Differences In Mutational Burden Research Articles

Patterns of the Predicted Mutation Burden in 19,778 Domesticated Barley Accessions Conserved Ex Situ.

Long-term conservation of more than 7 million plant germplasm accessions in 1750 genebanks worldwide is a challenging mission. The extent of deleterious mutations present in conserved germplasm and the genetic risk associated with accumulative mutations are largely unknown. This study took advantage of published barley genomic data to predict sample-wise mutation burdens for 19,778 domesticated barley (Hordeum vulgare L.) accessions conserved ex situ. It was found that the conserved germplasm harbored 407 deleterious mutations and 337 (or 82%) identified deleterious alleles were present in 20 (or 0.1%) or fewer barley accessions. Analysis of the predicted mutation burdens revealed significant differences in mutation burden for several groups of barley germplasm (landrace > cultivar (or higher burden estimate in landrace than in cultivar); winter barley > spring barley; six-rowed barley > two-rowed barley; and 1000-accession core collection > non-core germplasm). Significant differences in burden estimate were also found among seven major geographical regions. The sample-wise predicted mutation burdens were positively correlated with the estimates of sample average pairwise genetic difference. These findings are significant for barley germplasm management and utilization and for a better understanding of the genetic risk in conserved plant germplasm.

Abstract 4093: Modeling tumor immunoediting under immune selective pressure to inform neoantigen landscape dynamics for effective cancer vaccines

Abstract Tumor specific neoantigens, encoded by somatic mutations, are recognized by T cells, inducing anti-tumor immune responses. This renders neoantigens viable targets for personalized cancer vaccines. However, the identification of immunogenic neoantigens remains suboptimal. Understanding how immune selective pressure-mediated tumor evolution and neoantigen editing contribute to emergence of immunogenic neoantigens may improve prediction accuracy. In this study, we aimed to model tumor evolution and heterogeneity under immune pressure using three preclinical models of lung cancer with common driver mutations, Kras G12D/+ and p53 -/-, namely KPA, KPC, and HKP1, subcutaneously implanted in immunocompetent C57BL/6 (B6) and immune-deficient Rag1-/- mice. Our data suggest that despite shared driver mutations identified by whole exome and RNA-seq, tumor growth in vivo varied between tumor models. KPA tumors were immunogenic, as they were controlled in B6 mice but rapidly progressed in Rag1-/- mice, suggesting immunoediting of KPA-specific neoantigens. In contrast, HKP1 was non-immunogenic, with tumors progressing regardless of immune competency. KPC showed moderate tumor control in B6 mice, which was lost in Rag1-/- mice. We then estimated the cancer cell fraction on every mutation based on phylogenetic reconstruction. Immunogenic KPA tumors had significantly lower mutation burden in B6 than in Rag1-/- mice and the fraction of cell line mutations edited in vivo was significantly higher, suggesting active immunoediting in the former. Non-immunogenic HKP1 tumors did not show significant differences in mutation burden nor fraction of mutations edited between B6 and Rag1-/- mice. The immunogenicity was reflected in the immune infiltrate levels within tumors, with KPA being highly infiltrated by activated CD4 and CD8 T cells compared to KPC. HKP1 tumors showed increased infiltration of suppressive regulatory T cells. T cell receptor (TCR) sequencing on lung tumors showed that clonal expansion is strongly associated with immunogenicity. KPA tumors showed significantly higher TCR clustering with less diversity compared to KPC and more than HKP1. Following a simple approach to study the immunogenicity of shared neoantigens to elicit cross-protective anti-tumor responses, mice were immunized with whole irradiated (IR) tumor cell lines and implanted with live tumor cells (matched and unmatched). While immunization with IR-KPA protected against corresponding tumor implant and moderately against KPC, it did not protect against HKP1, indicating lack of immunogenicity of mutations shared between KPA and HKP1. Further studies testing the anti-tumor responses and characteristics of neoantigens shared between these three related tumor cell lines will inform the conditions under which tumors may escape or regress and help improve neoantigen identifying algorithms. Citation Format: Mariam Mathew George, Jayon Lihm, Hyejin Choi, Yuval Elhanati, Stephen Martis, Marta Luksza, Benjamin Greenbaum, Jedd D. Wolchok, Taha Merghoub. Modeling tumor immunoediting under immune selective pressure to inform neoantigen landscape dynamics for effective cancer vaccines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4093.

