Subclonal Heterogeneity Research Articles

Dissection of Clonal Evolution By Temporal Mutation Profiling in Chronic Lymphocytic Leukemia Patients Treated with Ibrutinib

Introduction: The irreversible Bruton tyrosine kinase (BTK) inhibitor ibrutinib is changing the paradigm of the treatment of chronic lymphocytic leukemia (CLL) with remarkable outcomes in first line as well as in relapsed CLL, including high risk patients with TP53 aberration. While the majority of patients demonstrate durable responses, with a median follow up of 3 years, approximately 18% of them develop resistance and relapse. Mutations in the BTK and PLCG2 genes emerged as predominant mechanisms conferring secondary ibrutinib resistance with BTK and/or PLCG2 mutations detected in the majority of resistant patients, often even 10 months before the clinical relapse. Given the very poor outcome of patients discontinuing ibrutinib, comprehensive understanding of the mechanisms of ibrutinib resistance and changes in the clonal architecture induced by the selective pressure of the drug are of major clinical importance. To dissect the clonal evolution in the context of all relevant mutation targets in CLL, we performed a temporal mutation profiling of 31 genes in paired pre- and post-treatment samples of patients receiving ibrutinib therapy.Methods: Sequential samples from 18 patients treated with single-agent ibrutinib were included in this study. Pre-treatment peripheral blood samples were available in all patients, with post-treatment samples at 18 months for 6 patients, at 12 months for 7 patients, at 8 months for 3 patients and at 3 months for 2 patients, with a median follow up of 12 months. Targeted deep NGS analysis of 31 recurrently mutated target genes, including ATM, BCOR, BIRC3, BRAF, BTK, CHD2, DDX3X, EGR2, EIF2A, EP300, FBXW7, HIST1H1E, IGLL5, KLHL6, KMT2D, LRP1B, MAPK1, MED12, MGA, MYD88, NFKBIE, NOTCH1, PLCG2, POT1, RIPK1, RPS15, SAMHD1, SF3B1, TP53, XPO1 and ZMYM3 was performed using the TruSeq Custom Amplicon approach (Illumina) on genomic DNA specimens extracted from peripheral blood mononuclear cells. Variant calling was performed using the VariantStudio3.0 application (Illumina) and only variants supported with more than 100 mutant reads were considered. Our cohort represented a heavily pretreated patient group with a median of 2.5 (range: 1-5) lines of prior therapies.Results: The ultra-deep NGS analysis revealed a total of 473 somatic mutations in the 18 paired samples with a median allelic depth of 25.550x across the 31 genes analyzed. The post-treatment samples carried a considerably higher number of mutations compared to the pre-treatment samples (114 vs 359 mutations) with more than half of the variants representing subclonal alterations with variant allele frequencies (VAF) of <10%. The most frequently mutated genes at baseline included TP53 (50%) , NOTCH1 (50%) and POT1 (28%) with similarly high mutation frequencies in the post-treatment samples. MYD88, BIRC3 and SF3B1 mutations were enriched in the post-ibrutinib samples with mutation frequencies of 11% vs 33%, 11% vs 39% and 11% vs 50%, respectively. Interestingly, XPO1 mutations emerged only in the post-treatment samples of five (28%) patients. As for the BTK and PLCG2 mutations, none of the pre-treatment samples carried a mutation in either of these genes, however, we detected emerging subclonal BTK and PLCG2 mutations (with VAFs between 1.5% and 11%) in 28% (5/18) and 33% (6/18) of the post-treatment samples, respectively. A subset of cases harbored multiple mutations in both genes with some of these representing novel BTK and PLCG2 mutations affecting previously unreported amino acid residues. Scrutiny of the clonal evolution revealed that all individual cases were characterized by almost unique combinations of mutations as well as patterns of emerging and disappearing mutant subclones upon the selective pressure of ibrutinib.Conclusions: The ultra-deep NGS analysis performed on paired samples of CLL patients treated with ibrutinib provided a significant insight into the mutational repertoire as well as subclonal dynamics of the leukemic compartment. Furthermore, it has revealed a profound subclonal heterogeneity that seems to be further enhanced by ibrutinib therapy, as demonstrated by a higher frequency of subclonal variants in the post-treatment samples. The clinical significance and the potential role of the novel BTK and PLCG2 variants identified in our study will require a longer follow up time, as currently all patients harboring these subclonal variants are still on ibrutinib therapy. DisclosuresNo relevant conflicts of interest to declare.

Identification of Effective Combinations of Targeted Drugs for DNMT3A and NPM1 Mutated Acute Myeloid Leukemia

