Whole-exon Sequencing Research Articles

1035P - Hybrid capture-based genomic profiling of circulating tumour DNA (ctDNA) from patients with ovarian cancer

BackgroundGenomic profiling of blood-derived circulating tumour DNA (ctDNA) which is considered as a minimally invasive biopsy, has several potential applications in the management of ovarian cancer, including selection of targeted therapy, drug resistance prediction and the evaluation of treatment response. Therefore, we conducted this study to characterize genomic alterations (GA) in ctDNA from patients with ovarian cancer and compare GA detected in ctDNA with those detected in tissue databases. MethodsHybrid capture-based next generation sequencing (NGS) genomic profiling of 150 genes was performed on ctNDA from 76 patients with ovarian cancer in a CAP-accredited laboratory. The GA detected in ctDNA were compared with the 3D Med tissue database tested by NGS (N=260) and TCGA database tested by whole-exon sequencing (N=587). The fraction of ctDNA was estimated using the maximum somatic allele frequency (MSAF). GAs including single nucleotide variation (SNV), insertions/deletions, copy number variations, gene rearrangement and fusions were assessed. Germline alterations were excluded. Results89.5% (68/76) of patients (median age 57) detected ≥1 GA in ctDNA. Frequently altered genes detected in ctDNA for SNV were TP53 (52.6%), AR (11.8%), KRAS (9.2%), NOTCH2 (6.6%) and PIK3CA (5.3%) compared to 3D Med tissue database (median age 55) including 260 samples with TP53 (74.2%), AR (6.9%), KRAS (24.6%) NOTCH2 (11.5%) and PIK3CA (3.8%). In TCGA database including 587 patients with early stage diseases, the frequencies of the commonly altered genes detected in ctDNA were largely different with TP53 (65.9%), AR (1.0%), KRAS (1.0%), NOTCH2 (1.2%) and PIK3CA (2.6%). Alterations in five DNA damage repair associated-genes including BRCA1, BRCA2, ATM, ATR, RAD50 and CHEK2 were detected in 14.5% (11/76) of patients in ctDNA database compared to 19.2% (50/260) of patients in 3D Med tissue database and 12.6% (74/587) of patients in TCGA database. ConclusionsOur results suggest that genomic profiling of ctDNA could detected GAs in a significant subset of patients with ovarian cancer. Hybrid capture- NGS based ctDNA liquid biopsy has the potential capability to serve as an alternative to tissue biopsy and further studies are warranted. Legal entity responsible for the studyNanJing GaoChun People’s Hospital. FundingHas not received any funding. DisclosureY. Zhang: Full / Part-time employment: 3D Medicines Inc. S. Cai: Full / Part-time employment: 3D Medicines Inc. All other authors have declared no conflicts of interest.

Abstract 1602: Genomic profiling of lung cancer associated with idiopathic pulmonary fibrosis

Abstract Idiopathic pulmonary fibrosis (IPF) is reportedly associated with an increased risk of lung cancer. However, optimal diagnosis and treatment for lung cancer associated with IPF remains unclear. In this study, we aimed to clarify the mechanism of carcinogenesis and progression in lung cancer with IPF. We used tumor tissues and adjacent non-tumor lung tissues from 29 lung cancer patients who had undergone complete surgical resection at Nippon Medical School Hospital between 2013 and 2017. Whole-Exon Sequencing was performed on 29 paired samples, consisted of 19 lung cancers with IPF (IPF group) and 10 lung cancers with normal lung (non-IPF group). The candidate gene alterations discriminating between the IPF and non-IPF group, were identified using the random-forest method, RelifF method along with the data from Catalogue Of Somatic Mutations In Cancer (COSMIC). Twenty-five gene alterations were specifically found in IPF tissues and/or tumor tissues with IPF. Whenever 5 of 25 genes including MUC2 and TTF1, were altered in both IPF tissues and tumor tissues, 20 of 25 genes including CADM1, were altered in only tumor tissues. We are undergoing the validation study using other tissues in independent cohorts. Among the candidate genes, Cell adhesion molecule genes (CADM1) gene alterations were frequently found in 15 out of 19 in only tumor tissues with IPF. The CADM1 protein expression was decreased in tumors than in non-tumor fibrotic tissues by western blotting. The CADM1 induced the epithelial-mesenchymal transition (EMT) on the multifunctional cell process involved in the pathogenesis of fibrosis in vitro study. Whenever the down-regulation of CADM1 was frequently detected in various human cancers through its allelic loss as well as hypermethylation within promoter region in addition to inactivate gene mutation, the up-regulation of CADM1 was frequently detected in paired IPF tissues. The CADM1 may be a target candidate in lung cancer associated with IPF. Citation Format: Rintaro Noro, Akihiko Miyanaga, Aya Fukuizumi, Shinobu Kunugi, Teppei Sugano, Miwako Omori, Yuji Minegish, Jitsuo Usuda, Masahiro Seike, Kaoru Kubota, Mamiko Hirao, Kuniko Matsuda, Akihiko Gemma. Genomic profiling of lung cancer associated with idiopathic pulmonary fibrosis [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 1602.

