BACKGROUND:Breast cancer is etiologically heterogeneous, but which risk factors differ in their associations across tumor subtypes remains unclear. We conducted a large, pooled analysis to evaluate independent, dose-response associations between breast cancer risk factors and quantitative tumor features. METHODS:Analyses of 15,731 invasive breast cancers from 24 studies evaluated associations (p-trend) between reproductive and hormonal factors, body mass index (BMI), alcohol, smoking, and family history in relation to quantitative immunohistochemistry measures on tissue microarrays (ER, PR, HER2, KI67, TP53) and tumor grade. Analyses in a subset of 10 population-based studies estimated subtype-specific odds ratios (ORs) comparing cases to controls. A Bayesian False Discovery Probability (BFDP) <0.2 was used to identify associations with strong statistical evidence. RESULTS:Nulliparity and later age at menopause were associated with higher ER-positivity (p-trend=0.021 and 0.001, respectively), with corresponding OR[ER+] (95% CI) = 1.49 (1.16-1.90) for nulliparous vs. parous and 1.07 (1.03-1.11) per 5 years. Current combined menopausal hormone therapy (MHT) use was associated with lower grade (p-trend<0.001), with OR [grade1] = 3.37 (2.69-4.21) for current vs. never users. Higher BMI was associated with lower ER-positivity and higher grade in premenopausal women (p-trend<0.001 and <0.001), with OR[ER+] = 0.80 (0.74-0.87) and OR[grade1] = 0.75 (0.63-0.88) per 5 units, and with higher PR-positivity and higher grade in postmenopausal women (p-trend<0.001 and <0.001), with OR[PR+] = 1.08 (1.03-1.14) and OR[grade 3] = 1.10 (1.04-1.17) per 5 units. CONCLUSION:This pooled analysis of 15,731 cases showed that nulliparity, age at menopause, MHT, and BMI have independent, dose-response associations with ER, PR, and grade, clarifying patterns of etiologic heterogeneity. Associations with HER2, KI67 and TP53, or other risk factors did not meet our threshold for strong evidence.

Tamoxifen's pharmacokinetics are strongly influenced by the highly polymorphic CYP2D6, while the influence of other genetic variants has been inconclusive. To further delineate this genotypic-phenotypic impact, we conducted a multi-ancestry genome-wide association study in 636 hormone-receptor-positive (HR+) breast cancer (BC) patients treated with 20 mg tamoxifen daily for ≥8 weeks and validated these genetic determinants in another 869 patients. Association with clinical outcomes was examined in 1326 non-metastatic HR+ patients receiving adjuvant tamoxifen. A genome-wide significant association with Z-endoxifen levels was observed at the CYP2D6 locus on chromosome 22 and its downstream region of TCF20 rs932376 A > G. Both CYP2D6 metabolizer status and TCF20 rs932376 A > G were independent predictors of endoxifen levels in multivariable analysis. CYP2D6 metabolizer status accounted for greater variability of mean endoxifen levels compared to TCF20 rs932376 A > G (91.2% vs 48.8%). These findings were replicated in validation cohorts. Neither TCF20 rs932376 nor CYP2D6 metabolizer status was significantly associated with BC outcomes after adjustment for known prognostic factors. Our study confirmed that CYP2D6 metabolizer status remains as the prime predictor of steady-state Z-endoxifen levels, while TCF20 rs932376 A > G has a smaller, independent effect. Both genetic factors were not associated with BC clinical outcomes.

