Expression In Small Cell Lung Cancer Research Articles

Synergistic alterations in the multilevel chromatin structure anchor dysregulated genes in small cell lung cancer

Small cell lung cancer (SCLC) is an aggressive form of lung cancer that uniquely changes the chromosomal structure, although the basis of aberrant gene expression in SCLC remains largely unclear. Topologically associated domains (TADs) are structural and functional units of the human genome. Genetic and epigenetic alterations in the cancer genome can lead to the disruption of TAD boundaries and may cause gene dysregulation. To understand the potential regulatory role of this process in SCLC, we developed the TAD boundary alteration–related gene identification in tumors (TARGET) computational framework, which enables the systematic identification of candidate dysregulated genes associated with altered TAD boundaries. Using TARGET to compare gene expression profiles between SCLC and normal human lungfibroblast cell lines, we identified >100 genes in this category, of which 24 were further verified in samples from patients with SCLC using NanoString. The analysis revealed synergistic chromatin structure alteration at the A/B compartment and TAD boundary levels that underlies aberrant gene expression in SCLC. TARGET is a novel and powerful tool that can be used to explore the relationship of chromatin structure alteration to gene dysregulation related to SCLC tumorigenesis, progression, and prognosis.

Profilin 2 promotes growth, metastasis, and angiogenesis of small cell lung cancer through cancer-derived exosomes

Small cell lung cancer (SCLC) is highly aggressive and prone to hypervascular metastases. Recently, we found profilin 2 (PFN2) expression in SCLC but not in normal tissues. Furthermore, PFN2 expression had been shown to promote angiogenesis through exosomes. However, it remains unclear whether PFN2 contributes to the progression and metastasis of SCLC through angiogenesis. We report here that overexpression (OE) of PFN2 increased, whereas its knockdown (KD) decreased the proliferation, migration, and invasion of SCLC cell H446. The exosomes from OE-H446 (SCLC-OE-exo) exhibited similar effects on H446 properties. Culturing of endothelial cells (ECs) in SCLC-OE conditioned medium (CM) or SCLC-OE-exo increased the migration and tube formation ability of ECs, whereas SCLC-KD-CM and SCLC-KD-exo had inhibitory effects. Interestingly, both SCLC- and EC-derived exosomes were internalized in H446 more rapidly than in ECs. More importantly, OE-PFN2 dramatically elevated SCLC growth and vasculature formation as well as lung metastasis in tumor xenograft models. Finally, we found that PFN2 activated Smad2/3 in H446 and pERK in ECs, respectively. Taken together, our study revealed the role of PFN2 in SCLC development and metastasis, as well as tumor angiogenesis through exosomes, providing a new molecular target for SCLC treatment.

Tumour PD-L1 Expression in Small-Cell Lung Cancer: A Systematic Review and Meta-Analysis.

Antibodies against programmed death-1 (PD-1), and its ligand, (PD-L1) have been approved recently for the treatment of small-cell lung cancer (SCLC). Although there are previous reports that addressed PD-L1 detection on tumour cells in SCLC, there is no comprehensive meta-analysis on the prevalence of PD-L1 expression in SCLC. We performed a systematic search of the PubMed, Cochrane Library and EMBASE databases to assess reports on the prevalence of PD-L1 expression and the association between PD-L1 expression and overall survival (OS). This meta-analysis included 27 studies enrolling a total of 2792 patients. The pooled estimate of PD-L1 expression was 26.0% (95% CI 17.0–37.0), (22.0% after removing outlying studies). The effect size was significantly heterogeneous (I2 = 97.4, 95% CI: 95.5–98.5, p < 0.0001).Positive PD-L1 expression was a favourable prognostic factor for SCLC but not statistically significant (HR = 0.86 (95% CI (0.49–1.50), p = 0.5880; I2 = 88.7%, p < 0.0001). Begg’s funnel plots and Egger’s tests indicated no publication bias across included studies (p > 0.05). Overall, there is heterogeneity in the prevalence of PD-L1 expression in SCLC tumour cells across studies. This is significantly moderated by factors such as immunohistochemistry (IHC) evaluation cut-off values, and assessment of PD-L1 staining patterns as membranous and/or cytoplasmic. There is the need for large size, prospective and multicentre studies with well-defined protocols and endpoints to advance the clinical value of PD-L1 expression in SCLC.

