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Background: The aim of the present work was to evaluate the prognostic significance in patients with T1 breast cancer of tissue expression of the two oncosuppressors phosphatase and tensin homolog (PTEN) and non-metastatic clone 23 (NM23) as detected by immunohistochemistry., Materials and Methods: We prospectively analyzed 62 patients who underwent surgery for a T1 stage breast cancer. Expression of PTEN and NM23 was tested for correlation with clinical characteristics and clinical outcome., Results: Of the 62 patients considered for our study, 16 underwent mastectomy and 46 underwent conservative surgical treatment. The surgery was considered radical (R0) in all cases described. PTEN and NM23 expression was higher in patients with no lymph node metastases and no recurrent cancer at a mean follow-up of 36 months (range=6-48 months). This correlation was more evident when both PTNE and NM23 expression were highly expressed (p<0.0001)., Conclusion: Low or lack of PTEN and NM23 immunohistochemical expression in cancer tissue is a risk factor for lymph node involvement and recurrent disease. It may represent a valid prognostic factor in planning therapy in patients who had surgery for T1 breast cancer., (Copyright© 2021, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: There is controversy about whether expression of nm23 is involved in suppression of metastases or contributes to tumour progression. Few studies have examined the role of nm23 in sarcomas. The aim of this study was to determine if expression of nm23 protein or its H1-subtype correlated with clinicopathological parameters in adult soft tissue sarcomas (STS)., Materials and Methods: Protein expression was examined by immunohistochemistry on paraffin-embedded sections of 46 STS patients, quantified using a colour video imaging system and correlated to histological grade. The monoclonal antibodies used were anti-nm23 (recognising both nm23-H1 and nm23-H2) and anti-nm23-H1 (recognising nm23-H1 only)., Results: High-grade tumours significantly overexpressed nm23 (including both nm23-H1 and nm23-H2) compared with both intermediate and low-grade tumours (ANOVA and Tukey, all p < 0.05). Multiple regression analysis confirmed the significance of the relationship and independence of the nm23 (p = 0.005), but not nm23-H1., Conclusion: Expression of nm23 protein, particularly nm23-H2, is an independent predictor of malignant potential of adult STS.

Background: Despite surgical removal of the primary tumor of breast cancer in patients with apparently localized disease, relapse at local or distant sites may occur because undetectable micrometastases were present at the time of diagnosis. Identification of molecules associated with breast cancer metastasis suggests possible new treatments. PCD1, a gene encoding a new member of the PDZ and LIM domain-containing protein family, recently was identified. We examined the relationships between PCD1 mRNA expression in breast cancers and metastasis., Materials and Methods: PCD1 mRNA expression in breast cancer tissues was examined using a quantitative reverse transcription polymerase chain reaction., Results: PCD1 mRNA expression was greater in cancers than in noncancerous tissues (p < 0.0001). In addition, high PCD1 gene expression was more frequent in patients with lymph node metastasis., Conclusion: PCD1 appears to contribute to breast cancer progression and nodal metastasis, thus representing a potential target molecule in breast cancer diagnosis and treatment.

This review focuses on molecular events with the progression of gynaecological malignancies. The association of genetic abnormalities in cell cycle regulator genes, tumor suppressor genes and gene coding for growth factor receptors, with the progression of gynaecological cancers is reviewed. The possibility that some of these genes, cell cycle regulator genes in particular, could be co-operating with human papilloma virus genes in the induction and progression of gynaecological cancers is also discussed. The clinical effectiveness and reliability of these genes as markers of cancer progression and predictors of disease prognosis have been evaluated.

Background: Telomerase activity is up-regulated in most hepatocellular carcinomas (HCCs). Nm23-H2 has been shown to promote the expression of the c-myc gene, a candidate transcriptional activator of the human telomerase reverse transcriptase gene. Therefore, this study was undertaken to clarify the relationship between nm23-H2 expression and telomerase activity in HCCs., Materials and Methods: Telomerase activity and nm23-H2 protein expression in 24 HCCs were analyzed by telomeric repeat amplification protocol (TRAP) assay and immunohistochemistry, respectively., Results: The incidence of positive telomerase activity was significantly higher in tumor tissues than in normal liver tissues (18 out of 24 vs. 7 out of 24, p = 0.0015), and nm23 -H2 protein was more abundantly expressed in tumors than in corresponding normal liver tissues. Tumor nm23-H2 immunoreactivity was associated positively with tumor telomerase activity (p = 0.0037)., Conclusion: These results demonstrate that tumor nm23-H2 expression might be associated positively with telomerase activity in HCC and suggest the value of tumor nm23-H2 linked to telomerase activity as a therapeutic target for HCC.

