Although the development of multiple primary tumors in smokers with lung cancer can be attributed to carcinogen-induced field cancerization, the occurrence of multiple tumors at presentation in individuals with EGFR-mutant lung cancer who lack known environmental exposures remains unexplained. In the present study, we identified ten patients with early stage, resectable, non-small cell lung cancer who presented with multiple, anatomically distinct, EGFR-mutant tumors. We analyzed the phylogenetic relationships among multiple tumors from each patient using whole-exome sequencing (WES) and hypermutable poly(guanine) (poly(G)) repeat genotyping as orthogonal methods for lineage tracing. In four patients, developmental mosaicism, assessed by WES and poly(G) lineage tracing, indicates a common non-germline cell of origin. In two other patients, we identified germline EGFR variants, which confer moderately enhanced signaling when modeled in vitro. Thus, in addition to germline variants, developmental mosaicism defines a distinct mechanism of genetic predisposition to multiple EGFR-mutant primary tumors, with implications for their etiology and clinical management.

Although BCL2 mutations are reported as later occurring events leading to venetoclax resistance, many other mechanisms of progression have been reported though remain poorly understood. Here, we analyze longitudinal tumor samples from 11 patients with disease progression while receiving venetoclax to characterize the clonal evolution of resistance. All patients tested showed increased in vitro resistance to venetoclax at the posttreatment time point. We found the previously described acquired BCL2-G101V mutation in only 4 of 11 patients, with 2 patients showing a very low variant allele fraction (0.03%-4.68%). Whole-exome sequencing revealed acquired loss(8p) in 4 of 11 patients, of which 2 patients also had gain (1q21.2-21.3) in the same cells affecting the MCL1 gene. In vitro experiments showed that CLL cells from the 4 patients with loss(8p) were more resistant to venetoclax than cells from those without it, with the cells from 2 patients also carrying gain (1q21.2-21.3) showing increased sensitivity to MCL1 inhibition. Progression samples with gain (1q21.2-21.3) were more susceptible to the com bination of MCL1 inhibitor and venetoclax. Differential gene expression analysis comparing bulk RNA sequencing data from pretreatment and progression time points of all patients showed upregulation of proliferation, B-cell receptor (BCR), and NF-kappa B gene sets including MAPK genes. Cells from progression time points demonstrated upregulation of surface immunoglobulin M and higher pERK levels compared with those from the preprogression time point, suggesting an upregulation of BCR signaling that activates the MAPK pathway. Overall, our data suggest several mechanisms of acquired resistance to venetoclax in CLL that could pave the way for rationally designed combination treatments for patients with venetoclax-resistant CLL.

Figure S1, radiographic assessment of key lesions of patient MGH-18; Figure S2, supplementary genomic analysis for patient MGH-18; Figure S3, supporting data for mechanism of TROP2 T256R; Figure S4, TROP2 immunohistochemistry for metastatic lesions of MGH-18; Table S1, detailed cohort characteristics; Table S2, patient treatment histories; Table S3, supplementary mutational data for MGH-18; Table S4, antibodies used.

Richter syndrome (RS) arising from chronic lymphocytic leukemia (CLL) exemplifies an aggressive malignancy that develops from an indolent neoplasm. To decipher the genetics underlying this transformation, we computationally deconvoluted admixtures of CLL and RS cells from 52 patients with RS, evaluating paired CLL-RS whole-exome sequencing data. We discovered RS-specific somatic driver mutations (including IRF2BP2, SRSF1, B2M, DNMT3A and CCND3), recurrent copy-number alterations beyond del(9p21)(CDKN2A/B), whole-genome duplication and chromothripsis, which were confirmed in 45 independent RS cases and in an external set of RS whole genomes. Through unsupervised clustering, clonally related RS was largely distinct from diffuse large B cell lymphoma. We distinguished pathways that were dysregulated in RS versus CLL, and detected clonal evolution of transformation at single-cell resolution, identifying intermediate cell states. Our study defines distinct molecular subtypes of RS and highlights cell-free DNA analysis as a potential tool for early diagnosis and monitoring.