Abstract Introduction: As the molecular heterogeneity underlying cancer becomes better understood, the capacity to define actionable targeted therapeutic strategies for patients appears essential to improving outcomes. These challenges are exemplified in patients with acute myeloid leukemia (AML), where a broad spectrum of driver mutations, emergence of genetically heterogeneous subclones, rescue signals in the microenvironment, and tumor-intrinsic feedback pathways frequently result in a lack of durable responses and rapid development of resistance to many single-agent targeted therapies. To identify biomarker-driven effective combination strategies for patients with AML, we built upon our previous ex vivo profiling of targeted drug combinations in primary patient specimens through integrated analysis of an expanded cohort of 133 AML patients. Methods: Freshly isolated primary mononuclear cells from patients were cultured in the presence of a panel of 48 drug combinations in fixed molar dose series encompassing different classes of targeted small-molecule inhibitors already approved by the FDA or in clinical development. For comparison, cells were also tested against graded concentrations of each single-agent, and sensitivity was assessed by MTS-based viability assay. IC50 and AUC values were derived using a probit regression model. Efficacy of each combination relative to its respective single-agents was calculated as a Combination Ratio (CR) value, defined as the combination IC50 or AUC divided by the lowest single-agent IC50 or AUC value, where a CR value Results: Analysis of our discovery cohort of 58 AML patients revealed several associations between molecular features and combination sensitivity. An intriguing finding was the significantly enhanced efficacy of combinations involving the bromodomain inhibitor, JQ1, in patients with mutations in NPM1 or DNMT3A. Specifically, the combination of JQ1 with the multi-kinase inhibitor sorafenib demonstrated superior efficacy compared to each inhibitor alone in patients with NPM1 mutations (median IC50 CR: 0.437; p=0.010). JQ1 paired with the CDK4/6 inhibitor, palbociclib, demonstrated superior efficacy compared with single-agents for patients with DNMT3A mutations (median IC50 CR: 0.119; p=0.017). These associations between the presence of NPM1 or DNTM3A mutations and increased JQ1-inclusive combination efficacy were also validated using our expanded AML cohort of 133 patients. For patients with NPM1 mutations, one-way ANOVA based comparison of AUC values for JQ1, sorafenib, and the combination showed JQ1 as the more effective of the two single agents, with the combination significantly reducing AUC beyond either drug alone (p Conclusions: Among the observed patterns of ex vivo drug combination efficacy associated with select, actionable diagnostic and genetic subsets in our study, our data suggest that AML patients harboring NPM1 or DNMT3A mutations demonstrate moderate sensitivity to the bromodomain inhibitor JQ1 and their viability is further inhibited by combinations of JQ1 with sorafenib or palbociclib, respectively. More broadly, these findings warrant clinical evaluation and highlight the utility of integrating drug sensitivity data with genomic information to identifying novel treatment strategies for improving disease control and patient outcomes. Disclosures Traer: ImmunoGen: Consultancy; Tolero: Consultancy; Notable Labs: Equity Ownership. Druker: Henry Stewart Talks: Patents & Royalties; MED-C: Membership on an entity's Board of Directors or advisory committees; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Beta Cat: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; McGraw Hill: Patents & Royalties; Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Aptose Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; The Leukemia & Lymphoma Society: Other: Joint Steering Committee of AML Master Protocol, Research Funding; Roche TCRC: Consultancy, Membership on an entity's Board of Directors or advisory committees; Third Coast Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Cylene: Consultancy, Membership on an entity's Board of Directors or advisory committees; ARIAD: Research Funding; Oregon Health & Science University: Patents & Royalties: #843 Mutated ABL Kinase Domains (licensed to various companies); #0996 Detection of Gleevec Resistant Mutations (licensed to various companies, including MolecularMD); #0606 Treatment of Gastrointestinal Stromal Tumors (exclusively licensed to Novartis); GRAIL: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Monojul: Consultancy; Millipore: Patents & Royalties: Royalties from Dana-Farber Cancer Institute, which has an exclusive commercial license with Millipore for monoclonal antiphosphotyrosine antibody 4G10, which I developed while employed at DFCI.; Baxalta US Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; CTI Biopharma: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Tyner: Gilead: Research Funding; Incyte Corporation: Research Funding; Syros: Research Funding; Seattle Genetics: Research Funding; Leap Oncology: Consultancy; Aptose Biosciences: Research Funding; Array Biopharma: Research Funding; Constellation Pharmaceuticals: Research Funding; Agios Pharmaceuticals: Research Funding; Genentech: Research Funding; Takeda Pharmaceutical Company: Research Funding; Janssen Pharmaceutica: Research Funding; AstraZeneca: Research Funding.

Hybrid Stomach-Intestinal Chromatin States Underlie Human Barrett’s Metaplasia

Tissue metaplasia is uncommon in adults because established cis-element programs resist rewiring. In Barrett's esophagus, the distal esophageal mucosa acquires a predominantly intestinal character, with notable gastric features, and is predisposed to developing invasive cancers. We sought to understand the chromatin underpinnings of Barrett's metaplasia and why it commonly displays simultaneous gastric and intestinal properties. We profiled cis-regulatory elements with active histone modifications in primary human biopsy materials using chromatin immunoprecipitation followed by DNA sequencing. Mutations in Barrett's esophagus were examined in relation to tissue-specific enhancer landscapes using a random forest machine-learning algorithm. We also profiled open chromatin at single-cell resolution in primary Barrett's biopsy specimens using the assay for transposase-accessible chromatin. We used 1- and 2-color immunohistochemistry to examine protein expression of tissue-restricted genes. Barrett's esophagus bears epigenome fingerprints of human stomach and intestinal columnar, but not esophageal squamous, epithelia. Mutational patterns were best explained as arising on the epigenome background of active gastric cis-elements, supporting the view that adjoining stomach epithelium is a likely tissue source. Individual cells in Barrett's metaplasia coexpress gastric and intestinal genes, reflecting concomitant chromatin access at enhancers ordinarily restricted to one or the other epithelium. Protein expression of stomach-specific mucins; CLDN18; and a novel gastric marker, ANXA10, showed extensive tissue and subclonal heterogeneity of dual stomach-intestinal cell states. These findings reveal mixed and dynamic tissue-restricted chromatin states and phenotypic heterogeneity in Barrett's esophagus. Pervasive intragland variation argues against stem-cell governance of this phenotype.

High sensitivity sanger sequencing detection of BRAF mutations in metastatic melanoma FFPE tissue specimens

Mutations in the BRAF gene at or near the p. V600 locus are informative for therapy selection, but current methods for analyzing FFPE tissue DNA generally have a limit of detection of 5% variant allele frequency (VAF), or are limited to the single variant (V600E). These can result in false negatives for samples with low VAFs due to low tumor content or subclonal heterogeneity, or harbor non-V600 mutations. Here, we show that Sanger sequencing using the NuProbe VarTrace BRAF assay, based on the Blocker Displacement Amplification (BDA) technology, is capable of detecting BRAF V600 mutations down to 0.20% VAF from FFPE lymph node tissue samples. Comparison experiments on adjacent tissue sections using BDA Sanger, immunohistochemistry (IHC), digital droplet PCR (ddPCR), and NGS showed 100% concordance among all 4 methods for samples with BRAF mutations at ≥ 1% VAF, though ddPCR did not distinguish the V600K mutation from the V600E mutation. BDA Sanger, ddPCR, and NGS (with orthogonal confirmation) were also pairwise concordant for lower VAF mutations down to 0.26% VAF, but IHC produced a false negative. Thus, we have shown that Sanger sequencing can be effective for rapid detection and quantitation of multiple low VAF BRAF mutations from FFPE samples. BDA Sanger method also enabled detection and quantitation of less frequent, potentially actionable non-V600 mutations as demonstrated by synthetic samples.

