荧光原位杂交技术在泌尿生殖肿瘤中的应用

随着分子细胞遗传学的快速发展,荧光原位杂交（fluorescence in situ hybridization,FISH）技术已广泛应用于临床和科学研究。FISH技术综合了荧光信号的高度灵敏性、无放射性、直观性及原位杂交的准确性,通过检测样本中基因或染色体的异常改变而应用于遗传性疾病、肿瘤的研究及临床诊断和治疗监测。本文就荧光原位杂交技术在常见的泌尿生殖肿瘤中的应用进行综述。     

以浆膜腔积液上清为标本检测ras基因第12密码子突变及其意义

有研究显示,体液中的游离核酸可直接用于肿瘤的分子诊断,且效果优于以体液中的脱落细胞为实验材料[1].近年来随着肿瘤发病率的上升,恶性浆膜腔积液的比例不断升高,但部分恶性积液的诊断一直困扰人们.ras基因第12密码子突变广泛存在于多种恶性肿瘤,是研究肿瘤基因诊断较为理想的"通用"靶基因之一.本研究以浆膜腔积液上清为标本对K-ras和H-ras基因第12密码子突变进行聚合酶链反应扩增及限制性片段长度多态性(PCRRFLP)分析,探讨它们对恶性积液的诊断价值.     

肺癌胸水细胞块与组织学EGFR基因扩增检测对比研究

目的：对比肺癌胸水细胞块与组织学中EGFR基因扩增情况,探讨非小细胞肺癌患者阳性胸水细胞块检测EGFR基因扩增情况及临床意义。方法：收集60例非小细胞肺癌患者阳性胸水离心获取肿瘤细胞制作成石蜡细胞块,其中有28例的组织学对照,免疫组化分型后进行FISH检测EGFR基因,荧光显微镜观察基因扩增情况。结果：28例细胞块有组织学EGFR基因检测对照,在有组织学对照细胞块EGFR基因检测相符率100％。肺腺癌31例中,EGFR基因簇状扩增4例（12．9％）,点状扩增14例（45．2％）,无扩增13例（41．9％）,肺腺癌扩增率为58．1％;肺鳞癌25例中,EGFR基因簇状扩增2例（8．0％）,点状扩增9例（36．0％）,无扩增14例（56．0％）,肺鳞癌扩增率为44．0％;其他类型NSCLC患者4例中2例点状扩增,扩增率为50％。结论：肺腺癌及肺鳞癌胸水细胞块EGFR检测结果与组织学一致,EGFR基因在肺腺癌的扩增率高于肺鳞癌。应用FISH技术检测胸水细胞块中的EGFR基因扩增情况具有临床应用价值。     

恶性心包积液细胞块病理检查的意义（附30例分析）

目的：通过心包积液中肿瘤细胞制作的细胞块进行疾病诊断和肿瘤来源的鉴别诊断,评价心包积液对临床诊断的价值。方法：30例恶性肿瘤并发恶性心包积液患者,心包穿刺引流积液送病理制作细胞块进行肿瘤来源的诊断,验证临床疾病的诊断。结果：30例恶性心包积液细胞块鉴别诊断病因中,肺癌21例,其中腺癌17例,鳞癌3例,小细胞未分化癌1例;乳腺癌4例;食管癌1例;结肠癌2例;红细胞太多,无法制作细胞块2例。结论：恶性心包积液细胞块留取对疾病的诊断有重要临床意义,对晚期肿瘤转移性心包积液确定原发灶提供病理诊断和临床肿瘤的治疗有积极的作用。     

恶性心包积液细胞块病理检查的意义(附30例分析)

目的:通过心包积液中肿瘤细胞制作的细胞块进行疾病诊断和肿瘤来源的鉴别诊断,评价心包积液对临床诊断的价值。方法:30例恶性肿瘤并发恶性心包积液患者,心包穿刺引流积液送病理制作细胞块进行肿瘤来源的诊断,验证临床疾病的诊断。结果:30例恶性心包积液细胞块鉴别诊断病因中,肺癌21例,其中腺癌17例,鳞癌3例,小细胞未分化癌1例;乳腺癌4例;食管癌1例;结肠癌2例;红细胞太多,无法制作细胞块2例。结论:恶性心包积液细胞块留取对疾病的诊断有重要临床意义,对晚期肿瘤转移性心包积液确定原发灶提供病理诊断和临床肿瘤的治疗有积极的作用。     

