乳腺癌前哨淋巴结微转移分子检测及其临床意义

目的探讨乳腺癌前哨淋巴结(SLN)定位和SLN微转移检测的临床意义。方法对66例乳腺癌患者行术前Y探测仪SLN定位,用RTPCR法检测SLN中CK19mRNA的表达。同时与常规病检法比较其检测敏感性。并比较转移组、微转移组、无转移组患者的临床病理资料。结果SLN定位成功率为97%,RTPCR法与常规病检法转移的检出率相比较差异有统计学意义(P<0.05)。在常规病检阴性的38例淋巴结中,RTPCR法检出8例有微转移。同时乳腺癌转移组与微转移组患者在肿物大小与淋巴管浸润上有相似性,而同无转移组差异有统计学意义(P<0.05)。结论RTPCR法较常规病理检查更为敏感,通过SLN定位和RTPCR的联合使用,可明显提高乳腺癌SLN微转移的检出率。同时也证明RTPCR法是可靠的,SLN微转移有可能作为肿瘤预后的指标。     

宫颈癌前哨淋巴结活检的假阴性原因分析

目的探讨染料法识别宫颈癌前哨淋巴结（SLN）活检时出现假阴性的原因。方法选择49例早期宫颈癌患者,术前宫颈瘤周注射亚甲蓝,行广泛子宫切除＋盆腔淋巴结清扫术;进行前哨淋巴结定位及病理学检查。结果SLN识别率为87．8％（43／49）,灵敏度为81．8％,准确率为92％,假阴性率为18．2％。结论本组假阴性与肿瘤大小、淋巴转移的途径、术前放疗、病理检测方法有关。     

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。     

hMAM在乳腺癌前哨淋巴结中的表达及其临床意义分析

探讨人乳腺球蛋白(human mammary globulin,hMAM)在乳腺癌前哨淋巴结(sentinel lymph node,SLN)中的表达及其临床意义。RT-PCR检测20例乳腺癌组织及正常淋巴结组织中hMAM的表达;SLN定位后通过RT-PCR与常规病理检测SLN转移情况,并进行比较;分析SLN转移与临床病理的关系。乳腺癌组织中有18例过表达,表达率为90.0%,而正常淋巴结中hMAM不表达,两者差异有统计学意义(P<0.01)。常规病理检查转移率为50.98%,RT-PCR法检测的阳性率提高到70.59%,RT-PCR法比常规病理检测的检出率高19.61%,两者之间差异显著(χ2=38.28,P<0.01)。SLN无转移组、微转移组、转移组与肿瘤位置、ER表达及病理类型之间差异均不显著(P>0.05)。hMAM可作为辅助判断SLN是否转移的标志。     

甲状腺乳头状癌CD151和CK19mRNA表达及意义

目的探讨甲状腺乳头状癌中CD151和CK19 mRNA表达及其意义。方法采用RT—PCR方法检测54例甲状腺的良恶性病变组织中CD151和CK19 mRNA的表达,并与临床及病理资料进行分析比较。结果CD151和CK19 mRNA在甲状腺乳头状癌组中的表达与滤泡性腺瘤组、结节性甲状腺肿组以及正常甲状腺组比较均差异具有显著性（P〈0．01）;CK19 mRNA表达与甲状腺癌淋巴结转移有关（P〈0．05）;在甲状腺乳头状癌中,CD151和CK19基因mRNA的表达呈正相关（P〈0．01）。结论CD151和CK19基因共同参与甲状腺癌的发生、发展,并对淋巴结转移的判断有意义。     

