Clonal Events Research Articles

Abstract PO-013: MTAP protein expression is lost in nearly one-third of primary pancreatic cancers and is associated with sensitivity to MAT2A inhibition in patient-derived organoid models

Abstract Background: Deletion of chromosome region 9p21 containing CDKN2A is an early clonal event in many cancers, including pancreatic ductal adenocarcinoma (PDAC). The MTAP gene, which is directly adjacent to CDKN2A and therefore frequently co-deleted, encodes methylthioadenosine phosphorylase, a crucial enzyme in the methionine salvage pathway. Pre-clinical studies suggest that MTAP deletion can confer selective sensitivity to inhibition of the PRMT5-MAT2A axis, highlighting a potential therapeutic vulnerability for PDAC. However, comprehensive MTAP and CDKN2A biomarker data to identify patients most likely to respond to targeted inhibition are lacking. Design: We developed a quantitative, multiplex immunofluorescence (mIF) assay to jointly measure MTAP and CDKN2A protein expression at a single cell level in PDAC and applied our assay to two cohorts with extensive genomic annotation data: 1) a multi-institutional cohort of over 300 primary resected formalin-fixed paraffin-embedded PDAC specimens and 2) a cohort of 56 human patient-derived organoid samples fixed in situ and assembled into an “organoid tissue microarray.” We also conducted drug-sensitivity testing with a MAT2A inhibitor (AGI-24512) using 18 patient-derived organoids with defined MTAP and CDKN2A genomic status. Results: MTAP protein expression was completely lost in 97 (31%) of 315 primary resected tumors. Loss of MTAP was accompanied by loss of CDKN2A in 94 of 97 cases, while 3 cases showed MTAP protein loss and intact CDKN2A protein expression. An additional 108 (34%) of the 315 tumors exhibited loss of CDKN2A and intact MTAP expression. CDKN2A loss has previously been associated with reduced patient survival, but MTAP loss was not an effect modifier of this association. Within the organoid cohort, homozygous MTAP deletion was detected in 7 (13%) cases and heterozygous deletion detected in 17 (30%) cases. Organoids with homozygous MTAP deletion showed complete loss of MTAP protein expression via mIF, whereas heterozygous MTAP deletion resulted in diminished MTAP protein expression compared to MTAP wild-type organoids. In drug-sensitivity testing, 5 of 5 MTAP homozygous deleted patient-derived organoids showed increased sensitivity to MAT2A inhibition compared with MTAP wild-type or heterozygous organoid models. However, one wild-type model and one model with MTAP heterozygous deletion demonstrated comparable sensitivity to models with homozygous MTAP deletion. RNA sequencing analysis of these organoids revealed MTAP RNA levels similar to those seen in MTAP homozygous deleted organoids. Conclusion: MTAP protein expression is lost in nearly a third of primary pancreatic cancers and can be quantitated using mIF in both human tissue and organoid models. Integrative analysis of organoid mIF, DNA and RNA sequencing data suggests that MTAP deletion is the predominant, but not sole determinant of sensitivity to MAT2A inhibition with important implications for patient selection in ongoing clinical trials. Citation Format: Sara A. Väyrynen, Annan Yang, Junning Wang, Jinming Zhang, Kristen Lowder, Kevin S. Kapner, Tim Bosse, Sri Raghavan, Andressa Dias Costa, Hannah Williams, Chen Yuan, Ashley Pelton, Vicente Morales-Oyarvide, Douglas A. Rubinson, Lauren Brais, Emma Reilly, Margaret M. Kozak, David C. Linehan, Richard F. Dunne, Daniel T. Chang, Albert C. Koong, Aram F. Hezel, Brian M. Wolpin, Jonathan A Nowak, Andrew J Aguirre. MTAP protein expression is lost in nearly one-third of primary pancreatic cancers and is associated with sensitivity to MAT2A inhibition in patient-derived organoid models [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-013.

Initial Whole Genome Sequencing of Plasma Cell Neoplasms in First Responders and Recovery Workers Exposed to the World Trade Center Attack of September 11, 2001

Initial Whole Genome Sequencing of Plasma Cell Neoplasms in First Responders and Recovery Workers Exposed to the World Trade Center Attack of September 11, 2001