Single-cell analysis reveals the intra-tumor heterogeneity and identifies MLXIPL as a biomarker in the cellular trajectory of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a globally prevailing cancer with a low 5-year survival rate. Little is known about its intricate gene expression profile. Single-cell RNA sequencing is an indispensable tool to explore the genetic characteristics of HCC at a more detailed level. In this study, we profiled the gene expression of single cells from human HCC tumor and para-tumor tissues using the Smart-seq 2 sequencing method. Based on differentially expressed genes, we identified heterogeneous subclones in HCC tissues, including five HCC and two hepatocyte subclones. We then carried out hub-gene co-network and functional annotations analysis followed pseudo-time analysis with regulated transcriptional factor co-networks to determine HCC cellular trajectory. We found that MLX interacting protein like (MLXIPL) was commonly upregulated in the single cells and tissues and associated with a poor survival rate in HCC. Mechanistically, MLXIPL activation is crucial for promoting cell proliferation and inhibits cell apoptosis by accelerating cell glycolysis. Taken together, our work identifies the heterogeneity of HCC subclones, and suggests MLXIPL might be a promising therapeutic target for HCC.

Genomic and phenotypic heterogeneity in prostate cancer.

From a clinical, morphological and molecular perspective, prostate cancer is a heterogeneous disease. Primary prostate cancers are often multifocal, having topographically and morphologically distinct tumour foci. Sequencing studies have revealed that individual tumour foci can arise as clonally distinct lesions with no shared driver gene alterations. This finding demonstrates that multiple genomically and phenotypically distinct primary prostate cancers can be present in an individual patient. Lethal metastatic prostate cancer seems to arise from a single clone in the primary tumour but can exhibit subclonal heterogeneity at the genomic, epigenetic and phenotypic levels. Collectively, this complex heterogeneous constellation of molecular alterations poses obstacles for the diagnosis and treatment of prostate cancer. However, advances in our understanding of intra-tumoural heterogeneity and the development of novel technologies will allow us to navigate these challenges, refine approaches for translational research and ultimately improve patient care.

Abstract IA35: Follicular lymphoma dynamics through single-cell analysis

Abstract Follicular lymphoma (FL) is the second most common non-Hodgkin lymphoma in the Western world, generally characterized by a disseminated disease at diagnosis, an indolent clinical course, and recurrent, increasingly chemoresistant relapses. Overt FL is preceded by an insidious phase of asymptomatic growth and might emerge from common precursor clones, evolving over decades, and which might participate in subsequent relapses. Consequently, the cell of origin remains ambiguous. Although FL results from the malignant transformation of germinal center B cells, FL precursors emerge much earlier in B-cell ontogeny with the hallmark t(14;18) translocation and ensuing constitutive expression of BCL2 in bone marrow pre-B cells. While t(14;18) is considered the necessary early first hit to transformation, BCL2 as such is a very weak driver, as evidenced by the detection of t(14;18) in peripheral blood from a large fraction of the adult healthy population and the long latency and low penetrance observed in various BCL2 mouse models. Recent data of clonal dynamics in human and mouse models strongly suggest that additional illegitimate events likely accumulate over iterative GC passages, generating a highly mutagenic dynamics linked with slow oncogenic progression. Combined with the power of next-generation sequencing, molecular interrogations of t(14;18)+ clones in healthy individuals and FL patients recently provided the extent of FL genetic diversity in space (distinct tissues) and time (diagnosis vs. relapse). Thus, the path to transformation appears as a complex multi-hit process occurring along B-cell ontogeny, escalating along successive derailments of B-cell receptor diversification mechanisms, and subversion of specific immunologic properties of B cells. This protracted clonal evolution generates a massive accumulation of subclones and an associated complex overall genetic heterogeneity. This raises several questions. While FL originate from germinal center B cells, it is unclear to what extent overt lymphoma B cells retain GC B-cell functional dynamics or are blocked in a particular stage of the GC reaction. Furthermore, although one might intuitively assume that distinct subclones likely have a different gene expression profile associated to specific functions (and potentially driving distinct capacity to respond to therapy), it is still unclear to which extent subclonal genetic heterogeneity imprints functional heterogeneity. Because subclonal heterogeneity is hidden in bulk, where all distinct subclones are mixed and averaged, we recently used integrative single-cell analysis of phenotype, gene expression, and IGH sequence to track the characteristic human GC B-cell program in FL B cells. We used pseudo-time approaches to model the cyclic continuum of GC B-cell transitional states and identified characteristic patterns of synchronously expressed gene clusters in GC B cells. Strikingly this GC-specific gene expression synchrony was lost in single lymphoma B cells. Yet, distinct and conserved FL-specific cell states co-existed within single patient biopsies. Our data show that lymphoma B cells are not blocked in a GC B-cell state but may adopt new dynamic modes of functional diversity, opening novel definitions of lymphoma identity. Citation Format: Bertrand Nadel. Follicular lymphoma dynamics through single-cell analysis [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr IA35.

