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Small cell lung cancer (SCLC) is characterized by morphologic, epigenetic and transcriptomic heterogeneity. Subtypes based upon predominant transcription factor expression have been defined that, in mouse models and cell lines, exhibit potential differential therapeutic vulnerabilities, with epigenetically distinct SCLC subtypes also described. The clinical relevance of these subtypes is unclear, due in part to challenges in obtaining tumor biopsies for reliable profiling. Here we describe a robust workflow for genome-wide DNA methylation profiling applied to both patient-derived models and to patients’ circulating cell-free DNA (cfDNA). Tumor-specific methylation patterns were readily detected in cfDNA samples from patients with SCLC and were correlated with survival outcomes. cfDNA methylation also discriminated between the transcription factor SCLC subtypes, a precedent for a liquid biopsy cfDNA-methylation approach to molecularly subtype SCLC. Our data reveal the potential clinical utility of cfDNA methylation profiling as a universally applicable liquid biopsy approach for the sensitive detection, monitoring and molecular subtyping of patients with SCLC.

Poorly differentiated neuroendocrine carcinomas (PD-NEC) are rare cancers garnering interest as they become more commonly encountered in the clinic. This is due to improved diagnostic methods and the increasingly observed phenomenon of “NE lineage plasticity,” whereby nonneuroendocrine (non-NE) epithelial cancers transition to aggressive NE phenotypes after targeted treatment. Effective treatment options for patients with PD-NEC are challenging for several reasons. This includes a lack of targetable, recurrent molecular drivers, a paucity of patient-relevant preclinical models to study biology and test novel therapeutics, and the absence of validated biomarkers to guide clinical management. Although advances have been made pertaining to molecular subtyping of small cell lung cancer (SCLC), a PD-NEC of lung origin, extrapulmonary (EP)-PD-NECs remain understudied. This review will address emerging SCLC-like, same-organ non-NE cancer-like and tumor-type–agnostic biological vulnerabilities of EP-PD-NECs, with the potential for therapeutic exploitation. The hypotheses surrounding the origin of these cancers and how “NE lineage plasticity” can be leveraged for therapeutic purposes are discussed. SCLC is herein proposed as a paradigm for supporting progress toward precision medicine in EP-PD-NECs. The aim of this review is to provide a thorough portrait of the current knowledge of EP-PD-NEC biology, with a view to informing new avenues for research and future therapeutic opportunities in these cancers of unmet need.

Purpose: Introduced in 1987, platinum-based chemotherapy remains standard of care for small cell lung cancer (SCLC), a most aggressive, recalcitrant tumor. Prominent barriers to progress are paucity of tumor tissue to identify drug targets and patient-relevant models to interrogate novel therapies. Following our development of circulating tumor cell patient–derived explants (CDX) as models that faithfully mirror patient disease, here we exploit CDX to examine new therapeutic options for SCLC.Experimental Design: We investigated the efficacy of the PARP inhibitor olaparib alone or in combination with the WEE1 kinase inhibitor AZD1775 in 10 phenotypically distinct SCLC CDX in vivo and/or ex vivo. These CDX represent chemosensitive and chemorefractory disease including the first reported paired CDX generated longitudinally before treatment and upon disease progression.Results: There was a heterogeneous depth and duration of response to olaparib/AZD1775 that diminished when tested at disease progression. However, efficacy of this combination consistently exceeded that of cisplatin/etoposide, with cures in one CDX model. Genomic and protein analyses revealed defects in homologous recombination repair genes and oncogenes that induce replication stress (such as MYC family members), predisposed CDX to combined olaparib/AZD1775 sensitivity, although universal predictors of response were not noted.Conclusions: These preclinical data provide a strong rationale to trial this combination in the clinic informed by prevalent, readily accessed circulating tumor cell–based biomarkers. New therapies will be evaluated in SCLC patients after first-line chemotherapy, and our data suggest that the combination of olaparib/AZD1775 should be used as early as possible and before disease relapse. Clin Cancer Res; 24(20); 5153–64. ©2018 AACR.

