CN104204223A - 用于睾丸癌的体外诊断或预后的方法 - Google Patents
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Abstract
本发明的主题是一种用于睾丸癌的体外诊断或预后的方法,其包括检测至少一种HERV核酸序列的至少一种表达产物的步骤,已经分离的所述核酸序列作为分子标记物的用途,以及包括对HERV核酸序列的至少一种表达产物特异的至少一种结合配偶体的试剂盒。
Description
内源逆转录病毒构成传染性逆转录病毒的后代,其以它们的原病毒形式整合入种系细胞,并通过这种方式传染入宿主后代的基因组。
人类基因组测序使显示极高丰度的转座因子或其衍生物成为可能。实际上,重复序列代表接近一半的人类基因组,而内源逆转录病毒和逆转录转座子组成所述基因组的8%,当前元件数目接近超过400,000个。
目前存在于人类基因组的内源逆转录病毒元件(ERVs)的丰度是约100代内生化(endogenizations)的结果,其成功地发生在人类品系的进化过程中。在我们的时代之前,内生化的不同波动跨越范围从200万年到9000万年的周期,并通过“拷贝/粘贴”型现象继之以拷贝数目的扩增,具有错误表型的可能性,起因于,源于HERVs家族(即显示序列同源性的一组元件)形成中的遗传原病毒。根据推测最古老的元件(HERV-L家族的那些元件)在哺乳动物出现之前已经整合。两个家族(HERV-F和HERV-H)在第一代灵长类动物塑造它们的外观时期已经出现。HERV-FRD和HERV-K(HML-5)家族(在4000到5500万年前整合)是对高等灵长类动物特异的。另一方面,HERV-W和HERV-E家族,例如,在500万年到1000万年后整合,在与新大陆猴(NewWorld monkeys)分离后,并且对狭鼻猴(似人猿的(Hominoids)和猕猴科(Cercopithecidae))是特异的。
ERV序列在全部染色体上呈现,具有根据家族的不同密度,在ERVs的物理邻近性与它们的种系发生邻近性之间不存在相关性。
很长一段时间内,ERVs被认为是寄生物或是简单的DNA废物。尽管如此,ERVs对生物体的影响不是仅仅限于它们过去在模式化基因组中的参与或仍能提供支持的有害重组。
ERVs的丰度和结构复杂性使它们的表达分析非常复杂,并且常常难于解释。HERV表达的检测可以反映相同家族内一个或更多个基因座的转录活化。此外活化的单个基因座或多个基因座可能根据组织和/或背景而不同。
本发明人现在已经发现和证明,对应于精确鉴定的内源逆转录病毒元件的基因座的核酸序列与睾丸癌有关,并且这些序列是病理性病症的分子标记物。鉴定的序列或者是原病毒,即序列包含位于长末端重复(LTRs)5'和3'位置侧翼的所有或部分gag、pol和env基因,或所有或部分的LTRs或所有或部分的分离的基因。鉴定的DNA序列在序列表中分别用SEQ IDNO:1到775表示,它们的染色体位置在下文表中(NCBI36/hg18)标出,而它们的表达、过表达或低表达用癌症样本和正常样本之间的“表达比值”来表示。当核酸的表达或核酸表达的改变是对睾丸组织特异时,该信息用靶组织列中的符号“x”来标示。这表示如果在除了睾丸组织之外的生物区室中检测到相关核酸的表达或核酸表达的改变,这表示不能作为睾丸癌的标记物。经鉴定对睾丸组织特异的DNA序列在序列表中分别表示为SEQID NOs:1,2,3,4,5,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,23,24,25,26,27,29,31,32,33,34,36,38,39,40,41,42,43,44,45,46,47,50,51,52,53,57,58,59,61,63,64,65,68,70,71,72,74,75,76,77,78,79,80,83,84,85,86,87,88,89,90,91,92,94,96,97,102,104,106,107,109,110,115,116,118,121,122,123,127,132,133,136,137,142,147,148,150,159,160,162,163,164,165,166,167,170,171,175,182,187,191,198,199,201,207,210,216,219,222,224,228,230,234,244,256,263,265,266,269,271,281,286,288,291,292,296,298,304,308,333,341,347,403,429,452,485,513,556,574和755。经鉴定对睾丸组织不特异的DNA序列在序列表中分别表示为SEQ ID NOs:6,22,28,30,35,37,48,49,54,55,56,60,62,66,67,69,73,81,82,93,95,98,99,100,101,103,105,108,111,112,113,114,117,119,120,124,125,126,128,129,130,131,134,135,138,139,140,141,143,144,145,146,149,151,152,153,154,155,156,157,158,161,168,169,172,173,174,176,177,178,179,180,181,183,184,185,186,188,189,190,192,193,194,195,196,197,200,202,203,204,205,206,208,209,211,212,213,214,215,217,218,220,221,223,225,226,227,229,231,232,233,235,236,237,238,239,240,241,242,243,245,246,247,248,249,250,251,252,253,254,255,257,258,259,260,261,262,264,267,268,270,272,273,274,275,276,277,278,279,280,282,283,284,285,287,289,290,293,294,295,297,299,300,301,302,303,305,306,307,309,310,311,312,313,314,315,316,317,318,319,320,321,322,323,324,325,326,327,328,329,330,331,332,334,335,336,337,338,339,340,342,343,344,345,346,348,349,350,351,352,353,354,355,356,357,358,359,360,361,362,363,364,365,366,367,368,369,370,371,372,373,374,375,376,377,378,379,380,381,382,383,384,385,386,387,388,389,390,391,392,393,394,395,396,397,398,399,400,401,402,404,405,406,407,408,409,410,411,412,413,414,415,416,417,418,419,420,421,422,423,424,425,426,427,428,430,431,432,433,434,435,436,437,438,439,440,441,442,443,444,445,446,447,448,449,450,451,453,454,455,456,457,458,459,460,461,462,463,464,465,466,467,468,469,470,471,472,473,474,475,476,477,478,479,480,481,482,483,484,486,487,488,489,490,491,492,493,494,495,496,497,498,499,500,501,502,503,504,505,506,507,508,509,755,511,512,514,515,516,517,518,519,520,521,522,523,524,525,526,527,528,529,530,531,532,533,534,535,536,537,538,539,540,541,542,543,544,545,546,547,548,549,550,551,552,553,554