应用RT-PCR并Southern杂交技术检测乳腺癌骨髓微转移

目的检测原发性乳腺癌骨髓微转移的发生及与其它临床参考指标的关系.方法应用逆转录-聚合酶链反应(RT-PCR)并Southern杂交技术,检测骨髓单个核细胞中细胞角蛋白19(CK-19)基因表达.结果52例骨髓标本共检出微转移19例(36.5%),其中17例淋巴结有转移者,9例CK-19表达阳性(52.9%);而无淋巴结转移的35例中,10例CK-19表达阳性(28.6%).微转移检出率与肿瘤大小有关(P＜0.05).结论以CK-19为标志物,RT-PCR并Southern杂交方法检测原发性乳腺癌骨髓微转移灵敏、特异,可做为临床判断预后的参考指标.     

美蓝染色法检测乳腺癌前哨淋巴结的临床研究

目的 寻找检测乳腺癌前哨淋巴结(SLN)方法，研究前哨淋巴结活检(SLNB)预测腋窝淋巴结状况的准确性。方法 对40例乳腺癌患者行美蓝染色法检测SLN，并行腋窝淋巴结清扫术(ALND)后，将腋窝淋巴结转移状况与SLN进行对比分析，分析SLN检出率及临床特征。结果 40例患者中成功检测出SLN35例，成功率为87．5％。检出SLN60个，平均每例检出SLNl．7个。2例出现假阴性，假阴性率为10.5％，无假阳性。35例患者中有16例SLN阳性，阳性率为45.7％。SLNB预测腋窝淋巴结(ALN)的敏感性为88．8％，特异性为100％，准确性为94．4％。结论 美蓝染色法检测乳腺癌SLN经济实用，可以较准确地预测腋窝淋巴结的转移状况。     

CK19mRNA测定在乳腺癌腋淋巴结微转移诊断中的应用

研究敏感的方法检测乳腺癌腋窝淋巴结微转移。方法:对15例乳腺癌者的61个腋淋巴结和5个正常对照淋巴结同时进行HE染色组织学检查和CKl9逆转录-聚合酶链式反应(CK19RT-PCR)检测。结果:15例乳腺癌组织均有CK19mRNA表达,而5个正常淋巴结中均末见表达。61个腋窝淋巴结中7个组织学检查证实转移,其CK19mRNA亦表达阳性。组织学检测无转移的54个淋巴结中有12个CK19RT-PCR表达阳性,42个阴性;提示该12个淋巴结存在微转移只能用CK19RT-PCR方法检测出。经统计学分析,CK19RT-PCR与组织学检查二者有显著差异(X~2检验,P<0.01)。结论:CK19表达于恶性乳腺细胞,正常淋巴细胞无表达,CK19能作为组织特异基因进行RT-PCR扩增以检测乳腺癌腋淋巴结微转移。CK19 RT-PCR方法比组织学方法更敏感,特别对在筛选组织学检查淋巴结阴性而具有高度复发危险性的病人将有实用价值。     

连续切片病理检查测定乳腺癌前哨淋巴结微小转移158例分析

近年来,不少学者观察到早期乳腺癌腋窝淋巴结转移率很低,进行腋窝淋巴结清扫(ALDN )并不是对所有患者都有意义[1].前哨淋巴结(SLN)可以代表腋窝淋巴结的转移状况,因此检测SLN的微小转移即可达到检测腋窝全部淋巴结的目的.我们应用连续切片方法检测SLN中的微小转移情况,探讨检测乳腺癌SLN微小转移的有效手段.     