Supplementary Figure 1. Combination treatment with olaparib/AZD1775 is effective in select SCLC CDX models. Mice bearing CDX3 (a), CDX4 (b), CDX8 (c), or CDX8p (d) tumors were treated with vehicles (black) AZD1775 (red), olaparib (green), or the combination (blue) for 21 days. Each line depicts an individual relative tumor volume from all mice. e) CDX4 tumors were treated with vehicles (black) AZD1775 (red), topotecan (green), or the combination (blue). Topotecan was administered on days 1-3 and AZD1775 was administered on days 1-21. Supplementary Figure 2. CDX8p is more chemoresistant than CDX8. a) The clinical timeline for the patient that gave rise to CDX8/8p. Mice bearing CDX8 (b) or CDX8p (c) tumors were treated with vehicles (black) or cisplatin/etoposide (red). Individual relative tumor volumes are depicted. Supplementary Figure 3. Olaparib/AZD1775 is less effective after treatment with cisplatin/etoposide. Mice bearing CDX3 tumors were treated with cisplatin/etoposide on days 1-3 and allowed to recover for 3 weeks, at which time they were randomised and treated with vehicles (black) AZD1775 (red), olaparib (green), or the combination (blue) for 21days. The treatment periods are marked by orange lines. Individual relative tumor volumes are depicted for each mouse. Cohort sizes > 7. Supplementary Figure 4. AZD1775 and olaparib monotherapy activity in representative SCLC CDX models. CDX3 (a, b), CDX4 (c, d), CDX8 (e, f), or CDX8p (g, h) ex vivo cultures were treated with increasing concentrations of either AZD1775 (a, c, e, g) or olaparib (b, d, f, h) for 1 week and relative cell viability was assessed. One representative experiment of at least three is shown. Supplementary Figure 5. Representative immunohistochemical images for phospho-histone H3 (a), cleaved caspase 3 (b), phospho-CHK1 (c), and �H2AX (d) from CDX3 tumours treated with vehicle, AZD1775, olaparib, or the combination. Supplementary Figure 6. Lack of a BRCAness signature in olaparib-sensitive models. a) Heatmap of average log2 RPKM values are depicted for CDX3, CDX4, CDX8, and CDX8p following supervised clustering of models based on olaparib sensitivity. b) The number of synonymous (black) and nonsynonymous (white) mutations as determined by whole exome sequencing are graphed for CDX3, CD8, and CDX8p. The lack of a matched germline sample precluded somatic mutation calling in CDX4. c) Filtered RNAseq reads for PALB2 were loaded into the Broad Integrative Genomics Viewer and the E178* was highlighted in green for each read. Supplementary Figure 7. Published mechanisms of olaparib resistance. a) Lysates from two representative CDX tumors were immunoblotted for indicated proteins. b) PARP1 RNA expression was assessed in tumor lysates from CDX3, CDX4, DX8, and CDX8p. Three representative tumors from each model were examined. c) Lysates from two representative CDX tumors were immunoblotted for indicated proteins. Supplementary Figure 8. Treatment with AZD1775, olaparib, or the combination does not alter p21 expression. Duplicate tumor lysates taken 24 hours after indicated treatments were immunoblotted for indicated proteins. Supplementary Figure 9. Published mechanisms of AZD1775 resistance. a) SETD2 and PKMYT1 RNA expression was assessed in tumor lysates from CDX3, CDX4, CDX8, and CDX8p. Three representative tumors from each model were examined, and mean values are plotted against the cohort average maximal in vivo response to AZD1775. b) RRM2 levels were measured by immunoblotting lysates from CDX tumors harvested 2 hours after a single dose of vehicle (white), AZD1775 (red), olaparib (green), or the combination (blue). Samples were quantified and normalised to vinculin in each sample. Cohort sizes > 4 tumors per condition. Supplementary Table 1. Mass Specrometer and UPLC system parameters Supplementary Table 2. Optimization parameters for mass spectrometry analysis

IntroductionVasculogenic mimicry (VM), the process of tumor cell trans-differentiation to endow endothelial-like characteristics supportingde novovessel formation, is associated with poor prognosis in several tumor types, including small cell lung cancer (SCLC). In genetically engineered mouse models (GEMMs) of SCLC, NOTCH and MYC co-operate to drive a neuroendocrine (NE) to non-NE phenotypic switch and co-operation between NE and non-NE cells is required for metastasis. Here, we define the phenotype of VM-competent cells and molecular mechanisms underpinning SCLC VM using circulating tumor cell-derived explant (CDX) models and GEMMs.MethodsWe analysed perfusion within VM vessels and their association with NE and non-NE phenotypes using multiplex immunohistochemistry in CDX and GEMMs. VM-proficient cell subpopulations inex vivocultures were molecularly profiled by RNA sequencing and mass spectrometry. We evaluated their 3D structure and defined collagen-integrin interactions.ResultsWe show that VM vessels are present in 23/25 CDX models and in 2 GEMMs. Perfused VM vessels support tumor growth and only Notch-active non-NE cells are VM-competentin vivoandex vivo, expressing pseudohypoxia, blood vessel development and extracellular matrix (ECM) organization signatures. On Matrigel, VM-primed non-NE cells re-model ECM into hollow tubules in an integrin β1-dependent process.ConclusionsWe identify VM as an exemplar of functional heterogeneity and plasticity in SCLC and these findings take significant steps towards understanding the molecular events that enable VM. These results support therapeutic co-targeting of both NE and non-NE cells to curtail SCLC progression and to improve SCLC patient outcomes in future.