The development of a molecular screening method for cancer patients is of great importance, since it would contribute to the selection of the most effective chemotherapy regimen for each patient. In the present study we applied such a method, semi-quantative RT-PCR analysis, and we examined the expression of the multidrug resistance gene MDR-1, the metastasis suppressor gene nm23-H1 and the non-MDR drug resistant gene H Sema E in 53 ovarian and breast cancer specimens. Moreover, we have correlated the expression profile of these genes with the histopathological findings and clinical outcome of the examined patients. The majority of specimens were found to be positive for MDR-1 and H Sema E gene expression, while nm23-H1 was detected in less than 50% of the patients. Correlation and statistical analysis of the molecular data with clinicopathological features showed that nm23-H1 could serve as a good prognostic factor for ovarian cancer patients. In breast cancer patients, nm23-H1 expression was associated with a 6.1 higher death risk. Ovarian cancer patients who express nm23-H1, but not MDR-1 and H Sema E, tend to have longer survival than patients with any other gene combination. Finally, breast cancer patients with advanced disease showed a better response when they were negative for all the three genes studied. In conclusion this work proposes that the combined study of the expression of different genes may be a useful approach for evaluating patients' response to therapy.

The nm23 gene is thought to play a role as an inhibitor of metastatic progression in several human cancers and its down-regulation has been associated with increased metastasis and reduced survival in some studies, though not in others. To better investigate the role of nm23 in gastric cancer (GC), the expression and prognostic impact of this gene was examined in 107 radically operated GC patients in a high risk area. The expression of nm23 was determined immunohistochemically by using the rabbit antibody anti-human nm23 protein. The expression of nm23 was detected in 40.2% (n = 43) of 107 gastric tumours and correlated with a poorer clinical outcome. In a survival analysis at 5 years, patients with nm23-positive tumours had significantly worse prognosis than patients (n = 64) with nm23-negative tumours (p < 0.05). The prognostic significance of nm23 expression was confirmed by multivariate analysis including terms for tumour stage and lymph node involvement. Our results suggest that the expression of the nm23 gene in gastric carcinoma is significantly related to tumour progression and poor prognosis at 5 years.

A new member of the nm23 gene family, designated nm23-H4, was cloned recently. Nm23-H4 is a mitochondrial protein. To determine whether nm23-H4 could have a function in tumour progression like nm23-H1 or nm23-H2, we analysed nm23-H4 expression in 18 renal tumours and 42 colorectal carcinomas obtained from patients who underwent curative resection. As compared to the corresponding healthy tissue, 14 renal tumours and 41 colorectal carcinomas showed increased nm23-H4 mRNA expression. While the increase was only moderate in renal cell carcinoma, a strong overexpression of nm23-H4 was noted in most colorectal carcinomas, possibly indicating a role of this gene in tumour genesis. The nm23-H4 transcript levels did not correlate with tumour stage, grade of differentiation or lymph node involvement.

Two metastasis-associated proteins, CD44v6 and NM23-H1, are expressed by normal lymphoid cells, the former serving as activation marker and the latter as a constitutive protein. CD44v6 is considered as a marker of poor prognosis of various hematological cancers but its expression was not demonstrated in childhood acute lymphoblastic leukaemia (ALL). On the other hand, NM23-H1 is considered as a differentiation inhibitory factor in various hematological cancers and as a marker of poor prognosis. Therefore we analyzed the expression of CD44v6 and NM23-H1 in the bone marrow of sixteen pediatric ALL patients using immunocytochemistry. For the first time, we have demonstrated the expression of CD44v6 protein epitopes on leukemic cells in a proportion of ALL cases (6 out of 16), primarily in the medium/high risk group (except one case), suggesting a possible association to an unfavourable outcome. On the other hand, NM23-H1 protein expression was maintained in leukaemic cells in 50% of both low and medium/high risk ALL cases. The majority of the pediatric ALL cases expressed only one of the metastasis-associated proteins (10 out of 16). This feature is similar to the observations made in several adult solid cancers. The potential of CD44v6 expression in leukaemic cells as prognosticator in pediatric ALL should be evaluated in a larger clinical trial.