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.

Long Noncoding RNA H19 Facilitates Small Cell Lung Cancer Tumorigenesis Through miR-140-5p/FGF9 Axis.

PurposeLong noncoding RNAs (lncRNAs) are emerging as gene regulators to drive many important cancer phenotypes through interaction with microRNAs. There have been numerous data about upregulation of H19 and its strong oncogenic function in progression of cancers. However, the function and detailed mechanisms of H19 on small cell lung cancer (SCLC) are still unclear.MethodsIn this study, we investigated H19 expression in SCLC and para-carcinoma tissues. We also explored the function and detailed mechanisms of H19 on SCLC cells via RT-PCR, transwell assay, Western blot, dual-luciferase report assay and RNA pull-down experiments.ResultsIn this study, we observed that H19 was upregulated in SCLC compared with para-carcinoma tissues or NSCLC tissues. We also uncovered that H19 could promote proliferation and migration of SCLC cells. Functional investigation illustrated that H19 acted as a sponge for miR-140-5p to regulate its expression in SCLC. Interestingly, we further found that H19 upregulated FGF9 expression to promote SCLC progression via sponging miR-140-5p. H19 and FGF9 were also revealed to have similar expression patterns in clinical SCLC samples.ConclusionThese data demonstrated that H19 might be a promising prognostic and therapeutic target for SCLC.

SOX2 mediates cisplatin resistance in small-cell lung cancer with downregulated expression of hsa-miR-340-5p.

BackgroundThis study is aimed to unravel the genetic factors associated with microRNA (miRNA) expression in regulating sex‐determining region Y‐box 2 (SOX2)‐mediated cisplatin resistance in small‐cell lung cancer (SCLC).MethodsThe relevance of SOX2 expression in SCLC was analyzed in a panel of SCLC cells by quantitative real‐time PCR (qPCR) and western blot (WB). We selected DMS114 cell line, in which SOX2 was amplified via lentiviral vector‐mediated transfection of the SOX2 genes and tested for the half‐maximal inhibitory concentration (IC50) by MTS assay. High‐throughput sequencing and screening of differentially expressed miRNAs between SOX2‐overexpressing and normal control cells were performed. Finally, miRanda software was used to verify the miRNAs bound with SOX2 and qPCR was used to identify the expression of miRNAs which were binding with SOX2.ResultsCisplatin‐resistant SOX2‐overexpressing DMS114 cell lines were successfully developed, showing a statistically significant increase in SOX2 expression by qPCR and WB. Our results showed a typically higher IC50 value in SOX2‐overexpressing cells compared with the negative controls. The high‐throughput sequencing analysis revealed that 68 miRNAs were upregulated and 24 miRNAs were downregulated in the SOX2‐overexpressing cells. The 24 downregulated miRNAs were further verified. Of them, a cancer‐related miRNA, hsa‐miR‐340‐5p, showed a higher binding affinity with SOX2 in network regulation mapping, which was also found to be markedly downregulated under qPCR analysis.ConclusionWe demonstrated that downregulated expression of hsa‐miR‐340‐5p may affect cisplatin resistance by mediating SOX2 expression in SCLC cells, which may provide a potential target for the therapy of chemoresistant SCLCs.

High expression of GLI1 is associated withbetter survival in advanced SCLC.

Aberrant Sonic hedgehog (Shh) pathway signaling has been described in small cell lung cancer (SCLC), as well discrepancies, when analyzing expression of pathway components in SCLC cell lines vs tumor biopsies. Shh key component GLI1was evaluated in advanced SCLC and data correlated with patient survival. GLI1expression was analyzed by quantitative real-time polymerase chain reaction in pre-treatment fresh frozen tumor biopsies of 12advanced SCLC patients and mRNA level of GLI1was compared in short-term vs long-term survivor's samples (stratified by median survival, independent samples t-test). Expression of GLI1 mRNA was significantly higher in long-term (> 9.6months, n = 6) survivor's biopsies than in short-term (≤ 9.6months, n = 6) survivors (p = 0.0196, 95%CI: 0.000016to0.000147, two-tailed independent samples t-test). High GLI1mRNA expression in SCLC was found to be positive prognostic marker associated with longer survival. Further research is needed for validation of these results due to the small number of patients in the study.

