Vessela N. Kristensen

• Torland, Anne Vold; Geisler, Jürgen; Azimzade, Youness; Frafjord, Astri; Nilsen, Hogne Røed & Buer, Linn [Show all 15 contributors for this article] (2026). Multi-platform analysis of the tumor immune microenvironment associated with breast cancer subtypes. Oncoimmunology. ISSN 2162-4011. 15(1). doi: 10.1080/2162402X.2025.2610535. Full text in Research Archive Show summary

  Breast cancer is genetically and histologically heterogenous, and is influenced by a variety of factors, including the tumor microenvironment (TME). The PAM50 subtypes; Luminal A, Luminal B, Normal-like, Basal-like and Her2-enriched, are associated with different tumor phenotypes and overall survival. The quantity and quality of immune cell infiltration in breast tumors play a key role in cancer development and progression and are associated with survival and treatment response. We used multiplex immunohistochemistry, single-cell RNA sequencing, and two deconvolution algorithms to explore the immune landscape across PAM50 subtypes. Immunostaining of CD3+ T cells, tryptase+ mast cells, CD20+ B cells, CD68+ CD163+ macrophages, and CD66b+ granulocytes revealed marked differences in tumor immune infiltrates according to breast cancer subtypes. Luminal tumors were relatively deprived of T cells and B cells, while exhibiting sparse to moderate amounts of macrophage infiltration. In contrast, Her2-enriched tumors exhibited a moderate immune presence, with sparse to moderate T cell infiltration and moderate infiltration of B cells and macrophages. At the other end of the spectrum, Basal-like tumors stood out for their strikingly rich immune environment and are heavily infiltrated by T cells, B cells, and macrophages. The results from the single-cell and deconvolution analyses confirmed subtype-specific immune microenvironments, which also allowed us to observe increased levels of natural killer (NK) and CD8+ T cells in Her2-enriched and Basal-like subtypes. In conclusion, our findings demonstrate significant differences in the immune tumor microenvironmentbetween the established breast cancer molecular subtypes.

• Azimzade, Youness; Haugen, Mads; Kristensen, Vessela N.; Frigessi, Arnoldo & Luque, Alvaro Kohn (2026). Explainable machine learning-guided integrated multiomics analysis reveals macrophage-driven immune suppression in breast cancer. Nature Communications. 17(1). doi: 10.1038/s41467-026-73617-9. Full text in Research Archive Show summary

  Despite thorough characterizations of cellular compositions within the breast tumor microenvironment (TME), their implications for disease progression and patient prognosis remain poorly understood. Unraveling these effects is vital for identifying potential targets to improve treatment outcomes. In this study, we devise an explainable machine learning (XML) pipeline to scrutinize the associations between TME cellular constituents and relapse-free survival (RFS). By applying this pipeline to estimated cell fractions in the METABRIC and TCGA datasets and comparing these results with associations to pathological complete response (pCR) after neoadjuvant chemotherapy (NAC), we create a comprehensive catalog of the TME’s role based on 5000 patient samples. Our findings reveal an unexpected dichotomy in which macrophages correlate positively with pCR but negatively with RFS, particularly within estrogen receptor-positive (ER+) and Luminal A and B (LumA/B) cancer subtypes. We show that this pattern is driven by heterogeneity in breast tumors characterized by increasing levels of macrophage infiltration. Through imaging mass cytometry (IMC) data analysis, we find that macrophages tend to accumulate in the vicinity of HLA-ABChi epithelial cells as their frequency increases in tumor tissues and that they also express elevated levels of HLA-ABC protein. In both IMC and single-cell RNA sequencing (scRNA-seq) data, we uncover a significant association between these HLA-ABChi macrophages and regulatory and exhausted T cells (TReg and TEx), suggesting their involvement in immune suppression, likely by creating a chronically activated immunosuppressive TME. Subsequent cell-cell communication analysis predicts interactions between HLA-ABChi macrophages and TEx cells via the ligands SIGLEC9, ALCAM, and CSF1, and with TReg cells through APP, ANGPTL4, and SIGLEC9 signaling. Considering the clinical relevance of macrophages in ER+ (LumA/B) subtypes, this work enhances the characterization of macrophage-associated immune suppression in these tumors and identifies potential targets for immunomodulatory strategies.

• Billaud, Amandine; Figlioli, Gisella; Mooser, Clémence; Casamassima, Irene; Azzoni, Violette & Srivatsa, Jahnavi [Show all 50 contributors for this article] (2025). Large-scale meta-analysis and precision functional assays identify FANCM regions in which PTVs confer different risks for ER-negative and triple-negative breast cancer. Breast. ISSN 0960-9776. 85, p. 1–13. doi: 10.1016/j.breast.2025.104619. Full text in Research Archive Show summary

  The breast cancer risk conferred by germline protein truncating variants (PTVs) in known and putative breast cancer genes has been extensively investigated. However, the effect of FANCM PTVs on breast cancer risk remains unclear. Our previous clinical, genetic and functional results on the N-terminal p.Arg658∗ and the two C-terminal p.Gln1701∗ and p.Gly1906Alafs∗12 variants suggested that FANCM PTVs may confer different risks for ER-negative (ER-neg) and triple-negative (TN) breast cancer subtypes. Here, we performed meta-analyses of seven studies totaling 144 681 breast cancer cases and 123 632 controls. FANCM PTVs were tested for association with breast cancer risk overall and the disease clinical subtypes by single variant and burden analyses. Two CRISPR-Cas9-based functional assays were also conducted to test the fitness of cells after knock-in of the p.Arg658∗, p.Gln1701∗ and p.Gly1906Alafs∗12 PTVs and the sensitivity of different FANCM regions to genome editing. Our results suggest that the N-terminal FANCM region upstream of p.Tyr725 harbors essential functions, whereas downstream regions appear dispensable. This is supported by our genetic data which indicate that all FANCM PTVs, excluding the two C-terminal p.Gln1701∗ and p.Gly1906Alafs∗12, are associated with an increased risk of ER-neg (OR = 1.41, P = 0.023) and TN (OR = 1.64, P = 0.0023). Notably, PTVs upstream of AA position 670 are associated with a moderate risk of developing TN breast cancer, and that even when the p.Arg658∗ carriers were excluded from the analysis. Importantly, our results confirm previous data indicating that p.Arg658∗ carriers are at moderate risk of developing ER-neg (OR = 2.08, P = 0.030) and TN (OR = 3.26; P = 0.0034), whereas carriers of p.Gln1701∗ and p.Gly1906Alafs∗12 should not be considered at increased risk. Our data are useful for counseling carriers of FANCM PTVs, but further analyses are warranted to obtain more precise risk estimates.

• Puig, Rafael Riudavets; Brorson, Ina Skaara; O’Mahony, Denise; Kristensen, Vessela N. & Mathelier, Anthony (2025). HiFIseek: gene-specific enrichment of high-impact mutations in associated genomic regions. bioRxiv (preprint article). doi: 10.1101/2025.06.12.659305. Full text in Research Archive Show summary

  Transcription is regulated through the sequence-specific binding of transcription factors (TFs) to cis-regulatory regions (CRRs). Although scattered along the genome, multiple CRRs are brought in close spatial proximity to the genes they regulate through the formation of DNA loops. The sequence component of transcriptional regulation suggests that DNA variants in CRRs could disrupt TF-DNA interactions and gene regulatory networks through a cascading effect. To date, only a few cases of recurrent cis-regulatory variants have been described. As an alternative to variant recurrence, some methods utilize genomic annotations that indicate the functional impact (FI) of a variant and the CRRs of each gene to detect the enrichment of high-impact cis-regulatory variants (CRVs). However, the agreement across the different associations and FI scoring methods remains unexplored. This work demonstrates that gene-CRR and FI scoring methods exhibit little consensus, highlighting the impact of the choice of a specific CRR-score combination in a given analysis. In addition, we demonstrate that cancer genes exhibit a higher frequency of FI variants compared to non-cancer genes. Based on this, we developed HiFIseek, a Snakemake pipeline exploring the enrichment of FI variants in associated genomic regions using all region-score combinations and two enrichment detection methods. We apply HiFIseek to detect genes exhibiting high FI CRVs in eight cancer cohorts and a set of breast cancer risk-associated single-nucleotide polymorphisms (SNPs). In the cancer cohorts, we observe a small degree of agreement across CRR-score combinations. Although HiFIseek detected known cancer-related genes with FI CRVs, most of them did not show significant changes in their expression. In the SNPs cohort, HiFIseek found small consensus across CRR-score combinations. However, one of the combinations returned 18 known cancer-related genes, including BRCA1 , showing an enrichment of high-impact CRVs.

• Fjørtoft, Marit Otterlei; Garred, Øystein; Lingjærde, Ole Christian; Johannesen, Karin Teien; Ottestad, Lars & Bergheim, Inger Riise [Show all 29 contributors for this article] (2025). Single-cell analysis reveals tumour size as a key driver of immune cell profile alterations in primary breast tumours and corresponding lymph nodes. BJC Reports. 3(1). doi: 10.1038/s44276-025-00152-3. Full text in Research Archive Show summary

  At diagnosis, 30–40% of women with breast cancer have metastases in sentinel (SN) or axillary lymph nodes (ALN). Nodal status is a strong prognostic factor and guides treatment decisions. Immune checkpoint inhibition has shown some efficacy, which can increase in the neoadjuvant setting. A better understanding of how tumour cells in primary tumours and metastatic lymph nodes shape the local immune microenvironment may provide clues for more individualized therapeutic interventions. Methods We conducted deep immunophenotypic analysis of 29 primary breast tumours and 36 lymph nodes from 38 patients with primary operable breast cancer. Results The immune profile of the primary tumour was not predictive of the lymph node immune profile or metastatic status. Primary tumours showed prominent CD8 T cell exhaustion and activated regulatory T cells, and the frequencies of these subsets were associated with tumour size. The immune cell profile in lymph nodes were different from the profile in primary tumours, except for the ALN+ nodes, which displayed a T-cell profile more similar to primary tumours. The frequencies of the T cell subsets in lymph nodeswere associated with metastatic size. Tumour cells from smaller metastases exhibited a distinct phenotype compared to those from larger tumour deposits, and the size of the tumour cell deposit impacted the local immune cell composition. Conclusion The tumour size of primary tumours and metastatic size in lymph nodes are the main drivers of changes in immune cell composition

• Campos-Segura, Anthony Vladimir; Alvarez, Karin; Carrasco, Alexis German Murillo; Rossi, Benedito Mauro; Bohorquez, Mabel & Spirandelli, Florencia [Show all 77 contributors for this article] (2025). Characterization of Screening Strategies for Lynch Syndrome in Latin America. Clinical Gastroenterology and Hepatology. ISSN 1542-3565. 23(9), p. 1642–1654. doi: 10.1016/j.cgh.2024.12.026. Full text in Research Archive Show summary

  Background & Aims In Latin America, genetic testing for Lynch syndrome (LS) has been partially implemented. Traditionally, LS diagnosis relied on the Amsterdam criteria and Bethesda guidelines, collectively known as traditional screening (TS). However, TS may miss up to 68% of LS cases. To improve detection rates, universal tumor screening (UTS) has been introduced. UTS involves screening all newly diagnosed patients with colorectal cancer for molecular markers to more effectively identify LS cases. Methods Clinical and molecular data on 1684 patients with colorectal cancer, collected between 1999 and 2020, were provided by 24 Latin American genetic cancer registries and centers. Germline genetic testing was not consistently performed across all cases. Results LS screening strategies were available for 72% (1209/1684) of cases, with germline testing conducted in one-quarter (304/1209) of these. Most cases (78%; n = 943) underwent UTS, primarily in Argentina, Chile, and Uruguay, whereas 22% (266/1209) were screened through TS. UTS identified deficient mismatch repair tumors in 29% (272/943) of cases. The rate of LS confirmed by sequencing was higher with UTS (53.3%; 65/122) compared with TS (47.8%; 87/182), although the difference was not statistically significant (P = .175). Conclusions UTS is widely implemented in Latin America; however, the low detection rate of LS demonstrated in this study raises concerns about the routine use of germline genetic testing in our region. Our study provides real-world outcomes that highlight disparities in screening uptake and counseling referrals, illustrating the challenges that Latin American countries face in hereditary cancer syndrome screening. These results contribute to the rationale for designing effective screening strategies for LS, which may also be applicable to other hereditary cancer syndromes, ultimately.

• Røssevold, Andreas Hagen; Tekpli, Xavier; Lingjærde, Ole Christian; Russnes, Hege Elisabeth Giercksky; Vallon-Christersson, Johan & Borgen, Elin [Show all 12 contributors for this article] (2025). High tumor expression of CTLA4 identifies lymph node-negative basal-like breast cancer patients with excellent prognosis. Communications Medicine. 5(1), p. 1–13. doi: 10.1038/s43856-025-00865-z. Full text in Research Archive Show summary

  Background Tumor immune cell infiltration is a favorable prognostic factor in triple-negative breast cancer. Most triple-negative tumors belong to the aggressive basal-like subtype. We hypothesized that immune gene expression may identify low-risk patients for whom adjuvant chemotherapy can be de-escalated. Methods The expression of 753 immune-related genes was analyzed in tumor biopsies from 45 patients with basal-like disease and no lymph node metastases (Oslo1 cohort) and evaluated for prognostic value. Findings were validated in two independent cohorts. Oslo1 biopsies were also analyzed for tumor-infiltrating lymphocytes (TIL) and tertiary lymphoid structures (TLS). Results Here we show that a high expression of CTLA4 (above 63rd percentile) is associated with an excellent prognosis in the Oslo1 cohort. None of the patients in the CTLA4high group suffered disease recurrence (median follow-up 7.4 years) or breast cancer-related death (median follow-up 17.7 years). Analysis of the SCAN-B (n = 233; 97% without distant recurrence in CTLA4high group) and METABRIC cohorts (n = 155; 93% disease-specific survival in CTLA4high group) validates this finding, which also applies to patients who did not receive chemotherapy. CTLA4 expression correlates with TIL score and TLS levels (Oslo1 cohort), but no TILlow/CTLA4high patients died from breast cancer, suggesting that the CTLA4 readout identifies low-risk patients not captured by TIL assessment. Conclusions A high primary tumor expression of CTLA4 identifies patients with an excellent prognosis, for whom standard chemotherapy may be de-escalated or omitted.

• Martiniussen, Marit Almenning; Larsen, Marthe; Hovda, Tone; SANDHAUG, MERETE; Dahl, Fredrik Andreas & Eikvil, Line [Show all 11 contributors for this article] (2025). Performance of Two Deep Learning–based AI Models for Breast Cancer Detection and Localization on Screening Mammograms from BreastScreen Norway. Radiology: Artificial Intelligence (RAI). 7(3). doi: 10.1148/ryai.240039. Full text in Research Archive Show summary

  Two deep learning–based artificial intelligence (AI) models, one commercially available and one in-house, showed good performance for stand-alone cancer detection on retrospective mammography screening data. AI markings on the mammograms corresponded well to the true cancer location. Purpose - To evaluate cancer detection and marker placement accuracy of two artificial intelligence (AI) models developed for interpretation of screening mammograms. Materials and Methods - This retrospective study included data from 129 434 screening examinations (all female patients; mean age, 59.2 years ± 5.8 [SD]) performed between January 2008 and December 2018 in BreastScreen Norway. Model A was commercially available and model B was an in-house model. Area under the receiver operating characteristic curve (AUC) with 95% CIs were calculated. The study defined 3.2% and 11.1% of the examinations with the highest AI scores as positive, threshold 1 and 2, respectively. A radiologic review assessed location of AI markings and classified interval cancers as true or false negative. Results - The AUC value was 0.93 (95% CI: 0.92, 0.94) for model A and B when including screen-detected and interval cancers. Model A identified 82.5% (611 of 741) of the screen-detected cancers at threshold 1 and 92.4% (685 of 741) at threshold 2. Model B identified 81.8% (606 of 741) at threshold 1 and 93.7% (694 of 741) at threshold 2. The AI markings were correctly localized for all screen-detected cancers identified by both models and 82% (56 of 68) of the interval cancers for model A and 79% (54 of 68) for model B. At the review, 21.6% (45 of 208) of the interval cancers were identified at the preceding screening by either or both models, correctly localized and classified as false negative (n = 17) or with minimal signs of malignancy (n = 28). Conclusion - Both AI models showed promising performance for cancer detection on screening mammograms. The AI markings corresponded well to the true cancer locations.

• Zanti, Maria; O'Mahony, Denise; Parsons, Michael T.; Dorling, Leila; Dennis, Joe & Boddicker, Nicholas J. [Show all 50 contributors for this article] (2025). Analysis of more than 400,000 women provides case-control evidence for BRCA1 and BRCA2 variant classification. Nature Communications. 16(1). doi: 10.1038/s41467-025-59979-6. Full text in Research Archive Show summary

  Clinical genetic testing identifies variants causal for hereditary cancer, information that is used for risk assessment and clinical management. Unfortunately, some variants identified are of uncertain clinical significance (VUS), complicating patient management. Case-control data is one evidence type used to classify VUS. As an initiative of the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) Analytical Working Group we analyze germline sequencing data of BRCA1 and BRCA2 from 96,691 female breast cancer cases and 302,116 controls from three studies: the BRIDGES study of the Breast Cancer Association Consortium, the Cancer Risk Estimates Related to Susceptibility consortium, and the UK Biobank. We observe 11,207 BRCA1 and BRCA2 variants, with 6909 being coding, covering 23.4% of BRCA1 and BRCA2 VUS in ClinVar and 19.2% of ClinVar curated (likely) benign or pathogenic variants. Case-control likelihood ratio (ccLR) evidence is highly consistent with ClinVar assertions for (likely) benign or pathogenic variants; exhibiting 99.1% sensitivity and 95.3% specificity for BRCA1 and 93.3% sensitivity and 86.6% specificity for BRCA2. This approach provides case-control evidence for 787 unclassified variants; these include 579 with strong or moderate benign evidence and 10 with strong pathogenic evidence for which ccLR evidence is sufficient to alter clinical classification.

• Hoppe, Reiner; Winter, Stefan; Lo, Wing-Yee; Michailidou, Kyriaki; Bolla, Manjeet K. & Keeman, Renske [Show all 50 contributors for this article] (2025). Lessons learned from a candidate gene study investigating aromatase inhibitor treatment outcome in breast cancer. NPJ breast cancer. 11(1), p. 1–15. doi: 10.1038/s41523-025-00733-y. Full text in Research Archive Show summary

  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.

• Schmiester, Leonard; Brasó-Maristany, Fara; González-Farré, Blanca; Pascual, Tomás; Gavilá, Joaquín & Tekpli, Xavier [Show all 11 contributors for this article] (2024). Computational Model Predicts Patient Outcomes in Luminal B Breast Cancer Treated with Endocrine Therapy and CDK4/6 Inhibition. Clinical Cancer Research. ISSN 1078-0432. 30(17), p. 3779–3787. doi: 10.1158/1078-0432.CCR-24-0244. Full text in Research Archive Show summary

  Purpose: Development of a computational biomarker to predict, prior to treatment, the response to CDK4/6 inhibition (CDK4/6i) in combination with endocrine therapy in patients with breast cancer. Experimental Design: A mechanistic mathematical model that accounts for protein signaling and drug mechanisms of action was developed and trained on extensive, publicly available data from breast cancer cell lines. The model was built to provide a patient-specific response score based on the expression of six genes (CCND1, CCNE1, ESR1, RB1, MYC, and CDKN1A). The model was validated in five independent cohorts of 148 patients in total with early-stage or advanced breast cancer treated with endocrine therapy and CDK4/6i. Response was measured either by evaluating Ki67 levels and PAM50 risk of relapse (ROR) after neoadjuvant treatment or by evaluating progression-free survival (PFS). Results: The model showed significant association with patient’s outcomes in all five cohorts. The model predicted high Ki67 [area under the curve; AUC (95% confidence interval, CI) of 0.80 (0.64–0.92), 0.81 (0.60–1.00) and 0.80 (0.65–0.93)] and high PAM50 ROR [AUC of 0.78 (0.64–0.89)]. This observation was not obtained in patients treated with chemotherapy. In the other cohorts, patient stratification based on the model prediction was significantly associated with PFS [hazard ratio (HR) = 2.92 (95% CI, 1.08–7.86), P = 0.034 and HR = 2.16 (1.02 4.55), P = 0.043]. Conclusions: A mathematical modeling approach accurately predicts patient outcome following CDK4/6i plus endocrine therapy that marks a step toward more personalized treatments in patients with Luminal B breast cancer.