Abstract IA26: Tumor evolution: From Darwin’s finches to breast cancer

Abstract Tumor evolution is driven by heterogeneity for heritable traits in combination with environmental selection forces including cancer therapies. We have been investigating intratumor cellular genetic and phenotypic heterogeneity in breast tumors and described that higher heterogeneity is associated with poor clinical outcomes. We have also developed experimental models of intratumor subclonal heterogeneity and demonstrated that more heterogeneous tumors grow faster and develop heterogeneous distant metastases. By analyzing the underlying mechanisms we defined that intratumor heterogeneity is maintained by non-cell-autonomous factors and clonal cooperation drives metastases by modulating the local and systemic microenvironments. Lastly, we have identified epigenetic enzymes as regulators of cellular transcriptomic heterogeneity and showed that by modulating the activity of these enzymes, we can decrease cellular heterogeneity and therapeutic resistance. Overall, our studies demonstrate the need to study cancer patients as a whole and to apply a combination of molecular profiling, including single cell assays, and mathematical modeling to understand how tumors evolved and to develop more effective cancer therapeutic strategies based on this knowledge. Citation Format: Kornelia Polyak. Tumor evolution: From Darwin’s finches to breast cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA26.

Xenograft and cell culture models of S\xe9zary syndrome reveal cell of origin diversity and subclonal heterogeneity

Sézary Syndrome (SS) is a rare aggressive epidermotropic cutaneous T-cell lymphoma (CTCL) defined by erythroderma, pruritis, and a circulating atypical CD4 + T-cell clonal population. The diversity of Sézary cell (SC) phenotype and genotype may reflect either plasticity or heterogeneity, which was difficult to evaluate dynamically until the achievement of long-term SC expansion. Therefore, we developed six defined culture conditions allowing for the expansion of SC defined by their phenotype and monoclonality in four of seven SS cases. Engraftment of SC through the intrafemoral route into immunodeficient NOD.Cg-Prkdc(scid)Il2rg(tm1Wjll)/SzJ (NSG) mice was achieved in 2 of 14 SS cases. Secondary xenograft by percutaneous injection mimicked most of the features of SS with dermal infiltration, epidermotropism, and blood spreading. These models also allowed assessing the intra-individual heterogeneity of patient SC. Subclones sharing the same TCR gene rearrangement evolved independently according to culture conditions and/or after xenografting. This clonal selection was associated with some immunophenotypic plasticity and limited genomic evolution both in vitro and in vivo. The long-term amplification of SC allowed us to develop eight new SC lines derived from four different patients. These lines represent the cell of origin diversity of SC and provide new tools to evaluate their functional hallmarks and response to therapy.

Phenotype-based single cell sequencing identifies diverse genetic subclones in CD133 positive cancer stem cells

Tumor heterogeneity is one of the ongoing huddles in the field of colon cancer therapy. It is evident that there are countless clones which exhibit different phenotypes and therefore, single cell analysis is inevitable. Cancer stem cells (CSCs) are rare cell population within tumor which is known to function in cancer metastasis and recurrence. Although there have been trials to prove intra-tumoral heterogeneity using single cell sequencing, that of CSCs has not been clearly elucidated. Here, we articulate the presence of heterogeneous subclones within CD133 positive cancer stem cells through single cell sequencing. As a proof of principle, we performed phenotype-based high-throughput laser isolation and single cell sequencing (PHLI-seq) of CD133 positive cells in a frozen tumor tissue obtained from a patient with colorectal cancer. The result proved that CD133 positive cells were shown to be heterogeneous both in copy number and mutational profiles. Single cancer stem cell specific mutations such as RNF144A, PAK2, PARP4, ADAM21, HYDIN, KRT38 and CELSR1 could be also detected in liver metastatic tumor of the same patient. Collectively, these data suggest that single cell analysis used to spot subclones with genetic variation within rare population, will lead to new strategies to tackle colon cancer metastasis.

Adaptations and Advancement of Biologic Immunotherapy in the Management of Immunologically Cold Solid Malignancies

Contemporary breakthroughs within cancer immunotherapy are frequently cited amongst the most promising of therapeutic directions for medical oncology and perioperative solid tumour management. However to date, the efficacy of treatment of immunologically derived therapeutic modalities is limited to a few highly selective malignancies, exemplified by leukaemia or renal cell carcinoma. Many solid tumours exhibiting low immune activity, i.e., immunologically ‘cold’, such as highly aggressive pancreatic cancers, have correspondingly become regarded as inappropriate for prospective immunotherapeutic modulation. Standard approach in these tumours therefore relies upon early-stage identification and curative surgical resection, an identifiably imperfect option in both progression temporality and deterrence of metastatic disease. Fundamentally predicated upon the therapeutic activation of existing systemic immune resources, selectively towards malignant transformed cellular subpopulations, current cancer immunotherapy heavily utilises monoclonal antibody checkpoint inhibitors (i.e., PD-1, PDL-L1, CTLA-4) influencing resultant upregulation of physiologic immune activation pathways. These correspondingly enhance immunologic function and interfere with carcinogenesis. With ongoing development in the scientific understanding of complex tumour microenvironment interactions and subclonal heterogeneity, increasingly promising investigations have developed. These include the effective management of low immune activity cold solid tumours with original immunogenic cofactor therapies as well as immune modulation in conjunction with co-operative chemotherapeutic, radiological, or surgical intervention. Advancements in novel combination immunotherapies as well as innovative downstream management courses offer great optimism for the applicability of emerging cancer immunotherapy to prospective treatment of cold tumours. This review comprehensively analyses and discusses notable current research directions in the field and underscores future directions for continued scientific progress alongside relevant clinical applications.

