c-myc oncogene alterations in human thyroid carcinomas

A possible role of the c-myc oncogene in the neoplastic transformation of the human thyroid has been investigated. The structure, the methylation status, and the copy number of this oncogene have been analyzed in normal and in tumor thyroid DNAs by Southern blotting technique and dot blot hybridization. Among six carcinomas, four presented abnormal c-myc DNA structure: Three cases showed a mutation in the 5' flanking region of the gene, originating a new EcoRI site; the other case showed a deletion of 5 kb involving the first exon of the gene. This deletion was also observed in the white blood cells of the same individual. In addition, in three carcinomas a double dose of the oncogene has been demonstrated. In all the carcinomas examined, undermethylation of the c-myc oncogene has been observed. These findings suggest that c-myc oncogene alterations might be involved in the malignant transformation of the human thyroids and can be considered as tumor markers.     

c-fos and c-myc oncoprotein expression in human hepatocellular carcinomas.

Amounts of the proteins encoded by the two oncogenes c-myc and c-fos have been compared in seven specimens of hepatocellular carcinoma and two normal liver samples using a Western blot procedure. It was found that with the exception of one tumour, the amount of these proteins was markedly increased in the tumours when compared to the normal specimens. Furthermore, there appeared to be elevated c-myc and c-fos mRNA concentrations in the tumours which correlated with the protein levels. This is the first report of such a correlation in human hepatocellular carcinoma. We propose that transactivation of these oncogenes may in part be responsible for transformation in hepatocellular carcinoma.     

Hormone synthesis by primary breast carcinomas and prognosis in human breast cancer

The ability of human breast carcinomas to convert pregnenolone to progesterone and dehydroepiandrosterone to delta 4-androstene-3,17-dione (delta 4) was investigated as a potential aid for prognosis, and the following observations were recorded. 1. Neither the amounts of progesterone or delta 4 synthesized nor delta 4/progesterone ratios correlated with tumour size or lymph node involvement. 2. delta 4 synthesis was lower in carcinomas from patients who had recurrences within 2 years of mastectomy than in carcinomas from those who remained free of metastases. 3. Life table analysis of the results indicated that these parameters appeared unlikely to be useful aids for prognosis.     

Over-expression of the c-myc proto-oncogene in colorectal carcinoma.

Alterations in the c-myc proto-oncogene in colorectal cancer were studied at the level of RNA expression, gene amplification and rearrangements. One hundred cases of colorectal cancer, stratified by Dukes'' stage were examined. The level of messenger RNA expression was measured in tumours and matched normal mucosa from the same patient. Between 5 and 400 fold over-expression was found in 66% of tumours. Neither the presence nor the level of over-expression correlated with tumour staging. A significant correlation (P < 0.01) was found between over-expression of c-myc in tumours and the presence of synchronous adenomas elsewhere in the colon. In contrast to other tumours, no rearrangements of the gene were found on Southern analysis of colorectal cancers. Similarly, amplification of the gene was not found in the cancers examined.     

Overexpression of c-met proto-oncogene but not epidermal growth factor receptor or c-erbB-2 in primary human colorectal carcinomas.

The epidermal growth factor receptor (EGFR) and the protein products of c-erbB-2 and c-met proto-oncogenes belong to a family of growth factor receptors with tyrosine kinase activity. In human colonic carcinomas, the expression of the EGFR and c-erbB-2 have been studied at the protein level only, while c-met expression has not been reported. We have examined the mRNA expression of these genes in human normal colorectal mucosa and primary carcinomas. The results demonstrate that the normal mucosa shows highly variable levels of EGFR and c-erbB-2 mRNAs, but expresses consistently low amounts of c-met mRNA. Colorectal carcinomas did not express significantly higher levels of the EGFR and c-erbB-2 mRNAs than the normal mucosa. In contrast, c-met was consistently and significantly overexpressed (mean sixfold) in carcinomas as compared with normal mucosa. Seventy percent of paired normal-tumor specimens showed a tumor to normal c-met mRNA ratio of greater than 4. The expression of c-met mRNA was also enhanced in the adenomas, suggesting that over-expression of this proto-oncogene may have mechanistic significance in the early stages of human colorectal carcinogenesis.     

