Nm23 Protein Expression Research Articles

Nm23 protein expression in fine‐needle aspirates from breast carcinoma

nm23 protein expression in fine‐needle aspirates from breast carcinoma

Prognostic significance of Nm23/NDPK expression in breast carcinoma, assessed on 10-year follow-up by automated and quantitative immunocytochemical assays.

The Nm23 gene has been described as an antimetastatic gene; in some studies, disease progression in patients with solid tumours is related to Nm23 protein expression, which can be detected by immunohistochemical procedures. Detection of Nm23-H1 protein in breast cancer may be relevant for the monitoring of patient therapy, provided that the technical procedures are reliable and cost-effective. The aim of the present study was to determine the prognostic significance of Nm23, assessed by quantitative immunocytochemical assays (Nm23 ICAs), under optimal technical conditions. Nm23-H1 ICAs were performed on frozen sections, using an automated immunoperoxidase technique (Ventana) and computer-assisted analysis of digitized colour microscopic images (SAMBA), in a series of 168 breast carcinomas. The results of automated quantitative ICAs were correlated with patients' follow-up (129 months). Nm23-H1 immunocytochemical expression in histological sections of tumours in which more than 3 per cent of the surface area was positively stained was significantly (0.012) correlated with longer metastasis-free survival in both node-positive and node-negative groups of patients (P = 0.032 and P = 0.036, respectively) (Kaplan-Meier log-rank test, NCSS 6.0.1 software). Nm23 expression (cut-point 3 per cent) did not, however, correlate with overall survival, or with the recurrence-free survival. In multivariate analysis (proportional hazards regression, Cox model), the prognostic significance of Nm23 in terms of metastasis-free survival was independent of tumour size and grade, and of histological grade, in the entire cohort of patients. It is concluded that Nm23 immunodetection is only of limited practical clinical relevance in breast carcinoma, even when assessed under optimal technical conditions.

Nm23 Protein expression in larynx cancer and the relationship with metastasis.

The nm23 gene, which encodes nucleoside diphosphate (NDP) kinase, is proposed as a metastatic suppressor gene and has been demonstrated to correlate inversely with metastatic potential in several tumours. To elucidate the role of nm23 in larynx carcinomas, we examined using immunohistochemistry the expression of the nm23 protein in matched sets of primary tumours and metastatic lymph nodes. nm23 Protein was expressed in all the carcinomas as well as in non-neoplastic larynx mucosa. Overexpression of nm23 protein was found in the majority of primary tumours compared with corresponding normal mucosa, while decreased expression was associated with poor differentiation and distant metastasis and/or recurrence. No significant difference in age, sex and stage was found between primary tumours with high and low nm23 protein expression. These results suggest that decreased nm23 protein expression may play a role in metastasis and/or recurrence in larynx cancer and therefore could be used as a prognostic factor.

Expression of nm23/Nucleoside Diphosphate Kinase-A Protein in Endometrial Carcinoma

A metastatic tumor suppressor role for the nm23 gene product in breast carcinoma has been proposed. The biologic significance of nm23/NDP kinase-A (NDPK-A) expression in endometrial carcinoma remains undetermined. We sought to (1) characterize the pattern and intensity of nm23 protein expression in endometrial carcinoma and (2) assess the relationship between intensity/pattern of nm23 protein immunostaining and treatment response assessed by progression-free survival and survival to death. Formalin-fixed paraffin-embedded sections from 234 patients with endometrial cancer were immunostained with a mouse monoclonal IgG to nm23/NDPK-A protein. In most specimens of endometrial carcinoma (67.5%), nm23 expression was strongly upregulated. No association was found between either intensity (0 vs 1, 2, 3) or pattern (nuclear membrane vs cytoplasmic) of immunostaining and FIGO stage, ploidy status, histologic subtype, myometrial invasion, progression-free survival, or survival to death. Absence of nm23 staining (0 vs 1, 2, 3) was significantly associated with lower tumor grades (P= 0.02). For stage I patients, moderate to strong nm23 immunostaining intensity (2>, 3) was associated with a trend toward diminished progression-free survival (P= 0.08). Our data imply a heterogeneity of nm23 protein expression and possible distinct biologic roles for nm23 in endometrial compared with breast or ovarian carcinoma.

