Bjørn Naume

• Vandraas, Kathrine Flørenes; Andersen, Sarah Hjorth; Trewin-Nybråten, Cassia Bree; Andreassen, Bettina Kulle; Blix, Egil Støre & Naume, Bjørn [Show all 9 contributors for this article] (2026). Translating CLEOPATRA into routine practice: National treatment patterns and survival for patients with HER2-positive metastatic breast cancer. Breast. ISSN 0960-9776. 89. doi: 10.1016/j.breast.2026.104852. Full text in Research Archive Show summary

  Introduction Amplification of the epidermal growth factor receptor 2 (HER2) gene occurs in 15-20% of breast cancers and was historically associated with poor prognosis. The CLEOPATRA trial established pertuzumab added to trastuzumab and chemotherapy as standard of care for metastatic HER2-positive breast cancer, demonstrating a 15.7-month improvement in median overall survival (OS). Strict trial inclusion criteria may limit generalizability to clinical practice. Population-based data can assess whether these survival benefits translate to real-world outcomes. Methods Women diagnosed with HER2-positive metastatic breast cancer in 2012–2021 were identified through the Cancer Registry of Norway and linked to health and treatment databases. Patients were categorized by treatment regimen and year of treatment initiation: pre-CLEOPATRA (2012–2014; trastuzumab ± chemotherapy) and post-CLEOPATRA (2015–2021; trastuzumab + pertuzumab ± chemotherapy). OS was estimated using Kaplan–Meier and Cox regression adjusted for clinical, and socioeconomic factors. Outcomes were benchmarked against CLEOPATRA. Results The cohort comprised 329 women, 23% were aged ≥65 years. Seventy-eight patients (24%) received pre-CLEOPATRA regimens and 251 (76%) post-CLEOPATRA regimens; 59% met minimum one CLEOPATRA exclusion criterion. First-line dual HER2 blockade increased from 0% in 2012 to 75% in 2021. Median OS improved from 69 months to 96 months post-CLEOPATRA. Four-year OS increased from 59% to 65% (adjusted hazard ratio 0.77, 95% CI 0.53–1.12). Real-world OS was consistent with CLEOPATRA outcomes. Conclusions Nationwide real-world data demonstrate favorable survival outcomes following adoption of dual anti-HER2 therapy. While these findings are broadly consistent with the CLEOPATRA trial, the observational design does not allow direct assessment of equivalence between studies.

• Svalheim, K. G.; Andresen, Nikolai Kragøe; Bjerre, C.; Gilje, Bjørnar; Jakobsen, E. H. & Falk, Ragnhild Sørum [Show all 9 contributors for this article] (2026). Patient-reported outcomes from the randomized ALICE trial evaluating the addition of atezolizumab to anthracycline-based chemotherapy in metastatic triple-negative breast cancer. Breast. ISSN 0960-9776. 86. doi: 10.1016/j.breast.2026.104704. Full text in Research Archive Show summary

  Background The ALICE trial demonstrated that adding atezolizumab to anthracycline-based immunomodulatory chemotherapy improved progression-free survival (PFS) in patients with metastatic triple-negative breast cancer (mTNBC), including those with PD-L1–negative tumors. Here, we report the patient-reported outcome measures (PROMs). Methods Patients were randomized to receive chemotherapy plus atezolizumab (atezo-chemo) or chemotherapy plus placebo (placebo-chemo). PROMs were collected at baseline and weeks 9, 17, 25, and 49 using the EORTC QLQ-C15-PAL, Chalder Fatigue Questionnaire (CFQ), and Numeric Rating Scale (NRS) for pain. Results PROMs were available from 64 of 68 patients. At week 9, mean changes from baseline favored the atezo-chemo arm across all QLQ-C15-PAL domains, CFQ scores, and NRS pain intensity. Time-to-deterioration analyses also favored atezo-chemo, with statistically significant hazard ratios for global quality of life (QoL; HR 0.24), emotional functioning (HR 0.30), and pain (HR 0.20). Pain—a pre-specified cardinal symptom—improved in the atezo-chemo group at all time points. At 12 months, PROMs indicated sustained tolerability. Better baseline PROM scores were associated with improved PFS and overall survival, especially among patients treated with atezolizumab. Patients with >median QoL score at baseline recorded improved PFS when treated with atezolizumab (HR 0.25), while patients with ≤median QoL score did not (HR 1.02). Conclusions Adding atezolizumab to the study-chemotherapy in mTNBC improves both PFS and patient-reported quality of life, emotional well-being and symptom control. These findings support continued development of this combination regimen and suggest that baseline quality of life may serve as a useful predictor of immunotherapy benefit.

• 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.

• Ullern, Andreas; Holm, Kristian; Andresen, Nikolai Kragøe; Røssevold, Andreas Hagen; Bang, Corinna & Naume, Bjørn [Show all 8 contributors for this article] (2025). Gut microbiota diversity is prognostic in metastatic hormone receptor‐positive breast cancer patients receiving chemotherapy and immunotherapy. Molecular Oncology. ISSN 1574-7891. doi: 10.1002/1878-0261.70117. Full text in Research Archive Show summary

  Immune checkpoint blockade (ICB) is standard treatment in several cancer types, despite not being proven efficacious in metastatic hormone receptor‐positive breast cancer (HR+ mBC). The gut microbiota is associated with patient outcome and toxicity from cancer therapy, although limited data are available for breast cancer. In the randomized phase 2b trial ICON, immunomodulating chemotherapy was investigated in combination with dual ICB in HR+ mBC. To determine whether gut microbiota could inform prognosis, we performed 16S (V3‐V4) rRNA sequencing on fecal samples collected at baseline and after 8 weeks of study treatment. We showed that high alpha diversity before treatment was associated with prolonged progression‐free survival (PFS; primary trial endpoint) and overall survival. Alpha diversity was lower in patients with prior chemotherapy in the metastatic setting. However, alpha diversity remained significantly associated with PFS after correcting for prior chemotherapy and other factors in bivariate analyses. High‐grade immune‐related toxicity was also associated with high alpha diversity. These findings suggest that high alpha diversity should be further investigated as a positive prognostic factor in HR+ mBC and approaches to increase alpha diversity could potentially improve clinical outcome.

• Vandraas, Kathrine Flørenes; Andersen, Sarah Hjorth; Trewin-Nybråten, Cassia Bree; Ursin, Giske; Botteri, Edoardo & Kulle, Bettina [Show all 10 contributors for this article] (2025). Anti‐Her2 therapy patterns in metastatic breast cancer—Real‐world data suggest undertreatment. International Journal of Cancer. ISSN 0020-7136. 158(3), p. 633–641. doi: 10.1002/ijc.70120. Full text in Research Archive Show summary

  Treatment efficacy of anti‐HER2 therapies for metastatic breast cancer (mBC) has been demonstrated in clinical trials, but real‐world data are lacking. In particular, it is unclear whether patients in clinical practice receive treatment as recommended. We took advantage of population‐based registries in Norway to assess anti‐HER2 therapy patterns in real‐world data, with specific attention to the treatment of vulnerable groups. We included 715 patients with HER2+ mBC diagnosed from 2012 to 2021. Median age was 60 years, 473 (66%) had relapsed from early‐stage BC, and 440 (62%) had hormone receptor positive (HR+) disease. Anti‐HER2 therapy patterns aligned with national recommendations. Median treatment duration for first line therapy was 7.2 months, where 261 patients (55%) used trastuzumab and pertuzumab (±chemotherapy), followed by monotherapy with trastuzumab (195 patients, 41%). Second line therapy was initiated by 206 patients (43%), with a median duration of 7 months, where trastuzumab emtansine was the most prevalent therapy, used by 84 patients (41%). Third line therapy was initiated in 119 patients (25%) and 55 patients (11%) proceeded to fourth line therapy. The 182 patients (28%) who did not receive any anti‐HER2 therapy were older (74 years vs. 55 years) and had more comorbidity compared to treated patients. Among patients ≤75 years and healthy, 15% did not receive anti‐HER2 therapy. Patient characteristics strongly influence anti‐HER2 treatment patterns, and although numerous treatment options are available, a substantial proportion of HER2+ mBC patients did not receive targeted therapy. Undertreatment may be present.

• 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

• 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.

• 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.

• Asphaug, Lars; Akslen, Lars Andreas; Blix, Egil Støre; Boge, Beate; Borgen, Elin Faye & Brekke, Marianne B. [Show all 28 contributors for this article] (2025). Cost-Effectiveness of Prosigna for Adjuvant Treatment Decisions in Early Breast Cancer: Healthcare Sector and Societal Perspectives. Clinical Breast Cancer. ISSN 1526-8209. 25(6), p. e721–e730. doi: 10.1016/j.clbc.2025.04.011. Full text in Research Archive Show summary

  Background Gene expression profiling tests such as the Prosigna-assay are used to aid adjuvant treatment decisions in hormone receptor positive (HR+) HER2 negative (HER2-) early breast cancer (EBC). In this economic evaluation, the cost-effectiveness of Prosigna against immunohistochemical (IHC) markers including Ki-67, was evaluated from the Norwegian healthcare- and societal perspective. Materials and methods The impact of Prosigna on the treatment decision was assessed in the prospective, observational EMIT-1 trial. Using individual data collected during the first 12 months post-surgery, a decision model was built to project the economic consequences of using the Prosigna compared to IHC-markers for adjuvant treatment decisions. A bottom-up costing analysis assessed the two strategies' material- and labour costs. Health benefits were measured by cost per quality-adjusted life-years (QALYs) using the EQ-5D-5L questionnaire and data on income and welfare benefits was obtained from Statistics Norway. Results Of 2,178 HR+/HER2- pN0 EBC patients in the EMIT-1 trial, 1,985 had available health economic data and 1,850 had complete income and welfare benefit records. Including all pN0 patients in the Prosigna-test strategy, the test was above the cost-effective threshold (€26,000; incremental cost-per QALY gained (ICER) €255,622) in a healthcare sector perspective. Applying a societal perspective incorporating productivity costs, Prosigna was cost saving (ICER €-435,677). Restricting Prosigna-testing to patients assessed as chemotherapy candidates/uncertain chemotherapy candidates, the strategy was cost-effective from both the healthcare and societal perspective (ICER €8,884 and €-620,170, respectively). For other subgroups (pT>1cm, ≤/>50 years), cost-effectiveness was only achievable from a societal perspective (ICER €-574,911, €-602,955 and €-336,832, respectively). Conclusions Using the Prosigna-assay for all HR+/HER2- pN0 EBC patients was not cost-effective from a healthcare perspective, but from the societal perspective it was cost-saving compared to the IHC-classification. Selecting patients who are clear/uncertain candidates for chemotherapy based on IHC-classification, Prosigna is cost-effective from both perspectives. Micro-abstract This economic evaluation assessed the cost-effectiveness of the Prosigna gene expression profiling test versus immunohistochemical (IHC) markers for adjuvant treatment decisions in hormone receptor positive (HR+) HER2 negative (HER2-) early breast cancer (EBC) from Norwegian healthcare and societal perspectives. The Prosigna test was not cost-effective from a healthcare perspective but was cost-saving from a societal perspective. When use was restricted to patients who were clear chemotherapy candidates or uncertain candidates, Prosigna was cost-effective from both perspectives.

• 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.

• 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.

• 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.

• Kildal, Wanja; Cyll, Karolina Urszula; Kalsnes, Joakim; Islam, Rakibul; Julbø, Frida Marie Ihle & Pradhan, Manohar [Show all 35 contributors for this article] (2024). Deep learning for automated scoring of immunohistochemically stained tumour tissue sections – Validation across tumour types based on patient outcomes. Heliyon. 10(13), p. 1–19. doi: 10.1016/j.heliyon.2024.e32529. Full text in Research Archive Show summary

  We aimed to develop deep learning (DL) models to detect protein expression in immunohistochemically (IHC) stained tissue-sections, and to compare their accuracy and performance with manually scored clinically relevant proteins in common cancer types. Five cancer patient cohorts (colon, two prostate, breast, and endometrial) were included. We developed separate DL models for scoring IHC-stained tissue-sections with nuclear, cytoplasmic, and membranous staining patterns. For training, we used images with annotations of cells with positive and negative staining from the colon cohort stained for Ki-67 and PMS2 (nuclear model), the prostate cohort 1 stained for PTEN (cytoplasmic model) and β-catenin (membranous model). The nuclear DL model was validated for MSH6 in the colon, MSH6 and PMS2 in the endometrium, Ki-67 and CyclinB1 in prostate, and oestrogen and progesterone receptors in the breast cancer cohorts. The cytoplasmic DL model was validated for PTEN and Mapre2, and the membranous DL model for CD44 and Flotillin1, all in prostate cohorts. When comparing the results of manual and DL scores in the validation sets, using manual scores as the ground truth, we observed an average correct classification rate of 91.5 % (76.9–98.5 %) for the nuclear model, 85.6 % (73.3–96.6 %) for the cytoplasmic model, and 78.4 % (75.5–84.3 %) for the membranous model. In survival analyses, manual and DL scores showed similar prognostic impact, with similar hazard ratios and p-values for all DL models. Our findings demonstrate that DL models offer a promising alternative to manual IHC scoring, providing efficiency and reproducibility across various data sources and markers.

• Ullern, Andreas; Holm, Kristian; Røssevold, Andreas Hagen; Andresen, Nikolai Kragøe; Bang, Corinna & Lingjærde, Ole Christian [Show all 9 contributors for this article] (2024). Gut microbiota diversity is prognostic and associated with benefit from chemo-immunotherapy in metastatic triple-negative breast cancer. Molecular Oncology. ISSN 1574-7891. doi: 10.1002/1878-0261.13760. Full text in Research Archive
• Andresen, Nikolai Kragøe; Røssevold, Andreas Hagen; Borgen, Elin; Schirmer, Cecilie; Gilje, Bjørnar & Garred, Øystein [Show all 14 contributors for this article] (2024). Circulating tumor cells in metastatic breast cancer patients treated with immune checkpoint inhibitors – a biomarker analysis of the ALICE and ICON trials. Molecular Oncology. ISSN 1574-7891. doi: 10.1002/1878-0261.13675. Full text in Research Archive Show summary

  Immune checkpoint inhibitors (ICIs) have been introduced in breast cancer (BC) treatment and better biomarkers are needed to predict benefit. Circulating tumor cells (CTCs) are prognostic in BC, but knowledge is limited on CTCs in the context of ICI therapy. In this study, serial sampling of CTCs (CellSearch system) was evaluated in 82 patients with metastatic BC enrolled in two randomized trials investigating ICI plus chemotherapy. Programmed death-ligand 1 (PD-L1) expression on CTCs was also measured. Patients with ≥ 2 CTCs per 7.5 mL at baseline had gene expression profiles in tumor suggestive of increased T-cell activity, including increased tumor inflammation signature (TIS) in both triple-negative (P = 0.010) and hormone receptor-positive (P = 0.024) disease. Patients with luminal A BC had higher CTC levels. The association between CTC status and outcome was most apparent 4 weeks into therapy. PD-L1 expression in CTCs was observed in 6/17 CTC-positive patients and was associated with inferior survival. In conclusion, our study indicates that CTC numbers may inform on tumor immune composition, as well as prognosis. These findings suggest a potential of using CTCs as an accessible biomarker source in BC patients treated with immunotherapy.

