WO2019202536A1

WO2019202536A1 - Genotyping assays to identify mutations in xaf1

Info

Publication number: WO2019202536A1
Application number: PCT/IB2019/053202
Authority: WO
Inventors: Emilia Modolo PINTO; Raul C. RIBEIRO; Gerard P. ZAMBETTI
Original assignee: St. Jude Children's Research Hospital
Priority date: 2018-04-18
Filing date: 2019-04-17
Publication date: 2019-10-24
Also published as: BR112020021218A2

Abstract

Methods and compositions are provided for monitoring, treating, and identifying subjects at risk of developing a cell-proliferative disorder. Mutations in XAF1 have been identified that can result in reduced XAF1 activity and a higher risk of developing cancer. Thus, by detecting mutations in an XAF1 polynucleotide that reduce XAF1 activity, subjects at risk of developing a cell-proliferative disorder can be identified so that risk-adaptive therapy can be implemented or surveillance of the subject increased in order to reduce symptoms of the disorder. Further provided are compositions and kits for the detection of mutations in a XAF1 polynucleotide.

Description

GENOTYPING ASSAYS TO IDENTIFY MUTATIONS IN XAF1

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was supported by funds received from the American Lebanese Syrian Associated Charities (ALSAC) and NIH/NCI Cancer Center support Grant CA21765. The United States government has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates generally to the identification of subjects at an increased risk of developing a cell-proliferative disorder, wherein the subjects have a mutation in &XAF1

polynucleotide.

BACKGROUND OF THE INVENTION

The TP53 gene encodes a tumor suppressor protein that plays a critical role in protection during the development of cancers. p53 is crucial in multi-cellular organisms where it regulates cell cycle, and thus acts as a tumor suppressor that is involved in preventing cancer. Further, it is a transcription factor that regulates multiple genes involved in cell cycle control, apoptosis, DNA repair and senescence. Tumor initiation and maintenance depend upon inactivation of p53 and therefore the pathways under its control that would otherwise deter uncontrolled cancer cell growth. Tumor suppressor protein p53 controls apoptosis and cell cycle arrest pathways through direct binding to p53 response elements in the promoters of target genes in those pathways.

Tumor suppressor protein p53 is a homotetramer, each chain of which is composed of a transactivation domain located in the N-terminal region, a DNA-binding core domain located in the central region, and tetramerization and regulatory domains located in the C-terminal region.

Approximately 50% of all human cancers have mutant p53. And approximately 75% of such cancers have a single missense mutation in the DNA-binding core domain. The six most frequently mutated amino acid residues in p53's DNA-binding core domain are Arg-248, Arg-273, Arg-l75, Gly-245, Arg-249 and Arg-282.

X-linked inhibitor of apoptosis (XIAP)-associated factor 1 {XAFP) is a putative tumor suppressor gene, which encodes a 33.l-kDa protein with seven zinc fingers. Loss or reduction of XAF1 expression due to aberrant promoter hypermethylation is associated with the advanced stage and high grade of many cancers. A recent study demonstrated that PTEN-mx\\ mouse prostate tumors showing resistance to androgen-deprivation therapy have reduced levels of XAF1, and its reduction is associated with recurrence and metastasis in human samples. Among at least five distinct XAF1 transcripts (XAF1A-E) expressed in normal tissues, the full-length transcript (XAF1A) is preferentially inactivated in human tumors, whereas truncated short isoforms are rather increased, suggesting that these variants may function differentially or elicit a dominant-negative action.

There remains a need for early prediction of cancer risk in order to better diagnose and treat subjects. The use of XAF1 mutations alone or in combination or not with TP53 mutations can serve as a surprising combination of markers in order to identify subjects at risk of developing a cell- proliferative disorder, such as cancer.

BRIEF SUMMARY OF THE INVENTION

Methods and compositions are provided for identifying a subject at risk of developing a cell-proliferative disorder by detecting a mutation of an XAF1 polynucleotide in a sample obtained from a subject having the R337H-ZP53 mutation or in a subject without the R337H-ZP53 mutation. While individuals harboring the R337H- TP53 mutation are often predisposed to a relatively low risk of contracting a cell-proliferative disorder, carriers of the R337H- TP53 mutation plus a mutation in th eXAFl polynucleotide are at a significantly higher risk of developing a cell- proliferative disorder. For example, a mutation in codon 134 of th eXAFl polynucleotide set forth in SEQ ID NO: 1 can introduce a stop codon that can lead to a truncated, non-functional XAF1 polypeptide. Without the tumor suppressor activity of XAF1, cancer development is more likely. Accordingly, by identifying subjects having a mutation in a XAF1 polynucleotide that results in a XAF1 polypeptide having reduced activity, the subject can be identified as at risk of developing a cell proliferative disorder and can undergo corresponding interventions or surveillance to prevent or reduce the onset of symptoms of the disorder. Likewise, by identifying subjects having any reduced expression of XAF1 or reduced XAF1 activity, the subject can be identified as at risk of developing a cell proliferative disorder and can undergo corresponding interventions or surveillance to prevent or reduce the onset of symptoms of the disorder.

Further provided are kits for identifying subjects at an increased risk of developing a cell proliferative disorder by detecting a mutation in XAF1 or by detecting reduced XAF1 expression or activity. Accordingly, various methods of instituting and altering surveillance and treatment protocols following identification of a subject at risk of developing a cell-proliferative disorder are also provided. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the chromosome 17 with the 2cM sequence evaluated in this study. The two haploblocks observed in this population study are represented in grey bars. The identification and location of SNPs and microsatellites markers used for haplotype determination in this study were also included.

Figure 2 presents Genotyping results for 7P53-R337H and XAF1-E134* mutation. Figure 2 A shows a exon 10 fragment of TP 53 gene encompassing the codon 337 in wild-type,

heterozygous abd R337H sequence. Figure 2B showing XAFl-E 134* mutation in homozygous, heterozygous and wild-type sequence. This variant was verified by site restriction analysis using Hpa I with the respective homozygous, heterozygous and wild-type control DNA. Six probands represented in this plot are heterozygous for the referred mutation.

Figure 3 shows a representative pedigree of a Spanish family harboring ZP53-R337H and XTF/-E134* mutations (+). The proband is a pediatric ACT (III- 1 ) harboring both mutations as his healthy father and both healthy siblings. Relatives with cancer and tested positive are prevalent in the carrier side. The pedigree represents the current patient’s age or age at deceased.

Figure 4 presents an illustrative pedigree showing the segregation pattern for both haploblocks. The yellow bar represents DNA sequence having both haploblocks (TP53 and XAF1 mutations) that was present in the mother and segregate in the proband. Patient IV-2 was found to have the TP53-R337H mutation but was negative for the XAF1-E134* mutation.

Figure 5A details transient transfection of wild-type and TP53-R337H into p53 negative Saos-2 cells. Figure 5B presents Western blot data for the transient transfection experiment.

Figure 6 presents the results of a coalescence analysis for TP53-R337H allele to estimate time to the most recent common ancestor for both mutations assuming generation time of 25 yrs (Fig. 6A) and with a strong bottleneck (Fig. 6B)

Figure 7 presents the results of a coalescence analysis to estimate time to the most recent common ancestor for the XAF1-E134* allele assuming generation time of 25 yrs (Fig. 7A) and with a strong bottleneck (Fig. 7B).

Figure 8 details transient transfection of p53 -responsive reporters with or without p53 and XAF1 expression vectors into p53 negative Saos-2 cells.

Figure 9 reports transient transfection data with an independent p53-responsive promoter reporter. DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the inventions are shown. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

I Overview

Methods and compositions are provided for monitoring, treating, and identifying subjects at risk of developing a cell-proliferative disorder. As discussed in further detail herein, mutations in XAF1 have been identified that can result in reduced XAF1 activity and a higher risk of developing cancer. Thus, by detecting mutations in an XAFl polynucleotide that reduce XAF1 activity, subjects at risk of developing a cell-proliferative disorder can be identified so that risk-adaptive therapy can be implemented or surveillance of the subject increased in order to reduce symptoms of the disorder.

II. Methods for detecting mutation in a XAF1 polynucleotide

Methods and compositions disclosed herein identify subjects at an increased risk of developing a cell-proliferative disorder when the subjects have an altered XAF1 genotype. In some embodiments, the subjects have an altered XAF1 genotype along with a mutation in the TP53 gene. The TP53 gene encodes a tumor suppressor protein that plays a critical role in protecting against cancer. As used herein,“p53,”“phosphoprotein p53,” and“tumor suppressor p53,” refer to the tumor suppressor protein encoded by the TP53 gene. Tumor suppressor p53 is crucial in multi- cellular organisms where it regulates cell cycle and other integral processes, and thus acts as a tumor suppressor that is involved in preventing cancer. Further, it is a transcription factor that regulates genes involved in cell cycle control, apoptosis, DNA repair and senescence. TP53 is one of the most commonly mutated genes in human cancer, but only a small number of mutations have been studied in depth for their contribution to cancer progression. In some cases, frameshift or nonsense mutations result in the loss of p53 protein expression, as seen with other tumor suppressors. However, more frequently, the tumor-associated alterations in p53 result in missense mutations, leading to the substitution of a single amino acid in the p53 protein that can be stably expressed in the tumor cell. Further, some mutations in p53 can give rise to a more aggressive tumor profile, indicating that they have acquired separate functions in promoting tumorigenesis.

The methods and compositions identified herein rely on detecting mutation of an XAF1 polynucleotide, or reduced expression or activity of XAF1, to identify subjects having that are at increased risk of developing a cell-proliferative disorder compared to a control group of subjects with a wild-type XAF1 genotype or phenotype. Thus, detection of a mutation of an XAF1 polynucleotide, or reduced expression or activity of XAF1, can identify patients at an increased risk of developing a cell proliferative disorder. In some embodiments, the patients further have a mutation of the TP53 gene. For example, the patient population can have a missense, nonsense, or frameshift mutation of TP53 that reduces wild type TP53 activity. The missense R337H mutation of p53 has been described as a founder mutation in a great majority of Brazilian patients with adrenocortical tumors. Pinto et al ., Arq Bras Endocrinol Metab (2004) 48(5): 647-650. Thus, in specific embodiments, subjects comprising a TP53 mutation comprise the R337H mutation on chromosome 17. In certain embodiments, the R337H mutation reduces TP53 activity.

As used herein reduced TP53 activity refers to a reduction in wild type activity or an alteration of the activity of wild type TP53 to have an activity other than wild type activity.

Accordingly, reduction in TP53 activity refers to a reduction of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 5-10%, 10-20%, 10-30%, 10- 40%, 20-30%, 20-40%, 30-40%, 30-50%, 40-50%, 40-60%, 50-60%, 50-70%, 60-70%, 60-80%, 70-80%, 70-90%, 80-90%, 80-100%, 90-100%, or 95-100% compared to wild type TP53 activity. TP53 activity can be identified, for example, by assaying the levels and/or activation status of TP53 or identifying mutations in the TP 53 gene, such as the R337H mutation. For example, a mutation in TP53 could indicate that TP53 activity is decreased. Assays that can detect the level or activation status of proteins are known in the art and include western blots and protein array analysis.

Patients harboring a TP53 mutation can be at an increased risk of developing a cell- proliferative disorder. Further, those patients harboring a TP53 mutation and a mutation in a XAIJ polynucleotide can be at a greater risk of developing a cell-proliferative disorder than subjects harboring the TP53 mutation alone. X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1 ) is a tumor suppressor gene encoding a 33.1 kDa protein with seven zinc fingers. XAF1 has been shown to act as a feedback activator of p53 and to play a role in p53 -mediated cell-fate decisions through the modulation of E3 ubiquitin ligases. As used herein,“XAF1 activity” can refer to the interference of E3 ubiquitin ligase MDM2 binding to p53 and/or the promotion of homeodomain-interactive protein kinase 2 (HIPK2)-mediated p53 phosphorylation.

As used herein a XAF1 polynucleotide refers to any polynucleotide encoding a functional XAF1 polypeptide. XAF1 polynucleotides are known in the art and available in public sequence databases. Examples oiXAFl polynucleotides include, but are not limited to sequences deposited under accession numbers: NP_059993.2 or NP_954590. l.

According to the methods and compositions disclosed herein, any mutation in anXAFl polynucleotide or XAF1 polypeptide can indicate that the subject harboring the mutation is at an increased risk for developing a cell-proliferative disorder. In some embodiments, the mutation in an XAF1 polynucleotide is a missense mutation, a nonsense mutation, or a frameshift mutation. For example, the mutation can be a missense mutation, a nonsense mutation, or a frameshift mutation that produces a truncated XAF1 polypeptide, a non-functional XAF1 polypeptide, or an XAF1 polypeptide with reduced activity. Any codon of the XAF1 polynucleotide can contain the missense, nonsense, or frameshift mutation disclosed herein. In particular embodiments, the mutation is a nonsense mutation at codon 134 or codon 115. For example, the mutation can be a mutation in codon 134 of SEQ ID NO: 1 or codon 115 of SEQ ID NO: 1. In specific embodiments, the mutation is a GAA to TAA mutation (rsl46752602) in codon 134 of SEQ ID NO: 1 or codon 115 of SEQ ID NO: 1. As used herein the XAF1 genotype refers to the genetic sequence of a XAF1 polynucleotide. Likewise, the XAF1 phenotype refers to the cellular effect of a functional XAF1 polypeptide, or XAFl activity.

Mutations in XAF1 can be detected by any means known in the art for detecting alterations in a genetic sequence. For example, mutations mXAFl polynucleotides can be detected by restriction enzyme-PCR based assays, genotyping assays, Taqman genotyping, hybridization assays, amplification assays, ELISA or antibody-based assays for detecting truncated proteins, Western blot analysis, immunohistochemistry, immunofluorescence and/or sequencing. Sequencing can be performed using any number of methods, kits or systems known in the art. One example is using dye terminator chemistry and an ABI sequencer (Applied Biosystems, Foster City, CA). Sequencing also may involve single base determination methods such as single nucleotide primer extension ("SNapShot" sequencing method) or allele or mutation specific PCR. In other embodiments, mutations mXAFl can be detected by hybridization methods such as dynamic allele- specific hybridization, molecular beacons, SNP microarrays; enzyme-based assays, such as restriction fragment length polymorphism, PCR-based methods, Flap endonuclease, primer extension, 5' nuclease, and oligonucleotide ligation assay; or post-amplification methods such as single strand conformation polymorphism, temperature gradient gel electrophoresis, denaturing high performance liquid chromatography, high resolution amplicon melting, use of DNA mismatch-binding proteins, SNPlex, and surveyor nuclease activity.

Mutations in aXTF/polynucleotide and XAF1 activity can also be determined by measuring the methylation status of th eXAFl polynucleotide. The terms "methylation status" or "methylation level" refer to the presence, absence, and/or quantity of methylation at a particular nucleotide, or nucleotides within a portion of DNA. The methylation status of a particular DNA sequence (e.g., a XAF1 polynucleotide as described herein) can indicate the methylation state of every base in the sequence or can indicate the methylation state of a subset of the base pairs (e.g., of cytosines or the methylation state of one or more specific restriction enzyme recognition sequences) within the sequence, or can indicate information regarding regional methylation density within the sequence without providing precise information of where in the sequence the

methylation occurs. The methylation status can optionally be represented or indicated by a

"methylation value" or "methylation level." A methylation value or level can be generated, for example, by quantifying the amount of intact DNA present following restriction digestion with a methylation dependent restriction enzyme. In this example, if a particular sequence in the DNA is quantified using quantitative PCR, an amount of template DNA approximately equal to a mock treated control indicates the sequence is not highly methylated whereas an amount of template substantially less than occurs in the mock treated sample indicates the presence of methylated DNA at the sequence. Accordingly, a value, i.e., a methylation value, represents the methylation status and can thus be used as a quantitative indicator of methylation status. This is of particular use when it is desirable to compare the methylation status of a sequence in a sample to a threshold value. A "methylation-dependent restriction enzyme" refers to a restriction enzyme that cleaves or digests DNA at or in proximity to a methylated recognition sequence, but does not cleave DNA at or near the same sequence when the recognition sequence is not methylated. Methylation-dependent restriction enzymes include those that cut at a methylated recognition sequence (e.g., Dpnl) and enzymes that cut at a sequence near but not at the recognition sequence (e.g., McrBC). It is further recognized that mutations in an XAF1 polynucleotide may be detected along with other markers in a multiplex or panel format. Markers are selected for their predictive value alone or in combination with the XAF1 marker locus. Ultimately, the information provided by the methods disclosed herein will assist a physician in choosing the best course of treatment for a particular patient.

As used herein, the use of the term“polynucleotide” is not intended to limit the present invention to polynucleotides comprising DNA. Those of ordinary skill in the art will recognize that polynucleotides can comprise ribonucleotides and combinations of ribonucleotides and

deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The polynucleotides of the invention also encompass all forms of sequences including, for example, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and any other nucleic acid form.

The term "gene" refers to a nucleic acid (e.g., DNA) sequence that comprises coding sequences necessary for the production of a polypeptide, precursor, or RNA (e.g., rRNA, tRNA). The term also encompasses the coding region of a structural gene and the sequences located adjacent to the coding region on both the 5' and 3' end which allow for the expression of the sequence. Sequences located 5' of the coding region and present on the mRNA are referred to as 5' non-translated sequences. Sequences located 3' or downstream of the coding region and present on the mRNA are referred to as 3' non- translated sequences. A genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns" or "intervening regions" or "intervening sequences." Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nuclear or primary transcript; introns therefore are absent in the mature messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

As used herein, the“nucleic acid complement” of a sample comprises any polynucleotide contained in the sample. The nucleic acid complement that is employed in the methods and compositions disclosed herein can include all of the polynucleotides contained in the sample or any fraction thereof. For example, the nucleic acid complement could comprise the genomic DNA and/or the mRNA and/or cDNA of a given biological sample. Thus, mutations in XAF1 can be detected in the genomic DNA or, alternatively, the level and/or activity of XAF1 mRNA or XAF1 protein can be detected through the transcribed products thereof. It is recognized that a biological sample can be processed differently depending on the assay being employed to detect the level and/or activity of XAF1 mRNA or XAF1 protein. For example, when detecting mutations in th eXAFl polynucleotide, preliminary processing designed to isolate or enrich the sample for the genomic DNA can be employed. A variety of techniques known to those of ordinary skill in the art may be used for this purpose. When detecting the level of XAF1 mRNA, different techniques can be used enrich the biological sample with mRNA. Various methods to detect the level of mRNA or the presence of &XAF1 polynucleotide mutation can be used.