子宫炎症性肌纤维母细胞瘤的临床病理学特征及分子病理学研究

目的∶ 探讨子宫炎症性肌纤维母细胞瘤（inflammatory myofibroblastic tumor，IMT）的临床病理及分子病理学特征。方法;收集 5例子宫 IMT，回顾性分析其临床资料，观察组织形态学改变，进行免疫组化染色和4K基因荧光原位杂交（fluorescence in situ hybridization，FISH）检测，并采用二代测序（nex对t-generation sequencing，NGS）方法检测包括AIK 在内的507个癌基因点突变、插入、缺失、基因融合情况。结果∶该组病例发病年龄为 32～47岁，平均（39.60±5.12）岁，3 例肿瘤位于子宫体肌壁间，2 例位于子宫内膜下。显微镜下见梭形细胞肿瘤，富于黏液基质或者局部间质黏液样变。初始诊断 3 例为平滑肌肿瘤，2 例为内膜间质肉瘤。5 例免疫组织化学染色均为ALK 阳性，ER，PR。 Desmin 阳性表达;4 例患者的 SMA 免疫组化染色呈阳性表达，1 例不表达;3 例 CD10 染色呈阳性表达，2 例不表达;5例患者均不表达 CD117 和 CK;Ki-67的增殖指数除一例为40% 外，其余均较低。AIK 分离探针 FISH 检测显示 4 例阳性，1 例阴性，后者经 NGS 检测证实存在 ALK 基因的融合。结论∶ALK 阳性是子宫 IMT 的一种高度特异的免疫组化标记，子宫 IMT 的免疫组化和 RNA 测序具有较高的一致性，可借此将 IMT 与子宫梭形细胞肿瘤鉴别。     

子宫炎症性肌纤维母细胞瘤的临床病理学特征及分子病理学研究

目的∶ 探讨子宫炎症性肌纤维母细胞瘤（inflammatory myofibroblastic tumor，IMT）的临床病理及分子病理学特征。方法;收集 5例子宫 IMT，回顾性分析其临床资料，观察组织形态学改变，进行免疫组化染色和4K基因荧光原位杂交（fluorescence in situ hybridization，FISH）检测，并采用二代测序（nex对t-generation sequencing，NGS）方法检测包括AIK 在内的507个癌基因点突变、插入、缺失、基因融合情况。结果∶该组病例发病年龄为 32～47岁，平均（39.60±5.12）岁，3 例肿瘤位于子宫体肌壁间，2 例位于子宫内膜下。显微镜下见梭形细胞肿瘤，富于黏液基质或者局部间质黏液样变。初始诊断 3 例为平滑肌肿瘤，2 例为内膜间质肉瘤。5 例免疫组织化学染色均为ALK 阳性，ER，PR。 Desmin 阳性表达;4 例患者的 SMA 免疫组化染色呈阳性表达，1 例不表达;3 例 CD10 染色呈阳性表达，2 例不表达;5例患者均不表达 CD117 和 CK;Ki-67的增殖指数除一例为40% 外，其余均较低。AIK 分离探针 FISH 检测显示 4 例阳性，1 例阴性，后者经 NGS 检测证实存在 ALK 基因的融合。结论∶ALK 阳性是子宫 IMT 的一种高度特异的免疫组化标记，子宫 IMT 的免疫组化和 RNA 测序具有较高的一致性，可借此将 IMT 与子宫梭形细胞肿瘤鉴别。     

Ki-67在浆膜腔积液鉴别诊断中的应用

目的 :探讨 Ki- 6 7抗体对鉴别浆膜腔积液中良恶性细胞的价值 ,解决伴浆膜腔积液的疑难病例的诊断。方法 :用 Ki- 6 7单克隆抗体标记 4 7例浆膜腔积液涂片标本 ,每张涂片计数 10 0 0个细胞中的阳性细胞数 ,用百分率表示阳性指数 ,同时对涂片作 HE染色。结果 :恶性积液组 Ki- 6 7指数为 34.89% ,良性组为 0 .72 % ,两者差异具有显著性 (P<0 .0 0 5 )。结论 :浆膜腔积液涂片 HE染色诊断结合 Ki- 6 7标记 ,可提高恶性浆膜腔积液细胞学的阳性诊断率 ,可作为临床鉴别良恶性浆膜腔积液的参考指标。     

荧光原位杂交技术在泌尿生殖肿瘤中的应用

随着分子细胞遗传学的快速发展,荧光原位杂交(fluorescence in situ hybridization,FISH)技术已广泛应用于临床和科学研究.FISH技术综合了荧光信号的高度灵敏性、无放射性、直观性及原位杂交的准确性,通过检测样本中基因或染色体的异常改变而应用于遗传性疾病、肿瘤的研究及临床诊断和治疗监测.本文就荧光原位杂交技术在常见的泌尿生殖肿瘤中的应用进行综述.     