乳腔镜前哨淋巴结活检术的临床应用

目的 探讨经乳腔镜前哨淋巴结活检的可行性及应用前景。方法 应用亚甲蓝染色法检测62例乳腺癌患者的前哨淋巴结(SLN)。在乳腔镜下切除SLN,随后行乳腔镜腋窝淋巴结清扫,SLN、腋窝淋巴结同时行HE染色,评价SLN检出率及假阴性率。结果 62例患者61例检出前哨淋巴结,成功率98．4％。无腋窝淋巴结转移者35例,转移27例,假阴性率0。结论 乳腔镜前哨淋巴结活检检出率高,美容效果好,并发症低,对于乳腺癌腋窝淋巴结转移有较高的敏感性,可以为绝大多数乳腺癌进行准确淋巴分期。     

高频B超与钼靶X线及不同生物学指标检测对诊断乳腺癌腋窝淋巴结转移准确性探讨

目的:探讨高频B超、钼靶X线摄影联合检测LOX、Ki-67、RSK4蛋白表达水平对诊断乳腺癌腋窝淋巴结转移准确性。方法:对2014年9月—2017年9月收治的80例经手术治疗、病理证实的乳腺癌腋窝淋巴结转移的患者临床资料进行回顾性分析,并结合血液样本进行生化检测,统计检测方法的准确性、敏感性及不同蛋白表达的相关性。结果:高频B超、钼靶X线摄影联合使用对诊断的准确性较两者单独使用明显提高。LOX、Ki-67、RSK4蛋白表达水平与乳腺癌腋窝淋巴结转移有关(P0.05)。高频B超与钼靶X线摄影联合检查以及LOX、Ki-67、RSK4蛋白水平检测与病理检测结果吻合度均较高(均к0.4,P0.05)。结论:高频B超与钼靶X线摄影联合使用能提高诊断乳腺癌淋巴结转移的准确性,LOX、Ki-67、RSK4蛋白的表达可作为诊断腋窝淋巴结转移的参考指标。     

乳腺癌前哨淋巴结活检对腋窝淋巴结状态的预测价值研究

为探讨乳腺癌前哨淋巴结（SLN）组织学特征对腋窝淋巴结状态的预测价值，笔者应用专利蓝或锝99m标记的大分子右旋糖苷（99mTc-DX）为示踪剂成功显示20例乳腺癌患者的SLN。术中先进行前哨淋巴结活检（SLNB），再行乳腺癌改良根治术。并应用常规病理（HE）、免疫组化（IHC）和逆转录聚合酶链反应（RT-PCR）方法检测腋窝淋巴结转移。结果示，20例患者共找到腋窝淋巴结254个，其中SLN48个，NSLN206个。常规病理证实3例患者7个前哨淋巴结有癌转移，NSLN206个均无癌转移。免疫组化染色检测到7例患者11个SLN CK-19表达阳性，2个NSLN表达阳性。RT-PCR检测CK-19mRNA14例患者30个SLN和22个NSLN表达阳性。提示乳腺癌前哨淋巴结活检能准确预测患者腋窝淋巴结的状态。     

Syk和SYK-mRNA在乳腺浸润性导管癌中的表达及其与腋窝淋巴结转移的关系

目的 探讨Syk及其编码基因SYK-mRNA在乳腺癌中的表达及其与腋窝淋巴结转移的相关性.方法 分别采用免疫组化SP法和原位RT-PCR方法 同步检测52例乳腺浸润性导管癌(其中有腋窝淋巴结转移者32例)和39例乳腺非癌组织中Syk蛋白和SYK-mRNA的表达.结果 Syk 和SYK-mRNA在乳腺癌中的阳性表达率分别为42.3%(22/52)和38%(20/52),均显著低于非癌组织.Syk和SYK-mRNA表达的一致率为91%.无淋巴结转移组的乳腺癌Syk和SYK-mRNA阳性表达率均显著高于有淋巴结转移组的乳腺癌,但两组的Syk和SYK-mRNA表达量差异均无统计学意义.结论 Syk(SYK-mRNA)的表达缺失与乳腺癌腋窝淋巴结转移有关,SYK-mRNA基因可能是乳腺癌的一种候选抑癌基因.     