P53, c-erbB-2 and nm23 expression have no prognostic significance in primary pulmonary adenocarcinoma.

We analysed nm23, c-erbB-2 and p53 protein expression in lung adenocarcinoma in relation to clinicopathological status and patient survival, to elucidate any potential prognostic value. Published reports suggest that high p53 and c-erbB-2 protein expression and loss of nm23 protein expression are associated with poor prognosis. A total of 162 pulmonary adenocarcinomas resected between 1980 and 1991 were stained using monoclonal antibodies to nm23 (NCL-nm23), c-erbB-2 (NCL-CB11) and p53 (DAKO Do7). Antigen retrieval was by microwave heating and bound antibody was visualised using standard immunohistochemical methods. Staining was scored by two observers blinded to tumour status and patient survival. Of the tumours, 101/162 (62.3%) exhibited high-level p53 expression, 30 (18.5%) showed high-level c-erbB-2 membrane staining, and 77 (47.5%) demonstrated loss of nm23 positivity. The influence of T and N status and disease stage on postoperative survival was as expected. The predicted effect on patient survival of nm23, c-erbB-2 or nm23 protein expression suggested by previous studies was not verified by our work. This was true both overall and for groups within the same T or N status or stage. Immunohistochemical assessment of the nm23, c-erbB-2 and p53 proteins using the above monoclonal antibodies does not have value as an independent prognostic indicator in pulmonary adenocarcinoma.

Expression of nm23 protein in pulmonary adenocarcinomas: inverse correlation to tumor progression

Immunohistochemical assessment was made of nm23 protein expression in pulmonary adenocarcinoma. Of the 147 adenocarcinomas 67% ( 99 147 ) were weakly and 33% ( 48 147 ) strongly positive for nm23 protein, nm23 protein expression in primary tumors was shown to correlate inversely with advancing pathologic stage and the degree of metastasis in regional lymph nodes ( P < 0.05). The staining of tumors without nodal metastasis was more intense than with nodal metastasis ( P < 0.02). Nodal metastasis was seen in 37% ( 55 147 ) cases examined. The immunoreactivity to nm23 protein in tumor cells of nodal metastasis was essentially the same as in those of primary tumors ( P < 0.01). Significant correlation between patient prognosis and immunoreactivity for nm23 in primary tumors ( P < 0.05) was demonstrated. But none could be found between immunoreactivity and other parameters such as histologic grading, distant metastasis, tumor size or disease-free survival. Neither was there any significant correlation between pathologic parameters examined and the expression of nm23 in any histologic subtupe. Multivariate analysis using Cox's proportional hazards regression model with five variables indicated nm23 and lymph node metastasis to contribute to overall patient survival. Based on risk ratio disadvantageous state/advantageous states, the gravity of prognostic factors was assessed for lymph node metastasis as 9.25, nm23 expression as 2.06, distant metastasis as 1.23, pathologic stage as 0.78 and tumor size as 0.77. The results suggested that in pulmonary adenocarcinoma a reduced expression of nm23 protein was associated with lymph node metastasis and poor patient survival.

Abnormal expression of four novel molecular markers represents a highly aggressive phenotype in breast cancer. Immunohistochemical assay of p53, nm23, erbB-2, and cathepsin D protein.

In view of the cumulative results to date, p53, nm23, erbB-2, and cathepsin D are the most promising investigational prognostic factors in breast cancer. The clinical utility of these molecular markers to predict recurrence was evaluated. Archival pathology tissues of 100 breast cancer patients were analyzed by immunohistochemical assay. Molecular biologic data were merged with clinicopathologic variables. Thirty-two patients (32%) had recurrence of disease at a median follow-up of 48 months (range 26-72 months). Investigational factor expression had statistical correlation for recurrence with increasing coexpression: one variable 20.6%, two variables 34.2%, three variables 47.1%, four variables 80.0% (P = 0.003). In univariate analysis, lymph node metastasis, tumor size, erbB-2 protein overexpression, and loss of nm23 protein expression were significant variables to determine recurrence; in multivariate analysis, node status and tumor size emerged as the most significant variables for recurrence. Coexpression of the studied investigational variables functioned as significant prognostic correlates for recurrence. These findings suggest that the studied investigational prognostic factors possess the ability to discriminate a highly aggressive phenotype in breast cancer.

