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Background Oncogenic mutations in BRAF genes are found in approximately 5–10% of colorectal cancers. The majority of BRAF mutations are located within exons 11–15 of the catalytic kinase domains, with BRAF V600E accounting for more than 80% of the observed BRAF mutations. Sensitivity to BRAF- and mitogen-activated protein kinase (MEK) inhibitors varies depending on BRAF mutations and tumor cell types. Previously, we newly identified, BRAF L525R-mutation, in the activation segment of the kinase in colorectal cancer patient. Here, we characterized the function of the BRAF L525R mutation. Methods HEK293 cells harboring a BRAF mutation (V600E or L525R) were first characterized and then treated with cetuximab, dabrafenib, and selumetinib. Cell viability was measured using WST-1 assay and the expression of proteins involved in the extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) signaling pathways was evaluated using western blot analysis. Results The MEK inhibitor selumetinib effectively inhibited cell proliferation and ERK phosphorylation in BRAF L525R cells but not in BRAF V600E cells. Further studies revealed that AKT phosphorylation was reduced by selumetinib in BRAF L525R cells but not in BRAF V600E cells or selumetinib-resistant BRAF L525R cells. Moreover, the AKT inhibitor overcame the selumetinib resistance. Conclusions We established a model system harboring BRAF L525R using HEK293 cells. BRAF L525R constitutively activated ERK. AKT phosphorylation caused sensitivity and resistance to selumetinib. Our results suggest that a comprehensive network analysis may provide insights to identify effective therapies.

Tertiary lymphoid structures (TLS) are observed in several cancers and are associated with favorable prognosis. This study aimed to examine the clinicopathological, genetic, and gene expression profiles of lung adenocarcinoma patients with TLS.A total of 112 patients with pathological stage IB lung adenocarcinoma who underwent complete resection between 2011 and 2015 were enrolled in this study. We investigated whether TLS correlated with prognosis and programmed death-ligand 1 (PD-L1) expression. Furthermore, the correlation of TLS with tumor mutation burden (TMB) and genetic mutations was evaluated in patients for whom whole-exon sequencing data were available. In addition, using the Cancer Genome Atlas Lung Adenocarcinoma (TCGA-LUAD) dataset, gene expression analysis according to the TLS status was performed.Among the 112 patients, 49 were TLS-positive (TLS+). TLS+ correlated with longer recurrence-free survival (RFS) than TLS-negative cases (TLS-) (hazard ratio [HR], 0.47; 95 % confidence interval [CI]: 0.23-0.88, p = 0.02). In the multivariate analysis, TLS was a better independent prognostic factor for RFS (HR 0.37, 95 %CI 0.18-0.72, p 0.01). PD-L1 expression was not significantly different between TLS+ and TLS- patients (p = 0.54). TMB in TLS+ was similar to that in TLS- patients (p = 0.39); however, it tended to be lower than that in TLS- especially among smokers (p = 0.07). In gene expression analysis, RNA expression of chemokines related to lymph node formation, such as CXCL13, CCL19 and CCL21, was significantly higher, and biological processes such as positive regulation of humoral immune response and regulation of antigen receptor-mediated signaling pathway were enhanced in TLS+.TLS was a favorable prognostic factor and was not associated with PD-L1 expression in patients with lung adenocarcainoma. Moreover, gene expression analysis indicated that TLS is a site for the generation and regulation of antitumor immune responses.

Supplementary Table S1. Mutation signature analysis. Supplementary Table S2. Baseline characteristics of the transdifferentiated SCLC patients included in this study. Supplementary Table S3. Intensity of EGFR and Ki67 staining in lung cancer tissues obtained from the indicated patients. Supplementary Table S4. Baseline characteristics of the primary SCLC patients included in this study. Supplementary Table S5. Genetic alterations of the indicated lung cancer cell lines. Supplementary Figure S1. FGF9 immunohistochemistry staining in lung cancer cell lines. Supplementary Figure S2. List of the mutated genes before and after transdifferentiation. Supplementary Figure S3. Gene ontology term analysis in the context of upregulated (A) and downregulated (B) genes. Supplementary Figure S4. Computed tomography images of the patients (Case2-4) during the course of gefitinib therapy. Supplementary Figure S5. EGFR, Ki67 and FGFR1 immunohistochemistry staining of tissues from Cases #5 and #6. Supplementary Figure S6. mRNA expression levels of FGF family genes in lung cancer cell lines. Supplementary Figure S7. Expression of FGF9 and FGF2 in human NSCLC and SCLC cells. Supplementary Figure S8. Functional evaluations of FGF9 in human SCLC cell lines. Supplementary Figure S9. Tumor volumes after MLE12-EGFR transplantation. Supplementary Figure S10. Expression levels of Fgfr family genes in MLE12-FGF9 cells in vitro and in vivo. Supplementary Figure S11. Characteristics of lung adenocarcinoma cell lines. Supplementary Figure S12. In vivo evaluations of FGF9 in H2087 and H2110 cells.

Supplemental Experimental Procedures

Transdifferentiation of lung adenocarcinoma to small cell lung cancer (SCLC) has been reported in a subset of lung cancer cases that bear EGFR mutations. Several studies have reported the prerequisite role of TP53 and RB1 alterations in transdifferentiation. However, the mechanism underlying transdifferentiation remains understudied, and definitive additional events, the third hit, for transdifferentiation have not yet been identified. In addition, no prospective experiments provide direct evidence for transdifferentiation. In this study, we show that FGF9 upregulation plays an essential role in transdifferentiation. An integrative omics analysis of paired tumor samples from a patient with transdifferentiated SCLC exhibited robust upregulation of FGF9. Furthermore, FGF9 upregulation was confirmed at the protein level in four of six (66.7%) paired samples. FGF9 induction transformed mouse lung adenocarcinoma-derived cells to SCLC-like tumors in vivo through cell autonomous activation of the FGFR pathway. In vivo treatment of transdifferentiated SCLC-like tumors with the pan-FGFR inhibitor AZD4547 inhibited growth. In addition, FGF9 induced neuroendocrine differentiation, a pathologic characteristic of SCLC, in established human lung adenocarcinoma cells. Thus, the findings provide direct evidence for FGF9-mediated SCLC transdifferentiation and propose the FGF9–FGFR axis as a therapeutic target for transdifferentiated SCLC.Significance:This study demonstrates that FGF9 plays a role in the transdifferentiation of lung adenocarcinoma to small cell lung cancer.

