Small Cell Lung Cancer Subtypes Research Articles

Abstract 5308: Analysis of extracellular vesicle surface markers as small cell lung cancer biomarkers

Abstract Small Cell Lung Cancer (SCLC) is an aggressive tumor type, usually metastatic at diagnosis, with a median survival of less than a year. SCLC tumors are heterogeneous and are composed of neuroendocrine (A, A2, N) and non-neuroendocrine (Y) subtypes, with the cooperation by heterogeneous cells being key to drive tumor survival. Unfortunately, the current SCLC treatment approach does not take tumor heterogeneity into consideration. Importantly, there are no consensus markers that can identify SCLC subtypes or early tumors. Extracellular vesicles (EVs), small vesicles known to promote cell to cell communication, have been investigated as non-invasive biomarkers for cancer diagnostics, staging, and treatment monitoring. EVs are attractive candidates for biomarker research due to the selective packaging of RNA, DNA, and proteins that are then secreted in the blood. Therefore, our aim was to identify new biomarkers based on EV surface proteins with potential for early detection and tumor characterization. We used differential ultracentrifugation to isolate EVs from four SCLC subtypes A, A2, N, and Y, which were submitted to label-free mass spectrometry (EV-proteomics). We identified a total of 3011 proteins, with the two most distinct subtypes being A2 and N, with 786 differentially expressed EV-proteins. Meanwhile, the 2 most similar subtypes were N and A, with only 75 differentially expressed EV-proteins. Gene set enrichment analysis of EVs derived from the A2 subtype showed an association with pathways in cancer, chemokine signaling, and neuronal systems. In addition, PANTHER analysis identified enrichment in nicotine pharmacodynamic pathways on these EVs, which corroborate the association of A2 cell lines with drug resistance. EVs derived from the N and A subtypes were enriched in spliceosome, RNA processing, and metabolism, while EVs from Y cell lines were enriched in extracellular matrix organization and metabolic pathways, similar to pathways from cell line RNAseq data. 13 proteins were differentially expressed in all group comparisons, including proteins involved in PI3K and nicotinic acetylcholine receptor signaling pathways. Further comparison of EV-proteomics with top genes from CIBERSORT analysis using cell line RNAseq data showed an overlap of 9 surface proteins, with 3 possible SCLC pan markers. In conclusion, EV-proteins isolated from SCLC cell lines presented a unique signature for each subtype, especially EVs derived from A2 subtype, which has a signature associated with drug resistance. We identified surface proteins on SCLC EVs with potential as SCLC pan and tumor characterization, which will be validated on EVs derived from cell lines and patient plasma by Western blotting and single-EV microflow cytometry. Citation Format: Patricia M. Ozawa, Sarah M. Groves, Vito Quaranta, Alissa M. Weaver. Analysis of extracellular vesicle surface markers as small cell lung cancer biomarkers. [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 5308.

Abstract 2300: Non-invasive liquid biopsy assay for the detection of ASCL1 expression in CTCs of patients with lung cancer

Abstract Background: Lung cancer (LC) is the deadliest cancer worldwide, with small cell LC (SCLC) and neuroendocrine (NE) LC being the most aggressive and lethal forms. The transcription factor, achaete-scute homolog 1 (ASCL1), regulates neuronal and NE cell development, and is a hallmark of the ASCL1(+) SCLC subtype. Further, investigating subtype has become increasingly important as new drug candidates emerge for SCLC that may be efficacious in only some patients. ASCL1 by IHC tissue biopsy is highly specific, detecting patients who have transformed from NSCLC to SCLC and have acquired resistance to EGFR tyrosine kinase inhibitor therapy in NSCLC. Quantification of ASCL1 on circulating tumor cells (CTCs) is a blood-based, non-invasive alternative to tissue biopsy that provides functional protein-level expression data to monitor lung cancer patients across the disease continuum, informing patient treatment. Methods: A 4-color immunofluorescence (IF) assay was developed to detect ASCL1 expression in CTCs from routine blood samples, comprising detection of nucleated cells (DAPI), cytokeratin positive CTCs, CD45(+) white blood cells (WBC), and ASCL1(+) cells. During validation, cancer cell lines (SHP-77 [ASCL(+)] and U2-OS [ASCL1(-)]) were spiked into normal blood at a ratio of 1:10,000 (CLC/WBC) and plated onto coated glass slides. Slides were stained, imaged, and CTC candidates were identified with a proprietary imaging algorithm. The threshold of ASCL1 positivity (316 MFI) included 99% of all ASCL1(-), U2-OS cells. Assay specificity, sensitivity, overall accuracy, and subcellular localization of biomarker expression were assessed. The ability of the assay to quantify ASCL1 expression in CTCs was confirmed using 31 samples from 11 Extensive Stage SCLC patients. Results: The ASCL1 CTC assay achieved an overall accuracy of 0.992 (95% CI 0.081, 0.993), and an analytical specificity and sensitivity of 0.990 and 0.995, respectively. The assay displayed a positive predictive value and a negative predictive value of 0.979 and 0.998, respectively, determined by evaluating ASCL1 expression in SHP-77 (N=4062) and U2-OS (N=8645). ASCL1 expression was detected in the nucleus of all SHP-77 and majority of SCLC patient CTCs. Of the 11 Extensive Stage SCLC patients tested, 9 (82%) showed at least one conventional CTC (0.02 - 9.63 CTCs/mL), and all 9 (100%) had at least 1 ASCL1(+) CTC (0.06 - 5.5 CTCs/mL). Conclusion: This blood based ASCL1 assay is a highly specific and sensitive approach to monitor ASCL1 expression on CTCs isolated from the blood of lung cancer patients. Citation Format: Luisa Fernandez, Andrew Kunihiro, Mark Frilles, Alisa Tubbs, Jason Christiansen, David Bourdon. Non-invasive liquid biopsy assay for the detection of ASCL1 expression in CTCs of patients with 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 2300.