Xeno-MCL: Genomic, Transcriptomic and Pathologic Landscape Associated with Disease Progression, Clonal Evolution and Tissue Tropism in Patient-Derived Xenografts of Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin lymphoma with poor diagnosis. Frequent relapse to frontline therapies is one of the major medical challenges in MCL. This is likely due to the extremely complex inter- and intra-tumor heterogeneity (ITH) in MCL patients. MCL patients display diverse clinicopathological profiles and tissue dissemination patterns, which can involve both nodal and extra nodal sites, including splenomegaly, hepatomegaly, lymphadenopathy, and involvement of bone marrow (BM), gastrointestinal (GI) tract, peripheral blood (PB), and the central nervous system. To date, it has not been reported how tissue tropism of MCL cells contribute to ITH, disease progression, and clonal evolution. This cannot be easily addressed using primary patient samples without simultaneous or sequential sampling from diverse dissemination sites, which is largely not feasible, especially for those requiring invasive collection. To this end, we established patient-derived xenograft (PDX) models from primary patient samples and characterize them in this study. Due to the distinct microenvironment, orthotopic PDX models have been shown to be much better platforms for preclinical and mechanistic studies than heterotopic or subcutaneous PDX (PDX_sc) models. Therefore, we established multiple MCL PDX models via intravenous (PDX_iv) or intraosseous routes and characterized them for the first time on how they recapitulate the disease in MCL patients in comparison to subcutaneous models when available. These PDX_iv models were successfully passed onto serious generations (up to 10) through IV or SC via PDX cells from spleen of PDX_iv or PDX_sc tumor, respectively (Figure 1). To understand the recapitulation of ITH in PDX models and their contribution to disease progression and clonal evolution, we collected specimens from both disseminated PDX_iv and PDX_sc cells from serious generations and subject them to a multi-dimensional investigation, including pathological, transcriptomic, and genetic profiling (Figure 1). Consistent in MCL patients, we observed a high degree of histopathological heterogeneity across PDX models originating from different patients, including splenomegaly, hepatomegaly, lymphadenopathy, and involvement of BM, GI tract and/or PB in our PDX_iv models. These models displayed similar dissemination patterns across generations when checked at 2 months after intravenous cell implantation, while additional dissemination sites may be reached at late disease stages, such as the stomach, colon, kidney and uterus. To understand the disease progression and clonal evolution, we performed whole exome sequencing (WES) on 62 PDX samples collected across tissues and generations, primary patient samples used to establish the models (n=6), and their matched germline samples (n=6). Our data revealed a high degree of recapitulation of PDX_iv to patients and genetic inter-patient heterogeneity. Clonal evolution events were investigated for both PDX_iv and PDX_sc models. We demonstrated that PDX_iv models passed by PDX cells from the spleen are genetically more stable than PDX_sc models, in which more frequent chromosomal gain/loss and/or gene mutations occurred toward later generations. Mutations in certain genes such as SVEP1 and STAB1, which play important roles in lymphocyte homing, were found to associate with tissue tropism. To further address this, we performed single-cell RNA sequencing on 41 PDX samples collected across tissues and generations together with primary patient samples (n=8) and PBMC samples from healthy donors (n=2). Transcriptomic profiling showed that G1 PDX_iv models recapitulated primary patient samples very well, and along generations, further upregulation of cancer hallmarks was observed, indicating disease progression into more aggressive stages along generations and late disease stages. Again, PDX_sc models were more transcriptionally dysregulated across generations. Moreover, in vivo efficacy studies showed that PDX cells responded differently to drug treatments such as venetoclax and rituximab based on their homing sites. In conclusion, we addressed the tissue tropism, therapeutic response, clonal evolution in MCL PDX models at multi-dimensional platforms, for the first time, and comparing the mutagenesis and disease progression of orthotopic and heterotopic PDX models at an unprecedented resolution. Figure 1 Disclosures Wang: Lu Daopei Medical Group: Honoraria; Beijing Medical Award Foundation: Honoraria; OncLive: Honoraria; Molecular Templates: Research Funding; Verastem: Research Funding; Dava Oncology: Honoraria; Guidepoint Global: Consultancy; Nobel Insights: Consultancy; Oncternal: Consultancy, Research Funding; InnoCare: Consultancy; Acerta Pharma: Research Funding; VelosBio: Research Funding; BioInvent: Research Funding; Juno: Consultancy, Research Funding; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; Pulse Biosciences: Consultancy; Loxo Oncology: Consultancy, Research Funding; Targeted Oncology: Honoraria; OMI: Honoraria, Other: Travel, accommodation, expenses; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; MoreHealth: Consultancy; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding.

A Multi-Center Open-Labeled Phase II Study of Intensive Salvage Therapy Followed By Enasidenib Maintenance for Patients with Relapsed/Refractory IDH2mutant AML

