Immunohistochemically detected micrometastasis in lymph nodes from superficial esophageal squamous cell carcinoma

BACKGROUND AND OBJECTIVES: This study was conducted to determine the incidence and clarify the patterns of nodal micrometastasis, to elucidate the histopathologic parameters of tumor extension correlating with micrometastasis, and to evaluate whether nodal micrometastasis has clinical significance in patients with superficial esophageal cancer. METHODS: Lymph nodes resected from 78 patients with superficial esophageal squamous cell carcinoma were examined immunohistochemically using the monoclonal antibody cocktail AE1/AE3 to define histologically undetectable micrometastasis. Clinical records and pathologic features of all cases were reviewed. RESULTS: Of the 78 patients, 34 had neither micro- nor overt disease in the lymph nodes, 12 had nodal micrometastasis only, and 32 had histologically overt metastasis. Nodal micrometastasis was found in carcinomas reaching the muscularis mucosae or deeper tissues of the esophagus. Multivariate analysis showed that intraesophageal multicentric cancer and venous invasion had significant correlation with nodal micrometastasis (P = 0.005 and 0.017, respectively). However, no clinical impact of nodal micrometastasis could be detected regarding patient outcome. CONCLUSIONS: Nodal micrometastasis is not rare in patients with superficial esophageal cancer, but it does not appear to have clinical significance in these patients. Nodal micrometastasis correlates with intraesophageal multicentric cancer and venous invasion.     

Clinical outcome of patients with lymph node-negative breast carcinoma who have sentinel lymph node micrometastases detected by immunohistochemistry

BACKGROUND: The ideal pathologic assessment of sentinel lymph nodes (SLNs) in patients with breast carcinoma remains controversial. The authors evaluated how detailed assessment of SLNs using immunohistochemistry (IHC) and serial sectioning would affect treatment decisions and outcomes in patients with breast carcinoma who had negative SLNs on standard hematoxylin and eosin staining. METHODS: The SLNs from patients who were treated between June 1998 and June, 1999 and who had negative lymph node status determined by hematoxylin and eosin staining (n = 84 patients) were evaluated further with serial sectioning and cytokeratin IHC. Patients were offered adjuvant therapy based on primary tumor factors. RESULTS: The median patient age was 57 years, and the median tumor size was 1.2 cm. At a median follow-up of 40.2 months, 81 patients (96%) were alive with no evidence of disease, 1 patient was alive with disease, 1 patient had died of disease, and 1 patient had died of other causes. Fifteen patients (18%) had micrometastases identified on IHC. Of the total 84 patients, information regarding adjuvant therapy was not available for 5 patients. Of the remaining 79 patients, 10 patients (13%) were not offered adjuvant chemotherapy but had positive SLN status determined by IHC. SLN status based on IHC evaluation did not correlate with age (P = 0.077), tumor size (P = 0.717), grade (P = 0.148), estrogen receptor status (P = 1.000), or lymphovascular invasion (P = 0.274). Furthermore, IHC-detected positive SLN status did not correlate with distant metastasis (P = 0.372) or overall or distant metastasis-free survival (P = 0.543 and P = 0.540, respectively). CONCLUSIONS: Although the finding of SLN micrometastases by IHC may change management in > 12% of patients, preliminary results suggested that such micrometastases do not affect outcomes significantly.     

常规检查淋巴结阴性胃癌的淋巴结微转移研究

目的 研究常规病理检查无淋巴结转移的胃癌之淋巴结转移的特点,分析微转移与各种临床病理因素及预后的关系。方法 选用抗低分子量细胞角蛋白抗体ＡＥ１,运用免疫组化方法,检测１０５例常规病理检查无淋巴结转移的胃癌根治根本的胃周淋巴结１２４５个,并进行统计处理。结果 ３１例（２９．５％）胃癌的８１个淋巴结（６．５％）出现微转移。弥漫型胃癌的淋巴结微转移阳性率（４１．５％,２２／５３）明显高于肠型胃癌（１７．     

Prognostic value of micrometastases in sentinel lymph nodes of patients with breast carcinoma: a cohort study