Nm23--relationship to the metastatic potential of breast carcinoma cell lines, primary human xenografts, and lymph node negative breast carcinoma patients.

Since the discovery of nm23 (nonmetastatic) by Steeg et al. in 1988, a number of tumor cohort studies have shown an inverse relationship between the levels of expression of the nm23-H1 protein and disease aggressiveness and tumor metastatic potential. The relationship between the expression of nm23 protein and the metastatic potential of human breast carcinoma was analyzed in cell lines, xenografts, and in a retrospective lymph node negative breast carcinoma population. The lymph node negative breast carcinoma study was comprised of 40 patients: 19 with nonrecurrent and 21 with recurrent disease. The 40 patients were matched according to age, cathepsin D, tumor size, percent S-phase, DNA ploidy, steroid receptor status, and tumor grade. Nm23-H1 protein levels in cell lines and xenografts were analyzed quantitatively using Western blot analyses and semiquantitatively in tissue sections using immunocytochemistry. Immunocytochemical analysis of lymph node negative breast tumors was graded as the percent of tumor staining positive for nm23 and the intensity of staining. The metastatic potentials of the cell lines and xenografts were assessed as the ability to form metastatic lesions in nude mice. In the lymph node negative breast carcinoma patients, the metastatic potential was characterized as the incidence of breast carcinoma recurrence. The MCF-7 cell line expressed four- and tenfold higher levels of nm23-H1 than the highly metastatic MDA-MB-435 and MDA-MB-231 cells, respectively. Among the xenografts and cell lines, there was an inverse correlation between nm23-H1 expression and metastatic potential in athymic nude mice (correlation coefficient [R] = -0.51). The differences between the levels of nm23-H1 among the metastatic and nonmetastatic cell lines and xenografts were not statistically significant. Statistical analyses indicated that neither the intensity nor the percent of tumor staining positive for nm23 expression was correlated to the recurrence of breast carcinoma in the lymph node negative patient population that had been matched for other clinical prognostic markers. There was an inverse correlation (R = 0.51) between the levels of nm23-H1 expression in cell lines and xenografts and the metastatic potential in nude mice. In the retrospective lymph node negative breast carcinoma population, no clear association was demonstrated between the expression of nm23 and breast carcinoma recurrence. This observation suggests the nm23 expression does not predict outcome in lymph node negative breast carcinoma patients.

Expression of NM23 protein in acquired melanocytic nevi, malignant melanoma and metastases of malignant melanoma: an immunohistological assessment in human skin.

It has been suggested that nm23 may exert metastasis suppressor function in human tumors. We have analyzed expression of nm23 polypeptide in acquired melanocytic nevi (n = 19), dysplastic nevi (n = 19), malignant melanomas (n = 22) and metastases of malignant melanomas (n = 47) in situ. Nm23 protein was detected immunohistochemically on paraffin sections using the highly sensitive labeled avidin-biotin technique. We found that (1) nm23 polypeptide is predominantly expressed in the cytoplasmic but also in nuclear and membrane compartments of melanocytic human cells, (2) expression of nm23 protein does not correlate with benign or malignant phenotype in melanocytic tumors of human skin. Our study challenges the hypothesis that nm23 may function in malignant melanomas as a tumor suppressor gene.

Early stage human colorectal cancer: prognostic value of nm23-H1 protein overexpression.

Nm23 gene codifies for a nucleoside diphosphate kinase allowing the intracellular transduction of the signals. In colorectal cancer nm23 protein expression seems related to the progression of the disease. By immunohistochemistry we have studied the intracytoplasmatic nm23 H1 protein expression in 20 patients affected by colorectal cancer at initial stage. In 12 cases it resulted elevated and in four the disease recurred. The overexpression was not correlated with other prognostic factors. Nm23 H1-positive patients affected by colorectal cancer at initial stage could be considered at risk for disease recurrence and included in a more frequent follow-up protocol.