Introduction: Patients with relapsed/refractory acute myeloid leukemia (R/R AML) have limited therapeutic options and effective management remains clinical challenge. For medically fit patients, salvage therapy with an intensive chemotherapy regimen is the standard of care with remission rates of about 40-50%. Long-term survival is achievable only with disease control followed by allogeneic hematopoietic stem cell transplantation (HCT). A majority of patients with R/R AML who achieve a morphologic clinical remission with salvage chemotherapy frequently harbor detectable clonal events that act as a reservoir for disease relapse. Relapsed/refractory isocitrate dehydrogenase 2 (IDH2) mutant AML patients who undergo cytarabine based salvage chemotherapy will largely retain detectable IDH2 mutations and enasidenib maintenance therapy offers an attractive and tolerable approach to maintenance for such patients. Enasidenib (AG221) is a first-in-class, selective, potent inhibitor of the neomorphic activity of mutant IDH2 enzyme. It is FDA approved as monotherapy for R/R AML with a time to response of approximately 2 months, but there is limited data on the use of this agent following salvage chemotherapy. In the upfront setting, the combination of enasidenib with induction chemotherapy has demonstrated high response rates, providing rationale for this approach (and tolerability with the combination) in the R/R population. Methods: To understand the impact of tailored consolidation following salvage chemotherapy in fit R/R AML we designed a multi-center, phase II trial with 2 cohorts. Cohort assignment is based upon the investigators assignment of patients eligibility (cohort 1.) for allogeneic HCT or (cohort 2) no allogeneic HCT. The main inclusion criteria are, (a.) age over 18 years (b.) received salvage cytarabine based chemotherapy or venetoclax combined with hypomethylating agents (HMA) or 2 or more cycles of HMA for confirmed R/R AML (c.) achieved at least >50% reduction in blast percentage in a nadir marrow from salvage chemotherapy and (d.) adequate organ function and lack of acute life threatening illness. Patients will have received the investigator's choice of salvage therapy and subsequently undergo bone marrow assessment with protocol sampling. Patients confirmed to have obtained a remission will then begin enasidenib 100mg once a day starting between 14 and 60 days after starting salvage therapy. Patients in cohort 1 will receive 1-4 cycles of enasidenib and proceed with HCT; enasidenib can be re-initiated between D30-100 following HCT for a planned total of 24 cycles. Cohort 2 will continue enasidenib maintenance until progression/death or relapse (Schema: Fig 1). Study Endpoints: The primary endpoint of the study to determine the effect of enasidenib maintenance therapy following salvage induction therapy for IDH2 R/R AML, we will evaluate EFS at 12 months separately in the two cohorts of the study. Secondary endpoints include (1) Rate of successful HCT of the subjects enrolled on the study (2) Median duration of maintenance therapy in both the study arms & (3) Overall survival at 12 and 24 months in each cohort. The efficacy of the treatment will be assessed using Simon (1989) optimal two-stage, single arm, unblinded Phase II trials allowing for early termination for futility. This design has a 90% power to detect a 20% improvement rate compared to historical data, while controlling to 10% probability of erroneously findings. A total of 39 (C1) + 25 (C2) = 64 evaluable patients will be enrolled. Interim safety analysis will be done after enrollment of the first 28 patient in C1 and 17 patients in C2. Conclusion: This study is currently is open and enrolling patients at multiple collaborative institutions to answer the key question of efficacy of enasidenib maintenance following salvage chemotherapy. Correlative studies are being performed to understand minimal residual disease in IDH2 mutant AML and its clonal architecture. Disclosures Carraway: BMS: Consultancy, Other: Research support, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; ASTEX: Other: Independent Advisory Committe (IRC); Abbvie: Other: Independent Advisory Committe (IRC); Takeda: Other: Independent Advisory Committe (IRC); Stemline: Consultancy, Speakers Bureau; Jazz: Consultancy, Speakers Bureau. Griffiths:Novartis: Honoraria, Research Funding; Boston Biomedical: Honoraria; Genentech Inc: Research Funding; Astex Pharmceuticals: Research Funding; Alexion Pharmaceuticals: Honoraria, Research Funding; Celgene/BMS: Honoraria, Research Funding; AbbVie Inc: Honoraria; Persimmune: Research Funding.

Macroscopic somatic clonal expansion in morphologically normal human urothelium.

Knowledge of somatic mutation accumulation in normal cells, which is essential for understanding cancer development and evolution, remains largely lacking. In this study, we investigated somatic clonal events in morphologically normal human urothelium (MNU; epithelium lining the bladder and ureter) and identified macroscopic clonal expansions. Aristolochic acid (AA), a natural herb-derived compound, was a major mutagenic driving factor in MNU. AA drastically accelerates mutation accumulation and enhances clonal expansion. Mutations in MNU were widely observed in chromatin remodeling genes such as KMT2D and KDM6A but rarely in TP53, PIK3CA, and FGFR3 KMT2D mutations were found to be common in urothelial cells, regardless of whether the cells experience exogenous mutagen exposure. Copy number alterations were rare and largely confined to small-scale regions, along with copy-neutral loss of heterozygosity. Single AA-associated clones in MNU expanded to a scale of several square centimeters in size.

Investigation of tigecycline resistant Escherichia coli from raw meat reveals potential transmission among food-producing animals

