Large Cell Carcinoma Research Articles

Distribution of KRAS, DDR2, and TP53 gene mutations in lung cancer: An analysis of Iranian patients.

PurposeLung cancer is the deadliest known cancer in the world, with the highest number of mutations in proto-oncogenes and tumor suppressor genes. Therefore, this study was conducted to determine the status of hotspot regions in DDR2 and KRAS genes for the first time, as well as in TP53 gene, in lung cancer patients within the Iranian population.Experimental designThe mutations in exon 2 of KRAS, exon 18 of DDR2, and exons 5–6 of TP53 genes were screened in lung cancer samples, including non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) using PCR and sequencing techniques.ResultsAnalysis of the KRAS gene showed only a G12C variation in one large cell carcinoma (LCC) patient, whereas variants were not found in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) cases. The Q808H variation in the DDR2 gene was detected in one SCC sample, while no variant was seen in the ADC and LCC subtypes. Variations in the TP53 gene were seen in all NSCLC subtypes, including six ADC (13.63%), seven SCC (15.9%) and two LCC (4.54%). Forty-eight variants were found in the TP53 gene. Of these, 15 variants were found in coding regions V147A, V157F, Q167Q, D186G, H193R, T211T, F212L and P222P, 33 variants in intronic regions rs1625895 (HGVS: c.672+62A>G), rs766856111 (HGVS: c.672+6G>A) and two new variants (c.560-12A>G and c.672+86T>C).ConclusionsIn conclusion, KRAS, DDR2, and TP53 variants were detected in 2%, 2.17% and 79.54% of all cases, respectively. The frequency of DDR2 mutation is nearly close to other studies, while KRAS and TP53 mutation frequencies are lower and higher than other populations, respectively. Three new putative pathogenic variants, for the first time, have been detected in Iranian patients with lung cancer, including Q808H in DDR2, F212L, and D186G in coding regions of TP53. In addition, we observed five novel benign variants, including Q167Q, P222P and T211T in coding sequence, and c.560-12A>G and c.672+86T>C, in intronic region of TP53. Mutations of KRAS and DDR2 were found in LCC and SCC subtypes, respectively, whereas mutations of TP53 were seen in SCC and ADC subtypes with higher frequencies and LCC subtype with lower frequency. Therefore, Iranian lung cancer patients can benefit from mutational analysis before starting the conventional treatment. A better understanding of the biology of these genes and their mutations will be critical for developing future targeted therapies.

Preoperative thrombocytosis in surgically treated patients with non-small cell lung cancer.

Introduction Lung cancer is the most common cause of cancer‑related death. Accurate and easy‑to‑use prognostic factors are necessary. Increased platelet count might be a potential prognostic factor. Objectives We aimed to investigate the relationship between thrombocytosis and stage of lung cancer and to assess the frequency and clinical importance of thrombocytosis in this patient group. Patients and methods We retrospectively analyzed hospital records of consecutive patients with non-small cell lung cancer (NSCLC) who underwent curative‑intent pulmonary resections. Results Of 323 patients, 285 patients with NSCLC were selected (mean [SD] age, 66.55 [8.52] years; men, 63.86%). Squamous cell carcinoma was diagnosed in 130 patients (45.61%); adenocarcinoma, in 128 (44.91%); large cell carcinoma, in 16 (5.61%); and adenosquamous carcinoma, in 11 (3.86%). The prevalence of preoperative thrombocytosis in the whole sample was 10.18% (n = 29). Anemia was more common in patients with thrombocytosis compared with those without thrombocytosis (65.52% vs 30.08%; P <0.001). Thrombocytosis was found in 22.41% of patients with stage III+IV cancer and in 3.82% of those with stage I (P <0.001). Moreover, in patients with no metastases (N0, M0 according to the 7th edition of the TNM classification), thrombocytosis was more frequent in the group with stage II than in that with stage I cancer (3.85% vs 20.00%; P = 0.002). Thrombocytosis was also more frequent in patients with N2 than with N1 disease (9.76% vs 23.81%; P = 0.09). Conclusions Thrombocytosis is often observed in patients with NSCLC and is significantly associated with the higher stage of disease.

Comprehensive analysis of marker gene detection and computed tomography for the diagnosis of human lung cancer.