,555,557,558,559,560,561,562,563,564,565,566,567,568,569,570,571,572,573,575,576,577,578,579,580,581,582,583,584,585,586,587,588,589,590,591,592,593,594,595,596,597,598,599,600,601,602,603,604,605,606,607,608,609,610,611,612,613,614,615,616,617,618,619,620,621,622,623,624,625,626,627,628,629,630,631,632,633,634,635,636,637,638,639,640,641,642,643,644,645,646,647,648,649,650,651,652,653,654,655,656,657,658,659,660,661,662,663,664,665,666,667,668,669,670,671,672,673,674,675,676,677,678,679,680,681,682,683,684,685,686,687,688,689,690,691,692,693,694,695,696,697,698,699,700,701,702,703,704,705,706,707,708,709,710,711,712,713,714,715,716,717,718,719,720,721,722,723,724,725,726,727,728,729,730,731,732,733,734,735,736,737,738,739,740,741,742,743,744,745,746,747,748,749,750,751,752,753,754,756,757,758,759,760,761,762,763,764,765,766,767,768,769,770,771,772,773,774和775。
表
因此本发明的主题是一种用于取自患者的生物样本中睾丸癌体外诊断或睾丸癌严重性体外预后的方法,其包括检测至少一种核酸序列的至少一种表达产物的步骤,所述核酸序列选自SEQ ID NOs:1到775所示序列或选自与SEQ ID NOs:1到775所示序列之一显示至少99%同一性,优选的至少99.5%同一性和有利地至少99.6%或至少99.7%同一性的序列。
所述诊断使确定个体是否患病成为可能。所述预后使确定疾病的严重程度(分级和/或阶段)(其影响个体的生存和/或生活质量)成为可能。在本发明的背景下,诊断可以是非常早的,就精原细胞瘤来说有可能在揭示肿瘤前的直至几个月。
通过考虑基因组的核苷酸多样性确定如上所述的百分比同一性。已经知道在富含重复序列的基因组区域中其核苷酸多样性要高于不包含重复序列的区域。举例来说,Nickerson D.A.等人(1)已经显示在包含重复序列的区域中大约0.3%(0.32%)的多样性。
通过使用SAM方法(5)的统计学分析,继之以通过假阳性率的校正(6)和清除低于26的值,已经证明了区分上面所示每一序列的癌性状态的能力。因此,上面所示的每一序列在肿瘤状态和正常状态之间的表达显示显著差异。由此可知,观察到上述序列之一的表达差异构成病理性病症的标记物。当然,有可能将上文标示的几种序列的表达差异进行组合,例如通过所示序列的2,3,4,5,6,7,8,9,10和更多直至775中的一种或更多种组合。具体来说,SEQ ID NOs:1、26和29所示序列,单独或组合的(配对或所有三个)构成一个或更多个优选的标记物。
因此,在本发明的方法中,检测至少两种核酸序列的相应的至少两种表达产物,所述核酸序列选自SEQ ID NOs:1到775所示序列或选自与SEQID NOs:1到775所示序列之一显示至少99%同一性,优选的至少99.5%同一性和有利地至少99.6%或至少99.7%同一性的序列。
在本发明方法的一个实施方案中,检测至少两种核酸序列的表达产物,所述至少两种核酸序列选自经鉴定对睾丸组织特异的序列,即选自如下所示序列组:SEQ ID NOs:1,2,3,4,5,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,23,24,25,26,27,29,31,32,33,34,36,38,39,40,41,42,43,44,45,46,47,50,51,52,53,57,58,59,61,63,64,65,68,70,71,72,74,75,76,77,78,79,80,83,84,85,86,87,88,89,90,91,92,94,96,97,102,104,106,107,109,110,115,116,118,121,122,123,127,132,133,136,137,142,147,148,150,159,160,162,163,164,165,166,167,170,171,175,182,187,191,198,199,201,207,210,216,219,222,224,228,230,234,244,256,263,265,266,269,271,281,286,288,291,292,296,298,304,308,333,341,347,403,429,452,485,513,556,574和755,或选自与下列所示序列之一显示至少99%同一性,优选的至少99.5%同一性和有利地至少99.6%或至少99.7%同一性的序列:SEQ ID NOs:1,2,3,4,5,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,23,24,25,26,27,29,31,32,33,34,36,38,39,40,41,42,43,44,45,46,47,50,51,52,53,57,58,59,61,63,64,65,68,70,71,72,74,75,76,77,78,79,80,83,84,85,86,87,88,89,90,91,92,94,96,97,102,104,106,107,109,110,115,116,118,121,122,123,127,132,133,136,137,142,147,148,150,159,160,162,163,164,165,166,167,170,171,175,182,187,191,198,199,201,207,210,216,219,222,224,228,230,234,244,256,263,265,266,269,271,281,286,288,291,292,296,298,304,308,333,341,347,403,429,452,485,513,556,574和755。
在本发明方法的另一个实施方案中,检测选自经鉴定对睾丸组织特异的序列的至少一种序列的表达产物,即选自如下所示的序列组:SEQ IDNOs:1,2,3,4,5,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,23,24,25,26,27,29,31,32,33,34,36,38,39,40,41,42,43,44,45,46,47,50,51,52,53,57,58,59,61,63,64,65,68,70,71,72,74,75,76,77,78,79,80,83,84,85,86,87,88,89,90,91,92,94,96,97,102,104,106,107,109,110,115,116,118,121,122,123,127,132,133,136,137,142,147,148,150,159,160,162,163,164,165,166,167,170,171,175,182,187,191,198,199,201,207,210,216,219,222,224,228,230,234,244,256,263,265,266,269,271,281,286,288,291,292,296,298,304,308,333,341,347,403,429,452,485,513,556,574和755,或选自与下列所示序列之一显示至少99%同一性,优选的至少99.5%同一性和有利地至少99.6%或至少99.7%同一性的序列:SEQ ID