Prevalence of ERα-397 PvuII C/T, ERα-351 XbaI A/G and PGR PROGINS polymorphisms in Brazilian breast cancer-unaffected women

Polymorphisms of hormone receptor genes have been linked to modifications in reproductive factors and to an increased risk of breast cancer (BC). In the present study, we have determined the allelic and genotypic frequencies of the ERα-397 PvuII C/T, ERα-351 XbaI A/G and PGR PROGINS polymorphisms and investigated their relationship with mammographic density, body mass index (BMI) and other risk factors for BC. A consecutive and unselected sample of 750 Brazilian BC-unaffected women enrolled in a mammography screening program was recruited. The distribution of PGR PROGINS genotypic frequencies was 72.5, 25.5 and 2.0% for A1A1, A1A2 and A2A2, respectively, which was equivalent to that encountered in other studies with healthy women. The distribution of ERα genotypes was: ERα-397 PvuII C/T: 32.3% TT, 47.5% TC, and 20.2% CC; ERα-351 XbaI A/G: 46.3% AA, 41.7% AG and 12.0% GG. ERα haplotypes were 53.5% PX, 14.3% Px, 0.3% pX, and 32.0% px. These were significantly different from most previously published reports worldwide (P < 0.05). Overall, the PGR PROGINS genotypes A2A2 and A1A2 were associated with fatty and moderately fatty breast tissue. The same genotypes were also associated with a high BMI in postmenopausal women. In addition, the ERα-351 XbaI GG genotype was associated with menarche ≥12 years (P = 0.02). ERα and PGR polymorphisms have a phenotypic effect and may play an important role in BC risk determination. Finally, if confirmed in BC patients, these associations could have important implications for mammographic screening and strategies and may be helpful to identify women at higher risk for the disease.

Prevalence of ERα-397 PvuII C/T, ERα-351 XbaI A/G and PGR PROGINS polymorphisms in Brazilian breast cancer-unaffected women.

Polymorphisms of hormone receptor genes have been linked to modifications in reproductive factors and to an increased risk of breast cancer (BC). In the present study, we have determined the allelic and genotypic frequencies of the ERα-397 PvuII C/T, ERα-351 XbaI A/G and PGR PROGINS polymorphisms and investigated their relationship with mammographic density, body mass index (BMI) and other risk factors for BC. A consecutive and unselected sample of 750 Brazilian BC-unaffected women enrolled in a mammography screening program was recruited. The distribution of PGR PROGINS genotypic frequencies was 72.5, 25.5 and 2.0% for A1A1, A1A2 and A2A2, respectively, which was equivalent to that encountered in other studies with healthy women. The distribution of ERα genotypes was: ERα-397 PvuII C/T: 32.3% TT, 47.5% TC, and 20.2% CC; ERα-351 XbaI A/G: 46.3% AA, 41.7% AG and 12.0% GG. ERα haplotypes were 53.5% PX, 14.3% Px, 0.3% pX, and 32.0% px. These were significantly different from most previously published reports worldwide (P < 0.05). Overall, the PGR PROGINS genotypes A2A2 and A1A2 were associated with fatty and moderately fatty breast tissue. The same genotypes were also associated with a high BMI in postmenopausal women. In addition, the ERα-351 XbaI GG genotype was associated with menarche ≥ 12 years (P = 0.02). ERα and PGR polymorphisms have a phenotypic effect and may play an important role in BC risk determination. Finally, if confirmed in BC patients, these associations could have important implications for mammographic screening and strategies and may be helpful to identify women at higher risk for the disease.

Prevalence of the STK15 F31I polymorphism and its relationship with mammographic density

Several studies have identified the single nucleotide polymorphism STK15 F31I as a low-penetrance risk allele for breast cancer, but its prevalence and risk association in the Brazilian population have not been determined. The goal of this study was to identify the frequency of this polymorphism in the Brazilian setting. Considering the high degree of admixture of our population, it is of fundamental importance to validate the results already reported in the literature and also to verify the relationship between this variant and breast cancer risk. A total of 750 women without breast cancer were genotyped using the TaqMan PCR assay for STK15 F31I polymorphism. Clinical information was obtained from review of the medical records and mammographic density from the images obtained using the BI-RADS System. The estimated risk of developing cancer was calculated according to the Gail model. The genotypic frequencies observed in this study were 4.5, 38.7, and 56.6 percent, respectively, for the STK15 F31I AA, AT and TT genotypes. The AT and AA genotypes were encountered significantly more often in premenopausal women with moderately dense, dense and heterogeneously dense breast tissue (P = 0.023). In addition, the presence of the TT genotype was significantly associated with age at menarche ≥12 years (P = 0.023). High mammographic density, associated with increased breast cancer risk, was encountered more frequently in premenopausal women with the risk genotypes STK15 F31I AA and AT. The genotypic frequencies observed in our Brazilian sample were similar to those described in other predominantly European populations.

