Small Cell Lung Cancer Specimens Research Articles

Abstract PR005: Leveraging mass cytometry for phenotyping CTCs in SCLC liquid biopsies: Tracking therapy resistance at a personalized level

Abstract Liquid biopsies present a promising, minimally invasive procedure through which clinicians can assess and monitor disease status by interrogating circulating tumor cell (CTC) and immune cell phenotypes. In the case of Small-cell lung cancer (SCLC) – a type of lung cancer characterized by aggressive growth, dismal prognosis and limited treatment options - leveraging liquid biopsies and CTCs for evaluating disease status would be of great benefit, given that biopsies are scarce. Furthermore, SCLC CTC numbers are higher compared to other cancers, presenting a unique opportunity to utilize them for assessing SCLC therapy resistance and identifying new targetable markers. In this study, we leverage mass cytometry (i.e. CyTOF) to phenotype SCLC CTC phenotypes in longitudinal clinical specimens to assess therapy resistance at a personalized level. By using CyTOF - a multiplex cytometric approach that enables interrogation of 30-50 protein markers per single cell – we show that we are able to identify and analyze a significant number of CTCs and their heterogeneous expression profiles. To achieve this, we first optimized a CyTOF panel of ∼25-30 antibodies by using SCLC cell lines and blood specimens from healthy donors and SCLC patients. We show that by using this approach, we are able to identify and phenotype SCLC CTCs by using the combination of CD45 (negative expression) and CD56 positive expression with further validation achieved by TTF1 and SCLC subtype transcription factor expression (NEUROD1, POU2F3 and ASCL1). This approach avoids enriching for specific CTC epithelial phenotypes that express surface markers that fluctuate during epithelial- mesenchymal transition (EMT) (e.g. EpCAM, a common marker for isolating CTCs). For parallel immune profiling we assessed among others CD8/CD4 (T cells) and CD11b/HLA-DR (Myeloid-derived suppressor cells, MDSCs) expression. We then proceeded to interrogate CTC phenotypic differences in the naïve vs relapsed setting as well as dynamic changes in longitudinal blood specimens from SCLC patients by evaluating markers that describe immune suppression activity (PD-L1) and EMT status (E-Cadherin, Vimentin, MUC1, Twist and Slug). Importantly, we show that with our approach we can track in real time therapeutic targets that are currently being tested in clinical trials or have been recently FDA approved (e.g. pYAP and DLL3) and found that MUC1 and pYAP are significantly increased in CTCs detected in blood specimens of SCLC relapsed patients. Our immune profiling showed an increase of MDSC percentages and a decrease in the CD4/CD8 T cell ratio in the relapsed setting. Finally, when we used PHENOSTAMP, a previously developed reference EMT map for assessing EMT phenotypes in lung cancer clinical specimens, we observed an increase of EMT heterogeneity in CTCs in relapsed patients. Our study highlights the translational power of our approach in utilizing CyTOF for longitudinally tracking CTCs directly in SCLC patient liquid biopsies towards assessing therapy response and resistance at a personalized level. Citation Format: Loukia G Karacosta, Sayantan Bhattacharyya, Allison Stewart, Ashley Victorian, Ester F Lujan, Shafqat Ehsan, Subin Kim, Alberto Duarte, Runsheng Wang, Benedict Anchang, Jing Wang, Lauren Byers. Leveraging mass cytometry for phenotyping CTCs in SCLC liquid biopsies: Tracking therapy resistance at a personalized level [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr PR005.

Abstract 6943: Evolutionary dynamics under chemotherapy in small cell lung cancer: Insights from patient-derived xenotransplant models

Abstract Introduction: The treatment of patients with small cell lung cancer (SCLC) represents a major medical challenge due to aggressive tumor growth and the poor 5-year overall survival rate of only 5%. SCLC tumors typically are remarkably sensitive to chemotherapy at the beginning; however, the subsequent rapid relapse of therapy-resistant tumors is a severe obstacle. We therefore aimed to decipher the molecular patterns guiding processes of evolutionary adaptation in SCLC as a consequence of therapeutic interventions. Methods: Over 100 patient-derived xenotransplant models were generated from clinical SCLC specimens capturing a wide range of genetic and molecular features as well as clinical phenotypes with regard to chemotherapy response. We recapitulated chemotherapy response in vivo for 32 models, and performed in-depth genome and single-cell transcriptome sequencing to thus dissect genomic evolutionary trajectories and transcriptional plasticity of therapy-naïve and relapsed tumors, which includes the study of multiregional and longitudinal samples. Results: The response to chemotherapy in vivo reflected the clinical response of the patients; however, several models of relapsed patients remained sensitive to treatment. Genomic studies of clonal dynamics indicated selection of subclones as a result of therapeutic pressure. In contrast, chemo-refractory tumors maintained their clonal composition after therapy. In agreement with genomic profiles, single-cell transcriptome data revealed a tremendous shift in expression profiles and composition of subpopulations. Furthermore, subclonal genomic events including copy number alterations were found to shape the transcriptional heterogeneity in tumors. Conclusion: Here, we demonstrate a comprehensive functional characterization of multi-regional tumor sites and longitudinal specimens acquired from patients with SCLC, in which we elucidate genomic and transcriptional heterogeneity as a consequence of therapy response. Overall, our data points to a complex interplay between sub-clonal genomic changes and transcriptional plasticity shaping therapy response in which pre-existing genomic alterations impact selection under therapy. Citation Format: Laura C. Kaiser, Marcel Schmiel, Agnieszka Ruminska, Nazanin Alavinejad, Christian Müller, Justinas Valiulis, Martin Peifer, Maria Cartolano, Roman K. Thomas, Julie George. Evolutionary dynamics under chemotherapy in small cell lung cancer: Insights from patient-derived xenotransplant models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6943.

