Are circulating levels of progesterone and progesterone metabolites associated with incident breast cancer in postmenopausal women?In this case-cohort study of 405 incident breast cancer cases, elevated circulating progesterone levels were associated with a 16% increase in the risk of breast cancer.This study’s findings suggest that further research should be undertaken to assess how postmenopausal breast cancer risk is associated with both endogenous progesterone and progesterone metabolite levels, as well as their interactions with estradiol levels. The role of endogenous progesterone in the development of breast cancer remains largely unexplored to date, primarily owing to assay sensitivity limitations and low progesterone concentrations in postmenopausal women. Recently identified progesterone metabolites may provide insights as experimental data suggest that 5α-dihydroprogesterone (5αP) concentrations reflect cancer-promoting properties and 3α-dihydroprogesterone (3αHP) concentrations reflect cancer-inhibiting properties.To evaluate the association between circulating progesterone and progesterone metabolite levels and breast cancer risk.Using a sensitive liquid chromatography–tandem mass spectrometry assay, prediagnostic serum levels of progesterone and progesterone metabolites were quantified in a case-cohort study nested within the Breast and Bone Follow-up to the Fracture Intervention Trial (n = 15 595). Participation was limited to women not receiving exogenous hormone therapy at the time of blood sampling (1992-1993). Incident breast cancer cases (n = 405) were diagnosed during 12 follow-up years and a subcohort of 495 postmenopausal women were randomly selected within 10-year age and clinical center strata. Progesterone assays were completed in July 2017; subsequent data analyses were conducted between July 15, 2017, and December 20, 2018.Circulating concentrations of pregnenolone, progesterone, and their major metabolites.Development of breast cancer, with hazard ratios (HRs) and 95% CIs was estimated using Cox proportional hazards regression adjusted for key confounders, including estradiol. Evaluation of hormone ratios and effect modification were planned a priori.P = .048). The association with progesterone was linear in a 5-knot spline and stronger for invasive breast cancers (n = 267) . Among women in the lowest quintile (Q1) of circulating estradiol (<6.30 pg/mL) elevated progesterone concentrations were associated with reduced breast cancer risk per SD increase in progesterone levels  and increased risk among women in higher quintiles of estradiol  .The present study included 405 incident breast cancer cases and a subcohort of 495 postmenopausal women; the mean (SD) age at the time of the blood draw was 67.2 (6.2) years. Progesterone concentrations were a mean (SD) of 4.6 (1.7) ng/dL. Women with higher circulating progesterone levels were at an increased risk for breast cancer per SD increase in progesterone levels (HR, 1.16; 95% CI, 1.00-1.35; In this case-cohort study of postmenopausal women, elevated circulating progesterone levels were associated with a 16% increase in the risk of breast cancer. Additional research should be undertaken to assess how postmenopausal breast cancer risk is associated with both endogenous progesterone and progesterone metabolites and their interactions with estradiol. This case-cohort study examines the association of circulating progesterone and progesterone metabolite concentrations with breast cancer in postmenopausal women. In addition to possible progestational effects, the progestins used in these menopausal hormone therapy formulations can have antiandrogenic, proandrogenic, glucocorticoid concentration, and antimineralocorticoid effects12 and are unlikely to provide direct evidence as to the potential association between endogenous progesterone and breast cancer risk. Mechanistic studies using cell culture, tissue culture, and preclinical models have associated progesterone with the development of breast cancer.13 To our knowledge, epidemiologic data are limited to 1 study reporting a null association between circulating progesterone levels and breast cancer risk among postmenopausal women.14 This study was limited by assay technology, with almost 30% of samples having undetectable levels of progesterone. Thus, the association of the presence of endogenous progesterone with breast cancer risk among postmenopausal women has been infrequently studied primarily owing to low levels of circulating progesterone, inadequate sensitivity of available assays,15 and/or large sample volumes required for clinical assays.Although it is well accepted that pharmacologic and physiologic concentrations of estradiol are associated with increased mitogenic activity of breast epithelial cells, the possible association with progesterone and breast cancer risk continues to be debated.16 and laboratory studies have confirmed20 that breast cancer promotion may be associated with relative concentrations of progesterone metabolites, in particular the cancer-inhibiting 4-pregnenes  and cancer-promoting 5α-pregnanes  .21 Existing data on circulating estrogens21 enabled evaluation of the relative concentration of progesterone to estradiol with breast cancer risk, given that, along with estrogen, progesterone is generally regarded as a proliferative agent in the healthy breast.22It has been hypothesizede [5αP]) . While t21 In brief, the B ~ FIT cohort (n = 15 595) is a longitudinal cohort of postmenopausal women screened for participation in the Fracture Intervention Trial (FIT)23 from 1992 to 1993 at 10 of the 11 FIT clinical centers (23 (2) women randomized to participate in FIT, and (3) women invited to participate in an extension of the FIT Long-term Extension Trial (FLEX).26 The B ~ FIT questionnaire was completed by 64% of eligible women. All participants provided written informed consent; no financial compensation is provided. The B ~ FIT trial was approved by the institutional review board at each participating site and the National Cancer Institute; the present study was included in this approval. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline for cohort studies.Details of the prospective case-cohort study on which this study is based have been described. centers . Women iCancer incidence was based on questionnaire responses, with subsequent requests for medical record verification and/or cancer registry linkage at clinical sites in states with cancer registries or in Surveillance Epidemiology and End Results registry areas. Seventy-three percent of B ~ FIT participants resided in registry linkage areas, of which 29% were Surveillance, Epidemiology, and End Results registry areas.21 More than 99% of these women had sufficient baseline serum remaining for the measurement of progesterone-related compounds , which composed the present study population. Of the breast cancers included in the present analysis, 267 were diagnosed as invasive breast cancer and 54 as in situ cancer, with 84 missing information on cancer behavior. Among all breast cancer cases included in the present study, the mean (SD) time from sample collection to diagnosis was 5.6 (3.0) years .Of the participants in the B ~ FIT cohort (n = 15 595), 1811 were excluded from possible selection into the case-cohort study . Of the 15 Total estradiol levels were previously quantified using an independent HPLC-MS/MS assay.21Stable isotope dilution high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) was used to quantify pregnenolone, progesterone, and their major metabolite levels in serum: 17-hydroxypregnenolone, 17-hydroxyprogesterone, 3αHP, 5αP, and 20α-dihydroprogesterone. Details of the assay method have been published.For all analytes in the assay, the lower limit of quantitation was 0.5 ng/dL and the lower limit of detection was 0.1 ng/dL. No samples in the present study had undetectable levels for any of the hormone concentrations measured. Laboratory coefficients of variation of deidentified duplicate samples within and across batches were less than 3.5% for all hormone concentrations measured. Intraclass correlation coefficients were greater than 0.97  and 95% CIs for the association between each hormone measurement or ratio and breast cancer risk. Age was used as the time measurement for all analyses. For the subcohort, women entered the analysis at age at baseline and contributed person-time until age at first diagnosis of breast cancer (event) or censoring (age at death or end of follow-up). Breast cancer cases not included in the subcohort entered the analysis 6 months before their age at diagnosis, contributing information only to their risk set. Exposure curves from survivor function plots were parallel, suggesting no deviation from proportional hazards.We evaluated possible deviations from linearity of the hormone-breast cancer association using a 5-knot spline with knots at the 10th, 25th, 50th, 75th, and 90th percentiles for each hormone based on the distribution in the subcohort. Hormones were modeled continuously, given that 5-knot splines did not suggest major deviations from linearity. Individual hormone measurements were scaled by their SD and ratio measures were analyzed without transformation. Cox proportional hazards regression with robust variance adjustmentModels were adjusted for a priori selected confounding factors, based on knowledge of the literature and study design considerations as follows: clinic (10 sites), trial participation status , body mass index (continuous), and duration of prior menopausal hormone therapy use .20 that relative concentrations of 5αP and 3αHP are associated with breast cancer risk, we explored associations modeling the cross-classification of 5αP and 3αHP in tertiles based on the distribution in the subcohort.To further test the hypothesis21 Hazard ratios for breast cancer risk associated with increasing hormone concentration across categories of potential effect modifiers were estimated from stratified models, with significance assessed by a likelihood ratio test comparing a model with a multiplicative interaction term with a model without.We evaluated potential effect modification of the progesterone and 5αP:3αHP associations with breast cancer risk by age (≤65 and >65 years at baseline), body mass index , prior oral contraceptive use , and circulating estradiol level quintile. We maintained estradiol level quintiles given a prior B ~ FIT publication reporting increased breast cancer risk with the fifth compared with the first quintile of estradiol.We conducted the following sensitivity analyses: censoring breast cancer cases that were not confirmed by medical record and/or cancer registry linkage (n = 48) and restricting the analytic study population to women who underwent screening (n = 694). Evaluation of hormone ratios, effect modification, and the stated sensitivity analyses were predefined in the analytic plan.P values were based on 2-sided tests and a nominal P value <.05 was considered statistically significant. Analyses were conducted in SAS, version 9.4 (SAS Institute Inc).All The current case-cohort study included 405 incident breast cancers and a subcohort of 495 postmenopausal women . ParticiThe mean (SD) progesterone concentration among women in the subcohort 4.6 ng/dL (1.7)  (eTable 2 in the P = .048) (P = .004) in models evaluating invasive breast cancer as the outcome (P = .03) and limited to women who were screened   . This as outcome . The incP = .85)  compared with the lowest tertile (eTable 4 in the Higher levels of 5αP relative to 3αHP (5αP/3αHP) were not associated with risk of postmenopausal breast cancer  . AssociaP = .04), while higher vs lower circulating progesterone levels were associated with elevated breast risk cancer among women with higher quintiles of estradiol HRs: Q2, 