also be apparent to a skilled artisan. Typically, inquiries are made about an individual's family history of pancreatic cancer. If two or more first-degree relatives (sibling-sibling or parent-child) or second-degree relatives (uncle/aunt-cousin, grandparent-grandchild, etc.) in a family have been diagnosed with pancreatic cancer, then individuals in the family can be identified as having a family history of pancreatic cancer and/or as having an increased risk of pancreatic cancer.
In the various methods of the present invention, both mutations in the PALB2 gene and PALB2 gene expression can be analyzed in a patient sample by any suitable techniques known in the art.
“Sample” as used herein refers to any biological specimen, including any tissue or bodily fluid, that can be obtained from, or derived from a specimen obtained from, a human subject. Such samples include, healthy or tumor tissue, bodily fluids (e.g., blood), waste matter (e.g., urine, stool, sputum), buccal swap, etc.
For purposes of detecting mutations in the PALB2 gene, both genomic DNA and mRNA/cDNA can be used, and both are herein referred to generically as “gene.” Numerous techniques for detecting mutations are known in the art and can all be used for the method of this invention. The techniques can be protein-based or DNA-based. In either case, the techniques used must be sufficiently sensitive so as to accurately detect the small nucleotide or amino acid variations. Very often, a probe is utilized which is labeled with a detectable marker. Unless otherwise specified in a particular technique described below, any suitable detectable marker known in the art can be used, including but not limited to, radioactive isotopes, fluorescent compounds, biotin which is detectable using strepavidin, enzymes (e.g., alkaline phosphatase), substrates of an enzyme, ligands and antibodies, etc. See Jablonski et al., Nucleic Acids Res., 14:6115-6128 (1986); Nguyen et al., Biotechniques, 13:116-123 (1992); Rigby et al., J. Mol. Biol., 113:237-251 (1977). The PALB2 gene is known in the art, and its genomic DNA sequence can be found under NCBI Reference Sequence (RefSeq) No. NG—007406.1. The Reference Sequence for PALB2 mRNA and protein sequences can be found under NCBI Reference Sequence (RefSeq) No. NM—024675.3 and NP—078951.2, respectively. Given such sequence information, methods of analyzing PALB2 genomic DNA, mRNA and protein should be apparent to skilled persons in the art. Particularly, skilled artisans would immediately be able to provide probes and primers for detecting PALB2 mutations and epitopes for developing antibodies for detecting PALB2 protein. The reference PALB2 cDNA sequence is provided in SEQ ID NO:6, and the coding sequence thereof is provided in SEQ ID NO:7. The genomic DNA sequence of the PALB2 gene is provided in SEQ ID NO:8.
In a DNA-based detection method, target DNA sample, i.e., a sample containing a genomic region of interest, or the corresponding cDNA or mRNA must be obtained from the individual to be tested. Any tissue or cell sample containing the relevant genomic DNA, mRNA, or cDNA or a portion thereof can be used. For this purpose, a tissue sample containing cell nucleus and thus genomic DNA can be obtained from the individual. Blood samples can also be useful except that only white blood cells and other lymphocytes have cell nucleus, while red blood cells are anucleate and contain only mRNA. Nevertheless, mRNA is also useful as it can be analyzed for the presence of mutations in its sequence or serve as template for cDNA synthesis. The tissue or cell samples can be analyzed directly without much processing. Alternatively, nucleic acids including the target sequence can be extracted, purified, and/or amplified before they are subject to the various detecting procedures discussed below. Other than tissue or cell samples, cDNAs or genomic DNAs from a cDNA or genomic DNA library constructed using a tissue or cell sample obtained from the individual to be tested are also useful.
Thus, preferably, all or parts of the PALB2 gene (genomic or cDNA) is amplified by any known nucleic acid amplification technique such as PCR, to a sufficient quantity and purity, and further analyzed to detect mutations. Preferably, genomic DNA is isolated from a sample, and all exonic sequences and the intron/exon junction regions including the regions required for exon/intron splicing are amplied into one or more amplicons, and further analyzed for the presence or absence of mutations.
To determine the presence or absence of mutations, one technique is simply sequencing the target genomic DNA or cDNA. Various sequencing techniques are generally known and widely used in the art including the Sanger method and Gilbert chemical method. The newly developed pyrosequencing method monitors DNA synthesis in real time using a luminometric detection system. Pyrosequencing has been shown to be effective in analyzing genetic polymorphisms such as single-nucleotide polymorphisms and thus can also be used in the present invention. See Nordstrom et al., Biotechnol. Appl. Biochem., 31(2):107-112 (2000); Ahmadian et al., Anal. Biochem., 280:103-110 (2000). The obtained sequence is then compared to the wild-type or consensus sequences such as the relevant reference sequences found in Genome Browser database.
Mutation scanning in a target gene can also be economically accomplished by the dHPLC method. Specifically, the target gene is first amplified by PCR into different amplicons, and each amplicon is analyzed by dHPLC to detect the presence or absence of heterozygosity in each amplicon. The heterozygous amplicons thus identified are further sequenced to detect mutations. See, e.g., Cao et al., Breast Cancer Res Treat., 114(3):457-62 (2009). Alternatively, high resolution melting analysis is becoming more and more popular in mutations scanning, and can also be used in the methods of the present invention. Like dHPLC, PCR amplification is used to produce amplicons from the target gene, and each amplicon is analyzed by high resolution melting analysis to detect the presence or absence of heterozygosity in each amplicon. The heterozygous amplicons thus identified are further sequenced to detect mutations. See, e.g., Jiménez et al., Clin Biochem., 42(15):1572-6 (2009).
