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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.

Background: Current tissue-based genomic sequencing datasets typically reflect early stage, mostly treatment-naïve cohorts, and fail to capture the genomic landscape of advanced disease, including mutations conferring acquired resistance to therapy. Plasma cell-free DNA (cfDNA) can provide information on somatic alterations non-invasively and can capture spatial tumor heterogeneity. Here, we studied cfDNA from 15,564 blood samples from 12, 827 patients (11.5% of patients had serial samples) with MBC, and analyzed the differences in mutational landscape between cohorts of heavily pre-treated and primary untreated breast cancers. Materials and methods: Deidentified aggregate genomic data from stage III/IV breast cancer clinical samples submitted for cfDNA next-generation sequencing (NGS) analysis with Guardant360 (Guardant Health, Inc; Redwood City, CA, USA) assay between 11/25/16 and 5/26/20. This clinical assay detects somatic single nucleotide variants (SNVs), indels, copy number gains, and fusions in either all or a subset of exons in up to 74 genes. We compare the observed alterations to events detected from whole-exome sequencing data representing ~1,000 untreated primary breast cancers from The Cancer Genome Atlas (TCGA) project. Results: In the overall cohort (N = 12, 827), the average patient age was 61 (range 18-100); 99% were female. Of the 11,290 pts with at least one samples with a non-synonymous alteration, the most frequently mutated genes were TP53 (53%), PIK3CA (38%), ESR1 (28%), ATM (13%), GATA3 (10%), and ARID1A (10%) at varying allelic frequencies with dynamic changes over time (in pts with serial specimens) highlighting clonal heterogeneity and temporal evolution. Copy-number gains were most frequent in FGFR1 (13%), CCND1 (11%), EGFR (9%) and PIK3CA (9%), but fusions were infrequent (below 1%), with fusions involving FGFR3 (n=22), FGFR2 (n=12), ALK (n=7), NTRK1 (n=6), RET (n=4), and ROS1 (n=1). Overall, patients had significantly higher frequency of actionable alterations based on ctDNA analysis, as compared to that observed in TCGA. Similarly, non-synonymous mutations were identified in genes associated with acquired resistance, including ESR1 (28%), ERBB2 (9%), PTEN (8.1%), RB1 (7.7%), NF1 (6.2%), and AKT1 (4%), mutated at frequencies significantly exceeding those observed in TCGA. Finally, we developed a novel classifier to identify the breast cancer receptor subtypes based on differential genomic profile, which was further validated in an independent genomic dataset. Conclusions: In the largest dataset to date, we provide the comparative genomic landscape of the various SNVs, indels, copy-number gains, and fusions identified by next generation sequencing of cfDNA in patients with MBC, compared to primary breast cancer. Interestingly, both targetable genomic alterations and resistance mutations occur at frequencies much higher than observed in primary breast cancer, highlighting the genomic complexity of MBC and potential need for combinatorial therapy. Finally, we describe a novel classifier for detection of HR+ and HER2+ tumors, which could be particularly valuable clinically given the receptor switch with tumor evolution and practical difficulty with serial tissue biopsies in MBC. Comparison of genomic signature profiles between MBC and primary breast cancer will be presented at the meeting. Citation Format: Aditya Bardia, Massimo Cristofanilli, Justin Cha, Lesli Kiedrowski, Dejan Juric, Ben H Park, Adam Brufsky, Joyce O’Shaughnessy, Becky Nagy, Leif Ellisen, Ignaty Leshchiner, Gaddy Getz. Genomic landscape of metastatic breast cancer (MBC): Comprehensive cell-free DNA analysis from over 10,000 patients and comparison with primary breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD9-07.

SUMMARY To elucidate the deregulated functional modules that drive clear cell renal cell carcinoma (ccRCC), we performed comprehensive genomic, epigenomic, transcriptomic, proteomic, and phosphoproteomic characterization of treatment-naive ccRCC and paired normal adjacent tissue samples. Genomic analyses identified a distinct molecular subgroup associated with genomic instability. Integration of proteogenomic measurements uniquely identified protein dysregulation of cellular mechanisms impacted by genomic alterations, including oxidative phosphorylation-related metabolism, protein translation processes, and phospho-signaling modules. To assess the degree of immune infiltration in individual tumors, we identified microenvironment cell signatures that delineated four immune-based ccRCC subtypes characterized by distinct cellular pathways. This study reports a large-scale proteogenomic analysis of ccRCC to discern the functional impact of genomic alterations and provides evidence for rational treatment selection stemming from ccRCC pathobiology., Graphical Abstract, In Brief Comprehensive proteogenomic characterization in 103 treatment-naive clear cell renal cell carcinoma patient samples highlights tumor-specific alterations at the proteomic level that are unrevealed by transcriptomic profiling and proposes a revised subtyping scheme based on integrated omics analysis.