Comprehensive genomic profiling identifies novel genetic predictors of response to anti-PD-(L)1 therapies in NSCLC.

e20536 Background: Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of many different cancer types, including non-small-cell lung cancer (NSCLC). However, our understandings of the molecular and immunologic determinants of response to ICIs remain incomplete. Methods: We performed whole-exon sequencing (WES) in 73 patients with advanced NSCLC who underwent anti-PD-(L)1 therapies to further explore predictive biomarkers for treatment efficacy. Novel somatic mutations and copy number alterations were identified to be predictive of response or resistance to anti-PD-(L)1 therapies. Results: We confirmed that high tumor mutation burden (TMB) was associated with improved clinical outcomes. Quantification of TMB by targeted next-generation sequencing using a customized panel including 422 cancer-relevant genes (Geneseeq) was strongly correlated with WES results (Spearman's Rho = 0.81, p < 0.001). Patients whose tumour harbored FAT1 mutant had higher durable clinical response (DCB) and objective response rate (ORR) compared with wild-type (62.5% vs 20%, p = 0.02, and 50% vs 12.3%, p = 0.02, respectively), whereas patients with activating mutations in EGFR or ERBB2 had significantly shorter mPFS compared with other patients (51 vs 70.5 days, p = 0.0037, HR = 2.47 [95% CI, 1.33-4.62]). Furthermore, We found copy number loss in specific 3p segments (mPFS, 61.5 vs 65 days; survival rates at 6 months, 0% vs 31%, p = 0.012, HR = 2.08 [95% CI, 1.09-4.00]), which contain the tumor suppressor ITGA9 (mPFS, 36.5 vs 64 days, p < 0.0001, HR = 9.09 [95% CI, 2.91-27.78]) and several chemokine receptor pathway genes, were highly predictive of poor clinical outcome. These findings were further validated in two independently published datasets comprising multiple cancer types. Conclusions: Our findings suggest that comprehensive profiling of TMB and the aforementioned novel genetic alterations could result in greater predictive power of response in NSCLC patients treated with ICI therapies.

A multidisciplinary approach to the functional and esthetic rehabilitation of dentinogenesis imperfecta type II: A clinical report

A 20-year-old woman presented with an unesthetic appearance and severe wear of the anterior teeth. Definitive expected treatment was designed by multidisciplinary combination therapy. Meanwhile, gene sequencing was used diagnostically, and digital smile design (DSD) was used to design the esthetics of the anterior teeth. The 3-month follow-up showed an acceptable outcome. The whole-exon sequencing and direct sequencing of polymerized chain reaction products of dentin sialophosphoprotein were performed, but further sequencing was needed in the repeat region of dentin sialophosphoprotein exon 5 (the data were not shown). DSD assisted and improved the esthetic design of the definitive restoration, communication with the patient, and predictability of the treatment. This treatment demonstrates that the diagnosis and differential diagnosis of patients with dentinogenesis imperfecta (DGI)-II are essential and effective for implementing the definitive treatment plan. Gene screening can be used as an auxiliary method of diagnosing DGI-II. In terms of esthetic design, DSD played an important role in ensuring a satisfactory result. Moreover, a multidisciplinary treatment protocol focusing on prosthodontics, which is different from the traditional treatment of DGI-II and consists solely of prosthodontic rehabilitation, proved highly effective in the esthetic restoration of a patient with DGI-II.

Progressive pseudorheumatoid dysplasia confirmed by whole-exon sequencing in a Chinese adult before corrective surgery

BackgroundProgressive pseudorheumatoid dysplasia (PPD) is a rare autosomal recessive skeletal dysplasia caused by mutations in the Wnt1-inducible signaling pathway protein 3 (WISP3) gene. Available literatures in PPD emphasized treatment strategy for polyarthritis, while few mentioned spinal deformity and related surgical intervention.MethodsHere, we present a Chinese man with PPD who underwent spinal surgery twice because of canal stenosis and related symptoms caused by the disease. Whole-exon sequencing (WES) was performed to confirm diagnosis before the second surgery.ResultsA homozygous missense mutation (c.395G>A/p.C132Y) in WISP3 was identified that co-segregated with affected family members.ConclusionsOur study illustrated a surgical outcome of PPD and highlighted the significance of early diagnosis and individualized surgical strategy, and also verified the value of WES in the diagnosis of PPD.

Whole-exon sequencing of human myeloma cell lines shows mutations related to myeloma patients at relapse with major hits in the DNA regulation and repair pathways