Identification of transforming growth factor alpha in human primary breast carcinomas

Primary human mammary carcinomas were found to contain an acid stable polypeptide which bound to the receptor for epidermal growth factor (EGF). This polypeptide which was antigenetically different from EGF and thus identified as Tumor Growth Factor (TGF) alpha, could be identified in 25% of the tumors. Among these tumors 40% contained measurable levels of the EGF receptor. Thus these tumors exhibit the molecular prerequisite for autocrine growth stimulation.     

Expression of c-erbB3 protein in primary breast carcinomas.

Expression of c-erbB3 protein was investigated in 104 primary breast carcinomas comprising nine comedo ductal carcinoma in situ (DCIS), 91 invasive ductal carcinomas and four invasive lobular carcinomas using two monoclonal antibodies, RTJ1 and RTJ2. Of the 91 invasive ductal carcinomas, seven contained the comedo DCIS component adjacent to the invasive component. An immunohistochemical technique was used to evaluate the association between expression of c-erbB3 and clinical parameters and tumour markers such as epidermal growth factor receptor (EGFR), c-erbB2, cathepsin-D and p53 in archival formalin-fixed paraffin-embedded tumour tissues. Our results indicated that RTJ1 and RTJ2 gave identical staining patterns and concordant results. It was found that the overexpression of c-erbB3 protein was observed in 67% (6/9) of comedo DCIS, 52% (44/84) of invasive ductal carcinomas, 71% (5/7) of carcinomas containing both the in situ and invasive lesions and 25% (1/4) of invasive lobular carcinomas. A significant relationship (P < 0.05) was observed between strong immunoreactivity of c-erbB3 protein and histological grade, EGFR and cathepsin-D, but not with expression of c-erbB2, p53, oestrogen receptor status, lymph node metastases or age of patient. However, we noted that a high percentage of oestrogen receptor-negative tumours (59%), lymph node-positive tumours (63%) and c-erbB2 (63%) were strongly positive for c-erbB3 protein. We have also documented that a high percentage of EGFR (67%), c-erbB2 (67%), p53 (75%) and cathepsin-D-positive DCIS (60%) were strongly positive for c-erbB3. These observations suggest that overexpression of c-erbB3 protein could play an important role in tumour progression from non-invasive to invasive and, also, that it may have the potential to be used as a marker for poor prognosis of breast cancer.     

Presence of somatomedin receptors on primary human breast and colon carcinomas

Competitive binding techniques were used to study the interaction of insulin-like growth factor I (IGF-I) with a plasma membrane-enriched subcellular fraction purified from primary breast and colon carcinoma specimens obtained at surgery. The presence of specific binding sites for IGF-I was detected in all tumour specimens studied. Scatchard analysis and competition studies with insulin and insulin-like growth factor-II (IGF-II) revealed the presence of specific IGF-I receptors, showing a Kd-value of approximately 2 nM. These results are consistent with the hypothesis that somatomedins play a role in determining the proliferative behaviour of human breast and colon tumors, and suggest that recent laboratory studies showing dependence of neoplastic cells on somatomedins for optimum proliferation may have clinical relevance.     

The relationship between estrogen receptors in primary and secondary breast carcinomas and in sequential primary breast carcinomas

Summary A review of over 2000 patients who had estrogen receptors (ER) assayed in the primary breast carcinoma identified 48 cases in whom a subsequent second primary breast carcinoma or concurrent or recurrent secondary tumour had been tested for ER status. The relationship between the ER in the two specimens was as follows: Of 14 concurrent primary and secondary breast carcinomas the ER concentration was the same in 11 cases; in 1 case it was significantly higher in the primary tumour, in 2 others the reverse was observed. There was no major discordance in ER status. In 14 sequential carcinomas (after an average disease free time of 21 months), 12 pairs had identical ER status. There was major discordance of ER status in 2 cases where the secondary tumours contained ER while the primary carcinoma did not. The ER concentrations in the primary and the secondary carcinomas were comparable in 8 cases, while 3 and 5 cases had significantly higher or lower concentrations respectively in the sequential secondary tumour. In 20 cases where breast cancer developed in the contralateral breast (after an average disease-free interval of 27.7 months), essential concordance of ER status was observed in 15 of 20 sequential carcinomas. In 5 patients the first carcinoma was ER – and the second ER +; in one additional patient the first carcinoma was ER± and the second ER –. The ER concentrations differed significantly in 14 of the 20 bilateral carcinomas. The literature on estrogen receptor variation in breast carcinoma was reviewed.     

