ecDNA heterogeneity in SCLC, related to Figure 4

ecDNA in SCLC, related to Figure 1

Background Profiling circulating cell-free DNA (cfDNA) has become a fundamental practice in cancer medicine, but the effectiveness of cfDNA at elucidating tumor-derived molecular features has not been systematically compared to standard single-lesion tumor biopsies in prospective cohorts of patients. The use of plasma instead of tissue to guide therapy is particularly attractive for patients with small cell lung cancer (SCLC), due to the aggressive clinical course of this cancer, which makes obtaining tumor biopsies exceedingly challenging. Methods In this study, we analyzed a prospective cohort of 49 plasma samples obtained before, during, and after treatment from 20 patients with recurrent SCLC. We conducted cfDNA low-pass whole genome sequencing (0.1X coverage), comparing it with time-point matched tumor characterized using whole-exome (130X) and transcriptome sequencing. Results A direct comparison of cfDNA and tumor biopsy revealed that cfDNA not only mirrors the mutation and copy number landscape of the corresponding tumor but also identifies clinically relevant resistance mechanisms and cancer driver alterations not detected in matched tumor biopsies. Longitudinal cfDNA analysis reliably tracks tumor response, progression, and clonal evolution. Sequencing coverage of plasma DNA fragments around transcription start sites showed distinct treatment-related changes and captured the expression of key transcription factors such as NEUROD1 and REST in the corresponding SCLC tumors. This allowed for the prediction of SCLC neuroendocrine phenotypes and treatment responses. Conclusions cfDNA captures a comprehensive view of tumor heterogeneity and evolution. These findings have significant implications for the non-invasive stratification of SCLC, a disease currently treated as a single entity.

Amplified genes identified from cell-free ChIP-seq of patient plasma samples.

Fusion landscape and ecDNA RLF-MYCL fusions, related to Figure 3.

Expression and copy-number status of recurrently highly amplified genes in patient and cell line samples. The tables summarize copy-number signal and AmpliconArchitect curation, expression of key genes and results of the multivariate analysis.

Enhancer-enhancer interaction analysis derived from H3K27ac HiChIP in the NCI-H889 and NCI-H524 cell lines. Interaction strength of proximal enhancers was calculated from the “.hic” files generated by the JuiceBox suite.

Fusion analysis results calculated with STARfusion and summary of RLF exon1 and exon2 expression for the CCLE cohort. Table summarizes the RLF-MYCL expression qPCR results.

Validation of ecDNA in SCLC, related to Figure 2

Enhancer-enhancer interaction results of MYC and MYCL ecDNAs in the DMS-273 cell line.

Patient sample AmpliconArchitect assemblies, related to Figure 1

Cell line AmpliconArchitect assemblies, related to figure 1 and Figure 2

Experimental validation of copy-number of the MYC locus in NCI-H889, DMS-114, NCI-H446 and RA022 patient derived cell lines and number of ecDNA and HSR positive cells in the RA022 patient derived cell lines

Amplicon design used for the targeted single-cell copy-number analysis. Normalized depth of MYC and MYCL amplicons in single-cells for the DMS-273 cell line are shown, as well as comparison of MYC amplicon depths in single cells between the patient derived Adrenal gland (ecDNA+) and Cerv.LN (HSR+) cell lines.