As used herein, a“probe” is an isolated polynucleotide attached to a conventional detectable label or reporter molecule, such as a radioactive isotope, ligand, chemiluminescent agent, enzyme, or the like. Such a probe is complementary to a strand of a target polynucleotide, such as a polynucleotide comprising a mutation in a XAF1 polynucleotide or a polynucleotide that can detect XAF1 mRNA. Deoxyribonucleic acid probes may include those generated by PCR using XAF1 mRNA/cDNA specific primers or XAF1 markers, oligonucleotide probes synthesized in vitro , or DNA obtained from bacterial artificial chromosome, fosmid, or cosmid libraries. Probes include not only deoxyribonucleic or ribonucleic acids but also polyamides and other probe materials that can specifically detect the presence of a target DNA sequence. For nucleic acid probes, examples of detection reagents include, for example, radiolabeled probes, enzymatic labeled probes (e.g., horse radish peroxidase and alkaline phosphatase), affinity labeled probes (e.g., biotin, avidin, and steptavidin), and fluorescent labeled probes (e.g., 6-FAM, VIC, TAMRA, MGB, fluorescein, rhodamine, and texas red [for BAC/fosmids]). One skilled in the art will readily recognize that the nucleic acid probes described in the present invention can readily be incorporated into one of the established kit formats which are well known in the art.

As used herein,“primers” are isolated polynucleotides that are annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand and then be extended along the target DNA strand by a polymerase (e.g., a DNA polymerase). Primer pairs of the invention refer to their use for amplification of a target polynucleotide (e.g., by the polymerase chain reaction (PCR) or other conventional nucleic-acid amplification methods).“PCR” or“polymerase chain reaction” is a technique used for the amplification of specific DNA segments (see U.S. Pat. Nos. 4,683,195 and 4,800,159, herein incorporated by reference).

Probes and primers are of sufficient nucleotide length to bind to the target DNA sequence and specifically detect and/or identify a mutation in &XAF1 polynucleotide. It is recognized that the hybridization conditions or reaction conditions can be determined by the operator to achieve this result. This length may be of any length that is of sufficient length to be useful in a detection method of choice. Generally, 8, 11, 14, 16, 18, 20, 22, 24, 26, 28, 30, 40, 50, 75, 100, 200, 300, 400, 500, 600, or 700 nucleotides or more, or between about 11-20, 20-30, 30-40, 40-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, or more nucleotides in length are used. Such probes and primers can hybridize specifically to a target sequence under high stringency hybridization conditions. Probes and primers according to specific embodiments may have complete DNA sequence identity of contiguous nucleotides with the target sequence, although probes differing from the target DNA sequence and that retain the ability to specifically detect and/or identify a target DNA sequence may be designed by conventional methods. Accordingly, probes and primers can share about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity or complementarity to the target polynucleotide. Probes can be used as primers, but are generally designed to bind to the target DNA or RNA and are not used in an amplification process.

Specific primers can be used to amplify any region within the XAF1 polynucleotide, and particularly codon 134 and 115 (depending on the reference sequence). When the probe is hybridized with the polynucleotides of a biological sample under conditions that allow for the binding of the probe to the sample, this binding can be detected and thus allow for an indication of the presence of the level of th e XAFl expression in the biological sample. For example, standard PCR or RT-PCR can be used to measure XAF1 mutation or expression. Such identification of a bound probe has been described in the art. The specific probe may comprise a sequence of at least 80%, between 80% and 85%, between 85% and 90%, between 90% and 95%, and between 95% and 100% identical (or complementary) to a specific region or mutated region of the XAF1 polynucleotide, mRNA, or cDNA.

As used herein,“amplified DNA” or“amplicon” refers to the product of polynucleotide amplification of a target polynucleotide that is part of a nucleic acid template. For example, to determine whether a biological sample comprises a mutation in &XAF1 polynucleotide, such as the GAA to TAA mutation in codon 134 of SEQ ID NO: 1, the nucleic acid content of the biological sample may be subjected to a polynucleotide amplification method using a primer pair that includes a first primer derived from the 5' flanking sequence adjacent to XAF1 marker locus and a second primer derived from the 3' flanking sequence adjacent to the XAF1 marker locus to produce an amplicon that is diagnostic for the presence of mutation of interest. By“diagnostic” for the mutation of interest is intended the use of any method or assay which discriminates between the wild type and mutation genotype of the marker locus. In some cases, the amplicon may range in length from the combined length of the primer pairs plus one nucleotide base pair to any length of amplicon producible by a DNA amplification protocol. A member of a primer pair derived from the flanking sequence may be located a distance from the junction or breakpoint. This distance can range from one nucleotide base pair up to the limits of the amplification reaction, or about twenty thousand nucleotide base pairs. The use of the term“amplicon” specifically excludes primer dimers that may be formed in the DNA thermal amplification reaction.

Methods for preparing and using probes and primers are described, for example, in

Molecular Cloning: A Laboratory Manual, 2.sup.nd ed, vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1989 (hereinafter,“Sambrook et al, 1989”); Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley- Interscience, New York, 1992 (with periodic updates) (hereinafter,“Ausubel et al., 1992”); and Innis et al, PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990. PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as the PCR primer analysis tool in Vector NTI version 10 (Informax Inc., Bethesda Md.); PrimerSelect (DNASTAR Inc., Madison, Wis.); and Primer3 (Version 0.4.0.COPYRGT., 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.). Additionally, the sequence can be visually scanned and primers manually identified using guidelines known to one of skill in the art.

As outlined in further detail below, any conventional nucleic acid hybridization or amplification or sequencing method can be used to specifically detect the presence of a mutation in a XAF1 polynucleotide and/or the level of the XAF1 polypeptide or mRNA. By“specifically detect” is intended that the polynucleotide can be used either as a primer to amplify a region of the XAF1 polynucleotide or the polynucleotide can be used as a probe that hybridizes under stringent conditions to a polynucleotide comprising the mutation marker locus or a polynucleotide comprising the XAF1 mRNA or cDNA. The probe sequence can share at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity or complementarity to a fragment of an XAF1 polynucleotide or across the full length of the XAF1 polynucleotide marker locus (rsl46752602) or to the XAF1 polynucleotide.

A variety of nucleic acid techniques known to those of ordinary skill in the art, including, for example, nucleic acid sequencing, nucleic acid hybridization, and nucleic acid amplification. Nucleic acid hybridization includes methods using labeled probes directed against purified DNA, amplified DNA, and cell preparations (fluorescence in situ hybridization). Illustrative examples of nucleic acid sequencing techniques include, for example, chain terminator (Sanger) sequencing and dye terminator sequencing. Chain terminator sequencing uses sequence-specific termination of a DNA synthesis reaction using modified nucleotide substrates. Extension is initiated at a specific site on the template DNA by using a short radioactive (or otherwise labeled) oligonucleotide primer complementary to the template at that region. The oligonucleotide primer is extended using a DNA polymerase, standard four deoxynucleotide bases, and a low concentration of one chain terminating nucleotide, most commonly a di-deoxynucleotide. This reaction is repeated in four separate tubes with each of the bases taking turns as the di- deoxynucleotide. Limited incorporation of the chain terminating nucleotide by the DNA polymerase results in a series of related DNA fragments that are terminated only at positions where that particular di-deoxynucleotide is used. For each reaction tube, the fragments are size-separated by electrophoresis in a slab polyacrylamide gel or a capillary tube filled with a viscous polymer. The sequence is determined by reading which lane produces a visualized mark from the labeled primer as you scan from the top of the gel to the bottom. Dye terminator sequencing alternatively labels the terminators. Complete sequencing can be performed in a single reaction by labeling each of the di-deoxynucleotide chain-terminators with a separate fluorescent dye, which fluoresces at a different wavelength.

The present disclosure further provides methods for identifying nucleic acids which do not necessarily require sequence amplification and are based on, for example, the known methods of Southern (DNA:DNA) blot hybridizations, in situ hybridization, and FISH of chromosomal material, using appropriate probes.

In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA or RNA strand as a probe to localize a specific DNA or RNA sequence in a portion or section of tissue {in situ ), or, if the tissue is small enough, the entire tissue (whole mount ISH). DNA ISH can be used to determine the structure of chromosomes. Sample cells and tissues are usually treated to fix the target transcripts in place and to increase access of the probe. The probe hybridizes to the target sequence at elevated temperature, and then the excess probe is washed away. The probe that was labeled with either radio-, fluorescent- or antigen-labeled bases is localized and quantitated in the tissue using autoradiography, fluorescence microscopy, or immunohistochemistry, respectively. ISH can also use two or more probes, labeled with radioactive or non-radioactive labels, to simultaneously detect two or more transcripts. In some embodiments, fluorescence in situ hybridization (FISH) is employed.

In specific embodiments, probes for detecting a mutation in a XAF1 polynucleotide, such as the rsl46752602 marker locus, or XAF1 polynucleotides are labeled with appropriate fluorescent or other markers and then used in hybridizations. The Examples section provided herein sets forth various protocols that are effective for detecting the genomic abnormalities, but one of skill in the art will recognize that many variations of these assays can also be used. Specific protocols are well known in the art and can be readily adapted for the present invention. Guidance regarding methodology may be obtained from many references including: In situ Hybridization: Medical Applications (eds. G. R. Coulton and J. de Belleroche), Kluwer Academic Publishers, Boston (1992); In situ Hybridization: hi Neurobiology; Advances in Methodology (eds. J. H. Eberwine, K. L. Valentino, and J. D. Barchas), Oxford University Press Inc., England (1994); In situ

Hybridization: A Practical Approach (ed. D. G. Wilkinson), Oxford University Press Inc., England (1992)); Kuo et al. (1991) Am. J. Hum. Genet. 42: 112-119; Klinger et al. (1992) Am. J. Hum. Genet. 51 :55-65; and Ward et al. (1993) Am. J. Hum. Genet. 52:854-865). There are also kits that are commercially available and that provide protocols for performing FISH assays (available from e.g., Oncor, Inc., Gaithersburg, MD). Patents providing guidance on methodology include U.S. 5,225,326; 5,545,524; 6,121,489 and 6,573,043. All of these references are hereby incorporated by reference in their entirety and may be used along with similar references in the art to establish procedural steps convenient for a particular laboratory.

Southern blotting can be used to detect specific DNA sequences. In such methods, DNA that is extracted from a sample is fragmented, electrophoretically separated on a matrix gel, and transferred to a membrane filter. The filter bound DNA is subject to hybridization with a labeled probe complementary to the sequence of interest. Hybridized probe bound to the filter is detected.

In hybridization techniques, all or part of a polynucleotide that selectively hybridizes to a target polynucleotide comprising the mutated XAF1 polynucleotide or the rsl46752602 marker locus is employed. “Stringent conditions” or“stringent hybridization conditions,” when referring to a polynucleotide probe, is intended to refer to conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of identity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length or less than 500 nucleotides in length.

As used herein, a substantially identical or complementary sequence is a polynucleotide that will specifically hybridize to the complement of the nucleic acid molecule to which it is being compared under high stringency conditions. Appropriate stringency conditions which promote DNA hybridization, for example, 6X sodium chloride/sodium citrate (SSC) at about 45° C., followed by a wash of 2X SSC at 50° C., are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Typically, stringent conditions for hybridization and detection will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37°C, and a wash in IX to 2X SSC (20X SSC = 3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55°C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C, and a wash in 0.5X to IX SSC at 55 to 60°C. Exemplary high stringency conditions include hybridization in 50%

formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0.1X SSC at 60 to 65°C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than 24 hours, usually about 4 to about 12 hours. The duration of the wash time will be at least a length of time sufficient to reach equilibrium.

In hybridization reactions, specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA- DNA hybrids, the T_m can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: T_m = 8l.5°C + 16.6 (log M) + 0.41 (% GC) - 0.61 (% form) - 500/L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The T_m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. T_m is reduced by about l°C for each 1% of mismatching; thus, T_m, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the T_m can be decreased l0°C. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (T_m) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at l°C, 2°C, 3°C, or 4°C lower than the thermal melting point (T_m); moderately stringent conditions can utilize a hybridization and/or wash at 6°C, 7°C, 8°C, 9°C, or l0°C lower than the thermal melting point (T_m); low stringency conditions can utilize a hybridization and/or wash at 1 l°C, l2°C, l3°C, l4°C, l5°C, or 20°C lower than the thermal melting point (T_m). Using the equation, hybridization and wash compositions, and desired T_m, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a T_m of less than 45°C (aqueous solution) or 32°C (formamide solution), it is optimal to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al. , eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A

Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York) and Haymes et al. (1985) In: Nucleic Acid Hybridization, a Practical Approach, IRL Press,

Washington, D.C.

A polynucleotide is said to be the“complement” of another polynucleotide if they exhibit complementarity. As used herein, molecules are said to exhibit“complete complementarity” when every nucleotide of one of the polynucleotide molecules is complementary to a nucleotide of the other. Two molecules are said to be“minimally complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under at least conventional“low-stringency” conditions. Similarly, the molecules are said to be

“complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under conventional“high-stringency” conditions.

Regarding the amplification of a target polynucleotide (e.g., by PCR) using a particular amplification primer pair,“stringent conditions” are conditions that permit the primer pair to hybridize to the target polynucleotide to which a primer having the corresponding sequence (or its complement) would bind and preferably to produce an identifiable amplification product (the amplicon) having a region of a XAF1 polynucleotide or the rs 146752602 marker locus in a DNA thermal amplification reaction. In a PCR approach, oligonucleotide primers can be designed for use in PCR reactions to amplify a region of the XAF1 polynucleotide or the rsl46752602 marker locus. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York). See also Innis et al., eds. (1990) PCR Protocols:

A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCRMethods Manual (Academic Press, New York). Methods of amplification are further described in US Patent No. 4,683,195, 4,683,202 and Chen et al. (1994) PNAS 91 :5695-5699. These methods as well as other methods known in the art of DNA amplification may be used in the practice of the embodiments of the present invention. It is understood that a number of parameters in a specific PCR protocol may need to be adjusted to specific laboratory conditions and may be slightly modified and yet allow for the collection of similar results. These adjustments will be apparent to a person skilled in the art. Following amplification, the amplification product can then be sequenced or digested with a restriction enzyme and run on agarose gel in order to determine mutation status of a XAF1 polynucleotide.

The amplified polynucleotide (amplicon) can be of any length that allows for the detection of the XAF1 polynucleotide or the rsl46752602 marker locus. For example, the amplicon can be about 5, 10, 50, 100, 200, 300, 500, 700, 100, 2000, 3000, 4000, or 5000 nucleotides in length or longer.

Any primer can be employed in the methods of the invention that allows the rs 146752602 marker locus or a region of the XAF1 polynucleotide to be amplified and/or detected. Methods for designing PCR primers are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,

Plainview, New York). See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies

(Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Other known methods of PCR that can be used in the methods of the invention include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, mixed DNA/RNA primers, vector-specific primers, partially mismatched primers, and the like.

Thus, in specific embodiments, a method of detecting the presence of the rs 146752602 marker locus in a biological sample is provided. The method comprises: (a) providing a sample comprising the nucleic acid complement of a subject; (b) providing a pair of DNA primer molecules that can amplify an amplicon having the rs 146752602 marker locus; (c) providing DNA amplification reaction conditions; (d) performing the DNA amplification reaction, thereby producing a DNA amplicon molecule; and (e) detecting the DNA amplicon molecule. In order for a nucleic acid molecule to serve as a primer or probe it need only be sufficiently complementary in sequence to be able to form a stable double-stranded structure under the particular solvent and salt concentrations employed. In other embodiments, a method of detecting the level of the XAF1 polynucleotide in a biological sample is provided. The method comprises: (a) providing a sample comprising the nucleic acid complement of a subject; (b) providing a pair of DNA primer molecules that can amplify an amplicon having the XAF1 polynucleotide; (c) providing DNA amplification reaction conditions; (d) performing the DNA amplification reaction, thereby producing a DNA amplicon molecule; and (e) detecting the DNA amplicon molecule. In order for a nucleic acid molecule to serve as a primer or probe it need only be sufficiently complementary in sequence to be able to form a stable double-stranded structure under the particular solvent and salt concentrations employed.

In still other embodiments, a mutation in a XAF1 polynucleotide, such as the rs 146752602 marker locus, or the XAF1 polynucleotide may be amplified prior to or simultaneous with detection. Illustrative examples of nucleic acid amplification techniques include, for example, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence based amplification (NASBA). The polymerase chain reaction (U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159 and 4,965,188, each of which is herein

incorporated by reference in its entirety), commonly referred to as PCR, uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase copy numbers of a target nucleic acid sequence. For other various permutations of PCR see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159; Mullis et al, (1987) Meth. Enzymol. 155: 335; and, Murakawa et al.,( 1988) DNA 7: 287, each of which is herein incorporated by reference in its entirety.

The ligase chain reaction (Weiss (1991) Science 254: 1292, herein incorporated by reference in its entirety), strand displacement amplification (Walker et al. (1992) Proc. Natl. Acad. Sci. USA 89: 392-396; U.S. Pat. No. 5,270,184; and U.S. Pat. No. 5,455,166; each of which is herein incorporated by reference in its entirety), and thermophilic SDA (tSDA) (EP Pat. No. 0 684 315, incorporated by reference in its entirety) are known methods which can be employed in detecting the level and/or activity of XAF1.

