肝癌免疫组化诊断谱的研究和应用

目的：探讨鉴别肝细胞癌（HCC）、肝内胆管癌（ICC）和肝转移性腺癌（MAC）的免疫组化诊断谱特点。方法：对手术切除的300例HCC、35例ICC和30例MAC分别进行AFP、Hep Pau 1、CK18、CK19、CA19－9、CD34和pCEA等7种免疫组化染色,将特异性和敏感性的综合性能计分（CCS）≥8分的抗体定为具有高度诊断价值。结果：CCS≥8分的抗体在HCC中为Hep Par 1和CD34,在ICC中为CK19,在MAC中无。Hep Par 1的CCS（9分）明显高于AFP（7分）,其对HCC的敏感性达到83.7%,特异性达到96．7％。结论：HCC的一线抗体由Hep Par 1和CD34组成,二线抗体由pCEA和AFP组成：ICC的一线抗体为CK19,二线抗体为CA19－9。由3种一线抗体组合成肝癌的核心免疫组化谱,酌情使用二线抗体,可以较好地解决对HCC、ICC和MAC之间的免疫病理诊断和鉴别诊断。     

肝肿瘤鉴别诊断中免疫组化应用的研究进展

论述肝细胞癌（HCC）器官特异性抗体HepPar-1、甲胎蛋白（AFP）,HCC相对特异性抗体多克隆癌胚抗原（pCEA）、氨肽酶N（CD13）、CD10（neprilysin）、绒毛蛋白、CD34,肝内胆管细胞癌（ICC）与转移性腺癌（MAC）相对特异性抗体细胞角蛋白（CK）7、CK19、CK20、MOC31、甲状腺转录因子-1（TTF-1）等单独及联合应用在肝肿瘤鉴别诊断中作用的研究进展。     

Combined hepatocellular andcholangiocarcinoma originating fromthe same clone：a pathomolecular evidence-based study

Background:Combined hepatocellular and cholangiocarcinoma (CHC) is a unique subtype of liver cancer com?prising both hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC); however, its cellular origin remains unclear. The purpose of this study was to investigate the clinicopathologic features and the clonal relation?ship between HCC and ICC in 34 patients with CHC. Methods:The clinicopathologic features and prognosis of the 34 CHC patients were compared with those of 29 patients with separated HCC and ICC (SHC). Loss of heterozygosity (LOH) at 10 highly polymorphic microsatellite markers was detected in 16 CHC and 10 SHC tissues for determination of the clonal origin of CHC. Expression of hepatocyte markers [hepatocyte paraffn 1 (Hep Par 1) and glypican 3 (GPC3)] and cholangiocyte markers [cytokeratin (CK)7 and 19] in tumor tissues was examined by immuno histochemical analysis. Results:In the 16 CHC specimens, the difference in LOH patterns between HCC and ICC was less than 30%, suggest?ing the same clonal origin of HCC and ICC. Consistent with this ifnding, immunohistochemical analysis revealed that hepatocyte markers (Hep Par 1 and GPC3) and cholangiocyte markers (CK7 and CK19) were simultaneously expressed in both the HCC and ICC components in 52.9% of CHC specimens, suggesting that the two components shared a similar phenotype with hepatic progenitor cells (HPCs). On the contrary, in all 10 SHC cases, the difference in LOH patterns between the HCC and ICC components was greater than 30%, suggesting different clonal origins of HCC and ICC. Overall survival and disease?free survival were shorter for patients with CHC than for patients with SHC (P<0.05). Conclusions:Our results suggest that the HCC and ICC components of CHC may originate from the same clone, hav?ing the potential for dual?directional differentiation similar to HPCs. CHC tended to exhibit the biological behaviors of both HCC and ICC, which may enhance the inifltrative capacity of tumor cells, leading to poor clinical outcomes for patients with CHC.     

