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Through the wide adaptation of next-generation sequencing (NGS) technology within clinical practice, molecular profiling of the tumor has been the principal component of personalized treatment. In our study, we have generated a large collection of cancer genomes on East Asian epithelial ovarian carcinoma (EOC) patients and demonstrate the feasibility and utility of NGS platforms to explore the dynamic interrelations of major cancer driver alterations and their impacts on clinical prognosis and management. A total of 652 EOC patients have undergone clinical NGS panels to determine the prevalence of germline and somatic mutations. Notably, TP53 was the most frequently altered event (73%), followed by both BRCA1 and BRCA2 (22% each) and MYC (19%) through pan-EOC analysis. When analyzed based on individual histopathological levels, TP53 mutation was highly dominant in high-grade serous and mucinous histology, whereas mutations in PIK3CA and ARID1A were mostly observed in clear cell carcinoma, and KRAS, BRAF, and CDKN2A mutations were enriched in endometrioid, low-grade serous, and mucinous tumors, respectively. The network-based probabilistic model showed significant co-occurrences of TP53 with BRCA1 and ALK with BRCA2, NOTCH1, and ROS1, whereas mutual exclusivity of TP53 with KRAS and PIK3CA was evident. Furthermore, we utilized machine-learning algorithms to identify molecular correlates that conferred increased sensitivity to platinum and olaparib treatments including somatic mutations in BRCA1, ATM, and MYC. Conversely, patients with ALK mutation were considerably resistant to both treatment modalities. Collectively, our results demonstrate the clinical feasibility of prospective genetic sequencing to facilitate personalized treatment opportunities for patients with EOC.

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.

The advent of receptor tyrosine kinase inhibitors (RTKi) are important contributions to treat NSCLC patients harboring genetic aberration effectively. Nevertheless, the emergence of acquired or intrinsic RTKi resistance by other RTK activation bypass finally limits their efficacy. First of all, in this study, we identified MED12 mutation in TKI-resistant NSCLC cells based-on NGS analysis. Although MED12 was known to controls the response to multiple cancer drugs through regulation of TGF-beta receptor signaling reportedly, we additionally found that MED12 mutation activates not only TGF-beta receptor not also other ones, such as EGFR, meaning that blocking TGF-beta receptor or EGFR alone may not effective to overcome MED12 mutation-induced RTKi resistance. To overcome the RTKi resistance in NSCLC harboring MED12 mutation, we elaborated the two downstream signaling of RTK in MED12 knock-out NSCLC cells and observed that PI3K/AKT signaling is downregulated but MEK/ERK signaling is upregulated. We observed that only MEK inhibitor (MEKi), not PI3K/mTOR inhibitor, shows effective anti-cancer effect. Therefore, we explored why MEKi alone is effective in RTKi resistant NSCLC cells harboring MED12 mutation and presented that MED12 mutation directly suppressed activation of YAP via blocking a large mediator complex of MED12, MED13, CDK8 and CCNC, thereby increasing PTEN expression by inhibition of miR-29 expression. Increased PTEN inhibits reactivation of PI3K/AKT pathway. In conclusion, we first identified that MED12 mutation induces RTKi resistance though activating other RTKs, such as TGF-beta receptor or EGFR, and blocks PI3K/AKT pathway via dysregulation of YAP/PTEN/AKT interaction. Therefore, among two downstream signaling of RTK, activated MEK/ERK pathway can be a definitive target enough to overcome RTKi resistant NSCLC patients harboring MED12 mutation and MEKi could be a primary treatment option for resistant patients harboring MED12 mutation among NSCLC patients with repetitive recurrence of RTKi resistance by another RTK activation bypass. Citation Format: Hyun-Min Ryu, Deokhoon Kim, Shinkyo Yoon, Ho-Su Lee, Eunjin Lee, Chang Hoon Lee, Wanlim Kim, Seyoung Seo, Kyung Hae Jung, Sook Ryun Park, Sang-We Kim, Kang-Seo Park, Dae Ho Lee. RTK inhibitor resistance in NSCLC harboring MED12 mutation is overcome by only blocking MEK signaling, not AKT due to mutated MED12-induced YAP-PTEN dysregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 424.