• Yildiz, Tugba Akman; Arendt, Lisa M.; Geisler, Jürgen; Kristensen, Vessela N.; Frigessi, Arnoldo & Luque, Alvaro Kohn (2024). Modeling of Mouse Experiments Suggests that Optimal Anti-Hormonal Treatment for Breast Cancer is Diet-Dependent. Bulletin of Mathematical Biology. ISSN 0092-8240. 86:42(4), p. 1–33. doi: 10.1007/s11538-023-01253-1. Full text in Research Archive Show summary

  Estrogen receptor positive breast cancer is frequently treated with anti-hormonal treatment such as aromatase inhibitors (AI). Interestingly, a high body mass index has been shown to have a negative impact on AI efficacy, most likely due to disturbances in steroid metabolism and adipokine production. Here, we propose a mathematical model based on a system of ordinary differential equations to investigate the effect of high-fat diet on tumor growth. We inform the model with data from mouse experiments, where the animals are fed with high-fat or control (normal) diet. By incorporating AI treatment with drug resistance into the model and by solving optimal control problems we found differential responses for control and high-fat diet. To the best of our knowledge, this is the first attempt to model optimal anti-hormonal treatment for breast cancer in the presence of drug resistance. Our results underline the importance of considering high-fat diet and obesity as factors influencing clinical outcomes during anti-hormonal therapies in breast cancer patients.

• Tomasova, Kristyna; Seborova, Karolina; Kroupa, Michal; Horak, Josef; Kavec, Miriam & Vodickova, Ludmila [Show all 15 contributors for this article] (2024). Telomere length as a predictor of therapy response and survival in patients diagnosed with ovarian carcinoma. Heliyon. 10:e33525(13), p. 1–16. doi: 10.1016/j.heliyon.2024.e33525. Full text in Research Archive Show summary

  Impaired telomere length (TL) maintenance in ovarian tissue may play a pivotal role in the onset of epithelial ovarian cancer (OvC). TL in either target or surrogate tissue (blood) is currently being investigated for use as a predictor in anti-OvC therapy or as a biomarker of the disease progression, respectively. There is currently an urgent need for an appropriate approach to chemotherapy response prediction. We performed a monochrome multiplex qPCR measurement of TL in peripheral blood leukocytes (PBL) and tumor tissues of 209 OvC patients. The methylation status and gene expression of the shelterin complex and telomerase catalytic subunit (hTERT) were determined within tumor tissues by High-Throughput DNA methylation profiling and RNA sequencing (RNA-Seq) analysis, respectively. The patients sensitive to cancer treatment (n = 46) had shorter telomeres in PBL compared to treatment-resistant patients (n = 93; P = 0.037). In the patients with a different therapy response, transcriptomic analysis showed alterations in the peroxisome proliferator-activated receptor (PPAR) signaling pathway (q = 0.001). Moreover, tumor TL shorter than the median corresponded to better overall survival (OS) (P = 0.006). TPP1 gene expression was positively associated with TL in tumor tissue (P = 0.026). TL measured in PBL could serve as a marker of platinum therapy response in OvC patients. Additionally, TL determined in tumor tissue provides information on OvC patients' OS.

• Davidson, Aimee L.; Michailidou, Kyriaki; Parsons, Michael T.; Fortuno, Cristina; Bolla, Manjeet K. & Wang, Qin [Show all 50 contributors for this article] (2024). Co-observation of germline pathogenic variants in breast cancer predisposition genes: Results from analysis of the BRIDGES sequencing dataset. American Journal of Human Genetics. ISSN 0002-9297. 111(9), p. 2059–2069. doi: 10.1016/j.ajhg.2024.07.004. Full text in Research Archive Show summary

  Co-observation of a gene variant with a pathogenic variant in another gene that explains the disease presentation has been designated as evidence against pathogenicity for commonly used variant classification guidelines. Multiple variant curation expert panels have specified, from consensus opinion, that this evidence type is not applicable for the classification of breast cancer predisposition gene variants. Statistical analysis of sequence data for 55,815 individuals diagnosed with breast cancer from the BRIDGES sequencing project was undertaken to formally assess the utility of co-observation data for germline variant classification. Our analysis included expected loss-of-function variants in 11 breast cancer predisposition genes and pathogenic missense variants in BRCA1, BRCA2, and TP53. We assessed whether co-observation of pathogenic variants in two different genes occurred more or less often than expected under the assumption of independence. Co-observation of pathogenic variants in each of BRCA1, BRCA2, and PALB2 with the remaining genes was less frequent than expected. This evidence for depletion remained after adjustment for age at diagnosis, study design (familial versus population-based), and country. Co-observation of a variant of uncertain significance in BRCA1, BRCA2, or PALB2 with a pathogenic variant in another breast cancer gene equated to supporting evidence against pathogenicity following criterion strength assignment based on the likelihood ratio and showed utility in reclassification of missense BRCA1 and BRCA2 variants identified in BRIDGES. Our approach has applicability for assessing the value of co-observation as a predictor of variant pathogenicity in other clinical contexts, including for gene-specific guidelines developed by ClinGen Variant Curation Expert Panels.

• Shahrouzi, Parastoo; Azimzade, Youness; Brankiewicz-Kopcinska, Wioletta; Bhatia, Sugandha; Kunke, David & Richard, Derek [Show all 9 contributors for this article] (2024). Loss of chromosome cytoband 13q14.2 orchestrates breast cancer pathogenesis and drug response. Breast Cancer Research. ISSN 1465-5411. 26. doi: 10.1186/s13058-024-01924-4. Full text in Research Archive Show summary

  Breast cancer (BCa) is a major global health challenge. The BCa genome often carries extensive somatic copy number alterations (CNAs), including gains/amplifications and losses/deletions. These CNAs significantly affect tumor development, drug response and patient survival. However, how individual CNAs contribute is mostly elusive. We identified loss of chromosome 13q14.2 as a key CNA in BCa, occurring in up to 63% of patients, depending on the subtype, and correlating with poor survival. Through multi-omics and in vitro analyses, we uncover a paradoxical role of 13q14.2 loss, promoting both cell cycle and pro-apoptotic pathways in cancer cells, while also associating with increased NK cell and macrophage populations in the tumor microenvironment. Notably, 13q14.2 loss increases BCa susceptibility to BCL2 inhibitors, both in vitro and in patient-derived xenografts. Thus, 13q14.2 loss could serve as a biomarker for BCa prognosis and treatment, potentially improving outcomes for BCa patients. Graphical abstract

• Sharma, Abhibhav; Debik, Julia; Naume, Bjørn; Ohnstad, Hege Oma; Sahlberg, Guro Kristine Kleivi & Borgen, Elin [Show all 25 contributors for this article] (2024). Comprehensive multi-omics analysis of breast cancer reveals distinct long-term prognostic subtypes. Oncogenesis. 13(1), p. 1–13. doi: 10.1038/s41389-024-00521-6. Full text in Research Archive Show summary

  Breast cancer (BC) is a leading cause of cancer-related death worldwide. The diverse nature and heterogeneous biology of BC pose challenges for survival prediction, as patients with similar diagnoses often respond differently to treatment. Clinically relevant BC intrinsic subtypes have been established through gene expression profiling and are implemented in the clinic. While these intrinsic subtypes show a significant association with clinical outcomes, their long-term survival prediction beyond 5 years often deviates from expected clinical outcomes. This study aimed to identify naturally occurring long-term prognostic subgroups of BC based on an integrated multi-omics analysis. This study incorporates a clinical cohort of 335 untreated BC patients from the Oslo2 study with long-term follow-up (>12 years). Multi-Omics Factor Analysis (MOFA+) was employed to integrate transcriptomic, proteomic, and metabolomic data obtained from the tumor tissues. Our analysis revealed three prominent multi-omics clusters of BC patients with significantly different long-term prognoses (p = 0.005). The multi-omics clusters were validated in two independent large cohorts, METABRIC and TCGA. Importantly, a lack of prognostic association to long-term follow-up above 12 years in the previously established intrinsic subtypes was shown for these cohorts. Through a systems-biology approach, we identified varying enrichment levels of cell-cycle and immune-related pathways among the prognostic clusters. Integrated multi-omics analysis of BC revealed three distinct clusters with unique clinical and biological characteristics. Notably, these multi-omics clusters displayed robust associations with long-term survival, outperforming the established intrinsic subtypes.

• Ankill, Jørgen; Zhao, Zhi; Tekpli, Xavier; Kure, Elin Wenche Hegland; Kristensen, Vessela N. & Mathelier, Anthony [Show all 7 contributors for this article] (2024). Integrative pan-cancer analysis reveals a common architecture of dysregulated transcriptional networks characterized by loss of enhancer methylation. PLoS Computational Biology. ISSN 1553-734X. 20:e1012565(11), p. 1–33. doi: 10.1371/journal.pcbi.1012565. Full text in Research Archive Show summary

  Aberrant DNA methylation contributes to gene expression deregulation in cancer. However, these alterations’ precise regulatory role and clinical implications are still not fully understood. In this study, we performed expression-methylation Quantitative Trait Loci (emQTL) analysis to identify deregulated cancer-driving transcriptional networks linked to CpG demethylation pan-cancer. By analyzing 33 cancer types from The Cancer Genome Atlas, we identified and confirmed significant correlations between CpG methylation and gene expression (emQTL) in cis and trans, both across and within cancer types. Bipartite network analysis of the emQTL revealed groups of CpGs and genes related to important biological processes involved in carcinogenesis including proliferation, metabolism and hormone-signaling. These bipartite communities were characterized by loss of enhancer methylation in specific transcription factor binding regions (TFBRs) and the CpGs were topologically linked to upregulated genes through chromatin loops. Penalized Cox regression analysis showed a significant prognostic impact of the pan-cancer emQTL in many cancer types. Taken together, our integrative pan-cancer analysis reveals a common architecture where hallmark cancer-driving functions are affected by the loss of enhancer methylation and may be epigenetically regulated.

• Ohnstad, Hege Oma; Blix, Egil Støre; Akslen, Lars Andreas; Gilje, Bjørnar; Raj, Sunil Xavier & Skjerven, Helle [Show all 45 contributors for this article] (2024). Impact of Prosigna test on adjuvant treatment decision in lymph node-negative early breast cancer—a prospective national multicentre study (EMIT-1). ESMO Open. 9(6), p. 1–12. doi: 10.1016/j.esmoop.2024.103475. Full text in Research Archive Show summary

  Background EMIT-1 is a national, observational, single-arm trial designed to assess the value of the Prosigna, Prediction Analysis of Microarray using the 50 gene classifier (PAM50)/Risk of Recurrence (ROR), test as a routine diagnostic tool, examining its impact on adjuvant treatment decisions, clinical outcomes, side-effects and cost-effectiveness. Here we present the impact on treatment decisions. Patients and methods Patients with hormone receptor-positive, human epidermal growth factor receptor 2-negative pT1-pT2 lymph node-negative early breast cancer (EBC) were included. The Prosigna test and standard histopathology assessments were carried out. Clinicians’ treatment decisions were recorded before (pre-Prosigna) and after (post-Prosigna) the Prosigna test results were disclosed. Results Of 2217 patients included, 2178 had conclusive Prosigna results. The pre-Prosigna treatment decisions were: no systemic treatment (NT) in 27% of patients, endocrine treatment alone (ET) in 38% and chemotherapy (CT) followed by ET (CT + ET) in 35%. Post-Prosigna treatment decisions were 25% NT, 51% ET and 24% CT + ET, respectively. Adjuvant treatment changed in 28% of patients, including 21% change in CT use. Among patients assigned to CT + ET pre-Prosigna, 45% were de-escalated to ET post-Prosigna. Of patients assigned to ET, 12% were escalated to CT + ET and 8% were de-escalated to NT; of those assigned to NT, 18% were escalated to ET/CT + ET. CT was more frequently recommended for patients aged ≤50 years. In the subgroup with pT1c-pT2 G2 and intermediate Ki67 (0.5-1.5× local laboratory median Ki67 score), the pre-Prosigna CT treatment decision varied widely across hospitals (3%-51%). Post-Prosigna, the variability of CT use was markedly reduced (8%-24%). The correlation between Ki67 and ROR score within this subgroup was poor (r = 0.25-0.39). The median ROR score increased by increasing histological grade, but the ROR score ranges were wide (for G1 0-79, G2 0-90, G3 16-94). Conclusion The Prosigna test result changed adjuvant treatment decisions in all EBC clinical risk groups, markedly decreased the CT use for patients categorized as higher clinical risk pre-Prosigna and reduced treatment decision discrepancies between hospitals.

• Yiangou, Kristia; Mavaddat, Nasim; Dennis, Joe; Zanti, Maria; Wang, Qin & Bolla, Manjeet K. [Show all 50 contributors for this article] (2024). Polygenic score distribution differences across European ancestry populations: implications for breast cancer risk prediction. Breast Cancer Research. ISSN 1465-5411. 26:189(1), p. 1–14. doi: 10.1186/s13058-024-01947-x. Full text in Research Archive Show summary

  Background The 313-variant polygenic risk score (PRS313) provides a promising tool for clinical breast cancer risk prediction. However, evaluation of the PRS313 across different European populations which could influence risk estimation has not been performed. Methods We explored the distribution of PRS313 across European populations using genotype data from 94,072 females without breast cancer diagnosis, of European-ancestry from 21 countries participating in the Breast Cancer Association Consortium (BCAC) and 223,316 females without breast cancer diagnosis from the UK Biobank. The mean PRS was calculated by country in the BCAC dataset and by country of birth in the UK Biobank. We explored different approaches to reduce the observed heterogeneity in the mean PRS across the countries, and investigated the implications of the distribution variability in risk prediction. Results The mean PRS313 differed markedly across European countries, being highest in individuals from Greece and Italy and lowest in individuals from Ireland. Using the overall European PRS313 distribution to define risk categories, leads to overestimation and underestimation of risk in some individuals from these countries. Adjustment for principal components explained most of the observed heterogeneity in the mean PRS. The mean estimates derived when using an empirical Bayes approach were similar to the predicted means after principal component adjustment. Conclusions Our results demonstrate that PRS distribution differs even within European ancestry populations leading to underestimation or overestimation of risk in specific European countries, which could potentially influence clinical management of some individuals if is not appropriately accounted for. Population-specific PRS distributions may be used in breast cancer risk estimation to ensure predicted risks are correctly calibrated across risk categories.

• Tinholt, Mari; Tekpli, Xavier; Torland, Lilly Anne; Tahiri, Andliena; Geisler, Jürgen & Kristensen, Vessela N. [Show all 8 contributors for this article] (2024). The breast cancer coagulome in the tumor microenvironment and its role in prognosis and treatment response to chemotherapy. Journal of Thrombosis and Haemostasis. ISSN 1538-7933. 22(5), p. 1319–1335. doi: 10.1016/j.jtha.2024.01.003. Full text in Research Archive Show summary

  Background The procoagulant phenotype in cancer is linked to thrombosis, cancer progression, and immune response. A novel treatment that reduces the risk of both thrombosis and cancer progression without excess bleeding risk remains to be identified. Objectives Here, we aimed to broadly investigate the breast tumor coagulome and its relation to prognosis, treatment response to chemotherapy, and the tumor microenvironment. Methods Key coagulation-related genes (n = 35) were studied in a Norwegian cohort with tumor (n = 134) and normal (n = 189) tissue and in the Cancer Genome Atlas (n = 1052) data set. We performed gene set variation analysis in the Norwegian cohort, and in the Cancer Genome Atlas cohort, associations with the tumor microenvironment and prognosis were evaluated. Analyses were performed with cBioPortal, Estimation of Stromal and Immune cells in Malignant Tumors Using Expression Data, Tumor Immune Estimation Resource, the integrated repository portal for tumor–immune system interactions, Tumor Immune Single-cell Hub 2, and the receiver operating characteristic plotter. Six independent breast cancer cohorts were used to study the tumor coagulome and treatment response to chemotherapy. Results Twenty-two differentially expressed coagulation-related genes were identified in breast tumors. Several coagulome factors were correlated with tumor microenvironment characteristics and were expressed by nonmalignant cells in the tumor microenvironment. PLAT and F8 were independent predictors of better overall survival and progression-free survival, respectively. F12 and PLAU were predictors of worse progression-free survival. The PROCR-THBD-PLAT signature showed a promising predictive value (area under the curve, 0.75; 95% CI, 0.69-0.81; P = 3.6 × 10−17) for combination chemotherapy with fluorouracil, epirubicin, and cyclophosphamide. Conclusion The breast tumor coagulome showed potential in prediction of prognosis and chemotherapy response. Cells within the tumor microenvironment are sources of coagulome factors and may serve as therapeutic targets of coagulation factors.

• Shahrouzi, Parastoo; Forouz, Farzaneh; Mathelier, Anthony; Kristensen, Vessela N. & Duijf, Pascal Hubertus Gerardus (2024). Copy number alterations: a catastrophic orchestration of the breast cancer genome. Trends in Molecular Medicine. ISSN 1471-4914. 30(8), p. 750–764. doi: 10.1016/j.molmed.2024.04.017. Full text in Research Archive Show summary

  Breast cancer (BCa) is a prevalent malignancy that predominantly affects women around the world. Somatic copy number alterations (CNAs) are tumor-specific amplifications or deletions of DNA segments that often drive BCa development and therapy resistance. Hence, the complex patterns of CNAs complement BCa classification systems. In addition, understanding the precise contributions of CNAs is essential for tailoring personalized treatment approaches. This review highlights how tumor evolution drives the acquisition of CNAs, which in turn shape the genomic landscapes of BCas. It also discusses advanced methodologies for identifying recurrent CNAs, studying CNAs in BCa and their clinical impact.

• Liu, Yuxi; Peng, Cheng; Brorson, Ina Skaara; O'Mahony, Denise; Kelly, Rebecca L. & Heng, Yujing J. [Show all 15 contributors for this article] (2024). Germline polygenic risk scores are associated with immune gene expression signature and immune cell infiltration in breast cancer. American Journal of Human Genetics. ISSN 0002-9297. 111(10), p. 2150–2163. doi: 10.1016/j.ajhg.2024.08.009. Full text in Research Archive
• Torland, Lilly Anne; Lai, Xiaoran; Kumar, Surendra; Riis, Margit; Geisler, Jürgen & Lüders, Torben [Show all 10 contributors for this article] (2024). Benign breast tumors may arise on different immunological backgrounds. Molecular Oncology. ISSN 1574-7891. 18(10), p. 2495–2509. doi: 10.1002/1878-0261.13655. Full text in Research Archive Show summary

  Benign breast tumors are a nonthreatening condition defined as abnormal cell growth within the breast without the ability to invade nearby tissue. However, benign lesions hold valuable biological information that can lead us toward better understanding of tumor biology. In this study, we have used two pathway analysis algorithms, Pathifier and gene set variation analysis (GSVA), to identify biological differences between normal breast tissue, benign tumors and malignant tumors in our clinical dataset. Our results revealed that one-third of all pathways that were significantly different between benign and malignant tumors were immune-related pathways, and 227 of them were validated by both methods and in the METABRIC dataset. Furthermore, five of these pathways (all including genes involved in cytokine and interferon signaling) were related to overall survival in cancer patients in both datasets. The cellular moieties that contribute to immune differences in malignant and benign tumors were analyzed using the deconvolution tool, CIBERSORT. The results showed that levels of some immune cells were specifically higher in benign than in malignant tumors, and this was especially the case for resting dendritic cells and follicular T-helper cells. Understanding the distinct immune profiles of benign and malignant breast tumors may aid in developing noninvasive diagnostic methods to differentiate between them in the future.

