Mingxin Zuo

Mutation Profiling in Cholangiocarcinoma: Prognostic and Therapeutic Implications

Background Cholangiocarcinoma (CCA) is clinically heterogeneous; intra and extrahepatic CCA have diverse clinical presentations. Next generation sequencing (NGS) technology may identify the genetic differences between these entities and identify molecular subgroups for targeted therapeutics. Methods We describe successful NGS-based testing of 75 CCA patients along with the prognostic and therapeutic implications of findings. Mutation profiling was performed using either a) NGS panel of hotspot regions in 46 cancer-related genes using a 318-chip on Ion PGM Sequencer or b) Illumina HiSeq 2000 sequencing platform for 3,769 exons of 236 cancer-related genes plus 47 introns from 19 genes to an average depth of 1000X. Clinical data was abstracted and correlated with clinical outcome. Patients with targetable mutations were referred to appropriate clinical trials. Results There were significant differences between intrahepatic (n = 55) and extrahepatic CCA (n = 20) in regard to the nature and frequency of the genetic aberrations (GAs). IDH1 and DNA repair gene alterations occurred more frequently in intrahepatic CCA, while ERBB2 GAs occurred in the extrahepatic group. Commonly occurring GAs in intrahepatic CCA were TP53 (35%), KRAS (24%), ARID1A (20%), IDH1 (18%), MCL1 (16%) and PBRM1 (11%). Most frequent GAs in extrahepatic CCA (n = 20) were TP53 (45%), KRAS (40%), ERBB2 (25%), SMAD4 (25%), FBXW7 (15%) and CDKN2A (15%). In intrahepatic CCA, KRAS, TP53 or MAPK/mTOR GAs were significantly associated with a worse prognosis while FGFR GAs correlated with a relatively indolent disease course. IDH1 GAs did not have any prognostic significance. GAs in the chromatin modulating genes, BAP1 and PBRM1 were associated with bone metastases and worse survival in extrahepatic CCA. Radiologic responses and clinical benefit was noted with EGFR, FGFR, C-met, B-RAF and MEK inhibitors. Conclusion There are significant genetic differences between intra and extrahepatic CCA. NGS can potentially identify disease subsets with distinct prognostic and therapeutic implications.

The UPF1 RNA surveillance gene is commonly mutated in pancreatic adenosquamous carcinoma.

Pancreatic adenosquamous carcinoma (ASC) is an enigmatic and aggressive tumor that has a worse prognosis and higher metastatic potential than its adenocarcinoma counterpart. Here we report that ASC tumors frequently harbor somatically acquired mutations in the UPF1 gene, which encodes the core component of the nonsense-mediated RNA decay (NMD) pathway. These tumor-specific mutations alter UPF1 RNA splicing and perturb NMD, leading to upregulated levels of NMD substrate mRNAs. UPF1 mutations are, to our knowledge, the first known unique molecular signatures of pancreatic ASC.

HER2/neu-directed therapy for biliary tract cancer

BackgroundBiliary cancers are highly aggressive tumors that are often diagnosed an advanced disease stage and have a poor outcome with systemic therapy. Recent efforts towards molecular characterization have identified a subset of biliary patients that have HER2/neu amplification or mutation. HER2/neu amplification is associated with response to HER2/neu-directed therapy in breast and gastric cancers. However, the efficacy of HER2/neu-targeted therapy in biliary cancers is unknown.Patients and methodsWe retrospectively reviewed cases of advanced gallbladder cancer and cholangiocarcinoma with HER2/neu genetic aberrations or protein overexpression who received HER2/neu-directed therapy between 2007 and 2014. Clinical data were retrieved from medical records, and imaging studies were independently reviewed.ResultsNine patients with gallbladder cancer and five patients with cholangiocarcinoma had received HER2/neu-directed therapy (trastuzumab, lapatinib, or pertuzumab) during the study period. In the gallbladder cancer group, HER2/neu gene amplification or overexpression was detected in eight cases. These patients experienced disease stability (n = 3), partial response (n = 4), or complete response (n = 1) with HER2/neu-directed therapy. One patient had HER2/neu mutation and experienced a mixed response after lapatinib therapy. The duration of response varied from 8+ to 168 weeks (median 40 weeks), and three patients are still on therapy. One patient developed HER2/neu amplification as a secondary event after FGFR-directed therapy for FGF3-TACC3 gene fusion. The cholangiocarcinoma cases treated in this series had a higher proportion of HER2/neu mutations, and no radiological responses were seen in these patients despite HER2/neu-directed therapy.ConclusionsHER2/neu blockade is a promising treatment strategy for gallbladder cancer patients with gene amplification and deserves further exploration in a multi-center study.

