Abstract High-throughput DNA sequencing is central to precision oncology, yet robust and interpretable methods for integrated analysis of copy number alterations and somatic variants across sequencing platforms remain limited. We present CANCAN (Copy number integrative ANalysis in CANcer), a platform-agnostic computational framework for high-resolution analysis of allele-specific copy number and variant data. CANCAN integrates novel normalization and segmentation strategies and enables inference of tumor purity, ploidy, subclonality and mutation multiplicity, while providing statistical confidence estimates and transparent evaluation of alternative solutions. Benchmarking across whole-genome, whole-exome and targeted sequencing datasets from TCGA and the IMPRESS-Norway study demonstrates high concordance with established methods, with particularly strong performance on targeted sequencing data. CANCAN accurately estimates global genomic features, including purity and ploidy, even at reduced sequencing coverage, and shows comparable or improved agreement relative to existing tools. In addition, it provides detailed visualization of the genomic context of clinically relevant biomarkers, supporting diagnostic interpretation. CANCAN constitutes a reproducible and interpretable approach for integrated genomic analysis, addressing key methodological and practical challenges in clinical cancer genomics.

Background: Comprehensive genomic profiling (CGP) identifies molecular alterations to support diagnosis and guide therapy in patients with cancer, yet real-world evidence on its clinical impact remains limited. We evaluated the clinical utility of equitable and population-based CGP implementation within a publicly funded healthcare system, aiming to identify actionable alterations and support treatment recommendations in patients with advanced cancer. Methods: IMPRESS-Norway is a nationwide precision medicine trial enrolling patients with treatment-refractory advanced malignancies across oncology centres. Tumour tissue was analysed using the TruSight Oncology 500 assay (TSO500, Illumina) at four regional laboratories within the Infrastructure for Precision Diagnostics (InPreD), a national programme for implementing advanced diagnostics. Results were reviewed by the national Molecular Tumour Board (MTB) to identify actionable alterations and provide biomarker-guided treatment recommendations. Findings: From April 2021 to August 2024, 1,964 patients referred for molecular profiling consented to IMPRESS-Norway, of whom 1,835 were analysed with TSO500 and evaluated by the MTB. Of these, 769 (42%) were identified with at least one actionable alteration, and 545 (30%) received a therapy recommendation through IMPRESS-Norway (24%), other clinical trials (3%), standard-of-care (2%), or off-label programmes (0·6%). The most common actionable alterations involved PIK3CA, BRAF, ERBB2, NRAS and high tumour mutational burden. Median turnaround time from requisition to MTB recommendation was 37 days (IQR 30-47). Interpretation: Nationwide implementation of CGP within a publicly funded healthcare system is feasible and supports equal access to molecular diagnostics, enabling targeted therapies and clinical trial enrolment for a substantial proportion of patients with advanced cancer. Ongoing collection of comprehensive, structured, and standardised real-world data provides evidence for clinical utility, supports translational research, and facilitates National evaluation of cost-effectiveness of CGP in routine practice.

Structural variation (SV) is a frequent category of genetic alterations important for understanding cancer genome evolution and revealing key cancer driver events. With the development of high-throughput sequencing technologies, the ability to detect SVs of various sizes and types has improved, at both the DNA and RNA levels. However, SV calls are still prone to a considerable fraction of false positives, which necessitates visual inspection and manual curation as part of the quality control process. Identification of reliable and recurrent SVs in larger cohorts lends strength to revealing the driving roles of SVs in cancer development and to the discovery of potential diagnostic and prognostic biomarkers. Here, we present FuSViz, an application for visualization, interpretation, and prioritization of SVs. The tool provides multiple data view approaches in a user-friendly interface, allowing the investigation of prevalence and recurrence of SVs and relevant partner genes in a sample cohort. It integrates SV calls from DNA and RNA sequencing datasets to comprehensively illustrate the biological impact of SVs on the implicated genes and associated genomic regions. The functionality of FuSViz is intended for interrogation of both recurrent and private SVs, effectively assisting with pathogenicity evaluation and biomarker discovery in cancer sequencing projects.

