OSTEOSARCOMA-ASSOCIATED RISK MARKERS AND USES THEREOF

BACKGROUND OF INVENTION

Osteosarcoma, a common bone malignancy, is an aggressive cancer characterized by early metastasis and high mortality. In dogs, osteosarcoma typically afflicts middle-age large and giant breeds. Osteosarcoma is common in both humans and dogs resulting in a major impact on human and canine health.

SUMMARY OF INVENTION

The invention is premised on the identification of germ-line risk markers (e.g., SNPs) that can be used singly or together (e.g., forming a haplotype) to predict elevated risk of osteosarcoma in subjects, e.g., canine subjects. As described herein, a genome-wide association study (GWAS) was performed in Greyhounds, Rottweilers and Irish wolfhounds and germ-line risk markers that correlate with canine osteosarcoma were identified. These germ-line risk markers were confirmed to correlate with canine osteosarcoma in a second, larger sample set. Accordingly, aspects of the invention provide methods for identifying subjects that are at elevated risk of developing osteosarcoma or subjects having otherwise undiagnosed osteosarcoma. Subjects are identified based on the presence of one or more germ-line risk markers shown to be associated with the presence of osteosarcoma, in accordance with the invention. Prognostic and theranostic methods utilizing one or more germ-line risk markers are also described herein.

In some aspects, the disclosure relates to a method, comprising a) analyzing genomic DNA from a canine subject for the presence of a single nucleotide polymorphism (SNP) selected from:

- i) one or more chromosome 1 SNPs,
- ii) one or more chromosome 2 SNPs,
- iii) one or more chromosome 3 SNPs,
- iv) one or more chromosome 5 SNPs,
- v) one or more chromosome 7 SNPs,
- vi) one or more chromosome 8 SNPs,
- vii) one or more chromosome 9 SNPs,
- viii) one or more chromosome 11 SNPs,
- ix) one or more chromosome 13 SNPs,
- x) one or more chromosome 14 SNPs,
- xi) one or more chromosome 15 SNPs,
- xii) one or more chromosome 16 SNPs,
- xii) one or more chromosome 17 SNPs,
- xiv) one or more chromosome 18 SNPs,
- xv) one or more chromosome 19 SNPs,
- xvi) one or more chromosome 21 SNPs,
- xvii) one or more chromosome 25 SNPs,
- xvii) one or more chromosome 26 SNPs,
- xix) one or more chromosome 32 SNPs,
- xx) one or more chromosome 35 SNPs,
- xxi) one or more chromosome 36 SNPs, and
- xxii) one or more chromosome 38 SNPs; and
  
  b) identifying a canine subject having the SNP as a subject at elevated risk of developing osteosarcoma or having an undiagnosed osteosarcoma.

In some embodiments, the SNP is selected from BICF2P133066, BICF2P1421479, BICF2S2308696, BICF2P508906, BICF2P508905, BICF2S23216058, BICF2S23216058, BICF2P266591, BICF2P1332375, BICF2S23231062, BICF2S22945043, BICF2P326880, BICF2P893664, BICF2P1420547, BICF2P698281, BICF2S22919383, BICF2S22947803, BICF2S22947803, BICF2S22959094, BICF2S23228287, BICF2S23036972, BICF2P51623, BICF2P1346510, BICF2P1323908, BICF2P1137984, BICF2P1115364, BICF2P58266, BICF2P627162, BICF2P1422910, BICF2P162782, BICF2P162782, BICF2P1342901, BICF2P868731, BICF2P768889, BICF2P1052528, BICF2P408119, BICF2P1468011, BICF2P219326, BICF2P1462759, BICF2P307386, BICF2P1010170, BICF2S23038485, BICF2G630672865, BICF2G630672813, BICF2P1369145, BICF2G630672770, BICF2P81989, BICF2P916235, BICF2G630672753, BICF2P1177075, BICF2P411325, BICF2P1210630, TIGRP2P407733, BICF2P341331, BICF2P318350, BICF2S2335735, BICF2P1003572, BICF2P1104551, BICF2S23550277, BICF2P870378, BICF2P866460, BICF2P1303772, BICF2S23738710, BICF2P344455, BICF2P825177, BICF2S23324500, BICF2S23544574, BICF2P119783, BICF2S23758510, BICF2S23724888, BICF2P1129874, BICF2S23535303, BICF2S23520119, G326F32S322, BICF2S23238674, BICF2P645758, BICF2P189890, BICF2P819174, BICF2P162666, BICF2P1366853, BICF2P775251, BICF2S23746532, BICF2P1162557, BICF2S23538747, BICF2S23538670, BICF2S23218055, BICF2P680751, BICF2S23510137, BICF2P849639, BICF2S22945333, BICF2S2298851, TIGRP2P238123, TIGRP2P238132, BICF2P1466354, BICF2P440326, BICF2P874005, BICF2P928021, BICF2P1182592, BICF2P1378069, TIGRP2P238162, TIGRP2P253880, BICF2P461252, BICF2P879737, BICF2P163146, BICF2S23259485, TIGRP2P253975, BICF2S23760612, TIGRP2P254013, TIGRP2P254028, BICF2S23750273, BICF2P228579, TIGRP2P254054, BICF2P531896, TIGRP2P254060, BICF2P766570, BICF2P1014267, BICF2P1006929, BICF2P1299781, BICF2P672676, BICF2S23761559, BICF2P15617, BICF2P439160, TIGRP2P254095, TIGRP2P254109, BICF2P477812, BICF2P1238318, BICF2P1354921, BICF2S23741435, BICF2P37118, TIGRP2P254175, BICF2P1123483, TIGRP2P254184, BICF2P825842, BICF2P243632, BICF2P1139856, BICF2P1376844, TIGRP2P254212, TIGRP2P254216, and TIGRP2P254223.

In some embodiments, the SNP is selected from BICF2P133066, BICF2S2308696, BICF2P508906, BICF2P508905, BICF2S23216058, BICF2S23216058, BICF2P266591, BICF2P1332375, BICF2S23231062, BICF2S22945043, BICF2P326880, BICF2P893664, BICF2P1420547, BICF2P698281, BICF2S22919383, BICF2S22947803, BICF2S22947803, BICF2S22959094, BICF2S23228287, BICF2S23036972, BICF2P51623, BICF2P1346510, BICF2P1323908, BICF2P1137984, BICF2P1115364, BICF2P58266, BICF2P627162, BICF2P1422910, BICF2P162782, BICF2P162782, BICF2P1342901, BICF2P868731, BICF2P768889, BICF2P1052528, BICF2P408119, BICF2P1468011, BICF2P219326, BICF2P1462759, BICF2P307386, BICF2P1010170, BICF2P229090, BICF2S23516022, and BICF2S22922837. In some embodiments, the SNP is BICF2P133066.

In some embodiments, the SNP is two or more SNPs. In some embodiments, the SNP is three or more SNPs.

Other aspects of the disclosure relate to a method, comprising (a) analyzing genomic DNA from a canine subject for the presence of a risk haplotype selected from:

a risk haplotype having chromosome coordinates chr11:44392734-44414985,

a risk haplotype having chromosome coordinates chr8:35433142-35454649,

a risk haplotype having chromosome coordinates chr13:14549973-14645634,

a risk haplotype having chromosome coordinates chr25:21831580-21921256,

a risk haplotype having chromosome coordinates chr14:48831824-49203827,

a risk haplotype having chromosome coordinates chr5:16071171-16152955,

a risk haplotype having chromosome coordinates chr19:33963105-34145310,

a risk haplotype having chromosome coordinates chr16:43665149-43737129,

a risk haplotype having chromosome coordinates chr15:63767963-63800415,

a risk haplotype having chromosome coordinates chr16:40883517-41081510,

a risk haplotype having chromosome coordinates chr25:43476429-43528145,

a risk haplotype having chromosome coordinates chr1:112977233-113081800,

a risk haplotype having chromosome coordinates chr3:5162058-6465753,

a risk haplotype having chromosome coordinates chr7:64631053-64703475,

a risk haplotype having chromosome coordinates chr1:115582915-116790630,

a risk haplotype having chromosome coordinates chr2:19212450-19542015,

a risk haplotype having chromosome coordinates chr1:122033806-122051988,

a risk haplotype having chromosome coordinates chr35:18326079-18345318,

a risk haplotype having chromosome coordinates chr9:47647012-47668054,

a risk haplotype having chromosome coordinates chr38:11252518-11739329,

a risk haplotype having chromosome coordinates chr21:46231985-46363479,

a risk haplotype having chromosome coordinates chr17:14465884-14482152,

a risk haplotype having chromosome coordinates chr32:25136302-25156153,

a risk haplotype having chromosome coordinates chr36:29637804-29663408,

a risk haplotype having chromosome coordinates chr15:37986345-39974762,

a risk haplotype having chromosome coordinates chr1:29405587-29914411,

a risk haplotype having chromosome coordinates chr26:32374093-32428448,

a risk haplotype having chromosome coordinates chr25:29658978-29767164,

a risk haplotype having chromosome coordinates chr26:3529343-3550075,

a risk haplotype having chromosome coordinates chr5:14720254-15466603,

a risk haplotype having chromosome coordinates chr18:4266743-5854451,

a risk haplotype having chromosome coordinates chr1:16768869-18150476,

a risk haplotype having chromosome coordinates chr9:18896060-19633155, and

a risk haplotype having chromosome coordinates chr11:44390633-44406002; and

(b) identifying a canine subject having the mutation as a subject at elevated risk of developing osteosarcoma or having an undiagnosed osteosarcoma.

In some embodiments, the risk haplotype is selected from a risk haplotype having chromosome coordinates chr11:44392734-44414985, a risk haplotype having chromosome coordinates chr8:35433142-35454649, a risk haplotype having chromosome coordinates chr1:115582915-116790630, a risk haplotype having chromosome coordinates chr2:19212450-19542015, a risk haplotype having chromosome coordinates chr1:122033806-122051988, a risk haplotype having chromosome coordinates chr35:18326079-18345318, a risk haplotype having chromosome coordinates chr9:47647012-47668054, a risk haplotype having chromosome coordinates chr38:11252518-11739329, a risk haplotype having chromosome coordinates chr5:14720254-15466603, and a risk haplotype having chromosome coordinates chr18:4266743-5854451. In some embodiments, the risk haplotype is selected from a risk haplotype having chromosome coordinates chr11:44392734-44414985, a risk haplotype having chromosome coordinates chr1:115582915-116790630, and a risk haplotype having chromosome coordinates chr5:14720254-15466603. In some embodiments, the risk haplotype is the risk haplotype having chromosome coordinates chr11:44392734-44414985.

In some embodiments, the mutation is two or more mutations. In some embodiments, the mutation is three or more mutations. In some embodiments, the genomic region is two or more genomic regions. In some embodiments, the genomic region is three or more genomic regions.

In yet another aspect, the disclosure relates to a method, comprising (a) analyzing genomic DNA from a canine subject for the presence of a mutation in a gene selected from:

one or more genes located within a risk haplotype having chromosome coordinates chr11:44392734-44414985,

one or more genes located within a risk haplotype having chromosome coordinates chr8:35433142-35454649,

one or more genes located within a risk haplotype having chromosome coordinates chr13: 14549973-14645634,

one or more genes located within a risk haplotype having chromosome coordinates chr25:21831580-21921256,

one or more genes located within a risk haplotype having chromosome coordinates chr14:48831824-49203827,

one or more genes located within a risk haplotype having chromosome coordinates chr5:16071171-16152955,

one or more genes located within a risk haplotype having chromosome coordinates chr19:33963105-34145310,

one or more genes located within a risk haplotype having chromosome coordinates chr16:43665149-43737129,

one or more genes located within a risk haplotype having chromosome coordinates chr15:63767963-63800415,

one or more genes located within a risk haplotype having chromosome coordinates chr16:40883517-41081510,

one or more genes located within a risk haplotype having chromosome coordinates chr25:43476429-43528145,

one or more genes located within a risk haplotype having chromosome coordinates chr1:112977233-113081800,

one or more genes located within a risk haplotype having chromosome coordinates chr3:5162058-6465753,

one or more genes located within a risk haplotype having chromosome coordinates chr7:64631053-64703475,

one or more genes located within a risk haplotype having chromosome coordinates chr1:115582915-116790630,

one or more genes located within a risk haplotype having chromosome coordinates chr2:19212450-19542015,

one or more genes located within a risk haplotype having chromosome coordinates chr1:122033806-122051988,

one or more genes located within a risk haplotype having chromosome coordinates chr35: 18326079-18345318,

one or more genes located within a risk haplotype having chromosome coordinates chr9:47647012-47668054,

one or more genes located within a risk haplotype having chromosome coordinates chr38:11252518-11739329,

one or more genes located within a risk haplotype having chromosome coordinates chr21:46231985-46363479,

one or more genes located within a risk haplotype having chromosome coordinates chr17: 14465884-14482152,

one or more genes located within a risk haplotype having chromosome coordinates chr32:25136302-25156153,

one or more genes located within a risk haplotype having chromosome coordinates chr36:29637804-29663408,

one or more genes located within a risk haplotype having chromosome coordinates chr15:37986345-39974762,

one or more genes located within a risk haplotype having chromosome coordinates chr1:29405587-29914411,

one or more genes located within a risk haplotype having chromosome coordinates chr26: 32374093-32428448,

one or more genes located within a risk haplotype having chromosome coordinates chr25:29658978-29767164,

one or more genes located within a risk haplotype having chromosome coordinates chr26:3529343-3550075,

one or more genes located within a risk haplotype having chromosome coordinates chr5:14720254-15466603,

one or more genes located within a risk haplotype having chromosome coordinates chr18:4266743-5854451,

one or more genes located within a risk haplotype having chromosome coordinates chr1:16768869-18150476,

- one or more genes located within a risk haplotype having chromosome coordinates chr9:18896060-19633155, and
- one or more genes located within a risk haplotype having chromosome coordinates chr11:44390633-44406002; and
  
  (b) identifying a canine subject having the mutation as a subject at elevated risk of developing osteosarcoma or having an undiagnosed osteosarcoma.

In some embodiments, the gene is selected from one or more genes located within a risk haplotype having chromosome coordinates chr11:44392734-44414985, one or more genes located within a risk haplotype having chromosome coordinates chr8:35433142-35454649, one or more genes located within a risk haplotype having chromosome coordinates chr1:115582915-116790630, one or more genes located within a risk haplotype having chromosome coordinates chr2:19212450-19542015, one or more genes located within a risk haplotype having chromosome coordinates chr1:122033806-122051988, one or more genes located within a risk haplotype having chromosome coordinates chr35:18326079-18345318, one or more genes located within a risk haplotype having chromosome coordinates chr9:47647012-47668054, one or more genes located within a risk haplotype having chromosome coordinates chr38:11252518-11739329, one or more genes located within a risk haplotype having chromosome coordinates chr5:14720254-15466603, and one or more genes located within a risk haplotype having chromosome coordinates chr18:4266743-5854451. In some embodiments, the gene is selected from one or more genes located within a risk haplotype having chromosome coordinates chr11:44392734-44414985, one or more genes located within a risk haplotype having chromosome coordinates chr1:115582915-116790630, and one or more genes located within a risk haplotype having chromosome coordinates chr5:14720254-15466603. In some embodiments, the gene is one or more genes located within the risk haplotype having chromosome coordinates chr11:44392734-44414985.