NOs:1,2,3,4,5,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,23,24,25,26,27,29,31,32,33,34,36,38,39,40,41,42,43,44,45,46,47,50,51,52,53,57,58,59,61,63,64,65,68,70,71,72,74,75,76,77,78,79,80,83,84,85,86,87,88,89,90,91,92,94,96,97,102,104,106,107,109,110,115,116,118,121,122,123,127,132,133,136,137,142,147,148,150,159,160,162,163,164,165,166,167,170,171,175,182,187,191,198,199,201,207,210,216,219,222,224,228,230,234,244,256,263,265,266,269,271,281,286,288,291,292,296,298,304,308,333,341,347,403,429,452,485,513,556,574和755,并检测选自经鉴定对睾丸组织不特异的序列的至少一种序列的表达产物,即选自如下所示的序列组:SEQID NOs:6,22,28,30,35,37,48,49,54,55,56,60,62,66,67,69,73,81,82,93,95,98,99,100,101,103,105,108,111,112,113,114,117,119,120,124,125,126,128,129,130,131,134,135,138,139,140,141,143,144,145,146,149,151,152,153,154,155,156,157,158,161,168,169,172,173,174,176,177,178,179,180,181,183,184,185,186,188,189,190,192,193,194,195,196,197,200,202,203,204,205,206,208,209,211,212,213,214,215,217,218,220,221,223,225,226,227,229,231,232,233,235,236,237,238,239,240,241,242,243,245,246,247,248,249,250,251,252,253,254,255,257,258,259,260,261,262,264,267,268,270,272,273,274,275,276,277,278,279,280,282,283,284,285,287,289,290,293,294,295,297,299,300,301,302,303,305,306,307,309,310,311,312,313,314,315,316,317,318,319,320,321,322,323,324,325,326,327,328,329,330,331,332,334,335,336,337,338,339,340,342,343,344,345,346,348,349,350,351,352,353,354,355,356,357,358,359,360,361,362,363,364,365,366,367,368,369,370,371,372,373,374,375,376,377,378,379,380,381,382,383,384,385,386,387,388,389,390,391,392,393,394,395,396,397,398,399,400,401,402,404,405,406,407,408,409,410,411,412,413,414,415,416,417,418,419,420,421,422,423,424,425,426,427,428,430,431,432,433,434,435,436,437,438,439,440,441,442,443,444,445,446,447,448,449,450,451,453,454,455,456,457,458,459,460,461,462,463,464,465,466,467,468,469,470,471,472,473,474,475,476,477,478,479,480,481,482,483,484,486,487,488,489,490,491,492,493,494,495,496,497,498,499,500,501,502,503,504,505,506,507,508,509,510,511,512,514,515,516,517,518,519,520,521,522,523,524,525,526,527,528,529,530,531,532,533,534,535,536,537,538,539,540,541,542,543,544,545,546,547,548,549,550,551,552,553,554,555,557,558,559,560,561,562,563,564,565,566,567,568,569,570,571,572,573,575,576,577,578,579,580,581,582,583,584,585,586,587,588,589,590,591,592,593,594,595,596,597,598,599,600,601,602,603,604,605,606,607,608,609,610,611,612,613,614,615,616,617,618,619,620,621,622,623,624,625,626,627,628,629,630,631,632,633,634,635,636,637,638,639,640,641,642,643,644,645,646,647,648,649,650,651,652,653,654,655,656,657,658,659,660,661,662,663,664,665,666,667,668,669,670,671,672,673,674,675,676,677,678,679,680,681,682,683,684,685,686,687,688,689,690,691,692,693,694,695,696,697,698,699,700,701,702,703,704,705,706,707,708,709,710,711,712,713,714,715,716,717,718,719,720,721,722,723,724,725,726,727,728,729,730,731,732,733,734,735,736,737,738,739,740,741,742,743,744,745,746,747,748,749,750,751,752,753,754,756,757,758,759,760,761,762,763,764,765,766,767,768,769,770,771,772,773,774和775,或选自与下列所示序列之一显示至少99%同一性,优选的至少99.5%同一性和有利地至少99.6%或至少99.7%同一性的序列:SEQ ID NOs:6,22,28,30,35,37,48,49,54,55,56,60,62,66,67,69,73,81,82,93,95,98,99,100,101,103,105,108,111,112,113,114,117,119,120,124,125,126,128,129,130,131,134,135,138,139,140,141,143,144,145,146,149,151,152,153,154,155,156,157,158,161,168,169,172,173,174,176,177,178,179,180,181,183,184,185,186,188,189,190,192,193,194,195,196,197,200,202,203,204,205,206,208,209,211,212,213,214,215,217,218,220,221,223,225,226,227,229,231,232,233,235,236,237,238,239,240,241,242,243,245,246,247,248,249,250,251,252,253,254,255,257,258,259,260,261,262,264,267,268,270,272,273,274,275,276,277