非小细胞肺癌患者外周血中CK19 mRNA、MUC-1 mRNA及LUNX mRNA的表达及意义

目的 应用巢式逆转录聚合酶链反应(Nested RT-PCR)技术检测非小细胞肺癌患者外周血中CK19 Mrna、MUC-1 Mrna及LUNX Mrna的表达,探讨其作为肺癌微转移检测分子标记物的可能性.方法 应用巢式RT-PCR技术检测60例非小细胞肺癌患者外周血中靶基因的表达,并以15例肺良性疾病及20例健康人外周血作为对照.结果 CK19 Mrna、MUC-1 Mrna及LUNX Mr-NA表达的阳性率为:51.7%(31/60)、80.0%(48/60)和55.0%(33/60).对照组中15例肺良性疾病及20例健康人外周血中无LUNX Mrna表达,20例健康人外周血中有1例CK19 Mrna表达,而MUC-1 Mrna表达的阳性率在肺良性疾病中为60.0%(9/15),在健康人外周血中为65.0%(13/20).结论 CK19 Mrna、LUNX Mrna作为检测非小细胞肺癌患者外周血微转移的分子标记物,具有良好的敏感性和特异性.而MUC-1 Mrna作为诊断非小细胞肺癌微转移的指标还需进一步探讨.     

GPC3、Hep-Par-1在肝细胞癌中的表达及其价值

目的探讨磷脂酰肌醇蛋白聚糖3(GPC3)、Hep-Par-1在原发性肝细胞癌(HCC)诊断与鉴别诊断中的应用价值。方法应用免疫组化EliVision法,对已确诊的64例HCC、12例肝内胆管细胞癌(ICC)、11例肝转移性腺癌(MAC)、6例局灶结节性增生(FNH)、2例肝细胞腺瘤(HA)及28例癌旁肝组织进行GPC3、Hep-Par-1抗体标记,检测2种抗体在上述组织中的表达情况。结果 GPC3在HCC中表达率为91%(58/64),其中75%(48/64)为强阳性(+++),在其他组织中无表达,表达差异具有统计学意义(P<0.05)。Hep-Par-1在HCC中表达率为81%(52/64),在FNH、HA及癌旁肝组织中表达率均为100%,在MAC中表达率为9%(1/11),在其余组织中无表达,表达差异具有统计学意义(P<0.05)。结论 GPC3和Hep-Par-1对HCC的诊断均具有较高的敏感性及特异性,GPC3可取代Hep-Par-1成为HCC诊断的一线抗体。     

FN和CD44v6在肝胞癌中的表达及临床意义

目的:观察纤维连接蛋白(Fibronectin,FN)和CD44v6在肝细胞癌(Hepatocellular carcinoma,HCC)组织中的表达,探讨其与HCC侵袭转移的关系.方法:采用FN、CD44v6抗体,对39例HCC标本和10例正常肝组织进行SP免疫组化染色,对结果进行统计分析.结果:HCC组织中FN表达的阳性率为35.8%,而在正常肝细胞中全部表达,两者差异明显(P＜0.05).FN分布特征还与HCC分化程度及生长方式相关.CD44v6在HCC中表达的阳性率为69.2%,在正常肝组织中全部未表达,两者差异显著(P＜0.05).CD44v6的阳性表达率和表达程度与HCC的包膜、分化程度、临床分期和有否转移也有密切关系.结论:肝癌细胞FN表达的减少或消失和CD44v6的高表达与HCC的侵袭转移密切相关,两者在侵袭转移中的作用相反.FN和CD44v6可以作为HCC侵袭转移的观察指标.     

