The clinical significance of flow cytometric c-myc oncoprotein quantitation in testicular cancer

A sensitive flow cytometric assay has been developed using a monoclonal antibody, Myc 1-6E10, to quantitate c-myc oncoprotein levels in nuclei isolated from wax embedded testicular tumours. The oncoprotein (p62c-myc) level increased significantly with increasing teratoma differentiation. Patients with intermediate and undifferentiated tumours who developed recurrence had lower p62c-myc levels than those who were disease free since their initial treatment. Such quantitative biochemical methods may provide new prognostic indices for cancer patients.     

Elevated expression of the c-myc oncoprotein correlates with poor prognosis in head and neck squamous cell carcinoma

We quantitated c-myc oncoprotein in 44 squamous cell carcinomas of the head and neck using an enzyme-linked immunosorbence assay. The clinicopathological parameters of these patients were followed up for between 3 and 60 months and analysed for any correlations with observed levels of c-myc protein using the Kruskal-Wallis one-way analysis of variance method. Although no statistical correlation was found between different clinicopathological parameters (patient age, sex, TNM staging, number of lymph nodes invaded, extracapsular rupture of the tumour, its histopathological differentiation, or its site), the survival periods of patients with tumours possessing elevated levels of c-myc protein were found to be statistically shorter than those with lower levels of c-myc expression, (P less than 0.02). This indicates that c-myc expression may be an effective prognostic indicator in head and neck cancer.     

Flow cytometric and immunohistochemical analysis of p62c-myc oncoprotein in the bronchial epithelium of lung cancer patients.

The expression of p62c-myc in bronchial resection lines (BRLs) from lung cancer and control patients, has been examined by immunohistochemistry and parallel flow cytometry using antibodies directed against the p62c-myc oncoprotein. Both methods indicated a marked increase in nuclear p62c-myc levels in BRLs from tumour cases as compared to control BRLs. Immunohistochemistry also revealed greater cytoplasmic positivity in BRLs from cancer patients than from control cases. Flow cytometric quantitation of nuclear p62c-myc confirmed the immunohistochemical findings demonstrating that the median level of nuclear p62-myc fluorescence in BRLs from tumour cases was 1919 fluorescence units (FU) (range:216-7367 FU) and 144 FU (range:0-1365 FU) for non-tumour control BRLs. No consistent difference in p62c-myc fluorescence was observed between BRLs from smokers and non smokers. Both methods indicated that in lung tumour cases, nuclear p62c-myc was increased in histologically normal and abnormal BRLs, suggesting that hyperexpression of this protein is an early event preceding detectable morphological change. These results suggest that increased p62c-myc levels may be an early event in the pathogenesis of lung cancer.     

Long term follow-up of c-myc, p53 and proliferation measurements in malignant melanoma.

AIMS: We report a prospective study examining the prognostic significance of the c-myc oncoprotein, p53 tumour suppressor gene and proliferation rate measurements in malignant melanoma. METHODS: Flow cytometry (FCM) was used to measure the expression of c-myc, p53 and proliferation parameters in patients who had received an injection of the thymidine analogue bromodeoxyuridine prior to surgery. RESULTS: Sixty-seven patients had successful FCM measurements of the three parameters. c-myc was detected in 97% of patients with a median cell positivity of 62%. The median p53 positivity was 13%. The median potential doubling time (T(pot)) of the tumours wasf 9.4 days. In univariate analysis, each of the parameters showed an association with survival in metatstatic disease with rapid proliferation (p=0.006) or overexpression of c-myc (p=0.038) related to poor survival whereas increased positivity for p53 predicted better survival (p=0.013). CONCLUSIONS: These data indicate that laser cytometric technology can be used to obtain quantitative data on oncoproteins expression and cell proliferation rates in clinical samples of malignant melanoma.     

The potential of oncogene products as tumour markers

This paper discusses the possibilities of using the altered expression of oncogenes and their products in neoplastic tissues as markers for the diagnosis, prognosis and monitoring of human malignant disease. Results from our studies of the c-myc oncogene and its product in human solid tumours, using DNA and RNA hybridization and a set of monoclonal antibodies (MCAs) raised against synthetic peptides, are presented. These illustrate three important principles. Firstly, the level of expression of the gene correlates closely with histological grading. Secondly, the increased expression of an oncogene in a tumour can be used as a marker for imaging with radiolabelled MCA. Thirdly, the detection of a 40,000 dalton serum protein, which is immunologically related to the p62c-myc oncoprotein, and its quantitative analysis in patients with cancer and normal controls, suggests that oncogene-related serum proteins may provide novel markers for monitoring tumour activity. These data support the view that the detailed analysis of oncogene expression has the potential to predict a tumour's behaviour and response to different therapeutic modalities.     