Detection of multiple primary lung cancers is increasing in frequency, with up to 15% of all non-small cell lung cancer (NSCLC) patients presenting with two or more anatomically separate tumor nodules on CT scans. Increased detection is in part due to expanded lung cancer screening criteria in an aging population. Distinguishing multiple primary tumors from intrapulmonary metastases can be challenging, yet is critical for determining clinical management. Current models predict development of multiple primary tumors out of a cancerized field, such as occurs due to smoking. The occurrence of multiple primary tumors is unexplained in patients with EGFR-mutant lung cancer (~15% of all NSCLC), lacking environmental exposures. We identified patients with non-small cell lung cancer (NSCLC) who presented with multiple primary EGFR-mutant tumors, in the absence of family history of lung cancer or heavy smoking. We subjected the macrodissected tumors and surrounding normal tissues to whole exome sequencing as well as to analysis of hypermutable poly-guanine (poly-G) repeats, which are two orthogonal methods of lineage tracing. An additional familial case with a germline EGFR-T790M mutation was used to establish parameters for timing of somatic mutations, and functional properties of novel germline variants were modeled in vitro. Of eleven non-familial NSCLC cases with two or more geographically distinct EGFR-mutant lung cancers, two patients harbored a germline EGFR variant allele, which confers moderately enhanced signaling in vitro, followed by a somatically acquired EGFR mutation. In an additional four cases, both whole exome sequencing and poly-G repeat analyses indicate a distant shared somatic cell-of-origin, consistent with embryonic mosaicism. Three cases revealed clinically unappreciated metastasis and two cases remain unexplained. Together, our data suggest that multiple primary lung cancers with somatic EGFR driver mutations may result from genetic susceptibility, attributable either to attenuated EGFR germline variants or to embryonic mosaicism resulting in geographically disparate patches of predisposed lung tissue. Such predisposed cases should be surveilled for early detection of future tumors, and surgical resection in these cases should consider the life-long risk of additional cancers. Citation Format: Risa Burr, Ignaty Leshchiner, Christina L. Costantino, Martin Blohmer, Tilak Sundaresan, Justin Cha, Karsen Seeger, Sara Guay, Brian P. Danysh, Ira Gore, Raquel A. Jacobs, Kara Slowik, Filippo Utro, Kahn Rhrissorrakrai, Chaya Levovitz, Jaimie L. Barth, Taronish Dubash, Brian Chirn, Laxmi Parida, Lecia V. Sequist, Mari Mino-Kenudson, Kamila Naxerova, Shyamala Maheswaran, Gad Getz, Daniel A. Haber. Mechanisms of genetic predisposition to multifocal lung cancer [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 771.

Covalent inhibitors of Bruton tyrosine kinase (BTK) have transformed the therapy of chronic lymphocytic leukemia (CLL), but continuous therapy has been complicated by the development of resistance. The most common resistance mechanism in patients whose disease progresses on covalent BTK inhibitors (BTKis) is a mutation in the BTK 481 cysteine residue to which the inhibitors bind covalently. Pirtobrutinib is a highly selective, noncovalent BTKi with substantial clinical activity in patients whose disease has progressed on covalent BTKi, regardless of BTK mutation status. Using in vitro ibrutinib-resistant models and cells from patients with CLL, we show that pirtobrutinib potently inhibits BTK-mediated functions including B-cell receptor (BCR) signaling, cell viability, and CCL3/CCL4 chemokine production in both BTK wild-type and C481S mutant CLL cells. We demonstrate that primary CLL cells from responding patients on the pirtobrutinib trial show reduced BCR signaling, cell survival, and CCL3/CCL4 chemokine secretion. At time of progression, these primary CLL cells show increasing resistance to pirtobrutinib in signaling inhibition, cell viability, and cytokine production. We employed longitudinal whole-exome sequencing on 2 patients whose disease progressed on pirtobrutinib and identified selection of alternative-site BTK mutations, providing clinical evidence that secondary BTK mutations lead to resistance to noncovalent BTKis.