8q24 Copy number gains and expression of the c-myc mRNA stabilizing protein CRD-BP in primary breast carcinomas

The coding region determinant binding protein (CRD-BP) was isolated by virtue of its high affinity to the c-myc mRNA coding region stability determinant and shown to shield this message from nucleolytic attack, prolonging its half-life. CRD-BP is normally expressed during fetal life but is also activated de novo in tumors. Considering that aberrant CRD-BP expression may represent an additional mechanism interfering with c-myc regulation, we screened 118 primary breast carcinomas for CRD-BP expression, 60 of which had also been analyzed by comparative genomic hybridization (CGH). Copy number gains encompassing 8q24, the chromosome band that contains the c-myc locus, were detected in 48.3% (29/60) of tumors, whereas gains involving band 17q21, which contains the CRD-BP locus, were observed in 18.3% (11/60) of tumors. CRD-BP expression was detected in 58.5% (69/118) of tumors, implying mechanisms of activation alternative to gene amplification. Altogether, some 75% of the tumors had alterations pertaining to c-myc since they either harbored 8q24 gains and/or expressed CRD-BP. Significant associations were detected between CRD-BP expression and the absence of estrogen receptors (p = 0.005) and between the presence of 8q24 gains and an increased number of genomic changes as measured by CGH (p = 0.0017). Tumors were divided into 4 groups according to CRD-BP expression and 8q24 gains. The odds for tumors having both characteristics to be classified as poorly differentiated (grade III vs. grade I and II) were 19.6 times the corresponding odds for tumors neither expressing CRD-BP nor harboring 8q24 gains. For tumors either harboring 8q24 gains only or expressing CRD-BP alone, the corresponding odds were 6.4 and 3, respectively.     

Amplification of the c-myc proto-oncogene in cervical carcinoma

Amplification without structural rearrangements of the c-myc proto-oncogene was found in 14 of 44 primary cervical neoplasms. There was no apparent correlation with stage of disease or degree of cellular differentiation. The prognostic significance of c-myc amplification cannot be determined from this study.     

Elevated expression of c-myc proto-oncogene in scleroderma fibroblasts

Scleroderma is a connective tissue disease characterized by the overproduction of extracellular matrix components. The mechanisms of fibrosis may involve increased fibroblast proliferation in the scleroderma lesion due to the presence of cells with abnormal growth properties, in addition to the well-known over-production of several matrix components. The c-myc proto-oncogene has been implicated in dysregulation of cell growth in neoplastic cells and as an essential element of the response to growth factors in normal cells. Therefore, to investigate the molecular basis of growth in scleroderma, we compared expression of c-myc gene in scleroderma and control cells. In this report, we show that under low serum conditions (1% serum), scleroderma fibroblasts express 2.5-3 higher level of c-myc message. Moreover, stimulation of c-myc after addition of fresh 10% serum is blunted in scleroderma compared to control cells. Observed c-myc expression in scleroderma is similar to c-myc expression in transformed cells. In addition, there is also increased proliferation of scleroderma cells in 1% serum as measured directly by a nuclear label assay. These data suggest the presence of fibroblasts with abnormal growth properties in the scleroderma lesion.     

Expression of endogenous granzyme B in a subset of human primary breast carcinomas

Granzyme B (GrB) is the prototypic member of a serine protease family primarily used by cytotoxic lymphocytes to kill target cells. We report here that, by immunohistochemical staining of paraffin-embedded tumour sections, GrB protein was unexpectedly detected in malignant cells of a subset of breast cancers and their adjacent reactive endothelial and mesenchymal cells in which endogenous retinoblastoma protein (pRB) is overexpressed. The identity of the endogenous GrB was further confirmed experimentally in RB-deficient breast carcinoma cell culture upon overexpression of ectopic pRB. Our finding extends the recent paradigm-shifting trend for a more diverse biological role of granzyme B, and might provide a rational basis for exploring its potential prognostic value in a variety of human cancers.     

Cell proliferation and vascularization in human breast carcinomas.

Cell proliferation and vascularization were studied in 10 human breast carcinomas by an immunoenzyme technique. The monoclonal antibody (MAb) Ki-67 was used as a marker for proliferating cells and a polyclonal antibody directed against human von-Willebrand factor to identify blood vessels. The proportion of Ki-67-labelled cells varied from 1% to 20%, the number of small blood vessels from 4.4/mm2 to 57.6/mm2. Within single histological sections of individual tumours the percentage of proliferating cells was not related to the number of small blood vessels. However, after evaluation of 5 sections of each tumour, the average values showed that tumours with a high grade of vascularization had a higher percentage of Ki-67-positive cells than poorly vascularized samples. The influence of vascular density on cell proliferation was investigated in a selected area of one of the tumours (in 2-dimensions) and with regard to the over- and underlying sections (in 3-dimensions). After 2-dimensional evaluation, distances from proliferating cells to the closest blood vessel between 10 and 390 microns were observed, and after 3-dimensional evaluation none of the proliferating cells measured was located more than 130 microns away from the closest vessel.     