1.32 ; Q3, 1.27 ; Q4, 1.19 ; Q5, 1.23  (P = .05 for interaction). Because we observed similar HRs across Q2 through Q5 (80% of women in our study), we combined these groups and found that higher circulating progesterone levels, specifically a 1-SD increase in progesterone level, was associated with an 18% increased risk of breast cancer  among women with circulating estradiol levels of 6.30 pg/mL or greater. The 5αP/3αHP-breast cancer association was not significantly modified by age, body mass index, oral contraceptive use, or circulating estradiol quintile.Progesterone associations with breast cancer and 5αP/3αHP-associations with breast cancer were not modified by age, body mass index, or prior oral contraceptive use . The proIn this prospective study, women with increased serum progesterone concentrations were at an increased risk of postmenopausal breast cancer. Higher levels of 5αP were not associated with breast cancer risk, except among women with the lowest levels of 3αHP. To our knowledge, this is the first population-based study to evaluate the association of novel progesterone metabolites with postmenopausal breast cancer risk. We noted effect modification of the progesterone-breast cancer association by estradiol level, whereby increased serum progesterone levels were associated with reduced breast cancer risks among women with very low total circulating estradiol levels and increased risks among women with higher circulating total estradiol levels.14 The mean concentration of progesterone in postmenopausal women in that study , was consistent with the mean circulating progesterone concentration in our study population . However, the lowest concentrations measured in the previous study were below the limit of detection (3 ng/dL) of the radioimmunoassay used and represented almost one-third of the study population. It is likely that the reduced sensitivity of the progesterone assay available at the time the previous study was conducted may have limited the study’s ability to find an association. In the present study, no measures were below the limit of detection of the HPLC-MS/MS assay.In what appears to be the only prior study of progesterone and postmenopausal breast cancer risk published to date, progesterone was not associated with breast cancer risk.16  in breast tissue and circulation comparing established breast cancers with controls. However, when exploring the cross-classification of tertiles of 5αP and 3αHP concentrations, we noted an almost doubling in breast cancer risk comparing the highest third with the lowest third of 5αP concentrations among women with the lowest third of 3αHP concentrations. It is plausible that, at higher prediagnostic concentrations of 3αHP, the association with increasing 5αP concentration is either masked or not present when measured in circulation. However, we cannot rule out that variability in enzymatic activity of the hormones and metabolites may be influencing the observed association.Several progesterone metabolites have been characterized in healthy breast and cancerous breast cancer tissue and broadly fall into 2 groups: 4-pregnenes, which are metabolites that retain their double bond, and 5α-pregnanes, which are metabolites in which 5α-reductase has reduced the double bond16 . Differe22; along with estrogen, progesterone is generally regarded as a proliferative agent in the healthy breast. Given that the women in our study were limited to those reporting no use of hormones within 4 months of the blood draw, our data provide support for a differential role of progesterone at physiologic levels that is possibly predicated on the level of circulating estradiol. In vivo studies support that both estradiol and progesterone may be responsible for cellular proliferation in the mammary gland and that progesterone is not sufficient to stimulate cellular proliferation in the absence of estradiol.28 Experimental data suggest the reliance on paracrine signaling for much of progesterone’s actions in breast tissue, but these data also show that PR transcription in mammary epithelium is dependent on interactions of estrogen with ER-α,29 further supporting the interdependency of progesterone and estradiol in mammary biology. We believe our data support that, at very low circulating estradiol levels, higher progesterone levels were associated with reduced breast cancer risk, suggesting that relatively higher progesterone levels may have a weak antimitotic effect on proliferation in the breast in this context. Although progesterone level was associated with increased breast cancer risk at higher circulating estradiol levels, further supporting that higher progesterone concentrations may increase risk via an enhancement of the mitotic effect of increased estradiol concentrations on breast tissue. While the interaction we observed is biologically plausible, replication in additional study populations is warranted.Progesterone’s actions are tissue specific and occur in concert with other hormonesOur study has several strengths. The most important strength is the use of a highly sensitive and reproducible HPLC-MS/MS assay that quantifies hormone levels at concentrations relevant in postmenopausal women. Additional strengths include the use of prediagnostic serum, the prospective case-cohort design, the ability to stratify by circulating estradiol concentrations, and the large sample size.P value of .005 (.05/10), only the positive association between progesterone concentrations and increased invasive breast cancer risk would have remained statistically significant.The study also has limitations. A single blood sample may provide an imprecise average of long-term levels; however, a separate reproducibility study demonstrated moderate to high stability over 2 years  in average intraclass correlation coefficients of all hormones measured . We measured 7 hormones and included a moderate number of comparisons, suggesting that replication in future studies will be critical. We did not formally correct for multiple comparisons; however, using a Bonferroni-corrected 21 the bone mineral density of women in our study is comparable to that of women from a nationally representative sample of US women. In addition, while we had information on tumor invasiveness for most cases, we were largely missing hormone receptor status, which may be relevant. However, 5αP and 3αHP are reported to act with equal efficacy regardless of the receptor status of the breast cancer cell line evaluated.20 Therefore, additional research to assess circulating associations by tumor characteristics  appears to be warranted.The generalizability of our study may be limited, as the population comprises primarily women who were white and volunteered to be screened for participation in a clinical trial of medication to reduce fracture risk. However, as previously reported,Our prospective data suggest that increased serum progesterone concentrations, measured using a highly sensitive HPLC-MS/MS assay, in postmenopausal women, appeared to be associated with an increased risk of breast cancer. There was a suggestion that increased risk was most apparent among women with moderate to high circulating total estradiol levels; at very low levels of estradiol, progesterone was associated with reduced breast cancer risk. While individual metabolites or their ratios were not associated with breast cancer risk, we noted an apparently increased risk with higher levels of 5αP among women with low levels of 3αHP. The possible identification of increased breast cancer risk with circulating levels of progesterone in postmenopausal women supports the need for additional research regarding its role in the cause of breast cancer overall and by tumor subtype. Further research is also needed to evaluate the role of progesterone metabolites and the interaction between progesterone and estradiol with breast cancer risk.


Logistic LASSO regression was used to examine the relationship between twenty-nine variables, including dietary variables from food, as well as well-established/known breast cancer risk factors, and to subsequently identify the most relevant variables associated with self-reported breast cancer. We observed that as the penalty factor (λ) increased in the logistic LASSO regression, well-established breast cancer risk factors, including age (β = 0.83) and parity (β = −0.05) remained in the model. For dietary macro and micronutrient intakes, only vitamin B12 (β = 0.07) was positively associated with self-reported breast cancer. Caffeine (β = −0.01) and alcohol (β = 0.03) use also continued to remain in the model. These data suggest that a diet high in vitamin B12, as well as alcohol use may be associated with self-reported breast cancer. Nonetheless, additional prospective studies should apply more recent statistical techniques to dietary data and cancer outcomes to replicate and confirm the present findings.A multitude of dietary factors from dietary fat to macro and micronutrients intakes have been associated with breast cancer, yet data are still equivocal. Therefore, utilizing data from the large, multi-year, cross-sectional National Health and Nutrition Examination Survey (NHANES), we applied a novel, modern statistical shrinkage technique, logistic least absolute shrinkage and selection operator (LASSO) regression, to examine the association between dietary intakes in women, ≥50 years, with self-reported breast cancer ( In 2019, existing/prevalent cases of breast cancer in the United States reached more than 3.8 million, and approximately 42,000 women are expected to die from the disease in 2019  was obtained from the body measures data set. Parity was ascertained via the following NHANES question, “How many times have you been pregnant? , live births, miscarriages, stillbirths, tubal pregnancies or abortions)”. Alcohol and caffeine consumption were expressed as grams and mg per day, respectively, and obtained via 24-h dietary recall data.Age and race/ethnicity were obtained from the demographic variables and sample weights data set. NHANES categorizes race/ethnicity into four groups: non-Hispanic white, Hispanic, African American and other/multi-racial. Body mass index [BMI . The “survey” package (version 4.0) in R was utilized for the univariate analyses accounting for the stratified, multistage probability cluster sampling design in NHANES. NHANES provides sampling weights for analytical purposes, which account for oversampling of certain subgroups, differences between the sample and the population due to nonresponse, and population sizes. More specifically, NHANES provides sampling weights to be used for dietary analyses, which also account for the fact that not all participants completed the dietary interview and that different days of the week were represented in the 24-h periods for which dietary intake was assessed. Our study sample consisted of 6 cycles of continuous NHANES data from 1999–2010, thus dietary weights were adjusted to reflect the U.S. population. Due to different reference populations, the 4-year dietary weights for 1999–2002 were adjusted, while the remaining cycles for 2003–2010 adjusted the 2-year dietary weights. The NHANES stratification variable (SDMVSTRA) and primary sampling unit variable (SDMVPSU) were incorporated according to the survey design to appropriately adjust the variance estimates.2). Univariate analyses, specifically t-test for continuous variables and chi-square for discrete variables, respectively, were performed for demographic data including age and ethnicity, as well as age at first menarche, parity, and BMI. Also, mean intakes for macro- and micronutrients (from food only) were calculated and t-test analyses, as well as respective 95% confidence intervals of the mean differences were conducted to examine differences in dietary nutrient