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To study the mechanisms of relapse in acute lymphoblastic leukemia (ALL), we performed whole-genome sequencing of 103 diagnosis-relapse-germline trios and ultra-deep sequencing of 208 serial samples in 16 patients. Relapse-specific somatic alterations were enriched in 12 genes (NR3C1, NR3C2, TP53, NT5C2, FPGS, CREBBP, MSH2, MSH6, PMS2, WHSC1, PRPS1, and PRPS2) involved in drug response. Their prevalence was 17% in very early relapse (36 months) groups. Convergent evolution, in which multiple subclones harbor mutations in the same drug resistance gene, was observed in 6 relapses and confirmed by single-cell sequencing in 1 case. Mathematical modeling and mutational signature analysis indicated that early relapse resistance acquisition was frequently a 2-step process in which a persistent clone survived initial therapy and later acquired bona fide resistance mutations during therapy. In contrast, very early relapses arose from preexisting resistant clone(s). Two novel relapse-specific mutational signatures, one of which was caused by thiopurine treatment based on in vitro drug exposure experiments, were identified in early and late relapses but were absent from 2540 pan-cancer diagnosis samples and 129 non-ALL relapses. The novel signatures were detected in 27% of relapsed ALLs and were responsible for 46% of acquired resistance mutations in NT5C2, PRPS1, NR3C1, and TP53. These results suggest that chemotherapy-induced drug resistance mutations facilitate a subset of pediatric ALL relapses.

Atherosclerosis is a progressive vascular disease triggered by interplay between abnormal shear stress and endothelial lipid retention. A combination of these and, potentially, other factors leads to a chronic inflammatory response in the vessel wall, which is thought to be responsible for disease progression characterized by a buildup of atherosclerotic plaques. Yet molecular events responsible for maintenance of plaque inflammation and plaque growth have not been fully defined. Here we show that endothelial transforming growh factor β (TGF-β) signalling is one of the primary drivers of atherosclerosis-associated vascular inflammation. Inhibition of endothelial TGF-β signalling in hyperlipidemic mice reduces vessel wall inflammation and vascular permeability and leads to arrest of disease progression and regression of established lesions. These proinflammatory effects of endothelial TGF-β signalling are in stark contrast with its effects in other cell types and identify it as an important driver of atherosclerotic plaque growth and show the potential of cell-type-specific therapeutic intervention aimed at control of this disease.

Blood and lymphatic vasculatures are intimately involved in tissue oxygenation and fluid homeostasis maintenance. Assembly of these vascular networks involves sprouting, migration and proliferation of endothelial cells. Recent studies have suggested that changes in cellular metabolism are of importance to these processes1. While much is known about vascular endothelial growth factor (VEGF)-dependent regulation of vascular development and metabolism2,3, little is understood about the role of fibroblast growth factors (FGFs) in this context4. Here we identify FGF receptor (FGFR) signaling as a critical regulator of vascular development. This is achieved by FGF-dependent control of c-MYC (MYC) expression that, in turn, regulates expression of the glycolytic enzyme hexokinase 2 (HK2). A decrease in HK2 levels in the absence of FGF signaling inputs results in decreased glycolysis leading to impaired endothelial cell proliferation and migration. Pan-endothelial- and lymphatic-specific Hk2 knockouts phenocopy blood and/or lymphatic vascular defects seen in Fgfr1/r3 double mutant mice while HK2 overexpression partially rescues the defects caused by suppression of FGF signaling. Thus, FGF-dependent regulation of endothelial glycolysis is a pivotal process in developmental and adult vascular growth and development.

Signet-ring cell carcinoma (SRCC) has specific epidemiology and oncogenesis in gastric cancer, however, with no systematical investigation for prognostic genomic features. Here we report a systematic investigation conducted in 1868 Chinese gastric cancer patients indicating that signet-ring cells content was related to multiple clinical characteristics and treatment outcomes. We thus perform whole-genome sequencing on 32 pairs of SRC samples, and identify frequent CLDN18-ARHGAP26/6 fusion (25%). With 797 additional patients for validation, prevalence of CLDN18-ARHGAP26/6 fusion is noticed to be associated with signet-ring cell content, age at diagnosis, female/male ratio, and TNM stage. Importantly, patients with CLDN18-ARHGAP26/6 fusion have worse survival outcomes, and get no benefit from oxaliplatin/fluoropyrimidines-based chemotherapy, which is consistent with the fact of chemo-drug resistance acquired in CLDN18-ARHGAP26 introduced cell lines. Overall, this study provides insights into the clinical and genomic features of SRCC, and highlights the importance of frequent CLDN18-ARHGAP26/6 fusions in chemotherapy response for SRCC., Signet-ring cell carcinoma (SRCC) is a unique type of gastric cancer with no prognostic features. Here, the authors report a CLDN18-ARHGAP26/6 gene fusion in patients with a high signet-ring cell content, poor survival outcomes, and who experience no benefit from platinum/fluoropyrimidines-based chemotherapy.

