定量RT—PCR法检测乳腺癌前哨淋巴结CK19的研究

目的探讨定量RT—PCR法检测细胞角蛋白（CK19）在乳腺癌前哨淋巴结（SLN）中的表达，提高前哨淋巴结活检中微转移的检出率。方法采用常规病理检查法（HE染色）和定量RT—PCR法检测了40例乳腺癌患者SLN的CK19的表达量，同时选取10例来源于胃肠道的良性病变淋巴结作为定量RT—PCR检测的对照组。结果CK19在良性病变的淋巴结中没有表达。常规病理检查的敏感度为42．9％（9／21），假阴性为57．1％（12／21）．准确率为70．0％（28／40）。定量RT—PCR法检测出常规病理未检出的微小转移病例12例，敏感度为95．2％（20／21），假阴性为4．8％（1／21），准确率为97．5％（39／40）。结论前哨淋巴结活检可有效判断乳腺癌腋淋巴结转移状态，应用定量RT—PCR法检测CK19在SLN中的表达，可提高敏感度及准确率。     

逆转录聚合酶链扩增与免疫组化在检测胃癌区域淋巴结微转移中的相关研究

目的：利用逆转录聚合酶链扩增检测胃癌常规病理检查阴性的淋巴结微转移的发生及与免疫组化结果的关系。方法：利用逆转录聚合酶链反应（RT—PCR）方法检测480枚胃癌胃周淋巴结癌胚抗原（CEA）mR-NA基因表达,同时比较RT—PCR与免疫组化方法的检测敏感性。结果：利用TT—PCR检测CEA mRNA是一种很敏感的方法。检测138例胃癌患者取材的480枚胃周淋巴结。免疫组化阳性率27．5％（132／480）。RT—PCR阳性率58．8％（282／480）,两组之间有显著性差异（P〈0．01）;RT—PCR阳性率随着病期进展而增大。结论：RT—PCR技术是比免疫组化更敏感的方法,可以预测胃癌病人淋巴结微转移,有效避免已有微小转移的患者被漏诊、误诊。     

荧光定量RT-PCR在流感病毒检测上的应用

目的　探讨实时荧光定量RT PCR检测方法在流行性感冒检测诊断上的意义。方法　收集集体暴发疑似流感、SARS患者的咽拭子标本 16起 2 0 7份和双份血清标本 10 4份 ,应用荧光定量RT PCR检测方法、MDCK细胞培养法和血凝抑制试验进行病原学和血清学检测。结果　 2 0 7份集体暴发疑似流感、SARS患者的咽拭子标本中用荧光定量RT PCR检测 ,阳性 79份。阳性率 38 16 % (79 2 0 7)。MDCK细胞培养 ,16起中有 13起共 14 5份咽拭子有 6 2份标本阳性。阳性率 2 9 95 % (6 2 2 0 7)。两者的阳性率差异有显著意义 (χ2 =8.6 4 ,P <0 0 0 5 )。有 9起 10 4份成功的采集了双份血清 ,经HI试验有 6 4份双份血清与H3N2亚型抗原产生血抑 4倍增长 ,阳性率 6 1 5 4 % (6 4 10 4 )。结论　荧光定量RT PCR方法检测流感病毒能够在 3～ 4h内得出结果、且操作方便、特异性强、灵敏度高。因此我们认为实时荧光定量RT PCR方法 ,可作为一种快速的流感病毒检测诊断方法。     