Any method can be used for detecting either the non-amplified or amplified polynucleotides including, for example, Hybridization Protection Assay (HP A) (U.S. Pat. No. 5,283,174 and Nelson et al. (1995) Nonisotopic Probing, Blotting, and Sequencing, ch. 17 (Larry J. Kricka ed., 2d ed.), each of which is herein incorporated by reference in its entirety); quantitative evaluation of the amplification process in real-time (U.S. Pat. Nos. 6,303,305 and 6,541,205, each of which is herein incorporated by reference in its entirety); and determining the quantity of target sequence initially present in a sample, but which is not based on a real-time amplification (U.S. Pat. No. 5,710,029, herein incorporated by reference in its entirety).

Amplification products may be detected in real-time through the use of various self- hybridizing probes, most of which have a stem-loop structure. Such self-hybridizing probes are labeled so that they emit differently detectable signals, depending on whether the probes are in a self-hybridized state or an altered state through hybridization to a target sequence. For example, "molecular torches" are a type of self-hybridizing probe that includes distinct regions of self- complementarity (referred to as "the target binding domain" and "the target closing domain") which are connected by a joining region (e.g., non-nucleotide linker) and which hybridize to each other under predetermined hybridization assay conditions. In a preferred embodiment, molecular torches contain single-stranded base regions in the target binding domain that are from 1 to about 20 bases in length and are accessible for hybridization to a target sequence present in an amplification reaction under strand displacement conditions. Under strand displacement conditions,

hybridization of the two complementary regions, which may be fully or partially complementary, of the molecular torch is favored, except in the presence of the target sequence, which will bind to the single-stranded region present in the target binding domain and displace all or a portion of the target closing domain. The target binding domain and the target closing domain of a molecular torch include a detectable label or a pair of interacting labels (e.g., luminescent/quencher) positioned so that a different signal is produced when the molecular torch is self-hybridized than when the molecular torch is hybridized to the target sequence, thereby permitting detection of probe:target duplexes in a test sample in the presence of unhybridized molecular torches. Molecular torches and a variety of types of interacting label pairs are disclosed in U.S. Pat. No. 6,534,274, herein incorporated by reference in its entirety.

Another example of a detection probe having self-complementarity is a "molecular beacon." Molecular beacons include nucleic acid molecules having a target complementary sequence, an affinity pair (or nucleic acid arms) holding the probe in a closed conformation in the absence of a target sequence present in an amplification reaction, and a label pair that interacts when the probe is in a closed conformation. Hybridization of the target sequence and the target complementary sequence separates the members of the affinity pair, thereby shifting the probe to an open conformation. The shift to the open conformation is detectable due to reduced interaction of the label pair, which may be, for example, a fluorophore and a quencher (e.g., DABCYL and EDANS). Molecular beacons are disclosed in U.S. Pat. Nos. 5,925,517 and 6,150,097, herein incorporated by reference in their entireties. Other self-hybridizing probes are well known to those of ordinary skill in the art. By way of non-limiting example, probe binding pairs having interacting labels, such as those disclosed in U.S. Pat. No. 5,928,862 (herein incorporated by reference in its entirety) might be adapted for use in the present invention. Probe systems used to detect single nucleotide polymorphisms (SNPs) might also be utilized in the present invention. Additional detection systems include "molecular switches," as disclosed in U.S. Publ. No. 20050042638, herein incorporated by reference in its entirety. Other probes, such as those comprising intercalating dyes and/or fluorochromes, are also useful for detection of amplification products in the present invention. See, e.g., U.S. Pat. No. 5,814,447 (herein incorporated by reference in its entirety).

Various methods can be used to detect the polynucleotide of interest, including, for example, Genetic Bit Analysis (Nikiforov et al. (1994) Nucleic Acid Res. 22: 4167-4175) where a DNA oligonucleotide is designed which overlaps both the adjacent flanking DNA sequence and the inserted DNA sequence. The oligonucleotide is immobilized in wells of a microwell plate.

Following PCR of the region of interest (using one primer in the inserted sequence and one in the adjacent flanking sequence) a single-stranded PCR product can be hybridized to the immobilized oligonucleotide and serve as a template for a single base extension reaction using a DNA polymerase and labeled ddNTPs specific for the expected next base. Readout may be fluorescent or ELISA-based. A signal indicates presence of the insert/flanking sequence due to successful amplification, hybridization, and single base extension.

Another detection method is the Pyrosequencing technique as described by Winge ((2000) Innov. Pharma. Tech. 00: 18-24). In this method, an oligonucleotide is designed that overlaps the junction. The oligonucleotide is hybridized to a single-stranded PCR product from the region of interest (one primer in the inserted sequence and one in the flanking sequence) and incubated in the presence of a DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5' phosphosulfate and luciferin. dNTPs are added individually and the incorporation results in a light signal which is measured. A light signal indicates the presence of the transgene insert/flanking sequence due to successful amplification, hybridization, and single or multi-base extension.

Fluorescence Polarization as described by Chen et al. ((1999) Genome Res. 9: 492-498) is also a method that can be used to detect an amplicon of the invention. Using this method, an oligonucleotide is designed which overlaps the inserted DNA junction. The oligonucleotide is hybridized to a single-stranded PCR product from the region of interest (one primer in the inserted DNA and one in the flanking DNA sequence) and incubated in the presence of a DNA polymerase and a fluorescent-labeled ddNTP. Single base extension results in incorporation of the ddNTP. Incorporation can be measured as a change in polarization using a fluorometer. A change in polarization indicates the presence of the genomic abnormality sequence due to successful amplification, hybridization, and single base extension.

Taqman® (PE Applied Biosystems, Foster City, Calif.) is a method of detecting and quantifying the presence of a DNA sequence and is fully understood in the instructions provided by the manufacturer. Briefly, a FRET oligonucleotide probe is designed which overlaps the junction. The FRET probe and PCR primers (one primer in the insert DNA sequence and one in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs.

Hybridization of the FRET probe results in cleavage and release of the fluorescent moiety away from the quenching moiety on the FRET probe. A fluorescent signal indicates the presence of the flanking/transgene insert sequence due to successful amplification and hybridization.

In one embodiment, the method of detecting a mutation in a XAF1 polynucleotide, such as the rsl46752602 marker locus or the XAF1 polynucleotide comprises: (a) contacting the biological sample with a polynucleotide probe that hybridizes under stringent hybridization conditions with a XAF1 polynucleotide and specifically detects the XAF1 polynucleotide; (b) subjecting the sample and probe to stringent hybridization conditions; and (c) detecting hybridization of the probe to the polynucleotide, wherein detection of hybridization indicates the level of the XAF1 polynucleotide or the presence of the G-allele or the T-allele of the rsl46752602 marker locus. In specific embodiments, the method for detecting a mutation in a XAF1 polynucleotide is performed in vitro. i. Optical Detection Methods

Detection of antibodies or polynucleotides specific for a mutation in a XAF1

polynucleotide, such as the rsl46752602 marker locus, can be facilitated by coupling (z.e., physically linking) the antibody or polynucleotide to a detectable substance (i.e., antibody labeling or polynucleotide labeling). Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials (fluorophores, fluorochromes), luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, b-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of

fluorophores/fluorochromes, include phycoerythrin (PE), fluorescein isothiocyanate (FITC), peridinin-chlorophyll (PerCP), allophycocyanin (APC), R-phycoerythrin conjugated with cyanine dye (PE-Cy7), allophycocyanin-cyanine tandem (APC-H7), coumarin dye (Horizon v450), sulphonyl chloride (Texas Red), cyanine (CY3, CY5, Cy7), FAM, JOE, TAMRA, TET, VIC, rhodamine; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I, 131 I, 35 S, or 3 H. The skilled artisan will understand that additional moieties may be suitable for the methods disclosed herein.

A detectable moiety generally refers to a composition or moiety that is detectable by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical or chemical means such as fluorescence, chemifluorescence, or chemiluminescence, or any other appropriate means. The terms“fluorophore” and“fluorochrome” are defined as a chemical group, or component of a molecule that causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different (but equally specific) wavelength. A fluorophore/fluorochrome can refer to various fluorescent substances, including dyes, used in fluorescence microscopy or flow cytometry to stain specimens. The terms fluorophore” and“fluorochrome” are herein used interchangeably.

Fluorochromes may be conjugated to antibodies, proteins, polypeptides, peptides, or nucleotide probes which specifically bind to antigens, proteins, polypeptides, peptides,

polysaccharides, DNA, or RNA sequences. Thus, binding of an antibody, protein, polypeptide, peptide, or nucleotide probe to an antigen, protein, polypeptide, peptide, polysaccharide, DNA, or RNA may be detected by measuring a signal generated from a fluorochrome by flow cytometry, or any suitable optical imaging technique. Detection of a signal may indicate binding, whereas lack of detection of a signal may indicate lack of binding.

Methods and compositions for detectably labeling nucleic acid probes, such as

oligonucleotides, DNA-RNA hybrids, etc. are well known in the art. See, e.g., U.S. Pat. Nos.

6,316,230; 6,297,016; 6,316,610; 6,060,240; 6,150,107; and 6,028,290, each of which is hereby incorporated by reference in their entirety.

An antibody or a fragment thereof can be conjugated with a detectable moiety, wherein the detectable moiety can be, for example, a fluorophore, a chromophore, a radionuclide, or an enzyme. In specific embodiments, a fluorophore can be, for example, phycoerythrin (PE), fluorescein isothiocyanate (FITC), peridinin-chlorophyll (PerCP), allophycocyanin (APC), R-phycoerythrin conjugated with cyanine dye (PE-Cy7), allophycocyanin-cyanine tandem (APC-H7), and coumarin dye (Horizon v450). Detection of complexes formed between an antibody probe and marker can be achieved by an optical detection technique, including, but not limited to flow cytometry and microscopy. “Cell staining” when used in reference to an antibody means that the antibody recognizes a marker and binds to a marker in the specimen forming a complex, thereby“labeling” or otherwise “staining” the cell expressing the marker to make it visible and/or detectable by microscopy or flow cytometry. Combinations of antibodies can be collectively added to a specimen and thereby“stain the cell” for later analysis by visualization with a flow cytometer or microscope, for example. One of skill in the art could determine whether a cell expressed a specific protein based on the level of antibody that bound to the cell using standard methods.

The methods disclosed herein can also be used in immunofluorescence histochemistry. This technique involves the use of antibodies labeled with various fluorophores to detect substances within a specimen. In exemplary embodiments a pathologist can derive a great deal of

morphological information of diagnostic value by examining a specimen from a subject by microscope. Combinations of fluorophores or other detectable labels can be used by the methods on this invention, thereby greatly increasing the number of distinguishable signals in multicolor protocols.

In another embodiment, the method employs flow cytometry. In another embodiment, in a peripheral blood sample or blood sample, lymphocyte, monocyte and granulocyte populations can be defined on the basis of forward and side scatter. Forward and side scatter are used in one embodiment to exclude debris and dead cells.

Flow cytometry is an optical technique that analyzes particles or cells in a fluid mixture based on their optical characteristics, via the use of a flow cytometer (See, for example, Shapiro, "Practical Flow Cytometry," Third Ed. (Alan R. Liss, Inc., 1995); and Melamed et al. "Flow Cytometry and Sorting," Second Ed. (Wiley-Liss 1990)). Flow cytometers hydrodynamically focus a fluid suspension of particles/cells into a thin stream so that they flow down the stream in substantially single file and pass through an examination zone. A focused light beam, such as a laser beam illuminates the particles as they flow through the examination zone. Optical detectors within the flow cytometer measure certain characteristics of the light as it interacts with the particles/cells. Commonly used flow cytometers such as the Becton-Dickinson Immunocytometry Systems "FACSCAN" (San Jose, Calif.) can measure forward light scatter (generally correlated with the refractive index and size of the particle/cell being illuminated), side light scatter (generally correlated with the cell granularity), and particle fluorescence at one or more wavelengths. Data acquisition and analysis can be done using FACSCALIBETEZ LSRII flow cytometers (Becton Dickinson), and CELLQEIEST Pro, BD FACSDIVA software (both from Becton Dickinson), FLOWJO software (Tree Star, Ashland, OR) and/or KALUZA software (Beckman Coulter, Miami, FL)(Campana, D. (2009) Hematol Oncol Clin North Am. 23; 1083-98, vii).

In some embodiments, antibodies can be directly conjugated for simultaneous detection. For example, a method of the invention can comprise antibodies directly conjugated to a detectable fluorochrome for simultaneous detection of a plurality of markers for identifying a mutation in a XAF1 polynucleotide, such as the rsl46752602 marker locus. The skilled artisan will understand that any one antibody marker can be coupled to any fluorochrome for use in combination with any other antibody, and that preferred combinations can be used simultaneously with other antibody markers by the selection of different combinations of antibodies labeled with different

fluorochromes. ii. Detecting XAF1 polypeptides

The level and/or activity of the XAF1 polypeptides can be detected using a variety of protein techniques known to those of ordinary skill in the art, including, for example, protein sequencing and immunoassays. In specific embodiments, mutated or truncated XAF1 polypeptides can indicate a lower activity of XAF1 and an increased risk of developing a cell-proliferative disorder. As used herein, a“reduction” or“decrease” in the expression of a XAF1 polypeptide refers to a reduction of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 5-10%, 10-20%, 10-30%, 10-40%, 20-30%, 20-40%, 30-40%, 30-50%, 40- 50%, 40-60%, 50-60%, 50-70%, 60-70%, 60-80%, 70-80%, 70-90%, 80-90%, 80-100%, 90-100%, or 95-100% compared to wild type XAF1 expression obtained from a control sample(s) or a standard value in the art. Likewise, a“reduction” or“decrease” in the activity of a XAF1 polypeptide refers to a reduction of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 5-10%, 10-20%, 10-30%, 10-40%, 20-30%, 20-40%, 30-40%, 30-50%, 40-50%, 40-60%, 50-60%, 50-70%, 60-70%, 60-80%, 70-80%, 70-90%, 80-90%, 80- 100%, 90-100%, or 95-100% compared to wild type XAF1 activity obtained from a control sample(s) or a standard value in the art.

Illustrative non-limiting examples of protein sequencing techniques include, for example, mass spectrometry and Edman degradation. Mass spectrometry can, in principle, sequence any size protein but becomes computationally more difficult as size increases. A protein is digested by an endoprotease, and the resulting solution is passed through a high pressure liquid chromatography column. At the end of this column, the solution is sprayed out of a narrow nozzle charged to a high positive potential into the mass spectrometer. The charge on the droplets causes them to fragment until only single ions remain. The peptides are then fragmented and the mass-charge ratios of the fragments measured. The mass spectrum is analyzed by computer and often compared against a database of previously sequenced proteins in order to determine the sequences of the fragments.

The process is then repeated with a different digestion enzyme, and the overlaps in sequences are used to construct a sequence for the protein.

In the Edman degradation reaction, the peptide to be sequenced is adsorbed onto a solid surface (e.g., a glass fiber coated with polybrene). The Edman reagent, phenylisothiocyanate (PTC), is added to the adsorbed peptide, together with a mildly basic buffer solution of 12% trimethylamine, and reacts with the amine group of the C-terminal amino acid. The terminal amino acid derivative can then be selectively detached by the addition of anhydrous acid. The derivative isomerizes to give a substituted phenylthiohydantoin, which can be washed off and identified by chromatography, and the cycle can be repeated. The efficiency of each step is about 98%, which allows about 50 amino acids to be reliably determined.

Illustrative examples of immunoassays include, for example, immunoprecipitation, Western blot, ELISA, immunohistochemistry, immunocytochemistry, flow cytometry, and immuno-PCR. Polyclonal or monoclonal antibodies detectably labeled using various techniques known to those of ordinary skill in the art (e.g., calorimetric, fluorescent, chemiluminescent or radioactive) are suitable for use in the immunoassays. Antibodies against XAF1 are known in the art.

Immunoprecipitation is a technique of precipitating an antigen out of solution using an antibody specific to that antigen. The process can be used to identify protein complexes present in cell extracts by targeting a protein believed to be in the complex. The complexes are brought out of solution by insoluble antibody-binding proteins isolated initially from bacteria, such as Protein A and Protein G. The antibodies can also be coupled to sepharose beads that can easily be isolated out of solution. After washing, the precipitate can be analyzed using mass spectrometry, Western blotting, or any number of other methods for identifying constituents in the complex.

A Western blot, or immunoblot, is a method to detect protein in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate denatured proteins by mass. The proteins are then transferred out of the gel and onto a membrane, typically polyvinyldifluoride or nitrocellulose, where they are probed using antibodies specific to the protein of interest. As a result, researchers can examine the amount of protein in a given sample and compare levels between several groups.

An ELISA, short for Enzyme-Linked ImmunoSorbent Assay, is a biochemical technique to detect the presence of an antibody or an antigen in a sample. It utilizes a minimum of two antibodies, one of which is specific to the antigen and the other of which is coupled to an enzyme. The second antibody will cause a chromogenic or fluorogenic substrate to produce a signal.

Variations of ELISA include sandwich ELISA, competitive ELISA, and ELISPOT. Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool both for determining serum antibody concentrations and also for detecting the presence of antigen.

Immunohistochemistry and immunocytochemistry refer to the process of localizing proteins in a tissue section or cell, respectively, via the principle of antigens in tissue or cells binding to their respective antibodies. Visualization is enabled by tagging the antibody with, for example, color producing or fluorescent tags. Typical examples of color tags include, for example, horseradish peroxidase and alkaline phosphatase. Typical examples of fluorophore tags include, for example, fluorescein isothiocyanate (FITC) or phycoerythrin (PE).

Flow cytometry is a technique for counting, examining, and sorting microscopic particles suspended in a stream of fluid. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of single cells flowing through an optical/electronic detection apparatus. A beam of light (e.g., a laser) of a single frequency or color is directed onto a hydrodynamically focused stream of fluid. A number of detectors are aimed at the point where the stream passes through the light beam; one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter (SSC) and one or more fluorescent detectors). Each suspended particle passing through the beam scatters the light in some way, and fluorescent chemicals in the particle may be excited into emitting light at a lower frequency than the light source. The combination of scattered and fluorescent light is picked up by the detectors, and by analyzing fluctuations in brightness at each detector, one for each fluorescent emission peak, it is possible to deduce various facts about the physical and chemical structure of each individual particle. FSC correlates with the cell volume and SSC correlates with the density or inner complexity of the particle (e.g., shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness).

