nm23-H1 expression and loss of heterozygosity in colon adenocarcinoma.

BACKGROUND: The discovery that genetic alterations in oncogenes and tumour suppressor genes accompany tumour formation in many human tumours has encouraged the search for genes that promote or suppress tumour spread and metastasis; nm23 is a promising candidate for a metastasis suppressing gene. AIMS: To evaluate whether expression of nm23-H1 protein or loss of heterozygosity (LOH) of the nm23-H1 gene is associated with colon cancer progression. MATERIALS/METHODS: Paraffin wax embedded tissue sections were analysed immunohistochemically. DNA isolated from normal and tumour tissue was used for LOH analysis using a variable nucleotide tandem repeat (VNTR) marker located in the untranslated 5' region of the nm23-H1 gene. RNA isolated from tumour and normal tissue was used for "real time" RT-PCR. RESULTS: Of 102 adenocarcinomas examined, 58.8% stained weakly for nm23-H1 protein. There was a negative correlation between nm23-H1 positivity and tumour histological grade. In VNTR analysis, 70.2% of patients were informative and 27.4% of tumours had nm23-H1 LOH. There was a positive correlation between nm23-H1 LOH and both tumour histological grade and Dukes's stage. Expression of nm23-H1 mRNA was increased in 22 of 30 colon tumours compared with normal tissue. No significant correlation was found between nm23-H1 mRNA expression and histological grade or Dukes's stage of tumours. CONCLUSIONS: These findings suggest that nm23-H1 protein expression in early stages may have a role in suppressing metastasis in sporadic colon cancer, whereas at a later stage both reduced nm23-H1 protein expression and LOH of the nm23-H1 gene may play role in colon cancer progression and metastasis.

Effect of the nonsteroidal anti-inflammatory drug indomethacin on proliferation and apoptosis of colon carcinoma cells.

PURPOSE: Nonsteroidal anti-inflammatory drugs lower the incidence of and mortality from colon cancer. In this paper, we present the effect of indomethacin on growth inhibition and alterations in the expression of several genes involved in cell cycle and apoptosis in CaCo-2 colon adenocarcinoma cells. METHODS: We used the MTT test to evaluate the effect of indomethacin on the proliferation rate of colon cancer and normal fibroblast cells in vitro. The expression of c-myc oncoprotein and p53 and p27 suppressor proteins was examined using the immunocytochemical method. RESULTS: We have shown that indomethacin reduces the proliferation rate of CaCo-2 colon cancer cells (up to 60% at the concentration of 4 x 10(-4) M), alters their morphology, and induces cell death by apoptosis. The most pronounced inhibitory effect was observed at the concentration of 6 x 10(-4) M where the growth was completely suppressed. However, the growth of normal fibroblasts (Hef 522) was much less inhibited (about 30% of inhibition at the concentration of 6 x 10(-4) M). Indomethacin reduces the proliferation rate and induces apoptosis in CaCo-2 colon cancer cells through enhanced expression of c-myc, p53, and p27 proteins. CONCLUSIONS: This is the first report about p27-increased expression in colon carcinoma cells induced by indomethacin treatment. Increased expression of p27 represents a new mechanism of apoptosis in cells treated with NSAIDs (indomethacin). This effect probably contributes to the anti-proliferative effect on colon cancer cells in vitro.

Expression of erbB-3 protein in colorectal adenocarcinoma: correlation with poor survival.

BACKGROUND/AIMS: The family of erbB receptors includes four transmembrane glycoproteins with tyrosine kinase activity. These receptors are widely expressed in normal tissues, but they also have been implicated in the development of several human adenocarcinomas. c-erbB-3/HER-3 has been detected to a greater or lesser extent in many tissues from the digestive, urinary, reproductive and respiratory tracts. The overexpression of c-erbB-3/HER-3 protein has also been shown in 53%-88% of colorectal adenocarcinomas. In this study we investigated the expression of the c-erbB-3/ HER-3 gene product in colorectal tumour samples, and compared the results obtained with several clinicopathological parameters, including the survival of patients. METHODS: Paraffin-embedded tissue sections were analysed immunohistochemically, using monoclonal antibody RTJ1 to human erbB-3 protein. Antibody RTJ1 specificity was confirmed by immunoprecipitation followed by Western blotting analysis. Amplification of the erbB-3 oncogene was tested by dot-blot hybridization. RESULTS: Adenocarcinomas of the colon were positive for erbB-3 protein in 78% of samples examined. Dot-blot analysis showed no amplification of the erbB-3 gene in colon adenocarcinomas. Statistical analysis showed that patients with tumours that could not be stained for erbB-3 protein survived significantly longer (P<0.05) than patients with tumours staining positive for the erbB-3 protein. A Cox proportional-hazards model with stepwise variable selection identified age, sex and erbB-3 expression as important prognostic factors. CONCLUSION: These findings demonstrate that erbB-3 protein expression could serve as a prognostic factor in colorectal malignancies.

