BRCA1/2 mutation carriers are recommended to undergo risk-reducing salpingo-oophorectomy (RRSO) at 35 to 45 years of age. RRSO substantially decreases ovarian cancer risk, but at the cost of immediate menopause. Knowledge about the potential adverse effects of premenopausal RRSO, such as increased risk of cardiovascular disease, osteoporosis, cognitive dysfunction, and reduced health-related quality of life (HRQoL), is limited.The aim of this study is to assess the long-term health effects of premenopausal RRSO on cardiovascular disease, bone health, cognitive functioning, urological complaints, sexual functioning, and HRQoL in women with high familial risk of breast or ovarian cancer.We will conduct a multicenter cross-sectional study with prospective follow-up, nested in a nationwide cohort of women at high familial risk of breast or ovarian cancer. A total of 500 women who have undergone RRSO before 45 years of age, with a follow-up period of at least 10 years, will be compared with 250 women (frequency matched on current age) who have not undergone RRSO or who have undergone RRSO at over 55 years of age. Participants will complete an online questionnaire on lifestyle, medical history, cardiovascular risk factors, osteoporosis, cognitive function, urological complaints, and HRQoL. A full cardiovascular assessment and assessment of bone mineral density will be performed. Blood samples will be obtained for marker analysis. Cognitive functioning will be assessed objectively with an online neuropsychological test battery.This study was approved by the institutional review board in July 2018. In February 2019, we included our first participant. As of November 2020, we had enrolled 364 participants in our study.Knowledge from this study will contribute to counseling women with a high familial risk of breast/ovarian cancer about the long-term health effects of premenopausal RRSO. The results can also be used to offer health recommendations after RRSO.ClinicalTrials.gov NCT03835793; https://clinicaltrials.gov/ct2/show/NCT03835793.DERR1-10.2196/24414 BRCA1 germline mutation carriers have a cumulative risk of 44% to develop ovarian cancer before the age of 80 years. For BRCA2 mutation carriers, this cumulative risk is 17% [BRCA1 mutation carriers and at the age of 40-45 years for BRCA2 mutation carriers) provided that the woman no longer has a wish to have children [BRCA1/2 mutation carriers in the Netherlands has increased greatly from 81% to 98% currently [Female children . During urrently . While Rurrently .BRCA1/2 mutation carriers may have been treated for breast cancer, which can be cardiotoxic and potentially have a negative impact on the brain [The relationship between early menopause  and noncancer health outcomes, including cardiovascular disease (CVD), osteoporosis, cognition, and health-related quality of life (HRQoL), has been examined previously. However, most of the published studies had a limited follow-up (median <5 years), which is too short to establish an association with long-term health effects -15. Addihe brain -19. Lasthe brain .surgical menopause compared with natural menopause [BRCA1/2 mutation carriers. Thus, information about the cardiovascular risk of early surgical menopause is still limited [cause of the higher cardiovascular disease risk in women with POI. The reverse causality hypothesis postulates that POI is the result of accelerated vascular aging, which would also explain a statistical  association between earlier natural menopause and increased CVD risk [Before the age of 50 years, female sex hormones, such as estrogen and progesterone, are likely to protect women against CVD as they have atheroprotective properties ,22. Prevenopause . However limited -29. FurtCVD risk . If thisReproductive hormones play a role in maintaining bone health. Estrogen suppresses bone resorption and stimulates bone formation . PostmenBRCA1/2 mutation carriership was not provided [BRCA1/2 mutation carriers is limited.The neuroprotective effects of estrogens, progestogens, and androgens have been described in several studies . Rocca eprovided . Additioprovided . Thus, wBRCA1/2 mutation carriers with a history of breast cancer.RRSO may have an important impact on HRQoL, including generic issues, such as physical, role, and emotional functioning, as well as cancer-related anxiety, sexual functioning, and menopause-specific symptoms ,35,36. HThe number of postmenopausal years is associated with an increased prevalence of vulvovaginal atrophy . AtrophyBRCA1/2 mutation stated that no conclusions can be drawn regarding bone fracture incidence and HRQoL, and that further research is needed. CVD and cognition were not included as outcomes in this review, probably because of the paucity of studies [A recent Cochrane systematic review on the benefits and harms of RRSO in women with a  studies . TherefoBRCA1/2 mutation. Our secondary study aims are to assess overall HRQoL, endocrine symptoms, such as hot flashes and night sweats, sexual functioning, urogenital problems, and the prevalence of cardiovascular risk factors.The primary aim of our study is to assess the long-term health effects of RRSO on subclinical CVD, bone mineral density (BMD), and cognitive functioning in women at increased familial risk of breast or ovarian cancer mainly due to a We will include 500 women who have undergone RRSO before the age of 45 years, with a follow-up period of at least 10 years (the early RRSO group). We will compare them with 250 women from the same cohort (frequency matched on current age) who have not undergone preventive RRSO or have undergone RRSO at the age of 55 years or above (the late/non-RRSO group). The exclusion criteria are a metastatic disease; a serious physical comorbidity or psychiatric disorder; a language barrier; a metal cardiac valve; a bare metal coronary stent, since the reflection of the stent would interfere with the computed tomography scan; and natural POI before the age of 40 years. A history of nonovarian cancer, including breast cancer, is not a reason to be excluded.Eligible women who are alive will be invited for a clinic visit and questionnaire survey that will assess CVD status, BMD, cognitive functioning, and HRQoL. For eligible women who have died, we will collect information on the causes of death to examine selection bias.BRCA1/2 germline mutations included since 1997 by all eight Dutch University Medical Centers (UMCs) and the Netherlands Cancer Institute (NKI). The cohort currently consists of over 3600 proven female BRCA1/2 germline mutation carriers. Participants have given informed consent for linkage with the Netherlands Cancer Registry and the Dutch Nationwide Pathology Database (PALGA), which can provide information on all prophylactic mastectomies and salpingo-oophorectomies performed in the Netherlands since 1991. This provides us with nearly complete information on the occurrence of RRSO. Data on lifestyle, family history, reproductive factors, and hormone use have been collected from HEBON participants through questionnaires [Participants will be selected from the dynamic nationwide HEBON study cohort, a national collaboration on HEreditary Breast and Ovarian cancer in the Netherlands, with women tested for onnaires .1c, high-sensitivity cardiac troponin, and high‐sensitivity C‐reactive protein. BMD is assessed by a dual-energy X-ray absorptiometry (DXA) scan and laboratory blood sampling, and cognitive functioning is assessed by the Amsterdam cognition scan (ACS).Participants will be asked to complete an online questionnaire and an online cognitive assessment, and they will be asked to visit the outpatient clinic for a cardiovascular and bone health assessment. We will assess several surrogate endpoints of cardiovascular health, that is, coronary artery calcium (CAC) score (CAC scoring), pulse wave velocity (PWV), advanced glycation end products (AGEs), anthropometric measurements, and blood sampling. We will determine nonfasting blood levels of lipids, glucose, HbAThe online questionnaire is divided into the following several topics: general cardiovascular history, family history of CVD, traditional CVD risk factors , lifestyle , female-specific risk factors , history of fractures, calcium intake, vitamin D supplement use, glucocorticoid use, family history of osteoporosis/fractures, history of inflammatory diseases (rheumatoid disease and thyroid disorders), and perceived cognitive problems. We will assess generic HRQoL with the SF-36 health survey ,46 and tMeasurements of the CAC score as calculated by coronary calcium scoring will be performed according to standardized local protocols at the various UMCs using the Agatston score (AS) and the Multi-Ethnic Study of Atherosclerosis (MESA) coronary heart disease risk score calculator ,57. A diThe PWV will be assessed with an ambulatory arteriograph (TensioMed Kft) by the research physician. The arteriograph uses an oscillometric occlusive method, with an upper arm cuff to measure the time interval between the peak of the first systolic wave and the peak of the reflected systolic wave (return time). The device has been validated in numerous studies -65.With aging, the end products of nonenzymatic modification of proteins, lipids, and nucleic acids (AGEs) accumulate endogenously in the serum and tissues. Levels of AGEs in the skin are measured noninvasively by standardized autofluorescence and can predict future cardiovascular events ,67. SkinBone health will be assessed by a DXA scan, a vertebral fracture assessment, and blood levels of bone turnover markers (BTMs) for osteoclast and osteoblast activity . For logistic reasons, it is not possible to get fasting blood samples for every woman. Since β-CTX values are only valid in fasting blood samples, we will assess this BTM only in those women for whom we have fasting blood samples. P1NP will be measured in all samples.Lastly, women will receive an invitation to fill out the online ACS, which has recently been developed and validated . The ACSWe will examine the effect of age at RRSO on the risk of breast cancer, contralateral breast cancer, and ovarian cancer, as well as associations between RRSO and the prognosis of subsequent breast cancer and ovarian cancer. Additionally, after completion of the cross-sectional study, we will perform longitudinal follow-up every 4 to 5 years to evaluate incidence rates of outcomes of major interest, such as ischemic heart disease, and evaluate changes in outcomes  over time between the early RRSO group and the late/non-RRSO group. Moreover, with the anticipated prospective follow-up of our study population, we will look at mortality of cardiovascular disease.All data, including blood and DNA samples, will be stored for 30 years to enable prospective longitudinal follow-up of the study population on the long-term health effects of RRSO. The stored blood samples will allow for future international studies, for example, those on the modifying effects of genetic