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This study was performed to systematically assess the prevalence, topography and prognostic impact of disseminated tumor cells (DTCs) in lymph nodes (LN) of patients with primary, regional and distant metastasis-free head and neck squamous cell carcinoma (HNSCC) who underwent resection with elective neck dissection. From the routinely processed resection specimen, we could prospectively analyze a total of 1.137 exactly mapped LNs of 50 pN0-HNSCC patients, classified as tumor free by routine histopathology. Three immunohistochemistry (IHC) assays using antibodies directed against CK5/14, a broad spectrum of CKs (1-8, 10, 14-16 and 19), and CD44v6, respectively, were applied on 4.190 LN sections to detect DTCs. The IHC results were correlated with clinicopathologic parameters and clinical follow-up data. We detected seven micrometastases (MM) in five patients and 31 DTCs in 12 patients. Overall, 15 (30%) patients were positive for DTCs or MMs. Strikingly, the anatomical distribution of LN affected with DTCs was not random, but was dependent on the lateralization of the primary tumor and clustered significantly most proximal to the primary tumor. None of the investigated patients developed loco-regional lymphatic or distant metastasis during the mean follow-up period of 71 months. Our results reveal clinically occult tumor cell dissemination as an early and frequent event in HNSCC. Considering that higher rates of recurrences in therapeutic LN dissection concepts have been reported than in elective neck dissection strategies, our DTC-data support to perform elective neck dissections, since they appear to be effective in preventing loco-regional lymphatic recurrence from LN DTCs or MMs.

BACKGROUND Skip metastasis to mediastinal lymph nodes is a prognostic factor for patients with nonsmall cell lung carcinoma (NSCLC). Little is known about the biologic behavior of tumors with noncontinuous spread to the mediastinal lymph nodes. In patients with pN2 skip metastases, micrometastases to N1 lymph nodes, which only mimic skip metastases, have not been investigated. METHODS In a retrospective study, the authors analyzed the primary tumor specimens from 45 patients with pN2 NSCLC (18 patients had squamous cell carcinomas, 23 had adenocarcinomas, and 4 had large cell carcinomas). They immunohistochemically evaluated the expression of p21, p53, MUC-1, Bcl-2, c-ErbB-2, and E-cadherin. Survival rates and biomarker expression levels were compared between patients with pN2 disease and infiltration of N1 lymph nodes (without skip metastasis [n = 28]) and patients with pN2 disease without N1 infiltration (with skip metastasis [n = 17]). To evaluate micrometastasis in the pN1 lymph nodes of 17 patients with skip metastases, lymph nodes were stained using the anticytokeratin antibody, AE1/AE3. RESULTS The 5-year survival rate of patients with skip metastases was 41%, compared with 14% for patients without skip metastases (P = 0.019). In a multivariate analysis, the incidence of skip metastases did not vary significantly according to gender, age, histology, pT status, or cM status. Three skip-positive patients (17.6%) had micrometastatic tumor involvement of pN1 lymph nodes. After adding these patients to the group of patients without skip metastases, there was still a significant difference in survival between the two groups. p53, MUC-1, c-ErbB-2, and E-cadherin expression levels in primary tumor specimens were not significantly different in patients with continuous metastasis and patients with skip metastases. Patients with skip metastases expressed lower levels of p21 (P = 0.026), whereas Bcl-2 expression levels were considerably higher (P = 0.019) compared with the corresponding levels in patients without skip metastases. CONCLUSIONS Patients with NSCLC and pN2 skip metastases have a more favorable prognosis than do patients with pN2 disease without skip metastases. Tumor specimens from these patients exhibit elevated expression of the antiapoptosis gene BCL2 and lower expression levels of p21 relative to patients with pN2 disease without skip metastases. Micrometastases occurred in 3 of 17 (17.6%) patients with pN2 disease and skip metastases diagnosed by routine histopathology. Cancer 2004. © 2004 American Cancer Society.