Immuno-polymerase chain reaction (IPCR) utilizes nucleic acid amplification techniques to increase signal generation in antibody-based immunoassays. Because no protein equivalence of PCR exists, that is, proteins cannot be replicated in the same manner that nucleic acid is replicated during PCR, the only way to increase detection sensitivity is by signal amplification. The target proteins are bound to antibodies which are directly or indirectly conjugated to oligonucleotides. Unbound antibodies are washed away and the remaining bound antibodies have their oligonucleotides amplified. Protein detection occurs via detection of amplified oligonucleotides using standard nucleic acid detection methods, including real-time methods. ill. Biological samples

Identification of a mutation in a XAF1 polynucleotide can be determined from any sample obtained from a subject having a XAF1 polynucleotide or mutated XAF1 polynucleotide. Likewise, identification of reduced XAF1 expression or activity can be determined from any sample obtained from a subject in which XAF1 expression or activity can normally be detected.

As used herein, a“biological sample” or“sample” from a subject can comprise any sample from a subject, such as a subject with or without a TP53 mutation. For example, a biological sample can comprise a sample from any organism, including a mammal, such as a human, a primate, a rodent, a domestic animal (such as a feline or canine) or an agricultural animal (such as a ruminant, horse, swine or sheep). The biological sample can be derived from any cell, tissue or biological fluid from the organism of interest. The sample may comprise any clinically relevant tissue, such as, but not limited to, bone marrow samples, tumor biopsy, fine needle aspirate, peripheral blood, umbilical cord blood, hematopoietic stem cells derived therefrom or a sample of bodily fluid, such as, blood, plasma, serum, lymph, ascitic fluid, cystic fluid or urine. The sample used in the methods of the invention will vary based on the assay format, nature of the detection method, and the tissues, cells or extracts which are used as the sample. It is recognized that the sample typically requires preliminary processing designed to isolate or enrich the sample for the genomic DNA, mRNA, or protein. A variety of techniques known to those of ordinary skill in the art may be used for this purpose.

The term“specimen” or“biological sample” is intended to include any clinically relevant tissue, such as, but not limited to, bone marrow samples, tumor biopsy, fine needle aspirate, or a sample of bodily fluid, such as, blood, plasma, serum, lymph, ascitic fluid, cystic fluid, urine, blood cells, bone marrow cells, and cellular products that are derived from blood and bone marrow cells. Cellular products can include, but are not limited to, expressed proteins, expressed RNA, and DNA. In embodiments, a specimen can include cells derived from a variety of sources including, but not limited to, single cells, a collection of cells, tissue, cell culture, bone marrow, blood, or other bodily fluids. A tissue or cell source may include a tissue biopsy sample, a cell sorted population, cell culture, or a single cell. The term“biological sample” can be used interchangeably with the term “sample” or“patient sample.”

A control biological sample can be either a positive or a negative control for the test biological sample. Often, the control biological sample contains the same types of tissues, cells and biological fluids as that of the test biological sample. The sample can be obtained by removing a sample of cells from a subject, but can also be accomplished by using previously isolated cells. In addition, the biological sample can be freshly collected or a previously collected sample.

In some embodiments, the test sample or the biological sample can be a frozen biological sample, e.g., a frozen tissue or cells. The frozen sample can be thawed before employing methods, assays and systems of the invention. After thawing, a frozen sample can be centrifuged before being subjected to the assays disclosed herein.

A biological sample may be processed to release or otherwise make available a nucleic acid or a protein for detection as described herein. Such processing may include, for example, steps of nucleic acid manipulation, e.g., preparing a cDNA by reverse transcription of RNA from the biological sample. Thus, the nucleic acid to be amplified in one embodiment by the methods of the invention may be DNA or RNA. Isolation of protein, RNA, and DNA from the aforementioned sources is known to those of skill in the art, and is discussed herein.

In some embodiments, a biological sample can be a nucleic acid product amplified after polymerase chain reaction (PCR). The nucleic acid product, such as DNA, RNA and mRNA, can be isolated from a particular biological sample using any of a number of procedures, which are well-known in the art, the particular isolation procedure chosen being appropriate for the particular biological sample. Methods of isolating and analyzing nucleic acid variants as described above are well known to one skilled in the art.

In some embodiments, the test sample or the biological sample can be treated with a chemical and/or biological reagent. Chemical and/or biological reagents can be employed to protect and/or maintain the stability of the sample, including biomolecules (e.g., nucleic acid and protein) therein, during processing. One exemplary reagent is a protease inhibitor, which is generally used to protect or maintain the stability of protein during processing. In addition, or alternatively, chemical and/or biological reagents can be employed to release nucleic acid or protein from the sample.

In one embodiment, the method comprises obtaining a peripheral blood sample from a subject and analyzing the XAF1 expression or activity or XAF1 polynucleotide genotype from the blood sample taken from the subject. To do blood tests, blood samples are generally taken from a vein in the subject’s arm.

In another embodiment, the method comprises obtaining a bone marrow sample from a subject and analyzing the XAF1 expression or activity or XAF1 polynucleotide genotype from the blood sample taken from the subject. Specimens of marrow cells are obtained by bone marrow aspiration and biopsy.

As used herein, a "subject,"“patient,” or an "individual" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the subject, patient, or individual is a human. As described elsewhere herein, the subject can harbor or carry a mutation in the TP53 gene. In specific embodiments, the subject harbors or comprises the R337H-TP53 mutation. In some embodiments, the subject is from 0-2yrs old, 0-4 yrs old, 2-4 yrs old, 2-6 yrs old, 4-8 yrs old, 6-10 yrs old, 10-15 yrs old, 15-18 yrs old, or over 18 yrs old. In some embodiments, the subject is an infant, toddler, adolescent, or adult. The subject can be of any origin. In some embodiments, the subject is a native of Brazil.

iv. Increased risk of developing a cell-proliferative disorder

Methods and compositions provided herein can identify subjects at an increased risk of developing a cell-proliferative disorder based on a mutation in XAF1 polynucleotide or the level or activity of a XAF1 polypeptide. In specific embodiments, a mutation in an XAF1 polynucleotide indicates that the subject harboring the mutation is at an increased risk of developing a cell- proliferative disorder, particularly when the subject also harbors the R337H-TP53 or any other TP53 mutation. Likewise, in some embodiments, a reduction in XAF1 expression or a reduction in CARΊ activity can indicate that the subject is at an increased risk of developing a cell-proliferative disorder, particularly when the subject also harbors the R337H-TP53 mutation. Based on the assessed risk, a personalized prophylaxis or treatment regimen can be administered to the subject.

As used herein, an“increased risk” of developing a cell-proliferative disorder indicated by a mutation in XAF1 or reduction in XAF1 expression or activity comprises a statistically significant increase in the risk of developing the cell proliferative disorder. The risk can be based on the presence of a particular risk indicator ( e.g ., a mutation in XAF1 polynucleotide) relative to risk in the absence of that risk indicator. The increased risk can include, for example, a risk that is at least about 10% higher, 15% higher, 20% higher, 25% higher, 30% higher, 35% higher, 40% higher,

45% higher, 50% higher, 55% higher, 60% higher, 65% higher, 70% higher, 75% higher, 80% higher, 85% higher, 90% higher, 95% higher, 100% higher, 110% higher, 120% higher, 130% higher, 140% higher, 150% higher, 160% higher, 170% higher, 180% higher, 190% higher, 200% higher, or greater. Statistical significance means p < 0.05.

The methods and compositions disclosed herein can be used to identify subjects at risk of developing any cell-proliferative disorder. A "disorder" is any condition that would benefit from treatment including, but not limited to, chronic and acute disorders or diseases including those pathological conditions which predispose the subject to the disorder in question. The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders that are associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

In one embodiment, the cell proliferative disorder is a tumor.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but are not limited to, adrenocortical tumor, such as an adrenocortical adenoma or adrenocortical carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include, but not limited to, squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer and

gastrointestinal stromal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, superficial spreading melanoma, lentigo maligna melanoma, acral lentiginous melanomas, nodular melanomas, multiple myeloma and B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chronic

lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), Meigs' syndrome, brain, as well as head and neck cancer, and associated metastases. In certain embodiments, cancers that are amenable to treatment by the antibodies of the invention include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkins lymphoma (NHL), renal cell cancer, prostate cancer, liver cancer, pancreatic cancer, soft-tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, mesothelioma, and multiple myeloma. In some embodiments, the cancer is selected from: small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast carcinoma, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma. In other embodiments, the cancer is selected from a class of mature B-Cell cancers excluding Hodgkin's Lymphoma but including germinal-center B- cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, follicular lymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), chronic lymphoid leukemia (CLL), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), Waldenstrom macroglobulinemia (WM), central nervous system lymphoma (CNSL), Burkitt's lymphoma (BL), B-cell prolymphocytic leukemia, Splenic marginal zone lymphoma, Hairy cell leukemia, Splenic lymphoma/leukemia, unclassifiable, Splenic diffuse red pulp small B-cell lymphoma, Hairy cell leukemia variant, Waldenstrom macroglobulinemia,

Plasma cell myeloma, Solitary plasmacytoma of bone, Extraosseous plasmacytoma, Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), Nodal marginal zone lymphoma, Pediatric nodal marginal zone lymphoma, Pediatric follicular lymphoma, Primary cutaneous follicle centre lymphoma, T-cell/histiocyte rich large B-cell lymphoma, Primary DLBCL of the CNS, Primary cutaneous DLBCL, leg type, EBV-positive DLBCL of the elderly, DLBCL associated with chronic inflammation, Lymphomatoid granulomatosis, Primary

mediastinal (thymic) large B-cell lymphoma, Intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, Plasmablastic lymphoma, Large B-cell lymphoma arising in HHV8- associated multicentric Castleman disease, Primary effusion lymphoma: B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, and B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma.

"Tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer", "cancerous", "cell proliferative disorder", "proliferative disorder" and "tumor" are not mutually exclusive as referred to herein.

Once a subject has been identified as at risk of developing a cell-proliferative disorder, one of skill in the art can determine the appropriate therapy or surveillance protocol to apply to the subject. As used herein, the term“therapy” can include any therapy for treating a cell-proliferative disorder including but not limited to induction chemotherapy, chemotherapy, radiation therapy, stem cell transplantation, and biological therapy ( e.g ., monoclonal antibody therapy). Depending on the cell-proliferative disorder, specific drugs or drug combinations, drug dosages, duration of treatment, and other types of treatment, may be administered to achieve optimal results.

III. Methods for altering treatment or surveillance

Identification of subjects having an increased risk of developing a cell-proliferative disorder can permit one of skill in the art to monitor, surveil, and alter the treatment planof the subject, accordingly. As used herein, "treatment" (and grammatical variations thereof such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, subjects identified as having an increased risk of developing a cell-proliferative disorder are treated to delay development of the cell-proliferative disorder or to slow the progression of the disorder.

As used herein, "delaying progression" of a disorder or disease means to defer, hinder, slow, retard, stabilize, and/or postpone development of the disease or disorder (e.g., a cell proliferative disorder, such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed. By "reduce" or "inhibit" is meant the ability to cause an overall decrease, for example, of 20% or greater, of 50% or greater, or of 75%, 85%, 90%, 95%, or greater. In certain embodiments, reduce or inhibit can refer a reduction or inhibition of a symptom of a cell-proliferative disorder.

In some embodiments, the treatment is administered to the subject upon identifying an increased risk of a cell-proliferative disorder. "Administering" is meant a method of giving a dosage of a compound or a composition (e.g., a pharmaceutical composition) to a subject. The compositions utilized in the methods described herein can be administered, for example, intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, by catheter, by lavage, in creames, or in lipid compositions. The method of administration can vary depending on various factors (e.g., the compound or composition being administered and the severity of the condition, disease, or disorder being treated).

The treatment administered to a subject can be administered prophylactically or following a finding that the subject has a cell proliferative disorder. In some embodiments, the dosage amount or schedule is increased after identifying that the subject has a mutation in a XAF1 polynucleotide or a decrease in expression or activity of XAF1. The treatment can be a cancer treatment, such as an anti-cancer therapy. In some embodiments, the method comprises administering one or more anti-cancer therapies to the subject. The anti-cancer therapy can be selected from the group consisting of surgery, chemotherapy, radiation therapy, thermotherapy, immunotherapy, hormone therapy, laser therapy, anti -angiogenic therapy, and any combinations thereof. In some embodiments, the therapeutic agent is an antibody (e.g., polyclonal or monoclonal antibody) or an antigen binding fragment thereof. In one embodiment, the therapeutic agent is an antibody (e.g., polyclonal or monoclonal antibody), or an antigen binding fragment thereof, conjugated with a lipid, e.g., cholesterol.

As used herein, the term "anti-cancer agent" or“anti-cancer therapy” refers to any compound (including its analogs, derivatives, prodrugs and pharmaceutically salts) or composition which can be used to treat cancer. Anti-cancer compounds for use in the present invention include, but are not limited to, inhibitors of topoisomerase I and II, alkylating agents, microtubule inhibitors (e.g., taxol), and angiogenesis inhibitors. Exemplary anti-cancer compounds include, but are not limited to, paclitaxel (taxol); docetaxel; germicitibine; Aldesleukin; Alemtuzumab; alitretinoin; allopurinol; altretamine; amifostine; anastrozole; arsenic trioxide; Asparaginase; BCG Live;

bexarotene capsules; bexarotene gel; bleomycin; busulfan intravenous; busulfanoral; calusterone; capecitabine; platinate; carmustine; carmustine with Polifeprosan Implant; celecoxib; chlorambucil; cladribine; cyclophosphamide; cytarabine; cytarabine liposomal; dacarbazine; dactinomycin;

actinomycin D; Darbepoetin alfa; daunorubicin liposomal; daunorubicin, daunomycin; Denileukin diftitox, dexrazoxane; docetaxel; doxorubicin; doxorubicin liposomal; Dromostanolone propionate; Elliott's B Solution; epirubicin; Epoetin alfa estramustine; etoposide phosphate; etoposide (VP- 16); exemestane; Filgrastim; floxuridine (intraarterial); fludarabine; fluorouracil (5-FU); fulvestrant; gemtuzumab ozogamicin; goserelin acetate; hydroxyurea; Ibritumomab Tiuxetan; idarubicin;

ifosfamide; imatinib mesylate; Interferon alfa-2a; Interferon alfa-2b; irinotecan; letrozole;

leucovorin; levamisole; lomustine (CCNU); mechlorethamine (nitrogenmustard); megestrol acetate; melphalan (L-PAM); mercaptopurine (6-MP); mesna; methotrexate; methoxsalen; mitomycin C; mitotane; mitoxantrone; nandrolone phenpropionate; Nofetumomab; LOddC; Oprelvekin;

pamidronate; pegademase; Pegaspargase; Pegfilgrastim; pentostatin; pipobroman; plicamycin; mithramycin; porfimer sodium; procarbazine; quinacrine; Rasburicase; Rituximab; Sargramostim; streptozocin; talbuvidine (LDT); talc; tamoxifen; temozolomide; teniposide (VM-26); testolactone; thioguanine (6-TG); thiotepa; topotecan; toremifene; Tositumomab; Trastuzumab; tretinoin

(ATRA); Uracil Mustard; valrubicin; valtorcitabine (monoval LDC); vinblastine; vinorelbine;

zoledronate; and any mixtures thereof. In some embodiments, the anti-cancer agent is a paclitaxel- carbohydrate conjugate, e.g., a paclitaxel-glucose conjugate, as described in U.S. Pat. No.

6,218,367, content of which is herein incorporated by reference in its entirety. In particular embodiments, the anti-cancer agent is a platinate selected from the group consisting of cisplatin, oxaliplatin, carboplatin, paraplatin, sartraplatin, and any combinations thereof. In some embodiments, the anti-cancer agent is an immunomodulator. The methods of these embodiments comprise co-administering the conjugate and the immunomodulator to the subject. In some embodiments, the conjugate and the immunomodulator are co-administered in separate pharmaceutical compositions and at different times. In some embodiments, the conjugate and the immunomodulator are co-administered at the same time in the same pharmaceutical composition.

In some embodiments, the immunomodulator activates and stimulates an immune response against cancer cells. An immunomodulator may increase immune response by greater than 5%, 10%, 25%, 50%, 75%, 90%, 100% or more. An immunomodulator may reduce cancer cell numbers and/or growth by greater than 5%, 10%, 25%, 50%, 75%, 90%, 100% or more. Exemplary

immunomodulators include, but are not limited to, immune cells (e.g., natural killer cells, lymphokine-activated killer cells, cytotoxic T cells and dendritic cells), antibodies (e.g., anti-PD-Ll and anti-PD-l antibodies, anti-CD52, anti-VEGF-A, anti-CD30, anti-EGFR, anti-CD33, anti- CD20, anti-CTLA4, and anti-HER-2 antibodies), and cytokines (e.g., interferons and interleukins). In some embodiments, the immunomodulator is an anti-PD-Ll, an anti-PD-l antibody, or a mixture thereof. In certain exemplary embodiments, the immunomodulator is conjugated with a lipid.