Hepatocellular carcinoma (HCC) is the fifth most common type of cancers worldwide. However, current therapeutic approaches for this epidemic disease are limited, and its 5-year survival rate hasn't been improved in the past decades. Patient-derived xenograft (PDX) tumor models have become an excellent in vivo system for understanding of disease biology and drug discovery. In order to identify new therapeutic targets for HCC, whole-exome sequencing (WES) was performed on more than 60 HCC PDX models. Among them, four models exhibited protein-altering mutations in JAK1 (Janus Kinase 1) gene. To explore the transforming capability, these mutations were then introduced into HEK293FT and Ba/F3 cells. The results demonstrated that JAK1S703I mutation was able to activate JAK-STAT (Signal Transducer and Activator of Transcription) signaling pathway and drive cell proliferation in the absence of cytokine stimulation in vitro. Furthermore, the sensitivity to the treatment of a JAK1/2 inhibitor, ruxolitinib, was observed in JAK1S703I mutant PDX model, but not in other non-activating mutant or wild type models. Pharmacodynamic analysis showed that phosphorylation of STAT3 in the Ruxolitinib-treated tumor tissues was significantly suppressed. Collectively, our results suggested that JAK1S703I is an activating mutation for JAK-STAT signaling pathway in vitro and in vivo, and JAK-STAT pathway might represent a new therapeutic approach for HCC treatment. Monotherapy using a more potent and specific JAK1 inhibitor and combinatory therapy should be further explored in JAK1 mutant PDX models.

Acute lymphoblastic leukemia (ALL) is a leading cause of cancer-associated death in children. To study the mechanisms of drug resistance in ALL, we performed whole-genome sequencing of diagnosis-relapse-germline trios from 103 Chinese patients and ultra-deep sequencing of 208 serial bone marrow samples from 17 of them. Relapse-specific somatic alterations were enriched in 12 genes (NR3C1, NR3C2, TP53, NT5C2, FPGS, CREBBP, MSH2, MSH6, PMS2, WHSC1, PRPS1, and PRPS2), which were predominantly involved in response to thiopurines, glucocorticoids, methotrexate, and other drugs. Four lines of evidence indicate that these resistance mutations frequently developed during treatment, rather than pre-existing at diagnosis. First, two novel, relapse-specific mutational signatures (novel signatures 1 and 2), most likely caused by chemotherapeutic regimens, were detected in 15% and 14% of relapsed cases, respectively. Drug resistance mutations frequently appeared at novel signature-associated trinucleotide contexts, indicating that chemotherapy may directly cause drug resistance mutations in ALL. The signatures were validated in NCI TARGET relapsed ALL samples, 2% and 23% of which harbored novel signatures 1 and 2, respectively. The varying signature prevalence between cohorts may reflect treatment differences. The novel signatures were not detected in >2,000 adult cancers from the PCAWG study. Novel signature 1 induced C>G transversions, particularly at GCC and TCT trinucleotides, and showed transcription-strand bias indicating guanine adducts. Novel signature 2 favored C>T and C>G mutations at CCG, and correlated with relapse-specific dinucleotide variants and structural variants, indicating an agent causing multiple mutation types. The drugs inducing these novel signatures are being explored in vitro. Second, mathematical modeling using growth curves of drug-resistant ALL indicated that drug resistance mutations occur, in some cases, long after diagnosis, during active treatment. Third, some patients acquired multiple drug resistance mutations sequentially through successive relapses, a finding inconsistent with their pre-existence at diagnosis. Indeed, 20% of relapses had multiple drug resistance mutations targeting different drug classes. Fourth, most relapsed ALLs derived from a subclone detected at diagnosis, which then evolved additional mutations, including drug resistance mutations, not detectable at diagnosis using 2000X targeted sequencing. Drug resistance mutations were often subclonal at relapse, suggesting later appearance. Together these data indicate that fully drug-resistant clones may not necessarily pre-exist at diagnosis in ALL, but may be acquired later during treatment. Thus, early intensive or targeted treatment strategies in slow responders may forestall the subsequent development of drug resistance mutations. Citation Format: Benshang Li, Samuel W. Brady, Xiaotu Ma, Shuhong Shen, Yingchi Zhang, Yongjin Li, Yu Liu, Ningling Wang, Diane Flasch, Matthew Myers, Heather Mulder, Lixia Ding, Yanling Lu, Liqing Tian, Kohei Hagiwara, Ke Xu, Edgar Sioson, Tianyi Wang, Liu Yang, Jie Zhao, Hui Zhang, Ying Shao, Hongye Sun, Lele Sun, Jiaoyang Cai, Ting-Nien Lin, Lijuan Du, Fan Yang, Michael Rusch, Michael Edmonson, John Easton, Xiaofan Zhu, Jingliao Zhang, Cheng Cheng, Benjamin Raphael, Jingyan Tang, James Downing, Bin-Bing Zhou, Ching-Hon Pui, Jun Yang, Jinghui Zhang. Acquisition of drug resistance mutations during chemotherapy treatment in pediatric acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2872.