Pleuropulmonary blastoma is a rare childhood malignancy of lung mesenchymal cells that can remain dormant as epithelial cysts or progress to high-grade sarcoma. Predisposing germline loss-of-function DICER1 variants have been described. We sought to uncover additional contributors through whole exome sequencing of 15 tumor/normal pairs, followed by targeted resequencing, miRNA analysis and immunohistochemical analysis of additional tumors. In addition to frequent biallelic loss of TP53 and mutations of NRAS or BRAF in some cases, each case had compound disruption of DICER1: a germline (12 cases) or somatic (3 cases) loss-of-function variant plus a somatic missense mutation in the RNase IIIb domain. 5p-Derived microRNA (miRNA) transcripts retained abnormal precursor miRNA loop sequences normally removed by DICER1. This work both defines a genetic interaction landscape with DICER1 mutation and provides evidence for alteration in miRNA transcripts as a consequence of DICER1 disruption in cancer.

Background: While great strides have been made in the treatment of ER+ MBC, therapeutic resistance is nearly universal. The genomic evolution of ER+ breast cancer in the metastatic setting under the selective pressure of multiple lines of therapies is not well understood. To address this, we analyzed the clonal dynamics of serial metastatic samples (mets) to evaluate how tumors evolve and to identify acquired resistance mechanisms. Methods: We performed WES on 462 clinically annotated samples from 208 patients (pts) with ER+ MBC, including 67 primary tumor biopsies, 229 metastatic biopsies and 160 blood samples (cfDNA). Pts with multiple mets included cases with temporally concordant metastatic tumor and blood samples (48 pts) and cases with serial mets obtained over the course of treatment in the metastatic setting (69 pts). Treatments given between the serial mets included CDK4/6 inhibitors (23 pts), and selective estrogen receptor degraders (19 pts), among others. Results: In the temporally-concordant mets, we found that cfDNA mutations (muts) largely overlap with muts found in tumor biopsies, capturing >85% of clonal tumor muts. However, we observed a higher level of heterogeneity in cfDNA compared to biopsies (p.value< 1.05e-19, Welch test) and a subset of high-confidence muts that were only detected in cfDNA, including in clinically important genes such as ESR1, PIK3CA, KRAS, and ERBB2. Analysis of serial mets was used to elucidate the evolutionary dynamics within the metastatic setting under the selective pressure of treatment. The median duration between mets was 112 days and the median number of inter-biopsy unique treatments was two. Most tumors continued to evolve within the metastatic setting, with 50 out of 69 pts (72%) acquiring a meaningful sub-clone (50% increase in relative cancer cell fraction) and 31 out of 69 (45%) acquiring muts in known cancer genes, including a subset acquiring a plausible resistance alteration such as alterations that dysregulate ER (5 out of 69 pts, 7%; ESR1 mut, FOXA1 amplification (amp), NCOR1 bi-allelic deletion (del)), ERBB (4%; ERBB2 amp, ERBB3 mut), RAS (4%; KRAS mut, NRAS amp, NF1 del), FGF/FGFR (12%; FGFR2 mut, FGFR1/2 amp, FGF3 amp), and cell cycle (13%; RB1 del, CDK4 amp, AURKA amp, CDKN2A del). Finally, in pts who had multiple mets, we observed several cases of evolutionary convergence toward equivalent resistance mechanisms including convergent RB1 loss as a mechanism of resistance to a CDK4/6 inhibitor and convergent BRCA2 reversion following resistance to a PARP inhibitor. Conclusions: This study demonstrates that ER+ MBC continues to evolve under the selective pressure of treatments in the metastatic setting. These findings elucidate the challenge of studying high complexity and heavily treated tumors, while also highlighting some commonalities in the evolutionary trajectories selected by these treatments. The multiplicity of clinically relevant genomic alterations acquired in these advanced stages highlights the need for serial biopsies and the potential to inform post-progression therapeutic choices through targeting the acquired dependencies in post-progression tumors. Citation Format: Cohen O, Buendia-Buendia J, Wander S, Nayar U, Mao P, Waks A, Kim D, Freeman S, Adalsteinsson V, Helvie K, Livitz D, Rosebrock D, Leshchiner I, Dellostritto L, Garrido-Castro A, Jain E, Periyasamy S, Mackichan C, Lloyd M, Marini L, Krop I, Garraway L, Getz G, Winer E, Lin N, Wagle N. Evolutionary analysis of 462 serial metastatic biopsies from 208 patients with estrogen receptor-positive (ER+) metastatic breast cancer (MBC) using whole exome sequencing (WES) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD9-02.