Abstract 6247: Lurbinectedin shows potent activity in all four molecular subtypes of small cell lung cancer (SCLC) and POU2F3 and SLFN11 are biomarkers for a better response

Abstract Background - SCLC is the most aggressive lung cancer type and with the worst prognosis. There are four molecular subtypes based on the high expression of distinct transcription factors and with different therapeutic vulnerabilities. However, all share transcriptional addiction as pathogenic mechanism. Lurbinectedin is a novel oncogenic transcription inhibitor, approved in the United States and other countries for the treatment of adult patients with metastatic SCLC with disease progression on or after platinum-based chemotherapy. The aim of this study was to analyze the activity of lurbinectedin in the different SCLC molecular subtypes and to investigate new biomarkers of response. Methods and Results - We have characterized a panel of 20 SCLC human cell lines based on the expression of ASCL1, NEUROD1, YAP1 and POU2F3, where lurbinectedin yielded a mean IC50 value of 6.52 nM, which is considerable greater than the activity exerted by topotecan, irinotecan, carboplatin, etoposide, olaparib, alisertib and navitoclax (IC50 100 µM-100 nM). Lurbinectedin potency is high in the four molecular subtypes, with IC50 values of 4.1, 14.9, 7.2, and 0.2 nM for SCLC-A, -N, -I and -P, respectively. However, high expression of POU2F3 correlated with better responses. In fact, mean IC50 was 0.28 nM for POU2F3high cells versus 12.1 nM for all POU2F3low cells (p=0.0443). Additionally, basal SLFN11 levels were evaluated, observing that SLFN11high cells responded better to lurbinectedin, with IC50 values of 1.1 nM versus 11.8 nM for SLFN11low (p=0.05). Likewise, lurbinectedin treatment induced greater antitumor activity in SLFN11high tumor-bearing mice (H526: T/C, 18% on day 11) than in SLFN11low tumor-bearing mice (H82: T/C, 65% on day 7) after intravenous treatment at 0.18 mg/kg (on days 0, 7 and 14). Finally, SLFN11 expression was evaluated by IHC in FFPE tumor samples from SCLC patients participating in a multicenter phase II clinical trial in advanced solid tumors (NCT02454972), which allowed lurbinectedin accelerated approval in this indication by FDA. Patients with higher expression of SLFN11 had a slightly better overall survival (OS at 6 months: 68.4% (<15%, N=20) vs. 98.7% (≥15%, N=20) p=0.0261)), especially in the refractory/resistant subgroup (OS at 6 months: 33.3% (<15%, N=9) vs. 100.0% (≥15%, N=6) p<0.0001). Conclusions - Lurbinectedin is highly effective in all molecular subtypes of SCLC in vitro and in vivo, with IC50 values at least two logs more potent than for other antitumoral agents and its activity is even greater in tumors with high POU2F3 expression and/or high SLFN11 expression. Citation Format: Marta Martínez Diez, Gema Santamaría Nuñez, María José Guillén, Daniel Rueda, Eva Maria Garrido-Martin, Pablo Avilés, Carmen Cuevas. Lurbinectedin shows potent activity in all four molecular subtypes of small cell lung cancer (SCLC) and POU2F3 and SLFN11 are biomarkers for a better response. [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 6247.