• 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.

• Andresen, Nikolai Kragøe; Røssevold, Andreas Hagen; Quaghebeur, Claire; Gilje, Bjørnar; Boge, Beate & Gombos, Andrea [Show all 17 contributors for this article] (2024). Ipilimumab and nivolumab combined with anthracycline-based chemotherapy in metastatic hormone receptor-positive breast cancer: A randomized phase 2b trial. Journal for ImmunoTherapy of Cancer (JITC). 12:e007990(1), p. 1–12. doi: 10.1136/jitc-2023-007990. Full text in Research Archive Show summary

  Background Immune checkpoint inhibitors have shown minimal clinical activity in hormone receptor-positive metastatic breast cancer (HR+ mBC). Doxorubicin and low- dose cyclophosphamide are reported to induce immune responses and counter regulatory T cells (Tregs). Here, we report the efficacy and safety of combined programmed cell death protein-1/cytotoxic T-lymphocyte- associated protein 4 blockade concomitant with or after immunomodulatory chemotherapy for HR + mBC. Methods Patients with HR+ mBC starting first-/second- line chemotherapy (chemo) were randomized 2:3 to chemotherapy (pegylated liposomal doxorubicin 20 mg/ m 2 every second week plus cyclophosphamide 50 mg by mouth/day in every other 2-week cycle) with or without concomitant ipilimumab (ipi; 1 mg/kg every sixth week) and nivolumab (nivo; 240 mg every second week). Patients in the chemo-only arm were offered cross-over to ipi/ nivo without chemotherapy. Co-primary endpoints were safety in all patients starting therapy and progression-free survival (PFS) in the per-protocol (PP) population, defined as all patients evaluated for response and receiving at least two treatment cycles. Secondary endpoints included objective response rate, clinical benefit rate, Treg changes during therapy and assessment of programmed death- ligand 1 (PD-L1), mutational burden and immune gene signatures as biomarkers. Results Eighty-two patients were randomized and received immune-chemo (N=49) or chemo-only (N=33), 16 patients continued to the ipi/nivo-only cross- over arm. Median follow-up was 41.4 months. Serious adverse events occurred in 63% in the immune-chemo arm, 39% in the chemo-only arm and 31% in the cross- over-arm. In the PP population (N=78) median PFS in the immune- chemo arm was 5.1 months, compared with 3.6 months in the chemo-only arm, with HR 0.94 (95% CI 0.59 to 1.51). Clinical benefit rates were 55% (26/47) and 48% (15/31) in the immune-chemo and chemo- only arms, respectively. In the cross-over-arm (ipi/nivo-only), objective responses were observed in 19% of patients (3/16) and clinical benefit in 25% (4/16). Treg levels in blood decreased after study chemotherapy. High-grade immune-related adverse events were associated with prolonged PFS. PD-L1 status and mutational burden were not associated with ipi/nivo benefit, whereas a numerical PFS advantage was observed for patients with a high Treg gene signature in tumor. Conclusion The addition of ipi/nivo to chemotherapy increased toxicity without improving efficacy. Ipi/nivo administered sequentially to chemotherapy was tolerable and induced clinical responses.

• Chauhan, Sudhir Kumar; Dunn, Claire; Andresen, Nikolai Kragøe; Røssevold, Andreas Hagen; Skorstad, Gjertrud & Sike, Adam [Show all 11 contributors for this article] (2024). Peripheral immune cells in metastatic breast cancer patients display a systemic immunosuppressed signature consistent with chronic inflammation. NPJ breast cancer. 10(1). doi: 10.1038/s41523-024-00638-2. Full text in Research Archive Show summary

  Immunotherapies blocking the PD-1/PD-L1 checkpoint show some efficacy in metastatic breast cancer (mBC) but are often hindered by immunosuppressive mechanisms. Understanding these mechanisms is crucial for personalized treatments, with peripheral blood monitoring representing a practical alternative to repeated biopsies. In the present study, we performed a comprehensive mass cytometry analysis of peripheral blood immune cells in 104 patients with HER2 negative mBC and 20 healthy donors (HD). We found that mBC patients had significantly elevated monocyte levels and reduced levels of CD4+ T cells and plasmacytoid dendritic cells, when compared to HD. Furthermore, mBC patients had more effector T cells and regulatory T cells, increased expression of immune checkpoints and other activation/exhaustion markers, and a shift to a Th2/Th17 phenotype. Furthermore, T-cell phenotypes identified by mass cytometry correlated with functionality as assessed by IFN-γ production. Additional analysis indicated that previous chemotherapy and CDK4/6 inhibition impacted the numbers and phenotype of immune cells. From 63 of the patients, fresh tumor samples were analyzed by flow cytometry. Paired PBMC-tumor analysis showed moderate correlations between peripheral CD4+ T and NK cells with their counterparts in tumors. Further, a CD4+ T cell cluster in PBMCs, that co-expressed multiple checkpoint receptors, was negatively associated with CD4+ T cell tumor infiltration. In conclusion, the identified systemic immune signatures indicate an immune-suppressed environment in mBC patients who had progressed/relapsed on standard treatments and is consistent with ongoing chronic inflammation. These activated immuno-suppressive mechanisms may be investigated as therapeutic targets, and for use as biomarkers of response or treatment resistance.

• Rugo, Hope S.; Poznak, Catherine H. Van; Neven, Patrick; Danielewicz, Iwona; Lee, Soo Chin & Campone, Mario [Show all 15 contributors for this article] (2023). Radium-223 in women with hormone receptor-positive bone-metastatic breast cancer receiving endocrine therapy: pooled analysis of two international, phase 2, randomized, double-blind, placebo-controlled trials. Breast Cancer Research and Treatment. ISSN 0167-6806. doi: 10.1007/s10549-023-07147-z. Full text in Research Archive Show summary

  Abstract Background Most women with advanced breast cancer have skeletal metastases. Radium-223 is an alpha-emitting radionuclide that selectively targets areas of bone metastases. Methods Two double-blind, placebo-controlled studies of radium-223 were conducted in women with hormone receptor-positive (HR+), bone-predominant metastatic breast cancer. All patients received endocrine therapy (ET), as a single agent of the investigator’s choice (Study A) or exemestane + everolimus (Study B). Patients were randomized to receive radium-223 (55 kBq/kg) or placebo intravenously every 4 weeks for six doses. Accrual was halted following unblinded interim analyses per protocol amendments, and both studies were terminated. We report pooled analyses of symptomatic skeletal event-free survival (SSE-FS; primary endpoint), radiologic progression-free survival (rPFS) and overall survival (OS; secondary), and time to bone alkaline phosphatase (ALP) progression (exploratory). Results In total, 382 patients were enrolled, and 196 SSE-FS events (70% planned total) were recorded. Hazard ratios (95% confidence intervals) and nominal p values for radium-223 + ET versus placebo + ET were: SSE-FS 0.809 (0.610–1.072), p  = 0.1389; rPFS 0.956 (0.759–1.205), p  = 0.7039; OS 0.889 (0.660–1.199), p  = 0.4410; and time to bone ALP progression 0.593 (0.379–0.926), p  = 0.0195. Radium-223- or placebo-related treatment-emergent adverse events were reported in 50.3% versus 35.1% of patients (grade 3/4: 25.7% vs. 8.5%), with fractures/bone-associated events in 23.5% versus 23.9%. Conclusions In patients with HR+ bone-metastatic breast cancer, numeric differences favoring radium-223 + ET over placebo + ET for the primary SSE-FS endpoint were suggestive of efficacy, in line with the primary outcome measure used in the underlying phase 2 studies. No similar evidence of efficacy was observed for secondary progression or survival endpoints. Adverse events were more frequent with radium-223 + ET versus placebo + ET, but the safety profile of the combination was consistent with the safety profiles of the component drugs. Clinical trial registration numbers Study A: NCT02258464, registered October 7, 2014. Study B: NCT02258451, registered October 7, 2014.

• 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.

• 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.

• 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

• 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.

• 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.

• Staaf, Johan; Häkkinen, Jari; Hegardt, Cecilia; Saal, Lao H.; Kimbung, Siker & Hedenfalk, Ingrid [Show all 25 contributors for this article] (2022). RNA sequencing-based single sample predictors of molecular subtype and risk of recurrence for clinical assessment of early-stage breast cancer. NPJ breast cancer. 8:94(1), p. 1–17. doi: 10.1038/s41523-022-00465-3. Full text in Research Archive Show summary

  Abstract Multigene assays for molecular subtypes and biomarkers can aid management of early invasive breast cancer. Using RNA-sequencing we aimed to develop single-sample predictor (SSP) models for clinical markers, subtypes, and risk of recurrence (ROR). A cohort of 7743 patients was divided into training and test set. We trained SSPs for subtypes and ROR assigned by nearest-centroid (NC) methods and SSPs for biomarkers from histopathology. Classifications were compared with Prosigna in two external cohorts (ABiM, n  = 100 and OSLO2-EMIT0, n  = 103). Prognostic value was assessed using distant recurrence-free interval. Agreement between SSP and NC for PAM50 (five subtypes) was high (85%, Kappa = 0.78) for Subtype (four subtypes) very high (90%, Kappa = 0.84) and for ROR risk category high (84%, Kappa = 0.75, weighted Kappa = 0.90). Prognostic value was assessed as equivalent and clinically relevant. Agreement with histopathology was very high or high for receptor status, while moderate for Ki67 status and poor for Nottingham histological grade. SSP and Prosigna concordance was high for subtype (OSLO-EMIT0 83%, Kappa = 0.73 and ABiM 80%, Kappa = 0.72) and moderate and high for ROR risk category (68 and 84%, Kappa = 0.50 and 0.70, weighted Kappa = 0.70 and 0.78). Pooled concordance for emulated treatment recommendation dichotomized for chemotherapy was high (85%, Kappa = 0.66). Retrospective evaluation suggested that SSP application could change chemotherapy recommendations for up to 17% of postmenopausal ER+/HER2-/N0 patients with balanced escalation and de-escalation. Results suggest that NC and SSP models are interchangeable on a group-level and nearly so on a patient level and that SSP models can be derived to closely match clinical tests.

• 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.

• Røssevold, Andreas Hagen; Andresen, Nikolai Kragøe; Bjerre, Christina Annette; Gilje, Bjørnar; Jakobsen, Erik Hugger & Raj, Sunil Xavier [Show all 17 contributors for this article] (2022). Atezolizumab plus anthracycline-based chemotherapy in metastatic triple-negative breast cancer: the randomized, double-blind phase 2b ALICE trial. Nature Medicine. ISSN 1078-8956. p. 1–28. doi: 10.1038/s41591-022-02126-1. Full text in Research Archive Show summary

  Immune checkpoint inhibitors have shown efficacy against metastatic triple-negative breast cancer (mTNBC) but only for PD-L1positive disease. The randomized, placebo-controlled ALICE trial (NCT03164993, 24 May 2017) evaluated the addition of atezolizumab (anti-PD-L1) to immune-stimulating chemotherapy in mTNBC. Patients received pegylated liposomal doxorubicin (PLD) and low-dose cyclophosphamide in combination with atezolizumab (atezo-chemo; n = 40) or placebo (placebo-chemo; n = 28). Primary endpoints were descriptive assessment of progression-free survival in the per-protocol population (>3 atezolizumab and >2 PLD doses; n = 59) and safety in the full analysis set (FAS; all patients starting therapy; n = 68). Adverse events leading to drug discontinuation occurred in 18% of patients in the atezo-chemo arm (7/40) and in 7% of patients in the placebo-chemo arm (2/28). Improvement in progression-free survival was indicated in the atezo-chemo arm in the per-protocol population (median 4.3 months versus 3.5 months; hazard ratio (HR) = 0.57; 95% confidence interval (CI) 0.33–0.99; log-rank P = 0.047) and in the FAS (HR = 0.56; 95% CI 0.33–0.95; P = 0.033). A numerical advantage was observed for both the PD-L1positive (n = 27; HR = 0.65; 95% CI 0.27–1.54) and PD-L1negative subgroups (n = 31; HR = 0.57, 95% CI 0.27–1.21). The progression-free proportion after 15 months was 14.7% (5/34; 95% CI 6.4–30.1%) in the atezo-chemo arm versus 0% in the placebo-chemo arm. The addition of atezolizumab to PLD/cyclophosphamide was tolerable with an indication of clinical benefit, and the findings warrant further investigation of PD1/PD-L1 blockers in combination with immunomodulatory chemotherapy.

• 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.

• Nilssen, Yngvar; Brustugun, Odd Terje; Eriksen, Morten Tandberg; Guren, Marianne; Haug, Erik Skaaheim & Naume, Bjørn [Show all 8 contributors for this article] (2022). Compliance with recommended cancer patient pathway timeframes and choice of treatment differed by cancer type and place of residence among cancer patients in Norway in 2015–2016. BMC Cancer. 22(220). doi: 10.1186/s12885-022-09306-9. Full text in Research Archive Show summary

  Background Cancer patient pathways (CPPs) were implemented in Norway to reduce unnecessary waiting times, regional variations, and to increase the predictability of cancer care for the patients. This study aimed to determine if 70% of cancer patients started treatment within the recommended time frames, and to identify potential delays. Methods Patients registered with a colorectal, lung, breast, or prostate cancer diagnosis at the Cancer Registry of Norway in 2015–2016 were linked with the Norwegian Patient Registry and Statistics Norway. Adjusting for sociodemographic variables, multivariable quantile (median) regressions were used to examine the association between place of residence and median time to start of examination, treatment decision, and start of treatment. Results The study included 20 668 patients. The proportions of patients who went through the CPP within the recommended time frames were highest among colon (84%) and breast (76%) cancer patients who underwent surgery and lung cancer patients who started systemic anticancer treatment (76%), and lowest for prostate cancer patients who underwent surgery (43%). The time from treatment decision to start of treatment was the main source of delay for all cancers. Travelling outside the resident health trust prolonged waiting time and was associated with a reduced odds of receiving surgery and radiotherapy for lung and rectal cancer patients, respectively. Conclusions Achievement of national recommendations of the CCP times differed by cancer type and treatment. Identified bottlenecks in the pathway should be targeted to decrease waiting times. Further, CPP guidelines should be re-examined to determine their ongoing relevance.

