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Background: Homologous recombination deficiency (HRD) is highly prevalent in triple-negative breast cancer (TNBC) and predictive of response to PARP inhibition in the primary setting (Eikesdal et al, Ann Oncol, 2021). However, the prevalence of HRD across breast cancer subtypes has not been established. Methods: Pretreatment tumor biopsies from 201 patients (32 TNBC and 169 non-TNBC) with primary breast cancer in the phase II PETREMAC trial (ClinicalTrials #NCT02624973) were examined. These samples underwent targeted cancer gene panel sequencing and BRCA1 promoter methylation analysis to assess HRD status defined by homologous recombination repair (HRR) gene mutations and/or BRCA1 promoter methylation. HRR genes included BRCA1, BRCA2, BRIP1, BARD1, and PALB2 by strict definition (HRR-S), and additionally ABL1, ATM, ATR, ATRX, BLM, CDK12, CHEK1, EMSY, ERCC4, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, MEN1, MRE11, NBN, PTEN, and SETD2 by wider definition (HRR-W). HRD strict (HRD-S) was defined as biallelic gene inactivation by HRR-S mutations or BRCA1 methylation. Finally, tumors underwent PAM50 gene expression subtyping and evaluation of functional HRD by RAD51 nuclear foci analysis, for which a low score has been associated with HRD. Results: HRD-S was present in 13% of the breast cancers (total: n= 27/201; TNBC: 15/32; 47%; non-TNBC: 12/169; 7%), whereas HRD-W (HRR-W or BRCA1 methylation) was observed in 29% (total: n=58/201; TNBC: 19/32; 59%; non-TNBC: 39/169; 23%). Among 190 tumors analyzed for PAM50 intrinsic subtype, HRD-S was detected in 3/60 and 4/48 (5% and 8%) of tumors classified as luminal A and B, respectively, 1/35 (3%) of HER2-enriched, 4/21 (19%) of normal-like, and 12/26 (46%) of basal-like tumors. Out of 58 non-TNBC biopsies examined by RAD51 staining, four (7%) were classified as HRD-S and all these were scored as RAD51 low. The remaining 54 non-TNBC samples were homologous recombination proficient, and none of these exhibited functional HRD by RAD51 low scores. All four HRD-S/RAD51 low tumors were hormone receptor-positive, HER2 negative, and belonged to the luminal A (n=1), luminal B (n=2), and basal-like (n=1) subtypes, with HRD caused by germline BRCA1 (gBRCA1), gBRCA2, somatic BRCA1 mutations and BRCA1 methylation, respectively. Conclusion: The prevalence of HRD across all breast cancer subtypes suggests that HRD analysis and therapy targeting such DNA repair defects should be tested in future clinical trials. Citation Format: Christina Engebrethsen, Synnøve Yndestad, Andrea Herencia-Ropero, Oleksii Nikolaienko, Olav Karsten Vintermyr, Reidun K. Lillestøl, Laura Minsaas, Beryl Leirvaag, Gjertrud Iversen, Bjørnar Gilje, Egil Blix, Helge Espelid, Steinar Lundgren, Jürgen Geisler, Liv Jorunn Vassbotn, Hildegunn S. Aase, Turid Aas, Alba Llop-Guevara, Violeta Serra, Per Eystein Lønning, Stian Knappskog, Hans Petter Eikesdal. Homologous recombination deficiency across subtypes of primary breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-04.

Background A common 30 kb deletion affecting the APOBEC3A and APOBEC3B genes has been linked to increased APOBEC activity and APOBEC-related mutational signatures in human cancers. The role of this deletion as a cancer risk factor remains controversial. Materials and Methods We genotyped the APOBEC3A/B deletion in a sample of 1,470 Norwegian endometrial cancer cases and compared to 1,918 healthy controls. For assessment across Caucasian populations, we mined genotypes of the SNP rs12628403, which is in strong linkage disequilibrium with the deletion, in a GWAS dataset of 4,274 cases and 18,125 healthy controls, through the ECAC consortium. Results We found the APOBEC3A/B deletion variant to be significantly associated with reduced risk of endometrial cancer among Norwegian women (OR = 0.75; 95% CI = 0.62–0.91; p = 0.003; dominant model). Similar results were found in the subgroup of endometrioid endometrial cancer (OR = 0.64; 95% CI = 0.51–0.79; p = 3.6 × 10−5; dominant model). The observed risk reduction was particularly strong among individuals in the range of 50–60 years of age (OR = 0.51; 95% CI = 0.33–0.78; p = 0.002; dominant model). In the different populations included in the ECAC dataset, the ORs varied from 0.85 to 1.05. Although five out of six populations revealed ORs

BackgroundNormal cellBRCA1epimutations have been associated with increased risk of triple-negative breast cancer (TNBC). However, the fraction of TNBCs that may haveBRCA1epimutations as their underlying cause is unknown.MethodsTo address this question, we analyzedBRCA1methylation status in breast cancer tissue and matched white blood cells (WBC) from 411 patients with primary breast cancer, including 66 TNBCs, applying a highly sensitive sequencing assay, allowing allele-resolved methylation assessment. Further, to assess the time of origin and the characteristics of normal cellBRCA1methylation, we analyzed umbilical cord blood of 1260 newborn girls.ResultsWe found concordant tumor and mosaic WBCBRCA1epimutations in 10 out of 66 patients with TNBC and in four out of six patients with estrogen receptor (ER)-low expression (BRCA1epimutations affected the same allele in normal and tumor cells. AssessingBRCA1methylation in umbilical WBCs from girls, we found mosaic, predominantly monoallelicBRCA1epimutations, with qualitative features similar to those in adults, in 113/1260 (9.0%) of individuals.ConclusionsOur findings reveal prenatalBRCA1epimutations to be the underlying cause of around 20% of TNBC and low-ER expression breast cancers.