Characterization of PD-L1 protein expression and CD8+ tumor-infiltrating lymphocyte density, and their associations with clinical outcome in small-cell lung cancer.

This study aimed to characterize programmed death ligand-1 (PD-L1) expression and CD8+ tumor-infiltrating lymphocytes (TILs) density, and their impact on survival in patients with surgically resected small-cell lung cancer (SCLC). Fifty-six patients with surgically resected SCLC were included. PD-L1 protein expression and CD8+ TILs were tested by immunohistochemistry. A meta-analysis of 15 articles with 1,505 patients that investigated the prevalence and prognostic significance of PD-L1 expression in SCLC was conducted. Twenty-two (39.3%) patients had positive PD-L1 protein expression and 42 (75.0%) had high CD8+ TILs density. PD-L1 expression level was not associated with CD8+ TILs density (P=0.528). No any association between clinicopathological features and PD-L1 expression level or CD8+ TILs density was observed. Positive PD-L1 expression [hazard ratio (HR) =0.374, P=0.002] and high CD8+ TILs density (HR =0.429, P=0.008) were independently associated with significantly longer overall survival (OS), which remain the statistical significance in multivariate analyses (P=0.007, P=0.002; respectively). Meta-analysis showed that the prevalence of positive PD-L1 expression was 0.35 [95% confidence interval (CI), 0.22-0.48] and positive PD-L1 expression was correlated with markedly longer OS (HR =0.61; 95% CI, 0.31-0.91) in patients with SCLC. The prevalence of PD-L1 expression in surgically resected SCLC is lower than that published for NSCLC. There was no association between PD-L1 expression or CD8+ TILs density and clinicopathological parameters. PD-L1 expression and CD8+ TILs density was independently correlated with better outcome in patients with SCLC.

Abstract B064: The Aurora kinase A-inhibitor, alisertib, is a potential candidate for combination with immunotherapy in small cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is characterized by a loss of TP53 and RB1 and by amplification of MYC in about 25% of patients. Our group and others have shown that MYC-driven SCLC cell lines are vulnerable to Aurora Kinase A (AURKA) inhibition. Alisertib, a selective AURKA inhibitor, has also shown clinical efficacy in MYC-positive relapsed SCLC patients in combination with paclitaxel in a phase II trial (NCT02038647). AURKA regulates DNA chromosome alignment during cell division and thus its inhibition results in mitotic stress and subsequently DNA damage. We have previously demonstrated that DNA damage response (DDR) inhibitors synergize with immune checkpoint inhibition in SCLC in vivo models by activating a STING-mediated innate immune response. Based on these findings, we hypothesized that AURKA inhibition may activate the STING pathway and induce PD-L1 expression in SCLC. Methods: We compared AURKA gene expression among a panel of SCLC and NSCLC cell lines and, similarly, in tumor versus normal tissue in SCLC clinical dataset (Sato et al.). In parallel, we explored the landscape of STING and immune genes expression in two SCLC clinical datasets (George et al. and Sato et al.).We tested human and GEMM-derived SCLC cell lines for changes in immune and DDR-related protein expression by Western blot analysis after treatment with the AURKA-i alisertib. Results: AURKA gene expression was higher in SCLC cell lines as compared to NSCLC cell lines (p=0.023) and in SCLC tumors compared to normal tissue (FC=1.6; p&amp;lt;0.001), thus making AURKA an attractive target in SCLC. Furthermore, in SCLC clinical datasets, we found that CXCL10 and CCL5, the two downstream chemokines of STING pathway, are highly expressed in about 30% SCLC tumors and their expression is significantly associated with CMYC (Spearman Rho&amp;gt;0.45, p&amp;lt;0.001), possibly due to high baseline levels of replication stress and also to CD274, CD8 and other markers and chemokines suggestive of an inflamed immune microenvironment. We observed that in vitro treatment with alisertib (1µM for 72 hours) further increases the level of phospho-H2AX protein, a marker of DNA damage, along with PD-L1. Furthermore, alisertib treatment resulted in STING pathway activation, as indicated by increased phospho-STING_S366 and phospho-TBK1_S172 protein levels, in both human and murine SCLC cells. Conclusions: Our results demonstrate the potential of alisertib, the selective AURKA inhibitor, in modulation of PD-L1 and STING pathway activation in SCLC cells, thus supporting the rationale for in vivo testing with immunotherapy drugs. In particular, evaluation of this strategy in MYC-positive SCLC models could open new therapeutic investigations in this highly resistant SCLC subgroup. Citation Format: Carminia M. Della Corte, Liz Lauren Ajpacaja, Robert J Cardnell, Carl M Gay, Lixia Diao, Qi Wang, Kavya Ramkumar, Allison C. Stewart, Rebecca B. Kow, Hannah E Stumpf, You-Hong Fan, Jing Wang, John V Heymach, Lauren A. Byers. The Aurora kinase A-inhibitor, alisertib, is a potential candidate for combination with immunotherapy in small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B064. doi:10.1158/1535-7163.TARG-19-B064