Abstract 931: Comprehensive analysis of 3,600 small cell lung cancer cases reveals rare genetic subtypes and multiple mechanisms of histological transformation

Abstract Background: Small cell lung cancer (SCLC) is an aggressive neuroendocrine carcinoma with limited treatment options and extremely poor survival outcomes. A major barrier in the field has been the relative paucity of human tumors studied and lack of access to longitudinal samples to understand tumor evolution. Methods: Here we provide a comprehensive analysis of 3,600 patients with SCLC who underwent targeted genomic profiling of at least 324 cancer-related genes as part of routine clinical care, including 678 cases with additional clinical and treatment information obtained from a US-based de-identified SCLC clinico-genomic database that originated from approximately 280 US cancer clinics. This large cohort allowed us to examine for new genetic subtypes, ancestry-associated genomic alterations, biopsy site-specific patterns, survival trends and histological transformation of SCLC from non-small cell lung cancer (NSCLC). Results: Consistent with prior studies, SCLCs were predominantly TP53/RB1 altered. Yet, 5.5% of the cases in our cohort were TP53/RB1 wild-type tumors. These tumors often lacked a tobacco mutational signature, exhibited alternate mechanisms of p53/Rb pathway inactivation (e.g., CDKN2A, CCND1, MDM2), and had a high fraction of human papillomavirus-positive cases (12.7%). Another rare subtype of SCLCs included STK11-altered tumors (1.7%), which were observed more frequently in patients of African ancestry, and were associated with a decreased overall survival (OS) compared with the STK11 wild-type cohort. In our cohort, gene amplifications on 4q12 (KDR, KIT, PDGFRA) were associated with increased OS while CCNE1 amplification was associated with decreased OS. Interestingly, alterations in PTEN were more common in brain metastases compared to lung biopsies and liver metastases, suggesting its potentially unique role in brain metastases of SCLCs. Profiling of over 100 putative transformed SCLCs demonstrated that lineage plasticity may occur at variable lengths of time from the original NSCLC diagnosis and include multiple distinct molecular cohorts of NSCLC, beyond EGFR-mutant NSCLC (e.g., kinase fusion+ tumors: RET, ALK, ROS1, NTRK1). Conclusion: Our work underscores the existence of genetic subtypes in SCLC, including rare subtypes with potential clinical utility. Findings from this study provide an improved understanding of genetic subtypes in SCLC and better inform mechanisms of transformation to SCLC from NSCLC, that may further guide the development of personalized therapies for subsets of patients with this fatal tumor. Citation Format: Smruthy Sivakumar, Jay A. Moore, Meagan Montesion, Douglas I. Lin, Zoe Fleischmann, Ericka M. Ebot, Justin Newberg, Jennifer M. Mills, Priti S. Hegde, Garrett M. Frampton, Julien Sage, Christine M. Lovly. Comprehensive analysis of 3,600 small cell lung cancer cases reveals rare genetic subtypes and multiple mechanisms of histological transformation [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 931.

Abstract 4546: Comprehensive analysis using transcriptional factor based molecular subtypes and correlation to clinical outcomes in small-cell lung cancer

Abstract Background: Several studies have reported the predictive and prognostic value of novel transcriptional factor-based molecular subtypes in small-cell lung cancer (SCLC). We conducted an in-depth analysis using multi-omics data to elucidate the underlying characteristics that lead to differences in clinical outcomes between subtypes. Patients and Methods: Immunohistochemistry (IHC, n=252), target exome sequencing (n=422), and whole transcriptome sequencing (WTS, n=189) data generated from 427 patients with SCLC patients were comprehensively analyzed. The differences in the mutation profile, gene expression profile were analyzed according to the IHC-based molecular subtype. Clinical implication was evaluated based on treatment outcomes of individual patients. Results: IHC based molecular subtyping revealed a high incidence of ASCL1 subtype (SCLC-A, 56.3%) followed by ASCL1/NEUROD1 co-expressed (SCLC-trans, 17.9%), NEUROD1 (SCLC-N, 12.3%), POU2F3 (SCLC-P, 9.1%), triple-negative (SCLC-TN, 4.4%) subtypes showing high concordance with WTS-based subtyping. We delineated the SCLC-trans subtype resembling SCLC-A rather than SCLC-N in terms of both gene expression profiles and clinical outcomes. SCLC-TN type was defined as non-significant expression of A, N, P. Favorable overall survival (OS) was observed in SCLC-A compared to SCLC-N (adjusted HR 2.4, 95% CI 1.5-3.9, p < 0.001) and SCLC-P (adjusted HR 1.7, 95% CI 0.9-2.9, p = 0.087). SCLC-TN showed a similar OS with SCLC-A (adjusted HR 1.2, 95% CI 0.6 -2.7, p = 0598). The clinical outcome based on inflamed phenotype, clustered by effector cell gene expression profile which are only found in 5% of SCLC-N but 60% of SCLC-P, was more likely to benefit from first-line immunotherapy treatment than non-inflamed phenotype (p = 0.013). Inflammed phenotype demonstrated longer progression-free survival to the first line immunotherapy compared to the non-inflammed phenotype. (10.5 vs 4.3, p = 0.013) Conclusions: This study provides fundamental data, including the incidence and basic demographics of molecular subtypes of SCLC using both IHC and WTS from a comparably large cohort including potential differences in the distribution of subtypes based on ethnicity. Additionally, our results reveal differences in the underlying biological pathway activities and immunogenicity based on molecular subtype, possibly related to the difference in clinical outcomes, including immunotherapy response. Citation Format: Sehhoon Park, Tae Hee Hong, Soohyun Hwang, Hyun-Ae Jung, Jong-Mu Sun, Jin Seok Ahn, Myung-Ju Ahn, Jong Ho Cho, Yong Soo Choi, Jhingook Kim, Young Mog Shim, Hong Kwan Kim, Yoon-La Choi, Se-hoon Lee, Keunchil Park. Comprehensive analysis using transcriptional factor based molecular subtypes and correlation to clinical outcomes in small-cell 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 4546.