Next-generation sequencing analysis of formalin-fixed, paraffin-embedded (FFPE) samples has the potential to lead to major advances in cancer treatment and prevention. However, whole-genome sequencing (WGS) of these samples has remained challenging due to the difficult process of isolating high-quality DNA and distinguishing true variant calls from artifacts. As evidence of these challenges, only four WGS studies of FFPE samples have been published to date. Previous work has attempted to overcome these challenges by using whole-exome sequencing or smaller exon panels at high coverage depths with publications reporting success from less than half to most samples. Thus, there has not been widespread adoption of using FFPE samples for next-generation sequencing. We developed a novel DNA extraction and library prep protocol for whole-genome sequencing of FFPE tissue samples. This method produces larger DNA fragments that can be successfully used for WGS. We previously presented results of a large esophageal cancer cohort where we sequenced over 1,000 samples in tumor-normal pairs with data equivalent to that generated from fresh-frozen tissues. We recently expanded the tumor types, ages, and sources and achieved similar results. We have assayed FFPE tissues derived from breast cancer, colorectal cancer, non-small cell lung cancer, prostate cancer, and melanoma. These samples were sourced from several different labs, which have used variations on the standard fixation protocol. Some of these FFPE samples were over 15 years old. Variant calling provides excellent results without excessive C>T mutations. The coverage uniformity was very good, and CNV calls were made with results comparable to published TCGA data. These samples have been analyzed for MSI and TMB, and the results are comparable to those using fresh tissues. Our method makes new types of clinical studies possible utilizing FFPE samples that have been generated as part of standard hospital procedures. This method also facilitates performing studies comparing primary and recurrent cancers easier to establish and run as recurrent cancer cases can be enrolled and stored FFPE primary samples used to establish a cancer baseline. Citation Format: Shannon T. Bailey, James Lund, Muhammad Ekram, Wanfeng Yu, Richard T. Williams, Hongye Sun, Jeffrey R. Gulcher. High-quality whole-genome sequencing and analysis of FFPE samples from multiplecancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1706.

e13016 Background: Whole-genome sequencing (WGS) of formalin-fixed, paraffin-embedded (FFPE) samples could enable novel insights from archival sample collections, yet robust FFPE WGS is challenged by fragmented DNA, uneven genomic coverage & sequencing artifacts attributed to FFPE fixation. We report our proprietary extraction & library preparation methodology (SeqPlus) with high quality, uniform WGS sequencing performance comparable to that from fresh-frozen samples. Methods: We analyzed 20 paired esophageal carcinoma (EC) samples i.e., primary tumors & matched germline samples to assess SeqPlus performance on 10-15-year-old FFPE tissues, measure variant concordance between WGS and a high-depth sequencing panel (269 genes, 400x coverage) & identify novel genomic features. Results: At a targeted 70x WGS tumor sequencing depth, 93% of the genome was covered by ³ 20 reads, 99% of bases had 10x coverage & average duplicate reads were 31%. We noted similar transition/transversion ratios & mutational spectra as from fresh-frozen EC specimens, suggesting that extraction & library preparation contributes to prior FFPE artifacts. Concordance of tumor-specific SNVs & indels derived from WGS & targeted panel was high at 86%. All 76 targeted panel-detected variants above the WGS limit of detection (mutant allele frequency [MAF] > 10%) were detected by WGS, 2 variants (2 tumors) were detected only by WGS, and 12 variants at MAF ≤ 6% (9 tumors) were only detected by the targeted panel. Tumor WGS yielded SNV, indels & CNV findings beyond variants detected by targeted sequencing. WGS enabled detection of 10.4 putative cancer variants per tumor compared to 12 variants per patient from frozen specimens and a median of 7 (up to 16) cancer-associated variants in genes outside the targeted panel. WGS copy number analysis revealed CCND1, EGFR, TP63, and SOX2amplification, CDKN2A/B deletion and additional unrecognized genomic aberrations. Conclusions: Our study reinforces the utility of high-quality, uniform WGS sequencing of archival FFPE cancer samples with SeqPlus and unlocks the potential for massive-scale retrospective genomic analysis of archived pathology samples with associated clinical & outcomes data.