In some embodiments, the gene is selected from CDKN2B-AS, OTX2, BMPER, GRIK4, EN1, MARCO, MTMR7, SGCZ, CCL20, CD3EAP, ERCC1, ERCC2, FOSB, PPP1R13L, FER, MAN2A1, PJA2, CHST9, ADCK4, AKT2, AXL, BLVRB, C19orf47, C19orf54, CNTD2, CYP2A7, CYP2B6, CYP2S1, DLL3, EGLN2, FBL, FCGBP, GMFG, HIPK4, HNRNPUL1, ITPKC, LEUTX, LTBP4, MAP3K10, MED29, NUMBL, PLD3, PLEKHG2, PSMC4, RAB4B, SAMD4B, SERTAD1, SERTAD3, SHKBP1, SNRPA, SPTBN4, SUPT5H, TIMM50, KIAA1462, C19orf40, CEP89, RHPN2, BLMH, TMIGD1, FAM5C, NELL1, EMCN, AMDHD1, CCDC38, CDK17, ELKS, FGD6, HAL, LTA4H, METAP2, NDUFA12, NEDD1, NR2C1, NTN4, SNRPF, USP44,VEZT, EYA4, TCF21, ARVCF, C22orf25, COMT, XKR6, FBRSL1, BLID, C7orf72, COBL, DDC, FIGNL1, GRB10, IKZF1, VWC2, ZPBP, BCL2, KIAA1468, PHLPP1, PIGN, RNF152, TNFRSF11A, ZCCHC2, ABCA5, KCNJ16, KCNJ2, MAP2K6, CDKN2A, and CDKN2B. In some embodiments, the gene is selected from CDKN2B-AS, OTX2, BMPER, EN1, DLL3, KIAA1462, FAM5C, NELL1, EMCN, TCF21, BLID, VWC2, BCL2, and TNFRSF11A. In some embodiments, the gene is selected from CDKN2B-AS, OTX2, ADCK4, AKT2, AXL, BLVRB, C19orf47, C19orf54, CNTD2, CYP2A7, CYP2B6, CYP2S1, DLL3, EGLN2, FBL, FCGBP, GMFG, HIPK4, HNRNPUL1, ITPKC, LEUTX, LTBP4, MAP3K10, MED29, NUMBL, PLD3, PLEKHG2, PSMC4, RAB4B, SAMD4B, SERTAD1, SERTAD3, SHKBP1, SNRPA, SPTBN4, SUPT5H, TIMM50, KIAA1462, C19orf40, CEP89, RHPN2, BLMH, TMIGD1, FAM5C, BLID, C7orf72, COBL, DDC, FIGNL1, GRB10, IKZF1, VWC2, and ZPBP. In some embodiments, the gene is selected from CDKN2B-AS, ADCK4, AKT2, AXL, BLVRB, C19orf47, C19orf54, CNTD2, CYP2A7, CYP2B6, CYP2S1, DLL3, EGLN2, FBL, FCGBP, GMFG, HIPK4, HNRNPUL1, ITPKC, LEUTX, LTBP4, MAP3K10, MED29, NUMBL, PLD3, PLEKHG2, PSMC4, RAB4B, SAMD4B, SERTAD1, SERTAD3, SHKBP1, SNRPA, SPTBN4, SUPT5H, TIMM50, and BLID. In some embodiments, the gene is selected from CDKN2B-AS, CDKN2A, and CDKN2B.

In some embodiments, the mutation is two or more mutations. In some embodiments, the mutation is three or more mutations. In some embodiments, the gene is two or more genes. In some embodiments, the gene is three or more genes.

In some embodiments of any method provided herein, the genomic DNA is obtained from a bodily fluid or tissue sample of the subject. In some embodiments of any method provided herein, the genomic DNA is obtained from a blood or saliva sample of the subject. In some embodiments of any method provided herein, the genomic DNA is analyzed using a single nucleotide polymorphism (SNP) array. In some embodiments of any method provided herein, the genomic DNA is analyzed using a bead array. In some embodiments of any method provided herein, the genomic DNA is analyzed using a nucleic acid sequencing assay.

In some embodiments of any method described herein, the canine subject is a descendent of a Greyhound, Rottweiler or Irish Wolfhound. In some embodiments, the canine subject is a Greyhound, Rottweiler or Irish Wolfhound.

Yet another aspect of the disclosure relates to a method, comprising (a) analyzing genomic DNA in a sample from a subject for presence of a mutation in a gene selected from:

one or more genes located within a risk haplotype having chromosome coordinates chr11:44392734-44414985 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr8:35433142-35454649 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr13:14549973-14645634 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr25:21831580-21921256 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr14:48831824-49203827 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr5:16071171-16152955 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr19:33963105-34145310 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr16:43665149-43737129 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr15:63767963-63800415 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr16:40883517-41081510 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr25:43476429-43528145 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr1:112977233-113081800 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr3:5162058-6465753 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr7:64631053-64703475 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr1:115582915-116790630 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr2:19212450-19542015 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr1:122033806-122051988 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr35:18326079-18345318 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr9:47647012-47668054 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr38:11252518-11739329 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr21:46231985-46363479 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr17:14465884-14482152 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr32:25136302-25156153 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr36:29637804-29663408 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr15:37986345-39974762 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr1:29405587-29914411 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr26:32374093-32428448 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr25:29658978-29767164 or an orthologue of such a gene,

on or more genes located within a risk haplotype having chromosome coordinates chr26:3529343-3550075 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr5:14720254-15466603 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr18:4266743-5854451 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr1:16768869-18150476 or an orthologue of such a gene,

one or more genes located within a risk haplotype having chromosome coordinates chr9:18896060-19633155 or an orthologue of such a gene, and

one or more genes located within a risk haplotype having chromosome coordinates chr11:44,390,633-44,406,002 or an orthologue of such a gene; and

(b) identifying a subject having the mutation as a subject at elevated risk of developing osteosarcoma or having an undiagnosed osteosarcoma.

In some embodiments, the subject is a human subject. In some embodiments, the subject is a canine subject.

In some embodiments, the genomic DNA is obtained from a bodily fluid or tissue sample of the subject. In some embodiments, the genomic DNA is obtained from a blood or saliva sample of the subject. In some embodiments, the genomic DNA is analyzed using a single nucleotide polymorphism (SNP) array. In some embodiments, the genomic DNA is analyzed using a bead array. In some embodiments, the genomic DNA is analyzed using a nucleic acid sequencing assay.

In some embodiments, the gene is two or more genes. In some embodiments, the gene is three or more genes. In some embodiments, the mutation is two or more mutations. In some embodiments, the mutation is three or more mutations.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows results from the genome wide association study (GWAS). (A) A graph showing that each breed clusters as a distinct population. (B) A graph of the inbreeding coefficient for each breed showing that Greyhounds (Greys) are the least inbred, followed by the Rottweilers (Rotts), Irish Wolfhounds (IWHs) and AKC Greys. (C) A graph showing the extent of linkage disequilibrium in each breed. The lines from top to bottom are IWH, Grey AKC, Rott, and Grey. (D) A graph of the regions of homozygosity in each breed. The lines from top to bottom are IWH, Grey AKC, Rott and Grey. (E) A graph of the regions of low relative heterozygosity in each breed. Rott and Grey essentially overlap and are the top two lines. IWH and Grey AKC essentially overlap and are the bottom two lines.

FIG. 2A is a series of graphs showing the significant SNPs identified for each breed across the genome. The approximate boundaries of each chromosome on the X axis are indicated by vertical black lines.

FIG. 2B is a series of graphs showing the variance explained and genotype relative risk for loci with P<0.0005.

FIG. 3 is a series of graphs showing the genome wide association on chromosome 11 region and the syntenic region of human chromosome 9 as well as functional data implicating specific variants as likely disease variants. FIG. 3A is a series of graphs showing the location of CFA11 on dog chromosome 11 and the corresponding syntenic region on human chromosome 9. Blue vertical lines indicate the boundaries of CFA11. Horizontal grey bars show human genomic regions tested for functionality. The bar (G) indicates the human genomic region with the highest expression in a luciferase assay. Several of the most significant SNPs were in high linkage disequilibrium (LD) with the top SNP. FIG. 3B is a graph of luciferase expression driven by human genomic regions A-G in human osteosarcoma cells. FIG. 3C is a diagram showing the location of BICF2P133066 (chr11:44405676) within human genomic region G (vertical lines) and the identity of the nucleotide at the corresponding location in several mammals.

FIG. 4 shows the results of GRAIL (Gene Relationships Across Implicated Loci) analysis used to identify non-random connectivity between genes in the associated loci described herein. The genes associated with each breed as separated by dashed black lines.