Targeted Quantitative Mass Spectrometry Analysis of Protein Biomarkers From Previously Stained Single Formalin-Fixed Paraffin-Embedded Tissue Sections

Formalin-fixed, paraffin-embedded tissues represent a majority of all biopsy specimens commonly analyzed by histologic or immunohistochemical staining with adhesive coverslips attached. Mass spectrometry (MS) has recently been used to precisely quantify proteins in samples consisting of multiple unstained formalin-fixed, paraffin-embedded sections. Here, we report an MS method to analyze proteins from a single coverslipped 4-μm section previously stained with hematoxylin and eosin, Masson trichrome, or 3,3'-diaminobenzidine–based immunohistochemical staining. We analyzed serial unstained and stained sections from non–small cell lung cancer specimens for proteins of varying abundance (PD-L1, RB1, CD73, and HLA-DRA). Coverslips were removed by soaking in xylene, and after tryptic digestion, peptides were analyzed by targeted high-resolution liquid chromatography with tandem MS with stable isotope-labeled peptide standards. The low-abundance proteins RB1 and PD-L1 were quantified in 31 and 35 of 50 total sections analyzed, respectively, whereas higher abundance CD73 and HLA-DRA were quantified in 49 and 50 sections, respectively. The inclusion of targeted β-actin measurement enabled normalization in samples where residual stain interfered with bulk protein quantitation by colorimetric assay. Measurement coefficient of variations for 5 replicate slides (hematoxylin and eosin stained vs unstained) from each block ranged from 3% to 18% for PD-L1, from 1% to 36% for RB1, 3% to 21% for CD73, and 4% to 29% for HLA-DRA. Collectively, these results demonstrate that targeted MS protein quantification can add a valuable data layer to clinical tissue specimens after assessment for standard pathology end points.

EP14.02-003 Clinical Significance of MYC Family Members in Surgically Resected Limited-Stage Small Cell Lung Cancer: A Multicenter Study

Various preclinical studies have stated that members of the MYC transcription factor family (C-myc, L-myc and N-myc) are involved in disease progression and may represent promising therapeutic targets in small cell lung cancer (SCLC). However, there is limited knowledge on the clinical significance of MYC family members in surgically resected SCLC specimens. Our aim was to evaluate the expression and prognostic value of MYC family members in an international cohort of surgically resected SCLC patients.

A non-metabolic function of hexokinase 2 in small cell lung cancer: promotes cancer cell stemness by increasing USP11-mediated CD133 stability.

BackgroundMaintenance of cancer stem‐like cell (CSC) stemness supported by aberrantly regulated cancer cell metabolism is critical for CSC self‐renewal and tumor progression. As a key glycolytic enzyme, hexokinase 2 (HK2) plays an instrumental role in aerobic glycolysis and tumor progression. However, whether HK2 directly contribute to CSC stemness maintenance in small cell lung cancer (SCLC) is largely unclear. In this study, we aimed to investgate whether HK2 independent of its glycolytic activity is directly involved in stemness maintenance of CSC in SCLC.MethodsImmunoblotting analyses were conducted to determine the expression of HK2 in SCLC CSCs and their differentiated counterparts. CSC‐like properties and tumorigenesis of SCLC cells with or without HK2 depletion or overexpression were examined by sphere formation assay and xenograft mouse model. Immunoprecipitation and mass spectrometry analyses were performed to identify the binding proteins of CD133. The expression levels of CD133‐associated and CSC‐relevant proteins were evaluated by immunoblotting, immunoprecipitation, immunofluorescence, and immunohistochemistry assay. RNA expression levels of Nanog, POU5F1, Lin28, HK2, Prominin‐1 were analyzed through quantitative reverse transcription PCR. Polyubiquitination of CD133 was examined by in vitro or in vivo ubiquitination assay. CD133+ cells were sorted by flow cytometry using an anti‐CD133 antibody.ResultsWe demonstrated that HK2 expression was much higher in CSCs of SCLC than in their differentiated counterparts. HK2 depletion inhibited CSC stemness and promoted CSC differentiation. Mechanistically, non‐mitochondrial HK2 directly interacted with CD133 and enhanced CD133 expression without affecting CD133 mRNA levels. The interaction of HK2 and CD133 promoted the binding of the deubiquitinase ubiquitin‐specific protease 11 (USP11) to CD133, thereby inhibiting CD133 polyubiquitylation and degradation. HK2‐mediated upregulation of CD133 expression enhanced the expression of cell renewal regulators, SCLC cell stemness, and tumor growth in mice. In addition, HK2 expression was positively correlated with CD133 expression in human SCLC specimens, and their expression levels were associated with poor prognosis of SCLC patients.ConclusionsThese results revealed a critical non‐metabolic function of HK2 in promotion of cancer cell stemness. Our findings provided new insights into the multifaceted roles of HK2 in tumor development.