To investigate the characteristics of tet(X4)-carrying E. coli from retail raw meat, three hundred and eleven retail raw meat samples including chicken, pork, duck, and beef were collected for screening for tigecycline resistant isolates in Sichuan Province, China. We detected 25 (8.0%, 95%CI: 5.3%–11.6%) tet(X4) positive E. coli isolates from 311 retail meat with the highest prevalence (13.4%, 95%CI: 7.3%–21.8%) observed in pork. All the tet(X)-carrying isolates were analyzed by antimicrobial susceptibility testing, S1 nuclease pulsed-field gel electrophoresis (S1-PFGE), conjugation experiments, and whole genome sequencing (WGS) to study the phylogenetic relationship and genomic characteristics. Conjugation and S1-PFGE profiling showed 6 isolates (24.0%) successfully transferred the resistant phenotype with frequencies from 3.63 × 10−5 to 7.68 × 10−2. Simultaneously, tet(X4), floR, and the characteristic structure of adjacent abh gene and cassette flanked by ISCR2 were identified in all 25 isolates, and a structure of abh-tet(X4)-ISCR2-virD2-floR was aligned as an important genetic pattern around tet(X4). Two dominant sequence types (STs) ST1638 (n = 4) and ST48 (n = 4) were identified by in silico MLST. Single nucleotide polymorphisms analysis revealed two potential clone-like transmission clusters which showed high concordance with the local spatio-temporal epidemiology, suggesting that clonal transmission events were very likely. The presence of tet(X4) in multiple different clone complexes (CCs) and the identification of two clone-like transmission indicates its potentially extensive and successful spread among different food-producing animals.

Pervasive chromosomal instability and karyotype order in tumour evolution.

Chromosomal instability in cancer consists of dynamic changes to the number and structure of chromosomes1,2. The resulting diversity in somatic copy number alterations (SCNAs) may provide the variation necessary for tumour evolution1,3,4. Here we use multi-sample phasing and SCNA analysis of 1,421 samples from 394 tumours across 22tumour types to show that continuous chromosomal instability results in pervasive SCNA heterogeneity. Parallel evolutionary events, which cause disruption in the same genes (such as BCL9,MCL1, ARNT (also known as HIF1B), TERT and MYC) within separate subclones, were present in 37% of tumours. Most recurrent lossesprobably occurred before whole-genome doubling, that was foundas a clonal event in 49% of tumours. However, loss of heterozygosity at the human leukocyte antigen (HLA) locus and loss of chromosome 8p to a single haploid copy recurred at substantial subclonal frequencies, even in tumours with whole-genome doubling, indicating ongoing karyotype remodelling. Focal amplifications that affected chromosomes 1q21 (which encompasses BCL9,MCL1 and ARNT), 5p15.33 (TERT), 11q13.3 (CCND1), 19q12 (CCNE1) and 8q24.1 (MYC) were frequently subclonal yet appeared to be clonal within single samples. Analysis of an independent series of 1,024 metastatic samples revealed that 13 focal SCNAs were enriched in metastatic samples, including gains in chromosome 8q24.1 (encompassing MYC) in clear cell renal cellcarcinoma and chromosome 11q13.3 (encompassing CCND1) in HER2+ breast cancer. Chromosomal instability may enable the continuous selection of SCNAs, which are established as ordered events that often occur in parallel, throughout tumour evolution.

Germline DNMT3A mutation in familial acute myeloid leukaemia

ABSTRACT Acute myeloid leukaemia (AML) is a heterogeneous myeloid malignancy characterized by recurrent clonal events, including mutations in epigenetically relevant genes such as DNMT3A, ASXL1, IDH1/2, and TET2. Next-generation sequencing analysis of a mother and son pair who both developed adult-onset diploid AML identified a novel germline missense mutation DNMT3A p.P709S. The p.P709S protein-altering variant resides in the highly conserved catalytic DNMT3A methyltransferase domain. Functional studies demonstrate that the p.P709S variant confers dominant negative effects when interacting with wildtype DNMT3A. LINE-1 pyrosequencing and reduced representation bisulphite sequencing (RBBS) analysis demonstrated global DNA hypomethylation in germline samples, not present in the leukaemic samples. Somatic acquisition of IDH2 p.R172K mutations, in concert with additional acquired clonal DNMT3A events in both patients at the time of AML diagnosis, confirms the important pathogenic interaction of epigenetically active genes, and implies a strong selection and regulation of methylation in leukaemogenesis. Improved characterization of germline mutations may enable us to better predict malignant clonal evolution, improving our ability to provide customized treatment or future preventative strategies.

Abstract 1116: Spatio-temporal analysis of the tumor microenvironment of colorectal cancer subtypes using an orthotopic organoid transplantation model