BackgroundHuman myeloma cell lines (HMCLs) are widely used for their representation of primary myeloma cells because they cover patient diversity, although not fully. Their genetic background is mostly undiscovered, and no comprehensive study has ever been conducted in order to reveal those details.MethodsWe performed whole-exon sequencing of 33 HMCLs, which were established over the last 50 years in 12 laboratories. Gene expression profiling and drug testing for the 33 HMCLs are also provided and correlated to exon-sequencing findings.ResultsMissense mutations were the most frequent hits in genes (92%). HMCLs harbored between 307 and 916 mutations per sample, with TP53 being the most mutated gene (67%). Recurrent bi-allelic losses were found in genes involved in cell cycle regulation (RB1, CDKN2C), the NFκB pathway (TRAF3, BIRC2), and the p53 pathway (TP53, CDKN2A). Frequency of mutations/deletions in HMCLs were either similar to that of patients (e.g., DIS3, PRDM1, KRAS) or highly increased (e.g., TP53, CDKN2C, NRAS, PRKD2). MAPK was the most altered pathway (82% of HMCLs), mainly by RAS mutants. Surprisingly, HMCLs displayed alterations in epigenetic (73%) and Fanconi anemia (54%) and few alterations in apoptotic machinery. We further identified mutually exclusive and associated mutations/deletions in genes involved in the MAPK and p53 pathways as well as in chromatin regulator/modifier genes. Finally, by combining the gene expression profile, gene mutation, gene deletion, and drug response, we demonstrated that several targeted drugs overcome or bypass some mutations.ConclusionsWith this work, we retrieved genomic alterations of HMCLs, highlighting that they display numerous and unprecedented abnormalities, especially in DNA regulation and repair pathways. Furthermore, we demonstrate that HMCLs are a reliable model for drug screening for refractory patients at diagnosis or at relapse.

Clinical Characteristic of "triple-negative" Essential Thrombocythaemia Patients and Mutation Analysis by Targeted Sequencing

Essential thrombocythemia is a subgroup of myeloproliferative neoplasms. Previous studies identified mutations of JAK2, CALR, and MPL that are closely related with the pathogenesis of myeloproliferative neoplasms. All these mutations contribute to the hyperactivation of JAK2/STAT pathway. However, a small proportion of essential thrombocythemia patients does not display such mutations. The pathogenesis of "triple-negative" form of essential thrombocythemia remains unknown. To investigate the clinical characteristics of triple-negative essential thrombocythemia and related mutation genes. To identify the mutations associated with triple-negative essential thrombocythemia, next-generation sequencing was used to conduct targeted sequencing of 360 genes in samples from 68 patients. At least one missense mutation was detected in all the patients and all the detected genes. After screening the data, it was observed that 10 genes with the 10 highest mutation were follows: FLT3, SH2B3, ASXL1, ADAMTS1, TET2, TP53, EGFR, CUX1, GATA2, and MPL.When only rare genes (i.e., with a frequency in Asian populations lower than 5%, as estimated by the 1000 Genomes Project) were analyzed, the most frequently mutated genes in the patients were TET2 (33.82%), SH2B3(29.41%), and ASXL1 (23.53%). Our study identified some mutations that did not previously reported. Although all these mutations need further validation, high incidence rates may indicate relevance of the respective mutations to essential thrombocythemia pathogenesis. Some of the detected mutations have been previously reported; these mutations were also found in a large proportion of our subjects. whole-exon sequencing can provide a higher level of accuracy for gene mutation analysis and assist in identifying mutations that contribute to illustrate the pathogenesis of essential thrombocythemia.

Abstract 3442: Genome-wide association study reveals potential biomarkers after pulmonary metastasectomy for head and neck cancer

Abstract Introduction: At least 10% of head neck cancer squamous cell carcinoma (HNSCC) progress to pulmonary metastases with overall survival (OS) around 12 months. Whether aggressively pulmonary metastasectomy (PM) improving survival or not is controversial. Method and materials: Different head neck cancer patients receiving PM from thoracic surgeon were enrolled from 2003 to 2012. Matched HNSCC and adenoid cystic carcinoma (ACC) samples as control were selected to perform whole-exon sequencing (WES) and identify potential biomarkers between short- and long- term surviving groups after metastasectomy. Identified mutant genes were mapped into possible signaling pathways by using DAVID ( https://david.ncifcrf.gov/). Result: A total of 31 head and neck cancer patients were enrolled and the survivals were different significantly among cancer types. Eight matched PM samples (3 short-term post-metastaectomy survival (PMS) HNSCC, 3 long-term HNSCC, and 2 ACC), with differently clinical outcomes (PMS, P = 0.016; OS, P = 0.015), were enrolled for WES. Eighteen percentages (18%, 306/1706) of identified genetic alternations were mapped into DAVID. Cancer-associated signal pathways were top-ranked related to short-term HNSCC; hematopoietic cell lineage associated pathways for long-term HNSCC; complement and coagulation cascades associated pathways for ACC. The overlapped genes between short-term HNSCC and ACC were relative to metabolic-associated pathway, such as branched-chain amino acid consumption (P < 0.001, fold enrichment (FE) = 7.97) and the metabolism of arginine and proline metabolism (P < 0.001, FE = 4.96). The overlapped genes between long-term HNSCC and ACC were relative to focal adhesion (P =0.008, FE = 2.84) and ECM-receptor interaction (P = 0.015, FE = 4.08). Conclusion: Genome-wide association study effectively separated clinical outcome of head neck cancer patients and successfully identified specific mutations for prognosis after PM. Basic characteristics of patients receiving whole-exon sequencing for pulmonary metastatic tumor.GroupAgeGenderPrimary tumor siteHistological typeInitial pathologic stagingBrief historyOverall survivalPost-metastasectomy survivalCase 1Short-term survival86.0MaleHypopharynxSquamous cell carcinomapT4aN2bSurgical resection followed by adjuvant radiotherapy; one metastatic nodule at right lower lung was resected.23.914.4Case 2Short-term survival60.9MaleOropharynxSquamous cell carcinomapT4aN1Surgical resection followed by adjuvant chemoradiotherapy; two metastatic nodules at right and left upper lung were resected.26.011.9Case 3Short-term survival47.1MaleOropharynxSquamous cell carcinomapT4aN2bInduction chemotherapy followed by definitive surgical resection and adjuvant chemoradiotherapy; one left upper lung metastasis was resected.22.87.1Case 4Long-term survival38.9MaleHypopharynxSquamous cell carcinomapT2N2bSurgical resection followed by adjuvant radiotherapy; multiple bilateral lung metastases with sequential wedge resection for left and right side lesions52.140.2Case 5Long-term survival51.3MaleHypopharynxSquamous cell carcinomapT3N2bSurgical resection followed by adjuvant chemoradiotherapy; 3 metastatic lesion at right upper, middle and left lower lung were resected.89.842.7Case 6Long-term survival54.4MaleHypopharynxSquamous cell carcinomapT4aN2bSurgical resection followed by adjuvant chemoradiotherapy; 2 metastatic lesions at bilateral lung apices with sequential wedge resection134.891.1Case 7Control39.6FemaleOral cavityAdenoid cystic carcinomapT4aNoSurgical resection followed by adjuvant chemoradiotherapy; 2 metastatic nodules at right upper and middle lung were resected130.187.0Case 8Control46.2FemaleRight maxillary sinus and nasal cavityAdenoid cystic carcinomaUnknownSurgical resection followed by adjuvant radiotherapy; 2 metastatic nodules at right upper and middle lung were resected195.079.9 Citation Format: Hsueh-Ju Lu, Chih-Cheng Hsieh, Chi-Chun Yeh, Yi-Chen Yeh, Muh-Hwa Yang, Chi-Ying F. Huang, Peter Mu-Hsin Chang. Genome-wide association study reveals potential biomarkers after pulmonary metastasectomy for head and neck cancer [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 3442.

