METHODS, PROBE SETS, AND KITS FOR DETECTION OF DELETION OF TUMOR SUPPRESSOR GENES BY FLUORESCENCE IN SITU HYBRIDIZATION

Abstract

Methods, probe sets, kits, and compositions for gene deletion assays are disclosed. In some embodiments, the methods relate to preparing probes for a deletion assay, performing a deletion assay, or optimizing a deletion assay. In some embodiments, the methods and probe sets can provide reduced artifactual deletion frequency, for example, when analyzing samples subject to truncation artifacts. In some embodiments, the methods and probe sets can distinguish between small and large deletions.

Claims

1-9. (canceled)

10. A method of conducting a fluorescence in situ hybridization-based assay for deletion of a tumor suppressor gene comprising: (a) performing fluorescence in situ hybridization (FISH) with a probe set on a cellular sample comprising a plurality of cells, wherein the probe set comprises at least one first flanking probe that hybridizes to a position centromeric to the tumor suppressor gene, at least one second flanking probe that hybridizes to a position telomeric to the tumor suppressor gene, and at least one target probe that hybridizes to the tumor suppressor gene; (b) enumerating FISH signals from the at least one first and at least one second flanking probes and the at least one target probe in the plurality of cells; (c) providing at least one artifactual deletion frequency for (1) a deletion that affects only the target probe, (2) a deletion that affects the target probe and the centromeric flanking probe closest to the target probe, (3) a deletion that affects the target probe and the telomeric flanking probe closest to the target probe, or (4) a deletion that affects the target probe, the centromeric flanking probe closest to the target probe, and the telomeric flanking probe closest to the target probe, the artifactual deletion frequency being chosen from (i) an artifactual hemizygous deletion frequency and (ii) an artifactual homozygous deletion frequency; (d) determining at least one apparent deletion frequency from the enumerated FISH signals of step (b), for the same type of deletion event as at least one artifactual deletion frequency of step (c), the apparent deletion frequency being chosen from (i) an apparent hemizygous deletion frequency and (ii) an apparent homozygous deletion frequency, wherein the at least one apparent deletion frequency comprises an apparent hemizygous deletion frequency if an artifactual homozygous deletion frequency was not provided in step (c), and wherein the at least one apparent deletion frequency comprises an apparent homozygous deletion frequency if an artifactual hemizygous deletion frequency was not provided in step (c); and (e) determining whether the sample comprises cells with a hemizygous deletion of the tumor suppressor gene based on whether the apparent hemizygous deletion frequency is significantly greater than the artifactual hemizygous deletion frequency, or determining whether the sample comprises cells with a homozygous deletion of the tumor suppressor gene based on whether the apparent homozygous deletion frequency is significantly greater than the artifactual homozygous deletion frequency.

11. The method of claim 10, wherein the probe set further comprises at least one third flanking probe that hybridizes to a position centromeric to the hybridization site of the first flanking probe and at least one fourth flanking probe that hybridizes to a position telomeric to the hybridization site of the second flanking probe; and wherein the method further comprises: (i) enumerating FISH signals from the at least one third and at least one fourth flanking probes in the plurality of cells; (ii) providing at least one first artifactual deletion frequency for deletions of the tumor suppressor gene with endpoints between the at least one first and at least one second flanking probes; (iii) providing at least one second artifactual deletion frequency for deletions of the tumor suppressor gene wherein at least one of the endpoints is not between the at least first and at least second flanking probes; (iv) determining, from the enumerated FISH signals of step (i), at least one first apparent deletion frequency for deletions of the tumor suppressor gene with endpoints between the at least one first and at least one second flanking probes; (v) determining, from the enumerated FISH signals of step (i), at least one second apparent deletion frequency for deletions of the tumor suppressor gene wherein at least one of the endpoints is not between the at least one first and at least one second flanking probes; and (vi) determining whether the sample comprises cells with a small deletion of the tumor suppressor gene based on whether the at least one first apparent deletion frequency is significantly greater than the at least one first artifactual deletion frequency, and determining whether the sample comprises cells with a large deletion of the tumor suppressor gene based on whether the at least one second apparent deletion frequency is significantly greater than the at least one second artifactual deletion frequency.

12. The method of claim 10, wherein (i) the tumor suppressor gene is PTEN, p16, RB1, p53, a tumor suppressor gene located on a human chromosome at a chromosome band chosen from 1 Oq23, 17p13, 13q14, 9q24, and 9p21, or a tumor suppressor gene located on a human chromosome arm chosen from 1 Oq, 17p, 13q, 9p, 1 p, 5q, 19q, 20q, 8p, 12p, and 16q, or wherein (ii) the tumor suppressor gene is PTEN and at least one first flanking probe comprises a probe that hybridizes to TSPAN15, BMPR1A, or WAPAL and the at least one second flanking probe comprises a probe that hybridizes to FAS.

13. The method of claim 10, wherein the apparent deletion frequency is significantly greater than the artifactual deletion frequency if p is less than or equal to 0.05 according to at-test, or the apparent deletion frequency is significantly greater than the artifactual deletion frequency if the apparent deletion frequency exceeds the artifactual deletion frequency by three standard deviations.

14. The method of claim 10, wherein the at least one first flanking probe hybridizes to a position within or centromeric to a boundary zone centromeric to the tumor suppressor gene, or the at least one second flanking probe hybridizes to a position within or telomeric to a boundary zone telomeric to the tumor suppressor gene.

15. The method of claim 10, wherein the hybridization sites of the at least one target probe and the at least first and second flanking probes have sizes ranging from 50 to 200 kb, or the hybridization site of the at least one target probe is separated from the hybridization sites of the at least first and second flanking probes by a distance ranging from 500 kb to 20 Mb.

16. The method of claim 10, wherein the cellular sample is fixed and preserved, or is a formalin-fixed, paraffin-embedded sample with a thickness ranging from 3 to 6 μm.

17. The method of claim 10, wherein the cellular sample is prepared by fixation with ethanol or methanol:acetic acid combined with cytocentrifugation, thin layer deposition, a smear, or pipetting onto a microscope slide.

18. A probe set comprising at least one probe that hybridizes to PTEN, at least one probe that hybridizes to FAS or SUFU, and at least one probe that hybridizes to WAPAL, wherein the WAPAL-hybridizing probe is derived from RP11-661010.

19. A composition comprising the probe set of claim 18, wherein the probes of the probe set are distinguishably labeled.

20. A kit for detecting a deletion of a tumor suppressor gene comprising the probe set of claim 18.

21. The method of claim 11, wherein (i) the tumor suppressor gene is PTEN, p16, RB1, p53, a tumor suppressor gene located on a human chromosome at a chromosome band chosen from 1 Oq23, 17p13, 13q14, 9q24, and 9p21, or a tumor suppressor gene located on a human chromosome arm chosen from 1 Oq, 17p, 13q, 9p, 1 p, 5q, 19q, 20q, 8p, 12p, and 16q, or wherein (ii) the tumor suppressor gene is PTEN and at least one first flanking probe comprises a probe that hybridizes to TSPAN15, BMPR1A, or WAPAL and the at least one second flanking probe comprises a probe that hybridizes to FAS.

22. The method of claim 11, wherein the apparent deletion frequency is significantly greater than the artifactual deletion frequency if p is less than or equal to 0.05 according to at-test, or the apparent deletion frequency is significantly greater than the artifactual deletion frequency if the apparent deletion frequency exceeds the artifactual deletion frequency by three standard deviations.

23. The method of claim 11, wherein the at least one first flanking probe hybridizes to a position within or centromeric to a boundary zone centromeric to the tumor suppressor gene, or the at least one second flanking probe hybridizes to a position within or telomeric to a boundary zone telomeric to the tumor suppressor gene.

24. The method of claim 11, wherein the hybridization sites of the at least one target probe and the at least first and second flanking probes have sizes ranging from 50 to 200 kb, or the hybridization site of the at least one target probe is separated from the hybridization sites of the at least first and second flanking probes by a distance ranging from 500 kb to 20 Mb.

25. The method of claim 11, wherein the cellular sample is fixed and preserved, or is a formalin-fixed, paraffin-embedded sample with a thickness ranging from 3 to 6 μm.

26. The method of claim 11, wherein the cellular sample is prepared by fixation with ethanol or methanol:acetic acid combined with cytocentrifugation, thin layer deposition, a smear, or pipetting onto a microscope slide.

Description

K. BRIEF DESCRIPTION OF THE DRAWINGS

[0260] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and together with the description, serve to explain the principles of the invention.

[0261] FIG. 1. Schematic depiction of large interstitial deletion of part of a chromosome arm. In this example several cytobands are lost after the deletion has taken place.

[0262] FIGS. 2A and 2B show a schematic depiction of probe set configurations (panel A) and typical interpretations (panel B) using three-color interphase FISH analysis of nuclei.

[0263] FIG. 2A shows the configuration of the hybridization sites of the probes on a schematic chromosome. Probe A is labeled with a first color, represented as hatching (in fluorescence microscopy, this color could be red, for example); the target probe (“Tum Sup”) is labeled with a second color, represented as white (in fluorescence microscopy, this color could be green, for example); and probe B is labeled with a third color, represented as black (in fluorescence microscopy, this color could be blue, for example).