Alternatively, the restriction fragment length polymorphism (RFLP) and AFLP method may also prove to be useful techniques. In particular, if a mutations in the target nucleic acid region results in the elimination or creation of a restriction enzyme recognition site, then digestion of the target DNA with that particular restriction enzyme will generate an altered restriction fragment length pattern. Thus, a detected RFLP or AFLP will indicate the presence of a mutation.
Another useful approach is the single-stranded conformation polymorphism assay (SSCA), which is based on the altered mobility of a single-stranded target DNA spanning the mutations of interest. A single nucleotide change in the target sequence can result in different intramolecular base pairing pattern, and thus different secondary structure of the single-stranded DNA, which can be detected in a non-denaturing gel. See Orita et al., Proc. Natl. Acad. Sci. USA, 86:2776-2770 (1989). Denaturing gel-based techniques such as clamped denaturing gel electrophoresis (CDGE) and denaturing gradient gel electrophoresis (DGGE) detect differences in migration rates of mutant sequences as compared to wild-type sequences in denaturing gel. See Miller et al., Biotechniques, 5:1016-24 (1999); Sheffield et al., Am. J. Hum, Genet., 49:699-706 (1991); Wartell et al., Nucleic Acids Res., 18:2699-2705 (1990); and Sheffield et al., Proc. Natl. Acad. Sci. USA, 86:232-236 (1989). In addition, the double-strand conformation analysis (DSCA) can also be useful in the present invention. See Arguello et al., Nat. Genet., 18:192-194 (1998).
The presence or absence of a mutation at a particular locus in a genomic region of an individual can also be detected using the amplification refractory mutation system (ARMS) technique. See e.g., European Patent No. 0,332,435; Newton et al., Nucleic Acids Res., 17:2503-2515 (1989); Fox et al., Br. J. Cancer, 77:1267-1274 (1998); Robertson et al., Eur. Respir. J., 12:477-482 (1998). In the ARMS method, a primer is synthesized matching the nucleotide sequence immediately 5′ upstream from the locus being tested except that the 3′-end nucleotide which corresponds to the nucleotide at the locus is a predetermined nucleotide. For example, the 3′-end nucleotide can be the same as that in the mutated locus. The primer can be of any suitable length so long as it hybridizes to the target DNA under stringent conditions only when its 3′-end nucleotide matches the nucleotide at the locus being tested. Preferably the primer has at least 12 nucleotides, more preferably from about 18 to 50 nucleotides. If the individual tested has a mutation at the locus and the nucleotide therein matches the 3′-end nucleotide of the primer, then the primer can be further extended upon hybridizing to the target DNA template, and the primer can initiate a PCR amplification reaction in conjunction with another suitable PCR primer. In contrast, if the nucleotide at the locus is of wild type, then primer extension cannot be achieved. Various forms of ARMS techniques developed in the past few years can be used. See e.g., Gibson et al., Clin. Chem. 43:1336-1341 (1997).
Similar to the ARMS technique is the mini sequencing or single nucleotide primer extension method, which is based on the incorporation of a single nucleotide. An oligonucleotide primer matching the nucleotide sequence immediately 5′ to the locus being tested is hybridized to the target DNA or mRNA in the presence of labeled dideoxyribonucleotides. A labeled nucleotide is incorporated or linked to the primer only when the dideoxyribonucleotides matches the nucleotide at the variant locus being detected. Thus, the identity of the nucleotide at the variant locus can be revealed based on the detection label attached to the incorporated dideoxyribonucleotides. See Syvanen et al., Genomics, 8:684-692 (1990); Shumaker et al., Hum. Mutat., 7:346-354 (1996); Chen et al., Genome Res., 10:549-547 (2000).
Another set of techniques useful in the present invention is the so-called “oligonucleotide ligation assay” (OLA) in which differentiation between a wild-type locus and a mutation is based on the ability of two oligonucleotides to anneal adjacent to each other on the target DNA molecule allowing the two oligonucleotides joined together by a DNA ligase. See Landergren et al., Science, 241:1077-1080 (1988); Chen et al, Genome Res., 8:549-556 (1998); Iannone et al., Cytometry, 39:131-140 (2000). Thus, for example, to detect a single-nucleotide mutation at a particular locus in a genomic region, two oligonucleotides can be synthesized, one having the genomic sequence just 5′ upstream from the locus with its 3′ end nucleotide being identical to the nucleotide in the variant locus, the other having a nucleotide sequence matching the genomic sequence immediately 3′ downstream from the variant locus. The oligonucleotides can be labeled for the purpose of detection. Upon hybridizing to the target nucleic acid under a stringent condition, the two oligonucleotides are subject to ligation in the presence of a suitable ligase. The ligation of the two oligonucleotides would indicate that the target DNA has a mutation at the locus being detected.
