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Transformation to aggressive disease histologies generates formidable clinical challenges across cancers, but biological insights remain few. We modeled the genetic heterogeneity of chronic lymphocytic leukemia (CLL) through multiplexed in vivo CRISPR-Cas9 B-cell editing of recurrent CLL loss-of-function drivers in mice and recapitulated the process of transformation from indolent CLL into large cell lymphoma [i.e., Richter syndrome (RS)]. Evolutionary trajectories of 64 mice carrying diverse combinatorial gene assortments revealed coselection of mutations in Trp53, Mga, and Chd2 and the dual impact of clonal Mga/Chd2 mutations on E2F/MYC and interferon signaling dysregulation. Comparative human and murine RS analyses demonstrated tonic PI3K signaling as a key feature of transformed disease, with constitutive activation of the AKT and S6 kinases, downmodulation of the PTEN phosphatase, and convergent activation of MYC/PI3K transcriptional programs underlying enhanced sensitivity to MYC/mTOR/PI3K inhibition. This robust experimental system presents a unique framework to study lymphoid biology and therapy.Significance:Mouse models reflective of the genetic complexity and heterogeneity of human tumors remain few, including those able to recapitulate transformation to aggressive disease histologies. Herein, we model CLL transformation into RS through multiplexed in vivo gene editing, providing key insight into the pathophysiology and therapeutic vulnerabilities of transformed disease.This article is highlighted in the In This Issue feature, p. 101

Non-uniform dynamics of single CpGs among CLL progression

Treatment with venetoclax does not result in a reset of the epigenome

Transformation to aggressive disease histologies generates formidable clinical challenges across cancers, but biological insights remain few. We modeled the genetic heterogeneity of chronic lymphocytic leukemia (CLL) through multiplexed in vivo CRISPR-Cas9 B-cell editing of recurrent CLL loss-of-function drivers in mice and recapitulated the process of transformation from indolent CLL into large cell lymphoma [i.e., Richter syndrome (RS)]. Evolutionary trajectories of 64 mice carrying diverse combinatorial gene assortments revealed coselection of mutations in Trp53, Mga, and Chd2 and the dual impact of clonal Mga/Chd2 mutations on E2F/MYC and interferon signaling dysregulation. Comparative human and murine RS analyses demonstrated tonic PI3K signaling as a key feature of transformed disease, with constitutive activation of the AKT and S6 kinases, downmodulation of the PTEN phosphatase, and convergent activation of MYC/PI3K transcriptional programs underlying enhanced sensitivity to MYC/mTOR/PI3K inhibition. This robust experimental system presents a unique framework to study lymphoid biology and therapy.Significance:Mouse models reflective of the genetic complexity and heterogeneity of human tumors remain few, including those able to recapitulate transformation to aggressive disease histologies. Herein, we model CLL transformation into RS through multiplexed in vivo gene editing, providing key insight into the pathophysiology and therapeutic vulnerabilities of transformed disease.This article is highlighted in the In This Issue feature, p. 101

Supplementary file contains a PDF version of supplementary figures S1-S7 and associated figure legends.

Table of correlation between time points within one sample and number of hyper- or hypomethylated CpGs for all CLL (top) and naïve and memory B cell samples (bottom)

Supplementary file contains all supplementary tables S1-S16 in Excel format in the order in which they appear in the text.

Cohort summary and details for CLL, MBL, and single cell samples

Supplementary Figures 1-7 and matching legends

Methylation landscape of CD5 positive and negative naïve and memory B cells is highly similar

Methylation dynamics across CLL progression

DMR methylation in MBL and CLL sampl

CLL and normal B cell comparisons

Low count MBL already show signs of the altered DNA methylation landscape

Most human cancers converge to a deregulated methylome with reduced global levels and elevated methylation at select CpG islands. To investigate the emergence and dynamics of the cancer methylome, we characterized genome-wide DNA methylation in preneoplastic monoclonal B-cell lymphocytosis (MBL) and chronic lymphocytic leukemia (CLL), including serial samples collected across disease course. We detected the aberrant tumor-associated methylation landscape at CLL diagnosis and found no significant differentially methylated regions in the high-count MBL-to-CLL transition. Patient methylomes showed remarkable stability with natural disease and posttherapy progression. Single CLL cells were consistently aberrantly methylated, indicating a homogeneous transition to the altered epigenetic state and a distinct expression profile together with MBL cells compared with normal B cells. Our longitudinal analysis reveals the cancer methylome to emerge early, which may provide a platform for subsequent genetically driven growth dynamics, and, together with its persistent presence, suggests a central role in disease onset.Significance:DNA methylation data from a large cohort of patients with MBL and CLL show that epigenetic transformation emerges early and persists throughout disease stages with limited subsequent changes. Our results indicate an early role for this aberrant landscape in the normal-to-preneoplastic transition that may reflect a pan-cancer mechanism.See related commentary by Rossi, p. 6.This article is highlighted in the In This Issue feature, p. 1

List of DMRs and specifications between normal B cells and CLL as returned from the DMR caller

Supplementary methods, Table S1, supplementary figures 1-9

While cancers evolve during disease progression and in response to therapy, temporal dynamics remain difficult to study in humans due to the lack of consistent barcodes marking individual clones in vivo. We employ mitochondrial single-cell assay for transposase-accessible chromatin with sequencing to profile 163,279 cells from 9 patients with chronic lymphocytic leukemia (CLL) collected across disease course and utilize mitochondrial DNA (mtDNA) mutations as natural genetic markers of cancer clones. We observe stable propagation of mtDNA mutations over years in the absence of strong selective pressure, indicating clonal persistence, but dramatic changes following tight bottlenecks, including disease transformation and relapse posttherapy, paralleled by acquisition of copy-number variants and changes in chromatin accessibility and gene expression. Furthermore, we link CLL subclones to distinct chromatin states, providing insight into nongenetic sources of relapse. mtDNA mutations thus mirror disease history and provide naturally occurring genetic barcodes to enable patient-specific study of cancer subclonal dynamics.Significance:Single-cell multi-omic profiling of CLL reveals the utility of somatic mtDNA mutations as in vivo barcodes, which mark subclones that can evolve over time along with changes in accessible chromatin and gene expression profiles to capture dynamics of disease evolution.See related commentary by Hilton and Scott, p. 2965.This article is highlighted in the In This Issue feature, p. 2945

