应用RT-PCR检测CK19表达提高乳腺癌前哨淋巴结微转移检出的研究

目的:评价乳腺癌前哨淋巴结活检的临床意义,应用细胞角蛋白cytokeratin19(CK19)RT-PCR检测法提高其的敏感性及准确率.方法:随机选取1999年10月至2003年3月住院的乳腺癌患者73例,其中2001年1月至2003年3月的29例,对前哨淋巴结(SLN)进行了CK19 RT-PCR检测.采用专利兰进行SLN识别.RT-PCR法检测CK19 mRNA表达.结果:SLN识别成功67例,成功率为91.8%(67/73).对上述67例的SLN转移的常规病理诊断的敏感度68.8%(22/32),准确率85.1%(57/67).在行CK19 RT-PCR的29例中,检测出常规病理未检出的微小转移病例8例.敏感度90.0%(18/20),准确率93.1%(27/29).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CK19 RT-PCR检测SLN的微转移,可提高敏感度及准确率.     

Ⅰa-Ⅱa期宫颈癌患者盆腔淋巴结中高危型HPV DNA及CK19 的检测及其意义

目的:分别采用原位杂交法和免疫组化法检测早期宫颈癌盆腔淋巴结中高危型HPV DNA和CK19的表达,探讨早期宫颈癌微小转移的检测率及检测方法.方法:选取28例早期宫颈癌患者常规病理光镜检查证实转移和未转移淋巴结共104个,分别采用原位杂交法和免疫组化法检测高危型HPV DNA和CK19的表达.结果:所有常规病理检查阴性的80个淋巴结中高危型HPV DNA的阳性检出率为45%(36/80);CK19的检出率为25%(20/80).常规病理光镜检查证实淋巴未转移患者的57个淋巴结中23个高危型HPV DNA阳性(43.5%),来自15名患者中的9 人(60%);14个淋巴结CK19阳性(24.6%), 来自15名患者中的7名(46.6%).患者的微转移检出率分别为60%和46.6%.两种检测方法的结果有较好的一致性.高危型HPV DNA的检出率高于CK19(P＜0.001).结论:原位杂交法检测高危型HPV DNA,免疫组化法测CK19均可检测出早期宫颈癌淋巴微转移 ,原位杂交法是分子水平的检测,在宫颈癌淋巴微转移的检测中可能更敏感可靠.     

结直肠癌前哨淋巴结微转移分子检测及临床意义

目的:探讨结直肠癌前哨淋巴结(sentinellymphnode,SLN)定位和前哨淋巴结微转移检测的临床意义。方法:对52例结直肠癌患者,术前用异硫蓝标记法标记SLN并定位,用RT-PCR法检测SLN中CK20mRNA的表达。同时与常规病检法比较其检测敏感性。并分析结直肠癌转移与各种病理因素关系。结果:SLN定位成功率为96%,SLN状态与非SLN的符合率为100%。RT-PCR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=0·039。在常规病检阴性的40例淋巴结中,RT-PCR法检出8例有微转移。结直肠癌转移与肿瘤侵袭深度、分化程度、Duke’s分期密切关系。结论:SLN技术运用于结直肠癌将更方便、更准确判断淋巴结转移情况。RT-PCR法较常规病理检查更为敏感,通过SLN定位和RT-PCR的联合使用,可明显提高结直肠癌SLN微转移的检出率。并提高结直肠癌临床分期,为结直肠癌进一步治疗提供理论依据。     

外周血CK19 mRNA在乳腺癌治疗中的意义

目的 探讨乳腺癌患者外周静脉血CK19 mRNA检测对判断乳腺癌微转移的应用价值.方法 采用RT-PCR法检测110例乳腺癌及45例乳腺良性疾病患者外周血中CK19 mRNA,并比较乳腺癌患者外周血CK19 mRNA阳性表达率与乳腺良性疾病患者的差异,以及不同分期及治疗前后乳腺癌患者外周血CK19 mRNA阳性表达率的差异.结果 乳腺癌患者外周血CK19 mRNA阳性表达率明显高于乳腺良性疾病者,Ⅲ、Ⅳ期乳腺癌患者阳性表达率明显高于Ⅰ、Ⅱ期患者;乳腺癌患者治疗前CK19 mRNA阳性表达率明显高于治疗后;有淋巴结转移者CK19 mRNA阳性表达率明显高于无淋巴结转移者,差异均有统计学意义(P<0.01).结论 外周血CK19 mRNA检测可以预测乳腺癌发生的远处转移及微转移.     

