Glutathione S-transferase M1 null genotype: lack of association with tumour characteristics and survival in advanced breast cancer

Abstract

gene in 50-60% of individuals. Several studies have demonstrated a possible link with the GSTM1-null genotype and susceptibility to cancer. Furthermore, a GSTM1 isoenzyme has been positively associated with protective effect against mutagenic drugs, such as alkylating agents and anthracyclines. To determine whether GSTM1 polymorphisms are associated with tumour characteristics and survival in advanced breast cancer patients, and whether it may constitute a prognostic factor. We genotyped 92 patients receiving primary chemotherapy, which included cyclophosphamide, doxorubicine and 5-fluorouracil. The relationships between allelism at GSTM1 and clinicopathological parameters including age, menopausal status, tumour size, grade hormone receptors, involved nodes and p53 gene mutations were analysed. Of the patients with GSTM1-positive genotype, tissue samples obtained before and after treatment were available from 28 cases, allowing RNA extraction and GSTM1 expression by reverse transcription polymerase chain reaction. Relationships with clinical response to chemotherapy, and disease-free and overall survival were also evaluated. The data obtained was analysed using logistic regression to estimate the odds ratio and 95% confidence interval. = 0.0086) were the only factors associated with reduced disease-free or overall survival. GSTM1-null genotype alone had no effect on tumour characteristics and outcome of patients with advanced breast cancers. The lack of correlation of GSTM1 genotype with clinical tumour features, clinical response to chemotherapy and survival exclude a role for GSTM1 polymorphism as a prognostic factor in advanced breast cancer.

Background:

gene in 50-60% of individuals. Several studies have demonstrated a possible link with the GSTM1-null genotype and susceptibility to cancer. Furthermore, a GSTM1 isoenzyme has been positively associated with protective effect against mutagenic drugs, such as alkylating agents and anthracyclines.

Objectives:

To determine whether GSTM1 polymorphisms are associated with tumour characteristics and survival in advanced breast cancer patients, and whether it may constitute a prognostic factor.

Methods:

We genotyped 92 patients receiving primary chemotherapy, which included cyclophosphamide, doxorubicine and 5-fluorouracil. The relationships between allelism at GSTM1 and clinicopathological parameters including age, menopausal status, tumour size, grade hormone receptors, involved nodes and p53 gene mutations were analysed. Of the patients with GSTM1-positive genotype, tissue samples obtained before and after treatment were available from 28 cases, allowing RNA extraction and GSTM1 expression by reverse transcription polymerase chain reaction. Relationships with clinical response to chemotherapy, and disease-free and overall survival were also evaluated. The data obtained was analysed using logistic regression to estimate the odds ratio and 95% confidence interval.

Results:

= 0.0086) were the only factors associated with reduced disease-free or overall survival.

Conclusions:

GSTM1-null genotype alone had no effect on tumour characteristics and outcome of patients with advanced breast cancers. The lack of correlation of GSTM1 genotype with clinical tumour features, clinical response to chemotherapy and survival exclude a role for GSTM1 polymorphism as a prognostic factor in advanced breast cancer.

Introduction

]. There are currently five putative α class genes encoding subunits GSTA1, GSTA2, GSTA3, GSTA4 and GSTω, whereas the GST π class contains a single gene encoding the GSTP1 protein, and the θ class consists of two genes encoding the GSTT1 and GSTT2 proteins.

].

].

], clinical response to chemotherapy, and disease-free and overall survival.

Patients and samples

gene alterations, 92 women who were diagnosed with locally advanced breast carcinoma and who underwent primary chemotherapy were included in this study. The median clinical follow up was 78months (range 10-120months). Three of these patients had bilateral lesions, and in these three cases both lesions were examined. No family history for breast cancer was recorded in the 92 women. The patients received chemotherapy treatment (four or six courses, each lasting 21days) with a regimen containing cyclophosphamide, doxorubicine and 5-fluorouracil. The criteria for inclusion were as follows: inflammatory carcinomas, positive nodes and/or large (T3, T4) tumours. Clinical response to primary chemotherapy was categorized according to World Health Organization criteria and was considered as objective response (complete or partial response) or no response (stabilization or progression). In all cases neither radiotherapy nor hormone therapy were applied before chemotherapy.

for none, one or two, and three or more involved nodes, respectively).

].

Polymerase chain reaction method

The GSTM1-null genotype was determined by coamplifi-cation with the interferon-β gene, which served as an internal control. Primers for amplification of the GSTM1 gene corresponding to exon 4, intron 5 and exon 5 were 5' -ctgccctacttgattgatggg-3' and 5' -ctggattgtagcagatcatgc-3' (amplified product size, 271 base pairs). Primers for amplification of a part of the interferon-β gene, producing a constant 170-base pair band in all samples, were 5' -ggcacaacaggtagtaggcg-3' and 5' -gccacaggagcttctgacac-3' . Because the primers for the GSTM1 locus anneal to sites inside the coding region of the gene, the presence of the gene was determined by the presence of the band, whereas the null-genotype was determined by the lack of the band, using agarose gel electrophoresis (2%).