Recently, we isolated a novel subline of the MAT-LyLu cell line from the metastasized lungs of MAT-LyLu-bearing Copenhagen rats (MLL cells). In this study, we compared the MLL cells to the parental MAT-LyLu cells with respect to invasive ability, mRNA expression level for the nm23 metastasis suppressor gene, and response to gossypol (GP), a natural compound with documented antiproliferative and antimetastatic activity, in an in vitro invasion assay. ML cells were isolated from mechanically dissociated metastasized lungs from MAT-LuLu-bearing Copenhagen rats. Comparisons of the invasive ability and steady-state levels of nm23 mRNA between MLL and MAT-LuLu cells were determined by in vitro invasion assay and RT-PCR, respectively. The results show that MLL cells display a higher penetration percentage than MAT-LyLu cells in the in vitro invasion assay. Furthermore, RT-PCR revealed that MLL cells possess lower steady state levels of nm23 mRNA than MAT-LyLu cells, suggesting a molecular basis for the observed differences in in vitro invasive ability. Finally, both MLL and MAT-LyLu cells were susceptible to gossypol, which induced dose-dependent inhibition of invasive activity. These results report the isolation of a novel, more highly invasive subline of the MAT-LyLu cell line that is as susceptible to the inhibitory effects of gossypol as the parental MAT-LyLu cells. The MLL cells, in combination with the parental MAT-LyLu cells, can be valuable tools for investigating the biology and behavior of metastatic cells and their response to chemotherapeutic/preventive agents.

Bladder cancer is clinically characterized by a high recurrence rate for superficial tumours up to 70% and by the invasiveness of advanced bladder cancer. To learn more about the biological behaviour of an individual bladder cancer different tumour markers have been investigated. The aim of our study was to compare the potential of aggression of both superficial and invasive bladder tumours by means of the proliferation marker Ki67, the tumour suppressor gene p53, the non metastasizing protein nm23 and the evaluation of DNA ploidy. We examined 36 patients, 28 with a bladder tumour (Ta-T4) and 8 without as a control group. For immunohistochemistry (Ki67, p53, nm23) we took paraffin sections and scored semiquantitatively under a microscope. The DNA cytophotometry was done on bladder washings by evaluating the DNA ploidy of single cells. The results showed that benign tissues were negative for Ki67 and p53 but positive for nm23. The DNA diagnosis was diploid for all benign samples. The superficial bladder cancer (Ta, T1) showed, in comparison to the invasive tumours, significantly lower numbers of aneuploid cells and a higher rate of p53 mutations. On the other hand the invasive tumours (T2-4) were correlated to significantly higher proliferation rates and higher potencies for metastasizing. The combination of the investigated tumour markers allowed a graduation of the biological behavior of an individual bladder cancer. Especially a high p53 mutation rate and a non aneuploid DNA diagnosis were indicators for the recurrence of superficial bladder tumours. Invasive growth of bladder cancer was characterized by high Ki67 proliferation and low nm23 protein binding.

Background: Prostate cancer is one of the main causes of morbidity and mortality among men. Several oncogenes and growth factors have been studied in an attempt to explain the molecular basis of carcinogenesis and progress of this carcinoma. In this study we correlated the immunohistochemical expression of antioncogene nm-23 H1 and transforming growth factor beta 1 (TGF-beta 1) with the clinical stage, PSA values, Gleason score and survival in prostate cancer., Materials and Methods: Fifty nine patients with prostate cancer were evaluated. PSA measurement, Gleason score determination and clinical staging were recorded for all the patients by the time of initial diagnosis and prior to any treatment. Follow-up ranged from 12 to 40 months. Tissue sections from representative areas of the tumors were immunohistochemically stained for nm-23 H1 and TGF-beta 1. The expression of these markers was correlated with stage, PSA values, Gleason score and survival., Results: There was a negative correlation between nm-23 H1 staining and tumor stage and grade. High grade (Gleason score 8-10) and stage D tumors showed weaker staining than low stage and grade tumors. There was a positive correlation between TGF-beta 1 staining, tumor stage and serum PSA levels. Additionally, TGF-beta 1 proved to be a negative predicting factor for patient survival. In tumors expressing both markers, TGF-beta 1 was the one to determine the aggressiveness of the carcinoma., Conclusions: nm-23 H1 appears to be a tumor suppressor gene in prostate cancer, while TGF-beta 1 may act as a stimulating agent provoking aggressive behavior and metastasis. Their immunohistochemical staining may constitute complementary information in the evaluation of prostate cancer patients.