Background: High body mass index (BMI) is an established risk factor for developing breast cancer (BC), especially estrogen receptor (ER)-positive, and also has been associated with adverse survival. Still, patients with BC are currently treated independently of their BMI given limited understandings of the association between BC biology and patient adiposity. In this study, using retrospective data retrieved from two large BC studies, we aimed to identify genomic alterations of primary BC that are associated with BMI in the most common histological BC subtype - invasive carcinoma of no special type (NST). Patients, Data and Methods: Clinicopathological and genomic alteration data were retrieved from two study cohorts: METABRIC (Pereira et al. 2016) and ICGC (Nik-Zainal et al. 2016), with BMI recorded at the time of diagnosis and represented as either a continuous variable or a categorical variable of three categories - lean, overweight and obese. Stratification according to ER and HER2 status resulted in two focused subgroups: NST ER+/HER2- (n=392) and NST ER-/HER2- (n=152). Mutations classified as oncogenic using a set of predefined criteria were used to determine gene-level mutation status. Copy number alteration (CNA) calls were distinguished into three event types: amplification, hemizygous deletion and homozygous deletion. We used multivariable Firth’s logistic regression models with the presence of a genomic alteration as the response variable, BMI as the predicting variable of interest, and data cohort (METABRIC vs ICGC), age group (≤50 vs >50) and tumor grade (I & II vs III) as covariates, to assess the associations between BMI and recurrent gene-level genomic alterations, including gene mutations and CNAs. In a similar manner, we performed multivariable linear regression analysis, adjusting for age and tumor grade, to evaluate the associations of BMI with mutational signatures (MS) and tumor mutational burden in the ICGC NST subsets where these data are available. Results: Considering BMI as a categorical variable, we observed in the NST ER+/HER2- subgroup that PIK3CA was significantly less frequently mutated in obese compared to lean patients (33% vs 46%, odds ratio (OR) = 0.57 (95% confidence interval = (0.33, 0.97)), p = .039), while PTEN and TBX3 showed an increased frequency in overweight (6% vs 1%, OR = 4.14 (1.1, 22.34), p = .034) and obese (8% vs 1%, OR = 7.41 (1.82, 70.65), p = .008) patients, respectively. Regression analyses with BMI as a continuous variable revealed an increased prevalence of mutations in CDH1 and TBX3 genes as BMI increases by 1kg/m2 (OR = 1.14 (1.05, 1.24), p = .002, and OR = 1.13 (1.04, 1.22), p = .005, respectively) in patients with NST ER+/HER2- BC. No associations between BMI and oncogenic mutations was observed in the NST ER-/HER2- subgroup. Interrogation of gene-level CNAs in both subgroups demonstrated differences according to BMI in the prevalence of CNAs affecting a number of genes, many of which are known or have been presented with evidence to be involved in regulation of or regulated by hallmark pathways of BC, such as the MAPK/ERK, JAK/STAT and Wnt/β-catenin signaling pathways. We report a strong positive association between the single-base substitution signature 1 (SBS1), an age-correlated MS, and both continuous (coefficient (coef) = 18.3 (7.7, 28.9), p < .001) and categorical BMI (obese vs lean, coef = 336.3 (187.9, 484.8), p < .001) in the ICGC NST ER+/HER2- subgroup. Conclusion: This exploratory retrospective study suggests that the genomic profiles of primary BC may differ according to BMI. Clinical implications of these differences, especially the decreased prevalence of PIK3CA mutations in obese patients in the context of alpelisib, warrant further investigation. These results however indicate that patient adiposity should be taken into account in the era of personalized medicine. Citation Format: Ha-Linh Nguyen, Tatjana Geukens, Marion Maetens, Karen Van Baelen, Maxim De Schepper, Sophia Leduc, Edoardo Isnaldi, Sam Aparicio, Ake Borg, Jane Brock, Annegien Broeks, Carlos Caldas, Andrew Green, Hazem Khout, Eyfjörð Jórunn, Stian Knappskog, Savitri Krishnamurthy, Sunil Lakhani, Anita Langerod, John WM Martens, Leigh Murphy, Serena Nik-Zainal, Colin Purdie, Emad Rakha, Andrea Richardson, Anne Salomon, Peter Simpson, Christos Sotiriou, Paul Span, Benita Kiat-Tee Tan, Alastair Thompson, Stefania Tommasi, Marc Van de Vijver, Steven Van Laere, Alain Viari, Giuseppe Floris, Elia Biganzoli, François Richard, Christine Desmedt. The association between genomic alterations and body mass index in patients with early breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-09-18.

Supplementary Figure from Mutational Signature 3 Detected from Clinical Panel Sequencing is Associated with Responses to Olaparib in Breast and Ovarian Cancers

Purpose:The identification of patients with homologous recombination deficiency (HRD) beyond BRCA1/2 mutations is an urgent task, as they may benefit from PARP inhibitors. We have previously developed a method to detect mutational signature 3 (Sig3), termed SigMA, associated with HRD from clinical panel sequencing data, that is able to reliably detect HRD from the limited sequencing data derived from gene-focused panel sequencing.Experimental Design:We apply this method to patients from two independent datasets: (i) high-grade serous ovarian cancer and triple-negative breast cancer (TNBC) from a phase Ib trial of the PARP inhibitor olaparib in combination with the PI3K inhibitor buparlisib (BKM120; NCT01623349), and (ii) TNBC patients who received neoadjuvant olaparib in the phase II PETREMAC trial (NCT02624973).Results:We find that Sig3 as detected by SigMA is positively associated with improved progression-free survival and objective responses. In addition, comparison of Sig3 detection in panel and exome-sequencing data from the same patient samples demonstrated highly concordant results and superior performance in comparison with the genomic instability score.Conclusions:Our analyses demonstrate that HRD can be detected reliably from panel-sequencing data that are obtained as part of routine clinical care, and that this approach can identify patients beyond those with germline BRCA1/2mut who might benefit from PARP inhibitors. Prospective clinical utility testing is warranted.

Supplementary Table from Mutational Signature 3 Detected from Clinical Panel Sequencing is Associated with Responses to Olaparib in Breast and Ovarian Cancers

Although normal breast tissue and breast cancer estrogens are known to be elevated compared with plasma estrogen levels, the mechanism behind this phenomenon has been an issue of debate for 2 decades. If local estrogen aromatization were to be confirmed as the main estrogen source in breast cancer tissue, tissue-specific inhibition of estrogen production, avoiding systemic side effects, would become a potentially attractive option for breast cancer treatment and prevention. Based on recent results from our groups exploring tissue estrogens, together with estrogen-synthesizing and estrogen-regulated gene expression levels, we propose a new model to explain elevated breast tissue estrogen levels. Although local estrogen production may be important, the local contribution is overruled by rapid plasma-to-tissue equilibration, including active uptake of circulating estrogens or enhanced tissue binding. As for breast cancer tissue levels, elevated levels of estradiol may be explained to a large extent by estrogen receptor binding and local conversion of estrone into estradiol. This model indicates that effective suppression of benign and malignant tissue estrogens as a treatment for ER+ breast cancer requires systemic suppression and will not be markedly affected by local enzyme targeting. Clin Cancer Res; 17(15); 4948–58. ©2011 AACR.

Supplementary Data from Gene Expression Profiling–Based Identification of Molecular Subtypes in Stage IV Melanomas with Different Clinical Outcome

Purpose: The incidence of malignant melanoma is increasing worldwide in fair-skinned populations. Melanomas respond poorly to systemic therapy, and metastatic melanomas inevitably become fatal. Although spontaneous regression, likely due to immune defense activation, rarely occurs, we lack a biological rationale and predictive markers in selecting patients for immune therapy.Experimental Design: We performed unsupervised hierarchical clustering of global gene expression data from stage IV melanomas in 57 patients. For further characterization, we used immunohistochemistry of selected markers, genome-wide DNA copy number analysis, genetic and epigenetic analysis of the CDKN2A locus, and NRAS/BRAF mutation screening.Results: The analysis revealed four distinct subtypes with gene signatures characterized by expression of immune response, pigmentation differentiation, proliferation, or stromal composition genes. Although all subtypes harbored NRAS and BRAF mutations, there was a significant difference between subtypes (P < 0.01), with no BRAF/NRAS wild-type samples in the proliferative subtype. Additionally, the proliferative subtype was characterized by a high frequency of CDKN2A homozygous deletions (P < 0.01). We observed a different prognosis between the subtypes (P = 0.01), with a particularly poor survival for patients harboring tumors of the proliferative subtype compared with the others (P = 0.003). Importantly, the clinical relevance of the subtypes was validated in an independent cohort of 44 stage III and IV melanomas. Moreover, low expression of an a priori defined gene set associated with immune response signaling was significantly associated with poor outcome (P = 0.001).Conclusions: Our data reveal a biologically based taxonomy of malignant melanomas with prognostic effect and support an influence of the antitumoral immune response on outcome. Clin Cancer Res; 16(13); 3356–67. ©2010 AACR.

The metabolic network of sphingolipids plays important roles in cancer biology. Prominent sphingolipids include ceramides and sphingosine-1-phosphate that regulate multiple aspects of growth, apoptosis, and cellular signaling. Although a significant number of enzymatic regulators of the sphingolipid pathway have been described in detail, many remained poorly characterized. Here we applied a patient-derived systemic approach to identify and molecularly define progestin and adipoQ receptor family member IV (PAQR4) as a Golgi-localized ceramidase. PAQR4 was approximately 5-fold upregulated in breast cancer compared with matched control tissue and its overexpression correlated with disease-specific survival rates in breast cancer. Induction of PAQR4 in breast tumors was found to be subtype-independent and correlated with increased ceramidase activity. These findings establish PAQR4 as Golgi-localized ceramidase required for cellular growth in breast cancer.Significance:Induction of and cellular dependency on de novo sphingolipid synthesis via PAQR4 highlights a central vulnerability in breast cancer that may serve as a viable therapeutic target.