Background: The prognostic meaning and thus indication for adjuvanttherapy of lymphogenic micrometastases in breast cancer patientsis still under debate. Patients and methods: From 1999 to 2007, 703 patients with _cT_1–2N₀breast cancer underwent surgery including sentinel lymph nodebiopsy. Examination of sentinel lymph nodes consisted of hematoxylinand eosin and immunohistochemistry staining following serialsectioning of the sentinel node. Patients were divided intofour groups: _pN₀ (n = 423), _pN_1micro (n = 81), _pN_1a (n = 130)and _pN_1b (n = 69). Median follow-up was 40 months. Results: At the end of follow-up, 53 patients had died and 64had recurrent disease. Compared with _pN₀ and following adjustmentfor possible confounders, including adjuvant systemic treatment,overall survival was not significantly different for _pN_1microwhile significantly worse for _pN_1a and _pN_1b {hazard ratio (HR)[95% confidence interval (CI)]: 0.59 [0.14–2.58], 4.31[1.85–10.01], 10.66 [4.04–28.14], respectively}.Likewise, disease-free survival was not significantly differentfor _pN_1micro and worse for _pN_1a and _pN_1b (HR [95% CI]: 1.43[0.67–3.02], 2.79 [1.37–5.66], 7.13 [3.27–15.54],respectively). Distant metastases were more commonly observedin the _pN_1micro than in the _pN₀ group, but still not as commonas in the _pN_1a or _pN_1b group (HR [95% CI]: 4.85 [1.79–13.18],10.34 [3.82–28.00], 23.25 [7.88–68.56], respectively). Conclusion: Although the risk of distant metastases was higherin patients in the _pN_1micro than in the _pN₀ group, no statisticallysignificant differences were observed in overall or disease-freesurvival between _pN₀ and _pN_1micro. Micrometastatic lymph nodeinvolvement in itself should not be an indication for adjuvantchemotherapy in breast cancer patients. Key words: breast cancer, micrometastases, prognosis, sentinel lymph node Received for publication March 11, 2008. Revision received June 25, 2008. Accepted for publication July 1, 2008.     

Prognostic relevance of immunohistochemically detected lymph node micrometastasis in patients with gastric carcinoma

BACKGROUND: Micrometastases consisting of one to a few cells in lymph nodes resected during gastrectomy are difficult to identify using conventional hematoxylin and eosin (H&E) stains. It has been shown that immunostaining for cytokeratins is effective in detecting lymph node micrometastasis in a variety of human tumors, but only a few previous reports demonstrated its use in the treatment of patients with early and advanced gastric carcinoma, and those reports had conflicting results. METHODS: In this study, 3625 regional lymph nodes that were dissected in gastrectomy specimens from 153 patients with early-stage gastric carcinoma (46 patients) and advanced gastric carcinoma (107 patients) were immunostained with the anticytokeratin cocktail AE1/3 for micrometastasis (median, 23 lymph nodes; range, 8-66 lymph nodes). Micrometastasis (MM) was defined as a single tumor cell or clusters of tumor cells that were missed on conventional examination with H&E stains but were detected by immunostaining with broad-spectrum anticytokeratin antibodies. RESULTS: Lymph node metastasis (LNM) was detected in 609 lymph nodes (17%) by H&E staining. MM was identified in another 191 of the remaining lymph nodes (6.3%) from 75 patients. Twenty-eight of those patients were up-staged. There was a significant correlation between MM and depth of tumor invasion (P < 0.01). Patients with MM had a decreased 5-year survival rate (49%) compared with patients without MM (76%) for both early and advanced gastric carcinoma. The effect of MM on survival was most pronounced for patients in the Stage I and LNM negative group. CONCLUSIONS: Immunohistochemical examination using broad-spectrum anticytokeratin antibodies increased the detection rate of LNM and had a significant impact on staging and survival in patients with gastric carcinoma.     