Abstract 610: Modeling tumor immunoediting coupled with T-cell repertoire dynamic

Abstract As tumors evolve, cancer cells acquire somatic mutations and these mutations generate tumor specific neoantigens that are different from self and can be recognized by T-cells, inducing anti-tumor immune responses. Such immunogenic antigens can be effective targets for neoantigen-based cancer vaccines. However, the identification of immunogenic neoantigens remains limited with the current prediction models. Here we designed mouse experiments to model tumor evolution under immune pressure and its association with T-cell clones to identify these antigens agnostically and improve current prediction models. We injected three lung cancer cell lines (KPA, KPC, and HKP1) in wildtype immune-competent C57BL/6 mice and immune-deficient Rag1 defective (Rag1 KO) mice, respectively. The three tumor cell lines have common driver mutations, Kras G12D/+ and p53 flox/flox. Two weeks post tumor implantation, whole exome sequencing and RNA-seq was performed on tumor samples. We also performed T cell receptor (TCR) sequencing to profile tumor infiltrated TCR clones. Despite the shared driver mutations, tumor growth patterns in vivo over time are very distinct for each cell line. Interestingly, KPA tumors show differential evolutionary patterns in wild type mice versus Rag1 KO mice, regressing in the former but rapidly progressing in the latter, suggesting that the difference can be attributed to immunoediting of KPA specific neoantigens. HKP1 tumors do not show substantial differences in that aggressive tumor progression is observed in both types of mice. Using WES data, we estimated the cancer cell fraction of each mutation based on phylogeny re-construction. We found that evolved KPA tumors (immunogenic) had significantly lower mutation burden in wild type mice compared to Rag1 KO mice (3410 vs 4199 on average), and the fraction of cell line mutations edited in vivo is significantly higher in wild type than in Rag1 KO (26% vs 14% on average), suggesting again the active on-going immunosurveillance in immune competent mice. In contrast, HKP1 tumors (non-immunogenic) do not show significant differences in mutation burden nor fraction of mutations edited between wild type and Rag1 KO mice. Furthermore, TCR-seq data show that the clonal expansion of T-cells is strongly associated with tumor immunogenicity. KPA tumor samples show significantly higher TCR clustering but with less diversity, quantified by modified Simpson’s Index, that is strongly correlated with the suppression of mutational burden (r=0.74). In summary, we found that tumor evolution in immune competent mice is dependent on mutational context of the tumor. By analyzing longitudinal tumor and TCR sequencing data, we showed that the immunogenicity of tumors is associated with significant decrease in mutational burden and higher T-cell clustering that is indicative of tumor mutation and TCR clone expansion co-evolution in immune competent mice. Citation Format: Jayon Lihm, Hyejin Choi, Mariam M. George, Yuval Elhanati, David Hoyos, Marta Luksza, Taha Merghoub, Benjamin D. Greenbaum. Modeling tumor immunoediting coupled with T-cell repertoire dynamic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 610.

The prognostic value of TMB in early-stage non-small cell lung cancer: a systematic review and meta-analysis.

Tumor mutation burden (TMB) has been validated as a predictive biomarker for immunotherapy response and survival in numerous cancer types. Limited data is available on the inherent prognostic role of TMB in early-stage tumors. To evaluate the prognostic role of TMB in early-stage, resected non-small cell lung cancer (NSCLC). Systematic review and meta-analysis of pertinent prospective and retrospective studies. Publication search was performed in PubMed, Embase, Cochrane Library, and Web of Science databases. Based on the level of heterogeneity, a random- or fixed-effects model was used to calculate pooled effects of hazard ratio (HR) for overall survival (OS) and disease-free survival (DFS). The source of heterogeneity was investigated using sensitivity analysis, subgroup analysis, and publication bias assessment. Ten studies comprising 2520 patients were included in this analysis. There was no statistically significant difference in OS (HR, 1.18, 95% CI, 0.70, 1.33; p 0.53, I2 = 80%; phet < 0.0001) and DFS (HR, 1.18, 95% CI, 0.91, 1.52; p = 0.53, I2 = 75%; phet = 0.0001) between the high-TMB and low-TMB group. Subgroup analyses indicated that East Asian ethnicity, and TMB detected using whole exome sequencing, and studies with <100 patients had poor DFS in the high-TMB group. The inherent prognostic role of TMB is limited in early-stage NSCLC. Ethnic differences in mutation burden must be considered while designing future trials on neoadjuvant immunotherapy. Further research in the harmonization and standardization of panel-based TMB is essential for its widespread clinical utility.Registration: CRD42023392846.

Neuroendocrine pathways and breast cancer progression: a pooled analysis of somatic mutations and gene expression from two large breast cancer cohorts

BackgroundExperimental studies indicate that neuroendocrine pathways might play a role in progression of breast cancer. We aim to test the hypothesis that somatic mutations in the genes of neuroendocrine pathways influence breast cancer prognosis, through dysregulated gene expression in tumor tissue.MethodsWe conducted an extreme case–control study including 208 breast cancer patients with poor invasive disease-free survival (iDFS) and 208 patients with favorable iDFS who were individually matched on molecular subtype from the Breast Cancer Cohort at West China Hospital (WCH; N = 192) and The Cancer Genome Atlas (TCGA; N = 224). Whole exome sequencing and RNA sequencing of tumor and paired normal breast tissues were performed. Adrenergic, glucocorticoid, dopaminergic, serotonergic, and cholinergic pathways were assessed for differences in mutation burden and gene expression in relation to breast cancer iDFS using the logistic regression and global test, respectively.ResultsIn the pooled analysis, presence of any somatic mutation (odds ratio = 1.66, 95% CI: 1.07–2.58) of the glucocorticoid pathway was associated with poor iDFS and a two-fold increase of tumor mutation burden was associated with 17% elevated odds (95% CI: 2–35%), after adjustment for cohort membership, age, menopausal status, molecular subtype, and tumor stage. Differential expression of genes in the glucocorticoid pathway in tumor tissue (P = 0.028), but not normal tissue (P = 0.701), was associated with poor iDFS. Somatic mutation of the adrenergic and cholinergic pathways was significantly associated with iDFS in WCH, but not in TCGA.ConclusionGlucocorticoid pathway may play a role in breast cancer prognosis through differential mutations and expression. Further characterization of its functional role may open new avenues for the development of novel therapeutic targets for breast cancer.

