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Despite the epidemiological association between intrahepatic cholangiocarcinoma (iCCA) and HBV infection, little is known about the relevant oncogenic effects. We sought to identify the landscape and mechanism of HBV integration, along with the genomic architecture of HBV-infected iCCA (HBV-iCCA) tumors.We profiled a cohort of 108 HBV-iCCAs using whole-genome sequencing, deep sequencing, and RNA sequencing, together with preconstructed data sets of HBV-infected HCC (HBV-HCC; n = 167) and combined hepatocellular cholangiocarcinoma (HBV-cHCC/CCA; n = 59), and conventional (n = 154) and fluke-related iCCAs (n = 16). Platforms based on primary iCCA cell lines to evaluate the functional effects of chimeric transcripts were also used. We found that HBV had inserted at multiple sites in the iCCA genomes in 45 (41.7%) of the tumors. Recurrent viral integration breakpoints were found at nine different sites. The most common insertional hotspot (7 tumors) was in the TERT (telomerase reverse transcriptase) promoter, where insertions and mutations (11 tumors) were mutually exclusive, and were accompanied by promoter hyperactivity. Recurrent HBV integration events (5 tumors) were also detected in FAT2 (FAT atypical cadherin 2), and were associated with enrichment of epithelial-mesenchymal transition-related genes. A distinctive intergenic insertion (chr9p21.3), between DMRTA1 (DMRT like family A1) and LINC01239 (long intergenic non-protein coding RNA 1239), had oncogenic effects through activation of the mammalian target of rapamycin (mTOR)/4EBP/S6K pathway. Regarding the mutational profiles of primary liver cancers, the overall landscape of HBV-iCCA was closer to that of nonviral conventional iCCA, than to HBV-HCC and HBV-cHCC/CCA.Our findings provide insight into the behavior of iCCAs driven by various pathogenic mechanisms involving HBV integration events and associated genomic aberrations. This knowledge should be of use in managing HBV carriers.

Background There has been a growing interest in the association between mitochondrial dysfunction and sepsis. However, most studies have focused on mitochondrial structural damage, functional aspects, or the clinical phenotypes in sepsis. The purpose of this study was to evaluate mitochondrial DNA (mtDNA) gene mutations in critically ill pediatric patients with septic shock. Method Thirteen patients with severe sepsis or septic shock admitted to the pediatric intensive care unit (PICU) of a tertiary children's hospital were enrolled in this prospective observational study. Clinical data from electronic medical records were obtained. Whole-blood samples were collected within 24 h of PICU admission to perform PBMC isolation, mtDNA extraction, and mtDNA sequencing using next-generation sequencing. Results mtDNA sequencing revealed mutations in 9 of the 13 patients, presenting 27 point mutations overall, with 15 (55.6%) located in the locus related to adenosine triphosphate production and superoxide metabolism, including electron transport. Conclusion In this pilot study, significant numbers of mtDNA point mutations were detected in critically ill pediatric patients with septic shock. These mutations could provide promising evidence for mitochondrial dysfunction in sepsis and a basis for further large-scale studies. Impact This study is the first to examine mitochondrial DNA mutations in pediatric patients with septic shock using next-generation sequencing. A high frequency of mitochondrial DNA mutations was detected in these patients indicating an association with septic shock. This pilot study may provide a potential explanation for the association between mitochondrial dysfunction and septic shock on a genetic basis.

Background/aim Somatic mutations were investigated in 21 patients with postmenopausal estrogen receptor (ER)-positive and human epidermal growth factor receptor-2 (HER-2)-positive (ER+HER2+) breast cancer (BC) treated with neoadjuvant letrozole and lapatinib, to identify their distinct molecular landscape. Patients and methods We used tissue samples of 21 patients from phase II Neo ALL-IN cohort, and somatic alterations were examined using targeted exome sequencing performed in Foundation Medicine, Inc. (FMI). Results TP53 (61.9%) and PIK3CA (57.1%) were the two most frequently mutated genes that were inter-correlated (p=0.026). They were associated with unfavorable clinical outcomes, particularly when accompanying PIK3CA mutations at exon 9 in helical domains. Meanwhile, MLL2 alteration was negatively associated with mutations of TP53 or PIK3CA, and it tended to be present in patients with low KI-67 levels and no initial nodal involvement. Moreover, patients with MLL2 mutations numerically showed more favorable overall response rates (ORR) (80% vs. 56.2%) and better 5-year event-free survival (EFS) rates (100% vs. 87.5%) compared to the wild-type. Conclusion Mutations in TP53 and PIK3CA hotspot at exon 9 may be potential negative predictors of ER+HER2+ BC treated with neoadjuvant letrozole and lapatinib, while MLL2 inactivating mutation might confer therapeutic benefit in these patients.

IntroductionMismatch repair (MMR)-deficient and DNA polymerase epsilon (POLE)-mutated tumors exhibit a high tumor mutation burden (TMB) and have been proven to be associated with good responses to immune checkpoint inhibitor treatments. However, the relationship between mutational characteristics of MMR-deficient and POLE-mutated tumors and the spatial architecture of tumor-infiltrating lymphocytes (TILs) has not been fully evaluated.MethodsWe retrieved microsatellite instability-high (MSI-high, N=20) and POLE-mutated (N=47) cases from the clinical next-generation sequencing cohort at Asan Medical Center. Whole-slide immunostaining for CD3, CD4, CD8, FoxP3 and PD-1 were performed with tissue samples of colorectal and gastric cancer (N=24) and the tumor-positive TIL cell densities were correlated with the tumor’s mutational features. The findings were compared with the results of similar analyses in The Cancer Genome Atlas-Colorectal Adenocarcinoma (TCGA-COADREAD) cohort (N=592).ResultsThe MSI-high group showed significantly higher overall TMBs with a number of insertion/deletion (indel) mutations relative to the POLE-mutated group (median TMB; 83.6 vs 12.5/Mb). Oncogenic/likely-oncogenic POLE mutations were identified with ultrahypermutations (≥100 mutations/Mb) (2/47, 4.3%). Concurrent POLE mutations of unknown significance and MSI-high cases were identified in eight cases (8/67, 11%), and two of these colorectal cancers had multiple POLE mutations, showing an ultramutated phenotype (378.1 and 484.4/Mb) and low indel mutation burdens with complete loss of MSH-6 or PMS-2, which was similar to the mutational profile of the POLE-inactivated tumors. Intratumoral CD3-positive, CD4-positive, CD8-positive, FoxP3-positive and PD-1-positive TIL cell densities were more strongly correlated with the indel mutation burden than with the total TMB (correlation coefficient, 0.61–0.73 vs 0.23–0.38). In addition, PI3K/AKT/mTOR pathway mutations were commonly found in MSI-high tumors (75%) but not in POLE-mutated tumors.ConclusionsIndel mutation burden rather than total TMB could serve as a predictor of high TILs in both MSI-high and POLE-mutated tumors. Multiple uncharacterized/non-pathogenic POLE mutations occurring via MMR deficiency within MSI-high tumors may have combined pathogenic roles. A mutated PI3K/AKT/mTOR pathway may be a biomarker that can be used to stratify patients with advanced MSI-high tumors for immune therapy.

