Aberrant hypermethylation of the promoter region of the CHFR gene is rare in primary breast cancer

Summary Taxanes are among the most active agents and they are now known to be an indispensable component in chemotherapy for breast cancer. However, some patients are resistant to taxanes and the identification of the molecular characteristics that can predict the sensitivity to taxanes would be useful in selecting the most appropriate patients to receive taxane therapy. Taxanes are antimicrotubular agents that promote microtubular assembly and stabilize microtubules by preventing depolymerization. They interfere with normal mitotic transition and causes cell cycle arrest in the G2-M metaphase. CHFR (checkpoint with forkhead-associated and ring finger) is a recently identified gene, which functions as an important checkpoint protein early in G2-M transition. Its activation delays the cell cycle in prophase and promotes cell survival in response to the mitotic stress induced by either nocodazole or taxane. CHFR is frequently downregulated in human cancers, mostly owing to the hypermethylation of its promoter region. CHFR downregulation has been found in primary cancers or in the established tumor cells of various origins, such as the lung, colon, esophagus, and stomach. The aberrant hypermethylation of CHFR promoter appears to be a good molecular marker to predict sensitivity to taxanes in gastric, lung, and colon cancer. A downregulation of CHFR was observed in breast cancer cells, however, no apparent promoter hypermethylation has yet been reported. In addition, an alteration of the CHFR expression or aberrant promoter hypermethylation in primary breast cancer has not been fully investigated. In this study, we examined the methylation status of the promoter region of CHFR gene in 110 primary breast cancers. We observed the hypermethylation of the CHFR promoter region in only one case (0.9%). We herein show that the aberrant hypermethylation of this region is quite a rare event in primary breast carcinoma.     

A prospective study of breast cancer risk using routine mammographic breast density measurements.

Mammographic breast density is a major risk factor for breast cancer but estimates of the relative risk associated with differing density patterns have varied widely. It is also unclear how menopausal status influences this association and to what extent the effects of density are due to its correlation with other risk factors. Most recent investigations of breast density have been case-control studies, which provide indirect estimates of relative risk. We have prospectively followed 61,844 women for an average of 3.1 years to directly estimate risk among women in the four mammographic breast density categories defined by the American College of Radiology's Breast Imaging Reporting and Data System (BI-RADS). The study was population-based and used density assessments routinely made by community radiologists. Cox regression was used to obtain age-adjusted relative risk estimates and to control for other risk factors. Risk increased with density and the risk associated with extremely dense breasts, relative to entirely fatty breasts, was 4.6 (95% confidence interval, 1.7-12.6) for premenopausal women and 3.9 (95% confidence interval, 2.6-5.8) for postmenopausal women. Estimates for pre- and postmenopausal women did not differ significantly. Although breast density was significantly related to body mass index, age at first childbirth, and postmenopausal hormone use (P < 0.001), adjustment for these variables only slightly altered the relative risk estimates. Our results correspond well to those from case-control studies using more quantitative measures of mammographic breast density and suggest that routine Breast Imaging Reporting and Data System density measurements may be useful in models for assessing breast cancer risk in individual women.     

Insulin-like growth factor-I, IGF-binding protein-3, and mammographic breast density.

