High-grade Neuroendocrine Carcinomas Of Lung Research Articles

Real-world multiomic characterization of small cell lung cancer subtypes to reveal differential expression of clinically relevant biomarkers.

8508 Background: The dominant expression of four lineage-defining transcription factors ( ASCL1, NEUROD1, YAP1, or POU2F3) has enabled the classification of small cell lung cancer (SCLC) into four subtypes (SCLC-A/N/Y/P, respectively). Emerging evidence suggests that YAP1 expression is associated with a T-cell inflamed phenotype, and SCLC has significant intra-tumor heterogeneity mediated by MYC-driven activation of NOTCH signaling. We performed a large-scale analysis of real-world SCLC patient samples to examine the expression of clinically relevant biomarkers across SCLC subtypes. Methods: Comprehensive molecular profiling of 437 small cell lung neuroendocrine tumors (including 7.3% high-grade neuroendocrine lung carcinomas) was performed using next-generation DNA sequencing (592-gene panel), RNA sequencing (whole transcriptome), and immunohistochemistry at Caris Life Sciences (Phoenix, AZ). Tumors were stratified into 5 subgroups (SCLC-A/N/Y/P and -mixed) based on the relative expression of the four transcription factors. RNA expression of key genes and previously validated immune signatures (T-cell inflamed, NK cell, and STING pathway signatures) were evaluated across subgroups. Significance was tested by Chi-square, Fisher’s exact test, or Mann-Whitney U test. Results: Median age of the study cohort was 66 years (IQR: 59-72) and 50.6% of patients were female. The majority (67.3%) of samples were derived from metastatic sites. Stratification of tumors by expression resulted in 35.7% SCLC-A, 17.6% SCLC-N, 21.1% SCLC-Y, 6.4% SCLC-P, and 19.2% SCLC-mixed samples. Compared to tumors from metastatic sites, YAP1 expression was significantly increased (p < 0.001) in primary tumors. Amongst the 14 tumors obtained from the CNS, SCLC-N (36%, n = 5) was the most common subtype identified. dMMR/MSI-high (negative MMR protein expression/ ≥46 altered loci per tumor) was rare overall (0.5%, n = 2); TMB (median of 9-10 mut/Mb) was similar between the SCLC subtypes. SCLC-Y was associated with the highest expression of T-cell inflamed, NK cell and STING pathway signatures (p < 0.0001 each). MYC and NOTCH gene expression ( NOTCH1/2/3/4) strongly correlated with YAP1 expression. Analysis of co-mutations revealed that EGFR-sensitizing mutations (L858R and Exon 19 deletions) were recurrent (5.2%, n = 4) in SCLC-N tumors. The expression of SNF11, SSTR2, and MYC varied significantly among SCLC subtypes (p < 0.001 each), with the highest median expression of SNF11 and SSTR2 observed in SCLC-N, while MYC expression was highest in SCLC-P. Conclusions: Our analysis represents the largest real-world dataset of human SCLC tumors profiled by whole transcriptomic sequencing. The differential expression of immune genes and predictive biomarkers across SCLC subtypes may inform therapeutic vulnerabilities for rational and personalized treatment approaches in SCLC.

Differential development of large-cell neuroendocrine or small-cell lung carcinoma upon inactivation of 4 tumor suppressor genes

High-grade neuroendocrine lung malignancies (large-cell neuroendocrine cell carcinoma, LCNEC, and small-cell lung carcinoma, SCLC) are among the most deadly lung cancer conditions with no optimal clinical management. The biological relationships between SCLC and LCNEC are still largely unknown and a current matter of debate as growing molecular data reveal high heterogeneity with potential therapeutic consequences. Here we describe murine models of high-grade neuroendocrine lung carcinomas generated by the loss of 4 tumor suppressors. In an Rbl1-null background, deletion of Rb1, Pten, and Trp53 floxed alleles after Ad-CMVcre infection in a wide variety of lung epithelial cells produces LCNEC. Meanwhile, inactivation of these genes using Ad-K5cre in basal cells leads to the development of SCLC, thus differentially influencing the lung cancer type developed. So far, a defined model of LCNEC has not been reported. Molecular and transcriptomic analyses of both models revealed strong similarities to their human counterparts. In addition, a 68Ga-DOTATOC-based molecular-imaging method provides a tool for detection and monitoring the progression of the cancer. These data offer insight into the biology of SCLC and LCNEC, providing a useful framework for development of compounds and preclinical investigations in accurate immunocompetent models.