,278,279,280,282,283,284,285,287,289,290,293,294,295,297,299,300,301,302,303,305,306,307,309,310,311,312,313,314,315,316,317,318,319,320,321,322,323,324,325,326,327,328,329,330,331,332,334,335,336,337,338,339,340,342,343,344,345,346,348,349,350,351,352,353,354,355,356,357,358,359,360,361,362,363,364,365,366,367,368,369,370,371,372,373,374,375,376,377,378,379,380,381,382,383,384,385,386,387,388,389,390,391,392,393,394,395,396,397,398,399,400,401,402,404,405,406,407,408,409,410,411,412,413,414,415,416,417,418,419,420,421,422,423,424,425,426,427,428,430,431,432,433,434,435,436,437,438,439,440,441,442,443,444,445,446,447,448,449,450,451,453,454,455,456,457,458,459,460,461,462,463,464,465,466,467,468,469,470,471,472,473,474,475,476,477,478,479,480,481,482,483,484,486,487,488,489,490,491,492,493,494,495,496,497,498,499,500,501,502,503,504,505,506,507,508,509,510,511,512,514,515,516,517,518,519,520,521,522,523,524,525,526,527,528,529,530,531,532,533,534,535,536,537,538,539,540,541,542,543,544,545,546,547,548,549,550,551,552,553,554,555,557,558,559,560,561,562,563,564,565,566,567,568,569,570,571,572,573,575,576,577,578,579,580,581,582,583,584,585,586,587,588,589,590,591,592,593,594,595,596,597,598,599,600,601,602,603,604,605,606,607,608,609,610,611,612,613,614,615,616,617,618,619,620,621,622,623,624,625,626,627,628,629,630,631,632,633,634,635,636,637,638,639,640,641,642,643,644,645,646,647,648,649,650,651,652,653,654,655,656,657,658,659,660,661,662,663,664,665,666,667,668,669,670,671,672,673,674,675,676,677,678,679,680,681,682,683,684,685,686,687,688,689,690,691,692,693,694,695,696,697,698,699,700,701,702,703,704,705,706,707,708,709,710,711,712,713,714,715,716,717,718,719,720,721,722,723,724,725,726,727,728,729,730,731,732,733,734,735,736,737,738,739,740,741,742,743,744,745,746,747,748,749,750,751,752,753,754,756,757,758,759,760,761,762,763,764,765,766,767,768,769,770,771,772,773,774和775。
具体来说,在本发明的方法中,检测至少一种核酸序列的表达产物,优选的至少两种核酸序列或三种核酸序列的,所述核酸序列选自SEQ IDNOs:1,26和29所示的序列组,或选自与SEQ ID NOs:1,26和29所示序列显示至少99%同一性,优选的至少99.5%同一性和有利地至少99.6%或至少99.7%同一性的序列。
检测的表达产物是至少一种RNA转录物,具体来说至少一种mRNA或至少一种多肽。
当表达产物是mRNA转录物时,可以通过任意合适的方法诸如杂交、测序或扩增对其进行检测。通过与至少一种探针和/或至少一种引物(其被设计以便在预定实验条件下与mRNA转录物杂交)接触,显示与mRNA杂交的存在或缺失并任选的定量mRNA,可以对mRNA进行直接检测。在优选的方法中,其可以由扩增(例如,RT-PCR,NASBA等等)、芯片杂交或测序来构成。也可以使用来源于所述转录物的核酸(诸如cDNA拷贝等等)来间接检测mRNA。
通常,本发明的方法包括从待分析样本提取mRNA的初始步骤。
因此,所述方法可以包括∶
(i)从待分析样本提取mRNA的步骤,
(ii)从待分析样本检测和定量mRNA的步骤,
(iii)从参照样本提取mRNA的步骤,所述参照样本可以是源于相同个体的健康样本,或
(iv)从健康样本检测和定量mRNA的步骤,
(v)将待分析样本和参照样本中表达的mRNA量进行比较的步骤;有可能确定与健康参照样本表达的mRNA量不同的待分析样本表达的mRNA量与睾丸癌严重性的诊断或预后有关(癌性睾丸组织中mRNA量相对于健康睾丸组织中表达的mRNA量的差异一般是表达、过表达或低表达);
和具体来说∶
(i)待分析样本中mRNA的提取,
(ii)在待分析的RNA中,所述样本至少一种RNA序列表达水平的测定,所述RNA序列是至少一种核酸序列的转录产物,所述至少一种核酸序列选自SEQ ID NOs:1到775所示的序列,或选自与SEQ ID NOs:1到775所示的序列之一显示至少99%同一性,优选的至少99.5%同一性和有利地至少99.6%或至少99.7%同一性的序列,和
(iii)将(ii)确定的RNA序列表达水平与参照表达水平进行比较;有可能确定相对于参照表达水平显示差异的待分析RNA的表达水平与睾丸癌的诊断或预后有关(如上所决定);或
(i)从待分析样本提取mRNA的步骤,
(ii)从待分析样本检测和定量mRNA的步骤,
(iii)对待分析样本中表达的mRNA量相对于参照mRNA量进行比较的步骤;有可能确定与参照mRNA量不同的待分析样本表达的mRNA量与睾丸癌的诊断或预后有关(待分析样本mRNA量相对于参照mRNA量的差异一般是表达、过表达或低表达)。
在本发明方法的一个实施方案中,制备mRNA的DNA拷贝,在允许杂交的预定条件下将所述DNA拷贝与至少一种探针和/或至少一种引物接触,并检测与所述DNA拷贝杂交的存在或缺失。
被检测的表达产物也可以是多肽,其是如上所述至少一种转录物的翻译产物。在这种情况下,通过与所述多肽的至少一种特异性结合配偶体(具体来说是抗体或抗体类似物或适体)接触来检测表达的多肽。结合配偶体优选的是抗体,例如单克隆抗体或高度纯化的多克隆抗体或抗体类似物,例如具有竞争特性的亲和蛋白(nanofitinTM)。
可以如下获得多克隆抗体:用合适的免疫原免疫动物,继之以回收纯化形式的所需抗体,通过抽取所述动物的血清,并将所述抗体与其他血清组分分离,具体来说通过柱(其上结合有被所述抗体特异性识别的抗原)的亲和层析。
可以通过杂交瘤技术获得单克隆抗体,其一般原理在下文概述。
首先,用合适的免疫原免疫动物(通常是小鼠),然后所述小鼠的B淋巴细胞能够产生针对该抗原的抗体。然后将这些抗体生成的淋巴细胞与“永生化的”骨髓瘤细胞(实施例中是小鼠)融合以产生杂交瘤。然后从如此获得的细胞异质混合物中挑选能够产生特定抗体并能无限增殖的细胞。每个杂交瘤以克隆的形式增殖,每种均导致单克隆抗体的产生,其中例如可以通过ELISA,一维或二维Western印迹,免疫荧光或使用生物传感器来检测针对蛋白的识别特性。随后将如此挑选的单克隆抗体纯化,具体来说根据如上所述的亲和层析技术。