Abstract Colorectal cancer (CRC) can be classified in several molecular, morphological and histological subtypes. Modelling of these subtypes in genetically engineered mice contributed to a mechanistic understanding of CRC development. However, these models exhibit a late tumor onset and predominantly develop tumors in the small intestine, which is a rare location of human cancers. Furthermore, murine tumors rarely progress to advanced stages and poorly recapitulate the adenoma-carcinoma sequence observed in human CRC. Preliminary data suggest that the mutational make-up of the tumor may dictate its specific immune microenvironment, but may also drive microbial alterations in the gut, which in turn determine tumor development and survival. In order to study CRC in its native anatomical location (colon) and to elucidate the cross-talk between different molecular subtypes and the microenvironment in a spatio-temporal manner, we used an orthotopic transplantation model that is based on colonoscopy-guided injections of intestinal organoids into the submucosa of the rectum/distal colon. To investigate the role of genes altered in human CRC and their interactions, we performed CRISPR/Cas9-based gene editing in organoids followed by orthotopic allogenic transplantation into the colon of recipient syngeneic mice. Notably, organoids derived from normal and tumor tissues, which harbor CRC-relevant mutations, engraft in the colon. Tissues with activated oncogenes/inactivated tumor suppressors display the ability to form invasive tumors locally and seed to distant sites. Moreover, tumors generated by orthotopic injections of tumor-derived organoids not only recapitulate the histology of the primary tumor, but also show signs of tumor progression (e.g. towards undifferentiated sarcomatoid phenotypes). Whole exome sequencing data showed that organoids derived from tumors after implantation share common mutations with the parental organoids isolated from the endogenous tumor. Interestingly, after transplantation of the parental line specific mutations are selected for, as seen by their increased variant allelic frequency (VAF). Additionally, exclusive mutations were detected in both the parental- and the xenograft-derived organoid lines suggesting that in vivo tumor progression might be fuelled by a clonal selection event taking place after engraftment of the parental organoid line. In summary, these orthotopic transplantations allow us to efficiently model CRC subtypes in different syngeneic immuno-competent and immuno-deficient models, to functionally analyze tumour development from initiation to metastasis and to assess the combinatorial effect of mutations on tumor evolution, histomorphology and the cross talk of cancer cells with their microenvironment. Citation Format: Markus Tschurtschenthaler, Miguel G. Silva, Moritz Jesinghaus, Ankit Sinha, Jatin Roper, Roland Rad, Dieter Saur. Spatio-temporal analysis of the tumor microenvironment of colorectal cancer subtypes using an orthotopic organoid transplantation model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1116.

Abstract 3536: Genetic validation of an ATM immunohistochemistry assay for genomic and clinical-pathologic characterization of primary prostate cancers with ATM loss

Abstract Background: ATM, a protein kinase involved in double-strand DNA break repair, is mutated in a significant fraction of prostate cancers, and targeted therapies such as ATR inhibitors appear promising for ATM-deficient tumors. However, few ATM-deficient primary prostate cancers have been clinically and genomically characterized. Design: We validated an automated and dichotomously-scored clinical-grade immunohistochemistry (IHC) assay to detect ATM protein loss using prostate cancer cell lines and 52 very high-grade prostate tumors (Gleason score 5+4=9 or 5+5=10) with known ATM genomic status. We examined the frequency of ATM protein loss among 29 tumors with known pathogenic germline ATM mutations or variants of unknown significance (VUS). Finally, we screened more than 800 additional primary prostate carcinomas using tissue microarrays (TMA) for ATM protein loss. Targeted somatic genomic sequencing using HRD Plus (Myriad Genetics) or UW-OncoPlex was conducted in a subset of immunostained tumors in each cohort. Results: ATM protein loss by IHC was found in 13% (7/52) of very high-grade (primary Gleason pattern 5) tumors with somatic genomic sequencing data. Of 7 cases with ATM protein loss, 4 had a deleterious ATM mutation and 3 had apparent single copy ATM genomic loss. Of the remaining 45 cases without ATM protein loss, none had ATM genomic alterations. In the cohort of tumors with pathogenic germline ATM mutations, 74% (17/23) had ATM protein loss, but only 45% (9/20) with evaluable sequencing had a second pathogenic alteration in ATM detected (all of these had protein loss). Of those with germline mutations and ATM protein loss, the majority (76% or 13/17) had an apparent clonal alteration with homogeneous protein loss in all tumor cells sampled. In contrast, among 6 cases with germline ATM VUS, only one had protein loss. On TMA analysis, ATM loss was present in 3% (25/824) of evaluable tumors overall, and was significantly more common in tumors from Grade Group 5 (12/130; 9.2%) compared to all other Grade Groups (13/694; 1.9%) (P<0.0001). Of tumors found by TMA screening with available sequencing, 80% (4/5) with homogeneous ATM protein loss and 58% (7/12) with heterogeneous ATM protein loss had detectable pathogenic alterations in ATM. Of these, 82% (9/11) were inferred to have somatic ATM variants. Conclusions: Validated ATM IHC is a sensitive assay for detecting underlying ATM alterations and may be useful to evaluate VUS in ATM. However, not all tumors with ATM protein loss have detectable genomic alterations. ATM protein loss is likely an early clonal event in tumors with germline mutations and is significantly enriched in high-grade prostate cancers. Citation Format: Harsimar Kaur, Jessica Hicks, Colin Pritchard, Angelo De Marzo, William Isaacs, Jerry Lanchbury, Kirsten Timms, Emmanuel Antonarakis, Tamara L. Lotan. Genetic validation of an ATM immunohistochemistry assay for genomic and clinical-pathologic characterization of primary prostate cancers with ATM loss [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3536.

The Molecular Landscape of Hürthle Cell Thyroid Cancer Is Associated with Altered Mitochondrial Function-A Comprehensive Review.