Analysis of the int-1, int-2, c-myc, and neu oncogenes in human breast carcinomas

We have examined the DNA obtained from 100 primary breast carcinomas for oncogene markers which might have predictive value for poor prognosis. Ninety-six of the tumors were analyzed for the presence of restriction fragment length polymorphisms (RFLPs) previously identified in the int-2 gene. An 8.4-kilobase BamHI fragment and a 3.9-kilobase PstI fragment specific for the int-2 gene, in the absence of other restriction fragments, was found in 17 of 50 (34%) lymph node-negative patients and in 27 of 44 (61%) lymph node-positive patients. This combination of int-2 RFLPs (8.4/3.9) was found in a significantly different proportion (P = 0.02) of patients with greater than 3 positive lymph nodes compared to patients with fewer positive lymph nodes, suggesting that these RFLPs may be valuable for distinguishing among node-negative patients for chemotherapy. In contrast, the observed low frequency of int-1, int-2, neu, and c-myc amplification limited their usefulness as clinical predictors of disease recurrence.     

Genetic alterations of c-myc, c-erbB-2, and c-Ha-ras protooncogenes and clinical associations in human breast carcinomas

We have analyzed genomic DNA sequences from 125 prospectively collected single unilateral primary breast carcinoma samples for the presence of alterations of c-myc, c-erbB-1, c-erbB-2, c-Ki-ras and c-Ha-ras protooncogenes. Amplification of the c-myc gene was found in 18% of the samples, and in one sample a non-germ line c-myc related DNA fragment or rearrangement was detected. We have found a significant association (P = 0.0010) between amplified c-myc gene and inflammatory carcinoma, a particularly aggressive breast cancer. The c-erbB-2 gene was amplified in 22% of the tumor samples and a rearrangement was observed once. Alteration of the c-erbB-2 gene was significantly linked to histological grade III tumors (P = 0.005) and the absence of estrogen and progesterone receptors (P = 0.036). No amplifications were observed for c-erbB-1, c-Ki-ras, and c-Ha-ras genes. About 40% of breast carcinomas contain either amplified c-myc or c-erbB-2 protooncogenes, whereas simultaneous amplification of both was seen in only one sample, suggesting the involvement of two distinct molecular mechanisms in breast cancer. Comparison of DNA from peripheral blood and tumor samples indicated loss of one c-Ha-ras allele in 29% of patients heterozygous for this polymorphism. A significant correlation (P = 0.016) between c-Ha-ras locus (11p14) allele loss and patient survival was found. These data suggest that 11p14 allelic loss plays a role in the evolution of human breast cancer, amplification of c-erbB-2 gene is associated with increasing stage of malignancy, and alteration of the c-myc gene in inflammatory breast carcinoma may contribute to the rapid progression of this human tumor subtype.     

Loss of a c-H-ras-1 allele and aggressive human primary breast carcinomas

The human H-ras protooncogene was shown to be expressed in 16 of 22 invasive ductal carcinomas of the breast. The K- and N-ras protooncogenes were either not expressed or expressed at low levels. No amplification or rearrangement of the three ras genes was detected among the 104 breast carcinoma DNAs tested. These results indicate that the overexpression of H-ras in human breast tumors is not correlated with alteration of the protooncogene. In addition, we did not find any point mutation at the codon 12 of the H-ras or K-ras protooncogenes in 32 and 64, respectively, tumor DNAs examined. However, in tumor DNAs from 14 of 51 patients, heterozygous for H-ras-1 related BamHI restriction fragments, one allele was lost. This allele loss did not alter ras Mr 21,000 protein expression. Correlation with clinicopathological data showed, however, that the loss of one H-ras-1 allele in breast carcinoma DNAs is significantly linked to histological Grade III tumors, the lack of estrogen and/or progesterone receptors, and the subsequent occurrence of distal metastasis. Our results thus indicate that the loss of one H-ras-1 allele correlates with the most aggressive primary carcinomas of the breast.