Introduction Relapsed childhood acute lymphoblastic leukemia (ALL) is a leading cause of cancer-related death in children and has poor prognosis due to acquired drug resistance. However, the clonal evolution leading to drug resistance at ALL relapse is incompletely understood. Methods We performed whole-genome sequencing (WGS) of samples at diagnosis and relapse from 103 Chinese patients, most of whom were enrolled on the Shanghai Children's Medical Center (SCMC) ALL2005 frontline treatment protocol. We also performed ultra-deep sequencing at 5,000-50,000X coverage of 211 serial bone-marrow samples from 17 of these patients (3-23 per case) collected during ALL therapy. Fifteen ALL subtypes were identified based on copy number variation, structural re-arrangement and gene fusion by WGS and RNA-Seq analysis. Cases were selected based on sample availability with no bias on age, gender, cytogenetics, or immunophenotype compared to the characteristics of all relapsed cases at SCMC; however only one CRLF2-rearranged case was observed suggesting that CRLF2-rearrangement could be a rare event in Asian patients. Functional impact of novel mutations was assessed by ectopic expression in ALL cell lines. Results Relapse-specific somatic alterations were significantly enriched in 12 genes (NT5C2, NR3C1/2, PRPS1/2, TP53, CREBBP, MSH2/6, PMS2, WHSC1, and FPGS) predominantly involved in drug metabolism and response pathways. These somatic alterations were present in over 50% (56/103) of relapsed ALLs and were enriched in patients with early relapse (9-36 months from diagnosis) compared to very early (36 months) patients. NR3C1 mutations resulted in loss of glucocorticoid receptor transcriptional activity and severely impaired glucocorticoid response in vitro, while FPGS mutations led to reduced enzymatic activation of methotrexate. Genome-wide analysis identified 9 mutational signatures, including two novel ones exclusive to relapse. Novel signature 1 was characterized by C>G mutations and was present in 15% of the relapsed cases with an enrichment for hyperdiploid ALL, while novel signature 2, present in 14% of the relapsed cases, was characterized by C mutations (C>T in particular) followed by a G. These novel signatures gave rise to relapse-specific drug resistance mutations, including PRPS1, TP53, NT5C2, and KRAS mutations. The majority (59%) of the cases underwent a selective sweep as the relapse clone arose from a subclone detectable at diagnosis; however, very early relapse cases were less likely to experience a clonal sweep and had multiple lineages present at relapse. In patients tracked serially through multiple relapses, the clonal composition of drug resistance variants evolved substantially in response to chemotherapy. For example, in one patient, two independent NT5C2-mutant clones appeared at relapse, manifesting convergent evolution (Fig. 1a). One of the NT5C2-mutant clones expanded after subsequent treatment while the other diminished in prevalence. Sequential acquisition of multiple drug resistance mutations was also evident, with one patient sequentially acquiring KRAS, FBXW7, NR3C1 and FPGS mutations over a period of 5 years through multiple relapses (Fig. 1b). Indeed, 17% (18/103) of patients acquired multiple drug resistance mutations at relapse. The median time from detecting resistant clones to relapse was 41 days, suggesting that ultra-deep sequencing offers a means for early detection of genetic lesions that could serve as an indicator of relapse to enable earlier therapeutic intervention. Conclusions Very early relapses likely have different resistance mechanisms than early or late relapses, perhaps due to increased intrinsic resistance or the presence of multiple drug-resistant clones. At least a subset of relapse-specific drug resistance mutations may be acquired de novo, rather than pre-existing, as evidenced by resistance mutations bearing the novel relapse-specific mutational signatures which are likely to be therapy-induced. Relapsed ALL can acquire multiple drug resistance mutations targeting different drug classes. Frequent monitoring of disease, such as via cell-free DNA sequencing, may enable earlier detection of relapse and facilitate timely treatment to avoid selection for drug-resistant clones. Disclosures No relevant conflicts of interest to declare.

e13500Background: Next-generation sequencing analysis of formalin-fixed, paraffin-embedded (FFPE) samples has the potential to lead to major advances in cancer treatment and prevention. However, wh...

