【摘要】：研究背景:混合型肝細(xì)胞癌一膽管細(xì)胞癌(CHC)是一種罕見的原發(fā)性肝癌,其同時(shí)具有肝細(xì)胞肝癌(HCC)和肝內(nèi)膽管細(xì)胞癌(iCCA)的臨床病理特點(diǎn)。然而,關(guān)于獨(dú)立腫瘤中同時(shí)存在肝細(xì)胞肝癌和膽管細(xì)胞癌成份的分子機(jī)制尚未清楚,因此,我們?cè)噲D研究混合型肝細(xì)胞癌—膽管細(xì)胞癌中兩種腫瘤成份之間的關(guān)系。實(shí)驗(yàn)方法:我們的研究共納入了 75例肝癌患者,其中包括15例混合型肝細(xì)胞癌—膽管細(xì)胞癌,32例肝細(xì)胞肝癌和28例肝內(nèi)膽管細(xì)胞癌,我們對(duì)所有的患者進(jìn)行了免疫組化,包括肝細(xì)胞樣標(biāo)志物(Hep和GPC3),膽管細(xì)胞樣標(biāo)志物(CK7和CK19)。其中,我們選擇了7例肝混合細(xì)胞癌樣本分別進(jìn)行肝細(xì)胞肝癌,膽管細(xì)胞癌成份及正常肝組織的顯微切割,然后提取DNA進(jìn)行全外顯子測(cè)序。我們對(duì)測(cè)序結(jié)果進(jìn)行了錯(cuò)義突變,拷貝數(shù)變異,驅(qū)動(dòng)基因,高頻突變基因,易感基因及乙肝病毒整合的分析,進(jìn)行了克隆分析以進(jìn)一步明確兩種腫瘤成份之間的關(guān)系。為了進(jìn)一步研究混合型肝細(xì)胞癌—膽管細(xì)胞癌的細(xì)胞起源,我們對(duì)所有樣本進(jìn)行了免疫組化,標(biāo)志物為(EpCAM和c-kit),然后我們根據(jù)EpCAM的表達(dá)情況進(jìn)行了相關(guān)生存分析。實(shí)驗(yàn)結(jié)果:我們通過形態(tài)學(xué)及免疫組化進(jìn)一步確認(rèn)了混合型肝細(xì)胞癌—膽管細(xì)胞癌的診斷,根據(jù)DNA降解程度最終入選了 7例CHC患者進(jìn)行了外顯子測(cè)序。對(duì)于全外顯子測(cè)序結(jié)果,我們將肝細(xì)胞肝癌和膽管細(xì)胞癌成份共有的體細(xì)胞突變定義為共有突變,而各自獨(dú)有的突變則定義為獨(dú)有突變。我們發(fā)現(xiàn)CHC樣本中存在大量獨(dú)有的體細(xì)胞單核苷酸變異(SNV)(33.1%-86.4%)及獨(dú)有拷貝數(shù)變異(79.3%-97.3),這些結(jié)果表明CHC具有明顯的腫瘤異質(zhì)性。然而,在CHC的肝細(xì)胞肝癌和膽管細(xì)胞癌中存在大量共有錯(cuò)義SNV及體細(xì)胞拷貝數(shù)變異,同樣兩種成份之間也發(fā)現(xiàn)了大量共同的錯(cuò)義突變(包括SNV,插入缺失),這些表明肝混合細(xì)胞癌中兩種不同腫瘤成份為同一起源,克隆分析也進(jìn)一步支持了該結(jié)論。我們發(fā)現(xiàn)一些突變的基因,如VCAN,ACVR2A及FCGBP等與干細(xì)胞狀態(tài)的維持及分化相關(guān),參與了干細(xì)胞多能性調(diào)節(jié)通路及WNT,Notch通路,這些突變基因可能導(dǎo)致了混合癌中肝細(xì)胞肝癌和膽管細(xì)胞癌表型的分化。再者,不同的肝癌類型EpCAM表達(dá)情況差異明顯,其中肝混合細(xì)胞癌80%的陽性表達(dá),CK19(+)及CK19(-)的肝細(xì)胞肝癌陽性表達(dá)分別為66.7%和17.2%,肝內(nèi)膽管細(xì)胞癌為71.4%。生存分析表明EpCAM與肝癌患者的預(yù)后呈負(fù)相關(guān)關(guān)系。實(shí)驗(yàn)結(jié)論:全外顯子測(cè)序分析表明混合型肝細(xì)胞癌—膽管細(xì)胞癌為單克隆起源,其可促進(jìn)原發(fā)性肝癌基于腫瘤細(xì)胞起源的分子分型。再者,混合型肝細(xì)胞癌—膽管細(xì)胞癌明顯的腫瘤內(nèi)部異質(zhì)性鼓勵(lì)我們進(jìn)行多區(qū)域取樣測(cè)序從而發(fā)現(xiàn)腫瘤發(fā)生過程中共有的驅(qū)動(dòng)突變基因,從而讓靶向藥物的篩選變得更加精準(zhǔn)而高效。
[Abstract]:Background: mixed hepatocellular carcinoma-bile duct carcinoma (CHC) is a rare primary liver cancer, which has the clinicopathological characteristics of both hepatocellular carcinoma (HCC) and intrahepatic bile duct carcinoma (iCCA). However, the molecular mechanism of the simultaneous presence of HCC and BCC in independent tumors is not clear. Therefore, we try to study the relationship between the two tumor components in mixed HCC and BCC. Methods: our study included 75 patients with liver cancer, including 15 cases of mixed hepatocellular carcinoma (HCC), 32 cases of hepatocellular carcinoma (HCC) and 28 cases of intrahepatic bile duct cell carcinoma (HCC). We studied all the patients with liver cancer by immuno-histochemical method, including 15 cases of mixed hepatocellular carcinoma (HCC), 32 cases of hepatocellular carcinoma (HCC) and 28 cases of intrahepatic bile duct carcinoma (HCC). Including hepatocyte-like markers (Hep and GPC3), bile duct cell-like markers (CK7 and CK19). Among them, 7 cases of mixed cell carcinoma were selected for microcutting of hepatocellular carcinoma (HCC), bile duct carcinoma (BCC) and normal liver tissue, and then DNA was extracted for sequencing of the whole exons. The results of sequencing were analyzed by missense mutation, copy number variation, driving gene, high frequency mutant gene, susceptible gene and hepatitis B virus integration, and the relationship between the two tumor components was further clarified. In order to further study the cellular origin of mixed hepatocellular carcinoma (HCC), we carried out immunohistochemistry with the markers (EpCAM and c-kit) in all samples, and then we analyzed the survival of mixed hepatocellular carcinoma (HCC) according to the expression of BCC. The results showed that the diagnosis of mixed hepatocellular carcinoma (HCC) and bile duct carcinoma (BCC) was further confirmed by morphology and immunohistochemistry. Seven patients with CHC were sequenced according to the degree of DNA degradation. For the sequencing results of total exons, we define the somatic mutations common to HCC and BCC as common mutations, while the unique mutations are defined as unique mutations. We found that there were a large number of unique somatic single nucleotides variation (SNV) (33.1% 鈮，

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