Expression of metastasis suppressor gene product, nm23 protein, is not inversely correlated with the tumour progression in human malignant melanomas.

An inverse correlation between the nm23 RNA level and tumour progression of melanocytes has been reported. To elucidate whether the expression of nm23 gene product in malignant melanoma is also inversely correlated with metastatic potential, conventional prognostic parameters or the tumour suppressor protein p53, immunohisto-chemical studies using a monoclonal antibody against nm23-H1 protein were performed on 138 benign and malignant melanocytic tumours. The expression of nm23 protein was compared with that of p53 protein and conventional clinicopathological prognostic factors. The nm23 protein level in benign melanocytes and metastatic melanoma cells was also studied by Western blot analysis. No significant difference regarding the protein was observed between naevi and melanomas, either at histological or protein levels. The expression correlated with local recurrence within 1 year after surgery, level of invasion and tumour thickness, but no parallels were observed between the nm23 and p53 proteins, suggesting that gene is regulated by independent mechanisms, although located on the same chromosome. There was no inverse correlation between the nm23 protein and melanoma metastasis which suggested that the nm23 protein does not appear to be lost during melanoma metastasis.

The significance of nm23 protein expression in human gastric carcinomas.

The clinical significance of nm23 protein (nm23) expression was studied in tissue samples from 110 patients with primary gastric cancer by immunohistochemical staining with the anti-nm23 antibody. Primary carcinomas with either lymph node involvement or liver metastasis expressed significantly reduced levels of nm23 compared to those without metastasis. This relationship was clearer in the more differentiated adenocarcinomas than in the poorly differentiated adenocarcinomas. However, there was no correlation between nm23 expression and depth of invasion, quantity of stroma, infiltrating growth pattern, or macroscopic type. The cumulative 5-year survival rates based on nm23 immunoreactivity within the primary tumor were significantly higher in the nonreduced expression group (72%) than in the reduced expression group (45%). A multivariate analysis revealed that nm23 expression levels influence the outcome of patients as strongly as depth of invasion and more strongly than the other clinicopathological factors. These results suggest that the degree of nm23 expression is closely related to the metastatic potential of gastric carcinoma cells and can be used as a prognostic indicator independent of the clinicopathological features.

Correlation between nm23 protein and several cell adhesion molecules in human gastric carcinoma.

The correlation between nm23 protein (nm23) expression and the expression of several cell adhesion molecules was studied immunohistochemically in 110 resected gastric carcinomas. Formalin‐fixed and paraffin‐embedded samples were serially sectioned and stained with antibodies against nm23, integrin β1 subfamily members (α2β1, α3β1 and α4β1), LFA‐1, ICAM‐1, sialyl Lewisx (sLex) and CD44h, ‐V3, and ‐V6. Primary carcinomas presenting with either lymph node involvement or liver metastasis expressed significantly reduced levels of nm23 compared to tumors without metastasis. The percent of tumors expressing each adhesion molecule was as follows: α2β1, 27.3%; α3β1, 20.0%; α4β1, 14.5%; LFA‐1, 14.5%; ICAM‐1, 12.7%; sLex, 67.3%; CD44h, 55.5%; CD44V3, 20.0%; and CD44V6, 4.5%. Expression of α2β1 integrin and high levels of sLex were significantly correlated with lymph node metastasis, and expression of α3β1 integrin and high levels of sLex were correlated with liver metastasis. Expression of ICAM‐1 was inversely correlated with liver metastasis. Comparing the expression of each cell adhesion molecule with nm23 immunoreactivity, expression of sLex was significantly associated with nm23 expression. Of tumors expressing high levels of sLex, 75% showed reduced nm23 expression, compared to 52% of tumors with weak or no sLex expression (P < 0.05). A similar tendency was also observed in the metastasized secondary tumors. These results suggest that reduced nm23 expression may promote the metastatic properties of cancer cells in concert with increased sLex expression.