Quantitative proteomic analysis of bronchoalveolar lavage fluids from patients with small cell lung cancers.

Small cell lung cancer (SCLC) is one of the malignant cancers with aggressive progression and poor prognosis. Bronchoalveolar lavage fluid (BALF) has been arising recently as a potential source of biomarkers for lung cancers. In this study, we performed quantitative BALF proteomic analysis to identify potential biomarkers for SCLC. BALF were collected from tumor-bearing lungs and non-tumor lungs of five SCLC patients. Then, BALF proteomes were prepared for a TMT-based quantitative mass spectrometry analysis. Differentially expressed proteins (DEP) were identified when considering individual variation. Potential SCLC biomarker candidates were validated by immunohistochemistry (IHC). A public database of multiple SCLC cell lines was used to evaluate the correlation of these markers with SCLC subtypes and chemo-drug responses. We identified 460 BALF proteins in SCLC patients and observed considerable individual variation among the patients. Immunohistochemical analysis and bioinformatics resulted in the identification of CNDP2 and RNPEP as potential subtype markers for ASCL1 and NEUROD1, respectively. In addition, CNDP2 was found to be positively correlated with responses to etoposide, carboplatin, and irinotecan. BALF is an emerging source of biomarkers, making it useful for the diagnosis and prognosis of lung cancers. We characterized the proteomes of paired BALF samples collected from tumor-bearing and non-tumor lungs of SCLC patients. Several proteins were found elevated in tumor-bearing BALF, and especially CNDP2 and RNPEP appeared to be potential indicators for ASLC1-high and NEUROD1-high subtypes of SCLC, respectively. The positive correlation of CNDP2 with chemo-drug responses would help to make decisions for treatment of SCLC patients. These putative biomarkers could be comprehensively investigated for a clinical use towards precision medicine.

Involvement of Epithelial-Mesenchymal Transition Genes in Small Cell Lung Cancer Phenotypic Plasticity.

Small cell lung cancer (SCLC) is an aggressive cancer recalcitrant to treatment, arising predominantly from epithelial pulmonary neuroendocrine (NE) cells. Intratumor heterogeneity plays critical roles in SCLC disease progression, metastasis, and treatment resistance. At least five transcriptional SCLC NE and non-NE cell subtypes were recently defined by gene expression signatures. Transition from NE to non-NE cell states and cooperation between subtypes within a tumor likely contribute to SCLC progression by mechanisms of adaptation to perturbations. Therefore, gene regulatory programs distinguishing SCLC subtypes or promoting transitions are of great interest. Here, we systematically analyze the relationship between SCLC NE/non-NE transition and epithelial to mesenchymal transition (EMT)-a well-studied cellular process contributing to cancer invasiveness and resistance-using multiple transcriptome datasets from SCLC mouse tumor models, human cancer cell lines, and tumor samples. The NE SCLC-A2 subtype maps to the epithelial state. In contrast, SCLC-A and SCLC-N (NE) map to a partial mesenchymal state (M1) that is distinct from the non-NE, partial mesenchymal state (M2). The correspondence between SCLC subtypes and the EMT program paves the way for further work to understand gene regulatory mechanisms of SCLC tumor plasticity with applicability to other cancer types.

Identification of lineage-specific transcription factor defined subtypes in small cell/neuroendocrine bladder cancer.

568 Background: Small cell/neuroendocrine bladder cancer (SCBC) is a rare and clinically aggressive disease. However, its molecular characteristics are poorly studied; hence, biology-based therapeutic options are not available. Strong pan-cancer similarity in gene expression signature of neuroendocrine (NE) tumors including SCBC and small cell lung cancer (SCLC) has been reported. Recent genomics studies discovered that SCLC can be grouped into lineage- specific transcription factor (TF)-defined subtypes. Therefore, we explored whether TF-defined molecular subtypes could also be observed in SCBC with the goal of developing subtype-specific therapeutic strategies. Methods: Whole transcriptome RNAseq on a cohort of n = 44 pure SCBCs collected at Johns Hopkins (n=20) and Tenon Hospital (Paris) (n=24) and DNA panel sequencing on subset of the tumors (n=24) were performed. Two expert genitourinary pathologists selected the cases for the analyses based on the histopathological characteristics of the tumor tissue. Results: SCBC tumors displayed overexpression of canonical NE markers and high mutation rates in TERT, TP53 and RB1 with 92%, 75% and 38%, respectively, confirming the concordance between genomic features and histopathologic characteristics.Lineage-specific TFs (ASCL1, NEUROD1, and POU2F3) defined three molecular subtypes of SCBC that resembled the previously reported SCLC subtypes. The three subtypes expressed variable levels of NE biomarkers and were heterogeneous with respect to distinct downstream therapeutic targets. The ASCL1 and NEUROD1 subtypes expressed the highest levels of NE lineage-specific gene expression and were associated with expression of known regulators of the neuroendocrine phenotype including FOXA2 and HES6, respectively. The ASCL1 subtype was also enriched with DLLs and other genes that control oncogenic Notch signaling, whereas tumors enriched with POU2F3, a master regulator of the NE-low SCBC subtype, expressed TRPM5, SOX9 and CHAT. Finally, an inverse association between NE lineage-specific gene expression and immune signatures associated with response to immunotherapy was observed. Conclusions: Three lineage-specific TFs (ASCL1, NEUROD1, and POU2F3) defined discrete molecular subtypes in SCBC. Each subtype was characterized by distinct therapeutic targets and gene expression signatures associated with response to immunotherapy. Future studies will be required to test the clinical relevance of these observations.