[0264] FIG. 2B. A normal nucleus (left) will have three pairs of each color signal. For simple hemizygous loss (center nucleus) of the Tum Sup gene, a “white” FISH signal is missing (i.e., only one signal is present), but probes A and B flanking the tumor suppressor gene remain present in duplicate. For homozygous loss (right nucleus) both “white” FISH signals are deleted whilst flanking probes are retained.

[0265] FIG. 3. Possible FISH signals from cells having undergone complex rearrangements. The FISH signals in this schematic illustration are generated by the same probe set as in FIG. 2. The pattern at left could be generated by a cell in which two duplications of a region comprising the hybridization site of flanking probe A and one duplication of a region comprising the hybridization site of flanking probe B has occurred. The pattern at right could be generated by a cell in which the tumor suppressor has been hemizygously deleted and three extra copies of a region comprising the hybridization site of probe B have resulted from duplication events.

[0266] FIG. 4. Illustration of thresholds for scoring deletions using control normal prostate cells and PTEN deletion detection using a two-color probe set (Vysis Inc.) in which one probe hybridizes to PTEN and the other probe is a chromosome enumeration probe for chromosome 10 with a centromeric hybridization site.

[0267] FIGS. 5A and 5B show a FISH assay design using probes hybridizing to TSPAN15, PTEN, and FAS. Panel A is an illustration of hybridization sites of probes that can be used to detect PTEN deletion events.

[0268] FIG. 5A. The three probe set comprises a red PTEN probe which is flanked by the violet TSPAN15 on the centromeric side, and by the green FAS gene on the telomeric side.

[0269] FIG. 5B. A three dimensional schematic depiction is shown of four color interphase FISH using normal cells that will be expected to have two red (PTEN; represented as white), green (FAS; represented as diagonal hatching), blue (chromosome 10 enumeration probe; cross hatching) and violet (TSPAN15, represented as black) spots per nucleus. The blue centromere probe is used to determine if monosomy 10 may be present. Because of the close proximity of the hybridization sites of the flanking probes to that of the target probe, truncations tend to impact the flanking probes and the target probe together; scoring algorithms for deletion can be designed with improved discrimination between real deletion events that do not affect the flanking probes and the target probe and the truncations caused by sectioning. In the assay analyses of normal nuclei such as in the schematic section shown here can be used to provide threshold levels of artifactual deletion frequency for each probe due truncation exclusion during sectioning.

[0270] FIG. 6. Three dimensional schematic depiction of four color interphase FISH using cells hemizygously deleted for PTEN that will be expected to have one red (PTEN; represented as white), two green (FAS; represented as diagonal hatching), two blue (chromosome 10 enumeration probe; cross hatching) and two violet (TSPAN15; represented as black) spots per nucleus.

[0271] FIG. 7. Three dimensional schematic depiction of four color interphase FISH using cells hemizygously deleted for a region comprising PTEN and FAS that will be expected to have one red (PTEN; represented as white), one green (FAS; represented as diagonal hatching), two blue (chromosome 10 enumeration probe; cross hatching) and two violet (TSPAN15; represented as black) spots per nucleus.

[0272] FIGS. 8A-8D show metaphase FISH with three probes on chromosomes from a cell with a small homozygous PTEN deletion.

[0273] FIG. 8A. FISH signals from a centromeric flanking probe derived from RP11-420K10, with a hybridization site at 10q23.2. Two doublet FISH signals were observed for this probe.

[0274] FIG. 8B. FISH signals from a telomeric flanking probe derived from RP11-246B13, with a hybridization site at 10q25.1. Two doublet FISH signals were observed for this probe as well.

[0275] FIG. 8C. Absence of FISH signals from RP11-846G17, which would hybridize to PTEN if present. Only some bleedthrough of nonspecific DNA staining from the DAPI channel was visible.

[0276] FIG. 8D. Overlay of the three channels shown in panels A-C and the DAPI channel.

[0277] FIG. 9. Shown are two nuclei comprising multiple FISH signals from BMPR1A, PTEN, FAS, and chromosome 10 centromeric probes. The upper nucleus shows two centromeric signals (C) and two clusters of BMPR1A/PTEN/FAS (B/P/F) signals. The lower nucleus appears to be a post-replication nucleus in which there are two clusters of BMPR1A/PTEN/FAS plus centromeric signals (B/P/F/C), and two B/P/F clusters, one of which is near a centromeric signal. It is possible that two centromeric signals are not resolved, or that one was lost due to a truncation event.

[0278] FIG. 10. Copy number profile for prostate cancer samples versus normal reference DNA from CGH data. The profile on the left indicates extent of loss in the cancer samples, while the profile on the right indicates extent of gain. The arrowhead indicates a peak in the loss profile corresponding to the position of PTEN. See Example 1 for further discussion.

[0279] FIG. 11. FISH results for 82 prostate cancer samples with a hemizygous PTEN deletion. Probes with 1 missing FISH signal are indicated by shading; thus a sample that lost only 1 PTEN signal has shading only in the PTEN row, while a sample in which both flanking probes were lost as well has shading in all three rows. See Example 3 for further discussion.

[0280] FIG. 12. FISH results for 50 prostate cancer samples with a homozygous PTEN deletion. Plotting is as in FIG. 11 except that shading indicates homozygous loss and diagonal hatching indicates hemizygous loss. See Example 3 for further discussion.

[0281] FIGS. 13A-13E show representative FISH microscopy images for various PTEN deletion statuses. White arrowheads indicate PTEN FISH signals. In addition to PTEN, probes used were as in FIG. 9.

[0282] FIG. 13A. The cells in panel A had 2 of each FISH signal (normal).

[0283] FIG. 13B. The cells in panel B frequently lacked one PTEN FISH signal (small hemizygous PTEN deletion).

[0284] FIG. 13C. The cells in panel C frequently lacked one PTEN FISH signal and one probe A FISH signal (large hemizygous PTEN deletion).

[0285] FIG. 13D. The cells in panel D frequently lacked both FISH signals for PTEN and probe B (large homozygous deletion).

[0286] FIG. 13E. The cells in panel E frequently lacked two PTEN FISH signals (small homozygous deletion).

[0287] FIG. 14. Circular ideogram of chromosome 10q in which segmental duplications are connected by the curved lines on the interior of the circle. The three arrowheads outside the circle indicate, from bottom to top, the hybridization sites of probe A, the PTEN probe, and probe B, respectively.

[0288] FIG. 15. Chromosome 10 is shown with the location of PTEN circled. A plot of losses and gains generated from the CGH copy number data of Example 1 is shown below the schematic of the chromosome. In the bottom two rows are CNV and segmental duplication sites.

[0289] FIG. 16. An enlargement of the data from FIG. 15 in the neighborhood of PTEN is shown. Locations of Probe A and Probe B from FIG. 13 et al. are indicated. CNV and segmental duplications are shown in the 3.sup.rd and 5.sup.th rows beneath the CGH data. Below that is a representation of deleted areas in individual prostate cancer samples (thick lines indicate deletions).

[0290] FIG. 17. Probe subsets for a 2-part method for distinguishing small and large PTEN deletions. In the first part of the method, FISH can be performed with a flanking probe hybridizing to TSPAN15, a target probe hybridizing to PTEN, and a flanking probe hybridizing to FAS. A centromeric probe can also be used. Boundary zones, where breakpoints of small deletions seem to occur most often, are indicated by dotted lines. The position of a segmental duplication cluster (SD) is also indicated. In the second part (“reflex assay”), the flanking probe hybridize to BMPR1A and SUFU. Each part of the assay interrogates whether a region relatively close to PTEN on one side is affected by a large deletion and provides a more distant probe on the other side that would be affected in even larger deletions; in the case of SUFU, losses could be caused by deletions extending from PTEN all the way to the telomere.

[0291] FIG. 18. Schematic representation of FISH signals from probes as in FIG. 17 bound to an intact chromosome for the two parts of the assay to distinguish small and large PTEN deletions.

[0292] FIGS. 19A-19B show a schematic representation of probe hybridization sites for the probes of the two part assay as in FIG. 17 along chromosome 10 and BACs from which the probes can be derived.

[0293] FIG. 19A. Panel A shows the probes of the first subset and

[0294] FIG. 19B. Panel B shows the probes of the second subset (for the reflex assay).

[0295] FIG. 20. Relative positions of CNV and segmental duplication loci near PTEN, BMPR1A, SUFU, TSPAN15, and FAS.

[0296] FIG. 21. Python scripting language source code for extraction of hits from a blast file with 95% identity and length >10000 bp.

[0297] FIG. 22. Schematic depiction of probe set configurations using four-color fish to detect smaller and larger deletions. This shows the hybridization sites of probes on a blown up section of region 21q22.2. Target Probe A shown with vertical lines (DSCAM) is labeled in a 1.sup.st color (Aqua, for example). Target Probe B shown with horizontal stripes (HMGN1) is labeled in a second color (Gold, for example). Flanking Probe C shown checkered (ERG) is labeled in a third color (for example, orange). Flanking probe D, shown in solid (5′ TMPRSS2) is labeled in a fourth color (for example, green). The diagram also shows centromeric flanking probe E in dots (DYRK1A, labeled in green fluor, for example) and telomeric flanking probe F in crosshatch (U2AF1-labeled in gold fluor, for example) that are used in cases where 3 signals of the first four probes are lost, and a larger deletion boundary is in effect. In a four-color test, if probes A and B are lost, Probes C and D (Orange and Green fluors) would remain. If Probes A, B, and C are lost, Probe D (Green fluor) remains, and a follow-up 3-color test with any of the further flanking Probes can be used to determine the deletion boundary.

