Ken-Hong Lim1, Huan-Chau Lin2, Caleb Gon-Shen Chen3, Wei-Ting Wang4, Yu-Cheng Chang2, Yi-Hao Chiang2, Ching-Sung Lin4, Nai-Wen Su5, Ying-Wen Su5, Johnson Lin6, Yi-Fang Chang5, Ming-Chih Chang5, Ruey-Kuen Hsieh2, Yuan-Yeh Kuo7, Wen-Chien Chou8. 1. Graduate Institute of Oncology, National Taiwan University College of Medicine, Taipei, Taiwan; Division of Hematology and Oncology, Department of Internal Medicine, Mackay Memorial Hospital, Taipei, Taiwan; Laboratory of Good Clinical Research Center, Department of Medical Research, Mackay Memorial Hospital, Tamsui District, New Taipei City, Taiwan; Department of Medicine, Mackay Medical College, New Taipei City, Taiwan. Electronic address: khlim@mmh.org.tw. 2. Division of Hematology and Oncology, Department of Internal Medicine, Mackay Memorial Hospital, Taipei, Taiwan; Laboratory of Good Clinical Research Center, Department of Medical Research, Mackay Memorial Hospital, Tamsui District, New Taipei City, Taiwan. 3. Division of Hematology and Oncology, Department of Internal Medicine, Mackay Memorial Hospital, Taipei, Taiwan; Laboratory of Good Clinical Research Center, Department of Medical Research, Mackay Memorial Hospital, Tamsui District, New Taipei City, Taiwan; Department of Medicine, Mackay Medical College, New Taipei City, Taiwan; Institute of Molecular and Cellular Biology, National Tsing-Hua University, Hsinchu, Taiwan. 4. Laboratory of Good Clinical Research Center, Department of Medical Research, Mackay Memorial Hospital, Tamsui District, New Taipei City, Taiwan. 5. Division of Hematology and Oncology, Department of Internal Medicine, Mackay Memorial Hospital, Taipei, Taiwan; Laboratory of Good Clinical Research Center, Department of Medical Research, Mackay Memorial Hospital, Tamsui District, New Taipei City, Taiwan; Department of Medicine, Mackay Medical College, New Taipei City, Taiwan. 6. Division of Hematology and Oncology, Department of Internal Medicine, Mackay Memorial Hospital, Taipei, Taiwan. 7. Graduate Institute of Oncology, National Taiwan University College of Medicine, Taipei, Taiwan. Electronic address: yykuo@ntu.edu.tw. 8. Division of Hematology, Department of Internal Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan; Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan.
Abstract
BACKGROUND: Somatic CALR exon 9 mutations have recently been identified in patients with JAK2/MPL-unmutated myeloproliferative neoplasm, and have become an important clonal marker for the diagnosis of essential thrombocythemia (ET) and primary myelofibrosis. In the present study, we sought to use high-resolution melting analysis (HRMA) as a screening method for the detection of CALR mutations. METHODS: 32 JAK2/MPL-unmutated ET patients were retrospectively enrolled and 8 healthy adults were used as wild-type control. CALR exon 9 mutation was independently screened by HRMA with the CFX Connect real-time system and Sanger sequencing. TA-cloning was used to detect CALR exon 9 mutations in patients suspected to have low mutant allele burden. RESULTS: The maximal sensitivity of HRMA in identifying both CALR type 1 and type 2 mutants from patients' genomic DNA was 2.5%. Twenty-two samples were found to have distinct melting curves from wild-type. The presence of CALR mutations in 16 of these 22 samples was confirmed by Sanger sequencing, while the other 6 samples were wild-type by sequencing. After TA-cloning, CALR mutations were detected in 5 of 6 patients from 1 (6%) of 16 clones to 1 (2%) of 50 clones. Therefore, HRMA identified CALR mutations in 21 (65.6%) of 32 ET patients compared to 16 (50%) patients by Sanger sequencing, with a false positive rate of 3% and no false negative. CONCLUSION: The HRMA developed in our system is a rapid and sensitive technique for the detection of CALR exon 9 mutations.
BACKGROUND: Somatic CALR exon 9 mutations have recently been identified in patients with JAK2/MPL-unmutated myeloproliferative neoplasm, and have become an important clonal marker for the diagnosis of essential thrombocythemia (ET) and primary myelofibrosis. In the present study, we sought to use high-resolution melting analysis (HRMA) as a screening method for the detection of CALR mutations. METHODS: 32 JAK2/MPL-unmutated ET patients were retrospectively enrolled and 8 healthy adults were used as wild-type control. CALR exon 9 mutation was independently screened by HRMA with the CFX Connect real-time system and Sanger sequencing. TA-cloning was used to detect CALR exon 9 mutations in patients suspected to have low mutant allele burden. RESULTS: The maximal sensitivity of HRMA in identifying both CALR type 1 and type 2 mutants from patients' genomic DNA was 2.5%. Twenty-two samples were found to have distinct melting curves from wild-type. The presence of CALR mutations in 16 of these 22 samples was confirmed by Sanger sequencing, while the other 6 samples were wild-type by sequencing. After TA-cloning, CALR mutations were detected in 5 of 6 patients from 1 (6%) of 16 clones to 1 (2%) of 50 clones. Therefore, HRMA identified CALR mutations in 21 (65.6%) of 32 ET patients compared to 16 (50%) patients by Sanger sequencing, with a false positive rate of 3% and no false negative. CONCLUSION: The HRMA developed in our system is a rapid and sensitive technique for the detection of CALR exon 9 mutations.
Authors: Larysa Poluben; Maneka Puligandla; Donna Neuberg; Christine R Bryke; Yahsuan Hsu; Oleksandr Shumeiko; Xin Yuan; Olga Voznesensky; German Pihan; Miriam Adam; Ernest Fraenkel; Roni Rasnic; Michal Linial; Sergiy Klymenko; Steven P Balk; Paula G Fraenkel Journal: Am J Hematol Date: 2018-10-31 Impact factor: 10.047
Authors: Ji Hun Jeong; Hwan Tae Lee; Ja Young Seo; Yiel Hea Seo; Kyung Hee Kim; Moon Jin Kim; Jae Hoon Lee; Jinny Park; Jun Shik Hong; Pil Whan Park; Jeong Yeal Ahn Journal: Ann Lab Med Date: 2016-07 Impact factor: 3.464