NM23 is considered to be a metastasis suppressor gene the role of which as prognosticator in the case of malignant skin melanoma (MM) is highly controversial due to different results on gene, and protein expressions. Accordingly, we analyzed the protein expression of NM23 in 32 primary skin melanomas with a follow-up period of 5 years minimum. We found that NM23 expression was the lowest in the thickest primary tumors (based on the % of the positive cells and the incidence of low expressor tumors) but the difference was not significant statistically due to the extreme heterogeneity of the tumors. When primary tumors were grouped according to their biological behavior (non-metastatic; lymph-node (LN) metastatic; organ and LN metastatic tumors) we observed that the lowest NM23 protein expression (based on the % of positive tumor cells as well as on the incidence of low expressor tumors) was found in the LN metastatic tumors compared to other groups (p < 0.05). The NM23 phenotype of the primary tumors remained stable in the corresponding LN metastases in the case of the 5 analyzed tumors. There was no difference in the 5-year survival between patients with low (< 50% positive cells) or high NM23 protein expressing primary tumors. Collectively, these data suggest that the NM23 protein expression in the primary tumors of MM predicted lymphatic spread but did not affect 5-year survival because it did not correlate with organ metastasis.

Background: Aggressiveness of neuroblastoma is associated with increased expression of the putative metastasis suppressor genes, nm23-1 and nm23-2. These genes encode nucleoside diphosphate kinases A and B that form free or bound homo- and heteromers, which are distributed between soluble and particulate fractions of cells and display catalytic and non-catalytic activities., Materials and Methods: In order to establish which forms and activities of nm23 proteins are operative in neuroblastoma we stably transfected IMR-32 human neuroblastoma cells with constructs encoding wild type and catalytically inactive nm23-1 and nm23-2 proteins., Results: Overexpression of wild type nm23-1 proteins stimulated spontaneous neurite outgrowth and enhanced differentiation in response to serum starvation and retinoic acid. In contrast, overexpression of the catalytically inactive nm23-1T mutant enhanced TPA-mediated inhibition of differentiation., Conclusion: Our findings suggest that differentiation associated functions of nm23 proteins in IMR-32 neuroblastoma cells are carried out by bound nm23-1 proteins docked in a limited number of nm23-1 specific sites.

Background: The different clinical evolution of breast cancer with similar pathological characteristics prompted the authors to investigate the prognostic significance of different biological markers., Methods: Seventy-one primary breast carcinoma specimens obtained by mastectomy or quadrantectomy were examined for the determination of the p53, nm23 and Ki67 expression, with immunohistochemistry, and the DNA content, with static cytometry., Results: p53 protein was expressed as nuclear staining in 58% of the cases and was associated with high levels of Ki67, non-diploid lesions and lymph-node status. Positive staining for nm23 was significantly correlated only with histologic grading. A predictive role in disease recurrence was demonstrated only in patients with a high Ki67 nuclear expression., Conclusions: From these data, we may conclude that, besides all the other traditional clinical morphological parameters, a panel of different biological markers, such as Ki67 and the determination of p53 expression, may be utilized to further characterize breast cancer and its biological behavior.