Non-small cell lung cancer (NSCLC) is one of leading causes of cancer-associated mortality, with a high number of cases caused by metastasis. The early diagnosis of cancer contributes to the successful treatment of patients with lung cancer. The aim of the present study was to analyze the efficacy of marker gene detection and computed tomography (CT) in diagnosing human lung cancer. Lung cancer marker genes, including carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), tissue polypeptide antigen (TPA), pro-gastrin-releasing peptide (ProGRB), cytokeratin fragment 21-1 (Cyfra21-1) and neuron-specific enolase (NSE), were analyzed in patients with lung cancer. The tumor size was evaluated using CT, and the association between lung serum levels of marker gene protein expression and tumor size was investigated. A total of 328 patients with lung cancer were identified, including 204 adenocarcinoma, 75 large cell carcinoma and 49 squamous cell carcinoma cases. All patients were indicated to have a high serum level of CEA, CA125, TPA, ProGRB, Cyfra21-1 and NSE, compared with the normal range. Immunohistochemistry demonstrated higher expression levels of CEA, CA125, TPA, ProGRB, Cyfra21-1 and NSE in lung tumor tissues, compared with the normal range. Results indicated that CT was able to diagnose tumor size for patients with lung cancer. The CEA and CA125 expression levels were associated with CT-diagnosed adenocarcinoma tumor size. Large cell carcinoma tumor size was associated with serum levels of CEA, TPA and ProGRB. Results indicated that Cyfra21-1 and NSE were associated with the squamous cell carcinoma cases, as demonstrated using CT. In conclusion, these results indicated that comprehensive analysis of marker gene detection and CT results may be used to diagnose human lung cancer.

The expression level of CSDAP1 in lung cancer and its clinical significance.

Expression level of messenger RNA (mRNA) of cold shock domain protein A intronless pseudogene (CSDAP1) in lung cancer tissues was studied. Fresh pathological specimens collected from 317 patients with primary lung cancer through surgical resection from January 2007 to January 2012 were selected. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis were used to detect the transcription and translation of CSDAP1 in lung cancer tissues and cancer-adjacent normal tissues, and the results were analyzed in combination with clinicopathological features and prognosis of lung cancer. Among 317 lung cancer specimens, 105 cases (33.1%) had high expression of CSDAP1. Among 138 cases of pulmonary adenocarcinoma, 59 cases had high expression of CSDAP1, and the high expression rate was 42.8%. Among 170 cases of lung squamous cell carcinoma, 46 cases had high expression of CSDAP1, and the high expression rate was 27.5% (P<0.05). Three cases of large cell carcinoma and 9 cases of small cell carcinoma had extremely low expression or had no expression of CSDAP1. Among the 127 lung cancer patients with regional lymph node metastasis, 53 cases (41.7%) had high expression of CSDAP1, while among the 190 lung cancer patients without regional lymph node metastasis, 52 cases (27.4%) had high expression of CSDAP1 (P<0.05). The results also revealed that the expression of CSDAP1 was also related to tumor-node-metastasis (TNM) staging of lung cancer. One-year, three-year and five-year survival rates of lung cancer patients who had no expression of CSDAP1 were relatively high (P<0.05). The results suggested that CSDAP1 may play an important role in the occurrence, development and judgement of prognosis of lung cancer.

MiR-23b suppresses lung carcinoma cell proliferation through CCNG1.

Lung carcinoma with high incidence rate could be divided into four subtypes, including small cell carcinoma, squamous cell carcinoma, adenocarcinoma and large cell carcinoma. miR-23b has been reported to have a low expression and play major roles in abundant tumors, however there is little research in lung carcinoma and hence the purpose of this study was to explore the impact of miR-23b in lung carcinoma. The RNA level of miR-23b and cyclin G1 (CCNG1) was measured by reverse transcription quantitative PCR. Luciferase activity reporter assay was used to verify that CCNG1 is a target of miR-23b. MTT and Transwell assays were utilized to test the functional studies of miR-23b in lung cancer cells. In lung carcinoma and lung cancer cells miR-23b expression is low compared with that in paracancerous tissues and normal lung cells. Low miR-23b expression inhibited lung cancer cell proliferation measured by MTT assay. We applied luciferase reporter to determine whether CCNG1 is a target of miR-23b and there was a negative correlation between them. Moreover, interference with CCNG1 reduced the cell proliferation ability, which partially reversed function of miR-23b. miR-23b inhibited cell proliferation of lung cancer by directly targeting CCNG1. It is suggested that miR-23b/CCNG1 axis may present a new target for the treatment of lung cancer.

Abstract 1208: Is the decrease in the incidence of large cell carcinoma of the lung due to changes in classification towards adenocarcinoma

Abstract Background: Data from many populations show a decrease in the incidence of large cell carcinoma (LCC) of the lung while incidence of lung adenocarcinoma (AC) is decreasing to a lower rate or even increasing from the 1990s. We compared the changes in incidence of lung LCC and AC worldwide to test the hypothesis that at least part of the decline in LCC incidence was due to classification of these tumors as AC. Methods: We analyzed the annual percent change (APC) in the age-standardized rates (ASRs) of lung LCC and AC incidence between 1996 and 2007 in 78 high-quality cancer registries from Europe, Asia, North America, and South America, using linear regression. Results: Among women, the median APC of LCC incidence was -5.42, and that of AC incidence was -0.96. There was a positive correlation between the two measures (β=0.29; p=0.01). The APC of LCC incidence was negative in 64 populations (82.1%); that of AC incidence was negative in 50 populations (63.3%, p of the χ2 distribution = 0.07). Among women, the median APC for LCC and AC incidence was -5.06 and +2.11, respectively. The correlation between the two measures was 0.17 (p=0.03). The APC of LCC incidence was negative in 59 populations (76.6%); that of AC incidence was negative in 23 populations (29.9%, p of the χ2 distribution = 0.41). Conclusions: The decrease in LCC may be, in part, due to changes in histologic classification and triaging towards a diagnosis of adenocarcinoma. Citation Format: Dana Hashim, Ariana Znaor, William Travis, Paolo Boffetta. Is the decrease in the incidence of large cell carcinoma of the lung due to changes in classification towards adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1208.

