Koo, S.Y. Captured DNA libraries were sequenced with the Illumina GAIIx Genome Analyzer, yielding 150 (2 × 75) base pairs from the final library fragments. KHYG-1 was obtained from the Japanese Collection of Research BioResources (28) and cultured in RPMI medium (Life Technologies) supplemented with heat-inactivated FBS (10%), equine serum (10%), and 200 IU/mL of recombinant human IL-2 (Novartis). By continuing to use Gov.sg, you accept our use of cookies. FFPE tissue blocks from 61 patients with NKTCLs were procured for mutation analysis. There has been little progress in basic science and clinical research in this subtype of lymphoma, which continues to constitute a major challenge in managing these patients as there is currently no accepted standard first-line treatment for NKTCLs. The relative p-JAK3, p-STAT5, JAK3, and STAT5 levels in these cells were detected by Western blotting (top), and proliferation assays using these cells were conducted for 48 hours with or without IL-2 (bottom). JAK-mutant (NK-S1) cells showed IL-2–independent growth (Fig. Gan Teng (Reno) Gan Teng. MIGR1 expression vectors containing full-length wild-type JAK3 or JAK3A572V mutant were generously provided by Dr. Brian Druker (Howard Hughes Medical Institute, Chevy Chase, MD; ref. Find contact's direct phone number, email address, work history, and more. Further validation of the prevalence of JAK3 mutations was determined by Sanger sequencing and high-resolution melt (HRM) analysis in an additional 61 cases. and was admitted to the Singapore Bar in 1978. Lim, Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc. To decline cookies at any time, you may adjust your browser settings. The cycling and melting conditions were as follows: 1 cycle of 98°C for 2 minutes; 39 cycles of 98°C for 5 minutes; 58°C for 10 minutes; 1 cycle of 95°C for 30 minutes; and a melt from 72°C to 95°C increasing at 0.2°C/s. Mr Gan first joined ComfortDelGro Corporation Limited as Group Corporate Planning Officer in February 2006. IL-2–independent growth and constitutive JAK3 and STAT5 phosphorylation in a JAK3A572V-mutant NKTCL cell line. He is a practicing lawyer and is a Partner at Infinitus Law Corporation and Head of its Corporate, Commercial and Technology Department. These results indicate that the JAK3-activating mutations are gain-of-function alleles and contribute to the constitutive activity of the JAK/STAT pathway in an IL-2–independent manner. 8). Only well-mapped reads (mapping quality ≥30, number of mismatches within a 40-bp window ≤3) were used as input to the genotyper. ISSN: 2159-8274, Sign In to Email Alerts with your Email Address. In summary, our studies identified, for the first time, frequent JAK3 mutations in NKTCLs. In parallel, 50% of extra-nasal cases possessed JAK3 mutations and 31.7% of nasal cases had JAK3 mutations. Lim, Writing, review, and/or revision of the manuscript: G.C. See the complete profile on LinkedIn and discover Gan’s connections and jobs at similar companies. 4A) and cell viability in a dose-dependent fashion (Fig. Join Facebook to connect with Choon Kiat Gan and others you may know. For FFPE samples, genomic DNA was extracted from one or two 10-μm slices from each sample by removal of paraffin followed by proteinase K digestion according to standard procedures. Known somatic mutations in NKTCLs, such as TP53, KRAS, and NRAS (4), identified by exome sequencing were further validated by Sanger sequencing in the same tumors (Supplementary Table S2). The molecular pathogenesis of natural killer/T-cell lymphoma (NKTCL) is not well understood. View Choon Chia's business profile as Infrastructure & Operation Director, Systems (Rail Asset O&M) at Land Transport Authority. Koo, S.Y. Contact Information. All 3 missense mutations were predicted by PolyPhen to be damaging (5). The coding exons of JAK3 were fully sequenced in these 3 cell lines, and we confirmed that only NK-S1 harbored a homozygous JAK3A572V mutation. Cancer Discov; 2(7); 591–7. S1). Cornejo and colleagues (21) showed that when JAK3A572V retroviral–transduced bone marrow cells were transplanted into C57BL/6 and BALB/c mice, there was a constitutive activation of JAK/STAT signaling which led to the development of fatal polyclonal T-cell lymphoproliferative disorder. After blocking, membranes were probed with primary antibodies against phospho-STAT5 (Cell Signaling, catalog number 9356), STAT5 (Cell Signaling, catalog number 9363), phospho-JAK3 (Cell Signaling, catalog number 5031), JAK3 (Cell Signaling, catalog number 3775), and β-actin followed by either peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody. Lim, C. Goh, W. Yu, C.C.Y. The men's 1000 metres competition at the 2010 Asian Games in Guangzhou was held on 18 November 2010 at the Zengcheng Dragon Boat Lake. Further validation of the prevalence of JAK3 mutations was determined by Sanger sequencing and high-resolution melt … Ng, S.T. Among the patients with JAK3 mutations, there were 17 heterozygous JAK3A572V, 2 homozygous JAK3A572V, 2 heterozygous JAK3A573V, 1 homozygous JAK3A573V, and 1 heterozygous with both JAK3A572V and JAK3A573V mutations. C, drug-induced apoptosis was evaluated