Abstract B15: Genomic classification and prognosis in rhabdomyosarcoma: A report from the Children’s Oncology Group, the Institute of Cancer Research, and the National Cancer Institute

Abstract Purpose: Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood. Despite aggressive therapy, the 5-year survival rate for patients with metastatic or recurrent disease remains poor and beyond PAX-FOXO1 fusion status, no genomic markers are available for risk stratification. We therefore performed a large-scale study through an international consortium to more accurately determine the incidence of driver mutations and their association with clinical outcome. Patients and Method: Formalin-fixed, paraffin-embedded material was collected from patients enrolled on Children’s Oncology Group trials and UK patients enrolled on MMT trials. Pathology was reviewed centrally and extracted DNA was subjected to targeted capture sequencing using a panel of 39 genes previously associated with RMS. Mutations, indels, deletions, gene amplifications, and copy number variation was called using analysis pipelines developed at the NCI. Results: DNA from six hundred and forty-one patients was suitable for analyses. A median of 1 variant call was found per tumor. Mutation of a RAS isoform was found in 29% of all fusion negative cases, mutation of a RAS pathway member was seen in greater than 50% of cases, and 24% had no putative driver mutation identified. BCOR (15%), NF1 (11%), and TP53 (12%) mutations were found at a higher incidence than previously reported. Interestingly, mutations in HRAS were notable in the infant population whereas those in NRAS were enriched in adolescents. Among infants &amp;lt; 1 year, 71% of cases harbored a mutation in HRAS or KRAS. In contrast, mutation of MYOD1 was associated with an older age and a head and neck primary site. Finally, 29% of the evaluated tumors harbored multiple driver mutations consistent with subclonal variation and tumor heterogeneity in fusion-negative RMS. Conclusion: This is the largest genomic characterization of clinically annotated RMS tumors to date and provides genetic features that refine risk stratification and can be incorporated into prospective trials. Citation Format: Jack Shern, Joanna Selfe, Rajesh Patidar, Young Song, Marielle Yohe, Jun Wei, Xinyu Wen, Erin Rudinski, Donald Barkauskas, David Hall, Corinne Linardic, Meriel Jenney, Julia Chisholm, Rebecca Brown, Anna Kelsey, Susanne Gatz, Stephen Skapek, Douglas Hawkins, Janet Shipley, Javed Khan. Genomic classification and prognosis in rhabdomyosarcoma: A report from the Children’s Oncology Group, the Institute of Cancer Research, and the National Cancer Institute [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B15.

Single-cell sequencing of genomic DNA resolves sub-clonal heterogeneity in a melanoma cell line

We performed shallow single-cell sequencing of genomic DNA across 1475 cells from a cell-line, COLO829, to resolve overall complexity and clonality. This melanoma tumor-line has been previously characterized by multiple technologies and is a benchmark for evaluating somatic alterations. In some of these studies, COLO829 has shown conflicting and/or indeterminate copy number and, thus, single-cell sequencing provides a tool for gaining insight. Following shallow single-cell sequencing, we first identified at least four major sub-clones by discriminant analysis of principal components of single-cell copy number data. Based on clustering, break-point and loss of heterozygosity analysis of aggregated data from sub-clones, we identified distinct hallmark events that were validated within bulk sequencing and spectral karyotyping. In summary, COLO829 exhibits a classical Dutrillaux’s monosomic/trisomic pattern of karyotype evolution with endoreduplication, where consistent sub-clones emerge from the loss/gain of abnormal chromosomes. Overall, our results demonstrate how shallow copy number profiling can uncover hidden biological insights.

Abstract A43: Plasma-derived circulating tumor DNA (ctDNA) as a surrogate biomarker for treatment response with the polo-like kinase 1 (PLK1) inhibitor, onvansertib, in combination with LDAC or decitabine in acute myeloid leukemia (AML)

Abstract Background: Cancer cells from solid tumors release ctDNA into the bloodstream, clinically enabling assessment, monitoring tumor mutational heterogeneity, treatment response, minimal residual disease (MRD), and disease progression. However, the clinical utility of ctDNA in AML has yet to be established. In AML, blast cells carrying a driver mutation originate in the bone marrow (BM) and migrate into circulation in peripheral blood (PB). Here, we aimed to determine the potential of ctDNA as a surrogate for response to treatment in the phase 1b/2 study (NCT03303339) testing the efficacy and safety of the PLK1 inhibitor, onvansertib, in combination with either decitabine or LDAC in relapsed or refractory AML (R/R AML). Methods: R/R AML patients were dosed for 5 days with onvansertib in combination with either decitabine (5 days) or LDAC (10 days) within 21 to 28-day cycle. BM aspirates and blood samples were collected pre-dose, at the end of cycles 1, 2 and every other cycle after. Genomic DNA (gDNA) was extracted from BM and PB mononuclear cells (BMMCs and PBMCs, respectively), while ctDNA was extracted from plasma. Clinically significant mutations were detected in gDNA from PBMCs and BMMCs using a next-generation sequencing panel targeting variants in 75 genes associated with AML. For each patient, a droplet digital PCR (ddPCR) assay was developed to assess mutant allele frequencies (MAF) of a single variant of interest across timepoints and three sample sources (BMMC, PBMC, and Plasma). MAF changes across all 3 sample sources were compared to the clinical response assessed by immunohistochemistry in bone marrow aspirate (BM-IHC). Results: As of October 16th, 16 patients were evaluated, totaling 33 timepoints with MAF measured in all 3 sources. MAF in plasma highly correlated with BMMCs MAF (R2 = 0.85), more so than PBMCs with BMMCs (R2 = 0.78) and over plasma with PBMCs (R2 = 0.68). Clinically, 4 patients had a complete response (with or without count recovery, CR+CRi) and showed a corresponding drop in ctDNA MAF at or before clinical diagnosis. Similarly, 3 of 4 evaluable patients with progressive disease showed an increase in ctDNA MAF before or at the timepoint when blast percentages rose in BM, with the fourth patient maintaining a stable MAF across timepoints. In 5 patients with stable disease, 2 showed a divergence between ctDNA MAF and BM-IHC, while 3 maintained congruency. Conclusions: Serial testing of plasma-derived ctDNA was representative of treatment response and relapse in R/R AML patients. The correlation between ctDNA MAF and BMMCs MAF supports the utility of ctDNA as a surrogate biomarker for disease status, and in some cases provides a more immediate diagnostic of response or nonresponse. More patients need to be evaluated to build an objective classification of clinical significance around ctDNA MAF, and, importantly, the biology of R/R AML subclonal heterogeneity and MRD likely necessitate interrogation and monitoring of multiple variants of interest. Citation Format: Errin Samuelsz, Maya Ridinger, Mark Erlander, Latifa Hassaine, Marion Luebbermann, Brittany Ross. Plasma-derived circulating tumor DNA (ctDNA) as a surrogate biomarker for treatment response with the polo-like kinase 1 (PLK1) inhibitor, onvansertib, in combination with LDAC or decitabine in acute myeloid leukemia (AML) [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A43.

