Concordance Between Fragment Analysis and Next-Generation Sequencing in Identification of Non-Standard CALR Indels

Identification of CALR indels is key in the diagnosis of myeloproliferative neoplasms (MPNs) that are JAK2 or MPL wild-type. Most patients harbor either type 1 (52-bp deletion) or type 2 (5-bp insertion) exon 9 indels, both of which produce a +1 frameshift and loss of the KDEL ER-retention motif. Additional indels have been characterized. However, many still present diagnostic challenges in interpretation, especially when relying on fragment size alone. Our goal was to assess the concordance of detection of rare CALR indels using two available molecular diagnostic assays in the diagnosis of MPN.

We reviewed 6 years of CALR exon 9 testing at our institution, focusing on patients with non-type 1/type 2 indels (non-standard). Fragment sizing was performed by PCR with capillary electrophoresis (PCR-CE), with a subset reflexed to next-generation sequencing (NGS) for clarification. We assessed reading frame shifts, KDEL motif status, variant allele frequencies (VAFs), and co-occurring mutations where available.

Over a 6-year period, a total of 3474 CALR tests were performed, with indels being detected in 125 patients. Forty patients with exon 9 CALR indels other than 52 bp-del and 5 bp-ins were identified (40/125; 32%). Among these, the most frequently detected indel was the 9-bp deletion (19/125; 15.2%). Parallel NGS reports were available in 11 patients. From these, five showed a discrepant indel size (5/11 = 45%) between PCR-CE and NGS by ±1 or -2-bp. These discrepancies resulted in novel frameshifts that were originally categorized as in-frame indels by PCR-CE.

PCR-CE should be used as a screening method, and indels other than 52-bp del or 5-bp ins should prompt reflex NGS testing to determine frame and clonal status. These findings establish an opportunity to standardize molecular testing for CALR testing in patients with suspected MPN.