Quantitative detection of micrometastases in the lymph nodes of gastric cancer patients with real-time RT-PCR: a comparative study with immunohistochemistry

Histologic examination lacks the sensitivity to detect micrometastases in gastric cancer lymph nodes. In the present study, we applied a real-time RT-PCR approach to the quantitative detection of micrometastases in gastric cancer lymph nodes and compared diagnostic power with routine histology and immunohistochemistry. We studied 392 lymph nodes from 21 gastric cancer patients who underwent curative surgery. Real-time quantitative RT-PCR was performed on a LightCycler instrument using a hybridization probe for carcinoembryonic antigen (CEA) and cytokeratin-20 (CK20) as marker genes. Immunohistochemistry with antibodies to wide-keratin was also performed in the lymph nodes to compare the sensitivity and specificity. Median (average) values of CEA mRNA in lymph nodes in patients with histology(+), immunohistochemistry(+)/histology(-), immunohistochemistry(-)/histology(-) and negative control results were 4600 (16000), 200 (400), 0 (9.8) and 0 (0.6), respectively. There were some false-negative results with simple CEA and CK20 real-time RT-PCR due to the presence of low gene-expressing gastric cancers as revealed by CEA and CK20 immunohistochemistry. CEA in combination with CK20 (duplex) real-time RT-PCR partially covered this weakness. Consequently, all 71 histology(+) lymph nodes were positive for duplex real-time RT-PCR as well as wide-keratin immunohistochemistry. Positivity rates by histology, wide-keratin immunohistochemistry and duplex real-time RT-PCR were 18.0% (71/392), 20.9% (82/392) and 25.8% (101/392), respectively. In 2 of 8 patients with pT1N0, positive lymph nodes were observed by real-time RT-PCR but not by immunohistochemistry. These results indicate that duplex quantitative real-time RT-PCR is the most sensitive method for detecting micrometastases and useful for evaluating the prognostic significance of lymph node micrometastasis in gastric cancer patients.     

Histologic detection and clinical implications of micrometastases in axillary sentinel lymph nodes for patients with breast carcinoma.

BACKGROUND: Sentinel lymph node (SLN) biopsy is used increasingly in patients with clinically lymph node negative, early-stage breast carcinoma, because it can spare axillary dissection when the sentinel lymph nodes are negative. The question arises, however, whether complete axillary lymph node dissection (ALND) also is necessary in patients with only micrometastases (< or = 2 mm in greatest dimension) in axillary SLNs. The authors carried out the current study to ascertain the risk of non-SLN axillary metastases in such patients and to assess the detection rate of SLN micrometastases in relation to the sectioning interval and the number of sections examined. METHODS: The authors examined 109 patients with micrometastatic SLNs from a series of 634 patients with carcinoma of the breast who underwent SLN biopsy and complete ALND as part of the surgical treatment for their disease. The SLNs were sectioned completely at 50-microm intervals, and the sections were examined intraoperatively. RESULTS: The overall frequency of metastases in axillary non-SLNs was 21.8%. The frequency was correlated significantly with the size of the SLN micrometastatic focus (P = 0.02): 36.4% of patients with foci > 1 mm had metastases in axillary lymph nodes--a percentage approaching 44.7% of patients with macrometastatic SLNs--whereas only 15.6% of patients with micrometastases < or = 1 mm had other involved axillary lymph nodes. CONCLUSIONS: Outside of clinical trials, patients with T1 and small T2 breast carcinoma and micrometastatic SLNs should undergo complete ALND for adequate staging. However, patients with SLN micrometastases up to 1 mm in greatest dimension have a significantly lower risk of additional axillary metastases, raising the question of whether ALND may be avoided in this subgroup of patients.     

Immunohistochemically detected hepatic micrometastases predict a high risk of intrahepatic recurrence after resection of colorectal carcinoma liver metastases