Heterogeneity in patient derived samples, related to Figure 5

TPS8130 Background: Small-cell lung cancer (SCLC) is the most fatal type of lung cancer characterized by exquisite chemo-sensitivity at diagnosis and chemoresistance at relapse. Despite a highly mutated genome, patients with SCLC derive little benefit from immunotherapy. EZH2 (enhancer of zeste homolog 2) is a master epigenetic regulator of SCLC neuroendocrine cell fate and plasticity. EZH2 inhibition 1) promotes upregulation of Schlafen 11 (SLFN11) which irreversibly blocks replication in response to DNA damaging agents and 2) enhances intrinsic immune signaling, leading to constitutive MHC I recovery, sensitizing resistant SCLC models to DNA damaging chemotherapy and immunotherapy. Tazemetostat is a selective oral EZH2 inhibitor. Methods: This is an investigator-initiated, NCI Cancer Therapy Evaluation Program (CTEP) sponsored, phase I dose escalation and dose expansion study which will evaluate safety and tolerability of combination of tazemetostat with topotecan, a selective TOP1 inhibitor, and programmed cell death protein 1 (PD-1) inhibitor antibody pembrolizumab. Adult patients with relapsed/recurrent SCLC after at least platinum doublet (limited stage-SCLC) or chemo-immunotherapy (extensive stage-SCLC) and ECOG performance 0-1 are eligible. The regimen design involves a 7-day “run-in” of oral tazemetostat BID followed by 21-day cycles of tazemetostat (1-21 days), intravenous (IV) topotecan (day 1-5) and IV pembrolizumab (Day 1). The dose escalation cohort aims to determine safety and optimal doses of tazemetostat and topotecan (with standard dose of pembrolizumab) using a 3+3 design by assessing for dose limiting toxicities. The dose expansion cohort aims to assess safety, tolerability and preliminary efficacy of the combination in 15 additional patients with relapsed SCLC. The study involves collection of mandatory biopsies at pre-treatment and post-treatment (cycle 1) to gain insights into mechanism of action and resistance of the combination using single cell and spatial transcriptomic approaches. For more questions regarding enrollment and eligibility please contact Rasa.vilimas@nih.gov or anish.thomas@nih.gov . Clinical trial information: NCT05353439 .

Small-cell lung cancer (SCLC) is the most fatal form of lung cancer. Intratumoral heterogeneity, marked by neuroendocrine (NE) and non-neuroendocrine (non-NE) cell states, defines SCLC, but the cell-extrinsic drivers of SCLC plasticity are poorly understood. To map the landscape of SCLC tumor microenvironment (TME), we apply spatially resolved transcriptomics and quantitative mass spectrometry-based proteomics to metastatic SCLC tumors obtained via rapid autopsy. The phenotype and overall composition of non-malignant cells in the TME exhibit substantial variability, closely mirroring the tumor phenotype, suggesting TME-driven reprogramming of NE cell states. We identify cancer-associated fibroblasts (CAFs) as a crucial element of SCLC TME heterogeneity, contributing to immune exclusion, and predicting exceptionally poor prognosis. Our work provides a comprehensive map of SCLC tumor and TME ecosystems, emphasizing their pivotal role in SCLC's adaptable nature, opening possibilities for reprogramming the TME-tumor communications that shape SCLC tumor states.

Abstract Background: Small cell lung cancer (SCLC) is the most aggressive type of lung cancer. Current therapies have limited efficacy in SCLC, and despite a highly mutated genome, SCLC is largely unresponsive to immunotherapy. We sought to leverage the immune surveillance triggered by DNA damage in patients with relapsed SCLC using bintrafusp alfa, a bifunctional fusion protein targeting both PD-L1 and TGF-beta. Hyperprogressive disease (HPD), characterized by the rapid acceleration of tumor growth, has been reported in approximately 13% of patients treated with immune checkpoint inhibitors. However, the mechanistic basis is poorly understood. Methods: This is a safety run-in and phase II clinical trial. Cohort 1 enrolled patients with relapsed SCLC across Arms A (bintrafusp alfa 2400 mg q3 wks), B (bintrafusp alfa 2400 mg on D1 plus topotecan 1 mg/m2/day on D1-5 q3 wks), and C (bintrafusp alfa 1200 mg on D1 q2 wks plus temozolomide 200 mg/m2/day on D1-5 q4 wks). The primary endpoint was objective response rate (ORR). Results: Thirty-seven patients enrolled. Grade 3 treatment-related adverse events (TRAE) included transaminitis (27.0%), anemia (24.4%), lymphopenia (10.8%), and maculopapular rash (5.4%). Grade 4 TRAE included thrombocytopenia (8.1%), lymphopenia (5.4%) and transaminitis (5.4%). One patient experienced grade 5 tumor hemorrhage. By RECIST 1.1, partial responses were observed in 5 (13.5%), stable disease in 11 (29.7%), and progressive disease in 21 (56.8%) patients. Importantly, 13 (35.1%) patients developed HPD (≥ 2x increase in tumor growth rate from pre-trial vs on-trial, time to treatment failure ≤ 2 months, and ≥ 50% increase tumor burden). Changes in circulating cell-free DNA (n=20) tumor fraction using DELFI-TF, a mutation-independent, low-coverage whole genome sequencing approach, correlated with treatment responses. Pre-treatment serum from HPD (n=10 vs. 9 non-HPD) patients showed significantly lower levels of inflammatory cytokines (CXCL10, CCL13, CCL8, CCL19, TRAIL) and Granzyme H, and higher levels of anti-inflammatory cytokine IL10. Consistently, pre-treatment tumor transcriptomes of HPD (n=5 vs. 7 non-HPD) patients revealed suppression of IFN-gamma response, allograft rejection, and inflammatory response pathways. Conclusions: Concomitant TGF-beta and PD-L1 blockade is associated with a high frequency of HPD in SCLC patients. cfDNA could be critical for monitoring HPD. Tumor and blood immune signatures may inform the likelihood of HPD, with immune excluded tumors more likely to develop HPD. NCT Number: NCT03554473 Bintrafusp alfa was provided by EMD Serono (CrossRef Funder ID: 10.13039/100004755) Citation Format: Brett Schroeder, Nobuyuki Takahashi, Howard Yang, Renee Donahue, Zachary Skidmore, Alissa Konicki, Michael Nirula, Max Greenberg, George Chrisafis, Yang Zhang, Yo-Ting Tsai, Linda Sciuto, Samantha Nichols, Melissa Abel, Parth Desai, Rajesh Kumar, Christopher Schultz, Danielle Pinkiert, Chante Graham, Ajit Sharma, Justin Malin, Manan Krishnamurthy, Sophie Zhuang, Maxwell Lee, Lorenzo Rinaldi, Jeffrey Schlom, Lalage Wakefield, Anish Thomas. Hyperprogressive disease following bintrafusp alfa and DNA damaging chemotherapy in relapsed small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT257.