PCR was performed using 250ng template DNA in 10mmol/l Tris-HCl (pH8.4), 50mmol/l potassium chloride, 1.5mmol/l magnesium chloride (Bioprobe Systems, Illkirch, France), 0.2mmol/l concentrations of each deoxynucleotide triphosphate (dNTP), 500nmol/l concentrations of each primer and 2.5units of Taq DNA polymerase (Bioprobe Systems). The reaction (total volume 50 μ l) was amplified on a Omnigene thermal cycler (Hybaid Ltd, Ashford, Kent, UK). After an initial denaturation at 94°C for 3 min the reaction proceeded for 25 cycles of 50s at 94°C, 50s at 55°C and 50s at 72°C, concluded by a final extension step of 10min at 72°C. To test for contamination, negative controls (tubes containing the PCR mixture without the DNA template) were included in every run.

Reverse transcription polymerase chain reaction

]. Of the total RNA 1 μ g was dissolved in 20 μ l reverse transcriptase buffer (Gibco/BRL, Cergy Pontoise, France) containing 200 μ mol/l dNTP, 500 ng random hexamer and 200U avian myeloblastosis virus (AMV) reverse transcriptase (Superscript; Gibco/BRL). The reaction was incubated at 42°C for 50min and heated to 70°C for 15min.

• microglobulin was calculated.

Determination of p53 mutations

gene.

Hormonal receptor assay

The oestrogen and progesterone receptor levels were determined in cytosolic tumours using enzyme immunoassay methods (Abbott Laboratories, Rungis, France). The cutoff level used for oestrogen and progesterone was 20fmol/mg cytosolic proteins.

Statistical analysis

test and log-rank test. For all tests, optimality of the selected models was verified by all-possible-subsets analyses.

Clinicopathological data

= 28) of the cases studied.

Glutathione S-transferase M1 genotype determination

expression was 1.38 (range 0.02-23.27) in the untreated tumours, and 1.16 (range 0.01-6.56) in samples obtained after chemotherapy administration.

Glutathione S-transferase M1 and clinicopathological characteristics of the patients

expression determined before or after treatment (data not shown).

= 0.0002, OR = 14.1, 95% CI = 2.52-78.50) remained the only factors linked to the clinical response.

Impact on survival of the patients

= 0.0138, OR = 2.36, 95% CI=1.22-4.59) remained significantly associated with metastasis recurrence risk.

= 0.0530, OR = 2.31, 95% CI = 1.02-5.26).

Discussion

gene was not a characteristic of breast tumour cells.

gene mutations. These results suggest that clinical tumour features are not associated with GSTM1-null genotype, not only in primary breast cancers, but also in advanced breast cancers.

].

= 92), the clinical characteristics were well known and the results strongly suggest the lack of correlation of GSTM1-null genotype alone with prognostic factors, clinical reponse to chemotherapy and survival. Genotypes of other enzymes (CYP1A1, GSTT1), as well as combinations of genotypes, might be of interest with regard to cancer and carcinogen metabolism.

Taken together, these results show that advanced breast cancers arising in patients with GSTM1-null genotype have no worse clinical tumour charactaristics and outcome than those of patients without such deletions. Thus, the lack of correlation of GSTM1-null genotype with clinical tumour features, clinical response to chemotherapy and survival exclude a role for GSTM1 polymorphism as a prognostic factor in advanced breast cancer.

Acknowledgements

This work was supported by the 'Comité Départemental de Saône et Loire', 'Ligue Bourguignonne Contre le Cancer' and the 'Association Régionale pour l'Enseignement et la Recherche Scientifique/Groupe Interrégional de Recherche en Cancérologie'. We thank Mrs L Hahnel and M Arnal for their excellent technical assistance.

Figures and Tables

Polymerase chain reaction product of paired lymphocyte (L) and tumour (T) DNA from coamplification of glutathione S-transferase (GST)M1 (271 base pair) and interferon-β (170 base pair) genes. Lane 1 shows a homozygously present GSTM1 allele. Lane 2 shows a homozygously null-GSTM1 allele. M1 is ΦX174-HaeIII digested DNA marker. M2 is ΦX174-HinfI digested DNA marker.

Association of glutathione S-transferase (GST)M1 genotype and clinicopathological data of breast cancer patients studied

CI, confidence interval; OR, odds ratio.

Clinical response to primary chemotherapy and breast cancer outcome

Estimated by log rank test, median± standard error. GST, glutathione S-transferase.

Keywords

• advanced breast cancer
• GSTM1 polymorphism
• p53 gene mutations
• prognosis
• response to chemotherapy