Carriers of germline BRCA1/2 pathogenic variants (g BRCA1/2 PVs) have elevated young-onset breast cancer risk. To define the pretreatment genomic landscapes of young-onset g BRCA -associated breast cancer, we evaluated 136 treatment-naive tumors diagnosed before age 50 in the prospective POSH study and 66 noncarriers from The Cancer Genome Atlas. Using whole-exome sequencing, we analyzed somatic variation, allele-specific loss of heterozygosity (asLOH), homologous recombination deficiency (HRD), and single-base substitution (SBS) signatures. gBRCA1 and gBRCA2 breast cancers had high rates of asLOH but differed significantly in average HRD scores and median SBS composition of signatures SBS1 (aging-associated), SBS18 (ROS-associated), and SBS3 (HRD-associated). Compared with g BRCA2 tumors, g BRCA1 tumors with asLOH were significantly enriched for alterations in hallmark ROS, DNA repair, and epithelial-mesenchymal transition pathways. In ER-positive, HER2-negative tumors from g BRCA1/2 carriers compared with noncarriers, we found significant enrichment of RB1 , TP53 , FAT1 , and MYC single-nucleotide variants, indels, and copy number variants associated with CDK4/6 inhibitor (CDK4/6i) resistance. Together, these findings demonstrate significant differences between g BRCA1 - and g BRCA2 -associated breast cancers, and preexisting CDK4/6i resistance mechanisms, supporting prospective trials comparing individualized therapy for g BRCA1 versus g BRCA2 carriers and comparing poly(ADP-ribose) polymerase inhibitors versus CDK4/6i for ER-positive g BRCA1/2 -associated breast cancer.

Studies have identified genetic and epidemiologic factors associated with mammographic density (MD) phenotypes. However, MD-associated genetic variants only account for a small proportion of the total estimated heritability. Interrogating interactions between genetic and epidemiologic factors could potentially identify additional MD-associated loci, expand our understanding of the genetic basis of MD phenotypes, and clarify how epidemiologic factors modulate relationships between genetic variants and MD. We conducted six separate genome-wide, gene-environment (GxE) interaction analyses, applying 2 degrees of freedom (df) and 1df interaction tests, for each of three MD phenotypes (percent density, dense area (DA), and nondense area (NDA)). The six epidemiologic factors considered were height, ever parous, parity, ever menopausal hormone therapy, ever breastfeeding, and months of breastfeeding. We included European ancestry participants from multiple studies within the Markers of Density consortium and the Breast Cancer Association Consortium (n = 4895-16 218 depending on specific analyses). We identified 11 loci with genome-wide significant (P < 5 × 10-8) interaction tests including two novel common genetic signals interacting with parity (8p21.2) and ever breastfeeding (19p13.2) for NDA. Our results suggest that epidemiologic risk factors might influence relationships between common genetic variants and MD phenotypes at particular genomic loci.

