卵巢上皮性癌相关抗原的筛选和血清学检测

目的构建卵巢上皮性癌（卵巢癌）腹水肿瘤细胞的cDNA文库,从中筛选能用于卵巢癌早期诊断和免疫治疗靶点的卵巢癌相关抗原。方法用3例卵巢浆液性囊腺癌、1例卵巢黏液性囊腺癌、1例卵巢子宫内膜样癌患者的腹水肿瘤细胞构建cDNA文库,采用改良的重组克隆表达抗原的血清学鉴定（SEREX）技术与抑制性消减杂交（SSH）方法相结合的策略从文库中筛选卵巢癌相关抗原基因,并对其进行酶切鉴定、核苷酸测序分析。采用重组肿瘤抗原的微型血清学检测（SMARTA）法检测筛选出的卵巢癌相关抗原与96例卵巢癌患者和96例正常妇女的血清中相应自身抗体的阳性反应情况。结果经两轮血清学筛选和核苷酸测序分析,最终得到55个候选的卵巢癌相关抗原基因片段,代表45个基因,分为6类：（1）与已知的卵巢癌相关基因同源的基因,如BARD1基因等;（2）与其他肿瘤相关基因同源的基因,如TM4SF1基因等;（3）在一些特殊组织中表达的基因,如ILF3、FXR1基因等;（4）与一些特殊功能蛋白基因同源的基因,如TIZ、C1D基因等;（5）与胚胎来源的基因同源的基因,如PKHD1基因等;（6）其余为在基因库GenBank中无同源序列可比对的未知基因,如OV-189基因等。TM4SF1、C1D、BARD1、FXR1、OV-189基因的噬菌体重组融合抗原与卵巢癌患者血清中相应IgG型自身抗体反应的阳性率分别为28％、21％、23％、23％、31％,与正常妇女血清反应的阳性率分别为9％、6％、5％、8％、13％,分别比较,差异均有统计学意义（P〈0．01）;TIZ、FXR1、OV-189基因的重组抗原与卵巢癌患者血清中相应IgM型自身抗体反应的阳性率分别为26％、28％、18％,与正常妇女血清反应的阳性率分别为8％、11％、7％,分别比较,差异均有统计学意义（P〈0．05）。FXR1、OV-189基因的重组抗原与Ⅰ～Ⅱ期卵巢癌患者血清中相应IgG型自身抗体反应的阳性率（分别为34％、46％）高于Ⅲ～Ⅳ期者（分别为16％、23％）,两者分别比较,差异均有统计学意义（P值分别为0．045、0．021）;OV-189基因的重组抗原与高分化卵巢癌患者血清中相应IgG型自身抗体反应的阳性率（为67％）高于中～低分化者（为26％）,两者比较,差异均有统计学意义（P=0．001）。TIZ、FXR1基因的重组抗原与Ⅰ～Ⅱ期卵巢癌患者血清中相应IgM型自身抗体反应的阳性率（分别为40％、46％）高于Ⅲ～Ⅳ期者（分别为18％、18％）,两者分别比较,差异均有统计学意义（P值分别为0．018、0．004）。当联合分析TM4SF1、C1D、TIZ、FXR1基因的重组抗原的相应IgG型自身抗体及TIZ、FXR1基因的重组抗原的相应IgM型自身抗体（即自身抗体谱）时,诊断卵巢癌的敏感度为66％,准确度为73％;将该自身抗体谱与CA125联合时,敏感度、准确度均有明显提高,分别为83％、80％。结论SEREX技术与SSH方法相结合筛选肿瘤抗原基因是一种行之有效的、能筛选出具有较高特异性肿瘤抗原基因的研究策略;TM4SF1、C1D、TIZ、BARD1、FXR1、OV-189基因的重组抗原在血清中的相应自身抗体可作为卵巢癌诊断的标志物,多个抗原的联合检测可提高诊断效率。

Screening and sero-immunoscreening of ovarian epithelial cancer associative antigens

OBJECTIVE: To explore epithelial ovarian cancer (EOC) antigens that are potentially useful for cancer early detection and therapy. METHODS: A high quality cDNA library derived from ascites tumor cells of EOC patients (3 cases of serous EOC, 1 case of mucinous EOC, and 1 case of endometrial carcinoma of ovary) was constructed, and the method of combining serological analysis of recombinant cDNA expression libraries (SEREX) and suppression subtractive hybridization (SSH) was used for screening cDNA library. All of the positive clones were sequenced and bioinformatics analysis with BLAST software in GenBank was performed. Serological mini-arrays of recombinant tumor antigens (SMARTA) was used to investigate the prevalence of autoantibodies to these antigens in both 96 ovarian cancer patients and 96 cancer-free controls. RESULTS: Fifty-five positive clones encoding different antigenic genes of EOC recognized by IgG and (or) IgM were obtained. It showed that these 55 clones derived from 45 distinct genes and these genes could be grouped into 6 classes as following according to homology with known expressed sequence tag (EST): (1) known ovarian carcinoma related genes: BARD1, et al; (2) homologous genes with other tumors: TM4SF1, et al; (3) homologous genes with special tissues: ILF3, FXR1, et al; (4) homologous genes with special function: TIZ, C1D, et al; (5) embryo originating genes: PKHD1, et al; (6) novel genes: OV-189, et al. SMARTA results showed that the positive ratio of five EOC antigens TM4SF1 (28% vs. 9%), C1D (21% vs. 6%), BARD1 (23% vs. 5%), FXR1 (23% vs. 8%), OV-189 (31% vs. 13%) which reacting with their IgG autoantibodies, three antigens TIZ (26% vs. 8%), FXR1 (28% vs. 11%), and OV-189 (18% vs. 7%) which reacting with their IgM autoantibodies in patients was higher than in controls (P < 0.05). The positive ratio of EOC antigens FXR1 (34% vs. 16%), and OV-189 (46% vs. 23%) reacting with their IgG autoantibodies and TIZ (40% vs. 18%), FXR1 (46% vs. 18%) which reacting with their IgM autoantibodies in stage I-II patients was higher than that in stage III-IV (P < 0.05). The positive ratio of OV-189 (67% vs. 26%) which reacting with its IgG autoantibodies in well differentiated cases was higher than in moderately-poorly differentiated cases (P < 0.01). Combination of the above antigens showed a 66% sensitivity and 73% accuracy in discriminating EOC. Combination of the autoantibody profile of TM4SF1, C1D, TIZ, BARD1, FXR1, OV-189 with CA125 showed an 83% sensitivity and 80% accuracy in discriminating EOC. CONCLUSIONS: The strategy of combination of SEREX and SSH is a potent tool in isolating tumor-associated antigen genes. Autoantibody profile of TM4SF1, C1D, TIZ, BARD1, FXR1, OV-189 are potential tumor markers in EOC detection.