While the development of multiple primary tumors in smokers with lung cancer can be attributed to carcinogen-induced field cancerization, the occurrence of multiple primary tumors in individuals with EGFR -mutant lung cancer who lack known environmental exposures remains unexplained. We identified ten patients with early-stage, resectable non-small cell lung cancer who presented with multiple anatomically distinct EGFR -mutant tumors. We analyzed the phylogenetic relationships among multiple tumors from each patient using whole exome sequencing (WES) and hypermutable poly-guanine (poly-G) repeat genotyping, as orthogonal methods for lineage tracing. In two patients, we identified germline EGFR variants, which confer moderately enhanced signaling when modeled in vitro . In four other patients, developmental mosaicism is supported by the poly-G lineage tracing and WES, indicating a common non-germline cell-of-origin. Thus, developmental mosaicism and germline variants define two distinct mechanisms of genetic predisposition to multiple EGFR -mutant primary tumors, with implications for understanding their etiology and clinical management.

Background. Despite substantial progress in the treatment of HER2+ MBC, most patients (pts) still experience disease progression and cancer-related death. HER2-directed TKIs are highly effective therapies for pts with HER2+ MBC; however, an understanding of resistance mechanisms is needed. Pts receiving HER2-directed TKIs with cell-free DNA (cfDNA) sampling across the treatment spectrum present a unique opportunity to examine genomic alterations. Methods. Pts with biopsy-proven HER2+ MBC were selected from DF/HCC approved protocols for ultra-low pass whole genome sequencing if ≥1 cfDNA and/or tissue sample had been collected prior to and after at least six weeks of TKI treatment. Only pts with ≥ 1 sample with tumor fraction (TFx) ≥ 9.5% and ≥ 1 additional sample with TFx ≥ 4.5% were analyzed. Whole exome sequencing (WES) was performed on 19 tumor biopsies and 64 cfDNA samples from 25 pts. WES samples were analyzed, variants called and annotated, copy number profiles inferred, and TFx estimated. PhylogicNDT was used to detect the phylogenetic architecture, and clones were identified as growing, stable, shrinking, or truncal; mutational signatures were called using SignatureAnalyzer. After filtering likely benign variants, mutations were labeled likely resistance mechanisms if there was known evidence of resistance to anti-HER2 therapy in a human breast cancer cell line, breast cancer mouse models, or in vivo. Findings were correlated with pt clinicopathologic data. Results. In 4/8 pts with acquired resistance (TKI treatment ≥ 180 days), we identified alterations in previously characterized pathways and genes that could explain treatment resistance (e.g. PIK3CA, ERBB2, FGFR2). Mutations in growing subclones identified potential novel mechanisms of resistance and included inactivating mutations in CDK12, KMT2D, KMT2C, CHEK2, BRCA2, and FAT1 genes. In 7/17 pts with intrinsic resistance, we identified mutations in overlapping pathways and genes such as ERBB2 and PIK3CA, which were present in both growing subclonal and truncal clones. Four pts with hormone receptor positive disease - two of whom had not received prior aromatase inhibitors - had an activating ESR1 hotspot mutation (D538G). This raises the possibility that activating ESR1 mutations may be involved in resistance in these cases. Mutational signature analysis revealed a subset of samples with widespread APOBEC activation (with and without hyper mutation), subclonal HR/MMR-related signature, and capecitabine-related 5FU signature. Conclusions. Genomic analysis of paired samples from pts with HER2+ MBC identifies candidate resistance mechanisms to anti-HER2 TKIs and clonal evolution over time in the context of heterogeneity in treatment and sample timing. Additional studies will determine the functional role and clinical utility of assessing these alterations to overcome resistance. Citation Format: Heather A. Parsons, Conor Messer, Katheryn Santos, Brian P. Danysh, Melissa E. Hughes, Ashka Patel, Raquel A. Jacobs, Kara Slowik, Julian Hess, Chip Stewart, Kristy Schlueter-Kuck, Kahn Rhrissorrakrai, Filippo Utro, Chaya Levovitz, Nikhil Wagle, Jose Pablo Leone, Rachel Freedman, Laxmi Parida, Ian E. Krop, Gad Getz, Nancy U. Lin. Genomic mechanisms of resistance to tyrosine kinase inhibitors (TKIs) in HER2+ metastatic breast cancer (HER2+ MBC). [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 3874.