Biliary cancer: Utility of next‐generation sequencing for clinical management

Biliary tract cancers (BTCs) typically present at an advanced stage, and systemic chemotherapy is often of limited benefit.

Molecular Characterization of Gallbladder Cancer using Somatic Mutation Profiling

Gallbladder cancer is relatively uncommon with high incidence in certain geographic locations, including Latin America, East and South Asia and Eastern Europe. Molecular characterization of this disease has been limited and targeted therapy options for advanced disease remain an open area of investigation. In the present study, surgical pathology obtained from resected gallbladder cancer cases (n=72) was examined for the presence of targetable, somatic mutations. All cases were formalin-fixed and paraffin-embedded (FFPE). Two approaches were used: a) mass spectroscopy-based profiling for 159 point (‘hot-spot’) mutations in 33 genes commonly involved in solid tumors and b) next-generation sequencing (NGS) platform that examined the complete coding sequence of in 182 cancer-related genes. Fifty-seven cases were analyzed for hotspot mutations and 15 for NGS. Fourteen hotspot mutations were identified in nine cases. Of these, KRAS mutation was significantly associated with poor survival on multivariate analysis. Other targetable mutations included PIK3CA (N=2) and ALK (N=1). On NGS, 26 mutations were noted in 15 cases. P53 and PI3 kinase pathway (STK11, RICTOR,TSC2) mutations were common. One case had FGF10 amplification while another had FGF3-TACC gene fusion, not previously described in gallbladder cancer. In conclusion, somatic mutation profiling using archival FFPE samples from gallbladder cancer is feasible. NGS, in particular may be a useful platform for identifying novel mutations for targeted therapy.

Effect of NR5A2 inhibition on pancreatic cancer stem cell (CSC) properties and epithelial-mesenchymal transition (EMT) markers

NR5A2 (aka LRH‐1) has been identified as a pancreatic cancer susceptibility gene with missing biological link. This study aims to demonstrate expression and potential role of NR5A2 in pancreatic cancer. NR5A2 expression was quantified in resected pancreatic ductal adenocarcinomas and the normal adjacent tissues of 134 patients by immunohistochemistry. The intensity and extent of NR5A2 staining was quantified and analyzed in association with overall survival (OS). The impact of NR5A2 knockdown on pancreatic cancer stem cell (CSC) properties and epithelial‐mesenchymal transition (EMT) markers was examined in cancer cells using RT‐PCR and Western Blot. NR5A2 was overexpressed in pancreatic tumors, the IHC‐staining H score (mean ± SE) was 96.4 ± 8.3 in normal versus 137.9 ± 8.2 in tumor tissues (P < 0.0001). Patients with a higher NR5A2 expression had a median survival time 18.4 months compared to 23.7 months for those with low IHC H scores (P = 0.019). The hazard ratio of death (95% confidence interval) was 1.60 (1.07‐2.41) after adjusting for disease stage and tumor grade (P = 0.023). NR5A2 was highly expressed in pancreatic cancer sphere forming cells. NR5A2‐inhibition by siRNA was associated with reduced sphere formation and decreased levels of CSCs markers NANOG, OCT4, LIN28B, and NOTCH1. NR5A2 knockdown also resulted in reduced expression of FGB, MMP2, MMP3, MMMP9, SNAIL, and TWIST, increased expression of epithelial markers E‐cadherin and β‐catenin, and a lower expression of mesenchymal marker Vimentin. Taken together, our findings suggest that NR5A2 could play a role in CSC stemness and EMT in pancreatic cancer, which may contribute to the worse clinical outcome.