The Dynamics of Somatic Mutagenesis During Life in Humans.

From conception to death, human cells accumulate somatic mutations in their genomes. These mutations can contribute to the development of cancer and non-malignant diseases and have also been associated with aging. Rapid technological developments in sequencing approaches in the last few years and their application to normal tissues have greatly advanced our knowledge about the accumulation of these mutations during healthy aging. Whole genome sequencing studies have revealed that there are significant differences in mutation burden and patterns across tissues, but also that the mutation rates within tissues are surprisingly constant during adult life. In contrast, recent lineage-tracing studies based on whole-genome sequencing have shown that the rate of mutation accumulation is strongly increased early in life before birth. These early mutations, which can be shared by many cells in the body, may have a large impact on development and the origin of somatic diseases. For example, cancer driver mutations can arise early in life, decades before the detection of the malignancy. Here, we review the recent insights in mutation accumulation and mutagenic processes in normal tissues. We compare mutagenesis early and later in life and discuss how mutation rates and patterns evolve during aging. Additionally, we outline the potential impact of these mutations on development, aging and disease.

MODL-25. REPLICATION REPAIR DEFICIENT MOUSE MODELS PROVIDE INSIGHT ON HYPERMUTANT BRAIN TUMOURS, MECHANISMS OF IMMUNE EVASION, AND COMBINATORIAL IMMUNOTHERAPY

Replication repair deficiency (RRD) is the leading cause of hypermutant brain tumours in children. RRD is caused by defects in one of four mismatch repair (MMR) genes and mutations in POLE or POLD1. Such tumours are resistant to common therapeutic agents and animal models are needed to study RRD in vivo and test novel therapies like immune checkpoint inhibitors (ICIs). To model RRD brain tumours specifically, we engineered a Pole mutant mouse model harbouring the S459F mutation (PoleS459F). We combined PoleS459F mice with conditional Msh2 knockout (Msh2LoxP) and Nestin-cre mice. All Nestin-cre+Msh2LoxP/LoxPPoleS459F/+ mice rapidly succumbed to posterior fossa brain tumours between 8.6 and 12.4 weeks. Importantly, tumours exhibited hallmark “ultrahypermutation” (~350 mutations/Mb) and the corresponding signatures characteristic of human combined MMR and POLE-proofreading glioblastoma. Interestingly, Nestin-cre+Msh2LoxP/LoxPPoleS459F/S459F mice failed to establish normal cerebella, suggesting such mutational loads may not support normal brain development. Furthermore, OLIG2-cre+Msh2LoxP/LoxPPoleS459F/+ mice failed to develop tumors. Tumors transplanted into syngeneic vs immunocompromised animals egrafted well orthotopically in the mouse hindbrain but significantly less efficiently when engrafted subcutaneously. Furthermore, immunocompromised and subcutaneous tumors revealed striking differences in mutational burden and clonal architecture, suggestive of nonautonomous immunoediting. Finally, anti-PD1 was sufficient to treat subcutaneously engrafted tumors in immunocompetent animals. This first mouse model of immunocompetent, hypermutant brain tumors can be used to uncover unique characteristics of RRD tumour evolution and allow for immune based therapeutic preclinical testing. Experiments to assess combinational ICIs and other therapeutic interventions in orthotopically transplanted tumors will also be presented.

Racial Variations in Mutational Profile in Newly Diagnosed Acute Myeloid Leukemia Patients

