Molecular in vitro diagnosis of breast cancer

Abstract

A method for determining the benign or malignant state of a breast tumor in a biological sample is presented. The benign or malignant state is determined by measuring the variation of expression of twelve genes and at least one variant of each of the twelve genes when the variants exist, using a combination of 50 polynucleotide probe sets. An expression profile of the sample is then compared with reference expression profiles of control benign and malignant breast tissues. The probe sets can be arranged in a microarray.

1 . A method for determining a benign or malignant state of a breast tumor in a biological sample from a subject, by determining a variation of expression of
 at least 12 determined genes belonging to a group of determined genes selected from the nucleotide sequences of SEQ ID NO: 5419 to SEQ ID NO: 5693 and   at least one variant of each of said at least 12 determined genes, when said variants exist,   said at least 12 genes being represented by the following nucleotide sequences: SEQ ID NOs 5421; 5423; 5432; 5434; 5486; 5491; 5525; 5552; 5599; 5652; 5654 and 5683,   the method comprising:   (1) contacting nucleic acid molecules obtained from said biological sample to a combination of 50 polynucleotide probe sets to allow formation of a nucleic acid complex, and detecting said formation;   (2) determining a sample expression profile of said 12 genes and of variants of said 12 genes from the detection of the nucleic acid complexes;   (3) comparing said sample expression profile with a reference expression profile of a control benign breast tumor, and/or a reference expression profile of a control malignant tumor, and   (4) determining, from the comparison, that:
 (a) when said profile of expression is similar to the reference expression profile of a control benign breast tumor, then the breast cancer is benign, and 
 (b) when said profile of expression is similar to the reference expression profile of a control malignant breast tumor, then the breast cancer is malignant, 
   wherein, said 50 polynucleotide probe sets comprise:   probe set #1 consisting of the nucleic acids consisting of SEQ ID NO: 333, 335 and 334;   probe set #2 consisting of the nucleic acids consisting of SEQ ID NO: 364, 365 and 366;   probe set #3 consisting of the nucleic acids consisting of SEQ ID NO: 364, 366 and 365;   probe set #4 consisting of the nucleic acids consisting of SEQ ID NO: 312, 348 and 313;   probe set #5 consisting of the nucleic acids consisting of SEQ ID NO: 312, 314 and 313;   probe set #6 consisting of the nucleic acids consisting of SEQ ID NO: 171, 172 and 173;   probe set #7 consisting of the nucleic acids consisting of SEQ ID NO: 315, 336 and 331;   probe set #8 consisting of the nucleic acids consisting of SEQ ID NO: 1198, 1199 and 1200;   probe set #9 consisting of the nucleic acids consisting of SEQ ID NO: 315, 317 and 316;   probe set #10 consisting of the nucleic acids consisting of SEQ ID NO: 1522, 1523 and 1524;   probe set #11 consisting of the nucleic acids consisting of SEQ ID NO: 339, 341 and 340;   probe set #12 consisting of the nucleic acids consisting of SEQ ID NO: 353, 361 and 360;   probe set #13 consisting of the nucleic acids consisting of SEQ ID NO: 1185, 1186 and 1194;   probe set #14 consisting of the nucleic acids consisting of SEQ ID NO: 315, 370 and 316;   probe set #15 consisting of the nucleic acids consisting of SEQ ID NO: 324, 326 and 325;   probe set #16 consisting of the nucleic acids consisting of SEQ ID NO: 1165 and 1166;   probe set #17 consisting of the nucleic acid consisting of SEQ ID NO: 177;   probe set #18 consisting of the nucleic acids consisting of SEQ ID NO: 1551, 1552 and 1553;   probe set #19 consisting of the nucleic acids consisting of SEQ ID NO: 315, 316 and 370;   probe set #20 consisting of the nucleic acids consisting of SEQ ID NO: 1185, 1186 and 1194;   probe set #21 consisting of the nucleic acids consisting of SEQ ID NO: 1523, 1524 and 1557;   probe set #22 consisting of the nucleic acid consisting of SEQ ID NO: 1158;   probe set #23 consisting of the nucleic acids consisting of SEQ ID NO: 345, 347 and 346;   probe set #24 consisting of the nucleic acids consisting of SEQ ID NO: 367, 369 and 368;   probe set #25 consisting of the nucleic acids consisting of SEQ ID NO: 315, 331 and 330;   probe set #26 consisting of the nucleic acids consisting of SEQ ID NO: 327, 329 and 328;   probe set #27 consisting of the nucleic acid consisting of SEQ ID NO: 1203;   probe set #28 consisting of the nucleic acids consisting of SEQ ID NO: 1262, 1263 and 1264;   probe set #29 consisting of the nucleic acids consisting of SEQ ID NO: 1534, 1535 and 1536;   probe set #30 consisting of the nucleic acids consisting of SEQ ID NO: 2179, 2180 and 2181;   probe set #31 consisting of the nucleic acids consisting of SEQ ID NO: 1159, 1160 and 1161;   probe set #32 consisting of the nucleic acids consisting of SEQ ID NO: 327, 329 and 328;   probe set #33 consisting of the nucleic acids consisting of SEQ ID NO: 157, 158 and 159;   probe set #34 consisting of the nucleic acids consisting of SEQ ID NO: 1267, 1268 and 1269;   probe set #35 consisting of the nucleic acids consisting of SEQ ID NO: 167, 168 and 169;   probe set #36 consisting of the nucleic acids consisting of SEQ ID NO: 167, 168 and 177;   probe set #37 consisting of the nucleic acids consisting of SEQ ID NO: 1167, 1168 and 1169;   probe set #38 consisting of the nucleic acids consisting of SEQ ID NO: 1544, 1545 and 1546;   probe set #39 consisting of the nucleic acids consisting of SEQ ID NO: 969, 972 and 981;   probe set #40 consisting of the nucleic acids consisting of SEQ ID NO: 1162, 1170 and 1171;   probe set #41 consisting of the nucleic acids consisting of SEQ ID NO: 790, 791 and 792;   probe set #42 consisting of the nucleic acids consisting of SEQ ID NO: 368, 374 and 375;   probe set #43 consisting of the nucleic acids consisting of SEQ ID NO: 1551, 1552 and 1553;   probe set #44 consisting of the nucleic acids consisting of SEQ ID NO: 1554, 1555 and 1556;   probe set #45 consisting of the nucleic acids consisting of SEQ ID NO: 2026, 2027 and 2028;   probe set #46 consisting of the nucleic acids consisting of SEQ ID NO: 1081, 1082 and 1083;   probe set #47 consisting of the nucleic acids consisting of SEQ ID NO: 1129, 1140 and 1141;   probe set #48 consisting of the nucleic acids consisting of SEQ ID NO: 1519, 1520 and 1521;   probe set #49 consisting of the nucleic acids consisting of SEQ ID NO: 136, 137 and 138; and   probe set #50 consisting of the nucleic acids consisting of SEQ ID NO: 793, 794 and 795,   as specified as probe set #1 to probe set #50 in Table 7.   
     