factors, such as single nucleotide polymorphisms, on the outcomes of interest, and the novel cardiovascular markers or BTMs for these outcomes. All blood samples will be frozen and stored at −80°C at the NKI.We will analyze differences in the cardiovascular risk profile, BMD, and neurocognitive functioning between the early RRSO group and the late/non-RRSO group using multivariable linear regression analyses and logistic regression analyses with clinically relevant cutoff points for the outcomes of interest. The analyses will be adjusted for confounders, including lifestyle, reproductive factors, and medication use, where applicable. For the analysis of patient-reported outcomes, such as HRQoL, sexual functioning, and menopausal symptoms, we will use mixed effect models.BRCA1/2 mutation status, age at RRSO (<40 years/≥40 years), and time since RRSO (10-15 years/>15 years).We expect approximately 40% of the women to have a breast cancer history and will perform the analyses separately in women with and those without a breast cancer history, as breast cancer treatment may affect outcomes. Additional subgroup analyses will be performed according to HRT use (yes/no), We based our sample size calculations for cardiovascular status on subclinical atherosclerosis by assessing the differences in the CAC score between women in the early RRSO group and women in the late/non-RRSO group. We chose a clinically relevant cutoff for the CAC score of 100 Agatston units (AU). According to the MESA trial, 10% of subjects between 55 and 65 years of age exceed this value . For sam2 in BMD between the early RRSO and late/non-RRSO groups, as observed between women with premature and normal menopause and assuming an SD of 0.15 [P value set at .05 (two tailed). To detect a relevant difference between groups for the various HRQoL measures, we will use a half SD (an effect size of 0.5) to define clinically relevant group differences (over time). This requires about 67 patients per group. The available sample will be more than adequate to conduct subgroup analyses as well.With 750 women in our study, we will be able to detect a difference of 0.07 g/cm of 0.15 . Statist of 0.15 . This stThis study was funded by the Dutch Cancer Society in 2016. The Institutional Review Board of the Netherlands Cancer Institute approved this nationwide multicenter study in July 2018, and local approval from the participating sites followed in the months thereafter.This study will be conducted according to the standards of Good Clinical Practice, in agreement with the principles of the Declaration of Helsinki and with the Dutch law as stated in the Medical Research Involving Human Subjects Act (WMO). The study has been approved in writing by the Medical Ethics Committee of the Antoni van Leeuwenhoek/Netherlands Cancer Institute (AVL/NKI) to be conducted in all nine University Medical Centers and the Antoni van Leeuwenhoek and has been registered at “CCMO Toetsingonline” from the Dutch Central Committee on Research involving Human Subjects (file number NL63554.031.17) and on ClinicalTrials.gov (NCT03835793). Written informed consent will be collected from the participants.In February 2019, we included our first participant, and as of November 2020, we had enrolled 364 participants in our study. Of these 364 participants, 228 are in the early RRSO group and 136 are in the late/non-RRSO group. Recruitment of participants is currently ongoing. Results will be disseminated through peer-reviewed publications and will be incorporated in follow-up guidelines.BRCA1/2 mutation carriers, with an estimated uptake in the Netherlands of 81% to 99% [To our knowledge, this study is the first to assess the long-term noncancer health outcomes of early surgical menopause in women with a high familial risk of ovarian cancer, focusing on CVD, bone health, cognition, and HRQoL. The issue is important as RRSO at the age of 35-45 years is an increasingly common procedure in At present, it is still unclear whether early surgical menopause can be considered as a clinically relevant risk factor for CVD. During normal menopausal transition, CVD risk increases ,26,78,80The association between early surgical menopause and long-term bone health has also been rarely examined. Studies were inconclusive regarding decreased BMD and increased fracture risk, and whether these risks remain increased over time ,84. In aAfter menopause, women more often report sexual and urogenital problems, such as urinary incontinence, recurrent urinary tract infections, impaired libido, and dyspareunia, which interfere with HRQoL. Women with POI experience these problems even more ,87. We dOne of the major strengths of our study is that it will be the first to examine the health effects of premenopausal RRSO at less than 45 years with a follow-up of over 10 years compared to controls with natural menopause. Additionally, owing to the large number of participants, we will have sufficient power for subgroup analyses. A limitation of our study could be survivor bias. Owing to the inclusion criterion of at least 10 years of follow-up, patients may already have died of relevant study outcomes before study inclusion. However, as we conduct the cross-sectional study within a well-characterized cohort, we can assess the extent of such bias by collecting cause of death information for potentially eligible but already deceased women.This initial cross-sectional study will provide prevalence rates of noncancer outcomes. Therefore, we will follow the study cohort prospectively to obtain incidence rates of outcomes of interest and evaluate changes in outcomes over time.BRCA1/2 mutation carriers considering RRSO and can be used to offer individualized counseling about additional screening and interventions for noncancer diseases or complaints after RRSO.To summarize, the current literature lacks information on long-term noncancer outcomes in women at increased risk of breast/ovarian cancer who have undergone early oophorectomy. With this study, we will be able to provide insights into the prevalence and severity of CVD, osteoporosis, cognitive impairment, and HRQoL effects after RRSO. This knowledge will yield evidence-based information for women and for health care providers about the long-term effects of early RRSO. The results can be incorporated in decision-support tools about risk-reducing surgeries in


CTLA-4, PDCD1, PD-L1, BTLA, TIM-3, and LAG-3 genes in order to select SNPs which can be used as predictive biomarkers in personalized evaluation of cancer risk development and outcomes as well as possible response to immunotherapy.In recent years, immunotherapy has been revolutionized by a new approach that works by blocking receptors called immune checkpoints (IC). These molecules play a key role in maintaining immune homeostasis, mainly by suppressing the immune response and by preventing its overactivation. Since inhibition of the immune response by IC can be used by cancer to avoid recognition and destruction by immune system, blocking them enhances the anti-tumor response. This therapeutic approach has brought spectacular clinical effects. The ICs present heterogeneous expression patterns on immune cells, which may affect the effectiveness of immunotherapy. The inherited genetic variants in regulatory regions of ICs genes can be considered as potential factors responsible for observed inter-individual differences in ICs expression levels on immune cells. Additionally, polymorphism located in exons may introduce changes to ICs amino acid sequences with potential impact on functional properties of these molecules. Since genetic variants may affect both expression and structure of ICs, they are considered as risk factors of cancer development. Inherited genetic markers such as SNPs may also be useful in stratification patients into groups which will benefit from particular immunotherapy. In this review, we have comprehensively summarized the current understanding of the relationship between inherited variations of  Immune checkpoints (ICs) are key receptors that inhibit the immune response and prevent from its overactivation. Under normal conditions, this mechanism is responsible for maintaining tolerance to its own antigens; however; it can be used by cancer cells to avoid recognition and destruction . A discoRecently, a number of receptors belonging to the immune checkpoint family have been discovered. However, only two of them CTLA-4 and PD-1 are primarily and broadly explored and their blockade is used as a therapeutic procedure in the routine treatment of several cancers: among others, in the treatment of advanced melanoma, non-small cell lung cancer, non-Hodgkin’s lymphoma, and kidney cancer .In spite of the evidence of considerable clinical relevance of anti-CTLA-4 and anti-PD-1 antibodies there is still a need for deeper understanding of the relation between the host’s genetics and mechanisms involved in the regulation of these pathways; especially when considering that only some patients (20–40%) respond to therapy, while a small proportion of patients experience rapid progression or an increased risk of death . In addiTherefore, in recent years new inhibitory pathways have started to be intensively studied as promising targets for immune checkpoint blocking therapy. Several potential molecules as well as their ligands are considered , among tCTLA-4 and other immune checkpoints molecules have been studied in the context of variety types of cancers.The immune status of cancer is modulated by many factors including patients’ immune reactivity which may be affected by single nucleotide polymorphisms (SNPs) of immune related genes . These SCTLA-4, PDCD1, PD-L1, BTLA, TIM-3, and LAG-3 on cancer risk. In case of the CTLA-4, PDCD1 as well as PD-L1 genes, a considerable number of meta-analyses combining the results of numerous studies are available in the literature and in the next paragraphs their results will be preferentially analyzed. In the case of other IC molecules, the results of individual studies in particular cancers will be presented. Of note, we omitted those reports that analyzed only a small number of individuals in case-control groups.This review will summarize current knowledge about the impact of the genetic variants of eff) upon activation, and constitutively by the regulatory T cells (Tregs)  was discovered in 1987 by Brunet and colleagues Figure 1 (Tregs) . Althoug (Tregs) –15]. The (Tregs) , 17.CTLA-4 engagement, upon TCR activation, decreases T cells response through the inhibition of co-stimulatory signals coming from CD28. This molecule is vital for regulation of T-cell homeostasis and self-tolerance .From an “oncological” point of view, the most important feature of CTLA-4 is its engagement in evading immune surveillance by cancer cells, since cancer cells may employ mechanisms leading to upregulation of CTLA-4 and as consequence repression of immune response toward tumor cells . The disAfter more than decade later the idea of Prof. Allison’s team achieved an enormous success, when it was proved that antibody against CTLA-4 – ipilimumab, was active in patients with metastatic melanoma. Moreover, a long-term response to this treatment was observed in patients with prolonged stable disease . FinallyCTLA-4 gene may have impact on the ratio between these two isoforms and in consequence on T cell activity (The human CTLA-4 exists in two main isoforms: the full length CTLA-4 (flCTLA-4) and soluble CTLA-4 (sCTLA-4), which is devoid of the transmembrane domain . The flCactivity , 23.CTLA-4 gene is located between the CD28 and ICOS genes on chromosome 2q33.2. The structure of CTLA-4 gene is presented in The CTLA-4 gene expression is not fully understood. It is regulated by the NFAT TF, since it has been shown that NFAT binds to the CTLA-4 promoter region  and rs733618 (CTLA-4c.