Loss of heterozygosity of APC and DCC tumor suppressor genes in human sporadic colon cancer.

We examined 36 cases of human sporadic colon carcinoma and corresponding normal tissue samples to evaluate loss of heterozygosity at the APC and DCC tumor suppressor genes loci using restriction fragment length polymorphism polymerase chain reaction and variable nucleotide tandem repeat analysis. Observed informativity was 83% for APC and 75% for DCC. DNA from 6 (20%) of 30 informative tumors exhibited loss of heterozygosity at the APC locus. Loss of heterozygosity at the DCC locus was observed in 7 (26%) of 27 informative tumor DNAs. Our results support the view that malignant progression is a consequence of more than one genetic change and suggest that inactivation of APC and DCC genes plays a role in a multistep process of colon tumor progression.

The expression and role of insulin-like growth factor II in malignant hemangiopericytomas.

Hemangiopericytoma is a rare soft tissue tumor originating from contractile pericapillary pericytes. To address the issue of molecular genetic events that participate in genesis and progression of hemangiopericytoma we analyzed insulin-like growth factor (IGF) II and IGF I receptor in 29 tumors collected from a human tumor bank network. Seven of these tumors were associated with severe hypoglycemia; six were retroperitoneal and one was located in the leg. Of 22 tumors tested 12 (54.5%) exhibited IGF II mRNA, while almost 90% (17 of 19) of hemangiopericytomas exhibited IGF I receptor mRNA. Sera from some patients whose tumors expressed IGF II mRNA contained elevated levels of IGF II. Removal of the tumor eliminated most of the IGF II immunoreactivity from the sera. The potential role of IGF II as a growth-promoting factor was examined on three malignant primary hemangiopericytoma cell cultures. Extracellular addition of IGF II significantly enhanced cell proliferation in a dose-dependent manner. Antisense oligodeoxynucleotides that specifically inhibit IGF II mRNA, at a concentration of 40 or 80 micrograms/ml, inhibited the growth of hemangiopericytoma cells significantly, by 40%. Simultaneous administration of antisense deoxyoligonucleotides to both IGF II and IGF I receptor inhibited tumor cell proliferation by even 80%. Our data suggest that tumor cells produce IGF II, and that this in turn stimulates their proliferation by autocrine mechanisms.

Molecular pathology of hemangiopericytomas accompanied by severe hypoglycemia: oncogenes, tumor-suppressor genes and the insulin-like growth factor family.

Relatively little is known about molecular genetic events that participate in the genesis and progression of hemangiopericytoma. In this study, we describe two cases of hemangiopericytoma accompanied by severe hypoglycemia. Tumor cells from patient 1 exhibited insulin-growth factor I (IGF I) and insulin-like growth factor I receptor (IGF IR) mRNA transcripts. Tumor cells from patient 2 exhibited IGF II, IGF IR and IGF binding proteins 1-3 mRNA. Serum from patient 2 contained IGF II, mostly in a large molecular form ("big" IGF II); the IGF II level did not change after the tumor removal. The presence of IGF IR in tumor cells was confirmed by immunoprecipitation with antibodies that recognize human IGF IR subunit (visualized as a 460-kDa band). The hemangiopericytoma cells derived from patient 1 expressed 210000 IGF I receptors/cell. Specific binding of IGF I to the tumor cell membrane fraction was higher in tissue from patient 1, while the tissue of patient 2 showed relatively low IGF I binding. In contrast, IGF II binding was much higher in tissue from patient 2. Both tumor tissues showed positive immunostaining for c-Jun; one tumor showed strong immunostaining for c-Myc, H-Ras and p53, while the other exhibited strong reaction with H-Ras antibodies only. No loss of the heterozygosity at the genes APC, NFI and nm23-H1 loci in tumor tissue obtained from patient 1 was found. In effect, our results suggest multiple molecular genetic changes in hemangiopericytoma -- activation of some oncogenes and the IGF growth factor family. IGF ligands together with IGF IR could be responsible for hypoglycemia and perhaps the transformed phenotype.