早期非小细胞肺癌前哨淋巴结微转移检测的应用研究

目的 目前,尚无检测技术可准确判断肺癌前哨淋巴结(sentinel lymph node,SLN)微转移.本研究探讨CK19和MAGE A3表达与非小细胞肺癌(non-small cell lung cancer,NSCLC) SLN微转移的相关性及临床价值.方法 选择山东大学附属山东省肿瘤医院胸外科32例接受手术治疗的临床Ⅰ～ⅡA期NSCLC患者,术中联合应用染色法(异舒泛蓝溶液)和放射同位素法(99 Tc硫胶体检测)找寻SLN,并采用免疫组化技术检测SLN及非前哨淋巴结(non-sentinel lymph node,nowSLN)中CK19和MAGE-A3抗体的表达.结果 32例患者均检测出SLN,共清除淋巴结598枚,其中SLN 103枚,non-SLN 495枚.平均每例患者清除淋巴结(18.69±8.13)枚,清除SLN(3.22±1.74)枚.免疫组化法检测到20例患者44枚SLN中CK19表达阳性,19例患者31枚SLN中MAGE-A3抗体表达阳性.SLN免疫组化检查阳性率为42.72％,明显高于常规HE染色的阳性率(25.24％),P=0.01.SLN的阳性表达率与临床病理分期有关,P＜0.05;而与性别、年龄、肿瘤部位、分化程度、肿瘤大小和肿瘤类型无关,P＞0.05.结论 CK19和MAGMA3是判断淋巴结微转移较好的分子标志物,通过免疫组化技术检测SLN中CK19和MAGE-A3表达有助于评估区域淋巴结微转移状况.     

非小细胞肺癌患者外周血CD44V6 mRNA表达与微转移的相关性研究

目的 探讨CD44V6 mRNA表达与非小细胞肺癌(NSCLC)外周血微转移的关系及临床意义,及其与CK19 mRNA在外周血中联合检测的临床意义.方法 应用免疫组化SP法检测56例NSCLC癌组织中CD44V6 mRNA的表达情况,应用逆转录聚合酶链反应(RT-PCR)技术检测外周血中CD44V6 mRNA与CK19 mRNA的表达情况.采集20例肺部良性病变患者正常肺组织和外周血作对照.结果 CD44V6 mRNA在NSCLC癌组织中表达明显高于良性病变正常肺组织(P<0.001),其阳性表达率在各病理分期间、有无淋巴结转移组间的差异具有统计学意义(P<0.05),而在各病理类型间差异无统计学意义(P>0.05).NSCLC患者外周血中CD44V6 mRNA阳性表达率高于对照组(P<0.05),其阳性表达率在各病理分期间、有无淋巴结转移间的差异具有统计学意义(P<0.05).CD44V6 mRNA在NSCLC组织中表达与外周血中表达呈正相关,NSCLC外周血中CD44V6 mRNA与CK19 mRNA表达呈正相关;CD44V6 mRNA与CK19 mRNA联合检测灵敏度为75.0%,优于单基因检测(P<0.05).结论 NSCLC外周血中CD44V6 mRNA的高表达与其侵袭转移有关,CD44V6 mRNA可作为检测NSCLC外周血微转移的分子肿瘤标志物,并有望成为判定NSCLC预后的分子标志物;CD44V6 mRNA与CK19 mRNA联合检测可提高NSCLC血行微转移诊断的敏感度.     