联合检测E-cadherin、CEA及Calretinin在浆膜腔积液细胞学鉴别诊断中的意义

背景与目的：浆膜腔积液中转移性腺癌细胞、恶性上皮型间皮瘤细胞和反应性间皮细胞的形态有不少相似之处，有时仅凭形态学特征不能做出准确诊断。近年来免疫细胞化学在这方面得到较多应用，但国内报道仅局限于用CK、EMA、CEA、Vim和HBME-1几种抗体，而且不能较好地进行细胞学的鉴别诊断。本研究旨在探讨联合检测E-cadherin、CEA及calretinin在浆膜腔积液鉴别诊断中的应用价值。方法：选用浆膜腔积液标本共93例，其中胸水66例、腹水24例、心包积液3例。经组织学检查或结合临床资料证实的转移性腺癌55例、恶性上皮型间皮瘤6例、间皮细胞反应性增生32例。每例均制备HE染色的涂片和细胞块，并用细胞块切片作免疫细胞化学染色。结果：E-cadherin、CEA对诊断转移性腺癌的敏感性分别为85．5％(47／55)、78．2％(43／55)，特异性分别为100％(38／38)、97．4％(37／38)。E-cadherin和CEA联合应用诊断浆膜腔积液转移性腺癌的阳性率为96．4％(53／55)。Calretinin对诊断间皮瘤和间皮细胞增生的敏感性和特异性分别为81.6％(31／38)和87．2％(48／55)。结论：E-cadherin、CEA和calretinin是鉴别浆膜腔积液转移性腺癌细胞和间皮源性细胞有价值的一组抗体。     

Association of epidermal growth factor receptor (EGFR) gene mutations with EGFR amplification in advanced non-small cell lung cancer

Somatic mutations in the epidermal growth factor receptor (EGFR) gene are associated with the response to EGFR tyrosine kinase inhibitors in patients with non-small cell lung cancer (NSCLC). Increased EGFR copy number has also been associated with sensitivity to these drugs. However, given that it is often difficult to obtain sufficient amounts of tumor tissue for genetic analysis from patients with advanced NSCLC, the relationship between these two types of EGFR alterations has remained unclear. We have now evaluated EGFR mutation status both by direct sequencing and with a high-sensitivity assay, the Scorpion-amplification-refractory mutation system, and have determined EGFR copy number by fluorescence in situ hybridization (FISH) analysis in paired tumor specimens obtained from 100 consecutive patients with advanced NSCLC treated with chemotherapy. EGFR mutations or FISH positivity (EGFR amplification or high polysomy) were apparent in 18% (18/100) and 32% (32/100) of patients, respectively. The Scorpion-amplification-refractory mutation system was more sensitive than direct sequencing for the detection of EGFR mutations. Furthermore, EGFR mutations were associated with EGFR amplification (P = 0.009) but not with FISH positivity (P = 0.266). Our results therefore suggest the existence of a significant association between EGFR mutation and EGFR amplification in patients with advanced NSCLC.     

