Detection of CALR Mutation in Clonal and Nonclonal Hematologic Diseases Using Fragment Analysis and Next-Generation Sequencing.
OBJECTIVE
To describe three methods used to screen for frameshift mutations in exon 9 of the CALR gene.


METHODS
Genomic DNA from 47 patients was extracted from peripheral blood and bone marrow using the EZ1 DNA Blood Kit (Qiagen, Valencia, CA) and quantified by the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, San Diego, CA). After clinical history, cytogenetics, and molecular tests, patients were diagnosed with either clonal or nonclonal hematologic diseases. CALR screening was primarily performed using fragment analysis polymerase chain reaction, then next-generation sequencing and Sanger sequencing.


RESULTS
Among the 18 patients diagnosed with clonal diseases, one had acute myeloid leukemia (positive for trisomy 8), and 17 had myeloproliferative neoplasms (MPNs), including chronic myeloid leukemia (CML), essential thrombocythemia (ET), primary myelofibrosis (PMF), and polycythemia vera (PV). Patients with CML were positive for the BCR-ABL1 fusion. Ten patients were positive for JAK2 (PMF, n = 1; ET, n = 2; PV, n = 7), and three were CALR positive (ET, n = 1; PMF, n = 2). Patients diagnosed with a nonclonal disease were negative for JAK2, BCR-ABL, and CALR mutations.


CONCLUSIONS
Screening for CALR mutations is essential in BCR-ABL-negative MPNs since it not only provides valuable diagnostic and prognostic information but also identifies potential treatment targets. Since this study describes the importance of screening for known and novel biomarkers, we described in detail three methods that could be easily integrated into a clinical laboratory.
From the Department of Pathology and Laboratory Medicine, Geisel School of Medicine at Dartmouth, Hanover, NH, and Dartmouth Hitchcock Medical Center and Norris Cotton Cancer Center, Lebanon, NH.
Juli-Anne
JA
Gardner
From the Department of Pathology and Laboratory Medicine, Geisel School of Medicine at Dartmouth, Hanover, NH, and Dartmouth Hitchcock Medical Center and Norris Cotton Cancer Center, Lebanon, NH.
Jason D
JD
Peterson
From the Department of Pathology and Laboratory Medicine, Geisel School of Medicine at Dartmouth, Hanover, NH, and Dartmouth Hitchcock Medical Center and Norris Cotton Cancer Center, Lebanon, NH.
Scott A
SA
Turner
Universidade Federal De Sao Joao Del Rei, Divinopolis, Minas Gerais, Brazil.
Barbara L
BL
Soares
From the Department of Pathology and Laboratory Medicine, Geisel School of Medicine at Dartmouth, Hanover, NH, and Dartmouth Hitchcock Medical Center and Norris Cotton Cancer Center, Lebanon, NH.
Courtney R
CR
Lancor
Universidade Federal De Sao Joao Del Rei, Divinopolis, Minas Gerais, Brazil.
Luciana L
LL
Dos Santos
From the Department of Pathology and Laboratory Medicine, Geisel School of Medicine at Dartmouth, Hanover, NH, and Dartmouth Hitchcock Medical Center and Norris Cotton Cancer Center, Lebanon, NH.
Prabhjot
P
Kaur
From the Department of Pathology and Laboratory Medicine, Geisel School of Medicine at Dartmouth, Hanover, NH, and Dartmouth Hitchcock Medical Center and Norris Cotton Cancer Center, Lebanon, NH.
Deborah L
DL
Ornstein
From the Department of Pathology and Laboratory Medicine, Geisel School of Medicine at Dartmouth, Hanover, NH, and Dartmouth Hitchcock Medical Center and Norris Cotton Cancer Center, Lebanon, NH, gregory.j.tsongalis@hitchcock.org.
Gregory J
GJ
Tsongalis
From the Department of Pathology and Laboratory Medicine, Geisel School of Medicine at Dartmouth, Hanover, NH, and Dartmouth Hitchcock Medical Center and Norris Cotton Cancer Center, Lebanon, NH.
Francine B
FB
de Abreu
D037282:Calreticulin / Q000235:genetics*; D004252:DNA Mutational Analysis; D016044:Fusion Proteins, bcr-abl / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D053614:Janus Kinase 2 / Q000235:genetics; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000175:diagnosis* / Q000235:genetics; D015470:Leukemia, Myeloid, Acute / Q000175:diagnosis* / Q000235:genetics; D009154:Mutation; D011087:Polycythemia Vera / Q000175:diagnosis* / Q000235:genetics; D055728:Primary Myelofibrosis / Q000175:diagnosis* / Q000235:genetics; D013920:Thrombocythemia, Essential / Q000175:diagnosis* / Q000235:genetics
D037282:Calreticulin; D016044:Fusion Proteins, bcr-abl; D053614:Janus Kinase 2
CALR gene; Clonal diseases; Fragment analysis; MPN; Next-generation sequencing; Routine clinical laboratory; Sanger sequencing; Somatic mutation