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.

We studied resistance mechanisms to hormone therapy and CDK4/6 cell cycle inhibitors in ER+ breast cancer by analyzing whole-exome, whole-genome, single-cell, and bulk transcriptomes in 120 autopsy samples from 13 patients obtained by the Massachusetts General Hospital Rapid Autopsy program. For each patient, we inferred the clonal structure of the samples and tracked the metastatic spread of different clones throughout the body. For 7 patients, we also analyzed serial cfDNA samples to identify clones that were selected for during treatment. We identified significantly recurrent and convergent (arising independently in distinct clones) acquired mutations in ESR1, KRAS, and chromatin modifier genes, in particular, mutations in KMT2C, which may represent mechanisms of drug resistance in this clinical setting. To experimentally study the role of KMT2C mutations, we used CRISPR/Cas9 to knock out KMT2C in the ER+ CAMA1 breast cancer cell line that is sensitive to both ER and CDK4/6 inhibition. KMT2C knock-out cells demonstrated significantly increased viability under treatment with fulvestrant (ERi), palbociclib (CDK4/6i), or their combination compared to the control cell lines. We show that this increased drug resistance is driven by downregulation of the ESR1 pathway, suggesting a decreased dependency on ER signaling for cell cycle progression. In addition to the early survival benefit, KMT2C knock-out resulted in a dramatic outgrowth of cells under long-term fulvestrant treatment. The KMT2C KO fulvestrant-resistant outgrown cells were highly resistant to the CDK4/6 inhibitors palbociclib, ribociclib, and abemaciclib compared to control cells, as well as to novel ERalpha inhibitors and a range of targeted therapies currently in clinical trials. By testing a panel of compounds on KMT2C KO and control cell lines, we propose potential novel therapeutic strategies that may help overcome the development of resistance in KMT2C-mutant cells. These findings suggest that KMT2C mutations may be a mechanism of acquired resistance to CDK4/6 inhibitor combinations, and subsequent treatment with therapies directed towards ER or CDK4/6 pathways may be ineffective and other treatment avenues need to be developed. Citation Format: Elizaveta Leshchiner, Ignaty Leshchiner, Elizabeth E. Martin, Christopher T. Chen, Thomas Zhang, Christopher Pinto, Kahn Rhrissorrakrai, Filippo Utro, Chaya Levovitz, Raquel A. Jacobs, Brian P. Danysh, Kara Slowik, Maida Broudo, Laxmi Parida, Dejan Juric, Gad Getz. Chromatin modifier alterations confer resistance to endocrine deprivation and CDK4/6 inhibitors in ER+ breast cancer and drive convergent evolution in patient autopsy lesions [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 1789.

