They are hypothesized to play a role in multiple physiologic processes and pathologies, including breast cancer development and progression.We evaluated circulating 27-HC and 25-HC, and outcomes following a breast cancer diagnosis in 2282 women from the MARIE study over median follow-up of 11.6 years. 27-HC and 25-HC were quantified by liquid chromatography–mass spectrometry. We calculated hazard ratios (HR) and 95% confidence intervals [CI] using multivariable Cox Proportional Hazards regression.log2 = 1.80 [1.20–2.71]) and breast cancer-specific mortality (HRlog2 = 1.95 [1.14-3.31]). No associations were observed in women with estradiol levels above the median. Higher 25-HC levels were associated with lower risk of recurrence (HRlog2 = 0.87 [0.77-0.98]).We observed no associations between 27-HC and breast cancer prognosis overall. Associations between 27-HC and survival differed by circulating estradiol concentrations and endocrine therapy, but not by hormone receptor status. Among women with estradiol levels below the median (0.08 nM), 27-HC was associated with higher risk of all-cause mortality (HRAssociations between 27-HC and breast cancer prognosis varied by circulating estradiol levels and endocrine therapy. Less consistent results were observed for 25-HC. Breast cancer is the most commonly diagnosed cancer in women and the leading cause of cancer death in women worldwide . Despiten = 58) [Previous research suggests that higher concentrations of circulating pre-diagnosis 27-HC are associated with lower risk of postmenopausal breast cancer . To our n = 58) , while sn = 58) , 11–13. n = 58) , 13, andn = 58) .Following the experimental evidence on oxysterols in breast cancer and the inconsistent findings in breast cancer patients, we aimed to investigate the association between circulating 27-HC, 25-HC, and breast cancer prognosis in a large breast cancer cohort to contribute to better understanding the link between cholesterol metabolism and breast cancer survival following a breast cancer diagnosis. Due to hypothesized differences in associations by estradiol concentrations and endocrine therapy, we investigated the associations between 27-HC, 25-HC, and breast cancer prognosis by circulating estradiol levels and exogenous SERM and aromatase inhibitor (AI) use.We conducted this study in the Mammary Carcinoma Risk factor Investigation (MARIE), a large population-based patient cohort. Details of the study have been published previously . In brieDue to the hypothesized role of 27-HC as an endogenous SERM, we evaluated associations of oxysterols with breast cancer prognosis by treatment-related endocrine therapy use. Use of these medications was assessed via questionnaires during follow-up (FU) 1 and FU2 for the preceding time interval . Overall, a total of 97% of participants provided information on ever SERM use (yes/no), and 96% of participants provided information on ever AI use (yes/no). If self-reported information on endocrine therapy was not available, data from medical records were used. Examples on SERMs and AIs, as listed in the questionnaires, are included in the supplements.Follow-up information from participants was obtained by telephone in 2009 (FU1) and in 2015 (FU2). Information on recurrences, second cancers, and metastatic events were confirmed via medical records or through contact with the treating physicians. Vital status was retrieved through population registries of the study regions up to the end of June 2015, and copies of the death certificates were requested from local health offices. Cause of death was coded according to the 10th revision of the International Classification of Diseases (ICD-10-WHO). The primary outcomes for this study are all-cause mortality, breast cancer (BC)-specific mortality, and risk of recurrence. In the statistical analyses, all-cause mortality was attributed to death by any cause, and BC-specific mortality was attributed to breast cancer (ICD-10-C50) with deaths from other causes censored at date of death. Risk of recurrence in this study corresponds to the outcome “recurrence-free interval” as described in the STEEP criteria  includinBlood samples collected at recruitment have been stored at –80 °C. Median time between diagnosis and blood collection was 3.8 months . Oxysterol concentrations were measured by blinded personnel in 2,282 blood samples , and samples were distributed randomly across study batches. 27-HC (systematic name (25 R),26-hydroxycholesterol) and 25-HC were quantified by Biocrates LifeSciences  using a UHPLC-MS/MS with multiple reaction monitoring (MRM) in positive mode using a mass spectrometer with electrospray ionization (ESI). The assay has been validated according to European Medicines Agency (EMA) guidelines. Inter-batch coefficients of variation (CV) were assessed by including 16 blinded replicate quality controls. Mean inter-batch CVs were 15.8% for 27-HC and 29.2% for 25-HC, however 25-HC levels were generally lower in the quality control samples (mean: 12.75 nM) than in the study population (mean: 29.91 nM). Estradiol concentrations were measured using an ELISA in the Division of Cancer Epidemiology laboratory at the German Cancer Research Center (DKFZ) with an inter-batch CV of 16.2%. The ELISA had a sensitivity of 0.03 nM and a standard range of 0.07–7.34 nM.2-transformed to normalize the distributions and to allow estimation of the effect of a doubling in concentrations. We detected one outlier for 27-HC and 18 outliers for 25-HC using the Generalized ESD Many-Outlier Procedure [Biomarker concentrations were logrocedure , and con2-transformed values. Tests for trend were calculated modeling the median of the biomarker quartiles as a continuous variable. We used date of diagnosis as start date for follow-up time (time-to-event) and date of blood draw as start date for participants at risk (time-at-risk). Follow-up time ended at the date of the defined outcome  or at the date of last contact or end of follow-up 2 , whichever came first. In further analyses, we used a competing-risk model for BC-specific survival; results were similar to those from the models not considering other cause of death as a competing risk. The reverse Kaplan-Meier method was utilized to estimate the median follow-up time for the entire duration of follow-up. We tested the proportional hazards assumption using Schoenfeld residuals and did not observe violation of the proportional hazards assumption.We used delayed-entry Cox proportional hazards regression to estimate hazard ratios (HR) and 95% confidence intervals (CI) for risk of all-cause mortality, BC-specific mortality, and risk of recurrence. HR were calculated using biomarker quartiles (higher vs. lowest quartile) and continuous logEstablished prognostic factors were selected a priori as potential covariates for the survival model: tumor size , nodal status , and tumor grade . All Cox proportional hazard models were additionally adjusted for age at diagnosis, BMI, smoking status , alcohol consumption , and Charlson Comorbidity Index (CCI), and stratified by ER/ progesterone receptor (PR)-status , and study region . Multicollinearity of covariates was low (variance inflation <1.2). Study participants with missing information for covariates were excluded from the survival analyses (n participants = 17). Endocrine therapy and chemotherapy were not included in the model due to their strong association with hormone-receptor status and tumor characteristics.Non-parametric restricted cubic splines were used to examine possible non-linearity, comparing models with linear and cubic terms to models with only the linear term . There wn = 22), women who had recurrences within one year after blood draw (n = 58), and women whose blood had been collected before breast cancer diagnosis . Our primary analyses included the full follow-up period of the cohort. As a separate analysis, we conducted 5-year survival analyses.We evaluated cross-classification of 25-HC and 27-HC at the median concentration using high 27-HC/ low 25-HC as reference due to the observed differential effects of these oxysterols. In sensitivity analyses, we excluded women who died within one year after blood draw .All statistical tests were two-tailed and considered significant at 2 (range: 15.5–49.6 km/m2) (Table Median age at diagnosis of study participants was 63 years (range: 50–75 years) and median BMI was 25.3 km/mThe median follow-up time for the cohort was 11.6 years. Median concentration of 27-HC was 210.0 nM, median concentration of 25-HC was 20.4 nM, and median concentration of estradiol was 0.08 nM (corresponding to 22.2 ng/ml). We observed weak correlations between 27-HC and 25-HC (r = 0.32), and between estradiol and 27-HC and 25-HC (r < |0.1|) (data not shown). Storage time and BMI were weakly correlated with oxysterol concentration (r < |0.16|) (data not shown).het = 0.76; BC-death, phet = 0.95; recurrences, phet = 0.55).We observed no association between circulating 27-HC and breast cancer outcomes in the overall cohort (Table het = 0.02) and BC-specific death (phet = 0.05) by circulating estradiol levels (below vs. above median). Among women in the low estradiol subgroup, a doubling of 27-HC concentration was associated with higher risk of all-cause death (HRlog2 = 1.80 (1.20–2.71), ptrend = 0.02) and BC-specific death (HRlog2 = 1.95 (1.14–3.31), ptrend = 0.04), whereas no significant associations were observed among women in the high estradiol subgroup.We observed heterogeneity in associations between 27-HC and all-cause death (plog2 = 2.42 (1.11–5.28)); when further stratifying non-endocrine therapy users, the effect was only observed in hormone-receptor negative participants ; BC-specific death, HRlog2 = 3.96 (1.23–12.77); recurrences, HRlog2 = 3.50 (1.42–8.62)), and in low-estradiol participants ; BC-specific death, HRlog2 = 5.27 (1.19–23.25); recurrences, HRlog2 = 3.82 (1.25–11.64)). Furthermore, higher 27-HC levels were associated with a poorer prognosis among SERM users ; recurrences, HRlog2 = 2.30 (1.28-4.14)), while no significant association was observed among AI users.We next investigated the association between 27-HC and breast cancer prognosis in subgroups stratified by endocrine therapy Table . Among tlog2 = 0.87 (0.77–0.98), ptrend = 0.19) in the overall cohort . In participants with low estradiol levels, 25-HC in the highest quartile was associated with a higher risk of BC-specific death (HRQ4vs.Q1 = 1.67 (1.00–2.78)) as compared to the lowest quartile, while