The expression of p185(HER-2/neu) correlates with the stage of disease and survival in colorectal cancer.

BACKGROUND & AIMS: HER-2/neu oncogene encodes a transmembrane tyrosine kinase receptor that is amplified and/or overexpressed predominantly in adenocarcinomas. This phenomenon has been most intensively studied in breast carcinoma where its amplification and overexpression correlate with the overall course of disease and poor prognosis. This study was designed to investigate HER-2/neu gene expression in benign and malignant colorectal lesions and to evaluate its prognostic importance in colorectal cancer. METHODS: Two hundred twenty-one samples of normal colon, benign lesions, and colorectal adenocarcinomas were studied for expression of HER-2/neu oncoprotein. Immunohistochemical staining of formalin-fixed, paraffin-embedded tissue sections of primary tumor and lymph nodes was performed. Immunoprecipitation followed by Western blotting of freshly frozen samples of the same tumors were also performed. RESULTS: Normal colon mucosa, benign lesions, and adenocarcinomas clearly differed in the expression levels and histological distribution of p185(HER-2/neu). Normal mucosa was mostly negative, but significant number of benign lesions and adenocarcinomas overexpressed HER-2/neu protein. Adenocarcinomas were significantly more positive than benign lesions. The results show significant correlation with the epithelial abnormality degree and clinical parameters including Dukes' classification and relapse-free and postoperative survival period. CONCLUSIONS: The p185(HER-2/neu) rate expression could serve as an independent prognostic factor in patients with p185(HER-2/neu)-positive colorectal malignancies.

Concomitant point mutation of tumor suppressor gene p53 and oncogene c-N-ras in malignant neuroendocrine pancreatic tumor.

Activation by point mutation of ras family genes as well as point mutations of the p53 tumor suppressor gene are found in many tumors. Here we describe a rare case of malignant neuroendocrine pancreatic tumor with multiple metastases in different organs showing strong positivity for synaptophysin, glucagon-like peptide 1, pan-cytokeratin, moderate positivity for chromogranin, Phe-5 and calcitonin and weak positivity for vasointestinal peptide. We found a point mutation at codon 61 of the c-N-ras oncogene, and point mutations in the p53 tumor suppressor gene in the primary tumor as well as in its metastases in liver. The mutation in the c-N-ras gene was a cytosine to adenine transversion, resulting in the amino-acid lysine. Allele specific hybridization showed that the mutation involved one of two c-N-ras alleles as the oligonucleotide for the normal codon also hybridized to amplified tumor DNA. Concomitant mutation of the p53 tumor suppressor gene at codons 248 and 249 was found. The mutation in codon 248 was a cytosine to guanine transversion resulting in the amino-acid glycine. The mutation in codon 249 was a third base, G- > T, transversion leading to a change from arginine to serine. This is the first time that concomitant point mutations in c-N-ras and p53 have been found in a neuroendocrine pancreatic tumor. Based upon these and our previous results, we concluded that these genetic changes may play a role in the development of this particular pancreatic tumor.

Molecular genetics of malignant insulinoma.

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.

Local interferon therapy for melanoma patients.

BACKGROUND: Melanoma, once considered a rare form of cancer, is increasing in incidence throughout the world. The prognosis of malignant melanoma is inversely related to the depth of tumor invasion. METHODS: Twenty-seven patients were treated with r.IFN alpha 2c. Four patients were treated with human natural leukocyte interferon (HNLI). Interferon was applied locally. Historical control groups were used for comparison in the statistical analysis. The data were evaluated taking into account the single risk factor Clark levels III and IV. In the control group there were 10 patients with Clark levels III and IV; in the group of r.IFN apha 2c-treated patients there were 20 patients. The data were analyzed by using the Kaplan-Meier and Cox methods. RESULTS: The percentage of survivals was higher in the interferon-treated groups with Clark levels III and IV, than in the control group, that is 60% compared to 25%, and 40% compared to 33%, respectively. The results of the statistical analysis after 60 months of follow-up are significantly better in the interferon group (P = 0.0175). CONCLUSIONS: The control group was not selected at random. Therefore, on the basis of our results, one can say that the treatment of the melanoma patients, Clark levels III and IV, with the r.IFN alpha 2c is promising and that further investigation is justified.