Women with BRCA1/2 pathogenic variants (PVs) have a high likelihood of developing young onset breast cancer (BC) compared to noncarriers. In women enrolled in the POSH prospective cohort study of young women diagnosed with BC under age 40 or under age 50 if known BRCA1/2 positive, no difference in overall survival was observed in BRCA1/2 carriers vs noncarriers. Treatment options for BC include PARP inhibitors (PARPi) for BRCA1/2 driven tumors and CDK4/6 inhibitors (CDK4/6i) plus endocrine therapy for ER-positive, HER2-negative tumors. In BC from BRCA1/2 carriers unselected for age, up to 10% BRCA1 and 46% BRCA2 carriers do not have loss of heterozygosity (LOH) at the germline variant locus, suggesting non-BRCA1/2 tumorigenesis. It is not known what the tumor molecular landscape, including rates of LOH and homologous recombination deficiency (HRD), is in young BRCA1/2 PV carriers with BC, and whether the tumor molecular features impact disease outcomes or predict therapy response. To elucidate the molecular landscape of breast tumors in young women with BRCA1/2 PVs, we evaluated treatment naïve primary breast tumors from 136 (86 [63.2%] BRCA1; 50 [36.8%] BRCA2) PV carriers in the POSH study. 71 (52.2%) of the tumors were ER-positive. We performed whole exome sequencing and called single nucleotide variants, indels, copy number variants, and BRCA1/2 allele specific LOH in the tumors, calculated HRD scores and tumor mutational burden (TMB), and evaluated single base substitution (SBS) signatures. We found high rates of LOH by gene: BRCA1 (93%) and BRCA2 (96%), and by ER-status: ER-negative (94.4%) and ER-positive (93.8%). Mean HRD scores were significantly higher in tumors with LOH compared to nonLOH tumors in both BRCA1 (57.4 vs 22.6, p<0.0001) and BRCA2 (43.7 vs 23.5, p=0.005) carriers as well as ER-negative (57.6 vs 22.8, p<0.0001) and ER-positive tumors (46.4 vs 22.8, p<0.001). LOH tumors had higher proportional contribution of HRD-associated SBS3 than nonLOH tumors (0.36 vs 0.22, p=0.048). BRCA1 LOH tumors had higher median TMB than BRCA2 LOH tumors (4.8 vs 2.5, p=0.034). Overall survival in women with nonLOH tumors was 100% throughout the follow-up period but was not significantly different from that of women with LOH tumors. Overall survival did not differ by tumor HRD status: analyses were limited by small numbers in the non-LOH and low HRD groups. We found statistically significant enrichment of AURKA (OR:4.2, 95%CI:1.3-16.6, p=0.015) and MYC (OR:2.4, 95%CI:1.0-5.7, p=0.043) amplification which are associated with resistance to CDK4/6i in ER-positive, HER2-negative tumors from POSH cohort PV carriers compared to noncarriers from the TCGA. Given the high levels of LOH and presence of CDK4/6i resistance associated alterations, our data suggest that PARPi may be preferable over CDK4/6i in young BRCA1/2 carriers with ER-positive, HER2-negative BC when both therapies are being considered in the adjuvant setting. Mwangala P. Akamandisa, Mingyi Xia, Wilson Cheah, Bradley Wubbenhorst, Kurt D'Andrea, Mengyao Fan, Jake Shilan, William J. Tapper, Ellen Copson, Ramsey I. Cutress, Diana M. Eccles, Susan M. Domchek, Katherine L. Nathanson. Tumor molecular landscape and therapy implications in young BRCA1/2 carriers with breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6596.

