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Background: Small cell lung cancer (SCLC) is a highly lethal malignancy with limited treatment options. Most SCLC are “immune-cold” and respond poorly to immunotherapy. Targeting SCLC cell surfaceome with cellular therapies have shown promising efficacy and tolerability in DLL3-targeting CAR T (AMG119) and Bispecific T-cell engager (tarlatamab). Recent work has defined subtypes of SCLC based on master transcription factors ASCL1 (SCLC-A), NEUROD1 (SCLC-N), POU2F3 (SCLC-P) and an immune cell-rich “inflamed” subtype(SCLC-I). Notably, SCLC-A and SCLC-N express neuroendocrine (NE) features while SCLC-P and SCLC-I are non-neuroendocrine (non-NE). We hypothesize that SCLC may have distinct surfaceome expressions based on their subtypes, which can be successfully targeted with cellular therapies such as CAR T. Methods: Transcriptional profiling of patient tumors identified several putative cell surfaceome targets in SCLC. We confirmed their expression level by Western blot and flow cytometry in SCLC cell lines stratified by subtypes. Top targets were selected based on abundant expression in SCLC and restricted normal tissue expression. We then constructed third-generation CAR T-cells against the top targets and tested their efficacy in cancer cell/CAR T co-culture models and in vivo. Results: We validated cell surface expression of top targets DLL3, CD56 and GD2; and generated CAR Ts against these targets to test cytotoxicity across a large panel of SCLC cell lines. Specifically, DLL3 is present predominantly in SCLC-A and SCLC-N (NE) subtypes. CAR T against DLL3 showed robust cytotoxicity in vitro against DLL3-expressing cell lines (5 out of 6 SCLC-A/N cell lines, versus 1 out of 6 SCLC-P/I cell lines; median cytotoxicity 94% vs. 43%, p=0.0082, Mann-Whitney U test), as well as promising in vivo activity. We also confirmed CD56 (NCAM1) was broadly expressed on the cell surface in SCLC A/P/N subtypes, and 8 out of 9 cell lines with high CD56 expression demonstrated over 50% cytotoxicity in co-culture models. GD2 was found to be enriched specifically in SCLC-N subtype of SCLC. As expected, GD2-targeting CAR T had higher cytotoxicity in SCLC-N subtypes of SCLC compared to other subtypes (median cytotoxicity 91% vs 22%, p=0.047). Moreover, given that novel therapeutics are often tested in the relapsed setting, we tested target expressions in SCLC cell lines after treatment with cisplatin and etoposide (standard frontline chemotherapy for SCLC) to see if target levels change. We found that surface target expressions increased instead of decreased in most cell lines, suggesting that they may be suitable targets in the relapsed setting also. Conclusion: This study demonstrated efficacy of CAR Ts targeting several cell surfaceome targets enriched in certain subtypes of SCLC. The data supports further investigation of a subtype-specific personalized treatment of SCLC using CAR T therapies against surfaceome targets. Citation Format: Bingnan Zhang, Yan Yang, C.Allison Stewart, Kavya Ramkumar, Runsheng Wang, Robert Cardnell, Lixia Diao, Qi Wang, Jing Wang, Lauren Byers, Carl Gay, John Heymach. Subtype-specific targeting of cell surfaceome with CAR T therapies 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 3196.

Background: High-grade lung neuroendocrine tumors are a heterogeneous group of cancers with aggressive common features and a lack of effective therapeutic options. Recently, efforts have been made to identify new targets and drugs to improve clinical outcomes in these patients. Because of its high expression in neuroendocrine tumors compared to normal tissue, delta-like ligand 3 (DLL3) has emerged as a new therapeutic target in this setting.1,2 Targeting DLL3 with the half-life extended bispecific T cell engager (HLE BiTE®) immune therapy, tarlatamab, in a phase 1 clinical trial has demonstrated promising anti-tumor activity in small cell lung cancer (SCLC) patients.3 In order to more precisely guide patient selection and clinical trial design, a better understanding and quantification of DLL3 expression is required. Thus, we investigated DLL3 immunohistochemical expression across SCLC, large cell neuroendocrine carcinoma (LCNEC), and neuroendocrine carcinoma with combined histology (CNEC), and its associations with clinicopathological characteristics. Design: Formalin-fixed and paraffin-embedded surgically resected lung neuroendocrine carcinomas (SCLC, n=17; LCNEC, n=20; CNEC, n=8) were selected based on tissue availability at MD Anderson Cancer Center and stained with a commercially available immunohistochemistry assay for DLL3 (clone SP347, Ventana). Total percentage and intensity of DLL3 expression of neuroendocrine carcinoma cells were scored and the results were presented as H-Score (0-300) and percentage (%) of DLL3 positive tumor cells. We correlated DLL3 expression with clinicopathological characteristics. Results: DLL3 expression was observed in 37/45 (82%) of all cases (SCLC: %, median=65%, 0-100%; H-score, median=95, 0-175; LCNEC: %, median=65%, 0-100%; H-score, median=100, 0-180; CNEC: %, median=55%, 0-95%; H-score, median=90, 0-175). In patients with LCNEC, DLL3 % was higher in male patients (p=0.0500) and former smokers (p=0.0298) compared to females and current smokers, respectively. No other clinicopathological associations were found with age, Tumor-Node-Metastasis (TNM) staging system, recurrence, neoadjuvant therapy or overall survival. Conclusion:Our work confirms that most high-grade neuroendocrine tumors express DLL3 across histology types and TNM stage. Our results suggest that a large subset of patients with high-grade lung neuroendocrine tumors may be a target population of interest for DLL3-targeted therapies. References 1. Saunders LR, et al. Sci Transl Med. 2015;7(302):302ra136. 2. Alì G, et al. Front Oncol. 2021;11:729765. 3. Owonikoko TK, et al. Abstract 8510. Presented at: ASCO Annual Meeting, June 4-8, 2021; Virtual. Citation Format: Cibelle F. Lima, Pedro S. Rocha, Junya Fujimoto, Allison Stewart, Robert Cardnell, Wei Lu, Khaja Khan, Beate Sable, Aaron R. Ellison, Ignacio I. Wistuba, Carl M. Gay, Neda Kalhor, Lauren A. Byers, Luisa M. Solis-Soto. Delta-like ligand 3 immunohistochemical expression landscape in high-grade lung neuroendocrine tumors [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 5305.