Nodal follicular helper T-cell (T FH) lymphoma of the angioimmunoblastic (AITL) subtype has a dismal prognosis. Using whole-exome sequencing (n = 124), transcriptomic (n = 78), and methylation (n = 40) analysis, we identified recurrent mutations in known epigenetic drivers (TET2, DNMT3A, IDH2 R172 ) and novel ones (TET3, KMT2D). TET2, IDH2 R172 , DNMT3A co-mutated AITLs had poor prognosis (p < 0.0001). Genes regulating T-cell receptor (TCR) signaling (CD28, PLCG1, VAV1, FYN) or activation (RHOA G17V ) or regulators of the PI3K-pathway (PIK(3)C members, PTEN, PHLPP1, PHLPP2) were mutated. CD28 mutation/fusion was associated with poor prognosis (p = 0.02). WES of purified, neoplastic T-cell (CD3+PD1+) demonstrated high concordance with whole tumor biopsies and validated the presence of TET2 and DNMT3A in tumor and non-lymphoid cells, but other mutations (CD28, RHOA G17V , IDH2 R172 , PLCG1) in neoplastic cells. Integrated DNA-methylation and mRNA expression analysis revealed epigenetic alterations in genes regulating TCR, cytokines, PI3K-signaling, and apoptosis. RNA-seq analysis identified fusion transcripts regulating TCR-activation (8%), revealed a restricted TCR-repertoire (α = 87%, β = 72%), and showed the presence of Epstein-Barr virus transcriptome (73%). GEP demonstrated the association of B-cells or dendritic cells in the tumor milieu with prognosis (p < 0.01). RNA-seq and WES analysis of 12 AITL-patient-derived-xenografts (PDX) showed that bi-allelic TET2 and DNMT3A mutations or sub-clonal mutations (PLCG1, PHLPP2) propagated in sequential passages, and gene signatures related to T FH and T CM (central-memory) were well-maintained through passages. Gene expression signatures associated with late PDX passages (3rd-5th) were enriched with proliferation and metabolic reprogramming-related genes and predicted prognosis in an independent AITL series. Low PHLPP2 mRNA expression predicted poor prognosis (p = 0.05) and engineered PHLPP2 or TET2 loss in CD4+ T-cells showed enhanced PI(3)K activation, thus uncovering a therapeutic target for clinical trials.

Patients with localised, high-risk gastrointestinal stromal tumours (GIST) benefit from adjuvant imatinib treatment. Still, approximately 40% of patients relapse within 3 years after adjuvant therapy and the clinical and histopathological features currently used for risk classification cannot precisely predict poor outcomes after standard treatment. This study aimed to identify genomic and transcriptomic profiles that could be associated with disease relapse and thus a more aggressive phenotype. Using a multi-omics approach, we analysed a cohort of primary tumours from patients with untreated, resectable high-risk GISTs. We compared patients who developed metastatic disease within 3 years after finishing adjuvant imatinib treatment and patients without disease relapse after more than 5 years of follow-up. Combining genomics and transcriptomics data, we identified somatic mutations and deregulated mRNA and miRNA genes intrinsic to each group. Our study shows that increased chromosomal instability (CIN), including chromothripsis and deregulated kinetochore and cell cycle signalling, separates high-risk samples according to metastatic potential. The increased CIN seems to be an intrinsic feature for tumours that metastasise and should be further validated as a novel prognostic biomarker for high-risk GIST.