Detection of mutation can also be accomplished by a variety of hybridization-based approaches. Allele-specific oligonucleotides are most useful. See Conner et al., Proc. Natl. Acad. Sci. USA, 80:278-282 (1983); Saiki et al, Proc. Natl. Acad. Sci. USA, 86:6230-6234 (1989). Oligonucleotide probes (allele-specific) hybridizing specifically to an allele having a particular mutation at a particular locus but not to other alleles can be designed by methods known in the art. The probes can have a length of, e.g., from 10 to about 50 nucleotide bases. The target DNA and the oligonucleotide probe can be contacted with each other under conditions sufficiently stringent such that the mutations can be distinguished from the alternative variant/allele at the same locus based on the presence or absence of hybridization. The probe can be labeled to provide detection signals. Alternatively, the allele-specific oligonucleotide probe can be used as a PCR amplification primer in an “allele-specific PCR” and the presence or absence of a PCR product of the expected length would indicate the presence or absence of a particular mutation.
Other useful hybridization-based techniques allow two single-stranded nucleic acids annealed together even in the presence of mismatch due to nucleotide substitution, insertion or deletion. The mismatch can then be detected using various techniques. For example, the annealed duplexes can be subject to electrophoresis. The mismatched duplexes can be detected based on their electrophoretic mobility that is different from the perfectly matched duplexes. See Cariello, Human Genetics, 42:726 (1988). Alternatively, in a RNase protection assay, a RNA probe can be prepared spanning the mutations site to be detected and having a detection marker. See Giunta et al., Diagn. Mol. Path., 5:265-270 (1996); Finkelstein et al., Genomics, 7:167-172 (1990); Kinszler et al., Science 251:1366-1370 (1991). The RNA probe can be hybridized to the target DNA or mRNA forming a heteroduplex that is then subject to the ribonuclease RNase A digestion. RNase A digests the RNA probe in the heteroduplex only at the site of mismatch. The digestion can be determined on a denaturing electrophoresis gel based on size variations. In addition, mismatches can also be detected by chemical cleavage methods known in the art. See e.g., Roberts et al., Nucleic Acids Res., 25:3377-3378 (1997).
A great variety of improvements and variations have been developed in the art on the basis of the above-described basic techniques, and can all be useful in detecting mutations in the present invention. For example, the “sunrise probes” or “molecular beacons” utilize the fluorescence resonance energy transfer (FRET) property and give rise to high sensitivity. See Wolf et al., Proc. Nat. Acad. Sci. USA, 85:8790-8794 (1988). Typically, a probe spanning the nucleotide locus to be detected are designed into a hairpin-shaped structure and labeled with a quenching fluorophore at one end and a reporter fluorophore at the other end. In its natural state, the fluorescence from the reporter fluorophore is quenched by the quenching fluorophore due to the proximity of one fluorophore to the other. Upon hybridization of the probe to the target DNA, the 5′ end is separated apart from the 3′-end and thus fluorescence signal is regenerated. See Nazarenko et al., Nucleic Acids Res., 25:2516-2521 (1997); Rychlik et al., Nucleic Acids Res., 17:8543-8551 (1989); Sharkey et al., Bio/Technology 12:506-509 (1994); Tyagi et al., Nat. Biotechnol., 14:303-308 (1996); Tyagi et al., Nat. Biotechnol., 16:49-53 (1998). The homo-tag assisted non-dimer system (HANDS) can be used in combination with the molecular beacon methods to suppress primer-dimer accumulation. See Brownie et al., Nucleic Acids Res., 25:3235-3241 (1997).
Dye-labeled oligonucleotide ligation assay is a FRET-based method, which combines the OLA assay and PCR. See Chen et al., Genome Res. 8:549-556 (1998). TaqMan is another FRET-based method for detecting mutations. A TaqMan probe can be oligonucleotides designed to have the nucleotide sequence of the human nucleic acid spanning the variant locus of interest and to differentially hybridize with different alleles. The two ends of the probe are labeled with a quenching fluorophore and a reporter fluorophore, respectively. The TaqMan probe is incorporated into a PCR reaction for the amplification of a target nucleic acid region containing the locus of interest using Taq polymerase. As Taq polymerase exhibits 5′-3′ exonuclease activity but has no 3′-5′ exonuclease activity, if the TaqMan probe is annealed to the target DNA template, the 5′-end of the TaqMan probe will be degraded by Taq polymerase during the PCR reaction thus separating the reporting fluorophore from the quenching fluorophore and releasing fluorescence signals. See Holland et al., Proc. Natl. Acad. Sci. USA, 88:7276-7280 (1991); Kalinina et al., Nucleic Acids Res., 25:1999-2004 (1997); Whitcombe et al., Clin. Chem., 44:918-923 (1998).
In addition, the detection in the present invention can also employ a chemiluminescence-based technique. For example, an oligonucleotide probe can be designed to hybridize to either the wild-type or a variant locus but not both. The probe is labeled with a highly chemiluminescent acridinium ester. Hydrolysis of the acridinium ester destroys chemiluminescence. The hybridization of the probe to the target DNA prevents the hydrolysis of the acridinium ester. Therefore, the presence or absence of a particular mutation in the target DNA is determined by measuring chemiluminescence changes. See Nelson et al., Nucleic Acids Res., 24:4998-5003 (1996).
The detection of mutations for the present invention can also be based on the “base excision sequence scanning” (BESS) technique. The BESS method is a PCR-based mutation scanning method. BESS T-Scan and BESS G-Tracker are generated which are analogous to T and G ladders of dideoxy sequencing. Mutations are detected by comparing the sequence of normal and mutant DNA. See, e.g., Hawkins et al., Electrophoresis, 20:1171-1176 (1999).