In some embodiments, the therapeutic agent is a chemotherapeutic anti-cancer agent. As used herein the term "chemotherapeutic agent" refers to any chemical or biological agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancer as well as diseases characterized by hyperplastic growth. These agents can function to inhibit a cellular activity upon which the cancer cell depends for continued proliferation. In some aspect of all the embodiments, a chemotherapeutic agent is a cell cycle inhibitor or a cell division inhibitor. Categories of chemotherapeutic agents that are useful in the methods of the invention include alkylating/alkaloid agents, antimetabolites, hormones or hormone analogs, and miscellaneous antineoplastic drugs. Most of these agents are directly or indirectly toxic to cancer cells. In one embodiment, a chemotherapeutic agent is a radioactive molecule. One of skill in the art can readily identify a chemotherapeutic agent of use (e.g. see Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, l4th edition; Perry et ak, Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2nd ed. 2000 Churchill Livingstone, Inc; Baltzer L, Berkery R (eds): Oncology Pocket Guide to

Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer D S, Knobf M F, Durivage H J (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). In some embodiments, the chemotherapeutic agent can be a cytotoxic chemotherapeutic. The term

"cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g. At2l 1, 1131, 1125, Y90, Rel86, Rel88, Sml53, BΪ212, P32 and radioactive isotopes of Lu),

chemotherapeutic agents, and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.

The term chemotherapeutic agent is a broad one covering many chemotherapeutic agents having different mechanisms of action. Generally, chemotherapeutic agents are classified according to the mechanism of action. Many of the available agents are anti-metabolites of development pathways of various tumors, or react with the DNA of the tumor cells. There are also agents which inhibit enzymes, such as topoisomerase I and topoisomerase II, or which are antimitotic agents.

Chemotherapeutic agents include, but are not limited to, an aromatase inhibitor; an antiestrogen, an anti-androgen (especially in the case of prostate cancer) or a gonadorelin agonist; a topoisomerase I inhibitor or a topoisomerase II inhibitor; a microtubule active agent, an alkylating agent, an anti -neoplastic anti-metabolite or a platin compound; a compound targeting/decreasing a protein or lipid kinase activity or a protein or lipid phosphatase activity, a further anti-angiogenic compound or a compound which induces cell differentiation processes; a bradykinin 1 receptor or an angiotensin II antagonist; a cyclooxygenase inhibitor, a bisphosphonate, a heparanase inhibitor (prevents heparan sulphate degradation), e.g., PI-88, a biological response modifier, preferably a lymphokine or interferons, e.g. interferon g, an ubiquitination inhibitor, or an inhibitor which blocks anti-apoptotic pathways; an inhibitor of Ras oncogenic isoforms or a farnesyl transferase inhibitor; a telomerase inhibitor, e.g., telomestatin; a protease inhibitor, a matrix metalloproteinase inhibitor, a methionine aminopeptidase inhibitor, e.g., bengamide or a derivative thereof; a proteasome inhibitor, e.g., PS-341 (bortezomib/Velcade); agents used in the treatment of hematologic malignancies or FMS-like tyrosine kinase inhibitors; an HSP90 inhibitors; histone deacetylase (HD AC) inhibitors; mTOR inhibitors; somatostatin receptor antagonists; integrin antagonists; anti-leukemic compounds; tumor cell damaging approaches, such as ionizing radiation; EDG binders; anthranilic acid amide class of kinase inhibitors; ribonucleotide reductase inhibitors; S-adenosylmethionine decarboxylase inhibitors; antibodies against VEGF or VEGFR;

photodynamic therapy; angiostatic steroids; AT1 receptor antagonists; ACE inhibitors; and the like.

Other chemotherapeutic agents include, but are not limited to, plant alkaloids, hormonal agents and antagonists, biological response modifiers, preferably lymphokines or interferons, antisense oligonucleotides or oligonucleotide derivatives; or miscellaneous agents or agents with other or unknown mechanism of action. In some embodiments, the methods described herein comprise administering an effective amount of anti-cancer agent herein to a subject in order to alleviate a symptom of a cancer or other cell-proliferative disorder following identification of the subject as having a risk of developing a cell proliferative disorder. As used herein, "alleviating a symptom of a cancer" is ameliorating any condition or symptom associated with the cancer. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering anti-cancer agents to subjects are known to those of skill in the art. Such methods can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, topical, injection, or intratumoral administration. Administration can be local or systemic.

The term "effective amount" as used herein refers to the amount of a composition described herein needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect. The term

"therapeutically effective amount" therefore refers to an amount of a composition described herein that is sufficient to provide a particular anti -tumor effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact "effective amount". However, for any given case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of a composition described herein, which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for tumor size and/or growth, among others. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.In addition, the methods of treatment can further include the use of radiation, radiation therapy and/or the use of surgical treatments.

As used herein, the term“monitoring” refers to the observation of a disease over time. Monitoring of a subject's disease state can be performed by continuously measuring certain parameters and/or by repeatedly performing a medical test. In some embodiments of the present invention, a subject's disease state is monitored by repeatedly visiting the office of a physician to determine the change in a disease state or visiting a clinical laboratory that obtains samples of bodily fluid or tissue. Monitoring can further refer to assaying the samples using the method disclosed herein and comparing the results of the assays with one another and/or with a reference value to identify any change in the subject's disease state. In specific embodiments, the disease state that is monitored is a cell-proliferative disorder, such as cancer. In specific embodiments, monitoring of the subject is increased. For example, following an identification that the subject is at an increased risk of developing a cell-proliferative disorder, the subject can undergo increased testing. In some embodiments, monitoring comprises an increasing testing of the subject 1 time per year, 2 times per year, 3 times per year, 4 times per year, 5 times per year, 6 times per year, 7 times per year, 8 times per year, 9 times per year, 10 times per year, or more times per year compared to testing in the previous year. In particular embodiments, monitoring comprises increasing visits to a physician’s office or diagnosis center at least 1 time per year, 2 times per year, 3 times per year, 4 times per year, 5 times per year, 6 times per year, 7 times per year, 8 times per year, 9 times per year, 10 times per year, or more times per year compared to visits in the previous year.

VI. Kits and Assay Systems

Kits and assay systems for practicing the screening, prognostic, and diagnostic methods described herein are further provided. As used herein,“kit” refers to a set of reagents for the purpose of performing the various methods provided herein, more particularly, identifying a subject at risk of developing a cell-proliferative disorder by detecting a mutation in XAF1 or by detecting a reduction in expression or activity of XAF1. The term“kit” is intended to mean any manufacture (e.g., a package or a container) comprising at least one reagent, e.g., an antibody, a nucleic acid probes, etc. for specifically detecting a XAF1 polynucleotide mutation or expression of XAF1. The kit can further include packaged probe and primer sets (e.g., TaqMan probe/primer sets), arrays/microarrays of nucleic acid molecules, and beads that contain one or more probes, primers, antibodies, or other detection reagents for detecting of the level and/or activity of XAF1 mRNA and/or XAF1 protein. The kits/systems can optionally include various electronic hardware components. For example, arrays (e.g., DNA chips) and microfluidic systems (e.g., lab-on-a-chip systems) provided by various manufacturers typically include hardware components. Other kits/systems (e.g., probe/primer sets) may not include electronic hardware components, but can include, for example, one or more of XAF1 mRNA and/or XAF1 protein and/or activity detection reagents along with other biochemical reagents packaged in one or more containers.

The kit may be promoted, distributed, or sold as a unit for performing the methods of the present invention. Additionally, the kits may contain a package insert describing the kit and methods for its use.

The kit can comprise a plurality of antibodies, antibody fragments, and/or molecular probes wherein each antibody, antibody fragment, or molecular probe is specific for identifying an individual mutation in an XAF1 polynucleotide. As discussed elsewhere herein, one or more of the antibodies, antibody fragments, or polynucleotide probes within the kit can comprise a detectable label. Such detectable labels can comprise a radiolabel, a fluorophore, a peptide, an enzyme, a quantum dot, or a combination thereof. The kit can further comprise instructions for use.

As used herein, an“assay system” refers to a set of reagents for the purpose of performing the method disclosed herein, more particularly, the reagents needed for the purpose of identifying a subject at risk of developing a cell-proliferative disorder by detecting a missense or nonsense mutation in XAF1 or by detecting a reduction in expression or activity of XAF1. The assay system may be promoted, distributed, or sold as a unit for performing the methods of the present invention. Additionally, the assay system may contain a package insert describing the system and methods for its use.

In specific embodiments, a kit for identifying the level of the XAF1 mRNA and/or XAF1 protein in a biological sample is provided. The kit comprises a first and a second primer, wherein the first and second primer amplify a polynucleotide comprising the rsl46752602 marker locus.

One of skill in the art will further appreciate that any or all steps in the screening and diagnostic methods of the invention could be implemented by personnel or, alternatively, performed in an automated fashion. For example, the methods can be performed in an automated, semi-automated, or manual fashion, and as one-step or multi-step processes. XAF1 Sequences

SEQ ID NO: 1 XAF1 polynucleotide sequence

atggaaggag acttctcggt gtgcaggaac tgtaaaagac atgtagtctc tgccaacttc

ccctccatg aggcttactg cctgcggttc ctggtcctgt gtccggagtg tgaggagcct

gtccccaagg aaaccatgga ggagcactgc aagcttgagc accagcaggt tgggtgtacg

atgtgtcagc agagcatgca gaagtcctcg ctggagtttc ataaggccaa tgagtgccag

gagcgccctg ttgagtgtaa gttctgcaaa ctggacatgc agctcagcaa gctggagctc

cacgagtcct actgtggcag ccggacagag ctctgccaag gctgtggcca gttcatcatg

caccgcatgc tcgcccagca cagagatgtc tgtcggagtg aacaggccca gctcgggaaa

ggggaaagaa tttcagctcc tgaaagggaa atctactgtc attattgcaa ccaaatgatt

ccagaaaata agtatttcca ccatatgggt aaatgttgtc cagactcaga gtttaagaaa

cactttcctg ttggaaatcc agaaattctt ccttcatctc ttccaagtca agctgctgaa

aatcaaactt ccacgatgga gaaagatgtt cgtccaaaga caagaagtat aaacagattt

cctcttcatt ctgaaagttc atcaaagaaa gcaccaagaa gcaaaaacaa aaccttggat

ccacttttga tgtcagagcc caagcccagg accagctccc ctagaggaga taaagcagcc

tatgacattc tgaggagatg ttctcagtgt ggcatcctgc ttcccctgcc gatcctaaat

caacatcagg agaaatgccg gtggttagct tcatcaaaaa ggaaaacaag tgagaaattt

cagctagatt tggaaaagga aaggtactac aaattcaaaa gatttcactt ttaacactgg

cattcctgcc tacttgctgt ggtggtcttg tgaaaggtga tgggttttat tcgttgggct

ttaaaagaaa aggtttggca gaactaaaaa caaaactcac gtatcatctc aatagataca

gaaaaggctt ttgataaaat tcaacttgac ttcatgttaa aaaccctcaa caaaccaggc

gtcgaaggaa catacctcaa aataataaga gccatctatg acaaaaccac agccaacatc

atactgaatg agcaaaagct ggagcattac tcttgagaag tagaacaagg cacttcagtc

ctattcaaca tagtactgga agtctcgcca cagcaatcag gcaagagaaa gaagtaaaag

gcaccc

SEQ ID NO: 2 XAF1 protein sequence

MEGDF S VCRN CKRH V V S ANF TLHE AY CLRFL VLCPECEEP VPKETMEEHCKLEHQ QVGCTMCQQSMQKSSLEFHKANECQERPVECKFCKLDMQLSKLELHESYCGSRTELCQG CGQFIMHRMLAQHRD VCRSEQ AQLGKGERIS APEREIY CHY CNQMIPENKYFHHMGKCCP D SEFKKHFP V GNPEILP S SLP S Q A AENQT S TMEKD VRPKTRS INRFPLHSE S S SKK APRSKNK TLDPLLM SEPKPRT S SPRGDK A A YDILRRC S QC GILLPLPILN QHQEKCRWL AS SKRKT SEK FQLDLEKERYYKFKRFH Summary of Sequence Disclosure

SEQ ID NO: 1 is the human XAF1 polynucleotide sequence.

SEQ ID NO: 2 is the human XAF1 polypeptide sequence.

SEQ ID NO: 3 is the forward primer specific for XAF1.

SEQ ID NO: 4 is the reverse primer specific for XAF1.

SEQ ID NO: 5 is the forward primer for amplification of El34*-XAFl cDNA

SEQ ID NO: 6 is the reverse primer for amplification of El34*-XAFl cDNA

Embodiments

1. A method for identifying a subject at risk of developing a cell-proliferative disorder, said method comprising,

detecting a mutation within a XAF1 polynucleotide in a sample obtained from a subject; and

identifying said subject as at risk of developing a cell-proliferative disorder when a mutation is detected within said XAF1 polynucleotide.

2. The method of embodiment 1, wherein said mutation within a XAF1 polynucleotide is a missense or nonsense mutation.

3. The method of embodiment 1, wherein said subject comprises the R337H-TP53 mutation.

4. The method of any one of embodiments 1-3, wherein said XAF1 polynucleotide comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 1.

5. The method of any one of embodimens 1-4, wherein said mutation within a XAF1 polynucleotide occurs within codon 134 of said XAF1 polynucleotide.

6. The method of any one of embodiments 1-5, wherein said mutation within a XAF1 polynucleotide is a nonsense mutation within codon 134 of said XAF1 polynucleotide.

7. The method of embodiment 6, wherein said nonsense mutation is a GAA to TAA mutation (rsl46752602) within codon 134 of said XAF1 polynucleotide.

8. The method of any one of embodiments 1-7, wherein said detecting comprises: detecting, in an amplification-based assay, amplification of a nucleic acid molecule comprising at least a portion of the XAF1 polynucleotide comprising the mutation. 9. The method of embodiment 8, wherein said detecting comprises detecting, in an amplification-based assay, amplification of a nucleic acid molecule comprising codon 134 of the XAF1 polynucleotide.

10. The method of any one of embodiments 1-7, wherein said detecting comprises: detecting, in a hybridization assay, hybridization of one or more oligonucleotide probes comprising a nucleotide sequence that is complementary to a nucleotide sequence comprising the mutation in the XAFl polynucleotide.

11. The method of embodiment 10, wherein said detecting comprises: detecting, in a hybridization assay, hybridization of one or more oligonucleotide probes comprising a nucleotide sequence that is complementary to a nucleotide sequence comprising codon 134 of the CARΊ polynucleotide,

wherein codon 134 of the CARΊ polynucleotide comprises the mutation.

12. The method of any one of embodiments 1-7, wherein said detecting comprises: sequencing the CARΊ polynucleotide, or a fragment thereof.

13. The method of embodiment 12, wherein said detecting comprises sequencing at least a portion of a nucleotide sequence of the CARΊ polynucleotide comprising the mutation in the CARΊ polynucleotide.

14. The method of embodiment 12 or 13, wherein said detecting comprises sequencing at least a portion of a nucleotide sequence of the CARΊ polynucleotide comprising codon 134, wherein codon 134 of the CARΊ polynucleotide comprises the mutation.

15. The method of any one of embodiments 1-7, wherein said detecting comprises: TaqMan genotyping of the CARΊ polynucleotide, or a fragment thereof, in the sample.

16. The method of embodiment 15, wherein said detecting comprises: TaqMan genotyping of at least a portion of a nucleotide sequence of the CARΊ polynucleotide comprising codon 134,

wherein codon 134 of the CARΊ polynucleotide comprises the mutation.

17. The method of any one of embodiments 1-7, wherein said detecting comprises performing a SNP analysis of a nucleotide sequence of the CARΊ polynucleotide comprising the mutation.

18. The method of embodiment 17, wherein said detecting comprises performing a SNP analysis of a nucleotide sequence of the CARΊ polynucleotide comprising codon 134,

wherein codon 134 of the CARΊ polynucleotide comprises the mutation. 19. A method for identifying a subject at risk of developing a cell-proliferative disorder, said method comprising,

detecting a decrease in the expression of a XAF1 polypeptide in a sample obtained from a subject, or

detecting a decrease in the activity of XAF1 in a sample obtained from a subject, identifying said subject as at risk of developing a cell-proliferative disorder when a decrease in expression of XAF1 is detected or a decrease in the activity of XAF is detected.

20. The method of embodiment 19, wherein said subject comprises the R337H-TP53 mutation.

21. The method of any one of embodiments 1-20, wherein the cell-proliferative disorder is a cancer.

22. The method of embodiment 21, wherein said cancer is a solid tumor, adrenocortical tumor (adenoma or carcinoma), sarcoma, melanoma, non-small cell lung cancer, renal cell carcinoma, renal cancer, a hematological cancer, prostate cancer, castration-resistant prostate cancer, colon cancer, rectal cancer, gastric cancer, esophageal cancer, bladder cancer, head and neck cancer, thyroid cancer, breast cancer, triple-negative breast cancer, ovarian cancer, cervical cancer, lung cancer, urothelial cancer, pancreatic cancer, glioblastoma, hepatocellular cancer, myeloma, multiple myeloma, leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, myelodysplastic syndrome, brain cancer, CNS cancer, malignant glioma, or any combination thereof.

23. The method of any one of embodiments 1-22, further comprising administering a treatment to said subject with an increased risk of developing a cell-proliferative disorder.

24. The method of embodiment 23, wherein said treatment is specific for the cancer detected.

25. The method of any one of embodiments 1-24, further comprising monitoring said subject with an increased risk of developing a cell-proliferative disorder.

26. The method of embodiment 25, wherein said monitoring occurs at a higher frequency than monitoring for subjects with a standard risk of developing a cell-proliferative disorder.

27. A kit for identifying a subject at risk of developing a cell-proliferative disorder, the kit comprising:

(a) an agent for detecting a mutation in a XAF1 polynucleotide; and (b) instructions for using said agent and correlating a mutation in a XAF1 polynucleotide with an increased risk of a cell-proliferative disorder.

28. The kit of embodiment 27, wherein the subject comprises the R337H-TP53 mutation

29. The kit of embodiment 28, wherein said mutation in a CARΊ polynucleotide comprises a nonsense mutation of GAA to TAA within codon 134.

30. The kit of embodiment 29, wherein said XAF1 polynucleotide comprises SEQ ID

NO: 1.

31. The kit of any one of embodiments 27-30, wherein said agent is an antibody or nucleic acid probe.

32. Use of XAF1 as a marker for identifying a subject as at risk of developing a cell- proliferative disorder, said use comprising detecting a missense or nonsense mutation within a XAF1 polynucleotide in a sample obtained from a subject.