• Fleischer, Thomas; Haugen, Mads; Ankill, Jørgen; Silwal-Pandit, Laxmi; Børresen-Dale, Anne-Lise & Hedenfalk, Ingrid [Show all 10 contributors for this article] (2024). An integrated omics approach highlights how epigenetic events can explain and predict response to neoadjuvant chemotherapy and bevacizumab in breast cancer. Molecular Oncology. ISSN 1574-7891. 18(8), p. 2042–2059. doi: 10.1002/1878-0261.13656. Full text in Research Archive Show summary

  Treatment with the anti-angiogenic drug bevacizumab in addition to chemotherapy has shown efficacy for breast cancer in some clinical trials, but better biomarkers are needed to optimally select patients for treatment. Here, we present an omics approach where DNA methylation profiles are integrated with gene expression and results from proteomic data in breast cancer patients to predict response to therapy and pinpoint response-related epigenetic events. Fresh-frozen tumor biopsies taken before, during, and after treatment from human epidermal growth factor receptor 2 negative non-metastatic patients receiving neoadjuvant chemotherapy with or without bevacizumab were subjected to molecular profiling. Here, we report that DNA methylation at enhancer CpGs related to cell cycle regulation can predict response to chemotherapy and bevacizumab for the estrogen receptor positive subset of patients (AUC = 0.874), and we validated this observation in an independent patient cohort with a similar treatment regimen (AUC = 0.762). Combining the DNA methylation scores with the scores from a previously published protein signature resulted in a slight increase in the prediction performance (AUC = 0.784). We also show that tumors receiving the combination treatment underwent more extensive epigenetic alterations. Finally, we performed an integrative expression–methylation quantitative trait loci analysis on alterations in DNA methylation and gene expression levels, showing that the epigenetic alterations that occur during treatment are different between responders and non-responders and that these differences may be explained by the proliferation–epithelial-to-mesenchymal transition axis through the activity of grainyhead like transcription factor 2. Using tumor purity computed from copy number data, we developed a method for estimating cancer cell-specific methylation to confirm that the association to response reflects DNA methylation in cancer cells. Taken together, these results support the potential for clinical benefit of the addition of bevacizumab to chemotherapy when administered to the correct patients.

• Kentistou, Katherine A.; Kaisinger, Lena R.; Stankovic, Stasa; Vaudel, Marc; Oliveira, Edson Mendes de & Messina, Andrea [Show all 49 contributors for this article] (2024). Understanding the genetic complexity of puberty timing across the allele frequency spectrum. Nature Genetics. ISSN 1061-4036. 56(7), p. 1397–1411. doi: 10.1038/s41588-024-01798-4. Full text in Research Archive Show summary

  Pubertal timing varies considerably and is associated with later health outcomes. We performed multi-ancestry genetic analyses on ~800,000 women, identifying 1,080 signals for age at menarche. Collectively, these explained 11% of trait variance in an independent sample. Women at the top and bottom 1% of polygenic risk exhibited ~11 and ~14-fold higher risks of delayed and precocious puberty, respectively. We identified several genes harboring rare loss-of-function variants in ~200,000 women, including variants in ZNF483, which abolished the impact of polygenic risk. Variant-to-gene mapping approaches and mouse gonadotropin-releasing hormone neuron RNA sequencing implicated 665 genes, including an uncharacterized G-protein-coupled receptor, GPR83, which amplified the signaling of MC3R, a key nutritional sensor. Shared signals with menopause timing at genes involved in DNA damage response suggest that the ovarian reserve might signal centrally to trigger puberty. We also highlight body size-dependent and independent mechanisms that potentially link reproductive timing to later life disease.

• Mo, Torgeir; Brandal, Siri Helene Bertelsen; Geier, Oliver; Engebråten, Olav; Nilsen, Line & Kristensen, Vessela N. [Show all 10 contributors for this article] (2023). MRI Assessment of Changes in Tumor Vascularization during Neoadjuvant Anti-Angiogenic Treatment in Locally Advanced Breast Cancer Patients. Cancers. 15(18). doi: 10.3390/cancers15184662. Full text in Research Archive Show summary

  Anti-VEGF (vascular endothelial growth factor) treatment improves response rates, but not progression-free or overall survival in advanced breast cancer. It has been suggested that subgroups of patients may benefit from this treatment; however, the effects of adding anti-VEGF treatment to a standard chemotherapy regimen in breast cancer patients are not well studied. Understanding the effects of the anti-vascular treatment on tumor vasculature may provide a selection of patients that can benefit. The aim of this study was to study the vascular effect of bevacizumab using clinical dynamic contrast-enhanced MRI (DCE-MRI). A total of 70 women were randomized to receive either chemotherapy alone or chemotherapy with bevacizumab for 25 weeks. DCE-MRI was performed at baseline and at 12 and 25 weeks, and in addition 25 of 70 patients agreed to participate in an early MRI after one week. Voxel-wise pharmacokinetic analysis was performed using semi-quantitative methods and the extended Tofts model. Vascular architecture was assessed by calculating the fractal dimension of the contrast-enhanced images. Changes during treatment were compared with baseline and between the treatment groups. There was no significant difference in tumor volume at any point; however, DCE-MRI parameters revealed differences in vascular function and vessel architecture. Adding bevacizumab to chemotherapy led to a pronounced reduction in vascular DCE-MRI parameters, indicating decreased vascularity. At 12 and 25 weeks, the difference between the treatment groups is severely reduced.

• Zanti, Maria; O'Mahony, Denise G.; Parsons, Michael T.; Li, Hongyan; Dennis, Joe & Aittomäkkiki, Kristiina [Show all 124 contributors for this article] (2023). A likelihood ratio approach for utilizing case-control data in the clinical classification of rare sequence variants: Application to BRCA1 and BRCA2. Human Mutation. ISSN 1059-7794. 2023. doi: 10.1155/2023/9961341. Full text in Research Archive Show summary

  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.

• Muranen, Taru A.; Morra, Anna; Khan, Sofia; Barnes, Daniel R.; Bolla, Manjeet K. & Dennis, Joe [Show all 69 contributors for this article] (2023). PREDICT validity for prognosis of breast cancer patients with pathogenic BRCA1/2 variants. NPJ breast cancer. 9:37(1), p. 1–15. doi: 10.1038/s41523-023-00546-x. Full text in Research Archive Show summary

  Abstract We assessed the PREDICT v 2.2 for prognosis of breast cancer patients with pathogenic germline BRCA1 and BRCA2 variants, using follow-up data from 5453 BRCA1/2 carriers from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) and the Breast Cancer Association Consortium (BCAC). PREDICT for estrogen receptor (ER)-negative breast cancer had modest discrimination for BRCA1 carrier patients overall (Gönen & Heller unbiased concordance 0.65 in CIMBA, 0.64 in BCAC), but it distinguished clearly the high-mortality group from lower risk categories. In an analysis of low to high risk categories by PREDICT score percentiles, the observed mortality was consistently lower than the expected mortality, but the confidence intervals always included the calibration slope. Altogether, our results encourage the use of the PREDICT ER-negative model in management of breast cancer patients with germline BRCA1 variants. For the PREDICT ER-positive model, the discrimination was slightly lower in BRCA2 variant carriers (concordance 0.60 in CIMBA, 0.65 in BCAC). Especially, inclusion of the tumor grade distorted the prognostic estimates. The breast cancer mortality of BRCA2 carriers was underestimated at the low end of the PREDICT score distribution, whereas at the high end, the mortality was overestimated. These data suggest that BRCA2 status should also be taken into consideration with tumor characteristics, when estimating the prognosis of ER-positive breast cancer patients.

• Chung, Felicia Fei-Lei; Maldonado, Sandra González; Nemc, Amelie; Bouaoun, Liacine; Cahais, Vincent & Cuenin, Cyrille [Show all 22 contributors for this article] (2023). Buffy coat signatures of breast cancer risk in a prospective cohort study. Clinical Epigenetics. 15:102, p. 1–10. doi: 10.1186/s13148-023-01509-6. Full text in Research Archive Show summary

  Background Epigenetic alterations are a near-universal feature of human malignancy and have been detected in malignant cells as well as in easily accessible specimens such as blood and urine. These findings offer promising applications in cancer detection, subtyping, and treatment monitoring. However, much of the current evidence is based on findings in retrospective studies and may reflect epigenetic patterns that have already been influenced by the onset of the disease. Methods Studying breast cancer, we established genome-scale DNA methylation profiles of prospectively collected buffy coat samples (n = 702) from a case–control study nested within the EPIC-Heidelberg cohort using reduced representation bisulphite sequencing (RRBS). Results We observed cancer-specific DNA methylation events in buffy coat samples. Increased DNA methylation in genomic regions associated with SURF6 and REXO1/CTB31O20.3 was linked to the length of time to diagnosis in the prospectively collected buffy coat DNA from individuals who subsequently developed breast cancer. Using machine learning methods, we piloted a DNA methylation-based classifier that predicted case–control status in a held-out validation set with 76.5% accuracy, in some cases up to 15 years before clinical diagnosis of the disease. Conclusions Taken together, our findings suggest a model of gradual accumulation of cancer-associated DNA methylation patterns in peripheral blood, which may be detected long before clinical manifestation of cancer. Such changes may provide useful markers for risk stratification and, ultimately, personalized cancer prevention.

• Figlioli, Gisella; Billaud, Amandine; Ahearn, Thomas U.; Antonenkova, Natalia N.; Becher, Heiko & Beckmann, Matthias W. [Show all 114 contributors for this article] (2023). FANCM missense variants and breast cancer risk: a case-control association study of 75,156 European women. European Journal of Human Genetics. ISSN 1018-4813. 31(5), p. 578–587. doi: 10.1038/s41431-022-01257-w. Full text in Research Archive Show summary

  Evidence from literature, including the BRIDGES study, indicates that germline protein truncating variants (PTVs) in FANCM confer moderately increased risk of ER-negative and triple-negative breast cancer (TNBC), especially for women with a family history of the disease. Association between FANCM missense variants (MVs) and breast cancer risk has been postulated. In this study, we further used the BRIDGES study to test 689 FANCM MVs for association with breast cancer risk, overall and in ER-negative and TNBC subtypes, in 39,885 cases (7566 selected for family history) and 35,271 controls of European ancestry. Sixteen common MVs were tested individually; the remaining rare 673 MVs were tested by burden analyses considering their position and pathogenicity score. We also conducted a meta-analysis of our results and those from published studies. We did not find evidence for association for any of the 16 variants individually tested. The rare MVs were significantly associated with increased risk of ER-negative breast cancer by burden analysis comparing familial cases to controls (OR = 1.48; 95% CI 1.07–2.04; P = 0.017). Higher ORs were found for the subgroup of MVs located in functional domains or predicted to be pathogenic. The meta-analysis indicated that FANCM MVs overall are associated with breast cancer risk (OR = 1.22; 95% CI 1.08–1.38; P = 0.002). Our results support the definition from previous analyses of FANCM as a moderate-risk breast cancer gene and provide evidence that FANCM MVs could be low/moderate risk factors for ER-negative and TNBC subtypes. Further genetic and functional analyses are necessary to clarify better the increased risks due to FANCM MVs

• Morra, Anna; Schreurs, Maartje A. C.; Andrulis, Irene L.; Anton-Culver, Hoda; Augustinsson, Annelie & Beckmann, Matthias W. [Show all 125 contributors for this article] (2023). Association of the CHEK2 c.1100delC variant, radiotherapy, and systemic treatment with contralateral breast cancer risk and breast cancer-specific survival. Cancer Medicine. 12(15), p. 16142–16162. doi: 10.1002/cam4.6272. Full text in Research Archive Show summary

  Background - Breast cancer (BC) patients with a germline CHEK2 c.1100delC variant have an increased risk of contralateral BC (CBC) and worse BC-specific survival (BCSS) compared to non-carriers.<p> <p>Aim - To assessed the associations of CHEK2 c.1100delC, radiotherapy, and systemic treatment with CBC risk and BCSS.<p> <p>Methods - Analyses were based on 82,701 women diagnosed with a first primary invasive BC including 963 CHEK2 c.1100delC carriers; median follow-up was 9.1 years. Differential associations with treatment by CHEK2 c.1100delC status were tested by including interaction terms in a multivariable Cox regression model. A multi-state model was used for further insight into the relation between CHEK2 c.1100delC status, treatment, CBC risk and death.<p> <p>Results - There was no evidence for differential associations of therapy with CBC risk by CHEK2 c.1100delC status. The strongest association with reduced CBC risk was observed for the combination of chemotherapy and endocrine therapy [HR (95% CI): 0.66 (0.55–0.78)]. No association was observed with radiotherapy. Results from the multi-state model showed shorter BCSS for CHEK2 c.1100delC carriers versus non-carriers also after accounting for CBC occurrence [HR (95% CI): 1.30 (1.09–1.56)].<p> <p>Conclusion - Systemic therapy was associated with reduced CBC risk irrespective of CHEK2 c.1100delC status. Moreover, CHEK2 c.1100delC carriers had shorter BCSS, which appears not to be fully explained by their CBC risk.

• Figlioli, Gisella; Billaud, Amandine; Wang, Qin; Bolla, Manjeet K.; Dennis, Joe & Lush, Michael [Show all 96 contributors for this article] (2023). Spectrum and Frequency of Germline FANCM Protein-Truncating Variants in 44,803 European Female Breast Cancer Cases. Cancers. 15(13). doi: 10.3390/cancers15133313. Full text in Research Archive Show summary

  FANCM germline protein truncating variants (PTVs) are moderate-risk factors for ER-negative breast cancer. We previously described the spectrum of FANCM PTVs in 114 European breast cancer cases. In the present, larger cohort, we report the spectrum and frequency of four common and 62 rare FANCM PTVs found in 274 carriers detected among 44,803 breast cancer cases. We confirmed that p.Gln1701* was the most common PTV in Northern Europe with lower frequencies in Southern Europe. In contrast, p.Gly1906Alafs*12 was the most common PTV in Southern Europe with decreasing frequencies in Central and Northern Europe. We verified that p.Arg658* was prevalent in Central Europe and had highest frequencies in Eastern Europe. We also confirmed that the fourth most common PTV, p.Gln498Thrfs*7, might be a founder variant from Lithuania. Based on the frequency distribution of the carriers of rare PTVs, we showed that the FANCM PTVs spectra in Southwestern and Central Europe were much more heterogeneous than those from Northeastern Europe. These findings will inform the development of more efficient FANCM genetic testing strategies for breast cancer cases from specific European populations.

• Levi, Hagai; Carmi, Shai; Rosset, Saharon; Yerushalmi, Rinat; Zick, Aviad & Yablonski-Peretz, Tamar [Show all 155 contributors for this article] (2023). Evaluation of European-based polygenic risk score for breast cancer in Ashkenazi Jewish women in Israel. Journal of Medical Genetics. ISSN 0022-2593. 60(12), p. 1186–1197. doi: 10.1136/jmg-2023-109185. Full text in Research Archive Show summary

  Background Polygenic risk score (PRS), calculated based on genome-wide association studies (GWASs), can improve breast cancer (BC) risk assessment. To date, most BC GWASs have been performed in individuals of European (EUR) ancestry, and the generalisation of EUR-based PRS to other populations is a major challenge. In this study, we examined the performance of EUR-based BC PRS models in Ashkenazi Jewish (AJ) women. Methods We generated PRSs based on data on EUR women from the Breast Cancer Association Consortium (BCAC). We tested the performance of the PRSs in a cohort of 2161 AJ women from Israel (1437 cases and 724 controls) from BCAC (BCAC cohort from Israel (BCAC-IL)). In addition, we tested the performance of these EUR-based BC PRSs, as well as the established 313-SNP EUR BC PRS, in an independent cohort of 181 AJ women from Hadassah Medical Center (HMC) in Israel. Results In the BCAC-IL cohort, the highest OR per 1 SD was 1.56 (±0.09). The OR for AJ women at the top 10% of the PRS distribution compared with the middle quintile was 2.10 (±0.24). In the HMC cohort, the OR per 1 SD of the EUR-based PRS that performed best in the BCAC-IL cohort was 1.58±0.27. The OR per 1 SD of the commonly used 313-SNP BC PRS was 1.64 (±0.28). Conclusions Extant EUR GWAS data can be used for generating PRSs that identify AJ women with markedly elevated risk of BC and therefore hold promise for improving BC risk assessment in AJ women.

• Levy-Jurgenson, Alona; Tekpli, Xavier Kokou Mawu Yram; Kristensen, Vessela N. & Yakhini, Zohar (2023). Analysis of Spatial Molecular Data. Methods in molecular biology. ISSN 1064-3745. 2614, p. 349–356. doi: 10.1007/978-1-0716-2914-7_20. Full text in Research Archive
• Morra, Anna; Mavaddat, Nasim; Muranen, Taru A.; Ahearn, Thomas U.; Allen, Jamie & Andrulis, Irene L. [Show all 84 contributors for this article] (2023). The impact of coding germline variants on contralateral breast cancer risk and survival. American Journal of Human Genetics. ISSN 0002-9297. 110(3), p. 475–486. doi: 10.1016/j.ajhg.2023.02.003. Full text in Research Archive Show summary

  Evidence linking coding germline variants in breast cancer (BC)-susceptibility genes other than BRCA1, BRCA2, and CHEK2 with contralateral breast cancer (CBC) risk and breast cancer-specific survival (BCSS) is scarce. The aim of this study was to assess the association of protein-truncating variants (PTVs) and rare missense variants (MSVs) in nine known (ATM, BARD1, BRCA1, BRCA2, CHEK2, PALB2, RAD51C, RAD51D, and TP53) and 25 suspected BC-susceptibility genes with CBC risk and BCSS. Hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated with Cox regression models. Analyses included 34,401 women of European ancestry diagnosed with BC, including 676 CBCs and 3,449 BC deaths; the median follow-up was 10.9 years. Subtype analyses were based on estrogen receptor (ER) status of the first BC. Combined PTVs and pathogenic/likely pathogenic MSVs in BRCA1, BRCA2, and TP53 and PTVs in CHEK2 and PALB2 were associated with increased CBC risk [HRs (95% CIs): 2.88 (1.70–4.87), 2.31 (1.39–3.85), 8.29 (2.53–27.21), 2.25 (1.55–3.27), and 2.67 (1.33–5.35), respectively]. The strongest evidence of association with BCSS was for PTVs and pathogenic/likely pathogenic MSVs in BRCA2 (ER-positive BC) and TP53 and PTVs in CHEK2 [HRs (95% CIs): 1.53 (1.13–2.07), 2.08 (0.95–4.57), and 1.39 (1.13–1.72), respectively, after adjusting for tumor characteristics and treatment]. HRs were essentially unchanged when censoring for CBC, suggesting that these associations are not completely explained by increased CBC risk, tumor characteristics, or treatment. There was limited evidence of associations of PTVs and/or rare MSVs with CBC risk or BCSS for the 25 suspected BC genes. The CBC findings are relevant to treatment decisions, follow-up, and screening after BC diagnosis.

• Mueller, Stefanie H.; Lai, Alvina G.; Valkovskaya, Maria; Michailidou, Kyriaki; Bolla, Manjeet K. & Wang, Qin [Show all 207 contributors for this article] (2023). Aggregation tests identify new gene associations with breast cancer in populations with diverse ancestry. Genome Medicine. 15(7). doi: 10.1186/s13073-022-01152-5. Full text in Research Archive Show summary

  Background Low‐frequency variants play an important role in breast cancer (BC) susceptibility. Gene‐based meth‐ ods can increase power by combining multiple variants in the same gene and help identify target genes. Methods We evaluated the potential of gene‐based aggregation in the Breast Cancer Association Consortium cohorts including 83,471 cases and 59,199 controls. Low‐frequency variants were aggregated for individual genes’ coding and regulatory regions. Association results in European ancestry samples were compared to single‐marker association results in the same cohort. Gene‐based associations were also combined in meta‐analysis across individu‐ als with European, Asian, African, and Latin American and Hispanic ancestry. Results In European ancestry samples, 14 genes were significantly associated (q < 0.05) with BC. Of those, two genes, FMNL3 (P = 6.11 × 10−6) and AC058822.1 (P = 1.47 × 10−4), represent new associations. High FMNL3 expression has previously been linked to poor prognosis in several other cancers. Meta‐analysis of samples with diverse ancestry discovered further associations including established candidate genes ESR1 and CBLB. Furthermore, literature review and database query found further support for a biologically plausible link with cancer for genes CBLB, FMNL3, FGFR2, LSP1, MAP3K1, and SRGAP2C. Conclusions Using extended gene‐based aggregation tests including coding and regulatory variation, we report identification of plausible target genes for previously identified single‐marker associations with BC as well as the discovery of novel genes implicated in BC development. Including multi ancestral cohorts in this study enabled the identification of otherwise missed disease associations as ESR1 (P = 1.31 × 10−5), demonstrating the importance of diversifying study cohorts.