Detection of the c-myc oncogene product in testicular cancer

A set of monoclonal antibodies was constructed by immunising mice with peptide fragments of the c-myc oncogene product. One such antibody, Myc 1-6E10 was shown to bind to a 62,000 dalton protein identifiable with the c-myc product (p62c-myc). The antigen recognised was not destroyed by paraffin wax embedding. Myc 1-6E10 was used to characterise the distribution of p62c-myc in archival testicular tumour material. Normal testes expressed only small amounts of p62c-myc. Seminomas showed increased nuclear and cytoplasmic staining. Undifferentiated teratoma showed little activity, whereas p62c-myc was abundant in the nuclei of differentiating epithelial structures, yolk sacs and embryoid bodies. Only small amounts of p62c-myc were seen in the tumours of 5 patients who subsequently died from their disease.     

Long-term prognostic significance of HSP-70, c-myc and HLA-DR expression in patients with malignant melanoma.

AIM: Use of molecular markers indicative of the tumour oncogenic potential and host response may enhance our prognostic information for more effective treatment of melanoma patients. The roles of HSP-70 protein, c-myc oncogene and HLA-DR antigen expression were examined in melanoma patients and related to prognostic factors, recurrence rate and long-term survival. METHODS: Forty patients with tumours thicker than 1 mm were included in this study. All had elective node dissection and were followed for at least 7 years. Twenty-two had microscopic nodal metastases. Both primary melanoma tumour and lymph nodes were examined for the immunohistochemical expression of HSP-70 protein, c-myc oncogene and HLA-DR antigen. RESULTS: Eighteen patients had a recurrence (45%) and 23 patients survived overall (57.50%). Positive HSP-70 expression was observed in 52.50% of the primary melanomas and was associated with improved overall survival, especially in the patient group with tumours > or = 1.5 mm (70%vs 26.70%, P=0.0159). C-myc oncogene was overexpressed in 47.50% and HLA-DR antigen in 42.50% of the primary melanomas, but no correlation with survival was observed. The expression profile of these molecular markers in the primary tumour did not predict the status of regional nodes. HLA-DR expression in lymph nodes was observed exclusively in the nodal tissue surrounding the metastatic melanoma tumour in five patients. CONCLUSIONS: The immunohistochemical expression profile of HSP-70 but not of c-myc oncogene or HLA-DR antigen in the primary melanoma tumour could be of certain value in the identification of patients with graver prognosis who may benefit from more aggressive therapeutic strategies. Copyright Harcourt Publishers Limited.     

Flow cytometric quantitation of DNA and c-myc oncoprotein in archival biopsies of uterine cervix neoplasia

The c-myc nuclear associated oncoprotein has been quantitated simultaneously with DNA in nuclei extracted from archival biopsies of uterine cervix neoplasia. The oncoprotein and DNA were measured fluorimetrically in a flow cytometer using a mouse monoclonal antibody (MYC 1-6E10) and propidium iodide. Normal biopsies exhibited higher oncoprotein levels than carcinomas (P less than 0.00001). Furthermore, the maximum fluorescence signal in the normal tissue occurred at a lower antibody concentration compared with tumour tissue. There was no correlation between oncoprotein levels and histological grade, stage of disease, age of the patients or prognosis in the carcinomas. Aneuploidy, defined as a distinct second peak separate from the diploid distribution, was not a significant feature. The c-myc oncoprotein nuclear content does not appear to be a prognostic indicator in carcinoma of the cervix from the results of these studies but there is clearly diagnostic potential, particularly for automated analysis of cervical screening.     