33. The use of embodiment 32, wherein said subject comprises the R337H-TP53 mutation.

34. The use of embodiment 33, wherein said missense or nonsense mutation is a nonsense mutation within codon 134 of said XAF1 polynucleotide.

35. The use of embodiment 34, wherein said nonsense mutation is a GAA to TAA mutation within codon 134 of said XAF1 polynucleotide.

The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES

Example 1. Methods for XAF1 (rsl46752602) detection

Briefly, genomic DNA was extracted from peripheral blood and tumor tissue using standard procedures. XAF1 was amplified by PCR to generate a fragment of 395 bp with the following primers: 5'- CCAGTGATCATGCCCTTCCT -3' (SEQ ID NO: 3) and 5'- CCACTCTGAGGCATGGTTTAG -3' (SEQ ID NO: 4) in a total volume of 50ul. PCR product (20ul) was treated with 5 U of Hpal (New England Biolabs) for one and half hours in a 37°C water bath. PCR products with and without treatment are separated side by side on a 2% agarose gel electrophoresis. Hpal, which specifically recognizes and cleaves a DNA sequence 5’ GTTAAC 3’, generated two fragments of 209 and 186 bp in the presence of mutant allele in heterozygous state while the normal allele remains uncut. The nature of the mutation was further confirmed by bi- directional sequencing of an independent PCR product. The XAF1 gene probe was isolated from the BAC clone RP11-609D21 (Sanger Institute, UK) and the centromere probe of chromosome 17 (rZ17-14) provided by Cytogenetic Unit. See, Fang et al., IntJ Cancer, 2006.

Genotyping was also performed by allelic discrimination using TaqMan® fluorescent probes. The wild type allele labeled with the VIC probe and the mutated allele labeled with the FAM probe were assayed in the same well. The reaction was carried out in a 10 pl solution (2 pl of the extracted DNA, 5 mΐ of TaqMan® Genotyping Master Mix (Applied Biosystems), 0.25 mΐ of TaqMan® SNP Genotyping Assays (rs: 146752602) and 2.75 mΐ of Ultra-pure water).

Genomic DNA extraction from 3mm paper filter disk

Dried peripheral blood stored in in 3mm paper filter disk using 5% Chelex® 100 resin (BioRad). Briefly, 100 mΐ of the resin was added to the sample, vortex homogenized and incubated at 56°C for 30 minutes, followed by incubation at 96°C for 20 minutes. Afterwards, the samples are centrifuged at 13,000 rpm for 5 minutes and the supernatant with the DNA stored. A pool of two blood samples per well (one 3mm disk with dried peripheral blood from each newborn) was incubated with 180 mΐ DNALab washing solution containing 2.7% proteinase K for 20 min at 58oC. After washing, the entire solution was discarded and the two disks were incubated in 180 mΐ ultrapure water for 10 min at 58°C. The disks were finally washed twice with ultrapure water, left to dry at 58°C and stored at -20°C.

XAF1-E134 * genotyping by TaqMan assay

The ready-to-use assay mixture was preloaded with a forward and reverse primers for amplification of the polymorphic sequence and the two allele-specific TaqMan probes descriptive of the SNP of interest, the wild type allele (G) labeled with VIC dye and the mutant allele (T) with 6FAM dye. Reactions were performed in 96-well plates and for measurements of efficiency and linearity, all PCRs were carried out in a reaction volume of 10 mΐ. A working master mix was prepared that contained 5 mΐ of TaqMan Genotyping Master Mix (Applied Biosystems, Foster City, CA, USA), 0.25 mΐ of TaqMan SNP Genotyping Assays (C_l64688507_l0, Applied Biosystems, Foster City, CA, USA) and 4.75 mΐ of water or 2.75 pL water and 2 pL of genomic DNA. All analyses were conducted on an Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). Thermal cycling conditions were 10 min at 95°C, followed by 40 cycles of 92°C for 15 s and 60°C for 1 min. All results were automatically called by 7500 software version 2.0.5.

Transfection assay construct details

Wild-type and El34*-XAFl cDNA were obtained from human fibroblasts and synthesized using the engineered primers with forward sequence containing an EcoRI site plus a FLAG tag sequence. Forward primer:

5’ TGGAATTCgccaccATGGACTAC AAAGACGATGACGAC AAGGAAGGAGACTTCTCGGT GTGC3’ (SEQ ID NO: 5) and reverse primer: 5’ GGGCCAAGATCATGGGGTT3’ (SEQ ID NO: 6). The 1.4 Kb insert was released with EcoRI and Xbal digestion and cloned into the pcDNA3 expression vector (ThermoFisher, Waltham, Massachusetts, EEGA).

Example 2. XAF1 as a modulator of TP53-R337H-driven tumorigenesis

Pediatric adrenocortical tumors (ACTs) remain an extremely rare malignancy but with high incidence in Southern Brazil (Marigo et al., Journal of the National Cancer Institute, 1969; Sandrini et al., JCEM, 1997). To date, the most significant and well characterized genetic risk factor for pediatric ACTs in this region is the presence of the germline TP53-R337H mutation (Ribeiro et al., PNAS, 2001; Latronico et al., JCEM, 2001). This founder mutation (Pinto et al., ABEM, 2004), located within the tetramerization domain of p53 was first reported in a pediatric ACT patient of Portuguese ancestry (Chompret et al., 2000). Later on, several epidemiologic studies implicated this mutation in Brazilian patients with choroid plexus carcinoma, osteosarcoma (Custodio et al., Plos one, 2011; Figueiredo et al., J Med Genet, 2006, Oliveira, Clinics, 2007, Seidinger et al., Cancer, 2011), gastric (Achatz et al., Cancer Lett, 2007), breast tumors (Palmero et al., Cancer Lett, 2008, Gomes et al., Hered Cancer Clin Pract, 2012, Cury et al., Hered Cancer Clin Pract, 2014), soft tissue sarcoma (Marcel et al., J Med Genet, 2009), neuroblastoma (Seidinger et al., PlosOne, 2015), thyroid carcinoma (Formiga et al., Jama Oncol, 2017) and non-small cell lung cancer (Couto et al., Carcinogenesis, 2017). Assessment of the family history of cancer in TP53-R337H carriers established families fulfilling the classic criteria for Li-Fraumeni syndrome (LFS), others showing an apparently sporadic pattern of cancer presentation and TP53-R337H families with no cancer history (Achatz and Zambetti, Cold Spring Harb Perspect Med. 2016), suggesting the presence of genetic and/or environmental modifiers for disease penetrance in TP53-R337H carriers.

Here we report the identification of a second independent founder haploblock, containing the E134* mutation in the putative tumor suppressor XAF1, in cis with the TP53-R337H founder haploblock in a subset of pediatric ACTs cases from Southern Brazil (Pinto et al., Nat Comm,

2015). XAF1 is frequently inactivated in many human cancers, mostly by loss or reduction of expression due to aberrant promoter methylation (Fong WG et al., Genomics, 2000; Georgina Victoria- Acosta, Cell reports, 2015) and functions in a positive feedback loop with p53 to promote apoptosis (Min-Goo Lee, PNAS, 2014).

Screening a cohort of TP53-R337H cancer patients demonstrated that carriers of both mutations were remarkably prone to LFS-associate core cancers and secondary tumors compared to single TP53-R337H carriers. In addition, our studies as well as others support the functional interaction of p53 and XAF1. Altogether, our study describes the synergistic effect of two founder mutations and provides novel insight into the etiology of cancer susceptibility suggesting XAF1 as a modifier of TP53 pathway.

Extended haplotype shared by TP53-R337H carriers

To investigate the extension of the common haplotype shared among the TP53-R337H carriers the genomic sequence covering a region of 2 centimorgan (cM) on chromosome 17r, including the TP53 locus (chrl7: 6,000,000 to 8,000,000, GRch37/hgl9) obtained from whole genomic sequence analysis of paired blood and tumor DNA from 10 unrelated Brazilian TP53- R337H ACT patients (Pinto et al., Nature Communications, 2015) was analyzed. In addition, whole exome data from 2 unrelated TP53-R337H ACT patients and single nucleotide polymorphisms (SNPs) data from 25 individuals (21 TP53-R337H carriers) from four unrelated families (3

Brazilian and 1 Spanish) obtained using Axiom Genome-wide CEU 1 Array (Affymetrix, Santa Clara, CA, USA), SNP data from a Portuguese duo (1 TP53-R337H carrier) and 13 unrelated TP53-R337H ACT carriers (GSE35066) obtained by Illumina Fhiman6l 0-Quad vl.O chips (Illumina, San Diego, CA, USA) (Letouze et al., JCEM, 2012) were included for haplotype reconstruction analysis.

Selected variants in Public database

The germline variants were analyzed at the TP53 and XAF1 locus in the Pediatric Genome Cancer Project (PCGP, 1,120 patients among 17 different diagnoses; (website at

pecan.stjude.org/home) and The Cancer Genome Atlas Research Network (TCGA, 11,000 individuals; website at cancergenome.nih.gov). Each patient was analyzed to determine the presence of the two specific variants of interest: TP53-R337H (rsl2l9l2664) and XAF1-E134* (rsl46752602). For PCGP, the analysis process has been previously described in Zhang et al.

NEJM, 2015. For TCGA dataset, the raw sequence reads near XAF1-E134* locus was extracted using the“bamslicing” function on NCI-GDC data portal. Then we analyzed the presence of XAF1- E134* variants using the same pipeline developed in house. In addition, the presence of these two variants was independently queried from the database Online Archive of Brazilian Mutations (ABraOM, 609 elderly healthy Brazilians from Sao Paulo state; website at

abraom.ib.usp.br/), The Exome Aggregation Consortium (ExAC, 60,706 individuals; website at exac.broadinstitute.org/) and Laboratorio de Sequenciamento em larga escala da FMUSP (SELA, 362 individuals with endocrinopathies, website at

www.premium. fm.usp.br/index. php?mpg=l l.42.00&lab=SELA, Lerario et al, unpublished data) as reference.

Study sites and sample collection

The present study includes genomic DNA from 171 unrelated cancer patients harboring the TP53-R337H mutation from geographically distinct locations (Brazil, Argentina, Portugal, Spain, France, Germany and United States). One additional of XX relatives from YY families were also included. Haplotype was determined by segregation analysis in genomic DNA from parents (zz) or by loss of heterozygosity in tumor samples (ww). In addition, genomic DNA from 114 newborns tested positive and 3000 newborns tested negative for TP53-R337H undergoing routine prenatal screening (Phase 2 study of neonatal screening at the Parana State, Brazil, January 2016 to July 2017) were also included. The study protocol was approved by the local Ethics Committee and approved at St. Jude Children’s Research Hospital.

TP53-R337H mutation analysis

Genomic DNA was extracted from peripheral blood and tumor tissue using standard procedures. The exon 10 of the TP53 gene was amplified by polymerase chain reaction and sequenced on a 3730x1 DNA Analyzer (Applied Biosystems, Foster City, CA, USA) as previously reported (Latronico et al., JCEM, 2001). For the newborn cohort, genomic DNA was obtained from capillary blood (heel puncture) stored in 3mm paper filter and TP53-R337H genotyped as previously described (Custodio et al., Plos One, 2011).

Genetic analysis of additional markers at the 17p region

Genomic DNA from TP53-R337H carriers was tested to to determine the nature of the founder allele using two highly informative polymorphic markers: VNTRp53 within intron 1 of the human TP53 gene and p53(CA)n located 30 kb upstream of the 5' start site of the gene. The forward oligonucleotide primers for these two markers were labeled with fluorescent dye (FAM). The PCR mixture was separated by capillary electrophoresis in a 3730x1 DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Data were analyzed by using GeneMapper v4.0 software (Applied Biosystems, Foster City, CA, USA) as previously reported (Pinto et al., Oncogenesis, 2012). In addition, the allelic variants of the SNP rs9894946 (chrl7: 7,570,830, GRch37/hgl9) downstream TP53 gene were determined by PCR and sequencing (Garritano et al., Hum Mut, 2009).

XAF1-E134* genotyping Polymerase-chain-reaction primers were designed to amplify a 395 -bp genomic DNA fragment encompassing XAF1 exon 4 (forward primer: 5'- CCAGTGATCATGCCCTTCCT - 3'(SEQ ID NO: 3); reverse primer: 5'- CCACTCTGAGGCATGGTTTAG -3' (SEQ ID NO: 4)). PCR products were incubated with 5 U of Hpa I (New England Biolabs, MA, ETSA) following the manufacturer’s recommendations and separated on a 2% agarose gel electrophoresis. Hpa I, which specifically recognizes and cleaves the DNA sequence 5’ GTTAAC 3’, generates two fragments of 209 and 186 bp in the presence of the mutant allele in heterozygous state while the normal allele remains uncut. DNA from patients with this mutation in homozygous, heterozygous, or wild-type sequence were used as control. The nature of the mutation was further confirmed by bi-directional sequencing of an independent PCR product. XAF1-E134* status was also determined by TaqMan allelic discrimination assay (Applied Biosystems, Foster City, CA, ETSA) in genomic DNA from newborns. p53 and XAF1 mRNA and protein expression in tumor samples

p53 and XAF1 mRNA expression data were obtained from European Genome-phenome archive (EGAS00001000257) containing the transcriptome profiling of 16 pediatric ACTs, 7 of them TP53-R337H carriers and from the Gene Expression Omnibus repository (GEO databases GSE76019 and GSE76021) including expression data from 63 pediatric ACTs, 10 of them TP53- R337H cases. Experiments were performed as previously described (Pinto et al., Nature

Communications, 2015; Pinto et al., Clin Can Res, 2016).

Total protein obtained from pediatric adrenocortical tumor samples submitted to transcriptome profiling (n=l6, 7 TP53-R337H) and normal adrenocortical tissue obtained during nephrectomy for Wilms tumor (n=3) were analyzed for p53 and XAF1 expression. Equal amounts of protein (30ug) were resolved on 4-12% Nupage gels (Invitrogen, Carlsbad, CA, USA), transferred to nitrocellulose filters, and probed with antibodies against p53 (clone DO-l, 1 :500, EMD Millipore, Burlington, MA, USA), XAF1 (E1E40, 1 : 1000, Cell Signaling, Danvers, MA, USA) and b-actin (clone A5441, 1 :3000, Sigma, St Louis, MO, USA). Signals were detected using Supersignal West Dura Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions and imaged using the Odyssey Fc imaging system (LiCor, Lincoln, NE, USA).

Immunostochemical analysis Immunohistochemical staining was performed on 4 um deparaffmized tissue sections from primary tumors using Benchmark XT (Ventana Medical) and BondMax (Leica Microsystems) automated Stainers with the reagents supplied by the manufacturers. The primary antibodies for p53 (1 :200 dilution, Zeta Corp), and CARΊ (1 :50 dilution, Cell Signaling) were used according to the recommendations of the suppliers. Appropriate positive and negative controls were included.

Staining was scored based on the percentage of p53 and XAF1 positive signal and classified as negative, weak, moderate or strong and cellular localization as nuclear or cytoplasmic

Transient transfections and p53 reporter assays

Wild-type and TP53-R337H cDNA were cloned into pCMV-Neo-Bam expression vector as previous described (Ribeiro, PNAS, 2001). Wild-type and XAF1-E134* cDNAs were obtained from human fibroblasts and subcloned into the pCR8/GW/TOPO vector (ThermoFisher, Waltham, Massachusetts, EUA). Human osteosarcoma cell line Saos2 was transiently transfected in duplicates with l25ng of p50-2 reporter construct containing consensus p53 element and lOOng pCMV-Neo-Bam plus lug of pcDNA3 empty vectors (1) or with pCMV-Neo-Bam expression vector containing wild-type TP53 (2) or TP53-R337H plus empty pcDNA3 expression vector (3) or co-transfected with pcDNA3 expression vector containing wild-type XAF1 (4) or XAF1-E134* plus empty pCMV-Neo-Bam (5) by calcium phosphate method as previously described (Zambetti et ak, Genes & Development, 1992). SaOs2 were also co-transfected with a combination of wild- type TP53 or TP53-R337H plus wild-type XAF1 or XAF1-E134* and wild-type XAF1 plus XAF1- E134*. Cells were harvested, protein extracts prepared and quantified and equal amounts of protein used in a standard luciferase assay (Promega, Madison, WI, ETSA) and analyzed by western blotting as described in the previous section.

Estimation of the time to most recent common ancestor (TMRCA)

To estimate the time to the most recent common ancestor (tMRCA) for the TP53-R337H and XAF1-E134* carrier haplotypes among the Brazilian patients we used a combined approach of coalescent theory calculations and simulation in an approximate Bayesian computation framework. Ms was used for the simulations, combining recombination and mutation rates to assess the probability of neither event in the region of interest, as previously described (ref). Based on the tMRCA estimates, we employed a forward simulation approach (ref) to estimate the current number of carriers per generation. RESULTS

Defining the common haplotype associated with the TP53-R337H founder mutation

Genomic DNA from 10 unrelated pediatric ACT patients from Southern Brazil harboring the TP53-R337H mutation submitted to whole genome sequencing (Pinto et al., Nature

Communications, 2015) was analyzed for haplotype inference. The 2 centimorgam (cM) haplotype (chrl7: 6,000,000 to 8,000,000, GRch37/hgl9) observed in tumor tissue with copy neutral loss of heterozygosity for chromosome 17 (Pinto et al., Nature Communications, 2015) was reconstructed in the correspondent diploid genome of the germline samples. Resulting analysis showed an identical 82,797 bp (chrl7: 7,491,978 to 7,574,775) haploblock that includes the TP53 locus shared among all TP53-R337H allele-carrying chromosomes (h=10). Furthermore, the analysis identified a second haploblock encompassing a region of 94,910 bp (chrl7: 6,612,244 to 6,707,154), in the same chromosome harboring the TP53-R337H allele in 4/10 (40%) ACT cases. This second haploblock contains the allele“T” for the variant rsl46752602 (chrl7:6,663,899) leading to a stop gain mutation (El 34*) in the putative tumor suppressor XAF1. In addition, it was verified that DNA sequence between both haploblocks (784,824 bp) in these 4 chromosomes harboring both mutations are quite distinct (Figure 1), suggesting that both haploblocks are independent and came together by recombination.