Follicular helper T-cell lymphoma of the angioimmunoblastic type (AITL) is associated with dismal prognosis. We performed functional genomic approaches including whole-exome sequencing (WES; n=119), transcriptomic (n=78) and methylation (n=40) analysis. We identified recurrent mutations in known epigenetic drivers ( TET2, DNMT3A, IDH2 R172), and also identified novel ones (TET3, KMT2D). Somatic mutation of all three epigenetic drivers ( TET2, IDH2, and DNMT3A) was associated with poor prognosis (p<.001). Mutations in genes regulating T-cell receptor (TCR) signaling ( CD28 VAV1, FYN, PLCG1) or activation ( RHOA G17V), and regulators of the PI3K pathway (PIK(3)C members, PTEN,PHLPP-1/-2) were also found. Genome-wide DNA-methylation analysis integrated with mRNA expression profiling also revealed epigenetic alterations in genes regulating TCR-RHOA/B/C or PI3K-signaling. TET2 loss was noted in 85% AITLs and was significantly associated with RHOA G17V, CD28 and IDH2 R172mutations. AITLs lacking RHOA G17V tended to have mutations regulating the JAK-STAT pathway ( JAK2, JAK3, STAT1, STAT3, SOCS1). RNA-seq analysis identified fusion transcripts in genes regulating TCR activation (8%), revealed a restricted TCR repertoire in the majority of cases (a=87%, b=72%), and showed the presence of Epstein-Barr virus transcriptome (73%). GEP demonstrated association of B-cells in the tumor-milieu with better prognosis (p=.006), while dendritic cells were associated with worse prognosis (p=.001), which was further validated by immunohistochemistry using CD20, CD68, and CD163 antibodies. RNA-seq and corresponding WES analysis of 12 AITL patient-derived-xenografts (PDX) showed that bi-allelic TET2 mutations, DNMT3A mutations or sub-clonal mutations ( PLCG1 PHLPP2) werepropagated in sequential passages. Gene signatures related to T FH (follicular helper) and T CM (central memory) were also well-maintained in secondary passages in PDX models. Gene signatures of late PDX passages (3 rd-5 th) were enriched with genes related to proliferation and metabolic reprogramming, and in an independent cohort of AITLs, high expression of T3/T5 related signatures was associated with worse outcome (p=0.02/p=0.009). Low mRNA expression of PHLPP2 predicted poor prognosis (p=.03) and engineered PHLPP2 loss showed enhanced PI(3)K activation and FOXO1 inactivation in CD4+ T-cells in-vitro. Thus, we defined the genomic landscape for AITL, which is largely characterized by epigenetic alterations, TCR signaling and PI3K/AKT dysregulation, which may be amenable for therapeutic targeting.

Introduction Multiple myeloma (MM) is a heterogeneous disease where cancer-driver mutations and aberrant signaling may lead to disease progression and drug resistance. Drug responses vary greatly, and there is an unmet need for biomarkers that can guide precision cancer medicine in this disease. Methods To identify potential predictors of drug sensitivity, we applied integrated data from drug sensitivity screening, mutational analysis and functional signaling pathway profiling in 9 cell line models of MM. We studied the sensitivity to 33 targeted drugs and their association with the mutational status of cancer-driver genes and activity level of signaling proteins. Results We found that sensitivity to mitogen-activated protein kinase kinase 1 (MEK1) and phosphatidylinositol-3 kinase (PI3K) inhibitors correlated with mutations in NRAS/KRAS , and PI3K family genes, respectively. Phosphorylation status of MEK1 and protein kinase B (AKT) correlated with sensitivity to MEK and PI3K inhibition, respectively. In addition, we found that enhanced phosphorylation of proteins, including Tank-binding kinase 1 (TBK1), as well as high expression of B cell lymphoma 2 (Bcl-2), correlated with low sensitivity to MEK inhibitors. Discussion Taken together, this study shows that mutational status and signaling protein profiling might be used in further studies to predict drug sensitivities and identify resistance markers in MM.

Follicular lymphoma (FL) is the most common indolent type of B-cell non-Hodgkin lymphoma. Advances in treatment have improved overall survival, but early relapse or transformation to aggressive disease is associated with inferior outcome. To identify early genetic events and track tumor clonal evolution, we performed multi-omics analysis of 94 longitudinal biopsies from 44 FL patients; 22 with transformation (tFL) and 22 with relapse without transformation (nFL). Deep whole-exome sequencing confirmed recurrent mutations in genes encoding epigenetic regulators (CREBBP, KMT2D, EZH2, EP300), with similar mutational landscape in nFL and tFL patients. Calculation of genomic distances between longitudinal samples revealed complex evolutionary patterns in both subgroups. CREBBP and KMT2D mutations were identified as genetic events that occur early in the disease course, and cases with CREBBP KAT domain mutations had low risk of transformation. Gains in chromosomes 12 and 18 (TCF4), and loss in 6q were identified as early and stable copy number alterations. Identification of such early and stable genetic events may provide opportunities for early disease detection and disease monitoring. Integrative analysis revealed that tumors with EZH2 mutations exhibited reduced gene expression of numerous histone genes, including histone linker genes. This might contribute to the epigenetic dysregulation in FL.