Ascl1-induced Wnt11 regulates neuroendocrine differentiation, cell proliferation, and E-cadherin expression in small-cell lung cancer and Wnt11 regulates small-cell lung cancer biology

The involvement of Wnt signaling in human lung cancer remains unclear. This study investigated the role of Wnt11 in neuroendocrine (NE) differentiation, cell proliferation, and epithelial-to-mesenchymal transition (EMT) in human small-cell lung cancer (SCLC). Immunohistochemical staining of resected specimens showed that Wnt11 was expressed at higher levels in SCLCs than in non-SCLCs; 58.8% of SCLC, 5.2% of adenocarcinoma (ADC), and 23.5% of squamous cell carcinoma tissues stained positive for Wnt11. A positive relationship was observed between Achaete-scute complex homolog 1 (Ascl1) and Wnt11 expression in SCLC cell lines, and this was supported by transcriptome data from SCLC tissue. The expression of Wnt11 and some NE markers increased after the transfection of ASCL1 into the A549 ADC cell line. Knockdown of Ascl1 downregulated Wnt11 expression in SCLC cell lines. Ascl1 regulated Wnt11 expression via lysine H3K27 acetylation at the enhancer region of the WNT11 gene. Wnt11 controlled NE differentiation, cell proliferation, and E-cadherin expression under the regulation of Ascl1 in SCLC cell lines. The phosphorylation of AKT and p38 mitogen-activated protein kinase markedly increased after transfection of WNT11 into the SBC3 SCLC cell line, which suggests that Wnt11 promotes cell proliferation in SCLC cell lines. Ascl1 plays an important role in regulating the Wnt signaling pathway and is one of the driver molecules of Wnt11 in human SCLC. Ascl1 and Wnt11 may employ a cooperative mechanism to control the biology of SCLC. The present results indicate the therapeutic potential of targeting the Ascl1–Wnt11 signaling axis and support the clinical utility of Wnt11 as a biological marker in SCLC.

Defining the role of RUNX1T1 in "combined" small cell lung cancer.

e20101 Background: The RUNX1T1 gene, also known as ETO, was first identified in the fusion transcript AML1/ETO, generated by the translocation between chromosome 8 and 21, which occurs in 12%-15% of AML. We recently discovered that RUNX1T1 was specifically amplified only in the small cell lung cancer (SCLC) but not the non-small cell lung cancer (NSCLC) component of combined SCLC tumors. Here, we investigated if RUNX1T1 expression plays a role in transforming NSCLC to SCLC in combined SCLC tumor formation. Methods: We examined tumors from 92 of our SCLC patients by targeted exome and CNV analyses. RUNX1T1 mRNA expression was determined in various cancers by analysis of the CCLE and TCGA databases. RUNX1T1 mRNA expression was detected by RNAscope analysis of another 22 patient SCLC tumors. Stable overexpression of RUNX1T1 in NSCLC and SCLC cell lines used commercial RUNX1T1 lentiviral constructs. The resulting stable cell lines were analyzed by RT-qPCR, western blotting, Affymetrix gene array, and E2F-luciferase assays. Results: We found that 2 of 2 patients with combined SCLC demonstrated RUNX1T1 amplification in the SCLC component. Notably, only 2 of the remaining 90 patients with pure SCLC demonstrated RUNX1T1 amplification. RUNX1T1 mRNA is highly expressed in SCLC compared to the majority of other cancers, including NSCLC, using either CCLE or TCGA databases. Western blots confirmed higher RUNX1T1 protein expression in SCLC vs NSCLC cell lines, and RNAscope analysis confirmed greater RUNX1T1 mRNA expression in SCLC vs NSCLC patient tumors. When we stably overexpressed RUNX1T1 in NSCLC cell lines, there was no increase in neuroendocrine protein expression, however we did find an increased sensitivity to etoposide. Bioinformatic analysis of microarray data demonstrated significantly altered E2F pathway activity, and we could show a significant increase in E2F-luciferase activity after RUNX1T1 overexpression. Interestingly, this was associated with decreased p21 expression, which is a putative suppressor of E2F activity. Conclusions: RUNX1T1 expression is specifically upregulated in SCLC compared to NSCLC. E2F pathway activity is elevated after RUNX1T1 overexpression, consistent with an increased SCLC phenotype.