Abstract 3980: Dual targeting mitochondrial and cytosolic one-carbon metabolism via the proton-coupled folate transporter with novel 5-substituted pyrrolo[3,2-d]pyrimidine antifolates

Abstract Cellular one-carbon (1C) metabolism generates a host of metabolites especially critical to cancer cells. Classical 1C inhibitors often primarily target a single enzyme, commonly leading to drug resistance and necessitating the development of novel compounds targeting multiple enzymes. We synthesized several novel 5-substituted pyrrolo[3,2-d]pyrimidine compounds (AGF291, AGF320, and AGF347) with transport by the proton-coupled folate transporter (PCFT), a folate transporter with a narrow physiological niche, but commonly expressed in many solid tumors. These analogs showed substantial inhibition in proliferation assays with Chinese hamster ovary sublines engineered to individually express PCFT or the ubiquitously-expressed reduced folate carrier (the major tissue folate transporter), as well as in the PCFT-expressing human cancer cell lines H460 (large cell lung carcinoma), HCT-116 (colorectal carcinoma), and MIA PaCa-2 (pancreatic ductal adenocarcinoma). Full abrogation of inhibitory effects for all compounds required co-treatment with both adenosine and glycine, suggesting dual-targeting of cytosolic de novo purine biosynthesis and mitochondrial 1C/glycine metabolism. For cytosolic de novo purine biosynthesis, lack of protection by 5-aminoimidazole-4-carboxamide (AICA) suggested AICA ribonucleotide formyltransferase (AICARFTase), the second folate-dependent enzyme, as the likely enzyme target. AICARFTase inhibition was confirmed in intact H460 and HCT-116 cells by measuring accumulation of AICAR by targeted metabolomics. Targeting of mitochondrial enzymes in H460 and HCT-116 cells was suggested by selective inhibition of incorporation of [3-14C]serine over [14C]formate into the purine intermediate [14C]formyl glycinamide ribonucleotide. [2,3,3-2H]Serine isotope label scrambling analysis confirmed the mitochondrial target to be serine hydroxymethyltransferase2 (SHMT2). SHMT2 is a potentially promising anticancer drug target as its expression is highly correlated with the malignant phenotype across a broad spectrum of cancers beyond lung and colon, including breast, glioma, and liver. Indeed, our initial in-vivo studies with AGF291 suggest potent efficacy toward both MIA PaCa-2 and H460 xenograft mouse models. Our studies demonstrate the potential for tumor-selective dual targeting of both mitochondrial and cytosolic 1C metabolism to overcome resistance to classical 1C inhibitors. Citation Format: Aamod S. Dekhne, Gregory S. Ducker, Josephine Frühauf, Khushbu Shah, Md. Junayed Nayeen, Adrianne Wallace-Povirk, Carrie O'Connor, Zhanjun Hou, Lisa Polin, Aleem Gangjee, Joshua D. Rabinowitz, Larry H. Matherly. Dual targeting mitochondrial and cytosolic one-carbon metabolism via the proton-coupled folate transporter with novel 5-substituted pyrrolo[3,2-d]pyrimidine antifolates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3980.

Abstract 2562: Affinity-enhanced T-cell receptor (TCR) for adoptive T-cell therapy targeting MAGE-A4