BACKGROUND: Hepatic metastases from colorectal carcinoma frequently recur after resection and hepatic micrometastases most likely are important in the development of such recurrences. The objectives of the current study were to assess the feasibility of the immunohistochemical detection of hepatic micrometastases from colorectal carcinoma and to determine their clinical significance. METHODS: Fifty-three patients underwent curative hepatic resection for colorectal carcinoma metastases. Multiple tissue sections were cut from the advancing margin of the largest hepatic metastasis in each patient and were stained with an antibody against cytokeratin-20 to detect hepatic micrometastases, which were defined as discrete microscopic cancerous lesions surrounding the dominant metastasis. RESULTS: Normal hepatocytes and intrahepatic bile duct epithelia stained negative for cytokeratin-20 in all patients, whereas the largest hepatic tumors stained positive in 46 patients (86.8%). Among the 46 patients with hepatic tumors that were positive for cytokeratin-20, hepatic micrometastases were found immunohistochemically in 32 patients (69.6%). The presence of hepatic micrometastases was associated with a larger number of macroscopic hepatic metastases (P = 0.047) and patients with hepatic micrometastases were found to demonstrate a higher probability of intrahepatic recurrence (P = 0.003) compared with those patients without hepatic micrometastases. In addition, patients with hepatic micrometastases demonstrated a worse survival (10-year survival rate of 21.9%) compared with those patients without hepatic micrometastases (10-year survival rate of 64.3%) (P = 0.017). CONCLUSIONS: Immunohistochemical detection of hepatic micrometastases is feasible in patients with colorectal carcinoma liver metastases. Hepatic micrometastasis indicates widespread hepatic involvement and thus predicts an increased risk of intrahepatic recurrence after hepatic resection and a poorer patient prognosis.     

Detection of sentinel lymph nodes in gastric cancers based on immunohistochemical analysis of micrometastases

BACKGROUND AND OBJECTIVES: The sentinel lymph node (SN) theory has the potential to change the trend of surgery for gastric cancer that is based on wide resection of the stomach with dissection of regional lymph nodes. However, feasibility tests of SN mapping procedures in gastric cancers with analysis of micrometastasis are rare. This study aimed to estimate the clinical usefulness of SN mapping using a dual procedure with dye- and gamma probe-guided techniques for gastric cancers, based on immunohistochemical staining (IHC) analysis. METHODS: SN mapping procedures were performed on 41 patients with T1-T2 gastric cancer, and gastrectomy with D2 lymphadenectomy followed. All SNs and non-SNs obtained from the patients were tested by IHC analysis using anti-cytokeratin antibodies. RESULTS: Using the dual mapping procedure, SNs were detected in all patients (100%). SN was positive in all patients with lymph node metastasis except in one with non-solid type poorly differentiated adenocarcinoma with marked lymphatic permeation, thus achieving an accuracy rate of 98%. CONCLUSIONS: The method was accurate in predicting nodal status and could be an indicator for less invasive treatment in patients with gastric cancer.     

Lymphokine-activated killer cell function of lymphocytes from regional lymph nodes in patients with gastric carcinoma

Lymphokine-activated killer (LAK) cells generated by culture of regional lymph node cells (LNC) with interleukin 2 (IL 2) for 4 and 11 days were examined for their functional capabilities in comparison with those of peripheral blood mononuclear cells (PBM) in 25 patients with gastric carcinoma. The cytotoxic activity of LAK cells induced from LNC for 4-day culture with IL 2 was significantly lower than that from PBM. However, the LNC-LAK cytotoxicity was markedly increased up to almost the same level as that of PBM after 11-day culture. The production of interferon-γ (INF-γ) and tumor necrosis factor-α (TNF-α) from nonadherent LAK cells in LNC was also significantly reduced as compared to that from PBM 4 days after culture, when stimulated with or without tumor target, Raji cells. After 11-day culture with IL 2, however, the levels of these cytokines produced by LNC-LAK cells either with or without stimulation by tumor target were comparable to those by PBM-LAK cells, although the release of these cytokines was markedly reduced when compared to that after 4-day culture. Phenotypic analysis revealed decreased proportion of cells mediating NK activity in LNC before and 4 days after culture. CD56⁺ and CD57⁺ cells in LNC were increased after 11-day culture, although the percentages of these cells were still low as compared to those in PBM. The proportions of OKIal⁺ and CD25⁺ cells were uniformly increased after 4 and 11-day culture in both cell populations. Changes in subpopulations of CD4⁺ and CD8⁺ cells in LNC were not apparently different from PBM. These results indicated the differential LAK cell function of cells from regional lymph nodes from PBM in patients with gastric carcinoma. © 1995 Wiley-Liss, Inc.     