Subclonal evolution during primary breast cancer treatment is largely unexplored. We aimed to assess the dynamic changes in subclonal composition of treatment-naïve breast cancers during neoadjuvant chemotherapy. We performed whole exome sequencing of tumor biopsies collected before, at therapy switch, and after treatment with sequential epirubicin and docetaxel monotherapy in 51 out of 109 patients with primary breast cancer, who were included in a prospectively registered, neoadjuvant single-arm phase II trial. There was a profound and differential redistribution of subclones during epirubicin and docetaxel treatment, regardless of therapy response. While truncal mutations and main subclones persisted, smaller subclones frequently appeared or disappeared. Reassessment of raw data, beyond formal mutation calling, indicated that the majority of subclones seemingly appearing during treatment were in fact present in pretreatment breast cancers, below conventional detection limits. Likewise, subclones which seemingly disappeared were still present, below detection limits, in most cases where tumor tissue remained. Tumor mutational burden (TMB) dropped during neoadjuvant therapy, and copy number analysis demonstrated specific genomic regions to be systematically lost or gained for each of the two chemotherapeutics. Sequential epirubicin and docetaxel monotherapy caused profound redistribution of smaller subclones in primary breast cancer, while early truncal mutations and major subclones generally persisted through treatment. ClinicalTrials.gov, NCT00496795 , registered on July 4, 2007.

Background Ipilimumab improves survival for patients with metastatic malignant melanoma. Combining a therapeutic cancer vaccine with ipilimumab may increase efficacy by providing enhanced anti-tumor immune responses. UV1 consists of three synthetic long peptides from human telomerase reverse transcriptase (hTERT). These peptides comprise epitopes recognized by T cells from cancer patients experiencing long-term survival following treatment with a first-generation hTERT vaccine, and generate long-lasting immune responses in cancer patients when used as monotherapy. The objective of this trial was to investigate the safety and efficacy of combining UV1 with ipilimumab in metastatic melanoma. Patients and Methods In this phase I/IIa, single center trial [NCT02275416], patients with metastatic melanoma received repeated UV1 vaccinations, with GM-CSF as an adjuvant, in combination with ipilimumab. Patients were evaluated for safety, efficacy and immune response. Immune responses against vaccine peptides were monitored in peripheral blood by measuring antigen-specific proliferation and IFN-γ production. Results Twelve patients were recruited. Adverse events were mainly diarrhea, injection site reaction, pruritus, rash, nausea and fatigue. Ten patients showed a Th1 immune response to UV1 peptides, occurring early and after few vaccinations. Three patients obtained a partial response and one patient a complete response. Overall survival was 50% at 5 years. Conclusion Treatment was well tolerated. The rapid expansion of UV1-specific Th1 cells in the majority of patients indicates synergy between UV1 vaccine and CTLA-4 blockade. This may have translated into clinical benefit, encouraging the combination of UV1 vaccination with standard of care treatment regimes containing ipilimumab/CTLA-4 blocking antibodies.