Abstract Melanoma-associated antigens-A (MAGE-A) are members of an extensive family of cancer/testis (CT) antigens. Although emerging studies highlight the potential role of MAGE-A family members in promoting cancer growth, the biological function of MAGE-A4 remains poorly understood. MAGE-A4 is an attractive target because it has a high frequency of expression in multiple solid tumors according to The Cancer Genome Atlas (https://cancergenome.nih.gov/). In an attempt to further characterize its expression in solid tumors, immunohistochemistry analysis was performed in 534 resected non-small cell lung carcinoma (NSCLC) cases, stage I to IV with clinicopathological information including overall survival and recurrence. MAGE-A4 expression was observed in 24% of all NSCLC cases, with higher frequency observed in squamous cell carcinoma (SCC) (51%) versus adenocarcinoma (8%). MAGE-A4 expression was observed in 23% of the primary tumors and 48% of the recurrent tumor samples. Log-rank (Mantel-Cox) analysis showed that histology diagnosis, pathological stage, and MAGE-A4 expression are independent prognostic factors in NSCLC. However, no MAGE-A4 prognostic value was observed when analyzing the SCC subtype. An HLA-A*02-restricted MAGE-A4 TCR specific for MAGE-A4230-239 peptide GVYDGREHTV was identified from a pool of parental TCRs and engineered to optimize specificity and activity (MAGE-A4c1032). TCR fine specificity was assessed initially by mapping the response of the MAGE-A4c1032T-cells to panels of synthetic peptides with combinatorial substitutions at each amino acid with every other possible amino acid (X-Scan), to identify potentially cross-reactive peptides in the human and common pathogen proteomes. Secondly, MAGE-A4c1032T-cells were screened against a wide panel of normal primary cells from multiple organ systems, induced pluripotent stem cell-derived cells (iCells), and autologous whole blood to test for off-target reactivity, and against a panel of EBV-derived B-lymphoblastic cell lines expressing a wide range of HLA molecules to assess the risk of alloreactivity. Alloreactivity was observed in antigen negative cells expressing HLA-A*02:05, and therefore this allele is exclusionary. MAGE-A4c1032T-cell potency was assessed by a variety of in vitro assays, including proliferation, IFN-γ release and cytotoxicity in response to antigen-positive tumor lines in 2D and 3D culture, and cytokine release in response to freshly prepared antigen-positive primary tumor material. In conclusion, MAGE-A4 is a promising target for SPEAR T-cell therapy, and clinical trial opened this year to treat patients with inoperable or metastatic (advanced) NSCLC (SCC, adenosquamous, or large cell carcinoma), ovarian, head and neck SCC, gastric, esophageal (SCC or adenocarcinoma), urothelial tumors and melanoma (NCT03132922). Citation Format: Joana Senra, Pamela Villalobos, Barbara Mino, Luisa Solis, Carmen Behrens, Joseph P. Sanderson, Manoj Saini, Andrew B. Gerry, Nicholas J. Pumphrey, Miguel Maroto, Boris Sepesi, Patrick Hwu, David S. Hong, Elizabeht A. Mittendorf, George S. Blumenschein, Ignacio I. Wistuba, Gwendolyn Binder-Scholl, Rafael Amado. Affinity-enhanced T-cell receptor (TCR) for adoptive T-cell therapy targeting MAGE-A4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2562.

Abstract 1187: Comparison of intra-tumor heterogeneity and clonal evolution across lung cancer subtypes by multi-region whole genome sequencing

Abstract Both lung squamous cell carcinoma (LUSC) and lung cancer with neuroendocrine features (LUNE; i.e., small cell carcinoma (SCLC), large cell carcinoma (LCNEC) and other subtypes) are associated with tobacco smoking and have a very poor prognosis. We sought to characterize their clonal architecture and illustrate how the evolutionary patterns differ across these lung cancer subtypes. We performed multi-region whole genome sequencing of 83 samples from 36 LUSC and 67 samples from 23 LUNE patients that had been resected before systemic therapy. Among somatic copy number alterations, we observed genome doubling intra-tumor heterogeneity (ITH) and massive genomic instability in both subtypes. For somatic nucleotide variants (SNVs), the total mutation burden was higher in LUSC (16.7 average number of mutations/Mb) than LUNE (12.7/Mb), but for SNVs ITH was higher in LUNE. Among driver genes, TP53, CDKN2A, CR1, and NRK in LUSC and TP53, KRAS, KMT2D, and RB1 in LUNE were frequently mutated and were always clonal. Evidence of positive selection was found for TP53, PTEN, KMT2D and KEAP1 in LUSC, and for TP53 and KRAS in LUNE. As expected, the dominant mutation signatures in LUSC clones included signatures 4, 1, and 5, while APOBEC signatures (2 and 13) were predominantly found in sub-clones. In contrast, no evidence of APOBEC signatures were found in LUNE. Among structural variants, LUNE had more events (339.25 average number of events/tumor) than LUSC (206.7/tumor), with considerable ITH in both subtypes, particularly in LUNE. In LUNE, the dominant mechanism responsible for SVs included fork stalling and template switching within the microhomology-mediated break-induced repair (MMBIR). We found recurrent fusion events with hotspots including those previously identified near TTC28/CHEK2 (42% LUSC and 35% LUNE) and LRP1B (42% LUSC). Additional hotspots were found in chromosome bands 20p12.1 (22% LUSC), 1p31.1 (19% LUSC) and 12p11.22 (22% LUNE). We found &amp;gt;10-fold higher transposon insertions (LINE1) in LUSC (71.9 average number of events/tumor) than LUNE (6.3/tumor). Our findings improve our evolutionary understanding of these lung cancer subtypes and warrant further investigations on the mechanisms involved in clonal genomic instability. Citation Format: Tongwu Zhang, Joshua Sampson, Pier Alberto Bertazzi, Angela Pesatori, Dario Consonni, Bin Zhu, Belynda Hicks, Xing Hua, Jianxin Shi, Kevin Brown, Stephen Chanock, Maria Teresa Landi. Comparison of intra-tumor heterogeneity and clonal evolution across lung cancer subtypes by multi-region whole genome sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1187.