Transcriptional Circuitry of NKX2-1 and SOX1 Defines an Unrecognized Lineage Subtype of Small-Cell Lung Cancer.

Rationale: The current molecular classification of small-cell lung cancer (SCLC) on the basis of the expression of four lineage transcription factors still leaves its major subtype SCLC-A as a heterogeneous group, necessitating more precise characterization of lineage subclasses. Objectives: To refine the current SCLC classification with epigenomic profiles and to identify features of the redefined SCLC subtypes. Methods: We performed unsupervised clustering of epigenomic profiles on 25 SCLC cell lines. Functional significance of NKX2-1 (NK2 homeobox 1) was evaluated by cell growth, apoptosis, and xenograft using clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-associated protein 9)-mediated deletion. NKX2-1-specific cistromic profiles were determined using chromatin immunoprecipitation followed by sequencing, and its functional transcriptional partners were determined using coimmunoprecipitation followed by mass spectrometry. Rb1flox/flox; Trp53flox/flox and Rb1flox/flox; Trp53flox/flox; Nkx2-1flox/flox mouse models were engineered to explore the function of Nkx2-1 in SCLC tumorigenesis. Epigenomic landscapes of six human SCLC specimens and 20 tumors from two mouse models were characterized. Measurements and Main Results: We identified two epigenomic subclusters of the major SCLC-A subtype: SCLC-Aα and SCLC-Aσ. SCLC-Aα was characterized by the presence of a super-enhancer at the NKX2-1 locus, which was observed in human SCLC specimens and a murine SCLC model. We found that NKX2-1, a dual lung and neural lineage factor, is uniquely relevant in SCLC-Aα. In addition, we found that maintenance of this neural identity in SCLC-Aα is mediated by collaborative transcriptional activity with another neuronal transcriptional factor, SOX1 (SRY-box transcription factor 1). Conclusions: We comprehensively describe additional epigenomic heterogeneity of the major SCLC-A subtype and define the SCLC-Aα subtype by the core regulatory circuitry of NKX2-1 and SOX1 super-enhancers and their functional collaborations to maintain neuronal linage state.

The Deubiquitinase USP13 Maintains Cancer Cell Stemness by Promoting FASN Stability in Small Cell Lung Cancer.

USP13 is significantly amplified in over 20% of lung cancer patients and critical for tumor progression. However, the functional role of USP13 in small cell lung cancer (SCLC) remains largely unclear. In this study, we found that the deubiquitinase USP13 is highly expressed in SCLC tumor samples and positively associated with poor prognosis in multiple cohorts. In vitro and in vivo depletion of USP13 inhibited SCLC cancer stem cells (CSCs) properties and tumorigenesis, and this inhibitory effect was rescued by reconstituted expression of wide type (WT) USP13 but not the enzyme-inactive USP13 mutant. Mechanistically, USP13 interacts with fatty acid synthase (FASN) and enhances FASN protein stability. FASN downregulation suppresses USP13-enhanced cell renewal regulator expression, sphere formation ability, and de novo fatty acids biogenesis. Accordingly, we found FASN expression is upregulated in surgical resected SCLC specimens, positively correlated with USP13, and associated with poor prognosis of SCLC patients. More importantly, the small molecule inhibitor of FASN, TVB-2640, significantly inhibits lipogenic phenotype and attenuates self-renewal ability, chemotherapy resistance and USP13-mediated tumorigenesis in SCLC. Thus, our study highlights a critical role of the USP13-FASN-lipogenesis axis in SCLC cancer stemness maintenance and tumor growth, and reveals a potential combination therapy for SCLC patients.

Abstract 5709: Transcriptional circuitry of NKX2-1 and SOX1 defines a previously unrecognized lineage subtype of small cell lung cancer