Multiple Assays to Determine Methylguanine-Methyltransferase Status in Lung Carcinoids and Correlation with Clinical and Pathological Features

Background: O6-methylguanine-methyltransferase (MGMT) is a key enzyme for the DNA repair machinery strongly associated with response to alkylating agents in different tumors. Data on its expression and related clinical impact in neuroendocrine tumors are limited to the gastro-entero-pancreatic system, with controversial results in terms of prognostic or predictive value. In lung carcinoids, although clinical efficacy of alkylating agents has been shown in small studies, very few data to date are available on MGMT status. Objective: To assess MGMT status in lung carcinoids using multiple assays and to compare data with major clinical and pathological features. Methods: A retrospective series of 95 lung carcinoids and 51 control cases of high-grade neuroendocrine lung carcinomas was analyzed for MGMT promoter methylation, MGMT gene expression, and MGMT protein expression using pyrosequencing, quantitative real-time PCR, and immunohistochemistry, respectively. Results: MGMT protein expression was inversely correlated with MGMT promoter methylation and positively with MGMT gene expression. MGMT promoter methylation progressively increased from carcinoids to high-grade carcinomas. In the carcinoid group, decreased MGMT gene expression was significantly associated with aggressive features (atypical histotype, grade G2, larger tumor size, higher T stage, and positive nodal status) but not with survival. MGMT promoter methylation was associated with lower stage and negative nodal status. Conclusions: Our study investigated MGMT status in a large series of lung carcinoids in the attempt to move forward a rational use of alkylating agents in these tumors. Interestingly, low MGMT gene expression defines a subgroup of lung carcinoids with aggressive features.

Abstract A83: Genes of epithelial-mesenchymal transition (EMT) reveal impact on progression and metastasis of neuroendocrine lung carcinomas: A preliminary study

Abstract Background: Metastasis are responsible for the death of 90% of patients with lung cancer, indicating the need to know the multiple signaling pathways involved. Neuroendocrine lung carcinomas (NELC) encompass a wide spectrum of tumors, from the low-grade typical carcinoid (TC) and atypical carcinoid (AC), to the high-grade large cell neuroendocrine carcinoma (LCNEC) and the small-cell lung carcinoma (SCLC). Low-grade NELC are indolent, while high-grade NELC invade and metastasize rapidly. Biomarkers of NELC aggressiveness remain to be determined. Epithelial to mesenchymal transition (EMT) genes profile show promise as indicator of invasion and metastasis. Our aim was to evaluate: (1) EMT gene expression in NELC, (2) its relationship with the histologic subtypes, and (3) its impact on behavior of the tumors. Methods: Patients with SCLC (n = 10), LCNEC (n=5), AC (n=2) and TC (n=7) were included. EMT gene expression was quantified with a quantitative real-time (RT)-PCR carried out on StepOnePlus™ Real-Time PCR System (Applied Biosystems) with RT2 Profiler PCR Array System (Qiagen, Dusseldorf, Germany). Associations of the gene signature and clinicopathologic features, as well as prognostic factors were evaluated. Results: A 13-gene signature (AHNAK, COL3A1, DSP, IL1RN, MSN, PDGFRB, SNAI1, SNAI3, TCF3, TGF-beta 1, TGF-beta 2, TGF-beta 3, and VIM) that was related to EMT was upregulated in tumor-tissue from all NELC patients, mainly in those with high-grade NELC. An increased expression of DSP, TCF3, and TGF-beta 2 genes were shown in high-grade when compared to low-grade NELC and associated with lymph node metastasis with statistical significance respectively for DSP (p<0.05), TGF-beta 3 (p<0.05), and marginal significance for TCF3 (p=0.08). TCF3 was also associated with tobacco history (p=0.04). Conclusion: The EMT analysis identified genes involved in cell proliferation, motility, invasion, and metastasis of NELC. We further inferred DSP, TCF3, and TGF-beta 3 as target against lung cancer metastasis and invasion, thus arising as a promising therapeutic agent. Citation Format: Tabatha Gutierrez Prieto, Vanessa Karen de Sá, Eloisa Helena Ribeiro Olivieri, Eduardo Caetano Albino da Silva, Rui Manuel Reis, Dirce Maria Carraro, Vera Luiza Capelozzi. Genes of epithelial-mesenchymal transition (EMT) reveal impact on progression and metastasis of neuroendocrine lung carcinomas: A preliminary study [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A83.