肝细胞癌中Hep Par 1、p53、HBsAg表达差异与细胞形态计量学变化的关系

目的 探讨原发性肝癌组织、癌旁组织和正常肝组织中肝细胞抗原（Hep Par1)，p53，乙型肝炎表面抗原（HBsAg)的表达差异及与细胞形态学变化的相关性。方法 用免疫组织化学方法（两步法－EnVision System-HRP)检测10对肝癌和癌旁组织，3例正常肝组织中Hep Par1,p53,HBsAg等蛋白的表达差异；用HPIAS－1000图像分析系统测量实质细胞的总面积（以细胞直径代替）和核质比。结果 1）Hep Par 1:正常肝组织中肝细胞中度表达，癌旁肝组织中肝细胞为弥漫性高表达，肝癌细胞中阳性率为24．53％（13／53）；2）p53：正常肝组织和癌旁肝组织中均呈阴性；肝癌组织，在被检的10例样品中肝癌细胞均为阳性；3）HBsAg:正常肝组织中肝细胞呈阴性，癌旁肝组织中肝细胞为片状弥漫性或散在性高表达，肝癌组织，在被检的10例样品中肝癌细胞仅有1例呈阳性；4）各种组织中实质细胞的平均直径为癌旁肝细胞＞正常肝细胞＞肝细胞癌细胞；5）正常肝细胞和癌旁肝细胞的核质比无差别（P＞0．05），但均明显＜肝细胞癌细胞（P＜0．01和P＜0．01）。结论 肝细胞抗原（Hep Par 1)在分化较高的肝细胞癌细胞中有不同程度的表达，可作为鉴别原发性肝细胞癌细胞来源的标志；原发性肝细胞癌细胞核质比明显＞正常肝细胞和癌旁肝细胞（P＜0.01和P＜0.01)。     

VEGF、COX-2在HBV感染型和非HBV感染型肝细胞癌中的表达差异及意义

目的:探讨血管内皮生长因子(VEGF)、环氧合酶-2(COX-2)蛋白表达水平和肿瘤血管形成在肝细胞癌(HCC)中表达的临床病理意义.方法:利用血清学指标检测56例HCC的乙型肝炎病毒(HBV)感染情况,采用快速免疫组化法检测肝细胞癌中VEGF、COX-2的蛋白表达,抗CD105单克隆抗体显示血管内皮细胞,根据CD105阳性的血管内皮细胞计数测定肿瘤微血管密度(MVD).结果:HBV感染组中VEGF、COX-2蛋白以及MVD的阳性表达率均高于非HBV感染组,有统计学差异(P＜0.05).VEGF和COX-2表达呈正相关(r=0.429,P＜0.05).结论:HBV可能通过上调VEGF、COX-2等血管形成因子上调表达,共同促进了肿瘤血管的生成,从而促进HCC的生长、浸润和转移.     

双表型肝细胞癌新亚型的临床病理学研究进展

双表型肝细胞癌（dual-phenotype hepatocellular carcinoma,DPHCC）是近年报道的肝细胞癌（hepatocellular carcinoma,HCC）的一种新亚型,以组织学上为典型的肝细胞癌但同时表达肝细胞癌和肝内胆管癌（intrahepatic cholangiocarcinoma,ICC）的基因标志物为特征,因其具有HCC和ICC的双重生物学行为,恶性程度更高,临床预后差,因而正确诊断十分重要。CK19是DPHCC诊断的重要标志物之一,可以通过多种途径参与DPHCC的发生和恶性生物学行为的调控。本文拟从DPHCC的临床病理特点、免疫组织化学标记、组织起源发生等方面对DPHCC的研究进展进行综述。      

HBME-1、 CD56、 CK19、 Gal-3及34βE12在PTC中的表达及诊断价值

目的 探讨Hector Battifora mesthelial cell-1(HBME-1)、CD56、galeetin-3(Gal-3)、cytokeratin19(CK19)及34βE12在甲状腺乳头状癌(papillary thyroid carcinoma,PTC)中的表达及鉴别诊断中的价值.方法 收集75例PTC、85例甲状腺良性乳头状病变及35例恶性潜能未定的甲状腺肿瘤(thyroid tumors of uncertain malignant poten-tial,TTs-UMP)患者的资料,免疫组织化学法检测HBME-1、CD56、Gal-3、CK19及34βE12的表达.结果 5种蛋白中,34βE12的敏感度最高,为88.0％(66/75),CD56敏感度次之,为81.3％ (61/75).CK19、Gal-3的敏感度最低,为46.7％ (35/75),其特异性最高,均为100.0％ (85/85).联合检测中,CD56和(或)34βE12的敏感度最高,为96.0％ (72/75),34βE12和(或)CK19/Gal-3的特异性最高,为98.8％(84/85).TTs-UMP的免疫组织化学表达类似于甲状腺良性病变.结论 PTC的诊断中,结合形态学特点及HBME-1、CD56、Gal-3、CK19及34βE12的检测,可以有效提高诊断率.     