Some studies have suggested that insulin-like growth factor (IGF) pathway is related to premenopausal breast density, one of the strongest known breast cancer risk factors. This study was designed specifically to test the hypothesis that higher levels of IGF-I and lower levels of IGF-binding protein (IGFBP)-3 are associated with high mammographic breast density among premenopausal but not among postmenopausal women. A total of 783 premenopausal and 791 postmenopausal healthy women were recruited during screening mammography examinations. Blood samples were collected at the time of mammography, and plasma IGF-I and IGFBP-3 levels were measured by ELISA. Mammographic breast density was estimated using a computer-assisted method. Spearman's partial correlation coefficients (r(s)) were used to evaluate the associations. Adjusted mean breast density was assessed by joint levels of IGF-I and IGFBP-3 using generalized linear models. Among premenopausal women, high levels of IGF-I and low levels of IGFBP-3 were independently correlated with high breast density (r(s) = 0.083; P = 0.021 and r(s) = -0.124; P = 0.0005, respectively). Correlation of IGF-I with breast density was stronger among women in the lowest tertile of IGFBP-3 than among those in the highest tertile of IGFBP-3 (r(s) = 0.138; P = 0.027 and r(s) = -0.039; P = 0.530, respectively). In contrast, the correlation of IGFBP-3 with breast density was stronger among women in the highest tertile of IGF-I than among those in the lowest tertile of IGF-I (r(s) = -0.150; P = 0.016 and r(s) = -0.008; P = 0.904, respectively). Women in the combined top tertile of IGF-I and bottom tertile of IGFBP-3 had higher mean breast density than those in the combined bottom tertile of IGF-I and top tertile of IGFBP-3 (53.8% versus 40.9%; P = 0.014). No significant association was observed among postmenopausal women. Our findings confirm that IGF-I and IGFBP-3 are associated with breast density among premenopausal women. They provide additional support for the idea that, among premenopausal women, these growth factors may affect breast cancer risk, at least in part, through their influence on breast tissue morphology as reflected on mammogram.     

Parity and mammographic breast density in relation to breast cancer risk: indication of interaction.

We examined whether the harmful influence of nulliparity on breast cancer risk could be mediated by high mammographic density. Another possibility is that mammographic density and nulliparity act independently or perhaps synergistically on breast cancer risk. Our study population consisted of 129 cases and 517 controls who had been participants in the Nijmegen breast cancer screening programme for 10 years. Breast density was classified with a fully automated technique on digitized mammograms from the screening examination 10 years before diagnosis. Classification was based on the proportion of the breast that was composed of high density: < 5%, 5-25% or > 25%. Data on parity and potential confounders were obtained using a questionnaire, administered at the same examination. We found that nulliparae with low breast density (< 5%) were not at increased risk compared to parous women with low density: OR 1.1 (95% CI 0.2-5.8). Parous women with < 5% density formed the reference category throughout all analyses. The risks for parous women with 5-25% or > 25% density were 2.7 (95% CI 1.3-5.6) and 3.6 (95% CI 1.7-7.7) fold increased, respectively. However, when both factors were present (nulliparity and > or = 5% density), breast cancer risk was 7.1 times higher (95% CI 3.2-15.9). This could indicate that nulliparity and high breast density might work synergistically and that breast density is not just an explanatory factor in the influence of nulliparity on breast cancer risk. It is hypothesized that high breast density (reflecting fibro-glandular tissue with increased epithelial cell proliferation) is more susceptible to carcinogenic effects in the undifferentiated epithelial breast tissue of nulliparae than in the differentiated tissue of parous women. Since there were few data, no firm conclusions can be drawn. If these findings can be confirmed in a larger study population, however, they may have important implications for the prevention and early detection of breast cancer.     

Changes in mammographic breast density and concomitant changes in breast cancer risk.

Among participants of the biennial Nijmegen breast cancer screening programme, we examined whether diminution of mammographic breast density lowered breast cancer risk. Post-menopausal breast cancer cases (n = 108), who had to have participated in all the five screening rounds prior to their diagnosis, were matched to 400 controls on year of birth and screening history. Controls had to be free of breast cancer at the time of the case's diagnosis. Changes in breast density were measured over a 10-year period, by a fully computerized method. Women in whom 5-25% or >25% of the breast was composed of fibro-glandular density showed a threefold increased 10-year risk compared to women with <5% density. In women with 5-25% density initially, we observed a trend of decreasing risk with diminishing density: when women with <5% density throughout the whole period formed the reference category, the odds ratio (OR) for those who decreased from 5-25% to <5% density was 1.9 [95% confidence interval (CI) = 0.6-6.1] in contrast to the OR of 5.7 (95% CI = 2.2-15.2) for those with persisting 5-25% density. In women who increased from 5-25% density to >25% density the OR was 6.9 (95% CI = 2.1-22.9). In women with >25% density initially, diminishing density was not clearly associated with lowering risk, which may be partly explained by the low number of women who decreased to <5% (n = 12). Due to the limited size of the study these results have to be interpreted with caution. Although the results are not conclusive, they could indicate a trend of decreasing risk with diminishing breast density. Should this effect be real, it may have great implications for the primary prevention of breast cancer or for the identification of high-risk groups who would benefit by more frequent screening. Therefore, large-scale, long-term follow-up studies on the effects of changes in breast density are needed.     