The 2cM haplotype was further verified in 42 Brazilian, Portuguese and Spanish individuals (37 of them TP53-R337H carriers) whose DNA sequence was obtained by whole exome sequencing (n=2) (Pinto et al., Nature Communications, 2015), AXIOM (n=25) or SNP arrays (n=l5, Letouze, JCEM, 2012). Although SNP analysis lacked the density to capture the full complement of genomic sequence in the haploblocks obtained by whole genome sequencing and did not include probes to detect either mutation, we could infer the genotypes and conclude that Brazilian, Spanish and Portuguese share a common TP53-R337H haplotype. However, only the Spanish and subset of Brazilian cases shared the XAF1-E134* allele.

General frequency of TP53-R337H and XAF1-E134* mutations

The TP53-R337H allele (rsl2l9l2664) is rare, virtually absent from a worldwide meta- cohort analysis with an allele frequency of 0.00000851 (http://exac.broadinstitute.org/variant/l7- 7574017-C-T). However, a neonatal screening study of 171,649 newborns in Southern Brazil, detected this variant in 461 individuals (3 homozygous carriers) revealing an estimated allelic frequency of 0.001 in this region (Custodio, JCO, 2013). Furthermore, we interrogate this mutation in two Brazilian databases with exome data from healthy individuals living in southeast Brazil (ABraOM and SELA, total 971 individuals, 1942 chromosomes) and this variant was not observed. 15 ACT patients in PCGP were identified carrying the germline TP53-R337H variants, all from Brazil. In TCGA, four patients were identified with TP53-R337H germline variants: 2 ACT patients (TCGA-OR-A5J4 and TCGA-OR-A5J7) one patient that developed Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma (TCGA-VS-A8QA), and one patient that developed Stomach Adenocarcinoma (TCGA-VQ-AA6D). Chromosome 17 LOH or partial LOH was observed in the tumor samples of all four TCGA patients. In addition, we identified one ACT patient (TCGA-OR-A5K4) and one chromophobe renal cell carcinoma patient (TCGA-KN-8424) carrying a somatic mutation TP53- R337H in their tumor samples but not in the germline samples.

The XAF1-E134* allele (rsl46752602) is uncommon in Europe (367 out of 65310 alleles) and globally (439 out of 117736 alleles) with an allele frequency of 0.003729 (website at exac.broadinstitute.org/variant/l7-6663899-G-T). To estimate the frequency of the XAF1-E134* allele in the Southern Brazilian population we genotyped 3000 newborns negative for the TP53- R337H mutation. We observed 23 positive cases, representing an allele frequency of 0.004 (23 out 6000 chromosomes). In addition, this variant was observed in 4 out 1218 (ABraOM) and in 3 out 724 chromosomes (SELA) from Brazilian databases, revealing an allelic frequency of 0.003 and 0.004 respectively. Notably, a general population screen of 67,359 newborns also from Southern Brazil identified 159 TP53-R337H carriers, 112 who also harbored the XAF1-E134* nonsense mutation (70.4% double positive). In PCGP, excluding the Brazilian ACT cases, we identified additional four patients (OS017, HYP0146, EWS001312 and TALL014) of Caucasian ancestry carrying XAF1-E134* variants, but with no TP53-R337H. A pathogenic RET mutation (V804M) and a like-pathogenic mutation in PAX7 (E201G) were the additional cancer predisposition mutations observed in HYP0146 and OS017 respectively. Therefore, the frequency of XAF1- E134* in PCGP is 0.002. Interestingly, the two ACT patients and the Stomach Adenocarcinoma TCGA patient who carried TP53-R337H variants also carried XAF1-E134* variants. Strikingly, the TP53-R337H and XAF1 E134* also showed partial LOH in their matched tumors, suggesting that they are likely to be on the same haplotype.

Prevalence ofTP53-R337H and XAF 1-El 34* in cancer

To assess the potential contribution of XAF1-E134* to human cancer in carriers of the founder TP53-R337H we determined the allele frequency and segregation pattern of these mutations in 171 unrelated patients. The TP53-R337H and XAF1-E134* genotypes were determined by PCR and sequence analysis and/or restriction site analysis (Figure 2). The TP53- R337H Founder haplotype was verified by microsatellite markers analysis (VNTRp53 and p53CA) (Pinto et al., ABEM, 2004) and rs9894946 (Garritano et al., Human Mutation, 2010) genotyping. Four patients, one from Germany (Hermann, JCEM, 2011) and three from the ETnited States, who did not share the TP53-R337H founder haplotype were excluded from further analysis. All remaining cancer patients (n=l67) revealed the same founder TP53-R337H haplotype (Table 1). A subset harbored the XAF1-E134* mutation (n=l34, 80%) and when present co-segregated with the TP53-R337H allele as evidenced by parental (n=xx) or tumor DNA (n=yy) analysis. Enrichment of the double mutant with respect to its frequency in the general population (112 of 159; 70%) is significant (P = 0.053) and suggests that XAF1-E134* cooperates with TP53-R337H in enhancing cancer risk. The allelic frequency of the germline XAF1-E134* mutation in childhood cancers based on the Pediatric Cancer Genome Project is 0.002 (4 of 2,240 chromosomes), indicating that the XAF1 nonsense mutation alone does not significantly increase tumor susceptibility.

TP53-R337H and XAF1-E134* in sarcoma

Considering TP53-R337H probands with sarcoma at diagnosis (n=24) XAF1-E134* mutation was detected in 22 (92%) (Table 1). Four double positive carriers developed a secondary tumor including a malignant breast phyllodes tumor, thyroid cancer, adrenocortical carcinoma (65 yo) and a case with uterine cancer. We have also observed first degree relatives in the vertical lineage who developed cancer (mother with uterine leiomyosarcoma, a father with malignant peripheral nerve sheath tumor, a son with ACT (26 yo) and two related daughters one with leukemia and another pre-menopausal breast cancer) all tested positive for both mutations

(Supplementary Table 1). The prevalence of double mutants in sarcoma cases is significantly higher than the general population (P = 0.027), suggesting XAF1-E134* cooperates with TP53- R337H in sarcoma tumorigenesis.

TP53-R337H and XAF 1-El 34* in breast cancer

We have also analyzed a group of TP53-R337H patients with breast tumors (n=46) (Table 1), 35 (76%) of which were positive for XAF1-E134* mutation. Bilateral cancer (n=3) and secondary tumors (h=10), including sarcomas (n=4), gastric (n=3), pancreas (n=2), uterine and lung carcinoma between two and 11 years post-diagnosis were seen in breast cancer probands with both mutations (Table 1). In contrast, 1 of 11 patients harboring TP53-R337H-only developed a secondary cancer (Castera et al., European Journal of Human Genetics, 2014) [breast (64 yo) and endometrial (77 yo) cancer] (Table 1). These results demonstrate a positive correlation between double mutants and a tumor profile that is more aggressive than single mutants.

TP53-R337H and XAF 1-El 34* in adrenocortical cancer

For the TP53-R337H ACT probands (n=88) XAF1-E134* mutation was observed in 68 (77%) (Table 1). Three patients with both mutations developed a second malignancy, one a metachronous adrenocortical tumor, (Lima et ah, ABEM, 2011), a cervical intraepithelial neoplasia, a third patient developed secondary tumors: teratoma (cystic, monodermic type) at age 26 and Breast (ER+, PR+, HER2-) at age 31 (Supplementary Table 1). We have also documented in this group, five first degree relatives in the vertical lineage who developed cancer (leiomyosarcoma, choroid plexus carcinoma, lung and 2 pre-menopausal breast tumor patients), all carriers of both mutations (Supplementary Table 1). An illustrative pedigree of a Spanish family with a pediatric ACT proband and affected family members harboring both mutations is shown in Figure 3.

TP53-R337H and XAF 1-El 34* in other tumor types

Seven TP53-R337H probands with cancer [4 choroid plexus carcinoma, 1 lung cancer (non- smoker, 55 yo), 1 kidney cancer and 1 prostate] were found to have both mutations (Table 1). The prostate patient (42 yo at diagnosis) later developed a myxoid liposarcoma at age 53 and is alive with a diagnosis of high grade dysplasia polyposis.

In the total cohort of 167 probands, we identified three patients homozygous for both mutations (2 pediatric ACT and 1 adult CPC) segregating from non-consanguineous parents (Supplementary Table 1). In one case, the mother, heterozygous for both mutations, developed early onset breast cancer and is deceased.

Tablel: Probands identified by the presence of P53-R337H and negative forX4E7-El34* or having both mutations

Proband Total TP53- R337H TP53-R337H

ACT pediatric 16 5

ACT adult 4 1

Sarcomas 2 2

Breast (equal or less than 45 years) 5 2

Breast (more than 45 years) 6 1

CPC 0

Prostate 0

Lung 0

Kidney 0

Total 33 134 Proband with one or more secondary

tumors

Table 2. Probands Identified by Tumor Type and Presence of TP53 p.R337H-Only or Extended Haplotype Segregating Both Mutations.

TP53

p.R337H

+

XAF1 P

Study Participants Total TP53 p.R337H p.E134 value OR* Cl

Control (newborn screening) 159 47 112

(0.677,

ACT (total cases[N])

102 26 76 0.572 1.226 2.249)

(0.625,

ACT (<5 years)

77 20 57 0.645 1.195 2.34)

(0.471,

ACT (>5 years)

25

19 0.642 1.327 4.321)

(1.323,

Sarcomas

29

27 0.01 5.628 50.764)

(0.600,

Breast cancer (total cases [N]) ^ 15 44 0.614 1.23 2.618)

(0.551,

Breast cancer (<45 years)

31

24 0.518 1.436 4.225)

(0.407,

Breast cancer (> 45 years)

28

20 1 1.049 2.954)

(0.894,

Other cancers

10

10 0.063 8.867 Inf)

(0.922,

Total

200^T 43 157 0.087 1.53 2.547)

(1.021,

Multiple tumors (total cases [N]) ^4

30 0.033 3.132 12.913)

Abbreviations: ACT, adrenocortical tumor; OR, odds ratio; Cl, confidence interval.

*OR was estimated by adding 0.5 to the continency table. ' Four probands (3 ACTs and 1 choroid plexus carcinoma [CPC]) homozygous for both variants were excluded from the analysis.

Uncoupling TP53-R337H and XAF 1-El 34* mutations by recombination

Recombination between these mutant alleles was observed in four independent families (74, 87, 114 and 126). As shown in Figure 4, the chromosome harboring both TP53-R337H and XAF1- E134* mutations in the ACC proband (IV-l) was inherited from his mother. Additional family members were carriers of both mutations. However, the proband’s sister (IV-2) diagnosed with Marfan syndrome was positive for the TP53-R337H mutation and negative for the XAF1-E134* mutation. These findings highlight the importance of determining the segregation pattern for both mutations in TP53-R337H carriers and their family members.

XAF1 cooperates with wild-type andR337H p53 protein

Transient transfection of wild-type and TP53-R337H into p53 negative Saos-2 cells demonstrated that the mutant activated the p53 responsive reporter p50-2 as efficiently as did the wild-type TP53 (Figure 5 A). Of interest, Saos-2 cells co-transfected with wild-type TP53 plus wild- type XAF1 or R337H-TP53 plus wild-type XAF1 resulted in a pronounced enhanced luciferase activity (Figure 5A). In contrast, El34*-XAFl was significantly less active in stimulating wild-type p53 and R337H transactivation. Furthermore, co-transfection of E134* interfered with wild-type XAF1 activation of p53 in an apparent dominant negative fashion. These results indicate that XAF1 functions as a modifier of p53 activity. Total cell lysates for each construct were analyzed by western blotting (Figure 5B).

Coalescence analysis - time to most recent common ancestor (tMRCA)

The presence of a second founder haploblock in 40% of Brazilian TP53-R337H ACT carriers that is also present in Spanish carriers but not in Portuguese ones suggest that both mutations are originate in Europe and then introduced and spread in Brazilian population due to founder effect. We performed coalescence analysis to estimate time to the most recent common ancestor (tMRCA) for both mutations. TP53-R337H is located on a short and therefore old haplotype. We obtained tMRCA estimates of xxx generations based on HapMap genetic distances. Assuming a generation time of 25 years, these estimates translate to 29000 years. However, if considered a strong bottleneck simulated by setting the effective population size to Ne=50 could be younger (2000y) (Figure 6). The XAF1-E134* allele is located on a slightly longer haplotype. The inferred tMRCA is approximately 8000y, or l400y when assuming a strong bottleneck (Figure 7). Altogether coalescence analysis reveals that both mutations are older than Brazil discovery and support the introduction of both mutations in Brazil more than the inverse migration. The region between both haploblocks in Brazilian population contains different DNA sequence suggest that both haploblocks came together as result of independent recombination events rather than descending from a common ancestor/founder.

DISCUSSION

By using whole genome sequencing (WGS) data from pediatric adrenocortical tumors (ACTs) harboring the TP53-R337H mutation (Pinto et ah, Nature Communications, 2015) we identified the presence of two independent founder haploblocks at chromosome 17r. One haploblock contains the TP53-R337H (Pinto et ah, ABEM, 2004) and the second haploblock, distant about 2cM and telomeric to the TP53, containing the XAF1-E134* mutation. The TP53- R337H mutation was first identified in association with pediatric adrenocortical tumors (Ribeiro et ah, PNAS, 2001; Latronico et al, JCEM, 2001) and has been documented in other tumor types including those LFS core cancers (ref) in Brazilian individuals. The TP53-R337H mutation was showed to have a functional activity like the wild-type protein (Ribeiro et al., PNAS, 2001;

Wasserman JCO, 2015) and the reduced penetrance explains why healthy individuals can harbor this variant in their genomes without suffering any obvious healthy effects during lifetime (Achatz and Zambetti, 2016). The TP53-R337H mutation can also exist as an independent mutational event and not related to the founder mutation observed in Southeast Brazil. We have studied four adult cancer patients with independent occurrence of this germline mutation. A panel of cancer predisposition genes was offered to three of them and the only pathogenic mutation observed was the TP53-R337H. Although we have documented only four cases, it is interesting to note that family history of cancer in those families it is not remarkable different if compared with families harboring the founder TP53-R337H mutation, excluding the fact that in southeast Brazil most of TP53-R337H families are identified from a proband in pediatric age.

Our genomic analysis allows us to verify that all TP53-R337H Brazilian individuals included in this study share the same haploblock with the Portuguese pediatric ACT, the first documented case associated with the germline TP53-R337H mutation (Varley paper) and both Spanish pediatric ACTs analyzed in the present cohort. The allelic frequency of the TP53-R337H, absent globally, but enriched in Southeast Brazil is due to founder effect (Pinto, ABEM, 2004) and our data confirm previous reports suggesting this mutation of European origin. Although with high frequency in southeast Brazil the penetrance is low, with families fulfilling the classic criteria for Li-Fraumeni syndrome (LFS), others showing an apparently sporadic pattern of cancer presentation and families with no cancer history over a lifetime. Penetrance can be influenced by environmental factors, epigenetic changes or additional genetic modifiers in cis or trans. The presence of a second haploblock containing the XAF1-E134* mutation in cis in 40% of pediatric ACTs harboring the TP53-R337H mutation analyzed by WGS, with loss of heterozygosity and maintenance of both mutated alleles in tumor tissue prompted us to investigate the frequency of this nonsense mutant allele in Brazilian individuals.

XAF1 acts as a tumor suppressor by mediating apoptosis and many cancer cells lines showed weak or no expression of XAF1 due to promoter methylation. Sequence analysis predicts a 30l-amino acid XAF1 protein containing 7 potential zinc fingers. The XAF1-E134* variant observed in our study has a stop gained functional consequence with a global minor allele frequency of 0.003 that was verified and confirmed in southeast Brazilian population.

It has been speculated that TP53-R337H mutation arisen in an individual of European ancestry and was introduced in Brazil by Portuguese migrants during the first decades of colonization (Prolla paper). Our coalescence analysis data supports a model that this mutation, located on a short and therefore old haplotype is, in fact, older than the discovery of Brazil by Europeans. In addition, XAF1-E134* mutation, located on a slightly longer haplotype was determined to be older than Brazil. Notably, the XAF1-E134* allele was found in Spanish families and absent in the Portuguese, suggesting that the double mutation may have been introduced by an early Spanish colonist.

The possibility of XAF1-E134* as a modifier of the TP53-R337H penetrance was verified in a cohort of 165 TP53-R337H cancer patients and a total of 133 cases (80%) were found to carry both mutations in contrast with 70% of the TP53-R337H carrier newborns and unknown family history of cancer (P<0.05). The implication of having one or both mutations was evident in the sarcoma group where 24/26 (92%) of patients were positive for both mutations. A similar association of both mutations was also observed in patients with breast tumor diagnosed at pre- menopausal age (20/24; 84%). Considering the patients with a secondary tumor the number is more expressive (17/19= 90%), supporting the hypothesis of XAF1 being a modifier of the TP53-R337H penetrance.

It’s important to note that XAF1-E134* mutation segregates in cis with the TP53-R337H in all probands included in this study with parental or tumor tissue available (n=xx). However, this pattern was not seen in four individuals from four distinct families where both mutations was present in the parental line, segregates in the descendants except one that was showed to have the TP53-R337H and was negative for XAF1-E134* (n=3) or vice-versa (n=l) showing that recombination events still operates and may be responsible for different genotypes in actual and successive generations. WGS determined that genomic sequence between both haploblocks were quite distinct in the 4 chromosomes with the TP53-R337H analyzed implying that both founder mutations came together because of independent recombination events rather than descending from a common ancestor/founder and due to frequency and recombination events it’s not difficult to find Brazilian individuals with one or both mutations in our population. The 2cM region studied that is so prone to recombination could constitute of a series of long interspersed nuclear elements (LINEs) that facilitates the recombination events (ref).