Abstract Introduction: The current molecular classification of small cell lung cancer (SCLC) based on expression of four lineage transcription factors, SCLC-A (ASCL1), SCLC-N (NEUROD1), SCLC-P (POU2F3), and SCLC-Y (YAP1) still leaves its major subtype SCLC-A as a large heterogeneous group, necessitating more precise characterization of lineage subclasses. Experimental procedure: To refine the current SCLC classification and to identify specific lineage features of the SCLC subtypes, we performed unsupervised hierarchical clustering of H3K27ac profiles on transcriptional regulators from 25 SCLC cell lines and determined the epigenomic features for each cluster. Functional significance of the transcriptional lineage regulators for the identified cluster was evaluated by cell growth, apoptosis and xenograft using CRISPR-Cas9-mediated deletion. The specific cistromic profiles by ChIP-seq and its functional transcriptional partners using co-immunoprecipitation followed by mass spectrometry were determined to reveal their functional output in the identified subtype. Rb1fl/flTrp53fl/fl and Rb1fl/flTrp53fl/flNkx2-1fl/fl genetic engineered mouse model were generated to explore the function of Nkx2-1 in tumor initiation and differentiation. H3K27ac profiles were further analyzed to reveal 6 human SCLC specimen and 20 mice tumors epigenomic landscapes. Summary: We identified previously uncharacterized epigenomic sub-clusters of the major SCLC-A subtype, named SCLC-A1 and SCLC-A2. SCLC-A1 was characterized by the presence of a super-enhancer at the NKX2-1 locus, which was observed in human SCLC specimens and a murine SCLC model. We found NKX2-1, a dual lung and neural lineage factor, is uniquely relevant in SCLC-A1, where it maintains neural lineage rather than pulmonary epithelial identity. We further found maintenance of this neural identity in SCLC-A1 is mediated by collaborative transcriptional activity with another neuronal transcriptional factor SOX1. ? Conclusions: We comprehensively describe an additional epigenomic heterogeneity of the major SCLC-A subtype, and define SCLC-A1 subtype by the core regulatory circuitry representing NKX2-1 and SOX1 super-enhancers and their functional collaborations to maintain a neuronal linage state. Citation Format: ranran kong, Ayushi S. Patel, Takashi Sato, Seungyeul Yoo, Abhilasha Sinha, Yang Tian, Feng Jiang, Charles A. Powell, Eric Snyder, Jiantao Jiang, Shaomin Li, Hideo Watanabe. Transcriptional circuitry of NKX2-1 and SOX1 defines a previously unrecognized lineage subtype of small cell lung cancer [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 5709.

Chemotherapy- and Immune-Related Gene Panel in Prognosis Prediction and Immune Microenvironment of SCLC.

Small-cell lung cancer (SCLC) is a highly proliferative, invasive lung cancer with poor prognosis. Chemotherapy is still the standard first-line treatment for SCLC, but many patients relapse due to chemoresistance. Along with advances in immunology, it is essential to investigate potential indicators of the immune response and the prognosis of SCLC. Using bioinformatics analysis, we identified 313 differentially expressed genes (DEGs) in SCLC and normal lung samples, and we found that four upregulated genes (TOP2A, CDKN2A, BIRC5, and MSH2) were associated with platinum resistance, while immune-related genes (HLA family genes) were downregulated in SCLC. Then, a prognostic prediction model was constructed for SCLC based on those genes. Immune cell infiltration analysis showed that antigen presentation was weak in SCLC, and TOP2A expression was negatively correlated with CD8+ T cells, while HLA-ABC expression was positively correlated with M1 macrophages, memory B cells, and CD8+ T cells. We also found that TOP2A was related to poor prognosis and inversely correlated with HLA-ABC, which was verified with immunohistochemical staining in 151 SCLC specimens. Our study findings indicated that TOP2A may be a potential prognosis indicator and a target to reverse the immunosuppressive tumor microenvironment of SCLC.

Genomic and transcriptomic analysis of a library of small cell lung cancer patient-derived xenografts

Access to clinically relevant small cell lung cancer (SCLC) tissue is limited because surgical resection is rare in metastatic SCLC. Patient-derived xenografts (PDX) and circulating tumor cell-derived xenografts (CDX) have emerged as valuable tools to characterize SCLC. Here, we present a resource of 46 extensively annotated PDX/CDX models derived from 33 patients with SCLC. We perform multi-omic analyses, using targeted tumor next-generation sequencing, RNA-sequencing, and immunohistochemistry to deconvolute the mutational landscapes, global expression profiles, and molecular subtypes of these SCLC models. SCLC subtypes characterized by transcriptional regulators, ASCL1, NEUROD1 and POU2F3 are confirmed in this cohort. A subset of SCLC clinical specimens, including matched PDX/CDX and clinical specimen pairs, confirm that the primary features and genomic and proteomic landscapes of the tumors of origin are preserved in the derivative PDX models. This resource provides a powerful system to study SCLC biology.

Clinical characteristics and patient outcomes of molecular subtypes of small cell lung cancer (SCLC)