Small-cell lung cancer (SCLC) is speculated to harbor complex genomic intratumor heterogeneity (ITH) associated with high recurrence rate and suboptimal response to immunotherapy. Here, using multi-region whole exome/T cell receptor (TCR) sequencing as well as immunohistochemistry (IHC), we revealed a rather homogeneous mutational landscape but extremely cold and heterogeneous TCR repertoire in limited-stage SCLC (LS-SCLC) tumors. Compared to localized non-small cell lung cancers (NSCLCs), LS-SCLCs had similar predicted neoantigen burden and genomic ITH, but significantly colder and more heterogeneous TCR repertoire associated with higher chromosomal copy number aberration (CNA) burden. Higher mutational burden, higher T cell infiltration and positive PD-L1 expression were associated with longer overall survival (OS), while higher CNA burden were associated with shorter OS in patients with LS-SCLC.

Background Small cell lung cancer (SCLC) is a highly aggressive neuroendocrine malignancy that accounts for 15% of lung cancer diagnoses. The severity of this disease is exacerbated by the fact that there are few therapeutic options, which mostly offer limited clinical benefit, culminating in a 5-year survival rate of Methods To identify transcriptional subtypes, we used non-negative matrix factorization of gene expression data from 81 SCLC tumors and identified four subtypes largely based on differential expression of the transcription factors ASCL1, NEUROD1, and POU2F3. We hypothesized that these subtypes may underlie unique therapeutic vulnerabilities. We examined differential expression of genes that encode surface-expressed proteins that may be targetable by reagents such as therapeutic antibodies or antibody-drug conjugates (ADCs). Results Our four subtypes are defined either by high expression of ASCL1 (SCLC-A), NEUROD1 (SCLC-N), POU2F3 (SCLC-P), or an absence of those transcription factors and instead a prevalence of immunological factors (SCLC-Inflamed, or SCLC-I). We curated a list of approximately 60 candidate genes encoding surface proteins that are differentially expressed across the four subtypes. Within these 60 candidates, we have identified a few specific to each subtype for which there exist clinically available, targeted ADCs. The most prevalent subtype, SCLC-A, showed high expression of targets such as DLL3 (SCLC-A) and CEACAM5 (SCLC-A). SCLC-N highly expressed SSTR2, a somatostatin receptor that is being actively targeted in SCLC clinical trials. The two non-neuroendocrine subtypes, SCLC-P and SCLC-I shared some common hits such as the NK cell ligand MICA and B7H6. All of the identified and highlighted hits have been or are actively being pursued in clinical trials, highlighting the importance of understanding their expression levels pre- and post-treatment so that novel therapies can be developed that will be effective over the course of disease progression Conclusions The underlying biology defining our four identified subtypes of SCLC has revealed a striking number of targetable, differentially expressed surface protein encoding genes many of which already have clinically available reagents that could be repurposed for treatment of SCLC on a subtype-specific basis.

Small-cell lung cancer (SCLC) is speculated to harbor complex genomic intratumor heterogeneity (ITH) associated with high recurrence rate and suboptimal response to immunotherapy. Here, we revealed a rather homogeneous mutational landscape but extremely suppressed and heterogeneous T cell receptor (TCR) repertoire in SCLCs. Higher mutational burden, lower chromosomal copy number aberration (CNA) burden, less CNA ITH and less TCR ITH were associated with longer overall survival of SCLC patients. Compared to non-small cell lung cancers (NSCLCs), SCLCs had similar predicted neoantigen burden and mutational ITH, but significantly more suppressed and heterogeneous TCR repertoire that may be associated with higher CNA burden and CNA ITH in SCLC. Novel therapeutic strategies targeting CNA could potentially improve the tumor immune microenvironment and response to immunotherapy in SCLC.

