【摘要】：研究目的 近年來，經(jīng)典的前列腺癌早期診斷及篩查方式——血清學(xué)PSA遭到了眾多的質(zhì)疑。主要是因?yàn)榱夹?正常前列腺腺體細(xì)胞均產(chǎn)生和分泌PSA，從而導(dǎo)致血清學(xué)PSA診斷的假陽性問題。大量研究證實(shí)，單純血清PSA指標(biāo)指導(dǎo)前列腺癌早期診斷的優(yōu)勢明顯不足，對(duì)于PSA處于灰區(qū)的患者，前列腺穿刺活檢陽性率只有17-32%，PSA假陽性率高達(dá)65%之多，有超過2/3的對(duì)象接受了不必要的穿刺[4,5]。然而，陰性的穿刺結(jié)果也并不能完全排除腫瘤的存在，反復(fù)穿刺陽性率也僅有10%左右[6]。伴隨著2012年2月美國FDA正式批準(zhǔn)首個(gè)尿液中長鏈非編碼RNA診斷試劑盒——PROGENSA PCA3用于臨床[7,8]，前列腺癌分子診斷進(jìn)入了尿沉渣RNA無創(chuàng)檢測時(shí)代。據(jù)報(bào)道尿液中PCA3的診斷敏感度67%、特異度83%[9]。而同期發(fā)現(xiàn)的TMPRSS2-ERG融合基因不僅也能在尿沉渣中被檢測到，而且僅在前列腺腫瘤中發(fā)生融合，，歐美人群中的發(fā)生頻率50%左右。因此我們亟需開發(fā)種新型的高特異性且兼顧敏感性的分子診斷標(biāo)記物，提高現(xiàn)如今“尷尬的”穿刺診斷陽性診斷率。本研究的目的，是針對(duì)課題組前期發(fā)現(xiàn)的新型非編碼RNA-lncRNA383及中國人群樣本中有效表達(dá)的融合基因USP9Y-TTTY15作為前列腺癌早期尿液診斷可行性的研究。 研究方法 本課題組前期應(yīng)用第二代基因轉(zhuǎn)錄本測序技術(shù)對(duì)14對(duì)來源于前列腺癌根治手術(shù)的腫瘤及癌旁正常組織樣本進(jìn)行測序，篩選在組織學(xué)層面明顯異常表達(dá)的非編碼RNA和融合基因，并擴(kuò)大組織學(xué)樣本含量，運(yùn)用qRT-PCR驗(yàn)證測序結(jié)果�；仡櫺赃x取腫瘤患者及穿刺陰性患者尿沉渣，并應(yīng)用qRT-PCR的方式探索上述差異性表達(dá)的非編碼RNA及融合基因是否具有診斷價(jià)值。前瞻性連續(xù)性入組因PSA異常計(jì)劃行前列腺穿刺活檢患者的尿沉渣，應(yīng)用根據(jù)測序結(jié)果入選的診斷指標(biāo)進(jìn)行前瞻性連續(xù)性驗(yàn)證，進(jìn)步明確其診斷能力，并應(yīng)用logistic回歸分析建立診斷模型，以期提高前列腺癌早期診斷敏感性及特異性。 結(jié)果 經(jīng)過前期14對(duì)前列腺腫瘤及癌旁正常組織的轉(zhuǎn)錄本測序，我們發(fā)現(xiàn)137個(gè)差異性表達(dá)的長鏈非編碼RNA,我們按照以下2個(gè)標(biāo)準(zhǔn)進(jìn)行篩選差異性表達(dá)最為明顯的目的基因進(jìn)行后續(xù)研究。標(biāo)準(zhǔn)：RNA-Seq結(jié)果中所有符合FDR≤0.001，且倍數(shù)差異≥2的差異性表達(dá)lncRNA；標(biāo)準(zhǔn)二：目的long non-coding RNA應(yīng)至少在10對(duì)以上的配對(duì)組織樣本中出現(xiàn)致的變化趨勢。最終，在篩選出的lncRNA中，我們發(fā)現(xiàn)FR0348383（lncRNA383）表達(dá)量在前列腺腫瘤組織中的表達(dá)量很高。在后續(xù)的組織學(xué)水平驗(yàn)證中，lncRNA383在腫瘤組織中的表達(dá)量明顯高于癌旁正常組織。在回顧性分析中我們發(fā)現(xiàn)，前列腺穿刺患者尿沉渣中均存在lncRNA383，且表達(dá)趨勢與組織學(xué)結(jié)果類似，具有良好的穿刺陽性結(jié)果預(yù)測能力（AUC=0.858，cut-off=0.877，敏感性=68.8%，特異性=97.8%，陽性預(yù)測值=98.19%，陰性預(yù)測值=64.17%）。在后續(xù)的前瞻性連續(xù)性研究中，雖然其診斷效能較回顧性分析的結(jié)果有所下降（AUC=0.631，cut-off=0.776，敏感性85.4%，特異性43.5%），但仍與尿液中PCA3的診斷效率相近，且敏感性優(yōu)于文獻(xiàn)報(bào)道的尿沉渣中TMPRSS2-ERG的診斷敏感性，為將來進(jìn)步建立多診斷指標(biāo)綜合應(yīng)用模型提供新的選擇。此外我們還發(fā)現(xiàn)，lncRNA383的表達(dá)水平與前列腺癌患者血清PSA，Gleason評(píng)分及DRE結(jié)果無明顯相關(guān)性，這在定程度上可能會(huì)限制lncRNA383在臨床上的應(yīng)用價(jià)值。 與此同時(shí)，我們還在前期的腫瘤測序樣本中發(fā)現(xiàn)了個(gè)已知的融合基因（TMPRSS2-ERG）和37個(gè)首次報(bào)道的僅在前列腺腫瘤組織中表達(dá)的腫瘤特異性融合基因，其中發(fā)生頻率最高的是TMPRSS2-ERG和USP9Y-TTTY15，均達(dá)到21.4%。USP9Y-TTTY15基因的融合發(fā)生在USP9Y基因的exon3和TTTY15基因的exon4上。在后續(xù)的54對(duì)癌及癌旁組織驗(yàn)證中，我們發(fā)現(xiàn)USP9Y-TTTY15基因融合的發(fā)生頻率高達(dá)35.2%。有趣的是，融合后的USP9Y-TTTY15不具有明顯的開放閱讀區(qū)，而是可能作為個(gè)非編碼RNA發(fā)揮生物學(xué)作用。在涵蓋63例前列腺癌患者及90例穿刺陰性（-）患者的回顧分析中，我們發(fā)現(xiàn)尿沉渣中均存在融合基因USP9Y-TTTY15的表達(dá)，且表達(dá)趨勢與組織學(xué)結(jié)果類似，并且具有良好的診斷能力（AUC=0.802，cut-off=0.894，敏感性=85.7%，特異性=73.3%，陽性預(yù)測值=69.20%，陰性預(yù)測值=87.98%）。在后續(xù)涵蓋172例患者的前瞻性連續(xù)性研究當(dāng)中，融合基因USP9Y-TTTY15繼續(xù)保持了良好的診斷學(xué)效能（AUC=0.783， cut-off=0.872，敏感性=66.7%，特異性=81.6%）。