Prevalence of the STK15 F31I polymorphism and its relationship with mammographic density.

Several studies have identified the single nucleotide polymorphism STK15 F31I as a low-penetrance risk allele for breast cancer, but its prevalence and risk association in the Brazilian population have not been determined. The goal of this study was to identify the frequency of this polymorphism in the Brazilian setting. Considering the high degree of admixture of our population, it is of fundamental importance to validate the results already reported in the literature and also to verify the relationship between this variant and breast cancer risk. A total of 750 women without breast cancer were genotyped using the TaqMan PCR assay for STK15 F31I polymorphism. Clinical information was obtained from review of the medical records and mammographic density from the images obtained using the BI-RADS System. The estimated risk of developing cancer was calculated according to the Gail model. The genotypic frequencies observed in this study were 4.5, 38.7, and 56.6%, respectively, for the STK15 F31I AA, AT and TT genotypes. The AT and AA genotypes were encountered significantly more often in premenopausal women with moderately dense, dense and heterogeneously dense breast tissue (P = 0.023). In addition, the presence of the TT genotype was significantly associated with age at menarche ≥12 years (P = 0.023). High mammographic density, associated with increased breast cancer risk, was encountered more frequently in premenopausal women with the risk genotypes STK15 F31I AA and AT. The genotypic frequencies observed in our Brazilian sample were similar to those described in other predominantly European populations.

Polybrominated diphenyl ethers and polychlorinated biphenyls in human breast adipose samples from Brazil.

Twenty five human breast adipose tissue samples were collected in Porto Alegre, Brazil during 2004-2005 and analyzed for polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs). SigmaPBDE concentrations (sum of tri- to hepta-BDEs) ranged from 0.19 to 132 ng/g lipid with a median of 1.51 ng/g lipid. These concentrations are 3- to 100-times lower than those reported from other countries, with the exception of Japan, probably reflecting lower usage of PBDE-containing products or lower exposures to these chemicals. The predominant congener was BDE-47, followed by BDEs 99, 183, 153 and 100. One individual in the dataset had about 70-times higher PBDE concentrations than the rest of the participants. SigmaPCB (sum of PCBs 118, 138, 153, 180) ranged from 30 to 339 ng/g lipid, with a median of 51 ng/g lipid. No age dependency was found for PBDEs (r=-0.800-0.374, p>0.05) or PCB 180 (r=0.278, p>0.05). On the other hand, PCBs 118, 138 and 153 did show age dependency (r=0.410-0.458, p<0.05). This is the first study to report levels of PBDEs in human breast adipose from Brazil.

Founder effect of the BRCA1 5382insC mutation in Brazilian patients with hereditary breast ovary cancer syndrome.

The 5382insC mutation in BRCA1 is a frequently reported mutation, being very prevalent in Central and Eastern Europe. This mutation was recurrently reported in Brazil and one case was reported Portugal, but not in Spain and other South-American countries,. We analyzed the haplotypic profile of seven Brazilian carriers of 5382insC to characterize a possible founder effect. The analyses indicated that mutation carriers shared an identical haplotype. The absence of this mutation in Spain, other South American countries, and sub-Saharan populations, as well as the patients' own ancestry, point to a significant Central or Eastern European contribution to the present genetic background of Brazilian population, different from the population structuring of remaining South American countries.

Cryoablation of benign breast tumors: evolution of technique and technology.

We report on improvements in cryoprobe design and techniques of cryoablation as a minimally invasive alternative to open surgery for the treatment of benign breast tumors. In the study, which was conducted in 12 centers, 124 lesions in 102 patients were monitored for a period of 12 months after cryoablation. Two different treatment techniques were used: Double HI FREEZE and Tailored Freeze. In patients treated with the Tailored Freeze technique significantly better results were recorded 12 months after the procedure: the median reduction in tumor volume was 91%, 73% of all tumors treated were nonpalpable, 84% of lesions less than 2.5 cm in maximum diameter were nonpalpable, and none of the 31 mammograms performed yielded abnormal findings. Patient satisfaction was good to excellent in 92% of the patients. The safety profile of this technique was excellent; all complications were minor. Evolution of cryoablation freezing techniques, coupled with improvements in cryoprobe design, has resulted in significant improvements in both safety and effectiveness.