Immunohistological evaluation of single small hepatocellular carcinoma with negative staining of monoclonal antibody Hepatocyte Paraffin 1

BACKGROUND AND OBJECTIVES: The sensitivity and specificity of the monoclonal antibody Hepatocyte Paraffin 1 (Hep Par 1) for hepatocellular carcinoma (HCC) are very high, and the usefulness for differential diagnosis of hepatic tumors has been reported. However, there are some cases of HCC with negative staining for Hep Par 1. We examined the histopathological features of HCC with negative staining for Hep Par 1. METHODS: We examined 69 samples of single nodular HCC less than 2 cm in greatest dimension, resected from 1985 to 1994 in our hospital, with immunohistological staining for Hep Par 1, cytokeratin 19 (CK 19), MUC-1 glycoprotein (MUC-1), and epithelial membrane antigen (EMA). RESULTS: Hep Par 1 staining was positive in 64 cases (93%) and negative in 5 cases (7%). With regard to the histological structure, 3 of the 5 negative cases were scirrhous HCC. With regard to the grade of histological differentiation, 2 cases were poorly differentiated HCC, 3 cases were moderately differentiated HCC, and no well-differentiated HCC was found in the negative cases. CK 19, MUC-1, and EMA staining were negative in all cases. CONCLUSIONS: It is necessary to recognize the existence of Hep Par 1 negative HCC, in particular scirrhous HCC. This may be due to a different mechanism in the earlier stage of hepatocarcinogenesis.     

Cyclooxygenase-2 pathway correlates with vascular endothelial growth factor expression and tumor angiogenesis in hepatitis B virus-associated hepatocellular carcinoma

Evidence indicates that cyclooxygenase (COX)-2-derived prostaglandins (PGs) contribute to tumor growth by inducing angiogenesis. We investigated the role of COX-2 in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC). COX-2 and vascular endothelial growth factor (VEGF) expressions were examined by immunohistochemistry in 24 HBV-associated HCC. Tumor micro-vessel density (MVD) was assessed using CD34 immunohistochemistry. Hep3B HCC cell line, which carries integrated HBV genome, was stably transfected with human COX-2 cDNA. COX-2 and VEGF expressions were determined by Western blot while PG level was determined by ELISA. The effects of PGs on VEGF expression were also investigated. Expression of COX-2 and VEGF in HCC cells were observed in 19 (79%) and 16 (67%) cases, respectively. Well-differentiated HCC expressed COX-2 more strongly than less-differentiated HCC (p<0.001). COX-2 expression was found to correlate with VEGF expression and MVD (p=0.003 and 0.004, respectively). COX-2 overexpressing Hep3B clone had higher VEGF expression as compared to non-COX-2 expressing clone and parental cells. Treatment of the COX-2 overexpressing cells with a COX-2-selective inhibitor, NS-398 (10 microM), decreased PGE2 level and attenuated VEGF expression. Addition of PGE2 (10 microM) and the stable analog of PGI2, carbaprostacyclin (5 microM), to Hep3B cells also increased VEGF expression. Up-regulation of COX-2 correlates with VEGF expression and tumor angiogenesis in HBV-associated HCC. Moreover, COX-2 up-regulates VEGF expression in HCC cells, possibly via PGs production. Selective inhibition of COX-2 may block HCC associated angiogenesis and thus provides a rational approach for treatment of this malignancy.     

