BRCA1/2 individuals in the Sri Lankans. Hence we performed linkage analysis to identify susceptibility loci related to the inherited risk of cancer in a cohort of Sri Lankans affected with hereditary breast cancer. The Illumina global screening array having 654,027 single nucleotide polymorphism markers was performed in four families, in which at least three individuals within third degree relatives were affected by breast cancer. Two-point parametric linkage analysis was conducted assuming disease allele frequency of 1%. Penetrance was set at 90% for carriers with a 10% phenocopy rate.Although linkage studies have been utilized for the identification of variants associated with cancer in the world, little is known about their role in non MYO16 on chromosome 13. The two most informative families also suggested several candidate linked loci in genes, including ERAP1, RPRM, WWOX, CDH1, EXOC1, HUS1B, STIM1 and TUSC1. This study provides the first step in identifying germline variants that may be involved in risk of cancer in cancer-aggregated non-BRCA1/2 families from the understudied Sri Lankan population. Several candidate linked regions showed suggestive evidence of linkage to cancer risk.Thirty-one variants exhibited genome-wide suggestive HLODs. The top overall HLOD score was at rs1856277, an intronic variant in The online version contains supplementary material available at 10.1186/s13104-022-06081-5. BRCA1 and BRCA2 [BRCA1 and BRCA2 pathogenic variants [Inheritance of cancers among individuals in high risk families can be explained by significant familial aggregation of high, moderate or low penetrance genetic variants in cancer predisposing genes (CPGs) that are transmitted down the generations in each family . Breast nd BRCA2 . Howevernd BRCA2 . It is avariants . So far variants . There iThis is the first linkage analysis study conducted in the families affected with cancer in the Sri Lankan population. A genome wide linkage (GWL) scan was performed using data from 48 individuals from 4 cancer families, aiming to evaluate the possibility of identifying susceptibility loci conferring breast and other cancer predisposition.Index cases recruited into this study were women affected with breast cancer who had a family history of breast cancer but who have been identified as having no variants in CPGs   using the Illumina Global Screening Array which has 654,027 single nucleotide polymorphism (SNP) markers on the array. Quality assessment of the samples was performed by QuantiFluor. The genome-wide content was selected for high imputation accuracy at minor allele frequencies of  > 1% across all 26 1000 genomes project populations.The software program PLINK  was usedWe performed two-point parametric linkage analysis on this data using Merlin , which uMYO16 on chromosome 13. There were 13 HLOD scores greater than 2.00. Family 1 was not particularly informative by itself. There was no individual LOD score  > 0.54 and nearly every chromosome had a LOD score of that value  gene has been associated with poor clinical outcomes of patients affected with triple negative breast carcinoma [RPRM) is a potential p53-dependent tumor suppressor gene [RPRM gene has been found to be frequently hypermethylated in several human cancers [WWOX) gene has been reported mainly in breast cancer but also including ovarian, esophageal, lung and stomach carcinoma, and multiple myeloma [WWOX gene has been identified as a genome wide suggestively linked variant in this cohort. The region on chromosome 16 where E-cadherin (CDH1) gene is located is frequently associated with loss of heterozygosity and loss of tumour suppressor function in several cancers, including gastric [CDH1 gene resides. Family 2 showed top linked variants in multiple genes that are interesting candidates for cancer susceptibility, including KCNJ3 and EXOC2.The present linkage study performed across 4 Sri Lankan non-arcinoma . Reprimo cancers . Loss of myeloma . An intr gastric , breast  gastric  and ovar gastric . We haveBRCA1/2 families have been conducted in other populations [Several studies on linkage analysis in non ulations –25. HoweThere are several strengths and weaknesses of this study. In a heterogeneous disease like cancers, it would be unsurprising to find novel candidate genes and variants in different populations and in different families within a population. Family-based linkage studies such as this are able to utilize the long, linked haplotypes shared by closely related affected individuals, allowing for identification of linked chromosomal regions that may harbor causal variants that might not have been genotyped in this study. Variants that are rare in the general population may also be enriched in individual families ascertained for a strong family history of cancer, particularly early-onset cancers.In conclusion, this study provides the first step in identifying germline causal variants that may be involved in risk of cancer in cancer-aggregated families from the understudied Sri Lankan population. Several candidate chromosomal regions showed suggestive evidence of linkage to cancer risk.There are some limitations in this study as well. First, we used a microarray chip, meaning that there were large numbers of variants that were not genotyped. Thus, it is possible that we may have not identified a causal variant in this study, but more likely, a variant(s) that is linked to the casual variant. Even in family 2 where we were able to identify candidate exonic variants in the index case that were within the linked regions, it is possible that a variant that is in this linkage region but not covered by WES is the causal variant. Targeted sequencing of the candidate regions will be needed to elucidate the true causal variants. It is of course also possible that all of these linkage results are false positives because of the relatively small number of biospecimens available on affected family members. The number of patients and their family members who undergo WES testing is very low in Sri Lanka due to the high cost of the diagnostic testing. Out of the very few patients in Sri Lanka who underwent NGS testing, many were found to have at least one SNV in high, moderate and low risk cancer predisposing genes hence we had to exclude them from our study. Only the breast cancer probands who visited our clinics for genetic screening and had negative results in the testing of known cancer syndrome genes were invited to join this study and SNP genotyping was done for the family members who we recruited for the study. We plan to address these issues in future studies. Power can be improved by attempting to enroll more relatives within these four families and by adding more families to the study. We also plan additional studies using multipoint linkage techniques and plan to seek funding for additional genotyping and sequencing of informative family members. A detailed analysis of the phenotypic and clinical characteristics of this cohort in relation to the genotypic results is the subject of a future study.Additional file 1: Figure S1. Family 1. Figure S2. Family 2. Figure S3. Family 3. Figure S4. Family 4. Figure S5. Plot of two-point LOD scores for Family 1. Figure S6. Plot of two-point LOD scores for Family 2. Figure S7. Plot of two-point LOD scores for Family 3. Figure S8. Plot of two-point LOD scores for Family 4.Additional file 2: Table S1. Details of the cancer in affected relatives of the breast cancer index patient in each family. Table S2. The health status and the number of family members who provided biospecimens for genotyping. Table S3. A description of top LOD scores of family 1. Table S4. A description of top LOD scores of family 2. Table S5. A description of top LOD scores of family 3. Table S6. A description of top LOD scores of family 4.


Breast density, or the amount of fibroglandular tissue in the breast, has become a recognized and independent marker for breast cancer risk. Public awareness of breast density as a possible risk factor for breast cancer has resulted in legislation for risk stratification purposes in many US states. This review will provide a comprehensive overview of the currently available imaging modalities for qualitative and quantitative breast density assessment and the current evidence on breast density and breast cancer risk assessment.To date, breast density assessment is mainly performed with mammography and to some extent with magnetic resonance imaging. Data indicate that computerized, quantitative techniques in comparison with subjective visual estimations are characterized by higher reproducibility and robustness.Breast density reduces the sensitivity of mammography due to a masking effect and is also a recognized independent risk factor for breast cancer. Standardized breast density assessment using automated volumetric quantitative methods has the potential to be used for risk prediction and stratification and in determining the best screening plan for each woman. Breast density, or breast composition, indicates the amount of fibroglandular tissue in relation to fatty tissue within the breast. Breast tissue composition exists in high variability among women, and is also subject to change during life, influenced by endogenous  and exogenous  factors  1–4]. B–4. B1–4]Although the masking effect of breast cancer in dense breast is important, it must be noted that the association between breast density and risk for breast cancer is not merely a masking bias and cannot be explained by the reduced sensitivity of mammography alone. Several studies have consistently shown that breast density is also an independent and strong risk factor for breast cancer –17. The In the past decade, breast density notification laws have been passed with the intent to inform women about their own breast density and possible benefits from supplemental screening methods, such as breast ultrasound. As of April 2018, 35 states in the USA have mandated that patients and her primary care physicians be notified with patients’ breast density status and the risk posed by the breast density . Breast This review will provide a comprehensive overview of the currently available modalities for qualitative and quantitative breast density assessment and the current evidence on breast density and breast cancer risk assessment.SXA, Cumulus V, Quantra, and Volpara Density.The assessment of breast density is usually performed on the mammogram and is defined as the amount of fibroglandular tissue relative to the fatty tissue. On mammography, the fatty components appear radiolucent, while the fibroglandular components, consisting of epithelial and stromal tissue, appear radiopaque. The mammogram is a 2D image as a 3D structure. There are several methods available for assessing the mammographic density, ranging from subjective visual assessment to semi-automated methods and automated methods. Currently, there are no recommendations or criteria for standardized breast density assessment , 20. TheThe