Background The intrinsic immuno-ge7nomic characteristics of colorectal cancer cells that affect tumor biology and shape the tumor immune microenvironment (TIM) are unclear. Methods We developed a patient-derived colorectal cancer organoid (CCO) model and performed pairwise analysis of 87 CCOs and their matched primary tumors. The TIM type of the primary tumor was classified as immuno-active, immuno-exhausted, or immuno-desert. Results The gene expression profiles, signaling pathways, major oncogenic mutations, and histology of the CCOs recapitulated those of the primary tumors, but not the TIM of primary tumors. Two distinct intrinsic molecular subgroups of highly proliferative and mesenchymal phenotypes with clinical significance were identified in CCOs with various cancer signaling pathways. CCOs showed variable expression of cancer-specific immune-related genes such as those encoding HLA-I and HLA-II, and molecules involved in immune checkpoint activation/inhibition. Among these genes, the expression of HLA-II in CCOs was associated with favorable patient survival. K-means clustering analysis based on HLA-II expression in CCOs revealed a subgroup of patients, in whom cancer cells exhibited Intrinsically Immunogenic Properties (Ca-IIP), and were characterized by high expression of signatures associated with HLA-I, HLA-II, antigen presentation, and immune stimulation. Patients with the Ca-IIP phenotype had an excellent prognosis, irrespective of age, disease stage, intrinsic molecular type, or TIM status. Ca-IIP was negatively correlated with intrinsic E2F/MYC signaling. Analysis of the correlation between CCO immuno-genotype and TIM phenotype revealed that the TIM phenotype was associated with microsatellite instability, Wnt/β-catenin signaling, APC/KRAS mutations, and the unfolded protein response pathway linked to the FBXW7 mutation in cancer cells. However, Ca-IIP was not associated with the TIM phenotype. Conclusions We identified a Ca-IIP phenotype from a large set of CCOs. Our findings may provide an unprecedented opportunity to develop new strategies for optimal patient stratification in this era of immunotherapy.

Purpose In biliary tract cancer (BTC), standard chemotherapy has limited benefit and no molecular targeted agents have been approved. This study investigated the genetic profile of BTC to identify potential new therapeutic targets and predictive biomarkers. Methods Targeted exome sequencing was performed for 124 patients with BTC [gallbladder cancer (GBC), 25; intrahepatic cholangiocarcinoma (ICC), 55; extrahepatic cholangiocarcinoma (ECC), 44]. Survival analysis was performed in 112 patients who received palliative chemotherapy for locally unresectable or metastatic disease. Results Genetic alterations were observed in 104 patients (83.8%); the most commonly mutated genes were TP53 (44.4%), KRAS (29.0%), ARID1A (12.1%) and IDH1 (9.7%). IDH1/2 mutations appeared more frequently in ICC (23.6%, P = 0.0002) than in GBC (4.0%) or ECC (2.3%), while ERBB2/3 mutations were found only in GBC (20.0%) and ECC (11.4%). Patients harbouring TP53 mutations had shorter overall survival (OS; median 15.2 vs. 37.8 months, P = 0.018), while IDH1 mutations showed a tendency for longer progression-free survival (PFS; 10.6 vs. 6.1 months, P = 0.124). Potentially actionable genetic alterations were found in 54.8%, and 7.1% received appropriate molecular targeted therapy in the clinical trial setting. Germline or somatic mutations in DNA damage repair (DDR) genes were found in 63.5% of patients and were significantly associated with longer PFS (6.9 vs. 5.7 months, P = 0.013) and OS (21.0 vs. 13.3 months, P = 0.009) in patients who received first-line platinum-containing chemotherapies (n = 88). Conclusions A subgroup of patients with BTC may benefit from targeted therapy by the aid of genetic information. In particular, DDR alterations may be a predictive biomarker for response to platinum-containing chemotherapy in patients with BTC.

Circular RNAs (circRNAs) represent potential biomarkers because of their highly stable structure and robust expression pattern in clinical samples. The aim of this study was to evaluate the expression of a recently identified circRNA, hsa_circ_0005986; determine its clinical significance; and evaluate its potential as a biomarker of hepatocellular carcinoma (HCC). We evaluated hsa_circ_0005986 expression in 123 HCC tissue samples, its clinical significance, and its association with patients’ clinicopathological characteristics and survival. Hsa_circ_0005986 expression was downregulated in HCC tissues. Low hsa_circ_0005986 expression was more common in tumors larger than 5 cm [odds ratio (OR), 3.19; 95% confidence interval (CI), 1.51–6.76; p = 0.002], advanced TNM stage (III/IV; OR, 2.39; 95% CI, 1.16–4.95; p = 0.018), and higher BCLC stage (B/C; OR, 2.71; 95% CI, 1.30–5.65; p = 0.007). High hsa_circ_0005986 expression was associated with improved survival and was an independent prognostic factor for overall [hazard ratio (HR), 0.572; 95% CI, 0.339–0.966; p = 0.037] and progression-free (HR, 0.573; 95% CI, 0.362–0.906; p = 0.017) survival. Moreover, the circRNA–miRNA–mRNA network was constructed using RNA-seq/miRNA-seq data and clinical information from TCGA-LIHC dataset. Our findings indicate a promising role for hsa_circ_0005986 as a prognostic biomarker in patients with HCC.

PurposeHeat shock protein-90 (HSP90) remains an important cancer target because of its involvement in multiple oncogenic protein pathways and biologic processes. Although many HSP90 inhibitors have been tested in the treatment of KRAS-mutant non–small cell lung cancer (NSCLC), most, including AUY922, have failed due to toxic effects and resistance generation, even though a modest efficacy has been observed for these drugs in clinical trials. In our present study, we investigated the novel mechanism of resistance to AUY922 to explore possible avenues of overcoming and want to provide some insights that may assist with the future development of successful next-generation HSP90 inhibitors.Materials and MethodsWe established two AUY922-resistant KRAS-mutated NSCLC cells and conducted RNA sequencing to identify novel resistance biomarker.ResultsWe identified novel two resistance biomarkers. We observed that both integrin Av (ITGAv) and β3 (ITGB3) induce AUY922-resistance via focal adhesion kinase (FAK) activation, as well as an epithelial-mesenchymal transition, in both in vitro and in vivo xenograft model. mRNAs of both ITGAv and ITGB3 were also found to be elevated in a patient who had shown acquired resistance in a clinical trial of AUY922. ITGAv was induced by miR-142 downregulation, and ITGB3 was increased by miR-150 downregulation during the development of AUY922-resistance. Therefore, miR-150 and miR-142 overexpression effectively inhibited ITGAvB3-dependent FAK activation, restoring sensitivity to AUY922.ConclusionThe synergistic co-targeting of FAK and HSP90 attenuated the growth of ITGAvB3-induced AUY922-resistant KRAS-mutated NSCLC cells in vitro and in vivo, suggesting that this combination may overcome acquired AUY922-resistance in KRAS-mutant NSCLC.