Ⅰ期非小细胞肺癌预后因素的研究

目的 探讨Ⅰ期非小细胞肺癌(NSCLC)的联合预后因素。方法 回顾性分析58例Ⅰ期NSCLC患者的临床资料、术后病理结果和免疫组化技术检测的9项基因表达指标(c-myc、MDM2、c-erbB-2、EGFR、p53、p14^ARF、p16^INK4、p21^WAF1、nm23)。观察患者的总生存率、局部区域性复发率和远处转移率。结果 全组5年生存率、局部区域性复发率和远处转移率分别为71．1％,11．1％和33．5％。单因素分析结果表明,肿瘤细胞的低分化是影响总生存率的不良预后因素(P=0.028);c—myc与c-erbB-2的高表达均为影响总生存率和远处转移率的不良预后因素;促进肿瘤增殖基因总分组的高分值(P=0．041)与综合肿瘤基因组的高分值(P=0．006),是总生存率的不良预后因素。多因素分析结果表明,肿瘤细胞的分化程度与综合肿瘤基因的联合表达,是影响总生存率的独立预后因素。本组结果还显示,已行化疗的高危组患者,其总生存率与无远处转移率均优于未行化疗者,但差异尚未见有显著性。结论 肿瘤细胞的分化程度与综合肿瘤基因的表达,可能是Ⅰ期NSCLC的预后因素。高危组患者进行术后化疗似有提高疗效的趋势。     

Structure and expression of nuclear oncogenes in multi-stage thyroid tumorigenesis

We have investigated the possibility that structural alterations of the 'nuclear' oncogene family (c-myc, N-myc, L-myc, fos, myb and p53) leading to aberrant expression might, as in several other tumour types, play a role in the multi-stage development of tumorigenesis in the human thyroid follicular cell. Direct analysis of expression by slot and Northern blot RNA hybridisation showed that normal thyroid expresses surprisingly high levels of fos, and to a lesser extent c-myc, c-myc expression was markedly increased in all tumours, both benign and malignant, but no increase was seen in any other nuclear oncogene. fos expression was reduced specifically in one type of malignant tumour-follicular carcinoma-in inverse correlation with differentiation. Southern blot analysis showed no evidence of rearrangement or amplification of c-myc, or of any other 'nuclear' oncogene in any thyroid tumour. We conclude that there is no evidence that a primary abnormality of these genes plays a role in thyroid follicular cell tumorigenesis and suggest that the observed changes in expression can be adequately explained as secondary consequences of the tumour phenotype.     

Modulation of c-myc oncogene expression by phorbol ester and interferon-gamma: appraisal by flow cytometry

A flow cytometric assay was developed to examine the expression of the cellular myc oncogene in relation to cell cycle in individual cells. C-myc-oncoprotein was detected by indirect immunofluorescence using a purified sheep polyclonal antibody, anti-human-myc. Specific binding of anti-human-myc was measured by flow cytometry. C-myc oncoprotein was detected in 90% of HL-60 and 75% of Daudi cells; human hematopoietic cell lines known to express high levels of c-myc oncogene. However, c-myc protein could not be detected in the REH cell line, normal human peripheral lymphocytes or thymocytes. Nuclear DNA content was measured simultaneously using propidium iodide staining. There was an equal level of c-myc protein in G0/G1, S and G2/M phases. The extent and kinetics of c-myc oncoprotein induction have been determined following phorbol ester, 12-O tetradecanoylphorbol 13 acetate (TPA) and interferon-gamma (IFN-gamma) exposure of both HL-60 and Daudi cells. TPA produced a gradual reduction in the level of c-myc protein and arrested the cells in G0/G1 phase in HL-60 cells. However, TPA failed to reduce c-myc protein or to change cell cycle distribution in Daudi cells. Interestingly, c-myc protein levels were stimulated by exposure of both HL-60 and Daudi cells to IFN-gamma. The results indicate that flow cytometric assay of oncogene expression is feasible, fast and requires relatively few cells. It also allows for the direct correlation of modulation of oncogene expression with cell kinetics.     