[0298] FIG. 23. Map of the 21q22.13-21q22.3 region showing gene and exemplary probe locations and an indication of a zone within which deletions may occur.

EXAMPLES

[0299] Reference will now be made in detail to embodiments of the invention, aspects and results of which are illustrated in the accompanying drawings.

Example 1. Analysis of Copy Number Variation, Segmental Duplication, and Comparative Genomic Hybridization Data for Chromosome 10; Probe Site Selection

[0300] CGH data from Liu et al., Nat. Med. 2009; 15:559-65 were analyzed in silico. In silico copy number analysis of the chromosome 10q region in 58 metastatic CaP samples from 14 patients (Liu W et al. Copy number analysis indicates monoclonal origin of lethal metastatic prostate cancer, Nat. Med. 2009; 15:559-65) was performed applying rank segmentation, with a significance threshold of 1.0×10.sup.−6 and a minimum of 5 probes per segment (Nexus Copy Number v.4; BioDiscovery, El Segundo, Calif.). Genomic imbalances were assigned as either gain [log(3/2) or threshold of 0.2] or loss [log(½) or threshold of −0.3], each determined by two Copy Number Transitions (CNTs), as defined by Ferreira B I et al., Array CGH and gene-expression profiling reveals distinct genomic instability patterns associated with DNA repair and cell-cycle checkpoint pathways in Ewing's sarcoma, Oncogene 2008; 27:2084-2090.

[0301] Data showing areas of loss near PTEN are shown in FIG. 16. The average abundance of loci in the population of metastatic CaP samples relative to the reference was also determined and plotted (FIGS. 15 and 16; see also FIG. 10). It was determined that the abundance declined by more than 20% in the region from 81.5 Mb to 89.67 Mb (i.e., the ˜8 Mb centromeric to PTEN), representing a copy number transition in the population.

[0302] Copy Number Variation (CNV) data was obtained from the Sanger Institute's CNV Project (available on the Sanger Institute website) in ASCII text format. The data available corresponded to 269 distinct samples collected by the international consortium HapMap (available on the HapMap NCBI web server). The downloaded files were then filtered and only regions pertaining to chromosome 10 were transferred to a spreadsheet. Overlapping regions were selected manually on Microsoft Excel. Segmental duplication data was obtained by employing Blast (Altschul S F et al., J. Mol. Biol. 1990; 215:403-410) alignments using the assembled chromosome 10 as the reference database.

[0303] Individual segmental duplications obtained from the Segmental Duplication Database (She X et al., Shotgun sequence assembly and recent segmental duplications within the human genome, Nature 2004; 431:927-930, available on the humanparalogy web server at the University of Washington) were then aligned to the chromosome in automatic fashion and the resulting hits tabulated for each segment in the database. Tables for the more than 9000 sequences in Segmental Duplication Database were filtered, and only hits (alignments) with more than 95% homology and longer than 10 kilobases were automatically selected by computer script (FIG. 21). Contiguous regions were then selected manually in the same fashion as the CNV segments. The cut off value considered to group contiguous alignments (hits) was 50 kilobases of distance. Another cutoff value of 100 kilobases of distance was employed in order to group the resulting clusters.

[0304] It was determined that chromosome 10 contained clusters of segmental duplications (SDs) and CNVs in the regions listed in Table 1.

TABLE-US-00004 TABLE 1 Chromosome 10 Segmental duplications and CNVs Start (bp) End (bp) Length (bp) High density regions CNV1 42,004,899 42,760,575 755,676 CNV2 45,341,719 49,121,538 3,779,819 CNV3 50,641,980 51,595,172 953,192 CNV4 76,904,943 77,440,455 535,512 CNV5 80,945,468 81,722,592 777,124 CNV6 88,505,038 89,299,742 794,704 CNV7 90,825,044 91,007,466 182,422 CNV8 98,779,953 98,952,462 172,509 CNV9 102,141,840 102,555,726 413,886 CNV10 107,519,652 107,743,529 223,877 CNV11 110,448,977 110,633,486 184,509 CNV12 122,625,702 122,891,863 266,161 CNV13 124,253,065 124,444,805 191,740 CNV14 125,047,207 125,259,149 211,942 CNV15 127,443,890 127,776,692 332,802 CNV16 134,076,220 135,240,498 1,164,278 High density region SD1 41,991,393 42,173,845 182,452 SD2 42,531,265 42,689,258 157,993 SD3 45,492,329 46,843,228 1,350,899 SD4 47,023,808 47,533,536 509,728 SD5 47,731,989 47,900,982 168,993 SD6 48,376,799 48,697,638 320,839 SD7 48,865,543 49,055,736 190,193 SD8 50,735,042 51,157,549 422,507 SD9 51,275,627 51,628,828 353,201 SD10 52,104,752 52,214,149 109,397 SD11 57,043,647 57,055,423 11,776 SD12 75,091,834 75,143,840 52,006 SD13 80,936,173 80,980,415 44,242 SD14 81,081,817 81,275,000 193,183 SD15 81,379,701 81,625,517 245,816 SD16 81,959,635 82,002,788 43,153 SD17 88,743,560 88,770,949 27,389 SD18 88,890,157 89,250,617 360,460 SD19 127,598,385 127,609,227 10,842 SD20 135,233,079 135,363,669 130,590

[0305] The coordinates in Table 1 refer to positions in chromosome 10 of UCSC version NCBI36/hg18 (Mar. 2006) of the human genome.

[0306] Based on the locations of the segmental duplications, CNV, and population copy number transition, the TSPAN15 and BMPR1A loci were selected as hybridization sites for centromeric flanking probes. The FAS and SUFU loci were selected as hybridization sites for telomeric flanking probes.

Example 2. Three-Color FISH with a Sample Having a Homozygous PTEN Deletion

[0307] Metaphase chromosomes from cells of the PC3 cell line (Beheshti B et al., Evidence of chromosomal instability in prostate cancer determined by spectral karyotyping (SKY) and interphase fish analysis, Neoplasia 2001; 3:62-9) were fixed and hybridized with three distinguishably labeled probes prepared using the RP11-420K10 BAC, with a hybridization site at 10q23.2 (labeled green); the RP11-246B13 BAC, with a hybridization site at 10q25.1 (labeled red); and RP11-846G17, with a hybridization site at PTEN (labeled aqua). The chromosomes were also counterstained with DAPI (blue). Images in the red, green, aqua, and blue channels were obtained by fluorescence microscopy. Images from a representative set of chromosomes are shown in FIG. 8, in which four FISH signals were visible in the green and red channels (panels A and B). There were 4 FISH signals since the diploid genome has been replicated but not yet segregated at metaphase. No aqua FISH signals were observed for the PTEN probe (panel C) although slight DAPI fluorescence was visible in this channel. Panel D shows an overlay of the three images of panels A-C and the DAPI fluorescence from the blue channel.

Example 3. Deletion Mapping by Four-Color Interphase FISH

[0308] Four color interphase FISH was performed on 132 samples of cancerous prostate tissue deleted for at least one copy of PTEN. The 132 samples were a subset of 330 cancerous prostate clinical tissue samples taken at McGill University and the University of Toronto. Details about these samples appear in the tables below.

[0309] Breakdown of the Total 330 Patients

TABLE-US-00005 Radical Prostatectomies 134 (41%) Hormone refractory/Metastatic tumors 196 (59%) Total 330

[0310] Breakdown of the 132 Samples with a Hemi- or Homozygous PTEN Deletion

TABLE-US-00006 Radical Prostatectomies 86 Hormone refractory/Metastatic tumors 46 Total 132

[0311] The probes used were a probe derived from the BACs RP11-141D8 and RP11-52G13 (“probe A”; hybridization site centromeric to PTEN); a PTEN probe derived from the BAC RP11-846G17; and a probe derived from the BACs RP11-399O19 and RP11-360H20 (“probe B”; hybridization site telomeric to PTEN). The probes were labeled with distinguishable fluorophores by nick translation. Positional information for the BAC clones can be obtained from the Human March 2006 assembly of the UCSC Genome Browser 1. Also used was the chromosome 10 centromeric probe SpectrumAqua labeled CEP10, Vysis Abbott Molecular, Des Plaines, Ill., USA.

[0312] Analysis of the 132 samples indicated that 82 of them had hemizygous PTEN deletions and 50 had homozygous PTEN deletions, based on presence of 1 or 0 PTEN probe FISH signals in at least 30% of the cells. Additionally, the presence or absence of probes A and B was enumerated to determine whether the deletion(s) encompassed the hybridization sites of these probes as well. Results are listed in Table 2 below and shown in FIGS. 11-13.

