To determine the association between vitamin D deficiency and breast cancer.This case control study included 94 female patients aged 20-75 years of any marital status and parity. Newly diagnosed 42 breast cancer patients who presented to surgical OPD of Dow University Hospital from Jan 2016 to June 2016 were included into the study as “cases” after informed consent. Age-matched 52 females who presented to OPD for complain other than breast pathology were included as the “control group”. The sociodemographic of both cases and controls and histopathological characteristics of cases were recorded. Serum 25-(OH)2D levels were studied by the ELISA technique and recorded in ng/ml. Vitamin D deficiency was considered at serum level less than 20 ng/ml.Mean age was 40.1 Years for controls and 47.6 Years for cases. Mean height, weight and BMI did not differ between cases and controls. Serum Vitamin D levels were significantly lower in cases (85.7%) than controls (55.8%). The unadjusted and adjusted ORs for breast cancer in cases and controls showed a statistically significantly increased risk of breast cancer with low vitamin D concentration . After adjustment for age, parity, BMI, sun exposure, economic status and education status the ORs (95% CIs) for breast cancer risk was7.8 (1.99 - 30.58) for women with vitamin D concentrations <20 ng/mL.Findings of our study conclude that vitamin D deficiency is associated with risk of breast cancer. Breast cancer poses a serious health risk for women throughout the world. It is the most common cancer among women in United States.2For the large magnitude of breast cancer, a lot of efforts are being made to identify such risk factors that can be modified to prevent breast cancer. Vitamin D is assumed to be one of such risk factors. It is a fat soluble vitamin. Its main sources are skin production (via exposure to ultraviolet light) and dietary intake. Vitamin D has an important role in calcium and bone homeostasis. Vitamin D has not only shown to strengthen our bones but it also has capability to modulate several features of cancer. Its anticarcinogenic properties include inhibition of cell proliferation, invasion, metastasis and angiogenesis and induction of apoptosis and differentiation.3In a study done in Shaukat Khanum Memorial Cancer Hospital and Research Centre, Lahore, Pakistan vitamin D deficiency was found in 95.6% of breast cancer patients and in 77% of control group.4Several studies have been done to evaluate the association of vitamin D deficiency and breast cancer risk. There is controversy in the literature about this association. Some studies have found that breast cancer is associated with low levels of vitamin D.This case control study was approved by the College of Physicians and Surgeons Pakistan. Sample size was calculated by using WHO sample size calculator with 80% power of test and 5% level of significance considering proportions (P1=95.6%)1. Age 20-75 years 2. Resident of Karachi for >5 years (confirmed by seeing NIC) Cases: Diagnosed with invasive breast cancer within 6 months of all grades and of stage I-III.Age matched females presenting in clinic with no breast pathology.1. Pregnant 2. Lactating 3. Males 4. Stage IV breast cancer 5.Patients receiving neoadjuvant chemotherapy. The sociodemographic were recorded by direct questioning on to the proforma for the whole study population. The histopathological diagnosis of breast cancer, grade, stage of the tumor, and hormone receptor status  was recorded from the pathology reports of breast cancer patients. Serum 25-(OH)2D levels were studied by the ELISA technique on the blood samples drawn of the study population at their initial presentation and the values were recorded in ng/ml. Vitamin D deficiency was considered at serum level less than 20 ng/ml.Data was analyzed by using SPSS version 20 on computer. Categorical variables in cases and controls like marital status, parity, grades, stages, histopathology, receptor status of tumor, exposure to sunlight, economic status, education status and vitamin D deficiency were presented in frequency and percentages. Mean+/-SD was computed for numerical variables like age, size of tumor, duration of carcinoma breast, height, weight and BMI. For comparing baseline characteristics, P-values were calculated with Chi-square analyses for categorical variables and Mann-Whitney test for numerical variables. A P-value of ≤0.05 was considered statistically significant. Logistic regression modeling was used for assessment of the association between vitamin D deficiency and risk of breast cancer. Potentially confounding variables that were evaluated were age, parity, BMI, sun exposure, education and economic status. P value ≤0.05 was taken as significant.2, mean duration was 6.14 months and mean size of breast cancer was 3.82 centimeters. Mean age was 40.1 year for controls and 47.6 year for cases. Mean height, weight and BMI did not differ between cases and controls.Data of 94 women, including 42 breast cancer cases and 52 controls, were included in analysis. The characteristics of study population are shown in Serum Vitamin D levels were significantly lowers in cases than controls. 85.7% of cases and 55.8% of controls had Vitamin D levels less than 20 ng/ml. Only 14.3% of cases and 44.2% of controls had Vitamin D levels above 20 ng/ml .The ORs for breast cancer in cases and controls by category of vitamin D status are shown in Vitamin D deficiency is very common as shown in our results that 55.8% of controls and 88.7% of breast cancer patients are vitamin D deficient. This finding is slightly different from a study done in Shaukat Khanum Memorial Cancer Hospital and Research Centre Lahore, that showed vitamin D deficiency in 76.7% of controls and 95.6% of cases.15In our study, parity, BMI, sun exposure, economic status and education status did not show any statistically significant association with breast cancer risk. In our study 71.4% of cases and 46.2% of controls were overweight that may link increased frequency of vitamin D deficiency in cases as compared to controls because of high BMI as shown in study of Imtiaz et al.Findings of this study show that vitamin D deficiency is associated with risk of breast cancer.Authors` contribution:NS: Conceived, designed, did data collection, statistical analysis, manuscript writing, revising critically, editing and final approval of manuscript.FJ: Designed, did data collection, editing, revising critically and final approval of manuscript.FAM and NAQ: Contributed in acquisition of data, revising the article critically and final approval of the article.


Cervical cancer is the second most common female cancer which Ethiopia put a strategic goal to reduce its incidence and mortality by 2020. Lack of knowledge and poor attitude towards the disease and risk factors can affect screening practice and development of preventive behavior for cervical cancer. The aim of this study was to assess knowledge, attitude, practices and factors for each domain for cervical cancer among women of child bearing age in Hossana town, Southern, Ethiopia.Community based cross sectional study was carried out in June 2015. A total of 583 participants were selected using systematic random sampling technique. Pretested structured interviewer administered questionnaire was used to gather the data. Data were entered in to Epi Info software version 3.5.4 and exported to SPSS version 16 for descriptive and logistic regression analysis.Two hundred seventy (46.3%) of the respondents had poor comprehensive knowledge. Only 58 (9.9%) of participants had been screed for the cervical cancer before the survey. Two hundred three (34.8%) of participants had negative attitude towards selected proxy variables. Not having health seeking behavior for cervical cancer , had not ever received information about cervical cancer and its prevention  and not actively seeking health information about cervical cancer  were significantly associated factors with poor knowledge. Poor knowledge score was associated with poor attitude . Had not ever received information about the disease from any source  was significantly associated factor with not to be screened for the disease.This study highlighted the importance of awareness creation, increasing knowledge, promoting active searching for health information and experiences of receiving information from any information sources regarding cervical cancer. Therefore, it will be essential to integrate cervical cancer prevention strategies with other reproductive health services at all level of health care delivery system. Cancer is increasingly growing as a major public health problem in both developed and developing countries amongst the chronic diseases . Cancer Breast and cervical cancers are the leading cancers among women in developing countries, with estimated annual new cases of 882,900 and 444,500 respectively. More than 324,300 and 230,400 women die from these cancers every year, respectively. Globally, the number of new cases and deaths from cervical cancer is projected to continue to rise 720,415 and 394,905 respectively in 2025. A marked increase has been predicted specifically in GAVI-eligible countries, with a 58% expected increase in the number of new cases and a 63% expected increase in the number of related deaths . Similarly, those who had never received information about cervical cancer and its prevention were about 2.63 times more likely to have poor knowledge score when compared to those who had ever received information from any sources . In the same manner, participants who were not actively seeking health information about cervical cancer were 6.25 times more likely for having poor knowledge score as compared to those who were actively searching health information about cervical cancer . However, other socio demographic factors were not statistically significant with poor knowledge score for cervical cancer prevention and control .Among variables entered in bivariate and multivariate logistic regression analysis, only knowledge score was associated with poor attitude with wide confidence interval i.e low precision. Participants who had poor knowledge score were 56.5 more likely to have negative attitude .Despite the fact that precision was very low with wide confidence interval, women who had not ever received information were 45.2 more likely not to be screened for cervical cancer than those who had ever received information about the disease from any source  .Knowledge, attitude and practice of the community about any disease including cervical cancer and its factors offer crucial opportunity for comprehensive prevention and control strategies of the disease. Therefore, this study addressed knowledge, attitude and practice as an entry point for the prevention and control of cervical cancer and its associated factors among women of child bearing age.The findings of this study showed that less than half (46.3%) of participating women had poor level of comprehensive knowledge score from the composite score regarding vulnerable groups, risk factors, signs and symptoms and methods of prevention of cervical cancer. This finding is lower than a similar the study done in Addis Ababa among female ART attendants . This coLess than half, 254 (43.6%) of the respondents believed that all women are at risk of getting cervical cancer while 216 (37.0%) of them did not know which women are at risk of getting the disease. This finding showed that participants had much lower awareness level about risk groups when compared to findings of other studies done in Ethiopia , 20 and As observed from the study, 223 (38.3%) of the participants had no idea what factors increased the chances of getting cervical cancer. Majority of the participants were not able to cite the risk factors for cervical cancer. Only about nine in hundred indicated infection with human papillomavirus as a risk factor, but more than a quarter of participants cited at least one risk factor related to having multiple sexual partnership. These misconceptions were more reflected in this study than the study done in North West of Ethiopia . This evAs indicated from this study, major risk factors reported include multiple sexual partners. Although having sex with one sexual partner is sufficient to acquire infection with HPV, having multiple partner is an important risk factors . This fiAs observed from the study, 336(57.6%) of participants reported that cervical cancer is a preventable disease. Our finding lower than similar study in semi urban part of India; 37 12.2%) . Our fin.2% . OurTwo hundred three (34.8%) of participants had negative attitude towards selected proxy variables. Our finding is lower than other findings in Ethiopia , althougOnly 58 (9.9%) of respondents had been screened for the cervical cancer before the survey. This finding is consistent with hospital based finding in Nigeria among women of reproductive age . This stWith the contextual categorization of health service related factors, less than a quarter of the respondents had mentioned that they had ever received information about cervical cancer screening from health professionals. Women who have not had an intention to be screened reported reasons like unavailability of the service, unaware of where to get the services and financial constraints. Findings of this study are supported by other studies done in low resource settings , 17, 15.Findings from the multivariate logistic regression in the study showed that not having health seeking behavior for cervical cancer, had never received information about cervical cancer and its prevention and not actively seeking health information about cervical cancer were significantly associated factors with poor knowledge. This finding was congruent with one systematic review finding  and findThis study highlighted the importance of awareness creation, increasing knowledge, promoting active searching for health information and experiences of receiving information from any information sources regarding cervical cancer. The findings also strongly indicated that different health information dissemination strategies may be required for women to increase knowledge about and attitude towards prevention and control of cervical cancer. We recommend greater attention to the adaptation of comprehensive prevention packages for cervical cancer by integrating with other reproductive health services like antenatal care services, health information education and communication at all level of health care delivery system in the study area. Further more, it’s important to use proven approaches in implementation of health extension programs like health development army to encourage screening behavior of women by all health care providers and other health sector stakeholders. Finally, research is needed to fully understand the issues relevant to women who had no screening behavior for prevention and control of cervical cancer.The study did not address the role of the partner the of the study participants. Those participants who scored for screening is very small so that the model of multivariate analysis may not be stable.S1 Dataset(SAV)Click here for additional data file.


To examine the associations between pharmaceutically treated anxiety and depression present in the year prior to breast cancer diagnosis and the risk of incident cardiovascular disease (CVD), while controlling for traditional cardiovascular risk factors and clinical characteristics in a population-based observational study.n = 7227), diagnosed between 01-01-1999 and 12-31-2010, with no history of CVD, were selected from the Netherlands Cancer Registry. Drug dispensing data were derived from the PHARMO Database Network and used as proxy for CVD, anxiety, and depression. By multivariable Cox regression analysis, we examined the risk associated with pharmaceutically treated anxiety and depression for developing CVD after cancer diagnosis, adjusting for age, pharmaceutically treated hypertension, hypercholesterolemia, and diabetes mellitus in the year prior to cancer diagnosis, tumor stage, and cancer treatment.Adult 1-year breast cancer survivors  breast cancer survivors developed CVD. Women pharmaceutically treated for anxiety in the year prior to their cancer diagnosis had a 48% increased hazard for CVD  after full adjustment. This association was restricted to breast cancer survivors who were 65 years or younger. Depression was not associated with CVD risk . Older age , hypertension , and hypercholesterolemia  were associated with an increased hazard for incident CVD, whereas hormone therapy  was protective.Anxiety present in the year prior to breast cancer diagnosis increases the risk of incident CVD in 1-year breast cancer survivors, after adjustment for depression, traditional cardiovascular risk factors, and clinical characteristics. Cardiovascular disease (CVD) is a common comorbidity in breast cancer survivors , 2 and eIncidence rates of CVD in breast cancer survivors vary depending on the type of treatment and dosage . Even inPsychological factors, such as anxiety and depression, may also increase the risk of CVD in breast cancer survivors. Several studies have shown that depression and anxiety are predictive for the development and progression of CVD in non-cancer patients –16. A meCVD is understudied, underdiagnosed, and undertreated among women, yet we know that there are sex differences in the pathophysiology of CVD . SimultaData from the Southern Region of the Netherlands Cancer Registry (NCR) were used in this observational cohort study. The NCR registers cancer diagnosis, stage, and primary cancer treatment for all newly diagnosed cancer patients and is maintained by the Netherlands Comprehensive Cancer Organization . The SouFemale adult breast cancer patients diagnosed between 01-01-1999 and 12-31-2010 were selected from the NCR. To obtain information on survival status and date of death, the NCR was linked to the municipal Personal Records database. As anonymous observational patient information was used, this study does not fall under the Medical Research Involving Human Subjects Act in the Netherlands; therefore, this study was exempted from medical ethics review and no informed consent was required. This study was performed in agreement with the Declaration of Helsinki.Breast cancer survivors who had a history of CVD medication use (see the definition of CVD in the next section) in the 12 months prior to their cancer diagnosis were excluded, as our aim was to examine the risk of incident CVD following cancer diagnosis. In addition, to exclude the effect of detecting CVD due to increased clinical checkups and the direct and sometimes reversible effects of receiving cancer treatment, breast cancer survivors who developed CVD in the first year after diagnosis were also excluded. Follow-up for a diagnosis of CVD began 12 months after the cancer diagnosis (which was set as the index date), as primary cancer treatment is generally finalized within the first year. Hence, we excluded those who died or were lost to follow-up during the first year. Follow-up time was measured until onset of CVD, death, loss to follow-up, or until the end of the study period (31-12-2010), whichever occurred first.cardiac therapeutics  at unique dates within 6 months. Survivors who dispensed two cardiac drugs with less than 15 days in between were classified as having CVD only when they had three cardiac dispenses at unique dates in a 6-month period. We used a strict definition based solely on using cardiac therapeutics (ATC = C01) to avoid false classifications of CVD. Therefore, usage of CVD-related drugs such as diuretics (C03) or beta-blockers was insufficient to be classified as having CVD, as these drugs have a broad treatment range including non-CVD indications.CVD was defined as having at least two drug dispenses of Drug dispense information for anxiety disorders (ATC = N05B) and depression (ATC = N06A) during the 12 months prior to the cancer diagnosis was included. Patients with one or more drug dispensings were categorized as anxious or depressed (yes/no).Drug dispense information on the traditional cardiovascular risk factors including hypertension , C07, C08, C09 (except C09X)), hypercholesterolemia (ATC = C10), and diabetes mellitus (ATC = A10) during the 12 months prior to the cancer diagnosis was captured . Having Survivors’ age and clinical information on tumor stage and treatment ) were obtained from the NCR.χ2 test, or Student’s t test for independent samples as appropriate.Differences in patient characteristics  between breast cancer survivors with and without incident CVD were analyzed using ANOVA, the The associations between pharmaceutically treated anxiety and depression with incident CVD risk were examined separately and simultaneously using multivariable Cox regression analyses. Analyses included covariates which were entered in separate steps. First, we adjusted for age (continuous) as a potential confounder. Second, traditional cardiovascular risk factors  were added to the model. Finally, clinical information on tumor stage, chemo-, radio-, and hormone therapy was entered, which were considered possible explanatory variables. Assumptions underlying the multivariable Cox regression analysis were met .We tested effect modifications for age, traditional cardiovascular risk factors, and cancer treatment  by adding interaction terms  to the fully adjusted model. We examined whether the effect of anxiety and depression on CVD risk differed by age (≤65 vs >65 years at the time of cancer diagnosis), traditional cardiovascular risk factors, chemotherapy, radiation, or hormonal treatment. Missing data were handled in previous steps and described elsewhere . All staOf the 7889 eligible breast cancer survivors, 515 were excluded as they received prescribed CVD medications in the 12 months before or after their cancer diagnosis, and 147 were excluded as they were deceased or lost to follow-up in the first year after cancer diagnosis. After exclusion, 7227 1-year breast cancer survivors were included in statistical analyses.p’s <0.05).The 1-year breast cancer survivors who developed CVD differed from those who did not  showed that anxiety in the year prior to cancer diagnosis was associated with an increased risk for incident CVD in all models: age-adjusted , partially adjusted (adjusted for age and CVD risk factors) , and fully adjusted model , taking medication for hypertension , and hypercholesterolemia  were associated with an increased hazard for incident CVD, whereas being treated with hormone therapy  was protective for CVD (Table There were no cancer treatment moderation effects for anxiety on CVD risk, nor were there significant interaction effects with traditional cardiovascular risk factors. There was an age-related significant interaction effect with anxiety , where there remained a significant main effect of anxiety  on CVD risk. Stratified analyses showed that the association was restricted to women who were ≤65 years at breast cancer diagnosis , as there was no increased CVD risk among older survivors  (Fig. This population-based observational study showed that pharmaceutical therapy for anxiety but not depression in the year prior to breast cancer diagnosis was associated with an increased hazard of incident CVD in 1-year breast cancer survivors. This relation remained significant after adjustment for depression, traditional cardiovascular risk factors, and clinical characteristics, and was restricted to younger breast cancer patients. Associations did not differ by type of cancer treatment or the presence of traditional cardiovascular risk factors in the year prior to breast cancer diagnosis.The association between pharmaceutical treatment for anxiety present in the year prior to cancer diagnosis and the increased risk for CVD found in the current study is consistent with studies in healthy individuals, showing that anxiety predicts the onset of CVD , 12. We The lack of an association between depression and CVD risk was unexpected, as previous studies have demonstrated that depression is a risk factor for both incident CVD and CVD progression , 13–15. Surprisingly, there was no effect of chemo- or radiotherapy on CVD risk, despite the known cardiotoxicity of these treatments , 4. ThisA general limitation inherent to the observational study design is the lack of information on residual confounders. Furthermore, we used drug dispense information as a proxy for anxiety, depression, and CVD. We did not have additional information on medical diagnosis or patient-reported information on anxiety or depression. Nevertheless, algorithms based on pharmacy drugs are known to be more specific, yet less sensitive than medical diagnoses . Also, wStrengths of our study include the large population-based sample of breast cancer survivors and the usage of high-quality databases of the Netherlands Cancer Registry and PHARMO enabling a 13-year follow-up period. Additionally, this is to our knowledge the first study that examined the association between pharmaceutically treated anxiety and depression with incident CVD among breast cancer survivors. Furthermore, we estimated CVD risk 1 year after breast cancer diagnosis, striking a compromise between not starting too late and missing incident CVD due to ongoing cancer treatment or increased clinical checkups.In conclusion, 1-year breast cancer survivors with pharmaceutically treated anxiety in the year prior to their cancer diagnosis had a 48% increased risk of incident CVD, after adjustment for depression, traditional cardiovascular risk factors, tumor stage, and cancer treatment. This increased risk seems to be limited to those breast cancer survivors who were 65 years or younger at cancer diagnosis. Depression was not related to an increased risk of incident CVD. Future studies unraveling these associations are warranted in order to provide the best optimal care for women treated for breast cancer.


Breast cancer survivors are at an increased risk of second primary cancers, and the risk factors for the latter may have clinical significance. The aims of our study were to evaluate the incidences and risk factors of second primary female genital cancers  in a large cohort of breast cancer survivors. Using the Surveillance, Epidemiology, and End Results (SEER) database, we examined the standardized incidence ratio (SIR) and risk factors for second primary female genital cancers observed between 2000 and 2014. Breast cancer survivors had increased SIRs for second corpus uteri cancers and second ovarian cancers and a decreased SIR for second cervical cancers . Risk factors of second corpus uteri cancers were the age at first cancer diagnosis, race , and progesterone receptor (PR) status . In addition, the risk of second ovarian cancer was positively associated with age while inversely associated with race  and estrogen receptor (ER) status . Age, race, and hormone receptor status are risk factors of developing second female genital cancers among breast cancer survivors. Older age, black race, and a PR+ status in survivors are associated with a higher risk of second corpus uteri cancers. Additionally, older age and an ER− status should increase vigilance for potential second ovarian cancers.The online version of this article (10.1007/s12672-018-0330-0) contains supplementary material, which is available to authorized users. According to statistics regarding cancer incidence and mortality, breast cancer is the most common malignancy diagnosed in females, accounting for nearly one in three cancers . Breast Previous studies have suggested a consensus in the increased risk of second primary female genital cancers among breast cancer survivors. A large cohort study reported that women with breast cancer had a 30% excess risk for second primary cancers, particularly for endometrial cancer and ovarian cancer . The proThe aims of our study were to systemically evaluate the incidence of second primary female genital cancers in a large cohort of breast cancer patients and to identify risk factors for second primary corpus uteri cancer and ovarian cancer. Using the Surveillance, Epidemiology, and End Results (SEER) database, we calculated the standardized incidence ratio (SIR) for second primary cancers after breast cancer diagnosed between 2000 and 2014. Meanwhile, we used multivariable analysis to examine the risk factors for the development of a second primary female genital cancer after breast cancer.The SEER program of the National Cancer Institute collects information on cancer incidence, patient survival, and patient characteristics from several geographically defined regions in the USA. Data were selected from the SEER 18 registry database from January 1, 2000, to December 31, 2014. The registries included San Francisco-Oakland SMSA, Connecticut, Detroit (Metropolitan), Hawaii, Iowa, New Mexico, Seattle (Puget Sound), Utah, Atlanta (Puget Sound), San Jose-Monterey, Los Angeles, Rural Georgia, California excluding SF/SJM/LA, Kentucky, Louisiana, New Jersey, and Greater Georgia. The International Classification of Diseases for Oncology, 3rd edition (ICD-O-3) histopathology codes were used to analyze the cases of malignancy.From this database, we collected data for women with breast cancer only and breast cancer survivors with multiple cancers who were diagnosed between 2000 and 2014. Second primary cancers that were diagnosed within 6 months of breast cancer diagnosis were excluded as these were likely to be pre-existing or synchronous cancers . Cases dThere were 615,581 women with breast cancer only and 50,681 breast cancer survivors with multiple cancers in this database. A total of 4740 patients developed a second female genital cancer after a primary breast cancer diagnosis, including 3033 women with second corpus uteri cancer, 295 women with cervical cancer, and 1412 with ovarian cancer. Additionally, analyses were conducted based on the characteristics of the first breast cancer, which included the year of breast cancer diagnosis , age at breast cancer diagnosis , latency period , race , subtype , and menopausal status  [P values were at 0.05 significance alpha levels and were two-sided based on Poisson exact methods. To avoid statistically unstable estimates, the SIRs and CIs of the 0–20 age group were not presented where the number of observed cases was zero [To compare the relative risk with the general population, we used the SEER*Stat Multiple primary-standardized incidence ratios (MP-SIR) tool (version 8.3.4) to calculate the SIRs by dividing the observed numbers of second primary cancers by the expected numbers of second primary cancers based on the rates of the general population, along with the 95% confidence interval (95% CI). CIs and was zero .p value < 0.05. All tests were two-sided.The characteristics of the women with breast cancer only and those with second primary female genital cancer were compared with chi-square tests. We used crude semi-parametric Cox proportional hazards regressions to evaluate the hazards ratios (HRs) and corresponding 95% CIs and to show the risk factors for the development of a second primary female genital cancer after breast cancer. The latency period was started at the first breast cancer diagnosis and censored at the second cancer diagnosis. All statistical analyses and charts were generated using SPSS 19.0  and GraphPad Prism 6.07. Significance levels were set at Table Table The clinical characteristics of the women who developed breast cancer only and those who developed second primary female genital cancer are shown in the P < 0.001; 56+ vs. ≤ 45 years, aHR = 3.218, P < 0.001) , and PR status  . Our study also demonstrated that the risk of second primary ovarian cancer was positively associated with the year of first cancer and age at first cancer   and ER status  .When we considered risk factors for second primary corpus uteri cancer after breast cancer Table , the risIn this study, we found a significantly decreased risk of second cervical cancer in patients with first breast cancer. Viral carcinogenesis is the main risk factor for cervical cancer. Moreover, a previous study showed the induction of aromatase expression in cervical carcinomas and opened the possibility that the use of aromatase inhibitors (AIs) for primary breast cancer may be a potential favorable factor in the development of subsequent cervical cancer . AdditioOur study showed that the SIR for second primary corpus uteri cancer was significantly increased in patients with first breast cancer regardless of ER and PR status. Previous studies have confirmed an increased risk of second corpus uteri cancer after ER-positive breast cancer in patients with tamoxifen therapy compared to the general population , 14. IntOur study suggested that survivors had a higher risk of developing second primary ovarian cancer after the first breast cancer than the general population, which was in line with previous studies , 18. TheIn contrast to previous studies, we had extensive information on cancer risk factors for second female genital cancers, including the year of breast cancer diagnosis, age at breast cancer diagnosis, race, and hormone receptor status. For breast cancer survivors, in the development of second primary female genital cancer, older age (45+ years), black race, and PR+ breast cancer tended to be associated with a higher risk of second corpus uteri cancer. Meanwhile, older age (56+ years), white race, and ER- breast cancer needed heightened vigilance for potential second ovarian cancer. Elevated risks were noted in patients who were perimenopausal or postmenopausal, which may indicate an age-related preference in the risk of second primary female genital cancer among breast cancer patients. However, ER+ breast cancer patients had a decreased risk of developing second ovarian cancer compared to ER− patients. This decreased risk may be due to hormone therapy for first breast cancer. Nevertheless, the SEER database does not contain complete treatment information to evaluate the impact of hormone therapy.Our study had strengths and weaknesses. This study was based on a large, well-established, and standardized population database. However, the heterogeneous population and retrospective analysis of the SEER data were the main limitations. In addition, patients with first primary breast cancer tend to be given more thorough attention than the general population, and a surveillance bias exists for patients with second primary cancer. To address these issues, we analyzed data from a 15-year period and data from 18 registries, making these systemic errors non-differential. Potential pre-existing or synchronous cancers were another concern. Therefore, patients with second primary cancers diagnosed within 6 months of breast cancer diagnosis were excluded from our cohort. Moreover, due to the composition of the dataset, the numbers of patients for second female genital cancers in some race and age groups of breast cancer patients were small. These relevant findings would need further confirmation.In conclusion, this large population-based study suggests that women with first primary breast cancer have higher risks of developing second primary corpus uteri cancer and second primary ovarian cancer than the general population. In contrast, breast cancer survivors have a decreased risk of developing second primary cervical cancer. These increased and decreased risks of developing second primary cancers suggest associations with breast tumor biology and shared genetic susceptibility. Meanwhile, several risk factors were associated with an increase  or a decrease (year of first cancer) of second primary corpus uteri cancer and with an increase  or a decrease  of second primary ovarian cancer. These findings are useful for health planning, including screening and the development of specific guidelines for both the general population and breast cancer patients. Further work is needed to explore possible reasons for this association and the survival outcomes of these patients.Supplementary Table 1Breast Cancer Patient Characteristics within Subgroups (DOCX 16 kb)Supplementary Table 2Breast Cancer Patient Characteristics within Subgroups (DOCX 15 kb)


Finally, for the entire population, combined hormonal contraceptives demonstrated to do not increase breast cancer risk. The results of our study suggest that women at high familial risk and mutation carries develop tumors with different clinical-pathological characteristics and, consequently, are influenced by different protective and risk factors.Reproductive history and exogenous hormonal exposures are acknowledged risk factors for breast cancer in the general population. In women at increased breast cancer risk for genetic predisposition or positive family history, data regarding these risk factors are limited or conflicting, and recommendations for these categories are unclear. We evaluated the characteristics of reproductive life in 2522 women at increased genetic or familial breast cancer risk attending our Family Cancer Center. Breast cancers in BRCA mutation carriers were more likely to be hormone receptor negative, diagnosed at 35 years or before and multiple during the lifetime than tumors in women at increased familial risk, while the distribution of invasive cancers and HER2 positive tumors was similar in the different risk groups. At least one full-term pregnancy (HR 0.27; 95% CI 0.12–0.58;  Breast cancer (BC) represents the most common form of cancer and the leading cause of cancer death among women worldwide, with an estimated 1.7 million cases and 521,900 deaths in 2012. BC alone accounts for 25% of all cancer cases and 15% of all cancer deaths among females [In the general population, acknowledged risk factors for BC are aging, white race, reproductive history , exposures  or hormone replacement therapy), alcohol, tobacco smoking, overweight and obesity, lack of exercise, dense breast tissue, previous chest radiation, precancerous lesions, personal history of BC, family history and mutations in BC predisposition genes  .These risk factors have also been investigated in the high-risk population of BRCA mutation carriers in a variety of studies. Particularly, a recent meta-analysis  showed tIn conclusion, it results unclear the extent to which these modifiers of risk influence BC risk in BRCA mutation carriers and in women with a family history of BC. Nonetheless, an accurate risk assessment in these high-risk populations remains fundamental, in order to establish personalized strategies for risk control .We collected the main reproductive factors through the revision of the medical records of 2527 women attending the MFCC between May 2010 and January 2016. One woman with a MLH1 mutation and 4 women who underwent bilateral prophylactic mastectomy have been subsequently excluded, thus 2522 women were finally analyzed. Characteristics of the entire population divided by subgroups and the distribution of reproductive variables are presented in Table The mean age ± SD of the entire population at the end of the period was 52 ± 13 years, ranging from 17 years to 92 years. Among these women, 113 (5%) had a pathogenic mutation in one of the BRCA genes (VHR Group). The mean age for the mutation carriers at the end of the period was 51 ± 14, years and they were 64 BRCA1 and 49 BRCA2. In addition, 1541 (61%) were at High Risk (HR) with a mean age of 52 ± 13 years and 868 (34%) women were at Intermediate Risk (IR) with a mean age of 52 ± 12 years, according to the Modena criteria. Fourteen women with a BRCA variant of unknown significance (VUS) have been classified according to their family history (2 at Intermediate Risk and 12 at High Risk).p < 0.001) . One hundred and sixty-three of these women developed BC before the first access at the MCFF. The Modena Criteria correctly identified 3 different categories at increasing cumulative BC risk (p = 0.024), diagnosed at 35 years or before (p = 0.002) and multiple during the lifetime (p = 0.017). Consequently, age at diagnosis was significantly lower in VHR compared to HR and IR  (p = 0.007). On the contrary, the distribution of invasive BC (p = 0.338) and HER2 positive tumors (p = 0.545) was similar in the three groups of patients. Nevertheless, it should be noted that BRCA1-related BC were significantly more likely to be hormone receptor negative than BRCA2-related tumors (p = 0.036). Moreover, somewhat surprisingly, we found a higher rate of HER2-negative tumors than that seen in the general population for all the three groups at increasing risk .The characteristics of tumors developed by each group are summarized in Table The cumulative risks at 60 years according to the characteristics of reproductive life and CHCs use for each risk category are presented in Table p = 0.001). In order to avoid the potential bias due to BC detected before pregnancy, in this analysis we included women that developed cancer before getting pregnant in the group of nulliparous. Notably, among parous women, an increasing number of full-term pregnancies seem to be associated with a decrease in BC risk . Interestingly, only in HR group, age at first live birth ≤ 30 showed to significantly reduce risk for BC (p = 0.048).Among BRCA mutation carriers, women with at least one full-term pregnancy compared with nulliparous women had a lower risk of BC (p = 0.005) or more (p = 0.022) than one year showed to significantly reduce the risk for BC.We observed no association between ever having breast-fed and BC risk, among women at increased familial risk (HR and IR groups). In these two groups, there was also no statistically significant association between duration of breastfeeding (more or less than 12 months) and BC risk. Interestingly, only in VHR group, breastfeeding either less (p = 0.384) and for the HR group (p = 0.987). Conversely, in the IR group a miscarriage or an abortion seems to have increased the risk of BC (p = 0.018).We found no association between having a miscarriage or abortion and the risk of BC for the VHR group (p = 0.004). For the analysis of menopausal age, we’ve considered only women diagnosed after their menopause. Interestingly, late menopause (> 50 years) seems to be even protective in BRCA mutation carriers (p = 0.033).In HR women, a late age at menarche (> 12 years) showed to significantly reduce risk for BC . In previous literature, age at first full-term pregnancy demonstrated to be a powerful denominator of future BC risk in the general population, as it determines the length of time the undifferentiated breast tissue remains particularly vulnerable –21. Intep < 0.0001) for every 12 months of breastfeeding [After pregnancy, an extended period of breastfeeding contributes to the functional ripening of the glandular tissue. On these bases, the duration of breastfeeding is believed to have an influence on BC risk. Particularly, the Collaborative Group on Hormonal Factors in Breast Cancer found that the RR for BC decreased by 4.3%  for every year younger at menarche, and independently by a smaller amount , for every year older at menopause. Particularly, exposure to endogenous ovarian hormones are more relevant for estrogen receptor positive disease than for estrogen receptor negative disease and for lobular than for ductal tumors [Several studies have found that women using oral contraceptives have a slightly greater risk of BC than women who have never used them. This risk seems to go back to normal over time once the pills are stopped. Nevertheless, these studies mostly refer to CHC used before 1975 and may be the result of exposure to higher dosages of estrogens , 32. MorOne of the main limitations of our study consists in the lack of data, because several medical records have not been entirely filled. As regards tumor characteristics, the immune-histochemical features were available only for women with invasive carcinomas, diagnosed or treated in the province of Modena in the last 20 years, or for women who provided their past documentation. Moreover, the systematic evaluation of HER2 status became available in our Institution only in 2006, when Trastuzumab has been approved in the adjuvant setting. Another limitation of our study is the non-availability of information regarding start age for CHC, breastfeeding and abortion, which showed in previous literature to play a role in the modulation of BC risk.Somewhat unexpectedly, we found different risk and protective factors between HR and IR women . These results may have three possible explanations. Firstly, the IR group is smaller and with fewer events than the HR group, and this might have affected the statistical power of the analyses. Secondly, we do not have any information regarding the presence of other possible pathogenic mutations in other susceptibility genes in this population of women. It is likely that a part of these women with positive family history, especially in the HR group, may carry an inherited mutation other than BRCA, which may have contributed to modify their risk of developing BC, irrespectively of the characteristics of reproductive life. Finally, women belonging to the IR group might have a lower risk perception compared to HR risk women, because of their slighter familiarity and less intensive surveillance program. Therefore, IR women might have worked less hard in order to change their modifiable risk factors, such as alcohol assumption, tobacco smoking, dietary habits and physical exercise. Overall, these missing genetic and lifestyle factors represent possible confounders in our analysis.Overall, in our study at least one full-term pregnancy, breastfeeding and late age (> 50 years) at menopause represent the principal protective factors in BRCA mutation carriers, while in women at increased familial risk early age at first full-term pregnancy and late menarche represent the principal protective factors. These data are relevant in particular for BRCA mutation carriers, which in our sample got fewer pregnancies than other women, maybe because of the concern of mutation inheritance. These results confirm that pregnancies and the reproductive lifespan play crucial roles in the definition of BC risk in women at increased familial and genetic risk. Moreover, women at high familial risk and mutation carries develop tumors with different clinical-pathological features and, consequently, are influenced by different protective and risk factors. Nevertheless, it is noteworthy that in our sample as well as in literature, hormone receptor negative tumors are mainly associated with BRCA1-mutation carriers, while patients with a BRCA2 mutation seem to develop tumors with histopathological profile more similar to that of tumors affecting the general population , 34. TheSince 1995, the MFCC classifies women with a family history of breast and/or ovarian cancer according to the Modena criteria , 18 and,Criterion 1: At least three relatives with BC in two different generations, regardless of family structure. Alternatively, two relatives with BC and one with OC or one with BC and two with OC in two different generations.Criterion 2: At least two family members affected by BC with a first-degree relationship. Alternatively, one relative with BC and one with OC. In the case of male interposition, a relationship of different degree (second or third) is allowed.Criterion 3: At least one BC diagnosed before 40 years of age or bilateral.The Modena criteria used to classify families are the following:According to these criteria, families are divided into main clusters Table : HereditTogether with these basic clusters, some further criteria have been added Table : Early OThe characteristics of reproductive life were obtained through the revision of the medical records of 2527 women that started their surveillance program at the MFCC between May 2010 and January 2016. Particularly, we evaluated number of pregnancies, age at first full-term pregnancy, breastfeeding, abortions, menarche, menopause, and use of combined hormonal contraceptives (CHC). Also women with a previous diagnosis of breast cancer are admitted to the Center and have been included in the present study. The aim of our study was to analyze the impact of reproductive history and exogenous hormonal exposures in the development of BC in a population of BRCA mutation carriers and in women with family history of breast and/or ovarian cancer.At first, women were divided according to a combination of the Modena Criteria and the Tyrer-Cuzick model in the following three groups:women with a recognized BRCA1/2 mutation at the genetic testing.women not tested for BRCA genes or with a non-informative result, with a 3-fold increased lifetime risk of developing BC according to the Tyrer-Cuzick model or belonging to HBC/HBOC, SHBC/SHBOC, BOC, HOC, and EOBC families according to the Modena criteria.women not tested for BRCA genes or with a non-informative result, not belonging to the High Risk Group, with a 2-fold increased lifetime risk of developing BC according to the Tyrer-Cuzick model or belonging to FBC/FBOC, SFBC/SFBOC, SFBC+/SFBOC+ and MBC families according to the Modena criteria.Women already diagnosed with BC at the time of the first access were classified according to the Modena Criteria, since the Tyrer-Cuzick model should not be applied to affected women.Interestingly, the combined classification did not show to correctly stratify the cumulative risk at 60 years of these women Figure  and HR wwomen with a recognized BRCA1/2 mutation at the genetic testing.women not tested for BRCA genes or with a non-informative result, belonging to HBC/HBOC, SHBC/SHBOC, BOC, HOC, and EOBC families.women not tested for BRCA genes or with a non-informative result, belonging to FBC/FBOC, SFBC/SFBOC, SFBC+/SFBOC+ and MBC families.Notably, women with a BRCA variant of unknown significance (VUS) have been classified according to their family history, as current Guidelines recommend. One woman with a MLH1 mutation and 3 women who underwent bilateral prophylactic mastectomy have been excluded from the analyses.Since the median age of these women was quite low, we decided to evaluate the cumulative risk at 60 years according to the characteristics of their reproductive life. Analyses were conducted separately for subjects with mutations and women with a family history of BC according to the three risk categories. Unknown data  has been excluded from the analyses.p-value < 0.05 were considered significant.In the descriptive analysis, the continuous variables were summarized by means of mean and standard deviation (SD), and the categorical variables were reported as absolute and percent values. Distributions of continuous covariates by group were compared using one-way ANOVA . The comparison of categorical variables between groups was performed using the Chi2 test or Fisher's exact test, when appropriate. The cumulative risk to develop BC was calculated from the age of menarche to the age of diagnosis of BC, or age of the last control for censored patients. The cumulative hazard rate was calculated and graphically reported by means of Nelson-Aalen estimator . The cum


PALB2, CHEK2 and ATM make it difficult to estimate precisely associated cancer risks. Population-based family studies have provided evidence that at least some of these mutations are associated with breast cancer risk as high as those associated with rare BRCA2 mutations. We aimed to estimate the relative risks associated with specific rare variants in PALB2, CHEK2 and ATM via a multicentre case-control study.The rarity of mutations in PALB2 c.1592delT, c.2816T>G and c.3113G>A, CHEK2 c.349A>G, c.538C>T, c.715G>A, c.1036C>T, c.1312G>T, and c.1343T>G and ATM c.7271T>G. We assessed associations with breast cancer risk (42 671 cases and 42 164 controls), as well as prostate (22 301 cases and 22 320 controls) and ovarian (14 542 cases and 23 491 controls) cancer risk, for each variant.We genotyped 10 rare mutations using the custom iCOGS array: PALB2 c.1592delT OR 3.44 , PALB2 c.3113G>A OR 4.21  and ATM c.7271T>G OR 11.0 . We also found evidence of association with breast cancer risk for three variants in CHEK2, c.349A>G OR 2.26 (95% CI 1.29 to 3.95), c.1036C>T OR 5.06 (95% CI 1.09 to 23.5) and c.538C>T OR 1.33 (95% CI 1.05 to 1.67) (p≤0.017). Evidence for prostate cancer risk was observed for CHEK2 c.1343T>G OR 3.03  for African men and CHEK2 c.1312G>T OR 2.21  for European men. No evidence of association with ovarian cancer was found for any of these variants.For European women, strong evidence of association with breast cancer risk was observed for This report adds to accumulating evidence that at least some variants in these genes are associated with an increased risk of breast cancer that is clinically important. PALB2, CHEK2 and ATM.The rapid introduction of massive parallel sequencing (MPS) into clinical genetics services is enabling the screening of multiple breast cancer susceptibility genes in one assay at reduced cost for women who are at increased risk of breast (and other) cancer. These gene panels now typically include the so-called ‘moderate-risk’ breast cancer susceptibility genes, including PALB2 c.1592delT (a Finnish founder mutation), PALB2 c.3113G>A and ATM c.7271T>G. These mutations have been estimated to be associated with a 40% (95% CI 17% to 77%), 91% (95% CI 44% to 100%) and 52% (95% CI 28% to 80%) cumulative risk of breast cancer to the age of 70 years, respectively.BRCA2: 45% (95% CI 31% to 56%).Estimation of the age-specific cumulative risk (penetrance) of breast cancer associated with specific mutations in these three genes has been limited to those that have been observed more frequently, such as  95% CI 2% to 80% Case-control studies provide an alternative approach to estimating cancer risks associated with specific variants. This design can estimate the relative risk directly, without making assumptions about the modifying effects of other risk factors. However, because these variants are rare, such studies need to be extremely large to provide precise estimates.PALB2, CHEK2 and ATM relate to protein truncating and splice-junction variants.et alATM, which is comparable to that for truncating variants. Specifically, ATM c.7271C>G has been associated with a more substantial breast cancer risk in several studies.et al,CHEK2 from similarly designed studies were 6.18 (95% CI 1.76 to 21.8) for rare protein-truncating and splice-junction variants and 8.75 (95% CI 1.06 to 72.2) for evolutionarily unlikely missense substitutions.11The clearest evidence for association, and the most precise breast cancer risk estimates, for rare variants in PALB2, CHEK2 and ATM could be associated with increased risk of cancers other than breast cancer, as has been observed for BRCA1 and BRCA2 and both ovarian and prostate cancers.PALB2 carriers, there is little evidence to support or refute the existence of such associations, although a few individually striking pedigrees have been observed.18–20It is plausible that monoallelic mutations in PALB2, CHEK2 or ATM. These included three rare variants in PALB2: the protein truncating variants c.1592delT (p.Leu531Cysfs)ATM c.7271T>G .22In this study we selected rare genetic variants on the basis that they had been observed in breast cancer candidate gene case-control screening projects involving Participants were drawn from studies participating in three consortia as follows:Breast Cancer Association Consortium (BCAC), involving a total of 48 studies: 37 of women from populations with predominantly European ancestry (42 671 cases and 42 164 controls), 9 of Asian women (5795 cases and 6624 controls) and 2 of African-American women (1046 cases and 932 controls). All cases had invasive breast cancer. The majority of studies were population-based or hospital-based case-control studies, but some studies of European women oversampled cases with a family history or with bilateral disease (see online The 10.1136/jmedgenet-2016-103839.supp1Supplementary dataProstate Cancer Association Group to Investigate Cancer Associated Alterations in the Genome (PRACTICAL) involving a total of 26 studies: 25 included men with European ancestry (22 301 cases and 22 320 controls) and 3 included African-American men (623 cases and 569 controls). The majority of studies were population-based or hospital-based case-control studies (see online The Ovarian Cancer Association Consortium (OCAC), involving a total of 46 studies. Some studies were case-only and their data were combined with case-control studies from the same geographical region (leaving 36 study groupings). Of these groupings, 33 included women from populations with predominantly European ancestry (16 287 cases (14 542 with invasive disease) and 23 491 controls), 25 included Asian women (813 cases (720 with invasive disease) and 1574 controls), 17 included African-American women (186 cases (150 with invasive disease) and 200 controls) and 29 included women of other ethnic origin (893 cases (709 with invasive disease) and 864 controls). The majority of studies were population-based or hospital-based case-control studies ATM We selected for genotyping 13 rare mutations that had been observed in population-based case-control mutation screening studies. These variants were PALB2 variants c.2323C>T (p.Gln775*), c.3116delA (p.Asn1039IIefs) and c.3549C>G (p.Tyr1183*) were unable to be designed for measurement on the custom Illumina iSelect genotyping array and were not considered further . p Values were determined by the likelihood ratio test comparing models with and without carrier status as a covariate. We also applied conditional logistic regression, defining risk sets by study, and found that this made no difference to the OR estimates, CIs or p values to two significant figures; since model convergence was a problem for this latter regression analysis, all subsequent analyses were based on unconditional logistic regression. For the main analyses of breast cancer risk in European women, we also included as covariates the first six principal components, together with a seventh component specific to one study (Leuven Multidisciplinary Breast Centre (LMBC)) for which there was substantial inflation not accounted for by the components derived from the analysis of all studies. Addition of further principal components did not reduce inflation further. Data from all breast cancer studies were included to assess statistical significance. Data from cases selected for inclusion based on personal or family history of breast cancer were excluded in order to obtain unbiased OR estimates for the general population of white European women (leaving 37 039 cases and 38 260 controls from 32 studies). Multiple testing was adjusted for using the Benjamini-Hochberg procedure to control the false discovery rate, with a significance threshold of 0.05.PALB2 c.1592delT (Leu531Cysfs) was only observed in 35 cases and 6 controls, all from four studies from Sweden and Finland (Helsinki Breast Cancer Study (HEBCS), Kuopio Breast Cancer Project (KBCP), Oulu Breast Cancer Study (OBCS) and Karolinska Mammography Project for Risk Prediction Breast Cancer (pKARMA); see online −5); the OR estimate was 4.52 (95% CI 1.90 to 10.8) based on all studies and 3.44 (95% CI 1.39 to 8.52) based on unselected cases and controls  was identified in 44 cases and 8 controls from nine BCAC studies. Only one carrier of the variant was of non-European origin. Strong evidence of association with breast cancer risk was observed (p=6.9×10−8), with an estimated OR of 5.93 (95% CI 2.77 to 12.7) based on all studies and 4.21 (95% CI 1.85 to 9.61) based on unselected cases and controls. There was no evidence of a differential association by ER status (p=0.15).PALB2 variants (c.1592delT or c.3113G>A) was 3.85 (95% CI 2.09 to 7.09).Based on unselected cases, the estimated OR associated with carrying either of these PALB2 c.2816T>G (p.Leu939Trp) was identified in 150 cases and 145 controls and there was no evidence of association with risk of breast cancer. There was no evidence of association with risk of prostate or ovarian cancer for any of the three PALB2 variants (see ants see  and 4.CHEK2 c.349A>G (p.Arg117Gly) was identified in 44 cases and 18 controls in studies participating in BCAC; all of these women were of European origin. We found evidence of association with breast cancer (p=0.003), with little change in the OR after excluding selected cases (OR 2.03 (95% CI 1.10 to 3.73)).CHEK2 c.538C>T (p.Arg180Cys) was identified in 158 breast cancer cases and 142 controls in studies of white Europeans. Evidence of association with breast cancer risk (p=0.016) was observed, with an unbiased OR estimate of 1.34 (95% CI 1.06 to 1.70). A consistent OR estimate was observed for Asian women, based on 45 case and 45 control carriers (OR 1.16 (95% CI 0.75 to 1.76)).CHEK2 c.715G>A (p.Glu239Lys) mutations were identified in 15 cases and 9 controls, all European women participating in BCAC and no evidence of association with risk of breast cancer was observed (p=0.21).CHEK2 c.1036C>T (p.Arg346Cys) was identified in nine cases from seven studies and two controls from two different studies in BCAC (neither control carrier was from a study that had case carriers), all of European origin. We found evidence of association with breast cancer risk (p=0.017) with reduced OR estimate of 3.39 (95% CI 0.68 to 16.9) after excluding selected cases.CHEK2 variants (CHEK2 c.349A>G (p.Arg117Gly); c.538C>T (p.Arg180Cys); c.715G>A (p.Glu239Lys) and c.1036C>T (p.Arg346Cys)) were found to be associated with an increased risk of prostate or ovarian cancer (CHEK2 variant c.1312G>T (p.Asp438Tyr) was not associated with risk of breast cancer for European women (p=0.91). Variant c.1343T>G (p.Ile448Ser) was not observed in any breast cancer cases of European or Asian origin. It was detected in 48 cases and 29 controls of African origin, giving weak evidence of association ). CHEK2 c.1312G>T (p.Asp438Tyr) was identified in 23 cases and 11 controls from PRACTICAL, all European, providing evidence of association with prostate cancer risk ). CHEK2 c.1343T>G (p.Ile448Ser) was observed in 35 cases and 11 controls, all African, participating in PRACTICAL and was also associated with an increased risk of prostate cancer ). There was no evidence that these CHEK2 variants were associated with risk of ovarian cancer  was identified in 12 cases and 1 control in studies participating in BCAC, all of European origin, giving evidence of association with breast cancer risk (p=0.0012). The OR estimate based on unselected studies was 11.0 (95% CI 1.42 to 85.7). There was no evidence of association of this variant with prostate or ovarian cancer risk see  and 4.24Gly wasrare variants in so-called ‘moderate-risk’ genes are associated with an increased risk of breast cancer that is of clinical relevance.The present report adds to an accumulating body of evidence that at least some PALB2, CHEK2 and ATM, variants generated from these new testing platforms is not being used in clinical genetics services due to lack of reliable estimates of the cancer risk associated with individual variants, or groups of variants, in each gene. Previous analyses have been largely based on selected families, relying on data on the segregation of the variant. The present study is by far the largest to take a case-control approach. Consistent with previous reports,PALB2 c.3113G>A (p.Trp1038*), PALB2 c.1592delT (p.Leu531Cysfs) and ATM c.7271T>G  were found to be associated with substantially increased risk of breast cancer all with associated relative risk estimates of 3.44 or greater.These findings are presented at a time when detailed information about variants in these genes is becoming more readily available via the translation of diagnostic genetic testing from Sanger sequencing-based testing platforms to MPS platforms that test panels of genes in single assays.PALB2 variants (c.1592delT and c.3113G<A) were consistent with each other and with estimates based on segregation analysis.PALB2 c.2816T>G (p.Leu939Trp), with an upper 95% confidence limit excluding an OR >1.5 which is notable given the Align-Grantham Variation Granthan Deviation (Align-GVGD) score and the observed impact on protein function.30The estimates for the two loss-of-function ATM c.7271T>G  was also consistent with that found by segregation analysis.ATM c.7271T>G  could be due to the reduction in kinase activity  observed for ATM p.Val2424Gly,32The estimate for PALB2 c.3113G>A and 1.9 for c.1592delT and c.3113G>A combined.In contrast, we found no evidence of an association with risk of prostate or ovarian cancer with any of these three variants: however, the confidence limits were wide; based on the upper 95% confidence limit we could exclude an OR of >1.4 for prostate cancer for the loss-of-function CHEK2. Two of these  and c.1036C>T (p.Arg346Cys)) had evidence of a significant impact on the protein based on in silico prediction. We proposed these variants for inclusion in the iCOGS design as they had been identified in 3/1242 cases and 1/1089 controls and 3/1242 cases and 0/1089 controls, respectively, in a population-based case-control mutation screening study of CHEK2.et al, estimated an OR of 8.75 (95% CI 1.06 to 72.2) for variants with an Align-GVGD score C65 (based on nine cases and one control). The current analysis provides confirmatory evidence of this association in a much larger sample (OR 2.18 (95% CI 1.23 to 3.85)) including 40 unselected case and 18 control carriers. The evidence that CHEK2 is a breast cancer susceptibility gene is largely based on studies of protein truncating variants, in particular CHEK2 1100delC.We analysed six rare missense variants in PALB2 c.1592delT, c.3113G>A and ATM c.7271T>G because these associations had previously been reported; our aim was to more precisely estimate the associated relative risks. All three associations with breast cancer risk reported for CHEK2 variants remained statistically significant after adjusting for the other tests conducted in relation to breast cancer risk, but not after correcting for all tests for all cancers. Nevertheless, the findings for CHEK2 c.349A>G and c.1036C>T confirmed those reported previously, although collectively. The association observed with CHEK2 c.538C>T requires independent replication.The p values reported above have not been adjusted for multiple testing. This was not considered appropriate for the associations with breast cancer risk of et al,PALB2 c.1592delT; c.3113G>A ATM c.7271T>G) would place the carrier in a high-risk group, especially if other risk factors, such as a family history, are present. The high level of breast cancer risk associated with PALB2 c.1592delT and c.3113G>A reported here is consistent with the penetrance estimate reported for a group of loss-of-function mutations in PALB2Do this approach and new data have an impact on clinical recommendations for women and families carrying these rare genetic variants? Although age-specific cumulate risks for cancer are more informative for genetic counselling and clinical management of carriers, our study provides information that is relevant to clinical recommendations. As discussed in Easton The consistency of the relative risk estimates with those derived through family based studies supports the hypothesis that these variants combine multiplicatively with other genetic loci and familial risk factors; this information is critical for deriving comprehensive risk models. Even with very large sample sizes such as those studied here, however, it is still only possible to derive individual risk estimates for a limited set of variants, and even for these variants the estimates are still imprecise. This internationally collaborative approach also has limited capacity to improve risk estimates for rare variants that are only observed in specific populations. Inevitably, therefore, risk models will depend on combining data across multiple variants, using improved in silico predictions and potentially biochemical/functional evidence to synthesise these estimates efficiently. It will also be necessary develop counselling and patient management strategies that can accommodate a multifactorial approach to variant classification.10.1136/jmedgenet-2016-103839.supp2Supplementary Financial Support


While translational genomic sequencing research is increasing, few studies have been limited to healthy individuals; most have focused on patients with a disease or a strong family history of a disorder. The limited studies that have included healthy individuals have focused on the disclosure of medically actionable secondary results, rather than carrier status, to assess reproductive risks. To address this important gap, we conducted the NextGen study, which focuses on carrier status and medically actionable secondary findings in a population of women planning a pregnancy.We assessed 310 participants’ motivations for receiving genome sequencing for expanded carrier screening and experiences with familial genetic conditions that may relate to study participation.Most participants reported that obtaining general health information from genome sequencing was their primary motivator, even though they were recruited to join a study to learn more about carrier status. Forty‐two percent of enrolled women became pregnant prior to obtaining sequencing results.Genomic carrier testing may need to be offered to women prior to active pregnancy efforts to be useful for reproductive planning. The majority of genetic testing related to reproduction occurs prenatally through screening tests such as blood tests and ultrasounds and diagnostic tests such as amniocentesis or chorionic villus sampling  consortium, for example, most studies disclosed carrier status as a secondary (not primary) finding  Institutional Review Board. All participants provided written consent for participation and received written information on study procedures. This research was conducted as part of the National Human Genome Research Institute (NHGRI) CSER consortium.The detailed study design has been published elsewhere ?”We conducted a content analysis of the recorded responses to the open‐ended questions to identify key themes , women who enrolled in the study were older, more likely to be white, and more likely to have higher education and higher income ; 95.5% of participants were <40 years old. Compared to the general population of women within KPNW who became pregnant during the recruitment period . While 49% reported knowing a family with a child with a participant‐defined genetic condition, it is unknown if the conditions were actually monogenic disorders.The 292 participant responses to the first open‐ended question were categorized into three key themes: general information, reproductive planning, and support of research , secondary findings (e.g. “autoimmune conditions for her”), unknown family history , conditions related to known family history of disorder , or no response Table . Of the BRCA, “the breast cancer gene”). Of the 54 women who listed female‐specific cancers, 36 (67%) indicated a family history of that cancer. Twenty of 140 participants (14%) responded with a secondary finding that did not fit into one of the predefined disease categories, including poor vision, mental health issues, gallbladder problems, and Ehlers‐Danlos syndrome. Of the 140 participants who reported an interest in secondary findings, 36 (26%) of these responses included more than one disease category .Interest in secondary findings was the most common response to this question 140/45%), and participants mentioned a wide variety of conditions related to the participants’ own health. These conditions were coded into disease categories and are displayed in Fig. 40/45%, aCarrier status was mentioned by 50 (16%) of participants. This included specific conditions  and more general genetic diagnosis concerns . Responses captured in all categories included genetic information disclosed through study genome sequencing and information not currently available via sequencing .This study illuminated healthy individuals’ motivation for receiving genome sequencing. Our participants reported strikingly similar motivations for joining compared to other translational genome sequencing studies, a result that is surprising since most other related studies recruited participants with a known disease who were seeking a diagnosis for a suspected genetic disorder based on phenotype  many genetic conditions are very rare, and outside of a handful of more well‐known conditions such as cystic fibrosis , most people cannot name any autosomal recessive or X‐linked disorder; (2) we primed women to be aware of cancer as a secondary finding during the recruitment and consent process, since Hereditary Breast and Ovarian Cancer (HBOC) mutations are fairly widely known and an easily accessible way to explain medically actionable results to lay participants; (3) in recent years, HBOC has been extensively discussed in the popular media  as our population, and had cardiomyopathies or were comprised of a population enriched for atherosclerotic heart disease , the reasons for participation documented here may not be comprehensive. In these cases, we used the most conservative coding  to ensure we were not over interpreting responses. While women may have been primed during the recruitment process to mention interest in HBOC, the majority (67%) of women who indicated they were interested in a female cancer as a secondary finding mentioned a specific family history in their response. This makes it unlikely that the recruitment priming was the sole motivator for the over‐representative interest in cancer as a secondary finding. Our participants consented to clinical carrier testing as an eligibility requirement for participation in the study and may not represent the views of those who had declined or had not been offered clinical testing. Our population lacks diversity in terms of race/ethnicity, education level, marital status, and income. Our ability to broadly apply our findings to a more diverse population is limited.Open‐ended responses from participants were obtained from three different genetic counselors and were not recorded verbatim. However, the genetic counselors are professionally trained and experienced in documenting patient comments in the electronic medical record as part of their role in standard care. Because the full depth of the response may not have been recorded or fully elicited from the participant (e.g., if they responded “breast cancer” it may be unclear if they are interested due to family history, an erroneous assumption that most breast cancer is due to Women planning a future pregnancy who are interested in genomic carrier screening have motivations for obtaining genomic screening that are very broad and not limited to pregnancy planning. Future research should strive to increase diversity in the populations receiving sequencing to see if their reasons for utilizing the technology differ. Additionally, future genomic carrier screening research should offer screening to women earlier in their childbearing years to allow for result disclosure and subsequent reproductive planning options prior to actively attempting a pregnancy.SAI reports previous, unrelated research support from Medimmune; no conflicts exist with the current work. TLK, MCL, MJG, PH, CKM, EM, BJW, and KABG do not have any conflicts to disclose. The study sponsor, National Human Genome Research Institute, approved the study design and encouraged use of measures common to other sites in the Clinical Sequencing Exploratory Research consortium, but did not have input as to the data analysis, interpretation, or writing of this manuscript.


Lifestyle factors may influence the risk of developing pancreatic cancer. Whereas cigarette smoking is an established risk factor, the effects of high alcohol intake and obesity are more uncertain. The aim of the present study was to examine the associations of pre-diagnostic anthropometry, alcohol consumption, and smoking habits with pancreatic cancer risk in a Swedish prospective, population-based cohort, with particular reference to potential sex differences.n = 163). Non-parametric and chi-squared tests were applied to compare the distribution of risk factors between cases and non-cases. Cox regression proportional hazards models were used to estimate the relationship between investigative factors and pancreatic cancer risk. Anthropometric factors included height, weight, body mass index (BMI), waist and hip circumference, waist-hip ratio (WHR), and body fat percentage.The studied cohort consists of 28,098 participants, including all incident cases of pancreatic cancer, in the Malmö Diet and Cancer Study up until December 31, 2013 (p for trend = 0.009). Regular smoking was a significant risk factor among both women  and men , whereas occasional smoking was a significant risk factor only in women . Passive smoking at work for >20 years was significantly associated with an increased risk in the entire cohort  and in women selectively . Alcohol consumption was not a significant risk factor. A significant interaction was found between female sex and age (p = 0.045), but no other factor, in relation to pancreatic cancer risk.BMI was not a significant risk factor for pancreatic cancer, but a higher WHR was significantly associated with an increased risk in the entire cohort (hazard ratio (HR) 2.36, 95% confidence interval (CI) 1.28–4.35, WHR was the only pre-diagnostic anthropometric factor associated with pancreatic cancer risk, with no sex-related differences. Regular smoking was confirmed as a significant risk factor in both sexes, whereas occasional and passive smoking were significant risk factors only in women. Despite the lack of a significant interaction between smoking and sex in relation with pancreatic cancer risk, potential sex differences should be considered in future epidemiological studies. In 2012, pancreatic adenocarcinoma represented 3% of all cancer cases in developed countries, non-melanoma skin cancer excluded –6, but dSeveral risk factors for pancreatic cancer have been reported; however, only a few of them have been confirmed more consistently. Tobacco smoking seems to be the only established modifiable risk factor –10, but and at work/at home in adult life was found to significantly increase the risk of pancreatic cancer among never smokers, compared to those never exposed. Exposure to ETS at home or at work in adult life was borderline significantly associated with pancreatic cancer risk, but if only exposed during childhood, there was no increased risk. In that study, potential differences between sexes regarding ETS were not considered.While active smoking is considered a well-established risk factor for pancreatic cancer, the risk associated with passive smoking is less clear. In a previous study based on The European Prospective Investigation into Cancer and Nutrition (EPIC) cohort , environThe association between alcohol consumption and risk of pancreatic cancer is also under debate, and the hitherto published results are inconclusive. Some studies demonstrate an increased risk of pancreatic cancer in heavy alcohol drinkers –27, whilThe aim of the present study was to examine the associations of anthropometric factors, smoking and alcohol consumption, with pancreatic cancer risk in the Malmö Diet and Cancer Study, with particular reference to potential differences between sexes.The Malmö Diet and Cancer Study (MDCS) is a large prospective, population-based study, which is also part of The European Prospective Investigation into Cancer and Nutrition (EPIC). MDCS/EPIC is a consecutive cohort including men and women between 44 and 73 years of age at baseline, recruited between 1991 and 1996, with a total of 28,098 participants . The cohort has been described in detail previously –37.From baseline until last follow-up on December 31, 2013, 172 cases of pancreatic cancer were reported from the Swedish Cancer Register among the participants. Following review of pathology records, five cases were classified as either endocrine pancreatic cancer or other types of non-adenocarcinoma and these cases were transferred to the group of non-cases. For another four cases, no pathology reports or medical records were available and the diagnosis could not be confirmed, and these were excluded from all analyses, rendering a total number of 163 cases with confirmed pancreatic cancer, 61 men  and 102 women . A flowchart of the cohort is shown in Fig. Cox regression proportional hazards models were applied to investigate the impact of age at baseline, sex (entire cohort), smoking habits, ETS, alcohol consumption, and diabetes with pancreatic cancer-free person-years. The analyses were performed with adjustment for age only or for all factors except ETS. The potential interaction with risk factor and sex was tested with the covariates age, sex, risk factor, and risk factor × sex, and also repeated with the fully adjusted model. Cox regression proportional hazards models were further applied to estimate the impact of anthropometric factors on pancreatic cancer-free person-years adjusted for age, sex, diabetes mellitus (DM), smoking habits, and alcohol habits. Person-years were calculated by subtracting date at baseline (date of study entry) from date at diagnosis/date at migration/date of study end .Included anthropometric variables were height, weight, BMI, hip circumference, waist circumference, waist-hip ratio (WHR), and body fat percentage, which were all divided into tertiles for the statistical analyses. Alcohol consumption habits were recorded in grams per day, and smoking habits denoted as regularly, occasionally, former, and never. ETS was recorded as exposure during childhood (yes/no), exposure at home (no/<10 years/10–20 years/>20 years), or exposure at work (no/<10 years/10–20 years/>20 years).In the adjusted analyses, diabetes was defined as diagnosis of diabetes more than 24 months prior to endpoint or to the date of pancreatic cancer diagnosis, separating these participants from the group of participants with new-onset diabetes, i.e., cases with confirmed diabetes less than 24 months prior to the endpoint or to the date of pancreatic cancer diagnosis.p values <0.05 were considered significant.All statistical tests were two sided. All statistical analyses were performed using IBM SPSS Statistics version 22.0 .The distribution of investigative factors for individuals who developed pancreatic cancer during follow-up (cases) and for those who did not (rest of cohort) is shown in Table Age-adjusted risk of incident pancreatic cancer in relation with sex, smoking, ETS at work, alcohol consumption, and diabetes is shown in Table High age was significantly associated with an increased risk of pancreatic cancer in the entire cohort  and among women separately , but not among men. Alcohol consumption was not significantly associated with risk of pancreatic cancer, neither in the entire cohort nor in subgroup analysis according to sex. Prevalent diabetes did not correlate significantly with risk of pancreatic cancer.Risk of pancreatic cancer in relation with sex, age, smoking, ETS at work, alcohol consumption, and diabetes in the fully adjusted model is shown in Table In order to assess potential dose-dependent effects, we also examined the risk of pancreatic cancer according to tertiles of alcohol consumption in both the age-adjusted and fully adjusted model, but found no significant associations (data not shown).p for interaction = 0.045) in relation with pancreatic cancer risk. There was no significant interaction with any of the other factors and sex, neither in the age-adjusted nor in the fully adjusted model.In the fully adjusted model Table , there wETS during childhood and at home were not significantly associated with pancreatic cancer risk, neither in the entire cohort nor in sex-stratified analysis (data not shown).Risk of incident pancreatic cancer in relation with anthropometric factors is shown in Table p for trend = 0.009). This significant difference was not observed in subgroup analysis according to sex. There was a borderline significant association of high waist circumference with pancreatic cancer risk in the entire cohort (p for trend = 0.063), but not in subgroup analysis according to sex.In the entire cohort, the risk of incident pancreatic cancer was significantly higher for individuals with a high WHR .Inclusion of WHR in the fully adjusted model in Table In this study, we have examined the associations of anthropometric factors, and two other major lifestyle-related risk factors, smoking and alcohol consumption, with risk of pancreatic cancer, with particular reference to potential differences between sexes, in the Malmö Diet and Cancer study.As expected, the results confirm smoking as an important risk factor for incident pancreatic cancer, overall and in both sexes. This association was particularly evident in regular smokers, but even occasional smoking was shown to be significantly associated with risk of pancreatic cancer in women, but not in men. An additional difference between sexes was observed in that exposure to ETS at work for more than 20 years was found to be significantly associated with an increased risk of pancreatic cancer in women, but not in men. This association remained significant in the group of never and former smokers; however, in contrast to the results in the study on the EPIC cohort, no significant correlations to risk were seen in the group of only never smokers regarding ETS . The finIt is noteworthy that former smokers were not shown to have an increased risk of incident pancreatic cancer in the present study. One could ponder that regular smoking would be a potential risk factor not only when ongoing but also for a period of time after quitting. A previous study on the EPIC cohort found that both regular smokers and former smokers, who had smoked for more than 25 years or quit less than 5 years prior to study entry, had an increased risk of incident pancreatic cancer . Those wIn the present study, alcohol consumption did not differ significantly between cases and non-cases and was not found to be significantly associated with pancreatic cancer risk, neither in the entire cohort nor in sex-stratified analysis. High alcohol consumption is a well-known risk factor for pancreatitis , 32, andPrevalent diabetes has in many studies been highlighted as a risk factor for pancreatic cancer –15. AccoThe observation that age at baseline was higher among cases compared with the remaining cohort is expected, since the incidence of pancreatic cancer in Sweden in 2014 peaked at the age of 70–74 years, and the median age at baseline for the entire cohort of this study was 57.8 years, with the oldest individual being 74 years. Hence, the individuals entering the study at a higher age than the median were closer to their age of peak incidence. Of note, this significant difference was only seen in the entire cohort, and among women, with a significant interaction between female sex and age in the fully adjusted model.Another purpose of this study was to investigate the relationship between pre-diagnostic anthropometry and risk of pancreatic cancer, overall and according to sex. Apart from the finding of a high WHR being significantly associated with pancreatic cancer risk in the entire cohort, but not in sex-stratified analysis, none of the other investigated anthropometric factors was significantly associated with pancreatic cancer risk, neither in the entire cohort nor in sex-stratified analyses. According to the existing literature, BMI has been proposed as the anthropometric factor with the strongest association to risk of pancreatic cancer, but yet there is no definite consensus , 18, 19.Of note, the number of female participants in the MDCS is higher than the number of male participants, which resulted in a higher number of female than male pancreatic cancer cases. However, the incidence rate of pancreatic cancer did not differ significantly between men and women, which confirms the contemporary equal incidence rate of pancreatic cancer between the sexes , 3.In this study, regular smoking was confirmed to be a strong risk factor for pancreatic cancer in both sexes, but the relationship between occasional smoking and long-term environmental exposure with pancreatic cancer risk was only observed in women. Despite the lack of a significant interaction with sex, these findings suggest that the carcinogenic effects of smoking may be more hazardous in women and emphasize the importance of taking potential sex differences into consideration in epidemiological studies and prevention efforts. Furthermore, the results from this study demonstrate that a high waist-hip ratio was the only pre-diagnostic anthropometric factor being significantly associated with pancreatic cancer risk, with no difference between sexes.