Background: Increasing evidence shows the impact of mutational burden in acute myeloid leukemia (AML) and impact on clinical response. Classifying these mutations into exclusive sub-types that are mutually exclusive was recently attempted. We sought to identify differences in mutational burden in AML patients based on race. Methods: We retrospectively reviewed the patient charts to distinguish the mutational markers of AML that substantially impact the outcome of AML. We categorized the mutations in seven functional groups with mutually exclusive mutations Signaling and kinase pathway (FLT3, KRAS, NRAS, KIT, PNPN1, JAK2, CBL), Epigenetic modifiers (DNMT3A, IDH1, IDH2, TET2, ASXL1, EZH2, and MLL/KMT2A), Nucleophosmin (NPM1), Transcription factors (CEBPA, RUNX1, and GATA2), Tumor suppressors (TP53), Spliceosome complex (SRSF2, U2AF1, SF3B1, and ZRSR2), and Cohesin complex (RAD21, STAG1, STAG2, SMC1A, and SMC3). For estimating racial distribution, we included only Whites and African Americans (AA) in the study as they represent 95% of the total population at our Cancer Center. Remission and relapse were defined per standard guidelines. We compiled data of all newly diagnosed AML patients treated at our institution between 2016 to the end of 2019. Both next generation sequencing (NGS) and Polymerase Chain Reaction (PCR) methods of genetic marker recognition techniques were included in the study. Results: 159 patients with AML were included in the analysis. We excluded seven patients of different race, including Asian (n=2), Hispanic (n=3), and unknown (n=2). The median age of the patients at diagnosis were 47 years (range 14 - 84 years), 73.3 % were white Caucasian, and 52.8% were female. The median age for white and African American (AA) patients was similar (47 vs 42 year respectively, p=0.55659), however, AAs have more female than Whites (65.9% vs. 47.8%, p=0.04164). In descriptive analysis of genetic marker mutation distributions between Whites and AA we observed signaling and kinase pathway 26.9% vs 25%, p=0.80231; epigenetic modifiers 14.8% vs 25%, p=0.13144; nucleophosmin 14.8% vs 13.6%, p=85460; transcription factors 5.2% vs 6.82%, p=0.69686; tumor suppressors 7.8% vs 0%; spliceosome complex 6.1% vs 2.3%, p=0.32647 and cohesin complex 1% vs 0%, respectively. Overall, 32.2% achieved complete remission (CR), 21.5% complete remission with incomplete hematologic recovery (CRi) and 45.6% Refractory. The CR + CRi rates of Whites and AA were not statistically significant (54.8% vs 52.3% respectively, p=0.77699). The median number of induction required for CR in both races was the same (2 and 2, respectively). We did not find any differences in number of induction for achieving CR by race. However, the rate of relapse was higher in white patients than in AA (49.1% vs 31.8%, respectively) (p=0.05039). Conclusion: This analysis suggests that there might be variations in functional categories of mutations markers in AML patients by race, tumor suppressors (TP53) found more frequently in whites and epigenetic modifiers in AA. This might be at least in part the reason for a higher relapse rate among whites. Additional studies and larger cohorts are needed to further explore the correlation between race, molecular markers and outcomes for AML. Figure Disclosures Cortes: Daiichi Sankyo:Consultancy, Research Funding;Astellas:Research Funding;BioPath Holdings:Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding;Takeda:Consultancy, Research Funding;Pfizer:Consultancy, Research Funding;Telios:Research Funding;Jazz Pharmaceuticals:Consultancy, Research Funding;Merus:Research Funding;Immunogen:Research Funding;BiolineRx:Consultancy, Research Funding;Bristol-Myers Squibb:Research Funding;Arog:Research Funding;Amphivena Therapeutics:Research Funding;Novartis:Consultancy, Research Funding;Sun Pharma:Research Funding.Kota:Novartis:Consultancy;Pfizer:Consultancy.

Clinical analysis of gene mutation characteristics and mutation burden in myelodysplastic syndrome

Objective: To investigate the relationship between gene mutation characteristics, mutation burden and general condition, disease subtype and karyotype of patients with myelodysplastic syndrome (MDS), and its clinical value. Methods: High-throughput sequencing was used to detect 65 blood tumor-related genes in 191 MDS patients and 9 secondary acute myelocytic leukemia patitents(SAML), and to analyze the characteristics of abnormal genes, mutation burden, as well as the relationship with disease subtypes, chromosome karyotypes and age. Results: Mutations were found in 148 patients (77.5%), including 47 abnormal genes and 186 mutation sites. And gene mutations were found in 9 SAML patients, the number of mutations was significantly higher than that in MDS patients (χ(2)=11.911, P=0.018). Among the abnormal genes, the mutation frequency of U2AF1 (37.3%) and ASXL1 (41.6%) were higher, and there were significant differences in mutation burden among different abnormal genes (F=91.946, P<0.001). There were differences in the number of gene mutations among different subtypes of MDS, and the number of EB-2 gene mutations was the highest (2.2±1.5). In SLD, MLD, EB-1 and EB-2, the proportion of carrying ≥ 3 mutations increased gradually (χ(2)=52.471, P=0.037). TP53 mutation was associated with abnormal karyotype (r(φ)=0.177, P=0.019), especially with complex karyotype (r(φ)=0.440, P<0.001), while NPM1 mutation is associated with normal karyotype (r(φ)=0.173, P=0.024). The number of mutations carried by patients under 30 years old was the least, and the number of mutations increased with the increase of age. The number of mutations was the most in patients aged 60 to 79 years old (P=0.017), and the mutation frequency of epigenetic related genes increased with the increase of age (P=0.041). Conclusions: The mutation characteristics and mutation load of MDS-related genes are closely related to clinical factors such as disease subtype, chromosome karyotype and patient age.

