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

Area of Science:

• Molecular diagnostics
• Hematology
• Oncology

Background:

• Calreticulin (CALR) indels are key diagnostic markers for JAK2/MPL wild-type myeloproliferative neoplasms (MPNs).
• Standard CALR indels (type 1 and type 2) cause +1 frameshifts and KDEL motif loss.
• Non-standard CALR indels present interpretation challenges, particularly with fragment size analysis alone.

Purpose of the Study:

• To evaluate the concordance of rare CALR indel detection between PCR-CE and NGS assays.
• To assess the diagnostic utility of different molecular assays for non-standard CALR indels in MPN diagnosis.

Main Methods:

• Retrospective review of 6 years of CALR exon 9 testing.
• Analysis of patients with non-type 1/type 2 CALR indels.
• Comparison of fragment sizing by PCR with capillary electrophoresis (PCR-CE) and next-generation sequencing (NGS).

Main Results:

• 32% of detected CALR indels were non-standard.
• Discrepancies in indel size (±1 or -2 bp) were observed in 45% of cases compared between PCR-CE and NGS.
• These size discrepancies led to misclassification of frameshifts, with some in-frame indels by PCR-CE being frameshifts by NGS.

Conclusions:

• PCR-CE is a suitable screening method for CALR indels.
• Non-standard CALR indels require reflex NGS testing for accurate frameshift and clonal status determination.
• Standardizing molecular testing for CALR is essential for MPN diagnosis.