Abstract A82: Epithelial-mesenchymal transition (EMT) genes are involved in the behavior and aggressiveness of neuroendocrine lung carcinomas (NELC) and non-small cell lung cancer (NSCLC): A comparative analysis

Abstract Background: Recurrence and metastasis are responsible for 90% of deaths of patients with lung cancer. Adenocarcinomas (ADc) primarily invade blood vessels with distant metastasis, whereas squamous cell carcinoma (SqCC) involves the mediastinal lymph nodes. Neuroendocrine carcinomas of low grade (typical and atypical carcinoid) are indolent, while high-grade NE carcinoma (large-cell NE and small-cell carcinomas) metastasize rapidly. Biomarkers of invasiveness in lung carcinomas still cannot be definitely determined. Epithelial to mesenchymal transition (EMT) genes profile have promise as indicator of invasion and metastasis. Our aim was to compare EMT gene expression in NELC, ADc, and SqCC and its impact on behavior of these tumors. Methods: EMT gene expression was quantified with a quantitative real-time (RT)- PCR carried out on StepOnePlus™ Real-Time PCR System (Applied Biosystems) with RT2 Profiler PCR Array System (Qiagen, Dusseldorf, Germany). Results: Younger patients expressed higher amount of AHNAK, MSN, and TGF-beta 3 than older (p<0.05), whereas SNAI3 and TGF-beta 2 were more expressed in smokers (p<0.05). ADc and SqCC presented significant higher expression of COL3A1, DSP, and MSN in tumor compared to normal tissue (p<0.05). 13-gene signature (AHNAK, COL3A1, DSP, IL1RN, MSN, PDGFRB, SNAI1, SNAI3, TCF3, TGF-beta 1, TGF-beta 2, TGF-beta 3, and VIM) was upregulated in tumor tissue from all NELC patients. In ADc and NELC, AHNAK, IL1RN, SNAI3, and VIM was significantly different (p<0.05). ADc and SqCC compared with high-grade NELC also presented differences in COL3A1, DSP, and PDGFR (p<0.01). Advanced tumors, usually with metastasis, showed higher expression of AHNAK, MSN, SNAI3, TGF-beta 3, and VIM (p<0.05), as well as association with poor outcome (p <0.01). Conclusion: Different expression of EMT gene signature in neuroendocrine lung carcinomas and non-small cell lung cancer, its relationship with histologic types, advanced stage, lymph node metastasis, and death suggest a possible role of these markers in this malignancy, but more importantly provide a potential biomolecular marker to predict outcome. The correlation between NELC, ADc, SqCC, and specific EMT genes involved in cell proliferation and motility provides a possible role of these genes in the development and aggressiveness in these tumors. Moreover, the difference of gene expression between NSCLC and NELC emerges as a promising biomarker of behavior. Further studies are needed to validate EMT gene expression to predict prognosis and tumoral aggressiveness. Citation Format: Tabatha Gutierrez Prieto, Vanessa Karen de Sá, Eloisa Helena Ribeiro Olivieri, Eduardo Caetano Albino da Silva, Rui Manuel Reis, Dirce Maria Carraro, Vera Luiza Capelozzi. Epithelial-mesenchymal transition (EMT) genes are involved in the behavior and aggressiveness of neuroendocrine lung carcinomas (NELC) and non-small cell lung cancer (NSCLC): A comparative analysis [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A82.

P3.15-006 Comprehensive Analysis of EMT Gene Signature in Primary and Metastatic Small Cell and Non-Small Cell Carcinomas of the Lung

Metastasis are responsible for the death of 90% of patients with lung cancer (LC) indicating the necessity to know the multiple signaling pathways involved. Among them, high-grade neuroendocrine lung carcinomas (NELC) invade and metastasize rapidly. Therefore, biomarkers of aggressiveness in LC remain to be determined, especially in NELC. Epithelial to mesenchymal transition (EMT) genes profile emerge promise as indicator of invasion and metastasis. The aim was to investigate the expression of EMT markers and assessed their relationship with the clinicopathological features and prognosis.