Selected Articles from This Issue

Highlights| February 01 2023 Selected Articles from This Issue Author & Article Information Online Issn: 1557-3125 Print Issn: 1541-7786 ©2023 American Association for Cancer Research2023American Association for Cancer Research Mol Cancer Res (2023) 21 (2): 89. https://doi.org/10.1158/1541-7786.MCR-21-2-HI Related Content A commentary has been published: BET Inhibitors Target the SCLC-N Subtype of Small-Cell Lung Cancer by Blocking NEUROD1 Transactivation A commentary has been published: Downregulation of iNOS/NO Promotes Epithelial–Mesenchymal Transition and Metastasis in Colorectal Cancer A commentary has been published: TWEAK–Fn14–RelB Signaling Cascade Promotes Stem Cell–like Features that Contribute to Post-Chemotherapy Ovarian Cancer Relapse View more A commentary has been published: XIAP and PHB1 Regulate Anoikis through Competitive Binding to TRAF6 View less Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Tools Icon Tools Get Permissions Cite Icon Cite Search Site Article Versions Icon Versions Version of Record February 1 2023 Citation Selected Articles from This Issue. Mol Cancer Res 1 February 2023; 21 (2): 89. https://doi.org/10.1158/1541-7786.MCR-21-2-HI Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest Search Advanced Search NEUROD1 is the master transcriptional regulator defining the small-cell lung cancer (SCLC) SCLC-N subtype, but NEUROD1 has proven difficult to therapeutically target. Targeting NEUROD1 transcriptional coactivators may be an effective therapeutic approach, but such coactivators have yet to be defined. To identify NEUROD1 transcriptional coactivators, Chen and colleagues elucidated NEUROD1 genomic localization regions using ChIP-Seq and searched for colocalizing coactivator candidates using a ChIP-Seq database. The authors discovered that BRD4, a member of the bromodomain and extraterminal domain (BET) protein family, significantly colocalizes to NEUROD1-inhabited genomic regions. Co-immunoprecipitation experiments demonstrated that BRD4 and NEUROD1 interact, and treatment with BET inhibitors (BETi) decreases NEUROD1 target gene expression, suggesting BRD4 is a NEUROD1 transcriptional coactivator. Accordingly, the authors found that SCLC-N cells are more sensitive to BETi than are other SCLC subtype cells, and that BETi significantly slow SCLC-N patient-derived xenograft tumor growth in mouse models. Using a siRNA screen, the authors... You do not currently have access to this content.

Slit2/Robo1 signaling inhibits small-cell lung cancer by targeting β-catenin signaling in tumor cells and macrophages.

Small-cell lung cancer (SCLC) is an aggressive neuroendocrine subtype of lung cancer with poor patient prognosis. However, the mechanisms that regulate SCLC progression and metastasis remain undefined. Here, we show that the expression of the slit guidance ligand 2 (SLIT2) tumor suppressor gene is reduced in SCLC tumors relative to adjacent normal tissue. In addition, the expression of the SLIT2 receptor, roundabout guidance receptor 1 (ROBO1), is upregulated. We find a positive association between SLIT2 expression and the Yes1 associated transcriptional regulator (YAP1)-expressing SCLC subtype (SCLC-Y), which shows a better prognosis. Using genetically engineered SCLC cells, adenovirus gene therapy, and preclinical xenograft models, we show that SLIT2 overexpression or the deletion of ROBO1 restricts tumor growth in vitro and in vivo. Mechanistic studies revealed significant inhibition of myeloid-derived suppressor cells (MDSCs) and M2-like tumor-associated macrophages (TAMs) in the SCLC tumors. In addition, SLIT2 enhances M1-like and phagocytic macrophages. Molecular analysis showed that ROBO1 knockout or SLIT2 overexpression suppresses the transforming growth factor beta 1 (TGF-β1)/β-catenin signaling pathway in both tumor cells and macrophages. Overall, we find that SLIT2 and ROBO1 have contrasting effects on SCLC tumors. SLIT2 suppresses, whereas ROBO1 promotes, SCLC growth by regulating the Tgf-β1/glycogen synthase kinase-3 beta (GSK3)/β-catenin signaling pathway in tumor cells and TAMs. These studies indicate that SLIT2 could be used as a novel therapeutic agent against aggressive SCLC.