intakes between women with and without self-reported reported breast cancer diagnoses. All statistical tests were two-sided with 0.05 significance levels.Descriptive and dietary variables were tested for normality and were log-transformed as appropriate, including all dietary variables. Specifically, statistical analyses were conducted on log transformed variables, including univariate tests and LASSO regression; however, mean data and 95% confidence intervals shown are on the raw/non-log transformed data. Height and weight data were used to calculate BMI  to fit the logistic LASSO regression. The dietary weights were normalized and incorporated in the same fashion as standard weighted regression . The covn = 279 (with self-reported breast cancer) and n = 1116 (without breast cancer), and analyses for these data were conducted on the respective sample size. We also examined time since breast cancer diagnoses (age at interview minus age at diagnoses), and conducted correlation analyses between the time since diagnoses variable and dietary intakes. Specifically, independent pairwise correlation tests were performed on the log scale of all dietary variables with time since diagnoses.For the logistic LASSO regression, self-reported breast cancer was included as the dependent variable, Y, and coded as 0 for no cancer and 1 for presence of breast cancer. Additionally, we included all 21 dietary variables from food, in addition to alcohol and caffeine consumption, available during the respective years of analyses, via the NHANES 24-h dietary recall data, including: energy , % energy from carbohydrate, % energy from fat, % energy from protein, % energy from fat, cholesterol (mg), fiber (g), folate (μg), vitamin B12 (μg), vitamin B6 (mg), thiamin , riboflavin , calcium (mg), phosphorous (mg), magnesium (mg), iron (mg), vitamin A (RE), vitamin C (mg), vitamin E (mg), zinc (mg), sodium (mg), potassium (mg), caffeine (mg), and alcohol (g). Energy dense macronutrients, including dietary fat, carbohydrate, and protein were adjusted for energy intakes and included in the logistic LASSO regression model as % energy of the respective macronutrient. All dietary variables were included as continuous variables in the model. All variables evaluated as potential confounders and specifically those shown to be previously associated with breast cancer risk (based on literature) were also included in the model: age (continuous), age at menarche (continuous), and parity (continuous). We also included BMI in our model, however BMI data were available only on a subset of participants, p ≤ 0.05) between women with self-reported breast cancer and women without were observed for age 68.46 (0.74) vs. 63.19 (0.36) years, age at first menarche (12.62 (0.13) vs. 12.89 (0.06) years), and ethnicity, where women with self-reported breast cancer were more likely to be older, had a younger age at menarche, were less parous, and were more likely to be to be non-Hispanic white compared with women without self-reported breast cancer . Correlation analyses with dietary intakes and time since diagnoses showed no correlation between variables, except for alcohol  (data not shown).Demographic and well-established breast cancer risk factor data (including respective % (±SD) or mean (± SD)) are shown in p = 0.08)). Women with self-reported breast cancer also had higher alcohol (g) consumption ((5.31 (1.01) vs. 3.17 (0.49)) as well as vitamin A (IU) intakes ((685.55 (75.15), 648.52 (18.85)), however these variables did not reach statistical significance (p = 0.19).Using a large, cross-sectional, nationally representative sample, in conjunction with modern robust statistical techniques, we applied logistic LASSO regression, which minimizes multicollinearity between dietary variables, to assess the relationship between dietary intakes and breast cancer diagnoses. Via LASSO, we also accounted for well-established breast cancer risk factors, while simultaneously selecting for relevant coefficients from a multitude of variables, ultimately removing all other unrelated variables. Our initial univariate analyses showed that age, age at first menarche, ethnicity, and also vitamin B12 from food, was related to breast cancer. In the ultimate logistic LASSO regression, well-established breast cancer risk factors, including older age and lower parity were associated with increased breast cancer, and vitamin B12, and alcohol and caffeine intakes were also related to self-reported breast cancer. Thus, we showed that increased alcohol consumption and reduced caffeine use were associated with an increase in breast cancer, with only vitamin B12 from diet remaining in the model. To the knowledge of the authors, this is the first study to utilize the powerful LASSO shrinkage technique to assess the relationship between the multitude of dietary variables and other risk factors with breast cancer diagnoses.Our results of the association of well-established breast cancer risk factors, including age, race/ethnicity, age at menarche, and parity have been observed previously ,25,26,27Alcohol use has been consistently shown to increase breast cancer risk ,30,31,32ptrend = 0.029 [ptrend = 0.03) [Previous studies have shown that caffeine and/or coffee consumption may be associated with reduced breast cancer risk, but data remain equivocal ,40,41,42 = 0.03) . However = 0.03) ,49. It i = 0.03) ,51,52,53ptrend = 0.06) [Our findings of a positive relationship between vitamin B12 and breast cancer diagnoses have been reported in previous studies ,55,56,57 = 0.06) . As a pl = 0.06) ,61,62,63 = 0.06) ,65. CompStrengths of our study include the large sample size available via NHANES, which provides sufficient power to detect clinically relevant differences, and the generalizability of results due to the nationally-representativeness of the NHANES survey data. Additionally, another strength is representation via oversampling of diverse racial/ethnic subgroups, such as Hispanics and African Americans, inherent in the NHANES survey methodology, enabling inclusion of groups that are often underrepresented in the scientific literature on diet and breast cancer. Limitations include the retrospective, cross-sectional design, which does not allow for causal inference, and self-reported data on diet and breast cancer. Further, it is feasible that women may have changed their diet post-diagnoses, which may influence findings, nonetheless, our correlation analyses of time since diagnoses found only one association, possibly due to multiple comparisons, between dietary intakes and time since diagnoses in women with self-reported breast cancer, potentially suggesting little to no change in diet from time since diagnoses. We also reported on dietary intakes from food only, and not on supplemental intake. Supplement data availability during the study time period were limited, and therefore only dietary data were examined in order to increase sample size and preserve power to detect associations.In conclusion, to the knowledge of the authors, we showed for the first time, via a powerful shrinkage technique, that LASSO regression can be a viable option to narrow and decipher the role of a multitude of dietary factors and their relation to breast cancer diagnoses. We showed that established breast cancer factors, including age and parity continue to be associated with breast cancer diagnoses, and that alcohol use was positively associated, while caffeine intake was inversely related to breast cancer diagnoses. Our shrinkage analyses findings also suggest a potential role of dietary vitamin B12 intake and breast cancer diagnoses, however LASSO applications and use in assessing dietary intakes and breast cancer need to be confirmed in other prospective studies and warrant further investigation.


However, no study has investigated their possible impact on mortality after breast cancer.Estrogen metabolite concentrations of 2-hydroxyestrone . We used multivariable-adjusted Cox proportional hazards to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for the estrogen metabolites-mortality association. We evaluated effect modification using likelihood ratio tests. All statistical tests were two-sided.This population-based study was initiated in 1996–1997 with spot urine samples obtained shortly after diagnosis (mean = 96 days) from 683 women newly diagnosed with first primary breast cancer and 434 age-matched women without breast cancer. We measured urinary concentrations of 2-OHE1 to 16-OHE1 ratio (>median of 1.8 vs ≤median) were inversely associated with all-cause mortality  among women with breast cancer. Reduced hazard was also observed for breast cancer mortality  and cardiovascular diseases mortality , although the 95% confidence intervals included the null. Similar findings were also observed for women without breast cancer. The association with all-cause mortality was more pronounced among breast cancer participants who began chemotherapy before urine collection  than among those who had not .Urinary concentrations of the 2-OHE1 to 16-OHE1 ratio may be inversely associated with long-term all-cause mortality, which may depend on cancer treatment status at the time of urine collection.The urinary 2-OHE Breast cancer remains the most common cause of cancer death for women worldwide . Accumul1), 4-hydroxyestrone (4-OHE1), and 16-hydroxyestrone (16-OHE1)  . Most stxclusive . These tinogenic ,10; convtrogenic ,12. Givetrogenic  proposed 16-OHE1 .The urinary estrogen metabolites collected close to the time of breast cancer diagnosis may be associated with prognosis after breast cancer through the progression mechanisms of cell proliferation and oxidative stress . However1, 16-OHE1, and their ratio, with all-cause, breast cancer, and CVD-specific mortality among women with breast cancer. Second, we explored whether these associations differed by the lifestyle and clinical factors. We also incorporated a comparison group of women without breast cancer to explore whether breast cancer status may modify metabolites-mortality associations.In our study reported here, first we examined the associations of 2-OHEWe used the resources from the Long Island Breast Cancer Study Project (LIBCSP), which is a population-based study that included 1508 women with breast cancer and an age-matched cohort of 1556 women without breast cancer who were residents of Nassau and Suffolk counties, Long Island, New York . The detThe analytic cohort for the study reported here consisted of LIBCSP participants with available data on urinary estrogen metabolites. Women with and without breast cancer  provided a spot urine sample (A 25 ml spot urine sample was obtained by LIBCSP trained field staff at the baseline interview (mean = 96 days after diagnosis for women with breast cancer) . RespondThe covariate information was primarily drawn from the LIBCSP baseline interviewer-administered questionnaire. These detailed, structured instruments included assessments of sociodemographic characteristics, reproductive and menstrual history, lifestyle factors, medical and medication history, and other factors at or prior to the date of diagnosis for women with breast cancer and date of identification for women without breast cancer ,28. DietVital status and date and cause of death were determined via the National Death Index (NDI) ,30. For 1 and 16-OHE1) and their ratio were dichotomized at the median. Women with metabolite levels no more than median value were chosen as the referent group. We used multivariable Cox proportional hazards regression to estimate hazard ratios (HRs) and 95% confidence intervals (CIs)  . Person-ls (CIs) ,32. ViolFindings are presented for the three outcomes from four distinct models, with varying adjustment sets. Model 1 includes adjustment for age. Model 2 includes the minimal sufficient