联合检测EGFR、CEA对恶性胸腔积液的诊断价值

目的:评价联合检测胸腔积液患者的胸水细胞块表皮生长因子受体（epidermal growth factor receptor,EGFR）基因拷贝数,以及胸水、血清CEA水平对良恶性胸腔积液鉴别诊断的价值。方法:应用荧光原位杂交技术（Fish法）检测恶性胸腔积液（n=35）、良性胸腔积液（n=30)组患者胸水细胞块EGFR基因拷贝数水平。采用电化学发光全自动生化分析仪检测胸水及血清中CEA水平,根据受试者工作特性曲线（ROC）选取最佳灵敏性和特异性的点作为临界值,评价CEA及联合检测EGFR基因拷贝数对良恶性胸腔积液的诊断价值。结果:35例恶性胸腔积液完成Fish检测。恶性胸腔积液中15例阴性,20例阳性,阳性率为57.1％。其中13例为EGFR基因高度多体性,7例为EGFR基因扩增。肺腺癌16例中,EGFR基因高度多体性、扩增14例,肺腺癌扩增率为87.5％;肺鳞癌14例中,EGFR基因簇状扩增3例(21.4％),点状扩增3例(21.4％),无扩增8例(57.1％),肺鳞癌扩增率为42.9％。腺癌Fish阳性率(87.5％)高于鳞癌(42.9％）,P<0．01。30例良性胸腔积液中有1例脓胸患者EGFR Fish检测阳性,阳性率为3.3%,余检测结果均阴性。恶性胸腔积液患者胸水及血清CEA分别为（220.9±71.65）ng/ml、(18.11±11.38）ng/ml,显著高于良性胸腔积液组（2.31±1.29)ng/ml、(1.67±1.06）ng/ml,差异有统计学意义（P<0.01）。其中,恶性胸腔积液胸水CEA明显高于血清CEA,而良性胸腔积液组中,胸水CEA与血清CEA无明显差异。肺腺癌所致胸水及血清CEA分别为（441.02±102.65)ng/ml、(32.87±28.66）ng/ml,鳞癌所致胸水及血清CEA分别为（28.75±21.39）ng/ml、（5.99±5.32）ng/ml,腺癌显著高于鳞癌,差异有统计学意义（P<0.01）。比较胸水EGFR、CEA对良恶性胸腔积液诊断的效能,两者之间无明显差异（P=0.453＞0.05）。Spearman相关性分析胸水EGFR同CEA之间存在显著正相关。结论:EGFR在恶性胸腔积液的形成中起重要作用,通过Fish技术检测胸水细胞块EGFR基因拷贝数可行,其敏感性为57.1%。对肺腺癌导致恶性胸腔积液的诊断敏感性为87.5%。CEA（临界值5.0ng/ml）在恶性胸腔积液及血清中显著高于良性,其中胸水CEA检测诊断敏感性为65.7%,而在腺癌中为87.5%,其在胸水及血清中的比值＞1.5有助于恶性胸腔积液的诊断。EGFR基因突变阳性与肿瘤标记物CEA阳性表达呈正相关,尤见于肺腺癌患者,两者联合检测可提高诊断性试验的准确性。     

非小细胞肺癌胸腔积液与配对肿瘤组织中EGFR基因突变检测的比较

目的:探讨非小细胞肺癌胸腔积液与配对肿瘤组织标本中EGFR基因突变检测结果的一致性,评价胸腔积液标本检测EGFR基因突变的应用价值.方法:收集非小细胞肺癌患者胸腔积液与配对肿瘤组织样本72例,采用ARMS方法,检测样本中EGFR基因第18~21外显子突变情况.结果:细胞学样本和组织学样本中EGFR基因突变阳性率分别为48.61%和51.39%,两者差异无统计学意义(P>0.05),二者一致率为92.11%,不一致率为7.89%.结论:二者的一致率较高,恶性胸水可以作为无法获得肿瘤组织的晚期非小细胞肺癌EGFR基因检测的有效样本.     

Selection pressures of TP53 mutation and microenvironmental location influence epidermal growth factor receptor gene amplification in human glioblastomas

Epidermal growth factor receptor (EGFR) gene amplification occurs in glioblastomas as so-called double minutes. Because double minutes are extrachromosomal fragments, selection pressures must operate to maintain high EGFR copy number over multiple cell divisions. In analyses of glioblastoma lysates, EGFR amplification has been observed almost exclusively in glioblastomas harboring wild-type TP53 genes, which raises the alternative hypotheses that TP53 mutation either prevents amplification or selects against maintenance of EGFR-amplified cells. To address these possibilities at the cellular level, we studied 14 glioblastomas for TP53 mutation and EGFR gene amplification status, using fluorescence in situ hybridization (FISH) for the latter. Remarkably, four of the six cases with TP53 mutation had isolated EGFR-amplified cells in different regions, demonstrating that EGFR amplification occurs frequently at the cellular level in TP53-mutant glioblastomas. Thus, TP53 mutation does not prevent EGFR amplification but does not facilitate selection of EGFR-amplified cells. Of the eight cases without TP53 mutation, five had widespread EGFR amplification. In four of these five cases, multiple regions of the tumor were available for examination; FISH demonstrated a gradation of EGFR amplification, with highly amplified cells, primarily at the invading edges rather than the relatively solid tumor centers, suggesting that EGFR overexpression, when selected for in vivo, may be related to tumor invasion.     

Application of non-small cell lung cancer pleural effusion cell blocks in molecular pathological detection

Objective:The tumor tissues used in molecular pathological detection were usual y obtained by surgery, which would cause trauma and may not be suitable for the terminal cancer patients. This paper evaluated the value of the non-smal celllung cancer (NSCLC) pleural ef usion cellblocks as tumor tissues replacement materials in the application of molecular pathological detection. Methods: Tumor cells were made into cellblocks through stratified centrifugal from 30 NSCLC pa-tients with the pleural ef usion. The immunohistochemistry, fluorescence in situ hybridization (FISH) and gene sequencing methods were employed in our experiments. Results:The tumor cells of cellblock section were rich and could keep part of histological structure. Immunohistochemistry staining could assist diagnosis and tumor parting. Epidermal growth factor receptor (EGFR) FISH-positive was found in 33.33%of the group, high polysomy in 6 cases, amplification in 4 cases. EGFR gene mutations were found in 8 cases of 30 samples, with an incidence of 26.67%, 6 cases were detected in the exon 19, and 2 cases were detected in the exon 21. Conclusion:The NSCLC pleural ef usion cellblocks are useful for the diagnosis and determining the primary source of tumor, instructed targeted therapy.     