// Qingyang Gu 1, * , Bin Zhang 1, * , Hongye Sun 2 , Qiang Xu 2 , Yexiong Tan 3 , Guan Wang 2 , Qin Luo 2 , Weiguo Xu 1 , Shuqun Yang 1 , Jian Li 1 , Jing Fu 3 , Lei Chen 3 , Shengxian Yuan 3 , Guibai Liang 1 , Qunsheng Ji 1 , Shu-Hui Chen 1 , Chi-Chung Chan 1 , Weiping Zhou 3 , Xiaowei Xu 4 , Hongyang Wang 3 , Douglas D. Fang 1, 5 1 Discovery Services, WuXi AppTec Co., Ltd., Waigaoqiao Free Trade Zone, Shanghai, 200131 China 2 Genome Center, WuXi AppTec Co., Ltd., Waigaoqiao Free Trade Zone, Shanghai, 200131 China 3 Eastern Hepatobiliary Surgery Hospital/Institute of Shanghai, Shanghai, 200131 China 4 Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, Philadelphia, PA 19104, USA 5 Current address: Cancer Translational Research and In Vivo Pharmacology, China Novartis Institute for Biomedical Research, Zhangjiang Hi-Tech Park, Pudong, Shanghai, 200131 China * These authors have contributed equally to this work Correspondence to: Douglas D. Fang, e-mail: douglasfang@yahoo.com Hongyang Wang, e-mail: hywangk@vip.sina.com Keywords: hepatocellular carcinoma (HCC), patient-derived xenograft (PDX) tumor model, copy number alterations (CNA), single nucleotide polymorphism (SNP), fibroblast growth factor receptor (FGFR) Received: January 25, 2015 Accepted: May 09, 2015 Published: May 21, 2015 ABSTRACT Lack of clinically relevant tumor models dramatically hampers development of effective therapies for hepatocellular carcinoma (HCC). Establishment of patient-derived xenograft (PDX) models that faithfully recapitulate the genetic and phenotypic features of HCC becomes important. In this study, we first established a cohort of 65 stable PDX models of HCC from corresponding Chinese patients. Then we showed that the histology and gene expression patterns of PDX models were highly consistent between xenografts and case-matched original tumors. Genetic alterations, including mutations and DNA copy number alterations (CNAs), of the xenografts correlated well with the published data of HCC patient specimens. Furthermore, differential responses to sorafenib, the standard-of-care agent, in randomly chosen xenografts were unveiled. Finally, in the models expressing high levels of FGFR1 gene according to the genomic data, FGFR1 inhibitor lenvatinib showed greater efficacy than sorafenib. Taken together, our data indicate that PDX models resemble histopathological and genomic characteristics of clinical HCC tumors, as well as recapitulate the differential responses of HCC patients to the standard-of-care treatment. Overall, this large collection of PDX models becomes a clinically relevant platform for drug screening, biomarker discovery and translational research in preclinical setting.

Hepatocellular carcinoma (HCC) is the fifth most common type of cancer worldwide. However, current therapeutic approaches for this epidemic disease are limited and its 5-year survival rate has not been improved in the past decades. Patient-derived xenograft (PDX) tumor models have become an excellent in vivo system for understanding of disease biology and drug discovery. In order to identify new therapeutic targets for HCC, whole-exome sequencing (WES) was performed on more than 60 HCC PDX models. Among them, four models exhibited point mutations in JAK1 (Janus Kinase) gene, which were confirmed by Sanger sequencing. To explore the transforming capability, these mutations were then introduced into 293T and Ba/F3 cells by transient transfection and retroviral infection, respectively. The results demonstrated that JAK1S703I mutation was able to activate JAK-STAT (Signal Transducer and Activator of Transcription) signaling pathway and drive cell proliferation in the absence of cytokine stimulation in vitro. Furthermore, a modest sensitivity to the treatment of a JAK1/2 inhibitor, Ruxolitinib, was observed in JAK1S703I mutant PDX model, but not in other mutant or wild type models. Pharmacodynamic analysis showed that phosphorylation of STAT3 in the Ruxolitinib-treated tumor tissues was significantly inhibited. Collectively, our results suggested that JAK1S703I is an activating mutation for JAK-STAT signaling pathway in vitro and in vivo. JAK-STAT pathway might represent a new therapeutic opportunity for HCC. Monotherapy using a more potent inhibitor and combinatory therapy may be further explored in JAK1S703I mutant PDX model. Citation Format: Shuqun Yang, Chonglin Luo, Qingyang Gu, Qiang Xu, Hongye Sun, Ziliang Qian, Yexiong Tan, Hao Wu, Yuxin Qin, Yuhong Shen, Xiaowei Xu, Shu-Hui Chen, Chi-Chung Chan, Hongyang Wang, Mao Mao, Douglas D. Fang. Activating JAK1-S703I mutation may predict the sensitivity of JAK-STAT inhibition in hepatocellular carcinoma patient-derived xenograft tumor models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 688. doi:10.1158/1538-7445.AM2015-688

Targeted drugs have been widely used in the treatment of lung cancer, especially for non small cell lung cancer (NSCLC) patients. NCCN guideline lists multiple drugs which are particularly effective in the subgroup of patients with specific gene mutation variants, including the fusion transcripts of ALK, ROS1 and RET, and the mutations of EGFR, KRAS, BRAF and HER2. Multiple techniques have been developed to detect fusion transcripts in tumor samples, such as FISH, IHC and PCR assays. FISH is the most accurate method for fusion detection currently. The split probe FISH assay, which does not require the pre-knowledge of what is the fusion partner with ALK for example, can detect unknown ALK fusion products as well. However, it fails to identify which gene is fused with ALK (or other oncogene) in the chimera products, therefore is not informative for the mechanism or drug resistance study. In addition, due to the limitation on the fluoresce dye, it is quite difficult to multiplex fusion FISH probes for different genes into one assay. We describe here a next generation sequencing assay to detect a panel of fusion transcripts and mutations in one single assay. Our single assay can detect the fusion products of ALK, ROS1 and RET with any known or unknown fusion partners, together with the hotspot mutations in EGFR, KRAS, BRAF and HER2. Our assay can identify not only the exact sequence of the unknown fusion partners, but also any mutations within the full coding regions of ALK, ROS1 and RET. This information is critical for the resistance mechanism study and the next generation inhibitor drug development, since about half of the ALK inhibitor resistance was caused by the mutations in ALK gene itself. We have identified a novel ALK fusion gene with a never-reported fusion partner in a lung cancer patient. The xenograft model derived from this patient responds to crizotinib well in the in vivo study (published on Journal of Thoracic Oncology). We have demonstrated that our assay can reliably detect both the novel fusion and the well known EML4-ALK fusion from only 1-2 5um FFPE slides of the lung tumor tissues, together with other possible fusions and mutations as well. Note: This abstract was not presented at the meeting. Citation Format: Qiang Xu, Guan Wang, Bo Li, Maoxiang Qian, Douglas Fang, Lele Sun, Yundi Chen, Hongye Sun. A next generation sequencing assay to detect the fusion products of ALK, ROS1 and RET with any fusion partner genes and hotspot mutations in FFPE samples. [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 4668. doi:10.1158/1538-7445.AM2014-4668

Hepatocellular carcinoma (HCC) is intrinsically resistant to conventional chemotherapies. Developing effective therapeutics becomes critical to tackle the unmet medical need. The fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) signaling axis plays an important role in liver development and homeostasis in adults. Deregulation of the FGF/FGFR signaling pathway through genetic modification or overexpression of the receptors (or their ligands) has been increasingly recognized in HCC. Accordingly, therapeutic intervention of the pathway has been extensively explored. In this study, analysis of a panel of patient-derived xenograft (PDX) HCC models by Affymetrix human gene expression arrays revealed overexpression of FGFR1 in 13 out of 43 (30%) models. Antitumor activities of FGFR inhibitors, including lenvatinib, dovitinib, ENMD-2076, and LY2874455, were then tested in five models. Lenvatinib exhibited the most potent activity in all models, whereas LY2874455 failed to show the effect in one of models. IC50s for FGFR1 inhibition of ENMD-2076, lenvatinib, dovitinib and LY2874455 were 145 nM, 134.5 nM, 53.5 nM and 0.6 nM, respectively. In SNU-16 and NCI-H520 cell lines, IC50s of dovitinib, lenvatinib, ENMD-2076, and LY2874455 were 76/1355 nM, 94/4770 nM, 306/2280 nM, and 0.3/664 nM, respectively. Furthermore, analyses of PDX tissues by Affymetrix SNP Array 6.0 arrays revealed no amplification of FGFR1/2 genes in four models except for amplifications of FGFR3 gene in two models. Whole exome sequencing did not detect nonsynonymous genetic alterations, including SNPs and indels, in FGFR1/2 genes in these xenografts except for a few genetic variations in FGFR3/4 genes. Overall, due to the multi-targeting mechanisms of these inhibitors, in vitro inhibitory effects of the compounds on FGFR1 enzyme and cell proliferation may not correlate with the in vivo anti-tumor activities. Overexpression of FGFR1 gene may serve as a predictive biomarker for therapeutic intervention of the FGF/FGFR pathway in HCC by using lenvatinib, dovitinib and ENMD-2076. Further studies are required to define additional biomarkers for LY2874455. Thus, our findings provide the rationale for the clinical development of FGFR inhibitors in a subset of HCC patients expressing a high level of FGFR1 gene. Citation Format: Douglas D. Fang, Bin Zhang, Weiguo Xu, Kang Yan, Qingyang Gu, Qiang Xu, Yexiong Tan, Hongye Sun, Qin Luo, Weifeng Mao, Chiho Li, Jiangpeng Liao, Guibai Liang, Shu-Hui Chen, Chi-Chung Chan. Antitumor activities of FGFR inhibitors in FGFR1-overexpressing hepatocellular carcinoma patient-derived xenograft tumor models. [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 2839. doi:10.1158/1538-7445.AM2014-2839

Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide and the third leading cause of cancer-related deaths. Half of the total HCC-related deaths and new cases occurred in China. Different from many cancers, HCC is intrinsically resistant to conventional chemotherapies, and many attempts to develop new systemic agents for HCC have failed in clinic. Lack of clinically relevant and genetically well-defined preclinical models critically accounts for the failures. In this study, we established an extensive panel of Chinese HCC patient-derived xenograft (PDX) models by directly transplanting surgically resected patient tumor tissues in BALB/c nude or SCID mice. Implantation of 254 patient samples resulted in 70 transplantable PDX models (27.6%) covering stage I, II, III, and IV tumors. Xenograft tissues could be serially passaged, cryopreserved, and revived with a success rate of over 94%. Early passages between p2-p4 of PDX tissues were used in the histological and molecular characterization. The histology of PDX tissues recapitulated the features of the original samples and maintained the heterogeneity of human tumors. Unsupervised hierarchical cluster analysis of gene expression profiles in 7 paired samples assessed by GeneChip® PrimeView™ Human Gene Expression Array revealed that PDX tumor tissues retained the global gene-expression patterns of patient samples. Using next-generation sequencing we performed exome sequencing of 43 of PDX models. Contaminated murine DNA reads were removed by a proprietary algorithm to eliminate homology bias. A wide variety of SNPs were identified and some may represent driver mutations, such as ones on JAK1 and JAK2 genes. Copy number variation analysis by Genome-Wide Human SNP Array 6.0 in 24 PDX models elucidated amplifications of a number of genes, including MET, AKT2, AKT3, mTOR, ALDH1A1, ALDH1A2, JUN and NOV. In vivo efficacy studies exhibited differential responses to standard-of-care compounds in the models. Collectively, with the current intense interest in development of targeted therapies, the use of patient liver cancer tissue-derived models with well genomic characterization provides an excellent, and perhaps more relevant, platform for preclinical studies of experimental therapeutics, and for biomarker discovery and translational research. Citation Format: Douglas D. Fang, Qiang Xu, Yexiong Tan, Lei Chen, Hongye Sun, Qingyang Gu, Bin Zhang, Qin Luo, Shu-hui Chen, Hongyang Wang, Chi-Chung Chan. Genetic characterization of an extensive panel of patient-derived xenograft tumor models of hepatocellular carcinoma for preclinical development. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2739. doi:10.1158/1538-7445.AM2013-2739