单克隆抗体也可以是利用本领域技术人员公知的技术通过基因工程获得的重组抗体。
NanofitinsTM是小蛋白,其像抗体一样能够结合生物靶标,从而使得在生物体内检测它、捕获它或非常简单的靶向它成为可能。它们尤其作为抗体类似物存在。
适体是能够结合特定配体的合成的寡核苷酸。
本发明还涉及已经分离的至少一种核酸序列作为睾丸癌体外诊断或预后的分子标记物的用途,其特征在于所述核酸序列由以下组成:
(i)至少一种DNA序列,其选自序列SEQ ID NOs:1到775,或
(ii)至少一种DNA序列,其与选自序列SEQ ID NOs:1到775的序列互补,或
(iii)至少一种DNA序列,其与(i)和(ii)限定的序列显示至少99%同一性,优选的至少99.5%同一性和有利地至少99.6%或至少99.7%同一性,或
(iv)至少一种RNA序列,其是选自(i)限定的序列的转录产物,或
(v)至少一种RNA序列,其是下述序列的转录产物:选自与(i)限定的序列显示至少99%同一性,优选的至少99.5%同一性和有利地至少99.6%或至少99.7%同一性的序列。
在一个实施方案中,用途由至少两种核酸序列构成,所述核酸序列由以下组成∶
(i)选自序列SEQ ID NOs:1到775的至少两种DNA序列,优选的选自如下所示序列:SEQ ID NOs:1,2,3,4,5,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,23,24,25,26,27,29,31,32,33,34,36,38,39,40,41,42,43,44,45,46,47,50,51,52,53,57,58,59,61,63,64,65,68,70,71,72,74,75,76,77,78,79,80,83,84,85,86,87,88,89,90,91,92,94,96,97,102,104,106,107,109,110,115,116,118,121,122,123,127,132,133,136,137,142,147,148,150,159,160,162,163,164,165,166,167,170,171,175,182,187,191,198,199,201,207,210,216,219,222,224,228,230,234,244,256,263,265,266,269,271,281,286,288,291,292,296,298,304,308,333,341,347,403,429,452,485,513,556,574和755,和具体来说序列SEQ IDNos:1,26和29,或
(ii)与至少两种序列分别互补的至少两种DNA序列,所述至少两种序列选自序列SEQ ID Nos:1到775,优选选自如下所示的序列:SEQ ID NOs:1,2,3,4,5,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,23,24,25,26,27,29,31,32,33,34,36,38,39,40,41,42,43,44,45,46,47,50,51,52,53,57,58,59,61,63,64,65,68,70,71,72,74,75,76,77,78,79,80,83,84,85,86,87,88,89,90,91,92,94,96,97,102,104,106,107,109,110,115,116,118,121,122,123,127,132,133,136,137,142,147,148,150,159,160,162,163,164,165,166,167,170,171,175,182,187,191,198,199,201,207,210,216,219,222,224,228,230,234,244,256,263,265,266,269,271,281,286,288,291,292,296,298,304,308,333,341,347,403,429,452,485,513,556,574和755,和具体来说选自序列SEQ ID Nos:1,26和29,或
(iii)至少两种DNA序列,其与(i)和(ii)限定的两种序列分别显示至少99%同一性,优选的至少99.5%同一性和有利地至少99.6%或至少99.7%同一性,或
(iv)至少两种RNA序列,其分别是选自(i)限定的两种序列的转录产物,或
(v)至少两种RNA序列,其是下述两种序列的转录产物:选自与(i)限定的序列显示至少99%同一性,优选的至少99.5%同一性和有利地至少99.6%或至少99.7%同一性的序列。
本发明的主题还是一种用于取自患者的生物样本中睾丸癌的体外诊断或预后的试剂盒,其包括至少一种核酸序列的至少一种表达产物的至少一种特异性结合配偶体,所述核酸序列选自SEQ ID NOs:1到775所示的序列或选自与SEQ ID NOs:1到775所示核酸序列显示至少99%同一性,优选的至少99.5%同一性和有利地至少99.6%或至少99.7%同一性的序列,并且包括不超过775种核酸序列表达产物的特异性结合配偶体,所述核酸序列如SEQ ID NOs:1到775所示,或所述核酸序列与SEQ ID NOs:1到775所示核酸序列之一显示至少99%同一性,优选的与SEQ ID NOs:1到775所示序列之一显示至少99.5%同一性和有利地至少99.6%或至少99.7%同一性。
优选的,所述试剂盒包括至少一种核酸序列的表达产物的特异性结合配偶体,所述核酸序列选自SEQ ID NOs:1,26和29所示的序列组,或与SEQ ID NOs:1,26和29所示序列显示至少99%同一性,优选的至少99.5%同一性和有利地至少99.6%或至少99.7%同一性的序列。
在一个实施方案中,试剂盒包括至少两种核酸序列的至少两种表达产物的至少两种相应的特异性结合配偶体,所述核酸序列选自SEQ ID NOs:1到775所示的序列,或选自与SEQ ID NOs:1到775所示核酸序列显示至少99%同一性,优选的至少99.5%同一性和有利地至少99.6%或至少99.7%同一性的序列,并且包括不超过775种核酸序列表达产物的特异性结合配偶体,所述核酸序列如SEQ ID NOs:1到775所示,或所述核酸序列与SEQID NOs:1到775所示核酸序列显示至少99%同一性,优选的至少99.5%同一性和有利地至少99.6%或至少99.7%同一性。
例如,所述试剂盒包括至少两种核酸序列的表达产物的至少两种相应的特异性结合配偶体,所述核酸序列选自如下所示序列组:SEQ ID NOs:1,2,3,4,5,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,23,24,25,26,27,29,31,32,33,34,36,38,39,40,41,42,43,44,45,46,47,50,51,52,53,57,58,59,61,63,64,65,68,70,71,72,74,75,76,77,78,79,80,83,84,85,86,87,88,89,90,91,92,94,96,97,102,104,106,107,109,110,115,116,118,121,122,123,127,132,133,136,137,142,147,148,150,159,160,162,163,164,165,166,167,170,171,175,182,187,191,198,199,201,207,210,216,219,222,224,228,230,234,244,256,263,265,266,269,271,281,286,288,291,292,296,298,304,308,333,341,347,403,429,452,485,513,556,574和755,或与如下所示序列显示至少99%同一性的序列:SEQ IDNOs:1,2,3,4,5,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,23,24,25,26,27,29,31,32,33,34,36,38,39,40,41,42,43,44,45,46,47,50,51,52,53,57,58,59,61,63,64,65,68,70,71,72,74,75,76,77,78,79,80,83,84,85,86,87,88,89,90,91,92,94,96,97,102,104,106,107,109,110,115,116,118,121,122,123,127,132,133,136,137,142,147,148,150,159,160,162,163,164,165,166,167,170,171,175,182,187,191,198,199,201,207,210,216,219,222,224,228,230,234,244,256,263,265,266,269,271,281,286,288,291,292,296,298,304,308,333,341,347,403,429,452,485,513,556,574和755。