TABLE-US-00007 TABLE 2 Extents of deletions Hemizygous PTEN deletions Homozygous PTEN deletions Probes Affected Frequency Probes Affected Frequency PTEN 37% PTEN (0) 48% PTEN and Probe B 32% Probe A (1), PTEN (0), 22% Probe B (1) Probe A and PTEN  3% PTEN (0) and Probe B (0) 12% Probe A, PTEN, 28% PTEN (0) and Probe B (1) 10% and Probe B Probe A (1), PTEN (0),  4% Probe B (0) Probe A (1) and PTEN (0)  4%

[0313] In the column for homozygous PTEN deletions, the parenthesized numbers indicate how many FISH signals from a given probe were present; thus, for example, “Probe A (1), PTEN(0), Probe B (1)” indicates that either one chromosome was deleted for PTEN only and the other chromosome was deleted for the whole region from Probe A to Probe B, or one chromosome was deleted for PTEN and Probe B, and the other chromosome was deleted for Probe A and PTEN.

[0314] The minimum size of deletions affecting only PTEN was estimated as 176 kb. The size range of the largest deletions, affecting Probe A, PTEN, and Probe B, was estimated as at least 2.5 Mb.

Example 4. Resolution of PTEN Status in Samples Difficult to Interpret by Two Color FISH

[0315] A set of 91 formalin-fixed paraffin embedded samples from radical prostatectomies with unknown clinical outcome at the time of study were analyzed using both two-color interphase FISH with the commercially available PTEN and centromeric probes from Abbott Inc., and by four color FISH, using a probe set in which probe A was prepared from the BACs RP11-141D8 and RP11-52G13 and the PTEN probe and probe B were as in Example 3. Six samples were identified in which results differed between the two assays. These results are listed in Table 3 below.

[0316] The analysis criteria for the two color assay were: at least 70% of nuclei with 2 PTEN signals and two chromosome 10 centromeric probes: no copy change. 25%-30% of nuclei with simultaneous loss of one PTEN signal missing but presence of two centromeric probes: inconclusive. Greater than 30% of nuclei with one PTEN signal missing and both centromeric probes retained: hemizygous deletion. 30%-100% of nuclei with two PTEN signals missing and centromeric probes retained: homozygous deletion.

[0317] The analysis criteria for the four color assay were established after truncation artifacts were established for each probe in a given set. They were: at least 80% of nuclei with 2 PTEN signals and retaining flanking probes: no copy change. 18%-20% of nuclei with one PTEN signal missing whilst retaining both flanking probes: inconclusive. Greater than 20% with one PTEN signal missing but simultaneously retaining both flanking probes: hemizygous deletion. 20%-100% of nuclei with two PTEN signals missing which may be accompanied by simultaneous losses of one, both or neither flanking probes: homozygous deletion.

[0318] These criteria were based on a requirement that, to call a deletion, the apparent frequency must have been greater than the artifactual deletion frequency plus three standard deviations; these values were 30% for the two color assay and 20% for the four color assay; see the tables below showing control results for the two color and four color probe sets. Artifactual deletion frequencies were measured using control samples from noncancerous prostate samples from biopsies and/or radical prostatectomies performed on patients with benign prostate hyperplasia. Use of three standard deviations to set a significance threshold in FISH assays is discussed in Ventura et al., J. Mol. Diagn. 2006; 8:141-151.

TABLE-US-00008 TABLE 3 Control results - artifactual deletion frequency with 4-color probe set 2 CEP10/2 BMPR1A/1 Prostate samples PTEN/2 FAS control 1 12 control 2 5 control 3 4 control 4 10 control 5 12 control 6 8 control 7 2 control 8 8 control 9 4 control 10 13 Average 7.8 St dev 3.9 3 st dev 11.7 Average + 3 st dev 19.5

TABLE-US-00009 TABLE 4 Control results - artifactual deletion frequency with 2-color probe set Prostate samples 2 CEP10/1 PTEN (Vysis) control 1 18 control 2 17 control 3 14 control 4 20 control 5 15 control 6 10 control 7 5 control 8 16 control 9 18 control 10 6 Average 13.9 St dev 5.2 3 st dev 15.6 Average + 3 st dev 29.5

TABLE-US-00010 TABLE 5 Detection of PTEN deletion using two and four color PTEN probe sets Four color assay with Sample Two color assay Ex. 3 probes CaP-1 No copy change Hemizygous del CaP-2 Hemizygous del No copy change CaP-3 No copy change Hemizygous del CaP-4 No copy change Homozygous del CaP-5 Hemizygous del No copy change CaP-6 Hemizygous del No copy change CaP-7 Hemizygous del No copy change CaP-8 Hemizygous del No copy change CaP-9 Inconclusive Hemi- and homozygous del CaP-10 Inconclusive Hemizygous del CaP-11 Inconclusive Hemizygous del CaP-12 Inconclusive No copy change CaP-13 Inconclusive No copy change CaP-14 Inconclusive No copy change CaP-15 Inconclusive No copy change CaP-16 Inconclusive No copy change CaP-17 Inconclusive No copy change CaP-18 Homozygous del No copy change CaP-19 Inconclusive Hemizygous del CaP-20 Hemizygous del No copy change

[0319] “Hemi- and homozygous del” indicates that significant numbers of cells were present with both types of deletion, consistent with an initial deletion of one copy of PTEN, followed by clonal expansion, with a second deletion event resulting in a homozygously deleted subpopulation.

[0320] Thus, the four color assay was able to resolve samples 9-17 and 19 that were inconclusive according to the two color assay. Additionally, several samples appear to have given a false positive result in the two color assay (presumably due to an above-average number of truncation effects), and samples 1, 3, and 4 appear to have given a false negative result. Finally, in sample 9, the two-color assay apparently did not detect the hemizygously deleted population of cells.

[0321] It is thought that the improved specificity and sensitivity of the four-probe assay results from the lower artifactual deletion frequency resulting from having the flanking probes A and B positioned closer to the target of the assay (PTEN) but still at locations where many deletions should not affect the FISH signals from the flanking probes.

Example 5. Boundary Zone Identification and FISH Probe Preparation for p16

[0322] CGH data comparing average genomic copy number from melanoma cell samples to reference cells is obtained from the Broad Institute web server, described in [Beroukhim R et al., The landscape of somatic copy-number alteration across human cancers, Nature 2010; 463:899-905]. The nearest copy number transition zone in which the average relative copy number of loci in the population of 111 melanoma samples declines by at least 20% over an interval of at most 15 Mb is identified on the centromeric side of the p16 gene (also known as CDKN2A) at 9p21.

[0323] Genome annotations from the Wellcome Trust Sanger Institute [available on the Wellcome Trust Sanger Institute web server] for copy number variation polymorphic loci (CNVs) [Casci T. Genome evolution: CNV evolution revisited, Nature Reviews Genetics 2008; 9:814-815] and from the Department of Genome Sciences, University of Washington [available on the humanparalogy server at the University of Washington] for segmental duplications [Rudd M K et al., Segmental duplications mediate novel, clinically relevant chromosome rearrangements, Hum. Mol. Genet. 2009; 18:2957-62] in the area of the copy number transition zone and the surrounding vicinity are obtained.

[0324] At least one CNV in the copy number transition zone or within the 5 Mb centromeric to it is identified. The annotated endpoints of the nearest of the at least one CNV to p16 are referred to below as the distal and proximal CNV endpoints (with respect to proximity to p16).

[0325] At least one cluster containing at least four annotated segmental duplications within a 1 Mb range of annotated segmental duplications in the copy number transition zone or within the 5 Mb centromeric to it is identified. The endpoints of the segmental duplication cluster nearest to p16 are defined either by the annotated endpoints of a high density segmental duplication region in the Segmental Duplication Database (She X et al., Shotgun sequence assembly and recent segmental duplications within the human genome, Nature 2004; 431:927-930, and the humanparalogy server at the University of Washington) or by the location of the two segmental duplication loci within the 1 Mb range most proximal and distal to p16.

[0326] The region bounded by (1) the more distal to p16 of the distal endpoints of the CNV and the segmental duplication cluster and (2) the more proximal to p16 of the proximal endpoints of the CNV and the segmental duplication cluster is identified as a boundary zone.

[0327] A centromeric flanking probe is prepared which has a hybridization site whose center is within the 1 Mb extending in the centromeric direction from the edge of the boundary zone distal to p16.

[0328] On the telomeric side of p16, at least one CNV and/or at least one segmental duplication cluster is identified within 2 Mb of the telomeric end of the p16 locus. A telomeric flanking probe is prepared which has a hybridization site whose center is within the 1 Mb extending in the telomeric direction from the telomeric end of either the CNV or the segmental duplication cluster.

[0329] A target probe is prepared which has a hybridization site whose center is within the p16 locus or within 100 kb of either end of the p16 locus.

[0330] A FISH probe set comprising the centromeric flanking probe, the telomeric flanking probe, and the target probe can be used to assay for deletions of p16 via interphase FISH. The assays have high accuracy with formalin fixed paraffin embedded samples because truncation artifacts affecting the FISH signals of centromeric flanking probe, the telomeric flanking probe, and the target probe are readily distinguished from cells affected by genetic deletions having at least one endpoint between the telomeric and centromeric flanking probes.

Example 6. Deletion Mapping of 21q22.13-21q22.3 Region by Four-Color Interphase FISH

[0331] Four-color interphase FISH is performed on (FFPE-FNA) samples of prostate tissue:

[0332] 1. Normal cells

[0333] 2. hemizygous PTEN deletions, and

[0334] 3. homozygous PTEN deletions

using the methods described above.