乳腺癌前哨淋巴结术中分子诊断的临床实用性初探

目的 探讨GeneSearchTM乳腺淋巴结检测试剂盒(以下简称GeneSearch)在乳腺癌前哨淋巴结(SLN)术中诊断的临床实用性.方法 对复旦大学附属肿瘤医院2009年2月至6月诊治的88例乳腺癌患者行SLN活检.首先垂直长轴将所得淋巴结切成数块厚约2 mm的组织块,对各切面进行术中细胞印片后,奇数号组织块用于术后连续切片检查,偶数号组织块采用GeneSearch进行检测,应用即时荧光定量逆转录聚合酶链反应检测SLN中CK19和乳腺球蛋白表达的Ct值.将GeneSearch以术后连续切片的诊断为准,与术中细胞印片、术后连续切片的病理结果分别进行比较.结果 88例共获得225枚SLN,其中宏转移淋巴结27枚,微转移淋巴结9枚,阴性淋巴结189枚(其中5枚为孤立肿瘤细胞).从切割淋巴结开始到最终形成报告,GeneSearch耗时范围为35～45 min(平均40 min).基于淋巴结数目,GeneSearch与术后连续切片的总体符合率为95.6%(215/225),其检测敏感度为86.1%(31/36),均高于术中细胞印片[分别为94.7%(213/225)和72.2%(26/36)].SLN转移灶大小与CKl9和乳腺球蛋白的Ct值存在统计学相关性(P＜0.01).结论 GeneSearch用于SLN术中诊断时,其检测敏感度高于术中细胞印片,达到比较满意的效果,但在应用中仍存在一些问题.     

实时荧光定量PCR检测乳腺癌SOCS2基因表达

目的检测乳腺良恶性组织中细胞因子信号转导抑制因子（supressors of cytokine signaling,SOCS2）的mRNA表达及其临床病理意义。方法以18SrRNA为内j对照采用实时荧光定量PCR法,定量测定新鲜乳腺癌组织、癌旁组织和乳腺良性增生组织中。SOCS2 mRNA的表达并分析其与临床病理指标的关系。结果各组阳性率：乳腺癌35.00％（14／40）、癌旁乳腺组织100％（15／15）和乳腺良性增生组织81.25％（13／16）,其表达之间的差异具有统计学意义（P〈0．05）;乳腺癌组织的SOCS2表达水平与TNM分期和淋巴结转移呈明显的相关性（P〈0,05）。结论SOCS2在乳腺癌中的表达可能提示患者具有较好的预后。     

乳腺癌组织蛋白酶D和PSA表达与同侧腋淋巴结转移的关系

目的：探讨乳腺癌中组织蛋白酶Ｄ（ｃａｔｈｅｐｓｉｎＤ，ＣＤ）和前列腺特异性抗原（ＰＳＡ）的表达与同侧腋淋巴结转移的关系。方法：应用ＬＳＡＢ免疫组化法检测ＣＤ和ＰＳＡ在７８例乳腺癌中的表达，其中伴同侧腋淋巴结转移者３６例。结果：（１）７８例乳腺癌ＣＤ阳性表达率４１．０３％（３２／７８），ＰＳＡ阳性表达率４３．５％（３４／７８）；（２）同侧腋淋巴结转移组ＣＤ阳性表达率６９．４４％（２５／３６），无同侧腋淋巴结转移组ＣＤ阳性表达率１６．６７％（７／４２），差异有高度显著性（Ｐ＜０．０１）；（３）同侧腋淋巴结转移组ＰＳＡ阳性表达率２５．０％（９／３６），无同侧腋淋巴结转移组ＰＳＡ阳性表达率５９．５２％（２５／４２），差异有高度显著性（Ｐ＜０．０１）。结论：ＣＤ和ＰＳＡ的表达可作为乳腺癌预后检测的参考指标。ＣＤ表达阳性率与同侧腋淋巴结转移呈正相关，与预后负相关；而ＰＳＡ表达阳性率与同侧腋淋巴结转移呈负相关，与预后正相关     