HE、IHC和RT-PCR方法检测乳腺癌腋窝淋巴结转移的对照研究

目的　探讨检测乳腺癌腋窝淋巴结转移较敏感的方法。方法　应用常规HE染色法、细胞角蛋白19(CK19)作为单抗的免疫组化(IHC)SP染色及CK19 mRNA的逆转录聚合酶链反应(RT PCR)3 种方法检测20 例乳腺癌患者共239 个腋窝淋巴结的转移情况。结果　239 个腋窝淋巴结中,HE染色发现3 例患者7 个(2.9%,7/239)有癌转移,均位于level Ⅰ; IHC检测到7例患者13个(5.4%,13/239)有癌转移,其中11个位于level Ⅰ,2个位于level Ⅱ; RT PCR检测到14例患者52 个(21.8%,52/239)有癌转移,其中30 个位于level Ⅰ,22 个位于level Ⅱ。IHC和RT PCR检测证实,HE染色阳性的7 个淋巴结均有癌转移; IHC染色阳性的淋巴结均出现了PCR阳性扩增产物。HE、IHC及RT PCR 3 种检测方法对腋窝淋巴结微转移的检出率分别是0(0/232)、2.6%(6/232)及19.4%(45/232),P<0.05。结论　IHC及RT PCR是较HE检测淋巴结微转移更敏感的方法,而RT PCR较IHC更为敏感,能更准确地反映乳腺癌患者腋窝淋巴结的状况。     

超声联合空芯针穿刺活检在原发性乳腺癌腋窝淋巴结转移中的诊断价值

目的研究术前超声引导下淋巴结空芯针穿刺(US-CNB)在检测乳腺癌患者腋窝淋巴结转移中的诊断价值。方法回顾性研究2016年1月至2017年7月在我院行超声检查及超声引导下空芯针穿刺活检,并有腋窝淋巴结术后病理诊断的所有乳腺癌病人。计算US-CNB的敏感性、特异性、阳性预测值(PPV)、阴性预测值(NPV)、准确性,同时统计Kappa值以明确一致性情况。分析US-CNB结果与N和T分期的关系。结果以术后病理结果为金标准,US-CNB诊断淋巴结转移的敏感性为91.8%(90/98),特异性为58.3%(21/36),PPV为85.7%(90/105),NPV为72.4%(21/29),误诊率为41.7%(15/36),漏诊率为8.2%(8/98),约登指数为50.1%,准确性82.8%(111/134),ROC曲线下面积为0.751。比较US-CNB与术后病理的一致性,Kappa=0.535。排除新辅助化疗患者14例,特异性、PPV和误诊率分别为95.5%(21/22)、98.9%(90/91)以及4.5%,约登指数87.3%,准确性92.5%(111/120),ROC曲线下面积为0.936(P0.0001),比较排除后US-CNB与术后病理的一致性,Kappa=0.777。随着T和N分期的增加,US-CNB的敏感性增加。结论乳腺癌患者术前腋窝淋巴结空芯针穿刺可作为诊断腋窝淋巴结转移的可靠方法,与术后病理有较高的一致性。淋巴结穿刺结果阳性可能与乳腺癌较高肿瘤负荷相关。     

乳腺癌前哨淋巴结微转移的研究

目的：提高乳腺癌前哨淋巴结（SLN）病理诊断的准确性，为手术彻底切除肿瘤提供依据。方法：应用亚甲蓝生物染色的方法确定60例Ⅰ、Ⅱ期乳腺癌SLN并活检，44（73．3％）例SLN取材成功。每一枚SLN均进行冰冻病理切片、石蜡病理和角蛋白Keratinl9（CK-19）逆转录聚合酶链反应（RT—PCR）检测。结果：44例SLN冰冻病理切片、石蜡病理切片和CK-19诊断的灵敏度和特异度分别是77．8％和100．0％、88．9％和100．0％、100．0％和82．9％，诊断符合率分别为95．5％、97．7％和86．4％，诊断指数分别为0．778、0．889和0．829。结论：CK-19检测可进一步提高乳腺癌SLN微转移的检出率，提高SLN活检的准确性。但CK-19检测与冰冻病理病理检查联合应用可提高诊断的准确率和临床的可操作性。     