Previous studies on the immunoreactivity of β-catenin, MUC1 and c-met in gastric carcinomas regarding survival and clinico-pathological features led to contradictory results. Therefore, a series of 94 diffuse-type and mixed-type subcardial gastric carcinomas according to the Laurén classification were investigated to elucidate possible correlations with clinico-pathological and prognostic data. An immunohistochemical study was performed to detect the expression of β-catenin, MUC1 and c-met. Loss of membranous/cytoplasmic β-catenin expression in the tumour centre correlated with pT, loss at the invasion front with pTNM stage. MUC1 expression in the tumour centre correlated with lymph node metastasis and pTNM stage. c-Met did not show such associations. In multivariate survival analysis, loss of membranous/ cytoplasmic β-catenin expression as well as a strong MUC1 expression at the tumour invasion front represent independent predictors of a worse prognosis. On the other hand, c-met expression did not exhibit any prognostic value in this study.

PIM kinases are mediators of cytokine signalling pathways in hematopoietic cells and contribute to the progression of certain types of leukemia and solid tumor. Here the prognostic impact of proto-oncogene PIM-1 was analyzed in gastric carcinoma.Cancer tissues of 117 patients with potentially curative (R0) resections for gastric cancer were immunohistochemically stained for PIM-1.Cytoplasmic immunoreactivity for PIM-1 in tumor (46%) was higher (p=0.003) compared to that in gastric glands (16%) and foveolae (1%). PIM-1 immunoreactivity in gastric carcinoma correlated with tumor grading (p0.05) and Laurén category (p0.02). Overexpression of PIM-1 in gastric glands (p0.001) was associated with formation of lymph node metastases (p=0.035) and survival (p=0.04). Multivariate analysis of PIM-1 expression in gastric glands confirmed its association with prognosis.Up-regulation of PIM-1 oncogene might be a tumor marker for gastric cancer. The correlation of PIM-1 overexpression in gastric glands with formation of lymph node metastases and survival proposes a prognostic role in gastric cancer.

The purpose of this study was to evaluate the significance of human telomerase reverse transcriptase (hTERT) mRNA expression and telomerase activity as prognostic markers in non-small cell lung cancer (NSCLC).In a series of 69 curatively resected NSCLC specimens, telomerase activity was analyzed with the telomeric repeat amplification protocol (TRAP) assay and expression of hTERT mRNA by quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Partitioning of gene expression levels and protein activities to construct prognostic groups was attempted.Human hTERT mRNA transcripts were detected in 62 (89.9%) cases of NSCLC. Seven (10.1%) tumors were completely negative for hTERT expression. Dichotomized hTERT levels (0.42 versusor =0.42) were associated with prognosis and Kaplan-Meier survival curves demonstrated a significant difference (log rank: p0.01) with 5-year survival rates of 44.3% (+/-7.1%) for low as compared to 80% (+/-8.9%) for high hTERT mRNA expression. Low hTERT expression was also significantly associated with squamous cell histology (p0.03). Telomerase activity was not associated with survival, stage, pT and pN categories, histological type or grading. Comparison of hTERT mRNA expression and telomerase activity was possible in 66 patients and showed a significant difference (p0.0001) by Wilcoxon rank test.This is the first study which demonstrates that high hTERT mRNA expression is associated with improved 5-year survival rates. Expression patterns are distinct among histopathological subtypes of NSCLC and telomerase activity (TRAP) is significantly higher than hTERT mRNA expression.

Survivin suppresses programmed cell death and regulates cell division. To evaluate the prognostic importance of the apoptosis inhibitor survivin for non-small cell lung cancer (NSCLC), a study was performed in 64 patients with R0 resections for NSCLC and histopathological stages I-IIIA.Analysis of survivin mRNA expression was performed on 64 paired tumor and normal tissues by quantitative real-time RT-PCR.Survivin expression in tumors (median 3.68, min. 0.19, max. 28.63) was significantly higher (p0.04) than in corresponding normal tissues (median 1.68, min. 0.03, max. 155.59). Survivin mRNA was up-regulated in tumors of 42 patients (66%). However, survivin overexpression did not correlate with survival of lung cancer patients. There was no significant association of survivin mRNA levels with histological type (p = 0.29), pT (p = 0.41) and pN categories (p = 0.57), grading of the primary tumor (p = 0.45), or histopathological stage (p = 0.87).Overexpression of survivin mRNA in NSCLC might be a marker for noninvasive tumor detection, but has no prognostic importance.