BackgroundRecent studies have shown that according to the expression levels of achaete-scute homolog 1 (ASCL1), neurogenic differentiation factor 1 (NEUROD1), and POU class 2 homeobox 3 (POU2F3), small cell lung cancer (SCLC) can be divided into four subtypes: SCLC-A (ASCL1-dominant), SCLC-N (NEUROD1-dominant), SCLC-P (POU2F3-dominant), and SCLC-I (triple negative or SCLC-inflamed). However, there are limited data on the clinical characteristics and prognosis of molecular subtypes of SCLC.MethodsImmunohistochemistry (IHC) was used to detect the expression levels of ASCL1, NEUROD1, and POU2F3 in 53 patient samples of resectable SCLC. The subtype was defined by the differential expression of the transcription factors for ASCL1, NEUROD1, and POU2F3 or the low expression of all three factors with an inflamed gene signature (SCLC-A, SCLC-N, SCLC-P, and SCLC-I, respectively). The clinicopathological characteristics, immunological features (programmed death ligand 1 [PD-L1] expression and CD8+ tumor infiltrating lymphocyte [TIL] density), and patient outcomes of the four subtypes of SCLC were analyzed.ResultsPositive ASCL1, NEUROD1, and POU2F3 staining was detected in 43 (79.2%), 27 (51.0%), and 17 (32.1%) SCLC specimens by IHC. According to the results of IHC analysis, SCLC was divided into four subtypes: SCLC-A (39.6%), SCLC-N (28.3%), SCLC-P (17.0%), and SCLC-I (15.1%). The 5-year overall survival (OS) rates of these four subtypes were 61.9%, 69.3%, 41.7%, and 85.7%, respectively (P=0.251). There were significant differences in smoking status among different subtypes of SCLC (P= 0.031). However, we did not confirm the correlation between subtypes of SCLC and other clinicopathological factors or immune profiles. Cox multivariate analysis showed that N stage (P=0.025), CD8+ TILs (P=0.024), Ki-67 level (P=0.040), and SCLC-P (P=0.023) were independent prognostic factors for resectable SCLC.ConclusionsOur IHC-based study validated the proposed classification of SCLC using the expression patterns of key transcriptional regulatory factors. We found that SCLC-P was associated with smokers and was one of the poor prognostic factors of limited-stage SCLC. In addition, no correlation was found between PD-L1 expression or CD8+ TIL density and SCLC subtypes.

Programmed Cell Death Ligand 1 Expression in Cytological and Surgical Non-Small Cell Lung Cancer Specimens in Association with EGFR Mutation and Overall Survival: A Single-Institution Experience.

The aim of this study was to evaluate programmed cell death ligand-1 (PD-L1) expression and the relationship between driver mutations and survival analysis in advanced-stage non-small cell lung carcinoma (NSCLC). A total of 122 advanced-stage NSCLC patients were included in this retrospective study. The patients were diagnosed based on cytological examination and histopathological analysis of biopsy or resection material that had undergone at least 1 molecular analysis. The expression of PD-L1 in tumors and tumor-infiltrating lymphocytes (TIL) was scored and compared with age, sex, organ, biopsy method, tumor subtype, driver mutation status, and overall survival data. There was no statistically significant difference between PD-L1-positivity and age, gender, location, pattern, or pathological diagnosis of the type of sample. When the threshold value for PD-L1 IHC evaluation was accepted as ≥1% and ≥50%, the rate of positivity was 19.7% and 7.4%, respectively. Since there is a wide range of positivity rates reported in the literature, we could not reach a conclusion as to whether the PD-L1-positivity rate we observed was high or low. There is a need for comparative studies where the technique, clones, threshold values, and phases are homogenized. There is an inverse correlation between the EGFR-mutant population and PD-L1 positivity. In terms of overall survival, no relationship was found between PD-L1 positivity, the presence of TIL, and EGFR mutation status.

Delta-Like Protein 3 Expression in Paired Chemonaive and Chemorelapsed Small Cell Lung Cancer Samples.

Rovalpituzumab tesirine (Rova-T), an antibody-drug conjugate directed against Delta-like protein 3 (DLL3), is under development for patients with small cell lung cancer (SCLC). DLL3 is expressed on the majority of SCLC samples. Because SCLC is rarely biopsied in the course of disease, data regarding DLL3 expression in relapses is not available. The aim of this study was to investigate the expression of DLL3 in chemorelapsed (but untreated with Rova-T) SCLC samples and compare the results with chemonaive counterparts. Two evaluation methods to assess DLL3 expression were explored. Additionally, we assessed if DLL3 expression of chemorelapsed and/or chemonaive samples has prognostic impact and if it correlates with other clinicopathological data. The study included 30 paired SCLC samples, which were stained with an anti DLL3 antibody. DLL3 expression was assessed using tumor proportion score (TPS) and H-score and was categorized as DLL3 low (TPS < 50%, H-score ≤ 150) and DLL3 high (TPS ≥ 50%, H-score > 150). Expression data were correlated with clinicopathological characteristics. Kaplan–Meier curves were used to illustrate overall survival (OS) depending on DLL3 expression in chemonaive and chemorelapsed samples, respectively, and depending on dynamics of expression during course of therapy. DLL3 was expressed in 86.6% chemonaive and 80% chemorelapsed SCLC samples without significant differences between the two groups. However, the extent of expression varied in a substantial proportion of pairs (36.6% with TPS, 43.3% with H-score), defined as a shift from low to high or high to low expression. TPS and H-score provided comparable results. There were no profound correlations with clinicopathological data. Survival analysis revealed a trend toward a more favorable OS in DLL low-expressing chemonaive SCLC (p = 0.57) and, in turn, in DLL3 high-expressing chemorelapsed SCLC (p = 0.42) as well as in SCLC demonstrating a shift from low to high expression (p = 0.56) without being statistically significant. This is the first study to investigate DLL3 expression in a large cohort of rare paired chemonaive-chemorelapsed SCLC specimens. Comparative analysis revealed that DLL3 expression was not stable during the course of therapy, suggesting therapy-based alterations. Unlike in chemonaive samples, a high DLL3 expression in chemorelapsed samples indicated a trend for a more favorable prognosis. Our results highlight the importance to investigate DLL3 in latest chemorelapsed SCLC tumor tissue.