The collection of information regarding family history of cancer represents a crucial step in identifying those individuals who will benefit from genetic screening and surveillance. However, how family history information is taken, either positive or negative and how this information is analyzed is not uniform, particularly in Institutions from Brazil, although an increasing number of physicians have a system in place to document family history of cancer. The actual and complex setting in Brazil with no registries, no comprehensive medical records and having a high prevalent founder mutation in a tumor suppressor gene limits the collection of detailed family history of cancer sometimes restricts to the carrier side. In the present study family history was not uniformly collected and was based on data submitted by the patient or caregiver. However, family members with cancer in some families were tested and showed an aggregate of cancer patients sharing the same mutation as the proband although we have documented cancer cases in the carrier side without these mutations. Some of our probands have been tested for a panel of cancer genes and one of them was tested positive for a deleterious mutation in MSH6 gene supporting the growing number of studies in cancer predisposition genes and overlapping syndromes.

In conclusion, we have described the presence of two independent founder mutations, involving two tumor suppressor genes with additive effect in cancer penetrance. The second mutation account for 80 % of the southeast Brazilian population harboring the prevalent TP53- R337H mutation, implying the screening for both mutation is this population.

Example 3. XAF1 as a Modifier of P53 Function and Cancer Susceptibility

Delineating the common haplotype shared by TP53 p.R337H carriers WGS and WES analysis of chromosomal region 17r13.1 from 12 unrelated Brazilian ACT patients carrying the TP53 p.R337H allelel4 revealed a 29,97l-bp haplotype (chrl7: 7,570,956 to 7,600,926, GRCh37/hgl9; corresponding to 0.046 cM based on HapMap) (hereafter R337H-only haplotype; Figure 1). Previous studies proposed that the TP53 p.R337H founder allele2 originated from an individual of recent European ancestry2l and bears one copy (non-duplicate) of the l6-bp polymorphism in intron 3 (rsl7878362) and arginine at codon 72 (rsl042522). Furthermore, a long- range haplotype encompassing a 1,868,598 bp region (chrl7: 6,140,570 to 8,009,167,

GRCh37/hgl9; corresponding to 4.44 cM based on HapMap) was identified in a subset of ACT cases (5 of 12; 42%) (hereafter extended haplotype). Estimated age of the extended haplotype was 577 years (assuming a 28-year generation time; 95% [Cl], 208-1853), 22 bracketing with colonization of Brazil by Europeans and its previously suggested dispersal route into Southern Brazil.21 This extended haplotype harbors the“T” allele for SNP rsl46752602 (chrl7: 6,663,899, GRCh37/hgl9), resulting in a stop-gain variant (p.Glul34Ter/p.El34*) in the putative tumor suppressor XAF1 (Figure 1). Rare variants observed in the 2-Mb region of chromosome 17r13.1 cases were annotated. Only variants p.R337H and r.E134* showed consistent segregation within this 2-Mb region.

This extended haplotype was further verified by phasing, using genome-wide SNP array data from five unrelated multi -generational TP53 p.R337H families. We inferred genotypes and concluded that p.R337H carriers in these families share the common extended haplotype that co segregates with affected family members. The extended haplotype was also shared by relatives not expressing the phenotype, suggesting that incomplete penetrance and variable expressivity still modulate the cancer phenotype to accommodate effects of these variants.

TP53 p.R337H allele frequency

The TP53 p.R337H allele (rsl2l9l2664) is rare and virtually absent in a world-wide meta cohort with an allele frequency of 0.000009151 (gnomAD V2.1 control set). However, a neonatal screening study of 171,649 newborns in Parana, Southern Brazil, found this variant in 461 individuals (three being homozygous carriers), corresponding to an estimated allele frequency of 0.001.24 The TP53 p.R337H allele was not identified in individuals without cancer based on the Southeastern Brazilian ABraOM and SELA exome sequence databases (1,348 individuals, 2,696 chromosomes) as well as the Global Biobank Engine dataset. The TP53 p.R337H variant is reported in ClinVar as pathogenic (allele ID 12379). XAF1 r.E134* allele frequency

The XAF1 r.E134* allele (rsl46752602) is observed in Europeans (non-Finnish; 762 of 128,578 alleles) and worldwide (978 of 281,940 alleles) at an allele frequency of 0.006 and 0.004, respectively (https://gnomad.broadinstitute.org/variant/l7-6663899-G-T). This variant occurred at an allele frequency of 0.007 in the Global Biobank Engine database and 0.004 in the ABraOM and SELA Brazilian databases.

The R337H-only and extended haplotype frequency in Southern Brazil control population

Frequency of the XAF1 r.E134* allele in Southern Brazil was verified by genotyping 3000 newborns negative for the TP53 p.R337H variant. We identified 23 XAF1 r.E134* positive individuals, corresponding to an allele frequency of 0.004 (23 of 6,000 chromosomes), consistent with the ABraOMl9 and SELA databases. Strikingly, in a general screening of 67,359 newborns from the same region, 159 were TP53 p.R337H carriers, of whom 112 (70.4%) also harbored the XAF1 r.E134* variant. Analysis of parental genomic DNA revealed that both variant alleles segregated from the same parent in all cases.

The R337H-only and extended haplotype frequency of the cancer population

Genotyping of 204 cancer patients (Table 2) carrying the TP53 p.R337H founder allele demonstrated that 161 also harbored the XAF1 r.E134* variant. Segregation analysis determined that probands positive for p.R337H and r.E134* had both mutated alleles in the same haplotype. Four homozygous p.R337H and r.E134* carriers (probands #4, 61, 90 and 194, Table 2) developed childhood tumors (3 ACTs, 1 CPC) and were excluded from further cancer risk analysis. For the remaining 200 probands (Table 2) the relative frequency of the extended haplotype, although enriched, was not significantly different from population-based controls (OR, 1.5; 95% [Cl], 0.92- 2.55; P=0.09).

When stratified by tumor type, the relative frequency of the extended haplotype was significantly increased in patients with a first diagnosis of sarcoma (OR, 5.6; 95% [Cl], 1.32 to 50.76; P=0.0l). The relative frequencies of the p.R337H-only and extended haplotypes in ACT and breast cancer patients were comparable to those of controls even when considering age at diagnosis (Table 2). Ten additional patients diagnosed with CPC (n=3, excluding the one homozygous carrier), prostate (n=2), lymphoma (n=2), thyroid (n=l), lung (n=l), and renal carcinoma (n=l)] (Table 2) were collectively analyzed as a group due to limited sample sizes. All harbored the extended haplotype co-segregating the p.R337H and r.E134* alleles (Table 2). Multiple primary tumors across p.R337H carriers

Thirty-four probands (30 females and 4 males) developed multiple primary malignancies (Table 2). Thirty of these patients (88%) segregated both variants, which represents a significant enrichment compared to the control population (OR, 3.13; 95% [Cl], 1.02 to 12.91; P = 0.03). Breast cancer patients constitute 56% of these cases (19 of 34) with 17 (including all contralateral breast cancers) harboring the extended haplotype. Patients developing a third (h=10) or fourth (n=3) primary tumor were exclusively associated with the extended haplotype. One proband was diagnosed with 14 primary tumors, including 12 sarcomas (proband #199).

Considering all tumor types after first diagnosis (n=47), sarcomas were most prevalent (n=l4; 30%) and included two cases of malignant phyllodes of the breast. Remaining cases included breast cancer (n=9, 19%), adrenocortical carcinoma (n=4, 8%) and gastric cancer (n=3,

Family cancer history

Inspection of the 204 pedigrees information identified six families that fulfilled classic LFS criteria, whereas the vast majority met the revised Chompret criteria (h=141). Most probands share the same p.R337H-only or extended haplotype with one parent. However, recombination events leading to the loss of r.E134* (n=3) or p.R337H (n=l) alleles were observed in four individuals from independent families (Table Sl). For instance, the proband #149 diagnosed with breast cancer at age 27 years carrying the p.R337H founder allele was negative for the r.E134* variant, whereas her sister who developed breast cancer at age 29 years, shared both derived alleles with their mother. The father tested negative for both variants.

XAF1 increases transcriptional activity of p53

XAF1 functions through a positive feedforward loop with p53.15. To study the effect of XAF1 on p53 transactivation function, p53-null Saos-2 cells were transiently transfected with p53- responsive promoter-luciferase reporters with or without p53 and XAF1 expression vectors (Figure 8). Wild-type TP53 strongly induced the promoter containing intact p53-binding consensus sites (PG13) but not mutant sites (MG15). Full-length wild-type XAF1 and XAF1-E134* had no effect individually on either reporter (Figure 8). However, full length wild-type XAF1 stimulated wild- type TP53 transactivation, leading to increased luciferase expression. The non-sense XAF1-E134* was significantly attenuated in modulating p53 function and interfered with full-length wild-type XAF1 by blocking its ability to increase p53 transactivation in a dominant-negative manner (Figure 8). Comparable transactivation results were also obtained using p53-R337H (Figure 8). Western blot analysis showed that changes in promoter-reporter activities in response to p53 due to co expression with wild-type XAF1 and/or XAF1-E134* were not due to altered protein levels.

Studies with an independent p53-responsive promoter-reporter (p50-2) (Figure 9) yielded essentially identical results, thereby establishing XAF1 as a modifier of p53 transactivation function.

Whole genome and exome sequencing analysis identified a r.E134* variant in the putative tumor suppressor XAF1 shared by a subset of pediatric ACT patients harboring the TP53 p.R337H allele. Based on our epidemiological and functional analysis we propose that XAF1 acts as a modifier of p53 activity and cancer phenotype.

XAF1 is frequently inactivated in human cancers, mostly by gene silencing due to aberrant promoter methylation. Gain- and loss- of function studies in cell lines and mouse xenograft models further support XAF1 as a tumor suppressor. In addition, XAF1 has been reported to function within a positive feed-forward loop with p53 that impacts cell cycle arrest and cell death responses. Consistent with these findings, our in vitro studies demonstrate that full length XAF1 enhances both WT and p53-R337H transactivation, whereas XAF1 r.E134* is significantly attenuated in this activity.

If the XAF1-E134* allele modifies the cancer phenotype, the extended haplotype could impact tumor type, age of onset and/or penetrance of carriers. Strikingly, patients who developed sarcomas at first diagnosis were significantly associated with the extended haplotype (93% of cases). In addition, sarcoma was the most prevalent cancer in subsequent primary tumor diagnoses (30%) and one of the most frequently reported tumor type in family members of probands with the extended haplotype. These findings raise the intriguing possibility that XAF1 and p53 may have tissue specific functions, consistent with other inherited inactivating mutations predisposing carriers to specific tumor types, such as BRCA1/BRCA2 in breast and ovarian cancer.

The frequency of the p.R337H-only or extended haplotype was not significantly different in patients with adrenocortical tumors and controls, indicating that p.R337H is a sufficiently strong driver of tumorigenesis in this tissue. Complementary somatic events required for adrenocortical tumorigenesis include concomitant chromosome 17 and 11 copy neutral LOH. Of note, 52% (105 of 204) of our study cohort is comprised by p.R337H adrenocortical tumors. Therefore, the high percentage of ACTs potentially dilutes the biological contribution of the r.E134* allele to global cancer risk. With the exclusion of adrenocortical tumors, the extended haplotype was enriched in remaining cancer patients (OR, 2.0; 95% Cl, 1.04 - 3.98; P = 0.04). Although there were no differences in haplotype frequency in breast cancer patients, even considering those diagnosed at an early age, those harboring the extended haplotype are at an increased risk for secondary

malignancies, including all six contralateral breast cancer cases (four synchronous) and two cases of rare malignant phyllodes of the breast.

Recombination events were observed whereby individuals from families associated the extended haplotype have lost either the p.R337H or r.E134* allele. These findings underscore the need to validate not only the TP53 status, but the constitutive chromosome 17r13.1 haplotype of all family members, which will be particularly important for future studies assessing penetrance and cancer risk for carriers of the single or extended haplotype.

In conclusion, we identified XAF1 r.E134* as a linked variant that acts in concert with the TP53 p.R337H allele in modulating the cancer phenotype. These findings suggest that p.R337H haplotype may impact cancer risk more than just the p.R337H allele alone. We also propose that the modifier function of XAF1 may be relevant not only to p.R337H, but also other low penetrant mutant TP53 alleles. ETnderstanding the nature of the germline TP53 variant and the contribution of other modifiers that influence its function, such as r.E134*, will have important implications for genetic counseling, surveillance, and clinical management of affected patients.

Claims

WE CLAIM:

2. The method of claim 1, wherein said mutation within a XAF1 polynucleotide is a missense or nonsense mutation.

3. The method of claim 1, wherein said subject comprises the R337H-TP53 mutation.

4. The method of any one of claims 1-3, wherein said CARΊ polynucleotide comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 1.

5. The method of any one of claims 1-4, wherein said mutation within a CARΊ polynucleotide occurs within codon 134 of said CARΊ polynucleotide.

6. The method of any one of claims 1-5 claim 3, wherein said mutation within a CARΊ polynucleotide is a nonsense mutation within codon 134 of said CARΊ polynucleotide.

7. The method of claim 6, wherein said nonsense mutation is a GAA to TAA mutation (rsl46752602) within codon 134 of said CARΊ polynucleotide.

8. The method of any one of claims 1-7, wherein said detecting comprises: detecting, in an amplification-based assay, amplification of a nucleic acid molecule comprising at least a portion of the CARΊ polynucleotide comprising the mutation.

9. The method of claim 8, wherein said detecting comprises detecting, in an amplification- based assay, amplification of a nucleic acid molecule comprising codon 134 of the CARΊ polynucleotide.

10. The method of any one of claims 1-7, wherein said detecting comprises: detecting, in a hybridization assay, hybridization of one or more oligonucleotide probes comprising a nucleotide sequence that is complementary to a nucleotide sequence comprising the mutation in the XAF1 polynucleotide.

11. The method of claim 10, wherein said detecting comprises: detecting, in a hybridization assay, hybridization of one or more oligonucleotide probes comprising a nucleotide sequence that is complementary to a nucleotide sequence comprising codon 134 of the XAF1 polynucleotide,

wherein codon 134 of the CARΊ polynucleotide comprises the mutation.

12. The method of any one of claims 1-7, wherein said detecting comprises: sequencing the CARΊ polynucleotide, or a fragment thereof.

13. The method of claim 12, wherein said detecting comprises sequencing at least a portion of a nucleotide sequence of the CARΊ polynucleotide comprising the mutation in the CARΊ

polynucleotide.

14. The method of claim 12 or 13, wherein said detecting comprises sequencing at least a portion of a nucleotide sequence of the XAF1 polynucleotide comprising codon 134,

wherein codon 134 of the XAF1 polynucleotide comprises the mutation.

15. The method of any one of claims 1-7, wherein said detecting comprises: TaqMan genotyping of the XAF1 polynucleotide, or a fragment thereof, in the sample.

16. The method of claim 15, wherein said detecting comprises: TaqMan genotyping of at least a portion of a nucleotide sequence of the XAF1 polynucleotide comprising codon 134,

wherein codon 134 of the XAF1 polynucleotide comprises the mutation.

17. The method of any one of claims 1-7, wherein said detecting comprises performing a SNP analysis of a nucleotide sequence of the XAF1 polynucleotide comprising the mutation.

18. The method of claim 17, wherein said detecting comprises performing a SNP analysis of a nucleotide sequence of the XAF1 polynucleotide comprising codon 134, wherein codon 134 of the XAF1 polynucleotide comprises the mutation.

19. A method for identifying a subject at risk of developing a cell-proliferative disorder, said method comprising,

20. The method of claim 19, wherein said subject comprises the R337H-TP53 mutation.

21. The method of any one of claims 1-20, wherein the cell-proliferative disorder is a cancer.

22. The method of claim 21, wherein said cancer is a solid tumor, adrenocortical tumor

(adenoma or carcinoma), sarcoma, melanoma, non-small cell lung cancer, renal cell carcinoma, renal cancer, a hematological cancer, prostate cancer, castration-resistant prostate cancer, colon cancer, rectal cancer, gastric cancer, esophageal cancer, bladder cancer, head and neck cancer, thyroid cancer, breast cancer, triple-negative breast cancer, ovarian cancer, cervical cancer, lung cancer, urothelial cancer, pancreatic cancer, glioblastoma, hepatocellular cancer, myeloma, multiple myeloma, leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, myelodysplastic syndrome, brain cancer, CNS cancer, malignant glioma, or any combination thereof.

23. The method of any one of claims 1-22, further comprising administering a treatment to said subject with an increased risk of developing a cell-proliferative disorder.

24. The method of claim 23, wherein said treatment is specific for the cancer detected.

25. The method of any one of claims 1-24, further comprising monitoring said subject with an increased risk of developing a cell-proliferative disorder.

26. The method of claim 25, wherein said monitoring occurs at a higher frequency than monitoring for subjects with a standard risk of developing a cell-proliferative disorder.

(a) an agent for detecting a mutation in a XAF1 polynucleotide; and

(b) instructions for using said agent and correlating a mutation in a XAF1 polynucleotide with an increased risk of a cell-proliferative disorder.

28. The kit of claim 27, wherein the subject comprises the R337H-TP53 mutation

29. The kit of claim 28, wherein said mutation in a XAF1 polynucleotide comprises a nonsense mutation of GAA to TAA within codon 134.

30. The kit of claim 29, wherein said XAF1 polynucleotide comprises SEQ ID NO: 1.

31. The kit of any one of claims 27-30, wherein said agent is an antibody or nucleic acid probe.

33. The use of claim 32, wherein said subject comprises the R337H-TP53 mutation.

34. The use of claim 33, wherein said missense or nonsense mutation is a nonsense mutation within codon 134 of said XAF1 polynucleotide.

35. The use of claim 34, wherein said nonsense mutation is a GAA to TAA mutation within codon 134 of said XAF1 polynucleotide.