Hürthle cell thyroid carcinoma (HTC) accounts for 3–5% of all thyroid malignancies. Widely invasive HTC is characterized by poor prognosis and limited responsiveness to standard therapy with radioiodine. The molecular landscape of HTC is significantly different from the genetic signature seen in other forms of thyroid cancer. We performed a comprehensive literature review on the association between the molecular features of HTC and cancer metabolism. We searched the Pubmed, Embase, and Medline databases for clinical and translational studies published between 1980 and 2020 in English, coupling “HTC” with the following keywords: “genomic analysis”, “mutations”, “exome sequencing”, “molecular”, “mitochondria”, “metabolism”, “oxidative phosphorylation”, “glycolysis”, “oxidative stress”, “reactive oxygen species”, and “oncogenes”. HTC is characterized by frequent complex I mitochondrial DNA mutations as early clonal events. This genetic signature is associated with the abundance of malfunctioning mitochondria in cancer cells. HTC relies predominantly on aerobic glycolysis as a source of energy production, as oxidative phosphorylation-related genes are downregulated. The enhanced glucose utilization by HTC is used for diagnostic purposes in the clinical setting for the detection of metastases by fluorodeoxyglucose positron emission tomography (FGD-PET/CT) imaging. A comprehensive metabolomic profiling of HTC in association with its molecular landscape might be necessary for the implementation of tumor-specific therapeutic approaches.

Abstract A04: Tumor development trajectories for exploring differences between cancer subtypes

Abstract We have employed PhylogicNDT, our computational method for timing somatic events and subclonal architecture reconstruction, to explore the association of tumor development trajectory (from normal cell to malignant state) with cancer subtype and genetic landscape. Previous attempts to understand the order of somatic events that lead to the formation of cancers are mostly based on studies of premalignant samples, which are often hard or impossible to obtain due to small size or inaccessible location. Our approach allows us to use information from whole-exome and whole-genome data to computationally reconstruct the order of events preceding malignant transformation of the tissue. We show that this approach yields accurate reconstruction of premalignant events in lung, colorectal, and head and neck cancers based on previous exploration of premalignant lesions. We explored the association between the developmental trajectories, treatment outcome, and survival in several cancer types. PhylogicNDT can be used to infer the order of clonal driver events (including in precancerous stages), subclonal populations of cells and their phylogenetic relationships, and dynamics of cell populations. This methodology will enable a systematic study of tumor initiation and progression across cancer types and therapies. Citation Format: Ignaty Leshchiner, Daniel Rosebrock, Oliver Spiro, Edmund Mroz, Dimitri Livitz, James W. Rocco, Gad Getz. Tumor development trajectories for exploring differences between cancer subtypes [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr A04.

Genetic structure of SARS-CoV-2 reflects clonal superspreading and multiple independent introduction events, North-Rhine Westphalia, Germany, February and March 2020.

We whole-genome sequenced 55 SARS-CoV-2 isolates from Germany to investigate SARS-CoV-2 outbreaks in 2020 in the Heinsberg district and Düsseldorf. While the genetic structure of the Heinsberg outbreak indicates a clonal origin, reflecting superspreading dynamics from mid-February during the carnival season, distinct viral strains were circulating in Düsseldorf in March, reflecting the city’s international links. Limited detection of Heinsberg strains in the Düsseldorf area despite geographical proximity may reflect efficient containment and contact-tracing efforts.

Insights on early mutational events in SARS-CoV-2 virus reveal founder effects across geographical regions

Here we aim to describe early mutational events across samples from publicly available SARS-CoV-2 sequences from the sequence read archive and GenBank repositories. Up until 27 March 2020, we downloaded 50 illumina datasets, mostly from China, USA (WA State) and Australia (VIC). A total of 30 datasets (60%) contain at least a single founder mutation and most of the variants are missense (over 63%). Five-point mutations with clonal (founder) effect were found in USA next-generation sequencing samples. Sequencing samples from North America in GenBank (22 April 2020) present this signature with up to 39% allele frequencies among samples (n = 1,359). Australian variant signatures were more diverse than USA samples, but still, clonal events were found in these samples. Mutations in the helicase, encoded by the ORF1ab gene in SARS-CoV-2 were predominant, among others, suggesting that these regions are actively evolving. Finally, we firmly urge that primer sets for diagnosis be carefully designed, since rapidly occurring variants would affect the performance of the reverse transcribed quantitative PCR (RT-qPCR) based viral testing.

Detection of somatic mutations in saliva of patients with oral cavity squamous cell carcinoma.