OA07.02 TAZ Regulates SCLC Phenotypic Transition and Metastasis

Small cell lung cancer (SCLC) remains as the most lethal form of lung cancer with very poor prognosis. High metastatic capability and strong plasticity is the hallmark of SCLC. However, the exact cell subpopulation responsible for SCLC metastasis and critical regulators in contribution to SCLC plasticity still remain largely unclear. Growing evidence has revealed the importance of epigenetic reprograming during cancer malignant progression. Mammalian SWI/SNF complex BAF is an adenosine triphosphate (ATP)-dependent multi-component machinery, essential for remodeling chromatin architecture. Smarca4 (Brg1), the core ATPase component of BAF complex is preferentially required for the progression of SCLC with MAX (Myc-associated factor) inactivation. Also, it still remains elusive about the exact function of Brg1 and SWI/SNF complex in SCLC metastasis. Using the well-established Rb1L/L/Trp53 L/L (RP) mouse model, we here identify the NCAMhiCD44lo/- subpopulation derived from RP model as SCLC metastasizing cells (SMC). Moreover, we find that the SMC is progressively transitioned from the NCAMloCD44hi cells (Non-SCLC metastasizing cells, Non-SMC) during malignant progression of SCLC. Through combined analysis of ATAC-seq and RNA-seq data of SMC vs. non-SMC, we observe a dramatic alteration in chromatin accessibility between these two subpopulations, and TAZ, the core transcription cofactor of the Hippo pathway, is epigenetically silenced by the SWI/SNF complex during SCLC malignant progression. Knockout of Brg1 in RP mouse promotes TAZ expression and abrogates the phenotype transition from Non-SMC to SMC and almost completely blocks SCLC metastasis. Allograft assay further confirms TAZ as the critical molecular switch during the phenotypic transition and SCLC metastasis. In this study, we identify the NCAMhiCD44lo/- subpopulation derived from RP model as SMC subpopulation, which is progressively transitioned from the Non-SMC during SCLC malignant progression. Through combined analysis of ATAC-seq and RNA-seq data, we observe a dramatic alteration in chromatin accessibility between these two subpopulations, which points to the alteration of SWI/SNF complex activation. We further find that genetic disruption of the SWI/SNF chromatin-remodeling complex through Brg1 knockout abrogates the phenotype transition from Non-SMC to SMC and blocks SCLC metastasis. Mechanistically, we find that TAZ, the core transcription cofactor of the Hippo pathway, is epigenetically silenced by the SWI/SNF complex. Down-regulation of TAZ promotes the transition from Non-SMC to SMC and facilitates SCLC metastasis. Conversely, ectopic TAZ expression or TAZ activation by digitoxin treatment reversibly promotes the transition from SMC to Non-SMC and thus alleviates SCLC metastasis. Importantly, the treatment of digitoxin, a clinically used drug for heart failure treatment, promotes TAZ activation and inhibits SCLC phenotypic transition and metastasis. Through the immunostaining analyses of 101 Chinese SCLC specimens, we further find that low TAZ expression significantly correlates with SMC features and poor patient prognosis. Collectively, these data uncover an important subpopulation in contribution to SCLC metastasis and highlights TAZ as the critical molecular switch in this process. Moreover, TAZ activation by digitoxin treatment provides an effective avenue to control SCLC phenotypic transition and metastasis, providing a potential therapeutic avenue for the metastatic SCLC management in clinic.

P66.02 The Prognostic Implication of hes1 Protein Expression in Resected Small Cell Lung Cancers of 247 Cases