Molecular features and evolutionary trajectory of ASCL1+ and NEUROD1+ SCLC cells.

Small cell lung cancer (SCLC) is the most aggressive subtype of lung cancer without recognised morphologic or genetic heterogeneity. Based on the expression of four transcription factors, ASCL1, NEUROD1, POU2F3, and YAP1, SCLCs are classified into four subtypes. However, biological functions of these different subtypes are largely uncharacterised. We studied intratumoural heterogeneity of resected human primary SCLC tissues using single-cell RNA-Seq. In addition, we undertook a series of in vitro and in vivo functional studies to reveal the distinct features of SCLC subtypes. We identify the coexistence of ASCL1+ and NEUROD1+ SCLC cells within the same human primary SCLC tissue. Compared with ASCL1+ SCLC cells, NEUROD1+ SCLC cells show reduced epithelial features and lack EPCAM expression. Thus, EPCAM can be considered as a cell surface marker to distinguish ASCL1+ SCLC cells from NEUROD1+ SCLC cells. We further demonstrate that NEUROD1+ SCLC cells exhibit higher metastatic capability than ASCL1+ SCLC cells and can be derived from ASCL1+ SCLC cells. Our studies unveil the biology and evolutionary trajectory of ASCL1+ and NEUROD1+ SCLC cells, shedding light on SCLC tumourigenesis and progression.

Clinical and prognostic implications of CD47 and PD-L1 expression in surgically resected small-cell lung cancer

Pharmacological inhibition of the immune-checkpoint molecule CD47 has shown promising results in preclinical small-cell lung cancer (SCLC) models, whereas anti-programmed death-ligand 1 (PD-L1) inhibitors have been recently implemented in the standard of care of advanced-stage SCLC patients. Nevertheless, the expression pattern, clinical relevance and prognostic implication of both CD47 and PD-L1 are rather controversial in surgically treated SCLC patients. In total, 104 Caucasian SCLC patients from two Central European thoracic centers were included in this study. CD47 and PD-L1 expression as well as the expression of the four major SCLC molecular subtype markers (ASCL1, NEUROD1, YAP1 and POU2F3) were measured by immunohistochemistry. Expression levels were independently evaluated and statistically correlated with clinicopathological data and survival. Positive CD47 and PD-L1 expressions were seen in 84.6% and 9.6% of the samples, respectively. Meanwhile, the tumor-associated stroma was positive for PD-L1 in 59.6% of the cases. Stromal PD-L1 expression correlated with longer overall survival (OS) (versus PD-L1-negative stroma; median OS was 42 versus 14 months, respectively, P= 0.003) and was confirmed as an independent predictor of favorable outcome upon multivariate analysis (hazard ratio 0.530, 95% confidence interval 0.298-0.943, P= 0.031). Notably, neither CD47 nor PD-L1 presence was related to a distinct molecular SCLC subtype. CD47 shows a remarkably high expression while tumoral PD-L1 expression is generally low in surgically treated SCLC. Importantly, stromal PD-L1 expression may indicate a favorable clinical outcome and serve as a novel prognostic factor in these patients. Additional studies are warranted to further investigate the clinical impact of CD47 and PD-L1 expression in SCLC.

BET Inhibitors Target the SCLC-N Subtype of Small-Cell Lung Cancer by Blocking NEUROD1 Transactivation.

Our findings suggest that targeting transcriptional coactivators could be a novel approach to blocking the master transcription factors in SCLC for therapeutic purposes.

Molecular subtyping of small-cell lung cancer based on mutational signatures with different genomic features and therapeutic strategies.

Small-cell lung cancer (SCLC) is an exceptionally lethal malignancy characterized by extremely high alteration rates and tumor heterogeneity, which limits therapeutic options. In contrast to non-small-cell lung cancer that develops rapidly with precision oncology, SCLC still remains outside the realm of precision medicine. No recurrent and actionable mutations have been detected. Additionally, a paucity of substantive tumor specimens has made it even more difficult to classify SCLC subtypes based on genetic background. We therefore carried out whole-exome sequencing (WES) on the largest available Chinese SCLC cohort. For the first time, we partitioned SCLC patients into three clusters with different genomic alteration profiles and clinical features based on their mutational signatures. We showed that these clusters presented differences in intratumor heterogeneity and genome instability. Moreover, a wide existence of mutually exclusive gene alterations, typically within similar biological functions, was detected and suggested a high SCLC intertumoral heterogeneity. Particularly, Cluster 1 presented the greatest potential to benefit from immunotherapy, and Cluster 3 constituted recalcitrant SCLC, warranting biomarker-directed drug development and targeted therapies in clinical trials. Our study would provide an in-depth insight into the genome characteristics of the Chinese SCLC cohort, defining distinct molecular subtypes as well as subtype-specific therapies and biomarkers. We propose tailoring differentiated therapies for distinct molecular subgroups, centering on a personalized precision chemotherapy strategy combined with immunization or targeted therapy for patients with SCLC.