adjustment set, which was selected a priori by use of a directed acyclic graph (DAG) . This seModels 3 and 4 were constructed to address the possibility of confounding by cancer treatment. Model 3 included the minimal sufficient adjustment set (from model 2), with additional adjustments for initiation of chemotherapy and/or endocrine therapy prior to urine collection. Model 4 also included the minimal sufficient adjustment set, with further adjustment for the first course of chemotherapy and endocrine therapy for the first primary breast cancer (but with no adjustment for treatment initiated before urine collection). Information on the first course of chemotherapy and endocrine therapy was missing for 28% of women with breast cancer. Thus, we performed multiple imputations by using PROC MI in SAS , which i2), menopausal status , smoking history (never vs ever), and chemotherapy (yes vs no) or endocrine therapy initiated before the urine collection (yes vs no). We also considered a model that included both women with and without breast cancer to evaluate modification by breast cancer status (yes vs no). We assessed effect modification on the multiplicative scale  by comparing nested models with and without the cross-product terms from the likelihood ratio test , BMI , women with levels above the median were less likely to be overweight and obese  but more likely to have smoked cigarettes  and consumed alcohol . Other characteristics were similar across levels of the 2-OHE1 to 16-OHE1 ratio.As shown in 1 to 16-OHE1 ratio did not vary substantially by menopausal status, although the two individual metabolites were lower in postmenopausal women than premenopausal women [as previously reported , chemotherapy , or endocrine therapy  initiation status at the time of urine collection  the median was associated with reduced risk of all-cause mortality following breast cancer, regardless of the adjustment set included in our models. For example, in model 4, where we adjusted for socioeconomic, lifestyle factors, and first course of chemotherapy and hormone therapy (after performing multiple imputation), hazards were reduced by 26% . For individual metabolites, in model 4, we observed an inverse association for 2-OHE1  but a positive association for 16-OHE1 , although all 95% confidence intervals included the null.Among women with breast cancer, as shown in 1 to 16-OHE1 ratio, regardless of the adjustment set considered. For example, in model 4, where we accounted for both the minimal sufficient adjustment set and first course of treatment (after performing multiple imputation), the hazard was reduced by 27% . For individual metabolites, all associations were essentially null.As shown in 1 to 16-OHE1 ratio was also inversely associated with CVD-specific mortality across different models. For example, the hazard from model 4 was reduced by 24% . However, for the individual metabolites, the hazards were decreased by 38% for 2-OHE1  and increased by 19% for 16-OHE1 .As also shown in 1 to 16-OHE1 ratio and all-cause mortality by chemotherapy initiated before urine collection. A 2-OHE1 to 16-OHE1 ratio above (vs below) the median value was strongly associated with a lower all-cause mortality only among women who had received chemotherapy before urine collection  but not among women who had not .As shown in 1 to 16-OHE1 ratio and the two individual metabolites with mortality by history of MHT use  were associated with 18–26% reduced hazards for all-cause mortality among women with breast cancer, regardless of the adjustment set considered. Among women who had initiated chemotherapy before urine collection, the risk reduction was 58%. Higher levels of the 2-OHE1 to 16-OHE1 ratio were also associated with a 15–29% reduction in breast cancer– and CVD-specific mortality, although the 95% confidence intervals of these estimates included the null value.In this population-based study with long-term follow-up, we observed that higher concentration levels of the 2-OHE1 to 16-OHE1 ratio has been associated with a lower risk of developing breast cancer in several epidemiologic studies, including our own  and the Babylon Breast Cancer Coalition.Role of the funder: The funders had no role in the design of the study; the collection, analysis, and interpretation of the data; the writing of the manuscript; and the decision to submit the manuscript for publication.Disclosures: AIN has consulted for Otsuka Pharmaceuticals, United Biosource Corporation, Hospira, Teva, and Eisai and is on the Scientific Advisory Board of EHE, Intl. The other authors have no potential conflicts of interest.Authors' contributions: Study concept and design: all authors; data acquisition: MG, GCK, HP, NKK, SLT, MBT, RMS, and AIN; Statistical analysis: TW in consultation with MG, HBN, SJN, PTB, and PGM; interpretation of data: all authors; drafting of the manuscript: TW and MG; critical revision of the manuscript for important intellectual content: all authors; study supervision: MG; approved the manuscript: all authors.pkaa014_Supplementary_DataClick here for additional data file.


Several genes have been confirmed as risk genes for epithelial ovarian cancer (EOC). There are five main types of EOC, with different molecular changes and clinical characteristics, suggesting they should be considered different diseases. This review summarises the contribution of rare inherited mutations to EOC susceptibility, focussing on the frequency in each EOC type. Susceptibility genes can have a major clinical impact, reducing ovarian cancer incidence by screening of family members to detect women at higher risk than the general population. They can also lead to the development of new targeted treatments.BRCA1 and BRCA2 genes confer EOC risks by age 80, of 44% and 17% respectively. The mismatch repair genes, particularly MSH2 and MSH6, are also EOC susceptibility genes. Several other DNA repair genes, BRIP1, RAD51C, RAD51D, and PALB2, have been identified as moderate risk EOC genes. EOC has five main histotypes; high-grade serous (HGS), low-grade serous (LGS), clear cell (CCC), endometrioid (END), and mucinous (MUC). This review examines the current understanding of the contribution of rare genetic variants to EOC, focussing on providing frequency data for each histotype. We provide an overview of frequency and risk for pathogenic variants in the known susceptibility genes as well as other proposed genes. We also describe the progress to-date to understand the role of missense variants and the different breast and ovarian cancer risks for each gene. Identification of susceptibility genes have clinical impact by reducing disease-associated mortality through improving risk prediction, with the possibility of prevention strategies, and developing new targeted treatments and these clinical implications are also discussed.A family history of ovarian or breast cancer is the strongest risk factor for epithelial ovarian cancer (EOC). Germline deleterious variants in the  Epithelial ovarian cancer (EOC) is the seventh most common cancer in women worldwide, with over 295,000 incident cases each year, and is the leading cause of mortality relating to gynaecological malignancies, with 184,000 deaths each year  polymerase) inhibitors, which improve progression-free survival in selected women with ovarian cancer. Approximately 50% of HGS cases present defects in DNA repair mechanisms due to pathogenic variants in the homologous recombination deficiency (HRD) genes, such as BRCA1/2, or due to functional inactivation through methylation [BRCA1/2 variants, or women with other homologous recombination deficiencies [BRCA status [BRCA1/2 status affects clinical management of affected women, current guidelines recommend BRCA1/2 testing in all non-mucinous ovarian cancer cases [Furthermore, understanding the role of hylation . PARPi hciencies ,42,43. IA status . As BRCAer cases .BRIP1 is part of the Fanconi anaemia group (FANCJ) and is involved in the repair of DNA double-strand breaks by homologous recombination. BRIP1 was described as a candidate risk gene by Walsh et al. that identified germline loss-of-function variants in 1% of ovarian cancer cases not selected for age or family history [BRIP1 are the most common mutation found in ovarian cancer after BRCA1/2 with a frequency of approximately 1% of the EOC cases [The protein encoded by  history . PathogeOC cases .BRIP1 have not yet been assessed in prospective studies. Ramus et al. described a relative risk associated with protein truncating variants in BRIP1 of 11.2 (95% CI 3.2–34.1), with an estimated cumulative risk of 5.8% (95% CI 3.6–9.1%) by the age of 80 years [BRIP1 and risk of ovarian cancer has been confirmed in other analyses  and 3.4 (95% CI 1.5–7.6) for RAD51C and 12.0 (95% CI 1.5–90) and 10.9 (95% CI 4.6–26) for RAD51D , OR 11.8 (95% CI 1.1–40), and SRR 6.34 (95% CI 3.1–11.3)   [26,27,2RAD51C and 60 families with pathogenic variants in RAD51D and confirmed an increased risk of ovarian cancer associated with pathogenic variants in both genes  [RAD51C and 13% (95% CI 7–23%) for RAD51D based on their segregation analysis [RAD51C/D were also shown to be associated with breast cancer with cumulative risk of 21% (95% CI 15–29%) and 20% (95% CI 14–28%) for these genes by the age of 80 years, respectively [More recently, Yang et al. performed a study of 125 families with pathogenic variants in RAD51C and RAD51D, women carrying pathogenic variants in these genes could be offered preventive surgery starting from ages 45 to 50, once childbearing is complete.There is no consensus about the EOC risk threshold for surgical prevention, although the acceptability of RRSO for women with a lifetime risk greater than 10% is well-established. It has been recently suggested that this threshold should be lower and was demonstrated that prophylactic surgery is cost-effective for women at lifetime risk of 5% ,50,51. CPALB2 is a confirmed breast cancer susceptibility gene with a cumulative risk estimated to be 44% by 80 years [PALB2 is needed to recruit BRCA2 in the HR DNA repair pathway [80 years , and the pathway .PALB2 was an ovarian cancer susceptibility gene (p 0.08) [PALB2 has been confirmed as a risk gene by targeted sequencing in 5123 HGS cases and 5202 controls, WES data from 829 cases and 913 controls, and genotyping data from an independent set of ~14,000 EOC cases and 29,000 controls [Initial analysis of 3227 EOC cases and 3444 matched-controls suggested that larger numbers of samples were required to determine if (p 0.08) . Evidenc(p 0.08) . Recentlcontrols . The oddcontrols   [An increased risk of ovarian cancer has recently been confirmed in a study of 976 individuals with protein truncating variants in 1.4–6.0)  (Table 31.4–6.0)  and 3.2%1.4–6.0) . DiscussPALB2 were associated with increased risk of female and male breast cancer  and pancreatic cancer (RR 2.37 95% CI 1.2–4.5) [Yang et al. also showed that protein truncating variants in 1.2–4.5) .MSH2 and MSH6 genes [Germline protein truncating and known deleterious missense variants in four MMR genes are associated with Lynch syndrome, which is an inherited disorder that increases the risk of many types of cancers including colon, endometrial, ovarian, pancreatic, small-bowel, and ureteric. In ovarian cancer cases from Lynch syndrome families, deleterious variants in the MMR genes were mostly seen in END and CCC cases and were more prevalent in the H6 genes . This hiH6 genes ,34.Germline deleterious MMR gene variants in ovarian cancer cases appear to be very rare, which may partly be because they are associated with the rare histotypes of disease . Three sMSH6 than the other genes, with CCC and END cases both having greater than 1% frequency.We have combined the data across studies to provide an estimate of the frequency in these rarer histotypes . DeleterMLH1 