Introduction Although osimertinib overcomes the T790M mutation acquired after traditional epidermal growth factor receptor (EGFR) gene tyrosine kinase inhibitor (TKI) treatment, resistance to osimertinib eventually occurs. We explored resistance mechanisms of second-line osimertinib and their clinical implications by comparing next-generation sequencing (NGS) results before and after resistance acquisition. Methods We enrolled 34 patients with advanced EGFR-mutant adenocarcinoma whose biopsied tumour tissues were subjected to targeted NGS at the time of progression on osimertinib. For comparison, NGS was also performed on archived tumour tissues from each patient excised before osimertinib initiation. Results The tumours of three patients’ were observed to have transformed to small-cell carcinoma and those of two patients to squamous cell carcinoma. Among the remaining 29 patients, T790M mutations were maintained in seven patients (24.1%), including four patients (13.8%) acquiring C797S mutations and one with MET amplification. Among the 22 patients (75.9%) with T790M loss, a variety of novel mutations were identified, including KRAS mutations, PIK3CA mutations, and RET fusion, but MET amplifications (n = 4, 18.2%) were most frequently identified variations. Progression-free survival (PFS) on osimertinib was shorter among patients with T790M loss than among those who maintained T790M (5.36 versus 13.81 months, p = 0.009), and MET-amplified patients were found to have much worse PFS among patients with T790M loss (2.10 versus 6.35 months, p = 0.01). Conclusions Loss of the T790M mutation was associated with early resistance to osimertinib, and this was exacerbated by MET amplification. Further work is needed to fully understand the implications of each resistance mechanism.

Aims Salivary duct carcinoma (SDC) is an aggressive salivary gland neoplasm with a poor prognosis. Morphologically, it has many variants including sarcomatoid SDC. We evaluated the morphological features, immunohistochemistry profile, and genomic alteration of the rare variant, sarcomatoid SDC. Methods and results We evaluated the clinicopathological and molecular pathology for rare variant of sarcomatoid SDC. Among 102 SDC patients, three had sarcomatoid SDC. Review of clinicopathological features and immunohistochemistry and targeted exome sequencing was performed according to carcinomatous and sarcomatoid areas, respectively. The tumors were present in two submandibular glands and one parotid gland. In one case, a SDC arose in carcinoma ex pleomorphic adenoma. All consisted of a conventional invasive ductal carcinoma area and sarcomatoid features including spindle cells and multinucleated giant cells. AR and epithelial membrane antigen (EMA) were positive in both carcinoma and sarcomatoid areas. Cytokeratin AE1/AE3 were negative in all sarcomatoid areas. Targeted exome sequencing revealed multiple heterogeneous alterations including PIK3CA and TP53. Genomic alterations were nearly identical between typical carcinoma and sarcomatoid areas. Conclusions Clinicopathological features of sarcomatoid SDCs were not different from typical SDC, and genomic alteration according to subtypes was also similar to that of the conventional type. Androgen receptor (AR) expression is helpful in the diagnosis of SDC. The findings indicate that EMA and AR are useful in diagnosing sarcomatoid SDC when the tumor is composed of predominantly sarcomatoid components.

Little is known about the pathogenesis or molecular profiles of idiopathic pulmonary fibrosis-associated lung cancer (IPF-LC). This study was performed to investigate the genomic profiles of IPF-LC and to explore the possibility of defining potential therapeutic targets in IPF-LC. We assessed genomic profiles of IPF-LC by using targeted exome sequencing (OncoPanel version 2) in 35 matched tumour/normal pairs surgically resected between 2004 and 2014. Germline and somatic variant calling was performed with GATK HaplotypeCaller and MuTect with GATK SomaticIndelocator, respectively. Copy number analysis was conducted with CNVkit, with focal events determined by Genomic Identification of Significant Targets in Cancer 2.0, and pathway analysis (KEGG) with DAVID. Germline mutations in TERT (rs2736100, n = 33) and CDKN1A (rs2395655, n = 27) associated with idiopathic pulmonary fibrosis risk were detected in most samples. A total of 410 somatic mutations were identified, with an average of 11.7 per tumour, including 69 synonymous, 177 missense, 17 nonsense, 1 nonstop and 11 splice-site mutations, and 135 small coding indels. Spectra of the somatic mutations revealed predominant C > T transitions despite an extensive smoking history in most patients, suggesting a potential association between APOBEC-related mutagenesis and the development of IPF-LC. TP53 (22/35, 62.9%) and BRAF (6/35, 17.1%) were found to be significantly mutated in IPF-LC. Recurrent focal amplifications in three chromosomal loci (3q26.33, 7q31.2, and 12q14.3) and 9p21.3 deletion were identified, and genes associated with the JAK-STAT signalling pathway were significantly amplified in IPF-LC (P = 0.012). This study demonstrates that IPF-LC is genetically characterized by the presence of somatic mutations reflecting a variety of environmental exposures on the background of specific germline mutations, and is associated with potentially targetable alterations such as BRAF mutations. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Objectives: Thymic epithelial tumors (TETs) are rare malignant tumors that exhibit heterogeneous histology and clinical behavior. As immune check point inhibitors, drugs targeting anti-programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) have shown remarkable results against many cancers; thus, the importance of PD-1/PD-L1 immunohistochemistry as a predictive or prognostic biomarker has grown. However, limited data on PD-L1 and PD-1 expression in TETs have been reported; moreover, these results have been variable. Here, we examined the expression of PD-1/PD-L1 proteins in TETs and analyzed the clinicopathologic significance of this expression.Patients and Methods: A tissue microarray was constructed using 368 samples of TETs, each in triplicate. Immunohistochemistry for PD-L1 (SP263 assay) and PD-1 in TETs and CD8 in thymic carcinoma (TC) was performed; next, correlations with clinicopathologic characteristics were analyzed. PD-L1high was designated as ≥50% of tumor proportion score; PD-1high and CD8high were defined as ≥5% and 1% of tumoral immune cells, respectively.Results: The cohort consisted of 308 patients with thymomas and 60 patients with TC. PD-L1 positivity was identified in 90.6% (328/362, ≥1%) of TETs, PD-1 expression of intra-/peritumoral T cells was identified in 53.6% (194/362) of TETs and CD8 positivity was identified in 11% (7/60, ≥1%) of TC. Of the 362 patients, 141 (39.0%) exhibited high PD-L1 expression (PD-L1high). The PD-L1high thymoma group was correlated with high Masaoka-Koga stage (p < 0.001), type B3 histology (p < 0.001), and myasthenia gravis (p < 0.001). This group exhibited poor overall survival (OS, p = 0.003, log-rank) and worse disease-free survival (DFS, p = 0.042, log-rank). No survival differences were detected between PD-L1high and PD-L1low groups in TC. Additionally, there was no correlation between PD-1 expression and survival in patients with TETs. Multivariate analysis revealed that PD-L1high expression was an independent poor prognostic factor (p = 0.047, HR 2.087, 95% CI, 1.009–4.318) in thymomas.Conclusions: To our knowledge, this is the largest study on TETs published in English literature. This study provides useful information regarding the prognosis of and potential therapeutic options for patients with TETs.