Despite diagnostic and therapeutic progress, the overall prognosis of oral squamous cell carcinoma remains poor and prognostic information is essential for the evaluation and optimal treatment of patients. The prognosis of oral carcinoma depends primarily on clinicopathological factors, that however, have showed a limited predictive value for the identification of patients with high risk of disease relapse. In recent years, the analysis of DNA ploidy, cellular proliferation and oncogenes amplification have been used in attempt to identify new prognostic indicators. The review of the literature shows that TNM stage, tumor grading evaluated at the deep invasive margins and maximal tumor thickness effect on the prognosis. Moreover, the assessment of DNA ploidy and proliferative activity can be used to obtain additional prognostic informations. In contrast, additional studies are needed to understand the prognostic value of oncogenes expression in oral carcinoma.

Objectives: To explain the overall survival (OS) and disease free survival (DFS) in relation to nm23-H1 protein, DNA-ploidy and S-phase fraction (SPF) in transitional cell carcinoma of the bladder., Patients and Methods: Ninety-four samples were obtained from patients with transitional cell carcinoma of the bladder examined between 1994 and 1996. The patients were underwent cistectomy or surgical biopsy and the material was histologically evaluated according to World Health Organization classification. Nm23-H1 protein expression in immunohistological staining and DNA ploidy, S-phase fraction by flow cytometric were performed., Results: The correlation between OS and staging, grading, DNA-ploidy and S-phase was significant; whereas the overall survival and nm23-H1 protein, was not significant. The relationship between DFS and stage, DNA-ploidy and S-phase had a significant value. The correlation between DFS and age, sex, grading and nm23-H1 protein was not significant. There was no significant difference in age, sex, stage, grading, DNA-ploidy and SPF distribution between patients with nm23-H1 positive bladder cancer and those with nm23-H1 negative tumours., Conclusion: In our study, multivariate analysis showed that stage, ploidy and SPF were the strongest prognostic factors in predicting disease-free survival and prolonged survival, while nm23-H1 expression was not related to disease progression and/or prolonged survival. This expression, therefore, does not appear to be an independent prognostic factor in bladder cancer, although a still larger number of patients and a longer follow-up period are now needed for a definitive assessment of the prognostic significance of nm23-H1 expression.

In an effort to identify genetic changes that may be the early hallmarks of epithelial cell overproliferation, we searched for p53 and nm23-H1 allelic deletions in oral benign epithelial lesions. In the study group were 25 benign epithelial lesions (lichen planus--17; leukoplakia--8; recurrent aphthous ulcers--2; one specimen diagnosed as benign migratory glossitis). Among 21 samples analysed for exon 4 (p53 gene) LOH, only 6 were informative, with no loss of either allele. OF 23 samples tested for LOH at intron 6 of p53 gene, 8 were informative, again with no presence of LOH. For nm23-H1 gene, the analysis was performed on a total of 24 cases. Of them, 16 were informative, however, none exhibited LOH at this locus. In conclusion, whereas the presence of gross gene alterations (LOH) would have been definitive evidence for the involvement of p53 and/or nm23 in the hyperproliferation process, the absence of LOH does not exclude the presence of either smaller mutations, altered regulation of normal gene, or dysfunction at the level of wild type protein. Alternatively, p53 and nm23-H1 may have no relation to oral lesion formation, and cannot presently be considered as an early step in benign, tissue transformation.

Defective DNA mismatch repair proteins fail to correct replication errors (RERs). These defects may lead to secondary, mutation of oncogenes and tumor suppressor genes. Microsatellite instability might be a marker of such replication errors. Eighteen rectal tumors were examined to evaluate genetic instability, in sporadic rectal cancer by PCR. RERs were observed in 27.8% of the cases. No significant difference was noticed between RER+ and RER- patients as far as prognosis, clinicopathological features and p53 gene mutation are concerned. The incidence of nm23 gene mutation was the only statistically significant difference between the 2 groups. Three patients with only one altered microsatellite showed advanced tumor and nm23 gene mutation. Two cases with 5 altered microsatellites and nm23 gene mutated are disease-free: in one of them the p53 gene was also mutated. Probably more than one altered microsatellite is necessary to protect from the effects of secondary mutations.

The two nm23 genes, nm23-H1 and nm23-H2, are implicated in the metastatic process and tumor progression in some human tumors. Until now no data exist about nm23 expression in the different types of human renal tumors. To investigate if the nm23 genes play a central role in the progression of renal tumors, we have examined nm23-H1 and nm23-H2 gene expression using Northern-blot analysis and immunohistochemistry. We analysed clear cell type RCC, chromophilic RCC, chromophobic RCC, collecting duct type RCC and renal oncocytomas. Our results indicate that the nm23 genes do not play a central role in the prognosis of renal cell carcinoma in the analysed tumors.