Association of diet and mammographic breast density in the Minnesota breast cancer family cohort.

Mammographic breast density is a significant risk factor for breast cancer. The present report analyzes the association of breast density and dietary factors in 1508 women in a historical cohort study of breast cancer families in Minnesota. Diet was assessed by a semiquantitative food frequency questionnaire. Percent breast density was estimated visually by a radiologist experienced in mammography. The association of percent breast density with quartiles of energy-adjusted dietary intakes was examined in analysis of covariance models adjusting for potential confounding effects of age, body mass index, and other covariates as well as correcting for familial correlation. Analyses were performed on all women combined and were also stratified by menopausal status. Among premenopausal women, percent breast density was positively associated with intakes of polyunsaturated fat, polyunsaturated:saturated fat ratio, and vitamins C and E and was inversely associated with saturated fat and total dairy intake. Among postmenopausal women, vitamin B12 was linearly associated with increased breast density. The positive associations for vitamin C and B12 were attributable to supplement intake only. There was a suggestive positive trend between breast density and daily alcohol consumption in both premenopausal and postmenopausal women. After adjustment for other sources of alcohol, only wine intake among postmenopausal women was significant such that white wine showed a positive association and red wine an inverse association with percent breast density. There was no association with other examined dietary factors. The cross-sectional differences in breast density across levels of dietary factors were small in magnitude but may have implications for breast cancer risk.     

Case-control study of increased mammographic breast density response to hormone replacement therapy.

Previous studies have demonstrated an association between current hormone replacement therapy (HRT) use and increased mammographic breast density. Many of these studies have also shown that only 20-35% of women initiating HRT respond in this manner. This subgroup of HRT responders may be at an increased risk of breast cancer. We performed a case-control study to investigate how women who experience increased density in response to HRT (cases) differ from women who do not experience an increase in density with HRT use (controls) with regard to breast cancer risk factors, type of HRT, weight change, and baseline breast density. Participants were female residents of Olmsted County, Minnesota who received routine screening mammograms at the Mayo Clinic. Cases included 172 women identified between the years 1998 and 1999 by Mayo radiologists as having a HRT response. Controls were women who did not experience an increase in mammographic density with HRT use and were matched to cases on age (+/-3 years), menopausal status, duration of HRT, month of initiation of HRT, and months between baseline and follow-up mammograms. Mammograms were obtained from cases and controls before and during HRT therapy. Breast density was read as a four-category Bi-Rads density grade measure and as a quantitative percentage estimate, using a computer-assisted method. Risk factor information was obtained from both chart review and a mammography database of patient-provided information. There was no association between HRT response and first-degree family history of breast cancer [odds ratio (OR), 0.8; 95% confidence interval (CI), 0.4-1.5], parity (OR, 0.8; 95% CI, 0.4-1.7), later age at first birth (OR, 0.8 for age >25 years versus nulliparous women; 95% CI, 0.4-1.8), or history of biopsy (OR, 0.9; 95% CI, 0.6-1.5). There was also no association with baseline weight or change in weight between a woman's baseline and follow-up mammograms. However, there was evidence of an association between HRT response and type of HRT used; women who experienced a mammographic increase in density with HRT had 2.3 greater odds (95% CI, 1.4-3.7) of having taken estrogen-progestin combined therapy than estrogen alone, compared with controls. This association was stronger among women with a baseline weight below the median (OR, 5.2; 95% CI, 1.6-17.6). Also, there was an inverse association between HRT response and baseline density. Because all risk factors examined accounted for only 26% of the variation in the HRT response, genes or other unmeasured factors are thought to be involved.     

Longitudinal trends in mammographic percent density and breast cancer risk.