21基因检测对ER阳性、淋巴结阴性乳腺癌患者术后复发转移的指导价值

目的探讨21基因检测在ER阳性、淋巴结阴性乳腺癌患者术后复发转移中的指导价值。方法选取2014年2月至2018年2月扬州友好医院及江南大学附属医院的ER阳性、淋巴结阴性乳腺癌患者172例,根据患者术后是否发生复发转移分为复发转移组(n=43)和未复发转移组(n=129)。所有患者均行手术治疗,根据AJCC第7版标准诊断患者的组织学分级,免疫组化法检测患者的癌转移抑制基因(PR)、细胞增殖核抗原Ki67(Ki67)、P53基因(P53)、基底细胞角蛋白CK5/6(CK5/6)、拓扑异构酶Ⅱα(TOPⅡα)以及表皮生长因子受体(EGFR)等指标。采用实时荧光聚合酶链反应(PCR)检测21基因(包括16个乳腺癌相关基因及5个参考基因)表达,计算21基因复发风险评分;对于复发转移患者查阅病历资料,记录并统计患者年龄、肿瘤直径、组织分型、Ki67、P53、TOPIIα、EGFR及CK5/6表达水平,分析不同病理组织下21基因复发风险评分,并进行单因素及多因素Logistic回归分析。结果所有ER阳性、淋巴结阴性乳腺癌患者均经病理结果最终确诊,确诊率为100. 0%,病理结果中43例发生复发转移。2组患者均顺利完成21基因检测,复发转移组21基因检测评分为(37. 93±6. 83)分,高于未复发转移组(21. 48±3. 25)分,差异有统计学意义(t=12. 193,P 0. 05);单因素及多因素结果表明,乳腺癌不同病理组织下21基因检测评分与年龄、病理类型、组织分级、P53、TOPIIα、EGFR、CK5/6、手术方式差异无统计学意义(P 0. 05);乳腺癌不同病理组织下21基因检测评分与肿瘤直径、Ki67、PR具有统计学意义(P 0. 05)。结论将21基因检测用于ER阳性、淋巴结阴性乳腺癌患者术后复发转移中效果理想,有助于评估患者预后,为临床诊疗提供依据和参考。     

PCR技术与实体瘤微转移的研究

肿瘤的浸润与转移是恶性肿瘤的重要特征，也是肿瘤病人死亡的主要原因。许多恶性肿瘤患者常规病理学检查未显示有肿瘤转移，但术后仍有部分患者死于肿瘤的复发与转移。这提示，临床常规方法尚不能检出已转移的少量肿瘤细胞。近年来发展起来的免疫学和分子生物学技术，已能在肿瘤患者的血液、骨髓和淋巴结中检出少量肿瘤细胞，如在临床常规检查转移阴性的患者称之为微转移（micrometstasis）。本文主要简介应用RT－PCR和PCR等技术在常见肿瘤检出的微转移及其临床意义，还讨论PCR技术检测微转移的局限性及其研究简史。     

荧光定量PCR检测查菲埃立克体

依据查菲埃立克体16SrRNA基因序列设计特异性引物和TaqMan-MGB探针,以克隆的查菲埃立克体16SrRNA基因片段作DNA模板,建立实时荧光定量PCR检测方法。与套式PCR相比较,荧光定量PCR检测的灵敏度是其30倍;用荧光定量PCR检测其他相关立克次体和细菌DNA样本,检出结果为0;对荧光定量PCR检测重复性进行分析,变异系数(CV)批内和批间误差在0·2%~2·0%之间。结果证明本研究建立的荧光定量PCR方法具有种特异性和良好的重复性,可用于检测感染样本中的微量查菲埃立克体DNA。     

胃癌中HP DNA定量检测与美兰染色半定量检测的相关性及优劣性

目的探讨胃癌患者HP DNA定量检测与美兰染色半定量检测两种检测HP方法的相关性及优劣性。方法应用实时荧光定量PCR及美兰染色检测118例胃癌患者HP感染情况。结果HP DNA定量分析与美兰染色半定量分析呈显著正相关(r=0.749,P0.001);美兰染色等级越高,HP相对感染量越多(P0.001);HP DNA定量分析阳性率(74.6%)显著高于美兰染色半定量分析(阳性率62.7%)。结论美兰染色操作简单、快速、结果可靠;实时荧光定量PCR检测精确、定量、客观、不易漏诊;两种检测HP方法呈显著正相关,实时荧光定量PCR较美兰染色检测阳性率更高,更敏感。     

间变性大细胞淋巴瘤患儿EEF1G/ALK融合基因的鉴定与检测

目的 明确1例伴少见ALK融合基因的间变性大细胞淋巴瘤患儿的分子诊断,建立该融合基因的实时荧光定量PCR检测方法。方法 采用基于锚定多重PCR的二代测序技术,鉴定患儿腹股沟肿大淋巴结组织中ALK基因的伙伴基因。建立EEF1G/ALK融合基因实时荧光定量PCR方法,并对方法的重复性、灵敏度和特异性进行性能评价。在此基础上检测此患儿肿瘤组织、骨髓、外周血和脑脊液EEF1G/ALK融合基因的表达。结果 经二代测序鉴定该患儿肿瘤细胞EEF1G/ALK融合基因阳性,Sanger测序验证为EEF1G Exon 6和ALK Exon 20的融合。建立的实时荧光定量PCR方法最低定量限为40拷贝/体系;弱阳性标本和强阳性标本的批内精密度CV分别为0.52%和0.17%;批间精密度CV分别为1.32%和1.14%,重复性好;检测其他ALK融合阳性标本结果为阴性,特异性好。复发时送检的淋巴结穿刺组织和外周血的融合基因定量检测结果为138.92%和0.0039%,其余检测为阴性。结论 明确此例间变性大细胞淋巴瘤为少见EEF1G/ALK融合基因阳性。建立的EEF1G/ALK融合基因定量检测方法特异性、重复性好...     