在PSA處于4-10ng/ml灰區(qū)內(nèi)，融合基因USP9Y-TTTY15具有良好的預(yù)測前列腺穿刺陽性結(jié)果的能力（AUC=0.774，cut-off值=0.845，敏感性=81.8%，特異性=62.2%，陽性預(yù)測值=35.59%，陰性預(yù)測值=93.32%），顯著的陰性預(yù)測值有利于提高PSA灰區(qū)內(nèi)前列腺穿刺的陽性率，具有很強(qiáng)的臨床轉(zhuǎn)化意義。此外，其還可以聯(lián)合PSA和fPSA形成多指標(biāo)綜合診斷模型Y=71.354（XUSP9Y-TTTY15）+0.213（XPSA）-1.754（XfPSA）-34.286，其診斷敏感性和特異性更分別高達(dá)66.7%和91.9%，明顯高于文獻(xiàn)記錄的PCA3與TMPRSS2-ERG聯(lián)合診斷效率，是種無創(chuàng)的，理想的早期前列腺癌診斷指標(biāo)，對(duì)于提高前列腺穿刺診斷陽性率具有積極的臨床意義。 結(jié)論 非編碼RNA-lncRNA383和融合基因USP9Y-TTT15Y均可以作為獨(dú)立的預(yù)測因子應(yīng)用于早期前列腺癌的組織學(xué)、尿沉渣的篩查與診斷中。USP9Y-TTT15Y還能有效預(yù)測PSA處于灰區(qū)內(nèi)的前列腺穿刺陽性結(jié)果。此外，USP9Y-TTT15Y還對(duì)于Gleason評(píng)分≤6的前列腺癌具有良好的預(yù)測效能，為臨床早期治療手段的選擇及治療后隨訪方案的制定提供了重要的診斷依據(jù)。在試圖聯(lián)合lncRNA383與融合基因USP9Y-TTT15Y及其他眾多診斷指標(biāo)的綜合診斷模型過程中，很可惜lncRNA383被剔除，入選指標(biāo)僅有USP9Y-TTT15Y、PSA和fPSA，最終形成多指標(biāo)綜合診斷模型Y=71.354（XUSP9Y-TTTY15）+0.213（XPSA）-1.754（XfPSA）-34.286，其診斷敏感性和特異性更是分別高達(dá)66.7%和91.9%，明顯高于文獻(xiàn)記錄的PCA3與TMPRSS2-ERG聯(lián)合診斷效率，是種無創(chuàng)的、理想的早期前列腺癌診斷指標(biāo)，對(duì)于提高前列腺穿刺診斷陽性率具有積極的臨床轉(zhuǎn)化意義。
[Abstract]:research objective
In recent years, the classic diagnosis and screening of prostate cancer, serological PSA, has been questioned. It is mainly due to the production and secretion of PSA in benign / normal prostate gland cells, which leads to the false positive problems of serological PSA diagnosis. A large number of studies have proved that the simple blood purity PSA index guides the early diagnosis of prostate cancer. The positive rate of PSA in the grey area was only 17-32%, the false positive rate of PSA was as high as 65%, and those with more than 2/3 received the unnecessary puncture [4,5].. However, the negative puncture results could not completely exclude the existence of the tumor, and the positive rate of repeated puncture was only about 10% [6]. accompanied by 2012. In February, FDA officially approved the first long chain non coded RNA diagnostic kit in urine - PROGENSA PCA3 for clinical [7,8]. The molecular diagnosis of prostate cancer entered the era of RNA noninvasive detection of urine sediment. It is reported that the diagnostic sensitivity of PCA3 in urine is 67%, the specificity 83%[9]. and the current TMPRSS2-ERG fusion gene can not only be in the urine. The slag was detected and fused only in the prostate tumor, and in the European and American population, the frequency was about 50%. Therefore, we urgently need to develop a new highly specific and sensitive molecular diagnostic marker to improve the positive diagnostic rate of "awkward" puncture. The present new non coded RNA-lncRNA383 and the fusion gene USP9Y-TTTY15, which are effectively expressed in the Chinese population samples, are used as a study of the feasibility of early urine diagnosis for prostate cancer.
research method