An immunohistochemical study of occult micrometastases in regional lymph nodes of patients with stage I non-small cell lung carcinoma]

739 regional lymph nodes from 94 patients with stage I non-small cell lung carcinoma (NSCLC) were studied by immunohistochemistry. These lymph nodes, contained no metastasis as assessed by conventional histopathology, were recut. A series consecutive sections from the original blocks were immunostained with polyclonal and monoclonal antibodies to keratins, carcinoembryonic antigen (CEA) and human milk fat globulin membrane antigen (HMFG-2). Single tumor cells or small clusters of tumor cells, not visible on routine examination, were readily detected. The actual number of lymph nodes that contained occult tumor cells was 123 (16.6%) from 53 patients (56.4%). The majority of 102 immunostaining positive nodes were distributed in the hilar (29%) and peribronchial (25%) regions. Our data indicate that: 1. a series consecutive sections and immunohistochemistry may greatly increase the diagnostic yield of occult micrometastases in lymph nodes. 2. the high incidence of occult metastases in NSCLC may be of importance in relation to their rapid dissemination and high death rate. 3. the high frequency of occult nodal metastases in NSCLC raises questions in regard to our presently used criteria for staging, prognosis and treatment of ostensibly stage I disease. 4. perhaps resections of hilar and peribronchial lymph nodes will have an important clinical significance in prevention of wide dissemination of tumor cells.     

The incidence and significance of micrometastases in lymph nodes of patients with ductal carcinoma in situ and T1a carcinoma of the breast.

AIM: To report the incidence and predictive value of positive axillary nodes in ductal carcinoma in situ (DCIS) and T1a carcinoma of the breast. METHODS: Cases from The Netherlands Cancer Institute were used to determine the incidence of lymph-node metastases. All consecutive patients with primary breast cancer that were treated between 1989 and 1998 and who had undergone axillary dissection were selected. Patients were identified with pure DCIS (n = 71), DCIS with small invasion (n = 12), invasive ductal/lobular carcinoma (IDC/ILC) < or =5 mm (n = 18) or tubular carcinoma < or =10 mm (n = 17). All archived lymph nodes of these patients were re-evaluated using immunohistochemistry (IHC). RESULTS: In DCIS the incidence increased from 1.4% with routine staining to 11% with IHC. For DCIS with small invasion it was 0 vs 27%, respectively. In IDC/ILC sized 2-5 mm the incidence rose from 6 to 12% and in tubular carcinoma < or =10 mm from 0 to 12%. All but one of the immunohistochemically detected metastases were isolated tumour cells (n = 9) or small (micro)metastases (n = 4). Maximally two nodes per patient were affected. None of the patients with positive lymph nodes died during follow-up (mean 102 months). CONCLUSIONS: Survival of our patients appeared not to be influenced by the finding of micrometastases in the lymph nodes by IHC. Immunohistochemistry of the sentinel node seems not contributive to further treatment in these patients.     

Ⅰ期乳腺癌淋巴结、骨髓微小转移的研究

目的　探讨Ⅰ期乳腺癌患者淋巴结、骨髓微小转移癌 (LNM、BMM )与临床预后因素的关系。方法　分别以抗上皮细胞膜单克隆抗体为探针 ,对 5 2例Ⅰ期乳癌中LNM ,BMM进行研究。结果　分别有 2 3%( 12 / 5 2 ) ,19.2 %( 10 / 5 2 )病例伴有LNM、BMM。在低分化乳癌组中 ,LNM( 4 5 .0 %,9/ 2 0 )或BMM( 35 .0 %,7/ 2 0 )阳性率明显高于高分化组 ( 9.3%,3/32 ;9.3%,3/ 32 ) (P <0 .0 1;<0 .0 5 )。在LNM阳性组中 ,BMM阳性率 ( 5 8.3%,7/ 12 )明显高于阴性组 ( 7.5 %,3/ 40 ) (P <0 .0 1)。结论　采用免疫组织化学技术可大大提高乳癌微小转移检出率 ,乳癌淋巴道 ,血道转移可同时发生。     

Immunohistochemical investigation of axillary lymph nodes for micrometastases in patients with breast cancer using E29

The axillary lymph nodes from 31 mammary carcinoma patients who had undergone radical mastectomy and were negative for metastases at routine histologic examination of hilar sections, were investigated with E29, an anti-epithelial monoclonal antibody, to detect the presence of neoplastic epithelial cells. In 4 of 433 lymph nodes examined (0.9%) this antibody revealed the presence of epithelial metastatic foci which had not been observed at routine histological examination or interpreted as histiocytes. The 4 lymph nodes belonged to 4 different patients.     