The nm23 gene family is widely documented as a suppressor of the metastatic phenotype in many human cancers. However, its role with respect to the invasive behavior of primary central nervous system tumours has scarcely been addressed. This review describes the members of this family identified to date and outlines the in vitro and in vivo data gathered from nearly ten years of nm23 research within the context of malignant progression in peripheral tumours. It is clear that this is an area which can no longer be ignored by the brain tumor community. Human nm23 bears strong sequence homology to the awd gene in Drosophila, mutations in which cause abnormal morphology of larval neural tissue. The functions and possible mechanisms underlying the ability of nm23 to suppress metastatic behavior are discussed.

It has been proposed that nm23-H1, a candidate suppressor gene for metastasis, plays an important role in the metastasis of human tumors. In order to investigate its role in the human hepatocellular carcinoma (HCC), 18 matched pairs of tumorous and adjacent nontumorous liver tissues of hepatectomy from patients with HCC were studied by quantitative reverse transcription-PCR. Further analyses of the nm23-H1 gene were also done. The results of these molecular studies were correlated with the clinicopathologic features of the patients. Nm23-H1 transcript was expressed in all neoplastic and adjacent nontumorous liver tissue. The level of expression, however, did not correlate well with the extension or metastatic potential of the tumors. Instead, in 15 (83.3%) of 18 HCC, nm23-H1 expression was higher in the tumorous tissues, compared with the adjacent nontumorous tissues; and significantly higher levels of nm23-H1 mRNA expression was detected in HCC with poor differentiation (Edmonson classification, III and IV) than those with moderate differentiation (I and II). Southern blot analysis of nm23-H1 gene revealed neither amplification nor loss of heterozygosity of all HCC tissues examined. Direct sequencing of the nm23-H1 gene in all HCC tissues detected no mutations. Our findings suggested that increased nm23-H1 mRNA expression is correlated with HCC tumor progression.

Formalin-fixed, paraffin-embedded tissue blocks from 25 surgical specimens of renal cell carcinoma (RCC) including 13 adjacent normal renal tissues were investigated. The specimens were examined by immunohistochemistry using the monoclonal antibody, nm23-H1. The positive immunostaining of nm23-H1 protein was confined primarily to the cytoplasm of both normal renal tubular epithelial cells and renal tumor cells. Immunostaining of nm23-H1 protein was reduced significantly in RCC as compared to the normal renal tissues (P = 0.003). The positive immunostaining of nm23-H1 protein was seen in 92% (12/13) of normal renal tissues and in 60% (15/25) of RCC. No relationship was found between immunostaining of nm23-H1 protein and the patient's clinicopathological factors including age, tumor size, tumor location, tumor grade and tumor stage. Furthermore, immunostaining of nm23-H1 protein was not correlated with patient survival. Although immunoreactivity of nm23-H1 protein in patients with RCC was not correlated with survival, nm23-H1 protein may play a role in human renal tubular tumorigenesis.

The expression of nm23-H1 has been demonstrated to be highly correlated with the metastatic potential of various tumors. In the present investigation, meningiomas of different pathological grades were used to study on their nm23-H1 expression. Immunohistochemistry showed that nm23-H1 was expressed mainly in the cytoplasm especially in the perinuclear region in explants under short-term culture. Western-blotting demonstrated the specific expression of nm23 protein in all tumor samples. The expression was also found to be sex-dependent on tumor progression in female, but not in male patients. RT-PCR results confirmed nm23-H1 expression was higher in benign tumors than in their normal counterpart. Our observations thus suggest that nm23-H1 may play an important role in the progression of meningiomas in female patients.