乳腺癌非前哨淋巴结转移的预测

目的 :预测非前哨淋巴结 (non SLN)转移 ,以筛选出转移局限于前哨淋巴结 (SLN)的乳腺癌患者。方法 :采用99mTc SC作为示踪剂 ,对 95例乳腺癌患者行前哨淋巴结活检 ,对乳腺癌非前哨淋巴结转移进行单因素和多因素分析。结果 :95例患者中成功发现 91例患者有SLN (95 8% ) ,其中 85例患者SLN能准确反映腋窝淋巴结的病理状况 (93 4% )。临床肿块大小(P =0 0 2 8)、肿瘤分级 (P =0 0 40 )和原发灶cyclinD1蛋白 (P =0 0 17)的表达与non SLN转移显著相关。而Logistic多因素分析证实 ,临床肿块大小、肿瘤分级为独立的预测非前哨淋巴结转移的因子。结论 :可根据临床病理学特征 ,筛选出乳腺癌转移只局限于前哨淋巴结的患者 ,也存在免除腋窝淋巴结清扫的可能性     

乳腺癌P-pg、MRP表达与腋窝淋巴结转移的关系

目的 探讨乳腺癌组织中P-gp、MRP表达与腋窝淋巴结转移的关系。方法 34例乳腺浸润性导管癌患者接受回顾性研究，其中18例经病理证实腋窝淋巴结转移。免疫组织化学技术分别检测术后癌组织标本中P-gp、MRP表达，比较P-gp阳性组和阴性组患者间、MRP阳性组和阴性组患者间腋窝淋巴结转移的差异。结果 34例乳腺癌患者中，P-gp阳性表达率64．71％（22／34），MRP阳性表达率38．24％（13／34），两者共表达率20．59％（7／34）。P-gp表达阳性22例中，腋窝淋巴结转移12例，P-gp阴性表达12例，腋窝淋巴结转移6例，两组间腋窝淋巴结转移差异无统计学意义（P=1．000，双侧）；MRP表达阳性13例中，腋窝淋巴结转移8例，MRP阴性表达21例，腋窝淋巴结转移10例，两组间腋窝淋巴结转移差异无统计学意义（P=0．433，双倒）。结论 乳腺浸润性导管癌P-gp、MRP表达与腋窝淋巴结转移无关；P-gp、MRP表达不能赋予恶性肿瘤细胞更强的侵袭能力。     

One-step nucleic acid amplification (OSNA) for intraoperative evaluation of sentinel lymph node status in breast cancer: a comparative study between CK19 protein expression and CK19 mRNA level in primary tumors and lymph node metastasis

The one-step nucleic acid amplification (OSNA) method is an increasingly used procedure for intraoperative analysis of sentinel lymph node (SLN) status in breast cancer patients. It measures cytokeratin19 (CK19) mRNA copy numbers in homogenized samples of SLN; CK19 has been chosen for identifying node metastasis because most breast cancers express this molecule. However, to avoid false-negative OSNA results, testing the preoperative needle core biopsy (NCB) of breast carcinomas for CK19 by immunohistochemistry (IHC) has been recommended. This procedure relies on the assumption that protein expression is strictly related to mRNA expression. We developed this study to evaluate if IHC gives similar result to the molecular assay. In a series of breast cancer patients with axillary metastasis, corresponding surgically resected tumor and metastatic lymph node specimens have been tested for CK19 protein by IHC and for CK19 mRNA by real-time PCR; furthermore, CK19 immunostaining has been performed in NCBs when available. Statistical analysis revealed that (1) the immunohistochemical evaluation of CK19 in NCB is a reliable test, reflecting protein expression in the whole tumor and in the metastatic lymph node; (2) there is no correlation between CK19 protein expression and CK19 RNA level neither within the primary breast cancer nor within the metastatic node; moreover, no correlation as well has been found between protein expression in NCB and mRNA level in metastatic lymph nodes. Thus, our results suggest that there is no evidence-based reason to stain every NCB for CK19 before performing OSNA in patients with breast cancer.     