by Annexin V-FITC (fluorescein isothiocyanate) staining, followed by flow cytometric analysis. We offer the most extensive selection of academic programmes in Singapore, collaborating with leading universities worldwide to provide our students with diverse opportunities for overseas exposure. These results were further confirmed by high-resolution melt (HRM) analysis (Supplementary Fig. In this study, we conducted whole-exome sequencing to identify somatic mutations in protein-coding genes of NKTCL tumors to shed light on their pathogenesis and to uncover potential new therapeutic targets, which are urgently needed. Ang Choon Kiat has 8 jobs listed on their profile. Gan Choon Beng is a practicing lawyer and is the Senior Director at Infinitus Law Corporation and head of its Corporate, Commercial and Technology Department.He graduated from the University of Singapore (now known as the National University of Singapore) in 1977 with an LLB (Hons.) Experiments were repeated at least 3 times.y. A research-intensive university with an entrepreneurial dimension, NUS is ranked consistently as one of the world's top universities. Lim, Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S.L. However, this phenomenon was not observed in the K562 cells in which STAT5 phosphorylation is dependent on BCR/ABL1 (8) and not JAK3 (Fig. I. Proliferative response and establishment of cloned cells. This study is dedicated to Dr. Han Mo Koo. Ong, K.-W. Yeoh, K.A. For Sanger sequencing, PCR was carried out with Platinum Taq Polymerase (Life Technologies, catalog number 10966-083) and cycled at 95°C for 10 minutes; 39 cycles of 95°C for 30 seconds; 60°C for 30 seconds, 72°C for 1 minute, and a final extension of 72°C for 10 minutes. This article is highlighted in the In This Issue feature, p. 569. K562 (CCL-234) was purchased from American Type Culture Collection and cultured in DMEM supplemented with 10% heat-inactivated FBS and 10% equine serum. Lim, Supplied pathologic diagnosis for case series: L. Tan. Whole-exome sequencing was successfully conducted on fresh-frozen NKTCLs and paired blood samples from 4 different patients. We used Burrows Wheeler Aligner to align the sequence reads to the human reference genome NCBI built 37.1 (hg19) and then we ran SamTools to remove PCR duplicates. Natural killer (NK) cells as a responder to interleukin 2 (IL 2). The diagnosis of NKTCL was made according to the 2008 World Health Organization classification of tumors of the hematopoietic and lymphoid tissues (24). These data are compelling and suggest a potential target for this otherwise fatal disease. Gan Chimeg. Our pipeline first recalibrates the base qualities and realigns the sequence reads around micro-indels. If you don’t like our faces, listen to our fortnightly podcast E-Junkies where we lepak one corner with famous people, Your daily good stuff - AsiaOne stories delivered straight to your inbox, AsiaOne Online Pte Ltd. Company Registration No. We conducted whole-exome sequencing and identified Janus kinase 3 (JAK3) somatic–activating mutations (A572V and A573V) in 2 of 4 patients with NKTCLs. View Gan Chin Kiat’s profile on LinkedIn, the world’s largest professional community. Tan, L. Tan, S.C. Chong, K. Tay, M. Tao, R. Quek, S. Loong, K.-W. Yeoh, S.P. All samples were centrally reviewed by our hematopathologists. Total proteins were extracted with lysis buffer, resolved by SDS-PAGE gels, and blotted onto a nitrocellulose membrane. DNA was then extracted using a DNeasy blood and tissue mini kit (Qiagen). "Every month or so we will be working with our public transport operators to have more themed trains in the next year," he said. To detect single-nucleotide variants (SNV), we used a discovery pipeline based on the Genome Analyzer Toolkit (GATK). Lee, I. Cutcutache, W. Yu, C.C.Y. Presently, Khai Choon Gan occupies the position of Non-Independent Non-Executive Chairman for HL Global Enterprises Ltd., Non-Executive Chairman of Beijing Fortune Hotel Co., Ltd. and Managing Director at Hong Leong International (Hong Kong) Ltd. These are some of the notable cases which SPD officers handled in 2011: In PP v Abdul Razak Bin Hamid and PP v Tan Chye Guan Martin, the accused persons were both heinous child molesters, dubbed the “Letterbox Molester” and “Heartbeat Molester” respectively.They engaged in ruses to con children into physical contact and committed the offences over 20 years and 11 years respectively. Poon, W.S. In parallel, these cells were harvested, and protein extracts were subjected to Western blotting with antibodies against phosphorylated JAK3 (p-JAK3), phosphorylated STAT5 (p-STAT5), JAK3, STAT5, or β-actin as a normalization control. Through exome sequencing, we identified activating mutations of JAK3 that may play a significant role in the pathogenesis of NKTCLs. Sanger sequencing and HRM (25, 26) were used to confirm the JAK3 and JAK1 mutations identified and validate their prevalence in our NKTCL patient population. The authors also thank the Lee Foundation for its support, Huang Dachuan and Waraporn Chan-on for proofreading the manuscript, and Sabrina Noyes for assistance in manuscript submission. To further