     
         2 . The method according to  claim 1 , comprising determining the variation of expression of:
 at least 20 determined genes belonging to a group of determined genes selected from the nucleotide sequences of SEQ ID NO: 5419 to SEQ ID NO: 5693, and   at least one variant of each of said at least 20 determined genes, when said variants exist,   said at least 20 determined genes being represented by the following nucleotide sequences: SEQ ID Nos 5421; 5423; 5432; 5434; 5457; 5486; 5491; 5513; 5516; 5525; 5532; 5552; 5599; 5616; 5624; 5643; 5652; 5654; 5673 and 5683,   said method comprising:   (1) contacting the nucleic acid molecules obtained from said biological sample to a combination of at least 100 polynucleotide probe sets to allow the formation of the nucleic acid complex,   wherein said 100 polynucleotide probe sets comprise probe set #1 to probe set #100 as specified in Table 7.   
     
     
         3 . The method according to  claim 1 , comprising determining the variation of expression of:
 at least 35 determined genes belonging to a group of determined genes selected from the nucleotide sequences of SEQ ID NO: 5419 to SEQ ID NO: 5693, and   at least one variant of each of said at least 35 determined genes, when said variants exist,   said at least 35 determined genes being represented by the following nucleotide sequences: SEQ ID Nos 5421; 5423; 5431; 5432; 5434; 5440; 5457; 5462; 5470; 5486; 5491; 5505; 5513; 5516; 5525; 5532; 5546; 5552; 5594; 5599; 5601; 5603; 5616; 5618; 5624; 5627; 5630; 5643; 5645; 5652; 5654; 5660; 5669; 5673 and 5683,   said method comprising:   (1) contacting the nucleic acid molecules obtained from said biological sample to a combination of at least 150 polynucleotide probe sets to allow the formation of the nucleic acid complex,   wherein said 150 polynucleotide probe sets comprise probe set #1 to probe set #150 as specified in Table 7.   
     