Background. Approximately 98% of CML patients express e13a2 and/or e14a2 BCR::ABL1 mRNA isoforms resulting from genomic breakpoints that fall within a ~2.9 Kb region in BCR and a ~140 Kb region in ABL1. Rarer isoforms result from breakpoints in other parts of BCR and/or ABL1.Additional BCR::ABL1-associated abnormalities have also been described that may be prognostically relevant. We set out to assess the ability of long read nanopore sequencing to detect genomic BCR::ABL1 fusions and any BCR::ABL1-associated rearrangements or other structural variants in patients with CML. Methods. DNA from patients with CML (n=30) were fragmented to 5-10 Kb and prepared for sequencing using the Oxford Nanopore Technologies (ONT) ligation sequencing kit v14. Adaptive sampling was used to target a panel of 240 genes recurrently involved in hematological malignancies in all 30 cases (37.1 Mb total), with 11kb of padding sequence at either end of each gene to maximise coverage. The full lengths of chr9 and chr22 (223.9 Mb total) was also included for 25/30 cases. Sequencing was performed on the MinION Mk1b, Dorado (ONT) and minimap2 were used for basecalling and alignment, SV were called with sniffles2. Selected rearrangements were confirmed by targeted PCR and sequencing. BCR::ABL1 transcript type was known for all cases and full karyotype for 18 cases. Results. Median on-target sequencing depth was 38X, representing a 7-fold enrichment over non-targeted sequence. Median read N50 was 7.8 Kb. Evidence for BCR::ABL1 was called by sniffles2 in 27/30 cases. In the remaining 3 cases, the fusion call was initially filtered in 2 samples due to a low fraction of fusion reads (0.16 and 0.17), and only 2 fusion supporting reads in 1 sample. Evidence for the fusion was found upon manual review. BCR::ABL1 transcript type was concordant with the genomic breakpoints in all cases, including 3 with atypical fusions (e14a2, n=14; e13a2, n=10; e14a2/e13a2, n=3; e1a2, n=2; e14a3, n=1). Cytogenetic results were available for 18/30 cases and a Ph chromosome was seen in 12/18 of these, including 11 where it was seen as the sole abnormality. Two of these 11 cases (cases 15 and 30) had unexpected additional rearrangements detected by nanopore sequencing: case 15 had a t(1;9)(q25.3;q34.12) with a 6.1 Kb inversion at chr9:130,709,000 within 1Kb of the t(9;22) reciprocal breakpoint. Case 30 had an additional t(3;22)(p21.31;q11.23). One patient (case 26) had a Ph chromosome plus complex additional chromosome abnormalities that were partially resolved by nanopore sequencing. A normal karyotype was seen in 1/18 cases, with ABL1 inserted into BCR as determined by FISH. A 2.17 Mb deletion between chr9:130,842,023-133,012,328 was detected by nanopore sequencing, with breakpoints for BCR::ABL1 and the reciprocal fusion found at either end. Complex rearrangements involving 9q34 and/or 22q11 were detected in 5/18 samples by cytogenetics. Genomic breakpoints were detected by nanopore sequencing in all 5 cases, 2 of which were found to have additional complexity. Case 1 had a 46,XY,t(9;22;9)(q34;q11;q13) by karyotyping, but nanopore suggested 2 distinct t(9;22) events, with the additional t(9;22) revealed to have breakpoints at 9q21 and 9q34, separated by a 58.5 Mb duplication. Case 3 had a 46,XY,t(3;9)(p14;q34) by cytogenetics but nanopore sequencing indicated a t(3;9)(p21.31;q34.12), t(3;9)(p21.2;q34.13) and an additional t(3;22)(22q11.23;p21.2). Of the 12 cases that did not have cytogenetic results, 2 had evidence for additional rearrangements. Case 11 had a t(7;9)(q21.2;q34.12) and t(7;22)(q21.2;q11.23) whereas case 17 had a 154 bp inversion close to the BCR::ABL1 junction and a reciprocal breakpoint approximately 1 Mb downstream of BCR in intron 19 of CABIN1. Conclusions. This study demonstrates nanopore sequencing is able to successfully detect typical and atypical BCR::ABL1 genomic breakpoints and can revealthe underlying complexity in samples with BCR::ABL1-associated rearrangements, as well as other structural variants. Further work is required to determine the clinical significance of this additional genomic complexity in CML.