Detecting cancer at early stages or upon recurrence is critical to decreasing cancer morbidity and mortality. We developed TuFEst (Tumor Fraction Estimator), a cost-effective computational approach for pan-cancer detection and tumor burden estimation from ultra-low coverage whole genome sequencing (~0.1x, ULP-WGS) of minimally invasive cell-free DNA (cfDNA). Current state-of-the-art methods estimate tumor fraction (TF) from ULP-WGS depending exclusively on total copy number variation, which loses tumor signal in either copy number-quiet tumors or tumors with copy-neutral loss-of-heterozygosity. Additionally, it is difficult in many cases to distinguish clonal from sub-clonal copy-number events, therefore complicating the ability to estimate tumor fraction. On the other hand, fragments shed into the blood from cancer cells, i.e., circulating tumor DNA (ctDNA), of various cancer types show significantly different length distribution than that from normal cells in healthy donors. By synergistically integrating both (i) copy number variation and (ii) altered fragment length signals, TuFEst successfully achieved higher sensitivity and more accurate TF estimation than current methods in >200 cfDNA samples across different cancer types, even in low tumor-fraction cases (TF < 0.1%). Application of TuFEst to serial cfDNA samples from blood biopsies demonstrate its utility in accurately estimating TF in ~100 cfDNAs, suggesting that TuFEst can be used to detect early cancer recurrence during different treatments. In one breast cancer patient receiving CDK4/6 therapy, TuFEst indicated disease progression 262 days earlier than routine imaging. Altogether, our work suggests that accurate TF estimation from cfDNA can not only aid in detecting cancer at early stages but also provide evidence of disease progression during treatment. We believe that such a non-invasive, cost-effective, pan-cancer detection method will benefit both initial cancer screening and monitoring of resistance to therapy in clinical applications. Citation Format: Ziao Lin, Chip Stewart, Elizabeth E. Martin, Brian P. Danysh, Raquel A. Jacobs, Kara Slowik, Lee Lawton, Elizabeth Lightbody, Kahn Rhrissorrakrai, Filippo Utro, Chaya Levovitz, Carrie Cibulskis, Irene M. Ghobrial, Margaret Shipp, Ryan B. Corcoran, Dejan Juric, Laxmi Parida, Heather A. Parsons, Gad Getz. TuFEst: a sensitive and cost-effective pan-cancer detection approach with accurate tumor fraction estimation [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 5162.

Pirtobrutinib is a non-covalent BTK inhibitor (BTKi) designed to maintain activity despite the most common resistance mutation to covalent inhibitors, at BTK C481. To investigate mechanisms of disease progression on pirtobrutinib, we evaluated the effect of pirtobrutinib in vitro on the BCR pathway in pre- and post-treatment patient samples (n=5), and on 2 of these patients, we performed whole exome sequencing longitudinally including prior to and at relapse on pirtobrutinib. Phylogenetics and subclonal dynamics associated with resistance were evaluated using the PhylogicNDT and Concerti tools. To investigate the impact of identified BTK mutations on BCR activation, we generated 6 single and 3 double mutants using site directed mutagenesis and expressed them in the BTK null DT40 B cell line. We demonstrate that primary CLL cells from responding patients on the pirtobrutinib trial show reduced BCR signaling, reduced