A phase II study of doxorubicin/paclitaxel plus G-CSF for metastatic breast cancer.

This phase II trial was conducted to evaluate the percentage of objective responses and the toxicity profile of combination doxorubicin (Adriamycin) and paclitaxel (Taxol) with granulocyte colony-stimulating factor as first-line therapy for patients with metastatic breast cancer (MBC) not previously exposed to anthracycline-containing regimens. Patients with measurable, visceral-dominant MBC and a performance status of 0 to 2 were included in the study. Doxorubicin 60 mg/m2 was administered as a short intravenous infusion, followed by paclitaxel 250 mg/m2 as a 3-hour intravenous infusion on day 1. Granulocyte colony-stimulating factor 5 micrograms/kg/d was given prophylactically as a subcutaneous injection from day 2 until granulocyte recovery to > or = 1,500/mm3. Treatment was repeated every 21 days for a maximum of six courses. Dose reductions (to doxorubicin 50 mg/m2 and paclitaxel 175 mg/m2) and/or treatment delay were applied in case of severe toxicity. All 25 women who entered were evaluable for response and toxicity. The main grade 3/4 toxicities observed were leukopenia, thrombocytopenia, and mucositis. Alopecia occurred in all patients. No clinically relevant cardiovascular toxicity was observed. Severe myelosuppression and/or mucositis necessitated dose reductions at courses 2 or 3 in all but one patient. The complete response rate was 28%, and the partial response rate was 52% for an overall objective response rate of 80%. Median progression-free survival for complete responders was 11 months (range, 3 to 24 months), while the progression-free survival was 7+ months (range 2 to 14+ months) for partial responders and 5 months (range, 3 to 9 months) for nonresponders. This combination produces a high objective response rate in women with MBC, but dose reductions were necessary in almost all cases. Toxicity was manageable after dose reduction, allowing patients to be re-treated for two to six courses without life-threatening toxicity or toxic deaths. Unfortunately, the duration of response was limited even among complete responders. Further trials of this combination in patients with MBC should explore improvements in this study regimen.

p53 gene mutations and steroid receptor status in breast cancer. Clinicopathologic correlations and prognostic assessment.

BACKGROUND: There is increasing evidence linking development and progression of cancer to an accumulation of mutations at the genomic level. The most frequently mutated gene known to date in sporadic breast cancer appears to be the tumor suppressor gene p53. This study was designed to determine the frequency of p53 gene mutations in primary breast cancer, to correlate the presence of p53 mutations with established clinicopathologic parameters, including the estrogen receptor (ER) and progesterone receptor (PR) status, and to assess the prognostic significance of p53 mutations regarding patient survival. METHODS: We examined the p53 gene in genomic DNA samples from 192 primary breast cancers. Using denaturant gradient gel electrophoresis, the authors analyzed exons 5-9 in all tumors for mutations and performed DNA sequencing in 20 tumors to identify the exact nature of the p53 mutations. RESULTS: p53 gene alterations were identified in 43 of the 192 tumors (22%), the majority localized in exons 5 and 6. DNA sequencing showed mostly missense mutations resulting from G or C substitutions. p53 mutations were found more often in tumors of younger women (P = 0.002), Afro-American women (P = 0.05), and in tumors lacking ER (P = 0.03), PR (P = 0.04), or both (P = 0.06). There were no significant correlations with family history, tumor size, histologic grade or type, nodal status, or disease stage. The overall survival rates showed no significant difference between patients with mutant and wild-type p53 tumors. The same was true when the comparison was limited to node-negative patients or patients with ER-positive or ER-negative tumors. Finally, there was no significant difference in survival between patients with tumors harboring mutations in exons 5 and 6 versus exons 7-9. CONCLUSIONS: The results of this and other studies demonstrate a consistent relationship between ER-positive tumors and wild-type p53 on one hand and ER-negative cancers and p53 mutations on the other. Our data do not support a significant prognostic role for p53 mutations in predicting survival.

Tamoxifen protects against steroid-induced bone loss.

As part of a clinical trial of adjuvant endocrine treatment in postmenopausal women with operable breast cancer serial bone density measurements have been performed by dual photon absorptiometry. Tamoxifen alone was given to 26 women, and 20 received additional prednisolone. By 24 months after entry there was no significant difference between mean bone density of the two groups, nor any significant change from baseline levels. There was a mean gain of 0.46% in the tamoxifen group and 1.95% in those given additional prednisolone. Thus the predicted steroid-induced bone loss was inhibited by tamoxifen. This may be of more general use in prevention of osteoporosis in patients requiring long-term steroid treatment.