Cytohistology of papillary carcinoid and emerging concept of pulmonary neuroendocrine neoplasms.

This timely review starts by reporting the clinical, cytologic and histologic features of a morphologic variant of pulmonary carcinoid tumor forming exclusively of papillae. This growth pattern is so rare that it was not included in 2014 WHO classification of pulmonary neuroendocrine neoplasms. The current concept is reviewed, and example of spindle cell carcinoid, atypical carcinoid, large cell neuroendocrine carcinoma, and small cell carcinoma are illustrated with fine needle aspiration cytology, surgical and clinical follow-up. Finally, the new findings in cell biology and molecular biology that led to the emerging concept that carcinoids and high-grade neuroendocrine lung carcinomas are separate biological entities are reviewed and summarized in a tumorigenic module.

Altered pattern of Cul‐1 protein expression and neddylation in human lung tumours: relationships with CAND1 and cyclin E protein levels

The Cul-1 protein is the scaffold element of SCF complexes that are involved in the proteasomal degradation of numerous proteins regulating cell cycle progression. Owing to this central role in cell growth control, aberrant expression of the components of SCF is thought to play a role during tumourigenesis. Nothing is known about Cul-1 expression in human tumours. In this study, we have analysed its status in a series of 128 human lung carcinomas, comprising 50 non-small cell lung cancers (NSCLCs; 29 squamous cell carcinomas and 21 adenocarcinomas) and 78 neuroendocrine (NE) lung tumours (24 typical and atypical carcinoids, 19 large cell NE carcinomas and 35 small cell lung carcinomas), using immunohistochemistry. We report for the first time an altered pattern of Cul-1 expression in human tumours; indeed, we show that Cul-1 expression is up-regulated in 40% (51/128) of all lung tumours as compared to normal lung tissues, including 34% (17/50), 75% (18/24) and 30% (16/54) of NSCLCs, carcinoids and high grade neuroendocrine lung carcinomas, respectively. Furthermore, we demonstrate that high levels of Cul-1 protein are associated with a low KI67 proliferative index (p = 0.005) and with a decrease in the cyclin E oncoprotein (p = 0.0003), one of the major targets of SCF complexes. These data suggest that up-regulation of Cul-1 could protect cells from hyperproliferative signals through cyclin E down-regulation. Cul-1 is modified by neddylation, a post-translational modification that grafts ubiquitin-like Nedd8/Rub1 residues and controls Cul-1 activity. We also provide evidence that neddylated forms of Cul-1 are specifically expressed in high-grade NE lung tumours and are associated with down-regulation of the Cul-1 inhibitor CAND1 (p = 0.03) and a high level of cyclin E (p = 0.0002). These data support the notion that alterations in the Cul-1 neddylation/deneddylation pathway could contribute to the development of these highly aggressive lung tumours.

E2F-1, Skp2 and cyclin E oncoproteins are upregulated and directly correlated in high-grade neuroendocrine lung tumors

The transcription factor E2F-1 plays a crucial role in the control of cellular growth. We previously reported its differential pattern of expression in human lung tumors. In this study, we have investigated the relationships linking the status of E2F-1 and a mediator of its proteasomal degradation, the S-phase kinase-associated protein 2 (Skp2) F-box protein. Using immunohistochemistry in a series of 129 lung tumors of all histological types, we demonstrate that Skp2 accumulates preferentially in high-grade neuroendocrine (HGNE) lung carcinomas (86%, P<0.0001), and show that Skp2 overexpression is associated with advanced stages (P<0.0001) and nodal metastasis (P<0.0001) in neuroendocrine (NE) lung tumors. Unexpectedly, we observe that Skp2 and E2F-1 expression directly correlates in NE lung tumors (P<0.0001). Moreover, using cellular models, we identify Skp2 as a new E2F-1 transcriptional target. Furthermore, we also provide evidence that Skp2 interacts physiologically with E2F-1 and stimulates its transcriptional activity toward the cyclin E promoter. Consistently, we demonstrate that cyclin E expression directly correlates with Skp2 (P<0.0001) and E2F-1 (P=0.0001) status in NE lung tumors. Overall, our data provide the first evidence of a direct and functional interconnection between the E2F-1, Skp2 and cyclin E oncoproteins in HGNE lung carcinomas.