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Worldwide, there is a growing proportion of women who are overweight or obese. While obesity has been associated with an increased risk of developing breast cancer (BC) and worse prognosis in BC patients, yet the impact of adiposity (abnormal or excess body fat) on BC biology remains understudied in humans. This retrospective study aimed to investigate how the biology of primary BC would differ according to patients’ body mass index (BMI). We examined clinicopathological data (including BMI at the time of diagnosis) and molecular data (including genomic, bulk and single-cell transcriptomic data) of treatment-naïve (early stage) BC patients from five cohorts (N = 2071). We identified several genomic alterations considered actionable or of potential clinical relevance which had a different prevalence in overweight or obese patients compared to lean patients, for instances, less PIK3CA gene mutations, and more CCND1, CCNE1 and IGFR1 amplifications. Moreover, we found evidence supporting an ageing accelerating effect of obesity at the genetic level, through its association with an age-associated mutational signature. We showed that BMI-associated differences in transcriptomic profile were subtle at the bulk resolution while single cell profiling allowed detection of more pronounced changes in different cell compartments. Investigation at the single cell resolution revealed an elevated and unresolved inflammation of the BC tumor microenvironment (TME) associated with obesity, which had distinct characteristics contingent on the estrogen receptor status. Collectively, analyses at both genomic and transcriptomic levels implied that obesity is associated with an inflammaging-like phenotype of the TME. Our results indicate that patient adiposity might play a significant role in the heterogeneity of BC and should be considered in the context of precision medicine.

High-grade (HG) gastroenteropancreatic (GEP) neuroendocrine neoplasms (NEN) are rare but have a very poor prognosis and represent a severely understudied class of tumours. Molecular data for HG GEP-NEN are limited, and treatment strategies for the carcinoma subgroup (HG GEP-NEC) are extrapolated from small-cell lung cancer (SCLC). After pathological re-evaluation, we analysed DNA from tumours and matched blood samples from 181 HG GEP-NEN patients; 152 neuroendocrine carcinomas (NEC) and 29 neuroendocrine tumours (NET G3). Based on the sequencing of 360 cancer-related genes, we assessed mutations and copy number alterations (CNA). For NEC, frequently mutated genes were TP53 (64%), APC (28%), KRAS (22%) and BRAF (20%). RB1 was only mutated in 14%, but CNAs affecting RB1 were seen in 34%. Other frequent copy number losses were ARID1A (35%), ESR1 (25%) and ATM (31%). Frequent amplifications/gains were found in MYC (51%) and KDM5A (45%). While these molecular features had limited similarities with SCLC, we found potentially targetable alterations in 66% of the NEC samples. Mutations and CNA varied according to primary tumour site with BRAF mutations mainly seen in colon (49%), and FBXW7 mutations mainly seen in rectal cancers (25%). Eight out of 152 (5.3%) NEC were microsatellite instable (MSI). NET G3 had frequent mutations in MEN1 (21%), ATRX (17%), DAXX, SETD2 and TP53 (each 14%). We show molecular differences in HG GEP-NEN, related to morphological differentiation and site of origin. Limited similarities to SCLC and a high fraction of targetable alterations indicate a high potential for better-personalized treatments.

PURPOSE Gastroenteropancreatic neuroendocrine carcinomas (GEP-NEC) are rare and have a poor prognosis. Most GEP-NEC are diagnosed with metastatic disease, with only minor biopsies available for molecular diagnostics. We assessed the applicability of liquid biopsies for molecular profiling of GEP-NEC. MATERIALS AND METHODS We performed massive parallel sequencing of 76 cancer-related genes in circulating tumor DNA from 50 patients with advanced GEP-NEC and compared findings to previous analyses of solid tumor biopsies from the same patients. Plasma samples were collected before therapy, and the median time span between blood and tissue sampling was 25 days. RESULTS We detected 178 somatic mutations in the liquid biopsies, 127 (71%) were also detected in the solid biopsies, whereas 51 (29%) were unique to the liquid biopsies. In the same 76 genes, we previously detected 199 somatic mutations (single nucleotide variants) in solid biopsies, of which 127 (64%) were also now detected in liquid biopsies. In exploratory subgroup assessments, concordance was higher in patients with liver metastases ( P = 1.5 × 10–5) and increasing with level of liver involvement ( P = 1.2 × 10–4). The concordance was similar between GEP-NEC with different primary sites, except being lower in esophageal cases ( P = .001). Concordance was not associated with tumor mutation burden. Tumor tissue mutations also detected in liquid biopsies was lower for MSI (40%) versus MSS tumors (70%; P = 7.8 × 10–4). We identified potentially targetable mutations in plasma of 26 (52%) of patients with GEP-NEC; nine patients (18%) had potentially targetable mutation detected only in liquid biopsies. CONCLUSION Liquid biopsy analyses may be an applicable alternative to solid biopsies in GEP-NEC. Liquid biopsies may add additional mutations compared with tumor biopsies alone and could be useful for biomarker assessment in clinical trials for these patients.

Motivation With recent advances in the field of epigenetics, the focus is widening from large and frequent disease- or phenotype-related methylation signatures to rare alterations transmitted mitotically or transgenerationally (constitutional epimutations). Merging evidence indicate that such constitutional alterations, albeit occurring at a low mosaic level, may confer risk of disease later in life. Given their inherently low incidence rate and mosaic nature, there is a need for bioinformatic tools specifically designed to analyze such events. Results We have developed a method (ramr) to identify aberrantly methylated DNA regions (AMRs). ramr can be applied to methylation data obtained by array or next-generation sequencing techniques to discover AMRs being associated with elevated risk of cancer as well as other diseases. We assessed accuracy and performance metrics of ramr and confirmed its applicability for analysis of large public datasets. Using ramr we identified aberrantly methylated regions that are known or may potentially be associated with development of colorectal cancer and provided functional annotation of AMRs that arise at early developmental stages. Availability and implementation The R package is freely available at https://github.com/BBCG/ramr and https://bioconductor.org/packages/ramr. Supplementary information Supplementary data are available at Bioinformatics online.

Introduction: Glioblastoma (GBM) is invariably resistant to temozolomide (TMZ) chemotherapy. Inhibiting the proteasomal pathway is an emerging strategy to accumulate damaged proteins and inhibit their lysosomal degradation. We hypothesized that pre-treatment of glioblastoma with bortezomib (BTZ) might sensitize glioblastoma to temozolomide by abolishing autophagy survival signals to augment DNA damage and apoptosis.Methods: P3 patient-derived glioblastoma cells, as well as the tumour cell lines U87, HF66, A172, and T98G were investigated for clonogenic survival after single or combined treatment with temozolomide and bortezomib in vitro. We investigated the requirement of functional autophagy machinery by utilizing pharmacological inhibitors or CRISPR-Cas9 knockout (KO) of autophagy-related genes -5 and -7 (ATG5 and ATG7) in glioblastoma cells and monitored changes in autophagic flux after temozolomide and/or bortezomib treatments. P3 wild-type and P3 ATG5−/− (ATG5 KO) cells were implanted orthotopically into NOD-SCID mice to assess the efficacy of bortezomib and temozolomide combination therapy with and without functional autophagy machinery.Results: The chemo-resistant glioblastoma cells increased autophagic flux during temozolomide treatment as indicated by increased degradation of long-lived proteins, diminished expression of autophagy markers LC3A/B-II and p62 (SQSTM1), increased co-localisation of LC3A/B-II with STX17, augmented and no induction of apoptosis. In contrast, bortezomib treatment abrogated autophagic flux indicated by the accumulation of LC3A/B-II and p62 (SQSTM1) positive autophagosomes that did not fuse with lysosomes and thus reduced the degradation of long-lived proteins. Bortezomib synergistically enhanced temozolomide efficacy by attenuating cell proliferation, increased DNA double-strand breaks, and apoptosis in an autophagy-dependent manner. Abolishing autophagy in ATG5 KOs reversed the bortezomib-induced toxicity, rescued glioblastoma cell death and reduced animal survival.Discussion: We conclude that bortezomib abrogates temozolomide induced autophagy flux through an ATG5 dependent pathway.