具体来说,所述试剂盒包括2种或3种核酸序列表达产物的相应的特异性结合配偶体,所述核酸序列如SEQ ID NOs:1,26和29所示,或与SEQID NOs:1,26和29所示序列显示至少99%同一性,优选的至少99.5%同一性和有利地至少99.6%或至少99.7%同一性。
具体来说,所述试剂盒包括1种,2种或3种核酸序列的表达产物的特异性结合配偶体,所述核酸序列如SEQ ID NOs:1,26和29所示,或所述序列与SEQ ID NOs:1,26和29所示序列显示至少99%同一性,优选的至少99.5%同一性和有利地至少99.6%或至少99.7%同一性。
表达产物的最低特异性结合配偶体对应于上文给出的定义。
本发明还涉及一种利用SEQ ID NOs:1到775所示序列之一或与SEQID NOs:1到775所示序列显示至少99%同一性,优选的至少99.5%同一性和有利地至少99.6%或至少99.7%同一性的序列之一来鉴定睾丸癌治疗效果和/或进展的方法,其包括:获得一系列生物样本的步骤,和在所述生物样本系列中检测至少一种核酸序列的至少一种表达产物的步骤,所述核酸序列选自SEQ ID NOs:1到775所示序列。优选的,检测至少一种核酸序列的表达产物,优选的至少两种核酸序列或三种核酸序列的,所述核酸序列选自SEQ ID NOs:1,26和29所示的序列组。
在一个实施方案中,检测至少两种核酸序列的至少两种表达产物,所述两种核酸序列选自SEQ ID NOs:1到775所示序列或选自与SEQ ID NOs:1到775所示序列分别显示至少99%同一性,优选的至少99.5%同一性和有利地至少99.6%或至少99.7%同一性的序列。
在所述方法的另一个实施方案中,检测至少一种核酸序列的表达产物,优选的至少两种核酸序列或三种核酸序列的,所述核酸序列选自SEQ IDNOs:12,26和29所示的序列组,或选自与SEQ ID NOs:1,26和29所示序列分别显示至少99%同一性,优选的至少99.5%同一性和有利地至少99.6%或至少99.7%同一性的序列。
术语“生物样本”旨在表示组织,体液,所述组织和体液的组分,诸如细胞或凋亡体,和分泌囊泡,具体来说包括外体和微泡。举例来说,生物样本可以来源于在怀疑患有睾丸癌的患者中事先进行的睾丸活检切片,或可以来源于在呈现转移瘤的患者中除了睾丸之外的器官上进行的活检切片。就第二种情况来说,当核酸(分子标记物)的表达改变对睾丸器官特异时,有可能回到原发癌,即睾丸癌。生物样本也可以是生物体液,诸如血液或血液组分(血清,血浆),尿液,唾液,脑脊液,淋巴,母乳,精子,以及所述体液的组分,具体来说是如上所述的分泌囊泡。例如,在来源于上皮细胞的外体或微泡中检测到睾丸组织特异性转录物,是原发癌或转移瘤存在的标记,无需在器官水平取样。
附图∶
图1和2显示睾丸癌中对一组HERV序列观察到的差异化表达。更具体地,图1(聚簇分析)以探索方式将HERV元件分组,所述HERV元件具有与所有对照组织相比与睾丸癌有关的表达趋向性,而图2显示正常睾丸和肿瘤性睾丸之间HERV元件表达的统计学差异。
图3和4显示两种生物体液∶尿液和血清中HERV序列的检测。
实施例∶
实施例1∶睾丸癌中显示差异化表达的HERV序列的鉴定
方法:
睾丸癌中显示差异化表达的HERV序列的鉴定是基于采用GeneChip形式的高密度DNA芯片(称为HERV-V2,由本发明人设计,其制造转包给Affymetrix公司)的设计和使用。该芯片包含对应于人类基因组中独特的HERV序列的探针。使用一组原型参照分割为功能区域(LTR,gag,pol和env),然后通过在整个人类基因组(NCBI36/hg18)范围的相似性检索,对这些序列进行鉴定,10035个不同HERV基因座被鉴定、注释并最终集合在称为HERVgDB3的数据库中。
基于HERVgDB3确定作为芯片组成一部分的探针,并利用杂交特异性标准来挑选,其目的是从制备过程排除具有与不需要的靶标杂交的高风险的探针。为此,首先将HERVgDB3序列分割成25个重叠核苷酸(25-mers)的组,从而产生候选探针组。然后使用KASH算法(2),通过对整个人类基因组进行比对,评估每个候选探针的非特异性杂交的风险。实验评分记录杂交结果,添加比对中误差的数量、类型和位置的影响。该评分值与靶标/探针杂交能力有关。知晓候选探针在整个人类基因组范围的杂交能力使评估其捕获特异性成为可能。保留显示良好捕获亲和力的候选探针,然后将其集合为“探针组”,并最终在HERV-V2芯片上合成。
使用HERV-V2高密度芯片分析的样本对应于从肿瘤提取的RNAs和从邻近于这些肿瘤的健康组织提取的RNAs。分析的组织是睾丸,利用乳腺,卵巢,子宫,前列腺,肺,结肠和胎盘作为对照。就胎盘而言,只使用健康组织。对于每个样本,使用50ng RNA用于利用称为WTO的扩增方案的cDNA合成。WTO扩增的原理如下:在逆转录步骤之前添加随机引物,以及靶向RNA转录物3'端的引物,继之以称为SPIA的线性、单链扩增。然后测定、表征并纯化cDNAs,将2μg片段化,并通过末端转移酶的作用在3'端用生物素进行标记。将如此制备的靶标产物与对照寡核苷酸混合,然后按照Affymetrix公司推荐的方案进行杂交。然后将芯片进行显影和读取以获得它们的荧光图像。进行基于标准对照的质量控制,使用一组指标(MAD,MAD-Med图,RLE)来排除不符合统计学分析的芯片。
芯片分析首先包括数据的预处理,通过使用基于色氨酸探针信号强度的背景噪声进行校正,继之以基于分位数方法的RMA标准化(3)。然后通过使用COMBAT方法(4)进行与实验批次有关的效果双校正,以便确保观察到的表达差异是生物学原因而非技术原因。在这个阶段,使用通过欧几里得分配(聚簇分析)集合数据的工具进行数据的探索性分析,并且最后,使用利用SAM方法(5)的统计学分析,继之以通过假阳性率的校正(6)并清除低于26的值,以便寻找在组织的正常状态和肿瘤状态之间显示差异化表达的序列。
结果∶
对利用该方法通过分析HERV-V2DNA芯片产生的数据进行处理,使得能够鉴定出在正常睾丸和肿瘤性睾丸之间显示统计上显著的表达差异的一组“探针组”。此外聚簇分析的结果以及寻找对照样本之间的差异化表达显示HERV元件的差异化表达是与肿瘤性睾丸特异性相关的。
在肿瘤性睾丸中显示差异化表达的HERV元件的核苷酸序列如SEQID NOs:1到775所示,每种序列的染色体位置在NCBI参考36/hg18中给出;并且“靶组织”信息(X)标示了只在正常睾丸和肿瘤性睾丸(与对照组织之间的比较相比)之间的比较中观察到差异化表达的元件。还提供了作为正常状态和肿瘤状态之间表达比值的指示值,其只是为了显示而排列序列。
实施例2∶生物体液中HERV序列的检测
原理∶
本发明人证明了在生物体液中检测到HERV序列,这尤其使通过依赖原发性器官的远程检测来表征睾丸癌成为可能。对来源于不同个体的20个尿液样本和38个血清样本进行研究。
在下列条件下对血清和尿液进行离心∶
血清∶4℃500g10分钟。回收上清液,再在4℃16500g离心20分钟。该第二次离心的上清液,不含细胞,但包含外体、微泡、核酸和蛋白,在芯片上进行分析。芯片是根据先前所述模式使用的HERV-V2芯片。
尿液∶收集后在4℃800g离心4分钟。利用RNA保护细胞试剂(RNAprotect cell reagentTM)回收沉淀物。然后,向沉淀物中添加裂解缓冲液之前,5000g离心5分钟。芯片是根据先前所述模式使用的HERV-V2芯片。
结果∶
如图3和4所示,在血清上清液和来源于尿液的细胞沉淀物中均检测到大量阳性信号,包括对应于上文表中所列序列的表达信号。这证明了生物体液(具体来说是血清和尿液)是用于HERV序列检测的有效的生物材料来源。通常可以接受的阳性阈值是约26,即64。
参考文献