Pancreatic adenocarcinoma is the most common histological subtype of pancreatic cancer, representing approximately 85% of all cases [...]

Although described more than a decade ago, the mechanism by which the JAK2 46/1 haplotype increases the risk of developing JAK2-mutated myeloproliferative neoplasms (MPN) remains unexplained. Inflammation and immunity are linked to MPN development and thus could be relevant to the mechanism by which 46/1 mediates its effect. Here, we show that PD-L1 expression is elevated in 46/1 haplotype both in healthy carriers and CD34+ cells from MPN patients. Using circular chromosome conformation capture (4C-seq) we observed that PD-L1 and the neighboring PD-L2 loci physically interact with JAK2 in a manner that differs between 46/1 and non-risk haplotypes. CRISPR/Cas9 genome editing identified a region within JAK2 intron 2 that influences both JAK2 and PD-L1 expression. We suggest that increased PD-L1 expression may be relevant to the mechanism by which 46/1 leads to an increased inherited risk of developing MPN.

Purpose: gBRCA1/2 pathogenic variant (PV) carriers have elevated young-onset breast cancer risk. Understanding the distinct genomic landscapes of gBRCA1- and gBRCA2-associated breast cancer, including presence of PARP and CDK4/6 inhibitor (PARPi; CDK4/6i) response-associated alterations, may inform treatment selection for these patients. Patients and methods: We evaluated 136 treatment-naïve primary tumors from POSH study participants diagnosed with breast cancer before age 50 years (92.6% diagnosed ≤40): gBRCA1 86(63.2%), gBRCA2 50(36.8%). We evaluated somatic mutational and copy number variations (CNV), allele-specific loss of heterozygosity (asLOH), homologous recombination deficiency (HRD), and single-base substitution signatures (SBS) from whole exome sequencing. Results: Both gBRCA1 (93%) and gBRCA2 (96%) breast cancers had high rates of asLOH. We found significant differences between gBRCA1 and gBRCA2 tumors in average HRD scores (57.4±1.3 vs 43.7±1.5, p<0.0001) and SBS composition: SBS1 (aging-associated) 12.9 vs 7.3, p=0.013; SBS18 (reactive oxygen species [ROS]-associated) 1.4 vs 0, p=0.007; SBS3 (HRD-associated) 27.3 vs 42.6, p=0.002; and SBS26 (mismatch repair-associated) 5.9 vs 9.4, p=0.049. Compared to gBRCA2 tumors, gBRCA1 tumors with asLOH were significantly enriched for gains of chr6q, and alterations in Hallmark ROS, DNA repair, and epithelial-mesenchymal transition pathways. In ER-positive, HER2-negative tumors from POSH gBRCA1/2 carriers compared to noncarriers from the TCGA, we found significant enrichment of RB1 (OR:6.3, 95%CI:2.8-15.4, padj=0.001), TP53 (OR:4.6, 95%CI:1.9-12.1, padj=0.017), FAT1 (OR:3.9, 95%CI:1.84-8.7, padj=0.013), and MYC (OR:4.0, 95%CI:1.8-9.1, padj=0.017) SNV/indels/CNVs, which are associated with CDK4/6i resistance. Conclusion: Our data suggest that PARPi may be preferable over CDK4/6i to treat ER-positive, HER2-negative breast cancer in young-onset gBRCA1/2-associated breast cancer when both therapies are considered in adjuvant and metastatic settings. Additionally, we identified significant differences between gBRCA1- and gBRCA2-associated tumors, which may inform therapeutic development. ### Competing Interest Statement ERC reports: Honorarium AstraZeneca, Eli-Lilly, Guardant, Menarini Stemline, Novartis, Pfizer, Roche; Advisory boards/consultation: AstraZeneca, Eli-Lilly, Nanostring, Pfizer, Roche; Conference fees/travel/accommodation: Roche, Novartis; Educational grant: Daiichi-Sankyo; ERC and RIC report research support from SECA. ### Funding Statement Supported by Breast Cancer Research Foundation (K.L.N., and S.M.D); the Basser Center for BRCA (K.L.N.; S.M.D.); Gray Foundation (KLN); Funding for the POSH study has been provided by the Wessex Cancer Trust, Cancer Research UK, Breast Cancer Now and Prevent Breast Cancer. The long-term follow-up of the POSH study is supported by an Institutional Grant from AstraZeneca (CI: ERC, Co-I: DE, REC& WT). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The South West Multi-center Research Ethics Committee gave ethical approval for this work (MREC 00/6/69) I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