CCL3/CCL4 chemokine secretion, as well as effective induction of apoptosis and inhibition of proliferation, in response to pirtobrutinib. At time of progression, these primary CLL cells show increasing resistance to pirtobrutinib in signaling inhibition and cytokine production, with reduced inhibition of proliferation and induction of apoptosis. In WES analysis, patient #1 had 16 samples evaluated prior to, during, and at relapse on acalabrutinib, vecabrutinib and pirtobrutinib. Clonal analysis of samples collected during acalabrutinib shows steady selection of a clone harboring BTK p.C481S mutation with CCF 92% at relapse but then steadily decreases during pirtobrutinib treatment. Concerti's time-scaled phylogenetic tree shows the birth of a new clone containing the BTK gatekeeper mutation, p.T474I, during acalabrutinib treatment, which then grows rapidly under pirtobrutinib treatment, taking over nearly the entire cancer cell population and replacing the prior p.C481S clone. This complete clonal shift during pirtobrutinib treatment suggests that pirtobrutinib effectively inhibits the p.C481S clone, while the p.T474I gatekeeper clone is likely driving resistance in this patient. We also observed an additional gatekeeper clone BTK p.T474L develop at low levels, as well as another previously undescribed BTK mutation at p.M477I. Manual inspection showed that BTK mutations p.M477I and p.T474I are in cis and therefore in the same clone. Patient #2 had 10 samples evaluated prior to, during and at relapse on ibrutinib and pirtobrutinib. During ibrutinib therapy, we observed a steady increase in a clone with TP53 p.S240G and SF3B1 p.K666N mutations, reaching CCFs >40% at relapse on ibrutinib. We also noted a significant increase in BTK p.C481R (CCF 33%), BTK p.C481S (CCF 11%) and TP53 p.R196* (CCF 5%) at progression on ibrutinib. Under pirtobrutinib treatment the clone carrying BTK p.C481R decreased to CCF 20%, while BTK p.C481S (28%) and TP53 p.R197* (35%) both increased. Concerti's phylogenetic tree captures the birth of a resistant clone, harboring BTK p.L528W which significantly increases to CCF 30% at progression on pirtobrutinib. Functional characterization of the identified BTK mutations demonstrated that only T474I, T474L and C481S mutants showed adequate kinase activity, while all the other mutants including M477I, C48IR and L528W essentially lacked kinase activity as judged by phosphorylation at BTK Y223 and at PLCG2 Y753. As expected, the C481S variant was resistant to ibrutinib, but not to pirtobrutinib, while the T474I/L variants were sensitive to ibrutinib but resistant to pirtobrutinib. Interestingly, phosphorylation of AKT and ERK were retained downstream, even with mutations that failed to activate proximal BCR signaling. Furthermore, phosphorylation of AKT and ERK was also observed in the B7.10 cell line which lacks endogenous BTK, demonstrating significant activation of these pathways independent of BTK. In this study, we demonstrate that ex vivo efficacy of pirtobrutinib declines as patients’ CLL starts to progress, in concert with the development of gatekeeper and alternative site BTK mutations that lead to resistance to pirtobrutinib. Interestingly, many of the second-site BTK mutations fail to activate BTK phosphorylation but are still associated with downstream activation of phospho-AKT and phospho-ERK; the mechanism of this activation remains to be elucidated.