Supplementary Table 2 - %CLL cells marked by mtDNA mutations

Purpose:Although local tissue-based immune responses are critical for elucidating direct tumor–immune cell interactions, peripheral immune responses are increasingly recognized as occupying an important role in anticancer immunity. We evaluated serial blood samples from patients with advanced epithelial ovarian cancer (EOC) undergoing standard-of-care neoadjuvant carboplatin and paclitaxel chemotherapy (including dexamethasone for prophylaxis of paclitaxel-associated hypersensitivity reactions) to characterize the evolution of the peripheral immune cell function and composition across the course of therapy.Experimental Design:Serial blood samples from 10 patients with advanced high-grade serous ovarian cancer treated with neoadjuvant chemotherapy (NACT) were collected before the initiation of chemotherapy, after the third and sixth cycles, and approximately 2 months after completion of chemotherapy. T-cell function was evaluated using ex vivo IFNγ ELISpot assays, and the dynamics of T-cell repertoire and immune cell composition were assessed using bulk and single-cell RNA sequencing (RNAseq).Results:T cells exhibited an improved response to viral antigens after NACT, which paralleled the decrease in CA125 levels. Single-cell analysis revealed increased numbers of memory T-cell receptor (TCR) clonotypes and increased central memory CD8+ and regulatory T cells throughout chemotherapy. Finally, administration of NACT was associated with increased monocyte frequency and expression of HLA class II and antigen presentation genes; single-cell RNAseq analyses showed that although driven largely by classical monocytes, increased class II gene expression was a feature observed across monocyte subpopulations after chemotherapy.Conclusions:NACT may alleviate tumor-associated immunosuppression by reducing tumor burden and may enhance antigen processing and presentation. These findings have implications for the successful combinatorial applications of immune checkpoint blockade and therapeutic vaccine approaches in EOC.

Supplementary Data from Mitochondrial DNA Mutations as Natural Barcodes for Lineage Tracing of Murine Tumor Models

Murine models are indispensable tools for functional genomic studies and preclinical testing of novel therapeutic approaches. Mitochondrial single-cell assay for transposase-accessible chromatin using sequencing (mtscATAC-seq) enables the dissection of cellular heterogeneity and clonal dynamics by capturing chromatin accessibility, copy-number variations (CNV), and mitochondrial DNA (mtDNA) mutations, yet its applicability to murine studies remains unexplored. By leveraging mtscATAC-seq in novel chronic lymphocytic leukemia and Richter syndrome mouse models, we report the detection of mtDNA mutations, particularly in highly proliferative murine cells, alongside CNV and chromatin state changes indicative of clonal evolution upon secondary transplant. This study thus demonstrates the feasibility and utility of multi-modal single-cell and natural barcoding approaches to characterize murine cancer models.Significance:mtDNA mutations can serve as natural barcodes to enable lineage tracing in murine cancer models, which can be used to provide new insights into disease biology and to identify therapeutic vulnerabilities.

Supplementary Figure from Improved T-cell Immunity Following Neoadjuvant Chemotherapy in Ovarian Cancer

Supplementary Table from Improved T-cell Immunity Following Neoadjuvant Chemotherapy in Ovarian Cancer

Supplementary Data from Improved T-cell Immunity Following Neoadjuvant Chemotherapy in Ovarian Cancer

Purpose: Although local tissue-based immune responses are critical for elucidating direct tumor–immune cell interactions, peripheral immune responses are increasingly recognized as occupying an important role in anticancer immunity. We evaluated serial blood samples from patients with advanced epithelial ovarian cancer (EOC) undergoing standard-of-care neoadjuvant carboplatin and paclitaxel chemotherapy (including dexamethasone for prophylaxis of paclitaxel-associated hypersensitivity reactions) to characterize the evolution of the peripheral immune cell function and composition across the course of therapy. Experimental Design: Serial blood samples from 10 patients with advanced high-grade serous ovarian cancer treated with neoadjuvant chemotherapy (NACT) were collected before the initiation of chemotherapy, after the third and sixth cycles, and approximately 2 months after completion of chemotherapy. T-cell function was evaluated using ex vivo IFNγ ELISpot assays, and the dynamics of T-cell repertoire and immune cell composition were assessed using bulk and single-cell RNA sequencing (RNAseq). Results: T cells exhibited an improved response to viral antigens after NACT, which paralleled the decrease in CA125 levels. Single-cell analysis revealed increased numbers of memory T-cell receptor (TCR) clonotypes and increased central memory CD8+ and regulatory T cells throughout chemotherapy. Finally, administration of NACT was associated with increased monocyte frequency and expression of HLA class II and antigen presentation genes; single-cell RNAseq analyses showed that although driven largely by classical monocytes, increased class II gene expression was a feature observed across monocyte subpopulations after chemotherapy. Conclusions: NACT may alleviate tumor-associated immunosuppression by reducing tumor burden and may enhance antigen processing and presentation. These findings have implications for the successful combinatorial applications of immune checkpoint blockade and therapeutic vaccine approaches in EOC.