• Hugenschmidt, Harald; Labori, Knut Jørgen; Borgen, Elin; Brunborg, Cathrine; Schirmer, Cecilie & Seeberg, Lars Thomas [Show all 8 contributors for this article] (2021). Preoperative CTC-Detection by CellSearch ® Is Associated with Early Distant Metastasis and Impaired Survival in Resected Pancreatic Cancer. Cancers. 13(3). doi: 10.3390/cancers13030485. Full text in Research Archive Show summary

  n patients with presumed pancreatic ductal adenocarcinoma (PDAC), biomarkers that may open for personalised, risk-adapted treatment are lacking. The study analysed the impact of CTCs-presence on the patterns of recurrence and survival in 98 patients resected for PDAC with 5–10 years of follow-up. Preoperative samples were analysed by the CellSearch® system for EpCAM+/DAPI+/CK+/CD45-CTCs. CTCs were detected in 7 of the 98 patients. CTCs predicted a significantly shorter median disease-free survival (DFS) of 3.3 vs. 9.2 months and a median cancer specific survival (CSS)of 6.3 vs. 18.5 months. Relapse status was confirmed by imaging for 87 patients. Of these, 58 patients developed distant metastases (DM) and 29 developed isolated local recurrence (ILR) as the first sign of cancer relapse. All patients with CTCs experienced DM. pN-status and histological grade >2 were other independent risk factors for DM, but only CTCs predicted significantly shorter cancer-specific, disease-free and post-recurrence survival. Preoperative parameters did not affect clinical outcome. We conclude that CTC presence in resected PDAC patients predicted early distant metastasis and impaired survival. Preoperative CTCs alone or in combination with histopathological factors may guide initial treatment decisions in patients with resectable PDAC in the future

• 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.

• Lien, Tonje Gulbrandsen; Ohnstad, Hege Oma; Lingjærde, Ole Christian; Vallon-Christersson, Johan; Aaserud, Marit & Sveli, My Anh Tu [Show all 12 contributors for this article] (2021). Sample preparation approach influences pam50 risk of recurrence score in early breast cancer. Cancers. 13(23). doi: 10.3390/cancers13236118. Full text in Research Archive Show summary

  The PAM50 gene expression subtypes and the associated risk of recurrence (ROR) score are used to predict the risk of recurrence and the benefits of adjuvant therapy in early-stage breast cancer. The Prosigna assay includes the PAM50 subtypes along with their clinicopathological features, and is approved for treatment recommendations for adjuvant hormonal therapy and chemotherapy in hormone-receptor-positive early breast cancer. The Prosigna test utilizes RNA extracted from macrodissected tumor cells obtained from formalin-fixed, paraffin-embedded (FFPE) tissue sections. However, RNA extracted from fresh-frozen (FF) bulk tissue without macrodissection is widely used for research purposes, and yields high-quality RNA for downstream analyses. To investigate the impact of the sample preparation approach on ROR scores, we analyzed 94 breast carcinomas included in an observational study that had available gene expression data from macrodissected FFPE tissue and FF bulk tumor tissue, along with the clinically approved Prosigna scores for the node-negative, hormone-receptor-positive, HER2-negative cases (n = 54). ROR scores were calculated in R; the resulting two sets of scores from FFPE and FF samples were compared, and treatment recommendations were evaluated. Overall, ROR scores calculated based on the macrodissected FFPE tissue were consistent with the Prosigna scores. However, analyses from bulk tissue yielded a higher proportion of cases classified as normal-like; these were samples with relatively low tumor cellularity, leading to lower ROR scores. When comparing ROR scores (low, intermediate, and high), discordant cases between the two preparation approaches were revealed among the luminal tumors; the recommended treatment would have changed in a minority of cases.

• Haugen, Mads Haugland; Lingjærde, Ole Christian; Hedenfalk, Ingrid; Garred, Øystein; Borgen, Elin & Loman, Niklas [Show all 12 contributors for this article] (2021). Protein signature predicts response to neoadjuvant treatment with chemotherapy and bevacizumab in HER2-negative breast cancers. JCO Precision Oncology (JCO PO). 5, p. 286–306. doi: 10.1200/PO.20.00086. Full text in Research Archive Show summary

  <p>PURPOSE: Antiangiogenic therapy using bevacizumab has proven effective for a number of cancers; however, in breast cancer (BC), there is an unmet need to identify patients who benefit from such treatment. <p>PATIENTS AND METHODS: In the NeoAva phase II clinical trial, patients (N = 132) with large (≥ 25 mm) human epidermal growth factor receptor 2 (HER2)-negative primary tumors were randomly assigned 1:1 to treatment with neoadjuvant chemotherapy (CTx) alone or in combination with bevacizumab (Bev plus CTx). The ratio of the tumor size after relative to before treatment was calculated into a continuous response scale. Tumor biopsies taken prior to neoadjuvant treatment were analyzed by reverse-phase protein arrays (RPPA) for expression levels of 210 BC-relevant (phospho-) proteins. Lasso regression was used to derive a predictor of tumor shrinkage from the expression of selected proteins prior to treatment. <p>RESULTS: We identified a nine-protein signature score named vascular endothelial growth factor inhibition response predictor (ViRP) for use in the Bev plus CTx treatment arm able to predict with accuracy pathologic complete response (pCR) (area under the curve [AUC] = 0.85; 95% CI, 0.74 to 0.97) and low residual cancer burden (RCB 0/I) (AUC = 0.80; 95% CI, 0.68 to 0.93). The ViRP score was significantly lower in patients with pCR (P < .001) and in patients with low RCB (P < .001). The ViRP score was internally validated on mRNA data and the resultant surrogate mRNA ViRP score significantly separated the pCR patients (P = .016). Similarly, the mRNA ViRP score was validated (P < .001) in an independent phase II clinical trial (PROMIX). <p>CONCLUSION: Our ViRP score, integrating the expression of nine proteins and validated on mRNA data both internally and in an independent clinical trial, may be used to increase the likelihood of benefit from treatment with bevacizumab combined with chemotherapy in patients with HER2-negative BC.

• Nobre, Ana Rita; Risson, Emma; Singh, Deepak K.; Martino, Julie S. Di; Cheung, Julie F. & Wang, Jiapeng [Show all 14 contributors for this article] (2021). Bone marrow NG2+/Nestin+ mesenchymal stem cells drive DTC dormancy via TGF-β2. Nature Cancer. doi: 10.1038/s43018-021-00179-8. 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.

• Krüger, Kristi; Silwal-Pandit, Laxmi; Wik, Elisabeth; Straume, Oddbjørn; Stefansson, Ingunn Marie & Borgen, Elin [Show all 10 contributors for this article] (2021). Baseline microvessel density predicts response to neoadjuvant bevacizumab treatment of locally advanced breast cancer. Scientific Reports. 11, p. 1–11. doi: 10.1038/s41598-021-81914-0. Full text in Research Archive Show summary

  A subset of breast cancer patients benefits from preoperative bevacizumab and chemotherapy, but validated predictive biomarkers are lacking. Here, we aimed to evaluate tissue-based angiogenesis markers for potential predictive value regarding response to neoadjuvant bevacizumab treatment in breast cancer. In this randomized 1:1 phase II clinical trial, 132 patients with large or locally advanced HER2-negative tumors received chemotherapy ± bevacizumab. Dual Factor VIII/Ki-67 immunohistochemical staining was performed on core needle biopsies at baseline and week 12. Microvessel density (MVD), proliferative microvessel density (pMVD; Factor VIII/Ki-67 co-expression), glomeruloid microvascular proliferation (GMP), and a gene expression angiogenesis signature score, were studied in relation to pathologic complete response (pCR), clinico-pathologic features and intrinsic molecular subtype. We found that high baseline MVD (by median) significantly predicted pCR in the bevacizumab-arm (odds ratio 4.9, P = 0.012). High pMVD, presence of GMP, and the angiogenesis signature score did not predict pCR, but were associated with basal-like (P ≤ 0.009) and triple negative phenotypes (P ≤ 0.041). pMVD and GMP did also associate with high-grade tumors (P ≤ 0.048). To conclude, high baseline MVD significantly predicted response to bevacizumab treatment. In contrast, pMVD, GMP, and the angiogenesis signature score, did not predict response, but associated with aggressive tumor features, including basal-like and triple-negative phenotypes.

• 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.

• Hartkopf, Andreas; Brucker, Sara Y.; Taran, Florin-Andrei; Harbeck, Nadia; Au, Alexandra von & Naume, Bjørn [Show all 37 contributors for this article] (2021). Disseminated tumour cells from the bone marrow of early breast cancer patients: Results from an international pooled analysis. European Journal of Cancer. ISSN 0959-8049. doi: 10.1016/j.ejca.2021.06.028. 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.

• Helland, Thomas; Naume, Bjørn; Hustad, Steinar; Bifulco, Ersilia; Kvaløy, Jan Terje & Sætersdal, Anna Barbro [Show all 18 contributors for this article] (2020). Low Z-4OHtam concentrations are associated with adverse clinical outcome among early stage premenopausal breast cancer patients treated with adjuvant tamoxifen. Molecular Oncology. ISSN 1574-7891. 15(4). doi: 10.1002/1878-0261.12865. Full text in Research Archive Show summary

  Abstract Low steady‐state levels of active tamoxifen metabolites have been associated with inferior treatment outcomes. In this retrospective analysis of 406 estrogen receptor‐positive breast cancer (BC) patients receiving adjuvant tamoxifen as initial treatment, we have associated our previously reported thresholds for the two active metabolites, Z‐endoxifen and Z‐4‐hydroxy‐tamoxifen (Z‐4OHtam), with treatment outcomes in an independent cohort of BC patients. Among all patients, metabolite levels did not affect survival. However, in the premenopausal subgroup receiving tamoxifen alone (n = 191) we confirmed an inferior BC ‐specific survival in patients with the previously described serum concentration threshold of Z‐4OHtam ≤ 3.26 nm (HR = 2.37, 95% CI = 1.02–5.48, P = 0.039). The ‘dose–response’ survival trend in patients categorized to ordinal concentration cut‐points of Z‐4OHtamoxifen (≤ 3.26, 3.27–8.13, > 8.13 nm) was also replicated (P‐trend log‐rank = 0.048). Z‐endoxifen was not associated with outcome. This is the first study to confirm the association between a published active tamoxifen metabolite threshold and BC outcome in an independent patient cohort. Premenopausal patients receiving 5‐year of tamoxifen alone may benefit from therapeutic drug monitoring to ensure tamoxifen effectiveness.

• 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.

• Nilssen, Yngvar; Brustugun, Odd Terje; Eriksen, Morten Tandberg; Haug, Erik Skaaheim; Naume, Bjørn & Møller, Bjørn (2020). Patient and tumour characteristics associated with inclusion in Cancer patient pathways in Norway in 2015-2016. BMC Cancer. 20(1), p. 1–11. doi: 10.1186/s12885-020-06979-y. Full text in Research Archive Show summary

  Background Cancer patient pathways (CPPs) were implemented in 2015 to reduce waiting time, regional variation in waiting time, and to increase the predictability of cancer care for the patients. The aims of this study were to see if the national target of 70% of all cancer patients being included in a CPP was met, and to identify factors associated with CPP inclusion. Methods All patients registered with a colorectal, lung, breast or prostate cancer diagnosis at the Cancer Registry of Norway in the period 2015–2016 were linked with the Norwegian Patient Registry for CPP information and with Statistics Norway for sociodemographic variables. Multivariable logistic regression examined if the odds of not being included in a CPP were associated with year of diagnosis, age, sex, tumour stage, marital status, education, income, region of residence and comorbidity. Results From 2015 to 2016, 30,747 patients were diagnosed with colorectal, lung, breast or prostate cancer, of whom 24,429 (79.5%) were included in a CPP. Significant increases in the probability of being included in a CPP were observed for colorectal (79.1 to 86.2%), lung (79.0 to 87.3%), breast (91.5 to 97.2%) and prostate cancer (62.2 to 76.2%) patients (p < 0.001). Increasing age was associated with an increased odds of not being included in a CPP for lung (p < 0.001) and prostate cancer (p < 0.001) patients. Colorectal cancer patients < 50 years of age had a two-fold increase (OR = 2.23, 95% CI: 1.70–2.91) in the odds of not being included in a CPP. The odds of no CPP inclusion were significantly increased for low income colorectal (OR = 1.24, 95%CI: 1.00–1.54) and lung (OR = 1.52, 95%CI: 1.16–1.99) cancer patients. Region of residence was significantly associated with CPP inclusion (p < 0.001) and the probability, adjusted for case-mix ranged from 62.4% in region West among prostate cancer patients to 97.6% in region North among breast cancer patients. Conclusions The national target of 70% was met within 1 year of CPP implementation in Norway. Although all patients should have equal access to CPPs, a prostate cancer diagnosis, older age, high level of comorbidity or low income were significantly associated with an increased odds of not being included in a CPP.