Recent lung adenocarcinoma (LUAD) grading system proposed by the International Association for the Study of Lung Cancer (IASLC) has emphasized the proportion of high-grade patterns (HGPs). We aimed to evaluate the clinicopathologic and genomic characteristics associated with HGP which has not yet been fully investigated.Tissue samples from 174 patients who underwent surgical resection of LUAD from January to December 2015 were histologically evaluated. Proportions of HGPs, including solid, micropapillary, cribriform, and complex glandular patterns, were individually quantified. Prognostic implications of HGP proportion, both as a continuous variable and as subclasses divided by cutoffs of 20%, 50%, and 90% (low-intermediate grade [LIG], HGP 20%; high grade 1 [HG1], 20-50%, HG2, 50-90%; HG3, ≥90%) were evaluated. Different clinicopathologic factors and genomic alterations according to the HGP subclasses were assessed.Relative hazards of the HGP gradually elevated as its proportion increased over 20%, the cut-off value established by the IASLC grading system, and the cancer-specific overall survival (OS) of HG1 subclass was not significantly decreased compared to the LIG subclass on univariate analysis. However, further subgrouping showed significantly increased frequencies of male, advanced stage tumors, lymphovascular invasion, and spread through alveolar space in higher HGP subclasses. Also, common LUAD driver mutations, particularly EGFR mutations, were less frequent, whereas alterations in TP53 and cell cycle pathway-related genes were more frequent. Higher HGP subclasses and TP53 gene alteration were associated with shorter cancer-specific OS and RFS in multivariate survival analysis.HGP subclasses of LUAD displayed distinct clinicopathological characteristics and genomic alterations, including TP53 and cell cycle pathway, emphasizing the clinical value of these subclasses in LUAD. Higher HGP subclass and alteration in TP53 may be markers of poor post-operative survival.

Purpose The phase 3 ATTRACTION-2 study demonstrated that nivolumab monotherapy improved survival compared to placebo in patients with pretreated advanced gastric cancer (AGC). However, the efficacy of nivolumab seems to be limited to a subset of patients. Materials and methods The predictive values of blood neutrophil–lymphocyte ratio (NLR), serum Na, PD-L1 expression, MSI status, tumor EBV infection, and tumor mutation burden (TMB) were investigated in patients with AGC refractory to ≥2 lines of chemotherapy enrolled from Asan Medical Center in ATTRACTION-2 study. Results All 45 patients were analyzed; nivolumab (n = 28) and placebo (n = 17) groups. The objective response rate, median progression-free survival (PFS), and overall survival (OS) were 16.7%, 1.6 months, and 8.1 months in nivolumab group and 0%, 1.6 months and 6.5 months in placebo group. When comparing nivolumab with the placebo group, tumor PD-L1 expression, blood NLR, and serum Na were significant predictive factors of PFS and OS. A multivariate analysis revealed that PD-L1 ( +) and low NLR (≤ 2.9, median) were associated with better PFS. In the nivolumab group, PD-L1 ( +), low NLR, and normal Na (≥ 135 mmol/L) were associated with higher response and disease control rates, while tumor EBV infection and TMB were not. Conclusion Tumor PD-L1 expression, blood NLR, and serum Na could be predictive biomarkers for the efficacy of nivolumab in previously treated cases of AGC.

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.

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.

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

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.

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

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.

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.

To identify potential microRNA (miRNA) links between Smad3, a mediator of TGF-β (transforming growth factor-β) signaling, and E-cadherin, we characterized the miRNA profiles of two gastric cancer cell lines: SNU484-LPCX, which does not express Smad3, and SNU484-Smad3, in which Smad3 is overexpressed. We found that among differentially expressed miRNAs, miR-200 family members are overexpressed in SNU484-Smad3 cells. Subsequent studies, including analysis of the effects of silencing Smad3 in SNU484-Smad3 cells and a luciferase reporter assay, revealed that Smad3 directly binds to a Smad-binding element located in the promoter region of miR-200b/a, where it functions as a transcriptional activator. TGF-β did not affect the regulatory role of Smad3 in transcription of miR-200 and expression of epithelial-mesenchymal transition markers. We conclude that Smad3 regulates, at the transcriptional level, miR-200 family members, which themselves regulate ZEB1 and ZEB2, known transcriptional repressors of E-cadherin, at the posttranscriptional level in a TGF-β-independent manner. This represents a novel link between Smad3 and posttranscriptional regulation by miRNAs in epithelial-mesenchymal transition in gastric cancer cells.