BACKGROUND: While large GWAS analyses have not found convincing associations between MDM2 promoter SNP55 and gynaecological cancers, SNP55 is in linkage disequilibrium with two other functional SNPs in the same promoter, likely to obscure associations between single SNPs and cancer risk. Here, we assessed the impact of SNP55 on risk of endometrial and ovarian cancer, including sub-analyses stratified for other functional SNPs in the region.MATERIAL AND METHODS: Using a custom LightSNiP assay, we genotyped SNP55 in two large hospital-based cohorts of patients with ovarian (n = 1,332) and endometrial (n = 1,363) cancer and compared genotypes to healthy female controls (n = 1,858).RESULTS: Among individuals harbouring the SNP309TT genotype, the minor SNP55T-allele was associated with a reduced risk of endometrial (dominant model: OR = 0.63; CI = 0.45-0.88; p = 0.01). Regardless of the genotype in neighbouring SNPs, the SNP55T-allele was also associated with a reduced risk of endometrial cancer before 50 years of age (dominant model: OR = 0.56; CI = 0.34-0.90; p = 0.02). No association between SNP55 status and ovarian cancer risk was observed.CONCLUSIONS: MDM2 SNP55T-allele may correlate with reduced risk for endometrial cancer in a SNP309T-, but not SNP309G, context.

Low-level mosaic methylation of theBRCA1gene promoter occurs in 5–8% of healthy individuals and is associated with a significantly elevated risk of breast and ovarian cancer. Similar events may also affect other tumour suppressor genes, potentially being a significant contributor to cancer burden. While this opens a new area for translational research, detection of low-level mosaic epigenetic events requires highly sensitive and robust methodology for methylation analysis. We here present epialleleR, a computational framework for sensitive detection, quantification and visualisation of mosaic epimutations in methylation sequencing data. Analysing simulated and real data sets, we provide in-depth assessments of epialleleR performance, and show that linkage to epihaplotype data is necessary to detect low-level methylation events. The epialleleR is freely available athttps://github.com/BBCG/epialleleRandhttps://bioconductor.org/packages/epialleleR/as an open source R/Bioconductor package.

Epidemiological studies have established a positive association between obesity and the incidence of postmenopausal breast cancer. Moreover, it is known that obesity promotes stem cell-like properties of breast cancer cells. However, the cancer cell-autonomous mechanisms underlying this correlation are not well defined. Here we demonstrate that obesity-associated tumor formation is driven by cellular adaptation rather than expansion of pre-existing clones within the cancer cell population. While there is no correlation with specific mutations, cellular adaptation to obesity is governed by palmitic acid (PA) and leads to enhanced tumor formation capacity of breast cancer cells. This process is governed epigenetically through increased chromatin occupancy of the transcription factor CCAAT/enhancer-binding protein beta (C/EBPB). Obesity-induced epigenetic activation of C/EBPB regulates cancer stem-like properties by modulating the expression of key downstream regulators including CLDN1 and LCN2. Collectively, our findings demonstrate that obesity drives cellular adaptation to PA drives tumor initiation in the obese setting through activation of a C/EBPB dependent transcriptional network., Obesity is linked to cancer risk in post-menopausal breast cancer. At the molecular level this is governed by cellular adaption to palmitic acid through epigenetic activation of a C/EBPB-dependent transcriptional network that drives tumor formation.

Introduction: Glioblastoma (GBM) is the most frequent treatment resistant and poor prognosis primary brain tumor. Age-adjusted incidence among Caucasians is approx. twice that of Africans. Genetic changes in ethnicity may represent druggable targets for development of new treatments for GBM. Chondroitin sulfate proteoglycan 4 (CSPG4) with sequence homology to neuron-glial-2 (NG2), henceforth CSPG4/NG2, is a transmembrane proteoglycan that is upregulated in GBM, resulting in leaky neovasculature and high cell proliferation, and is independently prognostic for poor survival. Mechanisms regulating CSPG4/NG2 expression remain to be elucidated. Experiments: Tumor-derived DNA whole exome sequences (WES) from mixed American GBM patients (n=300) collected into the TCGA database and blood-derived DNA WES from controls (n = 2504) in the 1000 Genome database were analyzed for mutations in the CSPG4 gene by GATK-3.5. We used prime editing (PE) to introduce a 13-base pair (bp) deletion through design of PE guide RNA (n=27), screening GBM cells (n=14) for CSPG4/NG2 expression, transfecting and expanding PE cells, confirming editing using Sanger sequencing. We functionally characterized the mutation in PE cells and transplanted in orthotopic PDX mouse models compared to wild type (WT) and Cas9 control cells. Western blotting, BrdU proliferation assays, clonogenic- and wound healing assays were used to investigate function. Gelatin zymography assessed CSPG4/NG2 matrix metalloproteinase (MMP) cross-talk during invasion on mixed extracellular matrix- or collagen IV- coated transwells. Results: A novel 13bp frameshift deletion in exon 3 of the CSPG4 gene was identified from WES of GBM samples deposited in TCGA. The mutation was cancer specific (p Conclusions: We identified a novel 13bp deletion in CSPG4/NG2 with phenotypic effect that could be used as a future molecular target in GBM treatment, and present the first successful application of PE in human GBM cells. Citation Format: Victoria Smith Arnesen, Susina Suntharalingam, Żaneta Matuszek, Shahin Sarowar, Stian Knappskog, Stein Atle Lie, David R. Liu, Mohummad Aminur Rahman, Martha Chekenya. A novel 13-basepair deletion in CSPG4/NG2 abrogates protein expression, glioblastoma proliferation and invasion in vitro and in vivo in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3996.

ImportanceAbout 25% of all triple-negative breast cancers (TNBCs) and 10% to 20% of high-grade serous ovarian cancers (HGSOCs) harbor BRCA1 promoter methylation. While constitutional BRCA1 promoter methylation has been observed in normal tissues of some individuals, the potential role of normal tissue methylation as a risk factor for incident TNBC or HGSOC is unknown.ObjectiveTo assess the potential association between white blood cell BRCA1 promoter methylation and subsequent risk of incident TNBC and HGSOC.Design, Setting, and ParticipantsThis case-control study included women who were participating in the Women’s Health Initiative study who had not received a diagnosis of either breast or ovarian cancer before study entrance. A total of 637 women developing incident TNBC and 511 women developing incident HGSOC were matched with cancer-free controls (1841 and 2982, respectively) in a nested case-control design. Cancers were confirmed after central medical record review. Blood samples, which were collected at entry, were analyzed for BRCA1 promoter methylation by massive parallel sequencing. The study was performed in the Mohn Cancer Research Laboratory (Bergen, Norway) between 2019 and 2022.Main Outcomes and MeasuresAssociations between BRCA1 methylation and incident TNBC and incident HGSOC were analyzed by Cox proportional hazards regression.ResultsOf 2478 cases and controls in the TNBC group and 3493 cases and controls in the HGSOC group, respectively, 7 (0.3%) and 3 (0.1%) were American Indian or Alaska Native, 46 (1.9%) and 30 (0.9%) were Asian, 1 (0.04%) and 1 (0.03%) was Native Hawaiian or Pacific Islander, 326 (13.2%) and 125 (3.6%) were Black or African, 56 (2.3%) and 116 (3.3%) were Hispanic, 2046 (82.6%) and 3257 (93.2%) were White, and 35 (1.4%) and 35 (1.0%) were multiracial. Median (range) age at entry was 62 (50-79) years, with a median interval to diagnosis of 9 (TNBC) and 10 (HGSOC) years. Methylated BRCA1 alleles were present in 194 controls (5.5%). Methylation was associated with risk of incident TNBC (12.4% methylated; HR, 2.35; 95% CI, 1.70-3.23; P P P P = .003). Across individuals, methylation was not haplotype-specific, arguing against an underlying cis-acting factor. Within individuals, BRCA1 methylation was observed on the same allele, indicating clonal expansion from a single methylation event. There was no association found between BRCA1 methylation and germline pathogenic variant status.Conclusions and RelevanceThe results of this case-control suggest that constitutional normal tissue BRCA1 promoter methylation is significantly associated with risk of incident TNBC and HGSOC, with potential implications for prediction of these cancers. These findings warrant further research to determine if constitutional methylation of tumor suppressor genes are pancancer risk factors.