[0335] The 21q22.13-21q22.3 (specifically, 21q22.2) region comprises several genes, including TMPRSS2, HMGN1, DSCAM, and ERG1. The probes to be used are derived from BACs RP11-476D17 (Probe A: centromeric to HMGN1), a HMGN1 probe derived from BACs RP11-137J13 and RP11-348C15; a DSCAM probe derived from BACs RP11-139D12, RP11-907P24, RP11-280O17, RP11-183I12, RP11-281F3, RP11-1113M13, and RP11-123A7 and a probe derived from RP11-3504 (probe B; telomeric to DSCAM). The probes are labeled distinguishably (e.g., with red, green, aqua, and gold fluorophores) by nick translation. Positional information for the BACs can be obtained from Human (February 2009) assemblies of the UCSC Genome Browser. A chromosome 21 centromeric or pericentric probe may also be used.

[0336] Analysis of the samples indicates that a subset of cells having hemizygous PTEN deletion also have a HMGN1 deletion or a DSCAM deletion, or both, as shown by the presence of hybridization signals from Probe A and Probe B but not one or both of the HMGN1 and DSCAM probes. Another subset of cells showing homozygous PTEN deletions shows additional or single deletions. Normal cells show the presence of all the genes.

[0337] The embodiments within the specification provide an illustration of embodiments of the invention and should not be construed to limit the scope of the invention. The skilled artisan readily recognizes that many other embodiments are encompassed by the invention. All publications and patents cited in this disclosure are incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material. The citation of any references herein is not an admission that such references are prior art to the present invention.

[0338] Unless otherwise indicated to the contrary, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification, including claims, are approximations and may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

[0339] Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0340] When method steps are recited, including with labels such as (a) or (i), it is to be understood that the order of steps in the claim is not necessarily the only possible order, for example, in cases where a later-listed step does not require the product or result of an earlier-listed step.

EXEMPLARY EMBODIMENTS

[0341] 1. A method of preparing a probe set for a FISH-based tumor suppressor deletion assay, the method comprising: [0342] (a) identifying at least one boundary zone on a chromosome, said chromosome comprising a tumor suppressor gene, wherein the at least one boundary zone comprises a first boundary zone centromeric to the tumor suppressor gene; [0343] (b) providing at least a first flanking probe that hybridizes to a nucleic acid sequence within the first boundary zone or to a nucleic acid sequence distal to the tumor suppressor gene relative to the first boundary zone; [0344] (c) providing at least a second flanking probe that hybridizes to a nucleic acid sequence telomeric to the tumor suppressor gene; and [0345] (d) providing at least one target probe that hybridizes to a nucleic acid sequence in the tumor suppressor gene between the boundary zones.

[0346] 2. The method of embodiment 1, wherein the at least one target probe and the at least first and second flanking probes are derived from bacterial artificial chromosomes (BACs), cosmids, or amplification products, or are provided as sets of synthetic oligonucleotides.

[0347] 3. The method of any one of embodiments 1 to 2, wherein the first boundary zone is a primary boundary zone.

[0348] 4. The method of any one of embodiments 1 to 3, wherein the tumor suppressor gene is PTEN.

[0349] 5. The method of any one of embodiments 1 to 3, wherein the tumor suppressor gene is chosen from p16, RB1, and p53.

[0350] 6. The method of any one of embodiments 1 to 5, wherein identifying the at least one boundary zone comprises identifying a region in which (1) a copy number transition occurs in a population of samples known to comprise deletions of the tumor suppressor gene, and (2) at least one of a copy number variation (CNV) and a cluster of segmental duplications occurs.

[0351] 7. The method of any one of embodiments 1 to 6, further comprising identifying at least a second boundary zone, wherein the second boundary zone is telomeric to the tumor suppressor gene, and the second flanking probe hybridizes to a nucleic acid sequence within the second boundary zone or to a nucleic acid sequence distal to the tumor suppressor gene relative to the second boundary zone.

[0352] 8. The method of embodiment 7, further comprising identifying at least a third boundary zone and providing a probe that hybridizes to a nucleic acid sequence within the third boundary zone or to a nucleic acid sequence distal to the tumor suppressor gene relative to the third boundary zone.

[0353] 9. The method of any one of embodiments 1 to 8, further comprising providing at least one chromosome enumeration probe.

[0354] 10. The method of any one of embodiments 1 to 9, wherein the at least one target probe and at least first and second flanking probes can be used in a FISH-based tumor suppressor deletion assay wherein the assay has a significance threshold calculated as an artifactual deletion frequency plus three standard deviations less than or equal to 30% on formalin-fixed, paraffin-embedded cellular samples with a 5 μm thickness and with an average nuclear diameter less than 5 μm.

[0355] 11. The method of any one of embodiments 1 to 10, wherein the hybridization sites of the at least one target probe and the at least first and second flanking probes have sizes ranging from 50 to 200 kb.

[0356] 12. The method of any one of embodiments 1 to 11, wherein the hybridization site of the at least one target probe is separated from the hybridization sites of the at least first and second flanking probes by a distance ranging from 500 kb to 20 Mb.

[0357] 13. The method of any one of embodiments 1 to 12, wherein the hybridization sites of the at least first and second flanking probes are in the at least first and second boundary zones, respectively.

[0358] 14. The method of any one of embodiments 1 to 13, wherein the at least first and second boundary zones have sizes ranging from 300 kb to 2 Mb.

[0359] 15. The method of any one of embodiments 1 to 14, wherein at least one of the at least first and second boundary zones is identified based on a copy number transition in comparative genomic hybridization data from cancerous or precancerous cells.

[0360] 16. The method of any one of embodiments 1 to 15, wherein at least one of the at least first and second boundary zones is identified based on FISH analysis of a plurality of cellular samples known to contain deletions of the tumor suppressor gene.

[0361] 17. A kit for detecting deletions in tumor suppressor genes comprising a probe set prepared by the method of any one of embodiments 1 to 16.

[0362] 18. A method of conducting a FISH-based assay for deletion of a tumor suppressor gene comprising: [0363] (a) performing FISH with a probe set on a cellular sample comprising a plurality of cells, [0364] wherein the probe set comprises at least a first flanking probe that hybridizes to a position centromeric to the tumor suppressor gene, at least a second flanking probe that hybridizes to a position telomeric to the tumor suppressor gene, and at least one target probe that hybridizes to the tumor suppressor gene; [0365] (b) enumerating FISH signals from the at least first and at least second flanking probes and the at least one target probe in the plurality of cells; [0366] (c) providing at least one artifactual deletion frequency chosen from (i) an artifactual hemizygous deletion frequency and (ii) an artifactual homozygous deletion frequency; [0367] (d) determining at least one apparent deletion frequency chosen from (i) an apparent hemizygous deletion frequency and (ii) an apparent homozygous deletion frequency from the enumerated FISH signals of step (b), wherein the at least one apparent deletion frequency comprises an apparent hemizygous deletion frequency if an artifactual homozygous deletion frequency was not provided in step (c), and wherein the at least one apparent deletion frequency comprises an apparent homozygous deletion frequency if an artifactual hemizygous deletion frequency was not provided in step (c); and [0368] (e) determining whether the sample comprises cells with a hemizygous deletion of the tumor suppressor gene based on whether the apparent hemizygous deletion frequency is significantly greater than the artifactual hemizygous deletion frequency, or determining whether the sample comprises cells with a homozygous deletion of the tumor suppressor gene based on whether the apparent homozygous deletion frequency is significantly greater than the artifactual homozygous deletion frequency.

[0369] 19. The method of embodiment 18, wherein an apparent frequency is significantly greater than an artifactual frequency if p is less than or equal to 0.05 according to a t-test.

[0370] 20. The method of embodiment 18, wherein an apparent frequency is significantly greater than an artifactual frequency if the apparent frequency exceeds the artifactual frequency by three standard deviations.

[0371] 21. The method of any one of embodiments 18 to 20, wherein the at least one first flanking probe hybridizes to a position within or centromeric to a boundary zone centromeric to the tumor suppressor gene.

[0372] 22. The method of any one of embodiments 18 to 21, wherein the at least one second flanking probe hybridizes to a position within or telomeric to a boundary zone telomeric to the tumor suppressor gene.

[0373] 23. The method of any one of embodiments 18 to 22, wherein the method comprises providing an artifactual hemizygous deletion frequency and an artifactual homozygous deletion frequency in step (c); determining an apparent hemizygous deletion frequency and an apparent homozygous deletion frequency in step (d); and determining both whether the sample comprises cells with a hemizygous deletion of the tumor suppressor gene and whether the sample comprises cells with a homozygous deletion of the tumor suppressor gene in step (e).

[0374] 24. The method of any one of embodiments 18 to 23, wherein the probe set further comprises at least one chromosome enumeration probe specific for the chromosome that comprises the tumor suppressor gene, and the method further comprises enumerating FISH signals from the at least one chromosome enumeration probe, determining an apparent aneuploid frequency, and determining whether cells in the sample are aneuploid for said chromosome based on whether the apparent aneuploid frequency is significantly greater than an artifactual aneuploid frequency.