The purpose of this study was to clarify the relationship of the immunohistochemical expression of c-met, p53 and p21 with clinicopathological parameters and prognosis in gastric carcinomas. We analyzed specimens from 114 gastric cancer patients (median age 64 years, range: 33-86) who underwent gastrectomy with lymphadenectomy. Specimens were categorized according to the tumor differentiation, based on UICC, WHO, Laurén, Ming and Goseki classifications. Specimens were examined immunohistochemically with antibodies against c-met, p53 and p21. The expression was evaluated semiquantitatively and correlated with the clinicopathological parameters. The c-met staining pattern was positive in 73.7%. P53 and p21 were positive in 86.8 and 67.5%, respectively. No significant correlation between c-met or p21 expression and the clinicopathological parameters was seen. A significant increase of p53 expression was observed in stage pT3 and -4. The overexpression of c-met and p53 was significantly associated with a poor prognosis in the univariate survival analysis. In the multivariate analysis this impact was maintained for c-met. P21 proved to be a significant prognostic factor in the multivariate analysis. Our data suggest that the overexpression of c-met and p21 may represent independent prognostic factors in gastric carcinoma.

The expression of the nm23 gene has been associated with the development of metastasis. Numerous studies have shown down-regulation of nm23 expression in metastatic breast and colon cancer. The expression of the putative metastasis-suppressor gene nm23 in gastric carcinoma is controversial. The aim of this study was the analysis of nm23 expression in a large series of gastric cancer patients.In a retrospective immunohistochemical study specimens obtained from 116 gastric cancer patients (mean age 64 years; range: 33-85) who had undergone gastrectomy with extended lymphadenectomy were analyzed. Nm23 expression in the tumor epithelium was studied by immunohistochemistry followed by a semi-quantitative (score 0-3) evaluation. Statistical analysis including Chi-square test, uni- and multivariate survival analyses were performed.The nm23 staining pattern was positive (score 2-3) in 100 (86.2%) specimens and negative (score 0-1) in 16 (13.8%) samples. Lymph node metastasis was found in 65% of the patients. No significant correlations could be determined between nm23 expression and other variables such as gender, age, tumor differentiation, WHO-, Laurén-, Goseki-, or Ming-classification. The intensity of nm23 staining in the tumor cells was not significantly correlated with depth of tumor infiltration (T-stage), lymph node metastasis (N-stage), distant metastasis (M-stage), UICC-stage, or prognosis.Our series did not show a correlation of nm23 expression in terms of lymph node and distant metastasis or prognosis in gastric cancer patients.

As demonstrated for different malignancies the detection of sentinel nodes (SNL) helps to individualize the indication for lymphadenectomy. In esophageal cancer, the lymphatic spread involves three distinct anatomical compartments. This study analyses the localization of single lymph node metastases in esophageal cancer and discusses the possible theoretical impact on SNL navigation.143 patients with esophageal carcinoma were included in this retrospective analysis. All patients underwent transthoracic en-bloc esophagectomy with 2-field lymphadenectomy. A meticulous work-up of all resected lymph nodes allowed a specific assignment to defined nodal groups of the abdominal and mediastinal compartment. Of 143 patients, 80 (55.9%) were classified as pN1.Twenty four of 80 patients had one single (n=15 patients) or two single (n=9 patients) lymph node metastases (total of 33 nodal metastases). Twenty four of these 33 lymph node metastases (73%) were located in the abdominal compartment. 9 of 13 patients (69%) with esophageal carcinomas above the tracheal bifurcation had isolated abdominal lymph node metastases. The overall rate of skip metastasis to the abdominal compartment was 55%.Isolated lymph node metastases in esophageal cancer are predominantly located in the abdominal compartment along the lesser curvature and the left gastric artery. Therefore, SNL navigation in esophageal cancer has to focus on this region and requires initial laparotomy/laparoscopy irrespective of the type of esophageal resection.