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; however, failure mechanisms are identified in only a fraction of cases. To gain new insights into the basis of clinical response, we performed single-cell transcriptome sequencing of 105 pretreatment and post-treatment peripheral blood mononuclear cell samples, and infusion products collected from 32 individuals with large B cell lymphoma treated with either of two CD19 CAR-T products: axicabtagene ciloleucel (axi-cel) or tisagenlecleucel (tisa-cel). Expansion of proliferative memory-like CD8 clones was a hallmark of tisa-cel response, whereas axi-cel responders displayed more heterogeneous populations. Elevations in CAR-T regulatory cells among nonresponders to axi-cel were detected, and these populations were capable of suppressing conventional CAR-T cell expansion and driving late relapses in an in vivo model. Our analyses reveal the temporal dynamics of effective responses to CAR-T therapy, the distinct molecular phenotypes of CAR-T cells with differing designs, and the capacity for even small increases in CAR-T regulatory cells to drive relapse.

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.

Mechanisms of resistance to venetoclax, a highly selective oral BCL2 inhibitor approved for therapy of CLL, remain poorly understood and we sought to characterize the clonal evolution of resistance in patients developing progressive disease on venetoclax. We performed whole-exome sequencing (WES) on 7 relapsed/refractory CLL patients with disease progressing on venetoclax. We observed del17p in 43% (3/7), TP53 mutation in 86% (6/7) and unmutated IGHV in 71% (5/7) of patients. A median of four longitudinal tumor samples were sequenced per patient (total Nsamples= 25). We performed digital droplet PCR (ddPCR) analysis to look at the emergence of BCL2 G101V mutations that have been previously linked with venetoclax resistance after long times on therapy. G101V mutation was detected by ddPCR in two patients at low variant allelic fractions (VAF) of 0.03% and 4.68% respectively, and not detected by WES. We further detected one droplet positive for G101V mutation in two other patients in venetoclax progression bone marrow samples. Due to the very low VAF of the BCL2 G101V mutations, we suspected that other mechanisms of acquired resistance were more significant in this cohort of relatively early relapses. Analysis of WES data showed no somatic single nucleotide variants (sSNVs) selected in more than one patient with resistance. However, copy number analysis revealed acquired del8p in 3 patients, resulting in large subclonal expansions. Furthermore, del 8p co-occurred with amp 1q21.2-21.3 affecting the MCL1 gene in 2 patients, and with amp 8q, del 17p, del 18q22.1-23 and del 9p23-21.2 (containing the MYC, TP53, BCL2 and CDKN2A/B genes respectively) in a third. Two other patients showed expansion of clones harboring del 10q23.31-24.1, that includes PTEN, while a third contains an expanding clone with two IRF8 mutations (S283C and SFF416fs). Resistance in the final patient is likely associated with marked expansion of clones with TP53 and SF3B1 mutations. RNA-seq analysis comparing pre-venetoclax to post-venetoclax resistant samples from 8 CLL patients showed downregulation of the BCR, FCGR and MAPK signaling pathways, with upregulation of mitochondrial translation, oxidative phosphorylation and the TCA/citric acid cycle at the time of progression. RNA-seq analysis focusing on the patients with del 8p shows significant downregulation of the TNFRSF10A/10B genes (TRAIL-Rs), with gene set enrichment analysis (GSEA) showing positive enrichment for WNT5A/FZD4 signaling and CREB signaling via the PKC and MAPK pathways, concomitant with downregulation of the BCR and FCGR pathways at progression. Our data suggest several mechanisms of venetoclax resistance in CLL, including loss of TNFRSF10A/B, sometimes with MCL1 upregulation, as well as WNT pathway upregulation and BCR pathway downregulation, which we are now validating in primary patient PBMCs and relevant cell line models. Citation Format: Ishwarya Murali, Justin Cha, Ignaty Leshchiner, Yanan Kuang, Kevin Vasquez, Jasneet Khalsa, Stacey M. Fernandes, Filippo Utro, Kahn Rhrissorrakrai, Chaya Levovitz, Brian P. Danysh, Kara Slowik, Raquel A. Jacobs, Cloud P. Paweletz, Laxmi Parida, Gad Getz, Jennifer R. Brown. Mechanisms of primary and acquired resistance to venetoclax in chronic lymphocytic leukemia (CLL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1097.

Abstract Sacituzumab govitecan (SG), the first antibody–drug conjugate (ADC) approved for triple-negative breast cancer, incorporates the anti-TROP2 antibody hRS7 conjugated to a topoisomerase-1 (TOP1) inhibitor payload. We sought to identify mechanisms of SG resistance through RNA and whole-exome sequencing of pretreatment and postprogression specimens. One patient exhibiting de novo progression lacked TROP2 expression, in contrast to robust TROP2 expression and focal genomic amplification of TACSTD2/TROP2 observed in a patient with a deep, prolonged response to SG. Analysis of acquired genomic resistance in this case revealed one phylogenetic branch harboring a canonical TOP1E418K resistance mutation and subsequent frameshift TOP1 mutation, whereas a distinct branch exhibited a novel TACSTD2/TROP2T256R missense mutation. Reconstitution experiments demonstrated that TROP2T256R confers SG resistance via defective plasma membrane localization and reduced cell-surface binding by hRS7. These findings highlight parallel genomic alterations in both antibody and payload targets associated with resistance to SG. Significance: These findings underscore TROP2 as a response determinant and reveal acquired SG resistance mechanisms involving the direct antibody and drug payload targets in distinct metastatic subclones of an individual patient. This study highlights the specificity of SG and illustrates how such mechanisms will inform therapeutic strategies to overcome ADC resistance. This article is highlighted in the In This Issue feature, p. 2355