前哨淋巴结微转移分子检测对保乳治疗的 预后判断价值

 目的　探讨乳腺癌前哨淋巴结(SLN) 定位活检及微转移检测对保乳治疗的预后判断价值。方 法　对83 例临床考虑保乳患者于术前3～4 h 在肿瘤处皮下或瘤床内注射入99m Tc2DX ,术中用γ探测仪 定位并切除SLN ,再行象限切除+ 腋淋巴结清扫(ALND) 。术后淋巴结行常规病检。RT2PCR 检测 CK19 mRNA 以判断SLN 有无微转移。术后均行辅助性全身化疗+ 放射治疗,随访患者的临床转归, 所有数据行χ2 检验。结果　83 例中腋窝SLN 定位失败4 例,79 例病人共定位SLN127 个, 常规病检 SLN 阳性率为26. 6 %(21/ 79) ,其中微转移检出率13. 9 %(11/ 79) ,RT2PCR 检测微转移均阳性。SLN (89 个) 阴性病人58 例,RT2PCR 检测,SLN 阳性病人14 例,阳性率为31. 6 %(25/ 79) 。常规病理检查与 RT2PCR 检测结果差异( P < 0. 01) 。79 例病人中微转移阳性组5 年远处转移率、生存率与阴性组之间 差异有统计学意义( P < 0. 05) ,但局部复发率和3 年生存率差异无统计学意义( P > 0. 05) 。结论　RT2 PCR 方法是检测淋巴结微转移的敏感方法;SLN 微转移有可能作为判断乳腺癌保乳治疗的一个独立预 后因素。     

肺癌术中肺血管结扎顺序对肺癌微转移影响的研究

目的:探讨肺癌患者术中肺血管结扎顺序对肺癌微转移影响及临床意义.方法:60例非小细胞肺癌手术患者,术前随机分为先扎动脉组和先扎静脉组,每组30例,术前、术中和术后取患者外周静脉血,巢式RT-PCR方法检测外周血中CK19 mRNA、LUNX mRNA表达.选取10名健康成年人(健康组)外周静脉血做阴性对照.结果:术前先扎动脉组患者外周血中CK19 mRNA、LUNX mRNA表达率与健康组比较,差异均有统计学意义(P<0.05),术前先扎静脉组患者外周血中CK19 mRNA、LUNX mRNA表达率与健康组比较,差异均有统计学意义(P<0.05),而先扎动脉组和先扎静脉组间术前比较差异均无统计学意义(χ2=0.000,P=1.000),但术中与术后比较差异均有统计学意义(P<0.05).CK19 mRNA、LUNX mRNA在术前、术中、术后间差异有统计学意义(P<0.05),而CK19 mRNA、LUNX mRNA两者之间差异无统计学意义(P>0.05).腺癌与鳞癌间差异无统计学意义(P>0.05).Ⅰ、Ⅱ和Ⅲ期肺癌间差异有统计学意义(P<0.05).结论:肺癌术中先结扎肺静脉可减少癌细胞的微转移.CK19 mRNA、LUNX mRNA可以作为检测肺癌外周血中癌细胞微转移的2个指标,预测肺癌微转移.     

免疫组织化学和RT-PCR法检测乳腺癌骨髓和前哨淋巴结微小转移的临床研究

目的探讨免疫组织化学(IHC)和RT-PCR法检测乳腺癌骨髓和前哨淋巴结(SLN)微小转移的灵敏度及临床意义。方法留取乳腺癌改良根治术腋窝淋巴结HE染色证实阴性的病人的胸骨骨髓血和SLN,分别采用IHC和RT-PCR方法检测其微小转移情况。结果62例中,骨髓样本RT-PCR检测15例阳性表达,其中9例IHC检测也为阳性,二者结果有较好一致性(kappa=0.6945),检出率有统计学差异(P=0.0412);SLN样本RT-PCR法有13例KT19mRNA表达,其中7例IHC检出KT19阳性细胞,二者结果一致性较好(kappa=0.6483),检出率有统计学差异(P=0.0412);骨髓和SLN同时表达KT19mRNA仅3例,无显著相关(P=0.796);原发肿瘤大小和骨髓KT19mRNA表达率有关联(P=0.003)。结论常规检查未发现远处和腋窝淋巴结转移,骨髓和SLN可检出微小转移,RT-PCR较IHC更灵敏,肿瘤大小与骨髓微小转移有关联。由于骨髓和腋窝淋巴结微小转移不一定同步出现,选用灵敏方法对不同组织同时进行检测可能更具临床价值。     

巢式RT-PCR检测乳腺癌前哨淋巴结微转移的研究

目的：探讨乳腺癌前哨淋巴站（SLN）定位和腋淋巴结微转移检测的临床意义,方法：对20例乳腺癌患者术中肿块周围注射美蓝定位前哨淋巴 结,用巢式RT－PCR法检测腋淋巴结中Mammaglobin mRNA的表达,结果：SLN定位成功率为85．0％（17／20）,SLN与非SLN组微转移的检出率具显著差异（P＜0．01）。在常规病检阴性的淋巴结中,巢式RT－PCR法微转移的检出率为15．6％（17／109）,结论：巢式RT－PCR法较常规病理检查更为敏感,通过SLN定位和巢式RT－PCR的联合使用,可明显提高乳腺癌腋淋巴结微转移的检出效率。     