Hypoxia-inducible factor-1alpha (HIF-1alpha) expression was reported to be associated with tumor growth, progression and resistance to radio-/chemotherapy. Whether HIF-1alpha mRNA or protein expression is associated with histomorphological response or prognosis following neoadjuvant chemoradiation and surgery in resectable, locally-advanced esophageal cancer was analyzed.Fifty-three patients received neoadjuvant chemoradiation (cisplatin, 5-fluorouracil, 36 Gy) followed by transthoracic en bloc esophagectomy. HIF-1alpha mRNA and protein expressions were analyzed by quantitative RT-PCR and immunostaining.In squamous cell carcinoma, HIF-1alpha mRNA expression was significantly higher than in paired normal epithelium (p0.001). Normal squamous epithelium showed significant elevated expression in adenocarcinomas, suggesting a field effect (p0.04). HIF-1alpha protein expression showed a significant regulation following chemoradiation. Neither HIF-1alpha mRNA nor protein expression was associated with histomorphological regression or prognosis.HIF-1alpha mRNA expression is differentially upregulated in esophageal squamous cell carcinoma compared to adenocarcinomas, but does not predict tumor regression or prognosis.

The detection of single tumor cells or tumor cell clusters represents an important issue in intraoperative frozen section analysis. For example, surgical margins may be evaluated in order to minimize the number of additional operations. Furthermore, intraoperative diagnosis of lymph node micrometastasis (LNM) may help to define the area of appropriate lymph node dissection. In addition to haematoxylin and eosin (HE)-stained sections, immunohistochemical detection of single tumor cells or cell clusters may be helpful in this context. The aim of this study was to evaluate the clinical significance, reliability and sensitivity of intraoperative rapid immunostaining of frozen sections. Therefore, we compared the results of rapid immunohistochemical staining of frozen sections and paraffin sections applying the EnVision and Histofine(R) detection systems. In a prospective immunohistochemical study, paraffin and frozen sections of 20 gastric cancer specimens were analyzed. Paraffin as well as frozen sections were stained immunohistochemically applying the EnVision and Histofine detection systems. As primary antibodies, AE1/AE3 (anti-cytokeratin), EMA (anti-MUC1) and B lymphocyte marker anti-CD20 were applied. The rapid immunostaining procedure was able to be completed within 10-13 min. Rapid immunohistochemical staining of frozen and paraffin sections of the same tumors resulted in comparable immunoreactivity. The rapid EnVision and Histofine procedures allowed immunostaining of frozen sections in less than 13 min. These methods can represent useful additional tools in routine surgical pathology and research, enabling a more accurate frozen section diagnosis compared to staining with HE alone. Intraoperative rapid immunostaining can be a simple and useful technique to detect LNM.

Evidence suggests that the growth arrest and DNA damage-inducible gene 45 (Gadd45) is an effective indicator of poor prognosis or malignant potential in solid tumors. The purpose of this study was to determine the gene expression patterns and clinical relevance of Gadd45 in tumor specimens of non-small cell lung cancer (NSCLC) patients.Using a quantitative real-time RT-PCR method, the mRNA expression of Gadd45 was analyzed in tumor and paired normal-appearing tissues of 66 patients with NSCLC. The gene expression data for each patient were matched to the clinicopathological parameters.Gadd45 mRNA expression was detectable in all (100%) specimens analyzed. The overall median mRNA expression level of Gadd45 was approximately 10-fold lower in tumor tissues than in matching normal lung tissues (p0.001). High intratumoral Gadd45 expression was significantly associated with a poorer histological grading (p = 0.041).The significant decrease in Gadd45 expression in tumors compared with normal tissue and its association with histological grading suggest a role for Gadd45 in the differentiation pathway of NSCLC.

Cancer of the papilla of Vater (CPV) has a significantly better outcome compared to pancreatic cancer (PC) after curative resection. Increasing evidence suggests that prognostic differences are influenced by a different tumor biology. Secreted protein acidic and rich in cystein (SPARC)/osteonectin is a multifunctional matricellular protein involved in cell-matrix interactions and might be involved in tumor pathogenesis and progression. We examined quantitative SPARC mRNA expression in CPV and PC to evaluate if varying expression might contribute to the different biologic behaviour of these entities. Quantitative real-time reverse transcription-PCR was performed to analyze expression of SPARC mRNA in a series of 31 PC and 8 CPV specimens and corresponding uninvolved pancreatic tissues. Relative mRNA levels (ratio tumor/normal) were calculated as (SPARC/beta-actin in tumor)/(SPARC/beta-actin in paired normal tissue). SPARC expression levels were associated with clinical and histopathological parameters. SPARC mRNA expression was detected in all tumor and normal tissues of the pancreas and papilla of Vater. In pancreatic cancer, 15/31 (48.4%) patients showed overexpression of SPARC (ratio tumor/normal1) whereas in CPV only 1/8 (12.5%) exhibited SPARC overexpression and this difference was statistically significant (p0.05, Mann-Whitney test). No associations were detected with T- and N-categories, grading or prognosis. In conclusion, SPARC mRNA overexpression is significantly more frequent in CP than CPV and adds further evidence that CP and CPV are biologically different tumor entities.