• Kyte, Jon A; Røssevold, Andreas Hagen; Falk, Ragnhild Sørum & Naume, Bjørn (2020). ALICE: A randomized placebo-controlled phase II study evaluating atezolizumab combined with immunogenic chemotherapy in patients with metastatic triple-negative breast cancer. Journal of Translational Medicine. 18:252, p. 1–7. doi: 10.1186/s12967-020-02424-7. Full text in Research Archive Show summary

  Background Immunotherapy with checkpoint inhibitors (CI) represents an important novel development in cancer treatment. Metastatic triple-negative breast cancer (mTNBC) is incurable, with a median survival of only ~ 13 months. We have initiated the randomized placebo-controlled phase IIb study ALICE, evaluating PD-L1 blockade combined with immunogenic chemotherapy in mTNBC patients (n = 75). Intriguingly, the host immune response is strongly predictive for the effect of chemotherapy in mTNBC. In the ALICE trial, we release the brake on the immune response by use of atezolizumab, an inhibitory antibody against PD-L1. We utilize anthracyclines, shown to trigger the immune system, and low-dose cyclophosphamide, which has been reported to counter immunosuppressive cells. Methods ALICE is a randomized, double-blind, placebo-controlled exploratory phase II study evaluating the safety and efficacy of atezolizumab when combined with immunogenic chemotherapy in subjects with mTNBC. The trial will enroll 75 evaluable subjects, randomized 2:3 into two arms (A:B). The patients receive identical chemotherapy, i.e. pegylated liposomal doxorubicin (PLD 20 mg/m2 intravenously every 2nd week) + cyclophosphamide (50 mg per day, first 2 weeks in each 4 week cycle). Patients in arm A receive placebo, while patients in arm B receive atezolizumab. The primary objectives are assessment of toxicity and progression-free survival. The secondary objectives include overall survival, tumor response rate, clinical benefit rate, patient reported outcomes, biomarkers and assessment of tumor-immune evolution during therapy. Discussion The question of how CI should be combined with chemotherapy, is a key challenge facing the field. There is a strong preclinical rationale for exploring if anthracyclines, which are considered to induce immunogenic cell death, synergize with PD-L1 blockade, and if low-dose cyclophosphamide counters tumor tolerance. However, the data from patients is as yet very limited, and the clinical evaluation of these hypotheses is among the key objectives in the ALICE trial. The study includes extensive biobanking and translational sub-projects, also addressing other clinically important questions. These analyses may uncover mechanisms of drug efficacy or tumor resistance, and identify biomarkers allowing personalized therapy. If the trial suggests acceptable safety of the ALICE therapy and provide a signal of clinical efficacy, further studies are warranted. Trial registration NCT03164993, May 24th 2017; https://clinicaltrials.gov/ct2/show/record/NCT03164993

• 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.

• Kyte, Jon A; Andresen, Nikolai Kragøe; Russnes, Hege Elisabeth Giercksky; Fretland, Signe Øien; Falk, Ragnhild Sørum & Lingjærde, Ole Christian [Show all 7 contributors for this article] (2020). ICON: A randomized phase IIb study evaluating immunogenic chemotherapy combined with ipilimumab and nivolumab in patients with metastatic hormone receptor positive breast cancer. Journal of Translational Medicine. 18(1). doi: 10.1186/s12967-020-02421-w. Full text in Research Archive Show summary

  Background Immunotherapy with checkpoint inhibitors (CPI) targeting PD-1 or CTLA-4 has emerged as an important treatment modality for several cancer forms. In hormone receptor positive breast cancer (HR + BC), this therapeutic approach is largely unexplored. We have started a clinical trial, ICON (CA209-9FN), evaluating CPI combined with selected chemotherapy in patients with metastatic HR + BC. The tumor lymphocyte infiltration is predictive for the effect of chemotherapy in BC. In ICON, we use anthracycline, which are considered as “immunogenic” chemotherapy, and low-dose cyclophosphamide, which has been reported to counter immunosuppressive cells. Methods ICON is a randomized exploratory phase IIb study evaluating the safety and efficacy of combining nivolumab (nivo; anti-PD-1) and ipilimumab (ipi; anti-CTLA-4) with chemotherapy in subjects with metastatic HR + BC. Primary objectives are aassessment of toxicity and progression-free survival. The trial will enrol 75 evaluable subjects, randomized 2:3 into two arms (A:B). Patients in Arm A receive only chemotherapy, i.e. pegylated liposomal doxorubicin (PLD 20 mg/m2 intravenously every 2nd week) + cyclophosphamide (cyclo; 50 mg per day, first 2 weeks in each 4 week cycle). Patients in Arm B receive PLD + cyclo + ipilimumab (1 mg intravenously every 6th week) + nivolumab (240 mg intravenously every 2nd week). Patients in arm A will be offered ipi + nivo after disease progression. Discussion ICON is among the first clinical trials combining chemotherapy with PD-1 and CTLA-4 blockade, and the first in BC. There is a strong preclinical rationale for exploring if anthracyclines, which are considered to induce immunogenic cell death, synergize with CPI, and for combining PD-1 and CTLA-4 blockade, as these checkpoints are important in different phases of the immune response. If the ICON trial suggests acceptable safety and provide a signal of clinical efficacy, further studies are warranted. The cross-over patients from Arm A receiving ipilimumab/nivolumab without concomitant chemotherapy represent the first BC cohort receiving this therapy. The ICON trial includes a series of translational sub-projects addressing clinically important knowledge gaps. These studies may uncover biomarkers or mechanisms of efficacy and resistance, thereby informing the development of novel combinatory regimes and of personalised biomarker-based therapy. Trial registration NCT03409198, Jan 24th 2018; https://clinicaltrials.gov/ct2/show/record/NCT03409198

• Hugenschmidt, Harald; Labori, Knut Jørgen; Brunborg, Cathrine; Verbeke, Caroline Sophie; Seeberg, Lars Thomas & Schirmer, Cecilie [Show all 10 contributors for this article] (2020). Cytokeratin-positive cells in the bone marrow from patients with pancreatic, periampullary malignancy and benign pancreatic disease show no prognostic information. BMC Cancer. 20:1107, p. 1–13. doi: 10.1186/s12885-020-07510-z. Full text in Research Archive Show summary

  Background: Pancreatic and periampullary carcinoma are aggressive tumours where preoperative assessment is challenging. Disseminated tumour cells (DTC) in the bone marrow (BM) are associated with impaired prognosis in a variety of epithelial cancers. In a cohort of patients with presumed resectable pancreatic and periampullary carcinoma, we evaluated the frequency and the potential prognostic impact of the preoperative presence of DTC, defined as cytokeratin-positive cells detected by immunocytochemistry (ICC). Methods: Preoperative BM samples from 242 patients selected for surgical resection of presumed resectable pancreatic and periampullary carcinoma from 09/2009 to 12/2014, were analysed for presence of CK-positive cells by ICC. The median observation time was 21.5 months. Overall survival (OS) and disease-free survival (DFS) were calculated by Kaplan-Meier and Cox regression analysis. Results: Successful resections of malignant tumours were performed in 179 of the cases, 30 patients resected had benign pancreatic disease based on postoperative histology, and 33 were deemed inoperable intraoperatively due to advanced disease. Overall survival for patients with resected carcinoma was 21.1 months (95% CI: 18.0-24.1), for those with benign disease OS was 101 months (95% CI: 69.4-132) and for those with advanced disease OS was 8.8 months (95% CI: 4.3-13.3). The proportion of patients with detected CK-positive cells was 6/168 (3.6%) in resected malignant cases, 2/31 (6.5%) in advanced disease and 4/29 (13.8%) in benign disease. The presence of CK-positive cells was not correlated to OS or DFS, neither in the entire cohort nor in the subgroup negative for circulating tumour cells (CTC). Conclusions: The results indicate that CK-positive cells may be present in both patients with malignant and benign diseases of the pancreas. Detection of CK-positive cells was not associated with differences in prognosis for the entire cohort or any of the subgroups analysed.

• 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.

• 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.

• Johansson, Henrik J.; Socciarelli, Fabio; Vacanti, Nathaniel M.; Haugen, Mads Haugland; Zhu, Yafeng & Siavelis, Ioannis [Show all 36 contributors for this article] (2019). Breast cancer quantitative proteome and proteogenomic landscape. Nature Communications. 10:1600, p. 1–14. doi: 10.1038/s41467-019-09018-y. Full text in Research Archive Show summary

  In the preceding decades, molecular characterization has revolutionized breast cancer (BC) research and therapeutic approaches. Presented herein, an unbiased analysis of breast tumor proteomes, inclusive of 9995 proteins quantified across all tumors, for the first time recapitulates BC subtypes. Additionally, poor-prognosis basal-like and luminal B tumors are further subdivided by immune component infiltration, suggesting the current classification is incomplete. Proteome-based networks distinguish functional protein modules for breast tumor groups, with co-expression of EGFR and MET marking ductal carcinoma in situ regions of normal-like tumors and lending to a more accurate classification of this poorly defined subtype. Genes included within prognostic mRNA panels have significantly higher than average mRNA-protein correlations, and gene copy number alterations are dampened at the protein-level; underscoring the value of proteome quantification for prognostication and phenotypic classification. Furthermore, protein products mapping to non-coding genomic regions are identified; highlighting a potential new class of tumor-specific immunotherapeutic targets.

• Hartmann-Johnsen, Olaf Johan; Kåresen, Rolf; Schlichting, Ellen; Naume, Bjørn & Nygård, Jan Franz (2019). Using clinical cancer registry data for estimation of quality indicators: Results from the Norwegian breast cancer registry. International Journal of Medical Informatics. ISSN 1386-5056. 125, p. 102–109. doi: 10.1016/j.ijmedinf.2019.03.004. Full text in Research Archive Show summary

  Introduction Increased focus on quality indicators and the use of clinical registries for breast cancer for real world studies have shown higher compliance to recommended therapy and better survival. In 2010, the European Society of Breast Cancer Specialist (EUSOMA) proposed quality indicators (QI) covering diagnosis, treatment and follow-up. To become a EUSOMA certified Breast Cancer Unit, 14 specified quality indicators, in addition to other requirements, need to be met. To evaluate the compliance and results of recommended treatment in breast cancer care in Norway and to improve the quality of epidemiological data, the Cancer Registry of Norway (CRN) in cooperation with the Norwegian Breast Cancer Group (NBCG) developed the Norwegian Breast Cancer Registry (NBCR). The objective of this study is to assess the feasibility of using the NBCR for estimating the EUSOMA QI individually for all hospitals diagnosing and treating breast cancer in Norway. Methods To provide researchers with high quality cancer data as well as for the purpose of national cancer statistics, the CRN employs a cancer registry system to 1) longitudinal capture data from all patients from all medical entities that diagnose and/or treat cancer patients (e.g., pathology, radiology and clinical departments) in Norway; 2) curate data, i.e. validate the correctness of collected data, and assemble the validated cancer data as cancer cases; 3) provide data for analytics and presentation. Estimates for 10 EUSOMA QI were calculated at national and hospital level. To compare hospitals, a summary score of QIs was defined for each hospital. Results All hospitals currently treating breast cancer patients have the technical ability to submit data to the NBCR for estimation of QIs defined by EUSOMA. Data from pathology and surgery are of high quality. However, data from oncological and radiological departments are incomplete, but improving. This currently hinders three QIs from being calculated. QI on benign to malign diagnosis needs to be calculated at the individual Breast Centre. Over time the adherence to guidelines have improved and the hospital variation for the respective QI have decreased. Two hospitals met all minimum standard on ten QIs in year 2016 and one hospital did not meet one minimum standard, but met all other targets. Conclusion The NBCR has since 2012 published annual reports on breast cancer care and for the year 2016 measured 10 of 14 QI defined by EUSOMA. Increased compliance of recommended treatment in Norway has been observed during the years the registry has been active.

• Nilssen, Yngvar; Brustugun, Odd Terje; Eriksen, Morten Tandberg; Gulbrandsen, Johanne; Haug, Erik Skaaheim & Naume, Bjørn [Show all 7 contributors for this article] (2019). Decreasing waiting time for treatment before and during implementation of cancer patient pathways in Norway. Cancer Epidemiology. ISSN 1877-7821. 61, p. 59–69. doi: 10.1016/j.canep.2019.05.004. Full text in Research Archive Show summary

  Background: In 2015, Norway implemented cancer patient pathways to reduce waiting times for treatment. The aims of this paper were to describe patterns in waiting time and their association with patient characteristics for colorectal, lung, breast and prostate cancers. Methods: National, population-based data from 2007 to 2016 were used. A multivariable quantile regression examined the association between treatment period, age, stage, sex, place of residence, and median waiting times. Results: Reduction in median waiting times for radiotherapy among colorectal, lung and prostate cancer patients ranged from 14 to 50 days. Median waiting time for surgery remained approximately 21 days for both colorectal and breast cancers, while it decreased by 7 and 36 days for lung and prostate cancers, respectively. The proportion of lung and prostate cancer patients with metastatic disease at the time of diagnosis decreased, while the proportion of colorectal patients with localised disease and patients with stage I breast cancer increased (p < 0.001). After adjusting for case-mix, a patient’s place of residence was significantly associated with waiting time for treatment (p < 0.001), however, differences in waiting time to treatment decreased over the study period. Conclusions: Between 2007 and 2016, Norway experienced improved stage distributions and consistently decreasing waiting times for treatment. While these improvements occurred gradually, no significant change was observed from the time of cancer patient pathway implementation.

• Cosimo, Serena Di; Campbell, Christine; Azim, Hatem A.; Galli, Giulia; Bregni, Giacomo & Curigliano, Giuseppe [Show all 21 contributors for this article] (2018). The use of breast imaging for predicting response to neoadjuvant lapatinib, trastuzumab and their combination in HER2-positive breast cancer: Results from Neo-ALTTO. European Journal of Cancer. ISSN 0959-8049. 89, p. 42–48. doi: 10.1016/j.ejca.2017.10.036. Full text in Research Archive
• Bidard, François-Clément; Michiels, Stefan; Riethdorf, Sabine; Mueller, Volkmar; Esserman, Laura J. & Lucci, Anthony [Show all 50 contributors for this article] (2018). Circulating tumor cells in breast cancer patients treated by neoadjuvant chemotherapy: A Meta-analysis. Journal of the National Cancer Institute. ISSN 0027-8874. 110(6), p. 560–567. doi: 10.1093/jnci/djy018. Full text in Research Archive
• Hugenschmidt, Harald; Labori, Knut Jørgen; Brunborg, Cathrine; Verbeke, Caroline Sophie; Seeberg, Lars Thomas & Schirmer, Cecilie [Show all 10 contributors for this article] (2018). Circulating Tumor Cells are an Independent Predictor of Shorter Survival in Patients Undergoing Resection for Pancreatic and Periampullary Adenocarcinoma. Annals of Surgery. ISSN 0003-4932. 271(3), p. 549–558. doi: 10.1097/SLA.0000000000003035. Full text in Research Archive
• Borgen, Elin; Rypdal, Maria Christine; Sosa, Maria Soledad; Renolen, Anne; Schlichting, Ellen & Lønning, Per Eystein [Show all 9 contributors for this article] (2018). NR2F1 stratifies dormant disseminated tumor cells in breast cancer patients 11 Medical and Health Sciences 1112 Oncology and Carcinogenesis. Breast Cancer Research. ISSN 1465-5411. 20(120), p. 1–13. doi: 10.1186/s13058-018-1049-0. Full text in Research Archive Show summary

  Background: The presence of disseminated tumor cells (DTCs) in bone marrow (BM) is an independent prognostic factor in early breast cancer but does not uniformly predict outcome. Tumor cells can persist in a quiescent state over time, but clinical studies of markers predicting the awakening potential of DTCs are lacking. Recently, experiments have shown that NR2F1 (COUP-TF1) plays a key role in dormancy signaling. Methods: We analyzed the NR2F1 expression in DTCs by double immunofluorescence (DIF) staining of extra cytospins prepared from 114 BM samples from 86 selected DTC-positive breast cancer patients. Samples collected at two or more time points were available for 24 patients. Fifteen samples were also analyzed for the proliferation marker Ki67. Results: Of the patients with detectable DTCs by DIF, 27% had ≥ 50% NR2F1high DTCs, chosen a priori as the cut-off for “dormant profile” classification. All patients with systemic relapse within 12 months after BM aspiration carried ≤ 1% NR2F1high DTCs, including patients who transitioned from having NR2F1high-expressing DTCs in previous BM samples. Of the patients with serial samples, half of those with no relapse at follow-up had ≥ 50% NR2F1high DTCs in the last BM aspiration analyzed. Among the 18 relapse-free patients at the time of the last DTC-positive BM aspiration with no subsequent BM analysis performed, distant disease-free intervals were favorable for patients carrying ≥ 50% NR2F1high DTCs compared with those with predominantly NR2F1low DTCs (p = 0.007, log-rank). No survival difference was observed by classification according to Ki67-expressing DTCs (p = 0.520). Conclusions: Our study translates findings from basic biological analysis of DTC dormancy to the clinical situation and supports further clinical studies of NR2F1 as a marker of dormancy.