The second generation gene transcriptional sequencing technology was used to sequence the 14 pairs of tumor and normal tissue samples from radical prostatic cancer surgery in the earlier period, to screen out the uncoded RNA and fusion genes that were clearly expressed at the histological level, and to enlarge the content of histology samples, and to use qRT-PCR to verify the sequencing results. The diagnostic value of the non coded RNA and fusion gene of the above differential expression was explored by using qRT-PCR to explore the diagnostic value of the non coded RNA and fusion gene of the above differential expression. The prospective continuous entry group was planned for the urine sediment of the patients with prostate biopsy, and the diagnostic indexes selected according to the sequencing results were prospectively evaluated. In order to improve the sensitivity and specificity of the early diagnosis of prostate cancer, we developed a diagnostic model based on logistic regression analysis.
Result
After 14 pairs of prostatic tumors and the transcriptional transcripts of normal tissues adjacent to the cancer, we found 137 differentially expressed long chain noncoding RNA. We performed the following 2 criteria for screening the most distinct target genes for differential expression. Standard: all RNA-Seq results were consistent with FDR less than 0.001, and the multiple difference was more than 2. Differential expression of lncRNA; standard two: target long non-coding RNA should be at least in 10 pairs of paired tissue samples. Finally, in the screened lncRNA, we found that the expression of FR0348383 (lncRNA383) expression in the prostate tumor tissue is very high. In subsequent histological examination, lncRNA383 The expression in the tumor tissue was significantly higher than that in the normal paracancerous tissue. In the retrospective analysis, we found that lncRNA383 was present in the urine sediment of the patients with prostatic puncture, and the expression trend was similar to that of the histology. It had good prediction ability of the positive results of puncture (AUC=0.858, cut-off=0.877, sensitivity =68.8%, specific =97.8%, positive preconditioning. =98.19%, negative predictive value =64.17%). In subsequent prospective continuity studies, although their diagnostic effectiveness was lower than that of retrospective analysis (AUC=0.631, cut-off=0.776, sensitivity 85.4%, specificity 43.5%), the diagnostic efficiency of PCA3 was similar to that in urine, and the sensitivity was better than that of TMPRSS2-ERG in the reported urine sediment. Diagnostic sensitivity can provide a new choice for future progress in the establishment of multiple diagnostic indicators. In addition, we also found that there is no significant correlation between the expression level of lncRNA383 and the serum PSA, Gleason score and DRE results of the prostate cancer patients, which may limit the clinical application of lncRNA383 to a certain extent.