血清学方法和实时荧光定量PCR方法在新型布尼亚病毒检测中的应用研究

目的探讨血清学检测方法和实时荧光定量PCR方法在新型布尼亚病毒检测中的应用。方法连续采集烟台地区莱州、蓬莱疑似发热伴血小板减少综合征患者血清,分别用胶体金实验方法、ELISA实验方法检测抗新型布尼亚病毒IgM抗体,实时荧光定量PCR方法检测新型布尼亚病毒核酸,SPSS18．0统计结果。结果胶体金方法和ELISA方法检测结果总符合率为96．67％,一致性KAPPA检验K系数为0．933;胶体金方法和实时荧光定量PCR方法检测结果均受病程影响（两者P值均小于0．05）;实时荧光定量PCR2疗法检测不受年龄、性别影响（P均〉0．05）。结论新型布尼亚病毒检测发病12天以前推荐使用实时荧光定量PCR方法,发病16天以后,可使用血清学方法检测抗新型布尼亚病毒IgM抗体。     

胃癌组织中SIRT1、Noxa的表达及其临床意义

目的探讨SIRT1与Noxa在胃癌组织中的表达水平相关性,分析两者表达与患者临床病理学特征及预后的关系。方法应用实时荧光定量PCR法和免疫组化EnVision两步法检测106例胃癌及癌周正常组织中SIRT1及Noxa的mRNA和蛋白表达,分析两者相关性及其与患者性别、年龄、肿瘤部位、肿瘤大小、浸润深度及转移等临床病理特征的关系。结果实时荧光定量PCR结果显示,胃癌组织中SIRT1 mRNA表达较高,Noxa mRNA表达较低。免疫组化结果显示,SIRT1在胃癌组织中的阳性率高于正常胃组织(79.2%vs 41.5%),Noxa在胃癌组织中的阳性率低于正常胃组织(43.3%vs 71.6%),差异有统计学意义(P0.05)。SIRT1表达与肿瘤分化程度、肿瘤直径、浸润深度、淋巴结和远隔器官转移情况相关(P均0.05),Noxa表达与肿瘤直径、浸润深度、淋巴结和远隔器官转移情况相关(P均0.05)。胃癌组织中SIRT1与Noxa表达呈负相关。SIRT1阳性、Noxa阴性的患者2年生存率较低,其是预后较差的独立危险因素。结论 SIRT1和Noxa在胃癌的发生、发展中可能具有协同作用,联合检测两者的表达有助于判断患者的临床表现和预后。     

实时荧光定量RT-PCR检测胃癌患者外周血hTERT mRNA表达水平

目的:检测胃癌患者外周血中端粒酶催化亚基hTRT mRNA的表达水平,探讨其作为胃癌早诊标志物的可行性.方法:应用实时荧光定量RT-PCR技术检测108例原发性胃癌、100例良性胃溃疡和120例健康献血员血清hTRT mRNA表达水平.结果:胃癌组hTRT mRNA表达水平(13.48±0.83 copies/ml)显...     

Management of peritoneal carcinomatosis

Gastrointestinal malignancies frequently recur with metastatic disease limited to the abdominal cavity. Due to full thickness penetration of tumor through bowel wall and spillage of tumor from lymphatic channels by surgical trauma, tumor cells are disseminated throughout the peritoneal surfaces either prior to at the time of surgical removal of the primary tumor. Diagnosis of recurrent cancer is difficult because no sensitive diagnostic test is available by which to image a small tumor volume present on peritoneal surfaces. Computerized tomography can not demonstrate small to moderate nodules. Intraperitoneal instillation of 131-1 labeled monoclonal antibody has allowed visualization of mucinous tumor on peritoneal surfaces not seen by any other radiologic test. Intraperitoneal chemotherapy has been shown to provide palliation in patients with small volume disease confined to peritoneal surfaces. Because of limited penetration of chemotherapy into large tumor nodules this treatment strategy has not been effective for bulky intraabdominal recurrent cancer. Cytoreductive surgery can make patients relatively disease free. New surgical technologies combined with postoperative intraperitoneal chemotherapy have been shown to be curative for selected patients with recurrent cystadenocarcinoma. The wider application of immediate postoperative intraperitoneal chemotherapy treatments for gastrointestinal patients in an adjuvant setting may be of value in preventing the occurrence of peritoneal carcinomatosis and in improving survival.     