KL-6 mucin is a useful immunohistochemical marker for cholangiocarcinoma

This study aimed to clarify the clinical significance of the expression of KL-6 mucin, a type of MUC1, in primary liver cancer. Tissue specimens were collected from 21 patients with cholangiocarcinoma (CC), 78 with hepatocellular carcinoma (HCC), and 12 with combined hepatocellular and cholangiocarcinoma (cHCC-CC). Immunohistochemical analysis was done using a monoclonal antibody for KL-6 mucin as well as antibodies for Hep1 or CK7. KL-6 staining was positive in all the CC tissues examined, while it was not positive in any of the HCC tissues. Similar selectivity of KL-6 staining was also observed in the cHCC-CC specimens, and the cholangiocellular tissue could be clearly delineated by KL-6 staining. In contrast, 79.5% of HCC specimens and 25.0% of cHCC-CC specimens were positive for Hep1 in the hepatocellular tissues, while none of the CC or cHCC-CC specimens were positive in the cholangiocellular tissues. Staining for CK7 was positive in 95.2% of CC and 35.9% of HCC specimens, while 58.3 and 25.0% of cHCC-CC specimens displayed positivity for CK7 in the cholangiocellular and hepatocellular tissues, respectively. These results suggest that KL-6 may be a useful tumor marker for distinguishing CC from HCC. In addition, the high selectivity of KL-6 for cholangiocallular tissue may help to provide information for deciding the clinical strategy in cHCC-CC patients.     

The significance of immunohistochemistry in the investigation of liver neoplasms: differential diagnosis, prognostic markers

The immunohistochemical investigation used 55 primary hepatic tumors (hepatocellular carcinoma (HCC)--32, cholangiocellular carcinoma (CCC)--23). Wide panels of such antibodies as hepatocytic marker (Hep Par--1) CK-8, CK-19, polyclonal CEA, CD10, alpha-fetoprotein, TTF-1 as well as proliferative features of HCC (Ki-67) including regulators of stage-to-stage transition through mitoses of tumor cells (cyclin-D1 and A, genes p53 and RB), unrestricted tumor cell mitosis (telomerases), and intercellular adhesion marker (beta-catenin) were employed for differential diagnosis of neoplasia. The most efficient marker HCC was Hep Par--l (sensitivity--100%, specificity--92%) while the sensitivity of CCC (CK-19) was 83% and specificity--78%. Of particular importance for differentiation between HCC and CCC were the nature of microcirculatory flow identifiable with the aid of CD31 and presence of pseudocapsule in HCC detected by means of calponin. CEA and CD10 played a part too while the remaining markers were either expressed very seldom (alpha-fetoprotein) or absent (TTF-1). Most nuclear antigens (Ki-67, cyclin-A, p53 and RB) were intensely expressed in poorly-differentiated HCC cells. Cyclin-D1 and mutated suppressor-gene p53 expression involved lowered overall and relapse-free survival.     

Lack of influence of cyclooxygenese-2 expression in hepatocellular carcinomas on patient survival

Recent studies suggest that cyclooxygenese-2 (COX-2) enzyme activation may play a role inhepatocarcinogenesis. However, the clinical significance of COX-2 expression in hepatocellular carcinoma (HCC)remains obscure. This study evaluated COX-2 expression in hepatitis B and hepatitis C virus related HCC andin HCC patients with an unknown etiology. Liver tissue samples of 31 patients with HCC (27 men and 4 women;age range, 48-75 years) were analyzed. COX-2 expression was evaluated by immunohistochemically in thetumor tissues. Patient data including age, sex, Child score, stage, grade of the tumor and survival were analyzed.Of these patients 19 were positive for hepatitis B virus (HBV), 6 were positive for hepatitis C virus (HCV) and 6patients were negative for all viral markers and other etiologic factors. COX-2 staining were evaluated in 2groups (group 1: COX-2 expression less than 25% (grades 1-2 COX-2 expression), and group 2: Cox-2 expression25% or more (grades 3-5 COX-2 expression). COX-2 expression was shown in all HCC samples with positive ornegative viral markers. No difference was found between degree of COX-2 expression and the etiology of HCC.COX-2 expression was not correlated with number of lesion or stage of the disease or grade of the tumor. COX-2 expression was not related with Child score of the patients. Median survival of all patients was 32 months.Median survival of patients did not differ according to patient’s viral marker status. No difference was observedin median survival of patients in group 1 and 2. As a result, COX-2 system seem to be shared part inhepatocarcinogenesis regardless factors that initiate the disease. Although COX-2 expression appears to beindependent of disease’s characteristics’, treatments that target this system appear to be feasible in themanagement of HCC.