To investigate therapy induced evolutionary patterns in glioblastoma (GBM), we completed whole exome sequencing of all samples, as well as transcriptome sequencing, DNA copy number, DNA methylation, and mRNA from at least one primary sample, and one (post-therapy) recurrent sample. Sample collection was enabled through The Cancer Genome Atlas Research Network (TCGA). Multiple spatially distinct samples were available for five primary and six recurrent cases, allowing the integration of intratumoral heterogeneity with the evolutionary processes that shape the tumor recurrence after therapeutic intervention. Sample unique mutations were common, but most of the somatic variants in driver genes (like TP53 and EGFR) were preserved across the tumor and after therapy. We found that the therapeutic bottleneck resulted in proportionally higher numbers of (mostly clonal) mutations, reduced clonal complexity and changes in the mutation spectrum. GBM recurrence could be grouped into two categories, characterized by drastically different clonal evolution patterns. Our findings suggest that multi-sector sequencing sharpens the analysis but is not essential for precise delineation of the tumor progression process. Projecting our results on genomic data from 253 primary GBMs, we observed that the number of clonal, but not subclonal, mutations increased with age at diagnosis, and the two categories differed in their mutation spectrum. This study represents an important advance toward comprehensive characterization of the genomic alterations of GBM before and after cytotoxic treatment and surgery. Our analysis of spatially and temporally distinct samples provides new insights into the relevance of intratumoral heterogeneity on disease progression in GBM. Acknowledgements: This work is supported by Award Numbers 5 P50 CA127001 and 5 P50 CA083639-12 from the National Cancer Institute (NCI) to RGWV. H.K. is supported in part by the Odyssey Program and Theodore N. Law Endowment for Scientific Achievement at The University of Texas MD Anderson Cancer Center. The results published here are in whole or part based upon data generated by The Cancer Genome Atlas project established by the NCI and NHGRI. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B130. Citation Format: Hoon Kim, Siyuan Zheng, Seyed S. Amini, Selene Virk, Tom Mikkelsen, Daniel J. Brat, Jonna Grimsby, Carrie Sougnez, Andrew E. Sloan, Mark L. Cohen, Erwin G. Van Meir, Lisa Scarpace, Peter W. Laird, John N. Weinstein, Eric S. Lander, Stacey Gabriel, Gaddy Getz, Matthew Meyerson, Lynda Chin, Jill S. Barnholtz-Sloan, Roeland GW Verhaak. The intratumoral heterogeneity of glioblastoma suggests a pivotal role for clonal evolution. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B130.

The landscape of somatic alterations in cancer is being rapidly uncovered by large-scale cancer genome sequencing projects in individual laboratories as well as consortia such as The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC). The decreasing cost of sequencing has resulted in an increase in the number of cancer genomes that are being sequenced, and thus the numbers of candidate somatic mutations being identified are rapidly expanding. A single cancer genome from a cancer with an average mutation background rate, such as ovarian or breast cancer, will have 3000-6000 mutations. A high mutation rate cancer such as melanoma or colon cancer can have as many as 100,000 mutations/genome. Multiplying these numbers by the dozens of sequenced genomes and hundreds of sequenced exomes across a wide spectrum of tumor types yields a staggering number of candidate mutations. However, approaches for rapidly validating these findings at scale have been lagging behind. Current approaches, such as custom designed hybrid capture arrays followed by sequencing, can take months to complete and cannot begin until the initial sequencing is complete and mutations are called. This can lead to long delays in the interpretation and publication of biological findings. Here we describe the results of several approaches for rapid and efficient validation on large-scale projects, such as TCGA. We will share our approach and results using Fluidigm and PacBio sequencing for validation and sample extension at a small scale with rapid turnaround where we have validated PIK3CA and TP53 mutations in breast cancer samples of varying purity as well as significantly mutated genes in medulloblastoma. Ideally one could validate mutations and gene fusions concurrent with their discovery. To that end, we will also describe our validation results using RNA-Seq data, commonly produced concurrently with genome and exome sequencing, in lung squamous and renal cancer. Finally, we will explore an approach for instant-validation through simultaneous sequencing of barcoded discovery and cross-validation libraries. 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 3960. doi:1538-7445.AM2012-3960