P40 expression in small cell lung cancer: The presence of p40-positive cells does not always indicate squamous differentiation.

BackgroundSmall cell lung cancer (SCLC) is normally diagnosed with hematoxylin and eosin stains, although some cases require immunohistochemistry (IHC). P40 is highly sensitive and specific for squamous cell carcinoma and is thus considered the best marker for this cancer. However, the status of p40 expression in SCLC is not well known. The aim of this study was to analyze p40 expression in resected SCLC using IHC.MethodsForty‐four surgically resected SCLC cases were enrolled. Clinical data were obtained from the patients’ medical records. Pathologists blinded to the patients’ clinical data reviewed the SCLC slides. IHC was performed on a representative slide of each case.ResultsAlthough p40 was not diffusely expressed in any of the SCLC cases, p40‐positive cells were observed in the tumors in 15 cases (34.1%). Ten of these exhibited p40 in < 1% of tumor cells. In the remaining five cases, 1–5% of tumor cells expressed p40, and in three of these, the cells expressing p40 also expressed TTF‐1 and neuroendocrine markers. There was no statistically significant relationship between p40 positivity and any other clinicopathological characteristics.ConclusionsSome resected SCLCs express p40 focally. This result suggests that the presence of positive p40 cells does not exclude a diagnosis of SCLC. Thus, small biopsy or cytology specimens with p40‐positive cells must be diagnosed carefully.

The Epithelial Sodium Channel (αENaC) Is a Downstream Therapeutic Target of ASCL1 in Pulmonary Neuroendocrine Tumors

Small cell lung cancer (SCLC) is an aggressive neuroendocrine carcinoma, designated as a recalcitrant cancer by the National Cancer Institute, in urgent need of new rational therapeutic targets. Previous studies have determined that the basic helix-loop-helix transcription factor achaete-scute homolog 1 (ASCL1) is essential for the survival and progression of a fraction of pulmonary neuroendocrine cancer cells, which include both SCLC and a subset of non-SCLC. Previously, to understand how ASCL1 initiates tumorigenesis in pulmonary neuroendocrine cancer and identify the transcriptional targets of ASCL1, whole-genome RNA-sequencing analysis combined with chromatin immunoprecipitation-sequencing was performed with a series of lung cancer cell lines. From this analysis, we discovered that the gene SCNN1A, which encodes the alpha subunit of the epithelial sodium channel (αENaC), is highly correlated with ASCL1 expression in SCLC. The product of the SCNN1A gene ENaC can be pharmacologically inhibited with amiloride, a drug that has been used clinically for close to 50 years. Amiloride inhibited growth of ASCL1-dependent SCLC more strongly than ASCL1-independent SCLC in vitro and slowed growth of ASCL1-driven SCLC in xenografts. We conclude that SCNN1A/αENaC is a direct transcriptional target of the neuroendocrine lung cancer lineage oncogene ASCL1 that can be pharmacologically targeted with antitumor effects.

Identification of candidate responders for anti-PD-L1/PD-1 immunotherapy, Rova-T therapy, or EZH2 inhibitory therapy in small-cell lung cancer.