Background: Androgen steroid hormones are key drivers of prostate cancer. Previous work has shown that androgens can drive the expression of alternative mRNA isoforms as well as transcriptional changes in prostate cancer cells. Yet to what extent androgens control alternative mRNA isoforms and how these are expressed and differentially regulated in prostate tumours is unknown. Methods: Here we have used RNA-Seq data to globally identify alternative mRNA isoform expression under androgen control in prostate cancer cells, and profiled the expression of these mRNA isoforms in clinical tissue. Results: Our data indicate androgens primarily switch mRNA isoforms through alternative promoter selection. We detected 73 androgen regulated alternative transcription events, including utilisation of 56 androgen-dependent alternative promoters, 13 androgen-regulated alternative splicing events, and selection of 4 androgen-regulated alternative 3′ mRNA ends. 64 of these events are novel to this study, and 26 involve previously unannotated isoforms. We validated androgen dependent regulation of 17 alternative isoforms by quantitative PCR in an independent sample set. Some of the identified mRNA isoforms are in genes already implicated in prostate cancer (including LIG4, FDFT1 and RELAXIN), or in genes important in other cancers (e.g. NUP93 and MAT2A). Importantly, analysis of transcriptome data from 497 tumour samples in the TGCA prostate adenocarcinoma (PRAD) cohort identified 13 mRNA isoforms Open Peer Review Reviewer Status Invited Reviewers 1 2 3 version 1 03 Aug 2018 report report report Sebastian Oltean, University of Exeter, Exeter, UK 1. Cyril F. Bourgeois , University of Lyon, Lyon, France 2. Jennifer Byrne , The Children's Hospital at Westmead, Westmead, Australia 3. Any reports and responses or comments on the article can be found at the end of the article. Page 1 of 35 F1000Research 2018, 7:1189 Last updated: 21 AUG 2021

Key Points Diagnostic and relapse diffuse large B-cell lymphoma (DLBCL) biopsies reveal increased mutational burden/loss of heterozygosity in HLA-A. Serially sampled tumor biopsies provide insight into therapeutic targets and evolutionary divergence in relapsed/refractory DLBCL.

Abstract Molecular analysis of circulating tumor DNA (ctDNA) has a large potential for clinical application by capturing mutations and heterogeneity through noninvasive sampling. The CircSarc studies aims to provide new insights into the clinical utility of liquid biopsies in soft tissue sarcomas (CircSarc STS) and gastrointestinal stromal tumors (CircSarc GIST). In CircSarc STS, we have enrolled 35 high-grade soft-tissue sarcoma patients with localized disease. Plasma from each patient has been collected longitudinally throughout the course of their disease, with a follow-up of up to 5 years. To identify patient-specific somatic mutations, the patients’ tumor and germline DNA have been exome sequenced. Somatic mutations in cfDNA are being identified using ultra-low-pass WGS or targeted NGS. At time of surgery and/or progression, we detect ctDNA in 30% of the first soft-tissue sarcoma samples with higher levels in larger or metastatic tumors. In a proof-of-concept study, we have shown that that levels of tumor-specific mutations in plasma correlated to clinical manifestation of metastatic disease. In CircSarc GIST, plasma has been collected for a cohort of 30 metastatic GISTs and at time of diagnosis for 50 GISTs. Somatic mutations in the tumor have been determined by targeted sequencing or whole-exome sequencing. Somatic mutations in ctDNA are identified using targeted NGS. The levels of somatic mutations in ctDNA are being correlated with clinicopathologic features. ctDNA was detected in 36% of treatment-naïve GIST patients, including all patients with metastatic disease, and the tumor burden was the most important detection determinant. Analysis of metastatic GISTs is ongoing, but repeated plasma samples in a progressing patient have demonstrated that clonal evolution can be monitored over time. ctDNA analysis of the patient revealed multiple resistance mutations, and these were spatially distributed in the primary tumor. Our work shows that detection of mutations in plasma is particularly feasible for patients with high tumor burden. Mutational analysis by use of liquid biopsies can capture the molecular heterogeneity of the tumor and guide treatment decisions during progression. Citation Format: Heidi M. Namløs, Kjetil Boye, Seyed H Moosavi, Daniel Vodak, Nitin Sharma, Susanne Lorenz, Bodil Bjerkehagen, Stine Næss, Eivind Hovig, Nina Jebsen, Anders Ståhlberg, Ola Myklebost, Leonardo A. Meza-Zepeda. Tracking the evolution of soft tissue sarcoma and GIST using liquid biopsies [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A31.