-1722T>C) are located in the upstream regulatory region and may impact the transcription-associated binding activity of CCAAT-enhancer binding protein C/EBPβ and nuclear factor 1 (NF-1) TFs, respectively. The in silico analysis suggested that the change of adenine to guanine at -1661 position may lead to creation of a new binding site for a C/EBPβ TF   . This as-1722 was predicted to destroy a binding site for a NF-1 TF. It has been suggested that the NF-1 TF and element surrounding -1772 position may regulate CTLA-4 alternative splicing, since CTLA-4c.-1722*C allele was associated with higher expression of sCTLA-4 .In contrast, the substitution of thymine to cytosine at position  sCTLA-4 . Hence, rs11571317C>T (CTLA-4c.-658C>T) is located in 5’ UTR region. In silico analysis revealed that this polymorphism is located in potential binding site for a SP1 TF , and that presence of thymine at this site may lead to disruption of the SP1 binding motif. This observation suggests that CTLA-4c.-658*T allele may be associated with lower expression of CTLA-4 molecule  may confer increased risk of cancer development.molecule . Hence, rs5742909C>T (CTLA-4c.-319C>T) is located in the promoter region. It has been demonstrated that this variant modified a binding site for a LEF1 TF. The presence of cytosine or thymine at position -319 has been shown to have an impact on regulatory potential of the LEF1, which led to differential activation of the -319C>T variant by LEF1. More precisely, it has been demonstrated in the reporter assay that in the presence of LEF1, the activity of the reporter gene was higher for thymine at -319 position in activated Jurkat cells  and CD4+ T cells, but not on CD8+ T cells  than PBMCs from individuals with CTLA-4c.-319 C/T or C/C genotypes. The authors concluded, that the carriers of CTLA-4c.-319*T allele “have stronger negative regulation of T-cell proliferation and function, which might be the underlying mechanisms conferring cervical cancer susceptibility”  is a non-synonymous SNP which leads to an amino acid change from threonine to alanine in position 17 of CTLA-4 leader peptide (Thr17Ala). The presence of the CTLA-4c.49 A/A (Thr17) genotype, as opposed to the G/G genotype (Ala17), was associated with significantly lower levels of T cells activation and lower proliferation of these cells. It has been also demonstrated that CTLA-4 molecule with (Thr17) had a higher capacity to bind CD80 and a stronger inhibitory effect on T cell activation compared to (Ala17)  . Moreove (Ala17) . It was fficking , 39. Takrs3087243G>A (CTLA-4CT60G>A) is situated in the 3’ UTR. The CTLA-4CT60 G/G genotype has been shown to be associated with lower expression of sCTLA-4 transcript  of healthy donors , CTLA-4c.-319C>T , CTLA-4CT60G>A , CTLA-4c.-1661A>G , and CTLA-4c.-1722T>C (in 14 studies). According to this analysis, three SNPs were significantly associated with overall cancer risk: CTLA-4c.-1661A>G ; CTLA-4c.-319C>T (T+ vs. C/C OR = 1.33); CTLA-4c.49A>G , while CTLA-4CT60G>A and CTLA-4c.-1722T>C were not associated with overall cancer risk. In analyses stratified by ethnicity, both CTLA-4c.49A>G and CTLA-4c.-1661A>G were significant susceptibility polymorphisms in Asians, but not in Caucasians, while CTLA-4c.-319C>T was associated with overall cancer susceptibility in Caucasians . When stratify by cancer type, the strong associations between the CTLA-4c.49A>G and bone, liver and pancreatic cancers  as well as breast and head and neck cancers  were observed. Additionally, CTLA-4c.-1661A>G was associated with significantly increased susceptibility to breast and head and neck cancers . Previous meta-analyses by Geng et al. (2014), Sun et al. (2008) and Zhang et al. (2011) . In a stratified analysis by ethnicity, a significant association was observed for CTLA-4c.-319*T allele in Asian, but not in Caucasian women. This meta-analysis confirmed the results of the previous studies . In the study by Xiong et al. (365 women - cases and controls) the carriage of CTLA-4CT60*G allele was associated with increased cervical cancer risk (OR = 1.92), and with more advanced stages of this disease   , 75. HowCTLA-4c.49A>G and breast cancer (BC) was evaluated in two meta-analyses . The most recent study examining relationship between CTLA-4c.49A>G and BC outcome was performed by Babteen and colleagues among Egyptian women .The n et al. , based on et al. , 91, 92.CTLA-4CT60G>A and the risk of BC. These studies revealed that A/A genotype was associated with lower risk of BC developing, while CTLA-4CT60*G allele increased the risk. Such association was not found in the study by Goske and colleagues   , howeverr growth . This grgenotype . Moreovegenotype  and by Cgenotype  revealedCTLA-4 SNPs: CTLA-4c.-1772T>C, CTLA-4c.-1661A>G, CTLA-4c.-319C>T, CTLA-4c.49A>G, CTLA-4CT60G>A and gastric cancer (GC) risk . This finding is inconsistent with results for other types of cancer, where the CTLA-4c.-319*T allele has been identified as a tumor risk factor. Additionally, this meta-analysis indicated that the carriage of the CTLA-4c.-1661*A allele may predispose to GC development (OR = 1.78)  risk , 95–100. = 1.78) .CTLA-4 gene polymorphisms and risk of hepatocellular cancer (HCC) available in the literature. One of such study was published in 2018 by Wang et al.  risk was the subject of several studies.The evaluation of association between CTLA-4c.49A>G and CRC risk. Importantly, most of the studies included in this work were performed on Caucasians.The meta-analysis by Jiang et al. (2013)   ders11568821C>T  is located in intron 4, in an enhancer-like structure where four imperfect tandem repeats are placed. This region contains binding sites for TFs involved in hematopoietic differentiation and inflammation . According to Prokunina et al.  is located in exon 5. This synonymous variation (Ala268Ala) does not modify the amino acid structure of the protein. Therefore, it is speculated that this SNP may be in LD with another polymorphism, which may alter PD-1 expression (+CD4+ T cells) was significantly lower in individuals with PD-1.5 C/C genotype than those with the PD-1.5 C/T and PD-1.5 T/T genotypes  is located in the 3’UTR region, in a putative miRNA binding site. According to a study by Zhang et al. (PD-1.6 G/G genotype (but not in lymphocytes from patients with PD-1.6 A/A) miR-4717 mimic significantly decreased PD-1 expression and increased TNF-α and IFN-γ production, whereas miR-4717 inhibitor acted in opposite way  is a nonsynonymous SNP, located in exon 5, causing an amino acid substitution from alanine to valine in the extracellular domain of PD-1 leading to a different structure and possibly altering the function of PD-1  is located in intron 1, where a number of regulatory elements and splicing control elements exist. Due to the disruption of the splice site or alteration of the mRNA secondary structure, PD-1 rs7421861T>C may induce aberrant splicing, and lead to translational suppression (rs41386349G>A (C>T REV) is located in intron 4. According to data presented by Zheng et al. . A similar conclusion concerning PD-1.5 was drawn by the authors of three earlier meta-analyses   .PD-1.5, also PD-1.3 was associated with overall cancer risk. On the basis of 9 articles . Two earlier meta-analyses: by Zhang et al.  indicated an association of PD-1.3 G/A genotype with lower overall cancer risk.The findings of the meta-analysis by Hashemi et al.  revealedarticles , 167–170g et al.  and by Dg et al.  (in the T/C genotype of rs7421861T>C had an increased risk of cancer (OR = 1.16). Given the results of this study, it can be assumed that higher risk of cancer concerned carriers of rs7421861*C allele (OR = 1.14)   revealed = 1.14) . Despite = 1.14)  being si = 1.14)  significPD-1.1 and overall cancer risk was observed in two additional meta-analyses (PD-1.1 A/A genotype in the Asian population (OR = 1.15). Of note, taking into consideration the completely different genotype distribution among populations (PDCD1 gene (quite broadly examined in the context of cancers)—PD-1.9 —PD-1.9  and PD-1 . Whereas, in the study by Li et al.  . However, given the fact that in the study by Li et al. genotype distributions both in controls and cases were not in HWE, these results should be treated cautiously. In the study by Guzman et al. /IFNG (rs2430561AA)/PDCD1(rs2227981CT), although a major contribution to the observed association was suggested for CD28 and IFNG SNPs.As far as we know, only n et al. , based oontrols)  PD-1.5 Cn et al.  PD-1.5 sPDCD1 have been studied also in breast cancer. Two meta-analyses , indicated an association between PD-1.5 and susceptibility to BC. A decreased risk of BC (OR = 0.8) was observed for carriers of the PD-1.5*T allele (increased for PD1.5 C/C genotype). This observation is in line with the results obtained for overall cancer risk, however it is worth mentioning that two subgroup analyses for BC  were conducted on the same two studies. Two original research articles on the basis of which the analyses were made , whereas in the study by Haghshenas et al.  . A similar association was observed in a subgroup analyses by cancer type (combining the results of two above mentioned studies) performed by Hashemi et al., as part of meta-analysis   , which i21861T>C , 172, PDPDCD1 polymorphisms and ovarian cancer. The results of a case-control study  by Tan et al. demonstrated that possession of PD-1.9*T allele was associated with increased risk of ovarian cancer (OR = 1.67). The same allele was also associated with higher FIGO stage and higher differentiation grade , as in the case of overall cancer risk in Asians. Additionally, from data obtained by Li et al. it can be concluded that the PD-1.5*T allele was associated with a reduced risk of ovarian cancer (OR = 0.82)  .inter alia at evaluation of association between PDCD1 polymorphisms and the risk of gastrointestinal cancer . Just as in the case of overall cancer risk, decreased risk of gastrointestinal cancer was observed for individuals with the PD-1.5 T/T genotype (OR = 0.60), whereas increased risk was noted for subjects with rs7421861 T/C genotype (OR = 1.19)   . A highe = 1.16) . It is nPDCD1 SNPs as potential risk factors for esophageal cancer, more precisely esophageal squamous cell carcinoma (ESCC). In all three studies the PD-1.6 was suggested to be associated with susceptibility to esophageal cancer. However, two of them  . Moreover, in this study individuals with PD-1.6 G/G genotype and carriers of PD-1.6*G allele had, respectively, higher TNM stage (OR = 1.81) and higher risk of distant metastasis (OR = 1.67)  and higher TNM stage (OR = 1.37) for rs7421861*T allele carriers     . Moreove = 1.48) . This ob of ESCC , 175, hoPDCD1 polymorphisms  and risk of esophagogastric junction adenocarcinoma (EGJA) . Increased risk of EGJA  was noted for carriers of rs7421861*C allele (OR = 1.43)  and this observation was in accordance with the results obtained for gastrointestinal cancer (PD-1.1 C/C decreased risk of EGJA was noted (OR = 0.86)  . Higher l cancer , ESCC inl cancer  as well l cancer . Moreove = 0.86) . No asso (PD-1.6 .PD-1.5 and gastric cancer indicated that the presence of PD-1.5 C/T genotype may be a risk factor for GC (OR = 1.77) . Given the inconsistency with the results of other research and the fact that only 122 GC patients and 166 controls were enrolled to this study, further examination of this SNP in the context of GC will be required.Savabkar et al. in their study aimed at determining the potential association between  = 1.77) . From thPD-1.9, PD-1.6, and rs7421861T>C were studied in gastric cardia adenocarcinoma (GCA)  (The ma (GCA) , however = 2.04) .PD-1.3 and PD-1.6 may be considered as risk factors for HCC. There was no evidence of association between the PD-1.3 and HCC risk in the study carried out by Bayram et al. on 236 patients and the same number of control subjects   .PDCD1 were also investigated in colorectal cancer  by Mojtahedi et al. However, after patients subdivision for cancer location, it was observed that PD-1.5 C/T genotype was significantly more frequent in colon cancer patients as compared with healthy controls (OR = 1.74)   examined by Ge et al. , only rs7421861T>C was shown to be associated with susceptibility to CRC, with the rs7421861 T/C genotype increasing risk of CRC development (OR = 1.31)    . Moreove = 1.31) . Additio = 2.18) , however = 1.47) .PDCD1 polymorphisms with hematological malignancies was evaluated in several studies including multiple myeloma and leukemia.The potential association of PD-1.1, rs41386349, PD-1.9) individually were associated with multiple myeloma (MM). However, the authors observed higher frequency of the haplotype combination (PD-1.1 rs41386349 PD-1.9/PD-1.1 rs41386349 PD-1.9) G C C/G C C in the group of MM patients (N = 124) as compared with controls (N = 211). In the study by Grzywnowicz et al.  and susceptibility to chronic lymphocytic leukemia (CLL) were found in the group of 114 patients and 150 controls (PD-1.9 C/T genotype as associated with a decreased risk of leukemia (OR = 0.43)  , howeverPDCD1 SNPs and lung cancer was performed only in few studies. In a subgroup analysis performed on 1,058 patients and 1,103 controls , includi et al. , 197,  were examined in NSCLC in the study performed on 320 patients and 199 control individuals by Du et al.  as well as for rs4742098 A/G heterozygotes (OR = 1.60) (rs2297136 G/G genotype were less likely to exhibit LN metastasis (OR = 0.27) and more likely to exhibit distant metastasis (OR = 3.83) in comparison to subject with the rs2297136 A/A genotype. In case of rs4742098A>G, carriers of rs4742098*G allele had greater depth of tumor infiltration as compared to individuals with rs4742098 A/A (OR = 2.30). Taking into consideration the fact that this is the only study considering 3’UTR polymorphisms and NSCLC, further investigations are needed .Additionally, three SNPs in 3’UTR of u et al. . In the  = 1.60) . Furtherrs4143815G>C did not show the association with NSCLC risk and progression in the aforementioned study by Du et al.  had shorter overall survival as compared to rs4143815*C allele carriers. Similar effect was also observed for two promoter polymorphisms of PD-L1 gene—rs822336G>C and rs822337T>A. In details, subjects with rs822336 C/C as well as subjects with rs822337 A/A genotypes showed shorter overall survival as compared to rs822336*G and rs822337*T allele carriers, respectively. The above described associations between PD-L1 polymorphisms and cancer risk are summarized in Although u et al.  it was du et al.  that indBTLA was discovered in 2003 by Gavrieli et al.  as a thiSimilarly, to CTLA-4 and PD-1, BTLA  is a type 1 transmembrane glycoprotein comprising 289 amino acids Figure 4+ and CD8+ T cells, however upon T cell activation expression progressively decreased. In contrast to CTLA-4 and PD-1 which are highly expressed on Treg cells, BTLA expression is weak, while it is highly expressed on anergic T cells generated by chronic exposure to antigens  secretion   , 210, sytis (RA) . Below wBTLA genetic variants in different populations is presented in The frequency of the described below rs1844089C>T (G>A REV) and rs2705535C>T (G>A REV) are located in first intron, the exact functional role of these SNPs was not established yet, however Ge at al. postulated that rs2705535 may affect splicing of BTLA gene (TLA gene .rs9288953C>T (G>A REV) is situated in the first intron. Ge et al.  is located in exon 4, and leads to the exchange of asparagine to threonine at position 197 in the intracellular domain. Oki and colleagues  indicated that IL-2 production was strongly inhibited in cells expressing BTLA with BTLAc.590*A, whereas in cells with BTLAc.590*C the IL-2 production was enhanced. Moreover, transfected cells were stimulated with anti-CD3 and anti-BTLA antibodies (Ab). Anti -BTLA Ab inhibited IL-2 production only in Jurkat expressing BTLAc.590*A. The authors suggest that BTLAc.590*C had not ability to inhibit IL-2 production by Jurkat cells.lleagues  performeBTLAc.590*A allele was associated with decreased inhibitory activity of BTLA in Jurkat cells it may potentially constitute cancer risk factor.Even though the amino acid exchange is not within the ITIM motifs of BTLA, it is hypothesized that this substitution may alter the posttranslational modifications of BTLA, such as glycosylation of asparagine, or phosphorylation of threonine by serine/threonine kinase, which may influence the strength of BTLA signaling by SHP1/SHP2 . Hence, rs9288952T>C (BTLAc.800G>A REV) is a non-synonymous SNP causing an amino acid substitution from proline to leucine at position 267 in exon 5, which was initially described as associated with susceptibility to RA (ty to RA .rs1982809T>C (A>G REV) is situated in 3’ nearby gene region of BTLA (73 bp). In silico analysis with application of SNPinfo . Further studies are needed to established the functional role of this SNP. SNPinfo  and Fast SNPinfo  databasepatients . Althougrs2705511A>C is located in the intragenic region between genes encoding CD200 and BTLA (-97820bp||-3334bp). Rs2705511 and rs1982809 are in moderate linkage disequilibrium with each other  in relation to malignant BC risk  GTTTT was associated with the three times increased risk of BC in this population. These authors analyzed also association between these SNPs and some clinical features. The frequency of rs1844089 C/T genotype was higher in patients with tumor size over 5 cm, while  GTCTC haplotype was significantly more frequent in patients with metastasis to LNs  and esophageal squamous cell carcinoma was investigated by Cao et al.  risk of ESCC development in males. In a stratified analysis by age, BMI, smoking status, and alcohol consumption it was noticed that rs3112270 T/C genotype may protect lean individuals with BMI<24 from ESCC . Opposite effect was observed for rs3112270 C/C genotype in subjects with BMI>24. In this group C/C genotype increased 2 times risk of ESCC (C/C vs. T/T). Additionally, the G/A genotype of rs2171513G>A (G/A vs. A/A) was associated with lower risk of ESCC (OR = 0.61) in individuals’, who overused alcohol  and EGJA risk in 1,234 patients and 1,540 control subjects  TAAG increased three times risk of EGJA development. Moreover, after adjustment for gender, age, alcohol consumption and smoking, the A/A genotype of rs1982809A>G was associated with two times higher risk of EGJA in heavy smokers.Tang et al. examined relationship between subjects . None ofBTLA gene were investigated: rs1844089G>A, rs2705535C>T, and 9288953C>T. The T/T genotype of rs2705535C>T was associated with 2 times increased risk of rectal cancer development (in comparison to C/T+C/C), while the presence of rs9288953 T/T genotype decreased 1.4 times the risk of rectal cancer (in comparison to C/T+C/C)  .BTLA polymorphisms and hematological malignancies is concerned, such evaluation was performed only for CLL. The association of BTLA gene polymorphisms and CLL risk were evaluated by Karabon et al. . Additionally, the T/T genotype of rs9288953C>T was associated with 1.7 times higher risk of CLL.As far as potential association between n et al.  in 321 pBTLA SNPs rs1844089G>A, rs2705535A>C, rs9288953C>T, rs9288952A>G, rs16859633T>C, rs1982809A>G, rs2705511A>C, and renal cell carcinoma (RCC). The presence of rs1982809*G allele (G/G + A/G) was associated with 1.4 times higher risk of RCC, while G/G genotype was associated with higher risk (OR = 2.75) of the clear cell RCC (ccRCC) high grade tumors  , metastasis to LNs (OR = 3.71), and development of adenocarcinoma subtype of lung cancer (OR = 2.8) (BTLA polymorphisms and cancer risk is shown in The association of the following genetic variants of ontrols) . The posR = 2.8) . The sum+ (Th1), CD8+ (Tc1), and Th17 cells (T Cell Immunoglobulin And Mucin-Domain Containing-3 was discovered in 2002 (TIM-3) . TIM-3 a17 cells . TIM-3 e+ TIM-3+ CD8+ T cells are considered to be the most dysfunctional T cells—a “deeply” exhausted T cell subset  in a TIM-3/galectin-9 dependent manner . The PD-1l subset . CD8+ T gression . As mentt manner . TIM-3 ise cells .Several ligands have been proposed for TIM-3: phosphatidyloserine (PtdSer) on apoptotic cells; high-mobility group box1 (HMGB-1) - a damage associated molecular pattern protein that is secreted by stressed innate immune cells; and galectin-9. It has been demonstrated that interaction between galectin-9 and TIM-3 resulted in inhibition of Th1 and Th17 response including peripheral tolerance , 223.HAVCR2 (minus strand) gene located on chromosome band 5q33.3 together with genes HAVCR1 and TIMD4 for other members of the TIM family, TIM-1 and TIM-4 respectively  in different populations is presented in The frequency of the rs891246256G>A (-1541C>T REV) and rs10053538C>A (-1516G>T REV) are the 2 kb upstream SNPs. The function for rs891246256G>A has not been established yet. The rs10053538 is situated at the putative binding site of p300 TF  of this SNP has been shown to be associated with higher expression of TIM-3 on liver infiltrating lymphocytes