carriers the OR was 3  [MSH2 carriers, OR data from different studies ranged from 2 to 14 [MSH6 carriers, ranged from 2 to 9 [Five other case-control studies have investigated MMR frequencies between 2000 and 7500 EOC cases, four of which included data from commercial laboratories where clinical grade testing was performed in women with hereditary cancer risk ,27,28,5747–6.59) , for MSH 2 to 14 ,27,28,29m 2 to 9 .MLH1 carriers by the age of 75 years, 17% (95% CI 5.7–28.0%) for MSH2 carriers, and 13% (95% CI 0.1–31.2%) for MSH6 carriers [A Lynch syndrome prospective study had estimated the cumulative risk of ovarian cancer as a unique disease to be 10% (95% CI 4.8–15.4%) for MLH1, MSH2, and MSH6 carriers, respectively [The cumulative risk of endometrial cancer by the age of 75 years was estimated to be 43% (95% CI 33.1–52.3%), 57% (95% CI 41.8–71.6%), and 46% (95% CI 27.3–65.0%) for ectively . Due to ectively .Protein truncating variants have been classified as deleterious, however a relatively large number of missense variants, including some rare predicted pathogenic missense variants have also been identified in these genes. Determining if any of these missense variants are also pathogenic is important for patient management and risk prediction in families.BRCA1/2 genes and the interpretation of the results of genetic testing for reporting and counselling [Over many years, due to efforts of commercial labs, independent research groups, and large consortia, many missense variants of unknown significance (VUS) or unclassified variants (UV) have been reclassified as either pathogenic or benign. However, there are still many variants that remain unclassified. Large-scale consortia efforts, such as the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) are in the process of investigating and classifying these variants in the nselling .BRCA1/2 genes  [To date, the National Institutes of Health (NIH) repository, ClinVar, has reported approximately 7000 pathogenic or likely pathogenic variants and 7400 VUS in iciency) . The samiciency) .BRCA1/2 and the MMR genes  and found little evidence for ovarian cancer risk association between these variants in any of the six genes [Individual studies may include analysis of missense variants using bioinformatic tools such as SIFT , PolyPheix genes .Missense variants in the moderate-risk genes recently confirmed as susceptibility genes in ovarian cancer need further investigation to determine if any are likely to be pathogenic. Some studies have included predicted deleterious missense variants in estimating mutation frequencies.BRIP1 and 26 in PALB2. They performed the RAML burden test and found an increased risk association for uncommon and rare missense variants in BRIP1, but not for PALB2 [Ramus et al. used SIFT, PolyPhen-2, and Provean scores to predict if uncommon  < 1%) and rare (MAF < 0.1%) missense variants were potentially deleterious and identified 35 for or PALB2 ,54.RAD51C/D, Song et al. performed the RAML test for 28 rare predicted deleterious missense variants (12 in RAD51C and 16 in RAD51D) [In  RAD51D) . Evidenc RAD51D)  . A case-control study was performed by Dicks et al. where FANCM targeted sequencing was performed in 3107 HGS cases, 1491 cases of other histotypes, and 3368 unaffected matched controls [p 0.008) and no evidence of association was observed with other histotypes (p 0.82) [controls . A signi(p 0.82) . The rel(p 0.82) . Lifetim(p 0.82) . Additio(p 0.82) .FANCM and moderate increase in risk of HGS ovarian cancer, larger and prospective studies are still needed to confirm this.Although this study had shown evidence of association between germline protein truncating variants in ATM is a candidate ovarian cancer susceptibility gene due to its role in breast [ATM variants are associated with fivefold higher risk of breast cancer by the age of 50 years [BRCA2 gene [n breast  and pancn breast . Germlin50 years  and someCA2 gene .ATM might be associated with moderate increased risk , howeveBARD1 and NBN genes were included in breast/ovarian cancer genetic panel tests, due to their breast cancer risk, despite the ovarian cancer risk for deleterious variants in these genes being unknown. The BARD1 encoded protein interacts closely with the BRCA1 protein due to sharing the N-terminal RING finger and the BRCA1 C-terminal domains. Interaction between these genes affects double-strand break repair and apoptosis suggesting that this protein may play an important role in BRCA1 tumour suppression [BARD1 was considered a potential candidate susceptibility gene for ovarian cancer. However, Ramus et al. found no significant differences in BARD1 deleterious variants frequency in a cohort of ~3200 ovarian cancer cases compared with ~3400 matched-controls (p 0.39) [BARD1  . Several.21–1.68 ; SSR 1.2.21–1.68 ; OR 1.4,.21–1.68 ). A mode.21–1.68 ,71.NBN encoded protein interacts with MRE11A and RAD50 encoded proteins as a large complex, which interacts with the protein produced by the ATM gene. These combined proteins have an important role in identifying broken strands of DNA and repairing it. Due to its essential function in the DNA repair pathway and because this gene is commercially available in gene testing panels for ovarian cancer, case-controls studies have examined if they are susceptibility genes. However, most studies have not found a higher frequency of pathogenic variants in NBN, MRE11A, or RAD50 in cases compared to controls  and theCHECK2 (a cell cycle checkpoint regulator) are associated with an increased risk of breast cancer [CHECK2 was proposed to be a candidate gene for ovarian cancer risk on that basis. A prospective study had shown that in estrogen receptor–positive breast cancer, CHEK2*1100delC heterozygosity was associated with increased risk of early death, breast cancer–specific death, and risk of a second breast cancer [CHECK2 and an increased risk of ovarian cancer [Germline pathogenic variants in t cancer  and CHECt cancer . Howevern cancer ,27,35.TP53 was suggested as a potential candidate susceptibility gene for ovarian cancer from a case only study [ly study  and fromly study ,57. Thisly study ,35.POLK, SLX4 , and FBXO10, but further studies are required to confirm this [FANCA, FANCB, FANCC, FANCD2, FANCE, FANCG, FANCI, and FANCL) have been tested in these case-control studies but a risk association has not been detected [Other candidate genes selected for targeted sequencing validation in cases and controls have shown weak evidence of association with ovarian cancer risk for protein truncating variants in irm this . Severaldetected . A largedetected ,57,66.We have presented a comprehensive review of the contribution of rare genetic variants to ovarian cancer, with a focus on the relationship between genetic variation and ovarian cancer histotypes, using data mostly only presented as supplementary in the original papers.BRCA1/2, deleterious germline variants are more common in HGS (8% for BRCA1 and 6% for BRCA2), but there is a significant frequency of variants in END (3% for both genes), CCC (3% for BRCA1 and 1% for BRCA2), and LGS (3% for BRCA1 and 2% for BRCA2) patients. Consequently, the current guidelines recommend BRCA1/2 testing in all non-mucinous ovarian cancer cases. For the MMR genes, the frequency of deleterious germline variants is higher in END and CCC patients, with variants in MSH6 being more common than variants in MLH1, MSH2, and PMS2 (greater than 1% in MSH6 and approximately or less than 0.5% in MLH1/MSH2/PMS2). For BRIP1, the frequency of protein truncating variants seems to be similar for HGS and END (approximately 1.2%), but slightly lower for LGS cases (0.8%). For RAD51C, a slightly higher frequency of variants in the HGS cases is observed (0.5%), and frequencies range from 0.2 to 0.4% in END, CCC, and LGS. No variants in MUC were found. In contrast, a higher frequency of protein truncating variants in END cases is seen for RAD51D (0.9%), compared to the HGS cases (0.5%). No variants were detected in CCC, LGS, or MUC. A higher frequency of protein truncating variants in CCC is seen in PALB2, compared to 0.4% in HGS and 0.2% in LGS. However, larger numbers of the non-HGS cases are needed to confirm these frequencies especially in the moderate risk genes that have been recently confirmed as susceptibility genes for ovarian cancer.For BRCA1 and BRCA2, and ongoing efforts will be required for other genes [The role of individual missense variants in the known ovarian cancer genes is not yet known. Work by the ENIGMA consortium is underway to identify deleterious missense variants in er genes .BRCA1/2 and the MMR genes [BRCA1 mutations, depending on the population [BRIP1, RAD51C, RAD51D, and PALB2 genes, and this could affect prevalence estimates. These types of changes cannot be detected with the methods currently used for targeted sequencing in clinical panel testing.The frequency of large genomic alternations  have been examined in MR genes ,73. Thespulation . We do nAs outcomes in ovarian cancer are linked to its stage at diagnosis, the ability to identify women at risk earlier or before diagnosis has important clinical implications. The use of multigene panel testing has allowed women to be tested for multiple genetic variants associated with increased cancer risk, enabling personalised risk estimates to be developed ,75. WhilBRCA1, BRCA2, or mismatch repair gene mutations, which confer lifetime ovarian cancer risk well in excess of 10% [BRIP1, RAD51C, and RAD51D may also be offered RRSO [There has been some debate as to what level of lifetime cancer risk justifies prophylactic surgical intervention. Traditionally, a threshold of greater than 10% lifetime ovarian cancer risk was used, allowing for RRSO in women carrying s of 10% . The mors of 10% . On thatred RRSO ,78.The best approach to managing patients with genetic variants that do not sufficiently increase ovarian cancer risk to justify RRSO but increase risk to higher than that of the general population is not yet known. While it is plausible that targeting these women for screening may allow for detection of cancers at an earlier stage, this has not been studied. Studies investigating the impact of ovarian cancer screening using ultrasonographic and biochemical methods in both the general population, and high-risk groups (greater than 10% risk on the basis of family history or presence of genetic variants) have been associated with stage-shift, but have not been shown to significantly reduce mortality ,79.BRCA1 and BRCA2, and colon cancer in Lynch syndrome. Furthermore, breast and ovarian cancer genes are often combined into a single panel test, potentially uncovering variants in genes for which pathogenicity is disputed. As an example, PALB2 is associated with a lifetime breast cancer risk of 53%, but also lifetime risks of 5% and 2–3% for ovarian and pancreatic malignancies, respectively [PALB2 exists in an area of clinical uncertainty. The opposite is seen for deleterious variants in BRIP1, with an increased risk of ovarian cancer but no increase in breast cancer risk. For these reasons, all women undergoing genetic testing for familial susceptibility to ovarian cancer should receive appropriate pre- and post-test counselling, and follow-up with relevant clinical services. Genetic information should be applied cautiously in the clinic, after careful evaluation of the available literature, as some genes included on so called “ovarian cancer” panel tests have now been shown not to increase ovarian cancer risk [Genetic testing is fraught with ethical, legal, and psychosocial implications for patients, and the rate of advancement in our understanding of cancer genetics often outstrips our ability to use information in the clinic ,81. It iectively . While acer risk ,83  in serous and endometrioid ovarian cancers ,90,91,92To