Introduction: Follicular lymphoma (FL) is an indolent malignancy, characterized by multiple relapses during the disease course. Annually around 2-3% of patients experience transformation to aggressive disease (tFL), most commonly to diffuse large B-cell lymphoma (DLBCL). Both transformation and progression of disease within 2 years (POD24) are associated with poor prognosis, yet the molecular events underlying these processes are not well understood. The existence of common progenitor cells (CPCs) has been inferred from genetic analyses of longitudinal biopsies. Improved characterization of genetic alterations associated with CPCs in cases with transformation and POD24 may improve our understanding of disease progression, and reveal molecular markers for high-risk disease. Methods: We performed whole-exome sequencing of 97 serial tumor biopsies and matched normal samples purified from peripheral blood from 44 FL patients. An average sequencing coverage of 700X was achieved for both tumor and normal samples, which ensured high data quality and ability to detect SNVs and InDels using our bench-marked bioinformatics pipeline. SNP6.0 data was available for 93 sequenced tumors and used to infer allele-specific copy number alterations. Several computational tools were applied to identify potential cancer driver genes (MutSig2CV, 2020plus), mutational signatures (MutationalPatterns), and to study clonal evolution (PyClone, ClonEvol). Results: Twenty-two of the 44 FL patients experienced relapses without transformation (referred to as the nFL group), and 22 patients experienced transformation (the tFL group). Nineteen patients (including both groups) experienced POD24. Both transformation and POD24 were associated with inferior overall survival. The median non-synonymous mutational burden was 96 per biopsy (range 10-326). Pre-treatment FL biopsies from the tFL group had significantly higher mutational burden compared to the nFL group (p<0.02). The application of two different approaches for driver gene discovery resulted in a total of 55 as putative drivers. Sixteen of these genes were identified by both tools, including the known FL driver genes CREBBP, KMT2D, TNFRSF14 and BCL2, all being mutated in more than 40 % of cases, and the novel cancer driver genes CTSS and VPS39. By applying MutSig2CV to different categories of biopsies, we identified RRAGC, ATP6V1B2, and HNRNPU as being significantly mutated in pre-treatment tumors of the nFL group, whereas TNFRSF14 and EZH2 were significantly mutated in pre-treatment tumors of the tFL group. When comparing pretreatment and relapsed FL biopsies, we identified CTSS, ZNF493 and HLA-A as significantly mutated in relapsed FL, thus potentially linking these genes to FL relapse. Analysis of mutational signatures demonstrated the presence of the same signatures for longitudinal samples, and no contrasting difference was found between nFL and tFL biopsies. Finally, we constructed clonal phylogenetic trees for 31 patients with serial biopsies and observed that both non-transformed and transformed tumors can evolve directly from CPCs. Contrary to previous findings, we did not find a preference for divergent evolution from FL to tFL, as compared to FL to relapsed FL. There was no significant correlation between evolution pattern and time intervals in the nFL and tFL groups. Based on the mutational history, we confirmed that CREBBP and KMT2D were the most frequently mutated genes seen in CPCs and PCLO, ATP6V1B2 and LRRN3 were also identified as early mutated genes. TNFRSF14, TBL1XR1 and GNAI2 were often mutated later, but before clonal expansion. Conclusions: We show that at diagnosis, the mutational burden in the tFL group is significantly higher than in the nFL group. By systematically applying driver discovery tools we have confirmed known driver genes and discovered novel genes that may be of importance for FL progression and transformation. Divergent evolution is a common feature both during relapse of FL and transformation to DLBCL, regardless of the time interval between the longitudinal biopsies. A better characterization of the CPCs may provide additional therapeutic opportunities towards a cure for FL patients. Disclosures Holte: Novartis: Honoraria, Other: Advisory board.