The expression of the MDR1 gene has been shown to correlate with tumor aggressiveness and oncogenic activation both in experimental tumor models and in human clinical specimens In order to verify whether this association also takes place in ovarian carcinoma, we studied tumor samples from 39 patients by means of immunohistochemistry for the overexpression of P-glycoprotein (MDR1), nm23, c-erb-B2 and p53. MDR1 (p = 0.023), nm 23 (p = 0.037) and c-erb-B2 (p = 0.015) were expressed significantly more in specimens from patients with advanced stage of disease. There were no differences in p53 expression between both groups of patients. Furthermore, we found a significant coexpression of MDR1 and nm23 (p = 0.028), and of MDR1 and c-erb-B2 (p = 0.0077). There was no association between the expression of the MDR1 gene and p53. These results parallel those previously reported by us for mammary carcinoma, and seem to indicate that expression of the multidrug resistance gene (MDR1) is inherent to the development of the malignant phenotype in several human tumors.

The nm23 and mts1 genes have been the focus of attention as regards the association of their expression with metastatic behaviour. The level of nm23 and mts1 gene products has been demonstrated to correlate with metastatic potential in some tumors, but not in all. Here we show that these two genes might be coregulated and the ratio of their expression correlated with metastatic behaviour. Western blot analysis showed that the expression of both NM23 and MTS1 proteins was reduced in human lung cancer CH27 cells by retinoic acid treatment, but the ratio of NM23: MTS1 increased in a dose-dependent manner. Results also exhibited that retinoic acid altered the microtubule assembly of CH27 cells and reduced the metastatic ability of the cells in vitro. These data suggest that the metastatic potential of CH27 cells may be related to the relative expression of these two genes, and that their pathway in regulating metastatsis might be linked.

The nm23 gene was originally identified in murine melanoma cell lines as a putative metastasis suppressor gene. 1a a limited number of studies in breast carcinomas nm23 mRNA and/or protein levels were found to correlate inversely with lymph node metastases, and positively with the survival of patients. Using a monoclonal antibody to nm23-Hl protein we have examined the immunohistochemical expression of nm-23 in breast ductal carcinomas of 44 lymph node-negative patients with similar tumor pathologic features. The mean follow-up period of the patients was 138 months. Thirty two out of 44 tumors (72%) disclosed high immunohistochemical expression of nm23 protein and 12 (28%) low or negative expression. No correlation was observed between nm23 expression and the relapse or death rate of the patients. Similarly, no association was found between nm23 protein levels and estrogen receptor status or p53 protein. Our results do not seem to agree with the proposed antimetastatic property of nm23 protein, and indicate that its immunohistochemical determination has no prognose significance in the management of node-negative breast cancer patients.

Malignant insulinoma is an rare form of cancer with poor prognosis and a reported 5-year survival of 35%. Relatively little is known about the etiology of this disease or of the oncogenes and tumor suppressor genes that participate in its genesis and progression. To address this issue, several protooncogenes, including K-ras, N-ras, erbB-2, erbB-3,c-myc, c-fos, c-jun were examined. Also analyzed was the expression of the growth factors TGF-alpha, EGF, and insulin as well as the EGF receptor (EGF-R), p53 and the putative anti-metastasis gene nm23-H1. These were examined in malignant insulinomas, benign insulinomas, pancreatic B cell hyperplasias and in normal endocrine pancreas. Normal endocrine pancreas showed moderate immunoreaction for c-myc and a strong reaction for insulin. All other parameters were negative. Benign pancreatic B cell hyperplasias were slightly or moderately positive for N-ras and TGF-alpha, and were weakly positive for EGF-R. They were strongly positive for c-myc and insulin. In malignant insulinomas there was strong immunoreaction for c-myc, TGF-alpha, N-ras, K-ras and p53. Insulin reaction was moderate or strong. Molecular genetic studies have been performed for the presence of activating point mutations in codon 12 of the c-K-ras oncogene. Mutations were detected using primer-mediated, mutant-enriched, polymerase chain reaction-restriction fragment length polymorphism analysis and were further characterized by allele-specific oligonucleotide hybridization. Four out of six patients with malignant insulinoma and two out of eight patients with benign insulinoma harbored K-ras point mutations at codon 12. All patients with mutated K-ras oncogene also had elevated levels of p53 protein as well as c-myc and TGF-alpha. In one extremely malignant case we found concomitant mutation at codon 12 of K-ras and codon 61 of the N-ras gene. Our data are consistent with the idea that malignant progression is accompanied by the progressive accumulation of multiple genetic lesions and suggest that activation of myc, TGF-alpha and ras genes may be early events in the development of insulinoma.