• Svartdal, Lisa Gregusson; Haugen, Mads; Hongisto, Vesa; Aure, Miriam Ragle; Leivonen, Suvi-Katri & Kristensen, Vessela N. [Show all 10 contributors for this article] (2023). High-throughput screen in vitro identifies dasatinib as a candidate for combinatorial treatment with HER2-targeting drugs in breast cancer. PLOS ONE. 18(1). doi: 10.1371/journal.pone.0280507. Full text in Research Archive Show summary

  Human epidermal growth factor receptor 2-positive (HER2+) breast cancer is an aggressive subtype of this disease. Targeted treatment has improved outcome, but there is still a need for new therapeutic strategies as some patients respond poorly to treatment. Our aim was to identify compounds that substantially affect viability in HER2+ breast cancer cells in response to combinatorial treatment. We performed a high-throughput drug screen of 278 compounds in combination with trastuzumab and lapatinib using two HER2+ breast cancer cell lines (KPL4 and SUM190PT). The most promising drugs were validated in vitro and in vivo, and downstream molecular changes of the treatments were analyzed. The screen revealed multiple drugs that could be used in combination with lapatinib and/or trastuzumab. The Src-inhibitor dasatinib showed the largest combinatorial effect together with lapatinib in the KPL4 cell line compared to treatment with dasatinib alone (p < 0.01). In vivo, only lapatinib significantly reduced tumor growth (p < 0.05), whereas dasatinib alone, or in combination with lapatinib, did not show significant effects. Protein analyses of the treated xenografts showed significant alterations in protein levels compared to untreated controls, suggesting that all drugs reached the tumor and exerted a measurable effect. In silico analyses suggested activation of apoptosis and reduced activity of survival pathways by all treatments, but the opposite pattern was observed for the combinatorial treatment compared to lapatinib alone.

• Daniel, Petr; Balušíková, Kamila; Václavíková, Radka; Šeborová, Karolína; Ransdorfová, Šárka & Valeriánová, Marie [Show all 14 contributors for this article] (2023). ABCB1 Amplicon Contains Cyclic AMP Response Element-Driven TRIP6 Gene in Taxane-Resistant MCF-7 Breast Cancer Sublines. Genes. 14:296(2), p. 1–23. doi: 10.3390/genes14020296. Full text in Research Archive Show summary

  A limited number of studies are devoted to regulating TRIP6 expression in cancer. Hence, we aimed to unveil the regulation of TRIP6 expression in MCF-7 breast cancer cells (with high TRIP6 expression) and taxane-resistant MCF-7 sublines (manifesting even higher TRIP6 expression). We found that TRIP6 transcription is regulated primarily by the cyclic AMP response element (CRE) in hypomethylated proximal promoters in both taxane-sensitive and taxane-resistant MCF-7 cells. Furthermore, in taxane-resistant MCF-7 sublines, TRIP6 co-amplification with the neighboring ABCB1 gene, as witnessed by fluorescence in situ hybridization (FISH), led to TRIP6 overexpression. Ultimately, we found high TRIP6 mRNA levels in progesterone receptor-positive breast cancer and samples resected from premenopausal women.

• Ankill, Jørgen; Aure, Miriam Ragle; Bjørklund, Sunniva; Langberg, Geir Severin Rakh Elvatun; Kristensen, Vessela N. & Vitelli, Valeria [Show all 8 contributors for this article] (2022). Epigenetic alterations at distal enhancers are linked to proliferation in human breast cancer. NAR Cancer. 4(1), p. 1–16. doi: 10.1093/narcan/zcac008. Full text in Research Archive Show summary

  Aberrant DNA methylation is an early event in breast carcinogenesis and plays a critical role in regulating gene expression. Here, we perform genome-wide expression-methylation Quantitative Trait Loci (emQTL) analysis through the integration of DNA methylation and gene expression to identify disease-driving pathways under epigenetic control. By grouping the emQTLs using biclustering we identify associations representing important biological processes associated with breast cancer pathogenesis including regulation of proliferation and tumor-infiltrating fibroblasts. We report genome-wide loss of enhancer methylation at binding sites of proliferation-driving transcription factors including CEBP-β, FOSL1, and FOSL2 with concomitant high expression of proliferation-related genes in aggressive breast tumors as we confirm with scRNA-seq. The identified emQTL-CpGs and genes were found connected through chromatin loops, indicating that proliferation in breast tumors is under epigenetic regulation by DNA methylation. Interestingly, the associations between enhancer methylation and proliferation-related gene expression were also observed within known subtypes of breast cancer, suggesting a common role of epigenetic regulation of proliferation. Taken together, we show that proliferation in breast cancer is linked to loss of methylation at specific enhancers and transcription factor binding and gene activation through chromatin looping.

• Seborova, Karolina; Hlavac, Viktor; Holy, Petr; Bjørklund, Sunniva; Fleischer, Thomas & Rob, Lukas [Show all 16 contributors for this article] (2022). Complex molecular profile of DNA repair genes in epithelial ovarian carcinoma patients with different sensitivity to platinum-based therapy. Frontiers in Oncology. 12:1016958, p. 1–16. doi: 10.3389/fonc.2022.1016958. Full text in Research Archive Show summary

  Epithelial ovarian carcinoma (EOC) is known for high mortality due to diagnosis at advanced stages and frequent therapy resistance. Previous findings suggested that the DNA repair system is involved in the therapeutic response of cancer patients and DNA repair genes are promising targets for novel therapies. This study aimed to address complex inter-relations among gene expression levels, methylation profiles, and somatic mutations in DNA repair genes and EOC prognosis and therapy resistance status. We found significant associations of DUT expression with the presence of peritoneal metastases in EOC patients. The high-grade serous EOC subtype was enriched with TP53 mutations compared to other subtypes. Furthermore, somatic mutations in XPC and PRKDC were significantly associated with worse overall survival of EOC patients, and higher FAAP20 expression in platinum-resistant than platinum-sensitive patients was observed. We found higher methylation of RAD50 in platinum-resistant than in platinum-sensitive patients. Somatic mutations in BRCA1 and RAD9A were significantly associated with higher RBBP8 methylation in platinum-sensitive compared to platinum-resistant EOC patients. In conclusion, we discovered associations of several candidate genes from the DNA repair pathway with the prognosis and platinum resistance status of EOC patients, which deserve further validation as potential predictive biomarkers.

• Dorling, Leila; Carvalho, Sara; Allen, Jamie; Parsons, Michael T.; Fortuno, Cristina & Kristensen, Vessela N. [Show all 50 contributors for this article] (2022). Breast cancer risks associated with missense variants in breast cancer susceptibility genes. Genome Medicine. 14(1), p. 1–17. doi: 10.1186/s13073-022-01052-8. Full text in Research Archive Show summary

  Background: Protein truncating variants in ATM, BRCA1, BRCA2, CHEK2, and PALB2 are associated with increased breast cancer risk, but risks associated with missense variants in these genes are uncertain. Methods: We analyzed data on 59,639 breast cancer cases and 53,165 controls from studies participating in the Breast Cancer Association Consortium BRIDGES project. We sampled training (80%) and validation (20%) sets to ana‑ lyze rare missense variants in ATM (1146 training variants), BRCA1 (644), BRCA2 (1425), CHEK2 (325), and PALB2 (472). We evaluated breast cancer risks according to fve in silico prediction-of-deleteriousness algorithms, functional protein domain, and frequency, using logistic regression models and also mixture models in which a subset of variants was assumed to be risk-associated. Results: The most predictive in silico algorithms were Helix (BRCA1, BRCA2 and CHEK2) and CADD (ATM). Increased risks appeared restricted to functional protein domains for ATM (FAT and PIK domains) and BRCA1 (RING and BRCT domains). For ATM, BRCA1, and BRCA2, data were compatible with small subsets (approximately 7%, 2%, and 0.6%, respectively) of rare missense variants giving similar risk to those of protein truncating variants in the same gene. For CHEK2, data were more consistent with a large fraction (approximately 60%) of rare missense variants giving a lower risk (OR 1.75, 95% CI (1.47–2.08)) than CHEK2 protein truncating variants. There was little evidence for an association with risk for missense variants in PALB2. The best ftting models were well calibrated in the validation set. Conclusions: These results will inform risk prediction models and the selection of candidate variants for functional assays and could contribute to the clinical reporting of gene panel testing for breast cancer susceptibility

• Pedersen, Christine Aaserød; Cao, Maria Dung; Fleischer, Thomas; Rye, Morten Beck; Knappskog, Stian & Eikesdal, Hans Petter [Show all 12 contributors for this article] (2022). DNA methylation changes in response to neoadjuvant chemotherapy are associated with breast cancer survival. Breast Cancer Research. ISSN 1465-5411. 24:43(1), p. 1–14. doi: 10.1186/s13058-022-01537-9. Full text in Research Archive Show summary

  Background Locally advanced breast cancer is a heterogeneous disease with respect to response to neoadjuvant chemotherapy (NACT) and survival. It is currently not possible to accurately predict who will benefit from the specific types of NACT. DNA methylation is an epigenetic mechanism known to play an important role in regulating gene expression and may serve as a biomarker for treatment response and survival. We investigated the potential role of DNA methylation as a prognostic marker for long-term survival (> 5 years) after NACT in breast cancer. Methods DNA methylation profiles of pre-treatment (n = 55) and post-treatment (n = 75) biopsies from 83 women with locally advanced breast cancer were investigated using the Illumina HumanMethylation450 BeadChip. The patients received neoadjuvant treatment with epirubicin and/or paclitaxel. Linear mixed models were used to associate DNA methylation to treatment response and survival based on clinical response to NACT (partial response or stable disease) and 5-year survival, respectively. LASSO regression was performed to identify a risk score based on the statistically significant methylation sites and Kaplan–Meier curve analysis was used to estimate survival probabilities using ten years of survival follow-up data. The risk score developed in our discovery cohort was validated in an independent validation cohort consisting of paired pre-treatment and post-treatment biopsies from 85 women with locally advanced breast cancer. Patients included in the validation cohort were treated with either doxorubicin or 5-FU and mitomycin NACT. Results DNA methylation patterns changed from before to after NACT in 5-year survivors, while no significant changes were observed in non-survivors or related to treatment response. DNA methylation changes included an overall loss of methylation at CpG islands and gain of methylation in non-CpG islands, and these changes affected genes linked to transcription factor activity, cell adhesion and immune functions. A risk score was developed based on four methylation sites which successfully predicted long-term survival in our cohort (p = 0.0034) and in an independent validation cohort (p = 0.049). Conclusion Our results demonstrate that DNA methylation patterns in breast tumors change in response to NACT. These changes in DNA methylation show potential as prognostic biomarkers for breast cancer survival.

• Bjørklund, Sunniva; Aure, Miriam Ragle; Häkkinen, Jari; Vallon-Christersson, Johan; Kumar, Surendra & Evensen, Katrine Bull [Show all 34 contributors for this article] (2022). Subtype and cell type specific expression of lncRNAs provide insight into breast cancer. Communications Biology. 5(1), p. 1–14. doi: 10.1038/s42003-022-03559-7. Full text in Research Archive Show summary

  Abstract Long non-coding RNAs (lncRNAs) are involved in breast cancer pathogenesis through chromatin remodeling, transcriptional and post-transcriptional gene regulation. We report robust associations between lncRNA expression and breast cancer clinicopathological features in two population-based cohorts: SCAN-B and TCGA. Using co-expression analysis of lncRNAs with protein coding genes, we discovered three distinct clusters of lncRNAs. In silico cell type deconvolution coupled with single-cell RNA-seq analyses revealed that these three clusters were driven by cell type specific expression of lncRNAs. In one cluster lncRNAs were expressed by cancer cells and were mostly associated with the estrogen signaling pathways. In the two other clusters, lncRNAs were expressed either by immune cells or fibroblasts of the tumor microenvironment. To further investigate the cis-regulatory regions driving lncRNA expression in breast cancer, we identified subtype-specific transcription factor (TF) occupancy at lncRNA promoters. We also integrated lncRNA expression with DNA methylation data to identify long-range regulatory regions for lncRNA which were validated using ChiA-Pet-Pol2 loops. lncRNAs play an important role in shaping the gene regulatory landscape in breast cancer. We provide a detailed subtype and cell type-specific expression of lncRNA, which improves the understanding of underlying transcriptional regulation in breast cancer.

• Lirussi, Lisa; Ayyildiz, Dilara; Liu, Yan; Montaldo, Nicola Pietro; Huroz, Sergio Carracedo & Aure, Miriam Ragle [Show all 16 contributors for this article] (2022). A regulatory network comprising let-7 miRNA and SMUG1 is associated with good prognosis in ER+ breast tumours. Nucleic Acids Research (NAR). ISSN 0305-1048. 50(18), p. 10449–10468. doi: 10.1093/nar/gkac807. Full text in Research Archive Show summary

  Abstract Single-strand selective uracil–DNA glycosylase 1 (SMUG1) initiates base excision repair (BER) of uracil and oxidized pyrimidines. SMUG1 status has been associated with cancer risk and therapeutic response in breast carcinomas and other cancer types. However, SMUG1 is a multifunctional protein involved, not only, in BER but also in RNA quality control, and its function in cancer cells is unclear. Here we identify several novel SMUG1 interaction partners that functions in many biological processes relevant for cancer development and treatment response. Based on this, we hypothesized that the dominating function of SMUG1 in cancer might be ascribed to functions other than BER. We define a bad prognosis signature for SMUG1 by mapping out the SMUG1 interaction network and found that high expression of genes in the bad prognosis network correlated with lower survival probability in ER+ breast cancer. Interestingly, we identified hsa-let-7b-5p microRNA as an upstream regulator of the SMUG1 interactome. Expression of SMUG1 and hsa-let-7b-5p were negatively correlated in breast cancer and we found an inhibitory auto-regulatory loop between SMUG1 and hsa-let-7b-5p in the MCF7 breast cancer cells. We conclude that SMUG1 functions in a gene regulatory network that influence the survival and treatment response in several cancers.

• Dixon-Suen, Suzanne C.; Lewis, Sarah; Martin, Richard M.; English, Dallas R.; Boyle, Terry & Giles, Graham G. [Show all 174 contributors for this article] (2022). Physical activity, sedentary time and breast cancer risk: a Mendelian randomisation study. British Journal of Sports Medicine. ISSN 0306-3674. 56(20), p. 1157–1170. doi: 10.1136/bjsports-2021-105132. Full text in Research Archive Show summary

  Objectives Physical inactivity and sedentary behaviour are associated with higher breast cancer risk in observational studies, but ascribing causality is difficult. Mendelian randomisation (MR) assesses causality by simulating randomised trial groups using genotype. We assessed whether lifelong physical activity or sedentary time, assessed using genotype, may be causally associated with breast cancer risk overall, pre/post-menopause, and by case-groups defined by tumour characteristics. Methods We performed two-sample inverse-variance-weighted MR using individual-level Breast Cancer Association Consortium case-control data from 130 957 European-ancestry women (69 838 invasive cases), and published UK Biobank data (n=91 105–377 234). Genetic instruments were single nucleotide polymorphisms (SNPs) associated in UK Biobank with wrist-worn accelerometer-measured overall physical activity (nsnps=5) or sedentary time (nsnps=6), or accelerometer-measured (nsnps=1) or self-reported (nsnps=5) vigorous physical activity. Results Greater genetically-predicted overall activity was associated with lower breast cancer overall risk (OR=0.59; 95% confidence interval (CI) 0.42 to 0.83 per-standard deviation (SD;~8 milligravities acceleration)) and for most case-groups. Genetically-predicted vigorous activity was associated with lower risk of pre/perimenopausal breast cancer (OR=0.62; 95% CI 0.45 to 0.87,≥3 vs. 0 self-reported days/week), with consistent estimates for most case-groups. Greater genetically-predicted sedentary time was associated with higher hormone-receptor-negative tumour risk (OR=1.77; 95% CI 1.07 to 2.92 per-SD (~7% time spent sedentary)), with elevated estimates for most case-groups. Results were robust to sensitivity analyses examining pleiotropy (including weighted-median-MR, MR-Egger). Conclusion Our study provides strong evidence that greater overall physical activity, greater vigorous activity, and lower sedentary time are likely to reduce breast cancer risk. More widespread adoption of active lifestyles may reduce the burden from the most common cancer in women.

• Tahiri, Andliena; Puco, Katarina; Naji, Faris; Kristensen, Vessela N.; Alfsen, G. Cecilie & Farkas, Lorant [Show all 10 contributors for this article] (2022). Kinase activity profiling in renal cell carcinoma, benign renal tissue and in response to four different tyrosine kinase inhibitors. OncoTarget. 13(1), p. 970–981. doi: 10.18632/oncotarget.28257. Full text in Research Archive Show summary

  Kinase activity is frequently altered in renal cell carcinoma (RCC), and tyrosine kinase inhibitors (TKIs) are part of the standard treatment strategy in patients with metastatic disease. However, there are still no established biomarkers to predict clinical benefits of a specific TKI. Here, we performed protein tyrosine kinase (PTK) profiling using PamChip® technology. The aim of this study was to identify differences in PTK activity between normal and malignant kidney tissue obtained from the same patient, and to investigate the inhibitory effects of TKIs frequently used in the clinics: sunitinib, pazopanib, cabozantinib and tivozanib. Briefly, our results showed that 36 kinase substrates differs (FDR < 0.05) between normal and cancer kidney tissue, where members of the Src family kinases and the phosphoinositide-3-kinase (PI3K) pathway exhibit high activity in renal cancer. Furthermore, ex vivo treatment of clear cell RCC with TKIs revealed that pathways such as Rap1, Ras and PI3K pathways were strongly inhibited, whereas the neurotrophin pathway had increased activity upon TKI addition. In our assay, tivozanib and cabozantinib exhibited greater inhibitory effects on PTK activity compared to sunitinib and pazopanib, implying they might be better suitable as TKIs for selected RCC patients.

• Dennis, Joe; Tyrer, Jonathan P.; Walker, Logan C.; Michailidou, Kyriaki; Dorling, Leila & Kristensen, Vessela N. [Show all 50 contributors for this article] (2022). Rare germline copy number variants (CNVs) and breast cancer risk. Communications Biology. 5, p. 1–15. doi: 10.1038/s42003-021-02990-6. Full text in Research Archive Show summary

  Germline copy number variants (CNVs) are pervasive in the human genome but potential disease associations with rare CNVs have not been comprehensively assessed in large datasets. We analysed rare CNVs in genes and non-coding regions for 86,788 breast cancer cases and 76,122 controls of European ancestry with genome-wide array data. Gene burden tests detected the strongest association for deletions in BRCA1 (P = 3.7E−18). Nine other genes were associated with a p-value < 0.01 including known susceptibility genes CHEK2 (P = 0.0008), ATM (P = 0.002) and BRCA2 (P = 0.008). Outside the known genes we detected associations with p-values < 0.001 for either overall or subtype-specific breast cancer at nine deletion regions and four duplication regions. Three of the deletion regions were in established common susceptibility loci. To the best of our knowledge, this is the first genome-wide analysis of rare CNVs in a large breast cancer case-control dataset. We detected associations with exonic deletions in established breast cancer susceptibility genes. We also detected suggestive associations with non-coding CNVs in known and novel loci with large effects sizes. Larger sample sizes will be required to reach robust levels of statistical significance.

• Vitelli, Valeria; Fleischer, Thomas; Ankill, Jørgen; Arjas, Elja; Frigessi, Arnoldo & Kristensen, Vessela N. [Show all 7 contributors for this article] (2022). Transcriptomic pan-cancer analysis using rank-based Bayesian inference. Molecular Oncology. ISSN 1574-7891. 00(0), p. 1–16. doi: 10.1002/1878-0261.13354. Full text in Research Archive Show summary

  The analysis of whole genomes of pan-cancer data sets provides a challenge for researchers, and we contribute to the literature concerning the identification of robust subgroups with clear biological interpretation. Specifically, we tackle this unsupervised problem via a novel rank-based Bayesian clustering method. The advantages of our method are the integration and quantification of all uncertainties related to both the input data and the model, the probabilistic interpretation of final results to allow straightforward assessment of the stability of clusters leading to reliable conclusions, and the transparent biological interpretation of the identified clusters since each cluster is characterized by its top-ranked genomic features. We applied our method to RNA-seq data from cancer samples from 12 tumor types from the Cancer Genome Atlas. We identified a robust clustering that mostly reflects tissue of origin but also includes pan-cancer clusters. Importantly, we identified three pan-squamous clusters composed of a mix of lung squamous cell carcinoma, head and neck squamous carcinoma, and bladder cancer, with different biological functions over-represented in the top genes that characterize the three clusters. We also found two novel subtypes of kidney cancer that show different prognosis, and we reproduced known subtypes of breast cancer. Taken together, our method allows the identification of robust and biologically meaningful clusters of pan-cancer samples.