Glioblastoma (GBM) is invariably resistant to temozolomide (TMZ) chemotherapy. Inhibiting the proteasomal pathway is an emerging strategy to accumulate damaged proteins and inhibit their lysosomal degradation. We hypothesized that bortezomib (BTZ) might sensitize GBM cells to TMZ. We examined change in autophagic flux after drug treatments and in combination with pharmacological inhibitors or CRISPR cas9 knockout of autophagy-related genes -5 and -7 (ATG5 and ATG7, respectively). Autophagic flux was increased in temozolomide resistant GBM cells as indicated by diminished levels of the autophagy markers LC3A/B-II and p62(SQSTM1), increased localisation of LC3A/B-II with STX17, higher long-lived protein degradation and no induction of apoptosis. In contrast, BTZ treatment abrogated autophagic flux by accumulation of LC3A/B-II and p62(SQSTM1) positive autophagosomes that did not fuse with lysosomes and reduced degradation of long-lived proteins. BTZ synergistically enhanced TMZ efficacy by attenuating cell proliferation, increased DNA damage and apoptosis. CRISPR Cas ATG5 knockout reversed BTZ-induced autophagy blockade and rescued the GBM treated cells from death. We conclude that bortezomib abrogates temozolomide induced autophagy through ATG5 dependent pathway.

MotivationWith recent advances in the field of epigenetics, the focus is widening from large and frequent disease- or phenotype-related methylation signatures to rare alterations transmitted mitotically or transgenerationally (constitutional epimutations). Merging evidence indicate that such constitutional alterations, albeit occurring at a low mosaic level, may confer risk of disease later in life. Given their inherently low incidence rate and mosaic nature, there is a need for bioinformatic tools specifically designed to analyse such events.ResultsWe have developed a method (ramr) to identify aberrantly methylated DNA regions (AMRs). ramr can be applied to methylation data obtained by array or next-generation sequencing techniques to discover AMRs being associated with elevated risk of cancer as well as other diseases. We assessed accuracy and performance metrics of ramr and confirmed its applicability for analysis of large public data sets. Using ramr we identified aberrantly methylated regions that are known or may potentially be associated with development of colorectal cancer and provided functional annotation of AMRs that arise at early developmental stages.Availability and implementationThe R package is freely available at https://github.com/BBCG/ramr

Epidemiological studies have established a positive association between obesity and the incidence of postmenopausal breast cancer. Moreover, it is known that obesity promotes stem cell-like properties of breast cancer cells. However, the cancer cell-autonomous mechanisms underlying this correlation are not well defined. Here we demonstrate that obesity-associated tumor formation is driven by cellular adaptation rather than expansion of pre-existing clones within the cancer cell population. While there is no correlation with specific mutations, cellular adaptation to obesity is governed by palmitic acid (PA) and leads to enhanced tumor formation capacity of breast cancer cells. This process is governed epigenetically through increased chromatin occupancy of the transcription factor CCAAT/enhancer-binding protein beta (C/EBPB). Obesity-induced epigenetic activation of C/EBPB regulates cancer stem-like properties by modulating the expression of key downstream regulators including CLDN1 and LCN2. Collectively, our findings demonstrate that obesity drives cellular adaptation to PA drives tumor initiation in the obese setting through activation of a C/EBPB dependent transcriptional network.SignificanceCellular adaptation to obesity-induced palmitic acid drives tumor initiation through activation of a C/EBPB-dependent transcriptional network. This highlights a cell-autonomous mechanistic connection between obesity and postmenopausal hormone receptor negative breast cancer.

We show that the quantitative PCR reaction volume can be reduced significantly compared to the standard procedures, without compromising data quality. By analyzing dilution series (100–10−7) we found that measurement in 1 μl reaction volume indeed gave valid results comparable to 10 μL, when using a routine pipetting robot. This may enable a significant cost reduction through cutback of both biological material as well as chemical reagents.

This abstract was withdrawn by the authors.

BACKGROUND: MDM2 is a negative regulator of p53 and is upregulated in numerous human cancers. While different MDM2 splice variants have been observed in both normal tissues and malignant cells, their functions are poorly understood. METHODS: We evaluated the effect of MDM2 splice variants by overexpression in MCF-7 cells and analyses of expression of downstream genes (qPCR and Western blot), subcellular localization (immunofluorescence), cell cycle assays (Nucleocounter3000), apoptosis analysis (Annexin V detection), and induction of senescence (β-galactosidase analysis). RESULTS: In a screen for MDM2 splice variants in MCF-7 breast cancer cells, extended with data from healthy leukocytes, we found P2-MDM2-10 and MDM2-Δ5 to be the splice variants expressed at highest levels. Contrasting MDM2 full-length protein, we found normal tissue expression levels of P2-MDM2-10 and MDM2-Δ5 to be highest in individuals harboring the promoter SNP309TT genotype. While we detected no protein product coded for by MDM2-Δ5, the P2-MDM2-10 variant generated a protein markedly more stable than MDM2-FL. Both splice variants were significantly upregulated in stressed cells ( P = 4.3 × 10 −4 and P = 7.1 × 10 −4 , respectively). Notably, chemotherapy treatment and overexpression of P2-MDM2-10 or MDM2-Δ5 both lead to increased mRNA levels of the endogenous MDM2-FL ( P = .039 and P = .070, respectively) but also the proapoptotic gene PUMA ( P = .010 and P = .033, respectively), accompanied by induction of apoptosis and repression of senescence. CONCLUSION: We found P2-MDM2-10 and MDM2-Δ5 to have distinct biological functions in breast cancer cells. GENERAL SIGNIFICANCE: Alternative splicing may influence the oncogenic effects of the MDM2 gene.

Anthracyclines are key components of human breast cancer chemotherapy. Here, we explored the role of Akt signaling in anthracycline resistance. The antitumor activity of doxorubicin and Akt inhibitor A-443654 alone or combined was examined in estrogen receptor (ER) positive and negative human breast cancer cell lines. Further, we examined mRNA changes induced by anthracyclines in locally advanced breast cancers biopsied before and after treatment in two clinical trials. Doxorubicin increased Akt phosphorylation in ER positive MCF7 and T47D cell lines, with no effect in ER negative MDA-MB231 breast cancer cells. A-443654 was significantly more cytotoxic in doxorubicin-resistant compared to doxorubicin-naïve MCF7. This difference was not observed in MDA-MB231. Among 24 patients, AKT1 gene expression increased 24 hrs after the initial epirubicin exposure in ER positive tumors responding to therapy (n=6), as compared to ER positive non-responders (n=7) or ER negative tumors (n=11). In contrast, AKT1 mRNA changes after 16 weeks of doxorubicin were unrelated to clinical response and ER status (n=30). In conclusion, rapid Akt activation was observed in ER positive breast cancers which responded to anthracyclines. Increased cytotoxicity of A-443654 in doxorubicinresistant MCF7 cells indicates a possible role for Akt inhibitors in ER positive breast cancers where chemoresistance evolves. publishedVersion