Background: Although non-small cell lung cancer (NSCLC) patients with EGFR sensitizing mutations shows dramatic responsiveness to EGFR-TKI, some of them still showed primary resistance. The loss of PTEN and NF-κB activation have known to contribute to intrinsic EGFR-TKI resistance in EGFR mutant lung cancer. Interestingly, Transglutaminase 2 (TG2) was well known to down-regulate PTEN and IκBα (NF-kB inhibitor) levels in tumor cells. Therefore, we investigated whether TG2 plays a role in the resistance and what kind of mechanisms involves in that resistance. Methods: We have established de novo and ectopic TG2 over-expressing EGFR mutant NSCLC cell lines. Subsequently, we have elaborated the resistance to EGFR-TKI therapy in these TG2-over-expressing cells. We have tried to investigate whether TG2 inhibitor could restore EGFR-TKI sensitivity with EGFR inhibitors in vitro and in vivo. Then, we evaluated TG2 expression in two intrinsic and eight sensitive clinical cases. Findings: The higher TG2 expression and lower PTEN and IκBα expression were evident in the intrinsic EGFR-TKI resistant NSCLC. EGFR-TKI sensitive NSCLC cells, harboring EGFR mutations, that stably expressed TG2 had reduced PTEN and IκBα and exhibited EGFR-TKI resistance. TG2 inhibition alone or co-treatment with TG2 inhibitor and EGFR-TKI in intrinsic EGFR-TKI resistant NSCLC cells harboring EGFR mutations overcame EGFR-TKI resistance via PTEN and IκBα restoration. Interpretation: TG2 elicits intrinsic EGFR-TKI resistance via PTEN loss and activation of the NF-κB pathway in NSCLC harboring EGFR sensitizing mutation. Therefore, TG2 may be a useful predictive marker and also be a target for overcoming the resistance. Funding Statement: This manuscript was supported by a grant from the National R&D Program for Cancer Control, Ministry of Health & Welfare, Republic of Korea (1520220) and the PostGenome Technology Development Program [10067758, Business model development driven by clinico-genomic database for precision immuno-oncology] funded by the Ministry of Trade, Industry and Energy (MOTIE, Korea). Declaration of Interests: D.H.L. declares honoraria from AstraZeneca, Boehringer-Ingelheim, Bristol-Myers Squibb, CJ Healthcare, ChongKunDang, Eli Lilly, Janssen, Merck, MSD, Mundipharma, Novartis, Ono, Pfizer, Roche, Samyang Biopharm and ST Cube. The other authors declare that they have no conflict of interest. Ethics Approval Statement: Animal procedures were approved by the institutional review board of Asan Medical Center

// Sung-Min Ahn 1, 2, * , Adnan Ahmad Ansari 3, 4, * , Jihun Kim 4, 5 , Deokhoon Kim 4 , Sung-Min Chun 4, 5 , Jiyun Kim 5 , Tae Won Kim 6 , Inja Park 7 , Chang-Sik Yu 7 , Se Jin Jang 4, 5 1 Division of Hematology-Oncology, Department of Internal Medicine, Gachon University Gil Hospital, Incheon, South Korea 2 Gachon Institute of Genome Medicine and Science, Gachon University Gil Hospital, Incheon, South Korea 3 Department of Biomedical Engineering, University of Ulsan College of Medicine, Seoul, South Korea 4 Asan Center for Cancer Genome Discovery, Asan Institute for Life Sciences, Asan Medical Center, Seoul, South Korea 5 Department of Pathology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, South Korea 6 Department of Oncology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, South Korea 7 Department of Surgery, University of Ulsan College of Medicine, Asan Medical Center, Seoul, South Korea * These authors have contributed equally to this work Correspondence to: Se Jin Jang, email: jangsejin@amc.seoul.kr Chang-Sik Yu, email: csyu@amc.seoul.kr Keywords: early-onset colorectal cancer, POLE, hypermutation, immunotherapy, PD-1 blockade Received: March 30, 2016 Accepted: August 24, 2016 Published: September 06, 2016 ABSTRACT Early-onset colorectal cancers (EOCRCs) may have biological or genomic features distinct from late-onset CRCs (LOCRCs). Previous studies have mostly focused on the germline predisposition conditions of EOCRCs, but we hypothesized that EOCRCs may have distinct somatic aberrations that accelerate cancer development. To identify the somatic aberrations that accelerate cancer development at an early age, we conducted whole exome sequencing for 28 polyposis-unrelated, microsatellite stable (MSS) EOCRCs with no known germline predisposition conditions. Surprisingly, we found two distinct groups in the context of mutational burden: 6 hypermutated cases with 2325 to 10973 mutations and 22 nonhypermutated cases with 47 to 154 mutations. Further analysis revealed that four of the six hypermutated cases had the same POLE P286R mutation. We validated this finding in 83 MSS EOCRCs and 27 MSS LOCRCs, which revealed that 7.2% of EOCRCs (6/83) had the POLE P286R mutation, which was not found in LOCRCs. Clinicopathologically, EOCRCs with POLE mutations occurred far more frequently in the right colon than in the left colon, affecting men more frequently than women. In summary, we have identified a unique subclass of colon cancer characterized by a hypermutation associated with the POLE mutation. The acquisition of the POLE mutation leading to hypermutation can accelerate cancer development. Clinically, this subset with hypermutation may be susceptible to immune checkpoint blockade.

Human epidermal growth factor receptor 2 (HER2) inhibitors, such as trastuzumab and lapatinib are used to treat HER2-positive breast and gastric cancers. However, as with other targeted therapies, intrinsic or acquired resistance to HER2 inhibitors presents unresolved therapeutic problems for HER2-positive gastric cancer. The present study describes investigations with AUY922, a heat shock protein 90 (HSP90) inhibitor, in primary lapatinib-resistant (ESO26 and OE33) and lapatinib-sensitive gastric cancer cells (OE19, N87, and SNU-216) harboring HER2 amplification/over-expression. In order to investigate whether AUY922 could overcome intrinsic and acquired resistance to HER2 inhibitors in HER2-positive gastric cancer, we generated lapatinib-resistant gastric cancer cell lines (OE19/LR and N87/LR) by continuous exposure to lapatinib in vitro. We found that activation of HER2 and protein kinase B (AKT) were key factors in inducing intrinsic and acquired lapatinib-resistant gastric cancer cell lines, and that AUY922 effectively suppressed activation of both HER2 and AKT in acquired lapatinib-resistant gastric cancer cell lines. In conclusion, AUY922 showed a synergistic anti-cancer effect with lapatinib and sensitized gastric cancer cells with intrinsic resistance to lapatinib. Dual inhibition of the HSP90 and HER2 signaling pathways could represent a potent therapeutic strategy to treat HER2-positive gastric cancer with intrinsic and acquired resistance to lapatinib. [BMB Reports 2018; 51(12): 660-665].

The immunogenomic characteristics of hepatocellular carcinomas (HCCs) with immune cell stroma (HCC-IS), defined histologically, have not been clarified. We investigated the clinical and molecular features of HCC-IS and the prognostic impact of Epstein-Barr virus (EBV) infection.We evaluated 219 patients with conventional HCC (C-HCC) and 47 with HCC-IS using in situ hybridization for EBV, immunohistochemistry, multiplex immunofluorescence staining, and whole exome and transcriptome sequencing. Human leukocyte antigen types were also extracted from the sequencing data. Genomic and prognostic parameters were compared between HCC-IS and C-HCC.CD8 T cell infiltration was more frequent in HCC-IS than C-HCC (mean fraction/sample, 22.6% vs. 8.9%, false discovery rate q 0.001), as was EBV positivity in CD20-positive tumor-infiltrating lymphocytes (TILs) (74.5% vs. 4.6%, p 0.001). CTNNB1 mutations were not identified in any HCC-IS, while they were present in 24.1% of C-HCC (p = 0.016). Inhibitory and stimulatory immune modulators were expressed at similar levels in HCC-IS and EBV-positive C-HCC. Global hypermethylation, and expression of PD-1 and PD-L1 in TILs, and PD-L1 in tumors, were also associated with HCC-IS (p 0.001), whereas human leukocyte antigen type did not differ according to HCC type or EBV positivity. HCC-IS was an independent factor for favorable recurrence-free survival (adjusted hazard ratio [aHR] 0.23; p = 0.002). However, a subgroup of tumors with a high density of EBV-positive TILs had poorer recurrence-free (aHR 25.48; p 0.001) and overall (aHR 9.6; p = 0.003) survival, and significant enrichment of CD8 T cell exhaustion signatures (q = 0.0296).HCC-IS is a distinct HCC subtype associated with a good prognosis and frequent EBV-positive TILs. However, paradoxically, a high density of EBV-positive TILs in tumors is associated with inferior prognostic outcomes. Patients with HCC-IS could be candidates for immunotherapy.Hepatocellular carcinomas with histologic evidence of abundant immune cell infiltration are characterized by frequent activation of Epstein-Barr virus in tumor-infiltrating lymphocytes and less aggressive clinical behavior. However, a high density of Epstein-Barr virus-positive tumor-infiltrating lymphocytes is associated with inferior prognostic outcomes, possibly as a result of immune escape due to significant CD8 T cell exhaustion.

