粘液样/圆细胞型脂肪肉瘤石蜡包埋组织中FUS-CHOP融合基因的检测

目的:探讨逆转录-聚合酶链反应(RT-PCR)方法用于检测甲醛固定、石蜡包埋组织中FUS-CHOP融合基因的可行性及其对粘液样/圆细胞型脂肪肉瘤诊断的价值.方法:收集粘液样/圆细胞型脂肪肉瘤石蜡包埋组织标本25例,以其它类型脂肪肉瘤(包括分化良好型、去分化型、多形型),滑膜肉瘤,低度恶性纤维肉瘤,恶性纤维组织细胞瘤,粘液脂肪瘤,脂肪母细胞瘤,神经鞘瘤伴粘液变性和粘液软骨肉瘤作为阴性对照,共18例.所有标本均经过甲醛固定、石蜡包埋.用嵌套式RT-PCR的方法检测FUS-CHOP融合基因mRNA并经测序证实,以β-actin基因作为内对照检测mRNA质量.结果:43例标本中,30例可检出β-actin,阳性率为69.8%,其中粘液样/圆细胞型脂肪肉瘤标本β-actin阳性18例(72%),对照组β-actin阳性12例(66.7%).25例粘液/圆细胞型脂肪肉瘤中15例检出Ⅱ型FUS-CHOP融合基因表达,阳性率60%(15/25),去除β-actin阴性病例的阳性率为77.8%(14/18).其中1例β-actin阴性表达而检出FUS-CHOP融合基因.对照组18例标本均未检出FUS-CHOP融合基因.结论:FUS-CHOP融合基因在粘液样/圆细胞型脂肪肉中呈特异性表达,可作为该肿瘤的特异性标志物用于诊断.嵌套式RT-PCR的方法可用于甲醛固定、石蜡包埋组织中FUS-CHOPmRNA的检测.     

基因表达谱在肿瘤病理诊断与鉴别诊断中的意义

利用基因芯片技术对肿瘤细胞基因表达谱进行分析。通过筛选出不同肿瘤个体及不同肿瘤类型具有特征性的差异表达基因,以达到利用差异表达基因对肿瘤进行诊断与鉴别诊断的目的。这些被筛选出的差异表达基因不仅与肿瘤组织的生长发育、增殖凋亡和信号传导有关,而且与肿瘤的发病机制、浸润转移、肿瘤的抗药性有关,尤其是一些肿瘤差异表达基因或基因组显示与肿瘤的组织学来源及组织学类型相关联。基因表达谱在临床肿瘤病理诊断与鉴别诊断中具有实际应用价值。     

膀胱尿路上皮癌石蜡包埋组织中miR-20a的表达与复发的相关性分析

目的 探讨膀胱尿路上皮癌(膀胱癌)石蜡包埋组织中提取microRNA的可行性,分析miR-20a在膀胱癌石蜡包埋组织中的表达情况与临床病理特征及术后复发的关联性。方法 荧光定量RT-PCR法检测50例新鲜膀胱癌组织和对应的石蜡包埋组织中miR-20a基因表达的相关性,分析不同年度间181例石蜡包埋膀胱癌组织中miR-20a基因的表达,并与患者术后复发进行相关性分析。结果 石蜡包埋膀胱癌组织与新鲜冷冻组织中miR-20a的表达密切相关（r=0.792,P＜0.001）;不同年度间石蜡组织的miR-20a表达稳定,差异无统计学意义。miR-20a表达量与肿瘤临床特征明显相关,高表达患者其术后复发明显增高（P﹤0.05）。结论 石蜡包埋膀胱癌组织与新鲜冷冻组织中miR-20a的表达有一致性,用石蜡组织提取microRNA是可行的,膀胱癌细胞中miR-20a的作用与其表达量相关,高表达是术后复发的独立因素。     

组织芯片技术在肿瘤标志物筛选中的应用

组织芯片技术是在 1张玻片上分析数百个肿瘤样本的新方法 ,可高通量的分析基因和蛋白。方法包括从石蜡包埋组织供体取组织芯 ,然后放入另一独立的石蜡块中 ,制成组织切片[1] 。它是在基因芯片的基础上发展而来 ,组织芯片技术可在数千种肿瘤中同时快速评价基因拷贝数量和表达。肿瘤的发生、演进过程中标志物的出现、改变对肿瘤的病理诊断、肿瘤分级、分期提供了重要依据。每年都有新的肿瘤标志物被发现 ,如何快速、准确、简便地检测肿瘤标志物的存在和这些标志物的特异性 ,以及在肿瘤不同发展阶段的变化 ,组织芯片技术在大规模、快速筛选特异…     