Detection of the c-myc oncogene product in colonic polyps and carcinomas

The c-myc oncogene has been implicated in the processes of normal cell proliferation and differentiation. Elevated levels of c-myc mRNA and its gene product (p62c-myc), have been detected in a variety of solid tumours and cultured cel lines. Its precise role in normal cell function and in neoplastic transformation and progression has yet to be elucidated. We have used a monoclonal antibody, raised by peptide immunisation, to determine the distribution by immunoperoxidase staining of the c-myc oncogene product in archival specimens of colonic polyps and carcinomas. Samples from 42 patients with colon carcinoma, 24 with benign polyps and 15 normal colon biopsies were examined. Normal colon revealed maximum staining in the mid-zone of the crypts, corresponding to the zone of differentiation and maturation. The staining was predominantly cytoplasmic. Adenomatous polyps revealed the most intense pattern of staining in areas of dysplastic change. Colonic tumours showed a wide range of staining. Well differentiated tumours contained more cytoplasmic p62c-myc than poorly differentiated tumours. These findings suggest that the c-myc oncogene product may play an important role in the evolution of colonic neoplasia.     

Analysis of c-myc oncogene in human esophageal carcinoma: immunohistochemistry, in situ hybridization and northern and Southern blot studies.

We have examined the c-myc gene expression and the gene organization in resected human esophageal squamous cell carcinomas, and in the adjacent normal esophageal mucosa from 20 patients undergoing radical surgery. Immunohistochemistry of p62c-myc was compared with that of proliferating cell nuclear antigen (PCNA) in order to examine the biologic significance of p62c-myc. Relative c-myc expression detected by Northern blot analysis ranged from 0.41 to 2.8, but the degree of c-myc expression did not correlate with other clinicopathological prognostic parameters. In sity hybridization localized the elevated c-myc mRNA expression to tumor cells and basal and parabasal cells of the adjacent normal mucosa. Immunohistochemistry showed altered localization of p62c-myc, i.e., both cytoplasmic and nuclear immunostaining in advanced carcinomas. c-myc immunoreactivity exhibited wider distribution compared with that of PCNA, a cell cycle related antigen, which may indicate induction of cell proliferation by p62c-myc. DNA hybridization showed mild amplification in one out of 17 tumors and no evidence of gene rearrangement. There was no distinct correlation between the results of Northern or Southern blot analysis and the results of in situ hybridization or immunohistochemistry. Gene alteration of the c-myc locus, as well as overexpression of the c-myc oncogene, appeared to be limited, and analysis of the c-myc gene yielded limited prognostic value in human esophageal carcinomas.     

Use of the OM-11-906 monoclonal antibody for determining p62 c-myc expression by flow cytometry in relation to prognosis in colorectal cancer

The expression of the c-myc protein product (p62 c-myc) and deoxyribonucleic acid (DNA) ploidy status was determined by a flow cytometric technique in 83 patients with colorectal cancer followed up for a median of 30 months (range 6-60 months). The OM-11-906 antibody, used to detect p62 c-myc, revealed a 62 kDa and 45 kDa band on Western blots in tumours. Correlation of quantitative dot blotting of tumour mRNA to flow cytometric p62 c-myc expression was good (r = 0.87, P less than 0.01). Levels of p62 c-myc varied in colorectal cancer and low levels (less than 20 fluorescein units) correlated with improved survival (log rank chi 2 = 4.69, df = 1, P = 0.03), and this was a better prognostic index than DNA ploidy (log rank analysis chi 2 = 2.38, df = 1, P less than 0.1). Although expression of the c-myc gene was found, using the OM-11-906 antibody, to be a prognostic feature in colorectal cancer, these and other results need to be interpreted with caution given the presence of two protein bands by Western blotting.     

int-2 Amplification in breast cancer: Association with decreased survival and relationship to amplification of c-erbb-2 and c-myc

Amplification of the int-2 oncogene was measured in a series of breast tumours and related to amplification of the c-myc and c-erbB-2 oncogenes, histopathological features and relapse-free and overall survival. int-2 was amplified in 11%, c-myc in 20% and c-erbB-2 in 27% of the tumours assessed. int-2 amplification was associated with large tumour size (p < 0.05) and reduced relapse-free (p < 0.05) and overall (p < 0.0005) survival. c-myc amplification was associated with poor tumour differentiation p < 0.05) but had no association with prognosis. c-erbB-2 amplification was associated with low levels of expression of oestrogen receptor mRNA (p < 0.05), poor tumour differentiation (p < 0.05) and shortened relapse-free (p < 0.OOOl) and overall survival (p < 0.0001). This is the first report of an association between amplification of the int-2 oncogene in breast tumours and a significantly increased risk of death from breast cancer, and suggests that int-2 may be useful for identifying breast-cancer patients having a poor prognosis.     