[0375] 25. The method of any one of embodiments 18 to 24, wherein the cellular sample is fixed and preserved.

[0376] 26. The method of embodiment 25, wherein the cellular sample is a formalin-fixed, paraffin-embedded sample with a thickness ranging from 3 to 6 μm.

[0377] 27. The method of any one of embodiments 18 to 26, wherein the tumor suppressor gene is PTEN.

[0378] 28. The method of embodiment 27, wherein the at least one first flanking probe comprises a probe that hybridizes to TSPAN15, and the at least one second flanking probe comprises a probe that hybridizes to FAS.

[0379] 29. The method of embodiments 27 or 28, wherein the at least one first flanking probe comprises a probe that hybridizes to BMPR1A or WAPAL, and the at least one second flanking probe comprises a probe that hybridizes to FAS.

[0380] 30. The method of any one of embodiments 18 to 26, wherein the tumor suppressor gene is chosen from p16, RB1, and p53.

[0381] 31. The method of any one of embodiments 18 to 30, wherein the hybridization sites of the at least one target probe and the at least first and second flanking probes have sizes ranging from 50 to 200 kb.

[0382] 32. The method of any one of embodiments 18 to 31, wherein the hybridization site of the at least one target probe is separated from the hybridization sites of the at least first and second flanking probes by a distance ranging from 500 kb to 20 Mb.

[0383] 33. A method of conducting a FISH-based assay for distinguishably detecting small and large deletions of a tumor suppressor gene comprising: [0384] (a) performing FISH on a cellular sample comprising a plurality of cells with a probe set, or performing FISH on a first cellular sample comprising a plurality of cells with a first probe subset comprised by a probe set and performing FISH on a second cellular sample comprising a plurality of cells from the same individual as the first cellular sample with a second probe subset comprised by said probe set, [0385] wherein the probe set comprises at least one target probe that hybridizes to the tumor suppressor gene, at least a first flanking probe that hybridizes to a position centromeric to the tumor suppressor gene, at least a second flanking probe that hybridizes to a position telomeric to the tumor suppressor gene, and at least one of at least a third flanking probe that hybridizes to a position centromeric to the hybridization site of the first flanking probe and at least a fourth flanking probe that hybridizes to a position telomeric to the hybridization site of the second flanking probe; [0386] (b) enumerating FISH signals from the at least one target probe and the at least first, at least second, and at least one of the at least third and at least fourth flanking probes in the plurality or pluralities of cells; [0387] (c) providing at least one first artifactual deletion frequency for deletions of the tumor suppressor gene with endpoints between the at least first and at least second flanking probes; [0388] (d) providing at least one second artifactual deletion frequency for deletions of the tumor suppressor gene wherein at least one of the endpoints is not between the at least first and at least second flanking probes; [0389] (e) determining, from the enumerated FISH signals of step (b), at least one first apparent deletion frequency for deletions of the tumor suppressor gene with endpoints between the at least first and at least second flanking probes; [0390] (f) determining, from the enumerated FISH signals of step (b), at least one second apparent deletion frequency for deletions of the tumor suppressor gene wherein at least one of the endpoints is not between the at least first and at least second flanking probes; and [0391] (g) determining whether the cellular sample comprises cells with a small deletion of the tumor suppressor gene based on whether the at least one first apparent deletion frequency is significantly greater than the at least one first artifactual deletion frequency, and determining whether the cellular sample comprises cells with a large deletion of the tumor suppressor gene based on whether the at least one second apparent deletion frequency is significantly greater than the at least one second artifactual homozygous deletion frequency.

[0392] 34. The method of embodiment 33, wherein the method comprises performing FISH on a first cellular sample comprising a plurality of cells with a first probe subset comprised by the probe set and performing FISH on a second cellular sample comprising a plurality of cells from the same individual as the first cellular sample with a second probe subset comprised by the probe set; the tumor suppressor gene is PTEN; the first probe subset comprises a flanking probe that hybridizes to TSPAN15 and a flanking probe that hybridizes to FAS; and the second probe subset comprises a flanking probe that hybridizes to BMPR1A or WAPAL and a flanking probe that hybridizes to SUFU.

[0393] 35. The method of any one of embodiments 33 to 34, wherein the at least one first flanking probe hybridizes to a position centromeric to a first boundary zone centromeric to the tumor suppressor gene, and the at least one second flanking probe hybridizes to a position telomeric to a second boundary zone telomeric to the tumor suppressor gene.

[0394] 36. The method of any one of embodiments 33 to 35, wherein the at least one third flanking probe hybridizes to a position centromeric to a first distal boundary zone centromeric to the hybridization site of the at least one first flanking probe, or the at least one fourth flanking probe hybridizes to a position telomeric to a second distal boundary zone telomeric to the hybridization site of the at least one second flanking probe.

[0395] 37. The method of embodiment 36, wherein the at least one first flanking probe hybridizes to a position centromeric to a first boundary zone centromeric to the tumor suppressor gene, and the at least one second flanking probe hybridizes to a position telomeric to a second boundary zone telomeric to the tumor suppressor gene.

[0396] 38. The method of any one of embodiments 33 to 37, wherein determining that the cells comprise two deletions of the tumor suppressor gene, at least one of the deletions being a large deletion, is indicative of a metastasizing or metastatic tumor.

[0397] 39. The method of any one of embodiments 33 to 38, wherein the cellular sample is fixed and preserved.

[0398] 40. The method of embodiment 39, wherein the cellular sample is a formalin-fixed, paraffin-embedded sample with a thickness ranging from 3 to 6 μm.

[0399] 41. The method of any one of embodiments 33 and 35 to 40, wherein the tumor suppressor gene is PTEN.

[0400] 42. The method of embodiment 41, wherein the at least one first flanking probe comprises a probe that hybridizes to BMPR1A or WAPAL, and the at least one second flanking probe comprises a probe that hybridizes to FAS.

[0401] 43. The method of any one of embodiments 41 to 42, wherein the method comprises providing at least one third flanking probe that hybridizes to TSPAN15.

[0402] 44. The method of any one of embodiments 41 to 43, wherein the method comprises providing at least one fourth flanking probe that hybridizes to SUFU.

[0403] 45. The method of any one of embodiments 33 and 35 to 40, wherein the tumor suppressor gene is chosen from p16, RB1, and p53.

[0404] 46. The method of any one of embodiments 33 to 45, wherein the hybridization sites of the at least one target probe and the at least first and second flanking probes have sizes ranging from 50 to 200 kb.

[0405] 47. The method of any one of embodiments 33 to 46, wherein the hybridization site of the at least one target probe is separated from the hybridization sites of the at least first and second flanking probes by a distance ranging from 500 kb to 20 Mb.

[0406] 48. A method of optimizing a FISH-based assay for deletion of a tumor suppressor gene, comprising: [0407] (a) providing a plurality of candidate probe sets, wherein each candidate probe set comprises at least a first flanking probe that hybridizes to a position centromeric to the tumor suppressor gene, at least a second flanking probe that hybridizes to a position telomeric to the tumor suppressor gene, and at least one target probe that hybridizes to the tumor suppressor gene; [0408] (b) for each candidate probe set, [0409] (i) performing FISH with the candidate probe set on at least one cellular sample comprising a plurality of cells comprising a euploid number of intact copies of the tumor suppressor gene; [0410] (ii) enumerating FISH signals from the at least first and at least second flanking probes and the at least one target probe of the candidate probe set in the plurality of cells of the at least one sample; and [0411] (iii) determining an artifactual deletion frequency from the enumerated FISH signals of step (ii); and [0412] (c) selecting a probe set from the candidate probe sets for use in the optimized FISH-based assay for deletion of a tumor suppressor gene, wherein the selected probe set was determined to have a favorable artifactual deletion frequency in step (iii).

[0413] 49. The method of embodiment 48, further comprising, before, after, or in parallel with step (b), for each candidate probe set, [0414] (iv) performing FISH with the candidate probe set on a plurality of cellular samples comprising a plurality of cells comprising a homozygous or hemizygous deletion of the tumor suppressor gene; [0415] (v) enumerating FISH signals from the at least first and second flanking probes and the at least one target probe of the candidate probe set in the plurality of cells; [0416] (vi) determining an apparent deletion frequency from the enumerated FISH signals of step (v) for each of the plurality of samples; and [0417] (vii) after step (vi) and step (iii), determining a sensitivity value of the candidate probe set based on how many of the plurality of samples were determined to have an apparent deletion frequency significantly greater than the artifactual deletion frequency of step (iii);
wherein the selected probe set of step (c) was determined to have a favorable sensitivity value in step (vii).

[0418] 50. The method of any one of embodiments 48 to 49, wherein the cellular sample is fixed and preserved.

[0419] 51. The method of embodiment 50, wherein the cellular sample is a formalin-fixed, paraffin-embedded sample with a thickness ranging from 3 to 6 μm.

[0420] 52. A probe set comprising at least one probe that hybridizes to PTEN, at least one probe that hybridizes to FAS or SUFU, and at least one probe that hybridizes to TSPAN15.

[0421] 53. A kit for detecting deletions in the PTEN gene comprising the probe set of embodiment 52.