BRCA-associated protein 1 mutant cholangiocarcinoma: an aggressive disease subtype

BACKGROUND BRCA-associated protein 1, an enzyme encoded by the BAP1 gene, is commonly mutated in uveal melanoma, mesothelioma, and renal cancers. Tumors with BAP1 mutation follow an aggressive course. BAP1 mutations have also been observed in cholangiocarcinoma (CCA). The clinical phenotype of BAP1 mutant CCA may yield useful prognostic and therapeutic information but has not been defined. METHODS The records of CCA patients who underwent next-generation sequencing (NGS) were reviewed, and data on clinical, histopathological, genetic, and radiological features; response to therapy; time to progression; and survival were analyzed. RESULTS Twenty-two cases of BAP1-mutation associated CCA were diagnosed from January 1, 2009, to February 1, 2015, at our center. Twenty patients had intrahepatic CCA and two had extrahepatic CCA. Tumor sizes (largest dimension) ranged from 2 to 16 cm (mean, 8.5 cm). Twelve patients had tumors that were poorly differentiated. Majority of the patients had advanced disease at presentation and 13 had bone metastases. Thirteen patients (59%) experienced rapidly progressive disease following primary therapy (chemotherapy or surgical resection). The mean time to tumor progression was 3.8 months after the first line chemotherapy. CONCLUSIONS BAP1 mutation in CCA may be associated with aggressive disease and poor response to standard therapies. Therefore, BAP1-targeted therapies need to be investigated.

Abstract 5361: Development of targeted inhibitors against RecQ1 helicase

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: RecQ helicases are a ubiquitous family of DNA unwinding enzymes involved in the maintenance of chromosome stability. RecQ1 helicase genomic variations have an important prognostic role and represents a therapeutic target in pancreatic cancer [JCO 2006; 24 (11) 1720-1728]. Pubchem data indicated potential RecQ1 activity >600 compounds using a qHTS RecQ1 assay (NIH Chemical Genomics Center). Methods: We applied a set of filters including molecular weight, potency and Hill curve fitting and also removed those interfering with biological assay to identify sixteen compounds for studying RecQ1 inhibition. Two compounds showed strong inhibition of RecQ1 helicase activity at 50 µM and at IC50 < 5 µM, respectively. From a large library of compounds, eight potential inhibitors were synthesized in three simple steps. First, sulfonylation of various amines with 3-Nitrobenzenesulfonyl chloride was performed followed by reduction of the nitro group into an amine. Finally, acylation with variety of acid chlorides resulted in the final library of RecQ1 analogs. These agents were studied for RecQ1 helicase activity assay with the radiolabeled partial duplex substrate in vitro. The RecQ1 inhibitor candidate compounds were investigated in pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-1, PANC-1, and Mia-Paca2) for anti-tumor efficacy using cell proliferation assay with CellTiter-Glo® luminescence. Combination studies with test compounds and poly (ADP ribose) polymerase (PARP) inhibitor olaparib and topoisomerase Inhibitor SN38 were performed. Results: Though none of the compounds showed the same level of activity of the parent compounds, we were able to demonstrate a modest level of helicase activity with several. Two compounds displayed strong inhibition of pancreatic cancer cell proliferation. Two other compounds displayed synergistic inhibition with SN38 or olaparib of pancreatic cancer cell proliferation and induced DNA damage. Conclusions: Synthesis of RecQ1 inhibitors is feasible and shows potential for anti-tumor efficacy. Note: This abstract was not presented at the meeting. Citation Format: Mingxin Zuo, David Maxwell, Basvoju A. Bhanu Prasad, Zhenghong Peng, William Bornmann, Milind M. Javle. Development of targeted inhibitors against RecQ1 helicase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5361. doi:10.1158/1538-7445.AM2014-5361