While cancers evolve during disease progression and in response to therapy, temporal dynamics remain difficult to study in humans due to the lack of consistent barcodes marking individual clones in vivo. We employ mitochondrial single-cell assay for transposase-accessible chromatin with sequencing to profile 163,279 cells from 9 patients with chronic lymphocytic leukemia (CLL) collected across disease course and utilize mitochondrial DNA (mtDNA) mutations as natural genetic markers of cancer clones. We observe stable propagation of mtDNA mutations over years in the absence of strong selective pressure, indicating clonal persistence, but dramatic changes following tight bottlenecks, including disease transformation and relapse posttherapy, paralleled by acquisition of copy-number variants and changes in chromatin accessibility and gene expression. Furthermore, we link CLL subclones to distinct chromatin states, providing insight into nongenetic sources of relapse. mtDNA mutations thus mirror disease history and provide naturally occurring genetic barcodes to enable patient-specific study of cancer subclonal dynamics.Significance:Single-cell multi-omic profiling of CLL reveals the utility of somatic mtDNA mutations as in vivo barcodes, which mark subclones that can evolve over time along with changes in accessible chromatin and gene expression profiles to capture dynamics of disease evolution.See related commentary by Hilton and Scott, p. 2965.This article is highlighted in the In This Issue feature, p. 2945

Small cell lung cancer (SCLC) is a high-grade neuroendocrine cancer that accounts for ~15% of lung cancers. While nearly all SCLCs are genetically driven by near universal loss of function (LOF) mutations in RB1 and TP53; several recent studies show that there are different phenotypic SCLC molecular subtypes characterized by expression of lineage transcription factors. These include the neuroendocrine ASCL1 and NEUROD1 subtypes which together comprise ~70-80% of SCLCs. Initially subtypes were thought to be mutually exclusive, but recent evidence shows intra-tumoral subtype heterogeneity and plasticity between subtypes. A recent study found that 35-40% of human SCLCs express both ASCL1 and NEUROD1, but the mechanisms driving ASCL1 and NEUROD1 intra-tumoral heterogeneity are not well understood. My laboratory previously developed an autochthonous CRISPR-based SCLC genetically-engineered mouse model (GEMM) generated by intratracheally injecting adenoviruses encoding Cre recombinase and sgRNAs targeting Rb1, Trp53, and Rbl2. Cre turns on Cas9 expression and allows for CRISPR/Cas9 editing of Rb1, Trp53, and Rbl2 in somatic cells in the lungs. The unique advantage of this model is that it allows the inclusion of sgRNAs targeting additional genes of interest in the same adenovirus. Using this CRISPR-based autochthonous SCLC GEMM approach, we studied the consequences of inactivating the epigenetic modifier KDM6A during SCLC tumorigenesis. KDM6A functions as an H3K27 histone demethylase and also exists in the COMPASS complex with KMT2C/D to promote H3K4 mono-/di-methylation at enhancers. KDM6A along with its protein binding partner KMT2D are mutated in SCLC and KDM6A has been implicated in controlling differentiation in other lineages. Strikingly, we found that KDM6A inactivation in SCLC GEMMs induced plasticity from ASCL1 to NEUROD1 resulting in SCLC tumors that expressed both ASCL1 and NEUROD1. ATAC-sequencing showed open chromatin at the promoters of NEUROD1 and NEUROD1 target genes in KDM6A inactivated tumors. Interestingly, KDM6A inactivated tumors showed a spectrum of ASCL1 to NEUROD1 heterogeneity where some KDM6A inactivated tumors completely lost ASCL1 and solely expressed NEUROD1, some tumors expressed ASCL1 and NEUROD1 in a mutually exclusive manner, while others primarily expressed ASCL1 with very few NEUROD1 positive cells. Mechanistically, KDM6A binds and maintains ASCL1 target genes in an active chromatin state with its loss increasing H3K27me3 near both promoters and enhancers, and decreasing H3K4me1/2 at enhancers together leading to a cell state primed for ASCL1 to NEUROD1 subtype switching. This work identifies KDM6A as an epigenetic regulator that controls ASCL1 to NEUROD1 subtype plasticity and provides an autochthonous SCLC GEMM to model ASCL1 and NEUROD1 subtype heterogeneity, which is found in 35-40% of human SCLCs. Citation Format: Leslie Duplaquet, Yixiang Li, Matthew A. Booker, Yingtian Xie, Radhika A. Patel, Deli Hong, Thomas Denize, Emily Walton, Yasmin N. Laimon, Roderick Bronson, Jackson Southard, Shuqiang Li, Sabina Signoretti, Michael Y. Tolstorukov, Paloma Cejas, Henry W. Long, Michael C. Haffner, Matthew G. Oser. Small cell lung cancer subtype plasticity is regulated by KDM6A. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5774.