Papillary thyroid microcarcinoma (PTMC) has excellent outcomes, but extensive lymph node (LN) metastasis can be associated with fatal outcomes. We evaluated the mutational profiles of primary tumors and their metastatic LNs of PTMCs with extensive lateral cervical LN metastases. Formalin-fixed, paraffin-embedded archival samples from 16 sets of normal thyroid tissue, the primary PTMC, and the largest metastatic LN were used for targeted sequencing. A total of seven somatic variants were confirmed in the PTMCs compared to the normal tissue. The BRAFV600E mutation was the most common and seen in 12 primary tumors (75%) and 11 metastatic LNs (69%). A nonsense mutation in AR and an in-frame deletion in ACVR2A were detected in one primary tumor and its metastatic LN (6%). Missense mutations in KMT2A, RAF1, and ROS1 were detected in one primary tumor (3%). A frameshift deletion mutation in JAK2 was detected in a metastatic LN (3%). In PTMCs without the BRAF mutation, an ALK and RET rearrangement (one PTMC and its metastatic LN, 6%) was detected. In one patient, the BRAF mutation was detected in the primary tumor, but only a RET rearrangement was detected in its metastatic LN. No mutations were detected in two patients. The mutational frequency of PTMCs was really low, even in those with extensive LN metastasis. The mutational status of the primary tumor and its metastatic LNs were not significantly different, and this suggests a minor role for genetic alterations in the process of LN metastasis in PTMC.

Molecular testing is increasingly important in cancer diagnosis. Targeted next generation sequencing (NGS) is widely accepted method but structural variation (SV) detection by targeted NGS remains challenging. In the brain tumor, identification of molecular alterations, including 1p/19q co-deletion, is essential for accurate glial tumor classification. Hence, we used targeted NGS to detect 1p/19q co-deletion using a newly developed deep learning (DL) model in 61 tumors, including 19 oligodendroglial tumors. An ensemble 1-dimentional convolution neural network was developed and used to detect the 1p/19q co-deletion. External validation was performed using 427 low-grade glial tumors from The Cancer Genome Atlas (TCGA). Manual review of the copy number plot from the targeted NGS identified the 1p/19q co-deletion in all 19 oligodendroglial tumors. Our DL model also perfectly detected the 1p/19q co-deletion (area under the curve, AUC = 1) in the testing set, and yielded reproducible results (AUC = 0.9652) in the validation set (n = 427), although the validation data were generated on a completely different platform (SNP Array 6.0 platform). In conclusion, targeted NGS using a cancer gene panel is a promising approach for classifying glial tumors, and DL can be successfully integrated for the SV detection in NGS data.

Pemetrexed and platinum (PP) combination chemotherapy is the current standard first-line therapy for treatment of malignant mesothelioma (MM). However, a useful predictive biomarker for PP therapy is yet to be found. Here, we performed targeted exome sequencing to profile somatic mutations and copy number variations in 12 MM patients treated with PP therapy. We identified 187 somatic mutations in 12 patients (65 synonymous, 102 missense, 2 nonsense, 5 splice site, and 13 small coding insertions/deletions). We identified somatic mutations in 23 genes including BAP1, TP53, NRAS, and EGFR. Interestingly, rare NRAS p.Q61K and EGFR exon 19 deletions were observed in 2 patients. We also found somatic chromosomal copy number deletions in CDKN2A and CDKN2B genes. Genetic alteration related to response after PP therapy was not found. Somatic mutation profiling in MM patients receiving PP therapy revealed genetic alterations in potential therapeutic targets such as NRAS and EGFR. No alterations in genes with potential predictive role for PP therapy were found.

Purpose: To better understand the complete genomic architecture of lung adenocarcinoma. Experimental Design: We used array experiments to determine copy number variations and sequenced the complete exomes of the 247 lung adenocarcinoma tumor samples along with matched normal cells obtained from the same patients. Fully annotated clinical data were also available, providing an unprecedented opportunity to assess the impact of genomic alterations on clinical outcomes. Results: We discovered that genomic alternations in the RB pathway are associated with significantly shorter disease-free survival in early-stage lung adenocarcinoma patients. This association was also observed in our independent validation cohort. The current treatment guidelines for early-stage lung adenocarcinoma patients recommend follow-up without adjuvant therapy after complete resection, except for high-risk patients. However, our findings raise the interesting possibility that additional clinical interventions might provide medical benefits to early-stage lung adenocarcinoma patients with genomic alterations in the RB pathway. When examining the association between genomic mutation and histologic subtype, we uncovered the characteristic genomic signatures of various histologic subtypes. Notably, the solid and the micropapillary subtypes demonstrated great diversity in the mutated genes, while the mucinous subtype exhibited the most unique landscape. This suggests that a more tailored therapeutic approach should be used to treat patients with lung adenocarcinoma. Conclusions: Our analysis of the genomic and clinical data for 247 lung adenocarcinomas should help provide a more comprehensive genomic portrait of lung adenocarcinoma, define molecular signatures of lung adenocarcinoma subtypes, and lead to the discovery of useful prognostic markers that could be used in personalized treatments for early-stage lung adenocarcinoma patients. Clin Cancer Res; 21(11); 2613–23. ©2014 AACR. See related commentary by Collisson, p. 2418

Dysfunction of T helper 17 (Th17) cells leads to chronic inflammatory disorders. Signal transducer and activator of transcription 3 (STAT3) orchestrates the expression of proinflammatory cytokines and pathogenic cell differentiation from interleukin (IL)-17-producing Th17 cells. However, the pathways mediated by STAT3 signaling are not fully understood. Here, we observed that Fos-related antigen 1 (FRA1) and JUNB are directly involved in STAT3 binding to sites in the promoters of Fosl1 and Junb. Promoter binding increased expression of IL-17 and the development of Th17 cells. Overexpression of Fra1 and Junb in mice resulted in susceptibility to collagen-induced arthritis and an increase in Th17 cell numbers and inflammatory cytokine production. In patients with rheumatoid arthritis, FRA1 and JUNB were colocalized with STAT3 in the inflamed synovium. These observations suggest that FRA1 and JUNB are associated closely with STAT3 activation, and that this activation leads to Th17 cell differentiation in autoimmune diseases and inflammation.