in tumor tissue of HCC patients in comparison to -1516 G/G genotype as determined by immunohistochemistry ( p300 TF . The prehemistry rs1036199A>C (+4259T>G REV) is located in exon 3. This is a missense polymorphism causing substitution from arginine to leucine in position 140 (R140L). The functional consequence of this change has not been reported so far but it can be postulated that this variant may affect the mucin domain (rs1036199 and autoimmune diseases (ADs) mainly in Asian populations pointed to the rs1036199*G allele as a risk factor increasing susceptibility to ADs , -574*T (OR = 1.99), and +4259*G (OR = 2.21) were associated with a higher overall cancer risk  (OR = 1.79) and for the -574*T  (OR = 1.77) alleles . This allele was also associated with digestive system cancer risk (OR = 1.61)   . The authors concluded that their study clearly demonstrated involvement of HAVCR2 gene polymorphisms  in conferring higher risk for development of different human cancers   [based o = 1.61) ]. As forcontrols , the car = 2.21) . In the  = 2.39) . This ste system  which wae system . The ass cancers .HAVCR2 genetic variants and the risk of gastric cancer (GC) development was published in 2010 by Cao at al , -822 C/T (OR = 3.19), and -547 G/T (OR = 2.74)  in GC patients  of PC development in comparison to subjects with the G/G genotype  genotype and +4259 G/G (OR = 5.11) and an increased risk of CRC  . Additiopatients . Tong etpatients  conductegenotype . To the g et al.  examinedk of CRC .HAVCR2 gene polymorphisms and susceptibility to breast cancer were carried out on 560 BC patients and 583 control subjects of Northwest China descent  in comparison to the G/G genotype. Among patients, possession of -1516*T allele was associated with increased risk of LN metastasis (OR = 1.68)  (of note G/G genotype was not detected in this study) as well as with the LN or distant metastasis in BC patients (OR = 3.16). The two remaining genetic variants were not associated with BC risk or clinical parameters (The first study attempting to examine relations between the  descent . The aut = 1.68) . Additiogenotype . In anotgenotype  assessedrameters .1516G>T, -574G>T, and +4259T>G polymorphisms were investigated in non-Hodgkin lymphoma (NHL). A case-control study was performed on 496 NHL patients and 512 control subjects enrolled from the Han Chinese Population. The study demonstrated that subjects with the -574 G/T or +4259 T/G genotypes had more than two times higher risk for NHL development  in comparison to subjects possessing wild type genotypes -574 G/G and +4259 T/T  genotype was associated with increased risk of NSCLC (OR = 2.81)  (To the best of our knowledge so far only one report assessing the association of lleagues  examined = 2.81) . Moreovectively) .-1516G>T, -574G>T, and +4259T>G polymorphisms in 322 RRC patients and 402 control subjects of Chinese descent. Of the examined SNPs, association with increased RCC cancer risk was found for the -574 G/T (OR = 2.85) genotype and +4259 T/G genotype (OR = 3.34) . Additionally, the +4259*G allele was associated in RCC patients with metastasis (OR = 2.18) (HAVCR2 genetic variants and cancer risk is shown in Cai and colleagues  investig = 2.18) . The sumLymphocyte activation gene 3 is a heavily glycosylated type I transmembrane protein which shares significant homology with the CD4 molecule—in particular in its extracellular part, which consists of four immunoglobulin superfamily-like domains (D1-D4) Figure 6+ and CD8+ T cells, B cells, NK cells, but also on plasmacytoid DCs  MM, whereas carriers of rs3782735*A allele were less likely to develop MM (OR = 0.69) (Lee and colleagues  performe = 0.69) .LAG-3 genetic variants (described below) in different populations is presented in The frequency of the As was mentioned in the Introduction, the association between inherited variants in genes for ICs and cancer risk has been extensively investigated for CTLA-4 and broadly for PD-1/PD-L1 in a wide spectrum of cancers. Whereas, the potential targets for immune checkpoint blockers, like BTLA, TIM-3, and LAG-3 have been significantly less frequently investigated in such context. Moreover, there has been no publication to date that summarizes the knowledge on this subject. Therefore, our publication aims to fill this gap and collect currently available data and present the state of the art on that topic.CTLA-4c.49*A allele is associated with higher overall cancer risk alongside the risk of developing particular types of cancers, including breast, bone, and cervical cancers. Similarly, the CTLA-4c.-319*T increased an overall cancer risk in Caucasians, and predisposed to breast, cervical and hepatocellular cancers and bone cancer in Asian. The CTLA-4CT60*G allele was associated with higher risk of breast and hepatocellular cancers, while the CTLA-4c.-1661*G allele with overall, breast and gastric cancer risk.Our general impression derived from analysing of available literature survey is that SNPs may be considered as useful risk biomarkers in terms of cancer. From numerous studies it might be concluded that the PDCD1 gene polymorphisms, the PD1.1*A allele seems to be associated with susceptibility to overall cancer in Asians as well as with susceptibility to ovarian, breast, and EGJA cancers . The available data suggest that the PD-1.5*T allele may protect from cancer development in general, and specifically against ovarian and breast cancers. The rs7421861*C allele increased overall cancer risk as well as the development of colorectal and EGJA cancers. Finally, it may be suggested that the PD-1.9*T allele may confer higher risk of ovarian and EGJA cancers development.As for PD-L1 gene, namely rs4143815G>C has been shown to be associated with cancer risk, in particular the rs4143815*G allele increased risk of overall, hepatocellular, ovarian, and gastric cancers.Only one polymorphism in the BTLA gene polymorphisms and cancer risk is limited and since a meta-analysis of published results has not been performed yet, future research in this field is needed. On the basis of available literature, it can be however postulated, that rs1982809*G confer higher risk of lung cancer, CLL and renal cancer. The rs9288953 G/G genotype can be considered as a factor increasing risk of CLL and colorectal cancer, whereas rs1844089*T allele as a risk factor of breast cancer. Finally, rs2705535*A allele seems to be associated with higher risk of breast cancer and rs2705511*C with higher susceptibility to colorectal cancer and CLL.Since the number of studies concerning BTLA gene polymorphisms, genetic variants of the HAVCR2 gene for TIM-3 have also been rarely investigated so far, however two meta-analyses have been published. According to them, the minor alleles of -1516G>T, -822C>T, -574G>T, and +4259T>G can be considered as overall cancer risk factors. In particular, the +4259*G allele was associated with the increased risk of lung cancer, colorectal cancer, and NHL; the -574*T allele with NHL and gastric cancer; -822*T allele with gastric cancer and colorectal cancer and -1516*T with gastric cancer. The associations between variants of genes encoding LAG-3 and TIGIT and cancer risk has not been evaluated yet and this area is almost unexplored. Similarly, to A limitation of any conclusion arising from this literature survey is that most of the individual studies and meta-analysis were performed on Asian populations. It is worth mentioning that significant differences in distribution of genotypes between populations are observed and some of the presented associations are true only for individuals of Asian origin Table 1.As association between particular ICs’ SNPs and cancer risk seems to be relevant to specific population there is the urgent need to perform well designed studies on large scale in different populations in order to elucidate variants important in terms of risk estimation and selection of patients to the immunotherapy for particular ethnicities.The next issue concerns the quality of some studies, as well as numbers of patients and controls in compared cohorts. We observed the following negligence in carrying out case-control studies and meta-analyses which may influence the reliability of published data. In several reports we noted deviation from HWE in control subject which was not mentioned, and even worst the authors stated that such deviation was not observed. We came across a meta-analysis which was published in the same form in another journal. Another issue which created considerable difficulties in proper interpretation of analyzed data was serious inconsistency in describing SNPs and their alleles. The authors use the old, common names of SNPs without providing SNP reference numbers. Some of analyzed genes are located in minus orientation and we noticed that authors do not pay attention to that describing alleles in relation to plus or minus strand even within one work. This is particularly valid for SNPs causing G to C or C to G substitution if their frequency is similar in the population .As a conclusion from this study, we propose future directions of the research broadening and complementing presented topic.We think that in the future the researchers should analyze SNPs which will cover the whole genes of interest. The analysis of constellation of SNPs will allow to evaluate the phenotypic effect of particular haplotypes on immune cells which may allow evaluate if particular haplotype is associated with higher, lower or neutral risk of cancer development . MoreoveIt is also worth to analyze the impact of variation within genes encoding IC in context of tumor progression .Very important, but relatively poorly investigated is functional relevance of inherited variations not only in context of gene transcription, but also on gene translation. Little is also known about influence of SNPs on epigenetic gene regulation.TIGIT is considered as a new target for immunotherapy due to its important role in maintenance of immunosuppressive phenotype of T cells in TM. Nonetheless, to the best of our knowledge, in the literature there is no data concerning TIGIT polymorphisms in relation to cancer risk. The future research will need to fill this gap in our knowledge.CTLA-4, PDCD1, PD-L1 genes, while more studies are needed for BTLA, TIM3, LAG3, and TIGIT.In conclusion, the variants in genes encoding the molecules which regulate the immune surveillance might be considered as low-risk variants (OR<2) for cancer development, which has been well documented by numerous reports for The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The authors participated equally in conception, writing, and revising the manuscript. MW prepared the figures. All authors contributed to the article and approved the submitted version.This work was supported by the National Science Centre, Poland, grant number 2019/33/B/NZ5/03029, as well as by the Hirszfeld Institute of Immunology and Experimental Therapy statutory program number 28/2020.