a better prognosis was observed in participants with higher estradiol levels (HRQ4vs.Q1 = 0.57 (0.32–1.00)).We observed no heterogeneity in associations by hormone receptor status, but by circulating estradiol levels ), and among those participant who were using both SERM and AI (HRlog2 = 0.76 (0.61–0.95). No associations were observed for AI users or non-endocrine therapy users. However, in participants not using endocrine therapy, we observed significant interactions in associations by estradiol levels , ptrend = 0.03; higher estradiol levels HRlog2 = 0.50 (0.27–0.95), ptrend = 0.07).In subgroup analyses stratified by endocrine therapy Table , we obseAdditional adjustment for estradiol concentration and biomarker concentration , mode of detection, chemotherapy, and endocrine therapy (SERM and AI) had minimal impact on the effect estimates.In exploratory analyses of 27-HC and 25-HC cross-classified, we observed that participants with high 27-HC/low 25-HC levels had a significantly higher risk of recurrences as compared to participants with low 27-HC/high 25-HC (HR = 1.65 (1.11–2.46)) Table . No statThe associations for the 5-year survival analysis between 27-HC, 25-HC, and breast cancer prognosis were similar to the associations of the main analyses Table .Excluding participants with recurrences within one year after blood draw attenuated the association between 27-HC and risk of recurrence (from HR = 1.30 (0.94–1.74) to HR = 1.16 (0.84–1.62)). Exclusion of outliers, women who died within one year after blood draw and women whose blood had been collection before breast cancer diagnosis, did not meaningfully change the risk estimates for any of the outcomes (<10%) (data not shown).We assessed associations between 27-HC, 25-HC, and survival after a breast cancer diagnosis in a well-characterized cohort of 2282 breast cancer patients, observing no association between 27-HC and breast cancer survival overall, while higher levels of 25-HC were associated with a lower risk of recurrence. Higher levels of 27-HC were associated with a poorer prognosis among participants with relatively low estradiol levels and among participants not using endocrine therapy. The findings of the main analyses were supported by the 5-year survival analyses reporting similar effect estimates. Our study aims were based on strong experimental data for 27-HC and breast cancer development and progression and a previous epidemiological study associating 27-HC with breast cancer risk. We investigated the outcomes of all-cause mortality and BC-specific mortality given that (1) 27-HC is suggested to have a role in multiple physiologic processes and (2), 27-HC binds to the estrogen receptor in tissues beyond breast cancer; evaluations stratified by estradiol were conducted given previous experimental and other epidemiological studies suggesting actions of oxysterols may depend on the background estrogenic environment.i) at 1.32 µM (=1320 nM) for the ERα and 0.42 µM (= 420 nM) for ERβ [d) of 0.1 nM (ERα) and 0.4 nM (ERβ) for estradiol [50 = 1 µM) [27-HC is the most extensively studied oxysterol with respect to breast cancer. It was identified as the first endogenous SERM, exhibiting agonistic or antagonistic effects depending on the target tissue . 25-HC, stradiol . Using a = 1 µM) . As circ = 1 µM)  of 27-HCn = 58 breast cancer patients), and reported, similar to our overall findings, no significant association between plasma levels of 27-HC, 25-HC, and disease-free survival [r = 0.63) and free and total 25-HC (r = 0.54) are modestly correlated [Q4vsQ1 = 0.56 (0.36–0.87)) [protein = 0.42 (0.21–0.84); HRmRNA = 0.38 (0.18–0.78)) [Clinical and epidemiologic studies on circulating oxysterols and breast cancer progression are sparse. To our knowledge, only one study directly assessed circulating free oxysterols ) . Larger 8–0.78))  and bett8–0.78)) , while a8–0.78)) . No sign8–0.78)) . CYP7B1 8–0.78))  and a be8–0.78))  or presu8–0.78)) . Further8–0.78)) . The finWe observed no heterogeneity in associations by hormone-receptor status. Beyond the ER-mediated pathway, another pathway linking oxysterols to breast cancer progression includes the liver X receptor (LXR), primarily expressed in liver, intestine, adipocytes, and macrophages with various functions including the regulation of the cholesterol metabolism. Oxysterols including 27-HC and 25-HC, but with lower potency, were reported to be the main ligands to the LXR , 25. It In further subgroup analyses, we assessed associations between 27-HC, 25-HC, and breast cancer prognosis by endocrine therapy use due to a potential interaction between the endogenous ER modulators 27-HC and 25-HC and endocrine therapy. SERMs are synthetic drugs that bind to the ER and exert estrogenic or anti-estrogenic effects depending on the target tissues. AI, mainly used in postmenopausal patients, block aromatase action, the enzyme synthesizing estrogen, resulting in lower systemic estrogen levels. While higher levels of 27-HC were generally associated with a poorer prognosis among SERM users, 25-HC levels were associated with a better prognosis in SERM users. Furthermore, we observed effect heterogeneity by estradiol levels among the no-endocrine therapy participants with a poorer prognosis in the low-estradiol subgroup and a better prognosis in the higher estradiol subgroup for higher 27-HC and 25-HC levels.The study of Dalenc et al. reported increased 27-HC levels after AI treatment and decreased 25-HC levels after tamoxifen treatment over 28 days, however the magnitude of association was not fully described . We straLastly, it needs to be noted that 27-HC plays a role in a number of other mechanisms including: reduction of P53 transcriptional activity , productp trend = 0.08) [r = 0.43) [We provide novel data on the association between circulating levels of the structurally similar oxysterols 27-HC, 25-HC, and breast cancer prognosis in a study with detailed data on disease characteristics, treatment characteristics and outcomes. Importantly, we were able to account for estradiol levels and endocrine therapy use in our analyses. Blood samples were collected non-fasting in this study; however, the impact of fasting status on 27-HC concentration was demonstrated to be relatively weak  . A limit = 0.08) . In addi = 0.08)  and mode = 0.43) . Data we = 0.43)  did not  = 0.43) . FurtherFinally, the CV of 25-HC was 29.2%, potentially resulting in exposure misclassification; however, it merits noting that the concentrations in the quality control samples were substantially lower than those in the study samples, and the CVs may not reflect the precision of the assay at higher analyte concentrations. Although, to our knowledge, decomposition of oxysterols at very low temperatures has not been reported, we cannot rule out the possibility that long-term storage could have had an effect on the sample concentrations , 46.Lastly, while the current study was hypothesis based, we carried out multiple analyses to examine associations and did not adjust the significance level for multiple testing. Thus, it is possible that some of our findings are due to chance.We provide the first data on circulating 27-HC, 25-HC, and breast cancer outcomes in a large breast cancer cohort suggesting differential associations between 27-HC, 25-HC, and prognosis in breast cancer patients depending on the underlying estrogenic environment. For a better characterization of the potential importance of these cholesterol metabolites in women with a breast cancer diagnosis further experimental studies better describing 27-HC, 25-HC, and estradiol and their overlapping and independent signaling pathways in breast cancer are required, together with epidemiological studies assessing 27-HC and 25-HC by estradiol levels and endocrine therapy.Supplemental material


Men born with pathogenic/likely pathogenic variants in genes associated with the Hereditary Breast and Ovarian Cancer Syndrome have a higher risk to develop breast cancer and other cancers (such as prostate cancer) and should undergo adequate surveillance protocols in highly specialized Centers.A retrospective study was conducted to assess these genetic variants’ epidemiological and phenotypical manifestations in male carriers, as well as the efficacy of the surveillance protocol and compliance toward it through a survey. During follow-up, a genetic panel for testing was implemented, the starting age for surveillance was delayed, and the six-month screening interval was extended to annual.BRCA1 (n = 31), BRCA2 (n = 55), both BRCA2 and another gene (n = 5), CDH1 (n = 2), CHEK2 (n = 4), NF1 (n = 1), RAD51C (n = 4), and an additional two men with no actionable genetic variant identified. The follow-up period ranged from 1 to 13 years, and only one man developed cancer. Survey responses from 48 men in active surveillance showed that more than half recognizes their carrier status and consequent surveillance impact on their life, including the risk of transmission to offspring, fear of future cancer, meaningful distress, and feeling of injustice. Biannual surveillance was not actively detecting more cancer disease cases, confirming the adequacy of the currently implemented protocolA total of 104 men from a tertiary hospital’s High-Risk Consultation were included, 102 with positive genetic testing for With support of Genetics to fulfill the current gaps in high-risk management, the proposed redefinition of surveillance protocol would adapt it to the population needs and concerns. Breast Cancer (BC) is the most frequently diagnosed cancer in women and one of the leading causes of cancer-related death. However, this disease can also appear in men, corresponding to less than 1% of all cases of breast carcinoma and about 0.5% of all malignancies in men in western countries   technique, other genes have been identified as possible contributors to the disease, such as PALB2.On the other hand, similarly to what happens in women, genetic predisposition is a significant risk factor in men. Despite its low incidence in the general population, men born with genetic variants associated with hereditary BC have a higher risk of developing it, as well as other cancers (such as prostate cancer (PC)). Carriers of pathogenic and likely pathogenic (P/LP) variants in these genes undergo surveillance protocols, and, to the best of our knowledge, in most cases, the development of cancer disease is rare. In this context, it is questionable whether the surveillance proposed for these asymptomatic carriers is the most appropriate, considering the psychosocioeconomic impact that cannot be underestimated, validated by the scarcity of personal and material resources, and also marked by a significant embarrassment that could be avoided  manifest epidemiologically and phenotypically in male carriers as well as the efficacy of the surveillance protocol. The compliance toward it was also evaluated by collecting the at-risk individuals’ opinions.A retrospective review was performed on male individuals carrying demonstrably identified  variants in genes associated with hereditary BC, followed between January 2008 and April 2022 in the Oncogenetics and the High-Risk Consultations of the Breast Center of Centro Hospitalar Universitário de São João (CHUSJ). The molecular study immediately provided variants’ pathogenicity, and ClinVar database was used to search for recent reclassification of variants of uncertain significance (VUS).By a retrospective analysis of the clinical records, demographic and epidemiological information was retrieved, including age, marital status, number of children, smoking habits, comorbidities, follow-up start date, genetic variant identified, and family history of cancer.Until 2016, molecular testing consisted in this study of a singular gene at a time. After that year, the Oncogenetic Department of CHUSJ started providing the NGS breast-specific panel, which simultaneously analyzed five–six genes by then and nowadays ~ 20 genes related to BC.To manage the genetic risk, every carrier had one first consultation to provide proper counseling and discuss the possibility of preimplantation genetic diagnosis (PGD). Then, at-risk individuals were submitted to the surveillance protocol proposed by the National Comprehensive Cancer Network (NCCN) guidelines in force at that time: recommendation of clinical breast examination, digital rectal examination, breast imaging, and PSA serum measurement every six months, starting at 35 years old. In 2016, the High-Risk Consultation medical team extended this period to annual, based on NCCN update guidelines version 2.2015, and began recommending surveillance starting at 40 years old.Considering these changes, there was interest in studying which differences would arise and which would be men’s behavior toward them.Among the subset of male carriers diagnosed with cancer, we evaluated the date of diagnosis and whether it had been prior to or after the genetic study, primary and secondary tumors, anatomopathological stage according to the TNM classification system, treatment performed, and health condition at last observation.To complement the investigation, a survey was conducted via telephone to men ≥ 40 years old in active surveillance for at least 2 years to assess the psychosocioeconomic impact of their carrier status and compliance with the protocol. In the absence of a known validated survey aimed at assessing this impact, we elaborated one, with questions directed at evaluating the emotional implications of their carrier status, interference in work and social activities, and satisfaction with the current surveillance protocol. It was performed in a semi-structured format, asking participants to classify statements according to the 5-point Likert scale and allowing them to freely express their opinions on the topic. The purpose of this survey was explained to participants at first, predicting tacit informed consent when answering the questions.Data analysis was subsequently performed using IBM SPSS version 28.0.1.0 statistical software, presenting absolute and relative frequency for categorical variables and the median and interquartile range for continuous variables.A total of 104 men followed in the High-Risk Consultation were included in our study. Personal history of cancer (non-HBOCS) was present in 7.7%, 50% of the carriers had no comorbidities, 31.7% had cardiovascular risk factors or cardiopulmonary disease, and 10.7% had other comorbidities . A detailed overview of the participants’ characteristics is presented in Table n = 9, 8.7%), asymptomatic carriers with an actionable variant identified , and men with no actionable variant identified , including one symptomatic and one asymptomatic , and there is no indication to search for VUS in healthy individuals , although the High-Risk Consultation medical team decided to continue the surveillance program based on his family history of cancer. Of the 103 males tested, 6 (5.8%) were index cases, three of which were studied before 2016 and the other 3 after. The remaining 97 (94.2%) males were family members, who were only checked for the specific family variant previously identified in the index case . Only one man (1.0%) from the study population had not performed genetic test, as a BRCA2 variants, 29.8% (n = 31) for BRCA1, and 4.9% (n = 5) for both BRCA2 and another gene. Variants in non-BRCA genes were identified in 10.5% (n = 11) of the men , BRCA2 , and CDH1 , while, after 2016, genetic variants in BRCA1 , BRCA2 , BRCA1 + BRCA2 , BRCA2 + ATM , BRCA2 + CDH1 , BRCA2 + MSH6 , CDH1 , CHEK2 , NF1 , and RAD51C  were identified. The classification of the identified genetic variants as pathogenic, likely pathogenic or VUS is described in Table BRCA2 gene alterations.A total of 55 men (52.9%) were positive for .8% n = 3 for BRCA.8% n = 3 for BRCAMen not old enough to start active surveillance joined the non-eligible group for surveillance and were discharged from the High-Risk Consultation until they reached the required age .The follow-up period ranged from 1 to 13 years, with a median period of 5 years. Two eligible men did not get to start the surveillance program, since one of them decided to proceed with risk management in his hometown region, and the other one was soon referred to Palliative Care due to the progression of his PC. Seven were lost to follow-up (Table n = 3) was the most diagnosed cancer, followed by PC (n = 2) and gastric cancer (n = 2). Other cancers found in this group included choroidal melanoma (n = 1), glioblastoma (n = 1), pheochromocytoma (n = 1), GIST (n = 1), and colorectal cancer (n = 1). The underlying genotypic and phenotypic characteristics of each symptomatic case are summarized in Table Of the 104 participants, 10 (9.6%) presented with cancer disease. MBC . The surveillance protocol allowed the detection of only one PC case (Patient I), diagnosed in advanced stage in 2010, throughout the investigation performed over imaging findings recorded in his first examination.The survey was completed by all 48 invited men, with a median age of 60 years old (ranging from 42 to 79). Figure Overall, the participants showed no significant embarrassment going to the surveillance consultations nor looking for information about MBC, even referring to feel comfortable at the Breast Center despite its feminine environment.About 70% of inquired men denied inconveniences attending the appointments. On the contrary, about 30% (most of them in active working age) showed a willingness to extend the screening interval and preference for less uncomfortable exams, highlighting the psychosocioeconomic impact of undergoing surveillance to manage their hereditary cancer risk.BRCA1/2 (87.5%) but also in non-BRCA genes (14.4%). Ten participants presented with cancer (MBC being the most common and PC the second most), with only one case (1.0%) detected during the follow-up period due to screening. Most participants did not have significant apprehension about attending surveillance appointments. However, some would prefer less uncomfortable exams and longer screening intervals, demonstrating the psychosocioeconomic impact associated with surveillance.This study examined 102 men with P/LP variants associated with hereditary cancer and 2 without any actionable variant identified. Most of these men had no significant comorbidities, although a third had cardiovascular risk factors or cardiopulmonary disease. At the time of genetic testing, P/LP variants and VUS were primarily identified in BRCA1 and BRCA2 genetic variants in the general population is estimated to be between 1 in 500 and 1 in 1000, respectively , it is our understanding that this man should be invited for retesting, according to NCCN (2023) recommendations, incorporating these recently MBC-associated genetic alterations, namely, the CHEK2 1100delC variant.Genetic variants in the CDH1 pathogenic variants appear only to be associated with lobular breast cancer and not ductal breast cancer or other rare types of breast cancer. Since lobular development does not occur in the male breast, men carrying CDH1 P/LP variants are at no increased risk of developing BC .NF1 gene and BC in men is a rare phenomenon . The one man in our study who had not performed genetic testing belongs to a family that would have been on hold in terms of investigation for several years. Since, nowadays, it is proven there are other genes associated with MBC risk, there is an indication to extend the genetic testing in the index case, with obvious implications for himself and his family, if an actionable variant is identified in one of the non-The only cancer detected during follow-up occurred in 2010, before the periodicity change in surveillance protocol. With the greater spacing of consultations from 2016, the fear was that more interval cancers would appear, yet it did not happen. This means that, even though in the first phase of our study patients were monitored biannually, a 6-months interval surveillance was not actively detecting more cases of cancer disease, confirming the adequacy of the NCCN protocol currently implemented—although, as practiced in this study’s follow-up period since 2016, we consider it is appropriate starting surveillance at 40 years old [instead of the 35 years proposed by NCCN (2023)].Nowadays, even with annual surveillance, patients are sometimes forced to go to the hospital more often than strictly necessary. Due to the widespread use of imaging techniques for more precise diagnosis, there is a consequent difficulty in scheduling and processing them in a timely manner, which significantly impacts patients’ daily activities. Besides, when it comes to favorable surveillance outcomes, the unnecessary expenses of human and material resources must be a matter of concern when improving the pathway of care.Because of the small number of men with cancer disease, there were insufficient data for a meaningful outcome analysis.BRCA1/2 pathogenic variants were the most frequent genetic variants found between symptomatic carriers. Men with BRCA1 variants have a lifetime breast cancer risk of 1–5%, 2–3% of pancreatic cancer, and 7–26% risk of PC. Altered BRCA2 carriers have a 5–10% lifetime breast cancer risk, 15–61% risk of PC, 3–5% risk of pancreatic cancer, and 3–5% risk of melanoma  and associated with worse survival compared to BRCA wild-type cancers  with Gleason 8 and bone metastasis at presentation and the other one (Patient I) with biochemical relapse treated with salvation radiation.The risk of PC is up to five-fold higher in BRCA2 P/LP variants and melanoma showed some evidence of an association, even though inconsistent conclusions have been drawn. The development of in situ choroidal melanoma (1.0%) in Patient J (positive for a BRCA2 pathogenic variant) reinforces the need for subsequent studies on this possible association.Emerging data have suggested a potential benefit of pancreatic cancer and melanoma screening in selected individuals at increased risk. Because of limited definite data on long-term screening results, each situation should be evaluated based on family history and jointly decided with the patient , it was possible to reach a large number of participants in our study. Our population was recruited from a tertiary ERN GENTURIS health care provider institution in a dedicated center certified by EUSOMA. In fact, the High-Risk Consultations were always performed by the same three medical care providers, following the same guidelines (NCCN). The second limitation is the small amount of novel MBC-associated genes carriers that precludes clinical information from being totally informative in elucidating the clinical phenotype associated with them. Regarding the uncommon nature of MBC, although we cannot draw robust conclusions, these data serve as a point of reference for national averages for many of the behavioral variables discussed. Another limitation is having a high percentage (32.7%) of asymptomatic carriers under 40 years old who are not yet eligible to start surveillance—although they were included in our sample, they are not helpful yet in retrieving important outcome information to support our conclusions. As strength of this study, we highlight very little research investigating the effective impact of P/LP variants associated with hereditary MBC and considering the implications of the surveillance itself from a holistic point of view of the patient.To the best of our knowledge, no studies have taken simultaneously into account all the issues considered in our study, using a survey to describe the effects of breast and prostate surveillance on high-risk men’s quality of life.These data may represent a step toward evidence-based guidelines specific for men with P/LP variants in genes associated with hereditary breast cancer syndromes. As surveillance protocol, we suggest clinical breast examination, digital rectal examination, breast imaging, and PSA serum measurement every 2 years starting at 40 years old (or 10 years before the earliest known MBC or PC case in the family) and annually from 50 years old beyond. Even though further national studies are needed to corroborate our results, this proposal could adapt the risk management to the national reality and, especially, to the population needs, lowering its psychosocioeconomic impact and most likely without an increase in the incidence of cancer.As important as screening at-risk men and diagnosing cancers in early stages, it is crucial to support and accompany these carriers who are going through a situation of uncertainty inherent to the carrier status itself and the still insufficient knowledge about HBOCS in men. Also, considering everything analyzed in this study, when referring exclusively to men, instead of designating HBOCS, we propose the designation of Hereditary Breast and Prostate Cancer Syndrome (HBPCS).Therefore, there is an urgent need to improve genetic education and training and create special units for specific diseases, as well as a better organization and composition of services in a multidisciplinary and user-centered approach, fulfilling the current gaps concerning the optimal management of high-risk individuals.


Introduction: Ultrasound is a non-invasive, low-cost technique that does not use ionising radiation and provides a “real-time” image, and for these reasons, this method is ideal in several situations. Purpose: To demonstrate breast ultrasound evaluation as a first-line diagnostic method and to evaluate the variation of breast characteristics with age. Material and Methods: A total of 105 women with a mean age of 30 years participated and were divided into three age groups: 18–39, 40–59, and 60–79 years, excluding participants subject to mastectomy. After completing the informed consent, all participants answered personal and sociodemographic questions, such as personal and family history, menstrual cycle, pregnancy, ultrasound, and mammography, among others. They were then submitted to a bilateral breast ultrasound examination. Subsequently, all the images and their data were analysed, and a technical report of the examination was given to all the participants. Results: A total of 105 women with a mean age of 30 years participated, 58 of whom underwent the examination for the first time. In 31, changes (of which only 7 were known) were diagnosed. It was verified that, according to age group, the density of the breast stroma varied; older women have less breast density. Conclusions: Ultrasound is a good method for breast evaluation and can be considered important for the early evaluation of breast pathology and follow-up of the pathology. The breast is composed of Cooper’s ligaments . This is the most prominent surface structure on the anterior chest wall, located between the second and sixth ribs on the vertical axis and between the outer edge of the sternum and the midaxillary line on the horizontal axis (in an adult) and can be divided into four quadrants—the upper outer quadrant, the lower outer quadrant, the upper inner quadrant, and the and lower inner quadrant—to make it easier to locate and describe tumours ,2,3. TheBreast size is determined by the amount of fat surrounding the glandular tissue. Glands are composed of lobules interspersed with glandular, connective, and adipose tissue. Lobes are subdivided into lobules composed of alveoli. The lobes connect to the nipple through the galactophore ducts. The nipple is surrounded by the areola ,2,3. ArtNowadays, there is a higher incidence of breast pathologies not only due to the advancement of technology that allows the early detection of pathologies but also due to the awareness of society at this level. Over time, norms have been created to try to change the panorama of the increase in pathologies associated with the breasts, with the emergence of screening tests: mammography , ultrasound (a complementary method to mammography), and physical examination . The last one is extremely important for the woman to know her body . Other iUltrasonography is a non-invasive imaging technique and may be repeated as often as necessary since it is painless, inexpensive, and does not involve exposure to ionising radiation ,9,10.Although mammography can detect breast pathology and thus reduce breast cancer mortality, it has been described in the literature as an imperfect tool that is not as effective, particularly in the female population with dense mammary glands ,12,13.Breast ultrasound is a more sensitive and accurate modality than mammography for the early diagnosis of breast cancer in Chinese women with suspected breast lesions. This might also be true for all women with dense breasts worldwide. For older or obese women, mammography can be used to supplement ultrasound to increase sensitivity, specificity, and accuracy. However, long-term follow-up is still needed to assess whether ultrasound reduces breast cancer mortality .Ultrasonography performs real-time detection of breast lesions and evaluates their morphological characteristics, such as shape, echogenicity, nodules, and cysts .The breast is composed of lobules interspersed with adipose tissue and connective tissue. These fat layers are heterogeneous and hypoechogenic. The stroma of the breast is much more hyperechogenic. Fat lobules, contained within the breast stroma, can be mistaken for masses. Planar sheets composed of fibrous connective tissue provide structure to the breast. Cooper’s ligaments are thin linear echogenic bands that ascend from the chest wall. The lactiferous ducts are distinct hypoechogenic tubular structures radiating from the nipple and should be a maximum of 2 mm in diameter (identified under the areola), and, subsequently, they drain into the lactiferous sinus (maximum 4 mm) ,17.Posterior to the breast, the pectoral muscles can be seen as a slightly hypoechogenic area with linear strands. The ribs are posterior to this musculature and have attenuating properties that cause artefacts. Other areas that cause similar artefacts are the nipple area and the areolar region. Compression is vital in differentiating a mass from the attenuating anatomy .Lymph nodes are usually found in the breast, but they are more predominant in the axillary region, are frequently isoechoic with the surrounding breast parenchyma, and may be difficult to identify . On the Pregnancy, due to the influence of prolactin on the growth of ductal, lobular, and alveolar structures in the last half of pregnancy, has a protective effect against the development of breast cancer. On the other hand, multiparous women who have their first child at an advanced age present a twofold higher risk of developing breast cancer ,23.Among the significant risk factors for the development of a second breast cancer, personal history of breast cancer stands out ,25.Women with a history of endometrial, ovarian, and colon cancer are also at an increased risk of developing breast cancer compared with the general population ,24. The According to one study, women with dense breasts are more predisposed to breast cancer, on the other hand, mammography in these women is less sensitive.; the addition of ultrasound screening can increase breast cancer detection rates by 1.9–4.2%, depending on the population . AutomatBreast cancer is the most frequently diagnosed pathology in women, and it is the second cause of death from cancer, and it represents one-third of all cancer cases. However, due to increasingly earlier detection associated with increased screening, awareness, and adherence, as well as better therapeutic strategies, the number of cases of breast cancer has been decreasing .Breast cancer incidence does not show a uniform geographic distribution. The incidence rate is higher in developed countries, but it has been shown to be increasingly prevalent in developing countries .In Portugal, breast cancer is the most frequently diagnosed malignant neoplastic pathology in the female population and is considered the second leading cause of death in women . In 2020That said, it would be beneficial if the word “early” was associated with breast ultrasound. Ultrasound has the potential to yield high sensitivity and specificity in the detection of breast cancer. Ultrasound is widely available, easy to maintain, inexpensive, durable, and easily transportable. In view of the increasing global burden of breast cancer and the lack of access to timely detection through imaging, ultrasound can be an effective primary detection tool and screening method for breast lesions, particularly in resource-poor settings where mammography is not available .Despite the enormous technological and scientific evolution in the last few years in the approach to breast cancer diagnosis and treatment, organised population screening remains a controversial topic, and there is no international consensus on the best way to implement it. Future research will involve the study and implementation of patient-centred models based on each patient’s clinical and genetic characteristics, allowing the definition of individualised recommendations for when to start, at what age to suspend, and how often to perform screening according to the individual risk determined; this will allow for better risk stratification and risk–benefit balance associated with breast cancer screening, which, ideally, will lead to increased early detection of breast cancer, a reduced mortality risk, and reduced rates of false positives and overdiagnosis .It is known that younger people are more easily predisposed to undergo this examination instead of mammography. On the other hand, by detecting the pathology earlier, that is, by detecting breast alterations at an early stage, the prognosis may be more favourable.Given the above, this study primarily aimed to highlight the early assessment of breast pathology by ultrasound and its importance. The secondary aim was to evaluate breast characteristics throughout the course of ageing.Also, this study aim was to confirm ultrasound as a screening test to raise awareness of its importance and the importance of breast assessment.In this study, 105 female participants aged between 18 and 79 years were evaluated. This sample was divided into three groups according to the following age division: 18 to 39 years old; 40 to 59 years old; and 60 to 79 years old.The division was fair considering the decrease in stromal density with increasing age in female patients and according to the variables available in the sociodemographic questionnaire regarding the participant’s general and personal information, such as family history, age, menopause, taking the pill, and pregnancy, among other factors.In the present study, participants who had already undergone mastectomy were not accepted. The entire procedure for data collection was explained in advance, and after answering a sociodemographic questionnaire, the participants signed an informed consent form. This study was approved by the Ethics Committee of the Polytechnic of Coimbra (No. 5/2018).To carry out this study, the acquisition protocol followed the European guidelines  with theEach participant was given a questionnaire assessing several essential details, such as age, smoking habits, alcohol consumption, menstrual cycle, pregnancy, and personal and family history, among others.During the breast ultrasound, the participant was positioned supine with a slight inclination to the contralateral side.Scanning was performed clockwise, and a total of 12 images (minimum) were acquired after bilateral evaluation. General Electric Healthcare’s Logiq E ultrasound equipment  and a 7–12 MHz linear probe was used.For better acoustic contact without causing too much pressure on the participants’ breasts, a water-based gel was used. In this way, it was possible to obtain a good characterisation and visualisation of the breast stroma.All images were acquired by students pursuing a degree in medical imaging and radiotherapy with the supervision and guidance of an experienced professional in the field of breast ultrasound.r < 0.2: very low linear correlation;0.2 < r < 0.39: low linear correlation;0.4 < r < 0.69: moderate linear correlation;0.70 < r < 0.89: high linear correlation;0.9 < r < 1: very high correlation.In this study, the Pearson correlation test was used with the aid of the IBM SPSS Statistics Version 22.0 software to measure the degree of association between two variables). The values can be interpreted as follows (by convention) :r < 0.2:Of the 105 participants, 30% (31 cases) had changes in the breast stroma, and the remaining 70% did not have any type of changes.Of the participants who had alterations (31 cases), only 21% (7 cases) were aware of them. Initially, participants were divided into three groups by age: 72% of participants were 18 to 39 years old, 15% were 40 to 59 years old, and 13% were 60 to 79 years old .Of the total number of participants surveyed, 33.4% responded that they had a family history of breast pathology, and 11.4% responded that they already had a personal history.When asked if they performed breast self-exams, 53.3% of the participants admitted that they did not.Only 30% of the study participants already had children. Of the 84% of menstruating participants, 61% used a contraceptive method.Regarding the consumption of alcohol, most participants denied drinking alcohol frequently (97%), and only 16% had a smoking habit.All responses to the sociodemographic questionnaire are shown in When analysing the presence or absence of alterations in the breast stroma by age group, it was possible to notice that among the participants of group 1 (18–39 years old), 28.9% had breast alterations; among those in group 2 (40–59 years old), 37.5% had breast lesions; and finally, among those in group 3 (60–79 years), 23.1% had changes .Of the total number of participants with breast alterations, lesions on the left (20.2%) were more common than on the right (17.2%). After three months, 18 of the participants who had breast changes were contacted to repeat the exam and find out which of the changes (if any) remained. Of these participants, nine maintained the changes, three cases were proven to be false positives (cycle phase or trichotomy), and the remaining six women did not repeat due to schedule incompatibilities.p < 0.05) and age presenting a positive correlation with other exams that the participants had already performed (p < 0.05). None of the other variables in this study were significantly correlated according to Pearson’s test.The correlation between the variables was analysed, with the presence/absence of breast alterations presenting a positive correlation with personal history  when the variables were the presence/absence of breast alterations and personal history, and they were moderate for age and other exams that the participants previously performed (r between 0.4 and 0.69—moderate linear correlation).In this study, it was possible to verify that breast alterations can occur in any age group and that there are several external factors that can act as risk or safeguard factors. Therefore, it is important to use breast ultrasound from an early age and not from a certain age.Currently, the first-line test for early diagnosis of breast pathology is mammography. However, it is an imperfect tool that is not equally effective for all women. Overall, the sensitivity of mammography for detecting breast cancer is 85%. However, in women with dense breast tissue, the sensitivity of mammography is lower (47.8–64.4%) .Although breast density tends to decrease with age, it is a significant problem in women of all ages; as breast density increases, so does the chance of developing breast cancer ,37,38.Ultrasound of the breast may be a solution for detecting breast cancer in women with dense breast tissue for whom mammography is less effective and in women who have an increased risk of breast cancer. Women with dense breast tissue constitute the largest group of intermediate-risk women for whom mammography may not be sufficient. Breast ultrasound detects small lesions that are clinically significant, invasive, and predominantly present with negative lymph nodes  that areAnalysing the obtained data in this study, it is possible to acknowledge that in group 2 40–59 years), there was a higher incidence of breast alterations. However, it was also observed that in group 1 18–39 years), the percentage of alterations was high. The incidence of breast lumps was relatively higher in childbearing-age women ,39,40. years, t–59 yearsIt was also confirmed that as participants’ ages increased, the density of the breast stroma decreased ,42.p < 0.05) suggests that with their increase, the incidence of breast alterations increases as well.The positive correlation between the presence or absence of breast alterations and personal history  were detected in the participants.It was thus concluded that it is extremely important to breast tissue changes early so that there can be a follow-up of the evolution of the alterations, which could have several advantages.The results were quite positive, as the female population generally participated freely, and there were only difficulties in the second evaluation phase due to the incompatibility of schedules and the fact that it was necessary to wait 3 months after the first evaluation.The present study found that the participants did not carry out breast self-examination as often as would be desirable, as this is the first step towards the early detection of any pathology associated with the breast.Finally, it was concluded that evaluation by breast ultrasound should be easily accessible because, in addition to its advantages, it is a technique more easily accepted by the population due to its painlessness.Therefore, the female population will be able to know their bodies better and achieve early diagnosis of malignant breast pathology at an early stage, consequently increasing the success of treatment and reducing the mortality rate due to breast cancer.


Few modifiable risk factors for epithelial ovarian cancer have been identified. We and other investigators have found that individual psychosocial factors related to distress are associated with higher risk of ovarian cancer. The present study examined whether co‐occurring distress‐related factors are associated with ovarian cancer risk.Five distress‐related factors were measured repeatedly over 21 years of follow‐up: depression, anxiety, social isolation, widowhood, and, in a subset or women, posttraumatic stress disorder (PTSD). Cox proportional hazards models estimate relative risks (RR) and 95% confidence intervals (CI) of ovarian cancer for a time‐updated count of distress‐related factors, in age‐adjusted models, then further adjusted for ovarian cancer risk factors and behavior‐related health risk factors.age‐adjusted = 1.71; 95% CI = 1.16, 2.52). No significant difference in ovarian cancer risk was observed in women with one or two versus no distress‐related psychosocial factors. In the subsample with PTSD assessed, ≥3 versus no distress‐related psychosocial factors was associated with twofold greater ovarian cancer risk . Further analysis suggested that women at highest ovarian cancer risk had PTSD co‐occurring with any other distress‐related factor . Adjusting for cancer risk factors and health behaviors minimally impacted risk estimates.Across 1,193,927 person‐years of follow‐up, 526 incident ovarian cancers occurred. Women with ≥3 versus no distress‐related psychosocial factors demonstrated increased ovarian cancer risk (HRPresence of multiple indicators of distress was associated with risk of ovarian cancer. When including PTSD as an indicator of distress, the association was strengthened. We assessed 5 types of distress in two large cohorts of women across up to 23 years. Experience of three or more types of distress was associated with 1.7 times greater risk of incident ovarian cancer. Adjusting for health behaviors and ovarian cancer risk factors had a minimal impact on risk estimates. For example, if anxiety has a causal effect on cancer incidence, studies of depression that do not account for anxiety would misestimate the association of depression with cancer incidence given the co‐morbidity of these conditions. Meta‐analyses of the relation between depression and cancer incidence have noted that studies typically account for few potential confounders and rarely account for other mental health symptoms or sources of distress.Thus, in the present study, we prospectively examined the combination of multiple forms of distress, including depression, anxiety, and PTSD, and experiences commonly associated with high levels of distress, including social isolation and widowhood, in relation to risk of developing ovarian cancer. We use two large prospective cohorts of women, the Nurses' Health Study (NHS) and the Nurses' Health Study II (NHSII) and examine risk overall, by menopausal status, and separately for high‐grade serous/poorly differentiated cancers, a common and aggressive form of the disease.Relevant psychosocial factors were selected based on prior work, including in NHS and NHSII, demonstrating increased risk of incident ovarian cancer with each factor examined separately.22.1The NHS is an ongoing longitudinal study of 121,700 female nurses enrolled in 1976 at ages 30–55 years. The NHSII is a companion study of 116,429 female nurses aged 24–42 years begun in 1989. Both studies queried participants at baseline about lifestyle factors, health behaviors, and medical history via questionnaire. Biennial and additional one‐time questionnaires were used to update health status and distress measures over follow‐up. The study protocol was approved by the institutional review boards of the Brigham and Women's Hospital and Harvard T.H. Chan School of Public Health, and those of participating registries as required. Return of questionnaires by mail constituted implied consent. Signed releases were obtained to collect medical records.2.2Five distress‐related psychosocial factors were measured repeatedly over follow‐up, including depression, anxiety, social isolation, widowhood, and, in a subset of participants, PTSD.The five‐item Mental Health Index (MHI‐5) assessed depressive symptoms .Symptoms of anxiety were assessed using the eight‐item Crown‐Crisp phobic anxiety index (CCI) .Every 2 years, we created a time‐updated count of number of distress‐related psychosocial factors based on the most recent data reported for each factor. As few women reported all four types of distress, our highest level included participants who reported three or four types.To examine the association of duration of distress‐related factors with ovarian cancer, we coded the number of distress‐related factors across each 4 years of follow‐up as: none (reference); high (3 or 4 in both 2‐year periods); or mixed .In a subsample of NHSII women, information on trauma exposure and PTSD was available. In 2008, lifetime exposure to trauma was assessed using the Brief Trauma Questionnaire.2.3Ovarian cancer diagnosis was self‐reported on each biennial questionnaire with follow‐up through 2015. We identified additional cases through report by family members and via the National Death Index. We obtained an adjudicated indicator by requesting pathology reports or linking to the relevant cancer registry, to obtain information by which a gynecologic pathologist, blinded to women's exposure status, abstracted information on morphology, histology, stage, and grade. In a sample of 459 ovarian cancer cases, concordance between a centralized review of slides and pathology report abstraction was 78% for histologic type, 79% for grade, and 94% for invasiveness.2.42), and past‐year leisure‐time physical activity . Self‐report of health‐related factors has good validity in the NHS cohorts.Ovarian cancer risk factors and relevant health risk factors were queried regularly on the biennial questionnaires and, for the current study, were time‐updated approximately every 2–4 years. These included duration of oral contraceptive use , history of tubal ligation or hysterectomy (ever/never), family history of ovarian cancer (any/none), menopausal status , parity (coded continuously), smoking status , body mass index . Participants were included in analyses if they completed each first assessment of the distress‐related psychosocial factors , had not been diagnosed with cancer at the time of these assessments, and had not had a bilateral oophorectomy or menopause due to pelvic irradiation prior to each respective baseline. After exclusions, 65,066 NHS and 50,628 NHSII participants were included. Participants accrued person‐time from baseline until the date of ovarian cancer diagnosis (cases), removal of ovaries, pelvic radiation, breast cancer diagnosis, death, non‐responsiveness to biennial questionnaires, or end of follow‐up in 2019 (non‐cases), whichever came first.I2 < 20%),We examined the distribution of covariates across the number of distress‐related factors in each sample. We then used Cox proportional hazards models to estimate relative risks (RR) and 95% confidence intervals (CI) of ovarian cancer for each time‐updated count of distress‐related factors, separately by cohort. To reduce the likelihood that ovarian cancer symptoms might have induced distress, we included a latency period of 4–6 years. For example, we examined distress‐related factors from 2002 to 2004 and risk of ovarian cancer from 2006 to 2008. We assessed statistical heterogeneity across the two cohorts using meta‐analysis, and, finding no substantive difference I2 < 20%,We conducted a secondary analysis in the subsample of NHSII participants who provided information on PTSD. First, to directly compare associations in this subsample with those in the main analyses, we examined number of distress‐related psychosocial factors, excluding PTSD, with ovarian cancer risk. Next, we added PTSD to the count of psychosocial factors and examined risk of ovarian cancer. Finally, as prior work in this cohort has demonstrated PTSD is strongly associated with ovarian cancer incidence,p‐value for heterogeneity.Sensitivity analyses examined risk of Type 2 ovarian cancers only . For these analyses, women with other types of ovarian cancer were censored at the date of their cancer diagnosis. As cancer risk factors vary by menopausal status, we also conducted analyses stratified by menopausal status using multiplicative interaction terms and a Wald test to assess the For all analyses, inverse probability weighting was used to adjust for the probability of each participant surviving from cohort enrollment until study baseline. Weights were calculated using variables available at enrollment that predicted survival, including age, BMI, height, exercise, pack‐years of cigarette smoking, alcohol consumption, body shape at age 5, cancer history, occurrence of menopause, ovary removal, use of beta blockers, parity, race, and ethnicity.3At baseline, dose‐dependent relations were observed between number of distress‐related psychosocial factors and current smoking, low physical activity, higher BMI, family history of breast cancer, being post‐menopausal, and never using oral contraceptives Table .age‐adjusted = 1.71; 95% CI = 1.16, 2.52; Table ptrend for increasing number of distress‐related psychosocial factors did not reach statistical significance (p = 0.11). Having persistently high distress‐related factors across 4 years was associated with risk of ovarian cancer similar to that found in the main analysis .In the two cohorts, 526 incident ovarian cancers occurred  across 1,193,927 person‐years of follow‐up. Women with ≥3 versus no distress‐related psychosocial factors demonstrated significantly increased ovarian cancer risk (HRN = 346 incident cases), women with ≥3 versus no indicators of distress were at greater risk for developing ovarian cancer  in the fully adjusted model, although this did not reach statistical significance with the limited cases ; formal analysis (lead); methodology ; writing – original draft (lead). Andrew Ratanatharathorn: Formal analysis ; investigation ; methodology ; writing – review and editing . Lori Chibnik: Formal analysis ; investigation ; methodology ; writing – review and editing . Laura Kubzansky: Conceptualization ; data curation ; methodology ; supervision ; writing – review and editing . Shelley Tworoger: Conceptualization ; funding acquisition ; investigation ; methodology ; supervision ; writing – review and editing .This research was supported by National Institutes of Health grants UM1 CA186107, P01 CA87969, and U01 CA176726, and US Department of Defense grant OC120441. The funders had no role in the conceptualization, drafting, or approval of the manuscript or its contents.The authors declare no potential conflicts of interest.The study protocol was approved by the institutional review boards of the Brigham and Women's Hospital and Harvard T.H. Chan School of Public Health, and those of participating registries as required. Return of questionnaires by mail constituted implied consent. Signed releases were obtained to collect medical records.Table S1.Table S2.Click here for additional data file.


Exploring the potential association between peripheral blood leukocyte counts and breast cancer risk by Mendelian randomization (MR) analysis methods. Genetic data related to peripheral blood sorting counts of leukocytes were collected from a genome-wide association study by Blood Cell Consortium (BCX). Single nucleotide polymorphic loci predicting peripheral blood sorting counts of these leukocytes were selected as instrumental variables according to the correlation assumption, independence assumption and exclusivity assumption of MR. The data on breast cancer and its subtypes were obtained from Breast Cancer Association Consortium (BCAC) and FinnGen Consortium. In this study, the Inverse-Variance Weighted (IVW), Weighted Median, MR-Egger, Maximum Likelihood (ML), MR-PRESSO and Constrained Maximum Likelihood and Model Averaging (cML-MA) methods of random effects models were used for MR analysis. Cochran’s Q analysis, and MR-Egger intercept analysis were applied for sensitivity analysis. IVW and cML-MA were considered the primary analytical tools, and the results of the other 4 MRs were used as complementary and validation. The results suggest that there is no significant causal relationship between leukocyte count and breast cancer risk . In addition, we analyzed whether there was a potential correlation between the five main types of categorized leukocyte counts and different breast cancer subtypes. We did not find significant evidence to support a significant correlation between leukocyte counts and breast cancer subtypes. Notably, in 2018, the United States reported the diagnosis of approximately 268,670 new BC cases5. Moreover, BC exhibits pronounced heterogeneity, showcasing varying clinical presentations among individual patients, thus posing intricate challenges in its therapeutic approach. Drawing upon both molecular and histological evidence, BC may be systematically categorized into three primary subtypes: hormone receptor-positive BC (estrogen receptor (ER+) or progesterone receptor (PR+)), human epidermal receptor 2-positive BC (HER2+), and triple-negative breast cancer (TNBC)8.Breast cancer (BC) is a common female cancer, and due to its high mortality and morbidity, BC has become a significant burden on women’s health10. Tumor-associated immune cells have exhibited prospective prognostic utility across diverse malignancies, with elevated quantities of tumor-infiltrating lymphocytes frequently linked to more favorable clinical outcomes in BC11. However, research in the realm of the association between circulating immune cell subtypes and the clinical attributes or risk profiles of BC patients remains notably underexplored.The significance of the immune microenvironment in the initiation and advancement of BC has garnered growing recognition12 suggests that the percentage of certain leukocyte types in a woman’s blood may predict her risk of being diagnosed with breast cancer in the short and long term. The immune system plays an important role in the occurrence and development of BC, and it protects the health of the human body. Gene mutations are not uncommon in normal human beings, but not every harmful mutation will develop into a malignant tumor. This is because the immune system eliminates these mutant cells in real time and resolves the threat of cancer at the initial stage. Only when the immune system malfunctions or the mutated cells establish an immune escape mechanism can the mutated cells, which were originally very small, have the opportunity to continue to divide and proliferate, and eventually form tumors14. Therefore, detecting the immune function status of the body may be beneficial to assess the risk of malignant tumors such as BC16. White blood cells are important immune cells in the body, including neutrophils, eosinophils, basophils, lymphocytes and monocytes18. Different types of white blood cells participate in the body's immune defense response in different ways. A previous retrospective study has provided preliminary evidence of a conceivable correlation between peripheral leukocyte and neutrophil counts and the clinical attributes of BC patients19. Larsson et al.20 reported significant associations between peripheral blood mononuclear cell populations and prognosis in patients afflicted with metastatic BC. Furthermore, there is emerging evidence suggesting potential links between peripheral blood lymphocyte levels and clinical characteristics, as well as chemotherapy responsiveness, in BC patients21. The level of peripheral blood leukocyte count may be one of the most important indicators for evaluating the risk characteristics of BC patients, while the leukocyte categorical count may better reflect the dynamic characteristics of the microenvironment of BC patients.A patient’s leukocyte levels may provide clues to BC risk. A study by Kresovich et al.23. This approach mitigates the confounding issues inherent in observational epidemiology, ultimately yielding more dependable and rigorous results. MR is now widely used in the study of BC26. In this study, we performed MR analysis of two samples to investigate the potential association between peripheral blood leukocyte counts and breast cancer risk. In addition, we further investigated the potential relationship between categorized leukocyte cell counts  and breast cancer subtypes . The flowchart of this study is shown in Fig. The majority of the research evidence regarding the association between peripheral blood leukocyte counts and BC risk is derived from observational studies. Nevertheless, these studies are susceptible to various limitations, including confounding factors, selection bias, and other biases that may compromise the validity of their findings. Mendelian randomization (MR) analysis employs genetic variants as instrumental variables (IVs) to enhance the causal inference in exposure-outcome relationships, particularly when pleiotropic effects are absent28. The study included 563,946 European ethnic subjects and provided GWAS data on five types of peripheral blood leukocyte counts: neutrophils, eosinophils, basophils, lymphocytes and monocytes 34. Specifically, all SNPs had to meet the following criteria: (1) Exposure correlation P-value less than 5 × 10–8, along with an F-statistic greater than 10, thus satisfying the correlation assumption of MR. (2) SNPs with significant linkage disequilibrium with the measured SNPs (r2 = 0.001) were removed in the range of 10,000 kb, thus satisfying the independence assumption of MR. (3) SNPs with significant associations with outcomes or confounders were manually removed via the PhenoScanner website, thus satisfying the exclusivity hypothesis. These eligible instrumental variables were then extracted from the outcome GWAS with the necessary coordination so that the effect of a single instrumental variable on exposure corresponded to the same allele as the effect on outcome. The data of the IVs are shown in Supplementary data Based on the relevance, independence and exclusivity assumptions of MR, single nucleotide polymorphism loci  that met the requirements were screened from the above exposure GWAS data and used as instrumental variables (IV) in this study Fig. A33,34. S38. IVW is one of the most commonly used MR analysis methods40. The IVW method has the strongest causal inference and provides the most reliable findings when the instrumental variables are not confounded by horizontal pleiotropy. The weighted median and MR-Egger methods, on the other hand, serve as complements to the IVW method42. Because these two methods can be used under a wider range of conditions and provide more conservative results. Of these, the weighted median method allows no more than 50% of the instrumental variables to be confounded by horizontal pleiotropy, whereas the MR-Egger method allows all instrumental variables to be pleiotropic, but this pleiotropy cannot interfere with the correlation of instrumental variables with exposure43. In contrast to the IVW method, the ML method offers the advantage of a reduced standard error, and its findings remain unbiased in the absence of heterogeneity or horizontal polymorphism. We also used the MR-PRESSO method to identify and remove any outlier variants44. This method regresses the genetic variance results on the genetic variance exposure and uses residual squares to identify outliers. The cML-MA method, a MR technique that incorporates ML and model averaging, is applied to address both correlated and uncorrelated pleiotropic effects. Importantly, it does not rely on the InSIDE (Instrument Strength Independent of Direct Effect) assumption, setting it apart from other MR approaches. cML-MA has better type I error control. Xue et al.45 demonstrated that, in certain scenarios, the cML-MA method might offer advantages over the IVW method. As such, in this research, the primary analytical tools employed were the cML-MA and IVW methods, which were complemented by results obtained from four additional methods. MR-Egger and MR-PRESSO methods were employed to identify horizontal pleiotropy. In the MR-PRESSO analysis, the number of distributions was specified as 1000. Furthermore, the detection of heterogeneity was carried out using the IVW method and MR-Egger regression, with the quantification of heterogeneity achieved through the Cochran Q statistic. A P < 0.05 was considered statistically significant, while Bonferroni-corrected adjusted p-values  in analyses involving multiple comparisons were considered statistically significant in order to minimize the potential risk of type I error and to improve the overall validity and interpretability of the results. The selection of all instrumental variables and the MR analysis process were done by applying the “TwoSampleMR” and “MRcML” R package (version 4.3.1)48.In this study, the Inverse-Variance Weighted (IVW), Weighted Median, MR-Egger, Maximum Likelihood (ML), MR-PRESSO and Constrained Maximum Likelihood and Model Averaging (cML-MA) methods of random effects models were used for MR analysisAnimal and human experiments were not conducted in this study.12. We further explored this by the method of MR analysis. IVW and cML-MA were considered the primary analytical tools, and the results of the other 4 MRs were used as complementary and validation. When neither IVW nor cML-MA was statistically significant, we considered that there was no causal relationship between exposure and outcome.Previous studies have suggested that leukocyte counts in breast cancer patients may play a key role in breast cancer riskThe results suggest that there is no significant causal relationship between leukocyte count and breast cancer risk . We did not find significant evidence to support a significant correlation between leukocyte counts and breast cancer subtypes . However, considering multiple comparisons, we extended the P value to BP. the IVW result did not fulfill the requirement of BP, and therefore, this result was also not significant.In conclusion, the results of MR analysis indicated that there was no significant correlation between the counts of peripheral blood leukocytes and their subtypes and the risk of breast cancer and its subtypes.49. Immune mechanisms play an obvious role in the occurrence and development of breast cancer. Therefore, in-depth studies targeting this area are beneficial to further reveal the pathogenesis of breast cancer and to improve its prevention and treatment measures. Peripheral blood leukocyte count is a routine clinical test, which is mainly used for the assessment of patients’ general condition and the diagnosis of acute bacterial infections, with the characteristics of being easy to perform and low cost51. As important immune cells in the organism, the levels of various leukocytes in peripheral blood can also reflect changes in immune function. Wei et al.52 recruited 140 Chinese women, 75 with breast cancer and 65 healthy controls. Their results showed that breast cancer patients had higher white blood cell counts, neutrophil counts and monocyte counts. Okutural et al.53 showed that neutrophil levels are associated with risk of breast cancer. A meta-analysis evaluated the association between neutrophil-to-lymphocyte ratio as a biomarker and breast cancer prognosis using leukocyte subtypes54. Therefore, the present study investigates the correlation between peripheral blood leukocyte sorting count levels and the risk of breast cancer development, giving researchers the opportunity to deepen their understanding of the immune mechanism of breast cancer development and clinicians the opportunity to find a simple method to assess the risk of breast cancer.Breast cancer is a malignant tumor that poses a serious health risk to womenIn this study we performed a large-scale MR analysis using six different MR analysis methods. We first investigated the potential relationship between leukocyte counts and the risk of breast cancer and its subtypes. The results of the MR analyses did not support a significant causal relationship between them. Then, we further investigated the relationship between leukocyte subtypes and breast cancer risk. The IVW results showed that there was a statistically significant correlation between an elevated peripheral blood monocyte count and a reduced risk of ER+ breast cancer . However, considering multiple comparisons, we extended the P-value to BP. The IVW results did not fulfill the requirement of BP, and therefore this result was also not significant. The results of our MR study showed no significant causal relationship between leukocyte count and breast cancer risk.55. In a recent study, Farrell et al.56 collected clinical characteristics of 37,052 men and 15,004 women, and their results indicated that the white blood cell count levels were approximately 6.0 ± 1.4 (109/L) in men and 5.9 ± 1.5 (109/L) in women. Gender may not be the main influencing factor of white blood cell count. The patient's body mass index and age may be more important influencing factors.The present study is a two-sample MR study, which has the greatest advantage of avoiding causal reversal and minimizing the effect of confounding factors by using instrumental variables in place of phenotype for causal inference. The subjects of the breast cancer GWAS data in this study were all female, whereas the leukocyte GWAS data in this study were from a population of both sexes. This may affect the reliability of the study results to some extent. Current data do not support making gender distinctions. The results of previous studies have shown that a patient's leukocyte level can be affected by a variety of factors, such as time of diagnosis, body weight, hormone levels, and menopausal statusOverall, the results of the MR study showed no significant correlation between white blood cell counts and breast cancer risk. The results of this paper need to be further validated in clinical trials and in larger patient cohorts.Supplementary Legends.Supplementary Table 1.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.Supplementary Information 8.Supplementary Information 9.Supplementary Information 10.Supplementary Information 11.Supplementary Information 12.Supplementary Information 13.Supplementary Information 14.Supplementary Information 15.


Personalized breast cancer screening has the potential to improve the accuracy and effectiveness of breast cancer screening by tailoring the screening protocol to an individual’s risk factors. Currently, most women undergo mammograms at regular intervals based on age and family history, regardless of their risk factors. We examined the applicability of the Gail model, a well-known breast cancer prediction tool including several risk factors, in an Asian population. While the tool predicts the risks of developing breast cancer in the next 2, 5, 10, and 15 years to some extent, the age at which the disease occurs cannot be estimated. In addition, the tool performs better for longer prediction horizons (10 or 15 years), limiting its utility for risk prediction for two-year screening intervals.long-term ranges: 0.86–1.71; E/Oshort-term ranges:1.24–3.36). Subgroup analyses show that the model underestimates risk in women with breast cancer family history, positive recall status, and prior breast biopsy, and overestimates risk in underweight women. The Gail model absolute risk does not predict the age of breast cancer occurrence. Breast cancer risk prediction tools performed better with population-specific parameters. Two-year absolute risk estimation is attractive for breast cancer screening programs, but the models tested are not suitable for identifying Asian women at increased risk within this short interval.Personalized breast cancer risk profiling has the potential to promote shared decision-making and improve compliance with routine screening. We assessed the Gail model’s performance in predicting the short-term (2- and 5-year) and the long-term (10- and 15-year) absolute risks in 28,234 asymptomatic Asian women. Absolute risks were calculated using different relative risk estimates and Breast cancer incidence and mortality rates . Using linear models, we tested the association of absolute risk and age at breast cancer occurrence. Model discrimination was moderate (AUC range: 0.580–0.628). Calibration was better for longer-term prediction horizons (E/O Breast cancer five-year survival of over 80% is expected with detection at the early stages ,2,3. An In terms of cost-effectiveness, age-targeted regular mammography screening for breast cancer is reported to be cost-effective, even when considering false positives and overdiagnosis . HoweverCurrent risk assessment tools estimate absolute risks for a time horizon of at least five years ,14,15,16The efficacy of population-based screening programs for breast cancer also depends on accurate risk assessment. Prediction models associated with high sensitivity and specificity would allow programs to focus screening efforts on high-risk individuals while limiting the overtreatment of other populations. However, before a model is used to predict risk, we have to show that the model is good at predicting risk by demonstrating accuracy, reliability, and generalizability . There aThe Gail model is a widely used breast cancer risk prediction model due to its simplicity. Self-assessments can be made by responding to a questionnaire . The modThe risk threshold at which women are identified to be at high risk of developing breast cancer determines what kind of personalized follow-up interventions are recommended for screening and prevention . A five-In this study, we assessed the calibration of the Gail model in a prospective cohort of 28,234 asymptomatic Asian women undergoing mammography screening. Two-, five-, ten-, and 15-year absolute risks were calculated and evaluated. In addition, this work seeks to identify relevant thresholds for classifying Asian women as high risk for developing breast cancer.n = 1744) and with unknown age at the point of screening (n = 14) were excluded. A total of 26,380 women were included in this study.The Singapore Breast Screening Project (SBSP) was created to test the effectiveness of mammography screening on women aged 50–64 years. It was a randomized trial that invited 69,473 (41.7%) women to participate in a single free screening . As partThis study was approved by the SingHealth Centralised Institutional Review Board (CIRB Ref: 2019/2210) and the A*STAR Institutional Review Board (A*STAR IRB Ref:2021-141). Informed consent was not obtained as we used existing data of participants recruited from 1994 to 1997 for the Singapore Breast Screening Project (SBSP) and some participants would have died.The first diagnosis of invasive breast cancer, occurring at least 6 months after recruitment and before age 80 years, was identified via linkage with the Singapore Cancer Registry with the latest date of occurrence set at 31 December 2019.2), age at menarche (years), age at menopause (years), age at first live birth , parity , prior biopsy , and family history of breast cancer. Women who underwent biopsy during screening were assigned a code of 1 for the risk factor of biopsy. Those who did not have a biopsy done in the past year were assigned a code of 0. Family history of breast cancer was determined by whether the woman’s mother, sister(s), or daughter(s) had a history of the disease, with a code of yes or no. The number of first-degree relatives with breast cancer was coded as 0 for no family history, 1 for having one relative with the disease , and 2 for having two first-degree relatives with the disease. Recall status (yes/no) from the first screen for any reason  was extracted from the radiology report. Missing values for BMI were replaced by the mean. Missing values for risk factors were coded as the reference level to obtain the absolute risks estimated by the Gail model .,34.23,34Using breast cancer incidence and mortality rates specific to the Singaporean population, as opposed to using published rates for “Whites”, the overestimations of absolute risks were observed to be smaller . The higMost models overestimate the risk of breast cancer, regardless of the population parameters used . Model cThe Gail model for the 15-year absolute risk prediction was the best-performing model, in terms of absolute risk distribution, discriminatory ability, and calibration . This mop-value < 0.05), using the Asian  or Singapore (beta10-year = −0.47 and beta15-year = −0.43) prediction models (10-year = 0.52). However, the relationship was reversed and no longer significant when we adjusted for age at screening and ethnicity (beta10-year = 0.14) (Higher absolute risk was significantly associated with an earlier occurrence of breast cancer (n models . These a = 0.14) .The absolute risk of developing breast cancer (6th decile) for a 50-year-old woman (recommended age to start subsidized mammography screening in Singapore) ranges from 0.25 to 0.39% for 2-year, 0.62–0.98% for 5-year, 1.46–2.14% for 10-year, and 2.16–3.50% for 15-year projections . The difUsing a large dataset comprising 28,234 asymptomatic Asian women living in Singapore who attended a mammography screening, we showed that using population-specific breast cancer incidence and mortality rates improved the performance of the Gail model. Our results suggest that breast cancer screening should be a long-term commitment as (1) current prediction models for a shorter time horizon do not perform optimally, and (2) a higher predicted risk of developing breast cancer does not necessitate an earlier diagnosis. Breast cancer risk prediction was most well-calibrated with the highest discriminatory ability in the long-term (10- or 15-year absolute risk). The absolute risks of developing breast cancer were 0.82%, 1.74%, and 2.79% in the next five, ten, and fifteen years, respectively, for an average 50-year-old Asian woman living in Singapore .A previous work by Chay et al. on the same cohort reported good calibration in the 10-year absolute risk model (the Gail model) when they used the relative risk of the closest ethnic-specific data (Asian American Breast Cancer Study model) . On the Gao et al. showed in the same dataset that the performance of the Gail model in estimating 5-year absolute risk improved with the use of Singaporean race-specific invasive breast cancer rates and mortality rates for deaths not due to breast cancer ,45. WithAnother desirable function of risk prediction models is to help inform when to start screening. Worldwide, mammography screening recommendations are based on age ,46. TherInstead of a one-size-fits-all age-based approach, individualized breast cancer risk predictions may aid in shared decision-making with clinicians on whether it is helpful to start screening earlier. A multi-modal approach is needed to maximize the utility of the risk-based screening strategy. For example, The BREAst screening Tailored for HEr (BREATHE) study, a prospective cohort study in Singapore, endeavors to change the current age-based screening paradigm in a prospective study for women aged 35–59 years . BrieflyAccording to data from the Surveillance, Epidemiology, and End Results (SEER) registry  . The corThe strengths and limitations of using SBSP to evaluate the Gail model have been elaborated by Chay et al. and Gao et al. . The souBreast cancer risk prediction tools perform better with population-specific parameters. Two-year absolute risk estimation is attractive for breast cancer screening programs, but current models are not tailored to identify women at increased risk within this short interval. The importance of routine screening should be emphasized, as it is not currently possible to predict the age of disease onset. Risk-threshold-dependent recommendations for interventions will need to be reconsidered for specific populations.