Germline pathogenic variants in CDKN2A are well established as an underlying cause of familial malignant melanoma. While pathogenic variants in other genes have also been linked to melanoma, most familial cases remain unexplained. We assessed pathogenic germline variants in 360 cancer‐related genes in 56 Norwegian melanoma‐prone families. The index cases were selected based on familial history of melanoma and/or multiple primary melanomas, along with previous negative tests for pathogenic CDKN2A variants. We found 6 out of 56 index individuals to carry germline pathogenic or likely pathogenic variants in BRCA2, MRE11, ATM, MSH2, CHEK2, and AR. One family member with melanoma (not index case) carried a pathogenic variant in MAP3K6. In addition, we found a high fraction of variants previously considered benign and/or as variants of uncertain significance in xeroderma pigmentosum‐related genes. In particular, XPCL48F was found in 8 indexes; thus, the allele fraction (0.07) was significantly higher than in comparable healthy populations (0.02–0.03; P‐values from 0.007 to 0.014). In conclusion, we found that several melanoma‐prone families have pathogenic variants in genes not usually linked to melanoma.

Abstract Background Constitutional BRCA1 epimutations (promoter hypermethylation) are associated with an elevated risk of triple-negative breast cancer and high-grade serous ovarian cancer. While MGMT epimutations are frequent in colon cancer, glioblastoma, and B-cell lymphoma, it remains unknown whether constitutional MGMT epimutations are associated with risk of any of these malignancies. Methods We designed a nested case–control study, assessing potential associations between MGMT epimutations in blood from healthy individuals and subsequent risk of incident cancer. The study cohort was drawn from postmenopausal women, participating in the Women’s Health Initiative (WHI) study, who had not been diagnosed with either colon cancer, glioblastoma, or B-cell lymphoma prior to study entry. The protocol included n = 400 women developing incident left-sided and n = 400 women developing right-sided colon cancer, n = 400 women developing diffuse large B-cell lymphomas, all matched on a 1:2 basis with cancer-free controls, and n = 195 women developing incident glioblastoma multiforme, matched on a 1:4 basis. All cancers were confirmed in centralized medical record review. Blood samples, collected at entry, were analyzed for MGMT epimutations by massive parallel sequencing. Associations between MGMT methylation and incident cancers were analyzed by Cox proportional hazards regression. Results Analyzing epimutations affecting the key regulatory area of the MGMT promoter, the hazard ratio (HR) was 1.07 (95% CI 0.79–1.45) and 0.80 (0.59–1.08) for right- and left-sided colon cancer, respectively, 1.13 (0.78–1.64) for glioblastoma, and 1.11 (0.83–1.48) for diffuse large B-cell lymphomas. Sensitivity analyses limited to subregions of the MGMT promoter and to individuals with different genotypes of a functional SNP in the MGMT promoter (rs16906252), revealed no significant effect on HR for any of the cancer forms. Neither did we observe any effect of rs16906252 status on HR for any of the cancer forms among individuals methylated or non-methylated at the MGMT promoter. Conclusions Constitutional MGMT promoter methylation in normal tissue is not associated with an increased risk of developing colon cancer, glioblastoma, or B-cell lymphoma.