Richter syndrome (RS), an aggressive lymphoma that develops in patients with chronic lymphocytic leukemia (CLL), is a striking example of histologic transformation. While recent therapeutic advances have transformed the treatment landscape of CLL and lymphoma, RS remains associated with dismal overall survival. Despite an advanced genomic and molecular characterization of CLL over the past decade, the current understanding of the genetic factors driving evolution of CLL to RS is limited. To decipher the genetics underlying this transformation, we have performed an integrative analysis of exome, genome and transcriptome data generated from matched RS and CLL samples from a discovery cohort of 53 patients with newly diagnosed RS of DLBCL histology. Through computational deconvolution of CLL and RS clones, we constructed phylogenetic relationships and traced evolution of CLL to RS, confirming both clonal related (87%) and unrelated cases (13%). In addition to identifying recognized RS-risk genetic lesions, we discovered novel RS-specific alterations, including 5 putative somatic driver genes (IRF2BP2, SRSF1, B2M, DNMT3A and EZH2), frequent copy number alterations beyond del(9p21)(CDKN2A/B), (including amp(7q21.2) (CDK6), amp(9p24) (PDL1/L2), and amp(1q23)(MCL1)), and recurrent whole genome duplication and chromothripsis. Integration of exome and genome sequencing data led to the identification of distinct molecular subtypes of RS with prognostic importance. To confirm these molecular subtypes, a validation cohort of 47 RS cases has been assembled with paired exome and transcriptome data. To further investigate the stepwise clonal evolution of CLL to RS, we performed single-cell RNA-sequencing on biopsy samples obtained at diagnosis from 5 individuals with clonally related transformation. Using a novel tool, CNVSingle, we inferred allele specific single-cell copy number alterations that enabled identification of the single-cell clusters representing distinct CLL and RS genetic subclones as well as intermediate, or transitional, evolutionary states. RS cells displayed gene expression enriched in pathways of MYC targets and cell cycle, in line with similar analysis on bulk transcriptomes. Finally, by ultra-low pass (ULP)-WGS sequencing of plasma samples from RS patients, we demonstrate detection of RS tumor DNA in plasma months prior to initial clinical diagnosis (n=3 of 6) or post-allogeneic stem cell transplant relapse (n=2 of 2). cfDNA is thus a promising tool for early detection of emerging RS and RS relapse as well as for non-invasive detection surrounding diagnosis. Altogether, our study defines RS-specific alterations and provides a molecular definition of RS, identifies distinct genetic subtypes of RS with prognostic significance, traces the evolutionary path to RS and suggests future strategies for improved detection. Citation Format: Erin M Parry, Ignaty Leshchiner, Romain Guieze, Connor Johnson, Eugen Tausch, Sameer A Parikh, Camilla K Lemvigh, Conor Messer, Filippo Utro, Chaya Levovitz, Kahn Rhrissorrakrai, Matthew S Davids, Julien Broseus, Shuqiang Li, Ziao Lin, Binyamin A Knisbacher, Christof Schneider, Laura Z Rassenti, Thomas J Kipps, Nitin Jain, William Wierda, Florence Cymbalista, Neil E Kay, Kenneth J Livak, Brian P Danysh, Chip Stewart, Donna Neuberg, Jennifer R Brown, Laxmi Paridi, Stephan Stilgenbauer, Gaddy Getz, Catherine Wu. Evolutionary history of transformation from chronic lymphocytic leukemia to Richter syndrome [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A13.

Chimeric Antigen Receptor (CAR)-T cell therapy has revolutionized the treatment of hematologic malignancies. Approximately half of patients with refractory large B-cell lymphomas achieve durable responses from CD19-targeting CAR-T treatment, across the commercially available CAR-T products with differing designs. Known failure mechanisms such as antigen loss account for only a fraction of cases without durable responses, and this knowledge gap has limited advances in CAR-T engineering and optimal targeting to patients. We hypothesized that characterization of the transcriptional programs and temporal evolution of CAR-T and host immune cell populations could provide novel insights into the basis of clinical response to CAR-T cell therapy for B cell lymphoma. We performed 10X single-cell RNA sequencing on serial samples collected from 32 individuals with high grade B cell lymphoma treated with the two first FDA-approved CD19 CAR-T products: axicabtagene ciloleucel (axi-cel, utilizing a CD28z costimulatory domain) and tisagenlecleucel (tisa-cel, with a 4-1BB domain). We analyzed 106 samples, including pre-infusion blood samples, infusion product, and post-infusion T cells sorted by flow cytometry into CAR+ and CAR- populations. Analyzing 602,577 single-cell transcriptomes, we discerned major differences in the dynamics of response of the two products. Tisa-cel responders showed dramatic expansion of CD8+ T cells at day 7 after infusion, which represented less than 10% of cells in the product. Conversely, CD8+ T cells in products of non-responders failed to expand to the same degree post-infusion and had a more effector- than memory-like T cell phenotype. In one tisa-cel–treated patient who had no CD8+ T cell expansion after initial infusion and relapsed at 6 months post-infusion, re-treatment with a second dose of the same product led to a durable response and was associated with greater CD8+ T cell expansion as well as a shift in CD4+ T phenotype from cytotoxic to helper. In contrast, axi-cel responders had pre-expanded effector populations distributed more heterogeneously among CD4+ and CD8+ T cells. Finally, we identified nominal elevations in CAR-T regulatory cells (CAR-Tregs) among both axi-cel and tisa-cel non-responders in our dataset, which we confirmed in an external dataset. These small increases in CAR-Tregs were sufficient to uniformly suppress conventional CAR-T cell expansion and drive late relapses in an in vivo mouse model of lymphoma after treatment with CARs with either CD28z or 4-1BB co-stimulatory domains. In summary, this represents the largest CAR-T scRNAseq cohort established thus far and provides important insights into (i) the temporal dynamics of a successful CAR-T response, (ii) the molecular phenotypes of CAR-T cells with different costimulatory domains, and (iii) the capacity for small increases in CAR-Tregs to drive relapse. Citation Format: Nicholas J. Haradhvala, Mark B. Leick, Katie Maurer, Satyen Gohil, Rebecca C. Larson, Estelle Yao, Matthew J. Frigault, Shuqiang Li, Kenneth J. Livak, Kahn Rhrissorrakrai, Filippo Utro, Chaya Levovitz, Raquel A. Jacobs, Kara Slowik, Brian P. Danysh, Laxmi Parida, Catherine J. Wu, Gad Getz, Marcela V. Maus. Differential dynamics of response at single cell resolution between axi-cel and tisa-cel CAR-T therapy in refractory B-cell lymphomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3575.