The initial rate of reduction of BCR::ABL1 mRNA on TKI therapy, usually measured as the halving time over the first 3 months of treatment, has prognostic value for both early and late responses as well as the likelihood of achieving TFR. Standardized measurement of pretreatment disease levels by RT-qPCR, however, is problematic due to the different properties of the 3 established reference genes (ABL1, GUSB, BCR) used for CML MRD analysis. These differences are not relevant for standard MRD assessments, but the use of ABL1 or BCR results in reference gene-specific distortions at high disease burdens that limit their utility for assessment of baseline disease levels. GUSB is completely independent of BCR::ABL1 and is thus theoretically the best reference gene to determine halving times. To date, however, no comparative analysis of the utility of different reference genes to assess early response kinetics has been performed. We have evaluated the utility of halving times derived using GUSB and ABL1 as reference genes in FASCINATION, a multicenter, prospective, open-label, interventional phase II trial designed to evaluate the efficacy and tolerability of asciminib following <6 weeks treatment with other TKIs or <4 weeks of hydroxyurea. Halving times in days were calculated by comparing BCR::ABL1/ABL1 and BCR::ABL1/GUSB levels at trial entry (63% and 23%, respectively, at the start of asciminib) to the corresponding measurements at 3 months on asciminib. Halving times and IS levels at 3 months were compared to molecular outcomes at 18 months. Of 103 cases with available data, 45 achieved DMR at 18 months (MR4 or better; median 0.0015% IS) and 58 did not achieve DMR (median 0.052% IS). Median halving times were shorter for patients who achieved DMR using both ABL1 (13.5 vs 20.9, P<0.001) and GUSB (11.2 vs 18.1, P<0.001) as reference genes. Similarly, IS values at 3 months were significantly lower for cases who achieved DMR at 18 months (0.23% vs 1.6%; P<0.001). Using ROC analysis, the predictive value of GUSB halving times (AUC GUSB = 0.792) for achievement of DMR at 18 months was superior to both ABL1 halving times (AUC ABL1 = 0.746) and IS values at 3 months (AUC = 0.776). Similarly, we found that GUSB halving times correlated better (r=0.58, Spearman's rank correlation) with month 18 IS levels considered as a continuous variable compared to both ABL1 halving time (r=0.47) and month 3 IS levels (r=0.51). Focusing on GUSB, 19/51 (37%) cases had a halving time shorter than the median for all cases (13.9 days) but were not in DMR at 18 months. 12 of these 19 cases also had lower than the median IS levels at 3 months (0.73%). Conversely 13/52 cases (25%) had a long halving time but achieved DMR at 18 months. 8/13 also had higher than median IS levels at 3 months. Of the 45 cases who achieved DMR at 18 months, 11 (24%) had IS levels at 3 months that were higher (median = 3.97%, range 0.77%-35.3%) than the median for all cases (0.73%). Only 1/11 had a shorter than median GUSB halving time. Conversely, 17/58 (29%) of cases who were not in DMR at 18 months had <0.73% IS at 3 months (median = 0.40; range 0.05-0.70); only 5/17 had long GUSB halving times. These data indicate that combining halving times with 3 month IS data adds limited additional predictive value. Since GUSB measurements remain largely unstandardized, we assessed the performance of the AcroMetrix™ BCR-ABL Panel (Thermo Fisher), recently calibrated to GUSB and BCR as well as ABL1, to standardize MRD results using GUSB as a reference gene in two centers. Established GUSB conversion factors (CF) derived by sample exchange were essentially indistinguishable from those derived using the panel [Lab 1: existing CF (0.99) / panel CF (1.02) = 0.97; Lab 2: existing CF (1.57) / panel CF (1.40) = 1.12, thus validating the panel to standardize GUSB measurements. We conclude that halving times over the first 3 months of asciminib treatment using GUSB or ABL1 as well as IS levels at 3 months are all predictive of molecular response at 18 months, but the predictive value of GUSB halving times are greatest. We therefore recommend that standardized baseline BCR::ABL1/GUSB measurements are incorporated into future studies to enable comprehensive assessment of the value of BCR::ABL1 halving times for routine management.

The role of germline genetics in adjuvant aromatase inhibitor (AI) treatment efficacy in ER-positive breast cancer is poorly understood. We employed a two-stage candidate gene approach to examine associations between survival endpoints and common germline variants in 753 endocrine resistance-related genes. For a discovery cohort, we screened the Breast Cancer Association Consortium database (n ≥ 90,000 cases) and retrieved 2789 AI-treated patients. Cox model-based analysis revealed 125 variants associated with overall, distant relapse-free, and relapse-free survival (p-value ≤ 1E-04). In validation analysis using five independent cohorts (n = 8857), none of the six selected candidates representing major linkage blocks at CELA2B/CASP9, NR1I2/GSK3B, LRP1B, and MIR143HG (CARMN) were validated. We discuss potential reasons for the failed validation and replication of published findings, including study/treatment heterogeneity and other limitations inherent to genomic treatment outcome studies. For the future, we envision prospective longitudinal studies with sufficiently long follow-up and endpoints that reflect the dynamic nature of endocrine resistance.