Analysis of nm23-H1 expression in breast cancer. Correlation with p53 expression and clinicopathologic findings.

Metastasis is the most frequent cause of death in patients with breast cancer. The nm23-H1 and p53 genes have been involved in the development of breast cancer metastasis. We have analyzed the correlation between the expression of nm23 protein and several established clinicopathologic factors. Our results show that the antimetastatic role of nm23-H1 is not related to the cell proliferative status or tumor grade and that it is not associated with the expression of p53. We also demonstrate a strong inverse relationship between the expression of nm23-H1 protein, lymph node metastasis and vascular invasion. These data support the antimetastatic role of the nm23-H1 gene and suggest that nm23-H1 and p53 genes may be involved in different steps of the metastatic process.

Elevated nm23 Protein Expression Is Correlated with Diminished Progression-Free Survival in Patients with Epithelial Ovarian Carcinoma

Objectives: The role of the candidate metastasis-suppressor gene nm23-H1 first characterized in breast cancer remains controversial, with both metastasis suppression and disease progression being linked to elevated nm23-H1 gene expression in different human tumor types. We sought to characterize (1) the pattern and intensity of nm23-H1/nucleoside diphosphate (NDP) kinase expression in human epithelial ovarian carcinoma (EOC) and (2) the relationship between nm23-H1/NDP kinase expression and tumor extent at diagnosis (FIGO stage) and response to treatment as defined by progression-free survival and actuarial survival. Methods: Twenty-four patients with EOC aged 61.1 ± 13.0 (mean ± SD) years were followed for 614.0 ± 289.7 days after a debulking procedure, cisplatin-based chemotherapy (19 of 24), and second-look laparotomy (9 of 24). After the primary debulking procedure, 63% of patients had no or microscopic residual disease. Overnight incubation of formalin-fixed tumor sections at 4°C with primary rabbit polyclonal IgG antibody to human nm23-H1/NDP kinase was followed by detection with standard ABC method. Nonimmune rabbit serum and normal breast tissue served as controls. Immunohistochemical staining was graded by a clinically blinded observer for intensity of staining (0, negative; 1, weak; 3, strong), pattern of staining (focal or diffuse), and histologic grade of tumor (1 through 4). Results: Of the EOCs, 54% were histologic grade 3 or 4 and 58% were FIGO stage III; 88% (21 of 24) stained positively, and 18 of 21 stained strongly and 14 of 21 stained diffusely. No correlation was found between either intensity or pattern of nm23-H1/NDP kinase immunostaining and histologic grade. No correlation was found between either staining pattern or intensity and FIGO stage. There was a trend toward decreased actuarial survival with both focal pattern (P= 0.12, log-rank test) and strong intensity (P= 0.15, log-rank test) of nm23-H1 staining. Decreased progression-free survival was likewise correlated with focal nm23-H1/NDP kinase immunostaining pattern (P= 0.02, log-rank test) and strong intensity of nm23-H1/NDP kinase staining (P= 0.08, log-rank test). Conclusions: Expression of nm23-H1/NDP kinase is strongly upregulated in most EOCs. Redundant overexpression of nm23-H1/NDP kinase may contribute to deranged cell cycle progression and EOC proliferation. Pattern and intensity of nm23-H1/NDP kinase immunostaining of EOC tissue retrieved at primary operation may identify patients at risk for tumor progression and help guide treatment strategies. These findings suggest that nm23-H1 gene expression may have distinct if not opposite biologic functions in EOC and breast carcinoma.

Nm23-H1 protein immunohistochemical expression in human breast cancer

A series of 76 patients undergoing surgery for primary breast carcinoma has been prospectively studied in order to evaluate the relative weight of nm23-H1 protein expression in disease-free survival. Expression of nm23 protein was immunohistochemically assessed. In all, 39% (29/74) of the turners showed positive staining for nm23-H1 protein expression. Negative nm23-H1 expression was found in poorly differentiated, tumors (p<0.02). There was no significant relationship between nm23-H1 and the other clinicopathological and biological features examined. In the univariate statistical analysis, node positivity, G3 histological grade and high flow cytometric S phase fraction (SPF) value proved to be significantly related to risk of relapse. In the multivariate analysis, only histological grade (G3) and high SPF values (>10.6) proved to be independently related to risk of relapse, with a hazard ratio of 9.84 and 7.98 respectively. Our preliminary study suggests that immunohistochemical nm23-H1 expression should not be considered a marker for predicting tumor progression and patient prognosis.