Another useful technique that is gaining increased popularity is mass spectrometry. See Graber et al., Curr. Opin. Biotechnol., 9:14-18 (1998). For example, in the primer oligo base extension (PROBETM) method, a target nucleic acid is immobilized to a solid-phase support. A primer is annealed to the target immediately 5′ upstream from the locus to be analyzed. Primer extension is carried out in the presence of a selected mixture of deoxyribonucleotides and dideoxyribonucleotides. The resulting mixture of newly extended primers is then analyzed by MALDI-TOF. See e.g., Monforte et al., Nat. Med., 3:360-362 (1997).
In addition, the microchip or microarray technologies are also applicable to the detection method of the present invention as will be apparent to a skilled artisan in view of this disclosure. For example, to genotype an individual, genomic DNA isolated from the individual can be prepared and hybridized to a DNA microchip having probes designed based on the target gene sequence.
As is apparent from the above survey of the suitable detection techniques, it may or may not be necessary to amplify the target DNA, i.e., the genomic region of interest, or the corresponding cDNA or mRNA to increase the number of target DNA molecule, depending on the detection techniques used. For example, most PCR-based techniques combine the amplification of a portion of the target and the detection of the mutations. PCR amplification is well known in the art and is disclosed in U.S. Pat. Nos. 4,683,195 and 4,800,159, both which are incorporated herein by reference. For non-PCR-based detection techniques, if necessary, the amplification can be achieved by, e.g., in vivo plasmid multiplication, or by purifying the target DNA from a large amount of tissue or cell samples. See generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989. However, even with scarce samples, many sensitive techniques have been developed in which small genetic variations such as single-nucleotide substitutions can be detected without having to amplify the target DNA in the sample. For example, techniques have been developed that amplify the signal as opposed to the target DNA by, e.g., employing branched DNA or dendrimers that can hybridize to the target DNA. The branched or dendrimer DNAs provide multiple hybridization sites for hybridization probes to attach thereto thus amplifying the detection signals. See Detmer et al., J. Clin. Microbiol., 34:901-907 (1996); Collins et al., Nucleic Acids Res., 25:2979-2984 (1997); Horn et al., Nucleic Acids Res., 25:4835-4841 (1997); Horn et al., Nucleic Acids Res., 25:4842-4849 (1997); Nilsen et al., J. Theor. Biol., 187:273-284 (1997).
In yet another technique for detecting mutations, the Invader® assay utilizes a novel linear signal amplification technology that improves upon the long turnaround times required of the typical PCR DNA sequenced-based analysis. See Cooksey et al., Antimicrobial Agents and Chemotherapy 44:1296-1301 (2000). This assay is based on cleavage of a unique secondary structure formed between two overlapping oligonucleotides that hybridize to the target sequence of interest to form a “flap.” Each “flap” then generates thousands of signals per hour. Thus, the results of this technique can be easily read, and the methods do not require exponential amplification of the DNA target. The Invader® system utilizes two short DNA probes, which are hybridized to a DNA target. The structure formed by the hybridization event is recognized by a special cleavase enzyme that cuts one of the probes to release a short DNA “flap.” Each released “flap” then binds to a fluorescently-labeled probe to form another cleavage structure. When the cleavase enzyme cuts the labeled probe, the probe emits a detectable fluorescence signal. See e.g. Lyamichev et al., Nat. Biotechnol., 17:292-296 (1999).
The rolling circle method is another method that avoids exponential amplification. Lizardi et al., Nature Genetics, 19:225-232 (1998) (which is incorporated herein by reference). For example, SniperTM, a commercial embodiment of this method, is a sensitive, high-throughput SNP scoring system designed for the accurate fluorescent detection of specific variants. For each mutation, two linear, allele-specific probes are designed. The two allele-specific probes are identical with the exception of the 3′-base, which is varied to complement the variant site. In the first stage of the assay, target DNA is denatured and then hybridized with a pair of single, allele-specific, open-circle oligonucleotide probes. When the 3′-base exactly complements the target DNA, ligation of the probe will preferentially occur. Subsequent detection of the circularized oligonucleotide probes is by rolling circle amplification, whereupon the amplified probe products are detected by fluorescence. See Clark and Pickering, Life Science News 6, 2000, Amersham Pharmacia Biotech (2000).
A number of other techniques that avoid amplification all together include, e.g., surface-enhanced resonance Raman scattering (SERRS), fluorescence correlation spectroscopy, and single-molecule electrophoresis. In SERRS, a chromophore-nucleic acid conjugate is absorbed onto colloidal silver and is irradiated with laser light at a resonant frequency of the chromophore. See Graham et al., Anal. Chem., 69:4703-4707 (1997). The fluorescence correlation spectroscopy is based on the spatio-temporal correlations among fluctuating light signals and trapping single molecules in an electric field. See Eigen et al., Proc. Natl. Acad. Sci. USA, 91:5740-5747 (1994). In single-molecule electrophoresis, the electrophoretic velocity of a fluorescently tagged nucleic acid is determined by measuring the time required for the molecule to travel a predetermined distance between two laser beams. See Castro et al., Anal. Chem., 67:3181-3186 (1995).
In addition, the allele-specific oligonucleotides (ASO) can also be used in in situ hybridization using tissues or cells as samples. The oligonucleotide probes which can hybridize differentially with the wild-type gene sequence or the gene sequence harboring a mutation may be labeled with radioactive isotopes, fluorescence, or other detectable markers. In situ hybridization techniques are well known in the art and their adaptation to the present invention for detecting the presence or absence of a mutation in a genomic region of a particular individual should be apparent to a skilled artisan apprised of this disclosure.