The Evolutionary Origins of Recurrent Pancreatic Cancer.

Surgery is the only curative option for stage I/II pancreatic cancer; nonetheless, most patients will experience a recurrence after surgery and die of their disease. To identify novel opportunities for management of recurrent pancreatic cancer, we performed whole-exome or targeted sequencing of 10 resected primary cancers and matched intrapancreatic recurrences or distant metastases. We identified that recurrent disease after adjuvant or first-line platinum therapy corresponds to an increased mutational burden. Recurrent disease is enriched for genetic alterations predicted to activate MAPK/ERK and PI3K-AKT signaling and develops from a monophyletic or polyphyletic origin. Treatment-induced genetic bottlenecks lead to a modified genetic landscape and subclonal heterogeneity for driver gene alterations in part due to intermetastatic seeding. In 1 patient what was believed to be recurrent disease was an independent (second) primary tumor. These findings suggest routine post-treatment sampling may have value in the management of recurrent pancreatic cancer. SIGNIFICANCE: The biological features or clinical vulnerabilities of recurrent pancreatic cancer after pancreaticoduodenectomy are unknown. Using whole-exome sequencing we find that recurrent disease has a distinct genomic landscape, intermetastatic genetic heterogeneity, diverse clonal origins, and higher mutational burden than found for treatment-naïve disease.See related commentary by Bednar and Pasca di Magliano, p. 762.This article is highlighted in the In This Issue feature, p. 747.

High-Dimensional Heterogeneity of Waldenström Macroglobulinemia within Its Immune Tumor Microenvironment

Waldenström macroglobulinemia (WM), a malignant B-cell lymphoplasmacytic lymphoma, is a rare subtype of non-Hodgkin lymphoma representing about 1% of all cases. To better understand the WM pathogenesis, we performed large-scale data-driven proteomic profile of WM tumor cells associated with tumor-driven immune changes in the tumor microenvironment of 66 bone marrow (BM) samples from WM patients compared to 10 age-matched healthy donors (HD) by time-of-flight mass cytometry (CyTOF) technology. Our workflow has been designed based on extensive 3 CyTOF antibody panels to evaluate WM tumor within B cell lymphopoiesis concurrently with immune landscape of the tumor microenvironment in WM by state-of-art technology CyTOF. To map B cell lymphomagenesis in WM, we defined whole spectrum of maturation of B cell development, from hematopoietic stem cells and B cell precursors through immature B cells, transitional B cells, and naïve B cells together with memory un-switched and switched B cells, plasmablasts and plasma cells in BM samples of WM patients by positive and negative co-expression of 13 B cell-stage specific markers. Various immunophenotyping aberrancies within WM B lymphomagenesis were associated with WM clones characterized by significant increase of 11 B subset clusters from un-switched and switched memory B cells to plasma cells. Interestingly, WM clusters differ in intra-clonal expression of activation surface molecules (CD23, CD24, CD25, CD81, CD329, CD200, and CD319); transcriptional factors and regulators controlling B cell development (MYD88, Bcl-6, IRF-4, sXBP-1, and FGFR-3) and stemness-related markers (Oct3/4, Nanog, Sox-2, c-Myc, and Notch-1) in WM supporting the idea of sub-clonal heterogeneity insight of WM tumor. Moreover, decrease in cell frequency of B cell precursors (pro-B and pre-BI), naive B cells, and plasmablasts were observed in WM patients versus HD. To generate a comprehensive view of the tumor microenvironment, we observed significant upregulation of g/dT cells, CD4+ and CD8+ T effector cells, CD8+ T effector memory cells, monocytes, and neutrophils immune subsets and downregulation of immature T cells, CD8+ T naïve cells, plasmacytoid dendritic cells, myelo/mono progenitor clusters. Ibrutinib (IBRU) treatment has been effective in relapsed/refractory WM patients; therefore highest numbers of WM patients were receiving IBRU therapy in our cohort. IBRU treated WM patients had decreased frequency of naive B, CD4+ T naive cells and specific clusters of un-switched and switched memory B cells. Moreover, responder versus non-responders to IBRU therapy revealed increase of CD8+T effector memory cells. In sum, correspondence analysis reflecting data of each patient and immune subsets revealed stratification of WM patients with reflection on their clinical outcome, therefore providing the rational for prediction of WM patient status. This study was supported by APVV-16-0484 and VEGA 2/0076/17. Disclosures Hunter: Janssen: Consultancy. Richardson:Karyopharm: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees. Kastritis:Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Honoraria; Prothena: Honoraria; Genesis: Honoraria. Treon:BMS: Research Funding; Janssen: Consultancy; Pharmacyclics: Research Funding. Anderson:Celgene: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

Intraclonal Heterogeneity Caused By Activation-Induced Cytidine Deaminase Is Not a Prognostic Biomarker in Untreated Advanced Stage Follicular Lymphoma: An Analysis of SWOG S0016