This file includes six supplementary Tables. Supplementary Table S1:List of primer sequences. Supplementary Table S2:The calculated IC50 values of PC9 parental and H1975 parental, and corresponding resistant clones. Supplementary Table S3:Summary of whole exome sequencing data. Supplementary Table S4:Number of tags in PIK3CA G118D position in H1975 parental and resistant clones. Supplementary Table S5:The results of the FISH analysis in PC9 and PC9-COR#9 cells. Supplementary Table S6:Number of tags in EGFR E746_A750del position in PC9 parental and resistant clones.

supplemental legend

Purpose: Although large-cell neuroendocrine carcinoma (LCNEC) of the lung shares many clinical characteristics with small-cell lung cancer (SCLC), little is known about its molecular features. We analyzed lung LCNECs to identify biologically relevant genomic alterations.Experimental Design: We performed targeted capture sequencing of all the coding exons of 244 cancer-related genes on 78 LCNEC samples [65 surgically resected cases, including 10 LCNECs combined with non–small cell lung cancer (NSCLC) types analyzed separately, and biopsies of 13 advanced cases]. Frequencies of genetic alterations were compared with those of 141 SCLCs (50 surgically resected cases and biopsies of 91 advanced cases).Results: We found a relatively high prevalence of inactivating mutations in TP53 (71%) and RB1 (26%), but the mutation frequency in RB1 was lower than that in SCLCs (40%, P = 0.039). In addition, genetic alterations in the PI3K/AKT/mTOR pathway were detected in 12 (15%) of the tumors: PIK3CA 3%, PTEN 4%, AKT2 4%, RICTOR 5%, and mTOR 1%. Other activating alterations were detected in KRAS (6%), FGFR1 (5%), KIT (4%), ERBB2 (4%), HRAS (1%), and EGFR (1%). Five of 10 cases of LCNECs combined with NSCLCs harbored previously reported driver gene alterations, all of which were shared between the two components. The median concordance rate of candidate somatic mutations between the two components was 71% (range, 60%–100%).Conclusions: LCNECs have a similar genomic profile to SCLC, including promising therapeutic targets, such as the PI3K/AKT/mTOR pathway and other gene alterations. Sequencing-based molecular profiling is warranted in LCNEC for targeted therapies. Clin Cancer Res; 23(3); 757–65. ©2016 AACR.

EGFR-mutated lung cancers account for a significant subgroup of non–small cell lung cancers overall. Third-generation EGFR tyrosine kinase inhibitors (TKI) are mutation-selective inhibitors with minimal effects on wild-type EGFR. Acquired resistance develops to these agents, however, the mechanisms are as yet uncharacterized. In this study, we report that the Src–AKT pathway contributes to acquired resistance to these TKI. In addition, amplification of EGFR wild-type alleles but not mutant alleles was sufficient to confer acquired resistance. These findings underscore the importance of signals from wild-type EGFR alleles in acquiring resistance to mutant-selective EGFR-TKI. Our data provide evidence of wild-type allele-mediated resistance, a novel concept of acquired resistance in response to mutation-selective inhibitor therapy in cancer treatment. Cancer Res; 77(8); 2078–89. ©2017 AACR.

This file includes eight supplementary Figures. Supplementary Figure S1:Flow cytometric data for PC9 and PC9-COR cells treated with DMSO or rociletinib. Supplementary Figure S2:Results of human phospho-RTK array experiments in PC9, H1975, and corresponding resistant cells. Supplementary Figure S3:The results of MTS cell proliferation assays for third-generation EGFR-TKIs resistant clones. Supplementary Figure S4:Copy number variants (CNVs) in PC9 and H1975 resistant cells analyzed from exome sequence data. Supplementary Figure S5:Sequence reads for KRAS in PC9-AZDR#4, PC9-AZDR#5, and PC9 parental cells. Supplementary Figure S6:Loss of heterozygosity (LOH) in H1975 resistant cells. Supplementary Figure S7:Efficacy of rociletinib and cetuximab or afatinib co-treatment with H1975 cells expressing wild type or C797S. Supplementary Figure S8:Summary of the mechanisms underlying acquired resistance to third-generation EGFR-TKIs.

Supplementary Table 1- List of the 244 cancer related genes of the target capturing panel Supplementary Table 2 - The coverage statistics for each sample of large cell neuroendocrine carcinoma (LCNEC) Supplementary Table 3 - The coverage statistics for each sample of advanced small cell lung cancer (SCLC) Supplementary Table 4 - Validation of the mutations detected in this study using the Oncomine® Cancer Research Panel (OCP) Supplementary Table 5- Primer Sequences: Sanger Sequencing Supplementary Table 6 - Antibody for immunohistochemical staining Supplementary Table 7 - Candidate somatic mutations of large cell neuroendocrine carcinoma (LCNEC) Supplementary Table 8 - Candidate somatic mutations in advanced small cell lung cancer (SCLC) Supplementary table 9 - Hotspot mutations detected in small cell lung cancer (SCLC) Supplementary Table 10- Variant frequency of candidate somatic mutations of large cell neuroendocrine carcinoma (LCNEC) combined with non-small cell lung cancer (NSCLC) Supplementary Table 11- Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) analysis for the top six most frequently mutated genes in large cell neuroendocrine carcinoma (LCNEC)

Supplementary figure legends

Supplementary Fig. S1. The mean baseline Levels of PIP3 (38:4) per mg tissue in xenograft models transplanted with SCLC cells Supplementary Fig. S2. Metabolic alteration in SCLC cells treated with gedatolisib in vitro and in vivo Supplementary Fig. S3. Comparative metabolic profiling in LAD and SCLC cell lines Supplementary Fig. S4. Gene expression alteration after treatment with PI3K/mTOR inhibitor Supplementary Fig. S5. Schematic diagram of metabolic linkage Supplementary Fig. S6. Addition of 5''-deoxy-5''-(methylthio)-adenosine (MTA) reversed drug sensitivity from a resistant to a sensitive phenotype Supplementary Fig. S7. HPRT1 expression was downregulated by an HPRT1-specific siRNA in SCLC cells

Supplementary Table S1. The pathway classification of metabolites used for the MSEA Supplementary Table S2. Genetic profiles in 42 SCLC cell lines Supplementary Table S3. Quantitative phospho-peptide analysis in H1048 cells with or without Gedatolisib treatment Supplementary Table S4. Quantitative phosphatidyl lipids analysis in SCLC cell Supplementary Table S5. Quantitative phosphatidyl lipids analysis in H1048 cells Supplementary Table S6. 116 metabolites in SCLC cells with or without Gedatolisib treatment Supplementary Table S7. 116 metabolites in H1048 and SBC5 with or without Gedatolisib treatment Supplementary Table S8. Metabolite set enrichment analysis (MSEA) to identify significantly enriched metabolic pathways related to gedatolisib Supplementary Table S9. Amount of 116 metabolites in SCLC tissues

Supplementary methods for Sanger sequencing, copy number analysis with quantitative PCR, and Immunohistochemical staining

Comprehensive genomic analysis has revealed that the PI3K/AKT/mTOR pathway is a feasible therapeutic target in small-cell lung carcinoma (SCLC). However, biomarkers to identify patients likely to benefit from inhibitors of this pathway have not been identified. Here, we show that metabolic features determine sensitivity to the PI3K/mTOR dual inhibitor gedatolisib in SCLC cells. Substantial phosphatidyl lipid analysis revealed that a specific phosphatidylinositol (3,4,5)-trisphosphate (PIP3) subspecies lipid product PIP3 (38:4) is predictive in assessing sensitivity to PI3K/mTOR dual inhibitor. Notably, we found that higher amounts of purine-related aqueous metabolites such as hypoxanthine, which are characteristic of SCLC biology, lead to resistance to PI3K pathway inhibition. In addition, the levels of the mRNA encoding hypoxanthine phosphoribosyl transferase 1, a key component of the purine salvage pathway, differed significantly between SCLC cells sensitive or resistant to gedatolisib. Moreover, complementation with purine metabolites could reverse the vulnerability to targeting of the PI3K pathway in SCLC cells normally sensitive to gedatolisib. These results indicate that the resistance mechanism of PI3K pathway inhibitors is mediated by the activation of the purine salvage pathway, supplying purine resource to nucleotide biosynthesis. Metabolomics is a powerful approach for finding novel therapeutic biomarkers in SCLC treatment.Significance: These findings identify features that determine sensitivity of SCLC to PI3K pathway inhibition and support metabolomics as a tool for finding novel therapeutic biomarkers. Cancer Res; 78(9); 2179–90. ©2018 AACR.

Supplementary Figure 1: Representative example of tissue microarray (TMA) punched out from a combined large-cell neuroendocrine carcinoma (LCNEC) with adenocarcinoma. Supplementary Figure 2: Validation of five mutations on the PI3K/AKT/mTOR pathway by Sanger sequencing (LCNEC 68c: PIK3CA E545K, LCNEC 9c: PTEN K342*, LCNEC 13c: PTEN S59*, LCNEC 4c: RICTOR R910H, LCNEC 11p: MTOR E2419K). Supplementary Figure 3: Correlation of gene copy number, calculated using the total depth on the covered region of each of 244 targeted genes and those obtained from the Oncomine® Cancer Research Panel (OCP). Supplementary Figure 4: Validation of the copy number gains or amplifications of 13 samples (8 LCNEC samples, 1 SCLC biopsy samples and 4 SCLC surgically resected cases) for MYCL1, MYC and FGFR1 by a quantitative real-time polymerase chain reaction (qPCR). Supplementary Figure 5: Overview of the key driver alterations and other activating alterations in small cell lung cancer (SCLC). Genetic alterations in the PI3K/AKT/mTOR pathway were detected in 24 (17%) of the tumors: PIK3CA (4%), PTEN (6%), AKT2 (2%) and RICTOR (6%). Copy number gains of each MYC family member were mutually exclusive. Supplementary Figure 6: Relationship between RB1 mutation and IHC staining of RB and p16. The mutual exclusivity of the protein expression between RB and p16 was distinct. Supplementary Figure 7: Relationship between genetic alteration and protein expression in large-cell neuroendocrine carcinoma (LCNEC) of the lung. Comparison of the over expression of receptor tyrosine kinases (RTKs) with the genetic alterations in resected LCNEC specimens (51 of 65 resected cases) revealed no significant relationship between strongly positive RTKs (KIT, EGFR, IGF1R, KDR, ERBB2) expression and genetic alterations.

The purpose of this study was to investigate the clinicopathological, gene expression and genetic features of stage I lung adenocarcinoma with necrosis.We retrospectively reviewed 521 cases with pathologic stage I lung adenocarcinoma resected by lobectomy and lymph node dissection. We calculated the ratio of tumor necrotic area by digital image analysis and investigated the relationship between tumor necrosis and prognosis. Furthermore, we analyzed the differentially expressed genes between cases with and without necrosis using The Cancer Genome Atlas Lung Adenocarcinoma (TCGA-LUAD) dataset. Using whole exon sequencing data (n = 97), we examined whether tumor necrosis correlates with single nucleotide variants (SNVs) and driver mutations.Eighty four (16%) cases of the study cohort had tumor necrosis. The presence of necrosis significantly correlated with poorer prognosis (5-year overall survival: 91.9% vs. 75.4%, p 0.001; 5-year recurrence-free survival: 86.0% vs. 59.0%, p 0.001); however, the ratio of necrotic area did not correlate with prognosis. In multivariable analysis, invasive component size, vascular invasion, and tumor necrosis were independently associated with a higher risk of recurrence (hazard ratio, 1.652; 95% confidence interval, 1.033-2.641; p = 0.036). Gene expression analysis of TCGA stage I lung adenocarcinoma revealed enrichment of biological processes, such as cell cycle and response to hypoxia, in cases with necrosis. The cases with tumor necrosis had more SNVs than those without tumor necrosis (p = 0.027), especially in smokers.Stage I lung adenocarcinoma with tumor necrosis has worse prognosis than that without, and has distinctclinicopathological features in terms of gene expression and genetic features.

Somatic mutations in human epidermal growth factor receptor 2 (HER2) are present in approximately 3% of breast cancers. Some HER2 mutations are activating, and they represent a mechanism of resistance to conventional anti-HER2 therapies such as trastuzumab and lapatinib. Consistently, in patients with HER2-amplified breast cancer, these mutations are predominantly observed in metastatic tumors obtained after exposure to anti-HER2 systemic therapies, possibly after clonal selection. Therefore, it is rare to find coexistent HER2 mutation and amplification in the early clinical course, and thus, the clinical relevance of HER2 mutation to the sensitivity to HER2-targeted drugs, particularly antibody-drug conjugates (ADCs) such as ado-trastuzumab emtansine (T-DM1) and the recently approved fam-trastuzumab deruxtecan (T-DXd), remains unclear. In this article, we describe a patient with de novo metastatic breast cancer who exhibited both HER2 amplification and the L755S mutation in the untreated primary breast tumor obtained at the initial diagnosis, and the lesion responded to T-DM1 and T-DXd after exhibiting clinical resistance to other HER2-targeted drugs. Our current case findings suggested that anti-HER2 ADCs should be prioritized over conventional trastuzumab- or lapatinib-based therapies for patients with HER2-amplified and comutated tumors. Key Points Although HER2 mutations were implicated in resistance to anti-HER2 monoclonal antibodies or HER2 tyrosine kinase inhibitors in preclinical studies, their clinical impact on sensitivity to anti-HER2 drugs is unclear owing to the rarity of concomitant HER2 mutation and HER2 amplification. A case of de novo metastatic breast cancer harboring both HER2 amplification and the L755S mutation in an untreated breast primary tumor displayed clinical resistance to standard trastuzumab- or lapatinib-based therapies but good responses to ado-trastuzumab emtansine (T-DM1) and fam-trastuzumab deruxtecan (T-DXd). Anti-HER2 antibody-drug conjugates such as T-DM1 and T-DXd may be prioritized over conventional trastuzumab- or lapatinib-containing therapies for patients with HER2-amplified and comutated tumors.

Tanaka et al. previously reported a case in which nivolumab for recurrent occupational cholangiocarcinoma resulted in complete response persisting for 26 months after discontinuation. Afterward, in that clinical trial, nivolumab was administered to two patients for recurrence. Both patients achieved complete response, suggesting that nivolumab is effective for occupational cholangiocarcinoma.

Mutational signatures associated with tobacco smoking (mutational smoking signatures: SS) are characterized mainly by C A mutations. The aim of this study was to characterize the association between the tumor immune microenvironment and the SS in lung adenocarcinoma.Lung adenocarcinomas surgically resected from 96 patients, for which whole exome sequencing data was available, were included in the study. We extracted the SS from whole exome sequencing data, calculated the weights of SS using deconstructSigs, and compared the clinicopathological features of SS positive (SS+) and negative (SS-) adenocarcinomas. We selected 18 tumor pairs from SS + and SS- adenocarcinomas (sex, EGFR mutation, and tumor size-matched) and examined the expression of five immune markers (CD20, CD8, FOXP3, CD204, and PD-L1) by immunohistochemistry.Of 96 specimens, there were 33 (34 %) SS + adenocarcinoma tumors. The smoking index significantly correlated with the weight of the SS (R = 0.43). Between SS + and SS- tumors, there was no significant difference in clinicopathological factors excluding smoking history. Immunohistochemistry revealed that the number of FOXP3 + T cells in SS + adenocarcinomas was significantly higher than that in the SS- adenocarcinomas (median number 58 vs. 36, p 0.01). Also, the number of CD20 + B cells in SS + adenocarcinomas was significantly higher than that in the SS- adenocarcinomas (median number 77 vs. 29, p 0.01); however; these phenomena could not be confirmed when stratified by smoking history.In lung adenocarcinoma, SS is associated with an immunosuppressive tumor microenvironment.

Next generation sequencing (NGS)-based tumor profiling identified an overwhelming number of uncharacterized somatic mutations, also known as variants of unknown significance (VUS). The therapeutic significance of EGFR mutations outside mutational hotspots, consisting of >50 types, in nonsmall cell lung carcinoma (NSCLC) is largely unknown. In fact, our pan-nation screening of NSCLC without hotspot EGFR mutations (n = 3, 779) revealed that the majority (>90%) of cases with rare EGFR mutations, accounting for 5.5% of the cohort subjects, did not receive EGFR-tyrosine kinase inhibitors (TKIs) as a first-line treatment. To tackle this problem, we applied a molecular dynamics simulation-based model to predict the sensitivity of rare EGFR mutants to EGFR-TKIs. The model successfully predicted the diverse in vitro and in vivo sensitivities of exon 20 insertion mutants, including a singleton, to osimertinib, a third-generation EGFR-TKI (R2 = 0.72, P = 0.0037). Additionally, our model showed a higher consistency with experimentally obtained sensitivity data than other prediction approaches, indicating its robustness in analyzing complex cancer mutations. Thus, the in silico prediction model will be a powerful tool in precision medicine for NSCLC patients carrying rare EGFR mutations in the clinical setting. Here, we propose an insight to overcome mutation diversity in lung cancer., LC-SCRUM-Japanで構築した日本最大のがん臨床ゲノムデータを活用しスーパーコンピュータで治療薬の効き目を予測 --がんゲノム医療における新たなツールの開発--. 京都大学プレスリリース. 2019-05-13.

Background In the era of genome-guided personalized cancer treatment, we must understand chemotherapy-induced genomic changes in tumors. This study evaluated whether adjuvant FOLFOX chemotherapy modifies the mutational profile of recurrent colorectal cancer (CRC). Methods Whole exome sequencing was performed on samples from primary CRC tumors, untreated metastatic tumors, and recurrent tumors following adjuvant FOLFOX chemotherapy. The samples were resected from four patients. Results The number of mutations or the mutation spectrum in individual patients was nearly identical. Copy number variants persisted regardless of FOLFOX therapy administration. The genomic signature of oxaliplatin exposure (G > T/C > A, T > A/A > T) was not enriched after FOLFOX chemotherapy. Overlapping single nucleotide variants (SNVs) and indels remained in 26–65% of the patient-matched tumor samples. One patient harbored an AKT1 E17K mutation in the recurrent tumor, whereas PIK3CA E542K and E88Q mutations were detected in the primary and untreated metastatic tumor samples. Genes related to intracellular Ca2+ homeostasis were enriched among the genes uniquely mutated after FOLFOX chemotherapy. Conclusions We found that the mutation rates, mutation spectrum, and copy number variants were nearly identical regardless of the administration of FOLFOX therapy in the four CRC cases. The mutational discordance between the patient-matched tumor samples is likely caused by tumor heterogeneity and chemotherapy-induced clonal selection. These findings might be useful as pilot data for larger studies to clarify the changes in the mutational landscape induced by adjuvant FOLFOX chemotherapy. Electronic supplementary material The online version of this article (10.1186/s12885-019-5479-6) contains supplementary material, which is available to authorized users.

Transdifferentiation of lung adenocarcinoma to small cell lung cancer (SCLC) has been reported in a subset of lung cancer cases that bear EGFR mutations. Several studies have reported the prerequisite role of TP53 and RB1 alterations in transdifferentiation. However, the mechanism underlying transdifferentiation remains understudied, and definitive additional events, the third hit, for transdifferentiation have not yet been identified. In addition, no prospective experiments provide direct evidence for transdifferentiation. In this study, we show that FGF9 upregulation plays an essential role in transdifferentiation. An integrative omics analysis of paired tumor samples from a patient with transdifferentiated SCLC exhibited robust upregulation of FGF9. Furthermore, FGF9 upregulation was confirmed at the protein level in four of six (66.7%) paired samples. FGF9 induction transformed mouse lung adenocarcinoma-derived cells to SCLC-like tumors in vivo through cell autonomous activation of the FGFR pathway. In vivo treatment of transdifferentiated SCLC-like tumors with the pan-FGFR inhibitor AZD4547 inhibited growth. In addition, FGF9 induced neuroendocrine differentiation, a pathologic characteristic of SCLC, in established human lung adenocarcinoma cells. Thus, the findings provide direct evidence for FGF9-mediated SCLC transdifferentiation and propose the FGF9–FGFR axis as a therapeutic target for transdifferentiated SCLC. Significance: This study demonstrates that FGF9 plays a role in the transdifferentiation of lung adenocarcinoma to small cell lung cancer.

Objectives Comprehensive genomic analysis of small-cell lung cancer (SCLC) revealed various genetic alterations. However, obtaining suitable samples for genetic analysis is difficult in advanced SCLC. Thus, the prognostic effect of genetic alterations on the outcome of SCLC patients has not been well investigated. Therefore, this study evaluated the effect of genetic alterations on the survival of SCLC patients. Materials and methods We collected samples obtained from 220 patients with advanced SCLC before cancer treatment. Genomic DNA extracted from the samples was subjected to a 1.499 Mb-sized custom panel that captured all exons of 244 cancer-related genes, and the captured DNA was analyzed through next-generation sequencing. The associations between genetic alterations and overall survival were evaluated. Results Genetic analysis was successful in 204 samples (93%). Genetic alterations in the PI3K/AKT/mTOR pathway and inactivating mutations inTP53 and RB1 were detected in 14 (7%), 150 (74%), and 85 (42%) of the tumors. In extensive disease (ED, N = 126) patients, multivariate analysis revealed that the presence of genetic alterations in the PI3K/AKT/mTOR pathway was significantly associated with unfavorable survival [hazard ratio (HR), 2.14; 95% CI 1.02–4.06; P = 0.04]. In limited disease (LD, N = 78) patients, the presence of TP53 mutation and the absence of RB1 mutation were significantly associated with unfavorable survival (HR, 2.41; 95% CI 1.21–5.34; P = 0.01, and HR, 0.45; 95% CI 0.25–0.79; P Conclusions Sequencing-based genetic profiling is feasible and useful to predict the prognosis in advanced SCLC. Genetic alterations in the PI3K/AKT/mTOR pathway, TP53 mutations and RB1 mutations were associated with prognosis in SCLC patients. The genetic alterations associated with the prognosis were different between ED-SCLC and LD-SCLC.

Purpose Usual interstitial pneumonia (UIP) is a risk factor for lung carcinogenesis. This study was performed to characterize mutagenesis and mutational target genes underlying lung carcinogenesis in patients with UIP. Patients and Methods A cohort of 691 Japanese patients with lung adenocarcinoma (LADC), of whom 54 had UIP and 637 did not, was studied for driver oncogene aberrations. Whole-exome analysis was performed for 296 cases, including 51 with UIP, to deduce mutagenic processes and identify commonly affected genes. Logistic regression analysis was used to detect associations of gene aberrations with clinicopathological factors. Results The EGFR mutation was markedly less prevalent in patients with LADC with UIP than in those without (1.9% [one of 54] v. 49.9% [318 of 637]; P < .001), even in heavy smokers (25.3% [38 of 150] of patients with > 40 pack-years; P < .001). Mutational signature analysis indicated that UIP-positive LADCs develop through accumulation of single-nucleotide and indel mutations caused by smoking. Pulmonary surfactant system genes (PSSGs) NKX2-1/TTF1, SFTPA1, SFTPA2, SFTPB, and SFTPC were identified as targets for mutations (preferentially indels), and mutations were specifically associated with shorter overall survival of patients with UIP-positive LADC, independent of pathologic stage (hazard ratio, 4.9; 95% CI, 1.7 to 14.4; P = .0037). Conclusion LADCs with UIP develop through mutational events caused by smoking, independently of EGFR mutation. PSSGs were identified as a mutational target and as a novel prognostic factor in UIP-positive LADC. PSSG deficiency might increase the malignancy of tumor cells by increasing the tumor-promoting effects of UIP.

Comprehensive genomic analysis has revealed that the PI3K/AKT/mTOR pathway is a feasible therapeutic target in small-cell lung carcinoma (SCLC). However, biomarkers to identify patients likely to benefit from inhibitors of this pathway have not been identified. Here, we show that metabolic features determine sensitivity to the PI3K/mTOR dual inhibitor gedatolisib in SCLC cells. Substantial phosphatidyl lipid analysis revealed that a specific phosphatidylinositol (3,4,5)-trisphosphate (PIP3) subspecies lipid product PIP3 (38:4) is predictive in assessing sensitivity to PI3K/mTOR dual inhibitor. Notably, we found that higher amounts of purine-related aqueous metabolites such as hypoxanthine, which are characteristic of SCLC biology, lead to resistance to PI3K pathway inhibition. In addition, the levels of the mRNA encoding hypoxanthine phosphoribosyl transferase 1, a key component of the purine salvage pathway, differed significantly between SCLC cells sensitive or resistant to gedatolisib. Moreover, complementation with purine metabolites could reverse the vulnerability to targeting of the PI3K pathway in SCLC cells normally sensitive to gedatolisib. These results indicate that the resistance mechanism of PI3K pathway inhibitors is mediated by the activation of the purine salvage pathway, supplying purine resource to nucleotide biosynthesis. Metabolomics is a powerful approach for finding novel therapeutic biomarkers in SCLC treatment. Significance: These findings identify features that determine sensitivity of SCLC to PI3K pathway inhibition and support metabolomics as a tool for finding novel therapeutic biomarkers. Cancer Res; 78(9); 2179–90. ©2018 AACR.

Podoplanin-positive cancer-associated fibroblasts (CAFs) play an essential role in tumor progression. However, it is still unclear whether specific genomic alterations of cancer cells are required to recruit podoplanin-positive CAFs. The aim of this study was to investigate the relationship between the mutation status of lung adenocarcinoma cells and the presence of podoplanin-positive CAFs. Ninety-seven lung adenocarcinomas for which whole exome sequencing data were available were enrolled. First, we analyzed the clinicopathological features of the cases, and then, evaluated the relationship between genetic features of cancer cells (major driver mutations and the number of single nucleotide variants, SNVs) and the presence of podoplanin-positive CAFs. The presence of podoplanin-positive CAFs was associated with smoking history, solid predominant subtype, and lymph node metastasis. We could not find any significant correlations between major genetic mutations (EGFR, KRAS, TP53, MET, ERBB2, BRAF, and PIC3CA) in cancer cells and the presence of podoplanin-positive CAFs. However, cases with podoplanin-positive CAFs had a significantly higher number of SNVs in cancer cells than the podoplanin-negative CAFs cases (median 84 vs 37, respectively; p = 0.001). This was also detected in a non-smoker subgroup (p = 0.037). Multivariate analyses revealed that the number of SNVs in cancer cells was the only statistically significant independent predictor for the presence of podoplanin-positive CAFs (p = 0.044). In lung adenocarcinoma, the presence of podoplanin-positive CAFs was associated with higher numbers of SNVs in cancer cells, suggesting a relationship between accumulations of SNVs in cancer cells and the generation of a tumor-promoting microenvironment.