The Notch pathway is a conserved cell-cell communication pathway that controls cell fate decisions. Here we sought to determine how Notch pathway activation inhibits the neuroendocrine cell fate in the lungs, an archetypal process for cell fate decisions orchestrated by Notch signaling that has remained poorly understood at the molecular level. Using intratumoral heterogeneity in small-cell lung cancer as a tractable model system, we uncovered a role for the transcriptional regulators REST and YAP as promoters of the neuroendocrine to non-neuroendocrine transition. We further identified the specific neuroendocrine gene programs repressed by REST downstream of Notch in this process. Importantly, we validated the importance of REST and YAP in neuroendocrine to non-neuroendocrine cell fate switches in both developmental and tissue repair processes in the lungs. Altogether, these experiments identify conserved roles for REST and YAP in Notch-driven inhibition of the neuroendocrine cell fate in embryonic lungs, adult lungs, and lung cancer.

NeuroEndocrine Carcinomas (NECs) are rare yet lethal diseases; they are frequently metastatic and have limited treatment options. Whilst progress has been made in understanding the biology of Small Cell Lung Cancer (SCLC), the most studied NEC, research on NECs from other anatomical sites lags behind. In SCLC, circulating tumor cells (CTCs) can be used to generate patient-relevant mouse models; termed CTC-Derived eXplants (CDX) which are faithful in biology and chemotherapy response to donor patients1,2. A pilot prospective study is on-going to assess the potential utility of cell-free DNA (cfDNA) and/or CellSearch (CS) CTCs (EpCAM/cytokeratin positive) as liquid biopsies and feasibility of CDX generation from patients with advanced stage extrapulmonary NECs undergoing palliative chemotherapy. CS CTCs are enumerated before and during treatment and marker-independent RosetteSep-enriched CTCs from parallel blood samples are implanted in immunodeficient mice to attempt CDX generation. Twenty-seven patients were recruited from Jun-19 to Nov-21. CS-CTCs were present in 19/27; 70% pre-treatment samples (mean 57, median 2, range 0-685 CTCs per 7.5mL of blood). CDX generation was successful from a pre-treatment CTC sample from a patient originally diagnosed with a NEC of unknown origin who responded to Platinum/Etoposide. Whole-exome sequencing of this CDX and matched pre-treatment cfDNA from the donor revealed largely overlapping copy number changes and common mutations including an activating PIK3CA mutation. In addition, there was no evidence of TP53 or RB1 loss (hallmarks of SCLC) in neither the CDX nor the donor’s cfDNA. Immunohistochemical (IHC) analysis of this CDX showed a small cell NEC morphology, 90% Ki-67 positive cells, expression of EpCAM, pan-cytokeratins and neuroendocrine (NE) diagnostic markers, concordant with the donor’s biopsy. Further IHC and RNA sequencing revealed expression of the NE transcription factor atonal homolog 1 (ATOH1)2, CK20 and abundant Merkel cell polyomavirus (MCPyV) transcripts. These characteristics prompted reclassification of the original patient diagnosis as Merkel cell carcinoma (MCC). There was only 1 CS-CTC in the parallel pre-treatment blood sample implying that EpCAM-negative CTCs may have given rise to the CDX. The CDX showed in vivo sensitivity to Platinum/Etoposide, mirroring treatment response of the donor. Ex vivo cultures from CDX-derived cells grow as suspension clusters similar to ‘classic’ SCLC and MCPyV-positive MCC cell lines. In summary, this first Merkel Cell Carcinoma CDX recapitulates the biology of the donor’s tumor. As the donor remains alive 25 months from diagnosis of advanced disease, their CDX can serve as an avatar to guide future treatment. 1. Hodgkinson et al., Nature Med 2014, 20: p. 897-903. 2. Simpson et al., Nature Cancer 2020, 1: p. 437-451. Citation Format: Melissa Frizziero, Elaine Kilgour, Kathryn L. Simpson, Dominic Rothwell, Kristopher Frese, Melanie Galvin, Yitao Chen, Alastair Kerr, Sam Humphrey, Mukarram Hossain, Juan W. Valle, Mairéad G. McNamara, Caroline Dive. The first circulating tumor cell-derived explant (CDX) model of a Merkel cell carcinoma [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 3098.

Legacy code used to generate all the figure in the paper Raw data is here:http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-8465 from