     
         4 . The method according to  claim 1 , comprising determining the variation of expression of:
 at least 43 determined genes belonging to a group of determined genes selected from the nucleotide sequences of SEQ ID NO: 5419 to SEQ ID NO: 5693, and   at least one variant of each of said at least 43 determined genes, when said variants exist,   said at least 43 determined genes being represented by the following nucleotide sequences: SEQ ID Nos 5421; 5423; 5431; 5432; 5434; 5440; 5455; 5457; 5462; 5470; 5475; 5486; 5491; 5499; 5505; 5513; 5516; 5520; 5525; 5532; 5546; 5549; 5552; 5594; 5599; 5601; 5603; 5616; 5618; 5623; 5624; 5627; 5630; 5643; 5645; 5651; 5652; 5654; 5660; 5669; 5673; 5683 and 5686,   said method comprising:   (1) contacting the nucleic acid molecules obtained from said biological sample to a combination of at least 200 polynucleotide probe sets to allow the formation of the nucleic acid complex,   wherein said 200 polynucleotide probe sets comprise probe set #1 to probe set #200 as specified in Table 7.   
     
     
         5 . The method according to  claim 1 , comprising determining the variation of expression of:
 at least 206 determined genes belonging to a group of determined genes selected from the nucleotide sequences of SEQ ID NO: 5419 to SEQ ID NO: 5693, and   at least one variant of each of said at least 206 determined genes, when said variants exist,   said at least 206 determined genes being represented by the following nucleotide sequences: SEQ ID Nos 5420; 5421; 5422; 5423; 5425; 5426; 5427; 5428; 5429; 5430; 5431; 5432; 5434; 5435; 5436; 5437; 5438; 5439; 5440; 5441; 5442; 5443; 5444; 5445; 5446; 5447; 5449; 5450; 5451; 5452; 5453; 5454; 5455; 5456; 5457; 5459; 5460; 5461; 5462; 5463; 5465; 5467; 5468; 5470; 5473; 5475; 5476; 5479; 5480; 5482; 5484; 5485; 5486; 5487; 5488; 5489; 5490; 5491; 5492; 5493; 5495; 5496; 5497; 5498; 5499; 5502; 5503; 5505; 5506; 5507; 5508; 5509; 5511; 5512; 5513; 5515; 5516; 5517; 5518; 5519; 5520; 5523; 5524; 5525; 5526; 5528; 5529; 5530; 5531; 5532; 5533; 5534; 5536; 5538; 5539; 5540; 5543; 5544; 5545; 5546; 5547; 5549; 5550; 5552; 5553; 5554; 5556; 5558; 5559; 5560; 5562; 5563; 5564; 5565; 5566; 5567; 5568; 5569; 5570; 5571; 5573; 5574; 5575; 5576; 5577; 5578; 5581; 5582; 5585; 5587; 5588; 5590; 5591; 5593; 5594; 5595; 5597; 5598; 5599; 5600; 5601; 5602; 5603; 5604; 5605; 5606; 5608; 5609; 5610; 5611; 5612; 5613; 5615; 5616; 5617; 5618; 5622; 5623; 5624; 5625; 5627; 5629; 5630; 5631; 5632; 5633; 5634; 5635; 5637; 5638; 5639; 5640; 5641; 5642; 5643; 5644; 5645; 5647; 5648; 5649; 5651; 5652; 5653; 5654; 5656; 5657; 5658; 5660; 5661; 5665; 5667; 5669; 5672; 5673; 5675; 5676; 5677; 5678; 5680; 5681; 5683; 5684; 5686; 5687; 5689 and 5692,   said method comprising:   (1) contacting the nucleic acid molecules obtained from said biological sample to a combination of at least 1228 polynucleotide probe sets to allow the formation of the nucleic acid complex,   wherein said 1228 polynucleotide probe sets comprise probe set #1 to probe set #1228 as specified in Table 7.   
     
     
         6 . The method according to  claim 1 , comprising determining the variation of expression of:
 at least 275 determined genes belonging to a group of determined genes selected from the nucleotide sequences of SEQ ID NO: 5419 to SEQ ID NO: 5693, and   at least one variant of each of said at least 275 determined genes, when said variants exist,   said method comprising:   (1) contacting the nucleic acid molecules obtained from said biological sample to a combination of at least 1640 polynucleotide probe sets to allow the formation of the nucleic acid complex,   wherein said 1640 polynucleotide probe sets comprise probe set #1 to probe set #1640 as specified in Table 7.   
     
     
         7 . The method according to  claim 1 , wherein determining the variation of expression comprises, determining an over representation or an under representation for each of said at least 12 determined genes and at least one variant of each of said at least 12 determined genes when said variants exist. 
     
     
         8 . The method according to  claim 1 , wherein the nucleic acid molecules obtained from said biological sample are mRNA or cDNA prepared from said mRNA, the mRNA resulting from the expression of the genes represented by the nucleotide sequences of SEQ ID NO: 5419 to SEQ ID NO: 5693, and/or at least one of said variants when present represented by the nucleotide sequences of SEQ ID NO: 4579 to SEQ ID NO: 5418 and SEQ ID NO: 5694. 
     
     
         9 . The method according to  claim 1 , wherein the 50 polynucleotide probe sets are arranged in a microarray.