date, ovarian cancer has largely been treated as a single entity. However, as outlined above, the five main histotypes of epithelial ovarian cancer appear to be characterised by distinct genetic mutations. The tumours also have different molecular profiles, and patients have different treatment responses . TherefoDespite significant advancements in our understanding of the genetic epidemiology of ovarian cancer, the known ovarian cancer risk variants explain less than half of the excess familial risk for ovarian cancer . EffortsBRCA1, BRCA2, and the mismatch repair genes, would provide more accurate risk estimates for moderate penetrance ovarian cancer genes, but are costly, and require long-term investment. Segregation analysis from large international consortia has been used to calculate ovarian cancer risks in RAD51C, RAD51D, and PALB2, and could be applied to other moderate risk ovarian cancer genes [Prospective population-based studies, which have been used to provide gold-standard estimates of lifetime cancer risk in er genes ,12.Genetic association studies, by their design, are unable to establish a causal relationship between germline variant status and cancer phenotype. The discovery of novel ovarian cancer risk genes should be followed-up by further efforts to characterise the functional mechanisms by which they contribute to cancer development and identify potential therapeutic targets.BRCA1, BRCA2, BRIP1, RAD51C, RAD51D, and the mismatch repair genes confer ovarian cancer risks that warrant the consideration of risk-reducing surgery. The best approach for managing women with deleterious variants in ovarian cancer genes that do not warrant prophylactic surgery requires further investigation.Ovarian cancer is the most lethal gynaecological malignancy, and an improved understanding of the contribution of rare genetic variants to the development of ovarian cancer allows for better clinical management of at-risk women. Based on the best available evidence, variants in


Current breast cancer risk models identify mostly less aggressive tumors, although only women developing fatal breast cancer will greatly benefit from early identification. Here, we evaluated the use of mammography features  as early markers of aggressive subtypes and tumor characteristics. Mammograms were retrieved from a population‐based cohort of women that were diagnosed with breast cancer from 2001 to 2008 in Stockholm‐Gotland County, Sweden. Tumor and patient characteristics were obtained from Stockholm Breast Cancer Quality Register and the Swedish Cancer Registry. Multinomial logistic regression was used to individually model each mammographic feature as a function of molecular subtypes, tumor characteristics and detection mode. A total of 4546 women with invasive breast cancer were included in the study. Women with microcalcification clusters in the affected breast were more likely to have human epidermal growth factor receptor 2 subtype  and potentially less likely to have basal subtype  compared to Luminal A subtype. High mammographic cBIRADS showed association with larger tumor size and interval vs screen‐detected cancers. Lack of density reduction was associated with interval vs screen‐detected cancers  and potentially of Luminal B subtype vs Luminal A subtype . In conclusion, microcalcification clusters, cBIRADS density and lack of breast density reduction could serve as early markers of particular subtypes and tumor characteristics of breast cancer. This information has the potential to be integrated into risk models to identify women at risk for developing aggressive breast cancer in need of supplemental screening. Current breast cancer risk models do not specifically identify women at risk of aggressive breast cancer. Using mammograms from a population‐based cohort of women diagnosed with invasive breast cancer, this study found that women with microcalcification clusters in the affected breast were more likely to have the HER‐2 subtype and potentially less likely to have the basal subtype compared to Luminal‐A subtype. Women with no breast density reduction had increased probability of interval versus screen‐detected cancer and possibly of Luminal‐B versus Luminal‐A subtype. These mammography features could potentially serve as early markers for aggressive breast cancer subtypes and tumor characteristics. BIRADSBreast Imaging Reporting and Data SystemBMIbody mass indexcBIRADScomputer‐generated Breast Imaging Reporting and Data SystemCIconfidence intervalsERestrogen receptorFISHfluorescence in situ hybridizationHER‐2human epidermal growth factor receptor 2HRThormone replacement therapyORodds ratioPPvaluePRprogesterone receptor1Breast cancer affects around 2 million individuals a year globally.To date, there remains a lack of early markers for potentially aggressive breast cancer from routine mammograms. Microcalcifications commonly found on mammographic screenings are routinely used for diagnosis of early breast cancer and in particular ductal carcinoma in situ.Furthermore, while earlier studies have found that mammographic density could be associated with aggressive breast cancer subtypes and tumor characteristics including interval breast cancer,In our study, we evaluated the associations of mammographic features  were sent invitations to participate in the LIBRO‐1 population cohort study. A total of 5715 women (61%) consented to participate—they provided blood, answered detailed questionnaire on lifestyle including hormonal and reproductive factors and consented to retrieval of mammography images. Detailed information on the cohort has been published previously.2.2Mammograms, both analogue and digital, were retrieved from Departments of Radiology and information on mammography screening history were retrieved from the Stockholm‐Gotland Regional Cancer Center mammography screening database.2.3We used a method developed in our group for the detection of microcalcification clusters that can be applied on different digital systems and vendors, enabling incorporation of both analogue and digital images for large population studies.2.4Percentage mammographic density was calculated using the area‐based STRATUS algorithm, which has been developed to analyze a range of image formats, including both analogue and digital images, with automation of density change measurements over time was used.In the statistical analysis of density, percent density measurements of the contralateral side to the breast cancer were used to ensure that the tumors did not affect image measurements. Examination closest to date of diagnosis was used. After our earlier study that showed increased probability of interval cancer with high mammographic density,d1 denotes area density at first mammography t1, d2 denotes area density at last mammography t2, and t are times on a yearly unit scale.For density change analysis, relative annual density area change (RDC) on the contralateral breast was computed by taking the difference in area density between two time points (defined as first and last mammography prediagnosis) per baseline density of each women using the equation2.5Tumor and patient characteristics were retrieved from linkages to Stockholm Breast Cancer Quality Registers and the Swedish Cancer Registry. Estrogen receptor (ER) and progesterone receptor (PR) status were determined by immunohistochemistry (IHC) or radioimmunoassay methods and categorized as positive or negative. HER‐2 status was determined by IHC/fluorescence in situ hybridization (FISH) and categorized as positive or negative.For classification of molecular subtypes, additional data on ER, PR, HER2 and Ki‐67 were obtained from medical and pathology records.Screening history from the mammography‐screening database at the Stockholm‐Gotland Regional Cancer Center was used to determine detection mode.2.6Multinomial logistic regression was used to individually model molecular subtype, tumor characteristics and mode of detection as a function of each mammographic feature, microcalcification clusters and mammographic density change. Microcalcification clusters were evaluated as a categorical covariate in terms of the presence vs absence of clusters in the cancerous breast and contralateral unaffected breast (separately). In addition, in women with available images from both breasts, we compared the number of microcalcification clusters between the affected and the unaffected sides by taking differences. We categorized the differences into two levels: higher number of microcalcification clusters in the affected breast than the unaffected breast (positive difference), no difference or fewer microcalcification clusters in the affected breast (no positive difference). Mammographic density was evaluated comparing cBIRADS B, C and D vs cBIRADS A. Density area change was evaluated in terms of no density reduction vs density reduction, with reduction defined as negative change in relative annual density area.For all analyses, models were adjusted for age at diagnosis, body mass index (BMI), hormone replacement therapy (HRT) use and postmenopausal status. For density and density change analyses, models were additionally adjusted for age at mammography. For density change analysis, the model was even adjusted for baseline density. Statistical analyses were performed using R 3.5.1.3A total of 4546 women with invasive breast cancer satisfying inclusion criteria were included in the study. The majority of women were aged 50 or more (79%) and postmenopausal (80%). For 3361 and 3303 women, images on the affected and unaffected side, respectively, were able to be retrieved from radiology departments for microcalcification analysis. Of which, 3036 women had images on the contralateral side for cBIRADS3.1Microcalcification clusters were present in the affected breast for 35% of the women and in the unaffected side for 26% of the women. Results indicated that women with microcalcification clusters in the affected breast were more likely to have a HER‐2 subtype breast cancer OR 1.78 (95% CI 1.24‐2.54) and possibly had a reduced probability of basal subtype OR 0.54 (95% CI 0.30‐0.96) compared to Luminal A subtype . We found a significant positive association between this difference and HER‐2 subtype  and a negative association with basal subtype  compared to the Luminal A subtype. Women with positive differences were less likely to be diagnosed as interval cancer compared to screen‐detected cancer . Detailed results are presented in Table 3.2Based on cBIRADS categories of the contralateral breast, the percentages of women were 9%, 38%, 47% and 6% in categories A, B, C and D, respectively. No significant associations between molecular subtypes and cBIRADS categories were found. Larger tumors were associated with higher density cBIRADS B, C and D, and interval vs screen‐detected cancers were associated with cBIRADS C and D compared to cBIRADS A. Detailed results are presented in Table 3.336% of the women considered in this analysis showed no reduction in relative density area. Results suggested that the lack of relative density area reduction over time was associated with Luminal B subtype vs Luminal A subtype OR 1.76 (95% CI 1.04‐2.99) . Prior studies in the literature have reported that mammographic feature asymmetry between breasts predicts individual near‐term breast cancer risk on the next sequential screening mammogram.5Mammographic features  could potentially be used as early markers to identify women at increased risk of developing aggressive breast tumors. Current breast cancer risk models identify women who will be at risk for breast cancer,PST consulted for AstraZeneca and Duke‐NUS. Other authors declare no potential conflicts of interest.All study participants gave informed consent and the study was approved by the Regional Ethical Review Board in Stockholm, Sweden .