FIG. 5 is a series of graphs and tables showing that pathways enriched in GWAS and fixed regions were also enriched for CGH changes in tumors. Numbers indicate the number of genomic loci overlapping between gene set (y axis) and region set (x axis). Those in bold have corrected p<0.05 as calculated by permutation analysis. RRV=regions of reduced variability.

FIG. 6 shows the p-value distribution of an allele frequency comparison between the osteosarcoma-prone racing greyhounds and AKC greyhounds, which rarely get osteosarcoma. SNPs in the extreme tail (p<1×10-9) are highly differentiated between the two populations and are candidate germ-line osteosarcoma risk variants.

FIG. 7 is a diagram showing highly significant overlap in the set of genes altered in canine osteosarcoma tumors and two human osteosarcoma cell lines.

FIG. 8 is a diagram showing the PDGFRB pathway genes implicated in canine osteosarcoma.

FIG. 9 is a quantile-quantile plot for the Leonberger study.

FIG. 10 is a graph showing significant SNPs identified for the Leonberger study across the genome. The approximate boundaries of each chromosome on the X axis are indicated by vertical black lines.

FIG. 11 is a graph showing clustering of significant SNPs and minor allele frequency (MAF) across a region of chromosome 11 from about 37 Mb to about 44 Mb.

FIG. 12 is a graph showing clustering of significant SNPs and MAF across a region of chromosome 24 from about 25 Mb to about 35 Mb.

FIG. 13 is a graph showing clustering of significant SNPs and MAF across a region of chromosome 35 from about 9 Mb to about 14 Mb.

DETAILED DESCRIPTION OF INVENTION

Osteosarcomas arise from mesenchymal stem cells, metastasize readily, and have widespread genetic abnormalities. Osteosarcoma in dogs is a spontaneously occurring disease with a global tumor gene expression signature indistinguishable from tumors from human pediatric patients and, while age of onset is higher in dogs, the clinical progression is remarkably similar. Both human and canine osteosarcomas most commonly arise at the ends of the long bones of the limbs and metastasize readily, usually to the lungs.

Aspects of the invention relate to germ-line risk markers (such as single nucleotide polymorphisms (SNPs), risk haplotypes, and mutations in genes) and various methods of use and/or detection thereof. The invention is premised, in part, on the results of a case-control GWAS of 304 Greyhounds, 155 Irish Wolfhounds, and 145 Rottweilers performed to identify germ-line risk markers associated with osteosarcoma. The study is described herein. Briefly, SNPs were identified that correlate with the presence of osteosarcoma in Greyhounds, Irish Wolfhounds, and/or Rottweilers. Significant SNPs were identified on chromosomes 1, 2, 3, 5, 7, 8, 9, 11, 13, 14, 15, 16, 17, 18, 19, 21, 25, 26, 32, 35, 36, and 38. These SNPs are listed in Table 1. Additionally, risk haplotypes having chromosomal regions on chromosomes 1, 2, 3, 5, 7, 8, 9, 11, 13, 14, 15, 16, 17, 18, 19, 21, 25, 26, 32, 35, 36, and 38 were identified that significantly correlated with osteosarcoma in Greyhounds, Irish Wolfhounds, and/or Rottweilers (chr11:44392734-44414985, chr8:35433142-35454649, chr13:14549973-14645634, chr25:21831580-21921256, chr14:48831824-49203827, chr5:16071171-16152955, chr19:33963105-34145310, chr16:43665149-43737129, chr15:63767963-63800415, chr16:40883517-41081510, chr25:43476429-43528145, chr1:112977233-113081800, chr3:5162058-6465753, chr7:64631053-64703475, chr1:115582915-116790630, chr2:19212450-19542015, chr1:122033806-122051988, chr35:18326079-18345318, chr9:47647012-47668054, chr38:11252518-11739329, chr21:46231985-46363479, chr17:14465884-14482152, chr32:25136302-25156153, chr36:29637804-29663408, chr15:37986345-39974762, chr1:29405587-29914411, chr26:32374093-32428448, chr25:29658978-29767164, chr26:3529343-3550075, chr5:14720254-15466603, chr18:4266743-5854451, chr1:16768869-18150476, chr9:18896060-19633155, and chr11:44390633-44406002). These germ-line risk markers were also found to correlate with canine osteosarcoma in a study involving a second, larger sample set. Additional regions were also identified in a third, follow-on study.

Accordingly, aspects of the invention provide methods that involve detecting one or more of the identified germ-line risk markers in a subject, e.g., a canine subject, in order to (a) identify a subject at elevated risk of developing osteosarcoma, or (b) identify a subject having osteosarcoma that is as yet undiagnosed. The methods can be used for prognostic purposes and for diagnostic purposes. Identifying canine subjects having an elevated risk of developing osteosarcoma is useful in a number of applications. For example, canine subjects identified as at elevated risk may be excluded from a breeding program and/or conversely canine subjects that do not carry the germ-line risk markers may be included in a breeding program. As another example, canine subjects identified as at elevated risk may be monitored, including monitored more regularly, for the appearance of osteosarcoma and/or may be treated prophylactically (e.g., prior to the development of the tumor) or therapeutically. Canine subjects carrying one or more of the germ-line risk markers may also be used to further study the progression of osteosarcoma and optionally to study the efficacy of various treatments.

In addition, in view of the clinical and histological similarity between canine osteosarcoma with human osteosarcoma, the germ-line risk markers identified in accordance with the invention may also be risk markers and/or mediators of cancer occurrence and progression in human osteosarcoma as well. Accordingly, the invention provides diagnostic and prognostic methods for use in canine subjects, animals more generally, and human subjects, as well as animal models of human disease and treatment, as well as others.

Elevated Risk of Developing Osteosarcoma

The germ-line risk markers of the invention can be used to identify subjects at elevated risk of developing osteosarcoma. An elevated risk means a lifetime risk of developing such a cancer that is higher than the risk of developing the same cancer in (a) a population that is unselected for the presence or absence of the germ-line risk marker (i.e., the general population) or (b) a population that does not carry the germ-line risk marker.

Osteosarcoma and Diagnostic/Prognostic Methods

Aspects of the invention include various methods, such as prognostic and diagnostic methods, related to osteosarcoma. Osteosarcoma is an aggressive malignant neoplasm arising from primitive transformed cells of mesenchymal origin. Osteosarcoma is the most common histological form of primary bone cancer in both dogs and humans. Osteosarcoma typically arises from the proximal humerus, the distal radius, the distal femur, and/or the tibia. Other sites include the ribs, the mandible, the spine, and the pelvis. In some instances, osteosarcoma may arise from soft-tissues (extraskeletal osteosarcoma). The tumor causes a great deal of pain, and can even lead to fracture of the affected bone. Metastasis of osteosarcoma tumors is very common and usually occurs in the lungs. It is to be understood that the invention provides methods for detecting germ-line risk markers regardless of the location of the osteosarcoma.

Currently available methods for diagnosis of osteosarcoma include X-ray, CT scan, PET scan, bone scan, MRI and bone biopsy. A bone biopsy may be, e.g., a needle biopsy or an open biopsy. Such methods for diagnosis may be used alone or in combination and may also be used to stage the cancer.

Osteosarcoma can be staged using, for example, the TNM system. This system uses three different codes to describe the size and location of the tumor, whether it has spread to the lymph nodes around the tumor, and whether it can be found in other parts of the body.

In the TNM system, “T” plus a letter or number (0 to 4) is used to describe the size and location of the tumor. The tumor stages for osteosarcoma are in the following table.

T0
There is no evidence of a primary tumor.

T1
The tumor is 8 cm (about 3 inches) or smaller.

T2
The tumor is larger than 8 cm.

T3
The tumor has spread to another site or sites on the same bone.

The “N” in the TNM system stands for node and is used to describe whether cancer has spread to regional lymph nodes. Lymph node stages are in the following table.

N0
The cancer has not spread into the regional lymph nodes.

N1
The cancer has spread to regional lymph nodes.

The “M” in the TNM system is used for cancer that has spread, or metastasized, to other parts of the body. The stages for metastatic osteosarcoma are in the following table.

M0
The cancer has not spread to the lymph nodes or other parts

of the body.