Introduction Relapses of diffuse large B-cell lymphoma typically occur within 2-3 years and only 10% of these patients reach a 3-year progression-free survival compared to 65% at diagnosis. Our ability to distinguish patients at risk for relapse remains based on clinical staging. We hypothesized that identifying genetic alterations in serial tumour biopsies at diagnosis and relapse would improve our ability to identify high-risk patients, make therapeutic selections and reveal molecular markers for chemo-immunotherapy resistant tumours. However, relatively few relapsed/refractory biopsies have been sequenced. A unique, clinically annotated, Nordic DLBCL cohort was used to identify significantly mutated genes, assess potential driver genes, comprehensively examine clonal evolution, and gauge the importance of clinical relapsed sampling. Methods To address the lack of information on the molecular foundations of relapsed/refractory DLBCL, we performed whole exome sequencing (WES) on 42 DLBCL cases, with 34% representing relapsed/refractory biopsies and 13 serially sample cases. Enriched with relapsed/refractory diffuse large B-cell lymphoma cases, we performed multiple computational analyses to identify significantly mutated genes (MutSig2CV), mutational signatures (NMF and DeConstructsSig), driver genes (IntOgen and CADD), clonal evolution architecture (SciClone and ClonEvol), druggable gene analysis (DGIdb), and HLA-inference and mutation calling (Polysolver). Results Clonal evolution analysis of 13 paired diagnostic and relapsed biopsies revealed that relapsed/refractory biopsies have remarkable similarities to diagnostic biopsies and often present with late divergent clonal evolution of the tumor. Mutational analysis of 18 serially sampled tumors determined that in the majority of cases druggable oncogenic variants do arise at relapse. In addition, time to relapse correlated with divergence of mutations from the diagnostic biopsy. In addition to being identified as a significantly mutated gene, mutations in HLA-A had an increased incidence in cases that ultimately relapsed. This result led to an in-depth investigation into the mutational prevalence, timing, impact on prognosis, and loss of heterozygosity in the human leukocyte antigen (HLA) haplotypes of relapsed/refractory DLBCL. HLA-A mutagenesis and loss of heterozygosity was discovered as mechanisms of immune evasion in cases that go on to relapse from R-CHOP like therapies (Figure 1). Conclusions Our results yield insight into the development of chemo-immunotherapy resistant diffuse large B-cell lymphoma, and highlight the clinical importance of sampling relapsed biopsies. Analysis of immune evasion through MHC Class I/II, specifically HLA-A, may provide better characterization of patients for relapse prediction. In the age of personalized medicine it will be instrumental to determine if relapsed biopsies offer additional insight for salvage therapy treatment. Divergence of biopsies, as characterized by shared genomic mutations, increase with time and the majority of cases present with new alterations in druggable genes post-therapy. Disclosures Leppa: Roche: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Celgene: Consultancy; Bayer: Research Funding. Holte:Novartis: Honoraria, Other: Advisory board.

Background: The genetic mechanisms for families who meet the clinical criteria for Lynch syndrome (LS) but do not carry pathogenic variants in the mismatch repair (MMR) genes are still undetermined. We aimed to study the potential contribution of genes other than MMR genes to the biological and clinical characteristics of Norwegian families fulfilling Amsterdam (AMS) criteria or revised Bethesda guidelines. Methods: The Hereditary Cancer Biobank of the Norwegian Radium Hospital was interrogated to identify individuals with a high risk of developing colorectal cancer (CRC) for whom no pathogenic variants in MMR genes had been found in routine diagnostic DNA sequencing. Forty-four cancer susceptibility genes were selected and analyzed by using our in-house designed TruSeq amplicon-based assay for targeted sequencing. RNA splicing-and protein-dedicated in silico analyses were performed for all variants of unknown significance (VUS). Variants predicted as likely to affect splicing were experimentally analyzed by resorting to minigene assays. Results: We identified a patient who met the revised Bethesda guidelines and carried a likely pathogenic variant in CHEK2 (c.470 T > C, p.I157T). In addition, 25 unique VUS were identified in 18 individuals, of which 2 exonic variants (MAP3K1 c.764A > G and NOTCH3 c.5854G > A) were analyzed in the minigene splicing assay and found not to have an effect on RNA splicing. Conclusions: Among high-risk CRC patients that fulfill the AMS criteria or revised Bethesda guidelines, targeted gene sequencing identified likely pathogenic variant and VUS in other genes than the MMR genes (CHEK2, NOTCH3 and MAP3K1). Our study suggests that the analysis of genes currently excluded from routine molecular diagnostic screens may confer cancer susceptibility.