• Mo, Torgeir; Brandal, Siri Helene Bertelsen; Luque, Alvaro Kohn; Engebråten, Olav; Kristensen, Vessela N. & Fleischer, Thomas [Show all 8 contributors for this article] (2022). Quantification of Tumor Hypoxia through Unsupervised Modelling of Consumption and Supply Hypoxia MR Imaging in Breast Cancer. Cancers. 14(5), p. 1–16. doi: 10.3390/cancers14051326. Full text in Research Archive Show summary

  Simple Summary Hypoxia in solid tumors is common in most solid cancers and is associated with treatment resistance to both chemo- and radiation-therapy. There is also reason to believe that hypoxia is an important determinant of metastic disease. Identifying hypoxia in solid tumors is important in treatment planning and decision making. In 2018 Hompland et al. proposed a method, based on quantifying consumption and supply of oxygen from diffusion weighted magnetic resonance imaging, to estimate the hypoxic fraction of a solid tumor. The method was based on training model parameters on a known hypoxia state in prostate cancer. In the present study we verified the validity of the consumption and supply concept in breast cancer. Furthermore, we developed and validated a new approach to the concept that does not require a ground truth to train the parameters. Abstract The purpose of the present study is to investigate if consumption and supply hypoxia (CSH) MR-imaging can depict breast cancer hypoxia, using the CSH-method initially developed for prostate cancer. Furthermore, to develop a generalized pan-cancer application of the CSH-method that doesn’t require a hypoxia reference standard for training the CSH-parameters. In a cohort of 69 breast cancer patients, we generated, based on the principles of intravoxel incoherent motion modelling, images reflecting cellular density (apparent diffusion coefficient; ADC) and vascular density (perfusion fraction; fp). Combinations of the information in these images were compared to a molecular hypoxia score made from gene expression data, aiming to identify a way to apply the CSH-methodology in breast cancer. Attempts to adapt previously proposed models for prostate cancer included direct transfers and model parameter rescaling. A novel approach, based on rescaling ADC and fp data to give more nuanced response in the relevant physiologic range, was also introduced. The new CSH-method was validated in a prostate cancer cohort with known hypoxia status. The proposed CSH-method gave estimates of hypoxia that was strongly correlated to the molecular hypoxia score in breast cancer, and hypoxia as measured in pathology slices stained with pimonidazole in prostate cancer. The generalized approach to CSH-imaging depicted hypoxia in both breast and prostate cancers and requires no model training. It is easy to implement using readily available technology and encourages further investigation of CSH-imaging in other cancer entities and in other settings, with the goal being to overcome hypoxia-induced resistance to treatment. Keywords: hypoxia; magnetic resonance imaging; diffusion weighted imaging; CSH

• Lemma, Roza Berhanu; Fleischer, Thomas; Martinsen, Emily; Ledsaak, Marit; Kristensen, Vessela N. & Eskeland, Ragnhild [Show all 8 contributors for this article] (2022). Pioneer transcription factors are associated with the modulation of DNA methylation patterns across cancers. Epigenetics & Chromatin. 15. doi: 10.1186/s13072-022-00444-9. Full text in Research Archive Show summary

  Methylation of cytosines on DNA is a prominent modification associated with gene expression regulation. Aberrant DNA methylation patterns have recurrently been linked to dysregulation of the regulatory program in cancer cells. To shed light on the underlying molecular mechanism driving this process, we hypothesised that aberrant methylation patterns could be controlled by the binding of specific transcription factors (TFs) across cancer types. By combining DNA methylation arrays and gene expression data with TF binding sites (TFBSs), we explored the interplay between TF binding and DNA methylation in 19 cancer types. We performed emQTL (expression–methylation quantitative trait loci) analyses independently in each cancer type and identified 13 TFs whose expression levels are correlated with local DNA methylation patterns around their binding sites in at least 2 cancer types. The 13 TFs are mainly associated with local demethylation and are enriched for pioneer function, suggesting a specific role for these TFs in modulating chromatin structure and transcription in cancer patients. Furthermore, we confirmed that de novo methylation is precluded across cancers at CpGs lying in genomic regions enriched for TF binding signatures associated with SP1, CTCF, NRF1, GABPA, KLF9, and/or YY1. The modulation of DNA methylation associated with TF binding was observed at cis-regulatory regions controlling immune- and cancer-associated pathways, corroborating that the emQTL signals were derived from both cancer and tumor-infiltrating cells. As a case example, we experimentally confirmed that FOXA1 knock-down is associated with higher methylation in regions bound by FOXA1 in breast cancer MCF-7 cells. Finally, we reported physical interactions between FOXA1 with TET1 and TET2 both in an in vitro setup and in vivo at physiological levels in MCF-7 cells, adding further support for FOXA1 attracting TET1 and TET2 to induce local demethylation in cancer cells.

• Ahearn, Thomas U.; Zhang, Haoyu; Michailidou, Kyriaki; Milne, Roger L.; Bolla, Manjeet K. & Dennis, Joe [Show all 205 contributors for this article] (2022). Common variants in breast cancer risk loci predispose to distinct tumor subtypes. Breast Cancer Research. ISSN 1465-5411. 24(1), p. 1–13. doi: 10.1186/s13058-021-01484-x. Full text in Research Archive Show summary

  Background Genome-wide association studies (GWAS) have identified multiple common breast cancer susceptibility variants. Many of these variants have differential associations by estrogen receptor (ER) status, but how these variants relate with other tumor features and intrinsic molecular subtypes is unclear. Methods Among 106,571 invasive breast cancer cases and 95,762 controls of European ancestry with data on 173 breast cancer variants identified in previous GWAS, we used novel two-stage polytomous logistic regression models to evaluate variants in relation to multiple tumor features (ER, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and grade) adjusting for each other, and to intrinsic-like subtypes. Results Eighty-five of 173 variants were associated with at least one tumor feature (false discovery rate < 5%), most commonly ER and grade, followed by PR and HER2. Models for intrinsic-like subtypes found nearly all of these variants (83 of 85) associated at p < 0.05 with risk for at least one luminal-like subtype, and approximately half (41 of 85) of the variants were associated with risk of at least one non-luminal subtype, including 32 variants associated with triple-negative (TN) disease. Ten variants were associated with risk of all subtypes in different magnitude. Five variants were associated with risk of luminal A-like and TN subtypes in opposite directions. Conclusion This report demonstrates a high level of complexity in the etiology heterogeneity of breast cancer susceptibility variants and can inform investigations of subtype-specific risk prediction.

• Grootes, Isabelle; Keeman, Renske; Blows, Fiona M.; Milne, Roger L.; Giles, Graham G. & Swerdlow, Anthony J. [Show all 55 contributors for this article] (2022). Incorporating progesterone receptor expression into the PREDICT breast prognostic model. European Journal of Cancer. ISSN 0959-8049. 173, p. 178–193. doi: 10.1016/j.ejca.2022.06.011. Full text in Research Archive Show summary

  Background Predict Breast (www.predict.nhs.uk) is an online prognostication and treatment benefit tool for early invasive breast cancer. The aim of this study was to incorporate the prognostic effect of progesterone receptor (PR) status into a new version of PREDICT and to compare its performance to the current version (2.2). Method The prognostic effect of PR status was based on the analysis of data from 45,088 European patients with breast cancer from 49 studies in the Breast Cancer Association Consortium. Cox proportional hazard models were used to estimate the hazard ratio for PR status. Data from a New Zealand study of 11,365 patients with early invasive breast cancer were used for external validation. Model calibration and discrimination were used to test the model performance. Results Having a PR-positive tumour was associated with a 23% and 28% lower risk of dying from breast cancer for women with oestrogen receptor (ER)-negative and ER-positive breast cancer, respectively. The area under the ROC curve increased with the addition of PR status from 0.807 to 0.809 for patients with ER-negative tumours (p = 0.023) and from 0.898 to 0.902 for patients with ER-positive tumours (p = 2.3 × 10−6) in the New Zealand cohort. Model calibration was modest with 940 observed deaths compared to 1151 predicted. Conclusion The inclusion of the prognostic effect of PR status to PREDICT Breast has led to an improvement of model performance and more accurate absolute treatment benefit predictions for individual patients. Further studies should determine whether the baseline hazard function requires recalibration.

• Normann, Lisa Svartdal; Haugen, Mads Haugland; Aure, Miriam Ragle; Kristensen, Vessela N.; Mælandsmo, Gunhild Mari & Sahlberg, Kristine Kleivi (2022). miR-101-5p Acts as a Tumor Suppressor in HER2-Positive Breast Cancer Cells and Improves Targeted Therapy. Breast Cancer: Targets and Therapy. 14, p. 25–39. doi: 10.2147/BCTT.S338404. Full text in Research Archive Show summary

  Purpose: Human epidermal growth factor receptor 2 positive (HER2+) breast cancers responding poorly to targeted therapy need improved treatment options. miR-101-5p has shown tumor-suppressive properties in multiple cancer forms, and we assessed the effect and mechanism of action of this miRNA in HER2+ breast cancer. Methods: Expression levels of miR-101-5p in two clinical datasets, TCGA and METABRIC, were compared between tumor and normal adjacent samples, and across molecular subtypes and HER2 status. The ability of miR-101-5p to sensitize for treatment with lapatinib, tucatinib and trastuzumab was explored in HER2+ breast cancer cells responding poorly to such targeted drugs. Proliferation and apoptosis assays and downstream protein analysis were performed. Results: Expression levels of miR-101-5p were significantly lower in tumor compared to normal adjacent tissue (p < 0.001), and particularly low in HER2+ tumors, both the HER2-enriched subtype (p ≤ 0.037) and clinical HER2-status (p < 0.001). In a HER2+ cell line (KPL4) responding poorly to targeted drugs, miR-101-5p overexpression inhibited proliferation (p < 0.001), and combinatorial treatment with lapatinib and trastuzumab significantly further decreased this inhibition (p = 0.004). Proteomic data and in silico analyses revealed PI3K/Akt- and HER2-pathways among the predicted regulated pathways. miR-101-5p alone (p = 0.018) and in combination with lapatinib and trastuzumab (p < 0.001) induced apoptosis, while the targeted drugs alone did not exert any significant effect neither on proliferation nor apoptosis. Conclusion: miR-101-5p acts as a tumor suppressor by inducing apoptosis in HER2+ breast cancer and sensitizes cells with initially poor response to lapatinib and trastuzumab.

• Mondragon, Jaime Abraham Castro; Aure, Miriam Ragle; Lingjærde, Ole Christian; Langerød, Anita; Martens, John WM & Børresen-Dale, Anne-Lise [Show all 8 contributors for this article] (2022). Cis-regulatory mutations associate with transcriptional and post-transcriptional deregulation of gene regulatory programs in cancers. Nucleic Acids Research (NAR). ISSN 0305-1048. 50(21), p. 12131–12148. doi: 10.1093/nar/gkac1143. Full text in Research Archive Show summary

  Abstract Most cancer alterations occur in the noncoding portion of the human genome, where regulatory regions control gene expression. The discovery of noncoding mutations altering the cells’ regulatory programs has been limited to few examples with high recurrence or high functional impact. Here, we show that transcription factor binding sites (TFBSs) have similar mutation loads to those in protein-coding exons. By combining cancer somatic mutations in TFBSs and expression data for protein-coding and miRNA genes, we evaluate the combined effects of transcriptional and post-transcriptional alterations on the regulatory programs in cancers. The analysis of seven TCGA cohorts culminates with the identification of protein-coding and miRNA genes linked to mutations at TFBSs that are associated with a cascading trans-effect deregulation on the cells’ regulatory programs. Our analyses of cis-regulatory mutations associated with miRNAs recurrently predict 12 mature miRNAs (derived from 7 precursors) associated with the deregulation of their target gene networks. The predictions are enriched for cancer-associated protein-coding and miRNA genes and highlight cis-regulatory mutations associated with the dysregulation of key pathways associated with carcinogenesis. By combining transcriptional and post-transcriptional regulation of gene expression, our method predicts cis-regulatory mutations related to the dysregulation of key gene regulatory networks in cancer patients.

• Mavaddat, Nasim; Dorling, Leila; Carvalho, Sara; Allen, Jamie; González-Neira, Anna & Keeman, Renske [Show all 94 contributors for this article] (2022). Pathology of tumors associated with pathogenic germline variants in 9 breast cancer susceptibility genes. JAMA Oncology. ISSN 2374-2437. 8(3). doi: 10.1001/jamaoncol.2021.6744. Full text in Research Archive Show summary

  IMPORTANCE Rare germline genetic variants in several genes are associated with increased breast cancer (BC) risk, but their precise contributions to different disease subtypes are unclear. This information is relevant to guidelines for gene panel testing and risk prediction. OBJECTIVE To characterize tumors associated with BC susceptibility genes in large-scale population- or hospital-based studies. DESIGN, SETTING, AND PARTICIPANTS The multicenter, international case-control analysis of the BRIDGES study included 42 680 patients and 46 387 control participants, comprising women aged 18 to 79 years who were sampled independently of family history from 38 studies. Studies were conducted between 1991 and 2016. Sequencing and analysis took place between 2016 and 2021. EXPOSURES Protein-truncating variants and likely pathogenic missense variants in ATM, BARD1, BRCA1, BRCA2, CHEK2, PALB2, RAD51C, RAD51D, and TP53. MAIN OUTCOMES AND MEASURES The intrinsic-like BC subtypes as defined by estrogen receptor, progesterone receptor, and ERBB2 (formerly known as HER2) status, and tumor grade; morphology; size; stage; lymph node involvement; subtype-specific odds ratios (ORs) for carrying protein-truncating variants and pathogenic missense variants in the 9 BC susceptibility genes. RESULTS The mean (SD) ages at interview (control participants) and diagnosis (cases) were 55.1 (11.9) and 55.8 (10.6) years, respectively; all participants were of European or East Asian ethnicity. There was substantial heterogeneity in the distribution of intrinsic subtypes by gene. RAD51C, RAD51D, and BARD1 variants were associated mainly with triple-negative disease (OR, 6.19 [95% CI, 3.17-12.12]; OR, 6.19 [95% CI, 2.99-12.79]; and OR, 10.05 [95% CI, 5.27-19.19], respectively). CHEK2 variants were associated with all subtypes (with ORs ranging from 2.21-3.17) except for triple-negative disease. For ATM variants, the association was strongest for the hormone receptor (HR)+ ERBB2− high-grade subtype (OR, 4.99; 95% CI, 3.68-6.76). BRCA1 was associated with increased risk of all subtypes, but the ORs varied widely, being highest for triple-negative disease (OR, 55.32; 95% CI, 40.51-75.55). BRCA2 and PALB2 variants were also associated with triple-negative disease. TP53 variants were most strongly associated with HR+ ERBB2+ and HR– ERBB2+ subtypes. Tumors occurring in pathogenic variant carriers were of higher grade. For most genes and subtypes, a decline in ORs was observed with increasing age. Together, the 9 genes were associated with 27.3% of all triple-negative tumors in women 40 years or younger. CONCLUSIONS AND RELEVANCE The results of this case-control study suggest that variants in the 9 BC risk genes differ substantially in their associated pathology but are generally associated with triple-negative and/or high-grade disease. Knowing the age and tumor subtype distributions associated with individual BC genes can potentially aid guidelines for gene panel testing, risk prediction, and variant classification and guide targeted screening strategies.

• Chen, Hongjie; Fan, Shaoqi; Stone, Jennifer; Thompson, Deborah J.; Douglas, Julie & Kristensen, Vessela N. [Show all 56 contributors for this article] (2022). Genome-wide and transcriptome-wide association studies of mammographic density phenotypes reveal novel loci. Breast Cancer Research. ISSN 1465-5411. 24:27(1), p. 1–15. doi: 10.1186/s13058-022-01524-0. Full text in Research Archive Show summary

  Background: Mammographic density (MD) phenotypes, including percent density (PMD), area of dense tissue (DA), and area of non-dense tissue (NDA), are associated with breast cancer risk. Twin studies suggest that MD phenotypes are highly heritable. However, only a small proportion of their variance is explained by identifed genetic variants. Methods: We conducted a genome-wide association study, as well as a transcriptome-wide association study (TWAS), of age- and BMI-adjusted DA, NDA, and PMD in up to 27,900 European-ancestry women from the MODE/ BCAC consortia. Results: We identifed 28 genome-wide signifcant loci for MD phenotypes, including nine novel signals (5q11.2, 5q14.1, 5q31.1, 5q33.3, 5q35.1, 7p11.2, 8q24.13, 12p11.2, 16q12.2). Further, 45% of all known breast cancer SNPs were associated with at least one MD phenotype at p<0.05. TWAS further identifed two novel genes (SHOX2 and CRISPLD2) whose genetically predicted expression was signifcantly associated with MD phenotypes. Conclusions: Our fndings provided novel insight into the genetic background of MD phenotypes, and further demonstrated their shared genetic basis with breast cancer. Keywords: Mammographic density, Breast cancer, Genome-wide association study (GWAS), Transcriptome-wide association study (TWAS)

• Escala-Garcia, Maria; Canisius, Sander; Keeman, Renske; Beesley, Jonathan; Anton-Culver, Hoda & Arndt, Volker [Show all 105 contributors for this article] (2021). Germline variants and breast cancer survival in patients with distant metastases at primary breast cancer diagnosis. Scientific Reports. 11:19787, p. 1–11. doi: 10.1038/s41598-021-99409-3. Full text in Research Archive Show summary

  Breast cancer metastasis accounts for most of the deaths from breast cancer. Identifcation of germline variants associated with survival in aggressive types of breast cancer may inform understanding of breast cancer progression and assist treatment. In this analysis, we studied the associations between germline variants and breast cancer survival for patients with distant metastases at primary breast cancer diagnosis. We used data from the Breast Cancer Association Consortium (BCAC) including 1062 women of European ancestry with metastatic breast cancer, 606 of whom died of breast cancer. We identifed two germline variants on chromosome 1, rs138569520 and rs146023652, signifcantly associated with breast cancer-specifc survival (P= 3.19 × ­10−8 and 4.42 × ­10−8). In silico analysis suggested a potential regulatory efect of the variants on the nearby target genes SDE2 and H3F3A. However, the variants showed no evidence of association in a smaller replication dataset. The validation dataset was obtained from the SNPs to Risk of Metastasis (StoRM) study and included 293 patients with metastatic primary breast cancer at diagnosis. Ultimately, larger replication studies are needed to confrm the identifed associations.

• Morra, Anna; Escala-Garcia, Maria; Beesley, Jonathan; Keeman, Renske; Canisius, Sander & Ahearn, Thomas U. [Show all 50 contributors for this article] (2021). Association of germline genetic variants with breast cancer-specific survival in patient subgroups defined by clinic-pathological variables related to tumor biology and type of systemic treatment. Breast Cancer Research. ISSN 1465-5411. 23:86(1), p. 1–18. doi: 10.1186/s13058-021-01450-7. Full text in Research Archive Show summary

  Background Given the high heterogeneity among breast tumors, associations between common germline genetic variants and survival that may exist within specific subgroups could go undetected in an unstratified set of breast cancer patients. Methods We performed genome-wide association analyses within 15 subgroups of breast cancer patients based on prognostic factors, including hormone receptors, tumor grade, age, and type of systemic treatment. Analyses were based on 91,686 female patients of European ancestry from the Breast Cancer Association Consortium, including 7531 breast cancer-specific deaths over a median follow-up of 8.1 years. Cox regression was used to assess associations of common germline variants with 15-year and 5-year breast cancer-specific survival. We assessed the probability of these associations being true positives via the Bayesian false discovery probability (BFDP < 0.15). Results Evidence of associations with breast cancer-specific survival was observed in three patient subgroups, with variant rs5934618 in patients with grade 3 tumors (15-year-hazard ratio (HR) [95% confidence interval (CI)] 1.32 [1.20, 1.45], P = 1.4E−08, BFDP = 0.01, per G allele); variant rs4679741 in patients with ER-positive tumors treated with endocrine therapy (15-year-HR [95% CI] 1.18 [1.11, 1.26], P = 1.6E−07, BFDP = 0.09, per G allele); variants rs1106333 (15-year-HR [95% CI] 1.68 [1.39,2.03], P = 5.6E−08, BFDP = 0.12, per A allele) and rs78754389 (5-year-HR [95% CI] 1.79 [1.46,2.20], P = 1.7E−08, BFDP = 0.07, per A allele), in patients with ER-negative tumors treated with chemotherapy. Conclusions We found evidence of four loci associated with breast cancer-specific survival within three patient subgroups. There was limited evidence for the existence of associations in other patient subgroups. However, the power for many subgroups is limited due to the low number of events. Even so, our results suggest that the impact of common germline genetic variants on breast cancer-specific survival might be limited.