Abstract 2534: Single-cell RNA sequencing reveals lung cancer-associated alterations in bronchial cell subpopulations in the mainstem bronchus

Abstract RATIONALE: We have previously shown that gene expression alterations in the mainstem bronchus epithelium reflects a physiologic response to cigarette smoke and can serve as a diagnostic biomarker for lung cancer. Furthermore, we have shown that single cell RNA sequencing (scRNA-Seq) can be used to detect smoking-associated gene expression alterations within specific airway epithelial and immune cell types. In this study, we use scRNA-Seq to profile the transcriptomes of single cells procured from the airways of patients with or without lung cancer in order to detect cancer-associated alterations within specific cell types. METHODS: We obtained bronchial brushings in the mainstem bronchus from ever smokers undergoing diagnostic bronchoscopy for suspect lung cancer. Patients were followed post-procedure until a final lung cancer diagnosis (n=5; 3 adenocaricinomas, 1 squamous cell carcinoma and 1 large cell carcinoma) or benign disease (n=7) was made. Single cells were isolated using FACS (n=190/donor) and RNA library preparation was performed using the CEL-Seq protocol followed by RNA-seq (75nt paired-end) on an Illumina Hiseq 2500. Latent Dirichlet allocation was used to identify transcriptomically distinct cellular subpopulations specific to the airways of patients with and without lung cancer. RESULTS: We detected distinct subpopulations of bronchial cells expressing known markers of basal (KRT5), ciliated (FOXJ1), club (SCGB1A1), and goblet (MUC5AC) epithelial cells. Cell type distributions differed between cancer status and within cancer sub-types. Specifically, patients with cancer had an increase in FOXJ1+ cells. Many of the genes in the previously reported diagnostic biomarker such as ATP12A and NKX3-1 are expressed in specific cell types. CONCLUSION: The scRNA-Seq data of the normal appearing airways of subjects with and without lung cancer suggests that the epithelial and immune cell types present and their relative proportions change with cancer status. Additionally, previously described lung cancer biomarker genes are expressed in a cell type specific manner indicating that future biomarker improvements may be made by examining subpopulations of cells. Analysis of a greater number of samples is needed to make robust conclusions regarding the changes associated with smoking, cancer, and cancer subtype in this sample population. Citation Format: Xingyi Shi, Grant Duclos, Joshua Campbell, Yaron Gesthalter, Patrick Autissier, Yves M. Dumas, Robert Terrano, Gang Liu, Marc E. Lenburg, Avrum Spira, Jennifer Beane. Single-cell RNA sequencing reveals lung cancer-associated alterations in bronchial cell subpopulations in the mainstem bronchus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2534.

Abstract 994: Cancer cells induce a protumorigenic senescent phenotype in fibroblasts through MMP1 but not autophagy in large cell carcinoma of the lung

Abstract Large cell carcinoma (LCC) is among the most aggressive histologic subtypes of non-small cell lung cancer, but the mechanisms underlying such aggressive nature remain unknown. We recently showed that fibroblasts from LCC patients exhibit premature senescence in vitro, and that co-culturing LCC cells (but not cancer cells from other lung cancer subtypes) with normal fibroblasts in transwells is sufficient to induce senescence in the latter in an oxidative stress-dependent manner, supporting that fibroblast senescence is induced by a secreted factor(s) from LCC cells. Remarkably, we also found that senescent fibroblasts secrete factors that stimulate the growth and invasion of LCC cells beyond the stimulation elicited by nonsenescent fibroblasts, revealing that fibroblast senescence may contribute to the aggressive nature of LCC. Whole-genome transcriptional profiling of a panel of lung cancer cell lines identified MMP1 among the genes with larger expression in LCC compared to other lung cancer subtypes. Since MMPs can induce oxidative stress, we examined whether the excessive MMP1 expression in LCC cells was involved in the induction of fibroblast senescence in co-cultures. Knocking down MMP1 in LCC cells was sufficient to abrogate fibroblast senescence in co-cultures as well as the growth and invasion enhancement elicited by the conditioned medium of fibroblasts in LCC cells. In contrast, autophagy, which has been previously associated with fibroblast senescence in breast cancer, was not upregulated in fibroblasts upon co-culture with LCC cells. These results support that the selective aberrant expression of MMP1 in LCC cells plays a major role in their ability to induce a protumorigenic senescent phenotype in adjacent fibroblasts through a mechanism that is independent of autophagy. Moreover, our observations identify MMP1 as a potential therapeutic target against the aberrant cancer cell-fibroblast crosstalk in LCC. Citation Format: Jordi Alcaraz, Marta Gabasa, Rafael Ikemori, Noemí Reguart, Evette Radisky, Derek Radisky. Cancer cells induce a protumorigenic senescent phenotype in fibroblasts through MMP1 but not autophagy in large cell carcinoma of the lung [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 994.