Richter’s syndrome (RS) arising from chronic lymphocytic leukemia (CLL) is a striking example of an aggressive malignant histology that emerges from indolent cancer. RS is a major barrier to disease control in CLL and is associated with poor clinical outcomes and limited survival. The genetic basis of RS is poorly understood, and its relationship to the antecedent CLL remains incompletely characterized. To study RS, we performed whole-exome sequencing (WES) on samples collected from 52 patients with RS of diffuse large B cell lymphoma (DLBCL) histology. For this genomic characterization, samples from 42 patients were analyzed as ‘trios’ (matched germline, CLL and RS tissue DNA) and those from 10 as ‘duos’ (matched CLL and RS DNA). Beyond addressing contamination of CLL DNA in the germline sample, we developed methods for discriminating between the RS and CLL clones which often coexist in the same samples. The discovery cohort revealed that RS and CLL were clonally-related in 45/52 (87%) cases based on WES analysis, with a distinct RS clone emerging from a CLL subclone. The remaining 13% were determined to be clonally unrelated. RS clones presented ~3x higher rates of additional mutations than the ancestral CLL clones from which they developed. We identified novel RS somatic driver mutations (in IRF2BP2, SRSF1, B2M, DNMT3A and others), a high rate of copy number variations with recurrent deletions (e.g., del(17p) [TP53], del(13q14.3), del(7q36), and del(15q13.11) [MGA], del(9p21.3) [CDKN2A/B], del(16q12.2)), focal amplifications (amp(7q21.2) [CDK6], amp(8q24.2) [RECQL4, MYC], amp(13q31.2) [ERCC5], and frequent whole genome duplication. To further investigate RS and CLL clonal evolution, we performed single-cell RNA-sequencing on biopsies at the time of RS diagnosis in 5 individuals with clonally related transformation. Using our novel tool, CNVSingle, we inferred allele specific single-cell copy number alterations, yielding cluster-specific copy number profiles that matched the WES results of individual subclones of the RS and CLL populations. This enabled mapping genetic clones to specific expression patterns. Finally, we devised and tested a methodology that uses cfDNA for early detection of emerging Richter’s disease and have successfully identified Richter‘s tumor DNA in the blood several months prior to the clinical diagnosis. Our study thus defines drivers, distinct molecular subtypes and evolutionary path to RS and suggests strategies for its improved detection. Citation Format: Erin M. Parry, Ignaty Leshchiner, Romain Guièze, Connor Johnson, Eugen Tausch, Sameer Parikh, Camilla Lemvigh, Conor Messer, Daniel Rosebrock, Filippo Utro, Chaya Levovitz, Kahn Rhrissorrakrai, Matthew Davids, Raquel A. Jacobs, Kara Slowik, Julien Broseus, Shanye Yin, Shuqiang Li, Geoff Fell, Ziao Lin, Binyamin A. Knisbacher, Neil Ruthen, Dimitri Livitz, Christof Schneider, Jialin Ma, Julian Hess, Laura Z. Rassenti, Thomas J. Kipps, Nitin Jain, William Wierda, Florence Cymbalista, Neil E. Kay, Kenneth J. Livak, Brian P. Danysh, Chip Stewart, Donna Neuberg, Jennifer R. Brown, Laxmi Parida, Stephan Stilgenbauer, Gad Getz, Catherine J. Wu. Evolutionary history of transformation from chronic lymphocytic leukemia to Richter’s syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4007.

Immune therapies have transformed the cancer therapeutic landscape but fail to benefit most patients. To elucidate the underlying mechanisms by which T cells mediate elimination of leukemia, we generated a high-resolution map of longitudinal T cell dynamics within the same tumor microenvironment (TME; bone marrow) during response or resistance to donor lymphocyte infusion (DLI), a widely used immunotherapy for relapsed leukemia. We analyzed 87,939 bone marrow-derived single T cell transcriptomes, along with chromatin accessibility and single T cell receptor clonality profiles, by developing novel machine learning tools for integrating longitudinal and multimodal data. We found that pre-treatment enrichment and post-treatment rapid, durable expansion of ‘terminal’ (TEX) and ‘precursor’ (TPEX) exhausted subsets, respectively, defined DLI response. In contrast to the common, shared pathways marking DLI response, a heterogeneous pattern of T cell dysfunction marked DLI resistance. Unexpectedly, TPEX cells that expanded in responders did not arise from the infusion product but instead from both pre-existing and novel clonotypes recruited to the TME. Further, we introduce a Bayesian method, Symphony, to define the T cell regulatory circuitry and master regulators underlying TEX and TPEX subsets that may be broadly relevant to other exhaustion antagonists across cancers. In conclusion, our data implicate the hierarchy of both TEX and TPEX subsets for immunotherapeutic responses in leukemia, extending the scope of their relevance beyond checkpoint blockade to adoptive cellular therapy. Moreover, our results provocatively suggest that immunologic ‘help’ from DLI, rather than direct transfer of anti-leukemic T cells, drove leukemic remission. Finally, we provide a general analysis paradigm for exploiting temporal single-cell genomic profiling for deep understanding of how immune therapies differentially shape the evolutionary trajectories of the TME in accordance with clinical outcome. Citation Format: Pavan Bachireddy, Elham Azizi, Cassandra Burdziak, Vinhkhang Nguyen, Christina Ennis, Zi- Ning Choo, Shuqiang Li, Kenneth Livak, Donna Neuberg, Robert Soiffer, Jerome Ritz, Edwin Alyea, Dana Pe'er, Catherine Wu. Mapping the evolution of T cell states during response and resistance to adoptive cellular therapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT008.