Immunotherapy for Uveal Melanoma - Current Knowledge and Perspectives.

Uveal melanoma is the most prevalent primary intraocular tumour in adults with the incidence between five and six cases per million people in the United States and Europe. The prognosis of patients with uveal melanoma is unfavourable with a 5-year survival rate of 50-70% despite significant advances in local tumour treatment using radiotherapy or surgical resection. Approximately 50% of the patients develop metastases within 15 years from initial diagnosis, mostly in the liver. The median survival rate after the onset of metastases is 6 months. Potential treatment options for metastatic uveal melanoma are chemotherapy, targeted therapy, and immunotherapy but no method showed satisfactory results. Immunotherapy with checkpoint inhibition showed promising results in the treatment of cutaneous melanoma; however, it did not appear to be equally effective with uveal melanoma. This may be due to differences in mutational burden, expression of neoantigens between these two types of tumour, immunosuppressive tumour microenvironment, and low immunogenicity and immune privilege of uveal melanoma. Considering the disappointing results of treatment with anti-CTLA-4 and PD-1/PD-L1 blockade in patients with advanced uveal melanoma several new forms of therapies are being developed. This may include immunotherapy with IMCgp100, glembatumumab vedotin and the infusion of autologous TILs, targeted therapy with selective MEK inhibitors, epigenetic therapy, and nanotherapy. Better insight into the molecular and genetic profile of uveal melanoma will facilitate detection of new prognostic biomarkers and thus enable a better modification of the existing immunotherapy methods and development of new forms of treatment specifically designed for uveal melanoma patients.

Pan-cancer characterization of immune-related lncRNAs identifies potential oncogenic biomarkers

Long noncoding RNAs (lncRNAs) are emerging as critical regulators of gene expression and they play fundamental roles in immune regulation. Here we introduce an integrated algorithm, ImmLnc, for identifying lncRNA regulators of immune-related pathways. We comprehensively chart the landscape of lncRNA regulation in the immunome across 33 cancer types and show that cancers with similar tissue origin are likely to share lncRNA immune regulators. Moreover, the immune-related lncRNAs are likely to show expression perturbation in cancer and are significantly correlated with immune cell infiltration. ImmLnc can help prioritize cancer-related lncRNAs and further identify three molecular subtypes (proliferative, intermediate, and immunological) of non-small cell lung cancer. These subtypes are characterized by differences in mutation burden, immune cell infiltration, expression of immunomodulatory genes, response to chemotherapy, and prognosis. In summary, the ImmLnc pipeline and the resulting data serve as a valuable resource for understanding lncRNA function and to advance identification of immunotherapy targets.

184PD - Neoadjuvant olaparib monotherapy in primary triple negative breast cancer

BackgroundNeoadjuvant 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. MethodsIn the phase 2 PETREMAC trial (NCT02624973), we assessed the efficacy of primary olaparib monotherapy for 10 weeks, followed by chemotherapy, in TNBC (T>2cm). 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. Results31 patients with TNBC were included (mean tumor size 59mm; 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. ConclusionsOlaparib yielded a high response rate when administered to treatment-naïve, large TNBC, indicating a potential role in sequential neoadjuvant therapy, for patients both with and without BRCA1/2 germline mutations. Clinical trial identificationNCT02624973; 2015-002816-34. Legal entity responsible for the studyHaukeland University Hospital, Bergen, Norway. FundingDet Regionale Samarbeidsorganet/Helse Vest, AstraZeneca. DisclosureH.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. Lønning: 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.

1328P - Continental differences in pathologic response with neoadjuvant ipilimumab (IPI) plus nivolumab (NIVO) in patients with macroscopic stage III melanoma in the phase II OpACIN-neo trial