• Jabeen, Shakila; Zucknick, Manuela; Nome, Marianne Eikhom; dannenfelser, ruth; Fleischer, Thomas & Kumar, Surendra [Show all 15 contributors for this article] (2018). Serum cytokine levels in breast cancer patients during neoadjuvant treatment with bevacizumab. Oncoimmunology. ISSN 2162-4011. 7(11), p. e1457598-1–e1457598-15. doi: 10.1080/2162402X.2018.1457598. Full text in Research Archive Show summary

  A high concentration of circulating vascular endothelial growth factor (VEGF) in cancer patients is associated with an aggressive tumor phenotype. Here, serum levels of 27 cytokines and blood cell counts were assessed in breast cancer patients receiving neoadjuvant chemotherapy with or without bevacizumab (Bev) in a randomized cohort of 132 patients with non-metastatic HER2-negative tumors. Cytokine levels were determined prior to treatment and at various time-points. The cytotoxic chemotherapy regimen of fluorouracil, epirubicin, and cyclophosphamide (FEC) had a profound impact on both circulating white blood cells and circulating cytokine levels. At the end of FEC treatment, the global decrease in cytokine levels correlated with the drop in white blood cell counts and was significantly greater in the patients of the Bev arm for cytokines, such as VEGF-A, IL-12, IP-10 and IL-10. Among patients who received Bev, those with pathological complete response (pCR) exhibited significantly lower levels of VEGF-A, IFN-γ, TNF-α and IL-4 than patients without pCR. This effect was not observed in the chemotherapy-only arm. Certain circulating cytokine profiles were found to correlate with different immune cell types at the tumor site. For the Bev arm patients, the serum cytokine levels correlated with higher levels of cytotoxic T cells at the end of the therapy regimen, which was indicative of treatment response. The higher response rate for Bev-treated patients and stronger correlations between serum cytokine levels and infiltrating CD8T cells merits further investigation. KEYWORDS: Bevacizumab, VEGF-A, neoadjuvant, cytokines, immunity

• Jabeen, Shakila; Espinoza, Jaime; Torland, Lilly Anne; Zucknick, Manuela; Kumar, Surendra & Haakensen, Vilde Drageset [Show all 15 contributors for this article] (2018). Noninvasive profiling of serum cytokines in breast cancer patients and clinicopathological characteristics. Oncoimmunology. ISSN 2162-4011. :e1537691. doi: 10.1080/2162402X.2018.1537691. Full text in Research Archive Show summary

  Cancers elicit an immune response by modifying the microenvironment. The immune system plays a pivotal role in cancer recognition and eradication. While the potential clinical value of infiltrating lymphocytes at the tumor site has been assessed in breast cancer, circulating cytokines – the molecules coordinating and fine-tuning immune response – are still poorly characterized. Using two breast cancer cohorts (MicMa, n = 131, DCTB, n = 28) and the multiplex Luminex platform, we measured the levels of 27 cytokines in the serum of breast cancer patients prior to treatment. We investigated the cytokine levels in relation to clinicopathological characteristics and in perspective of the tumor infiltrating immune cells predicted from the bulk mRNA expression data. Unsupervised clustering analysis of the serum cytokine levels in the MicMa cohort identified a cluster of pro-inflammatory, pro-angiogenic, and Th2-related cytokines which was associated with poor prognosis. Notably high levels of platelet derived growth factor BB (PDGF) reflected a more aggressive tumor phenotype and larger tumor size. A significant positive correlation between serum levels of interferon gamma-induced protein 10 (IP10) and its mRNA expression at the tumor site suggested that tumor-IP10-production may outflow to the bloodstream. High IP10 serum levels were associated with a worse prognosis. Finally, we found serum levels of both PDGF and IP10 associated with enrichment scores of specific tumor infiltrating immune cells. Our study suggests that monitoring cytokine circulating levels in breast cancer could be used to characterize breast cancers and the immune composition of their microenvironment through readily available biological material. KEYWORDS: Breast cancer, cytokines, pathological markers, immune response, prognosis

• Møller, Elen Kristine; Nord, Silje; Wedge, David C; Lingjærde, Ole Christian; Silwal-Pandit, Laxmi & Gythfeldt, Hedda [Show all 19 contributors for this article] (2018). Time series analysis of neoadjuvant chemotherapy and bevacizumab-treated breast carcinomas reveals a systemic shift in genomic aberrations. Genome Medicine. 10:92. doi: 10.1186/s13073-018-0601-y. Full text in Research Archive Show summary

  Background Chemotherapeutic agents such as anthracyclines and taxanes are commonly used in the neoadjuvant setting. Bevacizumab is an antibody which binds to vascular endothelial growth factor A (VEGFA) and inhibits its receptor interaction, thus obstructing the formation of new blood vessels. Methods A phase II randomized clinical trial of 123 patients with Her2-negative breast cancer was conducted, with patients treated with neoadjuvant chemotherapy (fluorouracil (5FU)/epirubicin/cyclophosphamide (FEC) and taxane), with or without bevacizumab. Serial biopsies were obtained at time of diagnosis, after 12 weeks of treatment with FEC ± bevacizumab, and after 25 weeks of treatment with taxane ± bevacizumab. A time course study was designed to investigate the genomic landscape at the three time points when tumor DNA alterations, tumor percentage, genomic instability, and tumor clonality were assessed. Substantial differences were observed with some tumors changing mainly between diagnosis and at 12 weeks, others between 12 and 25 weeks, and still others changing in both time periods. Results In both treatment arms, good responders (GR) and non-responders (NR) displayed significant difference in genomic instability index (GII) at time of diagnosis. In the combination arm, copy number alterations at 25 loci at the time of diagnosis were significantly different between the GR and NR. An inverse aberration pattern was also observed between the two extreme response groups at 6p22-p12 for patients in the combination arm. Signs of subclonal reduction were observed, with some aberrations disappearing and others being retained during treatment. Increase in subclonal amplification was observed at 6p21.1, a locus which contains the VEGFA gene for the protein which are targeted by the study drug bevacizumab. Of the 13 pre-treatment samples that had a gain at VEGFA, 12 were responders. Significant decrease of frequency of subclones carrying gains at 17q21.32-q22 was observed at 12 weeks, with the peak occurring at TMEM100, an ALK1 receptor signaling-dependent gene essential for vasculogenesis. This implies that cells bearing amplifications of VEGFA and TMEM100 are particularly sensitive to this treatment regime. Conclusions Taken together, these results suggest that heterogeneity and subclonal architecture influence the response to targeted treatment in combination with chemotherapy, with possible implications for clinical decision-making and monitoring of treatment efficacy.

• Bjørklund, Sunniva Maria Stordal; Panda, Anshuman; Kumar, Surendra; Seiler, Michael; Robinson, Doug & Gheeya, Jinesh [Show all 15 contributors for this article] (2017). Widespread alternative exon usage in clinically distinct subtypes of Invasive Ductal Carcinoma. Scientific Reports. 7(1). doi: 10.1038/s41598-017-05537-0. Full text in Research Archive Show summary

  Cancer cells can have different patterns of exon usage of individual genes when compared to normal tissue, suggesting that alternative splicing may play a role in shaping the tumor phenotype. The discovery and identification of gene variants has increased dramatically with the introduction of RNA-sequencing technology, which enables whole transcriptome analysis of known, as well as novel isoforms. Here we report alternative splicing and transcriptional events among subtypes of invasive ductal carcinoma in The Cancer Genome Atlas (TCGA) Breast Invasive Carcinoma (BRCA) cohort. Alternative exon usage was widespread, and although common events were shared among three subtypes, ER+ HER2−, ER− HER2−, and HER2+, many events on the exon level were subtype specific. Additional RNA-seq analysis was carried out in an independent cohort of 43 ER+ HER2− and ER− HER2− primary breast tumors, confirming many of the exon events identified in the TCGA cohort. Alternative splicing and transcriptional events detected in five genes, MYO6, EPB41L1, TPD52, IQCG, and ACOX2 were validated by qRT-PCR in a third cohort of 40 ER+ HER2− and ER− HER2− patients, showing that these events were truly subtype specific.

• Reinertsen, Kristin Valborg; Engebråten, Olav; Loge, Jon Håvard; Småstuen, Milada Cvancarova; Naume, Bjørn & Wist, Erik [Show all 10 contributors for this article] (2017). Fatigue During and After Breast Cancer Therapy—A Prospective Study. Journal of Pain and Symptom Management. ISSN 0885-3924. 53(3), p. 551–560. doi: 10.1016/j.jpainsymman.2016.09.011. Full text in Research Archive Show summary

  Context Chronic fatigue (CF) in breast cancer (BC) survivors is multifactorial and may be caused by immune activation triggered by BC or its treatment. In the Neoadjuvant Avastin in Breast Cancer study, BC patients received neoadjuvant chemotherapy (FEC100→taxane) ± bevacizumab, a monoclonal antibody with fatigue as a potential side effect. Objectives To examine fatigue levels and prevalence of CF before and during chemotherapy and at follow-up, and their associations with C-reactive protein (CRP) and clinical variables. Methods Eighty-four HER2-negative patients with cT2-4N0-3M0 BC responded to questionnaires and had CRP measured before treatment (T0), after FEC100 (T1), after taxanes before surgery (T2), and at two-year follow-up (T3). Results The prevalence of CF increased from 8% at T0 to 36% at T3, P < 0.0001. Fatigue levels peaked during chemotherapy from 12.0 at T0 to 20.0 at T2, and declined to 16.7 at T3, P < 0.001. Women with CF at T3 had higher fatigue levels at T0, T2, and T3 than those without CF (P ≤ 0.01). Psychological distress (P = 0.03) and pain (P = 0.04) at T3 were associated with CF at T3. Only psychological distress remained a significant predictor in multivariate analysis. CRP increased from T0 to T1 (P < 0.01) and declined to baseline values at T3, but changes were not associated with bevacizumab treatment. No association was found between bevacizumab or CRP, and fatigue levels or CF. Conclusion Neither bevacizumab treatment nor low-grade systemic inflammation as measured by CRP was associated with the increased fatigue levels and raised prevalence of CF, observed during and after BC therapy. Increased fatigue levels at baseline and psychological distress at T3 were associated with CF at T3.

• Ohnstad, Hege Oma; Borgen, Elin; Falk, Ragnhild Sørum; Lien, Tonje Gulbrandsen; Aaserud, Marit & Sveli, My Anh Tu [Show all 14 contributors for this article] (2017). Prognostic value of PAM50 and risk of recurrence score in patients with early-stage breast cancer with long-term follow-up. Breast Cancer Research. ISSN 1465-5411. 19(120). doi: 10.1186/s13058-017-0911-9. Full text in Research Archive Show summary

  Background The aim of this study was to investigate the prognostic value of the PAM50 intrinsic subtypes and risk of recurrence (ROR) score in patients with early breast cancer and long-term follow-up. A special focus was placed on hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2−) pN0 patients not treated with chemotherapy. Methods Patients with early breast cancer (n = 653) enrolled in the observational Oslo1 study (1995–1998) were followed for distant recurrence and breast cancer death. Clinicopathological parameters were collected from hospital records. The primary tumors were analyzed using the Prosigna® PAM50 assay to determine the prognostic value of the intrinsic subtypes and ROR score in comparison with pathological characteristics. The primary endpoints were distant disease-free survival (DDFS) and breast cancer-specific survival (BCSS). Results Of 653 tumors, 52.2% were classified as luminal A, 26.5% as luminal B, 10.6% as HER2-enriched, and 10.7% as basal-like. Among the HR+/HER2− patients (n = 476), 37.8% were categorized as low risk by ROR score, 22.7% as intermediate risk, and 39.5% as high risk. Median follow-up durations for BCSS and DDFS were 16.6 and 7.1 years, respectively. Multivariate analysis showed that intrinsic subtypes (all patients) and ROR risk classification (HR+/HER2− patients) yielded strong prognostic information. Among the HR+/HER2− pN0 patients with no adjuvant treatment (n = 231), 53.7% of patients had a low ROR, and their prognosis at 15 years was excellent (15-year BCSS 96.3%). Patients with intermediate risk had reduced survival compared with those with low risk (p = 0.005). In contrast, no difference in survival between the low- and intermediate-risk groups was seen for HR+/HER2− pN0 patients who received tamoxifen only. Ki-67 protein, grade, and ROR score were analyzed in the unselected, untreated pT1pN0 HR+/HER2− population (n = 171). In multivariate analysis, ROR score outperformed both Ki-67 and grade. Furthermore, 55% of patients who according to the PREDICT tool ( http://www.predict.nhs.uk/ ) would be considered chemotherapy candidates were ROR low risk (33%) or luminal A ROR intermediate risk (22%). Conclusions The PAM50 intrinsic subtype classification and ROR score improve classification of patients with breast cancer into prognostic groups, allowing for a more precise identification of future recurrence risk and providing an improved basis for adjuvant treatment decisions. Node-negative patients with low ROR scores had an excellent outcome at 15 years even in the absence of adjuvant therapy.

