Sten Wingren

Genotype of metabolic enzymes and the benefit of tamoxifen in postmenopausal breast cancer patients

BackgroundTamoxifen is widely used as endocrine therapy for oestrogen-receptor-positive breast cancer. However, many of these patients experience recurrence despite tamoxifen therapy by incompletely understood mechanisms. In the present report we propose that tamoxifen resistance may be due to differences in activity of metabolic enzymes as a result of genetic polymorphism. Cytochrome P450 2D6 (CYP2D6) and sulfotransferase 1A1 (SULT1A1) are polymorphic and are involved in the metabolism of tamoxifen. The CYP2D6*4 and SULT1A1*2 genotypes result in decreased enzyme activity. We therefore investigated the genotypes of CYP2D6 and SULT1A1 in 226 breast cancer patients participating in a trial of adjuvant tamoxifen treatment in order to validate the benefit from the therapy.MethodsThe patients were genotyped using PCR followed by cleavage with restriction enzymes.ResultsCarriers of the CYP2D6*4 allele demonstrated a decreased risk of recurrence when treated with tamoxifen (relative risk = 0.28, 95% confidence interval = 0.11–0.74, P = 0.0089). A similar pattern was seen among the SULT1A1*1 homozygotes (relative risk = 0.48, 95% confidence interval = 0.21–1.12, P = 0.074). The combination of CYP2D6*4 and/or SULT1A1*1/*1 genotypes comprised 60% of the patients and showed a 62% decreased risk of distant recurrence with tamoxifen (relative risk = 0.38, 95% confidence interval = 0.19–0.74, P = 0.0041).ConclusionThe present study suggests that genotype of metabolic enzymes might be useful as a guide for adjuvant endocrine treatment of postmenopausal breast cancer patients. However, results are in contradiction to prior hypotheses and the present sample size is relatively small. Findings therefore need to be confirmed in a larger cohort.

c-erbB-2 expression and benefit from adjuvant chemotherapy and radiotherapy of breast cancer

Frozen tissue from primary tumours of 152 premenopausal breast cancer patients, who participated in a trial comparing radiotherapy with adjuvant chemotherapy (cyclophosphamide, methotrexate, 5-fluorouracil, CMF), was analysed for c-erbB-2 protein expression, measured by flow cytometry. The relative risk of distant recurrence or death in the chemotherapy group as compared with the radiotherapy group was 3.0 (95% confidence interval (CI) 1.1-7.8) for patients whose tumours showed high c-erbB-2 levels and 0.87 (95% CI 0.43-1.7) for those with tumours with low levels of c-erbB-2 protein. Patients with highly proliferative tumours that did not overexpress c-erbB-2 benefited most, in terms of survival, from CMF. In addition, we found an increased risk of locoregional recurrence for tumours overexpressing c-erbB-2 when radiotherapy was replaced by chemotherapy.

Association between CYP17 gene polymorphism and risk of breast cancer in young women

Long‐term exposure to oestrogens is a well‐recognised risk factor for breast cancer, whereas little is known about the influence of polymorphisms of genes involved in oestrogen biosynthesis and metabolism. A candidate, containing a single bp polymorphism, T→C, (designated, A2 allele), might be the CYP17 gene, which codes for an enzyme involved in oestrogen synthesis. This polymorphism creates an additional Sp1‐type promoter site (CCACC box), which has been shown to be associated with increased serum oestrogen levels. We performed a case‐control study, to evaluate association of the CYP17 gene polymorphism with risk of breast cancer in young women (younger than 37 years). We found a statistically significant increased risk in carriers of at least 1 A2 allele [odds ratio (OR), 2.0; 95% confidence interval (CI), 1.1–3.5, p = 0.027], and a trend toward a gene‐dose effect illustrated by a slightly higher risk for A2‐homozygous subjects (OR, 2.8) than for heterozygous women (OR, 1.9). Furthermore, when we investigated the CYP17 genotype in relation to tumour characteristics, breast cancer patients with 1 or 2 A2 alleles tended to have lower oestrogen receptor levels (risk ratio, 0.70; CI, 0.41–1.2, p = 0.44). Our findings suggest that CYP17 gene polymorphism influences breast carcinogenesis in young women. Int. J. Cancer (Pred. Oncol.) 84:350–353, 1999.