BackgroundIn the multi-center investigator-initiated OpACIN-neo trial, patients (pts) with macroscopic stage III melanoma were randomized (stratified by center) to 3 different dosing schemes of neoadjuvant (neoadj) IPI+NIVO. Two cycles IPI 1mg/kg + NIVO3mg/kg was identified as the most favorable regimen with 20% grade 3-4 adverse events and a pathological response rate (pRR) of 77%. After a median follow-up (FU) of 8.3 months (mo) none (0/86) of the pts with a pathologic (path) response had relapsed, while 9/21 (43%) without a path response relapsed. Post-hoc analyses were conducted to investigate potential differences between pts treated in Europe (EU) and in Australia (AUS). MethodsBaseline patient characteristics, safety and efficacy in terms of path response were evaluated in pts treated in EU (n=48) and AUS (n=38). Multivariate analyses were performed using logistic regression method. Median FU was 9.3mo for EU pts and 6.9mo for AUS pts. ResultsBaseline characteristics (AUS vs EU) differed in age (median 60 vs 53 year [yr], p=0.017) and AUS pts were more likely to be male (65.8 vs 50.0%, p=0.142) and have an unknown primary melanoma (36.8 vs 20.8%, p=0.100); no difference in PD-L1 expression was observed. There was a trend to a higher pRR for AUS pts than for EU pts (84.2% vs 68.1%, OR 2.50, p=0.092). pRR was also higher for pts >60yr compared to £60yr (91.2% vs 64.7%, OR 5.64, p=0.010) and males vs females (83.7% vs 63.9%, OR 2.90, p=0.041). Multivariate analysis including continent, age and gender showed an adjusted OR for path response of 1.85 (p=0.289) for AUS vs EU pts, an OR of 4.89 (p=0.021) for pts >60yrs vs £60yrs and an OR of 2.50 (p=0.095) for males vs females. The frequency of high grade toxicity was the same in pts <60 compared to pts >60yr (42.3% vs 32.4%, p=0.353). ConclusionsThe continental difference in path response appears mostly driven by differences in age and gender. It remains to be elucidated whether the higher pRRs in elderly pts and pts from AUS can be explained by differences in mutational burden (analysis in progress and will be presented). Our data also indicate that neoadj IPI+NIVO is safe and highly effective in the elderly. Clinical trial identificationNCT02977052. Legal entity responsible for the studyNetherlands Cancer Institute. FundingBMS. DisclosureE.A. Rozeman: Travel / Accommodation / Expenses: MSD; Travel / Accommodation / Expenses: NanoString. A.M. Menzies: Advisory / Consultancy: BMS; Advisory / Consultancy: MSD; Advisory / Consultancy: Roche; Advisory / Consultancy: Novartis; Advisory / Consultancy: Pierre Fabre. R.A. Scolyer: Advisory / Consultancy: MSD; Advisory / Consultancy: Neracare; Advisory / Consultancy: Novartis. A.C.J. van Akkooi: Advisory / Consultancy, Research grant / Funding (institution): Amgen; Advisory / Consultancy, Research grant / Funding (institution): BMS; Advisory / Consultancy, Research grant / Funding (institution): Novartis; Advisory / Consultancy: Merck MSD; Advisory / Consultancy: Merck-Pfizer; Advisory / Consultancy: 4SC. J. Hansson: Advisory / Consultancy: BMS; Advisory / Consultancy: MSD; Advisory / Consultancy: Novartis. G.V. Long: Advisory / Consultancy: Aduro; Advisory / Consultancy: Amgen; Advisory / Consultancy: BMS; Advisory / Consultancy: Merck MSD; Advisory / Consultancy: Mass-Array; Advisory / Consultancy: Novartis; Advisory / Consultancy: Pierre Fabre; Advisory / Consultancy: Roche. C.U. Blank: Advisory / Consultancy, Research grant / Funding (institution): BMS; Advisory / Consultancy: MSD; Advisory / Consultancy: Roche; Advisory / Consultancy, Research grant / Funding (institution): Novartis; Advisory / Consultancy: Lilly; Advisory / Consultancy: Pierre Fabre; Advisory / Consultancy: Pfizer; Advisory / Consultancy: GSK; Advisory / Consultancy: GenMab; Research grant / Funding (institution): NanoString. All other authors have declared no conflicts of interest.

Somatic mutations in benign breast disease tissue and risk of subsequent invasive breast cancer

BackgroundInsights into the molecular pathogenesis of breast cancer might come from molecular analysis of tissue from early stages of the disease.MethodsWe conducted a case–control study nested in a cohort of women who had biopsy-confirmed benign breast disease (BBD) diagnosed between 1971 and 2006 at Kaiser Permanente Northwest and who were followed to mid-2015 to ascertain subsequent invasive breast cancer (IBC); cases (n = 218) were women with BBD who developed subsequent IBC and controls, individually matched (1:1) to cases, were women with BBD who did not develop IBC in the same follow-up interval as that for the corresponding case. Targeted sequence capture and sequencing were performed for 83 genes of importance in breast cancer.ResultsThere were no significant case–control differences in mutation burden overall, for non-silent mutations, for individual genes, or with respect either to the nature of the gene mutations or to mutational enrichment at the pathway level. For seven subjects with DNA from the BBD and ipsilateral IBC, virtually no mutations were shared.ConclusionsThis study, the first to use a targeted multi-gene sequencing approach on early breast cancer precursor lesions to investigate the genomic basis of the disease, showed that somatic mutations detected in BBD tissue were not associated with breast cancer risk.

The Subclonal Architecture of Metastatic Breast Cancer: Results from a Prospective Community-Based Rapid Autopsy Program "CASCADE".