A useful candidate for small-cell lung cancer (SCLC) therapy is immune checkpoint blockade therapy targeting programmed death-1 (PD-1) and its ligand, PD-L1. Furthermore, rovalpituzumab tesirine (Rova-T), a delta-like protein 3 (DLL3)-targeted antibody-drug conjugate, and enhancer of zeste homologue 2 (EZH2) inhibitor are expected to be the first targeted therapy for SCLC. The aim of the present study was to evaluate PD-L1, DLL3 and EZH2 expression in SCLCs to find a candidate responder to those therapies. Immunohistochemical (IHC) staining for PD-L1, DLL3 and EZH2 was performed in 20 patients with SCLC and the clinicopathological characteristics and IHC staining intensity were compared. It was demonstrated that 1/20 patients (5.0%) exhibited positive PD-L1 expression in the metastatic lesions, as well as in the primary lung tumor. DLL3 was highly expressed in 14/20 patients (70%) and EZH2 was positive in 17/20 patients (85%). None of these cases exhibited any correlation with age, sex, smoking, stage or treatment, whereas IHC staining was able to identify candidate responders to anti-PD-L1/PD-1 immunotherapy, Rova-T therapy, or EZH2 inhibitor therapy.

Transcription factor E2F1 promotes EMT by regulating ZEB2 in small cell lung cancer

BackgroundEpithelial-mesenchymal transition (EMT) is an early event in tumour invasion and metastasis, and widespread and distant metastasis at early stages is the typical biological behaviour in small cell lung cancer (SCLC). Our previous reports showed that high expression of the transcription factor E2F1 was involved in the invasion and metastasis of SCLC, but the role of E2F1 in the process of EMT in SCLC is unknown.MethodsImmunohistochemistry was performed to evaluate the expressions of EMT related markers. Immunofluorescence was used to detect the expressions of cytoskeletal proteins and EMT related markers when E2F1 was silenced in SCLC cell lines. Adenovirus containing shRNA against E2F1 was used to knock down the E2F1 expression, and the dual luciferase reporter system was employed to clarify the regulatory relationship between E2F1 and ZEB2.ResultsIn this study, we observed the remodelling of cytoskeletal proteins when E2F1 was silenced in SCLC cell lines, indicating that E2F1 was involved in the EMT in SCLC. Depletion of E2F1 promoted the expression of epithelial markers (CDH1 and CTNNB1) and inhibited the expression of mesenchymal markers (VIM and CDH2) in SCLC cell lines, verifying that E2F1 promotes EMT occurrence. Next, the mechanism by which E2F1 promoted EMT was explored. Among the CDH1 related inhibitory transcriptional regulators ZEB1, ZEB2, SNAI1 and SNAI2, the expression of ZEB2 was the highest in SCLC tissue samples and was highly consistent with E2F1 expression. ChIP-seq data and dual luciferase reporter system analysis confirmed that E2F1 could regulate ZEB2 gene expression.ConclusionOur data supports that E2F1 promotes EMT by regulating ZEB2 gene expression in SCLC.

68Ga]Pentixafor-PET/CT for imaging of chemokine receptor 4 expression in small cell lung cancer--initial experience.

Chemokine receptor CXCR4 is a key factor for tumor growth and metastasis in several types of human cancer. This study investigated the feasibility of CXCR4-directed imaging of small cell lung cancer (SCLC) with positron emission tomography/computed tomography (PET/CT) using the radiolabelled chemokine ligand [68Ga]Pentixafor.10 patients with primarily diagnosed (n=3) or pre-treated (n=7) SCLC (n=9) or large cell neuroendocrine carcinoma of the lung (LCNEC, n=1) underwent [68Ga]Pentixafor-PET/CT. 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG, n=6) and/or somatostatin receptor (SSTR)-directed PET/CT with [68Ga]DOTATOC (n=5) and immunohistochemistry (n=10) served as standards of reference.CXCR4-PET was positive in 8/10 patients and revealed more lesions with significantly higher tumor-to-background ratios than SSTR-PET. Two patients who were positive on [18F]FDG-PET were missed by CXCR4-PET, in the remainder [68Ga]Pentixafor detected an equal (n=2) or higher (n=2) number of lesions. CXCR4 expression of tumor lesions could be confirmed by immunohistochemistry.Non-invasive imaging of CXCR4 expression in SCLC is feasible. [68Ga]Pentixafor as a novel PET tracer might serve as readout for confirmation of CXCR4 expression as prerequisite for potential CXCR4-directed treatment including receptor-radio(drug)peptide therapy.