Abstract 2643: Increased copy number of EGFR gene is an independent outcome predictor in resected non-small cell lung cancer patients treated by adjuvant chemotherapy

Abstract Background: Lung cancers are the leading cause of cancer-related deaths worldwide. Surgical resection alone remains preferred treatment for early stage patients with non-small cell lung cancer (NSCLC), whereas surgery plus platinum-based adjuvant chemotherapy is recommended for stages II - IIIa. Despite wide spectrum of known genetic alterations, there is a lack of biomarkers predicting response to adjuvant therapy. The aim of our study was to determine the predictive role of selected gene alterations. Methods and patients: Formalin-fixed paraffin embedded tumor samples were obtained from 209 NSCLC patients with stage I to IIIa. Of these, 59 patients were treated by surgical resection only, the follow-up adjuvant chemotherapy was indicated in remaining 150 cases. There were 146 males and 63 females (mean age 66; range 29-82 years), tumor histology revealed 79 adenocarcinomas, 105 squamous cell carcinomas, 21 large cell carcinomas, two adenosquamous carcinomas and two sarcomatoid carcinomas. Fluorescent in situ hybridization (FISH) was used for evaluation of EGFR, CMYC, MET and FGFR1 gene copy numbers and ALK and ROS1 copy numbers and rearrangements, respectively. EGFR, KRAS and BRAF mutations were screened using Cobas EGFR Mutation Test (Roche), TheraScreen: KRAS Mutation kit (Qiagen) and BRAF p.Val600Glu kit (IntellMed). Results: In the group of 150 resectable NSCLC patients treated with platinum-based adjuvant therapy, increased copy number of EGFR gene predicted poor overall survival (OS) (p = 0.00001, hazard ratio (HR) 1.66, 95% confidence interval (CI) 1.32-2.10) as well as disease-free survival (DFS) (p = 0.0001, HR 1.54, 95% CI 1.23-1.91) in univariate analysis. In multivariate analysis adjusted to age and stage, the EGFR copy number was independent predictor of OS (p = 0.001) as well as DFS (p = 0.0004). In the group of 59 patients treated by surgical therapy only, no correlation of tested markers with clinical-pathological data was identified. Conclusions: Increased copy number of EGFR gene was found to be an independent predictor of outcome in NSCLC patients treated by adjuvant platinum-based therapy. Acknowledgment: This work was financially supported by NPU LO1304, TE02000058 and EATRIS-CZ. Citation Format: Vladimira Koudelakova, Radek Trojanec, Jana Potockova, Jiri Drabek, Jana Stranska, Petra Smickova, Pavla Kourilova, Ivona Grygarkova, Vitezslav Kolek, Jozef Skarda, Marian Hajduch. Increased copy number of EGFR gene is an independent outcome predictor in resected non-small cell lung cancer patients treated by adjuvant chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2643.

Molecular dissection of large cell carcinomas of the lung with null immunophenotype

The aim of this study was to subcategorise large cell carcinoma (LCC) with null immunophenotype according to the World Health Organization (WHO) Classification of 2015 into the existing groups of adenocarcinoma and squamous cell carcinoma by further molecular genetic analysis. Lineage-specific molecular alterations of these tumours could depict additional therapeutic approaches. We analysed a cohort of 35 LCC diagnosed according to the 2004 WHO classification and reclassified them according to the criteria of the 2015 WHO classification. Subsequently, tumours with a null immunophenotype were analysed by targeted next generation sequencing (42 marker genes including TP53, EGFR, KRAS, STK11 and SMARC4A) and fluorescence in situ hybridisation (ROS1, ALK). By applying the criteria of the 2015 WHO classification and subsequent molecular subtyping we could show that out of 35 previously diagnosed LCC, 16 cases could be reclassified into specific NSCLC subtypes using immunohistochemistry. Additionally, based on their mutational pattern, eight of the remaining 19 cases with null immunophenotype could be assigned as 'favour adenocarcinoma'. We demonstrate that molecular subtyping is helpful to further categorise LCC with null immunophenotype. Our findings argue for an algorithm including stratified molecular analysis of all respective cases.

Histologic subtyping of poorly-differentiated solid lung cancer with molecular testing for epidermal growth factor receptor mutation and ALK gene rearrangement: an analyses of 167 cases