BackgroundUnderstanding the cancer genome is seen as a key step in improving outcomes for cancer patients. Genomic assays are emerging as a possible avenue to personalised medicine in breast cancer. However, evolution of the cancer genome during the natural history of breast cancer is largely unknown, as is the profile of disease at death. We sought to study in detail these aspects of advanced breast cancers that have resulted in lethal disease.Methods and FindingsThree patients with oestrogen-receptor (ER)-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancer and one patient with triple negative breast cancer underwent rapid autopsy as part of an institutional prospective community-based rapid autopsy program (CASCADE). Cases represented a range of management problems in breast cancer, including late relapse after early stage disease, de novo metastatic disease, discordant disease response, and disease refractory to treatment. Between 5 and 12 metastatic sites were collected at autopsy together with available primary tumours and longitudinal metastatic biopsies taken during life. Samples underwent paired tumour-normal whole exome sequencing and single nucleotide polymorphism (SNP) arrays. Subclonal architectures were inferred by jointly analysing all samples from each patient. Mutations were validated using high depth amplicon sequencing.Between cases, there were significant differences in mutational burden, driver mutations, mutational processes, and copy number variation. Within each case, we found dramatic heterogeneity in subclonal structure from primary to metastatic disease and between metastatic sites, such that no single lesion captured the breadth of disease. Metastatic cross-seeding was found in each case, and treatment drove subclonal diversification. Subclones displayed parallel evolution of treatment resistance in some cases and apparent augmentation of key oncogenic drivers as an alternative resistance mechanism. We also observed the role of mutational processes in subclonal evolution.Limitations of this study include the potential for bias introduced by joint analysis of formalin-fixed archival specimens with fresh specimens and the difficulties in resolving subclones with whole exome sequencing. Other alterations that could define subclones such as structural variants or epigenetic modifications were not assessed.ConclusionsThis study highlights various mechanisms that shape the genome of metastatic breast cancer and the value of studying advanced disease in detail. Treatment drives significant genomic heterogeneity in breast cancers which has implications for disease monitoring and treatment selection in the personalised medicine paradigm.

Methylation Profiling Among Patients with TET2 Mutations in Myelodysplastic Syndromes Is Associated with Overall Survival

Background:Aberrant DNA methylation is an epigenetic hallmark of oncogenesis implicated in the pathogenesis of myelodysplastic syndromes (MDS). TET2 is one of the most frequently mutated genes in MDS, and is associated with net global hypermethylation in animal models. However, patients with MDS rarely present with a single somatic mutation, and thus methylation must be interpreted in the context of several mutated genes. We postulate that TET2 mutant MDS patients are not uniform and that epigenetic subgroups associated with clinical outcomes may exist. Methylation profiles at key regulatory regions could therefore serve as biomarkers in conjunction with somatic mutations. The aim of this study was to characterize the epigenetic heterogeneity of TET2 mutant MDS, and how specific methylation patterns relate to the molecular and clinical features of patients.Methods:To study methylation patterns in 96 patients with MDS, we utilized bisulfite padlock probes (BSPP), a novel enrichment technique (Deng et al. Nat Biotechnol 2009 and Diep et al. Nat Methods 2012) that targets ~500,000 unique CpGs in selected regions known to contain differentially methylated sites that have regulatory relevance, including transcription repressor CTCF binding sites, DNase I accessibility clusters, all microRNA genes, and all RefSeq gene promoters. BSPP was performed on genomic DNA purified from bone marrow mononuclear cells from untreated patients diagnosed with MDS that were genetically characterized in previous studies (Bejar et al. N Engl J Med 2011 and J Clin Oncol 2012). BSPP products were PCR amplified with primers containing individual barcodes as well as adaptors compatible with Illumina sequencers, allowing for massively parallel high throughput sequencing and digital quantification of CpG methylation.Results:Unsupervised clustering of the top 5% most variably methylated CpGs among 96 patients did not identify a correlation between gene mutations and methylation patterns. However, unsupervised analysis on the 26 patients with TET2 mutations (mean VAF 56.1%, not determined in 6 patients) sorted 20 patients into one of two discrete methylation clusters. Supervised analysis comparing these two clusters is shown in figure 1, highlighting the 9712 differentially methylated cytosines (DMCs) with >25% methylation difference and FDR-corrected p <0.05. The cluster with net hypermethylation of DMCs included 12 patients who had a lower mutational burden compared with the 8 patients in the relatively hypomethylated cluster (mean 1.8 vs. 3.4 mutations/patient, respectively). Patients in the hypomethylated cluster more frequently harbored mutations associated with poor prognosis (ASXL1 in 6, SRSF2 in 5, EZH2 in 2). In this cluster, 7775 DMCs were hypomethylated and 1937 were hypermethylated. DMCs were enriched in super enhancers, active promoters, DNase I accessibility clusters and intronic regions and underrepresented in CpG islands (Figure 2). Assessment of genes overlapping with DMCs compared to genes at all tested CpG sites were enriched for pathways involved in embryonic development and mesenchymal differentiation. Clinically, the hypomethylated patients were significantly more thrombocytopenic with a mean platelet count (1000/mm3) of 54.9±11.2 compared to a mean platelet count of 181.8±43.8 for the hypermethylated patients (p=0.045). The median follow up was 2.9 years, and the median overall survival was 0.48-1.1 years for the hypomethylated group versus 3.2-4.8 years for the hypermethylated group (Figure 3; HR 5.2, 95% CI 1.8-15.0, p=0.042).Discussion:We used BSPP to identify an epigenetic signature associated with survival among TET2 mutated patients with MDS. Unsupervised clustering identified methylation subgroups within TET2 mutants that are distinguished by differences in mutational burden, platelet count, and median overall survival. Between these two subgroups, DMCs are distributed in distinct patterns that identify regulatory regions that could serve as clinical biomarkers. Larger methylation profiling studies of MDS patients with various mutations and known treatment outcomes are ongoing. These will increase our ability to incorporate both epigenetic and genetic biomarkers into clinical practice. [Display omitted] [Display omitted] [Display omitted] DisclosuresBejar:Genoptix: Consultancy, Honoraria, Patents & Royalties: No royalties; Celgene: Consultancy, Honoraria; Foundation Medicine: Consultancy.