Mitotic arrest deficient-like 1 is correlated with poor prognosis in small-cell lung cancer after surgical resection

Mitotic arrest deficient-like 1 (MAD1L1) whose dysfunction is associated with chromosomal instability plays a pathogenic role in a few human cancers. However, the status of MAD1L1 expression in small-cell lung cancer (SCLC) remains unknown. Immunohistochemistry was used to determine the expression of MAD1L1 protein in 32 lymph node metastasis (LN-M) tissues and 88 primary SCLCs compared with 32 adjacent noncancerous tissues. The associations of MAD1L1 protein expression with the clinicopathologic features and clinical outcomes in patients with SCLC were analyzed. The ratio of MAD1L1 positive expression was higher in primary SCLC tissues (39.8%) and LN-M tissues (46.9%) compared with adjacent noncancerous tissues (9.4%). MAD1L1 positive expression was associated with tumor-node-metastasis (TNM) stage (P = 0.003), International Association for the Study of Lung Cancer (IASLC) stage (P = 0.004), tumor size (P = 0.015), lymph node metastasis (P = 0.014), and recurrence (P < 0.001). Multivariate analysis suggested that MAD1L1 positive expression was an independent factor for overall survival (hazard ratio (HR) 2.002; 95% confidence interval (CI) 1.065-3.763; P = 0.031) and recurrence-free survival (HR 2.263; 95% CI 1.197-4.276; P = 0.012). To sum up, MAD1L1 positive expression may be associated with tumour progression and metastasis in SCLCs and may thus serve as a new biomarker for prognosis in these patients.

Abstract 558: Correlations of FGFR1 with ligand signaling in small-cell lung cancer

Abstract Introduction: Small cell lung cancer (SCLC) accounts for 15% of all lung cancers and has been under-studied relative to novel therapies. Signaling through fibroblast growth factors (FGF2, FGF9) and their high-affinity receptor (FGFR1) has recently emerged as a contributing factor in the pathogenesis and progression of non-small cell lung cancer (NSCLC). However, knowledge of FGFR1 and ligand expression in SCLC is limited. Methods: FGFR1 gene copy number, mRNA levels and protein expression were determined by silver in situ hybridization (SISH), mRNA ISH and immunohistochemistry (IHC) in primary tumors from 78 SCLC patients. Expression of the ligands FGF2 and FGF9 mRNA and protein were determined by mRNA ISH and IHC, respectively. Spearman's correlations were conducted using SAS 9.4. Results: FGFR1 protein expression by IHC demonstrated a significant correlation with FGFR1 gene copy number (p = 0.03) and FGFR1 mRNA levels (p&amp;lt;0.0001). The positivity of FGFR1 mRNA in the SCLC cohort was 21%. FGFR1 mRNA expression correlated with both FGF2 (p = 0.0001) and FGF9 (p = 0.0018) mRNA levels, as well as with FGF2 (0.0105) and FGF9 (0.0012) protein levels. There was no significant association between FGFR1 and ligands with clinical characteristics or prognosis. Conclusions: FGFR1 amplification and mRNA levels correlate with FGFR1 protein expression. The SCLC cohort has a substantially higher proportion of FGFR1 mRNA positive cases (21%) compared to FGFR1 gene copy number positive cases (7.8%). Combined analysis of FGFR1 and ligand expression may be valuable to determine which SCLC patients may respond to FGFR1 inhibitor therapy. Key Words: small cell lung cancer; FGFR1; FGF2; FGF9 Citation Format: Hui Yu, Andrzej Badzio, Theresa Boyle, Xian Lu, Christopher J. Rivard, Ashley Kowalewski, Brad Rikke, Kim Ellison, Leslie Rozeboom, Biftu Hassan, Fred R. Hirsch. Correlations of FGFR1 with ligand signaling in small-cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 558. doi:10.1158/1538-7445.AM2015-558

Abstract 2961: Characterization of methylation profiles reveals distinct epigenomic patterns in SCLC and NSCLC