• Fleischer, Thomas; Tekpli, Xavier; Mathelier, Anthony; Wang, Shixiong; Nebdal, Daniel J.H. & Dhakal, Hari Prasad [Show all 16 contributors for this article] (2017). DNA methylation at enhancers identifies distinct breast cancer lineages. Nature Communications. 8(1379). doi: 10.1038/s41467-017-00510-x. Full text in Research Archive Show summary

  Breast cancers exhibit genome-wide aberrant DNA methylation patterns. To investigate how these affect the transcriptome and which changes are linked to transformation or progression, we apply genome-wide expression–methylation quantitative trait loci (emQTL) analysis between DNA methylation and gene expression. On a whole genome scale, in cis and in trans, DNA methylation and gene expression have remarkably and reproducibly conserved patterns of association in three breast cancer cohorts (n = 104, n = 253 and n = 277). The expression–methylation quantitative trait loci associations form two main clusters; one relates to tumor infiltrating immune cell signatures and the other to estrogen receptor signaling. In the estrogen related cluster, using ChromHMM segmentation and transcription factor chromatin immunoprecipitation sequencing data, we identify transcriptional networks regulated in a cell lineage-specific manner by DNA methylation at enhancers. These networks are strongly dominated by ERα, FOXA1 or GATA3 and their targets were functionally validated using knockdown by small interfering RNA or GRO-seq analysis after transcriptional stimulation with estrogen.

• Aure, Miriam Ragle; Vitelli, Valeria; Jernström, Sandra Johanna; Kumar, Surendra; Krohn, Marit & Due, Eldri Undlien [Show all 35 contributors for this article] (2017). Integrative clustering reveals a novel split in the luminal A subtype of breast cancer with impact on outcome. Breast Cancer Research. ISSN 1465-5411. 19(44). doi: 10.1186/s13058-017-0812-y. Full text in Research Archive Show summary

  Background Breast cancer is a heterogeneous disease at the clinical and molecular level. In this study we integrate classifications extracted from five different molecular levels in order to identify integrated subtypes. Methods Tumor tissue from 425 patients with primary breast cancer from the Oslo2 study was cut and blended, and divided into fractions for DNA, RNA and protein isolation and metabolomics, allowing the acquisition of representative and comparable molecular data. Patients were stratified into groups based on their tumor characteristics from five different molecular levels, using various clustering methods. Finally, all previously identified and newly determined subgroups were combined in a multilevel classification using a “cluster-of-clusters” approach with consensus clustering. Results Based on DNA copy number data, tumors were categorized into three groups according to the complex arm aberration index. mRNA expression profiles divided tumors into five molecular subgroups according to PAM50 subtyping, and clustering based on microRNA expression revealed four subgroups. Reverse-phase protein array data divided tumors into five subgroups. Hierarchical clustering of tumor metabolic profiles revealed three clusters. Combining DNA copy number and mRNA expression classified tumors into seven clusters based on pathway activity levels, and tumors were classified into ten subtypes using integrative clustering. The final consensus clustering that incorporated all aforementioned subtypes revealed six major groups. Five corresponded well with the mRNA subtypes, while a sixth group resulted from a split of the luminal A subtype; these tumors belonged to distinct microRNA clusters. Gain-of-function studies using MCF-7 cells showed that microRNAs differentially expressed between the luminal A clusters were important for cancer cell survival. These microRNAs were used to validate the split in luminal A tumors in four independent breast cancer cohorts. In two cohorts the microRNAs divided tumors into subgroups with significantly different outcomes, and in another a trend was observed. Conclusions The six integrated subtypes identified confirm the heterogeneity of breast cancer and show that finer subdivisions of subtypes are evident. Increasing knowledge of the heterogeneity of the luminal A subtype may add pivotal information to guide therapeutic choices, evidently bringing us closer to improved treatment for this largest subgroup of breast cancer.

• Helland, Thomas; Henne, Nina; Bifulco, Ersilia; Naume, Bjørn; Borgen, Elin & Kristensen, Vessela N. [Show all 15 contributors for this article] (2017). Serum concentrations of active tamoxifen metabolites predict long-term survival in adjuvantly treated breast cancer patients. Breast Cancer Research. ISSN 1465-5411. 19(1). doi: 10.1186/s13058-017-0916-4. Full text in Research Archive Show summary

  Background Controversies exist as to whether the genetic polymorphisms of the enzymes responsible for the metabolism of tamoxifen can predict breast cancer outcome in patients using adjuvant tamoxifen. Direct measurement of concentrations of active tamoxifen metabolites in serum may be a more biological plausible and robust approach. We have investigated the association between CYP2D6 genotypes, serum concentrations of active tamoxifen metabolites, and long-term outcome in tamoxifen treated breast cancer patients. Methods From an original observational study comprising 817 breast cancer patients, 99 women with operable breast cancer were retrospectively included in the present study. This cohort of patients were adjuvantly treated with tamoxifen, had provided serum samples suitable for measuring tamoxifen metabolites, and were relapse-free at 3 years after the primary treatment commenced. The median follow-up time from this entry point to breast cancer death was 13.9 years. Patients were CYP2D6 genotyped and grouped into four CYP2D6 phenotype groups (Ultra rapid, extensive, intermediate, and poor metabolizers). Tamoxifen and nine metabolites were quantified in serum (n = 86) and compared with CYP2D6 phenotype groups and outcome. Results Breast cancer patients with low concentrations of Z-4-hydroxy-tamoxifen (Z-4OHtam; ≤ 3.26 nM) had a breast cancer-specific survival (BCSS) of 60% compared to 84% in patients with Z-4OHtam concentrations > 3.26 nM (p = 0.020, log-rank hazard ratio (HR) = 3.56, 95% confidence interval (CI) = 1.14–11.07). For patients with Z-4-hydroxy-N-desmethyl-tamoxifen (Z-endoxifen) levels ≤ 9.00 nM BCSS was 57% compared to 84% for patients with concentrations > 9.00 nM (p = 0.029, HR = 3.73, 95% CI = 1.05–13.22). Low concentrations of Z-4OHtam and Z-endoxifen were associated with poorer survival also after adjusting for clinically relevant variables (HR = 4.27, 95% CI = 1.35–13.58, and HR = 3.70, 95% CI = 1.03–13.25, respectively). Overall survival analysis showed similar survival differences for both active metabolites. The Antiestrogen Activity Score showed comparable effects, but did not improve the prognostic information. Conclusions Patients with Z-4OHtam and Z-endoxifen concentrations lower than 3.26 nM or 9.00 nM, respectively, showed an adverse outcome. Our results suggest that direct measurement of active tamoxifen metabolite concentrations could be of clinical value. Validation in larger study cohorts is warranted.

• Egeland, Eivind Valen; Pedersen, Kjetil Boye; Park, Daehoon; Synnestvedt, Marit; Sauer, Torill & Naume, Bjørn [Show all 27 contributors for this article] (2017). Prognostic significance of S100A4-expression and subcellular localization in early-stage breast cancer. Breast Cancer Research and Treatment. ISSN 0167-6806. 162(1), p. 127–137. doi: 10.1007/s10549-016-4096-1. Full text in Research Archive Show summary

  Purpose: <br> Prognostic factors are useful in order to identify early-stage breast cancer patients who might benefit from adjuvant treatment. The metastasis-promoting protein S100A4 has previously been associated with poor prognosis in breast cancer patients. The protein is expressed in diverse subcellular compartments, including the cytoplasm, extracellular space, and nucleus. Nuclear expression is an independent predictor of poor outcome in several cancer types, but the significance of subcellular expression has not yet been assessed in breast cancer. <br>Methods: <br> Nuclear and cytoplasmic expression of S100A4 was assessed by immunohistochemistry in prospectively collected tumor samples from early-stage breast cancer patients using tissue microarrays. <br>Results: <br> In patients not receiving adjuvant systemic therapy, nuclear or cytoplasmic expression was found in 44/291 tumors (15%). Expression of either nuclear or cytoplasmic S100A4 was associated with histological grade III, triple-negative subtype, and Ki-67-expression. Patients with S100A4-positive tumors had inferior metastasis-free and overall survival compared to S100A4-negative. When expression was analyzed separately, nuclear S100A4 was a significant predictor of outcome, while cytoplasmic was not. In patients who received adjuvant treatment 23/300 tumors (8%) were S100A4-positive, but no tumors displayed nuclear staining alone. S100A4-expression was strongly associated with histological grade III and triple-negative subtype. Although not significant, metastasis-free and overall survival was numerically reduced in patients with S100A4-positive tumors. <br>Conclusion: <br> S100A4-expression was associated with poor outcome in early-stage breast cancer, but the low percentage of positive tumors and the modest survival differences imply that the clinical utility in selection of patients for adjuvant treatment is limited.

• Nordlund, Marianne Sparby; Westgaard, Arne; Paus, Elisabeth; Øijordsbakken, Miriam; Henanger, Karoline Johansen & Naume, Bjørn [Show all 7 contributors for this article] (2017). Specific antibodies and sensitive immunoassays for the human epidermal growth factor receptors (HER2, HER3, and HER4). Tumour Biology. ISSN 1010-4283. 39(6). doi: 10.1177/1010428317707436. Full text in Research Archive Show summary

  The use of trastuzumab in patients with breast cancer that overexpresses human epidermal growth factor receptor 2 has significantly improved treatment outcomes. However, a substantial proportion of this patient group still experiences progression of the disease after receiving the drug. Evaluation of the changes in expression of the human epidermal growth factor receptors could be of interest. Monoclonal antibodies against the extracellular domain of the human growth factor receptors, 2, 3, and 4, have been raised, and specific and sensitive immunoassays have been established. Sera from healthy individuals (Nordic Reference Interval Project and Database) were analyzed in the human epidermal growth factor receptor 2 assay (N = 805) and the human epidermal growth factor receptor 3 and 4 assays (N = 114), and reference limits were calculated. In addition, sera from 208 individual patients with breast cancer were tested in all three assays. Finally, the human epidermal growth factor receptor 2 assay was compared with a chemiluminescent immunoassay for serum human epidermal growth factor receptor 2/neu. Reference values were as follows: human epidermal growth factor receptor 2, <2.5 µg/L; human epidermal growth factor receptor 3, <2.8 µg/L; and human epidermal growth factor receptor 4, <1.8 µg/L. There were significant differences in human epidermal growth factor receptor 2 and human epidermal growth factor receptor 3 serum levels between the patients with tissue human epidermal growth factor receptor 2–positive and tissue human epidermal growth factor receptor 2–negative (p = 0.0026, p = 0.000011) tumors, but not in the serum levels of human epidermal growth factor receptor 4 (p = 0.054). There was good agreement between the in-house human epidermal growth factor receptor 2 assay and the chemiluminescent immunoassay. Our new specific antibodies for all the three human epidermal growth factor receptors may prove valuable in the development of novel anti-human epidermal growth factor receptor targeted therapies with sensitive immunoassays for measuring serum levels of the respective targets and in monitoring established treatment.

• Gingras, Isabelle; Holmes, Eileen; Azambuja, Evandro De; Nguyen, David H.A.; Izquierdo, Miguel & Zujewski, Jo Anne [Show all 16 contributors for this article] (2017). Regional Nodal Irradiation after Breast Conserving Surgery for Early HER2-Positive Breast Cancer: Results of a Subanalysis from the ALTTO Trial. Journal of the National Cancer Institute. ISSN 0027-8874. 109(8:djw331), p. 1–7. doi: 10.1093/jnci/djw331. Full text in Research Archive Show summary

  Background: Two randomized trials recently demonstrated that regional nodal irradiation (RNI) could reduce the risk of recurrence in early breast cancer; however, these trials were conducted in the pretrastuzumab era. Whether these results are applicable to human epidermal growth factor receptor 2 (HER2)–positive breast cancer patients treated with anti-HER2-targeted therapy is unknown. Methods: This retrospective analysis was performed on patients with node-positive breast cancer who were enrolled in the Adjuvant Lapatinib and/or Trastuzumab Treatment Optimization phase III adjuvant trial and subjected to BCS. The primary objective of the present study was to examine the effect of RNI on disease-free survival (DFS). A multivariable cox regression analysis adjusted for number of positive lymph nodes, tumor size, grade, age, hormone receptors status, presence of macrometastatis, treatment arm, and chemotherapy timing was carried out to investigate the relationship between RNI and DFS. Results: One thousand six hundred sixty-four HER2-positive breast cancer patients were included, of whom 878 (52.8%) had received RNI to the axillary, supraclavicular, and/or internal mammary lymph nodes. Patients in the RNI group had higher nodal burden and more frequently had tumors larger than 2 cm. At a median follow-up of 4.5 years, DFS was 84.3% in the RNI group and 88.3% in the non-RNI group. No differences in regional recurrence (0.9 % vs 0.6 %) or in overall survival (93.6% vs 95.3%) were observed between the two groups. After adjustment in multivariable analysis, there was no statistically significant association between RNI and DFS (hazard ratio = 0.96, 95% confidence interval = 0.71 to 1.29). Conclusions: Our analysis did not demonstrate a DFS benefit of RNI in HER2-positive, node-positive patients treated with adjuvant HER2-targeted therapy. The benefit of RNI in HER2-positive breast cancer needs further testing within randomized clinical trials.

• Silwal-Pandit, Laxmi; Nord, Silje; Gythfeldt, Hedda; Møller, Elen Kristine; Fleischer, Thomas & Rødland, Einar Andreas [Show all 26 contributors for this article] (2017). The longitudinal transcriptional response to neoadjuvant chemotherapy with and without bevacizumab in breast cancer. Clinical Cancer Research. ISSN 1078-0432. 23(16), p. 4662–4670. doi: 10.1158/1078-0432.CCR-17-0160. Full text in Research Archive Show summary

  Purpose: Chemotherapy-induced alterations to gene expression are due to transcriptional reprogramming of tumor cells or subclonal adaptations to treatment. The effect on whole-transcriptome mRNA expression was investigated in a randomized phase II clinical trial to assess the effect of neoadjuvant chemotherapy with the addition of bevacizumab. Experimental Design: Tumor biopsies and whole-transcriptome mRNA profiles were obtained at three fixed time points with 66 patients in each arm. Altogether, 358 specimens from 132 patients were available, representing the transcriptional state before treatment start, at 12 weeks and after treatment (25 weeks). Pathologic complete response (pCR) in breast and axillary nodes was the primary endpoint. Results: pCR was observed in 15 patients (23%) receiving bevacizumab and chemotherapy and 8 patients (12%) receiving only chemotherapy. In the estrogen receptor–positive patients, 11 of 54 (20%) treated with bevacizumab and chemotherapy achieved pCR, while only 3 of 57 (5%) treated with chemotherapy reached pCR. In patients with estrogen receptor–positive tumors treated with combination therapy, an elevated immune activity was associated with good response. Proliferation was reduced after treatment in both treatment arms and most pronounced in the combination therapy arm, where the reduction in proliferation accelerated during treatment. Transcriptional alterations during therapy were subtype specific, and the effect of adding bevacizumab was most evident for luminal-B tumors. Conclusions: Clinical response and gene expression response differed between patients receiving combination therapy and chemotherapy alone. The results may guide identification of patients likely to benefit from antiangiogenic therapy.