Flow cytometric measurements of DNA index and S-phase on paraffin-embedded early stage endometrial cancer: An important prognostic indicator

Paraffin-embedded curettage material from 120 patients with stage I and II endometrial cancer were analyzed by flow cytometry. The follow-up time in all cases was at least 5 years or until death. It was possible to determine DNA ploidy in 111 cases and S-phase was also evaluated in 92/111 cases. DNA ploidy and S-phase were then compared with the FIGO grading and clinical outcome. DNA ploidy results showed that 11% of grade 1 tumors were aneuploid, 14% of grade 2, 42% of grade 3, and 85% of the cases of uterine papillary serous carcinoma (UPSC) were aneuploid. Patients with tumors that showed an S-phase fraction below 5% had a cumulative 5-year cancer mortality of 7% whereas 49% of the patients with tumors having S-phase fraction above 10% died of their cancer. The prognostic significance of DNA and S-phase correlated fairly well with the FIGO grading as determined by a pathologist with special interest in gynecologic oncology and the DNA parameters added prognostic information independent of the FIGO grading.

C-erbB-2 overexpression and survival in early onset breast cancer

Young breast cancer patients have a decreased survival rate and it has been demonstrated that young age is an independent predictor of adverse prognosis. Overexpression of c-erbB-2 protein (also known as HER-2/neu) has been shown to be a prognostic indicator in breast cancer in general and especially among patients with axillary nodal metastases. The present study was initiated to determine the prognostic significance of c-erbB-2 protein overexpression in early onset breast cancer.A population consisting of 110 young breast cancer patients, ≤ 36-year-old at diagnosis, was analyzed with immunohistochemical staining for c-erbB-2 protein.Thirty patients (27%) were found to overexpress the c-erbB-2 protein. C-erbB-2 positivity was significantly associated with poor survival when all patients were included in the analysis (P = 0.002) and for patients with axillary nodal metastases (P = 0.0007). No such association was found for node-negative patients. Furthermore, the difference in prognosis in relation to c-erbB-2 among node-positive patients was maintained, when these were stratified in groups treated or not treated with adjuvant chemotherapy.The study indicates that overexpression of c-erbB-2 protein is a strong prognostic factor in young breast cancer patients with axillary nodal metastases. Moreover, the adverse prognosis associated with c-erbB-2 overexpression in node-positive patients was observed whether or not the patients had received adjuvant chemotherapy.

Prognostic value of DNA ploidy and S-phase fraction in relation to estrogen receptor content and clinicopathological variables in primary breast cancer.

Tumors from 472 women with primary breast cancer were analyzed by flow cytometry. Divided into four categories, DNA ploidy showed significant association with disease recurrence and mortality. When allowance was made for its correlation with nodal status and estrogen receptor (ER) content, DNA ploidy did not add prognostic information. S-phase fraction was estimated in 290 DNA histograms. In contrast, it was significantly related to recurrence and mortality when controlling for nodal status, tumor size and ER content. When the follow-up was divided into two periods DNA ploidy and S-phase fraction showed association with disease recurrence in the first period only (less than 2.5 years), while the association with mortality was valid for both periods. Light scatter was measured in 234 samples. A low light scatter variability for the stemline nuclei was related to a high recurrence rate during the early follow-up period. In conclusion, DNA flow cytometry adds prognostic information concerning breast cancer patients.