• Morra, Anna; Jung, Audrey Y.; Behrens, Sabine; Keeman, Renske; Ahearn, Thomas U. & Anton-Culver, Hoda [Show all 134 contributors for this article] (2021). Breast cancer risk factors and survival by tumor subtype: Pooled analyses from the breast cancer association consortium. Cancer Epidemiology, Biomarkers and Prevention. ISSN 1055-9965. 30(4), p. 623–642. doi: 10.1158/1055-9965.EPI-20-0924. Full text in Research Archive
• Dorling, Leila; Carvalho, Sara; Allen, Jamie; González-Neira, Anna; Luccarini, Craig & Wahlström, Cecilia [Show all 176 contributors for this article] (2021). Breast cancer risk genes - Association analysis in more than 113,000 women. New England Journal of Medicine. ISSN 0028-4793. 384(5), p. 428–439. doi: 10.1056/NEJMoa1913948. Full text in Research Archive
• Park, Hanla A.; Neumeyer, Sonja; Michailidou, Kyriaki; Bolla, Manjeet K.; Wang, Qin & Kristensen, Vessela N. [Show all 50 contributors for this article] (2021). Mendelian randomisation study of smoking exposure in relation to breast cancer risk. British Journal of Cancer. ISSN 0007-0920. 125(8), p. 1135–1145. doi: 10.1038/s41416-021-01432-8. Full text in Research Archive Show summary

  Abstract Background Despite a modest association between tobacco smoking and breast cancer risk reported by recent epidemiological studies, it is still equivocal whether smoking is causally related to breast cancer risk. Methods We applied Mendelian randomisation (MR) to evaluate a potential causal effect of cigarette smoking on breast cancer risk. Both individual-level data as well as summary statistics for 164 single-nucleotide polymorphisms (SNPs) reported in genome-wide association studies of lifetime smoking index (LSI) or cigarette per day (CPD) were used to obtain MR effect estimates. Data from 108,420 invasive breast cancer cases and 87,681 controls were used for the LSI analysis and for the CPD analysis conducted among ever-smokers from 26,147 cancer cases and 26,072 controls. Sensitivity analyses were conducted to address pleiotropy. Results Genetically predicted LSI was associated with increased breast cancer risk (OR 1.18 per SD, 95% CI: 1.07–1.30, P  = 0.11 × 10 –2 ), but there was no evidence of association for genetically predicted CPD (OR 1.02, 95% CI: 0.78–1.19, P  = 0.85). The sensitivity analyses yielded similar results and showed no strong evidence of pleiotropic effect. Conclusion Our MR study provides supportive evidence for a potential causal association with breast cancer risk for lifetime smoking exposure but not cigarettes per day among smokers.

• Baxter, Joseph S.; Johnson, Nichola; Tomczyk, Katarzyna; Gillespie, Andrea; Maguire, Sarah & Brough, Rachel [Show all 183 contributors for this article] (2021). Functional annotation of the 2q35 breast cancer risk locus implicates a structural variant in influencing activity of a long-range enhancer element. American Journal of Human Genetics. ISSN 0002-9297. 108(7), p. 1190–1203. doi: 10.1016/j.ajhg.2021.05.013. Full text in Research Archive Show summary

  A combination of genetic and functional approaches has identified three independent breast cancer risk loci at 2q35. A recent fine-scale mapping analysis to refine these associations resulted in 1 (signal 1), 5 (signal 2), and 42 (signal 3) credible causal variants at these loci. We used publicly available in silico DNase I and ChIP-seq data with in vitro reporter gene and CRISPR assays to annotate signals 2 and 3. We identified putative regulatory elements that enhanced cell-type-specific transcription from the IGFBP5 promoter at both signals (30- to 40-fold increased expression by the putative regulatory element at signal 2, 2- to 3-fold by the putative regulatory element at signal 3). We further identified one of the five credible causal variants at signal 2, a 1.4 kb deletion (esv3594306), as the likely causal variant; the deletion allele of this variant was associated with an average additional increase in IGFBP5 expression of 1.3-fold (MCF-7) and 2.2-fold (T-47D). We propose a model in which the deletion allele of esv3594306 juxtaposes two transcription factor binding regions (annotated by estrogen receptor alpha ChIP-seq peaks) to generate a single extended regulatory element. This regulatory element increases cell-type-specific expression of the tumor suppressor gene IGFBP5 and, thereby, reduces risk of estrogen receptor-positive breast cancer (odds ratio = 0.77, 95% CI 0.74–0.81, p = 3.1 × 10−31).

• Aure, Miriam Ragle; Fleischer, Thomas; Bjørklund, Sunniva; Ankill, Jørgen; Mondragón, Jaime Abraham Castro & Børresen-Dale, Anne-Lise [Show all 11 contributors for this article] (2021). Crosstalk between microRNA expression and DNA methylation drives the hormone-dependent phenotype of breast cancer. Genome Medicine. 13:72, p. 1–21. doi: 10.1186/s13073-021-00880-4. Full text in Research Archive Show summary

  Background Abnormal DNA methylation is observed as an early event in breast carcinogenesis. However, how such alterations arise is still poorly understood. microRNAs (miRNAs) regulate gene expression at the post-transcriptional level and play key roles in various biological processes. Here, we integrate miRNA expression and DNA methylation at CpGs to study how miRNAs may affect the breast cancer methylome and how DNA methylation may regulate miRNA expression. Methods miRNA expression and DNA methylation data from two breast cancer cohorts, Oslo2 (n = 297) and The Cancer Genome Atlas (n = 439), were integrated through a correlation approach that we term miRNA-methylation Quantitative Trait Loci (mimQTL) analysis. Hierarchical clustering was used to identify clusters of miRNAs and CpGs that were further characterized through analysis of mRNA/protein expression, clinicopathological features, in silico deconvolution, chromatin state and accessibility, transcription factor binding, and long-range interaction data. Results Clustering of the significant mimQTLs identified distinct groups of miRNAs and CpGs that reflect important biological processes associated with breast cancer pathogenesis. Notably, two major miRNA clusters were related to immune or fibroblast infiltration, hence identifying miRNAs associated with cells of the tumor microenvironment, while another large cluster was related to estrogen receptor (ER) signaling. Studying the chromatin landscape surrounding CpGs associated with the estrogen signaling cluster, we found that miRNAs from this cluster are likely to be regulated through DNA methylation of enhancers bound by FOXA1, GATA2, and ER-alpha. Further, at the hub of the estrogen cluster, we identified hsa-miR-29c-5p as negatively correlated with the mRNA and protein expression of DNA methyltransferase DNMT3A, a key enzyme regulating DNA methylation. We found deregulation of hsa-miR-29c-5p already present in pre-invasive breast lesions and postulate that hsa-miR-29c-5p may trigger early event abnormal DNA methylation in ER-positive breast cancer. Conclusions We describe how miRNA expression and DNA methylation interact and associate with distinct breast cancer phenotypes.

• Ben-Elazar, Shay; Aure, Miriam Ragle; Jonsdottir, Kristin; Leivonen, Suvi-Katri; Kristensen, Vessela N. & Janssen, Emiel [Show all 9 contributors for this article] (2021). miRNA normalization enables joint analysis of several datasets to increase sensitivity and to reveal novel miRNAs differentially expressed in breast cancer. PLoS Computational Biology. ISSN 1553-734X. 17(2). doi: 10.1371/JOURNAL.PCBI.1008608. Full text in Research Archive Show summary

  Different miRNA profiling protocols and technologies introduce differences in the resulting quantitative expression profiles. These include differences in the presence (and measurability) of certain miRNAs. We present and examine a method based on quantile normalization, Adjusted Quantile Normalization (AQuN), to combine miRNA expression data from multiple studies in breast cancer into a single joint dataset for integrative analysis. By pooling multiple datasets, we obtain increased statistical power, surfacing patterns that do not emerge as statistically significant when separately analyzing these datasets. To merge several datasets, as we do here, one needs to overcome both technical and batch differences between these datasets. We compare several approaches for merging and jointly analyzing miRNA datasets. We investigate the statistical confidence for known results and highlight potential new findings that resulted from the joint analysis using AQuN. In particular, we detect several miRNAs to be differentially expressed in estrogen receptor (ER) positive versus ER negative samples. In addition, we identify new potential biomarkers and therapeutic targets for both clinical groups. As a specific example, using the AQuN-derived dataset we detect hsa-miR-193b-5p to have a statistically significant over-expression in the ER positive group, a phenomenon that was not previously reported. Furthermore, as demonstrated by functional assays in breast cancer cell lines, overexpression of hsa-miR-193b-5p in breast cancer cell lines resulted in decreased cell viability in addition to inducing apoptosis. Together, these observations suggest a novel functional role for this miRNA in breast cancer. Packages implementing AQuN are provided for Python and Matlab: https://github.com/YakhiniGroup/PyAQN.

• Bahrami, Nazli; Jabeen, Shakila; Tahiri, Andliena; Sauer, Torill; Ødegård, Hilde Presterud & Geisler, Stephanie Beate [Show all 11 contributors for this article] (2021). Lack of cross-resistance between non-steroidal and steroidal aromatase inhibitors in breast cancer patients: the potential role of the adipokine leptin. Breast Cancer Research and Treatment. ISSN 0167-6806. 190, p. 435–449. doi: 10.1007/s10549-021-06399-x. Full text in Research Archive Show summary

  Abstract Purpose The aromatase inactivator exemestane may cause clinical disease stabilization following progression on non-steroidal aromatase inhibitors like letrozole in patients with metastatic breast cancer, indicating that additional therapeutic effects, not necessarily related to estrogen-suppression, may be involved in this well-known “lack of cross-resistance”. Methods Postmenopausal women with ER positive, HER-2 negative, locally advanced breast cancer were enrolled in the NEOLETEXE-trial and randomized to sequential treatment starting with either letrozole (2.5 mg o.d.) or exemestane (25 mg o.d.) followed by the alternative aromatase inhibitor. Serum levels of 54 cytokines, including 12 adipokines were assessed using Luminex xMAP technology (multiple ELISA). Results Serum levels of leptin were significantly decreased during treatment with exemestane ( p  &lt; 0.001), regardless whether exemestane was given as first or second neoadjuvant therapy. In contrast, letrozole caused a non-significant increase in serum leptin levels in vivo. Conclusions Our findings suggest an additional and direct effect of exemestane on CYP-19 (aromatase) synthesis presumably due to effects on the CYP19 promoter use that is not present during therapy with the non-steroidal aromatase inhibitor letrozole. Our findings provide new insights into the influence of clinically important aromatase inhibitors on cytokine levels in vivo that contribute to the understanding of the clinically observed lack of cross-resistance between non-steroidal and steroidal aromatase inhibitors in breast cancer patients. Trial registration Registered on March 23rd 2015 in the National trial database of Norway (Registration number: REK-SØ-84-2015).

• Graim, Kiley; Gorenshteyn, Dmitriy; Robinson, David G; Carriero, Nicholas J; Cahill, James A. & Chakrabarti, Rumela [Show all 14 contributors for this article] (2021). Modeling molecular development of breast cancer in canine mammary tumors. Genome Research. ISSN 1088-9051. 31(3), p. 337–347. doi: 10.1101/GR.256388.119. Full text in Research Archive
• Fores-Martos, Jaume; Cervera-Vidal, Raimundo; Sierra-Roca, Julia; Lozano-Asencio, Carlos; Fedele, Vita & Cornelissen, Sten [Show all 19 contributors for this article] (2021). Circadian PERformance in breast cancer: a germline and somatic genetic study of PER3 ^VNTR polymorphisms and gene co-expression. NPJ breast cancer. 7(1). doi: 10.1038/s41523-021-00329-2. Full text in Research Archive
• Rye, Inga Hansine; Huse, Kanutte; Josefsson, Sarah Elisabet; Kildal, Wanja; Danielsen, Håvard Emil Greger & Schlichting, Ellen [Show all 28 contributors for this article] (2021). Breast cancer metastasis: immune profiling of lymph nodes reveals exhaustion of effector T cells and immunosuppression. Molecular Oncology. ISSN 1574-7891. 16(1), p. 88–103. doi: 10.1002/1878-0261.13047. Full text in Research Archive Show summary

  Sentinel lymph nodes are the first nodes draining the lymph from a breast and could reveal early changes in the host immune system upon dissemination of breast cancer cells. To investigate this, we performed single-cell immune profiling of lymph nodes with and without metastatic cells. Whereas no significant changes were observed for B-cell and natural killer (NK)-cell subsets, metastatic lymph nodes had a significantly increased frequency of CD8 T cells and a skewing toward an effector/memory phenotype of CD4 and CD8 T cells, suggesting an ongoing immune response. Additionally, metastatic lymph nodes had an increased frequency of TIGIT (T-cell immunoreceptor with Ig and ITIM domains)-positive T cells with suppressed TCR signaling compared with non-metastatic nodes, indicating exhaustion of effector T cells, and an increased frequency of regulatory T cells (Tregs) with an activated phenotype. T-cell alterations correlated with the percentage of metastatic tumor cells, reflecting the presence of metastatic tumor cells driving T effector cells toward exhaustion and promoting immunosuppression by recruitment or increased differentiation toward Tregs. These results show that immune suppression occurs already in early stages of tumor progression.

• Normann, Lisa Gregusson Svartdal; Aure, Miriam Ragle; Leivonen, Suvi-Katri; Haugen, Mads Haugland; Hongisto, Vesa & Kristensen, Vessela N. [Show all 8 contributors for this article] (2021). MicroRNA in combination with HER2-targeting drugs reduces breast cancer cell viability in vitro. Scientific Reports. 11:10893, p. 1–11. doi: 10.1038/s41598-021-90385-2. Full text in Research Archive Show summary

  HER2-positive (HER2 +) breast cancer patients that do not respond to targeted treatment have a poor prognosis. The effects of targeted treatment on endogenous microRNA (miRNA) expression levels are unclear. We report that responsive HER2 + breast cancer cell lines had a higher number of miRNAs with altered expression after treatment with trastuzumab and lapatinib compared to poorly responsive cell lines. To evaluate whether miRNAs can sensitize HER2 + cells to treatment, we performed a high-throughput screen of 1626 miRNA mimics and inhibitors in combination with trastuzumab and lapatinib in HER2 + breast cancer cells. We identified eight miRNA mimics sensitizing cells to targeted treatment, miR-101-5p, mir-518a-5p, miR-19b-2-5p, miR-1237-3p, miR-29a-3p, miR-29c-3p, miR-106a-5p, and miR-744-3p. A higher expression of miR-101-5p predicted better prognosis in patients with HER2 + breast cancer (OS: p = 0.039; BCSS: p = 0.012), supporting the tumor-suppressing role of this miRNA. In conclusion, we have identified miRNAs that sensitize HER2 + breast cancer cells to targeted therapy. This indicates the potential of combining targeted drugs with miRNAs to improve current treatments for HER2 + breast cancers.

• Galili, Ben; Tekpli, Xavier; Kristensen, Vessela N. & Yakhini, Zohar (2021). Efficient gene expression signature for a breast cancer immuno-subtype. PLOS ONE. 16(1). doi: 10.1371/journal.pone.0245215. Full text in Research Archive
• Johnson, Nichola; Maguire, Sarah; Morra, Anna; Kapoor, Pooja Middha; Tomczyk, Katarzyna & Kristensen, Vessela N. [Show all 50 contributors for this article] (2021). CYP3A7*1C allele: linking premenopausal oestrone and progesterone levels with risk of hormone receptor-positive breast cancers. British Journal of Cancer. ISSN 0007-0920. 124, p. 842–854. doi: 10.1038/s41416-020-01185-w. Full text in Research Archive Show summary

  Abstract Background Epidemiological studies provide strong evidence for a role of endogenous sex hormones in the aetiology of breast cancer. The aim of this analysis was to identify genetic variants that are associated with urinary sex-hormone levels and breast cancer risk. Methods We carried out a genome-wide association study of urinary oestrone-3-glucuronide and pregnanediol-3-glucuronide levels in 560 premenopausal women, with additional analysis of progesterone levels in 298 premenopausal women. To test for the association with breast cancer risk, we carried out follow-up genotyping in 90,916 cases and 89,893 controls from the Breast Cancer Association Consortium. All women were of European ancestry. Results For pregnanediol-3-glucuronide, there were no genome-wide significant associations; for oestrone-3-glucuronide, we identified a single peak mapping to the CYP3A locus, annotated by rs45446698. The minor rs45446698-C allele was associated with lower oestrone-3-glucuronide (−49.2%, 95% CI −56.1% to −41.1%, P  = 3.1 × 10 –18 ); in follow-up analyses, rs45446698-C was also associated with lower progesterone (−26.7%, 95% CI −39.4% to −11.6%, P  = 0.001) and reduced risk of oestrogen and progesterone receptor-positive breast cancer (OR = 0.86, 95% CI 0.82–0.91, P  = 6.9 × 10 –8 ). Conclusions The CYP3A7*1C allele is associated with reduced risk of hormone receptor-positive breast cancer possibly mediated via an effect on the metabolism of endogenous sex hormones in premenopausal women.

• Park, JooYong; Choi, Ji-Yeob; Choi, Jaesung; Chung, Seokang; Song, Nan & Park, Sue K [Show all 50 contributors for this article] (2021). Gene-Environment Interactions Relevant to Estrogen and Risk of Breast Cancer: Can Gene-Environment Interactions Be Detected Only among Candidate SNPs from Genome-Wide Association Studies? Cancers. 13:2370(10), p. 1–21. doi: 10.3390/cancers13102370. Full text in Research Archive Show summary

  In this study we aim to examine gene–environment interactions (GxEs) between genes involved with estrogen metabolism and environmental factors related to estrogen exposure. GxE analyses were conducted with 1970 Korean breast cancer cases and 2052 controls in the case-control study, the Seoul Breast Cancer Study (SEBCS). A total of 11,555 SNPs from the 137 candidate genes were included in the GxE analyses with eight established environmental factors. A replication test was conducted by using an independent population from the Breast Cancer Association Consortium (BCAC), with 62,485 Europeans and 9047 Asians. The GxE tests were performed by using two-step methods in GxEScan software. Two interactions were found in the SEBCS. The first interaction was shown between rs13035764 of NCOA1 and age at menarche in the GE|2df model (p-2df = 1.2 × 10−3). The age at menarche before 14 years old was associated with the high risk of breast cancer, and the risk was higher when subjects had homozygous minor allele G. The second GxE was shown between rs851998 near ESR1 and height in the GE|2df model (p-2df = 1.1 × 10−4). Height taller than 160 cm was associated with a high risk of breast cancer, and the risk increased when the minor allele was added. The findings were not replicated in the BCAC. These results would suggest specificity in Koreans for breast cancer risk.

• Bergholtz, Helga; Kumar, Surendra; Wärnberg, Fredrik; Luders, Torben; Kristensen, Vessela N. & Sørlie, Therese (2020). Comparable cancer-relevant mutation profiles in synchronous ductal carcinoma in situ and invasive breast cancer. Cancer Reports. 3:e1248(3), p. 1–7. doi: 10.1002/cnr2.1248. Full text in Research Archive Show summary

  Background Ductal carcinoma in situ (DCIS) comprises a diverse group of preinvasive lesions in the breast and poses a considerable clinical challenge due to lack of markers of progression. Genomic alterations are to a large extent similar in DCIS and invasive carcinomas, although differences in copy number aberrations, gene expression patterns, and mutations exist. In mixed tumors with synchronous invasive breast cancer (IBC) and DCIS, it is still unclear to what extent invasive tumor cells are directly derived from the DCIS cells. Aim Our aim was to compare cancer‐relevant mutation profiles of different cellular compartments in mixed DCIS/IBC and pure DCIS tumors. Methods and results We performed targeted sequencing of 50 oncogenes in microdissected tissue from three different epithelial cell compartments (in situ, invasive, and normal adjacent epithelium) from 26 mixed breast carcinomas. In total, 44 tissue samples (19 invasive, 16 in situ, 9 normal) were subjected to sequencing using the Ion Torrent platform and the AmpliSeq Cancer Hotspot Panel v2. For comparison, 10 additional, pure DCIS lesions were sequenced. Across all mixed samples, we detected 23 variants previously described in cancer. The most commonly affected genes were TP53, PIK3CA, and ERBB2. The PIK3CA:p.H1047R variant was found in nine samples from six patients. Most variants detected in invasive compartments were also found in the corresponding in situ cell compartment indicating a clonal relationship between the tumor stages. A lower frequency of variants were observed in pure DCIS lesions. Conclusion Similar mutation profiles between in situ and invasive cell compartments indicate a similar origin of the two tumor stages in mixed breast tumors. The lower number of potential driver variants found in pure DCIS compared with the in situ cell compartments of mixed tumors may imply that pure DCIS is captured earlier in the path of progression to invasive disease.