Protein-based detection techniques may also prove to be useful, especially when the mutations causes amino acid substitutions or deletions or insertions or frameshift that affect the protein primary, secondary or tertiary structure. To detect the amino acid variations, protein sequencing techniques may be used. Alternatively, the recently developed HPLC-microscopy tandem mass spectrometry technique can be used for determining the amino acid sequence variations. See Gatlin et al., Anal. Chem., 72:757-763 (2000).
Other useful protein-based detection techniques include immunoaffinity assays based on antibodies selectively immunoreactive with mutant proteins or specifically with wild-type proteins. Antibodies can be used to immunoprecipitate specific proteins from solution samples or to immunoblot proteins separated by, e.g., polyacrylamide gels. Immunocytochemical methods can also be used in detecting specific protein in tissues or cells. Other well-known antibody-based techniques can also be used including, e.g., enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA), including sandwich assays using monoclonal or polyclonal antibodies. See e.g., U.S. Pat. Nos. 4,376,110 and 4,486,530, both of which are incorporated herein by reference.
The antibodies (or fragments thereof) useful in the present invention can be employed histologically—e.g., IHC, immunofluorescence or immunoelectron microscopy—for in situ detection of peptides encoded by nucleic acids of interest. In situ detection can be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody of the present invention. The antibody (or its fragment) is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample. Through the use of such a procedure, it is possible to determine not only the presence and amount of the expression product of a target gene, but also its distribution in the examined tissue. Using the present invention, a skilled artisan will readily perceive that any of a wide variety of histological methods (e.g., staining procedures) can be modified to achieve such in situ detection.
U.S. Pat. No. 5,965,377 discloses an antibody-based method for determining the presence of mutated protein in cells expressing the protein, wherein the normal protein contains amino-terminus and carboxy-terminus regions and wherein the mutated protein is typically a foreshortened protein from which carboxy-terminus regions are missing. This method can be adapted to detect truncation mutations in the target gene of the present invention. Specifically, an antibody reactive with the N-terminus of the target protein and an antibody reactive with the C-terminus of the target protein are used to react with a cell sample, and the ratio between the reactivity with the C-terminus and N-terminus can be obtained. If the reactivity with the C-terminus is about zero or no greater than about half of the reactivity with the N-terminus in the sample, it would indicate the presence of a truncation mutation in the gene. The antibody reactivity can be measured by any suitable immunoassays, e.g., immunohistochemistry (IHC) and ELISA.
The antibody based methods described above can also be used to determine generally the expression level of the target gene, PALB2, as will be apparent to skilled artisan.
For purposes of detecting a reduced level of gene expression, either mRNA or protein level in a sample from a patient can be determined by conventional methods known in the art. Protein expression level in a sample can be determined using an immunoassay described above. For mRNA level, typically hybridization of DNA probes or primers is utilized. For example, for mRNA expression level, qRT-PCT can be used. mRNA can be isolated from a particular sample, and the target gene mRNA, and preferably in addition, a reference gene mRNA (typically a housekeeping gene), are amplified by qRT-PCR, and the relative amount of the target gene mRNA is determined, which is compared to a predetermined reference standard level (e.g., an average level determined in a plurality of normal samples). Alternatively, digital PCR is also useful.
Additionally, gene expression level can also be detected indirectly by determining the methylation status of the target gene. If the target gene is methylated at a greater extent than normal, then the target gene expression is usually reduced. Methods for determining gene methylation status are well known in the art.
The present invention also provides kits for use in the methods of the present invention. The kits may include a carrier for the various components of the kits. The carrier can be a container or support, in the form of, e.g., bag, box, tube, rack, and is optionally compartmentalized. The carrier may define an enclosed confinement for safety purposes during shipment and storage. The kit also includes various components useful in detecting mutations, or determining gene expression (mRNA and protein) levels, using the above-discussed detection techniques. For example, the detection kit may include one or more oligonucleotides useful as primers for amplifying all or a portion of the PALB2 genomic or cDNA. The detection kit may also include one or more oligonucleotide probes for hybridization to the PALB2 genomic or cDNA or mRNA. Optionally the oligonucleotides are affixed to a solid support, e.g., incorporated in a microchip or microarray included in the kit.
In some embodiments of the invention, the detection kit contains one or more antibodies selectively immunoreactive with PALB2 protein, for example antibodies selectively immunoreactive with the N-terminus of PALB2 protein, and/or antibodies selectively immunoreactive with the C-terminus of PALB2 protein.
Various other components useful in the detection techniques may also be included in the detection kit of this invention. Examples of such components include, but are not limited to, Taq polymerase, deoxyribonucleotides, dideoxyribonucleotides other primers suitable for the amplification of a target DNA sequence, RNase A, mutS protein, and the like. In addition, the detection kit should include instructions on using the kit for the various methods of the present invention as described above.