S-phase fraction and survival benefit from adjuvant chemotherapy or radiotherapy of breast cancer

Cancer chemotherapy interacts with cell proliferation, but data on the relationship between cancer cell replication and the effect of adjuvant chemotherapy are scarce. We have investigated the S-phase fractions of the primary tumour from premenopausal breast cancer patients who participated in a randomised trial comparing 12 cycles of polychemotherapy (CMF) with post-operative radiotherapy. DNA flow cytometry was performed on frozen tissues from 208 primary breast carcinomas, of which the S-phase fraction was estimated in 176 cases. There was a significantly higher benefit from CMF among patients with a high S-phase fraction (P = 0.0033). The relative risk of distant recurrence or death in the chemotherapy group as compared with the radiotherapy group was 0.19 for patients whose tumours had an S-phase fraction of 10% or over (95% CI 0.07-0.51) and 1.55 (0.88-2.73) for patients whose tumours showed lower S-phase levels. The interaction was still significant in multivariate analysis (P = 0.0057), including lymph node metastases, tumour size and oestrogen receptor content. We conclude that the benefit from adjuvant chemotherapy compared with radiotherapy is largely confined to patients with highly proliferative tumours.

Catechol-O-Methyltransferase ( COMT ) gene polymorphism and breast cancer risk in young women

Oestrogen exposure has long been considered to be a main risk factor of breast cancer. More recently, interest has also focused on the possible carcinogenic influence from oestrogen metabolites, such as catechol oestrogens. O-methylation, catalysed by Catechol-O-Methyltransferase (COMT), is one pathway by which the potentially carcinogenic catechol oestrogens can be inactivated. The gene coding for COMT protein contains a single-nucleotide polymorphism (SNP), resulting in an amino acid shift Val → Met, which has been shown to determine high- and low-activity configuration of the enzyme. We hypothesized that the low-activity allele, COMTMet, may be implicated in early onset breast cancer. In the present case–control study, including 126 young breast cancer patients (≤ 36 years) and 117 healthy female blood donors, we analysed the association between COMTMet genotype and risk of breast cancer. No significant difference in the frequency of low-/high-activity alleles was found between cases and controls, indicating that the polymorphism, as a single factor, may not contribute to breast carcinogenesis in young women.

Candidate tumour suppressor genes at 11q23-q24 in breast cancer : evidence of alterations in PIG8, a gene involved in p53-induced apoptosis

One of the most consistently deleted chromosomal regions in solid tumours is 11q23–q25, which consequently has been postulated to harbour one or more tumour suppressor loci. Despite large efforts to identify the responsible genes, the goal remains elusive, but as knowledge accumulates new candidates are emerging. The present study was undertaken in an attempt to assess the possible implication of four genes residing at 11q23–q24, in a population of early onset breast cancer (n=41). The coding sequence of PIG8, CHK1, LOH11CR2A and PPP2R1B were screened for mutations using the protein truncation test or single-strand conformational polymorphism, in combination with direct DNA sequencing. Varying proportions of alterations were detected, ranging from 6% in PPP2R1B to 39% in PIG8. Many of these changes were deletions, in some cases corresponding to complete exons, thus likely to represent splice variants, while others were presumed to arise from aberrant splicing, since they occurred at sites with resemblance to exon/intron borders. Considering only bona fide mutations, the highest alteration frequency (17%) was again found in PIG8. Most of these alterations were likely to have an adverse impact on the translated protein as they either altered the reading frame or affected phylogenetically conserved residues. Our data represent the first evidence of alterations in the PIG8 gene in human malignancies, a finding that substantiates its role as a potential tumour suppressor gene as suggested by its involvement in p53-induced apoptosis.

Correlations of Ki-67 and PCNA to DNA ploidy, S-phase fraction and survival in uveal melanoma

In 79 patients with uveal melanoma, the tumours were investigated by DNA flow cytometry and immunohistochemical staining of PCNA and Ki-67. S-phase as a continuous variable was significantly correlated with Ki-67 (P = 0.033), but not with PCNA. DNA ploidy was not correlated with either of the two antigens. Ki-67 was significantly correlated with histopathological type (P < 0.001) and tumour size (P < 0.001). Large tumours and epithelioid cell type were associated with a high frequency of Ki-67 positive cells. A high level of Ki-67 positivity (> or = 6.5%) was also associated with a shorter survival (P = 0.0037), and when adjusted for DNA ploidy, histopathological type and tumour size, Ki-67 in the multivariate analysis remained an important prognostic factor (P = 0.017).