Background: Activation induced cytidine deaminase (AICDA or AID) causes somatic hypermutation (SHM) of the immunoglobulin heavy chain variable genes (IGHV). It has been widely hypothesized that aberrant targeting of AICDA creates additional driver mutations and intra-clonal heterogeneity which enables clonal evolution and thus disease progression. This hypothesis predicts that greater AICDA-mediated intraclonal heterogeneity will be associated with shortened progression free survival (PFS). To definitively test this hypothesis that AICDA-mediated heterogeneity is linked to risk of progression, we evaluated available samples from the largest US-based phase 3 trial of untreated follicular lymphoma patients treated with CHOP-based therapies, SWOG trial S0016. Methods: We have previously shown that ultradeep sequencing of two genes, the expressed IGHV and the 5' UTR of the translocated BCL2 allele, provide unrelated measures of AICDA-mediated genetic heterogeneity (Spence, J Immunol. 2014). Furthermore, the number of AICDA-mediated mutations in the 5' UTR of BCL2, but not in IGHV, correlates with the number of AICDA-mediated mutations at other known AICDA sites throughout the genome (such as PIM1, BCL6, RHOA, etc). Genomic DNA was prepared from all available paraffin-embedded specimens. Ultra-deep, amplicon-based sequencing (~10,000x) with internal controls to establish specimen-specific signal-to-noise parameters allowed sensitive and specific detection of sequence variants at 0.2% (FDR 1%). Results: For subjects with (152) and without (419) available specimens, the median duration of follow-up (11.3 years), the number of patients who progressed (60%), and other patient characteristics (demographics, histology, stage, FLIPI) were indistinguishable. Sequencing data that fulfilled strict quality metrics for low frequency variants were obtained for &gt;75% of the specimens (114 for BCL2; 120 for IGHV); further analysis was restricted to these subjects. The extent of intraclonal variation within both the IGHV and the 5' UTR of BCL2 was highly variable. For BCL2, a median of 7 mutations/case were detected (range 0-61); the mutations were highly skewed to the RCYW motif (p&lt;0.001), the preferred target of AICDA. These mutations defined a median of 5 subclones/case (range 1-22). The clonal IGHV was somatically mutated (&lt;0.98 germline identity) in 94% of samples (median 0.89). For IGHV, a median of 113 subclones/case were detected (range 0-1531). Consistent with our prior study, no correlation was seen between the number of mutations detected in the IGHV and BCL2 or the number of subclones defined by IGHV and BCL2. Neither the number of mutations in nor the number of subclones defined by BCL2 or IGHV were prognostic. Cox regression showed no relationship between PFS and mutation number or subclone count based on either IGHV or BCL2 (hazard ratios are indistinguishable from 1; parameters were treated both as a continuous variables and dichotomized as quartiles). Furthermore, receiver operator curves did not suggest a cut point for any of these measures that would predict disease progression at any time (areas under the curve are indistinguishable from 0.5). Conclusions: Several measures of AICDA-mediated mutation and resulting subclonal heterogeneity were not found to be prognostic in previously untreated, advanced stage FL treated with CHOP-based regimens. In contrast, AICDA-mediated mutations have been reported to accrue during transformation from low grade FL to large cell lymphoma. This paradox calls for a more refined model to explain the role of AICDA in the progression of FL. Support: NIH/NCI grants U10CA180888, U10CA180819, U10CA180821, R21CA198072(WRB); and in part by GlaxoSmithKline. Disclosures Rimsza: NanoSting: Patents &amp; Royalties: Named inventor on a patent licensed to NanoSting [Institution]. Leonard:Bayer Corporation: Consultancy; AstraZeneca: Consultancy; Genentech, Inc./F. Hoffmann-La Roche Ltd: Consultancy; Sandoz: Consultancy; ADC Therapeutics: Consultancy; Miltenyi: Consultancy; BeiGene: Consultancy; Sutro Biopharma: Consultancy; Celgene: Consultancy; Nordic Nanovector: Consultancy; Gilead: Consultancy; Karyopharm Therapeutics: Consultancy; Genentech, Inc./F. Hoffmann-La Roche Ltd: Consultancy; Bayer Corporation: Consultancy; Celgene: Consultancy; Epizyme, Inc: Consultancy; Merck: Consultancy; MorphoSys: Consultancy; AstraZeneca: Consultancy; Epizyme, Inc: Consultancy; Akcea Therapeutics: Consultancy; Miltenyi: Consultancy; ADC Therapeutics: Consultancy; BeiGene: Consultancy; Karyopharm Therapeutics: Consultancy; Merck: Consultancy; Sandoz: Consultancy; Gilead: Consultancy; Akcea Therapeutics: Consultancy; MorphoSys: Consultancy; Nordic Nanovector: Consultancy; Sutro Biopharma: Consultancy. Fisher:Celgene: Consultancy; Barclays: Honoraria; AstraZeneca: Consultancy; prIME: Honoraria. Smith:Portola Pharmaceuticals: Research Funding. Friedberg:Bayer: Honoraria, Other: Data &amp; Safety Monitoring Committee; Acerta: Other: Data &amp; Safety Monitoring Committee.

Dissection of Subclonal Evolution by Temporal Mutation Profiling in Chronic Lymphocytic Leukemia Patients Treated with Ibrutinib