As discussed above, the present invention provides a method of predicting an individual's response to therapy for pancreatic cancer, and a method of treating pancreatic cancer. For example, in one embodiment, once a patient is identified as having a germline or somatic mutation in a PALB2 gene or a reduced level of PALB2 gene expression, the patient is treated with a therapy that induces DNA double-strand breaks (DSB) or interferes with DNA double-strand breaks (DSB) repairs by homologous recombination in tumor cells. Many therapies are known in the art that “induce DNA double-strand breaks (DSB) or interfere with DNA double-strand breaks (DSB) repairs by homologous recombination in tumor cells”. For example, radiation therapy induces DNA damages including DNA double-strand breaks. Chemotherapeutics such as DNA damaging agents are also useful including, but not limited to, alkylating agents and topoisomerase inhibitors, which are designed to damage DNA in order to prevent cancer cells from reproducing.
Alkylating agents attach an alkyl group (CnH2n+1) to DNA causing DNA damages. Examples of alkylating agents include nitrogen mustards such as cyclophosphamide, mechlorethamine, uramustine, melphalan, chlorambucil, and ifosfamide; nitrosoureas such as carmustine, lomustine, streptozocin, alkyl sulfonates, and busulfan; platinum agents such as cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitrate and picoplatin; procarbazine; altretamine; tetrazines such as dacarbazine, mitozolomide and temozolomide; and antibiotics such as mitomycins, particularly mitomycin C.
Topoisomerase inhibitors inhibit the activity of topoisomerase enzymes (topoisomerase I and II), and interfere with the catalyzing the breaking and rejoining of the phosphodiester backbone of DNA strands during DNA replication and cell division. Examples of topoisomerase inhibitors include topoisomerase i inhibitors such as irinotecan, topotecan, camptothecin and D; topoisomerase ii inhibitors such as etoposide and doxorubicin.
In addition, inhibitors of DNA repair enzymes (e.g., PARP inhibitors) are also particularly effective in treating PALB2 defective pancreatic cancer. PARP (poly (ADP-ribose) polymerase) is a ubiquitous nuclear enzyme that is involved in DNA damage repairs. Examples of PARP inhibitors include BSI-201 by BiPar Sciences Inc. (Clin. Cancer Res., 15(20):6367-77 (2009)); olaparib (AZD2281) by AstraZeneca (N. Engl. J. Med., 361(2):123-34 (2009)), ABT-888 by Abbott Laboratories, Inc. (J. Clin. Oncol., 27(16):2705-11 (2009), and AG014699 by Pfizer Inc. (Clin. Cancer Res., 14(23):7917-23 (2008)).
Thus, in some embodiments of the present invention, a method of predicting an individual's response to therapy comprises detecting in a sample obtained from an individual, a mutation in the PALB2 gene, wherein the presence of the mutation would indicate that the individual has an increased likelihood of responding to a therapy that comprises radiation, an alkylating agent, a topoisomerase inhibitor, and/or a PARP inhibitor. In specific embodiments, the therapy comprises at least one mitomycin C, cisplatin, carboplatin, oxaliplatin, and a PARP inhibitor (e.g., BSI-201 or olaparib).
In some embodiments, the treatment method of the present invention comprises detecting, in a patient, a mutation in the PALB2 gene or a reduced PALB2 gene expression, and in the presence of such a mutation or reduced gene expression, treating the patient with a therapy that comprises radiation, an alkylating agent, a topoisomerase inhibitor, and/or a PARP inhibitor. In specific embodiments, the therapy comprises at least one mitomycin C, cisplatin, carboplatin, oxaliplatin, and a PARP inhibitor (e.g., BSI-201 or olaparib).
EXAMPLE 1
Exomic Sequencing Identifies PALB2 as a Pancreatic Cancer Susceptibility Gene
We have sequenced the entire genomes of five individuals. In addition, 68 patients have been evaluated for tumor-specific mutations in all exons of protein coding genes (exomic sequencing). This coincidentally yielded information about germline sequence variations in these individuals. To explore the utility of such information, we evaluated a pancreatic cancer patient (Pa10) whose tumor DNA had been sequenced. This patient had familial pancreatic cancer, as defined by the fact that his sister also had developed the disease.
Among the 20, 661 coding genes analyzed, we identified 15,461 germline variants in Pa10 not found in the reference human genome. Of these, 7318 were synonymous, 7721 were missense, 64 were nonsense, 108 were at splice sites, and 250 were small deletions or insertions (54% in-frame). Past studies have shown that tumors arising in patients with a hereditary predisposition harbor no normal alleles of the responsible gene: one allele is inherited in mutant form, often producing a stop codon, and the other (wild type) allele is inactivated by somatic mutation during tumorigenesis. In Pa10, only three genes met these criteria: SERPINB12, RAGE and PALB2. Of these, we considered PALB2 to be the best candidate because germline stop codons in SERPINB12 and RAGE, but not in PALB2, are relatively common in healthy individuals and because germline PALB2 mutations have previously been associated with breast cancer predisposition and Fanconi anemia although its function is not well understood. Pa10 harbored a germline deletion of 4 by (TTGT at c.172-175) producing a frameshift at codon 58; the pancreatic cancer that developed in Pa10 had also somatically acquired a transition mutation (C to T) at a canonical splice site for exon 10 (IVS10+2).