Introduction Lung cancer mimicking organizing pneumonia (LCOP) is a novel radiological entity of lung adenocarcinoma that could be misdiagnosed as inflammatory lesions. However, the characteristic biological and genetic features of LCOP are not fully clarified. Materials and methods We used thin-section CT images to select cases of (LCOP) among surgically resected lung adenocarcinoma patients. We compared the clinicopathological characteristics and the immunophenotypes of LCOP (n = 44) and other lepidic-predominant adenocarcinomas (non-LCOP, n = 56). We also analyzed the genomic mutation features of LCOP (n = 4) by whole-exome sequencing (WES). Results All LCOP lesions were lepidic-predominant invasive adenocarcinoma. Patients with LCOP had significantly superior recurrence-free survival, compared to non-LCOP patients (95.5% and 74.4%; P = 0.006, respectively). Vascular invasion and lymph node metastasis were less frequent in LCOP than in non-LCOP patients (P = 0.001 and P = 0.03, respectively). The cancer cell expression levels of aggressiveness-related molecules, including ezrin, ALDH-1, laminin-5 were similar between LCOP and non-LCOP. On the contrary, the number of tumor promoting stromal cells, i.e., podoplanin-positive cancer-associated fibroblasts and CD204-positive tumor associated macrophages, was significantly lower in LOCP (P = 0.021 and P = 0.037, respectively). WES revealed that ABCB1, DNAH3, MSI2, and SLITRK2 were specifically mutated in LCOP. Conclusions Our results indicate that LCOP is characterized by fewer tumor-promoting stromal cells, which may contribute to the better prognosis of LCOP patients. Moreover, recognition of specific somatic mutations of LCOP patients may provide information regarding the development and progression of this type of lung cancer.

Background: Patients with BRAFV600E-mutated metastatic colorectal cancer (mCRC) have a poorer prognosis as well as resistance to anti-EGFR antibodies. However, it is unclear whether BRAF mutations other than BRAFV600E (BRAFnon-V600E mutations) contribute to anti-EGFR antibody resistance. Methods: This study was composed of exploratory and inference cohorts. Candidate biomarkers identified by whole exome sequencing from super-responders and nonresponders in the exploratory cohort were validated by targeted resequencing for patients who received anti-EGFR antibody in the inference cohort. Results: In the exploratory cohort, 31 candidate biomarkers, including KRAS/NRAS/BRAF mutations, were identified. Targeted resequencing of 150 patients in the inference cohort revealed 40 patients with RAS (26.7%), 9 patients with BRAFV600E (6.0%), and 7 patients with BRAFnon-V600E mutations (4.7%), respectively. The response rates in RAS, BRAFV600E, and BRAFnon-V600E were lower than those in RAS/BRAF wild-type (2.5%, 0%, and 0% vs 31.9%). The median PFS in BRAFnon-V600E mutations was 2.4 months, similar to that in RAS or BRAFV600E mutations (2.1 and 1.6 months) but significantly worse than that in wild-type RAS/BRAF (5.9 months). Conclusions: Although BRAFnon-V600E mutations identified were a rare and unestablished molecular subtype, certain BRAFnon-V600E mutations might contribute to a lesser benefit of anti-EGFR monoclonal antibody treatment.

EGFR-mutated lung cancers account for a significant subgroup of non–small cell lung cancers overall. Third-generation EGFR tyrosine kinase inhibitors (TKI) are mutation-selective inhibitors with minimal effects on wild-type EGFR. Acquired resistance develops to these agents, however, the mechanisms are as yet uncharacterized. In this study, we report that the Src–AKT pathway contributes to acquired resistance to these TKI. In addition, amplification of EGFR wild-type alleles but not mutant alleles was sufficient to confer acquired resistance. These findings underscore the importance of signals from wild-type EGFR alleles in acquiring resistance to mutant-selective EGFR-TKI. Our data provide evidence of wild-type allele-mediated resistance, a novel concept of acquired resistance in response to mutation-selective inhibitor therapy in cancer treatment. Cancer Res; 77(8); 2078–89. ©2017 AACR.

To enable the widespread application of genomic medicine, the extraction of genomic DNA from thin sections of archived formalin-fixed and paraffin-embedded (FFPE) tissue blocks for next-generation sequencing (NGS) is often necessary. However, there are currently no guidelines available on which specific regions of the microtome sections to use for macrodissection with respect to the histopathological factors observed under microscopic examination. The aim of this study was to clarify the relationship between histopathological factors and DNA quality, and to standardize the macrodissection method for more efficient implementation of NGS. FFPE tissue specimens of 218 patients from the Biomarker Research for Anti-EGFR Monoclonal Antibodies by Comprehensive Cancer Genomics study were used to investigate the relationship between 15 histopathological factors and the quantitative ratio of double-stranded DNA (dsDNA) to total nucleic acids, as well as the ∆ crossing point value of each tissue specimen. Multivariate logistic regression analysis revealed that specimen storage of ≥3 years was negatively associated with dsDNA quality (P=0.0007, OR: 4.30, 95% CI: 1.85-10.04). In contrast, the presence of a mucus pool was positively associated with dsDNA quality (P=0.0308, OR: 0.23, 95% CI: 0.06-0.87). Metastatic tumors and longer specimen storage periods were significantly associated with lower ∆Cp values (P=0.0007, OR: 4.43, 95% CI: 1.87-10.49; and P=0.0003, OR: 5.51, 95% CI: 2.18-13.95, respectively). Therefore, macrodissection should not be performed on specimens exhibiting histopathological factors associated with poor DNA quality. In particular, the use of tissue blocks with a storage period of

Summary These occupational cholangiocarcinoma cases shared a high mutation burden, strand bias and unique trinucleotide mutational signatures, suggesting that the patients might have been exposed to a common strong mutagen. The underlying mechanisms of mutagenesis should be further investigated., Cholangiocarcinoma is a relatively rare cancer, but its incidence is increasing worldwide. Although several risk factors have been suggested, the etiology and pathogenesis of the majority of cholangiocarcinomas remain unclear. Recently, a high incidence of early-onset cholangiocarcinoma was reported among the workers of a printing company in Osaka, Japan. These workers underwent high exposure to organic solvents, mainly haloalkanes such as 1,2-dichloropropane (1,2-DCP) and/or dichloromethane. We performed whole-exome analysis on four cases of cholangiocarcinoma among the printing workers. An average of 44.8 somatic mutations was detected per Mb in the genome of the printing workers’ cholangiocarcinoma tissues, approximately 30-fold higher than that found in control common cholangiocarcinoma tissues. Furthermore, C:G-to-T:A transitions with substantial strand bias as well as unique trinucleotide mutational changes of GpCpY to GpTpY and NpCpY to NpTpY or NpApY were predominant in all of the printing workers’ cholangiocarcinoma genomes. These results were consistent with the epidemiological observation that they had been exposed to high concentrations of chemical compounds. Whole-genome analysis of Salmonella typhimurium strain TA100 exposed to 1,2-DCP revealed a partial recapitulation of the mutational signature in the printing workers’ cholangiocarcinoma. Although our results provide mutational signatures unique to occupational cholangiocarcinoma, the underlying mechanisms of the disease should be further investigated by using appropriate model systems and by comparison with genomic data from other cancers.