MUC1 represents a promising marker in breast cancer. However, due to the structural complexity of the MUC1 glycoprotein, multiple epitopes can be detected by monoclonal antibodies. This fact may be responsible for the contradictory results of previous investigations regarding the clinical and prognostic relevance of MUC1 expression in breast cancer. Therefore, we tried to evaluate the role of different glycosylated and non-glycoslyated MUC1 epitopes as well as other mucin-associated peptides (MUC2) and carbohydrates (Thomsen-Friedenreich antigen, sialyl-Lewisa, sialyl-Lewisx) as predictors of the clinical course and prognosis in mammary carcinomas. An immunohistochemical study applying numerous monoclonal antibodies (mabs) was performed to characterize the expression of a selected panel of MUC1 epitopes, and of MUC2, Thomsen-Friedenreich antigen, sialyl-Lewisa, and sialyl-Lewisx in a series of 140 patients with breast cancer. The results were correlated with clinicopathological variables as well as overall survival. Generally, more than 90% of the mammary cancers, were strongly stained with the MUC1-specific mabs. Especially ductal and lobular carcinomas were strongly MUC1- and sialyl Lewisa-positive, whereas MUC2 binding was significantly elevated in mucinous neoplasms. Associations between the immunoreactivity of any mab under study and tumor progression as reflected by pTNM staging could not be observed. However, expression of the sialylated MUC1 epitope detected by mab MY1.E12 was revealed as a favourable independent prognostic factor. These results confirm that MUC1 is generally strongly expressed in mammary carcinomas. As an exception, mucinous carcinomas are significantly less MUC1 reactive, but strongly express MUC2. Our data suggest that only the presence of a sialylated short-chain MUC1 glycoform is associated with a better prognosis, whereas the other molecules under study are not correlated with the course of disease and survival probability.

Enhanced expression of the apoptosis-preventing protein bcl-xL and the cell cycle-regulating protein p21 was observed in bone marrow infiltrates of systemic mastocytosis. Expression of bcl-2, Ki67, and p53 as well as ISEL apoptosis staining were comparable in patients with mastocytosis and in controls. An altered rate of apoptosis and cell cycling may contribute to accumulation of mast cells in mastocytosis.

The current discussion concerning the extent of resection in patients with adenocarcinoma of the gastroesophageal junction is focused beside the luminal extent of resection primarily on the value of lymphadenectomy.In order to clarify whether spread of lymph node metastasis shows different pathways in adenocarcinomas of the gastroesophageal junction, a prospective, morphological study of the topographical distribution of lymph node metastasis was performed. Transthoracic en bloc esophagectomy with two-field lymphadenectomy (n = 13) or transhiatal esophagectomy with lymphadenectomy of the lower mediastinum and compartment I (lymph node No. 1-3) as well as II (n = 4) was performed in type I carcinoma and transhiatally extended gastrectomy with D2-lymphadenectomy and lymphadenectomy of the lower mediastinum in type II and III carcinoma (n = 33).In 50 specimens, 1730 lymph nodes were evaluated with regard to metastatic infiltration. We found metastases in the lymph nodes of the lower mediastinum in 24% of type I carcinoma, in 11% of type II carcinoma and in 13% of type III carcinoma, whereas the lymph nodes of the upper mediastinum were tumor free in all patients with transthoracic en bloc resection and 2-field lymphadenectomy (n = 13). In all cases with lymph node metastasis abdominal lymph nodes were affected independently from the localization of the primary tumor.The main direction of lymph node spread in adenocarcinoma of the gastroesophageal junction is towards the abdomen and the lower mediastinum. For precise tumor staging standardized lymphadenectomy of the lower mediastinum and the abdominal compartments I and II is essential.