粘液样脂肪肉瘤TLS/FUS-CHOP融合基因的检测

目的:探讨在粘液样脂肪肉瘤石蜡包埋组织中检测TLS/FUS-CHOP特异性融合基因的可行性及TLS/FUS-CHOP融合基因表达在粘液样脂肪肉瘤诊断中的价值.方法:收集粘液样脂肪肉瘤石蜡包埋组织标本6例,以粘液样脂肪瘤、脂肪母细胞瘤、粘液瘤、骨外粘液样软骨肉瘤、粘液纤维肉瘤作为阴性对照,β-肌动蛋白和β2-微球蛋白作为内对照,用逆转录聚合酶链反应(RT-PCR)和巢式PCR检测TLS/FUS-CHOP融合基因的表达.结果:6例粘液样脂肪肉瘤中4例检出β-肌动蛋白mRNA表达,5例检出β2-微球蛋白mRNA表达,其中3例检出Ⅱ型TLS/FUS-CHOP融合基因mRNA表达.对照组均未检出TLS/FUS-CHOP融合基因的表达.结论:运用RT-PCR方法检测石蜡包埋组织中TLS/FUS-CHOP融合基因mRNA的表达可行,有助于粘液样脂肪肉瘤的诊断和鉴别诊断.     

组织微阵列(组织芯片)在肿瘤研究中的应用

 随着人类基因组计划的完成并进入后基因组时代 ,基因研究的重点已从结构转向功能[1] 。由于基因芯片 (gengchip)的广泛应用 ,基因表达谱和基因变异谱的研究会得出越来越多的与肿瘤分析相关的侯选标志基因。这些基因组学的早期发现 ,在临床诊断和治疗的实际应用之前需要进行进一步测试 ,需要在更多的临床肿瘤标本中通过原位杂交和免疫组织化学染色的验证 ,以筛选与某种肿瘤诊断、预后及治疗相关的基因[2 ] 。而目前传统的组织切片处理方法由于在一个蜡块内只能包埋一个组织标本 ,只能切出含有一份组织样本的切片 ,不利于快速高效的进行大量指标的筛选工作。     

EWS-ATF1融合基因在透明细胞肉瘤中的表达及其诊断意义

目的：探讨在透明细胞肉瘤（CCS）石蜡包埋组织中检测EWS-ATF1融合基因的可行性及EWS-ATF1融合基因表达在透明细胞肉瘤诊断中的意义。方法：收集1992年～2006年期间确诊的26例CCS石蜡包埋组织标本,20例非CCS石蜡包埋组织标本为对照组,β-肌动蛋白和β2微球蛋白作为内参照,用逆转录聚合酶链反应（RT-PCR）和巢式PCR检测EWS-ATF1融合基因的表达;过氧化物酶染色（SP）方法对26例CCS石蜡组织标本进行免疫组化染色,检测S-100、HMB-45蛋白的表达。结果：26例标本中,有22例β-肌动蛋白阳性;24例β2微球蛋白阳性,其中20例EWS-ATF1融合基因阳性（16例EWS-ATF1Ⅰ型,4为EWS-ATF1Ⅲ型）,余4例EWS-ATF1融合基因阴性。对照组均未检测到EWS-ATF1融合基因的表达。26例CCS石蜡组织标本中S-100及HMB-45的总阳性率分别为79.2%和70.8%。结论：在CCS石蜡包埋组织中运用RT-PCR检测EWS-ATF1融合基因是可行的,可作为辅助诊断和鉴别诊断的有力工具。     

SHOX2和RASSF1A基因启动子区甲基化检测在早期肺腺癌筛查和诊断中的应用价值

目的探讨SHOX2和RASSF1A基因启动子区甲基化检测对早期肺腺癌筛查及诊断的价值。方法下载癌症基因组图谱(TCGA)数据库中471例肺腺癌患者的mRNA测序数据及相应的413例甲基化数据分别计算两基因启动子区甲基化水平, 分析正常肺组织与肿瘤组织的甲基化水平差异。回顾性分析2018年1月至2019年1月南京大学医学院附属鼓楼医院接受手术的54例早期肺腺癌及31例肺良性肿瘤患者临床资料, 检测肿瘤组织及正常肺组织(距肿瘤>5 cm)(称为:临床样本)SHOX2及RASSF1A甲基化状态, 以任一基因启动子区甲基化阳性为两基因联合甲基化阳性。以病理诊断为金标准, 绘制受试者工作特征(ROC)曲线, 分析基因甲基化阳性诊断早期肺腺癌的效能。筛选出石蜡样本中肿瘤细胞比例>80%的患者, 对其肿瘤组织及正常肺组织进行mRNA高通量测序。分析两基因甲基化阳性与患者临床病理特征的关系, 采用Spearman法分析临床样本和TCGA数据库样本中两基因启动子区甲基化水平与mRNA表达水平的相关性。采用基因集变异分析(GSVA)法分析两基因甲基化阳性临床肺腺癌样本和对应的甲基化阴性肺腺癌样...     