Steroid hormones in lymph and blood from women with early breast cancer.

Postoperative levels of steroid hormones have been measured in lymph and blood of 28 patients undergoing iridium implants as part of conservative treatment for operable breast cancer. The aim was to establish whether there were higher levels of hormones in lymph draining from necrosing tumour cells compared with peripheral blood levels. No differences were found in the ratios of estradiol, dehydroxyepiandrosterone sulphate or sex hormone-binding globulin in those patients with complete compared with incomplete excision of the primary tumour. However, premenopausal patients who had an incompletely excised primary tumour had increased levels of free testosterone in lymph draining from the tumour site on the third and fourth postoperative days. Thus androgens may have an important role in the intracellular metabolism of some breast cancers.

LHRH analogue for treatment of recurrent and refractory mastalgia.

The LHRH analogue Zoladex was used to treat 21 premenopausal women with severe recurrent or refractory breast pain. Severity and pattern of mastalgia, whether cyclical or non-cyclical, was assessed using self-administered record cards. Symptom relief was achieved in 17 (81%) of the patients. This study showed Zoladex to be an effective short-term treatment for refractory and recurrent mastalgia.

Premenopausal bone loss in the lumbar spine and neck of femur: a study of 225 Caucasian women.

Two hundred and twenty-five premenopausal women were studied to evaluate age-related changes in trabecular bone mass. Measurements were made at the lumbar spine and femoral neck by dual photon absorptiometry. It was found that spinal bone density increased significantly from the 20s to reach a peak in the mid-30s. Identical trends were observed in total bone mass and bone mass normalized by length. Bone loss then proceeded at a rate of 1% per year, and by the early 50s, 10% of peak spinal density was lost. There was no peak in femoral neck density; loss commenced in the late 20s and continued at a rate of 0.4% per year. The cumulative premenopausal loss from the femur at 9% was comparable to that in the spine. It is concluded that significant amounts of trabecular bone are lost from both the spine and femoral neck before the menopause. The implications of these findings for the prevention of osteoporosis are discussed.

Bone mineral content of women receiving tamoxifen for mastalgia.

Dual photon absorptiometry (DPA) has been used to measure the effect of short and medium-term administration of tamoxifen on bone density in the axial skeleton of women with mastalgia. This provided a unique opportunity to monitor the effect of this 'anti-oestrogenic' agent in predominantly premenopausal women, not suffering from malignancy. In addition, plasma levels of calcium, phosphate, alkaline phosphatase and serum levels of oesteocalcin (GLA) have been assayed, both before and after 3 months of starting either tamoxifen or placebo treatment. No significant alterations in bone density were seen. Osteocalcin, alkaline phosphatase and electrolytes were unchanged and there was no dose response observed in women receiving either 10 mg or 20 mg of tamoxifen. Although possessing anti-oestrogenic properties, tamoxifen is also a partial agonist. Administration for the short periods does not measurably influence spinal or femoral bone density and thus the agent can probably be given safely for the short-term treatment of mastalgia.

Factors at presentation influencing the prognosis in breast cancer.

Presentation data on 607 breast cancer patients treated by a variety of modalities at Guy's Hospital, London, have been analysed using the Cox proportional hazards model, to identify factors associated with length of overall survival. When deaths attributable to causes other than breast cancer were treated as censoring events, the significant factors were found to be stage, mode of treatment, menstrual status and tumour size. The analysis was repeated for the subgroup of 326 patients treated by modified radical mastectomy. Four variables: stage, age at menarche, menstrual status and age were found to be significantly associated with both overall survival and length of distant recurrence-free interval. These factors have been combined to create a prognostic index which has been used to define subgroups of patients with different prognosis. The index has been validated on a separate group of 457 modified radical mastectomy patients treated at the same hospital.

Effect of tamoxifen on oestrogen binding, lipid and lipoprotein concentrations and blood clotting parameters in premenopausal women with breast pain.

As part of a controlled trial of the use of tamoxifen for the treatment of mastalgia, some of the metabolic and haematological effects of this agent were measured. A panel of haemostatic variables including prothrombin time, kaolin cephalin clotting time, fibrinogen, euglobulin lysis time, factor VII, factor VIII, protein C and anti-thrombin III were determined. In addition, levels of sex hormone-binding globulin and both total and free oestradiol were estimated. No alteration in clotting function was found during the administration of tamoxifen, although hepatic function did alter during this period with an increase in concentration of sex hormone-binding globulin. There was a significant increase in total oestradiol and free oestradiol although the percentage of biologically available free oestradiol fell slightly during the course of tamoxifen treatment. There was a slight reduction in low-density lipoprotein cholesterol with an increase in HDL2, a subclass of high-density lipoprotein (HDL) cholesterol, consistent with an oestrogen-agonist effect. These data suggest that tamoxifen administration does not adversely influence haemostatic mechanisms or lipoprotein metabolism in the short term.