Cross resistance to diverse anticancer nicotinamide phosphoribosyltransferase inhibitors induced by FK866 treatment.

Cross-resistance to drugs remains an unsolved problem in cancer chemotherapy. This study elucidates a molecular mechanism of cross-resistance to diverse inhibitors of nicotinamide phosphoribosyltransferase (NAMPT) with anticancer activity. We generated a variant of the human colon cancer cell line HCT116, HCT116RFK866, which exhibited primary resistance to the potent NAMPT inhibitor FK866, and was approximately 1,000-fold less sensitive to the drug than the parental HCT116. HCT116RFK866 was found to be cross-resistant to diverse NAMPT inhibitors, including CHS-828, GNE-617, and STF-118804. Whole-exon sequencing revealed two point mutations (H191R and K342R) in NAMPT in HCT116RFK866, only one of which (K342R) was present in the parental HCT116. Importantly, the protein level, NAMPT enzyme activity, and intracellular NAD+ level were similar between HCT116RFK866 and HCT116. Hence, we investigated NAMPT-binding partners in both cell lines by focused proteomic analyses. The amount of NAMPT precipitated with anti-NAMPT monoclonal antibody was much higher in HCT116RFK866 than in the parental. Furthermore, in HCT116, but not in HCT116RFK866, NAMPT was revealed to interact with POTE ankyrin domain family member E and beta-actin. Thus, these results suggest that NAMPT usually interacts with the two partner proteins, and the H191R mutation may prevent the interactions, resulting in resistance to diverse NAMPT inhibitors.

Assessment of basal-like breast cancer by circulating tumor DNA analysis.

Standardized methods for the detection and assessment of circulating tumor DNA (ctDNA) in breast cancer are not sufficient. In the present study, the method and the potential application of ctDNA in the diagnosis of breast cancer were explored. DNA was extracted from the tumor tissues, plasma and peripheral blood cells of 11 patients with early-stage invasive breast cancer. Primers were designed against the exons of phosphatidylinositol-4,5-biphosphate 3-kinase catalytic subunit α, p53, epidermal growth factor receptor, Akt and phosphatase and tensin homolog. The amplicon-based method for whole-exon sequencing was performed. The associations between the ctDNA mutant frequency with the tumor DNA mutant frequency, and the ctDNA concentration with clinical data were analyzed. A linear association was identified between the concentration of ctDNA and the tumor volume for the 3 patients with basal-like breast cancer, and not in other subtypes. The mutation frequency differed the least between ctDNA and tissue DNA in basal-like breast cancer. ctDNA retained the constituent ratio of gene mutations identified in the corresponding tumor tissue. The ctDNA detection rate depended to a certain extent on the mutation frequency in tumor tissue; for example, a mutant locus with a mutation frequency of >30% in tissues presented a detection rate of >40% in plasma samples, whereas a locus with a mutation frequency of <10% in tissue was associated with a detection rate of ≤1% in the plasma. Therefore, ctDNA may reflect the mutations observed in cancer. Compared with other subtypes, ctDNA may be a more sensitive biomarker for the assessment of mutation and cancer burden in basal-like breast cancer relative to other subtypes.

E3 Ubiquitin Ligase UBR5 Drives the Growth and Metastasis of Triple-Negative Breast Cancer.

Patients with triple-negative breast cancers (TNBC) are at high risk for recurrence and metastasis at an early time despite standard treatment, underscoring the need for novel therapeutic modalities. Here, we report for the first time a distinctive and profound role of the E3 ubiquitin ligase UBR5 in the growth and metastasis of TNBC. An analysis of primary TNBC specimen by whole-exon sequencing revealed strong gene amplifications of UBR5 associated with the disease. UBR5 overexpression in TNBC tissues was confirmed at mRNA and protein levels. CRISPR/Cas9-mediated deletion of ubr5 in an experimental murine mammary carcinoma model of TNBC dramatically abrogated tumor growth and metastasis in vivo, which could be reversed completely via reconstitution with wild-type UBR5 but not a catalytically inactive mutant. Loss of UBR5 caused an impairment in angiogenesis within the tumor, associated with increased apoptosis, necrosis, and growth arrest. Absence of UBR5 in the tumor triggered aberrant epithelial-to-mesenchymal transition, principally via abrogated expression of E-cadherin, which resulted in severely reduced tumor metastasis to secondary organs. Use of NOD/SCID mice revealed that tumor-derived UBR5 facilitated tumor growth in a manner completely dependent upon immune cells in the microenvironment, whereas it promoted metastasis in a tumor cell-autonomous fashion. Our findings unveil UBR5 as a novel and critical regulator of tumor growth, metastasis, and immune response and highlight the potential for UBR5 as an effective therapeutic target for the treatment of highly aggressive breast and ovarian cancers that fail conventional therapy. Cancer Res; 77(8); 2090-101. ©2017 AACR.

Abstract 12257: Calmodulin Interacting Genes as a Novel Candidate for Pathogenesis of Long-QT Syndrome

Background: Approximately 30% of long-QT syndrome (LQTS) cases remains genetically elusive. Here, we investigated usefulness of the whole exon sequencing (WES) by next-generation sequencing for identification of novel pathogenic candidates which directly or indirectly interact with proteins encoded by known LQTS genes. Methods: Of 1815 Japanese LQTS cohort patients, WES was performed in 59 LQTS patients and 61 unaffected individuals from 35 families and 138 unrelated LQTS cases, all were screened major LQTS genes such as KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2 and KCNJ2. After WES, the genes known as inherited arrhythmias were screened by the Human Gene Mutation Database, and the Sanger sequencing was also referred for validation of the mutations and common variants were excluded by the public (1000G, ESP6500 and dbSNP) and Riken database. Results: Total 92 candidate mutations including 11 de novo, 5 recessive (2 homozygous and 3 compound heterozygous) and 73 dominant mutations in 88 genes were identified in 23 of the 35 families. Protein-protein interaction network analyses revealed ten new pathogenic candidates (WDR26, RYR2, UBR5, UBR4, KIF21B, CIT, SIRT6, PIK3CG, PI4KA and RIMS1) that directly or indirectly interact with proteins encoded by known LQTS genes. Furthermore, gene based association studies identified an additional novel candidate; SLC2A5. Taken together, mutations in these new candidates or known LQTS genes were identified in 13 of the 35(37%) genotype-unknown LQTS families. Moreover, 5 of the 11 newly identified candidates directly interact with calmodulin (CaM). Subsequent variant analysis in the independent set of 138 cases identified 16 variants in the 11 genes, of which, 14 (87.5%) were in the CaM-interacting genes and 8 of them were in RYR2. Many of the patients with RyR2 mutation had similar exercise-induced cardiac events (3 syncope, 2 VF) as LQTS patients, while the QTc interval was shorter in patients with RyR2 mutation than those with genotype-negative LQTS (441±32 vs. 487±47ms; p=0.01). Conclusions: These findings suggest that CaM interacting genes have an important role on the pathogenesis of LQTS. In particular, RYR2 mutations may cause an overlap syndrome between catecholaminergic polymorphic ventricular tachycardia and LQTS.

Diagnosis of MDS: morphology, chromosome abnormalities and genetic mutations

Myelodysplastic syndromes (MDS) are a group of hematological neoplasms associated with ineffective hematopoiesis and that can transform into acute leukemia. The clinical classification of MDS which is defined by cytopenia, the rate of blasts in peripheral blood and bone marrow, dysplasia, and chromosomal abnormalities, has undergone continuous revision. To increase the accuracy of dysplastic evaluation, IWGM-MDS and the Research Committee for Idiopathic Hematopoietic Disorders, Ministry of Health, Labour and Welfare, Japan have proposed a quantitative and qualitative definition of dysplasia. Recently, refining the definition of dysgranulopoiesis was proposed by IWGM-MDS. Neutrophils with abnormal clumping of chromatin, and harboring more than 4 nuclear projections, were recognized as dysplastic features. At present, karyotypic abnormalities are detected in approximately 50% of de novo MDS and these remain the most critical prognostic factor. In the new cytogenetic scoring system, cytogenetic abnormalities were classified into five prognostic subgroups. This new classification was adopted by the revised IPSS. Approximately 80% to 90% of MDS patients have detectable mutations by whole-exon sequencing or whole genome sequencing. Many genetic mutations had biological and prognostic significance. It is important to further understand the utility of this factor in determining prognosis and in selecting among therapeutic options.