Small cell lung cancer (SCLC) is a highly aggressive malignancy prone to early recurrence and metastasis. Preclinical studies have found that Hes1, as a transcription repressor of the basic helix-loop-helix (BHLH) family, can not only prevent the proliferation and migration of SCLC tumor cells, but also inhibit the neuroendocrine transcription factor ASCL1. However, the prognostic implication of Hes1 protein on surgically resected SCLCs remains unclear. The current study aims to analyze the expression pattern and prognostic value of Hes1 protein in SCLC. Two hundred and forty seven surgically resected pure SCLC specimens were reviewed and included in this study by using tissue microarrays (TMA) for immunohistochemistry（IHC）analysis of Hes1 protein on a fully automatic Roche immunohistochemical instruments . And the corresponding clinicopathological features such as age, lymph node metastasis, major cell shape and tumor infiltrating lymphocytes(TILs), etc were reviewed and collected. Correlation analysis of Hes1 protein with clinic pathological features and survival analysis was performed using SPSS 25.0 and Graphpad Prism 5.0. Among the 247 surgically resected pure SCLC patients, 175 (71%) were male and 202 (82%) were less than 65 years. According to the AJCC Cancer Staging Manual (seventh edition), 78 (31.6%) were stage I, 68 (27.5%) were stage II, and 101 (40.9%) were stage III. Hes1 expression was localized in the nucleus of SCLC tumor cells. A total of 129 of the 247 enrolled SCLC patients showed high expression of Hes1 with a positive rate of 52.2%(129/247), and was found positively correlated with a lower age(≤65 yrs., p=0.014), no lymph node metastasis(P=0.003), main cell morphology of round cells(p=0.002), and TILs≤30% (p=0.010). Univariate survival analysis revealed a favorable survival in high expression group for a significant disease free survival (DFS, HR=1.477,95%CI 1.025-2.129, P=0.036 ) and a positive trend of overall survival (OS, HR=1.181,95%CI 0.778-1.792,P=0.435). In limited stage pure small cell lung cancer, high expression of Hes1 protein is related to age, lymph node metastasis, main cell morphology and TILs , which contributes as a potential biomarker for the prognosis of SCLC patients.

PTPN3 is a potential target for a new cancer immunotherapy that has a dual effect of T cell activation and direct cancer inhibition in lung neuroendocrine tumor

In our previous study, we found that inhibition of protein tyrosine phosphatase non-receptor type 3 (PTPN3), which is expressed in lymphocytes, enhances lymphocyte activation, suggesting PTPN3 may act as an immune checkpoint molecule. However, PTPN3 is also expressed in various cancers, and the biological significance of PTPN3 in cancer cells is still not well understood, especially for lung neuroendocrine tumor (NET).Therefore, we analyzed the biological significance of PTPN3 in small cell lung cancer and examined the potential for PTPN3 inhibitory treatment as a cancer treatment approach in lung NET including small cell lung cancer (SCLC) and large cell neuroendocrine cancer (LCNEC).Experiments in a mouse xenograft model using allo lymphocytes showed that PTPN3 inhibition in SCLC cells enhanced the anti-tumor effect of PTPN3-suppressed activated lymphocytes. In addition, PTPN3 was associated with increased vascularization, decreased CD8/FOXP3 ratio and cellular immunosuppression in SCLC clinical specimens. Experiments in a mouse xenograft model using autocrine lymphocytes also showed that PTPN3 inhibition in LCNEC cells augmented the anti-tumor effect of PTPN3-suppressed activated lymphocytes. In vitro experiments showed that PTPN3 is involved in the induction of malignant traits such as proliferation, invasion and migration. Signaling from PTPN3 is mediated by MAPK and PI3K signals via tyrosine kinase phosphorylation through CACNA1G calcium channel. Our results show that PTPN3 suppression is associated with lymphocyte activation and cancer suppression in lung NET. These results suggest that PTPN3 suppression could be a new method of cancer treatment and a major step in the development of new cancer immunotherapies.

Molecular Signature of Small Cell Lung Cancer after Treatment Failure: The MCM Complex as Therapeutic Target.

Simple SummarySmall cell lung cancer (SCLC) is a fatal malignant tumor with a poor prognosis for patients who relapse after first-line treatment. There are few effective treatments for SCLC patients with relapse. Elucidation of the molecular network related to treatment resistance is an important issue for this disease. In this study, the molecular signature of SCLC specimens after treatment failure was generated. Several pathways, e.g., “cell cycle”, “homologous recombination”, “DNA replication”, and “p53 signaling” were identified as the enriched pathways in this signature. Aberrant expression of MCM2, MCM4, MCM6, and MCM7 were detected in SCLC clinical specimens after treatment failure. This signature contains molecules involved in treatment resistance and will contribute to the study of SCLC molecular pathogenesis.Small cell lung cancer (SCLC) is a highly aggressive cancer, and patients who become refractory to first-line treatment have a poor prognosis. The development of effective treatment regimens is urgently needed. In this study, we identified a gene expression signature of SCLC after treatment failure using SCLC clinical specimens (GEO accession number: GSE162102). A total of 1,136 genes were significantly upregulated in SCLC tissues. These upregulated genes were subjected to KEGG pathway analysis, and “cell cycle”, “Fanconi anemia”, “alcoholism”, “systemic lupus erythematosus”, “oocyte meiosis”, “homologous recombination”, “DNA replication”, and “p53 signaling” were identified as the enriched pathways among the genes. We focused on the cell cycle pathway and investigated the clinical significance of four genes associated with this pathway: minichromosome maintenance (MCM) 2, MCM4, MCM6, and MCM7. The overexpression of these MCM genes was confirmed in SCLC clinical specimens. Knockdown assays using siRNAs targeting each of these four MCM genes showed significant attenuation of cancer cell proliferation. Moreover, siRNA-mediated knockdown of each MCM gene enhanced the cisplatin sensitivity of SCLC cells. Our SCLC molecular signature based on SCLC clinical specimens after treatment failure will provide useful information to identify novel molecular targets for this disease.