PP-9-2 The value of nm23 protein expression and tumor angiogenesis as a prognostic indicator in breast cancer of korean women

PP-9-2 The value of nm23 protein expression and tumor angiogenesis as a prognostic indicator in breast cancer of korean women

Immunohistochemical analysis of nm23 protein expression in malignant bone tumors.

Expression levels of nm23 protein in 72 malignant bone tumors comprising 41 osteosarcomas, 22 chondrosarcomas, 6 Ewing's sarcomas, and 2 malignant fibrous histiocytomas were examined immunohistochemically, using anti-nm23 protein polyclonal antibody, and compared with 51 cases of benign bone tumors or tumor-like lesions. Malignant bone tumors showed significantly higher nm23 protein expression than benign bone tumors or tumor-like lesions (P < 0.0001). In chondrosarcoma, nm23 expression increased in high-grade tumors (grade I versus grade II and III: P = 0.0229). In the cases of osteosarcoma, however, grade IV osteosarcomas showed decreased expression of nm23 compared with grade III tumors (P = 0.0122). There was no significant relationship between nm23 expression and histological type. nm23 expression had no correlation with metastatic potential in osteosarcoma, although the therapy was not uniform in our cases. Furthermore, in 6 cases of osteosarcoma and 1 case of Ewing's sarcoma, there was no clear tendency for a decrease of nm23 in the metastatic sites compared with primary sites, as reported in breast cancer. These results showed that, in contrast to reports on breast cancer and experimental models, nm23 protein expression in human bone tumors may be associated with malignant potentiality, except in cases of osteosarcoma.

Immunohistochemical analysis of nm23 gene product in human gallbladder carcinomas.

The expression of nm23, the product of a candidate suppressor gene for tumour metastasis, was examined immunohistochemically in human gallbladder carcinomas and compared with clinicopathological features. Seventy-eight (72%) of 107 carcinomas expressed nm23 protein regardless of histological type, while non-neoplastic mucosa occasionally showed very weak immunoreactivity to nm23. No obvious correlation was observed between nm23 protein expression and depth of tumour invasion or tumour stage. The expression of nm23 protein was detected in 60% and 74% of the cases with and without lymph node metastasis, respectively, indicating no relationship to metastatic ability. Fifty-eight percent of the cases showed reduction of nm23 immunoreactivity in tumour cells invading the stroma at the border of tumour cell nests compared with cells at the centre of the tumour. Only 7% of the cases showed increased nm23 expression in tumour cells at the border. These results suggest that in gallbladder carcinoma decreased expression of nm23 may not have implications for metastasis but may play a part in local invasion.

Reduced expression of nm23 protein is associated with advanced tumor stage and distant metastases in human colorectal carcinomas.

The nm23 gene, which encodes nucleoside diphosphate (NDP) kinase, is proposed as a metastatic suppressor gene. Loss of heterozygosity (LOH), and the expression of the nm23 gene were examined on matched sets of primary tumors and lymph node as well as distant metastases from colorectal carcinomas. Three (15%) of the 20 informative specimens examined showed LOH for the nm23 locus. nm23 was expressed in all the carcinomas as well as in nonneoplastic colonic mucosa at the mRNA and protein levels. Most of the carcinomas expressed the nm23 transcript at higher levels than the corresponding nonneoplastic colonic mucosa. By Western blotting, the level of nm23 protein expression in the tumors showed an inverse correlation with the tumor stage. Furthermore, distant metastatic tumors in the liver and lung showed reduced nm23 immunoreactivity in comparison with their primary tumor, although nm23 immunoreactivity was the same in the primary tumors and in local lymph node metastases. These results suggest that decreased nm23 expression may be associated with distant metastatic spread.