Ram Seshadri

Localization of the human multiple drug resistance gene, MDR1, to 7q21.1

SummaryMultiple drug resistance has been shown to be associated with amplification/increased expression of a gene designated MDR. The localization of one member of the MDR gene family, MDR1, to the long arm of chromosome 7 by in situ hybridization is reported.

Mutation frequency in human lymphocytes increases with age.

Several theories of ageing predict that somatic mutations should increase with age. This prediction was tested for human lymphocytes using a recently developed clonal technique for enumeration of mutations, and an increase of 1.6% per year in mutations with age was detected.

EMS1 amplification can occur independently of CCND1 or INT-2 amplification at 11q13 and may identify different phenotypes in primary breast cancer.

Chromosome 11q13 is amplified in about 13% of primary breast cancers. CCND1, encoding the cell cycle regulatory gene cyclin D1, and EMS1, encoding a filamentous actin binding protein, are favoured candidate onocogenes, whereas INT-2 is an unexpressed gene at this locus. In this study we tested the possibility that different regions of this large amplicon could be independently amplified and subsequently defined the phenotype of EMS1 amplified tumours in a series of 961 primary breast carcinomas. Using DNA slot blots, EMS1 was amplified in 15.2% of samples: 5.4% were coamplified for CCND1; 7.9% coamplified for INT-2 and 6.7% showed EMS1 amplification alone. The degree of amplification of CCND1 and INT-2 was highly correlated (P=0.0001). In contrast, no such relationship existed between EMS1 and CCND1 or INT-2 amplification, demonstrating independent amplification of EMS1 in 44% of amplified tumours. EMS1 amplification (⩾twofold increase in copy number) was positively correlated with patient age ⩾50 years (P=0.025), ER positivity (P=0.022), PgR positivity (P=0.018), and was negatively correlated with HER-2/neu (c-erbB2) amplification (P=0.01). In common with CCND1/INT-2, EMS1 amplification was associated with increased risk of relapse in patients with lymph node-negative disease (P=0.028). In contrast, EMS1 and CCND1/INT-2 amplification appeared to confer different phenotypes in ER positive and negative tumours. A ⩾threefold increase in EMS1 copy number was associated with an apparent increased risk of relapse and death in patients with ER negative tumours, but was without effect in ER positive tumours. In contrast, CCND1/INT-2 amplification had no effect in the patients with ER negative tumours but was associated with early relapse in ER positive patients. Thus EMS1 amplification may identify subgroups of breast cancer patients with increased probability of relapse and death distinct from those identified by CCND1/INT-2 amplification. Further studies are required to more clearly determine the functional consequences of EMS1 overexpression and a biological basis for the relationship between EMS1 amplification and phenotype in breast cancer.

Relationship between p53 gene abnormalities and other tumour characteristics in breast‐cancer prognosis

The prognostic significance of p53 gene abnormalities was investigated in 919 primary breast‐cancer patients. p53 expression and tumour‐cell proliferation fraction determined by MIB‐1 count, p53 exon 5 and 6 mutations and HER‐2/neu oncogene amplification were detected by immunohistochemistry, PCR‐SSCP and slot‐blot hybridization, respectively. Increased MIB‐1 count, p53 expression, HER‐2/neu oncogene amplification and p53 mutations were detected in 33%, 29%, 10% and 8% of tumours, respectively. Statistically significant associations were observed between p53 expression or MIB‐1 count and age below 50 years, high‐grade tumours, medullary carcinomas, and absence of hormone receptors. p53 mutations were associated with increased MIB‐1 count, HER‐2/neu oncogene amplification and absence of hormone receptors, but not with age, tumour size or grade, histological subtype, or the number of axillary nodes involved. After a median follow‐up of 66 months, p53 expression was observed to be associated with significant increases in risk of both relapse and death from breast cancer, but not after adjusting for the effect of other parameters. In these analyses, MIB‐1 count, and not HER‐2/neu oncogene amplification, was an independent predictor of prognosis. In node‐negative patients, only p53 exon 5 and 6 mutations and MIB‐1 count were associated with a statistically significant increase in risk of death from breast cancer, independent of tumour size and ER concentration. We conclude that tumour‐cell proliferation fraction, as measured by MIB‐1 count, is the most useful parameter of breast‐cancer prognosis, with the exception of ER, tumour size and the number of axillary nodes involved.

Sister-chromatid exchange (SCE) analysis in mothers exposed to DNA-damaging agents and their newborn infants

The incidence of SCE in the lymphocytes of mothers and their newborn infants was determined. A detailed antenatal history of parental habits such as smoking, alcohol consumption and possible exposure to DNA-damaging agents was documented. The results showed that the SCE rate in the newborn is significantly less than that of their mothers. Mothers who consumed alcohol, but not cigarette smokers, had a significantly increased SCE rate compared to control mothers. However, these maternal habits did not affect the SCE rate of their infants. Neonates with neural tube defects showed a significantly increased SCE rate compared to normal babies.