Importance of assessing CK19 immunostaining in core biopsies in patients subjected to sentinel node study by OSNA

Analysis of sentinel lymph node (SLN) by means of One-Step Nucleic Acid Amplification (OSNA) is being used increasingly as a very sensitive and quick method for intraoperative axillary staging in patients with breast cancer. This molecular diagnostic assay detects the expression level of cytokeratin 19 (CK19), a luminal epithelial cell marker broadly expressed in most breast carcinomas and not normally found in lymph nodes. Almost all breast cancers express this cytoskeleton protein, but some breast tumors have been found to lose the expression of CK19. CK19 immunostaining in core biopsies has been recommended in selecting patients eligible for OSNA analysis because SLNs with metastatic involvement by CK19-negative breast cancers may result in a false negative result by OSNA. However, the real frequency of CK19-negative breast cancer has to be elucidated. In this study, we have assessed the frequency and molecular profile of CK19-negative breast carcinomas in three series of cases. The first is a prospective series of 197 breast carcinomas, 111 of which were subjected to SLN evaluation by OSNA. The second is a retrospective series of 41 triple-negative (TN) breast carcinomas, and the third is a retrospective series of 68 breast cancer patients (matched core biopsies and metastatic lymph nodes) that had been evaluated by conventional procedures before the OSNA methodology was adopted in our institution. Our results not only demonstrate that lack of expression of CK19 is infrequent in breast cancers but also that performing CK19 immunohistochemical staining is important on diagnostic core biopsies in taking the decision of using OSNA methodology in the evaluation of sentinel nodes in breast cancer patients.     

Intraoperative frozen section examination of axillary sentinel lymph nodes in breast cancer

The study presents the results from intraoperative frozen section assessment of axillary sentinel lymph nodes (SLNs) in breast cancer. Routine histological frozen sections from one level were used, two sections stained with haematoxylin and eosin. Immunohistochemistry for cytokeratins was applied to the permanent SLN paraffin sections only. Axillary dissection was performed on all SLN-positive cases regardless of the size of the metastatic deposits. With a detection rate of 83%, 272 patients entered the study over a period of 46 months. A total of 61 cases were SLN positive by frozen section analysis. The paraffin sections gave an additional 23 SLN-positive cases. The false-negative rate for frozen sections was then 27% (23/84). Micrometastases were found in 28 of 84 cases, and macrometastases in 56. The false-negative rate of frozen sections for micrometastases was 71% (20/28), and for macrometastases 5% (3/56). A total of 73% (61/84) of the patients underwent axillary surgery as a one-step procedure.     

False-positive cells in sentinel lymph nodes

Melanoma sentinel lymph nodes (SLN) are carefully evaluated to maximize sensitivity. Examination includes hematoxylin and eosin (H+E) stained sections at multiple levels through the node, with subsequent immunohistochemical (IHC) stains for melanocytic markers if H+E sections are negative for melanoma. However, not all IHC-positive cells in SLN are metastatic melanoma, as evidenced by the presence of MART-1 positive cells in SLN from breast cancer patients with no history of melanoma (so-called 'false-positive' cells). These 'false-positive cells' could be nodal nevus, non-melanocytic cells with cross-reacting antigenic determinants, phagocytic cells containing melanocyte antigens, or possibly melanocytes or melanocyte stem cells liberated at the time of biopsy of the cutaneous melanoma. Examination of SLN requires careful correlation of H+E and IHC findings.     

mRNA markers of breast cancer nodal metastases: comparison between mammaglobin and carcinoembryonic antigen in 248 patients