The ability of tumor cells to invade and metastasize is relevant to the process of cancer progression and, as such, it represents an obstacle to cancer cure. So far, limited information is available on interactions between circulating tumor cells and blood cells. It is well-documented that galectin-4 is upregulated in many types of tumor cells and is involved in metastasis. Here, we address the hypothesis that tumor cells may interact with red blood cells (RBCs) via galectin-4. High galectin-4 expressing colon, normal pancreatic and pancreatic cancer-derived cell lines (n = 5) were incubated with peripheral blood cells from different donors. Their interactions and associated proteins were examined by immunostaining and live cell imaging. We found that (endogenous or exogenous) galectin-4 expressing tumor cells interact directly with RBCs. We also observed an accumulation of galectin-4 and human blood group antigens at the contact sites between these cells. By comparing the number of RBCs attaching to each tumor cell, we found that cells with high pre-incubation expression levels of galectin-4 attached significantly more RBCs than those with low expression levels (p

Purpose PTEN is an important tumor suppressor in breast cancer. Here, we examined the prognostic and predictive value of PTEN and PTEN pseudogene (PTENP1) gene expression in patients with locally advanced breast cancer given neoadjuvant chemotherapy. Methods The association between pretreatment PTEN and PTENP1 gene expression, response to neoadjuvant chemotherapy, and recurrence-free and disease-specific survival was assessed in 364 patients with locally advanced breast cancer given doxorubicin, 5-fluorouracil/mitomycin, or epirubicin versus paclitaxel in three phase II prospective studies. Further, protein expression of PTEN or phosphorylated Akt, S6 kinase, and 4EBP1 was assessed in a subgroup of 187 tumors. Results Neither PTEN nor PTENP1 gene expression level predicted response to any of the chemotherapy regimens tested (n = 317). Among patients without distant metastases (n = 282), a high pretreatment PTEN mRNA level was associated with inferior relapse-free (RFS; p = 0.001) and disease-specific survival (DSS; p = 0.003). Notably, this association was limited to patients harboring TP53 wild-type tumors (RFS; p = 0.003, DSS; p = 0.009). PTEN mRNA correlated significantly with PTENP1 mRNA levels (r s = 0.456, p

The metabolic network of sphingolipids plays important roles in cancer biology. Prominent sphingolipids include ceramides and sphingosine-1-phosphate that regulate multiple aspects of growth, apoptosis, and cellular signaling. Although a significant number of enzymatic regulators of the sphingolipid pathway have been described in detail, many remained poorly characterized. Here we applied a patient-derived systemic approach to identify and molecularly define progestin and adipoQ receptor family member IV (PAQR4) as a Golgi-localized ceramidase. PAQR4 was approximately 5-fold upregulated in breast cancer compared with matched control tissue and its overexpression correlated with disease-specific survival rates in breast cancer. Induction of PAQR4 in breast tumors was found to be subtype-independent and correlated with increased ceramidase activity. These findings establish PAQR4 as Golgi-localized ceramidase required for cellular growth in breast cancer. Significance: Induction of and cellular dependency on de novo sphingolipid synthesis via PAQR4 highlights a central vulnerability in breast cancer that may serve as a viable therapeutic target.

Background Epithelial to mesenchymal transition (EMT) is a well-characterized process of cell plasticity that may involve metabolic rewiring. In cancer, EMT is associated with malignant progression, tumor heterogeneity, and therapy resistance. In this study, we investigated the role of succinate dehydrogenase (SDH) as a potential key regulator of EMT. Methods Associations between SDH subunits and EMT were explored in gene expression data from breast cancer patient cohorts, followed by in-depth studies of SDH suppression as a potential mediator of EMT in cultured cells. Results We found an overall inverse association between EMT and the SDH subunit C (SDHC) when analyzing gene expression in breast tumors. This was particularly evident in carcinomas of basal-like molecular subtype compared to non-basal-like tumors, and a low SDHC expression level tended to have a prognostic impact in those patients. Studies in cultured cells revealed that EMT was induced by SDH inhibition through SDHC CRISPR/Cas9 knockdown or by the enzymatic inhibitor malonate. Conversely, overexpression of EMT-promoting transcription factors TWIST and SNAI2 caused decreased levels of SDHB and C and reduced rates of SDH-linked mitochondrial respiration. Cells overexpressing TWIST had reduced mitochondrial mass, and the organelles were thinner and more fragmented compared to controls. Conclusions Our findings suggest that downregulation of SDHC promotes EMT and that this is accompanied by structural remodeling of the mitochondrial organelles. This may confer survival benefits upon exposure to hostile microenvironment including oxidative stress and hypoxia during cancer progression. Electronic supplementary material The online version of this article (10.1186/s40170-019-0197-8) contains supplementary material, which is available to authorized users.

We determined prognostic impact of KRAS, BRAF, PIK3CA and TP53 mutation status and mutation heterogeneity among 164 colorectal cancer (CRC) patients undergoing liver resections for metastatic disease. Mutation status was determined by Sanger sequencing of a total of 422 metastatic deposits. In univariate analysis, KRAS (33.5%), BRAF (6.1%) and PIK3CA (13.4%) mutations each predicted reduced median time to relapse (TTR) (7 vs. 22, 3 vs. 16 and 4 vs. 17 months; p

In the Methods section of this Article, 'greater than' should have been 'less than' in the sentence 'Putative regions of clustered rearrangements were identified as having an average inter-rearrangement distance that was at least 10 times greater than the whole-genome average for the individual sample. '. The Article has not been corrected., SCOPUS: er.j, info:eu-repo/semantics/published

miRNAs are an important class of small non-coding RNAs, which play a versatile role in gene regulation at the post-transcriptional level. Expression of miRNAs is often deregulated in human cancers.We analyzed small RNA massive parallel sequencing data from 50 locally advanced breast cancers aiming to identify novel breast cancer related miRNAs. We successfully predicted 10 novel miRNAs, out of which 2 (hsa-miR-nov3 and hsa-miR-nov7) were recurrent. Applying high sensitivity qPCR, we detected these two microRNAs in 206 and 214 out of 223 patients in the study from which the initial cohort of 50 samples were drawn. We found hsa-miR-nov3 and hsa-miR-nov7 both to be overexpressed in tumor versus normal breast tissue in a separate set of 13 patients (p=0.009 and p=0.016, respectively) from whom both tumor tissue and normal tissue were available. We observedhsa-miR-nov3to be expressed at higher levels in ER-positive compared to ER-negative tumors (p=0.037). Further stratifications revealed particularly low levels in the her2-like and basal-like cancers compared to other subtypes (p=0.009 and 0.040, respectively).We predicted target genes for the 2 microRNAs and identified inversely correlated genes in mRNA expression array data available from 203 out of the 223 patients. Applying the KEGG and GO annotations to target genes revealed pathways essential to cell development, communication and homeostasis.Although a weak association between high expression levels ofhsa-miR-nov7and poor survival was observed, this did not reach statistical significance.hsa-miR-nov3expression levels had no impact on patient survival.

In this letter, we respond to and discuss the recent publication by Al-Moghrabi et al.: Methylation of BRCA1 and MGMT genes in white blood cells are transmitted from mothers to daughters. We discuss their findings with emphasis on two other recently published papers and argue that their data allows no conclusion regarding the transmission of BRCA1 methylation from parent to child.