The impact of mammography screening recall on quality-of-life (QOL) has been studied in women at average risk for breast cancer, but it is unknown whether these effects differ by breast cancer risk level. We used a vignette-based survey to evaluate how women across the spectrum of breast cancer risk perceive the experience of screening recall.Women participating in mammography or breast MRI screening were recruited to complete a vignette-based survey. Using a numerical rating scale (0–100), women rated QOL for hypothetical scenarios of screening recall, both before and after benign results were known. Lifetime breast cancer risk was calculated using Gail and BRCAPRO risk models. Risk perception, trait anxiety, and breast cancer worry were assessed using validated instruments.The final study cohort included 162 women at low , intermediate , and high-risk . Actual breast cancer risk was not a predictor of QOL for any of the presented scenarios. Across all risk levels, QOL ratings were significantly lower for the period during diagnostic uncertainty compared to after benign results were known (p < 0.05). In multivariable regression analyses, breast cancer worry was a significant predictor of decreased QoL for all screening scenarios while awaiting results, including scenarios with non-invasive imaging alone or with biopsy. High trait anxiety and family history predicted lower QOL scores after receipt of benign test results (p < 0.05).Women with high trait anxiety and family history may particularly benefit from discussions about the risk of recall when choosing a screening regimen. •Impact of screening recall on quality-of-life does not vary by breast cancer risk.•Breast cancer worry predicts lower quality-of-life ratings while awaiting results.•Quality-of-life ratings improve after receipt of benign results.•High trait anxiety predicts lower quality-of-life after benign results are known. HowevAlthough there are benefits to more intensive screening regimens in high-risk women, an inherent drawback to screening at younger ages and with multiple modalities is the increased frequency of recall for additional imaging and interventions in women who do not have cancer . StudiesWhile the impact of false positive examinations has been previously studied in average risk women, it is not known how these perceptions compare to those of women who are at high risk for breast cancer. As additional genetic and other risk factors for breast cancer are identified that qualify women for high-risk screening, it is important to understand the experience of false-positive screening examinations in this patient population to inform clinical guidelines and facilitate shared decision-making. In addition, this information is needed to inform cost-effectiveness analyses of high-risk screening strategies, as the cost-effectiveness of screening mammography has been shown to be highly sensitive to small, short-term effects on quality-of-life (QOL) related to the screening test .The purpose of this study was to better understand how women across a spectrum of breast cancer risk perceive the impact of false-positive screening examinations and biopsies on QOL. We hypothesized that perceived QOL during and after the experience of screening recall would vary by underlying breast cancer risk.2This study was HIPAA-compliant and approved by the Institutional Review Board of Massachusetts General Hospital.2.1We recruited women ages ≥25 scheduled for routine mammographic screening or high-risk breast MRI screening at Massachusetts General Hospital from July 2011 through December 2012. Women were eligible to participate regardless of history of prior screening recall and/or biopsy, or history of prior breast cancer. Eligible women received a written invitation by mail to participate in the study four weeks prior to their scheduled exam and were subsequently contacted by telephone after 10 days if no response was received. Participants were given the option to complete the survey online, by mail, or with assistance over the telephone with a study coordinator. Women could complete the survey at any time during the four weeks leading up to their screening exam appointment. On the day of the screening exam, the survey was closed.2.2We developed a vignette-based survey to assess women’s beliefs about breast cancer risk and the experience and consequences of false positive screening examinations leading to additional imaging and procedures. The survey instrument comprised of a series of vignettes describing temporary health state scenarios related to recall from screening mammography or MRI which participants were asked to rate using a numerical QOL rating scale, as deta2.3Participants were presented with a series of vignettes describing the temporary health states related to recall after a) abnormal screening mammography; or b) abnormal screening breast MRI. Descriptions of temporary health states such as vignettes can be used to elicit preferences and quality-of-life perceptions from the general population,22, and 2.4low/average risk (lifetime risk <15%); intermediate risk (lifetime risk 15–20%); and high risk (lifetime risk >20%) [Demographic information and breast cancer risk factors were used to calculate lifetime breast cancer risk using the Gail model  and the To assess perceived breast cancer risk, participants were asked to estimate their risk of developing breast cancer in their lifetime . Women who endorsed a personal history of breast cancer were asked to estimate their risk of developing a new or recurrent breast cancer in their lifetime .2.5high anxiety.The Spielberger State Trait Anxiety Index (STAI) is an established instrument used to assess general and trait anxiety . For the2.6Numeracy was assessed using the following multiple choice items taken from a previously validated instrument: 1) Whic2.7The Lerman Breast Cancer Worry Scale  is a 3-i2.8We performed t-tests, chi-square tests, univariate and multivariable regression to examine whether short-term quality of life ratings for each vignette were associated with the following variables: age (continuous), race, numeracy, personal and family history of breast cancer, breast cancer risk category (low/intermediate versus high), breast cancer worry and trait anxiety. Because breast cancer risk perception has been previously shown to be inaccurate among women,31, we p33.1Participant recruitment is summarized in The cohort included for final analysis included 162 women at low , intermediate  and high risk . Characteristics of the final cohort are summarized in 3.2The majority of women in the low/average and intermediate breast cancer risk groups overestimated their breast cancer risk (81% of low/average risk women and 68% of intermediate risk women). Perceived risk of breast cancer did not vary by actual risk category ; average perceived lifetime risk of breast cancer was 46%, 39%, and 48% in low/average, intermediate, and high-risk women, respectively . Among l3.3Univariate and multivariable regression results for QOL ratings during screening mammography recall are shown in QOL ratings did not vary by risk group for any of the presented scenarios across breast cancer risk groups . Across Multivariable regression analysis indicated that across all screening recall scenarios, breast cancer worry was a significant predictor of decreased QOL during the period of diagnostic uncertainty while awaiting results from non-invasive additional imaging or biopsy (mean QOL scores ranged from 25 to 33 in women with high worry versus 45–57 in women without high worry). High trait anxiety (top quartile STAI percentile score) was a predictor of persistently lower QOL scores in one scenario of diagnostic uncertainty after MRI-guided biopsy  and in all scenarios after receipt of benign results (mean QOL scores ranged from 50 to 60 in women with high anxiety versus 58–68 in women without high anxiety). Family history of breast cancer was also a predictor of persistently lower QOL scores after receipt of benign results in most (3 of 4) scenarios. Personal history of breast cancer and prior history of screening recall were not predictors of QOL ratings for any scenarios. Women who incorrectly answered one or more items on the numeracy measure had lower QOL ratings for screening mammography recall for additional imaging, but not for other scenarios. Age was a predictor of lower QOL score after MR-guided biopsy, but not in other scenarios. No other variables were predictors of QOL scores in multivariate analyses in any of the presented scenarios.4Our vignette-based survey of women across a spectrum of risk undergoing breast cancer screening suggests that perceived QOL during recall after screening mammography or MRI does not appear to vary by breast cancer risk level. Across a spectrum of risk, QOL was lower during the period of diagnostic uncertainty, and then improved after benign results were received. Breast cancer worry was a predictor of lower QOL during the period of diagnostic uncertainty, and trait anxiety and family history were significant predictors of persistently low QOL after receipt of benign results.Our finding that the perceived impact of screening recall does not differ between women at average and high risk of breast cancer may be explained by inaccurate breast cancer risk perceptions among the participants in our study. Interestingly, perceived breast cancer risk did not vary by actual risk category, with average and high-risk women estimating their lifetime risk of breast cancer to be 46% and 48%, respectively, and most (75%) average risk women incorrectly reporting >20% lifetime risk of breast cancer. These findings corroborate prior studies which have shown that women’s perceived risk of breast cancer is often inaccurate , althougOur findings are in keeping with the results of prior studies of women at average risk which have suggested that the period of diagnostic uncertainty is most distressing for women who have been recalled from screening, and that quality of life ratings improve on average after receipt of benign results . HoweverIn our study, trait anxiety was a predictor of persistently low QOL scores after receipt of benign results. In a prior study of Danish women recalled from screening mammography, trait aOur study has a few limitations worth noting. The participants in our study provided QOL ratings for hypothetical vignettes of the experience of screening recall for additional imaging and biopsy, and their perceptions may differ from women experiencing actual screening recall episodes. In addition, because of the nature of the scenario we could not assess short versus long-term consequences of screening recall. Finally, the women in our study received screening at a single institution and were predominantly non-Hispanic white women and highly educated, limiting generalizability.In conclusion, our vignette-based survey study suggests breast cancer risk affects QOL during breast cancer screening recall less than other factors such as breast cancer worry and trait anxiety. Across the spectrum of risk, breast cancer worry predicted lower QOL ratings for recall during the period of diagnostic uncertainty while awaiting test results. While quality of life generally improved after receipt of benign results, women with high trait anxiety levels reported persistently decreased quality of life even after results were known. Our results suggest that these women may particularly benefit from discussions regarding the potential for false-positive test results when choosing a breast cancer screening regimen.This study was HIPAA-compliant and approved by the Institutional Review Board of Massachusetts General Hospital.GE Healthcare. GSG was previously a consultant for GE Healthcare until 2018.JML and KPL receive research support from