The rates of shift work outside of daylight hours have increased in recent years, and nighttime shift work is now considered a potential carcinogenic occupational exposure. Light at night exposure, lower melatonin production, and the production of stress-related mediators disrupt normal sleep–wake cycles. Women who work lower-wage jobs and part-time workers whose shifts are determined entirely by their supervisors (rotating shifts) may be subject to stress related to efforts to align childcare and other needs with the unpredictable nature of rotating shift work. The causal link between breast cancer and the sleep cycle or circadian disruption are yet to be established; however, disruption of the circadian cycles by light at night exposure or chronic exposure to stress-related mediators have all been linked to the increased risk of breast cancer. We review the existing literature on shift work and breast cancer, identify knowledge gaps, and suggest future directions for research. Rates of shift work outside of daylight hours have increased in recent years, and nighttime shift work is now considered a potential carcinogenic occupational exposure as a result of decreased melatonin levels caused by light at night exposure ,2. In EuThe causal link between breast cancer and nighttime shift work or shift work rotating between day and night has been suggested, but has not yet been established . In vitrA separate line of research from a study at the National Toxicology Board, that focused on carcinogens in flight attendants and nurses, suggested an increased risk from night work .Our review of evidence for a link between shift work and breast cancer revels several gaps in the literature. Whereas both epidemiological and murine model studies suggest several routes by which work at night might predispose a woman to breast cancer, there is a lack of studies that follow changes in women who work primarily at night, including part- and full-time workers who have little or no control over the scheduling of their shifts, and the development of breast cancer. Likewise, the work linking carcinogens in nurses and flight attendants who work at night remains nascent.While many authors have posited a link between shift work and breast cancer and a separate body of investigation has looked at the psychological, behavioral, and broad health effects of shift work on women, the two bodies of research have failed to overlap over three decades of inquiry. Another important consideration is that breast cancer mortality is much higher among underrepresented minority and lower socioeconomic status women globally; women who are less likely to control the timing of their shifts. In the United States, for example, black women are 40% more likely to die from breast cancer than white women, although they are no more likely to develop the disease . LikewisNumerous epidemiological studies have suggested that night shift work may increase breast cancer risk, although few of these largely observational studies have clearly documented the necessary disruption of the circadian rhythm that is required by the International Agency for Cancer Research designation of shift work as a category 2a carcinogen. Lastly, it is also important to understand how social factors such as socioeconomic status and gender interact with shift work to contribute to breast cancer disparities.In 2007, the International Agency for Cancer Research (IARC) Monograph working group listed shift work that involves circadian disruption as a class 2a or probable carcinogen. This designation put shift work in the same category as anabolic steroids, nitrogen mustard, vinyl fluoride, and 62 other agents, considering them to be class 2a carcinogens . The 201Workers who are subject to shift rotations can present a more complex model for understanding the health impact of light at night on the sleep–wake cycle and melatonin production. In an attempt to understand the effect of shift rotations, light exposure, and melatonin levels, Dumont and Lanctôt measured the 24-h urinary 6-sulfatoxymelatonin (aMT6s) and ambulatory light exposure during night shift and day/evening shift periods of 13 full-time rotating shift workers . The autThe circadian timing mechanism in mammals is controlled by the suprachiasmatic nuclei. Filipski et al. created a mouse model to evaluate the effect of severe circadian dysfunction (ablation of the SCN) on tumor progression in mice with implants of Glasgow osteosarcoma and pancreatic adenocarcinoma. Both tumor types grew 2–3 times faster in mice with SCN lesions than in mice who underwent sham surgery. The survival of the mice with SCN lesions was significantly shorter than that of mice who underwent sham surgery. This led the authors to conclude that when the circadian rhythms of mice are disrupted, the growth of malignant tumors of two types is accelerated. This suggests that a host’s circadian clock may have a role in the endogenous control of tumor progression .PER3 was significantly associated with increased risk of breast cancer and posited it as a potential biomarker for breast cancer [NPAS2, CRY2 and CLOCK and breast cancer risk [Stevens and Zhu estimated that 2–10% of all mammalian genes are clock-controlled, which points to extensive circadian gene regulation . Just ast cancer . This cicer risk ,23,24,25An association between breast cancer and night work has been suggested by the authors of cohort studies, case-control studies, and meta-analyses . CausatiThe Breast Cancer Now Generations Study, which followed 113,000 women over a forty-year period, reported their findings on light at night exposure and breast cancer. When participants entered the study, from 2003 onwards, their exposure to light at night and sleeping patterns were established through questionnaires, classifying light levels in their bedroom as low , medium  or high . The study concluded that exposure to light at night does not increase breast cancer risk .However, no dose–response relationship emerged among workers exposed to fewer than 20 years of night work . Lee et Wegrzyn et al., using data from the Nurses’ Health Study I and II to examine associations between rotating night shift work and breast cancer risk, found a total of 9541 incident invasive breast malignancies over 24 years of follow-up . Women iIn summary, although numerous epidemiologic studies, including cohort studies, case-control studies, and meta-analysis, have reported a positive association between light at night exposure and breast cancer, other studies report no effect. In addition to the need to create coherence between study subpopulations and standardizing study methodology, the single biggest challenge may be the need to more precisely measure the actual disruption of the circadian rhythm and reflect that disruption against health outcome(s) in large cohorts.There remain significant observational epidemiologic data, biological (melatonin levels) and genetic  studies that support the theory that light at night disrupts the circadian rhythm in women and predisposes them to a higher risk of breast cancer.Although the causal link between shift work at night and increased risk of breast cancer is yet to be established, social factors such as socioeconomic status can contribute to breast cancer disparities. Coughlin completed an extensive literature review of the social determinants of breast cancer and found convincing evidence for a number of contributors to risk and survival . Census-Low socioeconomic status has also been associated with shift work. The report on the economics of workplace flexibility by the Council of Economic Advisors of the Executive Office of the President of the United States concluded that less skilled workers have less flexibility in terms of the scheduling of when they work their shifts than do more highly skilled workers . Less skIn July 2013, the Urban Institute released a report titled, “Nonstandard Work Schedules and the Well-Being of Low-Income Families” that described occupations and industries with the highest share of workers with non-standard schedules . OccupatSchedule unpredictability occurs when the hours or days of work are controlled solely by employers without input from workers. Schedules that vary from day to day or from week to week  and scheWhile men are only somewhat more likely than women to have flexible schedules , McMenamWomen who work inflexible shifts are more likely to also assume primary child care duties, which is another major source of physiological stress. Lambert, Fugiel, and Henly (2014) found that 45% of mothers with children below the age of 12 years reported that their employers decided their schedule without their input and concluded that women are disproportionately affected by the necessity to arrange childcare .In a seminal work on shift work and health, Finn was among the first to suggest that unpredictable shift work that is out of the control of workers interrupts endocrine and other physiological processes, and that rhythmic adjustments to new work schedules take days to weeks to occur. Consequently, the body is in a constant state of adjustment, with both physical and emotional consequences . This alThe physiological stress produced in low-wage workers by unpredictable work schedules and the work-life conflict that it produces  suggests another route to breast cancer beyond that which comes from light exposure during night shifts. Linnenbringer, Gehlert, and Geronimus and others posit a physiological route through repeated demands on stress hormone system and the ultimate physiological “weathering” that it brings ,44. ThesThe first direction for research suggested by this review is determining whether breast cancer risk is higher among low-wage hourly employees with histories of unpredictable shifts . This work will likely require the use of existing large datasets and secondary data analysis. Breast cancer data might come from state cancer registries. Employment data might come from the O*NET database, sponsored by the US Department of Labor and the Employment and Training Administration, contains detailed information on almost 1000 occupations. Workers’ responses to survey questions are used to create measures capturing a multitude of different aspects of an occupation, e.g., use of technology, physical demands, customer interaction, skills required—and many, many more. Researchers can add these measures to any dataset by matching on SOC  codes, which are commonly used to classify jobs. This allows researchers to incorporate different aspects of job quality into the inquiry. Data bases in other countries can similarly be combined.Should the link that is suggested by prior research be established, our review highlights the need for intervention(s) to address the known risk(s) associated with night shift work and test for intervention feasibility and acceptability. This likely will require original data collection and may require qualitative and quantitative approaches.Although no direct association has been established between shift work and breast cancer risk, mounting evidence suggests that shift work contributes to breast cancer risk. This evidence comes from studies that use a variety of methods, from epidemiologic studies like the Nurse’s Health Study I and II, murine studies, and environmental studies converge to establish the need for further work in the area. Likewise, additional study is needed on how stress from challenges to lower wage workers’ work and life balance might further contribute to breast cancer risk is warranted.