• Levy-Jurgenson, Alona; Tekpli, Xavier; Kristensen, Vessela N. & Yakhini, Zohar (2020). Spatial transcriptomics inferred from pathology whole-slide images links tumor heterogeneity to survival in breast and lung cancer. Scientific Reports. 10:18802, p. 1–11. doi: 10.1038/s41598-020-75708-z. Full text in Research Archive Show summary

  Abstract Digital analysis of pathology whole-slide images is fast becoming a game changer in cancer diagnosis and treatment. Specifically, deep learning methods have shown great potential to support pathology analysis, with recent studies identifying molecular traits that were not previously recognized in pathology H&amp;E whole-slide images. Simultaneous to these developments, it is becoming increasingly evident that tumor heterogeneity is an important determinant of cancer prognosis and susceptibility to treatment, and should therefore play a role in the evolving practices of matching treatment protocols to patients. State of the art diagnostic procedures, however, do not provide automated methods for characterizing and/or quantifying tumor heterogeneity, certainly not in a spatial context. Further, existing methods for analyzing pathology whole-slide images from bulk measurements require many training samples and complex pipelines. Our work addresses these two challenges. First, we train deep learning models to spatially resolve bulk mRNA and miRNA expression levels on pathology whole-slide images (WSIs). Our models reach up to 0.95 AUC on held-out test sets from two cancer cohorts using a simple training pipeline and a small number of training samples. Using the inferred gene expression levels, we further develop a method to spatially characterize tumor heterogeneity. Specifically, we produce tumor molecular cartographies and heterogeneity maps of WSIs and formulate a heterogeneity index (HTI) that quantifies the level of heterogeneity within these maps. Applying our methods to breast and lung cancer slides, we show a significant statistical link between heterogeneity and survival. Our methods potentially open a new and accessible approach to investigating tumor heterogeneity and other spatial molecular properties and their link to clinical characteristics, including treatment susceptibility and survival.

• Egeland, Nina Gran; Jonsdottir, Kristin; Aure, Miriam Ragle; Sahlberg, Kristine Kleivi; Kristensen, Vessela N. & Cronin-Fenton, Deirdre [Show all 10 contributors for this article] (2020). MiR-18a and miR-18b are expressed in the stroma of oestrogen receptor alpha negative breast cancers. BMC Cancer. 20. doi: 10.1186/s12885-020-06857-7. Full text in Research Archive Show summary

  Background Previously, we have shown that miR-18a and miR-18b gene expression strongly correlates with high proliferation, oestrogen receptor -negativity (ER−), cytokeratin 5/6 positivity and basal-like features of breast cancer. Methods We investigated the expression and localization of miR-18a and -18b in formalin fixed paraffin embedded (FFPE) tissue from lymph node negative breast cancers (n = 40), by chromogenic in situ hybridization (CISH). The expression level and in situ localization of miR-18a and -18b was assessed with respect to the presence of tumour infiltrating lymphocytes (TILs) and immunohistochemical markers for ER, CD4, CD8, CD20, CD68, CD138, PAX5 and actin. Furthermore, in two independent breast cancer cohorts (94 and 377 patients) the correlation between miR-18a and -18b expression and the relative quantification of 22 immune cell types obtained from the CIBERSORT tool was assessed. Results CISH demonstrated distinct and specific cytoplasmic staining for both miR-18a and miR-18b, particularly in the intratumoural stroma and the stroma surrounding the tumour margin. Staining by immunohistochemistry revealed some degree of overlap of miR-18a and -18b with CD68 (monocytes/macrophages), CD138 (plasma cells) and the presence of high percentages of TILs. CIBERSORT analysis showed a strong correlation between M1-macrophages and CD4+ memory activated T-cells with mir-18a and -18b. Conclusions Our study demonstrates that miR-18a and miR-18b expression is associated with ER- breast tumours that display a high degree of inflammation. This expression is potentially associated specifically with macrophages. These results suggest that miR-18a and miR-18b may play a role in the systemic immunological response in ER− tumours

• Tinholt, Mari; Stavik, Benedicte; Tekpli, Xavier; Garred, Øystein; Borgen, Elin & Kristensen, Vessela N. [Show all 9 contributors for this article] (2020). Coagulation factor V is a marker of tumor-infiltrating immune cells in breast cancer. Oncoimmunology. ISSN 2162-4011. 9:e1824644(1), p. 1–12. doi: 10.1080/2162402X.2020.1824644. Full text in Research Archive Show summary

  Background Factor (F) V is an essential cofactor in blood coagulation, however, F5 expression in breast tumors has also been linked to tumor aggressiveness and overall survival. The specific role of FV in breast cancer is yet unknown. We therefore aimed at dissecting the biological relevance of FV in breast cancer. Methods Gene expression data from a Scandinavian breast cancer cohort (n = 363) and the cancer genome atlas (TCGA) (n = 981) and 12 replication cohorts were used to search for F5 co-expressed genes, followed by gene ontology analysis. Pathological and bioinformatic tools were used to evaluate immune cell infiltration and tumor purity. T cell activation, proliferation and migration were studied in FV treated Jurkat T cells. Results F5 co-expressed genes were mainly associated with immune system processes and cell activation. Tumors with high expression of F5 were more infiltrated with both lymphoid (T cells, NK cells, and B cells) and myeloid cells (macrophages and dendritic cells), and F5 expression was negatively correlated with tumor purity (ρ = −0.32). Confirming a prognostic role, data from the Kaplan-Meier plotter showed that high F5 expression was associated with improved relapse-free survival. The strongest association was observed in basal-like breast cancer (HR = 0.55; 95% CI, 0.42–0.71). Exogenous FV did not substantially affect activation, proliferation or migration of human T cells. Conclusions F5 was identified as a novel marker of immune cell infiltration in breast cancer, and the prognostic role of F5 was verified. FV emerge as an interesting immunological biomarker with potential therapeutic relevance for the cancer-inflammation-thrombosis circuit.

• Liu, Jingjing; Smissen, Wendy J. C. Prager van der; Collee, Margriet J.; Bolla, Manjeet K.; Wang, Qin & Kristensen, Vessela N. [Show all 82 contributors for this article] (2020). Germline HOXB13 mutations p.G84E and p.R217C do not confer an increased breast cancer risk. Scientific Reports. 10:9688(1), p. 1–14. doi: 10.1038/s41598-020-65665-y. Full text in Research Archive Show summary

  In breast cancer, high levels of homeobox protein Hox-B13 (HOXB13) have been associated with disease progression of ER-positive breast cancer patients and resistance to tamoxifen treatment. Since HOXB13 p.G84E is a prostate cancer risk allele, we evaluated the association between HOXB13 germline mutations and breast cancer risk in a previous study consisting of 3,270 familial non-BRCA1/2 breast cancer cases and 2,327 controls from the Netherlands. Although both recurrent HOXB13 mutations p.G84E and p.R217C were not associated with breast cancer risk, the risk estimation for p.R217C was not very precise. To provide more conclusive evidence regarding the role of HOXB13 in breast cancer susceptibility, we here evaluated the association between HOXB13 mutations and increased breast cancer risk within 81 studies of the international Breast Cancer Association Consortium containing 68,521 invasive breast cancer patients and 54,865 controls. Both HOXB13 p.G84E and p.R217C did not associate with the development of breast cancer in European women, neither in the overall analysis (OR=1.035, 95% CI=0.859–1.246, P=0.718 and OR=0.798, 95% CI=0.482–1.322, P=0.381 respectively), nor in specifc high-risk subgroups or breast cancer subtypes. Thus, although involved in breast cancer progression, HOXB13 is not a material breast cancer susceptibility gene.

• Zhu, Qian; Tekpli, Xavier; Troyanskaya, Olga G. & Kristensen, Vessela N. (2020). Subtype-specific transcriptional regulators in breast tumors subjected to genetic and epigenetic alterations. Bioinformatics. ISSN 1367-4803. 36(4), p. 994–999. doi: 10.1093/bioinformatics/btz709. Full text in Research Archive Show summary

  Abstract Motivation: Breast cancer consists of multiple distinct tumor subtypes, and results from epigenetic and genetic aberrations that give rise to distinct transcriptional profiles. Despite previous efforts to understand transcriptional deregulation through transcription factor networks, the transcriptional mechanisms leading to subtypes of the disease remain poorly understood. Results: We used a sophisticated computational search of thousands of expression datasets to define extended signatures of distinct breast cancer subtypes. Using ENCODE ChIP-seq data of surrogate cell lines and motif analysis we observed that these subtypes are determined by a distinct repertoire of lineage-specific transcription factors. Furthermore, specific pattern and abundance of copy number and DNA methylation changes at these TFs and targets, compared to other genes and to normal cells were observed. Overall, distinct transcriptional profiles are linked to genetic and epigenetic alterations at lineage-specific transcriptional regulators in breast cancer subtypes. Availability and implementation: The analysis code and data are deposited at https://bitbucket.org/qzhu/breast.cancer.tf/.

• Fachal, Laura; Aschard, Hugues; Beesley, Jonathan; Barnes, Daniel R.; Allen, Jamie & Kristensen, Vessela N. [Show all 50 contributors for this article] (2020). Fine-mapping of 150 breast cancer risk regions identifies 191 likely target genes. Nature Genetics. ISSN 1061-4036. 52(1), p. 56–73. doi: 10.1038/s41588-019-0537-1. Full text in Research Archive Show summary

  Genome-wide association studies have identified breast cancer risk variants in over 150 genomic regions, but the mechanisms underlying risk remain largely unknown. These regions were explored by combining association analysis with in silico genomic feature annotations. We defined 205 independent risk-associated signals with the set of credible causal variants in each one. In parallel, we used a Bayesian approach (PAINTOR) that combines genetic association, linkage disequilibrium and enriched genomic features to determine variants with high posterior probabilities of being causal. Potentially causal variants were significantly over-represented in active gene regulatory regions and transcription factor binding sites. We applied our INQUSIT pipeline for prioritizing genes as targets of those potentially causal variants, using gene expression (expression quantitative trait loci), chromatin interaction and functional annotations. Known cancer drivers, transcription factors and genes in the developmental, apoptosis, immune system and DNA integrity checkpoint gene ontology pathways were over-represented among the highest-confidence target genes.

• Escala-Garcia, Maria; Abraham, Jean; Andrulis, Irene L.; Anton-Culver, Hoda; Arndt, Volker & Børresen-Dale, Anne-Lise [Show all 51 contributors for this article] (2020). A network analysis to identify mediators of germline-driven differences in breast cancer prognosis. Nature Communications. 11:312, p. 1–14. doi: 10.1038/s41467-019-14100-6. Full text in Research Archive Show summary

  Abstract Identifying the underlying genetic drivers of the heritability of breast cancer prognosis remains elusive. We adapt a network-based approach to handle underpowered complex datasets to provide new insights into the potential function of germline variants in breast cancer prognosis. This network-based analysis studies ~7.3 million variants in 84,457 breast cancer patients in relation to breast cancer survival and confirms the results on 12,381 independent patients. Aggregating the prognostic effects of genetic variants across multiple genes, we identify four gene modules associated with survival in estrogen receptor (ER)-negative and one in ER-positive disease. The modules show biological enrichment for cancer-related processes such as G-alpha signaling, circadian clock, angiogenesis, and Rho-GTPases in apoptosis.

• Pladsen, Arne Valebjørg; Nilsen, Gro; Rueda, Oscar M.; Aure, Miriam Ragle; Borgan, Ørnulf & Liestøl, Knut [Show all 18 contributors for this article] (2020). DNA copy number motifs are strong and independent predictors of survival in breast cancer. Communications Biology. 3. doi: 10.1038/s42003-020-0884-6. Full text in Research Archive Show summary

  Somatic copy number alterations are a frequent sign of genome instability in cancer. A precise characterization of the genome architecture would reveal underlying instability mechanisms and provide an instrument for outcome prediction and treatment guidance. Here we show that the local spatial behavior of copy number profiles conveys important information about this architecture. Six filters were defined to characterize regional traits in copy number profiles, and the resulting Copy Aberration Regional Mapping Analysis (CARMA) algorithm was applied to tumors in four breast cancer cohorts (n = 2919). The derived motifs represent a layer of information that complements established molecular classifications of breast cancer. A score reflecting presence or absence of motifs provided a highly significant independent prognostic predictor. Results were consistent between cohorts. The nonsite-specific occurrence of the detected patterns suggests that CARMA captures underlying replication and repair defects and could have a future potential in treatment stratification.

• Feng, Helian; Gusev, Alexander; Pasaniuc, Bogdan; Wu, Lang; Long, Jirong & Kristensen, Vessela N. [Show all 50 contributors for this article] (2020). Transcriptome‐wide association study of breast cancer risk by estrogen‐receptor status. Genetic Epidemiology. ISSN 0741-0395. 44(5), p. 442–468. doi: 10.1002/gepi.22288. Full text in Research Archive Show summary

  Previous transcriptome‐wide association studies (TWAS) have identified breast cancer risk genes by integrating data from expression quantitative loci and genome‐wide association studies (GWAS), but analyses of breast cancer subtype‐specific associations have been limited. In this study, we conducted a TWAS using gene expression data from GTEx and summary statistics from the hitherto largest GWAS meta‐analysis conducted for breast cancer overall, and by estrogen receptor subtypes (ER+ and ER−). We further compared associations with ER+ and ER− subtypes, using a case‐only TWAS approach. We also conducted multigene conditional analyses in regions with multiple TWAS associations. Two genes, STXBP4 and HIST2H2BA, were specifically associated with ER+ but not with ER– breast cancer. We further identified 30 TWAS‐significant genes associated with overall breast cancer risk, including four that were not identified in previous studies. Conditional analyses identified single independent breast‐cancer gene in three of six regions harboring multiple TWAS‐significant genes. Our study provides new information on breast cancer genetics and biology, particularly about genomic differences between ER+ and ER− breast cancer.

• Kramer, Iris; Hooning, Maartje J.; Mavaddat, Nasim; Hauptmann, Michael; Keeman, Renske & Steyerberg, Ewout W. [Show all 54 contributors for this article] (2020). Breast Cancer Polygenic Risk Score and Contralateral Breast Cancer Risk. American Journal of Human Genetics. ISSN 0002-9297. 107(5), p. 837–848. doi: 10.1016/j.ajhg.2020.09.001. Full text in Research Archive Show summary

  Previous research has shown that polygenic risk scores (PRSs) can be used to stratify women according to their risk of developing primary invasive breast cancer. This study aimed to evaluate the association between a recently validated PRS of 313 germline variants (PRS313) and contralateral breast cancer (CBC) risk. We included 56,068 women of European ancestry diagnosed with first invasive breast cancer from 1990 onward with follow-up from the Breast Cancer Association Consortium. Metachronous CBC risk (N = 1,027) according to the distribution of PRS313 was quantified using Cox regression analyses. We assessed PRS313 interaction with age at first diagnosis, family history, morphology, ER status, PR status, and HER2 status, and (neo)adjuvant therapy. In studies of Asian women, with limited follow-up, CBC risk associated with PRS313 was assessed using logistic regression for 340 women with CBC compared with 12,133 women with unilateral breast cancer. Higher PRS313 was associated with increased CBC risk: hazard ratio per standard deviation (SD) = 1.25 (95%CI = 1.18–1.33) for Europeans, and an OR per SD = 1.15 (95%CI = 1.02–1.29) for Asians. The absolute lifetime risks of CBC, accounting for death as competing risk, were 12.4% for European women at the 10th percentile and 20.5% at the 90th percentile of PRS313. We found no evidence of confounding by or interaction with individual characteristics, characteristics of the primary tumor, or treatment. The C-index for the PRS313 alone was 0.563 (95%CI = 0.547–0.586). In conclusion, PRS313 is an independent factor associated with CBC risk and can be incorporated into CBC risk prediction models to help improve stratification and optimize surveillance and treatment strategies.

• Ree, Anne Hansen; Nygaard, Vigdis; Pedersen, Kjetil Boye; Heinrich, Daniel; Dueland, Svein & Bergheim, Inger Riise [Show all 25 contributors for this article] (2020). Molecularly matched therapy in the context of sensitivity, resistance, and safety; patient outcomes in end-stage cancer?the MetAction study. Acta Oncologica. ISSN 0284-186X. p. 1–10. doi: 10.1080/0284186X.2020.1742377. Full text in Research Archive Show summary

  <i>Background</i>: In precision cancer medicine, the challenge is to prioritize DNA driver events, account for resistance markers, and procure sufficient information for treatment that maintains patient safety. The MetAction project, exploring how tumor molecular vulnerabilities predict therapy response, first established the required workflow for DNA sequencing and data interpretation (2014–2015). Here, we employed it to identify molecularly matched therapy and recorded outcome in end-stage cancer (2016–2019).<p><p> <i>Material and methods</i>: Metastatic tissue from 26 patients (16 colorectal cancer cases) was sequenced by the Oncomine assay. The study tumor boards interpreted called variants with respect to sensitivity or resistance to matched therapy and recommended single-agent or combination treatment if considered tolerable. The primary endpoint was the rate of progression-free survival 1.3-fold longer than for the most recent systemic therapy. The objective response rate and overall survival were secondary endpoints.<p><p> <i>Results</i>: Both common and rare actionable alterations were identified. Thirteen patients were found eligible for therapy following review of tumor sensitivity and resistance variants and patient tolerability. The interventions were inhibitors of ALK/ROS1-, BRAF-, EGFR-, FGFR-, mTOR-, PARP-, or PD-1-mediated signaling for 2–3 cases each. Among 10 patients who received treatment until radiologic evaluation, 6 (46% of the eligible cases) met the primary endpoint. Four colorectal cancer patients (15% of the total study cohort) had objective response. The only serious adverse event was a transient colitis, which appeared in 1 of the 2 patients given PD-1 inhibitor with complete response. Apart from those two, overall survival was similar for patients who did and did not receive study treatment.<p><p> <i>Conclusions</i>: The systematic MetAction approach may point forward to a refined framework for how to interpret the complexity of sensitivity <i>versus</i> resistance and patient safety that resides in tumor sequence data, for the possibly improved outcome of precision cancer medicine in future studies.

• Zhang, Haoyu; Ahearn, Thomas U.; Lecarpentier, Julie; Barnes, Daniel; Beesley, Jonathan & Qi, Guanghao [Show all 272 contributors for this article] (2020). Genome-wide association study identifies 32 novel breast cancer susceptibility loci from overall and subtype-specific analyses. Nature Genetics. ISSN 1061-4036. 52(6), p. 572–581. doi: 10.1038/s41588-020-0609-2. Full text in Research Archive
• Gythfeldt, Hedda von der Lippe; Lien, Tonje Gulbrandsen; Tekpli, Xavier; Silwal-Pandit, Laxmi; Borgen, Elin & Garred, Øystein [Show all 15 contributors for this article] (2020). Immune phenotype of tumor microenvironment predicts response to bevacizumab in neoadjuvant treatment of ER-positive breast cancer. International Journal of Cancer. ISSN 0020-7136. 147(9), p. 2515–2525. doi: 10.1002/ijc.33108. Full text in Research Archive Show summary

  Antiangiogenic drugs are potentially a useful supplement to neoadjuvant chemotherapy for a subgroup of patients with human epidermal growth factor receptor 2 (HER2) negative breast cancer, but reliable biomarkers for improved response are lacking. Here, we report on a randomized phase II clinical trial to study the added effect of bevacizumab in neoadjuvant chemotherapy with FEC100 (5‐fluorouracil, epirubicin and cyclophosphamide) and taxanes (n = 132 patients). Gene expression from the tumors was obtained before neoadjuvant treatment, and treatment response was evaluated by residual cancer burden (RCB) at time of surgery. Bevacizumab increased the proportion of complete responders (RCB class 0) from 5% to 20% among patients with estrogen receptor (ER) positive tumors (P = .02). Treatment with bevacizumab was associated with improved 8‐year disease‐free survival (P = .03) among the good responders (RCB class 0 or I). Patients treated with paclitaxel (n = 45) responded better than those treated with docetaxel (n = 21; P = .03). Improved treatment response was associated with higher proliferation rate and an immune phenotype characterized by high presence of classically activated M1 macrophages, activated NK cells and memory activated CD4 T cells. Treatment with bevacizumab increased the number of adverse events, including hemorrhage, hypertension, infection and febrile neutropenia, but despite this, the ECOG status was not affected.