As we age, cells accumulate somatic mutations, undergo epigenetic alterations, and experience telomere shortening. In older individuals, normal tissues are often overtaken by expanding clones of cells that have acquired mutations promoting proliferation. Whether epigenetic changes can also provide selective advantage and induce clonal expansion in ageing is less clear. Here, we sequenced 719 whole genomes and methylomes of single-cell colonies derived from HSCs of three healthy individuals and three breast cancer patients. Using somatic mutations, we built lineage trees of the HSCs for each individual. To establish whether loss or gain of methylation was heritable, we developed a method that accurately infers, for each CpG site, its zygotic methylation state as well as the number and timing of ancestral methylation changes that explain the observed methylation state of each somatic cell. We analysed ∼24 million CpG sites per individual and find that methylation states are, in general, stably heritable over decades of life. We find that during embryogenesis, when the embryo comprises a few thousand cells, developmental cells lay down unique methylation haplotypes, spanning hundreds of base pairs in size, that are stably inherited by descendants. This phase of rewiring the methylome has striking properties: (1) thousands of regions genome-wide show heterogeneity of methylation profiles laid down during this phase; (2) these regions are often conserved across different individuals; (3) it precedes gastrulation, so that the same changes are seen in all germ layers; (4) the process happens quickly, completing within a few generations of cell division; (5) it coincides with establishment of inactive X chromosome methylation in females; and (6) the methylation affects many key regulatory regions of the genome. To assess whether embryonic methylation rewiring can contribute to cancer development, we studied women with breast cancer who had BRCA1 promoter methylation. The tumour samples did not have germline or somatic mutation of BRCA1 (or other DNA repair genes), but had BRCA1 methylation along with the typical mutational signatures of homologous recombination deficiency. Intriguingly, we found that the same methylation change was present heterozygously in a subset of HSCs; that these HSCs all derived from one embryonic cell that pre-dated gastrulation; and that the somatic mutations defining this lineage of HSCs were also present clonally in the breast cancer. Thus, aberrant methylation of the BRCA1 promoter in embryogenesis was transmitted across germ layers, in blood and breast epithelium, and (with loss-of-heterozygosity) drove homologous recombination deficiency in a breast clone that transformed to cancer 4-6 decades later. By comparing normal healthy individuals of different ages with cancer patients, we have been able to unravel the process of normal ageing from cancer development at an unprecedented resolution. Lori D. Kregar, Nicholas Williams, Joe Lee, Michael Spencer-Chapman, Oleksii Nikolaienko, Emily Mitchell, Liv B. Gansmo, Per E. Lønning, Elisa Laurenti, Lucy Yates, Stian Knappskog, Jyoti Nangalia, Peter Campbell. Rewiring of methylation during embryogenesis can seed cancer decades later [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB308.

Abstract Background: Despite recent advances in personalized medicine, conventional chemotherapy remains a backbone in breast cancer therapy. Thus, identifying markers predicting sensitivity or resistance to individual chemotherapeutics is of great importance. Methods: In the EpiTax neoadjuvant trial, enrolling patients between 1997-2003, patients with primary breast cancers (T2 >4cm, T3/T4 and/or N2/N3) were randomized to epirubicin 90mg/m2/3W or paclitaxel 200mg/m²/3W monotherapy, with cross-over in case of inferior response. Pre-treatment snap-frozen tumor biopsies from 223 patients were analyzed by targeted NGS of a 360 gene panel. Endpoint for comparison was clinical response to the first regimen, since pCR was rare due to the large tumor sizes at inclusion. For validation purposes we performed targeted sequencing of tumor samples from a total of 478 patients included in the Gepar Trio (n=132), Quattro (n=171) and Quinto (n=175) trials, in which patients with >2cm tumors received neoadjuvant anthracycline/taxane combination regimens. Here, the primary endpoint was clinical response to combined treatment, but since these tumors were smaller than in the EpiTax-trial, pCR was included as a secondary endpoint. In addition, experimental validations were performed -by CDH1 knock-down and CRISPR/Cas9 knock-out in cell line models. Results: In samples from the EpiTax-trial, CDH1 mutations predicted an inferior response (trend across response groups; cPD, cSD, cPR and cCR) in the paclitaxel arm (p=0.01) as well as the epirubicin arm (p=0.04). The predictive value was observed within the subgroup of ER-positive cases (both for paclitaxel (p=0.005) and epirubicin (p=0.003)) but not among ER-negative tumors. The majority of CDH1 mutations (24/34=71%) were observed in lobular cancers. While lobular histology predicted resistance to paclitaxel (but not epirubicin), CDH1 mutations predicted resistance also within the subgroup of lobular cases (p=0.002), demonstrating CDH1 mutations to be an independent predictor and not only a co-variate to lobular histology. As assessing functionally linked genes, mutations in GATA3, a transcriptional regulator of CDH1, were predominantly observed in ductal cancers, and were not predictive of resistance to any compound. Yet, combining GATA3 and CDH1 mutations into a composite biomarker predicted resistance to both paclitaxel (p=0.007) and epirubicin (p=0.01), especially in ER-positive cases (p=0.002 and p=0.0004, respectively). While EMT-signatures had predictive value, this effect was largely dominated by CDH1, while other EMT-related genes had limited impact on response. In the independent validation cohort from the Gepar trials, selected with enrichment for lobular cancers (34%), CDH1 mutations were not significantly associated with resistance to therapy (p=0.19) although predicted lack of pCR (p=0.01). Combining GATA3 and CDH1 mutations predicted lack of clinical response (p=0.05) and lack of pCR (p=0.0007) respectively in this cohort. In the in vitro analyses, resistance to paclitaxel was observed in three different breast cancer cell lines upon siRNA mediated knock-down of CDH1, as well as in a CRISPR/Cas9 mediated CDH1 knock-out model, as measured by growth rate, induction of apoptosis, G2 arrest, mitochondrial respiration and tubulin stability. For anthracyclines, similar effects were observed for mitochondrial respiration. Conclusions: In conclusion, mutations in CDH1 predicted resistance to paclitaxel and epirubicin. Our data suggest that CDH1 mutations should be explored further as a predictive biomarker for potential application. Citation Format: Stian Knappskog, Reham Helwa, Sivaramakrishna Rachakonda, Liv B. Gansmo, Carsten Denkert, Lucy R. Yates, Christine Solbach, Michael Untch, Bruno V. Sinn, Anne-Sophie Litmeyer, Beyhan Ataseven, Jens Huober, David C. Wedge, Thomas Karn, Oleksii Nikolaienko, Frederik Marmé, Peter A. Fasching, Hans Petter Eikesdal, Elmar Stickeler, Christian Schem, Paul Jank, Marion van Mackelenbergh, Volkmar Müller, Baerbel Felder, Johannes Holtschmidt, Peter J. Campbell, Sibylle Loibl, Per Lonning. CDH1 mutations predict resistance to neoadjuvant taxane therapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-25-12.