Abstract Introduction: The irreversible Bruton tyrosine kinase (BTK) inhibitor ibrutinib is changing the paradigm of the treatment of chronic lymphocytic leukemia (CLL) with remarkable outcomes in first line as well as in relapsed CLL, including high risk patients with TP53 aberration. While the majority of patients demonstrate durable responses, with a median follow up of 3 years, approximately 18% of them develop resistance and relapse. Mutations in the BTK and PLCG2 genes emerged as predominant mechanisms conferring secondary ibrutinib resistance with BTK and/or PLCG2 mutations detected in the majority of resistant patients, often even 10 months before the clinical relapse. Given the very poor outcome of patients discontinuing ibrutinib, comprehensive understanding of the mechanisms of ibrutinib resistance and changes in the clonal architecture induced by the selective pressure of the drug are of major clinical importance. To dissect the clonal evolution in the context of all relevant mutation targets in CLL, we performed a temporal mutation profiling of 31 genes in paired pre- and post-treatment samples of patients receiving ibrutinib therapy. Methods: Sequential samples from 18 patients treated with single-agent ibrutinib were included in this study. Pre-treatment peripheral blood samples were available in all patients, with post-treatment samples at 18 months for 6 patients, at 12 months for 7 patients, at 8 months for 3 patients and at 3 months for 2 patients, with a median follow up of 12 months. Targeted deep NGS analysis of 31 recurrently mutated target genes, including ATM, BCOR, BIRC3, BRAF, BTK, CHD2, DDX3X, EGR2, EIF2A, EP300, FBXW7, HIST1H1E, IGLL5, KLHL6, KMT2D, LRP1B, MAPK1, MED12, MGA, MYD88, NFKBIE, NOTCH1, PLCG2, POT1, RIPK1, RPS15, SAMHD1, SF3B1, TP53, XPO1 and ZMYM3 was performed using the TruSeq Custom Amplicon approach (Illumina) on genomic DNA specimens extracted from peripheral blood mononuclear cells. Variant calling was performed using the VariantStudio3.0 application (Illumina) and only variants supported with more than 100 mutant reads were considered. Our cohort represented a heavily pretreated patient group with a median of 2.5 (range: 1-5) lines of prior therapies. Results: The ultra-deep NGS analysis revealed a total of 473 somatic mutations in the 18 paired samples with a median allelic depth of 25.550x across the 31 genes analyzed. The post-treatment samples carried a considerably higher number of mutations compared to the pre-treatment samples (114 vs 359 mutations) with more than half of the variants representing subclonal alterations with variant allele frequencies (VAF) of Conclusions: The ultra-deep NGS analysis performed on paired samples of CLL patients treated with ibrutinib provided a significant insight into the mutational repertoire as well as subclonal dynamics of the leukemic compartment. Furthermore, it has revealed a profound subclonal heterogeneity that seems to be further enhanced by ibrutinib therapy, as demonstrated by a higher frequency of subclonal variants in the post-treatment samples. The clinical significance and the potential role of the novel BTK and PLCG2 variants identified in our study will require a longer follow up time, as currently all patients harboring these subclonal variants are still on ibrutinib therapy. Disclosures No relevant conflicts of interest to declare.

Tumor heterogeneity of acute myeloid leukemia: insights from single-cell sequencing.

Individual tumors comprise genetically and epigenetically heterogeneous subclones, each of which is presumably associated with a distinct function, such as self-renewal or drug sensitivity. The dissection of such intratumoral heterogeneity is crucial to understand how tumors evolve during disease progression and under the selection of therapeutic intervention. As a paradigm of cancer intratumoral heterogeneity and clonal evolution, acute myeloid leukemia (AML) has been shown to possess complex clonal architecture based on karyotype studies, as well as deep sequencing of mixed cellular populations using next-generation sequencing (NGS) technologies. The recent development of single-cell sequencing (SCS) methods provides a powerful tool to allow analysis of genomes, transcriptomes, proteomes, and epigenomes at an individual cell level. The technologies applied in AML have broadened our understanding of AML heterogeneity and provided new insights for the development of novel therapeutic strategies. In this review, we summarize the progress in the research of AML heterogeneity using SCS technology and discuss the limitations and future direction regarding how SCS can contribute to AML prognosis and treatment.

Abstract 383: APOBEC3B is induced by activation of DNA repair pathway and modulates the survival and treatment response in human multiple myeloma

Abstract Constitutive genomic complexity, ongoing DNA damage, and accumulating mutations are observed with progression from monoclonal gammopathy of undetermined significance (MGUS) to active multiple myeloma (MM) to relapsed/refractory disease. Apolipoprotein B mRNA editing catalytic polypeptide-like 3B (APOBEC3B), a DNA cytosine deaminase in cancer mutagenesis, is recently linked to sub-clonal diversification, intra-tumor heterogeneity, and tumor evolution in MM. We here studied the upstream mechanisms of APOBEC3B dysregulation and further defined functional consequences of molecular manipulation of APOBEC3B in MM cells. We first asked whether APOBEC3B levels are altered in MM cells upon treatments with Melphalan (Mel), an alkylating agent used to treat MM which is known to induce replication stress, or with ionizing radiation (IR). Sub-lethal doses of Mel or IR induce APOBEC3B transcript and protein expression in a dose- and time-dependent manner in MM cell lines (n=7), associated with phosphorylation of γH2AX. Significantly, Bortezomib (btz), even at sub-lethal doses triggering DNA damage signaling, induced APOBEC3B expression in multiple MM cell lines. Next, inhibition of ATR or ATM activation pathway significantly decreased Mel- or IR or btz-induced APOBEC3B, suggesting that replication stress induced by Mel, IR, or btz activates ATM/ATR-dependent APOBEC3B induction. We used gene-specific CRISPR knock out (KO), shRNA knockdown (KD), and inducible-shRNA KD to study the functional impact of perturbation of APOBEC3B in MM cells. Both KO and KD of APOBEC3B decreased growth and survival in multiple MM cell lines sensitive or resistant to dexamethasone or lenalidomide. APOBEC3B inhibition significantly enhanced growth arrest followed by apoptosis in MM cells, suggesting that increased APOBEC3B levels contribute to MM cell survival. We next analyzed available data sources for MM cell lines from Cancer Cell Line Encyclopedia and the Genomics of Drug Sensitivity in Cancer, which include microarray gene expression and drug sensitivity information. APOBEC3B expression negatively correlates with MM cell sensitivity to JQ1, a BET inhibitor which inhibit MM cell growth and survival in vitroand in vivo. Importantly, in MM cell lines which are relatively resistant to pomalidomide and JQ1 than other cell lines, APOBEC3B KD by its shRNA enhances sensitivity to both drugs. Taken together, our findings provide new insights into the role of APOBEC3B in triggering cytidine deaminase-induced mutagenesis associated with progression of disease. Furthermore, DNA replication stress triggered by Mel, IR, or btz upregulates APOBEC3B expression, which in turn confers drug resistance. The role of APOBEC in disease pathogenesis and progression, coupled with its role mediating drug resistance, suggest potential utility of targeting APOBEC3B in novel MM therapies. Citation Format: Lijie Xing, Jiye Liu, Liang Lin, Shih-Feng Cho, Kenneth Wen, Tengteng Yu, Gang An, Ligui Qiu, Kenneth Anderson, Yu-Tzu Tai. APOBEC3B is induced by activation of DNA repair pathway and modulates the survival and treatment response in human multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 383.