Histological detection of axillary lymph node metastases is still the most valuable prognostic parameter for breast cancer, but about 30% of node-negative patients relapse within five years, suggesting that current methods are inadequate for identifying metastatic disease. More sensitive, PCR-based methods for the detection of metastatic cells are now available, enabling the amplification of cancer cell-specific mRNA messages by the RT-PCR assay. An ideal tumour marker, consistently expressed in tumour samples and not at all in normal lymph nodes, remains to be identified. The present study first investigated the expression of seven mRNA markers, CEA, CK19, c-Met, mammaglobin, MUC-1, beta1-->GalNAc-T and p97, selected on the basis of their previously reported specificity for breast cancer cells. Eighteen lymph nodes were examined from patients without tumours. Only mammaglobin mRNA and CEA mRNA were not expressed in normal nodes. All of the other markers showed a band of expression in 17%-55% of cases, indicating that they are not breast cancer-specific. CEA mRNA and mammaglobin mRNA expression could be detected in 15/20 (75%) and 19/20 (95%) primary breast carcinomas, respectively. The expression of mammaglobin mRNA and CEA mRNA was then compared in axillary lymph nodes from 248 consecutive breast cancer patients, 89 with histologically documented lymph node metastasis and 159 without histological evidence of metastatic disease. Ninety-seven per cent of the patients with histologically involved nodes showed expression of mammaglobin mRNA, whereas CEA mRNA was expressed in 79% of these cases. In the group of patients with histologically negative lymph nodes, 46 (29%) and 32 (20%) were found to be positive for mammaglobin and CEA expression, respectively, indicating the presence of metastases not detected by routine histological examination of one lymph node section. These results show that both mammaglobin RT-PCR and CEA RT-PCR are useful tools for the detection of breast cancer metastases in axillary lymph nodes. The detection sensitivity of the mammaglobin RT-PCR is far superior to that of the CEA RT-PCR, allowing the diagnosis of occult metastases in nearly one-third of cases.     

Rapid intraoperative immunohistochemical evaluation of sentinel lymph nodes for metastatic breast carcinoma.

OBJECTIVE: Sentinel lymph node (SLN) biopsy is an integral part of the surgical management of patients with breast cancer. Rapid immunohistochemistry (RIHC) has the potential to increase detection of metastatic carcinoma at the time of frozen section consultation. The authors assessed the accuracy and turnaround time of a newly developed RIHC method for pancytokeratin (RIHC-CK). METHODS: Sixty-six SLNs from 32 patients with breast carcinoma were examined for metastasis using the Zymed Sentinel Lymph Node Rapid IHC Kit. Intraoperative frozen sections (6 mum) of the SLNs were incubated with Zymed anti-pan-cytokeratin/HRP conjugate, diaminobenzidine (DAB), and stained with hematoxylin. Slides were ready within 8 minutes and were interpreted as positive or negative for metastatic carcinoma. Results were compared with previous intraoperative touch preparations, frozen sections, hematoxylin and eosin (Perm H&E), and AEl/3-immunostained permanent sections (Perm CK). RESULTS: Fourteen lymph nodes (19%) in 13 patients tested positive for metastatic carcinoma in Perm H&E, the gold standard. RIHC-CK had the highest sensitivity (92%) of the intraoperative tests, compared with touch preparations (64%) and frozen sections (80%). RIHC-CK showed 94% accuracy, compared with 96% (frozen section) and 93% (touch preparation). The RIHC technique took 8 minutes and was easy to perform and interpret. CONCLUSIONS: Zymed RIHC is a sensitive method for detecting breast cancer metastases in SLNs. The speed, accuracy, and ease of interpretation of the test allow for recognition of micrometastases (<2 mm) that might otherwise be undetectable by current methods of intraoperative evaluation. The prognostic significance and effect on surgical management of micrometastases in SLNs have yet to be determined.     

CK19表达及其在喉鳞癌淋巴结微转移诊断中的应用

目的:研究用免疫组化方法检测CK19及其在喉鳞癌淋巴结微转移诊断中应用与临床病理意义。方法:对40例喉鳞癌患者及清扫淋巴结163个(常规临床检查颈部淋巴结阴性pN₀)进行常规病理检查(HE染色)和抗角蛋白19抗体的免疫组化检测。结果:40例喉鳞癌组织中CK19均表达阳性,40例颈淋巴结常规病理检查(HE染色)发现5例有转移者(共23枚淋巴结),CK19检测也为阳性。19个淋巴结常规病理检查未发现转移者,CK19也表达阳性。随着肿瘤T分期的增加,淋巴结CK19表达阳性率亦增加。CK19表达阳性者预后较阴性者差。结论:CK19免疫组化法是检测喉鳞癌淋巴结微转移的敏感而便捷的方法,而检测喉鳞癌微转移有助于判断肿瘤进展程度与预后,特别对筛选组织学检查淋巴结阴性但存在微转移的病人有实用价值。