To determine whether PALB2 mutations occur in other patients with familial pancreatic cancer, we sequenced this gene in a cohort of 96 familial pancreatic cancer patients, 90 of which were of Caucasian ancestry. Sixteen of these patients had one first degree relative with pancreatic cancer and 80 had at least two additional relatives, at least one of which was first degree, with the disease. Truncating mutations were identified in three of the 96 patients, each producing a different stop codon (FIG. 1). The average age-of-onset of pancreatic cancer in these families was 66.7 years, similar to the mean age of onset of 65.3 years in the families without PALB2 mutations. We determined the germ-line sequence of an affected brother in one of these kindreds, and he harbored the same stop codon. Truncating mutations in PALB2′ are rare in individuals without cancer; none have been reported among 1,084 normal individuals in a previous study using a cohort of similar ethnicity to ours. While some families we identified with a PALB2 stop mutation had a history of both breast and pancreatic cancer, breast cancer was not observed in all families. From these data, PALB2 appears to be the second most commonly mutated gene for hereditary pancreatic cancer. Interestingly, the most commonly mutated gene is BRCA2, whose protein product is a binding partner for the PALB2 protein.
In summary, through complete, unbiased sequencing of protein-coding genes, we have discovered a gene responsible for a hereditary disease. We note that this approach is independent of classical methods for gene discovery, such as linkage analysis, which can be challenging in the absence of large families with monogenic diseases. We predict that variations of the approach described here will soon become a standard tool for the discovery of disease-related genes.
EXAMPLE 2
PALB2 Mutations and Response to DNA Damaging Agents in Pancreatic Cancer
We report here a patient with gemcitabine resistant pancreatic cancer for whom mitomycin C treatment, selected based on its preclinical activity in a personalized xenograft generated from the patient's surgically resected tumor, resulted in long lasting (36+ months) cancer control. Global genetic sequencing revealed biallelic inactivation of the PALB2 gene in this patient's cancer that disrupts BRCA1 and 2 interactions. This work suggests that inactivation of PALB2 is a determinant of response to DNA damage in pancreatic cancer and a new target for personalizing cancer treatment. Integrating personalized xenografts with unbiased genomic sequencing led to individualized treatment and the identification of a new biomarker of drug response.
The patient described in this report was enrolled in the J0507 Johns Hopkins Medical Institute clinical trial (NCT00276744). This is a pilot prospective clinical trial in which patients with resectable pancreatic cancer operated at the Johns Hopkins Hospital consent to have a portion of their resected tumor implanted and propagated in nude mice. These xenografted tumors are treated with a set of anticancer agents with the goal to identify the most effective agents that can be used to treat the patient's cancer.
Six-week-old female athymic nude mice (Harlan, Ind., US) are implanted with tumor tissues collected at the time of surgery. The research protocol was approved by the Johns Hopkins University Animal Care and Use Committee and animals were maintained in accordance to guidelines of the American Association of Laboratory Animal Care. The detail process for generation of xenografts and treatment protocols has been published elsewhere. Rubio-Viqueira et al., Clin. Cancer Res., 12(15):4652-61 (2006). Tumors were allowed to grow until reaching  ̃200 mm3, at which time mice were randomized to: 1) Control; 2) MMC 5 mg/Kg/ip single dose; or 3) cisplatin 6 mg/Kg single dose, with 5-6 mice (10 evaluable tumors) in each group. Tumor size was evaluated two times per week by caliper measurements using the following formula: tumor volume=[length X width2]/2. Relative tumor growth inhibition was calculated by relative tumor growth of treated mice divided by relative tumor growth of control mice since the initiation of therapy (T/C).
Genomic analysis. The sequences of 23,219 transcripts representing 20,661 protein-coding genes in the patient's cancer were determined. Whenever a variant was identified in the cancer, the patient's germline was also sequenced, revealing information about the germline variations in this patient.
Co-immunoprecipitation. To assess BRCA1 and BRCA2 nuclear binding a co-immunoprecipitation assay was performed using a commercially available kit (Thermo Scientific #23600, Waltham, Mass.). Samples from tumor JH033, sensitive to MMC and Panc185, resistant to MMC were used. Monoclonal antibody OP107 against BRCA1, purchased from Calbiochem (San Diego, La.), was used to immunoprecipitate the BRCA1/2 complex. After stabilization of the antibodies to the resin by a covalent union, the samples were added and incubated for 24 hours. Samples were eluted, electrophoresed and further immunoblotted with mAB against BRCA1 (OP107) and BRCA2 (OP95) purchased from Calbiochem (San Diego, La.).
Western Blot Analysis. Tumor tissues (75 mg/mouse) from control and treated with MMC were minced on ice in prechilled lysis buffer. 30 μg of sample was electrophoresed and further electrotransfered to Immobilon-P membranes (Millipore, Bedford, Mass.). Primary antibodies for BRCA1 (OP107), BRCA2 (OP95), or PALB2 (2134.00.02) from Strategic Scientific Inc. (Newark, Del.) and FANCD2 (4945) from Cell Signaling (Danvers, Mass.) were used to blot the membranes.
Results: Clinical Case. A 61 year-old male, with family history of pancreatic cancer, who had been previously tested and found to be wild-type for the BRCA2 gene, underwent a distal pancreatectomy and splenectomy for a pT3N1M0 infiltrating ductal adenocarcinoma of the pancreas. The patient had a 4 cm, poorly differentiated adenocarcinoma that had metastatized to 8 of 26 resected lymph nodes with prominent extranodal extension. Venous and perineural invasion were identified and the carcinoma extended to involve the celiac artery margin of resection. The patient was enrolled in J0507 and a portion of the surgically resected tumor, coded as JH033, was xenografted in nude mice. Two months after surgery, prior to initiating adjuvant treatment, the patient was found to have a biopsy proven metastasis to a supraclavicular lymph node and his CA 19-9 rose to 10,132 U/ml (FIGS. 2A and B). The patient was treated with single agent gemcitabine but developed significant disease progression after 4 months with pleural effusion, loco-regional progression in the abdominal cavity, and a CA 19-9 of 98,405 U/ml (FIGS. 2C and D).