6562 Background: Oral cavity squamous cell carcinoma (OCSCC) frequently presents as clinically advanced disease with poor prognosis. When diagnosed at early stages, survival rates approach 80%, underscoring the need for validated, cost-effective detection methods. OCSCC is driven by the serial acquisition of genetic alterations. Tumor-defining somatic mutations are attractive biomarkers and hence their presence in saliva may be associated with malignancy as shown in a few proof-of-concept studies, including our previous work. Based on this premise, we present a low-cost, accurate, next generation sequencing (NGS) test with high clinical utility aimed at detecting mutations in the saliva for early diagnosis and potential screening of OCSCC. Methods: We have designed a custom NGS panel that covers exons of 7 most frequently mutated genes in OSCC. This minimal gene set derived from the analysis from 3 public datasets, predicted incidence of at least one somatic aberration in 89% of patients. We recruited 91 treatment-naïve OCSCC patients and profiled DNA from tissue and matched pre-operative saliva using this test. We also tested DNA from 12 subjects with premalignant lesions with high-grade oral dysplasia and matched saliva. Results: Using stringent variant calling criteria, at least one somatic variant was detected in 88 (96%) of the 91 primary tumors. 90.9% of the matched saliva were concordant, with only a minor decrease in early stage disease. Tumor-specific mutations (≥5% AF) in driver genes were detected in 10 (83.3%) dysplastic lesions, suggesting that driving clonal events may occur early in disease development. Interestingly, in 3 matched saliva of the dysplastic samples, the same mutations were detected. To ensure a variant is not a false positive call, we performed a vigorous multistep analytical validation of this saliva-based test: (i) independent re-sequencing of 24 saliva confirmed 94% reproducibility; (ii) no functionally relevant variants were detected in saliva from 12 of 13 healthy subjects without history of tobacco and alcohol usage; (iii) reproducibility, sensitivity, and specificity were confirmed using a positive control with 7 loci at 0.25% AF across 8 independent saliva sequencing runs and a certified negative control and was found to be on par with droplet digital PCR. Conclusions: These data highlight the feasibility of saliva-based testing for early diagnosis of OCSCC and premalignant lesions.

IGHV sequencing reveals acquired N-glycosylation sites as a clonal and stable event during follicular lymphoma evolution

AbstractFollicular lymphoma B cells undergo continuous somatic hypermutation (SHM) of their immunoglobulin variable region genes, generating a heterogeneous tumor population. SHM introduces DNA sequences encoding N-glycosylation sites asparagine-X-serine/threonine (N-gly sites) within the V-region that are rarely found in normal B-cell counterparts. Unique attached oligomannoses activate B-cell receptor signaling pathways after engagement with calcium-dependent lectins expressed by tissue macrophages. This novel interaction appears critical for tumor growth and survival. To elucidate the significance of N-gly site presence and loss during ongoing SHM, we tracked site behavior during tumor evolution and progression in a diverse group of patients through next-generation sequencing. A hierarchy of subclones was visualized through lineage trees based on SHM semblance between subclones and their discordance from the germline sequence. We observed conservation of N-gly sites in more than 96% of subclone populations within and across diagnostic, progression, and transformation events. Rare N-gly-negative subclones were lost or negligible from successive events, in contrast to N-gly-positive subclones, which could additionally migrate between anatomical sites. Ongoing SHM of the N-gly sites resulted in subclones with different amino acid compositions across disease events, yet the vast majority of resulting DNA sequences still encoded for an N-gly site. The selection and expansion of only N-gly-positive subclones is evidence of the tumor cells' dependence on sites, despite the changing genomic complexity as the disease progresses. N-gly sites were gained in the earliest identified lymphoma cells, indicating they are an early and stable event of pathogenesis. Targeting the inferred mannose-lectin interaction holds therapeutic promise.

Clonal hematopoiesis in hematological disorders: Three different scenarios

Clonality studies can establish the single-cell origin of tumors and thus differentiate clonal malignant and premalignant processes from reactive polyclonal processes. Detection of clonal cells may be based on direct tracking of cell lineage-specific sequences or disease-specific somatic mutations identifying the clonal population. Historically, clonal hematopoiesis was defined using the principle of X-chromosome inactivation based on observation that in circulating clonal cells, only one of the active chromosomes was expressed. In myeloproliferative neoplasms (MPNs) virtually all circulating erythrocytes, platelets, and granulocytes are products of single mutated stem cells that preferentially differentiate into the myeloid rather than lymphoid lineage. Thus, clonal differentiated myeloid cells co-exist in circulation with polyclonal long-lived T lymphocytes that originated before the MPN-initiating somatic clonal event. Chronic lymphocytic leukemia (CLL) starts in a differentiating B cell, but other lymphoid lineages and myeloid cells remain polyclonal. Normal T and B cells co-exist with the CLL clone, but are diluted by the massively expanded CLL population, which outnumbers the residual normal cells. Clonal hematopoiesis of undetermined potential (CHIP) has been identified by whole-genome sequencing of healthy individuals. These clones contain a specific somatic mutation previously considered to be disease defining but are detected in only a small proportion of circulating leukocytes, and there is no obvious suppression of normal hematopoietic stem cells. However, more studies are needed to properly define these clones, their persistence or disappearance, and their relative propensity for transforming into leukemias, myeloproliferative neoplasms, or other clonal hematological malignancies.