• Seeberg, Lars Thomas; Brunborg, Cathrine; Waage, Anne; Hugenschmidt, Harald; Renolen, Anne & Stav, Ingunn [Show all 11 contributors for this article] (2017). Survival Impact of Primary Tumor Lymph Node Status and Circulating Tumor Cells in Patients with Colorectal Liver Metastases. Annals of Surgical Oncology. ISSN 1068-9265. 24(8), p. 2113–2121. doi: 10.1245/s10434-017-5818-2. Full text in Research Archive
• dannenfelser, ruth; Nome, Marianne Eikhom; Tahiri, Andliena; Ursini-Siegel, Josie; Vollan, Hans Kristian Moen & Haakensen, Vilde Drageset [Show all 12 contributors for this article] (2017). Data-driven analysis of immune infiltrate in a large cohort of breast cancer and its association with disease progression, ER activity, and genomic complexity. OncoTarget. 8(34), p. 57121–57133. doi: 10.18632/oncotarget.19078. Full text in Research Archive Show summary

  The tumor microenvironment is now widely recognized for its role in tumor progression, treatment response, and clinical outcome. The intratumoral immunological landscape, in particular, has been shown to exert both pro-tumorigenic and anti-tumorigenic effects. Identifying immunologically active or silent tumors may be an important indication for administration of therapy, and detecting early infiltration patterns may uncover factors that contribute to early risk. Thus far, direct detailed studies of the cell composition of tumor infiltration have been limited; with some studies giving approximate quantifications using immunohistochemistry and other small studies obtaining detailed measurements by isolating cells from excised tumors and sorting them using flow cytometry. Herein we utilize a machine learning based approach to identify lymphocyte markers with which we can quantify the presence of B cells, cytotoxic T-lymphocytes, T-helper 1, and T-helper 2 cells in any gene expression data set and apply it to studies of breast tissue. By leveraging over 2,100 samples from existing large scale studies, we are able to find an inherent cell heterogeneity in clinically characterized immune infiltrates, a strong link between estrogen receptor activity and infiltration in normal and tumor tissues, changes with genomic complexity, and identify characteristic differences in lymphocyte expression among molecular groupings. With our extendable methodology for capturing cell type specific signal we systematically studied immune infiltration in breast cancer, finding an inverse correlation between beneficial lymphocyte infiltration and estrogen receptor activity in normal breast tissue and reduced infiltration in estrogen receptor negative tumors with high genomic complexity.

• Demeulemeester, Jonas; Kumar, Parveen; Møller, Elen Kristine; Nord, Silje; Wedge, David C. & Peterson, April [Show all 20 contributors for this article] (2016). Tracing the origin of disseminated tumor cells in breast cancer using single-cell sequencing. Genome Biology. ISSN 1465-6906. 17(1). doi: 10.1186/s13059-016-1109-7. Full text in Research Archive Show summary

  Background Single-cell micro-metastases of solid tumors often occur in the bone marrow. These disseminated tumor cells (DTCs) may resist therapy and lay dormant or progress to cause overt bone and visceral metastases. The molecular nature of DTCs remains elusive, as well as when and from where in the tumor they originate. Here, we apply single-cell sequencing to identify and trace the origin of DTCs in breast cancer. Results We sequence the genomes of 63 single cells isolated from six non-metastatic breast cancer patients. By comparing the cells’ DNA copy number aberration (CNA) landscapes with those of the primary tumors and lymph node metastasis, we establish that 53% of the single cells morphologically classified as tumor cells are DTCs disseminating from the observed tumor. The remaining cells represent either non-aberrant “normal” cells or “aberrant cells of unknown origin” that have CNA landscapes discordant from the tumor. Further analyses suggest that the prevalence of aberrant cells of unknown origin is age-dependent and that at least a subset is hematopoietic in origin. Evolutionary reconstruction analysis of bulk tumor and DTC genomes enables ordering of CNA events in molecular pseudo-time and traced the origin of the DTCs to either the main tumor clone, primary tumor subclones, or subclones in an axillary lymph node metastasis. Conclusions Single-cell sequencing of bone marrow epithelial-like cells, in parallel with intra-tumor genetic heterogeneity profiling from bulk DNA, is a powerful approach to identify and study DTCs, yielding insight into metastatic processes. A heterogeneous population of CNA-positive cells is present in the bone marrow of non-metastatic breast cancer patients, only part of which are derived from the observed tumor lineages.

• Easton, Douglas F; Lesueur, Fabienne; Decker, Brennan; Michailidou, Kyriaki; Li, Jun & Allen, Jamie [Show all 149 contributors for this article] (2016). No evidence that protein truncating variants in BRIP1 are associated with breast cancer risk: Implications for gene panel testing. Journal of Medical Genetics. ISSN 0022-2593. 53(5), p. 298–309. doi: 10.1136/jmedgenet-2015-103529. Full text in Research Archive
• Jerusalem, Guy; Mariani, Gabriella; Ciruelos, E.M.; Martin, M.; Tjan-Heijnen, V.C.G & Neven, P. [Show all 18 contributors for this article] (2016). Safety of everolimus plus exemestane in patients with hormone-receptor–positive, HER2–negative locally advanced or metastatic breast cancer progressing on prior non-steroidal aromatase inhibitors: primary results of a phase IIIb, open-label, single-arm, expanded-access multicenter trial (BALLET). Annals of Oncology. ISSN 0923-7534. 27(9), p. 1719–1725. doi: 10.1093/annonc/mdw249. Full text in Research Archive Show summary

  Background This European phase IIIb, expanded-access multicenter trial evaluated the safety of EVE plus EXE in a patient population similar to BOLERO-2. Patients and methods Post-menopausal women aged ≥18 years with hormone receptor-positive, human epidermal growth factor-receptor-2–negative advanced breast cancer (ABC) recurring/progressing during/after prior non-steroidal aromatase inhibitors were enrolled. The primary objective was safety of EVE plus EXE based on frequency of adverse events (AEs), and serious AEs (SAEs). The secondary objective was to evaluate AEs of grade 3/4 severity. Results The median treatment duration was 5.1 months [95% confidence interval (CI) 4.8–5.6] for EVE and 5.3 months (95% CI 4.8–5.6) for EXE. Overall, 2131 patients were included in the analysis; 81.8% of patients experienced EVE- or EXE-related or EVE/EXE-related AEs (investigator assessed); 27.2% were of grade 3/4 severity. The most frequently reported non-hematologic AEs were (overall %, % EVE-related) stomatitis (52.8%; 50.8%) and asthenia (22.8%; 14.6%). The most frequently reported hematologic AEs were (overall %, % EVE-related) anemia (14.4%; 8.1%) and thrombocytopenia (5.9%; 4.6%). AE-related treatment discontinuations were higher in elderly (≥70 years) versus non-elderly patients (23.8% versus 13.0%). The incidence of EVE-related AEs in both elderly and non-elderly patients appeared to be lower in first-line ABC versus later lines. The incidence of AEs (including stomatitis/pneumonitis) was independent of BMI status (post hoc analysis). Overall, 8.5% of patients experienced at least one EVE-related SAE. Of the 121 on-treatment deaths (5.7%), 66 (3.1%) deaths were due to disease progression and 46 (2.2%) due to AEs; 4 deaths were suspected to be EVE-related. Conclusions This is the largest ever reported safety dataset on a general patient population presenting ABC treated with EVE plus EXE and included a sizeable elderly subset. Although the patients were more heavily pretreated, the safety profile of EVE plus EXE in BALLET was consistent with BOLERO-2. Clinical trial registration EudraCT Number: 2012-000073-23. This article has been accepted for publication in Annals of Oncology. Published by Oxford University Press.

• Sas-Chen, Aldema; Aure, Miriam Ragle; Leibovich, Limor; Carvalho, Silvia; Enuka, Yehoshua & Körner, Cindy [Show all 38 contributors for this article] (2016). LIMT is a novel metastasis inhibiting lncRNA suppressed by EGF and downregulated in aggressive breast cancer. EMBO Molecular Medicine. ISSN 1757-4676. 8(9), p. 1052–1064. doi: 10.15252/emmm.201606198. Full text in Research Archive Show summary

  Long noncoding RNAs (lncRNAs) are emerging as regulators of gene expression in pathogenesis, including cancer. Recently, lncRNAs have been implicated in progression of specific subtypes of breast cancer. One aggressive, basal‐like subtype associates with increased EGFR signaling, while another, the HER2‐enriched subtype, engages a kin of EGFR. Based on the premise that EGFR‐regulated lncRNAs might control the aggressiveness of basal‐like tumors, we identified multiple EGFR‐inducible lncRNAs in basal‐like normal cells and overlaid them with the transcriptomes of over 3,000 breast cancer patients. This led to the identification of 11 prognostic lncRNAs. Functional analyses of this group uncovered LINC01089 (here renamed LncRNA Inhibiting Metastasis; LIMT), a highly conserved lncRNA, which is depleted in basal‐like and in HER2‐positive tumors, and the low expression of which predicts poor patient prognosis. Interestingly, EGF rapidly downregulates LIMT expression by enhancing histone deacetylation at the respective promoter. We also find that LIMT inhibits extracellular matrix invasion of mammary cells in vitro and tumor metastasis in vivo. In conclusion, lncRNAs dynamically regulated by growth factors might act as novel drivers of cancer progression and serve as prognostic biomarkers.

• Fleischer, Thomas; Klajic, Jovana; Aure, Miriam Ragle; Louhimo, Riku; Pladsen, Arne Valebjørg & Ottestad, Lars [Show all 18 contributors for this article] (2016). DNA methylation signature (SAM40) identifies subgroups of the Luminal A breast cancer samples with distinct survival. OncoTarget. 8(1), p. 1074–1082. doi: 10.18632/oncotarget.13718. Full text in Research Archive
• Haakensen, Vilde Drageset; Steinfeld, Israel; Saldova, Radka; Shehni, Akram Asadi; Kifer, Ilona & Naume, Bjørn [Show all 9 contributors for this article] (2016). Serum N-glycan analysis in breast cancer patients - Relation to tumour biology and clinical outcome. Molecular Oncology. ISSN 1574-7891. 10(1), p. 59–72. doi: 10.1016/j.molonc.2015.08.002. Full text in Research Archive
• Seeberg, Lars Thomas; Waage, Anne; Brunborg, Cathrine; Hugenschmidt, Harald; Renolen, Anne & Stav, Ingunn [Show all 11 contributors for this article] (2015). Circulating Tumor Cells in Patients With Colorectal Liver Metastasis Predict Impaired Survival. Annals of Surgery. ISSN 0003-4932. 261(1), p. 164–171. doi: 10.1097/SLA.0000000000000580. Full text in Research Archive
• Brudvik, Kristoffer Watten; Seeberg, Lars Thomas; Hugenschmidt, Harald; Renolen, Anne; Schirmer, Cecilie & Brunborg, Cathrine [Show all 11 contributors for this article] (2015). Detection of Circulating Tumor Cells at Surgery and at Follow-Up Assessment to Predict Survival After Two-Stage Liver Resection of Colorectal Liver Metastases. Annals of Surgical Oncology. ISSN 1068-9265. 22(12), p. 4029–4037. doi: 10.1245/s10434-015-4482-7. Full text in Research Archive
• Bjørklund, Sunniva Maria Stordal; Kristensen, Vessela N.; Seiler, Michael; Kumar, Surendra; Grenaker, Grethe Irene & Ming, Yao [Show all 12 contributors for this article] (2015). Expression of an estrogen-regulated variant transcript of the peroxisomal branched chain fatty acid oxidase ACOX2 in breast carcinomas. BMC Cancer. 15(1). doi: 10.1186/s12885-015-1510-8. Full text in Research Archive Show summary

  Background Alternate transcripts from a single gene locus greatly enhance the combinatorial flexibility of the human transcriptome. Different patterns of exon usage have been observed when comparing normal tissue to cancers, suggesting that variant transcripts may play a role in the tumor phenotype. Methods Ribonucleic acid-sequencing (RNA-seq) data from breast cancer samples was used to identify an intronic start variant transcript of Acyl-CoA oxidase 2, ACOX2 (ACOX2-i9). Difference in expression between Estrogen Receptor (ER) positive and ER negative patients was assessed by the Wilcoxon rank sum test, and the findings validated in The Cancer Genome Atlas (TCGA) breast cancer dataset (BRCA). ACOX2-i9 expression was also assessed in cell lines using both quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blot analysis. Knock down by short hairpin RNA (shRNA) and colony formation assays were used to determine whether ACOX2-i9 expression would influence cellular fitness. The effect of ACOX2-i9 expression on patient survival was assessed by the Kaplan-Meier survival function, and association to clinical parameters was analyzed using a Fisher exact test. Results The expression and translation of ACOX2-i9 into a 25 kDa protein was demonstrated in HepG2 cells as well as in several breast cancer cell lines. shRNA knock down of the ACOX2-i9 variant resulted in decreased cell viability of T47D and MDA-MB 436 cells. Moreover, expression of ACOX2-i9 was shown to be estrogen regulated, being induced by propyl pyrazoletriol and inhibited by tamoxifen and fulvestrant in ER+ T47D and Mcf-7 cells, but not in the ER- MDA-MB 436 cell line. This variant transcript showed expression predominantly in ER-positive breast tumors as assessed in our initial set of 53 breast cancers and further validated in 87 tumor/normal pairs from the TCGA breast cancer dataset, and expression was associated with better outcome in ER positive patients. Conclusions ACOX2-i9 is specifically enriched in ER+ breast cancers where expression of the variant is associated with improved outcome. These data identify variant ACOX2 as a potential novel therapeutic biomarker in ER+ breast tumors.