Donor lymphocyte infusion (DLI) is a standard of care and potentially curative immunotherapy for relapsed leukemia after allogeneic hematopoietic stem cell transplant (allo-SCT). Despite low response rates for many leukemias, chronic myelogenous leukemia (CML) has historically exhibited an exquisite DLI sensitivity, and we previously reported that durable response to DLI was associated with reversal of exhaustion of bone marrow (BM) -infiltrating T cells (Bachireddy et al., Blood 2014). Critical questions remain, however, regarding the exact transcriptional states of those T cell subtypes mediating exhaustion, anti-leukemia responses, and resistance to DLI. To map evolving phenotypic T cell states in situ at single cell resolution, we profiled viable cells isolated from cryopreserved BM mononuclear cells from a median of 3 timepoints before and after DLI from 12 patients with relapsed CML after allo-SCT, including 6 long-term responders to DLI (R’s) and 6 nonresponders (NR’s), using single cell RNA sequencing (scRNA-seq). Using our computational tools for processing and analyzing scRNA-seq data (Azizi et al., Cell 2018), we detected 381,462 cells in total derived from 43 unique patient-timepoints that met our quality metrics. Because DLI’s anti-leukemic efficacy derives in large part from T cell activity, we sought a more refined characterization of T cells using our tool Biscuit (Azizi et al., Cell 2018) to merge, normalize and cluster T cells. We observed a marked increase in the number of T cell clusters in post-DLI samples compared to matched pre-DLI samples (p Comparing T cell exhaustion between R vs NR cases, we confirmed increased T cell exhaustion signatures in R-pre T cells. Using factor analysis techniques we found that anergy, dysfunction and tolerance are shared factors driving a subset of T cells that are enriched in NRs, suggesting multiple forms of T cell dysfunction in DLI resistance. We found that the clusters dominated by post-DLI R T cells were characterized by greater diversity of T helper subsets (Th1, Tfh, Th2, Th9, and Th22) and enrichment for exhaustion, type I and II IFN pathways, proinflammatory gene sets and CD8 T cell activation. Clusters dominated by NR T cells displayed increases in Th17 and Treg signatures, anergy and tolerance. These data suggest that (1) pretreatment T cell phenotypic diversity may be important for DLI response; (2) that DLI increases such diversity differently in R’s than in NR’s; (3) while T cell subsets exhibit some overlap pre-therapy, responders and non-responders become increasingly dissimilar post therapy. Citation Format: Elham Azizi, Pavan Bachireddy, Vinhkhang N. Nguyen, Shuqiang Li, Donna S. Neuberg, Robert J. Soiffer, Jerome Ritz, Edwin P. Alyea III, Dana Pe'er, Catherine J. Wu. Mapping the evolution of T cell states during DLI response and resistance using single-cell data and computational tools [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 3371.

Personal vaccines directed at neoantigens arising from tumor mutations can induce neoepitope-specific T cell responses in patients with highly mutated tumors such as melanoma. It remains unknown if this approach can be successfully applied in tumors with low mutation frequency. We conducted a phase 1/1b study to determine the safety and feasibility of patient-specific neoantigen-targeting vaccination in patients with newly diagnosed, methylguanine methyltransferase (MGMT) unmethylated glioblastoma, administered following maximum surgical resection and conventional radiotherapy. Tumor-specific mutations were identified by whole-exome sequencing. Each vaccine, composed of up to 20 synthetic long peptides containing predicted tumor neoepitopes admixed with poly-ICLC, was administered subcutaneously on a prime-boost schedule. Among 8 treated patients, adverse events were limited to mild injection site reactions. Seven patients (88%) received a vaccine with ≥10 neoepitope peptides (median 12, range, 7-20), with median time to vaccine initiation of 18.6 weeks from surgery. All patients died from progressive disease. Median progression-free and overall survival was 7.5 months (95% CI: 6.2, 9.7) and 16.8 months (90% CI: 9.6, 21.3), respectively. 3 patients dropped out of the study due to disease progression. We analyzed vaccine responses on 5 patients that got at least one booster immunization. All 3 patients who required dexamethasone during vaccine priming failed to generate interferon-γ responses in peripheral blood mononuclear cells. In contrast, 2 patients who did not receive dexamethasone during vaccine priming, generated robust de novo immune responses against multiple personal neoantigens. Circulating vaccine-induced polyfunctional neoantigen-specific CD4+ and CD8+ T cell responses were enriched for memory and activated phenotypes, and increased numbers of tumor-infiltrating CD4+ and CD8+ T cells were detected in these 2 patients. T cell receptor analysis identified identical clonotypes isolated from post-vaccination glioblastoma tissue and peripheral blood including a clonotype specific for ARHGAP35, a neoantigen targeted by vaccination. To our knowledge we provide the first evidence that tumor specific T cells can traffic from the periphery into glioblastoma tumors and that neoantigen-targeting vaccines can favorably alter the tumor immune milieu of glioblastoma. In conclusion, individualized, multi-neoepitope vaccination is feasible, safe, and generates neoantigen-specific T cell responses in the periphery and intracranial tumors of patients with glioblastoma. Citation Format: Derin B. Keskin, Itay Tirosh, Annabelle Anandappa, Jing Sun, Nathan D. Mathewson, Sachet A. Shukla, Evisa Gjini, Shuqiang Li, Letitia Li, Anita Giobbie-Hurder, Phuong M. Le, Zhuting Hu, Wandi Zhang, Oriol Olive, Christine McCluskey, Heather Daley, Patrick Y. Wen, Antonio E. Chiocca, Maegan Harden, Niall Lennon, Stacey Gabriel, Gad Getz, Donna Neuberg, Jerome Ritz, Eric S. Lander, Aviv Regev, Kai Wucherpfennig, Mario Suva, Edward F. Fritsch, Rodig Scott, Keith L. Ligon, Kenneth J. Livak, Hacohen Nir, Patrick A. Ott, Catherine J. Wu, David A. Reardon. Personal neoantigen-targeting vaccination generates neoepitope-specific T cell responses in tumors of patients with glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5631.