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Claims (18)
1.一种用于取自患者的生物样本中睾丸癌体外诊断或预后的方法,其包括检测至少两种核酸序列的相应至少两种表达产物的步骤,所述核酸序列选自SEQ ID NOs:1到775所示序列或选自与SEQ ID NOs:1到775所示序列之一显示至少99%同一性的序列。
2.如权利要求1所述的方法,其中检测至少两种核酸序列的表达产物,所述至少两种核酸序列选自如下所示的序列组:SEQ ID NOs:1,2,3,4,5,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,23,24,25,26,27,29,31,32,33,34,36,38,39,40,41,42,43,44,45,46,47,50,51,52,53,57,58,59,61,63,64,65,68,70,71,72,74,75,76,77,78,79,80,83,84,85,86,87,88,89,90,91,92,94,96,97,102,104,106,107,109,110,115,116,118,121,122,123,127,132,133,136,137,142,147,148,150,159,160,162,163,164,165,166,167,170,171,175,182,187,191,198,199,201,207,210,216,219,222,224,228,230,234,244,256,263,265,266,269,271,281,286,288,291,292,296,298,304,308,333,341,347,403,429,452,485,513,556,574和755,或选自与如下所示序列显示至少99%同一性的序列:SEQ ID NOs:1,2,3,4,5,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,23,24,25,26,27,29,31,32,33,34,36,38,39,40,41,42,43,44,45,46,47,50,51,52,53,57,58,59,61,63,64,65,68,70,71,72,74,75,76,77,78,79,80,83,84,85,86,87,88,89,90,91,92,94,96,97,102,104,106,107,109,110,115,116,118,121,122,123,127,132,133,136,137,142,147,148,150,159,160,162,163,164,165,166,167,170,171,175,182,187,191,198,199,201,207,210,216,219,222,224,228,230,234,244,256,263,265,266,269,271,281,286,288,291,292,296,298,304,308,333,341,347,403,429,452,485,513,556,574和755。
3.如权利要求2所述的方法,其中检测至少两种核酸序列的表达产物,优选三种核酸序列的表达产物,所述核酸序列选自SEQ ID NOs:1,26和29所示的序列组,或选自与SEQ ID NOs:1,26和29所示序列显示至少99%同一性的序列。
4.如权利要求1所述的方法,其中检测的表达产物是至少一种RNA转录物或至少一种多肽。
5.如权利要求4所述的方法,其特征在于所述RNA转录物是至少一种mRNA。
6.如权利要求4或5所述的方法,其中通过杂交,通过扩增或通过测序来检测RNA转录物,特别是mRNA。
7.如权利要求6所述的方法,其中在允许杂交的预定条件下,将mRNA与至少一种探针和/或至少一种引物接触,并检测与mRNA杂交的存在或缺失。
8.如权利要求5所述的方法,其特征在于制备mRNA的DNA拷贝,在允许杂交的预定条件下将所述DNA拷贝与至少一种探针和/或至少一种引物接触,并检测与所述DNA拷贝杂交的存在或缺失。
9.如权利要求4所述的方法,其中通过与所述多肽的至少一种特异性结合配偶体接触来检测表达的多肽,所述特异性结合配偶体特别是抗体或抗体类似物,亲和蛋白或适体。
10.已经分离的至少两种核酸序列作为睾丸癌体外诊断或预后的分子标记物的用途,其特征在于所述两种核酸序列由以下组成∶
(i)选自序列SEQ ID NOs:1到775的至少两种DNA序列,优选选自如下所示序列:SEQ ID NOs:1,2,3,4,5,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,23,24,25,26,27,29,31,32,33,34,36,38,39,40,41,42,43,44,45,46,47,50,51,52,53,57,58,59,61,63,64,65,68,70,71,72,74,75,76,77,78,79,80,83,84,85,86,87,88,89,90,91,92,94,96,97,102,104,106,107,109,110,115,116,118,121,122,123,127,132,133,136,137,142,147,148,150,159,160,162,163,164,165,166,167,170,171,175,182,187,191,198,199,201,207,210,216,219,222,224,228,230,234,244,256,263,265,266,269,271,281,286,288,291,292,296,298,304,308,333,341,347,403,429,452,485,513,556,574和755,和具体来说选自SEQ IDNos:1,26和29所示序列,或
(ii)与至少两种序列分别互补的至少两种DNA序列,所述至少两种序列选自序列SEQ ID Nos:1到775,优选选自如下所示序列:SEQ ID NOs:1,2,3,4,5,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,23,24,25,26,27,29,31,32,33,34,36,38,39,40,41,42,43,44,45,46,47,50,51,52,53,57,58,59,61,63,64,65,68,70,71,72,74,75,76,77,78,79,80,83,84,85,86,87,88,89,90,91,92,94,96,97,102,104,106,107,109,110,115,116,118,121,122,123,127,132,133,136,137,142,147,148,150,159,160,162,163,164,165,166,167,170,171,175,182,187,191,198,199,201,207,210,216,219,222,224,228,230,234,244,256,263,265,266,269,271,281,286,288,291,292,296,298,304,308,333,341,347,403,429,452,485,513,556,574和755,和具体来说选自SEQ ID Nos:1,26和29所示序列,或
(iii)至少两种DNA序列,其与选自(i)和(ii)限定的两种序列显示至少99%同一性,或
(iv)至少两种RNA序列,其分别是选自(i)限定的两种序列的转录产物,或
(v)至少两种RNA序列,其是至少两种序列的转录产物,所述至少两种序列选自与(i)限定的序列显示至少99%同一性的序列。
11.一种用于取自患者的生物样本中睾丸癌的体外诊断或预后的试剂盒,其包括至少两种核酸序列的至少两种表达产物的至少两种相应的特异性结合配偶体,所述核酸序列选自SEQ ID NOs:1到775所示的序列或与SEQ ID NOs:1到775所示核酸序列显示至少99%同一性的序列,并且包括不超过775种核酸序列表达产物的特异性结合配偶体,所述核酸序列如SEQ ID NOs:1到775所示,或所述核酸序列与SEQ ID NOs:1到775所示核酸序列显示至少99%同一性。