M1
The cancer has spread to other parts of the body.

M1a
The cancer has spread to the lung.

M1b
The cancer has spread to distant parts of the body, other

than the lung.

Tumor Grade

The TNM system also incorporates the tumor grade. The grade is generally determined by looking at cancer cells under a microscope. Tumor grades are in the following table.

G1
Low grade, cells are well differentiated

G2
Low grade, cells are moderately differentiated

G3
High grade, cells are poorly differentiated

G4
High grade, cells are not differentiated. The cells do not look

like any normal looking cells.

Stages I to IV

After the T, N, and M categories of the osteosarcoma have been identified, this information can be combined with the tumor grade to assign a stage (I to IV) to the osteosarcoma. Stages are in the following table.

Stage IA
The cancer is found only in the bone, is smaller than

8 cm, and is low grade (T1, N0, M0, G1-G2).

Stage IB
The cancer is found only in the bone, is larger than

8 cm, and is low grade (T2, N0, M0, G1-G2).

Stage IIA
The cancer is found only in the bone, is smaller than

8 cm, and is high grade (T1, N0, M0, G3-G4).

Stage IIB
The cancer is found only in the bone, is larger than

8 cm, and is high grade (T2, N0, M0, G3-G4).

Stage III
The cancer is found only in the bone but has spread to

other places on the bone (T3, N0, M0, any G).

Stage IVA
Stage IVA - The cancer has spread to the lung (any T,

N0, M1a, any G).

Stage IVB
The cancer has spread to lymph nodes and other parts of

the body, or the cancer has spread to distant parts of

the body other than the lung (Any T, N1, any M; or any T,

any N, M1b, any G).

Another staging system used is the Musculoskeletal Tumor Society (MSTS) staging system which was developed by Enneking at the University of Florida. The MSTS staging system characterizes nonmetastatic malignant bone tumors by grade (low-grade [stage I] versus high-grade [stage II]) and further subdivides these stages according to the local anatomic extent (intracompartmental [A] versus extracompartmental [B]). For bone tumors, the compartmental status is determined by whether the tumor extends through the cortex of the involved bone. The majority of high grade osteosarcoma are extracompartmental. Subjects with distant metastases are categorized as stage III.

Thus, in some embodiments, the prognostic or diagnostic methods of the invention may further comprise performing a diagnostic assay known in the art for identification and staging of osteosarcoma (e.g., x-ray, CT scan, PET scan, bone scan, MRI and/or bone biopsy).

Germ-Line Risk Markers

Aspects of the invention relate to germ-line risk markers and use and detection thereof in various methods. In general terms, a germ-line marker is a mutation in the genome of a subject that can be passed on to the offspring of the subject. Germ-line markers may or may not be risk markers. Germ-line markers are generally found in the majority, if not all, of the cells in a subject. Germ-line markers are generally inherited from one or both parents of the subject (i.e., were present in the germ cells of one or both parents). Germ-line markers as used herein also include de novo germ-line mutations, which are spontaneous mutations that occur at single-cell stage level during development. This is distinct from a somatic marker, which is a mutation in the genome of a subject that occurs after the single-cell stage during development. Somatic mutations are considered to be spontaneous mutations. Somatic mutations generally originate in a single cell or subset of cells in the subject.

A germ-line risk marker as described herein includes a SNP, a risk haplotype, or a mutation in a gene. Further discussion of each type of germ-line risk marker is provided herein. It is to be understood that a germ-line risk marker may also indicate or predict the presence of a somatic mutation in a genomic location in close proximity to the germ-line risk marker, as germ-line risk marks may correlate with a higher risk of secondary somatic mutations.

As used herein, a mutation is one or more changes in the nucleotide sequence of the genome of the subject. The terms mutation, alteration, variation, and polymorphism are used interchangeably herein. As used herein, mutations include, but are not limited to, point mutations, insertions, deletions, rearrangements, inversions and duplications. Mutations also include, but are not limited to, silent mutations, missense mutations, and nonsense mutations.

Single Nucleotide Polymorphisms (SNPs)

In some embodiments, a germ-line risk marker is a single nucleotide polymorphism (SNP). A SNP is a mutation that occurs at a single nucleotide location on a chromosome. The nucleotide located at that position may differ between individuals in a population and/or paired chromosomes in an individual. In some embodiments, a germ-line risk marker is a SNP selected from Table 1. In some embodiments, a germ-line risk marker is a SNP selected from Table 1 or Table 5. Table 1 provides the risk nucleotide identity for each SNP (see “allele” column). The risk nucleotide is the nucleotide identity that is associated with elevated risk of developing osteosarcoma or having an undiagnosed osteosarcoma. The position (i.e., the chromosome coordinates) and SNP ID for each SNP in Table 1 are based on the CanFam 2.0 genome assembly (see, e.g., Lindblad-Toh K, Wade C M, Mikkelsen T S, Karlsson E K, Jaffe D B, Kamal M, Clamp M, Chang J L, Kulbokas E J 3rd, Zody M C, et al.: Genome sequence, comparative analysis and haplotype structure of the domestic dog. Nature 2005, 438:803-819). The first base pair in each chromosome is labeled 0 and the position of the SNP is then the number of base pairs from the first base pair (for example, the SNP on chromosome 11 at position 44405676 is located 44405676 base pairs from the first base pair of chromosome 11).