Background: In kindreds carrying path_BRCA1/2 variants, some women in these families will develop cancer despite testing negative for the family's pathogenic variant. These families may have additional genetic variants, which not only may increase the susceptibility of the families' path_BRCA1/2, but also be capable of causing cancer in the absence of the path_BRCA1/2 variants. We aimed to identify novel genetic variants in prospectively detected breast cancer (BC) or gynecological cancer cases tested negative for their families' pathogenic BRCA1/2 variant (path_BRCA1 or path_BRCA2). Methods: Women with BC or gynecological cancer who had tested negative for path_BRCA1 or path_BRCA2 variants were included. Forty-four cancer susceptibility genes were screened for genetic variation through a targeted amplicon-based sequencing assay. Protein- and RNA splicing-dedicated in silico analyses were performed for all variants of unknown significance (VUS). Variants predicted as the ones most likely affecting pre-mRNA splicing were experimentally analyzed in a minigene assay. Results: We identified 48 women who were tested negative for their family's path_BRCA1 (n = 13) or path_BRCA2 ( n = 35) variants. Pathogenic variants in the ATM, BRCA2, MSH6 and MUTYH genes were found in 10% (5/48) of the cases, of whom 15% (2/13) were from path_BRCA1 and 9% (3/35) from path_ BRCA2 families. Out of the 26 unique VUS, 3 (12%) were predicted to affect RNA splicing (APC c. 721G > A, MAP3K1 c.764A > G and MSH2 c.815C > T). However, by using a minigene, assay we here show that APC c. 721G > A does not cause a splicing defect, similarly to what has been recently reported for the MAP3K1 c.764A > G. The MSH2 c.815C > T was previously described as causing partial exon skipping and it was identified in this work together with the path_ BRCA2 c.9382C > T (p.R3128X). Conclusion: All women in breast or breast/ovarian cancer kindreds would benefit from being offered genetic testing irrespective of which causative genetic variants have been demonstrated in their relatives.

Background: Androgen steroid hormones are key drivers of prostate cancer. Previous work has shown that androgens can drive the expression of alternative mRNA isoforms as well as transcriptional changes in prostate cancer cells. Yet to what extent androgens control alternative mRNA isoforms and how these are expressed and differentially regulated in prostate tumours is unknown. Methods: Here we have used RNA-Seq data to globally identify alternative mRNA isoform expression under androgen control in prostate cancer cells, and profiled the expression of these mRNA isoforms in clinical tissue. Results: Our data indicate androgens primarily switch mRNA isoforms through alternative promoter selection. We detected 73 androgen regulated alternative transcription events, including utilisation of 56 androgen-dependent alternative promoters, 13 androgen-regulated alternative splicing events, and selection of 4 androgen-regulated alternative 3′ mRNA ends. 64 of these events are novel to this study, and 26 involve previously unannotated isoforms. We validated androgen dependent regulation of 17 alternative isoforms by quantitative PCR in an independent sample set. Some of the identified mRNA isoforms are in genes already implicated in prostate cancer (including LIG4, FDFT1 and RELAXIN), or in genes important in other cancers (e.g. NUP93 and MAT2A). Importantly, analysis of transcriptome data from 497 tumour samples in the TGCA prostate adenocarcinoma (PRAD) cohort identified 13 mRNA isoforms (including TPD52, TACC2 and NDUFV3) that are differentially regulated in localised prostate cancer relative to normal tissue, and 3 (OSBPL1A, CLK3 and TSC22D3) which change significantly with Gleason grade and tumour stage. Conclusions: Our findings dramatically increase the number of known androgen regulated isoforms in prostate cancer, and indicate a highly complex response to androgens in prostate cancer cells that could be clinically important.

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.