Analysis of ultra-deep targeted sequencing reveals mutation burden is associated with gender and clinical outcome in lung adenocarcinoma.

Gender-associated difference in incidence and clinical outcomes of lung cancer have been established, but the biological mechanisms underlying these gender-associated differences are less studied. Recently we have characterized the genomic landscape of lung adenocarcinoma derived from Chinese population (Reference [1]). In this study we evaluated the clinical significance of mutation burden in lung adenocarcinoma and found that the male tumors harbored statistically greater burden of genetic alterations than female counterparts (Male median 3 (range 0–34) vs female median = 2 (0–24), male to female ratio = 1.636, 95% CI = 1.343–1.992) after adjustment of age at surgery, stage, smoking status. Kaplan-Meier survival analysis revealed that greater burden of genetic alterations was associated with worse overall survival. Moreover, multivariable analysis demonstrated mutation burden was an independent prognostic factor for the patients. Taken together, our analysis demonstrated gender disparity of mutation burden and their prognostic value in lung adenocarcinoma. This gender difference in mutation burden might provide an explanation for the distinct difference in the clinical outcomes between sexes in lung adenocarcinoma.

Gender Disparity and Mutation Burden in Metastatic Melanoma.

Gender differences in melanoma incidence and outcome have been consistently observed but remain biologically unexplained. We hypothesized that tumors are genetically distinct between men and women and analyzed the mutation spectra in 266 metastatic melanomas (102 women and 164 men) from The Cancer Genome Atlas (TCGA). We found a statistically significantly greater burden of missense mutations among men (male median 298 vs female median = 211.5; male-to-female ratio [M:F] = 1.85, 95% confidence interval [CI] = 1.44 to 2.39). We validated these initial findings using available data from a separate melanoma exome cohort (n = 95) and found a similar increase in missense mutations among men (male median 393 vs female median 259; M:F = 1.59, 95% CI = 1.12 to 2.27). In addition, we found improved survival with increasing log-transformed missense mutation count (univariate hazard ratio = 0.82, 95% CI = 0.69 to 0.98) for TCGA samples. Our analyses demonstrate for the first time a gender difference in mutation burden in cutaneous melanoma.

Calreticulin mutation burden — Is it a stable clone in patients with essential thrombocythemia and myelofibrosis?

Calreticulin mutation represents the second most frequent mutation after JAK2 V617F in myeloproliferative disorder and is considered to be a driving mutation. Herein the mutation burden was evaluated in patients with essential thrombocythemia or myelofibrosis and found to increase by 5.7% over time unrelated to the time elapsed from the initial to the final positive test. The longer the course of the disease when first tested (range 0–30years, mean 7.9years) the lower mutation burden was observed. The mutated clone was larger in type II in comparison with type I mutation when first tested but the difference in mutation burden from the final to the first positive test was significantly higher in those with type I. Similarly, the difference in mutation burden was higher in patients with essential thrombocythemia reaching almost 8% in comparison to 1.3% in post-essential thrombocythemia myelofibrosis. Thus a repeat calreticulin quantitative test is not warranted.

Decreased Mitochondrial DNA Mutagenesis in Human Colorectal Cancer

Genome instability is regarded as a hallmark of cancer. Human tumors frequently carry clonally expanded mutations in their mitochondrial DNA (mtDNA), some of which may drive cancer progression and metastasis. The high prevalence of clonal mutations in tumor mtDNA has commonly led to the assumption that the mitochondrial genome in cancer is genetically unstable, yet this hypothesis has not been experimentally tested. In this study, we directly measured the frequency of non-clonal (random) de novo single base substitutions in the mtDNA of human colorectal cancers. Remarkably, tumor tissue exhibited a decreased prevalence of these mutations relative to adjacent non-tumor tissue. The difference in mutation burden was attributable to a reduction in C∶G to T∶A transitions, which are associated with oxidative damage. We demonstrate that the lower random mutation frequency in tumor tissue was also coupled with a shift in glucose metabolism from oxidative phosphorylation to anaerobic glycolysis, as compared to non-neoplastic colon. Together these findings raise the intriguing possibility that fidelity of mitochondrial genome is, in fact, increased in cancer as a result of a decrease in reactive oxygen species-mediated mtDNA damage.