[0422] 54. The probe set of embodiment 52, wherein the at least one probe that hybridizes to PTEN, the at least one probe that hybridizes to FAS or SUFU, and the at least one probe that hybridizes to TSPAN15 are distinguishably labeled.

[0423] 55. The probe set of any one of embodiments 52 and 54, further comprising at least one probe that binds to BMPR1A or WAPAL.

[0424] 56. The probe set of any one of embodiments 52 and 54 to 55. wherein the probe set comprises a probe that binds to FAS.

[0425] 57. The probe set of any one of embodiments 52 and 54 to 56, further comprising a probe that binds to SUFU.

[0426] 58. The probe set of any one of embodiments 52 and 54 to 57, further comprising at least one chromosome enumeration probe.

[0427] 59. The probe set of any one of embodiments 52 and 54 to 58, wherein the probe set comprises at least one probe that hybridizes to PTEN, the at least one probe that hybridizes to FAS or SUFU, and the at least one probe that hybridizes to TSPAN15 that are derived from bacterial artificial chromosomes.

[0428] 60. The probe set of embodiment 59, wherein the at least one probe that hybridizes to PTEN comprises a probe derived from RP11-846G17, the at least one probe that hybridizes to FAS comprises a probe derived from at least one of RP11-399O19 and RP11-360H20, and the at least one probe that hybridizes to TSPAN15 comprises a probe derived from at least one of RP11-168B4 and RP11-124L5.

[0429] 61. The probe set of embodiment 60, wherein the probe set consists of probes derived from RP11-846617, at least one of RP11-399O19 and RP11-360H20, and at least one of RP11-168B4 and RP11-124L5.

[0430] 62. A composition comprising the probe set of any one of embodiments 52 and 54 to 61, wherein the at least one probe that hybridizes to PTEN, the at least one probe that hybridizes to FAS or SUFU, and the at least one probe that hybridizes to TSPAN15 are distinguishably labeled.

[0431] 63. A method of conducting a FISH-based assay for a deletion in bands 21q22.13-21q22.3 of chromosome 21 comprising: [0432] (a) performing FISH with a probe set on a cellular sample comprising a plurality of cells, [0433] wherein the probe set comprises: [0434] at least one target probe that hybridizes to at least one target gene located between TMPRSS2 and ERG, [0435] at least a first flanking probe that hybridizes to a position centromeric to the at least one target gene, [0436] and at least a second flanking probe that hybridizes to a position telomeric to the at least one target gene; [0437] (b) enumerating FISH signals from the at least first and at least second flanking probes and the at least one target probe in the plurality of cells; [0438] (c) providing at least one artifactual deletion frequency chosen from (i) an artifactual hemizygous deletion frequency and (ii) an artifactual homozygous deletion frequency; [0439] (d) determining at least one apparent deletion frequency chosen from (i) an apparent hemizygous deletion frequency and (ii) an apparent homozygous deletion frequency from the enumerated FISH signals of step (b), wherein the at least one apparent deletion frequency comprises an apparent hemizygous deletion frequency if an artifactual homozygous deletion frequency was not provided in step (c), and wherein the at least one apparent deletion frequency comprises an apparent homozygous deletion frequency if an artifactual hemizygous deletion frequency was not provided in step (c); and [0440] (e) determining whether the sample comprises cells with a hemizygous deletion of at least one of the target genes based on whether the apparent hemizygous deletion frequency is significantly greater than the artifactual hemizygous deletion frequency, or determining whether the sample comprises cells with a homozygous deletion of at least one of the target genes based on whether the apparent homozygous deletion frequency is significantly greater than the artifactual homozygous deletion frequency.

[0441] 64. The method of embodiment 63, wherein the at least one target gene is chosen from ETS2, FSMG1, B3GALT5, HMGN1, DSCAM, and BACE2.

[0442] 65. The method of any one of embodiments 63 to 64, wherein an apparent frequency is significantly greater than an artifactual frequency if p is less than or equal to 0.05 according to a t-test.

[0443] 66. The method of any one of embodiments 63 to 64, wherein an apparent frequency is significantly greater than an artifactual frequency if the apparent frequency exceeds the artifactual frequency by three standard deviations.

[0444] 67. The method of any one of embodiments 63 to 66, wherein the at least one first flanking probe hybridizes to a position within or centromeric to a boundary zone centromeric to the at least one target gene.

[0445] 68. The method of any one of embodiments 63 to 67, wherein the at least one second flanking probe hybridizes to a position within or telomeric to a boundary zone telomeric to the at least one target gene.

[0446] 69. The method of any one of embodiments 63 to 68, wherein the method comprises providing an artifactual hemizygous deletion frequency and an artifactual homozygous deletion frequency in step (c); determining an apparent hemizygous deletion frequency and an apparent homozygous deletion frequency in step (d); and determining both whether the sample comprises cells with a hemizygous deletion of the at least one target gene and whether the sample comprises cells with a homozygous deletion of the at least one target gene in step (e).

[0447] 70. The method of any one of embodiments 63 to 69, wherein the probe set further comprises at least one chromosome enumeration probe specific for chromosome 21, and the method further comprises enumerating FISH signals from the at least one chromosome enumeration probe, determining an apparent aneuploid frequency, and determining whether cells in the sample are aneuploid for said chromosome based on whether the apparent aneuploid frequency is significantly greater than an artifactual aneuploid frequency.

[0448] 71. The method of any one of embodiments 63 to 70, wherein the cellular sample is fixed and preserved.

[0449] 72. The method of embodiment 71, wherein the cellular sample is a formalin-fixed, paraffin-embedded sample with a thickness ranging from 3 to 6 μm.

[0450] 73. The method of any one of embodiments 63 to 72, wherein the at least one target gene comprises HMGN1. 74. The method of any one of embodiments 63 to 73, wherein the at least one target gene comprises DSCAM.

[0451] 75. The method of any one of embodiments 63 to 74, wherein the probe set comprises at least two target probes that hybridize to at least two target genes.

[0452] 76. The method of embodiment 75, wherein the at least two target genes comprise HMGN1 and DSCAM.

[0453] 77. The method of any one of embodiments 63 to 76, wherein the at least one first flanking probe comprises a probe that hybridizes to ERG.

[0454] 78. The method of any one of embodiments 63 to 77, wherein the at least one second flanking probe comprises a probe that hybridizes to TMPRSS2.

[0455] 79. The method of any one of embodiments 63 to 78, wherein the hybridization sites of the at least one target probe and the at least first and second flanking probes have sizes ranging from 50 to 200 kb.

[0456] 80. The method of any one of embodiments 63 to 79, wherein the hybridization site of the at least one target probe is separated from the hybridization sites of the at least first and second flanking probes by a distance ranging from 500 kb to 20 Mb.

[0457] 81. A method of conducting a FISH-based assay for distinguishably detecting small and large deletions in bands 21q22.13-21q22.3 of chromosome 21 comprising: [0458] (a) performing FISH on a cellular sample comprising a plurality of cells with a probe set, or performing FISH on a first cellular sample comprising a plurality of cells with a first probe subset comprised by a probe set and performing FISH on a second cellular sample comprising a plurality of cells from the same individual as the first cellular sample with a second probe subset comprised by said probe set, [0459] wherein the probe set comprises: [0460] at least two target probes that hybridize to at least two target genes located between TMPRSS2 and ERG, [0461] at least a first flanking probe that hybridizes to a position centromeric to the at least two target genes, [0462] at least a second flanking probe that hybridizes to a position telomeric to the at least two target genes; [0463] and, optionally, at least one of at least a third flanking probe that hybridizes to a position centromeric to the hybridization site of the first flanking probe and at least a fourth flanking probe that hybridizes to a position telomeric to the hybridization site of the second flanking probe; [0464] (b) enumerating FISH signals from the at least two target probes and the at least first, at least second, and, if present, at least one of the at least third and at least fourth flanking probes in the plurality or pluralities of cells; [0465] (c) providing at least one first artifactual deletion frequency for deletions of at least one of the target genes with endpoints between the at least first and at least second flanking probes, one of the endpoints being between two target genes; [0466] (d) providing at least one second artifactual deletion frequency for deletions of at least one of the target genes wherein (i) neither endpoint is between two target genes, or (ii) if at least one of the at least third and at least fourth flanking probes was used, at least one of the endpoints is not between the at least first and at least second flanking probes; [0467] (e) determining, from the enumerated FISH signals of step (b), at least one first apparent deletion frequency for deletions of at least one of the target genes with endpoints between the at least first and at least second flanking probes, one of the endpoints being between two target genes; [0468] (f) determining, from the enumerated FISH signals of step (b), at least one second apparent deletion frequency for deletions of at least one of the target genes wherein (i) neither endpoint is between two target genes, or (ii) if at least one of the at least third and at least fourth flanking probes was used, at least one of the endpoints is not between the at least first and at least second flanking probes; and [0469] (g) determining whether the sample comprises cells with a small deletion of at least one of the target genes based on whether the at least one first apparent deletion frequency is significantly greater than the at least one first artifactual deletion frequency, and determining whether the sample comprises cells with a large deletion of at least one of the target genes based on whether the at least one second apparent deletion frequency is significantly greater than the at least one second artifactual homozygous deletion frequency.