Abstract Mosaic epimutations (i.e., epigenetic gene inactivation) within the BRCA1 gene promoter occur in 5-8% of healthy individuals and are associated with a significantly elevated risk of breast and ovarian cancer. These epimutations arise prenatally and then serve as an underlying cause of cancer in adulthood, likely accounting for about 20% of triple-negative breast cancers. Similar mosaic epimutations have been detected in some other tumor suppressor genes as well, but the intraindividual and population frequencies of this phenomenon remain unknown. To address this gap, we have developed novel methodology for detection of mosaic epimutations. The experimental technique is a multiplexed, hybridization capture-based, targeted methylation sequencing assay covering gene regulatory areas. It allows cost-effective assessment of epimutations in established and emerging tumor suppressor genes. An accompanying tailored data analysis pipeline is designed to quantify epimutations. The pipeline is sensitive and robust to variance in methylation, sequencing errors and batch effects. We applied this method to a set of samples from healthy young males. Analysis uncovered significant interindividual epimutational heterogeneity and confirmed high sensitivity of the assay and its superiority compared to methylation profiling using microarrays. Here, we present details on the highly sensitive, scalable and robust methodology for analysis of mosaic epimutations together with the results on initial assessment of the prevalence of epimutations in healthy individuals. We anticipate that this method will become widely used for studies of epigenetic gene regulation and related cancer risk. Citation Format: Oleksii Nikolaienko, Per E. Lønning, Stian Knappskog. Studying mosaic epimutations at scale [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7012.

Obesity promotes a more aggressive breast cancer phenotype. Through spatial and single-cell- based analysis of hormone receptor-negative breast cancers, we identify a subset of tumor- associated neutrophils (TANs) positive for granzyme B (GZMB) enriched in the tumor microenvironment of obese patients. In breast tumors evolved in obese environments, TANs are in proximity of M2 polarized macrophages containing lipopolysaccharides (LPS) from gram- negative bacteria. Pyroptosis of macrophages releases bacterial LPS, activating local GZMB+ TANs. This induces release of the S100 family member S100A8 that promotes tumor progression. In sum, we describe an obesity associated cellular network of cancer cells, neutrophils and M2 polarized macrophages that promotes tumor growth. ### Competing Interest Statement The authors have declared no competing interest.