乳腺癌外周血CK-19mRNA的表达及临床意义

目的：研究外周血CK-19 mRNA表达与乳腺癌临床生物学指标的关系,探讨其作为乳腺癌微转移标志物的可能性,并判断手术和化疗对血液微转移的影响。方法：应用RT-PCR技术检测50例乳腺癌患者（乳腺癌组）术前、术后第2天、第1次化疗结束后以及术后9～12个月外周血中CK-19 mRNA的表达情况,并以20例乳腺良性疾病（良性对照组）和20例正常健康女性（正常对照组）作为对照。结果：术前乳腺癌组外周血CK-19 mRNA阳性率为24.00%（12/50）,其中0～Ⅰ期15.38%（2/13）Ⅰ,Ⅰ期20%（6/30）Ⅰ,Ⅱ期57.14%（4/7）Ⅰ,Ⅱ期乳腺癌CK-19 mRNA阳性表达率明显高于0～Ⅱ、Ⅰ期（P〈0.05）;有淋巴结转移者42.11%（8/19）表达阳性,无淋巴结转移者12.94%（4/31）表达阳性,二者差异有统计学意义（P〈0.05）。良性疾病组CK-19 mRNA的阳性率为5.00%（1/20）,正常对照组均阴性,乳腺癌组与良性疾病组和正常对照组比较差异有统计学意义（P均〈0.01）。乳腺癌组术后第2天CK-19 mRNA阳性率为26%（13/50）,与术前比较差异无统计学意义（P〉0.05）。术后首次化疗结束后CK-19 mRNA阳性率为12%（6/50）,化疗前后差异有统计学意义（P〈0.01）。综合治疗9～12个月后CK-19 mRNA阳性率为10%（5/50）,与术前比较差异有统计学意义（P〈0.01）。外周血CK-19 mRNA阳性率与雌激素受体（ER）、孕激素受体（PR）、CerbB2、p53状态无关。结论：CK-19 mRNA可作为检测乳腺癌患者外周血微转移的分子学标志物,有助于早期预测乳腺癌发生血道微转移,对指导治疗和评估预后具有重要意义。     

Do bone marrow micrometastases correlate with sentinel lymph node metastases in breast cancer patients?

BACKGROUND: Sentinel lymph node biopsies (SLNB) are used to detect axillary metastases as an important prognostic indicator for breast cancer patients. Bone marrow micrometastases (BMM) have also been shown to predict prognosis. This study examines whether SLNB and BMM are associated. STUDY DESIGN: A retrospective analysis was performed on 124 stages I to III breast cancer patients treated with mastectomy or lumpectomy, SLNB, and bone marrow aspiration between 1997 and 2003. SLNB were examined for the presence of metastases by hematoxylin and eosin (H&E) stains and also by immunohistochemistry (IHC) for lymph nodes negative by H&E. The kappa statistic was used to evaluate the association (agreement) between SLNB and BMM. RESULTS: In this study population, 36 patients (29%) had micrometastases detected in their bone marrow, and 51 patients (41%) had positive sentinel lymph nodes. Of the patients with positive BMM (n = 36), 53% (19 of 36) had positive SLNB (14 of 19 by H&E and 5 of 19 by IHC). In patients with negative BMM (n = 88), 36% (32 of 88) had a positive SLNB (27 of 32 by H&E and 5 of 32 by IHC). The kappa statistic and associated 95% confidence interval indicated poor agreement between SLNB and BMM (kappa = 0.15; 95% CI = -0.03, 0.32). CONCLUSIONS: There was poor agreement between axillary metastases and micrometastases detected in the bone marrow. This study suggests that BMM and axillary metastases are not concordant findings in most patients.     

The use of cytokeratin staining in sentinel lymph node biopsy for breast cancer.