Obtaining paired alpha- and beta-chain information of the T cell receptor (TCR) enables identification of which TCR binds to a particular antigen, providing critical knowledge for understanding how a tumor interacts with its immune microenvironment and for monitoring responses to immunotherapy. We have developed a rapid, cost-effective single-cell TCR sequencing protocol (scTCRseq) that processes single T cells flow sorted into 384-well plates. Exploiting the added specificity provided by RNase H-dependent PCR (rhPCR; Dobosy et al., BMC Biotechnology 11:80, 2011), our scTCRseq protocol mixes primers for all known productive alpha and beta TCR alleles and obtains specific amplification of only the alleles present in a single-cell lysate (typically one alpha allele and one beta allele) without the use of nested PCR. Applying this protocol to T cell samples from normal donors, we detect TCRs arising from all productive TRAV and TRBV genes, and observe the expected highly diverse repertoires. Our method also includes targeted sequencing of 96 (or more) marker transcripts in order to obtain cell phenotype information. Our scTCRseq protocol has been adapted to analyze low-input samples (100 pg-10 ng) of PBMC RNA for characterization of the TCR repertoire and to improve the sensitivity of paired TCR detection in Smart-seq2 libraries prepared from single-cell RNA. We recently applied this approach to analyze T cell clonality in the T cell infiltrate from a glioblastoma patient receiving neoantigen-specific peptide vaccination. Fresh tumor obtained post-vaccination at the time of recurrence was dissociated and single CD3+ T cells were isolated by flow cytometry. Smart-seq2 analysis suggested the presence of clones, but this was based largely on inference from one-chain data. Using our scTCRseq protocol to re-amplify the TCR segments, paired alpha- and beta-chain information was obtained from 277 of 363 (76%) single cells that passed quality control. Out of 231 distinct clonotypes detected, 25 were identified as clones, where a clone is defined as two or more cells with identical alpha- and beta-CDR3 segments. GLIPH analysis (Glanville et al., Nature 547:94-98, 2017) was used to cluster infiltrate TCRs by specificity. A representative TCR from each specificity cluster was cloned in a TCR-negative Jurkat cell line. Cellular assays were used to determine which specificity groups react with epitopes from peptides used in the vaccination. TCRs specific for neoantigens included in the vaccine were detected. We have demonstrated a robust workflow that combines sensitive detection of paired alpha- and beta-chain TCR sequences with TCR cloning and expression to determine which TCRs in a tumor infiltrate have neoantigen specificity. Citation Format: Shuqiang Li, Jing Sun, Annabelle Anandappa, Oliver Spiro, Ignaty Leshchiner, Krishnalekha Datta, Yun Bao, Zhuting Hu, Nir Hacohen, Nathan D. Mathewson, Itay Tirosh, Kai Wucherpfennig, Aviv Regev, Mario Suva, Derin B. Keskin, Catherine J. Wu, Kenneth J. Livak. Novel targeted single-cell TCR sequencing method used for analysis of a T cell infiltrate from a glioblastoma patient [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5387.

Intratumoral genetic heterogeneity has been characterized across cancers, including chronic lymphocytic leukemia (CLL), and presents challenges to therapy as presence of phenotypically diverse cell populations commonly fuels relapse and resistance to treatment. While insights have been previously gained from bulk analysis, further characterization on the single-cell level is needed to more accurately dissect the pathway and regulatory features associated with distinct genetic subclones. In order to more accurately identify subclones, define phylogenetic relationships, and probe genotype-phenotype relationships, we developed methods for targeted mutation detection in DNA and RNA isolated from thousands of single cells from five CLL samples. To precisely establish the subclonal architecture of the five CLL samples, we first applied targeted single-cell DNA sequencing for detection of mutation and copy number variation using CLL samples previously characterized by bulk whole exome sequencing (WES). Our results identified multiple genetically distinct subclones in four out of five CLL samples. Next, to understand the subclonal architecture and their associated gene expression pattern, we performed single-cell whole transcriptome sequencing (scRNA-seq) and pathway and geneset overdispersion analysis. Although scRNA-seq identified clear transcriptional heterogeneity, these data could neither be used to confidently resolve the genetic subclone structure nor assess the correspondence of genetic structure with the observed transcriptional heterogeneity due to sparseness of coverage at mutation sites of interest. Thus, we developed a targeted RNA approach to assess transcript expression profiles and mutational status in the same single cells. Our approach reliably detects subclonal mutations and enables recapitulation of single-cell DNA information, including phylogenetic structure. Integrative analysis to correlate genotype and phenotype reveals phenotypic convergence between distinct subclones and prompts the idea that convergent evolution generates phenotypically similar cells in distinct genetic branches, thus creating a cohesive expression profile in each CLL sample despite the presence of genetic heterogeneity. Specifically, by clearly resolving phylogenic relationships and simultaneously assessing DNA and RNA-level information from the same single cells, we uncovered mutated LCP1 and WNK1 as novel CLL drivers, supported by functional evidence demonstrating their impact on CLL pathways. Overall, we demonstrate the ability to robustly integrate DNA- and RNA-level information in order to dissect the impact of somatic mutations on cellular phenotype. Our study highlights the potential for single-cell RNA-based targeted analysis to sensitively determine transcriptional and mutational profiles of individual cancer cells leading to an increased understanding of driving events in malignancy. Citation Format: Jean Fan, Lili Wang, Joshua M. Francis, George Georghiou, Sarah Hergert, Shuqiang Li, Rutendo Gambe, Chensheng W. Zhou, Chunxiao Yang, Sheng Xiao, Paola Dal Chin, Michaela Bowden, Dylan Kotliar, Sachet A. Shukla, Jennifer R. Brown, Donna Neuberg, Dario R. Alessi, Cheng-Zhong Zhang, Peter V. Kharchenko, Kenneth J. Livak, Catherine J. Wu. Integrated analysis of targeted single-cell genetic and transcriptional heterogeneity suggests novel drivers of chronic lymphocytic leukemia [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 27.