More than 25% of non-small cell lung cancers (NSCLCs) carry mutations in KRAS, one of the most common oncogenic drivers in this disease. KRAS-mutant NSCLC responds poorly to currently available therapies; therefore, novel treatment strategies are needed. Here, we describe a particularly promising targeted therapeutic strategy against KRAS mutation-harboring NSCLC intrinsically resistant to treatment by PI3K inhibition. We found that intrinsic resistance to PI3K inhibition derived from RAF/MEK/ERK and RSK activation, bypassing blockage of the PI3K/AKT/mTOR pathway. The HSP90 inhibitor AUY922 suppressed both PI3K/AKT/mTOR and RAF/MEK/ERK signaling, rendering cells sensitive to a PI3K inhibitor (omipalisib, GSK458). Combining these two drugs achieved a synergistic effect, even using only sub-therapeutic concentrations. Dual inhibition of the HSP90 and PI3K signaling pathways with sub-therapeutic doses of these combined anticancer drugs may represent a potent treatment strategy for KRAS-mutant NSCLC with intrinsic resistance to PI3K inhibition.

Hepatic resection is the most curative treatment option for early-stage hepatocellular carcinoma, but is associated with a high recurrence rate, which exceeds 50% at 5 years after surgery. Understanding the genetic basis of hepatocellular carcinoma at surgically curable stages may enable the identification of new molecular biomarkers that accurately identify patients in need of additional early therapeutic interventions. Whole exome sequencing and copy number analysis was performed on 231 hepatocellular carcinomas (72% with hepatitis B viral infection) that were classified as early-stage hepatocellular carcinomas, candidates for surgical resection. Recurrent mutations were validated by Sanger sequencing. Unsupervised genomic analyses identified an association between specific genetic aberrations and postoperative clinical outcomes. Recurrent somatic mutations were identified in nine genes, including TP53, CTNNB1, AXIN1, RPS6KA3, and RB1. Recurrent homozygous deletions in FAM123A, RB1, and CDKN2A, and high-copy amplifications in MYC, RSPO2, CCND1, and FGF19 were detected. Pathway analyses of these genes revealed aberrations in the p53, Wnt, PIK3/Ras, cell cycle, and chromatin remodeling pathways. RB1 mutations were significantly associated with cancer-specific and recurrence-free survival after resection (multivariate P = 0.038 and P = 0.012, respectively). FGF19 amplifications, known to activate Wnt signaling, were mutually exclusive with CTNNB1 and AXIN1 mutations, and significantly associated with cirrhosis (P = 0.017). Conclusion: RB1 mutations can be used as a prognostic molecular biomarker for resectable hepatocellular carcinoma. Further study is required to investigate the potential role of FGF19 amplification in driving hepatocarcinogenesis in patients with liver cirrhosis and to investigate the potential of anti-FGF19 treatment in these patients. (Hepatology 2014;60:1971–1981)

4078 Background: Although many studies using whole-exome sequencing or targeted sequencing have reported the molecular profile of BTC, its clinical implications remains unclear. In this study, we assessed a predictive role of DDR gene mutations in advanced BTC patients treated with platinum-containing regimen. Methods: Eighty-eight patients with pathologically-confirmed BTC who received first-line gemcitabine-cisplatin combination (n = 69) or fluoropyrimidine-oxaliplatin combination (n = 19) were included in this analysis. Targeted exome sequencing was performed using Foundation Medicine T7 assay or in-house OncoPanel AMC. Germline or somatic mutations in ATM, ATR, BAP1, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, FAM175A, GEN1, MLH1, MSH2, MSH6, MRE11A, NBN, PALB2, PMS2, RAD50, RAD51, RAD51C, RAD51D, and XRCC2 were classified as DDR gene mutations. Data regarding baseline characteristics and treatment outcomes were retrospectively obtained from medical records. Results: The median age was 62 years (range, 25-78), with male comprising 64.8% (n = 57). By primary tumor site, 21 patients with GBC (23.9%), 44 with ICC (50.0%) and 23 with ECC (26.1%) were included. Most patients received palliative chemotherapy for their initially metastatic (50.0%) or recurred (44.3%) disease; the rest 5.7% had locally advanced disease. The median PFS and OS of overall patients were 7.1 and 16.1 months, respectively with median follow-up duration of 20.2 months. DDR gene mutations were found in 63.5% of patients. BRCA2 (18.2%) was most frequently mutated, followed by ATM (13.6%), and ATR (8.0%). DDR gene mutations were significantly associated with prolonged PFS (presence vs. absence; median, 6.9 vs. 5.7 months; P = 0.013) and OS (median, 21.0 vs. 13.3 months, P = 0.009). The impact of DDR gene mutations remained significant in multivariate analyses for PFS that included other prognostic factors (hazard ratio, 0.51; P = 0.009), but not for OS. Conclusions: The presence of DDR gene mutations might be a promising predictive biomarker for response to platinum-based chemotherapies in advanced BTC. Future investigation using novel agents targeting DDR gene alteration in BTC are warranted.

152 Background: ATTRACTION-2 phase III trial proved the clinical efficacy of nivolumab (Nivo) in AGC patients (pts) treated with ≥ 2 previous chemotherapy regimens. However, the benefits of Nivo seem to be limited to a subset of pts and there is a need to identify predictive markers to select pts who would benefit from Nivo. Methods: Clinical data and tumor samples of AGC pts enrolled from Asan Medical Center in ATTRACTION-2 study were retrospectively analyzed. PD-L1 (+) was defined as combined positive score of ≥ 1%. EBV and MSI status were determined by EBV-encoded small RNA in situ hybridization and IHC for MLH-1, MSH-2, PMS-2, and MSH-6, respectively. Tumor mutation burden (TMB) was acquired by targeted next-generation sequencing using the NextSeq platform with OncoPanel. Results: A total of 45 pts (28/17 in Nivo/placebo arms) were eligible for the analysis. Baseline neutrophil-lymphocyte ratio (NLR) was median 2.9 and 7 pts had hyponatremia ( < 135). In 36 pts with available tissues, there were PD-L1 (+) (N = 13, 36.1%), EBV (+) (N = 6, 16.7%), and no MSI-high. TMB data could be acquired in 29 pts and median TMB was 8.2/Mb (0.0-21.3). With a median follow-up of 28.3 months (mo) in surviving pts, objective response rate, median progression-free survival (PFS), and overall survival (OS) were 16.7%, 1.6 mo, and 8.1 mo in Nivo arm and 0%, 1.6 mo and 6.5 mo in placebo arm. In Nivo arm with measurable lesions, PD-L1 (+) pts had significantly higher disease control rate than PD-L1 (-) pts (87.5% vs. 20%, p = 0.015). In multivariate model adjusted for important factors (age, sex, Nivo vs. placebo, treatment line, NLR, Na, and PD-L1), NLR ≤ 2.9 and PD-L1 (+) were significant factors for PFS (Hazard Ratio [HR] 0.47, p = 0.037 and HR 0.32, p = 0.006, respectively) and PD-L1 (+) was a significant factor for OS (HR, 0.44, p = 0.012). With adjusting these factors with TMB, PD-L1 (+) remained favorable for PFS and OS. Pts with NLR ≤ 2.9 or PD-L1 (+) or Na > 135 significantly favored Nivo compared to placebo in terms of PFS and OS. Conclusions: Baseline NLR and PD-L1 status may be relevant predictive markers to select pts with AGC who would benefit from Nivo.