Ras, C-myc and C-erbB-2 oncoprotein expression in non-AIDS Mediterranean Kaposi's sarcoma

We have studied ras p21, c-myc p62 and c-erbB-2 oncogene expression in fourteen Greek patients with NON AIDS Mediterranean Kaposi's sarcoma using immunohistochemical analysis. Elevated expression of ras p21 expression was observed in all 14 cases studied (8 of which had intense levels of staining), whereas expression of c-myc and c-erbB-2 was less frequent, (6 out of 13 cases tested showed elevated c-myc expression and 6 out of 13 showed elevated c-erbB-2 expression). This report indicates that aberrant ras p21 oncogene expression is a feature of Kaposi's sarcoma and may be important in the early stages of this disease.     

C-MYC expression in medulloblastoma and its prognostic value

To identify prognostic factors in medulloblastoma, a common malignant brain tumor of childhood, expression of the oncogene c-myc was examined at the mRNA level by in situ hybridization. c-myc mRNA expression was observed in 30 of 72 tumors (42%). The c-myc gene copy number was determined by quantitative PCR from genomic DNA of paraffin-embedded tumors. c-myc gene amplification was present in 5 of 62 cases (8.3%). Therefore, c-myc amplification was obviously not the cause of c-myc mRNA expression in most samples. Kaplan-Meier estimation revealed a significant correlation between c-myc mRNA expression and survival (total mean follow-up 4.6 +/- 3.6 years, log-rank p = 0.02). Multivariate logistic regression analysis including sex, age, histological type, degree of surgical resection and expression of synaptophysin, GFAP and c-myc, was carried out on 54 patients who received both radiotherapy and chemotherapy. The analysis identified expression of c-myc as an independent predictive factor of death from disease.     

c-erbB-2 oncoprotein expression in primary and advanced breast cancer.

Immunoreactivity for c-erbB-2 oncogene product expression has been investigated in patients with breast cancer using the polyclonal antibody 21N. Three series of patients were studied, 602 presenting with primary operable cancer, 57 with stage 3 and 123 with stage 4 disease. Representative tissue sections of each primary tumour were stained using a standard immunoperoxidase technique. Invasive tumour membrane immunoreactivity was assessed and identified in 15% of patients with primary operable cancer and 20% in the advanced breast cancer group. The results demonstrate a relationship between poorer survival and oncogene expression in all three patient groups. Patients in the primary operable cancer group with membrane oncoprotein expression had a poorer outcome, 35% 10-year survival, compared with those in which membrane expression was absent, 55% 10-year survival. The median survival of patients with stage 3 disease with c-erbB-2 membrane positivity was 17 months compared to 24 months with membrane negativity. In stage 4 disease median survival with membrane expression was 8.8 months compared to 19.7 months with no membrane expression. In addition in the series of primary cancers a correlation existed between histological grade and membrane immunoreactivity. Multivariate analysis showed histological grade to be a more powerful prognostic factor than c-erbB-2 protein expression. In conclusion, this study demonstrates, in a large series of patients presenting to one centre, that c-erbB-2 protein expression is a prognostic indicator in patients with primary operable and advanced breast disease.     

The H-ras oncogene product p21 and prognosis in human breast cancer

Summary The protein product of the H-ras oncogene, p21, has been measured semiquantitatively in solubilized particulate fractions of 160 primary tumours from patients presenting without evidence of distant metastatic breast cancer. Levels of p21 have then been related to factors of established prognostic significance, and to clinical outcome after primary treatment in terms of disease-free interval and survival times. p21 was detected by Western blotting in all tumour fractions, but amounts varied markedly between different tumours. There was no significant relationship between levels of p21 and the menopausal status of the patient, tumour oestrogen receptors, grade, and clinical stage. However, there was a significant trend for tumours to be associated with lymph node involvement as p21 was increasingly expressed. Elevated levels of p21 were also significantly related to early disease recurrence and death from cancer. Multivariate stepwise analysis showed that both p21 and lymph node status were independent statistically significant factors for disease recurrence and survival, and that no other parameter was significant for clinical outcome after adjustment for p21 and lymph node status. These results indicate that tumour levels of p21 are an important prognostic variable in patients with early breast cancer.