Abstract Studies of therapeutic resistance in cancer have conventionally focused on identification of acquired exome mutations in tissue or circulating DNA at progression. However, this strategy has generated limited insights into resistance to CDK4/6 inhibitor and endocrine therapy combinations, which are the key first-line treatment modality in ER+ breast cancer. We utilized an alternative approach of integrated proteogenomic analysis of 8 pairs of pre- and post-treatment biopsies, 67 rapid autopsy samples, and 17 plasma samples from 12 ER+ breast cancer patients treated with CDK4/6 inhibitor combinations at a large academic center. In addition to whole exome sequencing, RNA sequencing, and immunohistochemistry (IHC) profiling on all tissue samples, we performed deep-scale mass spectrometry-based proteomics and phospho-proteomics on 35 rapid autopsy samples from 5 patients that had sufficient protein yields for analysis. We describe a patient with acquired Rb IHC loss after treatment progression without a Rb1 genetic alteration detected in exome sequencing or Rb transcript loss at her post-progression biopsy. Integrated proteogenomic analysis of ten autopsy lesions from this patient revealed convergent Rb protein loss across all tumor lesions, including lesions with and without Rb1 exome alterations. ESR1 mutations were frequently acquired at post-treatment biopsies at high cancer cell fractions, but a pre-existing ESR1-mutant subclone was nevertheless lost in a patient who acquired a concurrent Rb1-mutant subclone in the same tumor lesion. This suggests ESR1 mutations confer a relative fitness advantage that can nevertheless be mitigated in the presence of more potent synchronous resistance mechanisms. To investigate whether circulating tumor DNA reflects DNA shed specifically by progressing tumor lesions, we modeled the DNA shedding of tumor lesions into the plasma for 3 patients with both rapid autopsy and serial plasma samples available. In some instances, new lesions began shedding significant amounts of DNA months prior to their initial radiographic appearance. Together, these results illustrate the value of integrated multi-omics interrogation of different sample types to investigate therapeutic resistance and advance precision oncology. Citation Format: Christopher T. Chen, Ignaty Leshchiner, Liz Martin, Harry Kane, Kahn Rhrissorrakrai, Filippo Utro, Chaya Levovitz, Michael Gillette, Shankha Satpathy, Christopher Pinto, Daniel McLoughlin, Read Allen, Brian P. Danysh, Kara Slowik, Raquel A. Jacobs, Steven Carr, Laxmi Parida, Gad Getz, Dejan Juric. Proteogenomic characterization of CDK4/6 inhibitor-resistant ER+ breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P065.

Abstract Determining patterns and mechanisms of drug resistance is fundamentally required for improving clinical outcome of cancer treatments. The ability to study multiple samples from different metastatic sites of the same patient is a clinically challenging task, which has become possible with the advent of “rapid” autopsy procedures (<6 hours from death) conducted on the patients deceased from cancer. We have obtained and analyzed whole-exome, whole genome and transcriptome sequencing data from advanced breast cancer samples derived from multiple cancer lesions of the same patient post-treatment through the Massachusetts General Hospital Rapid Autopsy program. We have integrated genomic and transcriptomic data through advanced clonal reconstruction methods (PhylogicNDT) to investigate how the patients developed resistance to multiple lines of anti-cancer therapy. Additionally, we have used previously collected cell-free DNA samples to establish both a spatial and temporal picture of cancer drug resistance and progression. We have examined clonal heterogeneity and resistance to targeted therapies in 10 metastatic ER+ breast cancer patients over 120 distinct metastatic sites. The majority of patients in this cohort were treated with CDK4/6 cell cycle inhibitors in addition to hormone therapy. We discovered that multiple metastatic sites often share similar clonal structure, but overall progeny of several sibling clones spreads throughout the body, creating “families” of more closely related lesions. These distinct lesion populations often develop independent resistance mechanisms to the many lines of treatment that the patents have received. At least 8 out of 10 patients showed multiple known and potentially novel resistance mechanisms in separate branches of the phylogenetic tree, often converging on distinct mutations in the same resistance genes. Notably we have identified ESR1, KRAS and chromatin modifier recurrent and convergent mutations across the cohort. Temporal analysis using cfDNA taken over the course of treatment allows us to identify clones that expanded in response to a given treatment. Using RNA expression and pathway analysis, we identify unique transcriptional programs, differentially expressed genes and ER-signaling changes between distinct clones within one patient, as well as compare genetically similar clones across patents. Citation Format: Ignaty Leshchiner, Elizabeth E. Martin, Christopher T. Chen, Elizaveta Leshchiner, Thomas Zhang, Christopher Pinto, Filippo Utro, Kahn Rhrissorrakrai, Chaya Levovitz, Brian P. Danysh, Kara Slowik, Raquel A. Jacobs, Laxmi Parida, Gad Getz, Dejan Juric. Convergence of resistance patterns in breast cancer after multiple lines of treatment through analysis of rapid autopsy samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 37.