At this time, the results of the xenograft treatment studies became available (FIG. 2A), and based on the response of the patient's xenografted cancer to MMC, the patient was treated with MMC 8 mg/m2/28 days for a total of five courses. After treatment with MMC the CT scan findings improved and the CA 19-9 level normalized (FIG. 2D). This response was maintained for 22 months, after which the CA 19-9 rose to 392 U/ml and a new lung nodule developed in the left upper lobe (FIG. 2E). The patient was treated with 2 additional cycles of MMC with biochemical and CT scan response (FIGS. 2D and F) but developed incipient renal failure. Because the xenograft was also sensitive to cisplatin, platinum-based chemotherapy was initiated and the patient received three cycles of this agent. At his last follow up, three years after surgical resection, his CA 19-9 is 39 U/ml and the patient remains asymptomatic.
Mechanism underlying unique sensitivity to DNA damaging agents. This patient tumor was recently sequenced as a part of an effort to sequence the pancreatic cancer genome. The results of this sequencing allowed us to assess, in an unbiased fashion, potential genetic determinants of this patient's remarkable response to MMC. His carcinoma was found to have a somatically acquired transition mutation (C to T) at a canonical splice site for exon 10 (IVS10+2) in the PALB2 gene (FIG. 3A). A subsequent study identified a germline deletion of 4 base pairs (TTGT at  ̃172 to 175) that produced a frameshift mutation at codon 58 of PALB2. The PALB2 gene was therefore biallelically inactivated in this patient's cancer. Functional analysis demonstrated that this tumor has an intact FA complex 1 system leading to successful mono-ubiquitination of the FANCD2 protein (FIG. 3B, first lane) similar to the Panc 185 tumor used as a control that has a wild-type PALB2 gene and is resistant to MMC. The biallelic inactivation of PALB2 in our patient's tumor disrupts the interaction between the complex BRCA1/BRCA2, which is essential for double strand break repair (FIG. 3C).
Thus, we have described herein the remarkable clinical outcome of a patient with advanced, gemcitabine resistant, pancreatic cancer who was treated with DNA damaging agents based on the observation of significant activity of this class of agents against a personalized xenograft generated from the patients own tumor. Nearly complete sequencing of all of the coding genes in this patient's cancer revealed biallelic inactivation of PALB2, a DNA repair gene, loss of which mechanistically explains the observed sensitivities of the patient's cancer. This study highlights the potential power of global genomic sequencing for the discovery of novel markers of drug activity.
We report that an unbiased genomic sequencing of a patient's tumor led to the discovery of a genetic defect that explains the unique susceptibility of this patient's tumor to DNA damaging agents. B1allelic inactivation of the PALB2 gene alters the interaction of the BRCA1 and 2 proteins, required for proper functioning of the DNA damage repair pathway. Zhang et al., Curr. Biol., 19(6):524-9 (2009). Response of pancreatic cancer to DNA damaging agents can now be predicted by sequencing the PALB2 and BRCA2 genes. This situation is analogous to the EGFR gene mutations in lung cancer and response to EGFR inhibitors. It is likely that this is a generalizable phenomenon and extreme, clinically significant predictors of patient-specific responses to anticancer agents will be identified as additional cancers are sequenced. Importantly, the process presented here can be escalated and systematically used to discover rare, albeit clinically relevant, genetic defects that confer a vulnerability to therapeutic interventions. As the ability to obtain global genomic information from individual patient tumors becomes cheaper and more easily obtainable, living tumor xenografts with validated clinical response will become a viable platform to systematically explore “connections” between drug response and genetic determinants of response.
In summary, we describe here a patient with poor prognosis pancreatic cancer for whom a personalized xenograft model generated from the patient's own tumor, linked to global genomic sequencing, led to the discovery of a highly effective treatment regimen as well as the genetic defect explaining the observed sensitivity of this patient's cancer to DNA damaging agents. This approach forms the basis for linking personalized xenografts with global genomic sequencing for the development of personalized treatment and biomarker discovery.
All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The mere mentioning of the publications and patent applications does not necessarily constitute an admission that they are prior art to the instant application.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

## Claims

1. A method of administering a therapy to a human with a pancreatic cancer, comprising:
testing and detecting in a sample obtained from the human with a pancreatic cancer the presence of a mutation in the PALB2 gene selected from the group consisting of: 172-5 del TTGT; IVS5-1 G>T; 3116 del A; 3256 C>T, IVS10+2 C>T; and
administering to said human a therapy that induces DNA damage or interferes with DNA damage repair in tumor cells.

2. The method of claim 1 wherein said therapy is radiation therapy.

3. The method of claim 1 wherein said therapy is a DNA damaging agent.

4. The method of claim 1 wherein said therapy is an inhibitor of a DNA repair enzyme.

5. The method of claim 1 wherein said therapy comprises treatment with a PARP inhibitor.

6. The method of claim 1 wherein said sample is a normal tissue sample.

7. The method of claim 1 wherein the human has a family history of pancreatic cancer.