多肿瘤组织芯片在评价肿瘤细胞增殖与凋亡状态研究中的作用

目的：研究多种常见肿瘤组织芯片在观察癌细胞增殖和凋亡中的作用。方法：在病理档案材料中选取10种常见恶性肿瘤及其癌旁或正常组织的石蜡包埋标本，HE染色切片定位穿刺部位，每一蜡块穿取直径0．6mm大小组织柱，排布成196点列阵的芯片；用ki-67、p53、rasp21、bcl-2和bax单抗SP法进行免疫组化染色，对各切片阳性细胞进行计数观察。结果：组织芯片经染色后结构完整，在肿瘤组织中ki-67、p53和rasp21标记的阳性细胞比癌旁和正常组织明显增高；而大多数肿瘤中bcl-2和bax表达呈负相关，即bcl-2阴性或低表达者，则bax呈不同程度阳性表达，反之则相反。结论：组织芯片技术可以同时用于评价多种不同类型肿瘤样本的增殖活性和凋亡状态，为临床预后判断提供资料。     

非小细胞肺癌ALK基因重排检测

目的 通过荧光原位杂交(FISH)方法检测非小细胞肺癌(NSCLC)患者间变性淋巴瘤激酶(ALK)基因重排.方法 根据ALK断裂重排方式及特点,设计并制备红/绿双色荧光探针.以人外周血培养细胞为检测对象评价ALK融合基因检测探针的敏感性和特异性,以NSCLC石蜡组织样本为对象进行ALK基因重排检测以评价探针的性能.结果 本研究制备的荧光探针在EB病毒(EBV)转化的人淋巴细胞检测中的特异性和敏感性均可达到100％.在对2例NSCLC患者石蜡包埋组织样本检测中,检测阴性、阳性各1例,检测结果与免疫组织化学检测结果一致.结论 本研究中制备的双色荧光探针可用于NSCLC ALK基因重排的检测.     

An expression-based site of origin diagnostic method designed for clinical application to cancer of unknown origin

Gene expression profiling offers a promising new technique for the diagnosis and prognosis of cancer. We have applied this technology to build a clinically robust site of origin classifier with the ultimate aim of applying it to determine the origin of cancer of unknown primary (CUP). A single cDNA microarray platform was used to profile 229 primary and metastatic tumors representing 14 tumor types and multiple histologic subtypes. This data set was subsequently used for training and validation of a support vector machine (SVM) classifier, demonstrating 89% accuracy using a 13-class model. Further, we show the translation of a five-class classifier to a quantitative PCR-based platform. Selecting 79 optimal gene markers, we generated a quantitative-PCR low-density array, allowing the assay of both fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue. Data generated using both quantitative PCR and microarray were subsequently used to train and validate a cross-platform SVM model with high prediction accuracy. Finally, we applied our SVM classifiers to 13 cases of CUP. We show that the microarray SVM classifier was capable of making high confidence predictions in 11 of 13 cases. These predictions were supported by comprehensive review of the patients' clinical histories.     

PD-L1 Assay Concordance in Metastatic Renal Cell Carcinoma and Metastatic Urothelial Carcinoma

BackgroundImmune checkpoint inhibitors are now standard of care for many patients with metastatic renal cell carcinoma (mRCC) and metastatic urothelial carcinoma (mUC). Given real-world limitations in programmed death-ligand 1 (PD-L1) testing, concordance studies between PD-L1 assays are needed. We undertook comparisons of Dako 28-8 and Ventana SP142 assays in mRCC and Dako 22C3 and Ventana SP263 assays in mUC.Patients and MethodsThirty-two patients with mRCC and 18 patients with mUC who had received immune checkpoint inhibitor therapy were identified. Formalin-fixed paraffin-embedded tumor samples for patients with mRCC were evaluated with Dako 28-8 and Ventana SP142 PD-L1 immunohistochemistry assays. For patients with mUC, formalin-fixed paraffin-embedded tumor samples were evaluated with Dako 22C3 and Ventana SP263 PD-L1 immunohistochemistry assays.ResultsThe majority (29/32; 91%) of mRCC cases were concordant between assays. The majority (17/18; 94%) of mUC cases were also concordant between assays.ConclusionsThere was strong concordance between PD-L1 assays chosen for comparison in both mRCC and mUC, with similar performance characteristics. One limitation is the small number of cases in this study; larger comparison studies are needed for this biomarker in mRCC and mUC.     

Different assays for HER-2/neu evaluation in breast cancer.