Clinical, molecular, and prognostic correlates of number, type, and functional localization of TET2 mutations in chronic myelomonocytic leukemia (CMML)—a study of 1084 patients

Loss-of-function TET2 mutations (TET2MT) are frequent early clonal events in myeloid neoplasms and are thought to confer a fitness advantage to hematopoietic precursors. This large, multi-institutional study (n = 1084), investigated the TET2 mutational landscape and prognostic implications of the number, type, and location of TET2MT and the epistatic relationship with other somatic events in chronic myelomonocytic leukemia (CMML). Nine hundred and forty-two TET2MT were identified in 604 (56%) patients, of which 710 (75%) were predicted to be truncating (involving the catalytic domain). Three hundred and sixteen (29%) patients had ≥1 TET2MT, with 28%, 1%, and 0.2% harboring 2, 3, and 5 mutations, respectively. In comparison to TET2WT, TET2MT patients were older in age, more likely to have dysplastic CMML, a higher number of co-occurring mutations, and lower-risk stratification. Importantly, TET2MT were associated with a survival advantage (49 vs. 30 months, p < 0.0001), especially in the context of multiple TET2MT (≥2; 57 months, p < 0.001), and truncating TET2MT (51 months, p < 0.001). In addition, the adverse prognostic impact of ASXL1MT was partially mitigated by concurrent TET2MT, with the ASXL1WT/TET2MT genotype having better outcomes and resulting in further risk stratification of ASXL1 inclusive CMML prognostic models, in comparison to ASXL1MT alone.

Lack of Significant Differences in Somatic Alterations between MGUS, SMM and Symptomatic Multiple Myeloma: A Result from Comprehensive Genomic Profiling Study

Somatic alterations including single nucleotide variants (SNVs), copy number alterations (CNAs) and structural variants (SVs) are an important component and the hallmark of Multiple Myeloma (MM) with certain alterations having clinical implications. Recently, the chronological order of such events was explained in detailed studies however previous study has compared the frequency and clonality of such events in early stage plasma cell disorder (MGUS or SMM) and their comparison with MM.Here we performed whole genome (WGS) and RNA sequencing (RNAseq) on 67 samples (8 Unique paired samples from patients with MGUS and when progressed to MM, 10 paired samples at SMM stage and then progressed to MM, as well as non-progressor SMM (NP-SMM) patients who have not progressed after at least 5 years of follow-up. In addition, we profiled CNAs and their clonality level in 164 MGUS, 147 SMM and 336 newly diagnosed MM patients to construct the clonal structure and frequency of somatic alterations.We did not observe significant difference in overall frequency of mutations at MGUS (median=5662 SNVs, IQR= [5252 - 7864]) and SMM stage (median=5308 SNVs, IQR=[4236 - 6125] ) and at progression to MM (median= 5983 SNVs, IQR=[5097 - 8916]). Overall mutational load increased from precursor stage to MM by only 5% with no recurrent change and the difference was not statistically significant (p &gt; 0.05). Similar trend was also observed for SVs [median=36, IQR= [25.25 -40.75] at MGUS, median=34, IQR= [12-52] at SMM, median=27, IQR= [27-41] at MM]. In fact, some of the samples had lower SNV and SV load in MM compared to precursor stages even though that had increased cancer cell fraction. Copy number analysis on paired samples also showed &gt;90% similarity only with few non-recurrent focal CNAs. We have compared WGS data from non-progressor SMM with progressor SMM and newly-diagnosed MM. The non-progressor SMM patients showed identical CNAs and not significantly different Ig translocation frequencies to both other groups. Except for NRAS mutations, mutation frequency in NP-SMM was similar to others. Finally, by using data from 647 patients at three stages (MGUS, SMM and MM), we compared the frequency of CNAs at each time point. Similar to MM, we observed almost equal frequency of hyperdiploid and non-hyperdiploid SMM cases. In hyperdiploid SMM, clonal copy number gain was observed in 95% of patients involving two of the three chromosomes 9, 15 and 19. Moreover, a similar pattern was observed in other hyperdiploid chromosomes including chromosomes 3,5,7,11 and 21. The observed event frequency and clonality levels in SMM were identical to one observed in MM as well as MGUS. Non-hyperdiploid SMM group had early and clonal events targeting chromosome 13 deletion with gain of 1q, 11 and/or 9 at the same frequency level as in MM. The only significant difference was observed in subclonal deletions targeting some of the infrequent deletion events observed in HMM and NHMM. In conclusion, our data from longitudinal analysis of paired precursor and symptomatic MM samples as well as cross sectional data highlight lack of significant differences in genomic changes between precursor conditions and symptomatic myeloma. This lack of progressive and unique genomic change suggests the need for additional transcriptomic, epigenomic or tumor micro environment changes to accelerate the tumor development. Disclosures Bolli: Celgene: Honoraria; Gilead: Other: travel expenses; Novartis: Honoraria. Moreau:Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau. Munshi:Celgene: Consultancy; Oncopep: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Adaptive: Consultancy; Abbvie: Consultancy.

Comparative Somatic Mutational Profiling of CD34+ Hematopoietic Precursors (HSC) and Circulating Endothelial Cells (CEC) in Patients with Primary Myelofibrosis (PMF)

Comparative Somatic Mutational Profiling of CD34+ Hematopoietic Precursors (HSC) and Circulating Endothelial Cells (CEC) in Patients with Primary Myelofibrosis (PMF)