TABLE 1

List of SNPs associated with elevated risk of osteosarcoma

RE-

SNP
CHR
POSITION
ALLELE
BREED
GION

BICF2P133066
11
44405676
A
GREY
1

BICF2P1421479
8
35448126
C
GREY
2

BICF2S23118341
13
14588716
T
GREY
3

BICF2G630607426
13
14615683
T
GREY
3

BICF2G630607427
13
14616577
G
GREY
3

BICF2G630607427
13
14616577
A
GREY
3

BICF2G630607436
13
14628625
T
GREY
3

BICF2G630607436
13
14628625
A
GREY
3

BICF2P765580
25
21846080
A
GREY
4

BICF2P811222
25
21856115
T
GREY
4

BICF2P811227
25
21857586
G
GREY
4

BICF2P1340261
25
21870333
G
GREY
4

BICF2P1340261
25
21870333
A
GREY
4

BICF2S23541806
25
21873701
T
GREY
4

BICF2P729771
25
21894924
T
GREY
4

BICF2S23522137
25
21898097
C
GREY
4

BICF2S23325120
25
21912859
T
GREY
4

BICF2S23325120
25
21912859
A
GREY
4

BICF2P740200
25
21914402
G
GREY
4

BICF2S23117654
14
48841157
C
GREY
5

BICF2P412246
14
48863885
T
GREY
5

BICF2P257681
14
48907365
G
GREY
5

BICF2S23156412
14
48912477
A
GREY
5

BICF2S23246280
14
48969098
T
GREY
5

BICF2S23245388
14
49046791
A
GREY
5

BICF2S23715556
14
49085738
A
GREY
5

BICF2P66597
14
49193217
G
GREY
5

BICF2P1194727
5
16085937
G
GREY
6

BICF2P381887
5
16093715
T
GREY
6

BICF2P381179
5
16108115
G
GREY
6

BICF2P968528
5
16114280
A
GREY
6

BICF2P1248827
5
16145308
C
GREY
6

BICF2G63051679
19
33978816
A
GREY
7

BICF2G63051718
19
33999957
T
GREY
7

BICF2P1270504
19
34023257
G
GREY
7

BICF2G63051809
19
34134931
T
GREY
7

BICF2G630813090
16
43669044
C
GREY
8

BICF2G630813102
16
43676315
T
GREY
8

BICF2G630813102
16
43676315
T
GREY
8

BICF2P1152672
16
43712139
G
GREY
8

BICF2P1109206
16
43725055
A
GREY
8

BICF2G630418573
15
63780452
A
GREY
9

TIGRP2P215623
16
40896559
C
GREY
10

BICF2P357340
16
41073136
A
GREY
10

TIGRP2P331221
25
43485109
G
GREY
11

TIGRP2P331223
25
43495780
G
GREY
11

BICF2S23445991
25
43511232
T
GREY
11

TIGRP2P331236
25
43519306
C
GREY
11

BICF2P229090
1
112990217
A
GREY
12

BICF2S23516022
1
112990983
C
GREY
12

BICF2S22922837
1
113043126
G
GREY
12

BICF2P774726
3
5170412
A
GREY
13

BICF2P879101
3
5211310
C
GREY
13

BICF2P379530
3
5233667
A
GREY
13

BICF2P475099
3
5245090
C
GREY
13

TIGRP2P45104
3
5260931
A
GREY
13

BICF2P150216
3
5299580
G
GREY
13

BICF2P529456
3
5328933
C
GREY
13

TIGRP2P45112
3
5338620
A
GREY
13

BICF2P194703
3
5371137
A
GREY
13

BICF2P209319
3
5384473
T
GREY
13

BICF2P22627
3
5401853
G
GREY
13

TIGRP2P45129
3
5410018
C
GREY
13

BICF2P1038896
3
5426478
G
GREY
13

BICF2S2337294
3
5436506
C
GREY
13

BICF2P785126
3
5441183
G
GREY
13

BICF2P1209289
3
5456793
C
GREY
13

TIGRP2P45140
3
5471514
A
GREY
13

TIGRP2P45143
3
5475615
A
GREY
13

TIGRP2P45151
3
5487857
T
GREY
13

TIGRP2P45157
3
5508995
A
GREY
13

BICF2P112727
3
5521110
G
GREY
13

BICF2P148547
3
5534827
G
GREY
13

TIGRP2P45171
3
5564882
T
GREY
13

BICF2P761881
3
5573179
G
GREY
13

BICF2P363714
3
5581700
C
GREY
13

BICF2P1010972
3
5592544
A
GREY
13

TIGRP2P45176
3
5638043
G
GREY
13

TIGRP2P45178
3
5647032
C
GREY
13

BICF2P1026069
3
5700685
G
GREY
13

BICF2P327737
3
5709075
C
GREY
13

TIGRP2P45197
3
5719522
A
GREY
13

BICF2S22960989
3
5736343
G
GREY
13

TIGRP2P45218
3
5742823
G
GREY
13

BICF2P1190297
3
5752510
C
GREY
13

BICF2S2328630
3
5770706
G
GREY
13

BICF2P840628
3
5806192
C
GREY
13

TIGRP2P45242
3
5834019
T
GREY
13

TIGRP2P45246
3
5864745
G
GREY
13

TIGRP2P45249
3
5876747
A
GREY
13

BICF2P721123
3
5899710
A
GREY
13

TIGRP2P45259
3
5942967
C
GREY
13

TIGRP2P45264
3
5960454
G
GREY
13

BICF2P1018229
3
5970041
G
GREY
13

TIGRP2P45267
3
5988284
G
GREY
13

BICF2P569426
3
6000198
A
GREY
13

BICF2P238213
3
6009648
A
GREY
13

TIGRP2P45276
3
6048543
G
GREY
13

BICF2P1023047
3
6148587
T
GREY
13

BICF2P133199
3
6216098
G
GREY
13

BICF2P556026
3
6241715
A
GREY
13

BICF2P510160
3
6257542
C
GREY
13

TIGRP2P45301
3
6272593
C
GREY
13

BICF2P412371
3
6280200
G
GREY
13

BICF2P152439
3
6305511
T
GREY
13

TIGRP2P45306
3
6319525
G
GREY
13

BICF2P1458832
3
6417921
A
GREY
13

BICF2P158838
3
6433958
T
GREY
13

BICF2S23717124
3
6453902
G
GREY
13

BICF2G630564029
7
64646068
T
GREY
14

BICF2G630564047
7
64664054
T
GREY
14

BICF2P1090686
7
64672328
C
GREY
14

BICF2G630564076
7
64690186
T
GREY
14

BICF2S23535303
5
14740098
C
IWH
1

BICF2S23520119
5
14759439
T
IWH
1

G326F32S322
5
14786078
C
IWH
1

BICF2S23238674
5
14999278
T
IWH
1

BICF2P645758
5
15006237
T
IWH
1

BICF2P189890
5
15009903
G
IWH
1

BICF2P819174
5
15062440
T
IWH
1

BICF2P162666
5
15104598
G
IWH
1

BICF2P1366853
5
15123506
C
IWH
1

BICF2P775251
5
15254166
C
IWH
1

BICF2S23746532
5
15264066
A
IWH
1

BICF2P1162557
5
15333462
G
IWH
1

BICF2S23538747
5
15391207
G
IWH
1

BICF2S23538670
5
15395179
C
IWH
1

BICF2S23218055
5
15433708
G
IWH
1

BICF2P680751
5
15448667
C
IWH
1

BICF2S23510137
18
4281291
T
IWH
2

BICF2P849639
18
4315994
T
IWH
2

BICF2S22945333
18
4688275
T
IWH
2

BICF2S2298851
18
4705260
A
IWH
2

TIGRP2P238123
18
4824662
A
IWH
2

TIGRP2P238132
18
4929731
T
IWH
2

BICF2P1466354
18
4937944
C
IWH
2

BICF2P440326
18
4951290
C
IWH
2

BICF2P874005
18
4959653
C
IWH
2

BICF2P928021
18
4971847
G
IWH
2

BICF2P1182592
18
4980153
C
IWH
2

BICF2P1378069
18
4994445
T
IWH
2

TIGRP2P238162
18
5005071
G
IWH
2

TIGRP2P253880
18
5212336
T
IWH
2

BICF2P461252
18
5283915
T
IWH
2

BICF2P879737
18
5291054
G
IWH
2

BICF2P163146
18
5296804
T
IWH
2

BICF2S23259485
18
5338019
A
IWH
2

TIGRP2P253975
18
5362366
C
IWH
2

BICF2S23760612
18
5374587
A
IWH
2

TIGRP2P254013
18
5396556
C
IWH
2

TIGRP2P254028
18
5406832
A
IWH
2

BICF2S23750273
18
5422762
A
IWH
2

BICF2P228579
18
5430590
T
IWH
2

TIGRP2P254054
18
5449914
C
IWH
2

BICF2P531896
18
5457566
G
IWH
2

TIGRP2P254060
18
5467639
A
IWH
2

BICF2P766570
18
5479664
G
IWH
2

BICF2P1014267
18
5493249
A
IWH
2

BICF2P1006929
18
5512082
G
IWH
2

BICF2P1299781
18
5522052
T
IWH
2

BICF2P672676
18
5543603
G
IWH
2

BICF2S23761559
18
5562106
A
IWH
2

BICF2P15617
18
5572732
C
IWH
2

BICF2P439160
18
5587296
A
IWH
2

TIGRP2P254095
18
5594969
T
IWH
2

TIGRP2P254109
18
5605791
C
IWH
2

BICF2P477812
18
5619906
G
IWH
2

BICF2P1238318
18
5629193
T
IWH
2

BICF2P1354921