Archetype tasks link intratumoral heterogeneity to plasticity and cancer hallmarks in small cell lung cancer.

Small cell lung cancer (SCLC) tumors comprise heterogeneous mixtures of cell states, categorized into neuroendocrine (NE) and non-neuroendocrine (non-NE) transcriptional subtypes. NE to non-NE state transitions, fueled by plasticity, likely underlie adaptability to treatment and dismal survival rates. Here, we apply an archetypal analysis to model plasticity by recasting SCLC phenotypic heterogeneity through multi-task evolutionary theory. Cell line and tumor transcriptomics data fit well in a five-dimensional convex polytope whose vertices optimize tasks reminiscent of pulmonary NE cells, the SCLC normal counterparts. These tasks, supported by knowledge and experimental data, include proliferation, slithering, metabolism, secretion, and injury repair, reflecting cancer hallmarks. SCLC subtypes, either at the population or single-cell level, can be positioned in archetypal space by bulk or single-cell transcriptomics, respectively, and characterized as task specialists or multi-task generalists by the distance from archetype vertex signatures. In the archetype space, modeling single-cell plasticity as a Markovian process along an underlying state manifold indicates that task trade-offs, in response to microenvironmental perturbations or treatment, may drive cell plasticity. Stifling phenotypic transitions and plasticity may provide new targets for much-needed translational advances in SCLC. A record of this paper's Transparent Peer Review process is included in the supplemental information.

889MO Comparative expression of driver transcription factors in extra-pulmonary small cell carcinoma

Extra-pulmonary small cell carcinomas (EPSCC) are aggressive neuroendocrine tumors that are clinicopathologically distinct from small cell lung cancer (SCLC). Recent data suggest that transcriptionally-defined SCLC subtypes exhibit different underlying biology and therapeutic vulnerabilities. With limited data available on the EPSCC transcriptomic landscape, we analyzed gene expression profiles and its correlation with clinical outcomes across EPSCC anatomic sites. DNA (592 genes/whole-exome) and RNA (whole transcriptome) sequencing were performed for 1070 small cell carcinoma (SCC) patient (pt) samples that underwent molecular profiling at Caris Life Sciences (Phoenix, AZ). Samples were stratified into 5 subtypes based on the relative expression of key transcription factors (TFs): ASCL1, NEUROD1, YAP1, POU2F3, and Mixed. Real-world survival information was available for 111 pts treated with etoposide+platinum (EP) therapy. Overall survival (OS) was obtained from insurance claims & Kaplan-Meier estimates. Statistical significance is determined by Chi-square & Wilcoxon rank sum tests. EPSCC comprised 28.8% (n=308) of SCCs evaluated; most common primary sites included gynecologic (GYN, 21.8%, n=67), prostate (16.6%, n=51), bladder (15.9%, n=49) and colorectal (10.1%, n=31). Each primary site had a unique distribution of molecular subtypes relative to SCLC. ASCL1 was less frequent in GYN and bladder SCC (13.4% and 16.3%) compared to SCLC (35.7%), with bladder SCC enriched in NEUROD1 and POU2F3 (30.6% and 22.4% vs SCLC 17.5% and 6.4%, P<0.05). YAP1 was most common in GYN SCC (35.8% vs SCLC 20.7%, P<0.05). For both SCLC and EPSCC, low YAP1 was associated with improved OS (SCLC HR 2.1, P=0.05; EPSCC HR 4.3, P=0.02), and high NEUROD1 trended towards improved OS (SCLC HR 0.6, P=0.12; EPSCC HR 0.4, P=0.10) from start of EP therapy. Our analysis revealed differential expression of key lineage-defining TFs in EPSCCs from various anatomic sites that looked distinct from SCLC. EPSCC and SCLC OS was similarly associated with TF expression, suggesting the underlying biology of SCLC and EPSCC subtypes might predict comparable therapeutic vulnerabilities.

EP14.02-004 Barasertib Inhibits the Growth of SCLC Cell Lines and Transformed SCLC Patient Derived Model In Vitro and In Viv

Approximately 5% to 10% of EGFR (epidermal growth factor receptor) -mutant non-small cell lung cancers (NSCLCs) develops an acquired resistance to EGFR tyrosine kinase inhibitors with transformation into small-cell lung cancer (SCLC). After the transformation, clinical behavior mimics classic SCLC on many aspects, with frequent but transient responses to platinum-etoposide (EP) or taxanes, modest progression free survival and rare response to immune checkpoint inhibitors. Recently, four SCLC subtypes, defined largely by differential expression of transcription factors ASCL1, NEUROD1, and POU2F3 or low expression of all three transcription factor signatures, were suggested (Gay CM et al, Cancer Cell 2021) (SCLC-A, N, P and I, respectively).