Twenty-four advanced (surgical stage III and IV) ovarian carcinomas and 15 borderline ovarian tumours were studied for the overexpression of nm23 and HER-2/neu (c-erb-B2) by means of immunohistochemistry on sections from routinely processed, paraffin-embedded, archival tumour blocks, using the NCL-nm23 and the NCL-CB11 monoclonal antibodies and the streptavidin-biotin-peroxidase technique. Significantly more advanced ovarian carcinomas (p = 0.034) expressed high levels of nm23 when compared to borderline tumours. HER-2/neu (c-erb-B2) expression, as could be expected, was also significantly more frequent in advanced ovarian carcinomas (p = 0.006). We were not able to find the previously reported association between nm23 and HER-2/neu overexpression in our tumours. Our results on nm23 overexpression in ovarian cancer are coincident with those previously reported using nm23-mRNA measurements on fresh ovarian tissues. Thus, ovarian carcinoma seems to belong to the group of tumours, like colon carcinoma and neuroblastoma, in which nm23 overexpression is associated with a more malignant phenotype. Immunohistochemistry performed on archival samples from ovarian carcinomas seems adequate for the demonstration of nm23 overexpression in ovarian cancer. This opens the possibility for larger studies on series of patients with a closed follow-up, which could help to establish the role of this gene in this kind of tumour.

Ovarian carcinomas that have a distinctive natural history with early dissemination are particularly problematic. The aim of this immunohistochemical study was to assess whether the nm23-H1 gene product, which in some tumors shows inverse association with metastatic potential, could serve as a prognostic marker for ovarian carcinomas. The study, based on 73 benign and 54 malignant ovarian tumors, showed clear differences in the frequency of nm23-H1-positive samples, the intensity of staining and the histological localization of this protein. Differences were observed between normal ovary samples and benign lesions as well as between benign tumors and ovarian carcinomas and were highly significant. Furthermore, carcinomas that had detectable metastasis at the time of surgery were negative for nm23-H1 protein more frequently than those that did not. Although this is a prospective study in which collection of clinical data is ongoing, the results strongly suggest that nm23-H1 may serve as a potentially valuable marker for ovarian tumors.

Immunophenotypes of mammary (MPD) and extramammary Paget's disease (EPD) are still not well understood. Thirty-four formalin-fixed paraffin-embedded tissue sections from 33 patients with 6 MPD and 28 EPD were studied immunohistochemically with the use of polyclonal c-erbB-2 and pS2 antisera, and monoclonal nm23, B6.2, GCDFP-15, and p53 antibodies. Cases of MPD expressed a high incidence of c-erbB-2 and nm23 compared with those of EPD (100% vs. 29%; p < 0.01, and 83% vs. 29%; p < 0.05, respectively). Although high expression of B6.2 (> 83%) and moderate expression of GCDFP-15 (33-39%), pS2 (33-46%) and p53 (39-50%) were seen, the positivity was not significantly different between MPD and EPD. These findings indicate that MPD and EPD share immunohistochemical features but partially differ in their patterns of antigen expression.

The nm23 and mts1 genes are associated with the expression of the metastatic phenotype. We have shown previously that modulation of metastatic behaviour produces parallel changes in the expression of these genes and that the expression of the two genes is co-regulated. Here we show that modulation of gene expression affects the process of tubulin polymerisation. B16 melanoma cell lines F1 and ML8 were treated with alpha melanocyte stimulating hormone (MSH) and all-trans retinoic acid (RA) respectively. MSH reduced the proportion of nm23+ and increased mts1+ F1 cells, with a 55% decrease in the ratio nm23:mts1. In parallel, MSH increased the expression of depolymerised form of tubulin in these cells. Treatment of ML8 cells with RA decreased mts1 positivity to a greater extent that nm23 positivity and the nm23:mts1 ratio increased by 70% and, in parallel, reduced the expression of depolymerised form of tubulin. These data suggest that nm23 and mts1 gene expression regulates the biological behaviour of the tumour cell and confer on it invasive and metastasizing properties by affecting the state of tubulin polymerisation.