Abstract Background: Low level normal cell BRCA1 epimutations have been associated with an increased risk of triple-negative breast cancer (TNBC). However, the fraction of TNBCs that may have BRCA1 epimutations as their underlying cause is unknown. Neither are the time of occurrence and the potential inheritance pattern of BRCA1 epimutations established. Methods: To address these questions, we analyzed BRCA1 methylation status in breast cancer tissue and matched white blood cells (WBC) from 411 patients with primary breast cancer, including 66 TNBCs. Samples were analyzed by a highly sensitive next-generation sequencing (NGS) assay on an Illumina MiSeq sequencer, allowing allele-resolved methylation assessment. Further, to assess the time of origin and the characteristics of normal cell BRCA1 methylation, we analyzed umbilical cord samples from 1260 newborn girls and 200 newborn boys.To assess potential Mendelian heritage, we analyzed BRCA1 methylation status in WBCs from 575 mothers and 531 fathers of newborn girls with (n = 102) and without (n = 473) WBC BRCA1 methylation. Results: We found concordant tumor and mosaic WBC BRCA1 epimutations in 10 out of 66 patients with TNBC and in four out of six patients with estrogen receptor (ER)-low expression (< 10%) of tumors (combined 14 out of 72; 19.4%, CI: 11.1-30.5). These exceeded the number of tumors harboring germline (n = 5) or somatic (n = 4) BRCA1 mutations. Notably, BRCA1 methylation and BRCA1 mutations were mutually exclusive. Contrasting the findings in TNBC and ER-low exprssion tumors, we found WBC and tumor BRCA1 methylation concordance in only three out of 221 patients with ER >10+% tumors and zero out of 116 patients with HER2 positive tumors. Intraindividually, BRCA1 epimutations affected the same allele in normal and tumor cells. Assessing BRCA1 methylation in umbilical cord WBCs from newborn girls, we found mosaic, predominantly monoallelic BRCA1 epimutations, with qualitative features similar to those in adults, in 113/1260 (9.0%) of individuals. We found no correlation between WBC BRCA1 methylation in newborns and methylation status in their mothers, fathers, or any parent. Notably, WBC BRCA1 methylation occurred at a significantly lower frequency in newborn boys ( 9/200; 4.5%) as compared to newborn girls (p = 0.038). Similarly, WBC BRCA1 methylation was found less common among fathers (16/531; 3.0%), as compared to mothers (46/575; 8.0%; p = 0.0003). Conclusions: Our findings suggest prenatal BRCA1 epimutations might be the underlying cause of around 20% of TNBC and low-ER expressing breast cancers. Such constitutional mosaic BRCA1 methylation likely arise through gender-related mechanisms in utero, independent of Mendelian inheritance. Citation Format: Per Lonning, Hans Petter Eikesdal, Elisabet Ognedal, Bjornar Gilje, Steinar Lundgren, Egil Blix, Helge Espelid, Jürgen Geisler, Stephanie Geisler, Emiel Janssen, Synnøve Yndestad, Laura Minsaas, Beryl Leirvaag, Reidun Lillestol, Stian Knappskog, Oleksii Nikolaienko. Prenatal BRCA1 epimutations is a major cause of triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS07-09.

All Norwegian hospitals with cancer departments participate in a national cancer treatment study. It is relevant for patients with advanced cancer after standard treatment.

Germline pathogenic genetic variants in the BRCA1 and BRCA2 genes are the most frequent causes of familial breast and ovarian cancer. Contrasting BRCA2, epimutations in the BRCA1 gene are frequently detected in tissue from triple-negative breast (TNBC) and high-grade serous ovarian cancers (HGSOC). While studies over the last decade have reported BRCA1 epimutations in white blood cells (WBC) from breast and ovarian cancer patients, the potential hazard ratio for incident TNBC and HGSOC was not formally assessed until recently. Conducting a prospective nested case-control study on women participating in the American Women's Health Initiative Study, we provided firm evidence that mosaic WBC BRCA1 epimutations, even at allele frequencies < 0.1%, are associated with a significantly increased risk of both incident HGSOC and TNBC > 5 years after WBC collection. In a second study assessing BRCA1 epimutations in WBC and matched tumor samples from TNBC, our results indicated such epimutations to be the underlying cause of around 20% of TNBC, far exceeding the percentage of cases carrying BRCA1 germline pathogenic genetic variants. We detected primary constitutional BRCA1 epimutations in tissues derived from all three germ layers. They occur independently of BRCA1 promoter haplotypes but are present on the same allele in all WBC within affected individuals. Moreover, epimutations are consistently found on the same allele in normal and tumor breast tissue as well as in WBC. This finding, together with BRCA1 epimutations detected in WBC from newborns, strongly indicates an early embryonic event with clonal expansion affecting all germ layers. Future work in the field must lead to an understanding of exactly when and how the BRCA1 epimutations occur and, most importantly, whether primary constitutional epimutations in genes other than BRCA1 may cause an elevated risk of other cancer types.