Introduction. Male breast cancer (MBC) is a rare disease, whose main risk factor is genetic vulnerability. Despite care of men with MBC is modeled on care of women, men's experiences with the disease and concerns related to the status of genetic mutation carrier are unique. So far, little is known concerning the psychological impact in BRCA1/2 testing, especially with regard to specific subset of individuals, such as male subjects and the elderly. n = 7; high-risk unaffected subjects, n = 7; high-risk unaffected subjects,  We assessed self-reported anxiety and depression levels in 26 male subjects presenting at Unit of Breast Surgery in Breast Unit of AOUI Verona  received a positive test (either BRCA1 or BRCA2 pathogenic variant) and 10 (59%) a negative test. Of the 9 MBC patients tested, only one subject received a positive test result. No significant differences were observed in mean scores, mean change from baseline to follow-up, either for those with  Male breast cancer (MBC) is a rare disease, comprising only 0.1% of all men cancers , while fMBC tends to be diagnosed in later life, at a more advanced stage, and is considered similar to late-onset  FBC . GeneticDespite care of men with MBC is labeled on women's care, men's experiences with the disease and concerns related to the status of genetic mutation carrier are unique , 5.Several studies focused on the short- and long-term psychological impacts of breast cancer on women  and otheSince the introduction in clinical practice of genetic testing for BRCA1/2 gene mutations, there has been a rising interest in eventual psychological distress caused by testing . So far,Considering the gradual increase in MBC incidence , 2, it sThe aim of this retrospective study is to assess self-reported anxiety and depression levels in male subjects presenting at Unit of Breast Surgery in Breast Unit of AOUI Verona. We specifically examined the scores obtained by these subjects in the HADS questionnaire administered before the genetic testing for BRCA gene mutations and during a 6-month follow-up visit.This is a retrospective analysis of all male patients presenting with MBC and high-risk not-affected men, between 01/11/2015 and 31/01/2018, at the Unit of Breast Surgery at Verona AOUI. In our retrospective study were enrolled male subjects with a personal or family history of MBC, male subjects with a family member carrier of BRCA1/2 gene mutation, male subjects with a close family member diagnosed with female breast cancer (FBC) at 45 or younger, male subjects with a close family member diagnosed with bilateral breast cancer at any age, male subjects with three or more close family members diagnosed with breast cancer at any age, and male subjects with a close family member diagnosed with pancreatic cancer or metastatic PCa at any age or family history of ovarian cancer, pancreatic cancer, aggressive PCa, or metastatic PCa. Female subjects, male subjects affected by benign breast tumors, and healthy men with a family history not suggestive of genetic mutations linked to breast cancer were excluded.Clinicopathological, psychological, and genetic data were obtained from Breast Unit Database , which collects patients' and high-risk subjects' clinical records since January 1992. No additional tests were performed.We registered male subjects' following data: current age, age at breast cancer diagnosis, breast cancer stage and treatment, family history of breast, ovarian, or prostate cancer in firstdegree relatives and BRCA 1/2 mutation status (if available), and psychological distress . For MBC patients, we also recorded alcohol consumption and smoking habits, comorbidities, and history of psychological symptoms.Patients diagnosed with MBC and high-risk not-affected men were candidated to be examined by an expert geneticist, specifically dedicated to the study of hereditary breast, ovarian, and prostate cancer. During ambulatory interview, the geneticist drawn genealogical family trees based on participants' family history. The BRCA test was performed through a blood sample collected at the Brest Unit and sent to the Medical Laboratory Department, where it was validated by fluorimetry technique and multiplex polymerase chain reaction (PCR). BRCA1 and BRCA2 mutations were classified according to their potential functional effect as recorded in the Breast Cancer Information Core (BIC) database  as classParticipants' psychological distress was assessed through the HADS at the time of testing for BRCA1/2 gene mutation and during a 6-month follow-up visit . This isAll collected data were recorded in a Microsoft Office Excel spreadsheet.t-tests for independent and paired samples. Nonparametric tests were used when appropriate due to skewed distributions or low patient numbers. All statistical analyses were performed with IBM SPSS 10.0.Continuous variables are expressed as mean and standard deviation. When comparing two groups with normal distributions, we applied We collected data on 26 male subjects, who presented at our center between November 2015 and January 2018, were eligible for BRCA testing and received either a positive result (carriers) or a negative result (noncarriers).The mean age of participants was 58 years (SD = 12), seven subjects were >65 years of age. Thirty-five per cent of the subjects tested had been treated for MBC, while sixty-five per cent were unaffected subjects with strong family history for breast cancer; among them, 5 had other cancer than MBC (prostate cancer).All the nine subjects affected by MBC underwent modified radical mastectomy, before genetic testing. Among those with MBC, 3 subjects were diagnosed with early-stage MBC (Ia-Ib) and were administered ormonotherapy and 6 subjects were administered chemotherapy plus ormonotherapy due to advanced cancer stage at diagnosis.From January 2015 to January 2018, 26 men (7 MBC patients and 19 high-risk not-affected men) were engaged in genetic counseling and testing at our center and completed the HADS questionnaire in paper format prior to genetic testing and during a 6-month follow-up visit. Baseline characteristics of participants are shown in Between those with available data, namely, MBC patients, only one subject reported history of psychological symptoms. Most of the MBC patients (66%) had significant medical issues, such as hypertension, diabetes mellitus, or other cardiovascular diseases. Participants received genetic testing results between 2 and 4 weeks after completion of baseline assessment and were engaged in a 6-month follow-up visit. 8 subjects (30.8%) were found to be carriers and 18 noncarriers of BRCA1/2 gene mutation. Among the 17 unaffected men tested, 7 (41%) received a positive test (either BRCA1 or BRCA2 pathogenic variant) and 10 (59%) a negative test. Of the 9 MBC patients tested, only one subject received a positive test result.Self-rated levels of anxiety and depression were recorded with the HADS in 19 subjects, both prior to genetic testing and during follow-up visit 6 months after carrier status disclosure. 7 subjects failed to complete the HADS questionnaire after receiving genetic testing results, so only baseline assessment data were available.The affected and unaffected groups were divided into two subgroups (T+ and T−) according to their test results. No significant differences were observed in mean scores, mean change from baseline to follow-up, either for those with T+ or T− test results. Because of this lack of significant differences, only the findings on psychological distress for the unaffected group are shown .P=0.06), after receiving a negative test.In our sample, 5 men (19%) were scored as possible cases of anxiety disorder at baseline. These subjects obtained a nearly significant decrease in the anxiety score from 9 (SD 1.2) to 4.4 (SD 3.8)  –16. AccoThe finding that nearly 31% of participants received a positive test is not surprising because we specifically selected for oncogenetic counselling high-risk subjects, following the ASCO recommendations mentioned above . In thisNumerous studies claim that, although to a lesser extent than women, men also expresse a certain degree of psychological distress linked to neoplastic disease . Indeed,Both men with and without a personal history of MBC showed stable levels of depression and anxiety, measured by the HADS. These scores were either lower than or comparable to those of normative samples . The finWe found no significant variation in HADS-A and HADS-D scores from baseline to follow-up in men without cancer receiving either positive or negative BRCA1/2 test results. This finding seems to support the hypothesis that, in this population, test results do not influence the level of distress to any significant degree in short-term evaluation. This result is concordant with that of Reichelt et al. , who sugOur results support the main conclusions of other studies carried out in women, namely, that no adverse psychological consequences seem to arise from genetic testing for BRCA1/2 mutation , 22. ThiThis study has several limitations. Firstly, the small sample size does not allow an absolute generalization of the observed results. Secondly, the absence of short-term increase in anxiety and depression levels does not guarantee the absence of deterioration in long-term psychological conditions. Lastly, we believe that psychological evaluation in a genetic-testing setting is a complex topic, which should be carried out using diversified and numerous tools to grasp the facets of individual experiences.Further investigations are clearly needed in order to deepen knowledge upon psychological implication of genetic testing in male suffering from and at high-risk for MBC. We intend to expand the experience of our center by implementing the use of quantitative methods with focus groups and with the involvement of family members in psychological support interviews.