Residual marrow injury following cytotoxic drugs

Abstract A number of cytotoxic drugs were tested in mice for their ability to produce residual damage to the bone marrow. This was assessed by the ability of the drug to produce depletion of pluripotential stem cells and granulocytic progenitor cells persisting for at least two months after cessation of the drug. Evidence of residual damage was found after administration of mitomycin, chlorambucil and melphalan but not after nitrogen mustard, dimethyl-triazeno-imidazole-carboxamide or adriamycin. Residual damage has previously been found following busulphan and bischloro-nitrosurea and the features in common of the drugs causing residual damage suggest that the effect may require the ability to alkylate pluripotential stem cells. These experimental findings may explain a number of clinical observations in man.

Prospective study of the prognostic significance of epidermal growth factor receptor in primary breast cancer

Bearing in mind the continuing controversy over the prognostic significance of epidermal growth factor receptor (EGF‐r) expression, we investigated its clinical significance prospectively in 345 primary breast cancer patients. The prognostic significance of EGF‐r expression, as measured by a radioligand binding assay, was determined by Coxs multivariate analysis using EGF‐r concentration as a continuous or dichotomous variable. Increased EGF‐r expression was detected in 20–32% of tumours, depending on the cut‐off in concentration used. EGF‐r expression, irrespective of the cut‐off, was not associated with tumour size or grade or the number of axillary nodes involved. There was, however, a strong inverse association between EGF‐r expression and the absence of hormone receptors. After a median follow‐up period of 57 months, multivariate analysis suggested that EGF‐r expression was associated with increases in risk for both relapse and death from breast cancer, even after adjusting for oestrogen receptor (ER) concentration, tumour size and the number of axillary nodes involved. Patients with ER‐positive tumours, which also expressed EGF‐r, had increases in risk for both relapse and death from breast cancer compared with tumours without EGF‐r. Expression of EGF‐r was not a predictor of poor prognosis in either node‐negative or ER‐negative subgroups of patients.

Vimentin expression is not associated with poor prognosis in breast cancer

The clinical significance of vimentin intermediate filament (VIF) expression was studied in relation to other established prognostic parameters in primary breast cancer. Archival tumour samples embedded in paraffin were examined by immunohistochemistry with monoclonal antibodies (MAbs) to VIF, p53 protein and cell proliferation marker MIB‐1. The vimentin staining pattern was heterogeneous, but in vimentin‐positive areas > 80% of the tumour cells were positive. There was no association between vimentin expression and tumour size or the number of axillary lymph nodes involved. Vimentin expression was significantly associated with high‐grade tumours, absence of hormone receptors, increased p53 expression and high tumour proliferation fraction as estimated by MIB‐1 count. Despite these associations with several recognised features of tumour aggressiveness, vimentin expression was not associated with increases in risk of relapse or death from breast cancer.

RCH-ACV: A lymphoblastic leukemia cell line with chromosome translocation 1;19 and trisomy 8

A cell line (RCH-ACV) was established from a bone marrow sample of a child with acute lymphoblastic leukemia (ALL). The cell line lacked Epstein-Barr virus nuclear antigen and exhibited a recently described nonrandom chromosome translocation, 1;19, thought to be associated with pre-B-ALL and poor prognosis. Banding studies confirm that the breakpoint of chromosome #19 occurs at p13.3. Cell surface marker analysis using a panel of monoclonal antibodies revealed markers consistent with common ALL phenotype. Although the cells did not show cytoplasmic immunoglobulin, studies of the immunoglobulin gene rearrangement confirmed the pre-B phenotype. This cell line could be of great value to studies of the role of the specific translocation 1;19 in the etiology of pre-B-ALL.

A simple index using video image analysis to predict disease outcome in primary breast cancer

Image analysis was used to investigate the prognostic significance of immunostaining for oestrogen receptor (ER), p53 tumour‐suppressor protein and tumour cell proliferation (MIB‐1) in a random cohort of 200 primary breast cancer patients with between 4 and 7 years of clinical follow‐up. Image measurements of the percentage of immunopositive cancer cell nuclei (% positive nuclear area) were recorded for the above tumour features for each patient. Assessment of relative risk using Coxs univariate analysis indicated that tumour size, number of cancer‐involved nodes, MIB‐1 and ER % positive nuclear area were significantly associated with breast cancer disease outcome, i.e., relapse‐free survival and overall survival. In multivariate analysis, tumour size, number of involved nodes, ER and MIB‐1 % positive nuclear area were retained as independent predictors of prognosis, depending on the image measurement cut‐point used. A prognostic model, which can be used without reference to nodal involvement, was constructed for tumour size, ER cut‐point of 30% positive nuclear area and MIB‐1 cut‐point of 10% positive nuclear area. Kaplan‐Meier analysis of this image‐based prognostic index identified 4 risk groups with predicted 5‐year overall survival rates of 93%, 83%, 76.7% and 61.5%. We conclude that image measurements of ER and proliferative rate can be combined with tumour size to construct a prognostic index which reliably predicts disease outcome in primary breast cancer without knowledge of the nodal status of the patient. Int. J. Cancer (Pred. Oncol.) 84:203–208, 1999.