Abstract Background: Small cell lung cancer (SCLC) is a highly lethal malignancy characterized by rapid growth, early metastasis and poor prognosis. SCLC shows distinct molecular and clinical features when compared to other lung cancer subtypes. Previous analyses by us and others have identified genomic and proteomic differences between SCLC and Non-Small Cell Lung Cancer (NSCLC). Epigenetic alterations are some of the earliest events that could also lead to oncogenic changes and thus play an essential role in tumor initiation and progression. However, epigenetic differences between SCLC and NSCLC contributing to the alterations in gene and protein expression patterns, distinct biological features and therapeutic response have not been well characterized. Here, we investigate the differences in the methylation patterns of SCLC and NSCLC to provide novel insights into epigenetic associated gene alterations to identify potential therapeutic targets in SCLC. Material and Methods: A genome-wide DNA methylation profiling of SCLC and NSCLC cell lines was used for this investigation. We correlated DNA methylation status with gene expression and protein expression levels in 31 SCLC and 73 NSCLC lines to identify the relationship of epigenetic with genomic and proteomic features distinguishing SCLC from NSCLC. Results: SCLC and NSCLC lines exhibited different methylation profiles and we identified 484 genes that had a significant inverse correlation between methylation status and mRNA expression levels (Rho ≤ -0.5 and FDR = 0.01), (“genes regulated by methylation,” GRM) that distinguished SCLC from NSCLC. Ingenuity pathway analysis of the 484 genes identified significant associations with neuregulin signaling, immune trafficking, integrin signaling, glioma invasiveness canonical pathways. Proteomic profiling by Reverse Phase Protein Array (RPPA) validated the different expression of some of the 484 genes identifying nine that were hypermethylated and downregulated at protein levels in SCLC compared to NSCLC lines (PTEN, CyclinD1, Caveolin, Notch3, TAZ, HSP27, STAT6 and both total and phosphorylated levels of receptor tyrosine kinases such Her2 and, MET). Conclusions: Genome wide methylation, mRNA expression, and detailed proteomic analyses have identified specific epigenic differences between SCLC and NSCLC that impact on important signaling pathways including widespread loss of PTEN function and receptor tyrosine kinase (RTK) expression in SCLCs which need to be considered in developing new rationale therapies for SCLC. Citation Format: Seema Mukherjee, Bonnie S. Glisson, John D. Minna, Robert J. Cardnell, Luc Girard, Adi Gazdar, Lixia Diao, Jing Wang, Lauren A. Byers. Characterization of methylation profiles reveals distinct epigenomic patterns in SCLC and NSCLC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2961. doi:10.1158/1538-7445.AM2015-2961

Somatostatin receptors in bronchopulmonary neuroendocrine neoplasms: new diagnostic, prognostic, and therapeutic markers.

Gastroenteropancreatic neuroendocrine neoplasms are known for their overexpression of somatostatin receptors (SSTRs), which provide the molecular basis for diagnostic and therapeutic interventions. In contrast, few data on the SSTR expression profile exist for bronchopulmonary neuroendocrine neoplasms (BP-NEN). A total of 240 formalin-fixed, paraffin-embedded specimens from 26 typical carcinoid (TC), 30 atypical carcinoid (AC), and 34 small cell lung cancer (SCLC) patients were examined retrospectively by immunohistochemistry (IHC) using specific rabbit monoclonal antibodies and evaluated by the immunoreactive score. Adjacent slides from 20 samples of each tumor type were subjected to additional RT-quantitative PCR mRNA analysis. With different expression patterns, SSTRs were present in most of the tumor sections, at both the protein and mRNA levels. The RT-quantitative PCR data correlated with the IHC scores. SSTR1 was detected in approximately 65% of the TC and AC, but hardly in the SCLC, whereas both SSTR2A and SSTR5 were present in approximately 45% of each entity. Furthermore, the SSTR1 expression level was positively correlated with patient survival. Our results suggest that SSTRs can be used as novel diagnostic, prognostic, and therapeutic markers of BP-NEN. The differences in the SSTR expression profile between the three types of BP-NEN may help to set a diagnostic cutoff and predict patient prognosis. Similar to TC and AC, our results also revealed a previously unappreciated high level of SSTR2A expression in SCLC within a subgroup of patients. However, in most cases, pan-somatostatin analogs may represent an additional therapeutic option.