BACKGROUND: Mammographic density is a strong risk factor for breast cancer. However, whether changes in mammographic density are associated with risk remains unclear. MATERIALS AND METHODS: A study of 372 incident breast cancer cases and 713 matched controls was conducted within the Mayo Clinic mammography screening practice. Controls were matched on age, exam date, residence, menopause, interval between, and number of mammograms. All serial craniocaudal mammograms 10 years before ascertainment were digitized, and quantitative measures of percent density (PD) were estimated using a thresholding method. Data on potential confounders were abstracted from medical records. Logistic regression models with generalized estimating equations were used to evaluate the interactions among PD at earliest mammogram, time from earliest to each serial mammogram, and absolute change in PD between the earliest and subsequent mammograms. Analyses were done separately for PD measures from the ipsilateral and contralateral breast and also by use of hormone therapy (HT). RESULTS: Subjects had an average of five mammograms available, were primarily postmenopausal (83%), and averaged 61 years at the earliest mammogram. Mean PD at earliest mammogram was higher for cases (31%) than controls (27%; ipsilateral side). There was no evidence of an association between change in PD and breast cancer risk by time. Compared with no change, an overall reduction of 10% PD (lowest quartile of change) was associated with an odds ratio of 0.9997 and an increase of 6.5% PD (highest quartile of change) with an odds ratio of 1.002. The same results held within the group of 220 cases and 340 controls never using HT. Among the 124 cases and 337 controls known to use HT during the interval, there was a statistically significant interaction between change in PD and time since the earliest mammogram (P = 0.01). However, in all groups, the risk associated with the earliest PD remained a stronger predictor of risk than change in PD. CONCLUSION: We observed no association between change in PD with breast cancer risk among all women and those never using HT. However, the interaction between change in PD and time should be evaluated in other populations.     

Variation in the promoter region of the myeloperoxidase gene is not directly related to lung cancer risk among male smokers in Finland

In order to examine whether a polymorphism in the promoter region of the myeloperoxidase (MPO) gene is associated with lung cancer among male smokers, we conducted a case-control study nested within a Finnish clinical trial cohort. Although we found no evidence of an overall association between lung cancer risk and MPO genotype, the variant MPO genotype was associated with an increased risk of lung cancer among a subset of older men. These findings contrast with those from previous studies that report decreased lung cancer risk among MPO variant individuals.     

Relations of omega-3 and omega-6 intake with mammographic breast density

Purpose

Omega-3 (n-3) and n-6 fatty acids (FA) intake could influence the occurrence of certain diseases such as breast cancer but little is known about their relation to mammographic density (MD). The purpose of this study is to examine the association of the intake of n-3 FA and n-6 FA with MD among 777 premenopausal and 783 postmenopausal women.

Methods

In this cross-sectional study, FA intake was assessed with a self-administered food-frequency questionnaire and MD was measured using a computer-assisted method. Multivariate analyses were performed by using generalized linear models to evaluate the associations of quartiles of FA intake with MD.

Results

For increasing quartiles of total long-chain n-3 FA intake (< 0.11, 0.11–0.20, 0.21–0.32, and ≥ 0.33 g/day), adjusted mean MD was 29, 29, 27, and 25 %, respectively (P _trend = 0.005). This association remained significant among postmenopausal (P _trend = 0.006) but not among premenopausal (P _trend = 0.21) women. No significant association was found between n-6 FA intake and MD. However, for increasing quartiles of the n-6 FA/long-chain n-3 FA ratio intake (< 31.75, 31.75–52.28, 52.29–94.28, and ≥ 94.29), adjusted mean MD was 26, 27, 29, and 29 %, respectively (P _trend = 0.008).

Conclusions

Higher intake of long-chain n-3 FA was associated with lower MD, suggesting that increased long-chain n-3 FA intake could be a strategy for breast cancer prevention.     

A novel polymorphism in the promoter region of ERBB4 is associated with breast and colorectal cancer risk.