Dukes B 期大肠癌前哨淋巴结微转移的定位检测及临床意义*

目的:探讨大肠癌前哨淋巴结（sentinel lymph node ,SLN ）微转移（micrometastasis,MM）的检测方法及其临床意义。方法:前瞻性研究64例行根治性手术的Dukes B期大肠癌患者,对其中61例定位成功的122 枚SLN 应用常规HE染色联合免疫组化SP法,检测其前哨淋巴结中细胞角蛋白20（CK20）及端粒酶的表达,随访3 年,记录患者的临床病理参数和生存资料,分析其相关性。结果:1）61例中有6 例患者9 枚SLN 经常规HE检测阳性。2）免疫组化法检测SLN :CK20阳性表达27.3%（15/55）;端粒酶阳性表达21.8%（12/55）。 3）两者联合检测前哨淋巴结微转移（SLNMM）检出率为38.2%（21/55）。 4）Dukes B期患者SLNMM（+）组癌复发转移率明显高于同期SLNMM（-）组（P<0.05）,生存率较低（P<0.05）,而与Dukes C期对比,差异无统计学意义;SLNMM（-）组患者的复发转移率、生存率与同期Dukes C期患者对比,有显著性差异（P<0.05）。 结论:CK20、端粒酶免疫组化法均可检测出大肠癌SLN 中存在的微转移（MM）,两者联合可提高检出率;大肠癌SLNMM的检出使大肠癌的Dukes分期更加精细,从而有助于指导术后的辅助治疗和预后判断。      

美蓝染色法鉴别哨兵淋巴结及其临床意义

目的：探讨美蓝染色法鉴别哨兵淋巴结（SLN）的可行性及其活检的临床意义。方法：采用美蓝染色法，对50例乳腺癌患者行腋窝淋巴作图，所得SLN和非哨兵淋巴结（NSLN）均行常规HE染色。阴性SLN再行连续切片及免疫组化检查，结果：50例患者中SLN阳性45例，SLN鉴别成功率为90．0％，45例中常规病检16例SLN阳性，对29例SLN阴性者采用连续切片和免疫组化检查发现7例（24．1％）有微转移，硝兵淋巴结活检的准确率，灵敏度和假阴性率分别为91．1％，85．7％和8．9％，结论：采用美蓝染色法能准确鉴别SLN，反映乳腺癌患者腋窝淋巴结状况，采用连续切片和免疫组化检查可检测出NSLN中的微转移灶，降低假阴性率。     

结直肠癌前哨淋巴结体外定位检测的临床研究

目的:探讨用体外亚甲蓝作为染色剂寻找前哨淋巴结(sentinellymph node,SLN)的方法在结直肠癌SLN定位中的可行性及临床价值。方法:将行标准根治性切除的结直肠癌标本离体后,在肿块四周黏膜下注射亚甲蓝后追踪辨认。蓝染的淋巴结视为SLN,未蓝染的淋巴结被视为NSLN。SLN中无癌细胞转移者常规行细胞角蛋白(CK、AE1/AE3)免疫组化检查,CK阳性者视为有微转移病例。结果:82例患者成功标记SLN(96.47%),准确度为90.24%。通过CK检测,16例SLN阴性患者发现微转移,总转移率提升了18.30%,TNM分期得以提升的患者达18.82%。HE染色下SLN的假阴性率为23.17%,结直肠癌的假阴性率分别为6.38%和45.71%,有统计学意义（P=0.001）。结论:前哨淋巴结活检（SLNB）对结肠癌区域淋巴结转移情况的判断更有临床价值,但在直肠癌方面的应用值得进一步探讨。     

Possible predictive markers related to micro-metastasis in breast cancer patients