Objective: To study the histological subtyping of poorly differentiated solid lung cancer by using immunohistochemistry and mucin staining along with analysis of epidermal growth factor receptor (EGFR) mutation and anaplastic lymphoma kinase (ALK) gene rearrangement. Methods: Among 827 cases of non-small cell lung cancer at Beijing Hospital from April 2014 to April 2017, 167 cases of solid poorly differentiated lung cancer were identified and histopathologically subtyped by mucin staining (D-PAS) and immunohistochemistry using 10 antibodies (CK7, vimentin, Ki-67, CK5/6, p40, TTF1, Napsin A, CD56, chromogranin A, and synaptophysin). Paraffin embedded tumor samples were subjected to mutation analysis of exons 18, 19, 20 and 21 of the EGFR gene by amplification refractory mutation system (ARMS) method. Immunohistochemistry (Ventana D5F3) for ALK gene rearrangement was performed followed by ALK fluorescence in situ hybridization (FISH) verification. Results: There were 79 females and 88 males in the study cohort. The patient's age ranged from 35 to 77 years (mean 62 years). Cases with solid growth pattern (at least >10%) and without typical histological features of adenocarcinoma, squamous cell carcinoma or neuroendocrine carcinoma were further divided based on immunohistochemistry and mucin stain into 64 cases(38.32%)of adenocarcinoma, 34 cases(20.35%) squamous cell carcinoma, 21 cases(12.57%)large cell neuroendocrine carcinoma, 5 cases(2.99%)combined large cell neuroendocrine carcinoma, 2 cases(1.20%)adenosquamous carcinoma and 41 cases(24.55%)large cell carcinoma. The Ki-67 positive rate ranged from 5% to 65%. Mutations of EGFR were detected in 5 cases (2.99%, 5/167) of adenocarcinoma(19del in 3 cases and L858R in 2 cases). Two cases(1.20%, 2/167) with ALK-rearranged were identified by immunohistochemistry (Ventana D5F3) and confirmed by ALK FISH. Conclusions: Poorly differentiated solid lung cancer without distinct morphological features can be further histologically subtyped by mucin staining and immunohistochemistry. Molecular testing should be performed for accurate molecular target therapy to improve the prognosis.

Landscape of transcriptional deregulation in lung cancer

BackgroundLung cancer is a very heterogeneous disease that can be pathologically classified into different subtypes including small-cell lung carcinoma (SCLC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC) and large-cell carcinoma (LCC). Although much progress has been made towards the oncogenic mechanism of each subtype, transcriptional circuits mediating the upstream signaling pathways and downstream functional consequences remain to be systematically studied.ResultsHere we trained a one-class support vector machine (OC-SVM) model to establish a general transcription factor (TF) regulatory network containing 325 TFs and 18724 target genes. We then applied this network to lung cancer subtypes and identified those deregulated TFs and downstream targets. We found that the TP63/SOX2/DMRT3 module was specific to LUSC, corresponding to squamous epithelial differentiation and/or survival. Moreover, the LEF1/MSC module was specifically activated in LUAD and likely to confer epithelial-to-mesenchymal transition, known important for cancer malignant progression and metastasis. The proneural factor, ASCL1, was specifically up-regulated in SCLC which is known to have a neuroendocrine phenotype. Also, ID2 was differentially regulated between SCLC and LUSC, with its up-regulation in SCLC linking to energy supply for fast mitosis and its down-regulation in LUSC linking to the attenuation of immune response. We further described the landscape of TF regulation among the three major subtypes of lung cancer, highlighting their functional commonalities and specificities.ConclusionsOur approach uncovered the landscape of transcriptional deregulation in lung cancer, and provided a useful resource of TF regulatory network for future studies.

The role of prophylactic cranial irradiation in surgically resected combined small cell lung cancer: a retrospective study.

Combined small cell lung cancer (C-SCLC) is defined as small cell lung cancer (SCLC) combined with any of non-small cell lung cancer (NSCLC) histological types, such as large cell carcinoma, squamous cell carcinoma, or adenocarcinoma. Since C-SCLC is an increasingly recognized subtype of small cell carcinoma, we conducted a retrospective study in our institution to explore the value of prophylactic cranial irradiation (PCI) in patients with C-SCLC treated by surgery. Between 2005 and 2014, the records of all consecutive patients with pathologically diagnosed C-SCLC after surgery in our institution were reviewed. Overall survival (OS), disease-free survival (DFS), and brain metastasis free survival (BMFS) were estimated by Kaplan-Meier method. Survival differences were evaluated by log-rank test, while multivariate analysis was performed by a Cox proportional hazards model. Of the total 91 patients included in this analysis, 11 patients (12.1%) were in PCI group and 80 (87.9%) in non-PCI group. The 5-year cumulative incidence of brain metastasis in the whole group was 22.2% (26.3% in non-PCI group vs. 0% in PCI group), and 5-year OS rate was 44.1%. Patients treated with PCI had significantly longer OS (P=0.011) and DFS (P=0.013), also had the trend to live a longer BMFS with marginal significance (P=0.092) than non-PCI-treated patients. The multivariate analysis showed that PCI [hazard ratio (HR) =0.102, P=0.024] was one of independent prognostic factors of the OS in surgery-treated C-SCLC patients. C-SCLC patients have a relative high risk of developing brain metastases based on our study. These data showed that PCI could improve OS and DFS, as well as tend to decrease brain metastases in surgically resected C-SCLC. However, whether PCI could be part of comprehensive treatment modalities in C-SCLC should be assessed in prospective studies.