18
5652736
A
IWH
2

BICF2S23741435
18
5661990
A
IWH
2

BICF2P37118
18
5694792
C
IWH
2

TIGRP2P254175
18
5713966
C
IWH
2

BICF2P1123483
18
5726554
C
IWH
2

TIGRP2P254184
18
5728091
T
IWH
2

BICF2P825842
18
5765212
T
IWH
2

BICF2P243632
18
5783766
A
IWH
2

BICF2P1139856
18
5794059
A
IWH
2

BICF2P1376844
18
5796973
C
IWH
2

TIGRP2P254212
18
5826824
C
IWH
2

TIGRP2P254216
18
5834540
G
IWH
2

TIGRP2P254223
18
5845556
G
IWH
2

BICF2P426201
1
16782692
C
IWH
3

BICF2S2436535
1
16851821
C
IWH
3

BICF2P460868
1
16997009
A
IWH
3

BICF2P252171
1
17009485
A
IWH
3

TIGRP2P15921
1
17060348
G
IWH
3

TIGRP2P15926
1
17068084
A
IWH
3

BICF2S22943775
1
17098395
C
IWH
3

BICF2P300536
1
17109675
G
IWH
3

BICF2P69061
1
17130042
T
IWH
3

BICF2P931019
1
17133605
A
IWH
3

BICF2P172990
1
17142397
C
IWH
3

TIGRP2P15939
1
17163604
C
IWH
3

TIGRP2P15940
1
17180308
G
IWH
3

BICF2P1166345
1
17182762
G
IWH
3

TIGRP2P15943
1
17196849
G
IWH
3

BICF2S2334324
1
17226603
C
IWH
3

BICF2S23638404
1
17342812
T
IWH
3

BICF2P510074
1
17400867
A
IWH
3

TIGRP2P16004
1
17493415
T
IWH
3

TIGRP2P16009
1
17497796
G
IWH
3

BICF2P1225386
1
17742179
C
IWH
3

BICF2P976808
1
17746147
C
IWH
3

BICF2P572866
1
17780758
T
IWH
3

BICF2P51884
1
17795209
C
IWH
3

TIGRP2P16037
1
17806466
C
IWH
3

BICF2P230951
1
17814464
T
IWH
3

BICF2P674288
1
17830618
A
IWH
3

BICF2G630711956
1
18126292
G
IWH
3

BICF2P718275
9
18911590
A
IWH
4

BICF2S23223892
9
18996332
A
IWH
4

BICF2P328758
9
18998670
T
IWH
4

BICF2P158760
9
19014494
C
IWH
4

BICF2S23055739
9
19037490
T
IWH
4

BICF2S23516922
9
19084362
G
IWH
4

BICF2S2438924
9
19096254
T
IWH
4

BICF2S2294380
9
19121052
G
IWH
4

BICF2S2294380
9
19121052
G
IWH
4

BICF2S2439997
9
19128539
T
IWH
4

BICF2S2439997
9
19128539
T
IWH
4

BICF2P452889
9
19160959
T
IWH
4

BICF2P364366
9
19183929
C
IWH
4

BICF2P47724
9
19194063
C
IWH
4

BICF2P438701
9
19281859
A
IWH
4

BICF2P438701
9
19281859
A
IWH
4

BICF2P589810
9
19289821
C
IWH
4

BICF2P589810
9
19289821
C
IWH
4

BICF2P394578
9
19312506
T
IWH
4

TIGRP2P122861
9
19338752
T
IWH
4

TIGRP2P122861
9
19338752
C
IWH
4

BICF2P1079087
9
19349148
A
IWH
4

BICF2P579696
9
19381199
T
IWH
4

BICF2P497312
9
19396139
C
IWH
4

BICF2P1230700
9
19418665
T
IWH
4

TIGRP2P122831
9
19498086
C
IWH
4

BICF2P422703
9
19601839
G
IWH
4

BICF2P1125643
9
19623231
C
IWH
4

BICF2P1125643
9
19623231
C
IWH
4

BICF2P1125643
9
19623231
T
IWH
4

BICF2S2308696
1
115597438
A
ROTT
1

BICF2P508906
1
115630525
C
ROTT
1

BICF2P508905
1
115631080
T
ROTT
1

BICF2S23216058
1
115636142
C
ROTT
1

BICF2S23216058
1
115636142
T
ROTT
1

BICF2P266591
1
115646651
A
ROTT
1

BICF2P1332375
1
115759072
A
ROTT
1

BICF2S23231062
1
115892937
C
ROTT
1

BICF2S22945043
1
115944412
G
ROTT
1

BICF2P326880
1
115961693
T
ROTT
1

BICF2P893664
1
116008326
T
ROTT
1

BICF2P1420547
1
116028956
A
ROTT
1

BICF2P698281
1
116217014
A
ROTT
1

BICF2S22919383
1
116340675
G
ROTT
1

BICF2S22947803
1
116361619
A
ROTT
1

BICF2S22947803
1
116361619
A
ROTT
1

BICF2S22959094
1
116371940
C
ROTT
1

BICF2S23228287
1
116399288
A
ROTT
1

BICF2S23036972
1
116403385
A
ROTT
1

BICF2P51623
1
116415896
C
ROTT
1

BICF2P1346510
1
116479292
T
ROTT
1

BICF2P1323908
1
116492092
G
ROTT
1

BICF2P1137984
1
116508040
C
ROTT
1

BICF2P1115364
1
116524913
G
ROTT
1

BICF2P58266
1
116596060
G
ROTT
1

BICF2P627162
1
116619074
C
ROTT
1

BICF2P1422910
1
116627544
G
ROTT
1

BICF2P162782
1
116637538
A
ROTT
1

BICF2P162782
1
116637538
G
ROTT
1

BICF2P1342901
1
116649825
T
ROTT
1

BICF2P868731
1
116659732
C
ROTT
1

BICF2P768889
1
116667958
A
ROTT
1

BICF2P1052528
1
116688554
T
ROTT
1

BICF2P408119
1
116694244
A
ROTT
1

BICF2P1468011
1
116704630
A
ROTT
1

BICF2P219326
1
116735064
A
ROTT
1

BICF2P1462759
1
116744917
C
ROTT
1

BICF2P307386
1
116753371
T
ROTT
1

BICF2P1010170
1
116761963
C
ROTT
1

BICF2S23038485
2
19229091
C
ROTT
2

BICF2G630672865
2
19244386
A
ROTT
2

BICF2G630672813
2
19288113
G
ROTT
2

BICF2P1369145
2
19360520
C
ROTT
2

BICF2G630672770
2
19383308
A
ROTT
2

BICF2P81989
2
19402118
C
ROTT
2

BICF2P916235
2
19426763
G
ROTT
2

BICF2G630672753
2
19462778
A
ROTT
2

BICF2P1177075
2
19483009
C
ROTT
2

BICF2P411325
2
19515571
C
ROTT
2

BICF2P1210630
1
122048812
C
ROTT
3

TIGRP2P407733
35
18338700
A
ROTT
4

BICF2P341331
9
47659782
A
ROTT
5

BICF2P318350
38
11259655
C
ROTT
6

BICF2S2335735
38
11303710
T
ROTT
6

BICF2P1003572
38
11361700
A
ROTT
6

BICF2P1104551
38
11400039
A
ROTT
6

BICF2S23550277
38
11455402
G
ROTT
6

BICF2P870378
38
11480634
A
ROTT
6

BICF2P866460
38
11489608
T
ROTT
6

BICF2P1303772
38
11506377
G
ROTT
6

BICF2S23738710
38
11512589
C
ROTT
6

BICF2P344455
38
11526769
C
ROTT
6

BICF2P825177
38
11541386
A
ROTT
6

BICF2S23324500
38
11589162
G
ROTT
6

BICF2S23544574
38
11618856
C
ROTT
6

BICF2P119783
38
11641925
A
ROTT
6

BICF2S23758510
38
11673310
C
ROTT
6

BICF2S23724888
38
11684867
C
ROTT
6

BICF2P1129874
38
11714169
T
ROTT
6

BICF2P254147
21
46251007
C
ROTT
7

BICF2S2376247
21
46254772
G
ROTT
7

TIGRP2P286750
21
46283811
C
ROTT
7

BICF2P171066
21
46308873
A
ROTT
7

TIGRP2P286767
21
46334269
T
ROTT
7

TIGRP2P286795
21
46349601
G
ROTT
7

BICF2S23533459
17
14472761
C
ROTT
8

BICF2G630590368
32
25147661
A
ROTT
9

BICF2P92014
36
29651125
A
ROTT
10

BICF2S23339954
15
37998132
T
ROTT
11

TIGRP2P199934
15
38019697
A
ROTT
11

TIGRP2P199940
15
38033078
T
ROTT
11

TIGRP2P199942
15
38042634
C
ROTT
11

BICF2S23157944
15
38264190
T
ROTT
11

BICF2S22922723
15
38304593
T
ROTT
11

BICF2P752717
15
38499624
A
ROTT
11

BICF2P382393
15
38512016
T
ROTT
11

BICF2P334525
15
38516288
T
ROTT
11

BICF2P187948
15
38531916
T
ROTT
11

BICF2P360394
15
38551281
G
ROTT
11

BICF2S23032337
15
38597342
A
ROTT
11

BICF2P881582
15
38619239
G
ROTT
11

BICF2P178377
15
38646852
T
ROTT
11

BICF2S22912603
15
38674144
A
ROTT
11

BICF2P1031206
15
38746955
C
ROTT
11

TIGRP2P200003
15
38767028
C
ROTT
11

BICF2S23018355
15
38769763
A
ROTT
11

BICF2P1020099
15
38800344
C
ROTT
11

TIGRP2P200032
15
38904027
C
ROTT
11

TIGRP2P200033
15
38909750
G
ROTT
11

BICF2P217898
15
38919888
A
ROTT
11