[0470] 82. The method of embodiment 81, wherein the at least two target genes are chosen from ETS2, FSMG1, B3GALT5, HMGN1, DSCAM, and BACE2.

[0471] 83. The method of any one of embodiments 81 to 82, wherein the probe set comprises at least one of at least a third flanking probe that hybridizes to a position centromeric to the hybridization site of the first flanking probe and at least a fourth flanking probe that hybridizes to a position telomeric to the hybridization site of the second flanking probe.

[0472] 84. The method of any one of embodiments 81 to 83, wherein the method comprises performing FISH on a first cellular sample comprising a plurality of cells with a first probe subset comprised by the probe set and performing FISH on a second cellular sample comprising a plurality of cells from the same individual as the first cellular sample with a second probe subset comprised by the probe set; the at least two target genes comprise HMGN1 and DSCAM; the first probe subset comprises a flanking probe that hybridizes to DYRKIA and a flanking probe that hybridizes to TMPRSS2; and the second probe subset comprises a flanking probe that hybridizes to ERG and a flanking probe that hybridizes to U2AF1.

[0473] 85. The method of any one of embodiments 81 to 84, wherein the at least one first flanking probe hybridizes to a position centromeric to a first boundary zone centromeric to the at least two target genes, and the at least one second flanking probe hybridizes to a position telomeric to a second boundary zone telomeric to the at least two target genes.

[0474] 86. The method of any one of embodiments 81 to 85, wherein either (i) the at least one third flanking probe is present and hybridizes to a position centromeric to a first distal boundary zone centromeric to the hybridization site of the at least one first flanking probe, or (ii) the at least one fourth flanking probe is present and hybridizes to a position telomeric to a second distal boundary zone telomeric to the hybridization site of the at least one second flanking probe.

[0475] 87. The method of embodiment 86, wherein the at least one first flanking probe hybridizes to a position centromeric to a first boundary zone centromeric to the at least two target genes, and the at least one second flanking probe hybridizes to a position telomeric to a second boundary zone telomeric to the at least two target genes.

[0476] 88. The method of any one of embodiments 81 to 87, wherein the cellular sample is fixed and preserved.

[0477] 89. The method of any one of embodiments 88, wherein the cellular sample is a formalin-fixed, paraffin-embedded sample with a thickness ranging from 3 to 6 μm.

[0478] 90. The method of any one of embodiments 81 to 83 and 85 to 89, wherein the at least two target genes comprise HMGN1 and DSCAM.

[0479] 91. The method of embodiment 90, wherein the at least one first flanking probe comprises a probe that hybridizes to ERG, and the at least one second flanking probe comprises a probe that hybridizes to TMPRSS2.

[0480] 92. The method of any one of embodiments 81 to 83 and 85 to 91, wherein the method comprises providing at least one third flanking probe that hybridizes to DYRKIA.

[0481] 93. The method of any one of embodiments 81 to 83 and 85 to 92, wherein the method comprises providing at least one fourth flanking probe that hybridizes to U2AF1.

[0482] 94. The method of any one of embodiments 81 to 93, wherein the hybridization sites of the at least two target probes and the at least first and second flanking probes have sizes ranging from 50 to 200 kb.

[0483] 95. The method of any one of embodiments 81 to 94, wherein the hybridization sites of the at least two target probes are separated from the hybridization sites of the at least first and second flanking probes by distances ranging from 500 kb to 20 Mb.

[0484] 96. A probe set comprising at least one probe that hybridizes to at least one target gene located between TMPRSS2 and ERG, at least one probe that hybridizes to DYRKIA or ERG, and at least one probe that hybridizes to TMPRSS2 or U2AF1.

[0485] 97. The probe set of embodiment 96, wherein at least one probe that hybridizes to the at least one target gene, the at least one probe that hybridizes to DYRKIA or ERG, and the at least one probe that hybridizes to TMPRSS2 or U2AF1 are distinguishably labeled.

[0486] 98. The probe set of any one of embodiments 96 to 97, wherein the at least one target gene is chosen from ETS2, FSMG1, B3GALT5, HMGN1, DSCAM, and BACE2.

[0487] 99. The probe set of any one of embodiments 96 to 98, wherein the probe set comprises a probe that binds to HMGN1.

[0488] 100. The probe set of any one of embodiments 96 to 99, wherein the probe set comprises a probe that binds to DSCAM.

[0489] 101. The probe set of any one of embodiments 96 to 100, wherein the probe set comprises a probe that binds to DYRKIA.

[0490] 102. The probe set of any one of embodiments 96 to 101, wherein the probe set comprises a probe that binds to U2AF1.

[0491] 103. The probe set of any one of embodiments 96 to 102, wherein the probe set comprises a probe that binds to TMPRSS2.

[0492] 104. The probe set of any one of embodiments 96 to 103, wherein the probe set comprises a probe that binds to ERG.

[0493] 105. The probe set of any one of embodiments 96 to 104, further comprising at least one chromosome enumeration probe.

[0494] 106. The probe set of any one of embodiments 96 to 105, wherein at least three of the probes in the probe set are derived from bacterial artificial chromosomes.

[0495] 107. The probe set of embodiment 106, wherein the probe set comprises at least one probe that hybridizes to HMGN1 derived from at least one of RP11-137J13 and RP11-348C15, at least one probe that hybridizes to ERG derived from at least one of RP11-476D17 and RP11-951I21, and at least one probe that hybridizes to TMPRSS2 derived from at least one of RP11-35C4, CTD-3095D11, and RP11-671L10.

[0496] 108. The probe set of embodiment 106, wherein the probe set comprises at least one probe that hybridizes to DSCAM derived from at least one of RP11-139D12, RP11-907P24, RP11-280O17, RP11-183I12, RP11-281F3, RP11-1113M13, and RP11-123A7, at least one probe that hybridizes to ERG derived from at least one of RP11-476D17 and RP11-951I21, and at least one probe that hybridizes to TMPRSS2 derived from at least one of RP11-35C4, CTD-3095D11, and RP11-671L10.

[0497] 109. The probe set of embodiment 106, wherein the probe set comprises at least one probe that hybridizes to HMGN1 derived from at least one of RP11-137J13 and RP11-348C15, at least one probe that hybridizes to DSCAM derived from at least one of RP11-139D12, RP11-907P24, RP11-280O17, RP11-183I12, RP11-281F3, RP11-1113M13, and RP11-123A7, at least one probe that hybridizes to ERG derived from at least one of RP11-476D17 and RP11-951I21, and at least one probe that hybridizes to TMPRSS2 derived from at least one of RP11-35C4, CTD-3095D11, and RP11-671L10.

[0498] 110. The probe set of embodiment 106, further comprising at least one of (i) a probe that hybridizes to U2AF1 derived from at least one of RP11-446L19 and CTD-2601A22, or (ii) a probe that hybridizes to DYRKIA derived from at least one of RP11-105O24, RP11-777J19, and CTD-3140L2.

[0499] 111. The probe set of embodiment 96, wherein the probe set consists of probes derived from: [0500] (i) at least one probe that hybridizes to HMGN1 or DSCAM derived from at least one of RP11-137J13, RP11-348C15, RP11-139D12, RP11-907P24, RP11-280O17, RP11-183I12, RP11-281F3, RP11-1113M13, and RP11-123A7, [0501] (ii) a probe that hybridizes to ERG derived from at least one of RP11-476D17 and RP11-951I21, [0502] (iii) a probe that hybridizes to TMPRSS2 derived from at least one of RP11-35C4, CTD-3095D11, and RP11-671L10, and [0503] (iv) optionally at least one of (a) a probe that hybridizes to U2AF1 derived from at least one of RP11-446L19 and CTD-2601A22, or (b) a probe that hybridizes to DYRKIA derived from at least one of RP11-105O24, RP11-777J19, and CTD-3140L2.

[0504] 112. The probe set of embodiment 96, wherein the probe set consists of: [0505] (i) a probe that hybridizes to HMGN1 derived from at least one of RP11-137J13 and RP11-348C15, [0506] (ii) a probe that hybridizes to DSCAM derived from at least one of RP11-139D12, RP11-907P24, RP11-280O17, RP11-183I12, RP11-281F3, RP11-1113M13, and RP11-123A7, [0507] (iii) a probe that hybridizes to ERG derived from at least one of RP11-476D17 and RP11-951I21, [0508] (iv) a probe that hybridizes to TMPRSS2 derived from at least one of RP11-35C4, CTD-3095D11, and RP11-671L10, [0509] (v) a probe that hybridizes to U2AF1 derived from at least one of RP11-446L19 and CTD-2601A22, and [0510] (vi) a probe that hybridizes to DYRKIA derived from at least one of RP11-105O24, RP11-777J19, and CTD-3140L2.

[0511] 113. The probe set of embodiment 96, wherein the probe set consists of probes derived from RP11-139D12, at least one of RP11-476D17 and RP11-951I21, and at least one of RP11-35C4 and CTD-3095D11.

[0512] 114. A probe set comprising at least one probe that hybridizes to PTEN, at least one probe that hybridizes to FAS or SUFU, and at least one probe that hybridizes to WAPAL.

[0513] 115. A kit for detecting deletions in bands 21q22.13-21q22.3 of chromosome 21 comprising the probe set of any one of embodiments 96 to 114.