BI-RADS lexicon is the most widely used method clinically by radiologists. The current revised 5th edition of the BI-RADS atlas defines the four breast density/composition categories as follows: ACR-MG-a, the breasts are almost entirely fatty; ACR-MG-b, there are scattered areas of fibroglandular density; ACR-MG-c, the breasts are heterogeneously dense, which may obscure small masses; and ACR-MG-d, the breasts are extremely dense, which lowers the sensitivity of mammography . Women cTo overcome the limitations of subjective visual assessments, computer-aided semi-automated and fully automated measurement approaches are now available and can be either a 2D area-based or 3D volumetric-based methods for assessing breast density. The most commonly used 2D technique that had been regarded as the gold standard is the semi-automated interactive thresholding technique Cumulus™, which was introduced by Byng et al. and allows separation of mammographic dense from fatty tissue, and thus calculates percentage mammographic density . CumulusUltrasound (US) of the breast is a ubiquitous and cost-effective yet relatively operator-dependent imaging modality, which is easy to perform without the need for intravenous contrast application or ionizing radiation. To date, breast US cannot be reliably used for either a qualitative or quantitative assessment of breast density. However, the latest version of the US BI-RADS atlas recommends a classification of breast tissue composition with US in three descriptive categories: ACR-US-a, homogeneous background echotexture—fat; ACR-US-b, homogeneous background echotexture—fibroglandular; and ACR-US-c, heterogeneous background echotexture . To overBreast density assessment with mammography requires the use of ionizing radiation and compression of the breast, which is often uncomfortable or even painful for the patient. Mammography is a 2D method assessing the breast, which is a 3D structure, and therefore is likely not to provide an accurate absolute, but rather a rough estimate. Additionally, except for R2 Quantra, Volpara, and Densitas, most of the other methods require interaction from radiologists. To address the problems of mammography-based breast density assessment, promising approaches of volumetric, quantitative assessment of the amount of fibroglandular tissue on magnetic resonance imaging (MRI) have been developed and tested.In contrast to mammography, MRI allows radiation- and compression-free 3D imaging of the breasts and is also able to include breast areas near the chest wall and axilla. MRI provides images related to the fat and water composition of the breast. Since the water composition is highly correlated with the prevalence of fibroglandular tissue, these images can be used for slice-by-slice segmentation of fibroglandular and fatty tissues for quantitative breast density assessment . Many ofTo allow precise segmentation, one of the most important steps is the exact definition of breast and tissue borders. The boundaries for the segmentation usually are the anterior border of the major pectoral muscle and the ventral chest wall. As cranial and caudal boundaries, the inferior border of the manubrium sterni and the submammary fold are chosen. For the definition of the lateral borders, Nie et al.  proposedC-means clustering, and Wu et al. [C-means clustering. Wengert et al. introduced an iterative segmentation for the separation of the bivariate signal intensity values on Dixon sequences [Besides semi-automated approaches, there are already fully automated, volumetric measurement approaches available to assess the amount of fibroglandular breast tissue on MRI. Gubern-Merida et al.  used an u et al.  developeequences  Fig. 2)C-means cequences  and testequences , 53, 54.equences , 56•. Thequences , 57. Theequences , 57–59.However, it must be pointed out that while fully automated, volumetric MRI-based measurement approaches provide percentage values of breast density, such values are currently not included in the 5th edition of the BI-RADS lexicon. The MRI BI-RADS lexicon currently contains the recommendation to assess the amount of fibroglandular tissue with MRI similar to mammography with four classes : ACR-MRIBreast density impacts the risk for breast cancer in different ways:Breast density can mask breast cancer due to an overlap with normal breast, i.e., the masking effect reduces the sensitivity of mammography screening.Breast density is an independent risk factor for breast cancer.There is good evidence that the sensitivity of mammography decreases with increasing breast density. Increasing breast density leads to the effect of overlapping normal breast tissue resulting in coalescent areas of breast parenchyma and obliteration of tissue asymmetries and underlying tumors especially with 2D imaging modalities –8. This P = .016, in women aged 40–79 years; 95.2% vs 54.9%, sensitivity, P = .007, in women aged 40–49 years). These cancers are usually higher grade, carry a poorer prognosis and often manifest as interval cancers, and presumably constitute some proportion of the cancers masked at SFM screening in women with higher density categories.Most of the evidence on the reduced sensitivity of mammography in dense breasts is from studies that employed screen film mammography (SFM) , 6. WithRecently, digital breast tomosynthesis (DBT) has been implemented in breast imaging. Several large-scale studies worldwide that investigated DBT in the screening setting have demonstrated an increase in cancer detection as well as significant reduction in recall rates compared with FFDM, which is most likely attributable to a decreased masking effect –71. Howe3 reported as low as 3.2% [3 of 6–7%. Initial results with ABUS screening in women with dense breast are promising. The addition of ABUS to mammography resulted in the detection of 12.3 per 1000 breast cancers, compared to 4.6 per 1000 by mammography alone [An additional supplemental screening tool in women with higher density is the whole-breast ultrasound (ABUS). Although there is an incremental cancer detection rate with ABUS beyond that of mammography, this comes at the expense of higher false-positive rates with a PPV as 3.2% , 73–76.  as 3.2%  surmised as 3.2% . The rephy alone . HoweverTo date, there is no data available on the benefits of supplemental screening with MRI in women with higher breast density categories. The American Cancer Society (ACS) guidelines neither recommend for nor against screening MRI in women with the risk factor of a higher breast density –82. ScreAlthough the masking effect of breast cancer in dense breast is important, breast density is also an independent risk factor for breast cancer , 87, 88.There is debate as to what extent breast density constitutes an independent risk factor for breast cancer, yet numerous studies have demonstrated an association between breast density and risk for breast cancer that is more than merely a masking bias and cannot be explained by the reduced sensitivity of mammography alone.In the literature, it has been reported that a linear increase in breast density is associated with a four- to sixfold increase in the risk of breast cancer . In a meIn many of the studies published on the associations of the relative risk of breast cancer with mammographic density, women with almost entirely fatty breast were compared with women with extremely dense breast. As only approximately 10% of women have either extremely dense or almost entirely fatty breasts, the results are potentially misleading. Nevertheless, when comparing the relative risk for cancer in women with dense breasts to the risk in the average patient, i.e., a tissue density approximately halfway between the two middle categories of scattered areas of fibroglandular density and heterogeneously dense, the relative risk in women with heterogeneously dense breasts compared with the average woman is approximately 1.2 and in women with extremely dense breasts compared with the average woman approximately 2.1.Although the relative risk of breast density is much smaller than that of other major risk factors for breast cancer, such as age, family history, reproductive history, and genetic mutations, breast density is still a risk factor. Breast density can be easily assessed with mammography, and mammographically dense breasts are relatively common (approximately 50% of the screening population). It therefore stands to reason that the risk factor of breast density alone contributes far more cancer risk to the population than other much stronger risk factors that are less common, such as BRCA mutations or high-risk status , 31, 89.P = .5 and P = .4). Initial data indicate that with MRI, BPE might a more valuable imaging biomarker for breast cancer risk than fibroglandular tissue. However further studies are warranted to confirm these findings.Efforts have also been made to investigate the correlation of breast cancer risk with the amount of fibroglandular tissue on MRI. King et al. investigated the relationships between breast cancer and the amount of fibroglandular tissue and level of background parenchymal enhancement (BPE) on MRI . They foBreast density has become an important topic in breast imaging. Breast density impacts the risk for breast cancer in different ways. On one hand, breast density reduces the sensitivity of mammography due to a masking effect, and on the other hand, there is evidence that breast density is an independent risk factor for breast cancer. Although the extent to which it is an independent risk factor remains controversial, there is a consensus that the increased breast cancer risk is not solely attributable to the masking effect . This em


We applied logistic regression to estimate odds ratios (OR) and 95% confidence intervals (CIs) for the association of nighttime fasting duration and time of breakfast with breast cancer risk in all women and stratified by menopausal status. Models were adjusted for age, center, education, family history of breast cancer, age at menarche, number of children, breastfeeding, age at first child, body mass index (BMI), contraceptive use, and hormonal replacement therapy (HRT). A later time of breakfast was associated with a non-significant increased risk of breast cancer . This association was stronger among premenopausal women, among whom each hour later, the time of breakfast was associated with an 18% increase in breast cancer risk . The association was not observed in postmenopausal women. We did not observe an association between nighttime fasting duration and breast cancer risk after adjusting for the time of breakfast. In this study, late breakfast was associated with increased breast cancer risk, especially among premenopausal women, compared with early breakfast. Aside from nutritional quality, circadian nutritional behaviors should be further studied in relation to cancer.Circadian nutritional behaviors, defined by the daily eating/fasting cycle, have been linked with breast cancer. This study aimed to further disentangle the association of nighttime fasting duration and time of breakfast with breast cancer risk. We analyzed data from 1,181 breast cancer cases and 1,326 population controls from the Spanish multicase-control study (MCC-Spain), 2008–2013. We collected circadian nutritional behaviors at mid-age  In Some cross-sectional studies have suggested an association between a prolonged nightly fasting period and a reduction in potential breast cancer risk factors , 21. How1 is a large population-based case-control study of 5 common tumors, which was conducted in Spain between 2008 and 2013 , recreational physical activity, and smoking . Using tCases were classified into three subtypes based on pathology records, namely, (1) tumors with hormonal receptors either for estrogens or progesterone , (2) tumors with overexpression of the human epidermal growth factor 2 (HER2 +), and (3) tumors without hormonal receptors nor overexpression of HER2 (triple negative).In total, 6 months to 5 years after enrollment in the study (median time 3 years), a telephonic interview was performed to assess circadian nutritional behaviors, timing of physical activity, and sleep patterns . This interview also included a question on bedroom light during sleep assessed with a four-digit Likert scale (a) total darkness, (b) almost dark, (c) dim light, and (d) quite illuminated. Chronotype is the individual preference for the timing of circadian activity and has a genetic basis . This waWe compared basic characteristics among cases and controls and in premenopausal and postmenopausal women separately.2), contraceptive use , and hormonal replacement therapy (HRT) and menopausal status . All the covariates included in the main model had less than 3% of missing values; therefore, we applied a complete case analysis. We initially explored separate models for the two exposures (nighttime fasting duration and time of breakfast) and then a model mutually adjusting both circadian nutritional behaviors.To investigate the associations between nighttime fasting duration, time of breakfast, and breast cancer risk, we built logistic regression models and estimated odds ratios (ORs) and 95% confidence intervals (CIs). Models were adjusted for age , center , and educational level . We also adjusted all models for well-established breast cancer risk factors: family history of breast cancer , age at menarche , number of children , breastfeeding , age at the first child , BMI 1 year before inclusion to the study . To test for linearity, we conducted an ANOVA comparing two models with the exposure of interest included with or without the smoothing term. None of the models showed a significant departure from linearity; therefore, we considered exposure variables as continuous. We further categorized nighttime fasting duration and time of breakfast according to the median point in controls: 11.00 h  and 8:00 a.m. IQR 7:30–9:00 a.m.), respectively. We explored the correlation among both exposures and also with other circadian behaviors including those already examined in the previous MCC-Spain study on mistimed eating patterns  ratios and examining differences between subtypes with the Wald test.In sensitivity analyses, we explored adjustment for other lifestyle factors  and other potential risk factors for breast cancer including socioeconomic status, smoking, and age at menopause. We also explored further adjustment with other circadian behaviors including time of dinner, interval between dinner and sleep, indoor light-at-night, sleep duration, and chronotype. To investigate the potential influence of recall bias, we examined the association between nighttime fasting duration and time of breakfast with breast cancer risk using data reported for the year previous to diagnosis (or enrollment for controls) and checked the correlation with behaviors at mid-age. Finally, we explored the joint effects of nighttime fasting and breakfast timing in a model combining both exposures.2The statistical package R 4.0.5 was used to perform these analyses .SD) and of controls 58 years (12.5 SD). Overall, cases were more likely to have a family history of breast cancer, to be premenopausal women, to have less children, and to have a higher past consumption of alcohol and daily energy. Sleep differed between controls and cases with a duration of 6.9 h (1.3 SD) and 7.1 h (1.3 SD), respectively. Only 9 controls (0.7%) reported never having breakfast, whereas 21 cases (1.8%) skipped breakfast. Moreover, the time of breakfast was later for cases  compared with controls . Nighttime fasting duration was similar between both groups .The characteristics of our study population are shown in We found no correlation among controls between the time of breakfast and the time of last meal . Among premenopausal women, BMI was similar among both groups. Premenopausal cases had a longer nighttime fasting duration and a later breakfast compared with controls . In postmenopausal women, there were no differences in neither of these two nutritional circadian behaviors.We explored characteristics of cases and controls by menopausal status . PostmenIn all women, we observed no association between nighttime fasting duration and breast cancer risk after adjusting for the time of breakfast . Among pp-value from ANOVA test = 0.35). In postmenopausal women, the pattern was less clear with a slightly increased risk observed in the categorical analysis but without a clear dose response   . Figures85–1.11) . This wap-value for interaction = 0.021). We found no effect modification by chronotype  nor by HRT history (ever vs. never) among postmenopausal women .We found a statistically significant effect modification of the association between the time of breakfast and breast cancer risk by menopausal status . No differences were observed for postmenopausal women by subtype.In a multinomial logistic regression model, we explored the association of both nighttime fasting duration and time of breakfast with the RR of each breast cancer subtype. Among all women, we did not observe significant differences in nighttime fasting duration or time of breakfast and different cancer subtypes . Among pAdjustment for lifestyle factors and other potential breast cancer risk factors did not importantly change our estimates , 4. We eWe also investigated the associations of interest using the behaviors reported as corresponding to the year prior to baseline. We observed associations between a later time of breakfast (per hour increase) and breast cancer in all, premenopausal and postmenopausal women .We observed some differences in the correlation between data reported as corresponding to 40 years of age and to the previous year. For premenopausal women, the correlation was high for the time of breakfast (rho = 0.83), time of last meal (rho = 0.86), and nighttime fasting duration (rho = 0.82). For postmenopausal women, we found a low correlation for the time of breakfast (rho = 0.44), a moderate correlation for the time of last meal (rho = 0.52), and a low correlation for nighttime fasting duration (rho = 0.44).This is one of the first epidemiological studies to investigate the association between circadian nutritional behaviors and breast cancer risk. We found that having a late breakfast was associated with an increased risk of breast cancer compared with an earlier breakfast. This pattern was stronger among premenopausal women and was observed across all chronotypes. We observed an association between nighttime fasting and breast cancer, especially among premenopausal women, which disappeared after adjusting for the time of breakfast.p-value 0.07) (SD) compared with controls in this analysis . Results were not stratified for menopausal status, which according to our results might be an effect modifier in this association. These and other factors such as the prospective study design in the NutriNet-Santé cohort or the exposure assessment could explain the differences between the results from the NutriNet-Santé study and the results presented in this study.There is few evidence available on circadian timing of diet and cancer risk. To the best of our knowledge, only one other epidemiological study examined the association between the time of breakfast and breast cancer risk . The resue 0.07) . In thisSD).Two cross-sectional studies have suggested that prolonged nighttime fasting could reduce systemic inflammation and improve glycemic control, both potential breast cancer risk factors , 21. A pSeveral hypotheses could explain the association between the time of breakfast and breast cancer risk. Breakfast skipping might be compensated with a higher intake later on the day . This coThe association is biologically plausible. Delaying breakfast could be associated with worse glycemic control , lipid pThe differences in menopausal status could be explained by an increased susceptibility of breast tissue to circadian disruption in earlier life stages . It coulThe main strengths of this study are the large sample size of the study population, which enabled the stratification of our results by menopausal status, and the detailed information on circadian nutritional behaviors. This investigation gives new insights and future questions on the impact of chrononutrition in cancer, an emerging field of study that deserves more attention. The main limitation of this study is the potential for recall bias since women were asked for behaviors at 40 years of age. Circadian nutritional behaviors were assessed at one single time point, which may affect the validity of these exposures. Finally, given the observational nature of the study and its design, residual confounding cannot be completely ruled out, and causal interpretation of these findings should be taken with caution.Our results suggest that delaying circadian nutritional behaviors, specifically having a late breakfast, is associated with increased breast cancer risk, especially among premenopausal women. Together with our previous study on other circadian timing aspects, this study suggests that when to eat may also be an important aspect of healthy nutritional behaviors, influencing cancer risk. If these results are confirmed by prospective studies and clinical trials, public nutritional recommendations may consider including timing aspects aside from the quality and quantity components of the diet.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by CEIC 2008/3123/I. The patients/participants provided their written informed consent to participate in this study.BP-G, EA, TF, PA, IG-A, VM, JA, GF-T, AM-B, RM-G, NA, GC-V, MG, AM, MP, and MK performed the data acquisition. AP-C and AE performed the data curation. MP and MK carried funding acquisition. DR and MK performed the supervision of the study. AP-C wrote the first draft of the manuscript. All authors contributed to the study conception and design, commented on previous versions of the manuscript and read and approved the final manuscript.


Millions of women have given preference to the use of combined oral contraceptives (COCs) since its introduction in the 1960s. Both oestrogens and progestogens can regulate proliferation and it is plausible these effects may contribute to carcinogenesis. We aimed to review the accumulated knowledge to date to appreciate the modifying effects combined oral contraceptives may have on carcinogenesis. Our methodology involved a review of the current published literature, paying attention to studies published in the last 20 years. It has been noted that the overall cancer odds do not change with the use of COCs. Increased risk for breast cancer with COC use is not consistently backed in the literature; the results range from no increase in risk to a 20%–30% elevation in risk, and the risk seems to be temporary, limited to recent or current regular COC use. Also, diagnosed breast cancer cases seem to be clinically advanced in ever-users compared to never-users. Data show that the ongoing and prolonged use of COCs may provide diminished risk for endometrial, colorectal and ovarian cancers. Although studies do not clearly support increased risk with COC use in high-risk groups, such as women with family history of cancer or BRCA carriers, local and international guidelines are available for clinical decision-making. For cervical cancer, COCs seem to enhance the risk with more than 5 years of use, and in many studies, this enhanced risk diminishes after discontinuation and restores to those of never-users within 10 years. The relationship between COC use and liver malignancy risk assessments has provided conflicting findings. Some studies have suggested that hormonal contraceptives may increase the risk of not only hepatocellular carcinoma but also intrahepatic cholangiocarcinoma. Combined oral contraceptives are safe and effective and the effects are reversible. Patients who pursue family planning should be warned of possible carcinogenic outcomes, but it should also be explained that—in addition to sexual health advantages—preferring COCs may also decrease the risks of endometrial, colorectal and ovarian cancers. The useUp to now, there have not been any studies showing that overall cancer risk or mortality related to cancer in general has increased among COC users. Cohort studies are notably advantageous when it comes to analysing the general proportion of risks and advantages related to a COC exposure. Absolute risk of mortality from cancer was assessed in COC users in three large cohort studies, which are the Royal College of General Practitioners (RCGP) study, the Nurses’ Health Study and the Oxford Family Planning Association research; between ever-users and never-users, none of these studies found significant discrepancy –5. OveraLong-term malignancy risks and benefits were analysed in the RCGP oral hormonal pregnancy prevention study, which covered a cohort of more than 46,000 women who were followed up as far as 44 years [Long-term cancer risks were evaluated in a cohort of 267,400 female textile workers in China; no increased risk of cancers combined or of thyroid, liver, breast, lung, ovarian, colon, pancreatic, gall bladder, rectal, cervical or stomach cancers were detected. Only cancers of the uterine corpus were found to be reduced with COC use [Oestrogens and progestogens are among the modifiable factors that may increase the risk of breast cancer (BC) and this has been confirmed, for instance, in hormone replacement treatment (HRT) in the post-menopausal population. From the time of its introduction, a similar proposal has been suggested for COCs. In the breast, both oestrogen and progesterone have a proliferating effect, probably due to stem cell stimulation . InherenIn the Nurses’ Health research, the RCGP research and the Oxford Family Planning Association research, which are three large prospective cohort studies, neither long-lasting past COC use nor ongoing use was linked to an elevated risk of BC , 14, 17.In a population-based, case–control study, which consisted of more than 4500 women with breast cancer, BC risk did not vary significantly between ongoing (relative risk (RR) 1.0, 95% CI: 0.8–1.3) or previous COC users  . The RR On the contrary, some articles have proposed a relationship between COC use and BC , 19–21 . The resIn a meta-analysis by the Collaborative Group on Hormonal Factors in Breast Cancer consisting of 54 studies in 25 countries and including over 53,000 women, COC use was related to an elevated risk of BC , which faded after cessation  and ceased after 10 or more years . The len1.8 million women from a Danish registry were followed up for an average of 11 years in a prospective cohort study. In this research, ongoing or late COC users displayed an elevated risk of BC in contrast to counterparts who never gave preference to COCs  . DerivedFindings from the Nurses’ Health Study, which is the largest cohort to date , includiIn a recent meta-analysis, COC use and BC risk showed a significant linear dose–response relationship; 0.7% elevation in risk was reported with every 1 year increase of age .Since there is no consensus in the literature on whether breast cancer is associated with COC use, it is wise to consider the details if the latter is true. Diagnosed breast cancer cases were less clinically advanced in ever-users compared to never-users . AdditioBRCA) or a family history of BC may use COCs at least for short term durations. Beside the point in a meta-analysis by Moorman et al [BRCA1 and BRCA2 mutation carriers, COC use had an elevated but non-statistically significant relationship with BC .Combined oral contraceptives are not recommended for women with a personal history of BC  category 4 (unacceptable risk) for current breast cancer, category 3 (risks outweigh the benefits) for past and no evidence of disease for 5 years) . Howeveran et al , studyinOvarian cancer is the fifth leading cause of cancer death and the eight most common cancer in women . DespiteCOCs show a likely promising initial preventive measure for ovarian cancer. Researches have consistently demonstrated that continued use of COCs lowers the risk of ovarian cancer. Various broad pooled studies advocate that COCs provide a protective effect on ovarian malignancy risk, with a risk decrease of up to 50% with longer periods of COC use –62. The In a meta-analysis, which pooled 24 studies of oral hormonal contraception as primary protection for ovarian malignancy, ever-use was correlated with a decrease in ovarian malignancy in comparison with never-use , with more than 50% decrease among participants with ≥10 years of usage . RelatioIn the aforementioned reanalysis by a collaborative group, risk reductions per 5 years of COC use were different for different histological subtypes; for serous, 22.1%; for endometrioid, 27.1%; for clear cell carcinomas, 21.3%; and lastly, 6.7% for mucinous tumours, which was non-significant . SimilarTo understand the underlying mechanisms for how COCs prevent ovarian cancer, the process of ovarian cancer itself should be evaluated foremost. However, the aetiology of epithelial ovarian cancer has been debated for over more than half a century. It is known that ovarian cancer may develop through mutations with step-wise fashion  or much more aggressive mutations without precursor lesions causing widespread metastasis  . Carcinode novo [damage to the flat mesothelium was ending with entrapment of its cells during the healing process. Undergoing Müllerian metaplasia, they gave rise to mucinous, serous and endometrioid cystadenomas, which are cancer precursor lesions. But perplexities are born on the coelomic ancestry; most encountered mucinous, serous and endometrioid types are identical to intestinal/endocervical, fallopian tube and endometrium carcinomas, morphologically. Moreover, Müllerian cells are not present in ovaries. In addition to this, studies have failed to show a coelomic precursor lesion for these cancers, and therefore epithelial ovarian cancers are accepted as de novo .Novel theories propose that the plantation of tubal and endometrial cells on ovary epithelium is the initial step for the process for these cancers of Müllerian origin , 72, 73 There has been uneasiness that oral hormonal pregnancy prevention might elevate the risk of BC in susceptible populations like mutation carriers. Today, all women who are susceptible to gene mutation are proposed risk-reducing salpingo-oophorectomy (RR-BSO) at an age before which they are at the highest risk for ovarian malignancy as a routine practice; thus, COCs as protective measures are not required. Prophylactic surgeries are the standard of preventive care for these individuals. However, for women who must use COCs for their hormonal diseases or who have decided against RR-BSO, data advocate a decreased risk of ovarian malignancy in women with mutated susceptibility genes who take COC pills, although the hypothetical risk of elevated BC continues to remain. As discussed earlier, this risk was suggested by many studies but has not been consistently backed in the literature.Women who possess the BRCA1 and BRCA2 genes compose a high-risk population and correspond to 10–15% of all BC cases. In a case–control study which consisted of nearly 2500 matched pairs of women who carried BRCA1 gene, oral hormonal contraception usage was related with an elevated risk of early onset BC, granted that medication commenced under the age of 20  ; and the18 comparative, retrospective studies of oral hormonal contraceptive use in BRCA1/2 mutation carriers were analysed. This meta-analysis included 1503 cases of ovarian malignancy and 2855 cases of BC . The resIn the study outlined above, there was no indication of a significantly elevated BC risk in COC users in general, for ongoing users or in the first decade after discontinuance of use .In a meta-analysis deliberated beforehand, which studied COC use among BRCA1/2 mutation carriers, relationships between ever-use of oral contraceptives and ovarian and breast cancer among women who are carriers are akin to those shown for the common populace .In a recent study, a prospective cohort study which comprised 6030 BRCA1 and 3809 BRCA2 carriers, retrospective and prospective analyses were inconsistent. In the prospective arm of BRCA1 carriers, there was no elevation in the risk of BC, but retrospective results were consistent between full-cohort (HR: 1.39) and left-truncated (HR: 1.26) analyses. The BRCA2 carriers’ prospective arm and full-cohort showed elevations in the risk of BC  but retrospective results were inconsistent between full-cohort and left-truncated analyses (HR: 1.06) .In a recent systematic review, overall data on BRCA mutation carriers were found to be very limited to decide the use of COCs in this population. BRCA1/2 mutation carriers also benefitted from the protective effects of COC use, which decreases the risk of ovarian cancer . On the GLOBOCAN predicted 570,000 cases and 311,000 deaths for cervical malignancy in 2018 and these numbers rank it to be the fourth most frequently diagnosed cancer and the fourth leading cause of cancer death in women .Human papillomavirus (HPV) is the essentially fundamental (but not enough) factor of cervical malignancy , which iThe age-specific incidence rate of cervical cancer increases after 25 years of age and peaks around 40 years in high-resource countries, although it continues to rise until 55–69 years of age in low-resource countries and so the contribution of COCs to the life-long incidence of cervical malignancy will revolve around on mostly the consequences later in life, when majority are former users. Long-term cervical malignancy risk does not seem to scale up among ever-users in comparison to never-users  [In the EPIC study, which consisted of a cohort of over 300,000 participants who were followed up for 9 years, the relationship between hormonal cofactors and risk of acquiring cervical malignancy was assessed . SimilarIn a recent cohort study conducted in Denmark, nearly two million women of reproductive age were evaluated. Increased risk of cervical cancer was observed among recent and current users of COCs (RR 1.40 (95% CI: 1.28–1.53)). Risk analysis was similar for both adenocarcinoma and squamous cancer. This effect was observed more with the longer duration of use and declined after discontinuation. In this cohort, most women were unvaccinated against HPV; therefore, currently used COCs may have a similar risk profile compared to older formulations .in-situ malignancy, and in high-risk HPV carriers [In a meta-analysis, which consisted of 24 studies comprising almost 17,000 patients with cervical malignancy and approximately 36,000 controls support this effect, the risk of cervical invasive malignancy was elevated among ongoing users of COCs with a prolonged period of use ). This enhanced risk diminished after discontinuation and was restored to those of never-users within 10 years. An identical model of risk was detected for both invasive and carriers .The cervical cancer effects of COC usage is now known and elevated absolute risk in users is the valid measure of this consequence; however, it does not tell much about how exactly the HPV reliant carcinogenetic process is enhanced. COC use might, for instance, enhance the cervical vulnerability to HPV transmission and resultant infection or it might alter persistence or disposition of virus or the advancement or reversion of malign and pre-malign lesions. A plausible pathway to elucidate the relationships between oral contraceptive use and cervical cancer risk is that cervical tissue hormone receptors, especially progesterone, may be affected and the course of HPV may be altered. Particularly, hormones used in contraceptives are assumed to intensify the expression of HPV 16 E6 and E7 oncogenes, prompting the deterioration of p53 tumour suppressor genes and augmenting the capability of the viral DNA to mutate cells and promote neoplasticity –111 Fig. Data frin situ and invasive carcinoma.The latest 2015 WHO guidelines for MEC for contraceptive use categorises the recommendations into four groups. For HPV positivity, cervical intraepithelial neoplasies and cervical cancer awaiting treatment, the WHO recommendation group is 2; the advantages of usage outweigh the risks . While uThe use of COCs provides prevention against endometrial neoplasia. Consistently, studies have found that the longer the period of usage, the more significant is the decrease in the risk of endometrial neoplasia. On average, every 5 years of use was related to a RR of 0.76; consequently, after approximately 10–15 years, the risk lowers by 50%. After discontinuation, the preventive influence goes on beyond 30 years, and does not give the impression to rely much on the oestrogen dose in formulations or individual traits like parity, body mass index or menopausal condition . Since tp = 0·02)[The decrease in cancer rate related to COC use appears to vary by malignancy form, being more effective for carcinomas  than sarcomas .Findings from recent (2021) The Nurses’ Health Cohort Study II, including 107,069 women, showed oral hormonal contraception usage is connected to lower endometrial cancer risk  . These fThe use of COCs reduced the risk of endometrial cancer by 30%–40% in broad epidemiologic studies, and the risk decrease persevered for years after cessation , 115. ThColorectal cancer (CRC) is another common cancer in women, with relatively good survival rates that are at around 65%. The number of studies reporting on the relationship between COCs and CRC risk are voluminous and IARC has noted that the use of COCs may decrease the risk of colorectal cancer. For the past 20 years, still, there is no consensus between the results of epidemiological studies.In a meta-analysis, which consisted of 23 studies comprising 14 case–control and 9 cohort studies, relative CRC risk for ever-use versus never-use was 0.8 . In thisIn another meta-analysis, which consisted of 29 studies comprising nearly 16,000 colorectal cancer cases, relative CRC risk for ever-use versus never-use was 0.8 . In thisIn a recent population-based case–control study conducted in Northern Israel, nearly 3,000 CRC cases were evaluated. It was found that COC use was very inversely associated with CRC risk among people with Jewish and Arabic origin  and 0.14 (0.04–0.47), respectively) .However, in some recent high-quality cohort studies, including the Nurses’ Health Study, neither long-lasting past COC use nor ongoing use was linked to an elevated risk of CRC , 129–134In a cohort of 1.3 million women, which was followed up for 13 years, ever-use of COCs was found to be associated with an elevation in risk of anal cancer  . This maSeveral mechanisms, direct and indirect, were suggested for how COCs modify the risk for CRC  Figure . InsulinCOC use may decrease the CRC risk by at least 15% and this effect seems to not be dose-related, lasting for more than 30 years and not changing with duration of use.Primary liver malignancy is the sixth most common diagnosed cancer and the ninth most common cancer in women, and it is mostly an issue in less advanced countries . HepatocThe relationship between COC use and liver malignancy risk assessments has provided conflicting findings. In a meta-analysis of 12 case–control studies, which included over 700 women, the results relating to cancers, like HCC, are varied . In one Regarding the incidence of HCC, the effect of sex has been well demonstrated. Men are developing cancer more often than women, and this has brought about the hypothesis that female sex hormones may prevent hepatocellular carcinoma. Backing this, a case–control study, which consisted of 234 patients with treated HCC and 282 controls, demonstrated that post-menopausal HRT was decreasing the incidence of HCC (148). In a meta-analysis, it has been shown that people with differences in oestrogen receptor 1 gene had different odds of developing HCC (149). Furthermore, in a study which pooled nearly 800.000 women from 11 cohorts, surgical excision of both ovaries was significantly related to elevated odds of developing HCC , after calculating for other individual and clinical cofactors and the length of HRT . In the In a meta-analysis comprising 14 case–control studies and 3 cohort studies, a significant difference could not be detected; however, a detached investigation of these 14 case–control studies, contrary to the cohorts of the same meta-analysis, revealed a significant elevation in odds ratio (1.55) . In a caThere is some evidence regarding the probable biochemical pathways through which hormones may promote carcinogenesis in liver. Hepatocytes carry oestrogen receptors, which are found to be upregulated in HCC, probably because of their proliferative and mutagenic effects , 152. ThThe proliferation of cholangiocytes in the intrahepatic bile duct is upregulated by oestrogens. Cholangiocytes express both oestrogens receptor-α and -β and can develop intrahepatic cholangiocarcinoma (ICC), which is the most prevalent liver malignancy after HCC , 154. LaCombined oral contraceptives are safe and effective and the effects are reversible. Patients who pursue family planning should be warned of possible carcinogenic outcomes, but it should also be explained that—in addition to sexual health advantages—preferring COCs may also decrease risks for endometrial, colorectal and ovarian cancers. To this day, in no study has cancer been shown as an enhanced mortality factor among COC users.The WHO MEC published in 2015 and US MEC for contraceptive use published in 2016 agree that the evidence to date does not propose an elevated risk for BC among women with either a family history of BC or BC susceptibility genes. Therefore, women with breast cancer susceptibility genes (such as BRCA) or a family history of BC may use COCs with safety , 114.The European Society of Contraception and Reproductive Health Care published in 2018 has advocated against long-term use in BRCA carriers . The UK BC risk elevation with COC use in this high-risk group is reported in some studies. On the other hand, others show no increase in risk. That being said, BRCA1/2 carriers should be notified that COC use may enhance BC risk. Early detection strategies for BC should be discussed with patients and periodic inspection should not be omitted; unfortunately, these strategies are not as effective for ovarian cancer and effective treatment is less likely to be achieved. The respective 5-year survival rates are 90% and 48% published by the American Cancer Society .COCs can be used for contraceptive purposes in this population, but different forms of contraceptives should be elucidated. COC use for avoidance of ovarian cancer in cases where there is no need for birth control is still not advocated.Since the development and approval of the first COC pill, formulations have evolved from comprising high-dose oestrogen (150 mcg) to very low doses (10 and 20 mcg). Oestrogens and progestogens are among the factors that may cause BC, and this has been confirmed, for instance, in HRT in post-menopausal population. Similar concerns were raised for reproductive women using COCs, but these claims were not given much attention because the evidence was on women using high-dose formulations of old. However, some recent high-quality studies on women using modern formulations found increased BC risk , 165. ThScreening for cervical malignancy should not be a precondition for the planning of pregnancy prevention strategies. The WHO MEC guideline acknowledges COCs as category 2 in women with HPV, CINs and cervical cancer, claiming that the advantages of COC use outweigh the risks in these groups. COCs enhance the risk with longer than 5 years of use and this enhanced risk diminishes after discontinuation and restores to those of never-users within 10 years.Usually, cervical cancer progression is very slow, taking nearly a decade. Today, with comprehensive vaccination, widespread access to cervical cancer scanning and wider coverage of health services, elimination of cervical cancer is sought after. All women should be informed about sexually transmitted diseases, cervical cancer risk and screening strategies in polyclinic visits including family planning.For a quarter century, combined oral contraceptive carcinogenicity has been held under a microscope. Recent studies reviewed support that, in addition to their effective contraceptive effect, COCs have a strong and long-lasting suppressive effect on endometrial, ovarian and colorectal cancers. Conversely, we see that some studies report the risk of cervical and breast cancer in recent use. Although the carcinogenic effects reported on breast and cervical cancer are reversible, the overall risk can further be reduced by methods such as lifestyle changes  or HPV vaccines. In addition, since cancers are more common in advancing ages, the cumulative protective effect of drugs used in the reproductive age may become more important than the reversible effect seen in the interim period.In the same quarter century, medical practice shunned the paternalistic approach and shifted towards co-decision-making in counselling. It is important to discuss the benefit/harm balance with patients and make a decision specific to that patient. Once the patient population that should avoid combined oral contraceptives has been identified, it is not appropriate to make decisions on the remaining women solely for fear of cancer. Up-to-date and clear information flow will both strengthen the patient–doctor relationship and increase the patient’s compliance and confidence in the method chosen.The authors declare no conflicts of interest.This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.


Background: Previous experimental studies showed that limiting methionine in the diet of animals or in cell culture media suppresses mammary cancer cell proliferation or metastasis. However, no previous study has investigated the associations of changes in methionine intake with survival among breast cancer survivors. We aimed to examine the association between changes in dietary intake of methionine, folate/folic acid, and vitamin B12 from before to after diagnosis of breast cancer, and mortality among breast cancer survivors. Methods: We included 1553 postmenopausal women from the Women’s Health Initiative who were diagnosed with invasive breast cancer and completed a food frequency questionnaire both before and after breast cancer diagnosis. Multivariable Cox proportional hazards regression models were used to estimate adjusted hazard ratios (HRs) and 95% confidence (CIs) of all-cause and breast cancer mortality associated with changes in methionine intake and changes in folate/folic acid and vitamin B12 intake. Results: Relative to pre-diagnosis, 28% of women decreased methionine intake by ≥20%, 30% of women increased methionine intake by ≥20%, and 42% of women had a relatively stable methionine intake (±19.9%) following breast cancer diagnosis. During a mean 16.1 years of follow up, there were 772 deaths in total, including 195 deaths from breast cancer. Compared to women with relatively stable methionine intake, women with decreased methionine intake had lower risks of all-cause  and breast cancer mortality  in fully adjusted models. In contrast, increased methionine intake or changes in folate/folic acid or vitamin B12 intake were not associated with all-cause or breast cancer mortality. Conclusions: Among breast cancer survivors, decreased methionine intake after breast cancer diagnosis was associated with lower risk of all-cause and breast cancer mortality. Breast cancer is the most common malignancy in women worldwide , accountCancer cells proliferate rapidly compared to normal cells and require larger quantities of glucose and amino acids to sustain this proliferation . TherefoBreast cancer survivors may be motivated to make lifestyle changes , includin = 68,132), or an Observational Study (OS) component . The CT included the dietary modification (DM) trial, the hormone therapy (HT) trial, and the calcium and vitamin D (CaD) trial. The CT and OS were closed in 2004–2005 and the participants were invited to continue being followed in the WHI Extension Study since 2005. In the WHI, food frequency questionnaires (FFQs) were evaluated for more than one time only among the DM and OS participants  5.6) years of follow-up, there were 772 deaths in total, which included 195 from breast cancer and 577 from causes other than breast cancer. The average time from pre-diagnosis FFQ to breast cancer diagnosis was 1.9 (SD 1.2) years and the average time from diagnosis to post-diagnosis FFQ was 1.5 (SD 1.3) years.After breast cancer diagnosis, 42% of the participants maintained relatively stable dietary methionine intake (±19.9% change), 28% decreased dietary methionine intake (≥20% decrease), and 30% increased dietary methionine intake (≥20% increase) compared with their pre-diagnosis dietary methionine intake . CompareCompared with participants with relatively stable dietary methionine intake, participants with decreased dietary methionine intake post-diagnosis had significantly lower risk of all-cause mortality  . Similarp values ≥ 0.07).The results were similar when women who completed the post-diagnosis FFQ within six months of diagnosis were excluded, women who died within two years after the collection of the post-diagnosis FFQ were excluded, or women with stage four breast cancer were excluded . The assThis large prospective cohort study has shown that a decrease in dietary methionine intake post-diagnosis of invasive breast cancer was associated with lower risk of mortality from all-cause and breast cancer. However, increased dietary methionine intake was not associated with survival among breast cancer survivors. Dietary intake of folate/folic acid or vitamin B12 was not associated with breast cancer survival.PIK3CA genomic mutation and decreased expression of SLC7A11, which is a gene that encodes a cystine transporter also known as xCT that is correlated with increased methionine dependency in breast cancer cells [PIK3CA mutant cells use homocysteine through the transsulfuration pathway to synthesize cysteine. Consequently, less homocysteine is available to produce methionine, thus contributing to methionine dependency [To our knowledge, our study is the first to evaluate the relation between changes in methionine intake and breast cancer survival in humans. The findings are biologically plausible. Methionine is an essential amino acid derived from the diet. Aside from serving as a building block for proteins, methionine is also imbedded in the pathways pivotal for DNA methylation and polyamine synthesis . In breaer cells . Concurrpendency .Although no previous study has examined changes in diet methionine intake in relation to breast cancer survival in humans, several previous studies examining the association between methionine and breast cancer risk have shown inconsistent findings. A meta-analysis of seven prospective and six case-control studies found that premorbid high vs. low methionine intake was significantly associated with breast cancer risk among postmenopausal women but not among pre-menopausal women . A separStrengths of this study include the large sample size, prospective design, and long-term follow-up. In WHI, there is also detailed data on covariates that potentially cofound the associations between changes in dietary methionine intake and survival; thus, the role of methionine in mortality after diagnosis of breast cancer was examined thoroughly. This study also has limitations. First, dietary assessment is susceptible to measurement error. Second, a change in dietary methionine intake may not lead to a similar change in systemic methionine levels, as there is often a lag between the changes in dietary methionine intake and the change in circulating methionine concentrations. However, this study included FFQ collection on average 1.5 years after diagnosis, such that any temporary adaptations may be minimized. Third, it is possible that cancer survivors with a poorer prognosis would be more likely to change their diet post diagnosis. However, we excluded participants who had cancer at baseline, controlled for breast cancer stage, ER status, and PR status, and excluded persons who died within 2 years of collection of the post-diagnosis FFQ. Fourth, cancer treatment information was not available. However, we controlled for cancer stage, which is correlated with treatment in survivors in other studies of breast cancer survivors , althougThese findings provide evidence for possible development of low-methionine therapeutic diet or methionine lowering drugs by depleting methionine from cancer cells to enhance response to breast cancer chemotherapy or radiation therapy ,29,35. FIn conclusion, among postmenopausal invasive breast cancer survivors, decrease in dietary methionine intake after diagnosis of breast cancer was associated with lower risk of all-cause and breast cancer mortality. Further studies, including both observational studies and clinical trials, are needed to fully understand how dietary methionine intake changes after breast cancer diagnosis affect long-term health among breast cancer survivors. Lastly, studies exploring the underlying mechanisms are needed.


Genetic compositions of distinct human populations are different. How genomic variants influence many common and rare genetic diseases is always of great medical and anthropological interest, and understanding of genetic architectures of population groups in relation to diseases can advance our knowledge of medicine. Here, we have studied the genomic architecture of a group of Xavante Indians, an indigenous population in Brazil, and compared them with normal populations from the 1000 Genomes Projects. Principal component analysis (PCA) indicates that the Xavante Indians are genetically distinctive when compared to other ethnic groups. No incidence of breast cancer cases has ever been reported in the population, and polygenic risk analysis indicates extremely low breast cancer risk in this population when compared with germline TCGA (The Cancer Genome Atlas) breast cancer normal control samples. Low germinal mutation burden among this population is also observed. Our findings will help to deepen the understanding of breast cancer and might also provide new approaches to study the disease. This is, therefore, a global health issue, with incidences worldwide varying in accordance with life expectancy, cultural behavior and level of development of the corresponding countries or regions.According to the World Health Organization, in 20202 brought attention to an unusual observation3, that there are no reported incidences of breast cancer among Xavante Brazilian Indians. But other types of cancers do exist in this population such as cervical, lung, stomach and leukemia. The Xavante are Brazilian Indians, a Macro-Jê linguistic trunk of the Tupy branch, who were linked to the Asiatic population that migrated to the Americas through the Bering Strait more than 15,000 years ago by tracking Y chromosome studies4. These primitive people, after descending the Pacific coast along the Andes, later populated the Caribbean Islands and reentered the continent by the coast of Guyana. From here, this group gradually diverged from the other people of the Americas, such as the Mayas and Aztecs from Central America, and Incas from South America. The Brazilian Indians then penetrated the Amazon rainforest, which 12,000 years ago was a large savanna in its central area5. At that point they divided into two branches: the Tupy living across the Atlantic coast, and the Macro-Jê branch settling in the Central Region of Brazil who maintained the lifestyle of nomadic hunter-gatherers until today. The latter maintained a well-preserved culture6. Currently, they are the largest indigenous group7 with almost 22 thousand Xavante, 50% being female, living in 11 reserves in Mato Grosso State, with a population growth of more than 5% per year8. In 1993, Dr. Castro made an observation that this population group does not appear to develop breast cancer during their lifetime after working with the Xavante for 10 years. Since then, there have been no occurrences contradicting our observation9, and no study about breast cancer in Brazilian indigenous women has been published in the country to the present day.Indeed, an observation by Guilherme Bezerra de CastroAlthough analysis of the genetic makeup of the different populations in relation to cancer incidence is yet to be fully discerned, we carried out the current study to investigate the genetic characteristics of the Xavante to attempt to understand the observation on the lack of breast cancer incidence in this population and to provide comparisons with other ethnic groups from the 1000 Genomes Project, which include European, African, Asian and Hispanic populations.Authorization from Fundação Nacional do Índio (FUNAI) was acquired after approval from the Research Ethics Committee of the Faculty of Medicine in the Federal University of Mato Grosso (UFMT), and the National Commission of Research Ethics (authorization #1004/2001). Written consents, which were recorded and archived, were acquired from all subjects prior to the study either by the individual or their legal guardian in the case of minors, as well as the use of any documents necessary for the study of the Xavante population.10. We then distributed a questionnaire about comorbidity, number of offspring, breastfeeding, height, weight, and breast pathology. Blood samples from 14 of these women were used for exome-sequencing for study of this homogenous ethnic population. This cohort was selected because they are the least mixed ethnic group among those living close to cities, providing good access for the researchers to support the study, and their demographic information is listed in Supplementary Table Out of approximately 500 women in the Sangradouro Reserve , 182 volunteered for the project with a median age of 27 and life expectancy of 61.7 yearsAll DNA samples were extracted from 250 μl of whole blood using a commercially available kit according to the manufacturer's instructions . After extraction, the determination of the concentration of all DNA samples was carried out using the PicoGreen dsDNA quantification reagent  in an Anthos Zenyth 3100 . The linearity of the method was verified for the high-range standard curve (2 μg/ml of Lambda DNA standard) according to the manufacturer's recommendations prior to determination the DNA concentration.11, HaplotypeCaller from GATK best practices workflow12 was used for variant calling, and ANNOVAR13 was used for variant annotation and effect prediction. Only variants classified as “PASS” were considered for analyses. Variants that were not reported in dbSNP v147 and did not have population frequencies reported in 1000 Genome phase 3 data were considered novel. The functional consequences of nonsynonymous SNVs and splice variants were predicted using PolyPhen-2 (Polymorphism Phenotyping v2), SIFT, LRT, Mutation Taster, Mutation Assessor, and CADD14.Genomic DNA from 14 female Xavante from the Mato Grosso State was prepared. A total amount of 1.0 μg genomic DNA per sample was used as input material for the DNA library preparation. Sequencing libraries were generated using Agilent SureSelect Human All Exon kit  and were sequenced on an Illumina NextSeq 500 system  by Novogene Co. Ltd . Reads were mapped to the human genome reference (GRCh38) with BWA15, which includes 2548 samples from 26 populations were used for population admixture analysis together with our 14 samples. A total of 291,984 SNPs shared among these populations and also present in at least 7 of our samples were used to generate principal component analysis (PCA) plots after applying a linkage disequilibrium based variant pruner as implemented in PLINK16. Supplementary Fig. 17. The fit of different values of K was assessed using cross-validation (CV) procedures, and K = 9 showed the lowest CV error  breast cancer dataset as comparison. To minimize potential confounding from whole-genome sequencing performed in separate batches for the Xavante and TCGA cohorts, we assembled a single joint variant call set across all samples in this analysis starting from raw reads. After application of stringent sequencing quality control parameters, 2171 out of the 5218 (41.6%) variants were available for scoring, and this polygenic score was calculated in each of our samples as well as the TCGA breast cancer normal control samples.Khera et al. have developed a genome-wide polygenic score for breast cancer risk estimation which comprises 5218 common  variants20, which includes ATM, BARD1, BRCA1, BRCA2, BRIP1, CASP8, CDH1, CHEK2, CTLA4, CYP19A1, FGFR2, H19, LSP1, MAP3K1, MRE11A, NBN, PLAB2, PTEN, RAD51, STK11, TERT and TP53 genes.Genomic germline variation data which include SNVs and short indels from our cohort, 1296 normal female samples from the 1000 Genomes Project, and 200 randomly selected TCGA normal blood samples  were analyzed and annotated together. Exonic or splicing variants which have less than 1% population frequency and which were predicted to be damaging by at least 2 methods listed above were retained. We then compared the number of genes which carry these potenitally damaging variants among all sample populations. Since we were also interested in looking at relatedness to breast cancer risk, we further examined the mutation status of known breast cancer risk genes in all samples using the list recommended by National Breast Cancer Foundation2-sided Wilcoxon–Mann–Whitney tests (5% type I error) were used to compare of polygenic risk score and mutation load analysis between groups. Analyses were performed using R.21 about this population. Blood tests were also performed for biochemistry, hormonal and complete blood count which revealed an abnormal low hormonal level and high level of glycemia (data not shown) which is also observed by a recent publication22.The data we have collected showed that these women, on average, begin menarche at 12 years old, have their first child at age 15, breastfeed for two years, have seven children during the course of their reproductive lives (data not shown) and have low mammary pathology which is consistent with published demographic and behavioral dataThe mean coverage for all sequenced genomes was ~ 100×  when compared to the 1000 Genomes reference populations and TCGA breast cancer samples . Six of these variants were detected only in one of our samples, and 1 variant in the DSP gene (rs368802003) was detected in 2 samples. These 7 variants either lacked ClinVar annotation or were annotated as having benign or unknown effect by ClinVar. A MYBPC3 variant (rs11570112) was detected in 5 of our samples with possible pathogenic effect 27. Over the past decades, more than 4000 GWAS studies have been published, reporting over 200,000 associations to different phenotypes. We used the most recent version of the GWAS catalog which was mapped to GRCh38.p13, with a significance cutoff of 10–5. A total of 22,903 variants were present in at least one of the 14 Xavante exomes and 973 variants were shared by all 14 Xavante samples. We then looked at significant SNP-trait associated variants among these 973 SNPs which also have different frequencies from the 1000 Genomes reference populations  catalogThe carcinogenic process involves a multiplicity of mechanisms consisting of both somatic and germinal pathogenic or susceptibility variants, whose balance and complexity in each type of cancer are different.28, but can also be acquired and accumulated during one’s lifetime, as a result of errors that occur as cells divide (1 × 10−10 mutations/bp/cell division), or from exposure to environmental carcinogenic substances that damage DNA29. The International Agency for Research on Cancer (IARC) of WHO has identified and classified more than 500 agents representing potential threats for human beings as carcinogens. Some 88% of those potential agents classified by IARC are DNA-damaging agents, i.e. they carry an unmistakable mutagenic or clastogenic activity. However, aside from somatic mutations, we still face a scarcity of data on the role the mutational germinal load has on the carcinogenesis process30. By analyzing a population of Xavante with no observed incidence of breast cancer, we explored if and how the low mutation germline load could play a role in this pathological process when compared to populations with high mutational load and high breast cancer incidence.The vast majority of breast cancer is sporadic, representing on average 90% of all cases. Cancer-causing genetic changes can be inherited31. The impact of the higher mutational load in African populations in the context of breast cancer warrants further investigation and studies. Breast cancer is a complicated and multi-factor disease with different subtypes, and disease risk and mechanism vary in different ethnic groups. One major research area in assessing breast cancer risk is in the heritability of the disease. But known risk genes (BRCA1/BRCA2 and others) are insufficient to explain all inherited breast cancer risk. This is why more recent studies have pursued polygenic risk assessment32 in order to acquire a better estimates. We have shown in our study that the genomic signatures of the Xavante cohort appear to be rather unique. We believe that both the genetic distinctiveness of this population and low germinal mutational load are important in giving them the protection against breast cancer.The study of Xavante exomes provides useful insight into the role of germline mutation germinal load in these populations, indicating that their low germinal mutation load may explain, at least in part, the absence of breast cancer in Xavante women. Among all 1000 Genomes control populations, the African populations seems to have more mutated genes overall than all other normal populations which is also observed by Auton et al.33. This population has an overall low germline mutation load when compared to other populations, which could be the result of its unique environmental, cultural, demographic and genetic histories. The specific genomic profile of the Xavante could indicate that lifestyle and environmental factors played an important role in shaping the genetic structure of this population and might have been the reason for the observed absence of mutations34.The demographic and adaptive processes experienced by this indigenous group may have shaped their genetic architecture, and this might have important implications in biological and health related processes of the population18. Polygenic risk score (PRS) is an estimate of an individual’s genetic liability to a trait or disease, calculated according to their genotype profile and relevant genome-wide association study data35. It provides a quantitative metric of individual inherited risk based on the cumulative impact of many common polymorphisms. Weights are assigned to each genetic variant according to the strength of their association with disease risk18. The Xavante, when compared to breast cancer germline normal control samples, displayed an extremely low polygenic risk score for breast cancer development.Polygenic inheritance, involving many common genetic variants of moderate to low penetrance, plays a greater role than rare monogenic mutations36.The findings in this study could contribute to understanding the mechanisms of initialization in sporadic breast cancer and promote new approaches for early prevention and detection of the disease. The low number of mutagenic changes seen in the Xavante population may assist in the identification of novel loci of initialization of the process that triggers the changes linked to breast cancer, in addition to one that have already been identified37. Our study is unique in reporting the possible role played by germinal mutational events in the development of breast cancer. This is the first effort in trying to characterize whole exomes of the Xavante to look for genetic connections to the absence of breast cancer incidences in this population. This work will shed light onto identification of genetic variants associated with biological and clinical traits in this indigenous group.Although the number of samples in our study is small, the Xavante constitute a population with extremely low rate of interethnic relationships and has been shown to be genetically uniform38, the data presented applies solely to female breast cancer.This study concerns and is centered on female Xavante Indians. Since there are grounds to believe that male breast cancer may display different genetic determinants39 since the scoring system has been derived from samples of primarily European ancestry. But this is the sample set in which the most genetic studies have been done to date and this scoring system has covered the largest number of genetic markers related to breast cancer risk. We anticipate in the very near future there will be genomic studies expanded to other non-European ethnic groups since the whole biomedical community are more aware of crucial equity issues.We also want to emphasize the limitation of our PRS analysis system we adapted in this paper. These specific genome-wide polygenic scores will not give optimal predictive power for other ethnic groups40, the germinal mutational load may play a central role in cancer risk and carcinogenesis: since the latter may vary among families and population groups such as the Xavante, this may explain the age-dependence of cancer initiation and thus breast cancer incidence. Indeed, mutation accumulation in somatic cells is a time-dependent process and a stochastic one, adding to mutations inherited as the germinal mutation load. Considering the gold standard number of 7–11 driver mutations in a somatic cell to trigger and sustain a cancer process41, one can be led to accept that a germinal mutational load would increase the risk of cancer by adding to mutations acquired during a lifetime due to environmental carcinogens.This work does not intend to supply a complete set of data on the genetic mechanisms associated with breast cancer, but to add data on the possible role of germinal mutated genes in breast carcinogenesis. Indeed, it becomes increasingly clear that besides somatic mutations which further evolve during the carcinogenic process leading to oligogenic clones that may determine the prognosis of the tumor and its drug resistanceThe data obtained in the genetic analysis of the Xavante samples in this study reveals that there is significant difference between women of this group and of other ethnicities, showing possible existence of unstudied data to be found in the human genome to be found related to breast cancer. Many other factors could be implicated in the development of breast cancer or in protection against it. Our findings in this study may bring a new insight into human carcinogenesis, highlighting the importance of germinal mutational load beyond environmentally-driven or somatic mutational changes.More studies in this unique group of people will be needed to further understand their genetic characteristics in relation to breast cancer risk. Furthermore, how other factors, including reproduction history (age at first full term pregnancy for example) and environmental exposures, may also play a role in the differences found and will need to be investigated in order to gain a more complete understanding.Supplementary Information.


Family history, and genetic and non-genetic risk factors can stratify women according to their individual risk of developing breast cancer. The extent of overlap between these risk predictors is not clear.ATM, BRCA1, BRCA2, CHEK2, PALB2, BARD1, RAD51C, RAD51D, or TP53), and polygenic risk score (PRS) 5yAR above 1.3%.In this case-only analysis involving 7600 Asian breast cancer patients diagnosed between age 30 and 75 years, we examined identification of high-risk patients based on positive family history, the Gail model 5-year absolute risk [5yAR] above 1.3%, breast cancer predisposition genes (protein-truncating variants [PTV] in r=0.27). Fifty-three percent of breast cancer patients (n=4041) were considered high risk by one or more classification criteria. Positive family history, PTV carriership, PRS, or the Gail model identified 1247 (16%), 385 (5%), 2774 (36%), and 1592 (21%) patients who were considered at high risk, respectively. In a subset of 3227 women aged below 50 years, the four models studied identified 470 (15%), 213 (7%), 769 (24%), and 325 (10%) unique patients who were considered at high risk, respectively. For younger women, PRS and PTVs together identified 745 (59% of 1276) high-risk individuals who were not identified by the Gail model or family history.Correlation between 5yAR (at age of diagnosis) predicted by PRS and the Gail model was low (Family history and genetic and non-genetic risk stratification tools have the potential to complement one another to identify women at high risk.The online version contains supplementary material available at 10.1186/s12916-022-02334-z. Multiple randomized controlled trials have shown that screening mammography reduces mortality from breast cancer for women who are over 50 years old . ScreeniThe benefit of screening mammography for younger women aged 40 to 49 years is less clear . RecommeWith advances made in disease prediction, the approach to breast screening is now leaning towards a tailored, individual risk-based approach. For example, mammographic breast density, which refers to the proportion of fibroglandular breast tissue compared to fat seen on mammography, is a risk factor for breast cancer and is increasingly communicated to screening participants , 5. WomeThe risk of breast cancer is multifactorial. Apart from mammographic density, other known conventional risk factors include family history, menarche age, menopause age, height, body mass index, age at first childbirth, menopausal hormone therapy, and benign breast disease . Many ofATM, BRCA1, BRCA2, CHEK2, PALB2, BARD1, RAD51C, RAD51D, and TP53 : 45 to 59). Fifteen percent of the patients had first-degree relatives with breast (n=1133) and 2% with ovarian cancer (n=151). Five percent (n=385) of our breast cancer patients were PTV carriers with one or more of the nine known breast cancer predisposition genes [Table on genes  (Additior=0.27)   by PRS and 0.9 (IQR: 0.7 to 1.2) by the Gail model (Table n=4041) breast cancer patients were considered high risk by any of the four risk classification criteria studied ) ), positive family history (n=1247 (16%)), and PTV carriership (n=385 (5%)) , the breast cancer risk stratification tools studied identified 40% (n=1276) of the breast cancer patients to be high risk. Risk stratification by positive family history, PTV carriership, and 5-year absolute risk ≥1.3% by PRS or the Gail model identified 470 (15%), 213 (7%), 769 (24%), and 325 (10%) unique breast cancer patients who were considered at high risk of breast cancer, respectively  Fig. . The 5-yMore than 50% in the older age groups (50 years and above) of breast cancer were identified as at high risk when we added information of each criterion sequentially (starting with family history → above 1.3% 5-year absolute risk by the Gail model → above 1.3% 5-year absolute risk by PRS → PTV carriership)  developed breast cancer over a median follow-up of 20 years (IQR: 18 to 21), of which 19% (n=81) occurred within 5 years of recruitment. Among the 81, 38% (n=31) were above 1.3% 5-year absolute risk by PRS, while only 6% were above 1.3% 5-year absolute risk by the Gail model, suggesting a higher performance of the PRS than the Gail model for breast cancer risk stratification for middle-aged or older Chinese women.Summary characteristics and allele frequencies corresponding to the 313 variants included in the PRS of the 10,213 women from SCHS are presented in Additional file Due to the small number of events within 5 years of recruitment, we studied all breast cancer patients ignoring time to event. The proportion of patients with above 1.3% 5-year absolute risk by PRS (in SCHS’s breast cancer patients) was higher than that observed in SGBCC and MyBrCa’s patients   at above 1.3% 5-year absolute risk by PRS resulted in a larger proportion of high-risk women  as compared to using the Gail model (6%)  of these high-risk women identified by PRS developed breast cancer, which was similar to the 5%  by the Gail model, and 6%  by family history. Adding PRS to risk stratification identified 165 more breast cancer cases. Figure Identifying women  . The conIn terms of mammography screening, PRS has been reported to perform well at identifying the women who are most likely to benefit from this mode of detection . The assThe main strength of our study is that this is one of the largest and most well-characterized breast cancer cohorts of Asian women. However, many of the breast cancer risk assessment tools and the PRS are established based on European populations and their utility in Asian breast cancer populations remains unclear. Approximately half of the breast cancer population were identified as high risk, suggesting that other factors not considered in the risk prediction models  studied may be responsible. We classified breast cancer patients into risk categories based on 5-year absolute risks at the age of breast cancer diagnosis; this may not be representative of women without breast cancer. While we are likely to overestimate the 5-year absolute risk, results from the prospective cohort (SCHS) support the use of genetic factors on top of family history and the Gail model. As this is a case-only cohort, the proportions from the risk classification analysis are not representative of the general population, where most women will be classified as low risk. Nonetheless, this will not affect the comparison of how different criteria identify women at high risk.While this study’s main focus was to highlight the lack of an overlap between high-risk women identified by genetic and non-genetic risk factors, it is worth noting that other works in the field have studied the potential improvements in risk prediction by combining different risk factors. For instance, Choudhury et al. explored the value of adding mammographic density and PRS to classical risk factors in a population of women of European ancestry . In a moIn summary, our assessment shows that family history and genetic and non-genetic risk stratification tools have the potential to complement one another to identify women at high risk in breast screening programs. The results add to the growing body of evidence to support a paradigm shift from an approach that is age-based to risk-based.Additional file 1. Details on the study populations, DNA isolation and genotyping, polygenic risk score (PRS) calculation, and methods to obtain absolute risk by PRS and the Gail model.Additional file 2: Table S1. Targeted regions of gene panel (hg19). Table S2. List of predicted protein-truncating variants. Table S3. Carriers of protein truncating variants. Table S4. Characteristics of cohorts. Tables S5 and S6. Agreement between the five criteria in identifying breast cancer patients at high risk of breast cancer. Table S7. List of 313 variants in the polygenic risk score. Figure S1. Comparing the five-year absolute risk prediction. Figures S2 and S5. Venn diagram of breast cancer patients at high risk of breast cancer. Figures S3 and S4. Proportion of breast cancer patients identified as being at high risk.Additional file 3. The incidence and mortality rates of Singapore.Additional file 4: Table SM1 and SM2. List of 313 variants in the polygenic risk score. Table SM3. Mean and standard deviation of polygenic risk scores. Table SM4. Relative risk of the percentile corresponding to Figure SM4. Figure SM1-SM3. Flowcharts for individuals selected. Figure SM4 and SM5. Five-year absolute risk of developing breast cancer.