TIGRP2P200071
15
38987072
T
ROTT
11

TIGRP2P200088
15
39001982
T
ROTT
11

BICF2P1083029
15
39024671
A
ROTT
11

BICF2S23034677
15
39042745
T
ROTT
11

BICF2S2375443
15
39161851
G
ROTT
11

BICF2P1149468
15
39169562
G
ROTT
11

BICF2P742493
15
39188468
T
ROTT
11

BICF2P1309224
15
39210513
G
ROTT
11

BICF2S23720644
15
39215533
T
ROTT
11

BICF2S23113349
15
39241402
G
ROTT
11

TIGRP2P200144
15
39271970
T
ROTT
11

BICF2P1278650
15
39273742
T
ROTT
11

BICF2P1093819
15
39323300
G
ROTT
11

TIGRP2P200166
15
39346724
G
ROTT
11

BICF2P459854
15
39353529
T
ROTT
11

BICF2P587000
15
39367943
T
ROTT
11

BICF2P778010
15
39492981
T
ROTT
11

BICF2P325024
15
39516234
T
ROTT
11

BICF2P472555
15
39553988
T
ROTT
11

BICF2P307089
15
39561516
A
ROTT
11

BICF2P307089
15
39561516
G
ROTT
11

TIGRP2P200235
15
39605173
C
ROTT
11

TIGRP2P200235
15
39605173
A
ROTT
11

BICF2S23344310
15
39622801
A
ROTT
11

BICF2S23344310
15
39622801
G
ROTT
11

BICF2P1143365
15
39634571
G
ROTT
11

BICF2P1162997
15
39652018
A
ROTT
11

TIGRP2P200259
15
39690799
G
ROTT
11

BICF2P1197030
15
39698607
C
ROTT
11

TIGRP2P200265
15
39712997
G
ROTT
11

BICF2P244321
15
39760788
C
ROTT
11

BICF2P244321
15
39760788
A
ROTT
11

BICF2P654662
15
39790921
A
ROTT
11

BICF2P690478
15
39858245
A
ROTT
11

TIGRP2P200321
15
39903893
G
ROTT
11

BICF2P647591
15
39940786
T
ROTT
11

BICF2S23719922
15
39962039
T
ROTT
11

BICF2P1206600
15
39964571
C
ROTT
11

BICF2P848519
1
29420845
A
ROTT
12

BICF2P1096901
1
29442608
T
ROTT
12

BICF2P270493
1
29444774
G
ROTT
12

BICF2P378388
1
29542070
C
ROTT
12

BICF2P104206
1
29560184
T
ROTT
12

BICF2S2343850
1
29668952
A
ROTT
12

BICF2P1164085
1
29775073
G
ROTT
12

BICF2P806301
1
29785710
T
ROTT
12

BICF2P1433886
1
29902244
A
ROTT
12

BICF2S23712115
26
32385934
G
ROTT
13

BICF2S23712114
26
32386262
T
ROTT
13

BICF2G63095567
25
29671618
G
ROTT
14

BICF2G63095567
25
29671618
G
ROTT
14

BICF2G63095608
25
29693487
G
ROTT
14

BICF2G63095608
25
29693487
G
ROTT
14

BICF2G63095630
25
29713336
A
ROTT
14

BICF2G63095630
25
29713336
A
ROTT
14

BICF2G63095645
25
29736474
G
ROTT
14

BICF2G63095650
25
29758499
G
ROTT
14

BICF2G63095650
25
29758499
G
ROTT
14

BICF2P841536
26
3537143
A
ROTT
15

CHR = chromosome, ALLELE = risk nucleotide, BREED = breed identified in Table 4 in Examples (GREY = Greyhound, IWH = Irish Wolfhound, ROTT = Rottweiler), REGION refers to column 1 of Table 4 in Examples.

In some embodiments, the SNP may be one or more of:

- i) one or more chromosome 1 SNPs,
- ii) one or more chromosome 2 SNPs,
- iii) one or more chromosome 3 SNPs,
- iv) one or more chromosome 5 SNPs,
- v) one or more chromosome 7 SNPs,
- vi) one or more chromosome 8 SNPs,
- vii) one or more chromosome 9 SNPs,
- viii) one or more chromosome 11 SNPs,
- ix) one or more chromosome 13 SNPs,
- x) one or more chromosome 14 SNPs,
- xi) one or more chromosome 15 SNPs,
- xii) one or more chromosome 16 SNPs,
- xii) one or more chromosome 17 SNPs,
- xiv) one or more chromosome 18 SNPs,
- xv) one or more chromosome 19 SNPs,
- xvi) one or more chromosome 21 SNPs,
- xvii) one or more chromosome 25 SNPs,
- xvii) one or more chromosome 26 SNPs,
- xix) one or more chromosome 32 SNPs,
- xx) one or more chromosome 35 SNPs,
- xxi) one or more chromosome 36 SNPs, and
- xxii) one or more chromosome 38 SNPs, all of which are provided in Table 1.

In some embodiments, a SNP may be used in the methods described herein. In some embodiments, the method comprises:

a) analyzing genomic DNA from a canine subject for the presence of a SNP selected from:

- i) one or more chromosome 1 SNPs,
- ii) one or more chromosome 2 SNPs,
- iii) one or more chromosome 3 SNPs,
- iv) one or more chromosome 5 SNPs,
- v) one or more chromosome 7 SNPs,
- vi) one or more chromosome 8 SNPs,
- vii) one or more chromosome 9 SNPs,
- viii) one or more chromosome 11 SNPs,
- ix) one or more chromosome 13 SNPs,
- x) one or more chromosome 14 SNPs,
- xi) one or more chromosome 15 SNPs,
- xii) one or more chromosome 16 SNPs,
- xii) one or more chromosome 17 SNPs,
- xiv) one or more chromosome 18 SNPs,
- xv) one or more chromosome 19 SNPs,
- xvi) one or more chromosome 21 SNPs,
- xvii) one or more chromosome 25 SNPs,
- xvii) one or more chromosome 26 SNPs,
- xix) one or more chromosome 32 SNPs,
- xx) one or more chromosome 35 SNPs,
- xxi) one or more chromosome 36 SNPs, and
- xxii) one or more chromosome 38 SNPs; and

b) identifying the canine subject having one or more of the SNPs as a subject (a) at elevated risk of developing osteosarcoma or (b) having an undiagnosed osteosarcoma.

In some embodiments, the SNP is BICF2P133066.

It is to be understood that any number of SNPs (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more SNPs) may be detected and/or used to identify a subject.

Risk Haplotypes

In some embodiments, a germ-line risk marker is a risk haplotype. A risk haplotype, as used herein, is a chromosomal region containing at least one mutation that correlates with the presence of or likelihood of developing osteosarcoma in a subject. A risk haplotype is detected or identified and/or may be defined by one or more mutations. For example, a risk haplotype may be a chromosomal region with boundaries that are defined by two or more SNPs that are in linkage disequilibrium and correlate with the presence of or likelihood of developing osteosarcoma in a subject. Such SNPs may themselves be disease-causative or may, alternatively or additionally, be indicators of other mutations (either germ-line mutations or somatic mutations) present in the chromosomal region of the risk haplotype that correlate with or cause osteosarcoma in a subject. Thus, other mutations within the risk haplotype may correlate with presence of or likelihood of developing osteosarcoma in a subject and are contemplated for use in the methods herein. Accordingly, in some embodiments, methods described herein comprise use and/or detection of a risk haplotype. In some embodiments, the risk haplotype is selected from: