{'fraction_non_alphanumeric': 0.03489531405782652, 'fraction_numerical': 0.02567298105682951, 'mean_word_length': 5.280125195618154, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Comparison of real‐time PCR vs PCR with fragment length analysis for the detection of CALR mutations in suspected myeloproliferative neoplasms

Mutations in CALR, the gene which encodes calreticulin, are highly recurrent in essential thrombocytosis (ET) and primary myelofibrosis (PMF), being found in the majority of cases lacking the JAK2 V617F point mutation.1-4 More than 50 different CALR mutations have been described to date, with the two most common being a 52 base pair deletion (type 1 mutation) and a 5 base pair insertion (type 2 mutation). Additional mutations include variably sized deletions or insertions, which have been divided into type 1‐like, type 2‐like, or other mutations based on the precise mutated amino acid sequence.3‐5 Each of these various insertion and deletion mutations leads to a +1 frameshift and a common, novel C‐terminal amino acid sequence that lacks the KDEL endoplasmic reticulum localization signal and at least one stretch of negative amino acids that function in calcium binding in the normal protein. The diversity of CALR mutations presents methodological challenges for routine diagnostic laboratories. A variety of techniques have been employed including Sanger sequencing, PCR with fragment length analysis, melting curve analysis, and next‐generation sequencing studies.3,4,6-8 While sequencing methods offer the potential for the most comprehensive analysis, these studies are time‐consuming and not available at all institutions. There therefore remains a need in many laboratories for a rapid, inexpensive method to determine CALR mutation status. We have previously described a fragment length PCR assay (FL‐PCR) employed at our institution.9 Real‐time PCR assays, however, are also rapid, widely available, and offer the potential for increased specificity compared with fragment length analysis. In this report, we compare the results of the FL‐PCR assay with a commercially available real‐time PCR assay. 146 consecutive samples submitted to the Cleveland Clinic Molecular Diagnostics laboratory for CALR mutation analysis between March 2016 and February 2017 with sufficient residual DNA for analysis were retrospectively selected for inclusion in this study. CALR exon 9 was amplified by FL‐PCR as previously described in detail.9 Real‐time PCR was performed using the CALR RGQ PCR Kit (Qiagen, Germantown, MD) on the Rotor‐Gene Q 5Plex HRM platform (Qiagen), following the manufacturer's instructions. The CALR RGQ PCR Kit employs 7 separate reactions in a single run. Type 1 and type 2 CALR mutations are detected using allele‐specific amplification achieved by allele refractory mutation system (ARMS) technology. Less common mutations are detected via 5 reactions utilizing a series of 3′‐block (clamped) oligonucleotide probes to suppress amplification of wild‐type sequence. The presence of amplification in any of the 5 clamped reactions identifies the presence of a non‐type 1, non‐type 2 CALR mutation. Wild‐type and mutant CALR controls were analyzed in each run. Results of prior FL‐PCR were not available to individuals performing real‐time PCR during analysis. Results of 146 samples (144 peripheral blood, 2 bone marrow) are detailed in Table 1. Using FL‐PCR analysis, size altering CALR mutations were identified in 22 (15%) cases, including 14 type 1 mutations, 4 type 2 mutations, and 4 cases with atypically sized alterations. The 4 atypically sized deletions including one case with a 2 bp deletion, two cases with a 4 bp deletion, and one case with a 34 bp deletion. Using real‐time PCR, CALR mutations were identified in 20 (14%) cases, including 14/14 (100%) type 1 mutations, 4/4 (100%) type 2 mutations, and 2/4 (50%) cases with atypically sized alterations. All 124 cases negative by the FL‐PCR assay were also negative by the real‐time PCR assay. Using FL‐PCR as a gold standard, the real‐time PCR assay showed a sensitivity of 91%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 98%.