DNA methylation profiling of bladder cancer specific gene promoters has been shown to be a sensitive and specific diagnostic tool for early- and late-stage bladder cancer. We have recently shown that a urine-based assay that combines DNA and protein biomarkers can accurately triage patients undergoing evaluation for bladder cancer. As part of this assay, hypermethylation of Twist1 and Nid2 is performed by conventional methylation-specific PCR (cMSP). To increase throughput and lower cost, we have now developed an approach to determine the methylation at single-base resolution of CpG sites using next generation bisulfite sequencing. In addition to Twist1 and Nid2, methylation of the Vimentin promoter, which has been shown to be associated with epithelial to mesenchymal transition (EMT) in bladder cancer, was also evaluated using this technique. To determine the sensitivity of this approach, amplification was performed on control bisulfite converted genomic DNA that was either enzymatically methylated or chemically unmethylated at CpG sites using non-methylation specific PCR. Twist1, Nid2 and Vimentin PCR products were subsequently used as template for emulsion PCR and sequenced using the Ion Torrent PGM. Methylation status was determined by aligning sequencing reads to unmethylated reference sequences. In this assay, 0.4% methylated DNA could be reliably detected in 10 ng of bisulfite converted DNA, more than doubling the sensitivity of the current assay as well as potentially generating a single-base resolution mapping of additional CpG sites not yet quantified for Twist1, Nid2 and Vimentin. The clinical performance of the Next-Gen bisulfite sequencing assays for Twist1, Nid2 and Vimentin is currently being tested using urine samples from patients with bladder cancer as well as from age matched individuals who present with hematuria but who are found to be cancer-free. The technique we have developed could be used to detect other hypermethylated genes in a variety of bodily fluids where percent methylation may be much lower than in tumor tissue samples. Citation Format: Andrew R. Dunn, Shuqiang Li, Cecilia A. Fernandez, Anthony P. Shuber. Detection of bladder cancer-associated gene methylation using next-gen bisulfite sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 43. doi:10.1158/1538-7445.AM2013-43

FGFR3 mutations have been identified in ∼60-70% of low-stage, non-invasive tumors. Our group and others have developed assays to detect FGFR3 mutations in the urine of bladder cancer patients. However, urine-based assays have been limited by the technical ability to detect rare events in a dilute medium where there is a high background of normal DNA. In these assays, FGFR3 mutations are generally found in ∼30% of the urine samples, which is < 50% concordance with the expected detection in tissue. We have now developed an ultra-deep amplicon sequencing technique that increases FGFR3 mutation detection in urine to ∼67%, near the expected detection if every mutation found in tissue could be detected in urine. Amplicons were designed against FGFR3 exons 7, 10, and 15 using PCR primers containing the adapter sequences for unidirectional sequencing. Taqman probes were used to determine if sufficient DNA was present in each sample. Primary amplification was performed from DNA isolated from 4 ml of urine. The resulting PCR products were used as template for emulsion PCR and these were then sequenced using the Roche 454 GS Junior. Samples were analyzed for total DNA reads per sample and number of mutant sequencing reads to determine percent mutation. Urine samples from 29 patients with stage Ta bladder cancer were analyzed by both our previously described qPCR method and the new ultra-deep sequencing approach. Of the 29 samples, 2 did not have sufficient DNA for analysis by sequencing. Using ultra-deep amplicon sequencing, 18 out of 27 (66.7%) were positive for FGFR3 mutations, while only 3 out of 27 (11.1%) were positive for mutations by qPCR. The urine samples from the 15 newly identified mutations using deep sequencing contained FGFR3 mutations as low as 0.05%. Using matched tissue and urine samples, we demonstrate that the sensitivity in urine approximates the FGFR3 mutations observed in tissue. We have developed a highly sensitive non-invasive urine based assay that can detect FGFR3 mutant DNA when present at < 1% of the sample and that achieves > 90% concordance with tumor tissues. To our knowledge, this is the first practical application of next generation sequencing technology for diagnostic use. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2093. doi:1538-7445.AM2012-2093

We have recently developed a non-invasive assay that combines protein and DNA biomarkers for triaging bladder cancer patients and demonstrated that this multi-analyte approach improves clinical performance. This assay combines detection in urine of Matrix Metalloproteinase-2 (MMP-2) protein, hypermethylation of two DNA markers (Twist1 and Nid2) and mutation detection of the FGFR3 gene. To eliminate the need to perform separate analytical procedures for protein and DNA, and to decrease assay complexity and cost while increasing assay throughput, we are now developing a method for simultaneous detection of protein and DNA biomarkers on a qPCR platform. To this end, we have identified and characterized six DNA aptamers that specifically bind Matrix Metalloprteinase-2 (MMP-2). DNA aptamers are single stranded DNA oligos that can bind specifically to their target molecules, including proteins. Once bound to the protein, these aptamers can then be used as templates for quantitative PCR-mediated protein detection. We have developed qPCR assays for each of the six MMP-2-specific aptamers, and demonstrate detection limits for each aptamer as low as 5x102 copies. Kinetic and equilibrium analysis was used to determine the KD constant for the aptamers. High affinity interactions were observed for all aptamers, with two aptamers having the highest affinity and calculated KD values of 15 and 10nM. Using one of these aptamers, we bound MMP-2 protein to the aptamer in solution and then immunoprecipitated the protein/aptamer complexes using biotin-labeled MMP-2-specific antibodies. Immunoprecipitated complexes were then quantitated using the aptamer-specific qPCR assay. In this study, we show that by qPCR, we can efficiently detect 10-12 mol/L of MMP-2 using the aptamer as the detection molecule. This level of MMP-2 protein detection is equivalent to commercially available ELISA assays. To further improve performance and to take advantage of the known specificity of aptamers, we intend to develop a system that uses one aptamer for detection and a second biotin-labeled aptamer for immunoprecipitation. We subsequently demonstrate the ability to multiplex the aptamer qPCR assay with our qPCR assay for FGFR3 and intend to add the other DNA markers into this multiplex system in the future. This is the first demonstration of simultaneous detection of protein and DNA biomarkers using a single analytical platform. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-457. doi:1538-7445.AM2012-LB-457