12.如权利要求11所述的试剂盒,其包括至少两种核酸序列的表达产物的至少两种相应的特异性结合配偶体,所述核酸序列选自如下所示的序列组:SEQ ID NOs:1,2,3,4,5,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,23,24,25,26,27,29,31,32,33,34,36,38,39,40,41,42,43,44,45,46,47,50,51,52,53,57,58,59,61,63,64,65,68,70,71,72,74,75,76,77,78,79,80,83,84,85,86,87,88,89,90,91,92,94,96,97,102,104,106,107,109,110,115,116,118,121,122,123,127,132,133,136,137,142,147,148,150,159,160,162,163,164,165,166,167,170,171,175,182,187,191,198,199,201,207,210,216,219,222,224,228,230,234,244,256,263,265,266,269,271,281,286,288,291,292,296,298,304,308,333,341,347,403,429,452,485,513,556,574和755,或选自与如下所示序列显示至少99%同一性的序列:SEQ ID NOs:1,2,3,4,5,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,23,24,25,26,27,29,31,32,33,34,36,38,39,40,41,42,43,44,45,46,47,50,51,52,53,57,58,59,61,63,64,65,68,70,71,72,74,75,76,77,78,79,80,83,84,85,86,87,88,89,90,91,92,94,96,97,102,104,106,107,109,110,115,116,118,121,122,123,127,132,133,136,137,142,147,148,150,159,160,162,163,164,165,166,167,170,171,175,182,187,191,198,199,201,207,210,216,219,222,224,228,230,234,244,256,263,265,266,269,271,281,286,288,291,292,296,298,304,308,333,341,347,403,429,452,485,513,556,574和755。
13.如权利要求12所述的试剂盒,其包括至少两种核酸序列的表达产物的至少两种相应的特异性结合配偶体,所述核酸序列选自SEQ ID NOs:1,26和29所示的序列组,或选自与SEQ ID NOs:1,26和29所示序列显示至少99%同一性的序列。
14.如权利要求12所述的试剂盒,其包括核酸序列的表达产物的2种或3种相应的特异性结合配偶体,所述核酸序列如SEQ ID NOs:1,26和29所示,或与SEQ ID NOs:1,26和29所示序列显示至少99%同一性的序列。
15.如权利要求11至14中任一项所述的试剂盒,其中表达产物的至少两种相应的特异性结合配偶体分别是至少一种杂交探针和/或至少一种扩增引物,或至少一种抗体,或至少一种抗体类似物,或至少一种亲和蛋白,或至少一种适体。
16.一种用于评估睾丸癌的治疗效果和/或进展的方法,其包括分别检测至少两种核酸序列的至少两种表达产物的步骤,所述两种核酸序列选自SEQ ID NOs:1到775所示的序列,或选自与SEQ ID NOs:1到775所示的序列分别显示至少99%同一性的序列。
17.如权利要求16所述的方法,其中检测至少两种核酸序列的表达产物,所述至少两种核酸序列选自如下所示的序列组:SEQ ID NOs:1,2,3,4,5,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,23,24,25,26,27,29,31,32,33,34,36,38,39,40,41,42,43,44,45,46,47,50,51,52,53,57,58,59,61,63,64,65,68,70,71,72,74,75,76,77,78,79,80,83,84,85,86,87,88,89,90,91,92,94,96,97,102,104,106,107,109,110,115,116,118,121,122,123,127,132,133,136,137,142,147,148,150,159,160,162,163,164,165,166,167,170,171,175,182,187,191,198,199,201,207,210,216,219,222,224,228,230,234,244,256,263,265,266,269,271,281,286,288,291,292,296,298,304,308,333,341,347,403,429,452,485,513,556,574和755,或选自与如下所示序列显示至少99%同一性的序列:SEQ ID NOs:1,2,3,4,5,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,23,24,25,26,27,29,31,32,33,34,36,38,39,40,41,42,43,44,45,46,47,50,51,52,53,57,58,59,61,63,64,65,68,70,71,72,74,75,76,77,78,79,80,83,84,85,86,87,88,89,90,91,92,94,96,97,102,104,106,107,109,110,115,116,118,121,122,123,127,132,133,136,137,142,147,148,150,159,160,162,163,164,165,166,167,170,171,175,182,187,191,198,199,201,207,210,216,219,222,224,228,230,234,244,256,263,265,266,269,271,281,286,288,291,292,296,298,304,308,333,341,347,403,429,452,485,513,556,574和755。
18.如权利要求17所述的方法,其中检测至少两种核酸序列的表达产物,优选的三种核酸序列的表达产物,所述核酸序列选自SEQ ID NOs:1,26和29所示的序列组,或选自与SEQ ID NOs:1,26和29所示序列显示至少99%同一性的序列。
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PCT/FR2012/053017 WO2013093356A2 (fr) | 2011-12-20 | 2012-12-20 | Procede pour le diagnostic ou le pronostic, in vitro, du cancer du testicule |
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FR2928658B1 (fr) * | 2008-03-12 | 2012-01-13 | Biomerieux Sa | Procede pour le diagnostic ou pronostic in vitro du cancer du testicule |
FR2984364A1 (fr) * | 2011-12-20 | 2013-06-21 | Biomerieux Sa | Procede pour le diagnostic ou le pronostic in vitro du cancer de l'ovaire |
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US11473147B2 (en) | 2022-10-18 |
CN104204223B (zh) | 2018-06-15 |
WO2013093356A2 (fr) | 2013-06-27 |
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ES2675270T3 (es) | 2018-07-10 |
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