BACKGROUND: Controversy exists regarding the routine use of cytokeratin immunohistochemistry (IHC) in the histopathologic examination of breast cancer sentinel lymph nodes (SLN) because the clinical significance of micrometastases detected by IHC is unclear. This analysis was performed to determine the frequency of IHC-detected micrometastases. METHODS: All patients underwent SLN biopsy, followed by completion axillary dissection. This analysis included patients who had SLN evaluated by IHC. SLN were examined by hematoxylin and eosin (H&E) stain at 2-mm intervals, with IHC in 2 sections. The axillary dissection specimen was evaluated by routine H&E staining. RESULTS: IHC was performed in SLNs from 973 patients. Of the 869 patients with negative nodes by H&E, 58 (6.7%) were "upstaged" by IHC. In 6 of 58 patients (10.3%) who had IHC-only positive SLN, nodal metastases were found in the axillary dissection specimen. CONCLUSIONS: IHC resulted in upstaging of 6.7% of patients who had negative SLN on H&E staining. These patients had a 10.3% risk of residual axillary nodal metastases. However, the clinical significance of IHC-only positive SLN requires further study.     

逆转录-聚合酶链反应检测乳腺癌腋淋巴结微转移的研究

目的探讨乳腺癌患者淋巴结内癌微转移灶的临床意义。方法采用逆转录－聚合酶链反应技术,扩增细胞骨架角蛋白１９（ＣＫ１９）,对１５例乳腺癌患者的癌组织及其６１个腋窝淋巴结进行检测。结果１５例患者乳腺癌组织均有ＣＫ１９ｍＲＮＡ表达,５个正常淋巴结均无表达。６１个淋巴结同时进行逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）及组织学检查,７个淋巴结病理证实有转移,其ＣＫ１９ｍＲＮＡ亦都表达阳性;病理未发现转移的５４个淋巴结中有１２个ＣＫ１９ｍＲＮＡ表达阳性。结论ＣＫ１９ＲＴ－ＰＣＲ方法检测乳腺癌腋淋巴结癌微转移比组织学检查敏感（χ２检验,Ｐ＜０．０１）,特别在筛选组织学检查淋巴结阴性而具有高度复发危险性的患者具有实用价值。     

骨髓与外周血中角蛋白19与乳腺癌微小转移的关系

目的探讨骨髓与外周血中角蛋白19与乳腺癌微小转移的关系。方法对26例乳腺癌患者的骨髓及外周血采用RT-PCR方法检测角蛋白19（keratin,KT19）mRNA的表达,同时以免疫组化法检测其骨髓涂片中上皮膜抗原（epithelial membrane antigen,EMA）。结果 26例患者KT19 mRNA表达中,外周血阳性4例（15.4%）,骨髓阳性10例（38.4%）,其中3例外周血与骨髓同时阳性,二者之间差异具有显著性意义（P＜0.05）。免疫组化结果显示26例患者骨髓中有7例（26.9%）EMA阳性,其KT19 mRNA均为阳性,有3例（11.5%）免疫组化结果阴性而KT19 mRNA表达阳性。结论 RT-PCR方法检测KT19 mRNA是一种比较敏感的方法,同时免疫组化还是一种比较可靠的检测方法。外周血中KT19 mRNA的检测结果还不能完全取代骨髓的微小转移情况。     

Immunohistochemistry analysis of micrometastasis in pretreatment lymph nodes from patients with esophageal cancer