The Outcomes and Next Landscapes of Project HOPE (High-Tech Omics-Based Patient Evaluation) for Hepatocellular Carcinoma

Introduction: Project HOPE (High-tech Omics-based Patient Evaluation) aims to provide the multiomics data of cancer patients for the promotion of individualized medicine and the practice of prophylaxis. To reveal the relationship between the clinical data and genetic expression, genetic analyses based on the results of project HOPE were performed for 24 patients who underwent hepatectomy for hepatocellular carcinoma (HCC). Methods: Fresh surgical specimens obtained from HCC patients who underwent surgery at the Shizuoka Cancer Center Hospital, were analyzed by whole-exon sequencing (WES, Life Technologies) and gene expression profiling (GEP, Agilent Technologies). As of April 1, 2015, 24 patients have been analyzed, and the aberrant genes were identified from the results of the WES and GEP analyses. Results: The present study included 17 males and 7 females, with a median age of 67 years (range: 42-81 years). Six of the patients were positive for hepatitis B surface antigen, 9 were positive for hepatitis C antibody and 9 patients were negative for hepatitis virus infection. The WES analysis showed that specific single nucleotide variants (SNVs) were identified in the genes of CTNNB1 and TP53 at a frequency of 15% and 11%, respectively. The GEP analysis revealed the upregulation of the HIST1H3B (59%), EZH2 (33%) and MYCN (22%) genes in tumor tissue (in comparison to normal tissue), while the expressions of CDH1 (11%) and SOCS1 (11%) were found to be downregulated in tumor tissue. In the analysis of the association between the clinical data and gene expression, the rate of advanced tumor staging (UICC stage > 2) was significantly higher in the patients with upregulated HIST1H3B expression in tumor tissue than in those with normal HIST1H3B expression (57.1% vs. 10%, P=0.033). Moreover, the relapse-free survival rate of the patients with upregulated HIST1H3B expression of tumor tissue was significantly lower than that of patients with normal HIST1H3B expression (estimated median relapse-free survival time: 5.8 vs. 7.7 months; P=0.009). Conclusion: Our genetic analysis based on the results of Project HOPE revealed HIST1H3B to be novel cancer-related gene and a predictive factor for HCC. In the future, we plan to accumulate the results of 150 HCC patients, and it is hoped that the additional novel cancer-related genes will be identified.

Alternative Mammalian Target of Rapamycin (mTOR) Signal Activation in Sorafenib-resistant Hepatocellular Carcinoma Cells Revealed by Array-based Pathway Profiling

Sorafenib is a multi-kinase inhibitor that has been proven effective for the treatment of unresectable hepatocellular carcinoma (HCC). However, its precise mechanisms of action and resistance have not been well established. We have developed high-density fluorescence reverse-phase protein arrays and used them to determine the status of 180 phosphorylation sites of signaling molecules in the 120 pathways registered in the NCI-Nature curated database in 23 HCC cell lines. Among the 180 signaling nodes, we found that the level of ribosomal protein S6 phosphorylated at serine residue 235/236 (p-RPS6 S235/236) was most significantly correlated with the resistance of HCC cells to sorafenib. The high expression of p-RPS6 S235/236 was confirmed immunohistochemically in biopsy samples obtained from HCC patients who responded poorly to sorafenib. Sorafenib-resistant HCC cells showed constitutive activation of the mammalian target of rapamycin (mTOR) pathway, but whole-exon sequencing of kinase genes revealed no evident alteration in the pathway. p-RPS6 S235/236 is a potential biomarker that predicts unresponsiveness of HCC to sorafenib. The use of mTOR inhibitors may be considered for the treatment of such tumors.