Background Uterine serous carcinoma (USC) is a rare but aggressive subtype of endometrial carcinoma. Large-scale comprehensive efforts have resulted in an improved molecular understanding of its pathogenesis, and the p53 pathway has been proposed as a key player and is potentially targetable. Here we attempt to further portray the p53 pathway in USC by assessing p53 isoform expression. Methods We applied quantitative Real-Time PCRs (RT-qPCR) for expression analyses of total p53 mRNA as well as quantitative distinction of p53β, p53γ, and the total mRNA of amino-terminal truncated Δ40p53 and Δ133p53 in a retrospective cohort of 37 patients with USC. TP53 mutation status was assessed by targeted massive parallel sequencing. Findings were correlated with clinical data. Results The p53 isoform expression landscape in USCs was heterogeneous and dominated by total Δ133p53, while the distinct p53β and p53γ variants were found at much lower levels. The isoform expression profiles varied between samples, while their expression was independent of TP53 mutation status. We found high relative p53γ expression to be associated with reduced progression-free survival (PFS). Conclusions This is the first indication that elevated p53γ expression is associated with reduced PFS in USC. This single-center study may offer some insight in the landscape of p53 isoform expression in USC, but further validation studies are crucial to understand the context-dependent and tissue-specific role of the p53 isoform network in gynecological cancer. Electronic supplementary material The online version of this article (10.1186/s12885-018-4591-3) contains supplementary material, which is available to authorized users.

Background Optimal neoadjuvant therapy for luminal A breast cancer remains a topic of controversy. Methods In the phase 2 PETREMAC trial (NCT02624973), patients with large T2 (>4cm) or locally advanced breast cancers and luminal A characteristics (ER > 50%, HER2- and TP53 WT) received neoadjuvant endocrine therapy (NET) and CDK4/6 inhibition in concert. NET consisted of letrozole (postmenopausals) or tamoxifen + goserelin (premenopausals). Palbociclib (P) was added if Ki67 had decreased Results 88 patients were enrolled; mean tumor diameter 55 mm (range 20-95). 2 patients lacked Ki67 data for analysis, 1 was a screening failure. 47/85 (55%) had a drop >50% on NET alone. Among the remaining 38 patients, 31 had P added in concert, 5 had Ki67 50% in 22/31 patients (71%). NAC was required in 28/84 patients (33%) after NET +/- P. Thus far, 84 patients have completed neoadjuvant treatment, and 75 have surgery results ready. The clinical OR rate (ORR) before surgery was 24/32 for NET alone, 12/15 for NET + P and 24/28 for NAC after NET +/- P. The overall ORR before surgery was 85% (60/75, 13 w/missing data), and ORR 77% (36/47) for NET +/- P. Pathological complete response (pCR) has been observed in 4/75 patients at surgery, where 3/4 with pCR received NAC after NET +/- P. 40/88 tumors (45%) had mutations in the PI3K pathway; PIK3CA (33%), PTEN (6.8%) and AKT1 (4.5%). 5/15 lobular carcinomas (33%) and 16/73 other carcinomas (22%) harbored CDH1 mutations. CDH1 mutations were associated with a higher probability of Ki67 reduction >50% on NET alone (CDH1 mutated: 15/20 vs CDH1 WT: 32/66; p = 0.042). Conclusions NET +/- P was effective at reducing cell proliferation and yielded an ORR of 77% in these ER+, HER2 negative breast cancers. NAC was required only among 33% of the patients. CDH1 mutations seem predictive of response to NET in this setting. Clinical trial identification NCT02624973; 2015-002816-34. Legal entity responsible for the study Haukeland University Hospital, Bergen, Norway. Funding Det Regionale Samarbeidsorganet/Helse Vest, Pfizer. Disclosure P.E. Lonning: Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): AstraZeneca; Honoraria (self), Travel / Accommodation / Expenses: Pierre Fabre; Honoraria (self): Roche; Advisory / Consultancy: Laboratorios Farmaceuticos Rovi; Research grant / Funding (self): Pfizer; Research grant / Funding (self): Novartis; Licensing / Royalties: Cytovation. E.S. Blix: Honoraria (self): Pfizer. B. Gilje: Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Roche; Honoraria (self), Research grant / Funding (institution): AstraZeneca; Advisory / Consultancy, Research grant / Funding (institution): Astellas Oncology; Research grant / Funding (institution): Cellgene; Research grant / Funding (institution): Pfizer; Travel / Accommodation / Expenses: Pierre Fabre. E.A. Janssen: Honoraria (self): AstraZeneca; Honoraria (self): Pfizer. J. Geisler: Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony: Novartis; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony: MSD; Honoraria (self), Advisory / Consultancy: Lilly; Honoraria (self), Speaker Bureau / Expert testimony: Pierre Fabre; Honoraria (self), Speaker Bureau / Expert testimony: Brystol-Myers-Squibb; Advisory / Consultancy, Speaker Bureau / Expert testimony: AstraZeneca; Speaker Bureau / Expert testimony: Pfizer. T. Aas: Honoraria (self): AstraZenaca; Honoraria (self): Roche. S. Knappskog: Honoraria (self), Research grant / Funding (institution): AstraZenaca; Honoraria (self), Research grant / Funding (institution): Pfizer; Licensing / Royalties: Patent EP2389450 A1,Patent WO 2012/010661. H.P. Eikesdal: Honoraria (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: AstraZenaca; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Novartis; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Pfizer; Honoraria (self), Travel / Accommodation / Expenses: Pierre Fabre; Honoraria (self): Amgen; Honoraria (self), Advisory / Consultancy: Roche; Honoraria (self): HAI Interaktiv AS; Honoraria (self): Dagens Medisin; Honoraria (self): Brystol-Myers-Squibb; Advisory / Consultancy: Lilly; Research grant / Funding (institution): Abbvie. All other authors have declared no conflicts of interest.

Genomic alterations occurring during melanoma progression and the resulting genomic heterogeneity between metastatic deposits remain incompletely understood. Analyzing 86 metastatic melanoma deposits from 53 patients with whole-exome sequencing (WES), we show a low branch to trunk mutation ratio and little intermetastatic heterogeneity, with driver mutations almost completely shared between lesions. Branch mutations consistent with UV damage indicate that metastases may arise from different subclones in the primary tumor. Selective gain of mutated BRAF alleles occurs as an early event, contrasting whole-genome duplication (WGD) occurring as a late truncal event in about 40% of cases. One patient revealed elevated mutational diversity, probably related to previous chemotherapy and DNA repair defects. In another patient having received radiotherapy toward a lymph node metastasis, we detected a radiotherapy-related mutational signature in two subsequent distant relapses, consistent with secondary metastatic seeding. Our findings add to the understanding of genomic evolution in metastatic melanomas., As melanoma progresses, it evolves. Here, in advanced melanoma the authors study genomic evolution, highlighting trunk mutations dominated by the ultraviolet damage signature, common late truncal whole-genome duplication events, as well as selective copy number gain of mutant BRAF.

Loss of TP53 and RB1 function have both been linked to poor response to DNA damaging drugs in breast cancer patients. We inactivated TP53 and/or RB1 by siRNA mediated knockdown in breast cancer cell lines varying with respect to ER/PgR and Her-2 status as well as TP53 and RB1 mutation status (MCF-7, T47D, HTB-122 and CRL2324) and determined effects on cell cycle arrest, apoptosis and senescence with or without concomitant treatment with doxorubicin. In T47D cells, we found the cell cycle phase distribution to be altered when inactivating TP53 (P=0.0003) or TP53 and RB1 concomitantly (P≤0.001). No similar changes were observed in MCF-7, HTB-122 or CRL2324 cells. While no significant change was observed for the CRL2324 cells upon doxorubicin treatment, MCF-7, T47D as well as HTB-122 cells responded to knockdown of TP53 and RB1 in concert, with a decrease in the fraction of cells in G1/G0-phase (P=0.042, 0.021 and 0.027, respectively). Inactivation of TP53 and/or RB1 caused no change in induction of apoptosis. Upon doxorubicin treatment, inactivation of TP53 or RB1 separately caused no induction of apoptosis in MCF-7 and HTB-122 cells; however, concomitant inactivation leads to a slightly reduced activation of apoptosis. Interestingly, upon doxorubicin treatment, concomitant inactivation of TP53 and RB1 caused a decrease in senescence in MCF-7 cells (P=0.027). Comparing the effects of concomitant knockdown on apoptosis and senescence, we observed a strong interaction (P=0.001). We found concomitant inactivation of TP53 and RB1 to affect various routes of response to doxorubicin treatment in breast cancer cells.