At the same time, we found a known fusion gene (TMPRSS2-ERG) and 37 first reported tumor specific fusion genes expressed in the prostate tumor tissue for the first time, with the highest frequency of TMPRSS2-ERG and USP9Y-TTTY15, which all reached the fusion of 21.4%.USP9Y-TTTY15 gene. USP9Y gene exon3 and TTTY15 gene Exon4. In the subsequent 54 pairs of cancer and para cancerous tissue validation, we found that the frequency of USP9Y-TTTY15 gene fusion is as high as 35.2%., and the fusion USP9Y-TTTY15 does not have a clear open reading area, but may play a biological role as a non coded RNA. It covers 63 cases before it is covered. In the retrospective analysis of patients with adenocarcinoma and 90 cases of puncture negative (-), we found that the expression of fusion gene USP9Y-TTTY15 was found in urine sediment, and the expression trend was similar to that of histology, and had good diagnostic ability (AUC=0.802, cut-off=0.894, sensitivity = 85.7%, specific =73.3%, positive predictive value =69.20%, negative predictive value = = 87.98%. The fusion gene USP9Y-TTTY15 continued to maintain a good diagnostic efficiency (AUC=0.783, cut-off=0.872, sensitivity =66.7%, specific =81.6%) in a prospective continuous study covering 172 patients. The fusion gene USP9Y-TTTY15 had a good prediction of prostatic positive results in the 4-10ng/ml grey area of PSA. Ability (AUC=0.774, cut-off value =0.845, sensitivity =81.8%, specific =62.2%, positive predictive value =35.59%, negative predictive value =93.32%), significant negative predictive value is beneficial to improve the positive rate of prostate puncture in PSA gray area, and has a strong clinical significance. Furthermore, it can also combine PSA and fPSA to form a multi index comprehensive diagnostic model Y=71.. The diagnostic sensitivity and specificity of 354 (XUSP9Y-TTTY15) +0.213 (XPSA) -1.754 (XfPSA) -34.286 are up to 66.7% and 91.9% respectively. It is obviously higher than the combined diagnostic efficiency of PCA3 and TMPRSS2-ERG recorded in the literature. It is a noninvasive and ideal diagnostic index for early prostate cancer. It has positive clinical significance in improving the positive rate of prostate biopsy.
conclusion
Both non coded RNA-lncRNA383 and fusion gene USP9Y-TTT15Y can be used as independent predictors for early histology of prostate cancer. In the screening and diagnosis of urine sediment,.USP9Y-TTT15Y can also effectively predict the positive results of prostate biopsy in the gray area of PSA. In addition, USP9Y-TTT15Y is also used for prostate cancer with a Gleason score of less than 6. Good predictive effectiveness provides an important diagnostic basis for the selection of early clinical treatments and the formulation of follow-up follow-up programs. In the process of combining lncRNA383 with fusion gene USP9Y-TTT15Y and many other diagnostic indicators, it is a pity that lncRNA383 is eliminated, and only USP9Y-TTT15Y, PSA, and PSA are selected. FPSA, finally formed a multi index comprehensive diagnostic model, Y=71.354 (XUSP9Y-TTTY15) +0.213 (XPSA) -1.754 (XfPSA) -34.286, and its diagnostic sensitivity and specificity are up to 66.7% and 91.9% respectively. It is significantly higher than the combined diagnostic efficiency of PCA3 and TMPRSS2-ERG recorded in the literature. It is a noninvasive, ideal early diagnostic index for prostate cancer. The positive rate of diagnosis by row biopsy is of positive clinical significance.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.25

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