Epidemiological evidence on the association of soy intake with breast cancer risk is still inconsistent due to different soy intake levels across previous studies and small number of breast cancer cases. We aimed to investigate this issue by analyzing data from the China Kadoorie Biobank (CKB) study and conducting a dose–response meta-analysis to integrate existing evidence. The CKB study included over 300,000 women aged 30–79 from 10 regions across China enrolled between 2004 and 2008, and followed-up for breast cancer events until 31 December 2016. Information on soy intake was collected from baseline, two resurveys and twelve 24-h dietary recalls. We also searched for relevant prospective cohort studies to do a dose–response meta-analysis. The mean (SD) soy intake was 9.4 (5.4) mg/day soy isoflavones among CKB women. During 10 years of follow-up, 2289 women developed breast cancers. The multivariable-adjusted relative risk was 1.00  for the fourth (19.1 mg/day) versus the first (4.5 mg/day) soy isoflavone intake quartile. Meta-analysis of prospective studies found that each 10 mg/day increment in soy isoflavone intake was associated with a 3% (95% CI 1–5%) reduced risk of breast cancer. The CKB study demonstrated that moderate soy intake was not associated with breast cancer risk among Chinese women. Higher amount of soy intake might provide reasonable benefits for the prevention of breast cancer.The online version of this article (10.1007/s10654-019-00585-4) contains supplementary material, which is available to authorized users. Breast cancer is the most common cancer and one of the leading causes of cancer death among women throughout the world . Its incEpidemiological evidence on the relationship between soy foods and breast cancer is still limited and inconclusive , despiteSeveral cohort studies or nested case–control studies related to soy and incident breast cancer gave inconsistent results. Studies conducted in Western countries where dietary soy intake levels were low –24 or moUsing the data from the China Kadoorie Biobank (CKB), a large scale prospective cohort study involving over 300,000 women from 10 geographically and economically diverse regions in China, we evaluated the relationship between soy intake and risk of incident breast cancer. Subgroup analyses were also conducted to assess whether risk estimates varied by baseline characteristics like menopausal status. Participants’ soy consumption levels and cut-off values of soy categories varied across previous studies. In order to provide a clearer picture on the soy-breast cancer relationship, we also did a dose–response meta-analysis to integrate results of prospective cohort studies.n = 1610), with missing values for key variables , leaving 300,852 female participants in the main analyses. Approval of the study was obtained from ethics committees or institutional review boards at the University of Oxford, the Chinese Center for Disease Control and Prevention, the Chinese Academy of Medical Sciences, and all participating centers.The CKB study recruited participants from 5 urban areas and 5 rural areas in China. The study design and methods have been described in detail elsewhere . Brieflynd resurvey. Using 24-HDRs as the gold standard, the weighted Kappa statistic was 0.67 for validity of baseline food frequency and 0.74 for validity of soy consumption amount (excluding soy milk) in 2nd resurvey.In the baseline questionnaire, one item asked participants about the overall frequency of soy foods  during the past 12 months: never/rarely, monthly, 1–3 days per week, 4–6 days per week, or daily. After participants completed the baseline survey, two resurveys were conducted in 2008 and 2013, respectively, each involving about 5% of the randomly selected participants from each of the 10 study regions. The first resurvey questionnaire asked exactly the same question on soy consumption as the baseline one, whereas the question was split into 2 items in the second resurvey. One item asked about the frequency and amount of soymilk consumption and the other item about soy foods other than soymilk. To evaluate the reproducibility and validity of food frequency questionnaires (FFQs) used in baseline and resurveys, an intensive dietary study of 432 CKB participants (254 women) was conducted from 2015 to 2016. These participants completed two FFQs  and twelve 24-h dietary recalls (24-HDR). The 24-HDRs covered all kinds of soy foods in China and were conducted in three seasons separately, with 3 weekdays and 1 weekend day in each season. For female participants, the weighted Kappa statistic was 0.66 for reproducibility of baseline soy food frequency and 0.77 for reproducibility of soy consumption amount (excluding soy milk) in 2We combined intake frequency from baseline and 1st resurvey and the intake amount from the 2nd resurvey to estimate the usual amount of soy intake for each woman. Then, we converted the usual amount of soy intake into amount of soy isoflavone by taking into account the proportions that different kinds of soy foods contributing to total soy food amount among women in the 24-HDRs and the soy isoflavone content of different soy foods (Appendix).2).Information on socio-demographic characteristics , lifestyle factors , reproductive characteristics , and family history of cancer were obtained from the baseline questionnaire. Daily energy intake was calculated by taking into account the 12 groups of foods available in the present study. A participant was considered as having a family history of cancer if at least one of their parents or siblings was diagnosed with cancer. At baseline, body weight and height were measured by trained staff using calibrated instruments and BMI was calculated by weight (kg)/height squared  or percentages across 4 categories of baseline soy consumption, standardized for age and study region if appropriate, via logistic regressions for categorical variables or multiple linear regressions for continuous variables.Hazard ratios (HRs) and 95% confidence intervals (95% CIs) were calculated for breast cancer risk by both soy frequency and usual amount quartiles using Cox proportional hazard regression models. Categorical soy intake variables were treated as continuous variables to evaluate the linear trend. In the Cox models, age was used as the underlying time scale and baseline characteristics as covariates. Each Cox model was stratified by baseline age groups  and study region. Potential confounding factors adjusted for in the multivariable models were: education attainment, household income, smoking status, alcohol consumption, physical activity, baseline BMI, standing height, age at menarche, parity, average breastfeeding duration, menopausal status and age at menopause, use of oral contraceptives, family history of cancer, total energy intake, and consumption frequency of fresh fruit, fresh vegetables, preserved vegetables, red meat, poultry, fish and dairy products at baseline. Information on hormone replacement therapy was not collected at baseline and thus was not adjusted for in the models. However, we believed it would not influence the risk estimates since < 1% of the females have ever used hormone replacement treatment according to data from the second resurvey. The method of Schoenfeld residuals found no violation of the proportional hazards assumption for soy intake in fully-adjusted model.A stratified analysis was further performed separately among pre-menopausal and post-menopausal women to assess any modifying effect of menopause on the association of soy consumption and breast cancer. Besides, stratified analyses were conducted according to other baseline characteristics such as BMI. Multiplicative interactions were tested using likelihood ratio tests comparing models with and without the cross product terms between stratifying variables and soy food consumption categories.Several sensitivity analyses were done separately: (1) by excluding females who developed breast cancer during the first 2 years of follow-up; or (2) by excluding those with a family history of cancer.We searched on PubMed, Embase and Cochrane library from their dates of inception to March 2019 for prospective studies examining the association between soy intake and breast cancer. Studies using concentrations of isoflavones or their metabolites in biological samples were not included owing to the difficulty in converting biological concentration into amount of soy isoflavone intake. However, we believed that this had no great impact on the result because studies using biological concentrations as exposure assessment were mainly conducted among populations with pretty low level of soy intake , 16, 19.2 statistic and the Egger test was used to detect publication bias.In the dose–response meta-analysis, HRs were considered as effect sizes and the median or mean soy isoflavone intake of each consumption category was regarded as the consumption dose of corresponding category. To test for potential non-linear relationship between soy isoflavone intake and incident breast cancer, a non-linear dose–response meta-analysis was done by coding soy isoflavone intake as a restricted cubic spline (RCS) function. If there was no evidence of non-linear association, we firstly estimated study-specific log HRs and 95% CIs for each 10 mg/day increment in soy isoflavone intake using the method introduced by Greenland and Longnecker . Then weSeveral sensitivity analyses were done to assess the robustness of dose–response meta-analysis: (1) dropping one of the studies included in the main dose–response meta-analysis each time; (2) dropping studies grouping participants according to soy frequency rather than amount of soy intake; (3) including only those studies which assessed soy intake in a more precise way ; or (4) including all the studies in the synthetic review, i.e. additionally including studies with extremely low level of soy isoflavone intake.P values were two-sided and statistical significance was defined as P < 0.05.All statistical analyses were performed with Stata (version 15). All Of the 300,852 female participants, 44.5% resided in urban areas, and the mean (SD) age at baseline was 50.9 10.5) years  age at breast cancer diagnosis was 56.6 (9.8) years. Table P = 0.856 for frequency-menopausal status interaction and P = 0.972 for quartile-menopausal status interaction). In the subgroup analysis according to BMI, higher soy intake seemed to be inversely associated the risk of breast cancer in women with BMI < 24 kg/m2, with a HR (95% CI) of 0.76 (0.60–0.96) for the highest soy frequency consumption group and a HR (95% CI) of 0.75 (0.54–1.04) for the highest quartile of soy consumption (Appendix Table 2). But no statistically significant interaction between soy intake and BMI was detected . In sensitivity analyses, neither the exclusion of cases occurring during the first 2 years of follow-up, nor excluding those with a family history of cancer materially altered the results (data not shown).Baseline menopausal status did not appear to modify the results . Of these, 11 studies were eligible with their characteristics and results of quality assessment presented in Appendix Tables 5 and 6.In the dose–response meta-analysis, we further excluded three studies , 18, 20 P for non-linearity = 0.142; Fig. 2 = 19.8%, P for heterogeneity = 0.267). The Egger test including the 9 studies in the dose–response meta-analysis found no publication bias (P = 0.606).The dose–response meta-analysis using a RCS function failed to reject the linearity assumption between soy isoflavone intake and risk of breast cancer . Sensitivity analysis by including the five studies [2 = 35.9%, P for heterogeneity = 0.182). The dose–response meta-analysis of all 12 studies gave the same risk estimate  as the main dose–response meta-analysis of nine studies.Sensitivity analyses by excluding one of the nine studies each time from the dose–response meta-analysis found that the risk estimates remained stable (Appendix Table 7). Sensitivity analysis by excluding two studies , 28 whic studies –32 that The CKB study, which enrolled over 300,000 women from 10 diverse regions from China, found no association between soy intake and incident breast cancer overall. The dose–response meta-analysis integrating the CKB study and other prospective studies from Asia and Western countries found that each 10 mg/day of soy isoflavone intake was associated with a 3% reduced breast cancer risk.Findings from prospective studies on soy-breast cancer association were inconsistent, which may be due to different soy intake levels across different studies. Cohort studies conducted among women with low (< 5 mg/day in the highest consumption groups) , 18, 20 The soy intake level  was much lower among the CKB women than that assessed in the study conducted in Shanghai, China  , which mWe found an inverse association between higher soy food frequency and breast cancer among CKB women with lower BMI while no statistically significant association was observed among women with higher BMI. The Singapore Chinese study also reported different risks estimates among women with different BMI . HoweverThe present dose–response meta-analysis observed a 3% (95% CI 1–5%) reduced risk of breast cancer for each 10 mg/day increment in soy isoflavone intake. Most of the cohort studies included in the present dose–response meta-analysis used validated FFQs to assessed soy intake. Most studies were of high quality according to the NOS scores which evaluated studies in terms of exposure and outcome measurement, confounding adjustment, follow-up duration and so on. Sensitivity analysis by excluding the study  which waAccording to the Dietary Guidelines for Chinese Residents in 2016, the recommended daily amount of soy food intake for adults were 15–25 g soybean equivalents . The isoCould soy isoflavone supplement help? Two Western studies assessed soy supplement or soy isoflavone supplement, and neither of them found association of soy or soy isoflavone supplement with overall breast cancer risk , 39, thoAs the largest cohort study on soy-breast cancer association, the CKB study has several strengths, including large sample size, population from diverse areas across China, prospective cohort design, long duration of follow-up, unified method of exposure assessment across study regions, and stringent quality control of data. Our meta-analysis included only prospective cohort studies, which minimized recall bias.There are several limitations, nevertheless. Firstly, the baseline questionnaire asked CKB participants about soy intake frequency rather than intake amount, and thus the usual amount of soy isoflavone was estimated by combing information from baseline surveys, two resurveys and the 24-HDRs. More large scale prospective studies using more precise exposure measurement methods are warranted to verify findings from the CKB study. Secondly, some energy-providing food items such as oil were not assessed in the CKB study, and therefore the calculated total energy was lower than the actual total energy intake. However, the risk estimates in the CKB study remained stable before and after adjustment for the calculated total energy intake. Thirdly, data on hormone receptor status of breast cancer were unavailable in the CKB study, so we were unable to assess the association of soy intake with breast cancer subtypes. Existing prospective evidence of soy-breast cancer subtype association has been scarce and of low statistical power due to small sample sizes , 32, 39.The CKB study demonstrated that moderate soy intake was not associated with breast cancer risk among Chinese women. Higher amount of soy intake might provide reasonable benefits for the prevention of incident breast caner. More large scale and well-designed prospective studies are needed to verify our findings.Supplementary material 1 (DOCX 102 kb)Below is the link to the electronic supplementary material.