乳腺癌前哨淋巴结微小转移的检测

目的探讨检测乳腺癌前哨淋巴结(SLN)微小转移的最佳方法,研究临床病理因素与微小转移的相关性。方法应用同位素法检测乳腺癌SLN;对常规病理检查阴性的SLN,以100μm为间隔,进行多层间隔连续切片,并做HE和免疫组化染色检测微小转移;取肿瘤标本进行连续切片,并行免疫组化染色。结果共检测59例患者的121枚SLN和44份肿瘤标本,有14例(23.7%)患者的17枚(14.O%)SLN有微小转移。用HE染色法,切片数量从1层增加到3层时,微小转移的检出例数分别为3、7和10例;在3个层面上行间隔连续切片,HE分别与AE1/3、CK19和muc1联合检测时,微小转移的检出例数分别为14、12和16例。增加切片数量或采用联合检测的方法,可以提高微小转移的检出数量,微小转移与原发肿瘤大小、c-erbB2、MMP-2和血管内皮生长因子(VEGF)的表达相关。结论检测SLN微小转移的最佳方法为间隔100μm、在2个层面上行间隔连续切片,同时进行HE和muc1染色,可以检出绝大多数的微小转移。     

Evaluation of immune response according to the metastatic status in the regional lymph nodes in patients with gastric carcinoma

The immunological response in the regional lymph nodes (LN) of gastric cancer patients who had metastasis was studied by investigating both the degree of metastasis and the maturation status of dendritic cells (DCs). A total of 732 LNs was obtained from 29 gastric cancer patients, including 25 patients whose LNs were pathological metastasis-negative and 4 patients with metastasis-positive LNs. Micrometastasis (MM) in the LNs was analyzed by immunohistochemical staining of cytokeratin (CK) (IHC-MM) and by amplifying CEA and CK19 mRNA by quantitative real-time RT-PCR (PCR-MM). Distribution and density of mature DCs were evaluated in 119 LNs from pathological metastasis-positive cases by examining CD83 expression immunohistochemically. Then, following the examination of PCR-MM, immunological responses were analyzed by amplifying CD83, CD86, CD1a and IFNγ mRNA by quantitative real-time RT-PCR in 613 LNs from 25 pathological metastasis-negative cases. Among 119 LNs from 4 patients with LN metastasis, 20 LNs had histological metastasis and 6 histological metastasis-negative LNs had IHC-MM. The distribution and density of mature DCs were identical in the LNs regardless of metastasis status. Among 613 LNs from 25 patients without histological LN metastasis, 15 LNs (2.45%) from 6 cases (24%) were PCR-MM positive. The expression levels of CD83 and CD86 mRNA were significantly higher in the 15 PCR-MM-positive LNs than in the 598 PCR-MM-negative LNs. The expression levels of CD83 and CD86 mRNA in 136 PCR-MM-negative LNs from 6 PCR-MM-positive cases were also higher than those in 462 LNs from 19 PCR-MM-negative cases. In gastric cancer patients, activation of the immune response by maturation of DCs is wide-spread in regional lymph nodes at a stage before metastasis is detected histologically, including detection by IHC-MM.     

N0期31例胃癌患者区域淋巴结组织中CK19的表达

目的探讨常规染色显示无淋巴结转移的胃癌患者淋巴结微转移发生情况以及预后意义.方法回顾性分析31例常规染色提示无淋巴结转移的胃癌患者(T1-3N0M0),将常规检查阴性的淋巴结用CK19单克隆抗体进行免疫组织化学染色.结果31例常规病理检查无淋巴结转移的患者中位随访期为50个月,所切除淋巴结的中位数为8个,总的5年生存率为53.7%,其中通过免疫组化发现的微转移检出率为32.3%(10/31).淋巴结微转移患者5年生存率为30%,低于无微转移患者的66.5%,P=0.036.结论CK19的免疫组化染色是检测胃癌淋巴结微转移的敏感易行的方法,且微转移的检测有助于明确分期、判断预后及指导治疗.