PURPOSE: The receptor tyrosine kinase ERBB4/HER4 plays a role in cell division, migration, differentiation, as well as apoptosis, and is frequently overexpressed in breast and colorectal tumors. To understand the role of genetic variations in the regulation of ERBB4 expression, we identified new polymorphisms and investigated their functional implication and risk association with breast and colorectal cancer. EXPERIMENTAL DESIGN: We screened colorectal tumors from 92 patients for genetic variants at the ERBB4 ATG -1000 bp 5'-regulatory region by denaturing high-performance liquid chromatography and sequencing. Variants were subjected to DNA-protein interaction analyses (electrophoretic mobility shift assay), reporter gene assays in breast cancer cell lines MDA134 and MDA157, and immunohistochemical analyses of breast tumors. We established genotype frequencies within a breast cancer case-control collection (1,021 cases, 1,015 population-based controls) and a colorectal cancer case-control collection (459 cases, 569 blood donors) using matrix-assisted laser desorption ionization/time of flight mass spectrometry. Adjusted odds ratios (OR) and 95% confidence intervals (CI) were assessed by multivariate logistic regression. RESULTS: We identified five new germ line variants -815 A>T, -782 G>T, -638 insTC, -267 C>G, and -219 del10bp. Two variants showed in vitro functional effects. The -782T allele showed lower protein binding affinity and lower promoter activity compared with the -782G allele, however, the -815T allele showed higher protein binding affinity and higher promoter activity. The -782T variant was identified as a risk allele for breast and colorectal cancer (OR, 1.59; 95% CI, 1.06-2.34 and OR, 2.21; 95% CI, 1.22-3.99, respectively). CONCLUSION: The ERBB4 -782 G>T polymorphism, by virtue of its in vitro functional implication and incidence, is a risk factor for breast and colorectal cancer.     

The promoter and the enhancer region of the KLK 3 (prostate specific antigen) gene is frequently mutated in breast tumours and in breast carcinoma cell lines

KLK3 or prostate specific antigen (PSA) is a serine protease, which is an established tumour marker of prostatic adenocarcinoma. PSA is now used widely for the diagnosis and monitoring of patients with prostate cancer. Recent studies have demonstrated that about 70% of breast cancers produce PSA. In this study, we examined the molecular mechanism underlying the expression of the PSA gene in breast cancer and breast cancer cell lines. We analysed nine breast tumours categorized on the basis of high- or low-PSA expression in tumour cytosols and four breast cancer cell lines. To determine abnormalities associated with PSA expression in breast tumours, genomic DNA was extracted and all five exons of the PSA gene were polymerase chain reaction (PCR) amplified and sequenced on both strands. PCR amplification was also performed for the promoter and enhancer elements of the PSA gene. No mutations were observed in the coding portion of the gene. A polymorphism was observed in exon 2 from three breast tumours. However, sequencing of the promoter and the enhancer elements of the PSA gene reveals several point mutations. Within a 5.8-kb promoter/enhancer region of the PSA gene, we detected 16 different mutational hotspots (appearing more than once in the nine tumours). Among these hotspots, two appeared in seven out of nine tumours. Most importantly, the androgen response element (ARE I) in the proximal promoter was found mutated in four tumours and in the breast carcinoma cell line MCF-7. Mutations associated with the ARE I have been shown previously to result in an 80% decrease in PSA gene expression. The mutations in the core enhancer and promoter region probably contribute to the aberrant expression of the PSA gene in breast tumours, possibly by altering the regulation of the gene by steroid hormones.     

Dietary and lifestyle determinants of mammographic breast density. A longitudinal study in a Mediterranean population