MYLK gene and PTGS1 gene variants with osteoporosis and benign breast tumor risk and the impact of ovariectomy on osteoporosis in Korean women. We performed a genome-wide association study (GWAS) of women with osteoporosis and benign breast tumors. There were 60 single nucleotide polymorphisms (SNPs) and 12 SNPs in the MYLK and PTGS1 genes, associated with benign breast tumors and osteoporosis. Our study showed that women with homozygous MYLK rs12163585 major alleles had an increased risk of osteoporosis following ovariectomy compared to those with minor alleles. Women carrying the minor PTGS1 rs1213265 allele and not treated via ovariectomy carried a higher risk of osteoporosis than those who underwent ovariectomy with a homozygous genotype at the major alleles. Our results suggest that both the MYLK and PTGS1 genes are genetic factors associated with the phenotypes, and these associations appear to be modulated by ovariectomy.Osteoporosis, characterized by reduced bone mass and increased bone fragility, is a disease prevalent in women. Likewise, breast cancer is a multifactorial disease and considered the major cause of mortality in premenopausal and postmenopausal women worldwide. Our data demonstrated the association of the  Osteoporosis is defined by low bone mass and deterioration in bone architecture ,2. It isBenign breast disease, which proliferates in epithelial tissue, is a breast cancer precursor associated with an increased risk of breast cancer ,9. WomenThus, biochemical and genetic links between postmenopausal osteoporosis and breast disease are of great interest. Both bone and breast tissue are regulated not only by estrogen, which is a hormone that controls bone density, but also receptor activator of nuclear factor-κB ligand (RANKL), thereby restoring the equilibrium between bone formation and resorption ,15,16,17To prevent and treat heavy menstrual bleeding, dysmenorrhea, chronic pelvic pain, endometriosis, uterine prolapse, and gynecologic cancer, ovariectomy, a major gynecologic procedure, has been performed in premenopausal women . Breast MYLK and PTGS1 genes in the risk of osteoporosis and benign breast tumor. Furthermore, few/no study has examined the impact of ovariectomy on gene-disease risk for osteoporosis among Korean women. We identified the SNPs in our genome-wide association study (GWAS) that simultaneously increased the risk of osteoporosis and benign breast tumors.To the best of our knowledge, no study has demonstrated an association between both The present study was performed with data obtained from the Korean Genome and Epidemiology Study (KoGES) , which wn = 18,183). A control group of 6518 healthy participants was used in the present study, which consisted of individuals who were not diagnosed with hypertension, diabetes mellitus, hyperlipidemia, transient ischemic attack, myocardial infarction, chronic gastritis, gastric ulcer, intestinal polyps, acute liver disease, fatty liver, cholelithiasis, chronic bronchitis, chronic obstructive pulmonary disease (COPD), asthma, allergy, thyroid disease, arthritis, osteoporosis, gout, cataracts, glaucoma, periodontal disease, chronic nephritis, renal failure, malignant tumor, or fractures. Since the disease history was included in the KoGES project, it was selected as the criterion for healthy controls. Of the 6518 healthy controls, 2162 males were excluded and a total of 4356 healthy female controls were used. However, because benign breast tumors were not considered when the healthy controls were identified, the healthy control group for benign breast tumors included a final number of 3922 after subtracting 9080 subjects with other diseases from the 13,002 participants who stated the absence of tumors. The diagnosis of benign breast tumors and/or osteoporosis was made by a medical doctor. Consequently, 4356 healthy controls and 1382 osteoporosis cases were identified, and 3922 healthy controls and 1126 benign breast tumor cases were analyzed.We used GWAS to test the association between SNPs and the genetic risk of benign breast tumors and osteoporosis. Participants with missing information on osteoporosis and benign breast tumors were excluded from the 28,445 participants with accessible SNP information. In addition, since we analyzed the association between osteoporosis and ovariectomy, only females were evaluated in this study . The genotype data were obtained from the K-CHIP designed by the Center for Genome Science at the Korea National Institute of Health. Additional information regarding this protocol has been presented elsewhere [p-value of less than 0.001. The location of the genes and SNPs was identified using the genome reference consortium human build 37 (GRCh37).Genotype data were obtained from the Center for Genome Science, Korea National Institute of Health. DNA samples isolated from the participants were genotyped with an Axiomlsewhere ,23. Subjhttps://www.cog-genomics.org/plink2, accessed on 3 March 2021) and predictive analytics software (PASW) version 18.0  were used for most statistical analyses. We investigated the interaction between SNP and the risk of diseases using logistic regression models with the additive genetic model. The multivariable model was adjusted for age and residence [t-test, and p-values < 0.05 were considered significant.PLINK version 1.90 beta  was also used to predict the potential functional effects of the MYLK rs12163585 and PTGS1 rs1213265 genotypes. The geography of genetic variants (GGV) browser  was utilized to report the worldwide frequency of the minor alleles of SNPs. HaploReg database  and Hoseo University . Written informed consent was obtained from all subjects.In this study, 18,183 females from the HEXA cohort were included in the association study. Age and the number of subjects with each disease are listed in p < 0.001) were filtered based on the HEXA data. Of them, 464 and 469 SNPs associated with benign breast tumor and osteoporosis were found respectively. Only five SNPs were common between the two diseases . Two (rs3732486 and rs3732487) of the five SNPs were found in the MYLK gene, rs58154051 in the RPS6KA2 gene, rs2293973 in DLGAP2, and rs1213265 in PTGS1  and osteoporosis . In addition, rs1213265 in PTGS1 showed significant associations with benign breast tumors  and osteoporosis .In the GWAS, the SNPs  were associated with benign breast tumors and osteoporosis  and osteoporosis , the rs12163585 variant was associated with a decreased risk of benign breast tumors and osteoporosis   and osteoporosis  (MYLK gene showed association with benign breast tumors or osteoporosis (p < 0.05), but the association was significant in only one of the two diseases.We analyzed the association between benign breast tumors and osteoporosis and ectively . In addioporosis . The assctively) . In the ctively) . Nine otn = 16), among the 11,613 participants, there were 200 ovariectomized participants with minor alleles and 85 with homozygous genotypes in the major alleles. In contrast, in the non-ovariectomized participants, the number of minor allele carriers was 8218, and 3110 carried a homozygous genotype in the major allele. In the presence of a minor rs12163585 allele, the risk of osteoporosis was increased 1.86-fold in women who underwent an ovariectomy. However, in individuals carrying the homozygous genotype in the major alleles, the risk of osteoporosis was significantly increased by 2.36-fold (Excluding the participants with missing genotype data (.36-fold a. The ren = 15), the 11,614 participants included nine minor allele carriers and 276 homozygous genotype major allele carriers who had ovariectomies. In contrast, in the non-ovariectomized participants, the number of minor allele carriers was 344, and 10,985 carried homozygous genotypes in the major allele. With a homozygous rs1213165 genotype in the major allele, the risk of osteoporosis was increased 1.83-fold in women who underwent ovariectomy  women with a homozygous genotype in the MYLK rs12163585 major alleles had an increased risk of osteoporosis following ovariectomy than those with minor alleles, and (2) women who had the minor PTGS1 rs1213265 allele and were not ovariectomized carried a higher risk of osteoporosis than those with homozygous genotype of the major alleles and undergoing ovariectomy. The GWAS of osteoporosis and benign breast tumors revealed that five common SNPs in four genes were significantly associated with the two diseases. SNP rs58154051, located on chromosome 6 and belonging to the RPS6KA2 gene, did not show a statistically significant relationship with ovariectomy or osteoporosis  [MYLK was a candidate gene engaged in acute lung injury susceptibility and disease [MYLK pathogenic variants [MYLK gene significantly influenced the progression of prostate cancer, which is a sex hormone-dependent disease in males, and the MYLK variants found in this study were associated with benign breast tumors related to female hormones, in line with the previous study. Furthermore, androgen, involved in maintaining bone mass density and preventing osteoporosis, is transformed into endogenous estrogen. The ovaries generate enormous amounts of androgen for years in postmenopausal women, helping to retain bone mass. Ovariectomy, which reduces androgen production, may increase the risk of osteoporosis [MYLK gene and ovariectomy and demonstrate that the risk of osteoporosis in women who underwent ovariectomy was significantly higher and nearly double the risk of those without  . Gao et  disease . Anothervariants . Recentlvariants . The MYLoporosis . Our res without a.PTGS1, also known as cyclooxygenase 1 (COX1), is ubiquitously found in tissues and involved in the biosynthesis of prostaglandin (PG), which regulates renal, gastrointestinal, and platelet function [PTGS1 has been connected with multiple pathological disorders including inflammation, arthritis, and cancer. One study had compared whole-genome expression data of breast tissue samples with serum hormone levels using data from healthy women and breast cancer patients using microarrays. PTGS1 was found differentially expressed dependent on estradiol levels, which was downregulated in breast samples from women with high serum estradiol [PTGS1 that controls osteogenesis of adipose-derived stem cells via regulating the NF-κB signaling pathway is required for the osteogenic differentiation of adipose-derived stem cells [PTGS variants and nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit the biosynthesis of PG by PTGS and reduce inflammation [PTGS1 as a key candidate based on the differential expression in arthritis [PTGS1 promoted osteogenesis in adipose-derived stem cells and suppressed the NF-κB pathway. Additionally, the knockdown of PTGS1 may regulate the inflammatory microenvironment during bone remodeling [function . Besidesstradiol . In bothem cells . Nagao eammation . Anotherammation  mice cohrthritis . Wang etmodeling .Most hysterectomies performed for gynecologically benign conditions, preserve the ovaries. However, occasionally, some physicians suggest bilateral ovariectomies (BOs) along with hysterectomy to prevent the development of cancer . The hysMYLK rs12163585 and PTGS1 rs1213265 with a geographic genome variants (GGV) browser. The GGV browser uses maps of allelic frequencies in populations distributed across the globe based on 1000 genomes (hg19). While rs12163585 was mostly seen in Southeast Asia, rs1213265 was mostly detected in Africa (MYLK rs12163585 minor allele was associated with a decreased risk of osteoporosis and benign breast tumors in Asia, while the PTGS1 rs1213265 minor allele detected mostly in Africa was associated with an increase in both diseases. In the Haploview results, MYLK rs12163585 was predicted to change the motif factor binding site of GATA (MYLK, was suggested to sensitive triple-negative breast cancer marker along with GATA-3 [PTGS1, develops long bones and involves in their morphogenesis [Smad4 gene that has interaction with estrogen receptor α is required for TGF-β-induced epithelial to mesenchymal transition and bone metastasis of breast cancer cells [We analyzed the minor allele frequency of n Africa . The MYL of GATA . GATAs (h GATA-3 . Expressogenesis . Smad4 ger cells ,48,49.MYLK and PTGS1 are associated with both benign breast tumors and osteoporosis. Analysis of the differences in the risk of osteoporosis and ovariectomy in the MYLK rs12163585 and PTGS1 rs1213265 genotypes showed significant associations with each genotype. Consequently, pathological factors such as ovariectomies substantially affect the association between gene variants and osteoporosis in Korean women.Genetic variants in