Interferon reduces recurrences of basal cell and squamous cell cancers.

BACKGROUND: Interferon is considered to be an important curative agent for dermatologic diseases. We report the follow-up experience of patients with basal cell or squamous cell carcinomas treated with human natural leukocyte interferon (HNLI). RESULTS: Among 52 patients with basal cell carcinoma (BCC) treated with HNLI more than 10 years ago, and among 58 treated more than 5 years ago, only 2 recurrences were observed. There were no recurrences in 75 patients who had a complete response to HNLI treatment, nor were there any in 20 patients with either a partial or complete response to r.IFM alpha 2c treatment. Of 52 patients with squamous cell carcinoma (SCC), 31 had been treated more than 10 years earlier, two recurrences of the disease at the site of the original lesion were observed. CONCLUSION: Interferon treatment makes it possible to achieve a persistent cure in patients with BCC and SCC in a high proportion of cases. The potential advantage of nonsurgical treatment are an enhancement of cosmetic results through the prevention of destruction of important anatomic structures.

Prognostic significance of transforming growth factor alpha TGF-alpha) in human lung carcinoma: an immunohistochemical study.

Despite emerging data relating oncogene expression, growth factors and/or their receptors to the etiology of lung cancer, standard clinicopathological evaluation is still used for the diagnostic and prognostic purposes. Recent studies have shown that expression of some oncogenes and growth factors/receptors may be useful as markers in routine diagnostic and prognostic processes. For example, EGF/erb-B family of peptides may play a role in lung carcinogenesis. Similarly, expression of TGF-alpha mRNA and peptide has been shown to occur in various human lung carcinomas in vivo and in vitro. However, results concerning the role of TGF-alpha in lung carcinoma are conflicting and therefore its clinical value still remains obscure. To better evaluate the potential value of TGF-alpha in clinical application we have investigated the relationship between TGF-alpha expression in 51 lung carcinomas and 26 different clinical and clinicopathological parameters. The only significant correlation noted was between TGF-alpha and venous blood erythrocytes and eosinophils. This study suggests a relationship between metastasis and aggressive behavior of lung cancer. This data shows that TGF-alpha expression can not serve as an independent tumor marker for lung cancer.

Immunohistochemical detection of TGF-alpha, EGF-R, c-erbB-2, c-H-ras, c-myc, estrogen and progesterone in benign and malignant human breast lesions: a concomitant expression.

The expression of transforming growth factor-alpha (TGF-alpha), epidermal growth factor receptor (EGF-R) and oncogenes c-erbB-2, c-H-ras, c-myc, as well as estrogen (ER) and progesterone (PR) receptors were studied immunohistochemically in the tissue of 21 benign and 58 malignant human breast lesions. Twenty nine (50%) of 58 carcinomas were positive for EGF-R and c-erbB-2 product, 55 (94.8%) for c-myc product, 9 (15.5%) for c-H-ras product and 17 (29%) for TGF-alpha. Eighteen of 58 (31%) carcinomas were estrogen receptor positive and 22 (38%) were positive for progesterone receptor. No correlation was found between expression of each investigated parameter and the clinical stage or degree of histological differentiation of the carcinomas. However, a significant positive correlation was observed between lymph node involvement and c-erbB-2 and EGF-R/c-erbB-2 positive tumors. A strong correlation was also observed between high levels of EGF-R and low levels of estrogen receptor. In 15 of 17 cases we found simultaneous expression of EGF-R and TGF-alpha. We also found interesting patterns in concomitant expression of the investigated parameters suggesting a possible cascade of events that occur in breast cancer cells.

High c-erbB-2 protein level in colorectal adenocarcinomas correlates with clinical parameters.

