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Clin Chim Acta. 2018 Aug;483:183-191. doi: 10.1016/j.cca.2018.05.001. Epub 2018 May 1.

Pyrosequencing-based quantitative measurement of CALR mutation allele burdens and their clinical implications in patients with myeloproliferative neoplasms.

Author information

1
Department of Laboratory Medicine, Chungnam National University School of Medicine, Chungnam National University Hospital, Daejeon, Republic of Korea; Green Cross Laboratories, Yongin, Republic of Korea.
2
Division of Hematology/Oncology, Department of Internal Medicine, Chungnam National University School of Medicine, Chungnam National University Hospital, Daejeon, Republic of Korea.
3
Department of Laboratory Medicine, Chungnam National University School of Medicine, Chungnam National University Hospital, Daejeon, Republic of Korea.
4
Department of Laboratory Medicine, Chungnam National University School of Medicine, Chungnam National University Hospital, Daejeon, Republic of Korea; Cancer Research Institute, Chungnam National University School of Medicine, Daejeon, Republic of Korea. Electronic address: ksuny55@cnu.ac.kr.

Abstract

BACKGROUND:

We developed a pyrosequencing-based method for the quantification of CALR mutations and compared the results using Sanger sequencing, fragment length analysis (FLA), digital-droplet PCR (ddPCR), and next-generation sequencing (NGS).

METHODS:

Method validation studies were performed using cloned plasmid controls. Samples from 24 patients with myeloproliferative neoplasms were evaluated.

RESULTS:

Among the 24 patients, 15 had CALR mutations (7 type 1, 2 type 2, and 6 other mutations). The type 1 or type 2 mutation-positive results from pyrosequencing exhibited 100% concordance with the Sanger sequencing results. One novel CALR mutation was not detected by pyrosequencing. The CALR mutation allele burdens measured by pyrosequencing were slightly lower than those measured by FLA but slightly higher than the results obtained using ddPCR. Pyrosequencing exhibited high correlations with both methods. The mutation allele burdens estimated by NGS were significantly lower than those measured by pyrosequencing. An increased CALR mutation allele burden was associated with overt primary myelofibrosis. Patients with >70% mutation allele burdens in myeloid cells had a significantly longer time from diagnosis (P = 0.007), more bone marrow fibrosis (P = 0.010), and lower hemoglobin (P = 0.007).

CONCLUSIONS:

Pyrosequencing was a useful rapid sequencing method to determine the burden of CALR mutations.

KEYWORDS:

Allele burden; Calreticulin; Myeloproliferative neoplasms; Pyrosequencing; Somatic mutation

PMID:
29727699
DOI:
10.1016/j.cca.2018.05.001
[Indexed for MEDLINE]

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