A commentary on the promise of whole-exome sequencing in medical genetics

The dawn of next-generation sequencers (NGSs) and innovative sequencing technologies have brought a paradigm shift in medical research and clinical practice. Furthermore, the cost reduction of NGSs enables personalized medicine to come to fruition. However, whole-genome sequencing (WGS) remains expensive when applied to personal genome analysis. WGS generates a large amount of data that requires highperformance computer processing. Targeted whole-exon capture and sequencing [wholeexome sequencing (WES)] is more costeffective when compared with WGS because exons represent only B1–2% of the genome and also higher sequence coverage can be achieved by NGSs. In addition, most Mendelian disorders are caused by exonic mutations or splice-junction mutations, and protein-coding genes harbor B85% of the mutations that have large effects on diseaserelated traits.1 Thus, WES will provide many advantages and lower costs than WGS when analyzing personal genomes. WES was first successfully used in 2010 to discover the gene responsible for Miller syndrome, a Mendelian disorder.2 Since then, WES has been increasingly used as a fast and accurate genomic discovery approach to investigate both rare genetic disorders and common diseases. WES is widely applied across different areas of medicine, because it has the added advantage of reduced cost and requires analysis of a much smaller but essential dataset when compared with WGS. In addition, recent clinical molecular diagnostics have used WES to detect heterogeneous Mendelian diseases.3,4 A recent review of WES approaches in medical genetics describes the usefulness of WES in medicine and medical research and the impact of WES on clinical diagnoses.5 WES approaches have greatly facilitated the discovery of candidate genes or gene variants in Mendelian disorders and rare variants in common diseases and genomic characterization in cancer. Currently, WES is increasingly being applied to disease gene discovery, cancer typing and molecular diagnosis.5 Presently, WES is an essential tool in medical genetics, especially in the research of Mendelian disorders. WES or multigene tests using NGSs are widely applied to heterogeneous disorders including deafness or ciliopathy.5,6 WES is also being increasingly applied to genetic testing for undiagnosed patients.4,5 Yang et al.4 performed WES in undiagnosed patients whose phenotypes were suggestive of potential genetic disorders and achieved a molecular diagnosis for 62 of 250 (25%) patients. Because WES detects individual genetic variation, it can be used to construct a variation database of anthropic and ethnic populations. At the same time, because WES can detect groups of genetic variations that are unrelated to the indication for the first diagnostic purpose but are of medical value for individual patient care, such ‘incidental findings’ pose potential ethical problems that should be strongly considered and discussed in clinical practice.5,7 WES is a widely applied technique in medical genetics that is capable of detecting variations in whole exons. However, in practical use, understanding WES methodology and limitations are important. Current WES techniques are not capable of detecting all of the variations surrounding exons. Detecting variation by WES is limited by the experimental methods, probe coverage and/or platforms used.8–10 Hence, WES may not always detect pathogenic or causative variations in a genetic disease. In addition, because WES is a method to detect genomic sequence variations, when a candidate of causative variation in the disease is detected, it requires verification or support by secondary analyses. In particular, further functional analyses are important to confirm whether the variant is pathogenic or benign. Nevertheless, WES enables the unprecedented low cost and highly efficient analysis of whole exons. WES can be easily used to comprehensively detect individual variations in exons. It is without doubt that WES is a powerful tool in genome analysis, and it greatly progresses medical genetics. Although WESs’ limitations need to be overcome, we anticipate that WES will be used not only in medical research but also in clinical practice for example, molecular diagnosis (whole-gene test) and personal genomics before WGS becomes a common place in medical genetics. Thus, a paradigm shift in medicine by advancement in both WES and WGS is expected to continue.

Paradigm shifts in the genetics of inherited arrhythmias: Using next-generation sequencing technologies to uncover hidden etiologies

Long QT syndrome (LQTS), a rare inheritable arrhythmia first described by Romano andWard et al. in the 1960s, is characterized by the prolongation of the QT interval on surface ECGs and an increased risk of potentially fatal ventricular arrhythmias, especially torsade de pointes [1]. In 1995, after years of extensive clinical investigation and linkage analysis, several research groups, including Keating et al., successfully identified three distinct LQTS phenotypes (LQT1, LQT2, and LQT3) associated with mutations in genes encoding plasma membrane ion channels (KCNQ1, KCNH2, and SCN5A, respectively) [2–4]. These seminal studies motivated further extensive genetic screening in LQTS patients using a candidate gene approach and functional analyses of the mutant genes. These efforts provided evidence that ion channel genes represent the genetic basis for several other arrhythmogenic syndromes that occur in the structurally intact heart, often referred to as idiopathic ventricular fibrillation. At present, 13 genes responsible for LQTS have been identified. Approximately 90% of the genotyped LQTS subjects belong to the three major subtypes (LQT1-3) [5] in which numbers of distinct genotype-specific clinical characteristics have been demonstrated, including T-wave morphology [6], triggers for cardiac events [7], response to the epinephrine provocation test [8] and drug therapy [9]. Genetic testing for known arrhythmia susceptibility genes has become the standard-of-care for a number of disorders, including LQTS. Considering the remarkable progress of research in this area, there is no doubt that 1995 was the year in which genetic technologies experienced a paradigm shift with respect to both the understanding and clinical management of inherited arrhythmias. However, it should be noted that despite the rapid progress in understanding the genetic basis, the etiology still remains unknown in approximately 20% of LQTS conditions [10]. Therefore, additional studies are needed to reveal the missing heritable factors associated with these syndromes. Traditionally, DNA sequence information has been elucidated using Sanger sequencing, but this method is limited by the amount of DNA that can be processed at a given time and by the read length (average 800 base pairs). Although the Human Genome Project completed the sequencing of the entire human genome in 2001 using Sanger sequencing, it took 13 years of effort at an estimated cost of $2.7 billion. Now, next-generation sequencing (NGS), a revolutionary new genetic technology, has enabled whole genomes to be sequenced over a period of a few days at projected costs of less than $10,000. This technology involves massively parallel sequencing of clonally amplified or single DNA molecules that are spatially separated in a flow cell. To date, the genes responsible for disease in more than 3000 Mendelian disorders have yet to be identified. NGS opens up exciting new possibilities of discovering the genes associated with these monogenic disorders. Classical strategies involve linkage analysis in families with known shared genetic heritage by identifying candidate genomic regions encompassing the gene

Abstract B13: Modeling BRAF inhibitor resistance in genetically engineered mouse models of melanoma