Multiregion gene expression profiling reveals heterogeneity in molecular subtypes and immunotherapy response signatures in lung cancer

AbstractIntra-tumor heterogeneity may be present at all molecular levels. Genomic intra-tumor heterogeneity at the exome level has been reported in many cancer types, but comprehensive gene expression intra-tumor heterogeneity has not been well studied. Here, we delineated the gene expression intra-tumor heterogeneity by exploring gene expression profiles of 35 tumor regions from 10 non-small cell lung cancer tumors (three or four regions/tumor), including adenocarcinoma, squamous cell carcinoma, large-cell carcinoma, and pleomorphic carcinoma of the lung. Using Affymetrix Gene 1.0 ST arrays, we generated the gene expression data for every sample. Inter-tumor heterogeneity was generally higher than intra-tumor heterogeneity, but some tumors showed a substantial level of intra-tumor heterogeneity. The analysis of various clinically relevant gene expression signatures including molecular subtype, epithelial-to-mesenchymal transition, and anti-PD-1 resistance signatures also revealed heterogeneity between different regions of the same tumor. The gene expression intra-tumor heterogeneity we observed was associated with heterogeneous tumor microenvironments represented by stromal and immune cells infiltrated. Our data suggest that RNA-based prognostic or predictive molecular tests should be carefully conducted in consideration of the gene expression intra-tumor heterogeneity.

Klotho expression and nodal involvement as predictive factors for large cell lung carcinoma.

IntroductionKlotho has been recently described as a carcinogenesis suppressor. Large cell neuroendocrine lung carcinoma (LCNEC) is a rare, highly malignant neoplasm. In the light of increasing incidence of neuroendocrine tumours, biomarkers predicting survival are needed. We consider that Klotho might be one.Material and methodsWe analysed records of all patients diagnosed with LCNEC, atypical carcinoid and typical carcinoid operated on in our institution between 2007 and 2015. Initially, we found 134 cases. Forty-six specimens were unattainable and thus excluded from research. All patients diagnosed with LCNEC according to the WHO classification were included in the study. Immunohistochemical staining for Klotho was performed. We retrospectively reviewed patient charts and analysed multiple variables.ResultsPositive staining for Klotho was present in 36 tissue specimens, while 12 patients were Klotho-negative. Survival length was significantly higher in Klotho-positive cases (p = 0.024), while advanced nodal status (N1 and N2) represented a marker of poor outcome (p = 0.011). In multivariate analysis, both Klotho presence (p = 0.015; HR = 0.37; 95% CI: 0.17–0.86) and nodal involvement (p = 0.007; HR = 3.04; 95% CI: 1.37–6.82) were independent prognostic factors. Tumour vessel invasion and visceral pleura infiltration were not associated with worse treatment results. Klotho presence predicted a favourable prognosis in these groups (p = 0.018; p = 0.007).ConclusionsOur results suggest that Klotho might be a positive factor for predicting survival in LCNEC and nodal involvement a negative one. Thus, these two markers may assist in the selection of subjects with unfavourable prognosis and to personalise therapy regimens.

Epigenetic diagnostic biomarkers for non-small cell lung cancer: present and future perspectives

Non-small cell lung cancer (NSCLC), of which the main histological types are squamous cell carcinoma (SCC), adenocarcinoma, and large cell carcinoma (LCC), continues to be a major health problem worldwide. Despite several crucial breakthroughs in treatment, the 5-year overall survival rate of NSCLC patients (less than 15%) is still far from satisfactory. The poor prognosis is due in part to the lack of early diagnostic biomarkers. However, progress is being made in this area, particularly with respect to epigenetic markers. Epigenetic regulation of gene expression, which is defined as regulation that occurs without altering the DNA sequence, is involved in the pathology of numerous cancers, including NSCLC. Thus, specific aberrant epigenetic changes are potential biomarkers for the early detection and diagnosis of this disease. In this review, we provide insight into the clinical application of two types of epigenetic regulators: DNA methylation and non-coding RNAs (ncRNAs), both of which play crucial roles in NSCLC tumorigenesis and could be useful diagnostic markers and/or therapeutic targets for NSCLC.

Thin-Walled Cavitary Lung Adenocarcinoma: A Rare Unusual Case

: Thin-walled cavitary lung adenocarcinoma is not common. When cavitary lung cancer was classified as a histologic subtype, squamous cell carcinoma was the most common, followed by adenocarcinoma and finally large cell carcinoma. Cavitation was not found in small cell lung cancer. Some cases of thin-walled cavitary lung adenocarcinoma have been reported. Thin-walled cavitary lung cancer can be detected by the following radiological signs: irregular thickening of the cavity wall, wall nodule formation, the presence of compartments in the cavity, increased standard uptake values (SUVs) on positron emission tomography (PET). A thin‑walled cavity accompanied by mediastinal lymph node or distant metastasis, or enlargement of the lesion on long term follow-up. We report on a case of thin walled cavitary lung cancer whose cavity tended to enlarge during a 10-month follow-up, but no other evidence of imaging finding indicated potential lung cancer. It suggests that the unidirectional check-valve mechanism could make enlarging thin walled cavitary lesion due to lepidic growth pattern.