Peritoneal involvement in stage II colon cancer

A pathologist (K.S.) reviewed histologic slides for peritoneal involvement by tumor cells for 118 patients with stage II colon cancer. Patients were followed up for a median of 6 years. Tumor cells were found free in the peritoneal space in 16 cases (13.6%). The presence of cancer cells free in the peritoneal space was associated with lymphovascular invasion (P = .001) and neural invasion (P < .001). The overall 5-year survival was 80% in the patient population, but was 39% and 86% for those with and without tumor cells free in the peritoneal space, respectively (P < .0001). Multivariate analysis confirmed that free tumor cells within the peritoneal space (P < .0001) and lymphovascular invasion (P = .007) were related independently to outcome. Peritoneal involvement with tumor cells free in the peritoneal space in stage II colon cancer is a powerful indicator of outcome; patients have a survival similar to that for patients with stage III disease.     

Enhanced adherence activity of OK-432-induced peritoneal neutrophils to tumor cells correlates to their increased expression of CD11b/CD18.

We previously found that activated peritoneal neutrophils adhered to tumor cells and destroyed them in the cancer ascites of patients who had received intraperitoneal (ip) OK-432 injection therapy. Since tight adhesion to the tumor cell is essential for effective neutrophil-mediated tumor cell destruction, we investigated the mechanism of peritoneal neutrophil adhesion to tumor cells, using a microplate adhesion assay. An in vitro study demonstrated that the adherence activity of the peritoneal neutrophils of patients who received OK-432 injection therapy to tumor cells increased greatly compared to that of blood neutrophils. The expression of the adhesion molecules (CD11a,b,c/CD18) of peritoneal neutrophils, which was determined by an immunofluorescence study, was about four times as much in CD11b and twice as much in CD11c and CD18 compared to that in blood neutrophils. In vitro OK-432 stimulation of normal blood neutrophils increased neither the adhesion to PLC nor the CD11b expression. The enhanced adherence activity of peritoneal neutrophils to tumor cells was significantly inhibited by pretreatment of the neutrophils with anti-CD11b and anti-CD18 monoclonal antibodies (mAb), but not by pretreatment with anti CD11a or anti-CD11c mAb. These results indicated that the increased adhesiveness of OK-432-induced peritoneal neutrophils to tumor cells was due to the enhanced expression of CD11b/CD18. We concluded that CD11b/CD18 molecules on OK-432-induced peritoneal neutrophils play a crucial role in the neutrophil adherence activity against tumor cells, and these results are the first demonstration in the field of human neutrophil function.     

Molecular analysis of peritoneal fluid in ovarian cancer patients.

To determine whether genetic abnormalities present in primary ovarian tumors can be used to detect cancer cells in peritoneal fluid, we tested 14 ovarian cancers and 1 benign tumor of the ovary for loss of heterozygosity (LOH) at chromosomal arms 13q, 17p, 17q, and 22q and for mutations in the p53 and K-ras genes. In each case, matched primary tumor, normal tissue, and peritoneal fluid were analyzed. The highest frequency of LOH was found on chromosomal arm 17p (42%), followed by chromosomal arm 17q (36%), 22q (30%), and 13q (21%). Identical alterations were detected in matched peritoneal fluid (either peritoneal wash or ascitic fluid) in 3 of the 8 patients with LOH in the tumor (38%). Direct sequence analysis detected p53 mutations in 3 of the 14 malignant tumors (21%) and no (0) K-ras mutations. Identical mutations were detected in matched peritoneal fluid from all 3 patients with p53 mutations. All 8 of the 14 (57%) malignant tumors that showed at least one genetic abnormality were serous adenocarcinoma and identical alterations were detected in 5 of the 8 (62%) matched peritoneal fluid samples. Our findings indicate that molecular abnormalities can be detected in peritoneal fluid from patients with ovarian cancer and may be used to complement current conventional diagnostic procedures for detection of primary ovarian cancer.     