Dosage and duration of tamoxifen treatment for mastalgia: a controlled trial.

A controlled trial has been conducted in which 60 women with mastalgia were randomly allocated to receive tamoxifen at a dosage of either 10 mg or 20 mg daily for either 3 or 6 months. All eligible patients had self-rated moderate or severe mastalgia present for at least 6 months, for which no specific therapy had been given for the previous 3 months. End points of the study were pain control, measured by linear analogue scales, relapse rate and side-effects. Pain relief was achieved in 90 per cent of those receiving 10 mg daily and 86 per cent of those given 20 mg daily. The relapse rate was also similar for both dosages, being 48 per cent and 39 per cent respectively and usually occurred within 2-3 months of discontinuing treatment. However, side-effects were reported less frequently among those receiving 10 mg daily (21 per cent versus 64 per cent; chi 2 = 11.1, P less than 0.001). Prolongation of treatment from 3 months to 6 months did not materially improve the response rate (85 per cent versus 90 per cent). Side-effects were similar, as was the relapse rate among the patients receiving the two durations of treatment. The agent proved to be significantly more effective in the relief of cyclical rather than non-cyclical pain (94 per cent versus 56 per cent). Use of tamoxifen for the treatment of mastalgia is still experimental. Nevertheless, for the majority of women with mastalgia, pain relief can be achieved using tamoxifen 10 mg daily, given for a 3-month course. As almost half these patients will develop relapse of breast pain it may be necessary to give longer courses of therapy, although the safety of this more protracted treatment has yet to be determined.

Osteocalcin: a potential marker of metastatic bone disease and response to treatment.

Serum osteocalcin (BGP) is an osteoblast product that probably reflects the rate of bone formation. It is a potential marker of skeletal metastases and, to investigate this, BGP was measured by radioimmunoassay in the serum of normal subjects and patients with breast or prostate cancer. Significantly higher levels were found in patients with metastatic bone disease in comparison to both normal subjects (P less than 0.001) and patients with non-metastatic cancer (P less than 0.05 for breast cancer and less than 0.001 for prostate cancer). The range of values was wide. Levels were higher in sclerotic than lytic bone metastases (P less than 0.01) and lower in patients with hypercalcaemia (P less than 0.001). Serial measurements of BGP were made in 53 patients with skeletal metastases from breast cancer receiving systemic therapy. At 1 month BGP rose by greater than 0.5 ng/ml in 15/16 responding patients compared with 7/23 patients with progressive disease (P less than 0.01). Responding patients also showed a rise in the bone isoenzyme of alkaline phosphatase and a paradoxical deterioration in the bone scan appearance, both reflecting a flare in osteoblast activity. The early increase in responding patients was followed by a gradual decrease over subsequent months as the osteoblast reaction induced by systemic therapy subsided. We conclude that BGP measurements reflect a wide variability of bone formation rates in metastatic bone disease. Bone formation was usually increased, particularly when metastases were sclerotic in appearance, but in patients with hypercalcaemia the low BGP levels suggest uncoupling of bone resorption and formation. Serial measurements of BGP may be useful in monitoring response to treatment.

Immunohistochemical study of D5 antigen (an oestrogen receptor related protein) in normal breast, benign breast disease, and mammary carcinoma in situ.

Patterns of staining with a monoclonal antibody which recognises D5 antigen (a 29,000 kD oestrogen receptor-related protein) were studied in seven normal and 76 benign breast biopsy specimens as well as in 12 cases of pure in situ mammary carcinoma. Staining in benign breast lesions was weak and heterogeneous when compared with that seen in most infiltrating carcinomas. In situ carcinomas showed an intermediate pattern of staining. The finding of only small foci of weak positivity for D5 antigen in normal and benign breast disease indicated that there are similarities between the expression of D5 antigen and the presence of oestrogen receptor protein in these tissues. A further similarity was seen with in situ carcinomas, which have been shown to have lower oestrogen receptor content than infiltrating carcinomas and a more heterogeneous staining pattern with D5 than is seen in infiltrating tumours. The importance of these findings remains to be evaluated because the precise nature of D5 antigen and its association with the oestrogen receptor molecule is not fully understood.