Importance:Patients with relapsed small cell lung cancer (SCLC), a high replication stress tumor, have poor prognoses and few therapeutic options. A phase 2 study showed antitumor activity with the addition of the ataxia telangiectasia and Rad3-related kinase inhibitor berzosertib to topotecan. Objective:To investigate whether the addition of berzosertib to topotecan improves clinical outcomes for patients with relapsed SCLC. Design, Setting, and Participants:Between December 1, 2019, and December 31, 2022, this open-label phase 2 randomized clinical trial recruited 60 patients with SCLC and relapse after 1 or more prior therapies from 16 US cancer centers. Patients previously treated with topotecan were not eligible. Interventions:Eligible patients were randomly assigned to receive topotecan alone (group 1), 1.25 mg/m2 intravenously on days 1 through 5, or with berzosertib (group 2), 210 mg/m2 intravenously on days 2 and 5, in 21-day cycles. Randomization was stratified by tumor sensitivity to first-line platinum-based chemotherapy. Main Outcomes and Measures:The primary end point was progression-free survival (PFS) in the intention-to-treat population. Secondary end points included overall survival (OS) in the overall population and among patients with platinum-sensitive or platinum-resistant tumors. The PFS and OS for each treatment group were estimated using the Kaplan-Meier method. The log-rank test was used to compare PFS and OS between the 2 groups, and Cox proportional hazards models were used to estimate the treatment hazard ratios (HRs) and the corresponding 2-sided 95% CI. Results:Of 60 patients (median [range] age, 59 [34-79] years; 33 [55%] male) included in this study, 20 were randomly assigned to receive topotecan alone and 40 to receive a combination of topotecan with berzosertib. After a median (IQR) follow-up of 21.3 (18.1-28.3) months, there was no difference in PFS between the 2 groups (median, 3.0 [95% CI, 1.2-5.1] months for group 1 vs 3.9 [95% CI, 2.8-4.6] months for group 2; HR, 0.80 [95% CI, 0.46-1.41]; P = .44). Overall survival was significantly longer with the combination therapy (5.4 [95% CI, 3.2-6.8] months vs 8.9 [95% CI, 4.8-11.4] months; HR, 0.53 [95% CI, 0.29-0.96], P = .03). Adverse event profiles were similar between the 2 groups (eg, grade 3 or 4 thrombocytopenia, 11 of 20 [55%] vs 20 of 40 [50%], and any grade nausea, 9 of 20 [45%] vs 14 of 40 [35%]). Conclusions and Relevance:In this randomized clinical trial, treatment with berzosertib plus topotecan did not improve PFS compared with topotecan therapy alone among patients with relapsed SCLC. However, the combination treatment significantly improved OS. Trial Registration:ClinicalTrials.gov Identifier: NCT03896503.

Supplementary Data from PLX038: A Long-Acting Topoisomerase I Inhibitor With Robust Antitumor Activity in ATM-Deficient Tumors and Potent Synergy With PARP Inhibitors