To evaluate different methodologic approaches for HER-2/neu analysis, we performed Southern, Northern, Western blot and histochemical assay on 112 samples from 86 primary tumors and 26 synchronous axillary metastatic lymph nodes of patients affected by operable breast cancer. Simultaneous statistical analysis of data obtained with the four methods (31 samples) showed that Western blot detected a higher percentage of alterations than the other assays (Cochran and Victor tests, 0.01 less than p less than 0.05). The same result was emphasized by pair analysis (McNemar, p less than 0.05), which evaluated the assay data two by two. Immunohistochemical evaluations were more in accord with immunoprecipitation data when performed on frozen or Bouin-fixed, paraffin-embedded tissues than on formalin-fixed, paraffin-embedded tissues.     

Loss of pRb2/p130 expression is associated with unfavorable clinical outcome in lung cancer.

Altered expression of cell cycle regulators represents a frequent event in both small cell and non-small cell lung cancer (NSCLC). Despite several studies that reported involvement of tumor suppressor genes, such as p53 and pRb, in the development and progression of lung cancer, contrasting opinions exist about the prognostic role of this protein in this neoplasm. We developed an immunohistochemical assay suitable for the detection of pRb2/p130, the last discovered member of the retinoblastoma gene family, on formalin-fixed and paraffin-embedded sections. We evaluated the immunohistochemical expression of pRb2/p130 in 135 lung cancer specimens, and performed Western blot analysis in a subset of 30 corresponding tumor lysates. A high correlation between immunohistochemical data and Western blot results (P = 0.0004) was found. We statistically analyzed the relationship between overall survival (OS) time and pRb2/p130 expression according to the different histological types in 105 patients. We did not find any correlation between pRb2/p130 expression and OS in small cell lung cancers, whereas in NSCLCs a direct relationship between pRb2 and OS was found in both adenocarcinoma (P = 0.0002) and squamous cell carcinoma (P = 0.0002) histotypes. According to univariate analysis, pRb2/p130 was a prognostic factor of which the lost or reduced expression correlated with a shorter OS (P < 0.0000). At multivariate analysis, pRb2/p130 expression was an independent predictor of OS (P = 0.0001) when considered together with histotype. This study demonstrates for the first time the potential independent prognostic value of pRb2/p130 expression on formalin-fixed, paraffin-embedded sections from lung cancer patients. pRb2/p130 immunoreactivity can be used to predict OS in patients with NSCLC and, therefore, may represent a new prognostic marker.     

Development and Validation of a Scalable Next-Generation Sequencing System for Assessing Relevant Somatic Variants in Solid Tumors

Next-generation sequencing (NGS) has enabled genome-wide personalized oncology efforts at centers and companies with the specialty expertise and infrastructure required to identify and prioritize actionable variants. Such approaches are not scalable, preventing widespread adoption. Likewise, most targeted NGS approaches fail to assess key relevant genomic alteration classes. To address these challenges, we predefined the catalog of relevant solid tumor somatic genome variants (gain-of-function or loss-of-function mutations, high-level copy number alterations, and gene fusions) through comprehensive bioinformatics analysis of >700,000 samples. To detect these variants, we developed the Oncomine Comprehensive Panel (OCP), an integrative NGS-based assay [compatible with < 20 ng of DNA/RNA from formalin-fixed paraffin-embedded (FFPE) tissues], coupled with an informatics pipeline to specifically identify relevant predefined variants and created a knowledge base of related potential treatments, current practice guidelines, and open clinical trials. We validated OCP using molecular standards and more than 300 FFPE tumor samples, achieving >95% accuracy for KRAS, epidermal growth factor receptor, and BRAF mutation detection as well as for ALK and TMPRSS2:ERG gene fusions. Associating positive variants with potential targeted treatments demonstrated that 6% to 42% of profiled samples (depending on cancer type) harbored alterations beyond routine molecular testing that were associated with approved or guideline-referenced therapies. As a translational research tool, OCP identified adaptive CTNNB1 amplifications/mutations in treated prostate cancers. Through predefining somatic variants in solid tumors and compiling associated potential treatment strategies, OCP represents a simplified, broadly applicable targeted NGS system with the potential to advance precision oncology efforts.Abbreviations: AOHC, AcroMetrix Oncology Hotspot Control; CNAs, copy number alterations; FFPE, formalin-fixed paraffin-embedded; GoF, gain-of-function; indels, insertions/deletions; LoF, loss-of-function; LU, lung cohort; MCR, minimal common region; MO, molecular cohort; NCCN, National Comprehensive Cancer Network; NGS, next-generation sequencing; OCP, Oncomine Comprehensive Panel; PGM, Personal Genome Machine; PR, prostate cohort; QMRS, Quantitative Multiplex Reference Standard; SCC, small cell carcinoma; TCGA, The Cancer Genome Atlas