confirm the involvement of JAK/STAT signaling in the survival of NKTCLs, we next evaluated the effect of a pan-JAK inhibitor, CP-690550, in NK-S1, KHYG-1, and K562 cells. Facebook gives people the power to share and makes the world more open and connected. The molecular pathogenesis of natural killer/T-cell lymphoma (NKTCL) is not well understood. A, NK-S1, KHYG-1, and K562 cells were treated with CP-690550 for 48 hours, and the effect on STAT5 phosphorylation was evaluated by Western blotting. Lim, Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): G.C. Both NK-S1 and KHYG-1 cells showed a dose-dependent reduction in STAT5 phosphorylation. Gan Teng. The JAK1Y652D mutation was located in the JH2 domain as well. Tan, T. Tang, S.L. Reciprocally, KHYG-1 cells transiently overexpressing a mutated JAK3 (JAK3A572V) cDNA showed IL-2–independent proliferation and autophosphorylation of JAK3 and STAT5 (Fig. In total, 23 of 65 (35.4%) cases harbored JAK3 mutations. Identification and characterization of JAK3-activating mutations. In total, we found mutations in JAK3 in 23 of 65 (35.4%) cases (Supplementary Table S3) and for JAK1, besides the case with concomitant JAK1Y652D and JAK3A572V mutations described above, no additional mutations were identified. Furthermore, Annexin V staining revealed that the reduction of NK-S1 viability was due to an increase in cellular apoptosis (Fig. Choon Kiat Gan is on Facebook. We are glad to play a part in our nation's 48th birthday celebration. Conception and design: G.C. The extent of drug-induced apoptosis was evaluated by Annexin V-FITC (BD Biosciences) staining. Welcome to Singapore's directory of Specialist Doctors and Surgeons in private practice. Land Transport Authority (LTA) Singapore is a entity based in 1 Hampshire Road, Singapore, 219428, Singapore, which employs 908 executives. We also conducted Epstein-Barr virus–encoded RNA (EBER) testing on all cases. Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/). Ong, P. Tan, B.T. Teh, S.T. ©2012 AACR. See the complete profile on LinkedIn and discover Ang Choon Kiat’s connections and jobs at similar companies. Thus, it is conceivable that the JAK3 mutation may play an important role in the pathogenesis of NKTCLs. Ang Choon Kiat has 4 jobs listed on their profile. Our findings have important implications for the management of patients with NKTCLs. Lim, D. Tan, C. Goh, C.C.Y. To determine the prevalence of JAK1 and JAK3 mutations in NKTCLs, we Sanger sequenced an additional 61 NKTCL formalin-fixed, paraffin-embedded (FFPE) cases. In contrast, JAK3 wild-type KHYG-1 cells were clearly IL-2–dependent (Fig. Apart from 4 older cases that cannot be interpreted, all but one case were positive for EBER, regardless of JAK3 mutation status. In line with these observations, we identified the presence of activating JAK3 mutations in 35% of NKTCL tumors. This study was approved by the SingHealth Centralized Institutional Review Board (CIRB), study number 2004/407/B. By continuing to use Gov.sg, you accept our use of cookies. Jak-STAT pathway is involved in the induction of TNF-beta gene during stimulation by IL-2, Activating alleles of JAK3 in acute megakaryoblastic leukemia, Mutations in severe combined immune deficiency (SCID) due to JAK3 deficiency, Somatically acquired JAK1 mutations in adult acute lympho-blastic leukemia, Mutations of JAK2 in acute lymphoblastic leukaemias associated with Down’s syndrome, FERM domain mutations induce gain of function in JAK3 in adult T-cell leukemia/lymphoma, Jak3- and JNK-dependent vascular endothelial growth factor expression in cutaneous T-cell lymphoma, Activation status of the JAK/STAT3 pathway in mantle cell lymphoma, JAK3 mutations occur in acute megakaryoblastic leukemia both in Down syndrome children and non-Down syndrome adults, A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera, Somatic mutations of JAK1 and JAK3 in acute leukemias and solid cancers, Somatic mutations affect key pathways in lung adenocarcinoma, Activating mutations in human acute megakaryoblastic leukemia, Array-based genomic resequencing of human leukemia, Constitutive JAK3 activation induces lymphoproliferative syndromes in murine bone marrow transplantation models, The JAK kinase inhibitor CP-690,550 suppresses the growth of human polycythemia vera cells carrying the JAK2V617F mutation, CP-690,550, a therapeutic agent, inhibits cytokine-mediated Jak3 activation and proliferation of T cells from patients with ATL and HAM/TSP, The 2008 WHO classification of lymphoid neoplasms and beyond: evolving concepts and practical applications, KRAS mutation detection in paired frozen and formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissues, An Epstein-Barr virus positive natural killer lymphoma xenograft derived for drug testing, A novel natural killer cell line (KHYG-1) from a patient with aggressive natural killer cell leukemia carrying a p53 point mutation, Lung Microbiome and Lung Cancer Prognosis, Pan-Cancer Analysis of HLA LOH as a Method of Immune Evasion, Cancer Epidemiology, Biomarkers & Prevention, Disclosure of Potential Conflicts of Interest. 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