Lung cancer with usual interstitial pneumonia (UIP) pattern is a disease with poor prognosis. This study aimed to characterize the tumor microenvironment of lung adenocarcinoma associated with UIP (UIP-ADC).A total of 1341 consecutive patients with ADC who had undergone complete surgical resection were enrolled in this study, and the clinicopathological features of UIP-ADC were examined. Further, we selected 17 cases of UIP-ADC and non-UIP ADC each (adjusted for age, smoking status, pathological stage, and invasive size of lesion) for immunohistochemical analysis, and the biological differences between UIP-ADC and non-UIP ADC groups were analyzed.UIP-ADC was detected in 18 patients (1.3%). Patients with UIP-ADC had shorter cancer-specific survival (CSS) (5 yrs CSS; UIP-ADC 52.9% vs non-UIP ADC 81.8%, p 0.01). Evaluation of tumor-infiltrating lymphocytes (TILs) in cancer stroma showed that the number of CD8In the current study, we have demonstrated that the tumor microenvironment of UIP-ADC acquires an immunosuppressive state, and this could be one of the possible explanations for poor prognosis of this disease.

Resistance to vandetanib, a type I RET kinase inhibitor, developed in a patient with metastatic lung adenocarcinoma harboring a CCDC6-RET fusion that initially exhibited a response to treatment. The resistant tumor acquired a secondary mutation resulting in a serine-to-phenylalanine substitution at codon 904 in the activation loop of the RET kinase domain. The S904F mutation confers resistance to vandetanib by increasing the ATP affinity and autophosphorylation activity of RET kinase. A reduced interaction with the drug is also observed in vitro for the S904F mutant by thermal shift assay. A crystal structure of the S904F mutant reveals a small hydrophobic core around F904 likely to enhance basal kinase activity by stabilizing an active conformer. Our findings indicate that missense mutations in the activation loop of the kinase domain are able to increase kinase activity and confer drug resistance through allosteric effects., Mechanisms of acquired resistance to RET tyrosine kinase inhibitors in lung cancers are largely unknown. Here, the authors report in a lung adenocarcinoma patient harboring a CCDC6-RET mutation in the RET kinase (S904F) that results in resistance to the kinase inhibitor vandetanib by increasing the ATP affinity and autophosphorylation activity of RET kinase.

Adenosquamous carcinoma of the lung (Ad-Sq) is an uncommon subtype with poor prognosis. We analyzed the clinicopathological characteristics of Ad-Sq, focusing the correlation between Epidermal Growth Factor Receptor (EGFR) mutation and clinicopathological factors. A total of 67 cases were selected from September 1992 to May 2011. EGFR mutational analysis (n = 59) was performed by direct sequence. We also performed immunohistochemical staining for EGFR mutated cases using the two mutation-specific antibodies for deletion and L858R. Postoperative 3-year survival rate of Ad-Sq was 58.7%, statistically worse in comparison with adenocarcinoma (58.7% vs. 78.1%, P = 0.038). Twenty-four percent (14/59) were positive for EGFR mutations. Patients who had never been smokers and who were lymphatic permeation positive were seen more frequently in the mutation positive group (P = 0.035, 0.027, respectively). Moreover, the EGFR mutated group tended to have a more positive prognosis than negative. Focusing on the pathological features, the lepidic growth pattern was more frequently seen in the positive group (P = 0.018). Immunoreactivity for the DEL-specific and L858-specific antibody were observed in both adenocarcinoma and squamous cell carcinoma components. Our study demonstrated that EGFR mutated Ad-Sq had similar clinicopathological features as EGFR mutated adenocarcinoma.

Gastrointestinal stromal tumors represent the most common mesenchymal tumor of the digestive tract, driven by gain-of-function mutations in KIT. Despite its proven benefits, half of the patients treated with imatinib show disease progression within 2 years due to secondary resistance mutations in KIT. It remains unclear how the genomic and transcriptomic features change during the acquisition of imatinib resistance. Here, we performed exome sequencing and microarray transcription analysis for four imatinib-resistant cell lines and one cell line briefly exposed to imatinib. We also performed exome sequencing of clinical tumor samples. The cell line briefly exposed to imatinib exhibited few single-nucleotide variants and copy-number alterations, but showed marked upregulation of genes related to detoxification and downregulation of genes involved in cell cycle progression. Meanwhile, resistant cell lines harbored numerous genomic changes: amplified genes related to detoxification and deleted genes with cyclin-dependent kinase activity. Some variants in the resistant samples were traced back to the drug-sensitive samples, indicating the presence of ancestral subpopulations. The subpopulations carried variants associated with cell death. Pre-existing cancer cells with genetic alterations promoting apoptosis resistance may serve as a basis whereby cancer cells with critical mutations, such as secondary KIT mutations, can establish full imatinib resistance. © 2017 The Authors Genes, Chromosomes and Cancer Published by Wiley Periodicals, Inc.

Purpose: Although large-cell neuroendocrine carcinoma (LCNEC) of the lung shares many clinical characteristics with small-cell lung cancer (SCLC), little is known about its molecular features. We analyzed lung LCNECs to identify biologically relevant genomic alterations. Experimental Design: We performed targeted capture sequencing of all the coding exons of 244 cancer-related genes on 78 LCNEC samples [65 surgically resected cases, including 10 LCNECs combined with non–small cell lung cancer (NSCLC) types analyzed separately, and biopsies of 13 advanced cases]. Frequencies of genetic alterations were compared with those of 141 SCLCs (50 surgically resected cases and biopsies of 91 advanced cases). Results: We found a relatively high prevalence of inactivating mutations in TP53 (71%) and RB1 (26%), but the mutation frequency in RB1 was lower than that in SCLCs (40%, P = 0.039). In addition, genetic alterations in the PI3K/AKT/mTOR pathway were detected in 12 (15%) of the tumors: PIK3CA 3%, PTEN 4%, AKT2 4%, RICTOR 5%, and mTOR 1%. Other activating alterations were detected in KRAS (6%), FGFR1 (5%), KIT (4%), ERBB2 (4%), HRAS (1%), and EGFR (1%). Five of 10 cases of LCNECs combined with NSCLCs harbored previously reported driver gene alterations, all of which were shared between the two components. The median concordance rate of candidate somatic mutations between the two components was 71% (range, 60%–100%). Conclusions: LCNECs have a similar genomic profile to SCLC, including promising therapeutic targets, such as the PI3K/AKT/mTOR pathway and other gene alterations. Sequencing-based molecular profiling is warranted in LCNEC for targeted therapies. Clin Cancer Res; 23(3); 757–65. ©2016 AACR.