RNA sequencing-based analysis of gallbladder cancer reveals the importance of the liver X receptor and lipid metabolism in gallbladder cancer

Gallbladder cancer (GBC) is an aggressive malignancy. Although surgical resection may be curable, most patients are diagnosed at an advanced unresectable disease stage. Cholelithiasis is the major risk factor; however the pathogenesis of the disease, from gallstone cholecystitis to cancer, is still not understood. To understand the molecular genetic underpinnings of this cancer and explore novel therapeutic targets for GBC, we examined the key genes and pathways involved in GBC using RNA sequencing. We performed gene expression analysis of 32 cases of surgically-resected GBC along with normal gallbladder tissue controls. We observed that 519 genes were differentially expressed between GBC and normal GB mucosal controls. The liver X receptor (LXR)/retinoid X receptor (RXR) and farnesoid X receptor (FXR) /RXR pathways were the top canonical pathways involved in GBC. Key genes in these pathways, including SERPINB3 and KLK1, were overexpressed in GBC, especially in female GBC patients. Additionally, ApoA1 gene expression suppressed in GBC as compared with normal control tissues. LXR and FXR genes, known to be important in lipid metabolism also function as tumor suppressors and their down regulation appears to be critical for GBC pathogenesis. LXR agonists may have therapeutic value and as potential therapeutic targets.

Abstract B132: Next generation sequencing yields promising targets in advanced cholangiocarcinoma (CCA).

Background: The incidence of CCA is rising and the clinical efficacy of systemic therapy is suboptimal. Next generation sequencing (NGS) technology offers potential for targeted therapeutics against genetically heterogenous solid tumors including cholangiocarcinoma (CCA). Methods: DNA was extracted from biopsy specimens of 56 patients of CCA seen at MD Anderson Cancer Center Houston, TX. DNA sequencing was performed for 3,769 exons of 236 cancer-related genes plus 47 introns from 19 genes to an average depth of 1000X using the Illumina HiSeq 2000 platform.Results: 162 genomic variations (GV) were identified from 56 patient samples with an average of 2.89 GVs/patient (range 0-10). GVs identified were mutations (72%), amplifications (17%), loss/deletions (7%) and others (4%). 6 (11%) tumors showed no GVs. Most frequent GVs were TP53 (35%), KRAS (31%), ARID1A (16%), PBRM1 (11%), BAP1 (9%), ERBB2 (9%), FBXW7 (9%), SMAD4 (9%) and IDH1 (7%). ERBB2 GVs included 4 mutations and 1 amplification. KRAS mutation was associated with a statistically significant reduction in overall survival (OS). Mean OS in patients with KRAS mutation was 32 weeks vs. 63 weeks in KRAS wt (t = -2.126, p = 0.039). Ingenuity Pathway Analysis indicated disruption in cell cycle, proliferation, development, death and DNA repair pathways. Targetable signaling pathways from this study are described in Table 1. Targetable GVs were noted in 59% of patients. These are potentially targetable by inhibitors to ERBB2, FGF, mTOR, MEK, BRAF and PARP-1. Conclusion: These data are the single largest compilation of NGS analysis on CCA patients and demonstrate the range of GVs that are eligible for investigational targeted therapies. These results can be used as a basis to develop personalized treatments for CCA patients based on individual genetic profiles. ![Figure][1] Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B132. Citation Format: Chaitanya R. Churi, Asif Rashid, Rachna Shroff, Lopa Mishra, Mingxin Zuo, Ahmed Kaseb, Filip Janku, Thomas A. Aloia, Jean-Nicholas Vauthey, Steven Curley, Siraj Ali, Gary Palmer, Milind Javle. Next generation sequencing yields promising targets in advanced cholangiocarcinoma (CCA). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B132. [1]: pending:yes