In-depth proteomic analysis reveals unique subtype-specific signatures in human small-cell lung cancer.

BackgroundSmall‐cell lung cancer (SCLC) molecular subtypes have been primarily characterized based on the expression pattern of the following key transcription regulators: ASCL1 (SCLC‐A), NEUROD1 (SCLC‐N), POU2F3 (SCLC‐P) and YAP1 (SCLC‐Y). Here, we investigated the proteomic landscape of these molecular subsets with the aim to identify novel subtype‐specific proteins of diagnostic and therapeutic relevance.MethodsPellets and cell media of 26 human SCLC cell lines were subjected to label‐free shotgun proteomics for large‐scale protein identification and quantitation, followed by in‐depth bioinformatic analyses. Proteomic data were correlated with the cell lines’ phenotypic characteristics and with public transcriptomic data of SCLC cell lines and tissues.ResultsOur quantitative proteomic data highlighted that four molecular subtypes are clearly distinguishable at the protein level. The cell lines exhibited diverse neuroendocrine and epithelial–mesenchymal characteristics that varied by subtype. A total of 367 proteins were identified in the cell pellet and 34 in the culture media that showed significant up‐ or downregulation in one subtype, including known druggable proteins and potential blood‐based markers. Pathway enrichment analysis and parallel investigation of transcriptomics from SCLC cell lines outlined unique signatures for each subtype, such as upregulated oxidative phosphorylation in SCLC‐A, DNA replication in SCLC‐N, neurotrophin signalling in SCLC‐P and epithelial–mesenchymal transition in SCLC‐Y. Importantly, we identified the YAP1‐driven subtype as the most distinct SCLC subgroup. Using sparse partial least squares discriminant analysis, we identified proteins that clearly distinguish four SCLC subtypes based on their expression pattern, including potential diagnostic markers for SCLC‐Y (e.g. GPX8, PKD2 and UFO).ConclusionsWe report for the first time, the protein expression differences among SCLC subtypes. By shedding light on potential subtype‐specific therapeutic vulnerabilities and diagnostic biomarkers, our results may contribute to a better understanding of SCLC biology and the development of novel therapies.

MA01.03 Exploiting DNA Methylation for Classification of SCLC Subtypes from Liquid Biopsies Using a Robust Machine Learning Approach

Small cell lung cancer (SCLC) is a highly aggressive cancer with limited treatment options and a generally poor prognosis that has not appreciably changed despite recently approved therapies. Importantly, therapeutic options for SCLC patients have been focused on unselected populations as SCLC was thought to be a relatively homogenous malignancy in the past. Recently, our group and others identified four major distinct subgroups of SCLC (Gay CM et al. Cancer Cell 2021). Three of the four subtypes are defined by the predominant expression of a specific transcription factor, ASCL1 (SCLC-A), NEUROD1 (SCLC-N) and POU2F3 (SCLC-P) while the fourth subtype is defined by an inflamed phenotype (SCLC-I).

ASCL1 regulates super-enhancer-associated miRNAs to define molecular subtypes of small cell lung cancer.

Small cell lung cancer (SCLC) is a highly aggressive neuroendocrine tumor with dismal prognosis. Recently, molecular subtypes of SCLC have been defined by the expression status of ASCL1, NEUROD1, YAP1, and POU2F3 transcription regulators. ASCL1 is essential for neuroendocrine differentiation and is expressed in the majority of SCLC. Although previous studies investigated ASCL1 target genes in SCLC cells, ASCL1-mediated regulation of miRNAs and its relationship to molecular subtypes remain poorly explored. Here, we performed genome-wide profiling of chromatin modifications (H3K27me3, H3K4me3, and H3K27ac) by CUT&Tag assay and ASCL1 knockdown followed by RNA sequencing and miRNA array analyses in SCLC cells. ASCL1 could preferentially regulate genes associated with super-enhancers (SEs) defined by enrichment of H3K27ac marking. Moreover, ASCL1 positively regulated several SE-associated miRNAs, such as miR-7, miR-375, miR-200b-3p, and miR-429, leading to repression of their targets, whereas ASCL1 suppressed miR-455-3p, an abundant miRNA in other molecular subtypes. We further elucidated unique patterns of SE-associated miRNAs in different SCLC molecular subtypes, highlighting subtype-specific miRNA networks with functional relevance. Notably, we found apparent de-repression of common target genes of different miRNAs following ASCL1 knockdown, suggesting combinatorial action of multiple miRNAs underlying molecular heterogeneity of SCLC (e.g., co-targeting of YAP1 by miR-9 and miR-375). Our comprehensive analyses provide novel insights into SCLC pathogenesis and a clue to understanding subtype-dependent phenotypic differences.