Abstract Resistance to BRAF inhibitors in melanoma poses a serious clinical challenge to this otherwise potent therapy. Acquired resistance has been shown to arise from a wide variety of genetic and epigenetic sources including NRAS or MEK mutations, elevated PDGFRB or IGF1R levels, and BRAF splice variants. Many of these mechanisms were discovered in vitro and validated in patients, demonstrating the faithfulness of cell culture data. Nevertheless, cell culture may not be able to capture all sources of resistance, particularly if they involve interaction with the stroma. We have therefore undertaken the generation of de novo BRAF inhibitor resistance in a novel mouse model of melanoma, the iBIP (inducible Braf, Ink/Arf, Pten) model. Similar to existing BRAF mouse models, topical tamoxifen restricts BRAFV600E expression and PTEN loss to targeted melanocytes. The added utility of this model is the ability to extinguish BRAF transgene expression via withdrawal of doxycycline from the diet. We show that the molecular profile and tumor regression phentoype of BRAF inhibitor treatment in this model faithfully mirrors genetic BRAF extinction. Treatment with the BRAF inhibitor caused stable and complete tumor regression. However, long-term treatment with the inhibitor failed to generate de novo resistant melanomas. We hypothesized that the relatively stable mouse genome was not generating potentially resistance-conferring mutations or alterations at a fast enough rate. We therefore generated genomic instability in the iBIP model by crossing it onto a telomerase-deficient background. Melanomas in this model acquired resistance at a rate of approximately 30% after a short interval of sensitivity. Currently, characterization of these tumors is being carried out, including whole-exon sequencing, RNA-sequencing, and array comparative genomic hybridization. Preliminary aCGH data show a recurrent, whole-chromosome gain in two resistant tumors that is not present in untreated or sensitive tumors. Other efforts are focused on generating a large bank of resistant tumors, checking cross-resistance among BRAF inhibitors, and identifying any stromal and immune changes. This in vivo model of acquired BRAF inhibitor resistance provides an important complement to known resistance mechanisms and may also serve as a preclinical platform to test anti-resistance countertherapies.

Final “Perspectives on the News”

### Genomics and diabetes At a plenary session of the American Association of Clinical Endocrinology (AACE) Annual Meeting in April 2011, Eric Topol, La Jolla, California, discussed the concept that genomics can be used to create “a revolution in medicine” for diabetes prevention and management. Although the decoding of the human genome was greeted with much fanfare in 2000, this information “has failed so far to produce medical miracles.” There has, however, been remarkable progress in the use of the human genome to understand the underpinning of many common diseases. We are progressing from genome-wide association studies (GWASs), involving 106 single nucleotide polymorphisms (SNPs) and providing insight into common gene variations with population frequency >5%, to whole-exon sequencing of 1.5% of the genome, finding rare and low frequency elements, to whole genome sequencing, allowing an understanding of regulatory as well as coding sequences. More than one thousand GWASs have been carried out, identifying several hundred traits tracking with important diseases. The discovery of a variant of transcription factor 7-like 2 (TCF7L2) associated with diabetes may lead to the greatest understanding of its pathogenesis, and a number of other polymorphisms have been found to be related to diabetes. A meta-analysis of GWAS data from over 100,00 individuals confirms 38 gene loci (1), and a recent review cites 67 loci for nonautoimmune diabetes (2), suggesting that there are many different genetic pathways to its development. Topol suggested that diabetes risk is associated with “a combination of lower variants below the 5% threshold … down to <1%” (3). He reviewed a number of interesting candidates, including regions of the genome that do not appear to code for specific peptides but, rather, regulate transcription of other genes (4). Another potential cause is variation in the melatonin receptor 1b, which, like TCF7L2, is associated with impairment in insulin …

Case report: a p.C618S RET proto-oncogene germline mutation in a large Chinese pedigree with familial medullary thyroid carcinoma

We report a Chinese pedigree with familial medullary thyroid carcinoma. Direct sequencing of the entire coding sequences of Rearranged during Transfection (RET) identified a recurrent c.T1852A (p.C618S) mutation in 13 of 23 members. The polymorphisms c.A135G (p.A45A), c.A1296G (p.A432A), c.T2307G (p.L769L) and IVS19 + 15T > C were also found in 13 carriers, and c.G2073A (p.G691S) was found in 1 carrier. Of the 13 carriers, seven (mean age: 42.6 years, range: 27-64) presented MTC as the isolated clinical phenotype, with elevated basal serum calcitonin (average: 1077.9 ng/L, range: 504-2,652) and a mean diameter of thyroid nodules of 2.97 cm (range: 1.6-4.3); they underwent a total thyroidectomy with modified bilateral/unilateral neck dissection and/or level VI lymph node dissection. The other 6 carriers did not accept surgery (4 rejected, 2 awaited). These were 2 older patients (63 and 32 years) with elevated calcitonin (1359 and 41.4 ng/L) and multi-centric hypoechoic nodules (1.5 and 0.6 cm) with calcifications in both/left thyroid lobes; and Doppler ultrasound showed normal bilateral thyroids in 4 younger carriers (median age: 8.3 years, range: 4-12) but with increased calcitonin (average: 9.7 ng/L, range: 7.87-12.2) in 3 of them. The phenotype here is consistent with the clinical symptoms reported worldwide. We recommend that screening of hotspot regions of RET should be preferentially carried out, while whole-exon sequencing should be performed when clinical signs fail to reveal hotspot mutations or different phenotype discrepancies. Moreover, we strongly suggest prophylactic thyroidectomy should be performed before age 5 in carriers with p.C618S to prevent the occurrence and metastasis of MTC.