• Tahiri, Andliena; Tekpli, Xavier; Somisetty, Venkata Satheesh; DeWijn, Rik; Luders, Torben & Bukholm, Ida Rashida Khan [Show all 9 contributors for this article] (2020). Loss of progesterone receptor is associated with distinct tyrosine kinase profiles in breast cancer. Breast Cancer Research and Treatment. ISSN 0167-6806. 183, p. 585–598. doi: 10.1007/s10549-020-05763-7. Full text in Research Archive Show summary

  Abstract Purpose The aim of this study was to assess protein tyrosine kinase profles in primary breast cancer samples in correlation with the distinct hormone and growth receptor profles ER, PR, and HER2. Experimental design Pamchip® microarrays were used to measure the phosphorylation of 144 tyrosine kinase substrates in 29 ER+breast cancer samples and cell lines MCF7, BT474 and ZR75-1. mRNA expression data from the METABRIC cohort and publicly available PR chip-sequencing data were used for validation purposes, together with RT-PCR. Results In ER+ breast tumors and cell lines, we observed that the loss of PR expression correlated to higher kinase activity in samples and cell lines that were HER2−. A number of kinases, representing mostly proteins within the PI3K/AKT pathway, were identifed as responsible for the diferential phosphorylation between PR− and PR+ in ER+/HER2− tumors. We used the METABRIC cohort to analyze mRNA expression from 977 ER+/HER2− breast cancers. Twenty four kinaseencoding genes were identifed as diferentially expressed between PR+ and PR−, dividing ER+/HER2− samples in two distinct clusters with signifcant diferences in survival (p<0.05). Four kinase genes, LCK, FRK, FGFR4, and MST1R, were identifed as potential direct targets of PR. Conclusions Our results suggest that the PR status has a profound efect on tyrosine kinases, especially for FGFR4 and LCK genes, in ER+/HER2− breast cancer patients. The infuence of these genes on the PI3K/AKT signaling pathway may potentially lead to novel drug targets for ER+/PR− breast cancer patients

• Jiang, Xia; Finucane, Hilary K.; Schumacher, Fredrick R.; Schmit, Stephanie L.; Tyrer, Jonathan P. & Han, Younghun [Show all 333 contributors for this article] (2019). Shared heritability and functional enrichment across six solid cancers. Nature Communications. 10:431, p. 1–23. doi: 10.1038/s41467-018-08054-4. Full text in Research Archive Show summary

  Quantifying the genetic correlation between cancers can provide important insights into the mechanisms driving cancer etiology. Using genome-wide association study summary statistics across six cancer types based on a total of 296,215 cases and 301,319 controls of European ancestry, here we estimate the pair-wise genetic correlations between breast, colorectal, head/neck, lung, ovary and prostate cancer, and between cancers and 38 other diseases. We observed statistically significant genetic correlations between lung and head/neck cancer (rg = 0.57, p = 4.6 × 10−8), breast and ovarian cancer (rg = 0.24, p = 7 × 10−5), breast and lung cancer (rg = 0.18, p =1.5 × 10−6) and breast and colorectal cancer (rg = 0.15, p = 1.1 × 10−4). We also found that multiple cancers are genetically correlated with non-cancer traits including smoking, psychiatric diseases and metabolic characteristics. Functional enrichment analysis revealed a significant excess contribution of conserved and regulatory regions to cancer heritability. Our comprehensive analysis of cross-cancer heritability suggests that solid tumors arising across tissues share in part a common germline genetic basis.

• Tekpli, Xavier; Lien, Tonje Gulbrandsen; Røssevold, Andreas Hagen; Nebdal, Daniel J.H.; Borgen, Elin & Ohnstad, Hege Oma [Show all 19 contributors for this article] (2019). An independent poor-prognosis subtype of breast cancer defined by a distinct tumor immune microenvironment. Nature Communications. 10:5499, p. 1–14. doi: 10.1038/s41467-019-13329-5. Full text in Research Archive Show summary

  How mixtures of immune cells associate with cancer cell phenotype and affect pathogenesis is still unclear. In 15 breast cancer gene expression datasets, we invariably identify three clusters of patients with gradual levels of immune infiltration. The intermediate immune infiltration cluster (Cluster B) is associated with a worse prognosis independently of known clinicopathological features. Furthermore, immune clusters are associated with response to neoadjuvant chemotherapy. In silico dissection of the immune contexture of the clusters identified Cluster A as immune cold, Cluster C as immune hot while Cluster B has a pro-tumorigenic immune infiltration. Through phenotypical analysis, we find epithelial mesenchymal transition and proliferation associated with the immune clusters and mutually exclusive in breast cancers. Here, we describe immune clusters which improve the prognostic accuracy of immune contexture in breast cancer. Our discovery of a novel independent prognostic factor in breast cancer highlights a correlation between tumor phenotype and immune contexture.

• Lindholm, Evita Maria; Aure, Miriam Ragle; Haugen, Mads Haugland; Sahlberg, Kristine Kleivi; Kristensen, Vessela N. & Nebdal, Daniel J.H. [Show all 9 contributors for this article] (2019). miRNA expression changes during the course of neoadjuvant bevacizumab and chemotherapy treatment in breast cancer. Molecular Oncology. ISSN 1574-7891. 13(10), p. 2278–2296. doi: 10.1002/1878-0261.12561. Full text in Research Archive Show summary

  One of the hallmarks of cancer is sustained angiogenesis. Favorable results have been reported in some breast cancer (BC) patients receiving antiangiogenic therapy with bevacizumab (Bev) in combination with chemotherapy, and further knowledge on how Bev can be optimally combined with conventional treatment to increase efficacy is strongly needed. In this randomized, neoadjuvant phase II clinical trial, 132 patients with HER2‐negative, nonmetastatic BC were treated with Bev in combination with sequential chemotherapy. Biopsies were sampled before treatment, after 12 weeks with anthracycline and after taxane therapy at week 25. MicroRNA (miRNA) expression profiling was performed on biopsies from each time point. Altogether, 241 biopsies were analyzed with the aim of identifying miRNA‐based biomarkers of response to therapy. Results from the miRNA analyses were reported for the ER‐positive cohort, which were previously demonstrated to benefit from antiangiogenic therapy in this study. For both treatment arms of this cohort, significantly different expression was observed for 217 miRNAs between objective responding and nonresponding patients before treatment initiation. These miRNAs have been linked to regulation of epithelial–mesenchymal transition, metastasis, and tumor growth, among other processes. Bev in combination with chemotherapy resulted in similar miRNA changes to chemotherapy alone. However, the deregulation of miRNA expression occurred earlier in the Bev arm. In both arms, tumor suppressor miRNAs were found upregulated after treatment, while oncogenic miRNAs were downregulated in the Bev arm. Patients responding to Bev showed a strong correlation between deregulated miRNAs and decreased proliferation score during the course of treatment, with downregulation of miR‐4465 as the strongest indicator of reduced proliferation. Integrative analyses at miRNA‐, gene‐, and protein expression further indicated a longitudinal decrease in proliferation. Altogether, the results indicate that proliferation might represent a predictive factor for increased Bev sensitivity, which may aid in the identification of patients who could potentially benefit from Bev.

• Nguyen, Chinh BKrong; Kumar, Surendra; Zucknick, Manuela; Kristensen, Vessela N.; Gjerstad, Johannes & Nilsen, Hilde Loge [Show all 7 contributors for this article] (2019). Associations between clinical symptoms, plasma norepinephrine and deregulated immune gene networks in subgroups of adolescent with Chronic Fatigue Syndrome. Brain, Behavior, and Immunity. ISSN 0889-1591. 76, p. 82–96. doi: 10.1016/j.bbi.2018.11.008. Full text in Research Archive Show summary

  Background Chronic Fatigue Syndrome (CFS) is one of the most important causes of disability among adolescents while limited knowledge exists on genetic determinants underlying disease pathophysiology. Methods We analyzed deregulated immune-gene modules using Pathifier software on whole blood gene expression data (29 CFS patients, 18 controls). Deconvolution of immune cell subtypes based on gene expression profile was performed using CIBERSORT. Supervised consensus clustering on pathway deregulation score (PDS) was used to define CFS subgroups. Associations between PDS and immune, neuroendocrine/autonomic and clinical markers were examined. The impact of plasma norepinephrine level on clinical markers over time was assessed in a larger cohort (91 patients). Results A group of 29 immune-gene sets was shown to differ patients from controls and detect subgroups within CFS. Group 1P (high PDS, low norepinephrine, low naïve CD4+ composition) had strong association with levels of serum C-reactive protein and Transforming Growth Factor-beta. Group 2P (low PDS, high norepinephrine, high naïve CD4+ composition) had strong associations with neuroendocrine/autonomic markers. The corresponding plasma norepinephrine level delineated 91 patients into two subgroups with significant differences in fatigue score. Conclusion We identified 29 immune-gene sets linked to plasma norepinephrine level that could delineate CFS subgroups. Plasma norepinephrine stratification revealed that lower levels of norepinephrine were associated with higher fatigue. Our data suggests potential involvement of neuro-immune dysregulation and genetic stratification in CFS.

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• Lee, Stephanie; Tahiri, Andliena; Gjesdal, Kjell Inge; Lüders, Torben; Reitsma, Laurens Cornelus & Lyngra, Marianne [Show all 10 contributors for this article] (2025). Radiogenomics during neoadjuvant therapy with aromatase inhibitors: integrating dynamic magnetic resonance data and molecular biology. Clinical Cancer Research. ISSN 1078-0432. 31(12_Supplement), p. 01–01. doi: 10.1158/1557-3265.sabcs24-p1-01-07. Full text in Research Archive Show summary

  Abstract Background: Neoadjuvant treatment of locally advanced breast cancer (LABC) is now well-established and considerable progress has been made concerning drug development for subgroups of patients. However, reliable predictive markers are still lacking for many patients. Thus, radiological measures like repeated MRI of the breast and axilla, after 3 and 6 months of therapy, are used to follow the patients during neoadjuvant therapy to confirm successful downstaging. Although not established in clinical routine, dynamic MRI contains quantitative information that can provide valuable insight into the tumour microenvironment and, when combined with clinical and molecular data, has the potential to enhance the prediction of tumor responses. Here, we analyze split-dynamic MRI data in relation to clinicopathological and molecular data to further identify disease heterogeneity that can be used to improve prognostic accuracy and inform treatment decisions in breast cancer patients. Methods: This study reports results from eligible patients with ER pos., HER2 neg. LABC that were enrolled in the NEOLETEXE-trial (1). Clinicopathological, molecular, and advanced MRI data were acquired from this trial, collected before, during, and after neoadjuvant endocrine therapy (NET) with aromatase inhibitors given in sequence. MR images were generated using a split-dynamic technique that simultaneously acquires high spatial resolution and high temporal resolution images, providing information on tissue permeability and vascularity. The images underwent post-processing on the nordicICE software where parametric values were extracted based on whole-tumour volumes of interest. These parametric values were analysed in correlation with clinico-pathological data and with molecular findings from bulk RNA-seq and single cell data from a subset of the same patients. Results: From the kinetic MRI data, the volume transfer constant (Ktrans), a parameter reflecting capillary permeability, was significantly reduced after NET (p = 0.005). Bulk RNA-seq data was analysed for gene expression, pathway involvement, and cell type abundance before, during, and after treatment. Gene expression analysis revealed significant dysregulation of genes related to cell proliferation and cell cycle regulation following treatment. Biological pathway analysis revealed significant down-regulation of hemostatic activity and upregulation of cell cycle and interleukin signaling following treatment. Cell type analysis revealed a higher abundance of CD4+ T-cells and T-helper cells following treatment. These results will be further correlated with single cell data as well as measures of treatment response and clinical outcome. Conclusions: Our findings suggest that the addition of MRI data to clinicopathological and molecular data enhances the characterization of tumor responses during NET for locally advanced breast cancer. 1. The NEOLETEXE trial: a neoadjuvant cross-over study exploring the lack of cross resistance between aromatase inhibitors. Bahrami N. et al., Future Oncoloy 15 (32), 3675-3682, 2019. Citation Format: Stephanie Lee, Andliena Tahiri, Kjell-Inge Gjesdal, Torben Lüders, Laurens Reitsma, Marianne Lyngra, Alvaro Köhn-Luque, Vessela Kristensen, Jürgen Geisler, Jonn Terje Geitung. Radiogenomics during neoadjuvant therapy with aromatase inhibitors: integrating dynamic magnetic resonance data and molecular biology [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-01-07.

• O’Mahony, Denise G.; Lesluyes, Tom; Brorson, Ina S.; Vernhoff, Patrik; Sokolova, Ksenia & Aure, Miriam Ragle [Show all 21 contributors for this article] (2025). Clonal selection supported by single cell Dna sequencing reveals hormonal adaptation and resistance in locally advanced breast cancer During Neoadjuvant Aromatase Inhibition. bioRxiv - the preprint server for biology. doi: 10.1101/2025.09.24.678208. Full text in Research Archive Show summary

  Abstract Background The aromatase inhibitors (AI) letrozole and exemestane, are often used in sequence in targeting ER+ breast cancers. However resistance to AI poses a major barrier to sustained clinical benefit, while the biological mechanisms underlying the phenomenon remain largely unknown. In this study, we build on our clinical NeoLetExe trial, with the aim to investigate the molecular basis of resistance to AI, by analysing subclonal evolutionary dynamics during sequential treatment. Methods We use whole-exome sequencing (WES) data from 11 ER+ breast cancer patients and 3 timepoints of the Neoletexe trial to reconstruct cancer cell fraction–based subclonal composition. Single-cell DNA sequencing from matched tumour samples is used for validating identified clones and variants. Subclonal variants were annotated to genes by integrating evidence from public data and ExpectoSc. Pathway enrichment analysis using Human Base was conducted. Results Higher cancer cell fraction clone trajectories were significantly associated with reduced treatment response (p = 0.023). Clones reconstructed by WES were validated at 81% using single-cell DNA sequencing. Clones resistant to both letrozole and exemestane demonstrated PIK3CA/AKT/mTOR signaling activation, KRAS pathway dysregulation, hedgehog signaling, and androgen receptor pathways, alongside extensive immune activation and metabolic reprogramming. Drug-specific resistance patterns showed exemestane-resistant clones enriched for epigenetic control and miRNA-mediated silencing, while letrozole-resistant clones displayed metabolic dysregulation but notably lacked immune pathway activation. In contrast, treatment-sensitive clones maintained coordinated cell cycle control, preserved DNA damage responses, and retained immune signaling capacity. Analysis of FDA-approved breast cancer targets identified actionable alterations in PIK3CA (4 patients) and AKT1 (1 patient) that persisted through AI treatment, with RNA expression analysis revealing 48 additional therapeutic targets spanning PI3K/AKT/mTOR, CDK4/6, DNA repair (BRCA1/2, ATM), and immune checkpoint pathways. Conclusion WES-based cancer cell fraction analysis successfully captured subclonal evolutionary trajectories during AI treatment, revealing drug-specific mechanisms and identifying key molecular players in endocrine therapy resistance. This work establishes a framework for precision oncology approaches by providing actionable therapeutic targets and advancing our understanding of resistance mechanisms to improve clinical outcomes in sequential AI therapy.

• Nawaz, Meh Sameen; Minh, Hang; Jabeen, Shakila; Bjørnetrø, Tonje; Geisler, Jürgen & Kristensen, Vessela N. [Show all 7 contributors for this article] (2025). Extracellular Vesicle-Associated Cytokines as Novel Biomarkers in ER+ Breast Cancer. Full text in Research Archive
• Kentistou, Katherine A.; Kaisinger, Lena R.; Stankovic, Stasa; Vaudel, Marc; Oliveira, Edson Mendes de & Messina, Andrea [Show all 221 contributors for this article] (2024). Correction to: Understanding the genetic complexity of puberty timing across the allele frequency spectrum (Nature Genetics, (2024), 56, 7, (1397-1411), 10.1038/s41588-024-01798-4). Nature Genetics. ISSN 1061-4036. 56(8), p. 1763–1764. doi: 10.1038/s41588-024-01857-w. Full text in Research Archive
• Fjermeros, Kamilla; Ghannoum, Salim; Geisler, Stephanie; Bhargava, Sameer; Tahiri, Andliena & Klajic, Jovana [Show all 32 contributors for this article] (2024). The NEOLETRIB trial: neoadjuvant treatment with Letrozole and Ribociclib in ER-positive, HER2-negative breast cancer. Future Oncology. ISSN 1479-6694. 20(32), p. 2457–2466. doi: 10.1080/14796694.2024.2377531. Full text in Research Archive Show summary

  Chemotherapy is used as neoadjuvant therapy for all subgroups of breast cancer, including ER-positive, and HER2-negative cases. However, studies have suggested that using aromatase inhibitors combined with CDK4/6-inhibitors might be an appropriate alternative in selected patients. Thus, the NEOLETRIB trial evaluates the response of ER-positive, HER2-negative luminal A/B breast cancer to the combination of letrozole and ribociclib in the neoadjuvant setting. Comprehensive molecular biology procedures, including sequential single-cell RNA-sequencing of tumor biopsies, are performed during 6 months of treatment with extensive biobanking of blood samples, tumor biopsies and gut microbiome specimens. Our findings will hopefully contribute to an improved selection of patients who may benefit from this drug combination and give new insights into the intra-tumoral changes during this treatment.

• Liu, Aoxing; Genovese, Giulio; Zhao, Yajie; Pirinen, Matti; Zekavat, Seyedeh M. & Kentistou, Katherine A. [Show all 115 contributors for this article] (2024). Correction to: Genetic drivers and cellular selection of female mosaic X chromosome loss (Nature, (2024), 631, 8019, (134-141), 10.1038/s41586-024-07533-7). Nature. ISSN 0028-0836. 636(8043), p. E7–E7. doi: 10.1038/s41586-024-08215-0. Full text in Research Archive
• Rattalma, Arnoldo Frigessi Di; Köhn-Luque, Alvaro; Kristensen, Vessela N.; Gørbitz, Carl Henrik & Engebråten, Olav (2020). Kan statistikk og matematikk redde like mange liv som nye medisiner? Aftenposten (morgenutg. : trykt utg.). ISSN 0804-3116. Full text in Research Archive
• Jiang, Xia; Finucane, Hilary K.; Schumacher, Fredrick; Schmit, Stephanie L.; Tyrer, Jonathan P. & Han, Younghun [Show all 333 contributors for this article] (2019). Publisher Correction: Shared heritability and functional enrichment across six solid cancers (Nature Communications, (2019), 10, 1, (431), 10.1038/s41467-018-08054-4). Nature Communications. 10(1). doi: 10.1038/s41467-019-12095-8. Full text in Research Archive
• Rapp, Emma; Alnæs, Grethe Irene Grenaker; Kristensen, Vessela N. & Carlsen, Harald (2023). Characterization of single cells from breast tumors with mutations implicated in treatment response. Norges miljø- og biovitenskapelige universitet (NMBU). Full text in Research Archive Show summary

  Breast cancer has high tumor heterogeneity which can be challenging for individual patients. It is of importance to characterize the mutations in the tumors of breast cancer patients that affect the eradication of the malignant cells because of the treatment. By studying individual cells in the tumor instead of the entire tumor’s genetic material, cells of resistance at a single cell level can be identified. It is seen that the mutations which occur simultaneously in each cell, identify clones and the pseudo-times when they arise, and how these clones develop during the treatment. In the clinical trial NeoLetExe breast cancer patients are treated neoadjuvant with Letrozole and Exemestane sequentially in the following manner: group 1 first receives Letrozole and switches to Exemestane after 2 months, and group 2 first receives Exemestane before switching to Letrozole after 2 months. Biopsies are taken before starting treatment, when changing medication and during surgery. Letrozole and Exemestane are hormone therapy drugs that are used as treatment for breast cancer in postmenopausal patients. To study individual tumor cells, we employ a unique platform for single-cell analysis. The Mission Bio Tapestri platform is an instrument where single cells are separated and mixed with reagents to perform the analysis in isolated oil droplets, and results in specific parts of the genes being marked at a single cell level. A custom panel of 497 amplicons covering 528 sequence variants was designed in a pilot study. Tumor tissue from three patients at one timepoint (baseline), one patient with three time points (baseline, 2 months, and 4 months) and one patient with two time points (baseline and 4 months) in the NeoLetExe clinical trial was analyzed and shown in this thesis. It was discovered that some genes like ZNF717 were mutated in several or all the samples, and it was observed that clones with certain sequence variants were altered during treatment. Basic software and analysis strategies to analyze the complex data achieved with targeted single-cell sequencing is presented in this thesis, however more efficient analysis strategies should be investigated.

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