Background Neoadjuvant treatment of triple negative breast cancer (TNBC) in general implies different chemotherapy regimens administered in sequence. PARP inhibitors like olaparib have revealed clinical benefit in TNBC harboring BRCA germline mutations, but were ineffective when tested as monotherapy for BRCA WT metastatic breast cancer. While combining a PARP inhibitor with chemotherapy may be beneficial, such therapy is limited by bone marrow toxicity. Methods In the phase 2 PETREMAC trial (NCT02624973), we assessed the efficacy of primary olaparib monotherapy for 10 weeks, followed by chemotherapy, in TNBC (T > 2 cm). Tumor biopsies and blood leucocytes for targeted DNA sequencing of 360 cancer-related genes were collected prior to therapy and after 10 weeks on olaparib. Results 31 patients with TNBC were included (mean tumor size 59 mm; range 33-97). Olaparib monotherapy was well tolerated, yielding a clinical CR in 5 and a PR in 15 patients (all-over objective response rate; ORR 64%). Olaparib had no major impact on subsequent chemotherapy toxicity. The average pretreatment mutation rate was 3.3 indels/point mutations per tumor with no differences in mutation burden recorded between responders and non-responders or BRCA carriers vs. non-carriers. Among five patients harboring pathogenic BRCA1/2 mutations (4 germline, 1 somatic) all responded to olaparib, and n = 3/5 obtained pathological complete response. Interestingly, mutations in genes associated with DNA damage repair (ATRX, BRCA1/2, EMSY, MSH6, PARP10, PPM1D) occurred in n = 9/20 olaparib responders, but in none of the 11 non-responders (p = 0.011). Non-responders were characterized by mutations in oncogenic pathways (PIK3CA, AKT1, KRAS, IGF2R, NF2 and TGFBR2) in 7 out of 11 tumors (P = 0.00013). No significant association was observed between TP53 mutations (recorded in 23/31 tumors) and response to olaparib. Targeted DNA sequencing is ongoing for biopsies taken after 10 weeks of olaparib monotherapy; the results will be presented. Conclusions Olaparib yielded a high response rate when administered to treatment-naive, large TNBC, indicating a potential role in sequential neoadjuvant therapy, for patients both with and without BRCA1/2 germline mutations. Clinical trial identification NCT02624973; 2015-002816-34. Legal entity responsible for the study Haukeland University Hospital, Bergen, Norway. Funding Det Regionale Samarbeidsorganet/Helse Vest, AstraZeneca. Disclosure H.P. Eikesdal: Honoraria (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: AstraZeneca; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Novartis; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Pfizer; Honoraria (self), Travel / Accommodation / Expenses: Pierre Fabre; Honoraria (self): Amgen; Honoraria (self), Advisory / Consultancy: Roche; Honoraria (self): HAI Interaktiv AS; Honoraria (self): Dagens Medisin; Honoraria (self): Brysol-Mayers-Squibb; Advisory / Consultancy: Lilly; Research grant / Funding (institution): Abbvie. E.S. Blix: Honoraria (self): Pfizer. B. Gilje: Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): Roche; Honoraria (self), Research grant / Funding (institution): AstraZeneca; Advisory / Consultancy, Research grant / Funding (institution): Astellas Oncology; Research grant / Funding (institution): Celgene; Research grant / Funding (institution): Pfizer; Travel / Accommodation / Expenses: Pierre Fabre. E.A. Janssen: Honoraria (self): AstraZeneca; Honoraria (self): Pfizer. J. Geisler: Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony: Novartis; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony: MSD; Honoraria (self), Advisory / Consultancy: Lilly; Honoraria (self), Speaker Bureau / Expert testimony: Pierre Fabre; Honoraria (self), Speaker Bureau / Expert testimony: Brystol-Myers-Squibb; Advisory / Consultancy, Speaker Bureau / Expert testimony: AstraZeneca; Speaker Bureau / Expert testimony: Pfizer. T. Aas: Honoraria (self): AstraZeneca; Honoraria (self): Roche. S. Knappskog: Honoraria (self), Research grant / Funding (institution): AstraZeneca; Honoraria (self), Research grant / Funding (institution): Pfizer; Licensing / Royalties: Patent EP2389450 A1,Patent WO 2012/010661.. P.E. Lonning: Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): AstraZeneca; Research grant / Funding (self): Pfizer; Research grant / Funding (self): Novartis; Honoraria (self), Travel / Accommodation / Expenses: Pierre Fabre; Honoraria (self): Roche; Advisory / Consultancy: Laboratorios Farmaceuticos Rovi; Licensing / Royalties: Cytovation. All other authors have declared no conflicts of interest.

DNA methylation is an epigenetic mechanism that can alter gene expression without modifying the DNA sequence. Reactivation of tumor suppressor genes that have been epigenetically silenced in cancer could be a potential therapeutic approach. In this study, we performed long-term treatment of the breast cancer cell line SKBR3, with Olaparib or Doxorubicin in order to assess the methylation status of tumor suppressor genes, after the cell cultures had become resistant to these drugs. Using bisulphite conversion and massive parallel sequencing we examined CpG islands in promoter regions of 283 tumor suppressor genes at several time points under the two treatment regiments. Methylation analysis was carried out for each sample using our in-house bioinformatic pipeline established in collaboration with Roche and publicly available tools. We assessed differentially methylated CpG’s for each drug, compared to control samples treated with dimethyl sulfoxide (DMSO) using the R package methylKit. Gene ontology and pathway analysis revealed hypermethylation in genes belonging to homologous recombination pathway and hypomethylation in genes responding to cellular stress and protein kinase signaling in Olaparib-treated samples. In Doxorubicin-treated samples, we identified genes involved in inter-strand cross-link repair to be hypomethylated, while other genes associated with more general cellular response to DNA damage were hypermethylated. While in Olaparib-treated samples, differential methylation seemed to be reversible and dependent on the dosage of Olaparib, methylation patterns in Doxorubicin-treated samples appeared to change in an incremental manner, as a function of exposure time to the drug. Citation Format: Zuzana Sichmanova, Asta Ottesen, Per Eystein Lonning, Elisabet Ognedal Berge, Stian Knappskog. Changes in tumor suppressor methylation in SKBR3 cells during treatment with Olaparib or Doxorubicin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 818.

Resistance to chemotherapy remains a main cause of death among cancer patients. The primary aim of the present study was to identify genetic alterations predicting resistance to chemotherapy by whole exome sequencing (WES) of tumors subject to neoadjuvant sequential treatment with epirubicin and docetaxel. A long term goal is to identify biomarkers that may be applicable in testing for drug sensitivity prior to commencement of therapy for individual patients, leading to early administration of optimal treatment and sparing patients from side-effects of inefficient treatment. From a total of 100 patients treated with sequential monotherapy in the neoadjuvant setting, we performed WES on 146 tumor samples and matching blood samples from 51 patients. Biopsies for research were taken before treatment start, at the time of treatment switch and at the time of surgery. In the present work, we have analysed WES data to assess the mutational- and the copy number landscape and compared these results between patients having a good or poor response to the two administered drugs. SNV analysis revealed imbalance in several well established breast cancer related genes, between the different response groups. Copy number assessments revealed a general increase in copy number gains in chromosome 16 and increase of copy number losses in chromosomes 1 and X after treatment with epirubicin, indicating that subclones harbouring different types of CNVs are selected for during treatment. Correspondingly, after treatment with docetaxel we found a general increase of copy number gains in chromosome 8 and an increase of copy number losses in chromosomes 1 and 8. Finally, we assessed overall mutational signatures and observed shifts in these profiles during the two treatments. Citation Format: Andreas Venizelos, Stian Knappskog, Inger Marie Loees, Hans Petter Eikesdal, Per Eystein Lønning. Genetic alterations affecting treatment response in locally advanced breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2507.