Background Cetuximab has shown clinical benefit in patients with metastatic colorectal cancer (mCRC) harboring wild-type RAS . Human epidermal growth factor receptor 2 ( HER2 ) amplification may be a mechanism of cetuximab resistance. We evaluated the association between HER2 amplification and cetuximab efficacy in patients with mCRC harboring wild-type RAS and BRAF . Patients and Methods Between December 2003 and June 2013, we identified 142 patients with mCRC whose tumors harbored both wild-type exons 2, 3, and 4 in KRAS and NRAS , and wild-type exon 15 in BRAF using high throughput sequencing (OncoMap version 4.0). All patients received cetuximab after oxaliplatin, irinotecan, and fluoropyrimidine failure. HER2 status was determined using immunohistochemistry and silver in situ hybridization (SISH) and correlated with cetuximab efficacy. Results Of 142 RAS and BRAF wild-type tumors, we observed 7 cases (4.9%) of HER2 amplification by SISH. After a median follow-up of 13.2 months (range, 1.4-78.1 months), median progression-free survival (PFS) was significantly different according to HER2 status: 3.1 months in patients with HER2 amplification compared with 5.6 months in those with non-amplified HER2 (hazard ratio, 2.73; 95% confidence interval, 1.18-6.31; P = .019). Overall survival (OS) was not significantly different between groups, although there was a tendency towards shorter OS in patients with HER2 -amplified tumors (hazard ratio, 1.31; 95% confidence interval, 0.61-2.82; 10.1 vs. 13.5 months; P = .488). Conclusions HER2 amplification is predictive of shorter PFS after cetuximab treatment in patients with mCRC harboring wild-type RAS and BRAF . Further study is warranted for this patient population.

Anaplastic thyroid carcinoma (ATC), the most aggressive type of thyroid cancer, has no effective therapy. Due to its dismal prognosis, it is vital to understand the genetic alterations of ATC and identify effective molecular targets. Targeted next-generation sequencing was performed to investigate the mutational profile of ATC using a massive parallel sequencing approach.DNA from formalin-fixed, paraffin-embedded archival samples of 11 ATCs and normal matched pairs were used. A total of 48 genetic alterations were identified by targeted exome sequencing. These alterations were validated by mass spectrometric genotyping and direct Sanger sequencing.The most commonly mutated gene was BRAF, identified in 10 samples (91%), all showing the V600E point mutation. A KRAS point mutation was observed in the one sample (9%) without the BRAF(V600E) mutation. All 11 ATCs harbored BRAF or RAS mutations, reflecting the possibility that differentiated thyroid carcinomas progress to ATCs after the accumulation of mutations. A loss of function mutation of TP53 was observed in eight samples (73%), a PIK3CA mutation was observed in two samples (18%), and a frameshift mutation of PTEN was observed in one sample (9%). Twenty-eight novel mutated genes were found that had not previously been associated with ATC. Of these, loss of function mutations of NF2, KMT2D, and PKHD1 were repeatedly seen in three samples (27%), two samples (18%), and two samples (18%), respectively. Using direct Sanger sequencing, two samples (18%) were also found with a RASAL1 mutation. KMT2D and RASAL1 mutations were significantly associated with shorter ATC patient survival.This comprehensive analysis of ATCs using targeted massive parallel sequencing identified several novel mutations in ATCs, such as loss of function mutations of NF2 or KMT2D. Future studies are needed to confirm the role of these novel mutations as independent drivers of ATC development.

Background: Biliary tract cancer (BTC) is a heterogeneous group of cancers anatomically divided into the gallbladder cancer (GBC), intrahepatic cholangiocarcinoma (ICC) and extrahepatic cholangiocarcinoma (ECC). None of molecular target agents has been proven to improve the prognosis of BTC yet. To gain a deeper understanding of BTC's pathophysiology and find its new potential therapeutic targets, this study is aimed to investigate genetic profiles of BTC and their clinical implications. Methods: A total of 69 patients who had been pathologically diagnosed as adenocarcinoma of biliary tract from January 2014 to November 2016 and had available surgical or biopsy specimen were enrolled for this study. With the DNA specimen extracted from previously-collected tumor tissue, somatic mutations and copy number variation (CNV) analyses were performed using targeted exome sequencing. Data regarding the baseline patient characteristics and treatment outcomes were retrospectively obtained by reviewing the patients' medical records. Results: Nineteen patients with GBC (28%), 24 patients with ICC (35%) and 26 patients with ECC (37%) were included in this analysis. Genetic mutation and copy number alteration (CAN) were observed in 59 (85%) and 35 (51%) patients, respectively while 5 patients (7%) did not have any genetic alterations. The most commonly mutated gene was TP53 (n=33, 47.8%), followed by KRAS (n=18, 26.1%), ARID1A (n=10, 14.5%) and IDH1 (n=9, 13.0%). IDH1 mutation appeared more frequently in patients with ICC (n=7, 29.2%, P=0.015) compared to those with GBC (n=1, 5.3%) or ECC (n=1, 3.8%) while ERBB2 and ERBB3 mutation were found only in GBC and ECC. HER2 amplification was observed in 7 patients (4 with GBC and 3 with ICC). Among those, one patient showed 3+ for HER2 test by immunohistochemistry (IHC) while four patients were 2+. All somatic mutations and CNVs documented in the present study were classified into five pathway modules which were identified by Nakamura et al. (Nature Genetics 2015). Majority of genetic alterations (n=45, 65.2%) belonged to Kinase-RAS module, followed by TP53 (n=41, 59.4%), TGF-β-SWI/SNF-MYC (n=22, 31.9%), Epigenetic (n=16, 23.2%) and RB-cell cycle (n=10, 14.5%) modules. Alterations in Epigenetic module were most common in ICC (vs. GB vs. ECC, 50.0% vs. 15.8% vs. 3.8%, P=0.002) while alterations in TGF-β-SWI/SNF-MYC module were most frequent in ECC (vs. GB vs. ICC, 46.2% vs. 3.6% vs. 31.6%, P=0.031). Conclusion: Genetic profile of BTC is heterogeneous according to its anatomic location. Our results indicate that subgroup of BTC patients may have benefit from targeted therapy such anti-IDH and anti-HER2 inhibitors. Citation Format: Heejung Chae, Changhoon Yoo, Deokhoon Kim, Seonmin Lee, Kim Dan Bi, Jae Ho Jeong, Heung-Moon Chang, Baek-Yeol Ryoo, Kyu-pyo Kim. Evaluation of genetic mutations and copy number alterations in biliary tract cancer using targeted exome 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 3431.

We have sequenced the genomes of 110 small cell lung cancers (SCLC), one of the deadliest human cancers. In nearly all the tumours analysed we found bi-allelic inactivation of TP53 and RB1, sometimes by complex genomic rearrangements. Two tumours with wild-type RB1 had evidence of chromothripsis leading to overexpression of cyclin D1 (encoded by the CCND1 gene), revealing an alternative mechanism of Rb1 deregulation. Thus, loss of the tumour suppressors TP53 and RB1 is obligatory in SCLC. We discovered somatic genomic rearrangements of TP73 that create an oncogenic version of this gene, TP73Deltaex2/3. In rare cases, SCLC tumours exhibited kinase gene mutations, providing a possible therapeutic opportunity for individual patients. Finally, we observed inactivating mutations in NOTCH family genes in 25% of human SCLC. Accordingly, activation of Notch signalling in a pre-clinical SCLC mouse model strikingly reduced the number of tumours and extended the survival of the mutant mice. Furthermore, neuroendocrine gene expression was abrogated by Notch activity in SCLC cells. This first comprehensive study of somatic genome alterations in SCLC uncovers several key biological processes and identifies candidate therapeutic targets in this highly lethal form of cancer.