The MYC Paralog-PARP1 Axis as a Potential Therapeutic Target in MYC Paralog-Activated Small Cell Lung Cancer.

Poly (ADP-ribose) polymerase 1 (PARP1) is highly expressed in small cell lung cancer (SCLC) and has emerged as an attractive target for treatment of SCLC. However, the clinical significance of PARP1 expression in SCLC remains elusive. In this study, we showed that high PARP1 expression was associated with better overall survival (OS), and was positively correlated with the expression of MYC paralogs in patients with SCLC. We demonstrated that PARP1 was transcriptionally regulated by MYC paralogs. Integrative analysis of multiple RNA-seq data sets indicated that DNA damage response (DDR) genes involved in the replication stress response (RSR) and homologous recombination (HR) repair pathways were highly enriched in MYC paralog-addicted SCLC cell models and in human SCLC specimens. Targeting the MYC paralog-PARP1 axis with concomitant BET and PARP inhibition resulted in synergistic effects in MYC paralog-activated SCLC. Our study identified a critical PARP1 regulatory pathway, and provided evidence for a rational combination treatment strategy for MYC paralog-activated SCLC.

The significance of immune-regulatory molecule B7-H4 in small cell lung cancer.

B7-H4, a member of the B7 family, is detected in various cancers and is closely related to tumor development and prognosis. However, little is known about the clinical value of B7-H4 in small cell lung cancer (SCLC). Immunohistochemical analysis was carried out on 103 SCLC specimens. The relationship between B7-H4 staining and the major clinical parameters of SCLC was analyzed, and the two-year survival rates were investigated by chi-square test. Only 5.83% of the SCLC specimens tested positive for B7-H4. B7-H4 was detected on the membrane and in the cytoplasm of tumor cells. In contrast, B7-H4 expression was not detected in normal lung tissue samples. B7-H4 was not found to be associated with major clinical parameters, such as tumor size, gender, age, smoking status, limited/extensive stage, tumor node metastasis, Karnofsky Performance Status, lymph node metastasis status, distant metastasis, or ki-67. Moreover, no obvious differences were observed in the two-year survival rates of B7-H4 positive or B7-H4-negative SCLC patients. There is no correlation between B7-H4 expression and the proliferation or progression of SCLC. Therefore, B7-H4 is not a useful biomarker for SCLC prognosis.

Detection and comparison of EGFR mutations from supernatants that contain cell-free DNA and cell pellets from FNA non-small cell lung cancer specimens.

Epidermal growth factor receptor (EGFR) is an important marker for targeted therapy in patients with advanced non-small cell lung cancer (NSCLC). The samples obtained with minimally invasive biopsy techniques are usually small, and this limits their application in tissue subtyping or molecular profiling. The supernatants obtained after centrifugation of fine-needle aspiration (FNA) samples are typically discarded. However, these fractions might contain cell-free DNA that could be tested for EGFR mutations by genotyping methods that are normally used for plasma analysis. In this study, 214 patients with known or suspected NSCLC who underwent FNA were enrolled. The workflow of the supernatants before molecular detection was as follows. The discarded FNA samples (15 mL) were stored in CytoLyt, a cleaning, fixation solution, and 10 mL of each sample was placed in a preservation solution for separation by low-speed centrifugation. The primary supernatants (8 mL) were then separated by high-speed centrifugation to obtain secondary supernatants. DNA was extracted from the supernatants with QIAamp circulating nucleic acid kits (Qiagen) and circulating DNA kits (AmoyDx), and EGFR mutations were assessed with Super-ARMS EGFR detection kits (AmoyDx). The DNA was then extracted from corresponding cell pellets with tissue DNA kits (AmoyDx), and the EGFR status was analyzed with the amplification refractory mutation system and next-generation sequencing methods. All 214 samples yielded an adequate amount of cell-free DNA for EGFR detection. The use of different DNA commercial extraction kits and the DNA contents of tumor cells did not affect the yield of DNA from the supernatants. The external controlled cycle threshold value of the EGFR test was affected by the concentration of the DNA in the supernatants (P<.05). However, the difference in the concentrations of the DNA in the supernatants did not affect the EGFR mutation status. The EGFR-positive rate was 57.5% (123 of 214) in both the supernatants and the pellets from the 214 FNA samples. The concordance between EGFR variants in the supernatants and the corresponding pellets was 97.2%. EGFR mutations were also detected in 3 pellets but not in their corresponding supernatants and in 3 supernatants but not in their corresponding pellets. The supernatants of FNA biopsy samples might represent a new source for gaining information regarding the molecular characteristics of patients for targeted therapy. Discarded supernatants provided an adequate amount of cell-free DNA for EGFR detection, and this means that the pellets can be reserved for additional morphological and molecular analyses or to avoid repeat biopsies. Analyzing the EGFR status in cell supernatants and pellets might improve detection sensitivity and confer benefits to patients.