BACKGROUND: With recent advances in neoadjuvant therapy in esophageal cancer, pretreatment lymph node staging has become increasingly important in stratifying patients to appropriate treatment regimens and for prognostication. Immunohistochemical analysis (IHC) using epithelial markers has been shown to identify micrometastases in histologically negative lymph nodes. We performed this study to evaluate if IHC analysis in thoracoscopic/laparoscopic (Ts/Ls) pretreatment staging lymph nodes can reveal additional diagnostic information to routine histopathology. METHODS: Specimens of 106 patients with esophageal cancer who had pretreatment Ts/Ls staging were retrospectively studied. Lymph node biopsies were obtained for IHC staining using cytokeratin (CK) of AE1/AE3. IHC staining for p53, an apoptosis protein associated with poor prognosis in esophageal cancer, was also performed. RESULTS: 331 Ts/Ls staging lymph node biopsies were collected from 106 patients. A total of 15.4% (51/331) of the lymph nodes or 34.9% (37/106) of patients were found to have metastatic deposits by routine histology. All the histologically positive lymph nodes were CK positive. Among the remaining 280 histologically negative lymph nodes, 11(3.9%) were found to have micrometastasis by CK staining. Three patients (4.3%, 3/69) were upstaged from N0 to N1. They died of early recurrences after treatment. A total of 67.6% (25/37) of the patients with histologically positive lymph node were p53 positive. No histologically negative lymph node was found to be p53 positive in this series. CONCLUSIONS: Immunohistochemical analysis for CK can detect micrometastatic involvement of lymph nodes that are missed on routine pathologic examination, and, therefore, can improve lymph node staging. Its clinical significance in esophageal cancer warrants further study.     

Does the Method of Biopsy Affect the Incidence of Sentinel Lymph Node Metastases?

More detailed examination of the sentinel lymph node (SLN) in breast cancer has raised concerns about the clinical significance of micrometastases, specifically isolated tumor cells detected only through immunohistochemical (IHC) staining. It has been suggested that these cells do not carry the same biologic implications as true metastatic foci and may represent artifact. A retrospective institutional review board-approved review was conducted on clinically node-negative breast cancer patients who underwent SLN biopsy (SLNB) between 1997 and 2003. Retrospective analysis of tumor characteristics and the method of the initial diagnostic biopsy were correlated with the presence and nature of metastatic disease in the SLN. Of 537 SLNBs, 123 (23%) were hematoxylin-eosin (H&E) positive. SLN positivity strongly correlated with tumor size (p<0.001) and tumor grade (p=0.025), but not with the method of biopsy (needle versus excisional biopsy). Prior to July 2002, we routinely evaluated H&E-negative SLNs with IHC (n=381). Of the 291 H&E-negative patients, 26 had IHC-only detected micrometastases (9%). The likelihood of detecting IHC-only metastases did not correlate with tumor size or grade, but was significantly higher in patients undergoing excisional biopsy than core needle biopsy. While the method of biopsy has no demonstrable effect on the likelihood of finding metastases in the SLN by routine serial sectioning and H&E staining, it may significantly impact the likelihood of finding micrometastases by IHC. IHC should not be used routinely in the evaluation of the SLN and caution should be used when basing treatment decisions (completion axillary lymph node dissection or adjuvant therapy) on IHC-only detected micrometastases.     

Comparison of Immunohistochemistry with Conventional Histopathology for Evaluation of Sentinel Lymph Node in Breast Cancer

The best method of pathological evaluation of sentinel lymph node in breast cancer has not been agreed upon. Immunohistochemical (IHC) techniques have shown a greater sensitivity over conventional histology for the detection of micrometastais. The aim of the study was to determine whether IHC for Epithelial Membrane Antigen (EMA) on the sentinel node could be more sensitive than conventional histology for diagnosing micrometastasis in sentinel lymph nodes. Eighty-four clinically node negative breast cancer patients underwent sentinel node biopsy at time of surgery for breast cancer. The node was subjected to conventional histopathology as well as IHC for EMA. The sensitivity of histology viz a viz IHC for EMA for detection of sentinel node metastasis was 88% and the specitficity was 96%. The overall diagnostic accuray of histology viz a viz IHC was 93%. There were 4 patients with micrometastasis (<2.0 mm), which were positive on IHC but negative on histology. Two patients with poorly differentiated breast cancer had a false negative IHC for EMA result as compared to histology. Immunohistochemistry for Epithelial Membrane Antigen can increase the detection rate of micrometastasis in sentinel lymph node. This can have important bearing on deciding the need of adjuvant systemic therapy. A false negative result for EMA may be seen in patients with poorly differential cancer. Therefore the best policy seems to employ both histopathology and IHC for EMA for the comprehensive evaluation of sentinel lymph node in breast cancer.