Hepatocellular carcinoma (HCC) remains a significant clinical challenge with few therapeutic options available to cancer patients. MicroRNA 21-5p (miR-21) has been shown to be upregulated in HCC, but the contribution of this oncomiR to the maintenance of tumorigenic phenotype in liver cancer remains poorly understood. We have developed potent and specific single-stranded oligonucleotide inhibitors of miR-21 (anti-miRNAs) and used them to interrogate dependency on miR-21 in a panel of liver cancer cell lines. Treatment with anti–miR-21, but not with a mismatch control anti-miRNA, resulted in significant derepression of direct targets of miR-21 and led to loss of viability in the majority of HCC cell lines tested. Robust induction of caspase activity, apoptosis, and necrosis was noted in anti–miR-21-treated HCC cells. Furthermore, ablation of miR-21 activity resulted in inhibition of HCC cell migration and suppression of clonogenic growth. To better understand the consequences of miR-21 suppression, global gene expression profiling was performed on anti–miR-21-treated liver cancer cells, which revealed striking enrichment in miR-21 target genes and deregulation of multiple growth-promoting pathways. Finally, in vivo dependency on miR-21 was observed in two separate HCC tumor xenograft models. In summary, these data establish a clear role for miR-21 in the maintenance of tumorigenic phenotype in HCC in vitro and in vivo. Implications: miR-21 is important for the maintenance of the tumorigenic phenotype of HCC and represents a target for pharmacologic intervention. Mol Cancer Res; 13(6); 1009–21. ©2015 AACR.

Approximately 50% of patients with primary colorectal carcinoma develop liver metastases. Understanding the genetic differences between primary colon cancer and their metastases to the liver is essential for devising a better therapeutic approach for this disease. We performed whole exome sequencing and copy number analysis for 15 triplets, each comprising normal colorectal tissue, primary colorectal carcinoma, and its synchronous matched liver metastasis. We analyzed the similarities and differences between primary colorectal carcinoma and matched liver metastases in regards to somatic mutations and somatic copy number alterationss. The genomic profiling demonstrated mutations in APC(73%), KRAS (33%), ARID1A and PIK3CA (6.7%) genes between primary colorectal and metastatic liver tumors. TP53 mutation was observed in 47% of the primary samples and 67% in liver metastatic samples. The grouped pairs, in hierarchical clustering showed similar somatic copy number alteration patterns, in contrast to the ungrouped pairs. Many mutations (including those of known key cancer driver genes) were shared in the grouped pairs. The ungrouped pairs exhibited distinct mutation patterns with no shared mutations in key driver genes. Four ungrouped liver metastasis samples had mutations in DNA mismatch repair genes along with hypermutations and a substantial number of copy number alterations. Our results suggest that about half of the metastatic colorectal carcinoma had the same clonal origin with their primary colorectal carcinomas, whereas remaining cases were genetically distinct from their primary carcinomas. These findings underscore the need to evaluate metastatic lesions separately for optimized therapy, rather than to extrapolate from primary tumor data.

Understanding stem cell-differentiation at the molecular level is important for clinical applications of stem cells and for finding new therapeutic approaches in the context of cancer stem cells. To investigate genome-wide changes involved in differentiation, we have used immortalized neural stem cell (NSC) line (HB1.F3) and Olig2-induced NSC differentiation model (F3.Olig2). Using microarray analysis, we revealed that Olig2-induced NSC differentiation involves downregulation of Wnt pathway, which was further confirmed by TOPflash/FOPflash reporter assay, RT-PCR analysis, immunoblots, and immunocytochemistry. Furthermore, we found that Olig2-induced differentiation induces the expression of Dickkopf-1(Dkk1), a potent antagonist of Wnt signaling. Dkk1 treatment blocked Wnt signaling in HB1.F3 in a dosage-dependent manner, and induced differentiation into astrocytes, oligodendrocytes, and neurons. Our results support cancer stem cell hypothesis which implies that signaling pathway for self-renewal and proliferation of stem cells is maintained till the late stage of differentiation. In our proposed model, Dkk1 may play an important role in downregulating self-renewal and proliferation pathway of stem cells at the late stage of differentiation, and its failure may lead to carcinogenesis.

Albumin, an abundant plasma protein with multifunctional properties, is mainly synthesized in the liver. Albumin has been implicated in Alzheimer's disease (AD) since it can bind to and transport amyloid beta (Abeta), the causative agent of AD; albumin is also a potent inhibitor of Abeta polymerization. Despite evidence of non-hepatic transcription of albumin in many tissues including kidney and pancreas, non-hepatic synthesis of albumin at the protein level has been rarely confirmed. In a pilot phase study of Human Brain Proteome Project, we found evidence that microglial cells in brain may synthesize albumin. Here we report, for the first time, the de novo synthesis of albumin in human microglial cells in brain. Furthermore, we demonstrate that the synthesis and secretion of albumin from microglial cells is enhanced upon microglial activation by Abeta(1-42)- or lipopolysaccharide (LPS)-treatment. These data indicate that microglial cells may play a beneficial role in AD by secreting albumin that not only inhibits Abeta polymerization but also increases its clearance.

Bone mineral density (BMD) is the major factor for determining bone strength, which is closely correlated to osteoporotic fracture risk and is largely determined by multiple genetic factors. The RANK (TNFRSF11A), receptor for RANKL, is a member of the tumor necrosis factor receptor (TNFR) superfamily and plays a central role in osteoclast development.In order to investigate the effects of RANK polymorphism on BMD and osteoporosis, we directly sequenced the RANK gene in 24 Korean individuals and identified 25 sequence variants. Eleven of these polymorphisms were selected and genotyped in a larger-scale study of postmenopausal women (n = 560). Areal BMD (g/cm(2)) of the anterior-posterior lumbar spine and the nondominant proximal femur were measured using dual-energy X-ray absorptiometry.We found that two intronic polymorphisms in the RANK gene [RANK + 34863GA (rs12458117) and RANK + 35928insdelC (new polymorphism found in this study) in intron 6] were significantly associated with the BMD of the lumbar spine, i.e., rare alleles were significantly associated with low BMD of the lumbar spine among Korean postmenopausal women (p = 0.04 and 0.02, respectively). These polymorphisms were also associated with low BMD of proximal femur sites, including Ward's triangle, trochanter, and total femur. Our results suggest that +34863GA and +35928insdelC polymorphisms in RANK are possible genetic factors for low BMD in postmenopausal women.

The carbohydrate response element binding protein (ChREBP), a basic helix-loop-helix/leucine zipper transcription factor, plays a critical role in the control of lipogenesis in the liver. To identify the direct targets of ChREBP on a genome-wide scale and provide more insight into the mechanism by which ChREBP regulates glucose-responsive gene expression, we performed chromatin immunoprecipitation-sequencing and gene expression analysis. We identified 1153 ChREBP binding sites and 783 target genes using the chromatin from HepG2, a human hepatocellular carcinoma cell line. A motif search revealed a refined consensus sequence (CABGTG-nnCnG-nGnSTG) to better represent critical elements of a functional ChREBP binding sequence. Gene ontology analysis shows that ChREBP target genes are particularly associated with lipid, fatty acid and steroid metabolism. In addition, other functional gene clusters related to transport, development and cell motility are significantly enriched. Gene set enrichment analysis reveals that ChREBP target genes are highly correlated with genes regulated by high glucose, providing a functional relevance to the genome-wide binding study. Furthermore, we have demonstrated that ChREBP may function as a transcriptional repressor as well as an activator.