Small cell lung cancer (SCLC) is an aggressive form of neuroendocrine carcinoma, notable for early metastases and rapid relapse despite initial response to frontline platinum-based chemotherapy. To date, there are no validated predictive biomarkers in SCLC, hence all patients are treated the same way. Preclinical studies identified SLFN11, a putative DNA/RNA helicase that blocks replication at stressed replication fork, as a predictive biomarker to a wide range of agents targeting DNA damage such as platinum, topoisomerase I/II inhibitors and PARP inhibitors. Based on these observations, pre-specified biomarker analyses in a clinical trial demonstrated SLFN11 predicts better clinical outcomes in SCLC patients when treated with PARP inhibitor combinations such as temozolomide and veliparib. To better characterize the prevalence, heterogeneity and predictive value of SLFN11 in SCLC, we developed and validated a SLFN11 immunohistochemistry (IHC) assay meeting Clinical Laboratory Improvements Amendments (CLIA) standards, and a novel circulating tumor cell (CTC) assay (Epic Sciences®) to detect the expression level of SLFN11 in SCLC tumors or CTCs and correlated with clinical outcomes. We found that SLFN11 was expressed by IHC in roughly 50% of the SCLC clinical tumor samples, from three separate clinical trial cohorts (total of 207 extensive-stage SCLC patient samples). There was a wide range of H-scores by IHC which suggests heterogeneity in SLFN11 expression (H-score range 1.5-235). Similarly, analyses of patient CTCs from blood samples confirmed that SLFN11 is expressed in about 50% of treatment-naïve patients, however SLFN11 expression decreased significantly in patients on platinum treatment and at the time of relapse. Most patients had CTCs with pathologic features consistent with SCLC (i.e., were small, round, had high nuclear-to-cytoplasm ratios, and had salt-and-pepper like chromatin textures), although inter- and intra- patient heterogeneity was observed and SLFN11 expression was observed independent of morphologic subtype. Interestingly, SLFN11 expression was also found in white blood cells in the blood samples and was highest in platinum-naïve patients and lowest in patients while on platinum. Together, these data highlight the potential of SLFN11 as a predictive biomarker in SCLC. Based on our group and others' previous work, SLFN11 positivity by IHC is being used for selection of patients in an ongoing clinical trial (NCT04334941). In addition, given the substantial challenge of obtaining adequate tumor tissue in SCLC either at initial diagnosis or with re-biopsies, blood-based CTC analyses are an important tool to detect SLFN11 expression. Because of the dynamic nature of SLFN11 expression, CTC analyses can be especially valuable for longitudinal monitoring and may have real-time implications for treatment choice and response. Citation Format: Bingnan Zhang, C. Allison Stewart, Carl M. Gay, Qi Wang, Robert Cardnell, Junya Fujimoto, Luisa Fernandez, Adam Jendrisak, Cole Gilbertson, Joseph Schonhoft, Joshua Jones, Amanda K. Anderson, Ignacio I. Wistuba, Jing Wang, Rick Wenstrup, Lauren A. Byers. Detection of DNA replication blocker SLFN11 in tumor tissue and circulating tumor cells to predict platinum response in small cell lung cancer [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 384.

6011 Background: Epithelial-mesenchymal transition (EMT) has been associated with EGFR inhibitor resistance in preclinical studies of head and neck squamous cell carcinoma (HNSCC). Recently, we developed an EMT signature that predicts EGFR inhibitor resistance in lung cancer. Using this signature, we explored the association between EMT and drug response in HNSCC, focusing on the tyrosine kinase Axl as a potential therapeutic target. Methods: We conducted an integrated molecular and drug response analysis in HNSCC. A 76-gene EMT signature previously developed and validated in lung cancer was tested in HNSCC cell lines (n>50) and patient tumors from The Cancer Genome Atlas (TCGA) (n=113) and a MDACC cohort (n=105). Reverse phase protein array (RPPA) and proliferation assays were used to measure protein expression and sensitivity to erlotinib and the Axl inhibitors SGI-7079 and TP-0930. Results: The EMT signature identified distinct epithelial and mesenchymal subsets of HNSCC among cell lines and patient tumors. RPPA experiments revealed higher protein levels of the receptor tyrosine kinase Axl, vimentin, and N-cadherin and lower expression of E-cadherin and beta-catenin in mesenchymal HNSCC (p-values 10μM); however, we discovered that mesenchymal HNSCC were highly sensitive to two Axl inhibitors, SGI-7079 and TP-0930 (IC50s ≤1.2μM and 0.2uM, respectively). Conclusions: Our EMT gene expression signature identified discrete epithelial and mesenchymal subgroups of HNSCC. Mesenchymal HNSCC cells expressed higher levels of Axl protein and exhibited sensitivity to Axl inhibition, but resistance to erlotinib. These results highlight differences in drug response between epithelial and mesenchymal cancers and support Axl as a potential therapeutic target and predictive marker of EGFR inhibitor resistance in HNSCC. (Funded in part by 5 P50 CA097007-10)