Despite significant advances in micro-metastasis detection methods, little is known about the relationship between micro-metastasis and primary tumors. The purpose of this study was to assess the ability of expression of the breast cancer-related markers, HER-2/neu, COX-2, VEGF and PDGF-B, as a predictor for micro-metastasis. As destination sites for micro-metastasis, we examined the peripheral blood (BD), bone marrow (BM) and sentinel lymph node (SLN) from 53 breast cancer patients. Protein and gene expression of the markers at the primary site were determined by immunohistochemistry (IHC) and quantitative RT-PCR. BD and BM samples were processed using magnetic-activated cell separation and immunocytochemistry. SLNs were examined by hematoxylin and eosin (H&E) staining and IHC. The percentages of patients with micro-metastasis were 24.5% in BD, 56.6% in BM, 26.4% in SLN by H&E and 41.5% in SLN by IHC. COX-2 gene amplification was significantly associated with SLN micro-metastasis by H&E (P=0.03). Overexpression of HER-2/neu predicts the presence of SLN micro-metastasis as detected by H&E (P=0.005) and COX-2 overexpression predicts the presence of micro-metastasis in BM (P=0.005) and SLN by H&E (P<0.001) and IHC (P<0.001). Similarly, PDGF-B overexpression predicts micro-metastasis in BD (P=0.002), BM (P=0.003) and SLN by H&E (P=0.017), whereas VEGF overexpression predicts only the presence of SLN micro-metastasis by IHC (P=0.001). Our results indicate the possible value of using these markers to predict the risk of micro-metastasis in breast cancer.     

One-step nucleic acid amplification for intraoperative detection of lymph node metastasis in breast cancer patients.

PURPOSE: Detection of sentinel lymph node (SLN) metastasis in breast cancer patients has conventionally been determined by intraoperative histopathologic examination of frozen sections followed by definitive postoperative examination of permanent sections. The purpose of this study is to develop a more efficient method for intraoperative detection of lymph node metastasis. EXPERIMENTAL DESIGN: Cutoff values to distinguish macrometastasis, micrometastasis, and nonmetastasis were determined by measuring cytokeratin 19 (CK19) mRNA in histopathologically positive and negative lymph nodes using one-step nucleic acid amplification (OSNA). In an intraoperative clinical study involving six facilities, 325 lymph nodes (101 patients), including 81 SLNs, were divided into four blocks. Alternate blocks were used for the OSNA assay with CK19 mRNA, and the remaining blocks were used for H&E and CK19 immunohistochemistry-based three-level histopathologic examination. The results from the two methods were then compared. RESULTS: We established CK19 mRNA cutoff values of 2.5 x 10(2) and 5 x 10(3) copies/muL. In the clinical study, an overall concordance rate between the OSNA assay and the three-level histopathology was 98.2%. Similar results were obtained with 81 SLNs. The OSNA assay discriminated macrometastasis from micrometastasis. No false positive was observed in the OSNA assay of 144 histopathologically negative lymph nodes from pN0 patients, indicating an extremely low false positive for the OSNA assay. CONCLUSION: The OSNA assay of half of a lymph node provided results similar to those of three-level histopathology. Clinical results indicate that the OSNA assay provides a useful intraoperative detection method of lymph node metastasis in breast cancer patients.     

Intraoperative examination of sentinel lymph nodes by ultrarapid immunohistochemistry in breast cancer

BACKGROUND: The ultrarapid immunohistochemistry (IHC) technique was applied to the intraoperative examination of sentinel lymph nodes (SLNs) because routine SLN frozen section examinations sometimes produce false-negative results. The present study was undertaken to develop a reliable protocol for the ultrarapid IHC of SLNs. METHODS: SLNs from 79 breast cancer patients with clinically negative axillary node were examined intraoperatively by frozen hematoxylin-eosin (H&E) stain and by ultrarapid cytokeratin IHC assay. On the basis of the result of serially sectioned permanent study, the sensitivity and accuracy of each intraoperative technique were compared. RESULTS: The total number of dissected SLNs was 178 with a mean of 2.3 (1-5) per patient. The mean turnaround time for ultrarapid IHC was 20 min. The sensitivity rates of frozen H&E staining and ultrarapid IHC were 70.0 and 85.0%, respectively (P = 0.083). Each method had a specificity of 100%. The accuracy rates for frozen H&E staining and rapid IHC were 92.4 and 96.2%, respectively (P = 0.083). Ultrarapid IHC detected one additional patient with sentinel node micrometastasis and two additional patients with isolated tumor cells (ITCs). In those patients, two underwent completion axillary dissection simultaneously and could avoid a second operation. CONCLUSIONS: Ultrarapid cytokeratin IHC enhanced the intraoperative detection of sentinel node micrometastasis and ITCs in breast cancer without consuming much time. In patients who need completion axillary dissection after sentinel node biopsy, this technique could be helpful in avoiding a second operation.