High mammographic breast density (H-MBD) has been associated with increased breast cancer (BC) risk, even after adjustment for established BC risk factors. Only a few studies have examined the influence of diet on MBD. In a longitudinal study in Florence, Italy, we identified about 2,000 women with a mammogram taken 5 years after enrollment, when detailed information on dietary and lifestyle habits and anthropometric measurements had been collected. Original mammograms have been identified and retrieved (1,668; 83%), and MBD was assessed by 2 experienced readers, according to Wolfe's classification and a semiquantitative scale. By logistic analysis, we compared women with H-MBD (P2 + DY according to Wolfe's classification) with those with low-MBD (N1 + P1). H-MBD was confirmed to be inversely associated with BMI, number of children and breast feeding, while it was directly associated with higher educational level, premenopausal status and a previous breast biopsy. In multivariate analyses adjusted for non-dietary variables, H-MBD was inversely associated with increasing consumption of vegetables (p for trend = 0.005) and olive oil (p for trend = 0.04). An inverse association was also evident between H-MBD and frequent consumption of cheese and high intakes of beta-carotene, vitamin C, calcium and potassium (p for trend < or = 0.05). On the other hand, we found a positive association with increasing consumption of wine (p for trend = 0.01). This large longitudinal study, the first carried out in Mediterranean women, suggests that specific dietary components may play a key role in determining MBD in this population, thus possibly modulating BC risk.     

Interactions of alcohol and postmenopausal hormone use in regards to mammographic breast density

Purpose

We investigated the association of alcohol intake with mammographic breast density in postmenopausal women by their hormone therapy (HT) status.

Methods

This study included 2,100 cancer-free postmenopausal women within the Nurses’ Health Study and Nurses’ Health Study II cohorts. Percent breast density (PD), absolute dense (DA), and non-dense areas (NDA) were measured from digitized film mammograms using a computer-assisted thresholding technique; all measures were square root transformed. Alcohol consumption was assessed with a food frequency questionnaire (0, <?5, and ≥?5 g/day). Information regarding breast cancer risk factors was obtained from baseline or biennial questionnaires closest to the mammogram date. We used generalized linear regression to examine associations between alcohol and breast density measures in women with no HT history, current, and past HT users.

Results

In multivariable analyses, we found no associations of alcohol consumption with PD (p trend?=?0.32) and DA (p trend?=?0.53) and an inverse association with NDA (β?=???0.41, 95% CI ??0.73, ??0.09 for ≥?5 g/day, p trend?<?0.01). In the stratified analysis by HT status, alcohol was not associated with PD in any of the strata. We found a significant inverse association of alcohol with NDA among past HT users (β?=???0.79, 95% CI ??1.51, ??0.07 for ≥?5 g/day, p trend?=?0.02). There were no significant interactions between alcohol and HT in relation to PD, DA, and NDA (p interaction?=?0.19, 0.42, and 0.43, respectively).

Conclusions

Our findings suggest that associations of alcohol with breast density do not vary by HT status.

     

Codon 201(Gly) polymorphic type of the DCC gene is related to disseminated neuroblastoma

The deleted in colorectal carcinoma (DCC) gene is a potential tumor-suppressor gene on chromosome 18q21.3. The relatively high frequency of loss of heterozygosity (LOH) and loss of expression of this gene in neuroblastoma, especially in the advanced stages, imply the possibility of involvement of the DCC gene in progression of neuroblastoma. However, only few typical mutations have been identified in this gene, indicating that other possible mechanisms for the inactivation of this gene may exist. A polymorphic change (Arg to Gly) at DCC codon 201 is related to advanced colorectal carcinoma and increases in the tumors with absent DCC protein expression. In order to understand whether this change is associated with the development or progression of neuroblastoma, we investigated codon 201 polymorphism of the DCC gene in 102 primary neuroblastomas by polymerase chain reaction single-strand conformation polymorphism. We found no missense or nonsense mutations, but a polymorphic change from CGA (Arg) to GGA (Gly) at codon 201 resulting in three types of polymorphism: codon 201(Gly) type, codon 201(Arg/Gly) type, and codon 201(Arg) type. The codon 201(Gly) type occurred more frequently in disseminated (stages IV and IVs) neuroblastomas (72%) than in localized (stages I, II, and III) tumors (48%) (P=.035), and normal controls (38%) (P=.024). In addition, the codon 201(Gly) type was significantly more common in tumors found clinically (65%) than in those found by mass screening (35%) (P=.002). The results suggested that the codon 201(Gly) type of the DCC gene might be associated with a higher risk of disseminating neuroblastoma.