ABSTRACT:To identify genetic variants that influence myeloproliferative neoplasm (MPN) phenotypes, we undertook a 2-stage patient-only genome-wide association study. MPN subtypes (essential thrombocythemia [ET]; polycythemia vera [PV]) were compared with each other to healthy controls and stratified analyses was performed for chromosome 9p aberrations, JAK2 V617F mutation burden, and sex. The ET vs PV analysis identified known associations: (1) at HBS1L-MYB that increased ET risk (Pmeta = 7.93 × 10-6, odds ratio [OR] = 1.28) and reduced PV risk (Pmeta = 9.43 × 10-5, OR = 0.81) and (2) at GFI1B-GTF3C5 that predisposed to PV only (Pmeta = 1.43 × 10-9, OR = 1.38). Two further linked intronic variants, rs2425786 and rs2425788, at CDH22/CD40 were significant in females only (Pmeta = 2.67 × 10-8), with predisposition to PV (Pmeta = .0006, OR = 1.3) and reduction of ET risk (Pmeta = 7.82 × 10-5, OR = 0.75). A polygenic risk score consisting of 48 variants from 31 loci demonstrated moderate discriminative performance for ET and PV (area under the curve [AUC] = 0.718) and was improved by optimization for disease subtype (AUCET = 0.724 and AUCPV = 0.755). Overall, our results reveal that multiple germline variants influence MPN phenotype, with HBS1L-MYB and a novel sex-specific association with CDH22/CD40 being the strongest determinants.

Background The immune response in breast tumors has an important role in prognosis, but the role of spatial localization of immune cells and of interaction between subtypes is not well characterized. We evaluated the association between spatially resolved tissue infiltrating immune cells (TIICs) and breast cancer specific survival (BCSS) in a large multicenter study. Patients and methods Tissue microarrays with tumor cores from 17,265 breast cancer patients of European descent were stained for CD8, FOXP3, CD20, and CD163. We developed a machine learning based tissue segmentation and immune cell detection algorithm using Halo to score each image for the percentage of marker positive cells by compartment (overall, stroma, or tumor). We assessed the association between log transformed TIIC scores and BCSS using Cox regression. Results Total CD8+ and CD20+ TIICs (stromal and intra-tumoral) were associated with better BCSS in women with ER-negative (HR per standard deviation = 0.91 [95% CI 0.85 - 0.98] and 0.89 [0.84 - 0.94] respectively) and ER-positive disease (HR = 0.92 [95% CI 0.87 - 0.98] and 0.93 [0.86 - 0.99] respectively) in multi-marker models. In contrast, CD163+ macrophages were associated with better BCSS in ER-negative disease (0.94 [0.87 - 1.00]) and a poorer BCSS in ER-positive disease 1.04 [0.99 - 1.10]. There was no association between FOXP3 and BCSS. The observed associations tended to be stronger for intra-tumoral than stromal compartments for all markers. However, the TIIC markers account for only 7.6 percent of the variation in BCSS explained by the multi-marker fully-adjusted model for ER-negative cases and 3.0 percent for ER-positive cases. Conclusions The presence of intra-tumoral and stromal TIICs is associated with better BCSS in both ER-negative and ER-positive breast cancer. This may have implications for the use of immunotherapy. However, the addition of TIICs to existing prognostic models would only result in a small improvement in model performance. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement BCAC was supported by Cancer Research UK grant: PPRPGM-Nov20\100002 and by core funding from the NIHR Cambridge Biomedical Research Centre (NIHR203312). The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care. The B-CAST project was supported by the Horizon 2020 Research and Innovation Programs of the European Union B (grant number: 633784) and the NIHR Cambridge Biomedical Research Centre. AJB was supported by the NIH/Oxcam doctoral programme. The funding of the contributing studies is listed in Supplementary Table 8. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All participants provided written informed consent. The ethics committees or institutional review boards responsible for oversight of the individual studies are listed in Supplementary Table 9. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The tissue segmentation and TIIC scores generated by the Halo algorithm together with the phenotype data, the imputed datasets and the analysis code will be available at the European Genome Phenome Archive on publication (https://ega-archive.org/).

Legends to Supplementary Figures S1 to S6. This document comprises the legends to the supplementary figures.