Optimization of the peritoneal lavage

Review of 275 consecutive peritoneal lavages and concurrent histologic material from gynecologic operations suggested that cytologic evaluation was clinically indicated for only 60.7% of the lavages, representing 46% of the patients in the study. More than one concurrent lavage was received from 21.6% of all patients in the study, comprising 50% of patients with malignant lavages, 18.7% of patients with benign lavages, and 5.3% of patients for whom cytologic evaluation of peritoneal lavage was not clinically indicated. Malignant cells were diagnosed in 15% of the 167 lavages for which cytologic examination was clinically indicated. In this series of patients, identification of malignant cells in peritoneal lavages did not increase the tumor stage beyond that obtained solely from examination of the concurrent histologic material. There were no false-positive cytologic diagnoses and no lavages in which neoplastic cells were misinterpreted as benign. A significant number of lavages, including several from patients with histologically confirmed peritoneal tumor, were sparsely cellular and/or excessively bloody. It is suggested that although peritoneal lavages might be collected during all gynecologic operations, only specimens from selected cases should be submitted for cytologic evaluation, and greater attention should be given to specimen collection to ensure that only well-preserved and representative material from the peritoneum is submitted for cytologic evaluation. Diagn. Cytopathol. 1998;18:265–269. © 1998 Wiley-Liss, Inc.     

Matrix metalloproteinase-1 expression is a prognostic factor for patients with advanced gastric cancer.

Proteolytic activity of cancer cells is an important factor in metastasis. This study examined the relationship between MMP-1 expression of gastric cancer cells and peritoneal metastasis. MMP-1 expression was found in 76/103 (75.2%) cases examined and was significantly associated with both peritoneal metastasis and lymph node metastasis (p<0.05, respectively). The prognosis of patients with MMP-1 positive tumor was significantly worse than that of patients with MMP-1 negative tumor (p<0.05). These findings suggested that MMP-1 might be a prognostic factor in case of advanced gastric cancer and might be useful in determining whether or not adjuvant therapy was indicated for patients at high risk of peritoneal recurrence.     

Peritoneal washing is an adequate source for somatic BRCA1/2 mutation testing in ovarian malignancies

Genetic screening for BRCA mutations should be offered to all women diagnosed with epithelial ovarian, fallopian tube, and/or peritoneal cancers given the implications for treatment options and cancer risk assessments. Yet, while germline breast cancer susceptibility gene 1 (BRCA1) and breast cancer susceptibility gene 2 (BRCA2) testing is commonly performed, BRCA1/2 somatic mutations testing is rather challenging since the poor quality of DNA extracted from formalin fixed paraffin embedded (FFPE) samples can significantly impair this process. Peritoneal lavage is routinely performed in surgeries of suspected ovarian malignancies. We have analyzed fresh tumor, peritoneal lavage and blood samples from ten patients and we have found an excellent agreement (88%) between fresh tumor and peritoneal lavage for BRCA mutation testing. Importantly, 112 of the 114 genomic alterations detected in fresh tumor samples were also found in peritoneal lavage fluids. Our data suggest that peritoneal washings can indeed streamline BRCA genes mutation testing procedures.     

腹腔镜下先天性无阴道合并盆腔包块切除腹膜代阴道成型术研究

目的探讨腹腔镜下畸形子宫合并肌瘤、卵巢囊肿切除、腹膜代阴道治疗先天性无阴道的可行性研究。方法先天性无阴道畸形子宫合并子宫肌瘤、子宫腺肌病、宫腔积血2例,卵巢肿瘤3例。在腹腔镜下子宫肌瘤、卵巢囊肿切除术,同时行腹膜代阴道成形术进行分析,观察手术中出血、手术时间、手术并发症及手术后疗效。结果5例患者中,1例腹膜部分撕裂,用前庭粘膜替代,1例直肠前壁损伤,术中修补。5例手术均获成功,术中平均出血量60．15±27ml,手术平均时间101．24min±40．16min,住院平均时间9．2天。术后随访6～24个月,人工阴道壁呈淡粉色,柔软,滑润,弹性好,具有正常阴道外观。结论腹腔镜下切除子宫肌瘤、卵巢囊肿,同时行腹膜代阴道成形术是一种安全、手术效果好并具有康复快的优点,是先天性无阴道畸形子宫合并肌瘤等切除术同时腹膜代阴道成形理想的手术方式。