• Rye, Inga Hansine; Lundin, Pär; Månér, Susanne; Fjelldal, Renathe; Naume, Bjørn & Wigler, Michael [Show all 10 contributors for this article] (2015). Quantitative multigene FISH on breast carcinomas identifies der(1;16)(q10;p10) as an early event in luminal A tumors. Genes, Chromosomes and Cancer. ISSN 1045-2257. 54(4), p. 235–248. doi: 10.1002/gcc.22237. Full text in Research Archive
• Silwal-Pandit, Laxmi; Russnes, Hege Elisabeth Giercksky; Borgen, Elin; Vang, Veronica Okkenhaug; Vollan, Hans Kristian Moen & Schlichting, Ellen [Show all 11 contributors for this article] (2015). The sub-cellular localization of WRAP53 has prognostic impact in breast cancer. PLOS ONE. 10:e0139965(10). doi: 10.1371/journal.pone.0139965. Full text in Research Archive Show summary

  WRAP53 protein controls intracellular trafficking of DNA repair proteins, the telomerase enzyme, and splicing factors. Functional loss of the protein has been linked to carcinogenesis, premature aging and neurodegeneration. The aim of this study was to investigate the prognostic significance of WRAP53 protein expression in breast cancer. A tissue microarray was constructed from primary breast tumors and immunostained by a polyclonal WRAP53 antibody to assess the protein expression pattern. Two different patient cohorts with long term follow-up were studied; a test- and a validation set of 154 and 668 breast tumor samples respectively. Breast cancer patients with tumor cells lacking the expression of WRAP53 in the nucleus had a significantly poorer outcome compared to patients with tumor cells expressing this protein in the nuclei (HR = 1.95, 95%CI = 1.09–3.51, p = 0.025). Nuclear localization of WRAP53 was further shown to be an independent marker of prognosis in multivariate analysis (HR = 2.57, 95%CI = 1.27–5.19, p = 0.008), and also significantly associated with better outcome in patients with TP53 mutation. Here we show that the sub-cellular localization of the WRAP53 protein has a significant impact on breast cancer survival, and thus has a potential as a clinical marker in diagnostics and treatment.

• Sahlberg, Kristine Kleivi; Bottai, Giulia; Naume, Bjørn; Burwinkel, Barbara; Calin, George A. & Børresen-Dale, Anne-Lise [Show all 7 contributors for this article] (2014). A Serum MicroRNA Signature Predicts Tumor Relapse and Survival in Triple-Negative Breast Cancer Patients. Clinical Cancer Research. ISSN 1078-0432. 21, p. 1207–1214. doi: 10.1158/1078-0432.CCR-14-2011. Full text in Research Archive
• Reiche, Kristin; Kasack, Katharina; Schreiber, Stephan; Luders, Torben; Due, Eldri Undlien & Naume, Bjørn [Show all 12 contributors for this article] (2014). Long non-coding rnas differentially expressed between normal versus primary breast tumor tissues disclose converse changes to breast cancer-related protein-coding genes. PLOS ONE. 9(9). doi: 10.1371/journal.pone.0106076. Full text in Research Archive
• Gilje, Bjørnar; Nordgård, Oddmund; Tjensvoll, Kjersti; Borgen, Elin; Synnestvedt, Marit & Smaaland, Rune [Show all 7 contributors for this article] (2014). Comparison of molecular and immunocytochemical methods for detection of disseminated tumor cells in bone marrow from early breast cancer patients. BMC Cancer. 14(1). doi: 10.1186/1471-2407-14-514. Full text in Research Archive
• Naume, Bjørn; Synnestvedt, Marit; Falk, Ragnhild Sørum; Wiedswang, Gro; Weyde, Kjetil & Risberg, Terje [Show all 15 contributors for this article] (2014). Clinical Outcome With Correlation to Disseminated Tumor Cell (DTC) Status After DTC-Guided Secondary Adjuvant Treatment With Docetaxel in Early Breast Cancer. Journal of Clinical Oncology. ISSN 0732-183X. 32(34), p. 3848–3857. doi: 10.1200/JCO.2014.56.9327. Full text in Research Archive Show summary

  Purpose: The presence of disseminated tumor cells (DTCs) in bone marrow (BM) predicts survival in early breast cancer. This study explores the use of DTCs for identification of patients insufficiently treated with adjuvant therapy so they can be offered secondary adjuvant treatment and the subsequent surrogate marker potential of DTCs for outcome determination. Patients and Methods: Patients with early breast cancer who had completed six cycles of adjuvant fluorouracil, epirubicin, and cyclophosphamide (FEC) chemotherapy underwent BM aspiration 2 to 3 months (BM1) and 8 to 9 months (BM2) after FEC. Presence of DTCs in BM was determined by immunocytochemistry using pan-cytokeratin monoclonal antibodies. If one or more DTCs were present at BM2, six cycles of docetaxel (100 mg/m2, once every 3 weeks) were administered, followed by DTC analysis 1 and 13 months after the last docetaxel infusion (after treatment). Cox regression analysis was used to evaluate disease-free interval (DFI). Results: Of 1,066 patients with a DTC result at BM2 and available follow-up information (median follow-up, 71.9 months from the time of BM2), 7.2% were DTC positive. Of 72 docetaxel-treated patients analyzed for DTCs after treatment, 15 (20.8%) had persistent DTCs. Patients with remaining DTCs had markedly reduced DFI (46.7% experienced relapse) compared with patients with no DTCs after treatment (adjusted hazard ratio, 7.58; 95% CI, 2.3 to 24.7). The docetaxel-treated patients with no DTCs after treatment had comparable DFI (8.8% experienced relapse) compared with those with no DTCs both at BM1 and BM2 (12.7% experienced relapse; P = .377, log-rank test). Conclusion: DTC status identifies high-risk patients after FEC chemotherapy, and DTC monitoring status after secondary treatment with docetaxel correlated strongly with survival. This emphasizes the potential for DTC analysis as a surrogate marker for adjuvant treatment effect in breast cancer.

• Coleman, Robert; Aksnes, Anne-Kirsti; Naume, Bjørn; Garcia, Camilo; Jerusalem, Guy & Piccart, Martine [Show all 9 contributors for this article] (2014). A phase IIa, nonrandomized study of radium-223 dichloride in advanced breast cancer patients with bone-dominant disease. Breast Cancer Research and Treatment. ISSN 0167-6806. 145(2), p. 411–418. doi: 10.1007/s10549-014-2939-1. Full text in Research Archive
• Fleischer, Thomas; Edvardsen, Hege; Solvang, Hiroko Kato; Daviaud, Christian; Naume, Bjørn & Børresen-Dale, Anne-Lise [Show all 8 contributors for this article] (2014). Integrated analysis of high-resolution DNA methylation profiles, gene expression, germline genotypes and clinical end points in breast cancer patients. International Journal of Cancer. ISSN 0020-7136. 134(11), p. 2615–2625. doi: 10.1002/ijc.28606. Full text in Research Archive
• Fleischer, Thomas; Frigessi, Arnoldo; Johnson, Kevin C.; Edvardsen, Hege; Touleimat, Nizar & Klajic, Jovana [Show all 15 contributors for this article] (2014). Genome-wide DNA methylation profiles in progression to in situ and invasive carcinoma of the breast with impact on gene transcription and prognosis. Genome Biology. ISSN 1474-7596. 15, p. 435–435. doi: 10.1186/s13059-014-0435-x. Full text in Research Archive Show summary

  Background Ductal carcinoma in situ (DCIS) of the breast is a precursor of invasive breast carcinoma. DNA methylation alterations are thought to be an early event in progression of cancer, and may prove valuable as a tool in clinical decision making and for understanding neoplastic development. Results We generate genome-wide DNA methylation profiles of 285 breast tissue samples representing progression of cancer, and validate methylation changes between normal and DCIS in an independent dataset of 15 normal and 40 DCIS samples. We also validate a prognostic signature on 583 breast cancer samples from The Cancer Genome Atlas. Our analysis reveals that DNA methylation profiles of DCIS are radically altered compared to normal breast tissue, involving more than 5,000 genes. Changes between DCIS and invasive breast carcinoma involve around 1,000 genes. In tumors, DNA methylation is associated with gene expression of almost 3,000 genes, including both negative and positive correlations. A prognostic signature based on methylation level of 18 CpGs is associated with survival of breast cancer patients with invasive tumors, as well as with survival of patients with DCIS and mixed lesions of DCIS and invasive breast carcinoma. Conclusions This work demonstrates that changes in the epigenome occur early in the neoplastic progression, provides evidence for the possible utilization of DNA methylation-based markers of progression in the clinic, and highlights the importance of epigenetic changes in carcinogenesis.

• Ignatiadis, Michail; Riethdorf, Sabine; Bidard, François-Clement; Vaucher, Isabelle; Khazour, Mustapha & Rothé, Françoise [Show all 42 contributors for this article] (2014). International study on inter-reader variability for circulating tumor cells in breast cancer. Breast Cancer Research. ISSN 1465-5411. 16(2). doi: 10.1186/bcr3647. Full text in Research Archive

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• Matikas, Alexios; Naume, Bjørn; Wildiers, Hans; Sonke, Gabe; Dieci, Maria Vittoria & Karakatsanis, Andreas [Show all 32 contributors for this article] (2025). Randomised trial of trastuzumab deruxtecan and biology-driven selection of neoadjuvant treatment for HER2-positive breast cancer: A study protocol of ARIADNE. doi: 10.1136/bmjopen-2025-102626. Full text in Research Archive Show summary

  Introduction Neoadjuvant therapy is the standard of care for the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer (BC). Studies on first-generation antibody-drug conjugates, such as trastuzumab emtansine (T-DM1), showed equal or slightly lower efficacy than chemotherapy combined with dual HER2 blockade. Trastuzumab deruxtecan (T-DXd) is a next-generation conjugate approved for the treatment of metastatic HER2-positive and HER2-low BC, with greatly improved efficacy compared to T-DM1. Methods and analysis ARIADNE is an academic, international, open-label, randomised, comparative phase IIB trial. A total of 370 patients with non-metastatic HER2-positive BC and an indication for neoadjuvant therapy will be included and randomised 1:1 to receive either (1) docetaxel (or paclitaxel), carboplatin, trastuzumab (H) and pertuzumab (P) for three cycles or (2) T-DXd for three cycles. Further treatment is based on the intrinsic molecular subtype determined by the Prosigna assay: patients with HER2-enriched disease (estimated 60%) continue the same treatment for three more cycles. Patients with oestrogen receptor (ER)-positive and luminal (estimated 30%) disease receive H and P for three cycles, combined with letrozole and ribociclib for two cycles. Patients with ER-negative and luminal, basal-like or normal-like disease (estimated 10%) either continue the same treatment for three more cycles in the case of a radiologic complete response, or, in the case of no complete response, they receive four cycles of dose-dense epirubicin and cyclophosphamide. The primary endpoint of ARIADNE is locally assessed rate of pathologic complete response in the molecularly HER2-enriched population, defined as ypT0/Tis, ypN0, as determined by a pathologist blinded to treatment assignment (intention-to-treat (ITT) analysis). Key secondary endpoints include time-to-event endpoints (event-free, recurrence-free, distant recurrence-free and overall survival), safety, health-related quality of life and translational studies. Ethics and dissemination The study has been approved by the Swedish Medical Products Agency (Läkemedelsverket), the Swedish Ethical Review Authority (Etikprövningsmyndigheten) and the Norwegian Ethics Committee for Clinical Trials on Medicinal Products and Medical Devices, as well as by the review boards at all participating centres. Applications for ethical approval in Belgium, the Netherlands and Italy are ongoing. We intend to publish the results of the study in a scientific journal. The study results will be submitted to the European Union (EU) database within 1 year after the end of the clinical trial (CT).

• Vandraas, Kathrine Flørenes; Reinertsen, Kristin Valborg; Gudim, Hilde Beate; Bowen, Camilla Anette; Gjerdalen, Guri & Schlichting, Ellen [Show all 8 contributors for this article] (2024). Brystkreft under graviditet. Tidsskrift for Den norske legeforening (Tidsskriftet). ISSN 0029-2001. 144(12). doi: 10.4045/tidsskr.24.0182. Full text in Research Archive
• Vandraas, Kathrine Flørenes; Smedsland, Solveig Katrine; Engan, Harald Kåre; Kiserud, Cecilie E.; Naume, Bjørn & Brekke, Mette [Show all 7 contributors for this article] (2023). Seneffekter etter behandling av brystkreft. Tidsskrift for Den norske legeforening (Tidsskriftet). ISSN 0029-2001. 143(11). doi: 10.4045/tidsskr.23.0017. Full text in Research Archive Show summary

  Brystkreft er den vanligste kreftsykdommen blant norske kvinner. Ni av ti vil bli langtidsoverlevere. Å være kreftfri er ikke ensbetydende med å føle seg frisk, og mange opplever plagsomme seneffekter, som fatigue, smerter og frykt for tilbakefall. Fastlegen er den viktigste medisinske støttespilleren for majoriteten av disse kvinnene. Denne kliniske oversiktsartikkelen oppsummerer oppdatert kunnskap om seneffekter etter behandling av brystkreft. Ikke-farmakologiske tiltak kan ha positiv virkning på mange av de vanligste seneffektene.

• Sørbye, Sveinung Wergeland; Sundstrøm, Stein Harald; Stensvold, Andreas & Naume, Bjørn (2014). Funnene som vil endre kreftbehandlingen. [Journal]. Dagbladet. Full text in Research Archive Show summary

  Kreftbehandlingen blir stadig mer kompleks. Flere nye forskningsfunn som gir håp for livsforlengende behandling er nylig blitt lagt fram. På konferansen for American society of clinical oncology (ASCO), den årlige kreftkonferansen i USA, er flere nye behandlingsformer for kreft blitt presentert. Konferansen ble avsluttet tirsdag. - Tradisjonell cellegiftbehandling er kanskje forbi. Immunterapi kommer mot alle kreftformer. Dette er midler som stimulerer kroppens eget immunforsvar, slik at det bedre angriper kreftcellene. Slike medisiner kan brukes mot en rekke kreftformer, som lunge-, nyre- og blærekreft. TKI- hemmere blokkerer mutasjoner som styrer kreftutvikling, og her er det også mange nye behandlingsformer på vei.

• Mathiesen, Randi R.; Borgen, Elin; Sætersdal, Anna Barbro; Fangberget, Anne; Schirmer, Cecilie & Stav, I [Show all 10 contributors for this article] (2013). Detection and monitoring of circulating endothelial cells, circulating tumor cells and disseminated tumor cells during neoadjuvant breast cancer treatment including bevacizumab. Full text in Research Archive

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