Sections of normal colon, adenomas, and adenocarcinomas were examined by immunohistochemistry for the expression of c-erbB-2 proto-oncogene product in order to assess its potential diagnostic value in predicting the malignant potential of these lesions. We compared the degree of epithelial abnormality and clinical parameters, including Dukes' classification and survival time with the extent of immunoperoxidase staining. Sections of normal colon and tubular adenomas examined demonstrated a weak immunostaining localized to the luminal surface cells. However, higher level of c-erbB-2 expression was observed in dysplastic areas of one tubular and one villous adenoma. Out of 40 adenocarcinomas, only 2 samples showed weak immunoreaction, while 38 samples were moderately or strongly positive for c-erbB-2 protein. The intensity of staining correlated positively with the stage of disease and postoperative survival time.

Evidence for a role of EGF receptor in the progression of human lung carcinoma.

Sixty-three primary lung carcinomas were examined immunohistochemically for the presence of epidermal growth factor receptors (EGF-R). Of these tumors 35 (55.5%) were positive for EGF-R. A positive correlation between overexpression of EGF-R, on the one hand, and high metastatic rate, poor tumor differentiation and high rate of tumor proliferation, on the other hand, was found. From one tumor with an extremely high amount of EGF-R a primary cell culture was established. The addition of anti-EGF-R serum to this culture decreased cell proliferation. Our results support the evidence for a positive relationship between overexpression of EGF-R and invasiveness, poor differentiation and high growth rate of tumor cells. The blockade of EGF-R with specific antibodies suppressed tumor cell proliferation, suggesting a role of EGF-R in tumor progression.

Human lung cancers growing on extracellular matrix: expression of oncogenes and growth factors.

The goal of this study was to evaluate the extracellular matrix (ECM) as a model for growing human lung cancers and to study the feasibility of its application for cellular and molecular studies of tumor biology. Bovine corneal endothelial cell ECM coated dishes were evaluated as a growth substrate for tumor cultures. Growth success, morphology and oncoprotein/growth factor expression for 74 different lung cancers (adenocarcinoma, epidermoid carcinoma and small cell carcinoma) were compared after seeding fresh surgical explants onto bovine corneal endothelial cell ECM and plastic culture substrate. Nineteen out of 74 tumors (26%) plated on ECM demonstrated measurable growth. Growth on ECM was superior to growth on plastic for the lung tumors. All 19 tumor cultures showed malignant morphology and functions. They were examined under the light microscope, and in all cases pre- and post-cytology confirmed malignancy. Tumor cells seeded on ECM retained their malignant phenotype in comparison to tumors grown on plastic. Several oncoproteins (c-myc, c-Ha-ras, c-erbB-2) and growth factors/receptors (EGF, EGF-R, TGF alpha) were immunostained. These analyses were performed immediately after disaggregation of tumor cells obtained surgically and after seeding on ECM or plastic. Strong expression of oncoproteins/growth factors was detected in tumor cells immediately after surgery or when the cells were plated on ECM. On the other hand, moderate or no expression was observed in the same type of cells on plastic.

Collagenase derived from human fibrosarcoma is responsible for degradation of basement membranes.

A collagenase-like enzyme with the ability to degrade the proteins of artificial basement membranes (BM) was isolated from human fibrosarcoma. Secretion of the same peptide was observed from the primary fibrosarcoma cell cultures. This peptide degrades the artificial basement membranes derived from bovine corneal endothelial cells. Using electrophoretic methods it was found that the isolated and partially purified enzyme consists of eight bands of different molecular mass corresponding to the collagenase standard from Cl. histolyticum. Only two bands with molecular masses of 22,000 (pI 5.5) and 63,000 (pI 5.9) degrade BM.

Comparison of technetium-99m and iodine-123 imaging of thyroid nodules: correlation with pathologic findings.

Three hundred and sixteen patients with solitary or dominant thyroid nodules were imaged both with technetium-99m- (99mTc) pertechnetate and iodine-123 (123I). The images were preferred, but differences were small and in 27%-58% of the cases there was no difference in quality between the two radionuclides. Discrepancies between 99mTc and 123I images were found in 5%-8% of cases, twice as often in multinodular goiters as in single nodules. Cytologic/histologic examination was performed on all nodules but no correlation was found between the pathology and the type of discrepancy. Twelve carcinomas were found (4%) but none in nodules showing a discrepancy. There was great variation among the observers about the preference for radionuclides and about the existence or type of discrepancies. The slightly better overall quality of 123I scans is probably not of diagnostic significance and does not justify the routine use of 123I instead of 99mTc. Routine reimaging of 99mTc hot nodules with radioiodine for cancer detection does not appear to be necessary.