Prognostic Outcome of Mesenchymal Transition Biomarkers in Correlation with EGFR Expression in Epithelial Ovarian Carcinoma Patients

Background: CD44 is an epithelial-mesenchymal transition (EMT) surface receptor that regulates the interactivity between the cells and the extracellular matrix, thereby promoting cell migration. The epidermal growth factor receptor (EGFR) family is a trans-membrane kinase-related protein. It regulates cell adhesion proteins, which may promote cell proliferation and invasiveness. Mesenchymal epithelial transition (MET) is another EMT receptor that stimulates cell proliferation, invasion, survival, and angiogenesis. This study aimed to evaluate the prognostic impact of CD44, EGFR expressions, and MET gene amplification in epithelial ovarian cancer (EOC). Methods: This is a retrospective cohort study, including 85 cases of EOC. CD44 and EGFR expressions were evaluated in both epithelial and stromal cells by immunohistochemistry. Tumor cells also underwent a cytogenetic analysis using fluorescent in situ hybridization (FISH) to detect MET gene amplification. Results: High CD44 expression in tumors was significantly associated with serous subtypes (P=0.001), peritoneal deposits (P=0.002), and advanced stage (P=0.002). EGFR high tumor expression demonstrated a significant association with lymph node metastasis (P=0.038) and the advanced stage of EOC (P=0.016). Increased copy number of the MET gene was significantly associated with partial therapy response (P=0.030).  CD44 and EGFR tumor high expression was associated with poor overall survival (OS). In addition, MET gene gain in tumors was associated with a shorter OS (P=0.000). Conclusion: EMT biomarkers (CD44 and MET) and EGFR expression in EOC are independent prognostic factors for OS. MET gene increase copy number was detected in cases of serous neoplasm and associated with poor survival and minimal therapy response.     

肺腺癌中表皮生长因子受体基因突变与拷贝数的检测分析

目的检测肺腺癌组织中表皮生长因子受体（EGFR）19、21外显子基因突变和拷贝数,分析EGFR基因突变和拷贝数变化的相关性。方法应用荧光定量PCR技术和荧光原位杂交（FISH）技术分别检测58例肺腺癌患者肿瘤组织中的EGFR基因突变和基因扩增,用X^2检验进行数据分析。结果58例肺腺癌患者组织中,EGFR19、21外显子突变率为43．1％（25／58）,其中2例存在两种类型的突变。EGFR基因拷贝数增加阳性率为51．7％（30／58）,包括8例扩增,22例高多体扩增。不同分化程度的肺腺癌组织间,扩增阳性率差异无统计学意义（P〉0．05）,低分化癌的突变率低于高、中分化癌（P〈O．05）。EGFR基因突变与EGFR基因拷贝数之间显著相关（P〈0．01）。结论肺腺癌组织具有较高的EGFR基因突变率和扩增阳性率;联合检测EGFR基因拷贝数和基因突变,更有利于靶向药物的筛选。     

冀东满族肺腺癌患者胸液与相应肿瘤组织EGFR基因突变检测对比分析

目的:分析冀东满族肺腺癌患者胸液与相应肿瘤组织EGFR基因突变检测结果。方法:选取2010年9月至2014年9月间在我院胸外科治疗的74例有胸液的冀东满族肺腺癌患者为研究对象,采用变性高效液相色谱法(DHPLC)检测肺腺癌患者胸液及相应肿瘤组织样本中是否发生EGFR基因突变。结果:肿瘤组织EFGR基因突变检出率为50.00%,胸液样本EGFR基因突变检出率为52.70%,两者间无统计学差异(P＞0.05)。胸液上清、胸液沉淀、胸液上清与沉淀以及肿瘤组织EGFR基因检测结果一致性均良好。结论:肺腺癌患者胸液与相应肿瘤组织EGFR基因突变检测结果一致性良好,临床可通过胸液检测代替肿瘤组织检测,以期能够持续监测患者EGFR基因突变状态,以指导患者添加或者实施EGFR-TKIs治疗。