Recent genome-wide association studies (GWASs) have identified polymorphisms in several genes associated with lung cancer risk. Nevertheless, functional polymorphisms in DNA repair and metabolic genes that had been reported as being associated with risk for lung cancer, particularly for lung squamous cell carcinoma (SQC), were not examined in those studies. Therefore, significance of these functional polymorphisms was evaluated in a population, in which polymorphisms in the GWAS genes showed associations with lung SQC risk.Polymorphisms in three DNA repair genes, TP53, MDM2, and OGG1, and two metabolic genes, CYP1A1 and GSTM1, were examined for associations with lung SQC risk in a hospital-based case-control study consisting of 377 cases and 325 controls, which had been previously subjected to association studies on GWAS genes, CHRNA3, TERT, and HLA-DQA1.Genotypes for two DNA repair genes, TP53 and OGG1, showed significant associations with SQC risk (p0.05), and those for two GWAS genes, CHRNA3 and HLA-DQA1, showed significant associations with SQC risk (P0.05) with odds ratios between 1.65 (95% confidence interval = 1.06-2.57 for OGG1) and 2.57 (95% confidence interval = 1.03-6.87 for CHRNA3). Marginally significant associations were also observed for MDM2 and CYP1A1 genes. Interactions among these polymorphisms on SQC risk were not observed.Association of functional polymorphisms in DNA repair and metabolic genes with lung SQC risk was appreciated. This result indicates the necessity of reevaluation for the significance of functional polymorphisms in DNA repair and metabolic genes on lung cancer risk in other populations subjected to GWASs.

Targeted therapy against oncogenic ALK and ROS1 fusion using type I tyrosine kinase inhibitors (TKIs), which bind to the ATP-binding cleft of kinases, is highly effective in lung adenocarcinoma (LADC); however, such cancers inevitably acquire resistance, which severely limits the efficacy of cancer treatments. The identification of these resistance mutations led to the development of novel TKIs to overcome acquired resistance. Here we report resistance to vandetanib, a type I RET kinase inhibitor, developed in a patient with metastatic LADC harboring a CCDC6-RET fusion that initially exhibited a response to treatment. The resistant tumor acquired a secondary mutation resulting in substitution of an amino acid located outside the nucleotide binding cleft. The mutation conferred resistance to vandetanib by increasing the ATP affinity and auto-phosphorylation activity of RET kinase. A crystal structure of the mutant revealed a small hydrophobic core around the mutated amino acid likely to enhance basal kinase activity by stabilizing an active conformer. In addition, a thermal shift assay suggested that the mutation was less able to bind vandetanib. Molecular dynamics simulation supported the mutant underwent conformational changes decreasing the binding affinity of vandetanib. Our findings indicate that the missense mutation is able to increase kinase activity and confers drug resistance through allosteric effects. Citation Format: Takashi Nakaoku, Takashi Kohno, Mitsugu Araki, Seiji Nino, Rakhee Chauhan, Phillip P. Knowles, Katsuya Tsuchihara, Shingo Matsumoto, Yoko Shimada, Sachiyo Mimaki, Genichiro Ishii, Hitoshi Ichikawa, Yasushi Okuno, Kiyotaka Yoh, Neil Q. McDonald, Koichi Goto. A secondary RET mutation allosterically conferring resistance to vandetanib [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 918.

Fifty single-nucleotide polymorphisms (SNPs) associated with amino acid changes in 36 genes involved in diverse DNA repair pathways were assessed for associations with risk for small cell lung carcinoma (SCLC) by a case-control study consisting of 211 SCLC cases and 685 controls. An SNP, Val83Met, in the MTH1 (microtT homolog 1) gene encoding a triphosphatase that hydrolyzes pro-mutagenic oxidized nucleoside triphosphates, such as 8-hydroxy-dGTP and 2-hydroxy-dATP, showed the strongest and a significant association with SCLC risk [odds ratio (OR)=1.6, 95% confidence interval (CI): 1.2-2.2, P=0.004], while three other SNPs in the TP53, BLM and SNM1 genes, respectively, also showed marginal associations (0.05

Purpose: Third-generation EGFR-tyrosine kinase inhibitors (TKIs) are mutation-specific inhibitors that generally have minimal effects on wild type protein. The mechanisms underlying acquired resistance to third-generation EGFR-TKIs remain relatively uncharacterized. The purpose of this study is to clarify the mechanisms of acquired resistance to third-generation EGFR-TKIs in order to improve the prognosis of lung cancer patients harboring EGFR mutations. Experimental design: We established third-generation EGFR-TKIs resistant cells using a dose-escalation method. The PC9 (EGFR E746-A750del) and H1975 (EGFR L858R+T790M) human NSCLC cell lines were cultured with rociletinib (CO-1686) or osimertinib (AZD9291) for several months. Their resistance to a third-generation EGFR-TKIs was confirmed by the MTS cell proliferation assay. To clarify the heterogeneity of potential resistance mechanisms, those resistant cell lines were subcloned to isolate resistant clones. DNA isolated from the parental and resistant cell clones was then subjected to whole exome sequencing. Copy number alterations and several genetic alterations potentially relevant to EGFR-TKIs sensitivity were detected. Results: We identified Src-AKT pathway contribute to acquired resistance. In addition, we identified that EGFR wild type allele, but not mutation allele, amplification induced acquired resistance. It mirrors the decreased inhibitory pressure for wild type EGFR by mutation-specific EGFR-TKIs. Conclusion: We provide evidence of wild type allele-mediated resistance, a novel concept of acquired resistance, occurring in response to mutation-specific inhibitor therapy in cancer treatment. 1833 /2600characters Citation Format: Keigo Kobayashi, Shigenari Nukaga, Hiroyuki Yasuda, Keita Masuzawa, Jyunko Hamamoto, Ichiro Kawada, Katsuhiko Naoki, Sachiyo Mimaki, Shingo Matsumoto, Koichi Goto, Katsuya Tsuchihara, Tomoko Betsuyaku, Kenzo Soejima. EGFR wild type allele amplification induces acquired resistance to mutation-specific EGFR tyrosine kinase inhibitors in non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4111. doi:10.1158/1538-7445.AM2017-4111

A p53-inducible gene, the growth arrest and DNA damage-inducible gene 45 (gadd45), is associated with cell growth inhibition, DNA damage response and DNA repair. Here we report that Delta12-prostaglandin J2 (Delta12-PGJ2), an in vivo metabolite of arachidonic acid, which inhibits cell proliferation, induces gadd45 mRNA in HeLa cells. Because the p53 protein in HeLa cells is inactivated by papilloma virus E6, this type of gadd45 induction appears to be p53-independent. The induction was dose-dependent, and the maximum induction was observed at a concentration of 7.5 microgram/ml. In a time course study, gadd45 mRNA was induced 3 h after the addition of 7.5 microgram/ml Delta12-PGJ2. To investigate the transcriptional mechanism of gadd45 mRNA induction, we cloned a human genomic DNA fragment containing the gadd45 promoter region, and investigated the effect of Delta12-PGJ2 on the gadd45 promoter activity. In HeLa cells, 7.5 microgram/ml Delta12-PGJ2 markedly stimulated the gadd45 gene promoter about 20-fold or more. On the other hand, Delta12-PGJ2 did not stimulate the promoter activity of a reporter plasmid containing only p53 binding sites in HeLa cells, indicating that the gadd45 promoter activation by Delta12-PGJ2 was not mediated by p53. These results suggest that one of the mechanisms of cell growth arrest by Delta12-PGJ2 is mediated through a p53-independent induction of gadd45.