Background: While soft tissue sarcomas affect younger patients, few studies have assessed the distribution of underlying pathogenic germline variants. Patients and methods: We retrospectively identified all pediatric and young adult patients (0–22 years) at Haukeland University Hospital, Norway (1981–2019), through clinical and pathological records. We identified n = 46 eligible patients. From these 46 patients, adequate material representing normal tissue was available for n = 41 cases (n = 24 diagnosed with rhabdomyosarcoma, 9 with synovial sarcomas, 2 with Ewing sarcomas, and 6 without further classification), with matching tumor tissue for n = 40. Normal tissue samples were analyzed for germline pathogenic variants (PVs) by targeted sequencing of 360 cancer genes. Results: Out of the 41 analyzed cases, we found PVs or likely PVs in 7 (17%). These variants were found in TP53, MUTYH, FANCC, DICER1, FANCA, MYO3A, and MYO5B. Supporting the causality of these PVs, four cases revealed loss of heterozygosity (LOH) of the wild-type allele in the tumor tissue, one patient with a PV in DICER1 had a second somatic variant in DICER1, and a patient with a PV in TP53 had the altered allele amplified in the tumor. For three out of five with available family history, a history of other cancers in relatives was recorded. Among genes with variants of uncertain significance, CHD1L was of particular interest, revealing a stop-gain and a missense variant. Interpretation: A high fraction of young patients with soft tissue sarcoma harbor PVs. Among the genes affected, we substantiate a potential role of MYO5B and propose a potential role for MYO3A.

Background: To date, only two studies have compared the outcomes of patients with liver-limited BRAF V600E-mutated colorectal liver metastases (CRLMs) managed with resection versus systemic therapy alone, and these have reported contradictory findings. Methods: In this observational, international, multicentre study, patients with liver-limited BRAF V600E-mutated CRLMs treated with resection or systemic therapy alone were identified from institutional databases. Patterns of recurrence/progression and overall survival were compared using multivariable analyses of the entire cohort and a propensity score-matched cohort. Results: Of 170 patients included, 119 underwent hepatectomy and 51 received systemic treatment. Surgically treated patients had a more favourable pattern of recurrence with most recurrences limited to a single site, whereas diffuse progression was more common among patients who received systemic treatment (19 versus 44%; P = 0.002). Surgically treated patients had longer median overall survival (35 versus 20 months; P < 0.001). Hepatectomy was independently associated with better OS than systemic treatment alone (HR 0.37, 95% c.i. 0.21 to 0.65). In the propensity score-matched cohort, surgically treated patients had longer median overall survival (28 versus 20 months; P < 0.001); hepatectomy was independently associated with better overall survival (HR 0.47, 0.25 to 0.88). Conclusion: BRAF V600E mutation should not be considered a contraindication to surgery for patients with resectable, liver-only CRLMs.

We present, to our knowledge, the first methodological study aimed at enhancing the prognostic power of Cox regression models, widely used in survival analysis, through optimized data selection. Our approach employs a novel two-stage mechanism: by framing the prognostic stratum matching problem intuitively, we select prognostically representative patient observations to create a more balanced training set. This enables the model to assign equal attention to distinct prognostic subgroups. We demonstrate the methodology using an observational dataset of 1,799 patients with resected colorectal cancer liver metastases, 1,197 of whom received adjuvant chemotherapy and 602 who did not. In our study, as is current standard practice, the comparator was training prognostic models on the entire cohort (referred to as "model 1"). Models trained on the untreated and treated subgroups, matched through our approach (referred to as "model 3"), showed an improvement of up to 20% in bootstrapped C-indices compared to model 1. Notably, model 3 exhibited superior calibration, with a 6- to 10-fold improvement over model 1. Additional performance metrics aligned with these findings, and robustness was confirmed through bias-corrected bootstrapping. Given the ongoing development of numerous linear prognostic models and the general applicability of our approach to any observational data, this method holds significant potential to impact biomedical research and clinical practice where prognostic models are utilized.

Supplementary Figure 1 from GSTP1 Promoter Haplotypes Affect DNA Methylation Levels and Promoter Activity in Breast Carcinomas

Supplementary Table 4 from GSTP1 Promoter Haplotypes Affect DNA Methylation Levels and Promoter Activity in Breast Carcinomas