A large number of variants identified through clinical genetic testing in disease susceptibility genes are of uncertain significance (VUS). Following the recommendations of the American College of Medical Genetics and Genomics (ACMG) and Association for Molecular Pathology (AMP), the frequency in case-control datasets (PS4 criterion) can inform their interpretation. We present a novel case-control likelihood ratio-based method that incorporates gene-specific age-related penetrance. We demonstrate the utility of this method in the analysis of simulated and real datasets. In the analysis of simulated data, the likelihood ratio method was more powerful compared to other methods. Likelihood ratios were calculated for a case-control dataset of BRCA1 and BRCA2 variants from the Breast Cancer Association Consortium (BCAC) and compared with logistic regression results. A larger number of variants reached evidence in favor of pathogenicity, and a substantial number of variants had evidence against pathogenicity-findings that would not have been reached using other case-control analysis methods. Our novel method provides greater power to classify rare variants compared with classical case-control methods. As an initiative from the ENIGMA Analytical Working Group, we provide user-friendly scripts and preformatted Excel calculators for implementation of the method for rare variants in BRCA1, BRCA2, and other high-risk genes with known penetrance.

Background Rhabdomyosarcomas (RMS) are predominantly paediatric sarcomas thought to originate from muscle precursor cells due to impaired myogenic differentiation. Despite intensive treatment, 5-year survival for patients with advanced disease remains low (< 30%), highlighting a need for novel therapies to improve outcomes. Differentiation therapeutics are agents that induce differentiation of cancer cells from malignant to benign. The histone methyltransferase, Enhancer of Zeste Homolog 2 (EZH2) suppresses normal skeletal muscle differentiation and is highly expressed in RMS tumours. Results We demonstrate combining inhibition of the epigenetic modulator EZH2 with the differentiating agent retinoic acid (RA) is more effective at reducing cell proliferation in RMS cell lines than single agents alone. In PAX3-FOXO1 positive RMS cells this is due to an RA-driven induction of the interferon pathway resulting in apoptosis. In fusion negative RMS, combination therapy led to an EZH2i-driven upregulation of myogenic signalling resulting in differentiation. In both subtypes, EZH2 is significantly associated with enrichment of trimethylated lysine 27 on histone 3 (H3K27me3) in genes that are downregulated in untreated RMS cells and upregulated with EZH2 inhibitor treatment. These results provide insight into the mechanism that drives the anti-cancer effect of the EZH2/RA single agent and combination treatment and indicate that the reduction of EZH2 activity combined with the induction of RA signalling represents a potential novel therapeutic strategy to treat both subtypes of RMS. Conclusions The results of this study demonstrate the potential utility of combining EZH2 inhibitors with differentiation agents for the treatment of paediatric rhabdomyosarcomas. As EZH2 inhibitors are currently undergoing clinical trials for adult and paediatric solid tumours and retinoic acid differentiation agents are already in clinical use this presents a readily translatable potential therapeutic strategy. Moreover, as inhibition of EZH2 in the poor prognosis FPRMS subtype results in an inflammatory response, it is conceivable that this strategy may also synergise with immunotherapies for a more effective treatment in these patients.

Sphingosine-1-phosphate (S1P) is generated intracellularly and, when transported to the extracellular compartment, predominantly signals through S1P receptors. The S1P signalling pathway has been implicated in the pathophysiology of neurological injury following aneurysmal subarachnoid haemorrhage (aSAH). In this review, we bring together all the available data regarding the role of S1P in neurological injury following aSAH. There is agreement in the literature that S1P increases in the cerebrospinal fluid following aSAH and leads to cerebral artery vasospasm. On the other hand, the role of S1P in the parenchyma is less clear cut, with different studies arguing for beneficial and deleterious effects. A parsimonious interpretation of this apparently conflicting data is presented. We discuss the potential of S1P receptor modulators, in clinical use for multiple sclerosis, to be repurposed for aSAH. Finally, we highlight the gaps in our knowledge of S1P signalling in humans, the clinical challenges of targeting the S1P pathway after aSAH and other research priorities.