The genomes of all cancer cells carry somatic mutations, including both passenger and driver mutations. Driver mutations, which are implicated in oncogenesis, are most likely preserved in xenografted tumor cells in immunedeficient mice. Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive carcinomas in the gastrointenstinal tract. It is prevalent in the developing world and associated with substantial morbidity and mortality. Molecular mechanisms contributing to initiation and progression of ESCC are still poorly understood. Systematic identification of genetic variations and putative driver mutations may lead to understanding of pathogenesis of the disease. In this study, using next-generation sequencing we performed exome sequencing of eight paired Chinese esophageal squamous cell carcinoma and xenograft. Comparing with control genomes obtained from case-matched peripheral blood cells, we identified a wide variety of somatic variations in both primary and xenograft tumors, including TP53, RGMA, MUC4, TCEAL6, PRSS3, etc. Moreover, we found that mouse sequences accounted for a significant fraction of sequence reads from xenograft samples. Excluding these mouse sequences significantly improved the accuracy of mutation detection in xenograft samples. Overall, our findings shed a light on important somatic mutations involved in ESCC. In addition, comparison between primary and xenograft tumors may lead to identification of driver mutations that confer growth advantage. These mutations represent therapeutic opportunities for development of targeted therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2351. doi:1538-7445.AM2012-2351

The double hits theory suggests a major mechanism of tumorigenesis. However, compared to the somatic mutations, the loss of heterozygosity (LOH) was not frequently identified in many sequencing projects. Unlike the somatic mutations that were detected by the evidence of novel alleles in tumor tissue, the alleles involving in the LOH are present in normal tissues as well. Given the high level of tissue heterogeneity of primary tumors, including both tumorous cells and normal stroma cells, at whole tissue or million cells level LOH can only be detected as a quantitative change between tumor and normal samples, which are not easily distinguished from potential sequencing errors or other artifacts especially with low depth sequencing. By applying whole exome capture deep sequencing on case-matched primary tumor, blood and xenograft samples, we found that xenografts represent a much purer human tumor cell population than primary tumor tissues. These tumor cells growing in mice not only contain the majority of somatic mutations in the primary tumors (>95%), but also reveal a >20 folds enrichment of somatic LOH events, which were not categorized as LOH in primary tumors, versus somatic mutations. All these findings were detected after removing mouse sequence contaminations, which if included will induce about half of the total variations observed in the xenograft samples. We found that these LOH events affect both tumor suppressor genes and oncogenes. Given that driver mutations or other variations that are implicated in oncogenesis are most likely preserved in xenografted tumor cells in immunedeficient mice, we hypothesize that the bigger range of somatic LOH events that we observed in xenograft samples play a potential ever important role in tumorigenesis than what we thought previously. Our finding also sheds a new light on a novel approach for the discovery of potential drug targets by sequencing xenograft samples. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-397. doi:1538-7445.AM2012-LB-397

Introduction: The aim of this study was to identify anaplastic lymphoma kinase ( ALK ) rearrangements in lung cancer patient-derived xenograft (PDX) models and to explore their responses to crizotinib. Methods: Screening of 99 lung cancer PDX models by the NanoString ALK fusion assay identified two ALK -rearranged non–small-cell lung cancer (NSCLC) tumors, including one harboring a previously known echinoderm microtubule-associated protein-like 4 ( EML4 )– ALK fusion and another containing an unknown ALK fusion variant. Expression array, RNA-Seq, reverse transcription polymerase chain reaction, and direct sequencing were then conducted to confirm the rearrangements and to identify the novel fusion partner in the xenograft and/or the primary patient tumor. Finally, pharmacological studies were performed in PDX models to evaluate their responses to ALK inhibitor crizotinib. Results: Two ALK -rearranged NSCLC PDX models were identified: one carried a well-known EML4–ALK variant 3a/b and the other harbored a novel huntingtin interacting protein 1 ( HIP1 )– ALK fusion gene. Exon 28 of the HIP1 gene located on chromosome 7 was fused to exon 20 of the ALK gene located on chromosome 2. Both cases were clinically diagnosed as squamous cell carcinoma. Compared with the other lung cancer PDX models, both ALK -rearranged models displayed elevated ALK mRNA expression. Furthermore, in vivo efficacy studies demonstrated that, similar to the EML4–ALK-positive model, the HIP1–ALK -containing PDX model was sensitive to treatment with crizotinib. Conclusions: Discovery of HIP1 as a fusion partner of ALK in NSCLC is a novel finding. In addition, the HIP1–ALK -rearranged tumor is sensitive to treatment with crizotinib in vivo, implicating HIP1–ALK as an oncogenic driver of lung tumorigenesis. Collectively, our results indicate that HIP1–ALK -positive NSCLC may benefit from clinical applications of crizotinib.