High Resolution Melting Analysis for Rapid JAK2 Exon 14 and Exon12 Mutation Detection In Formalin Fixed Paraffin-Embeded Trephine Bone Marrow Biopsies of Patients with Polycythemia Vera. Detection of Novel JAK2 mutations
Authors: Burjanivova Tatiana , Nussenzveig Roberto H , Salama Mohamed E , Aboud Naser , Marcinek Juraj , Plank Lukas , Prchal Josef T. 
Published: October 2019

Abstract
Abstract 1984

Polycythemia vera (PV) belongs to the group of Ph1 negative myeloproliferative neoplasms and is characterized by the presence of JAK2V617F somatic mutation in >95% of PV patients. In some patients lacking this mutation, alternative mutations in exon 12 of the JAK2 gene have been reported. Identification of JAK2 mutations is a major WHO criterion for diagnosis of PV. Therefore, reliable and sensitive methods are needed for detection of these mutations. High resolution melting analysis (HRM) for mutation detection in JAK2 exons 12 and 14 have been previously described using DNA isolated from fresh patient samples. We report here the development of an HRM screening method for both JAK2 exons 12 and 14. We have applied these methods for retrospective screening of archived paraffin-embedded BM biopsies from PV patients.
A cohort of 101 PV patients was screened for JAK2 exons 12 and 14 mutations in DNA isolated from paraffin-embedded tissues. All included patients had bone marrow trephine biopsies exhibiting features consistent with or suspicious for PV. In addition, 6 samples with previously confirmed exon12 mutation by allele-specific PCR were tested by this HRM method. DNA was isolated from paraffin-embedded tissues after deparaffinization process in graded alcohols and xylene. Briefly, deparaffinized samples were digested with proteinase K for 3days and DNA was isolated using the DNeasy Micro Kit (Qiagen, Valencia, CA). Following DNA isolation, samples were analyzed by HRM for JAK2 exons 12 and 14. Short exon-specific PCR amplicons (90-100 bp) were generated, which allowed the detection of the mutation in degradated material. All abnormal samples were bi-directionally sequenced. All 6 known exon 12 mutated samples were identified by HRM. Moreover, we repeatedly identified an abnormal exon 12 melting curve in 1/101 paraffin embedded samples. Sequencing results for this sample indicate the presence of a large (~32 bp) duplication. All 7 positive samples were confirmed by direct sequencing (see table 1). Interestingly, all three delH538K539 insL samples identified used a distinct codon for L and also had reproducibly different melting profiles.
Ninety-two of 101 patients were positive for JAK2V617F mutation. One patient sample exhibited an abnormal HRM profile from wild-type and V617F positive control analysis. Two mutations were found in this patient by direct sequencing including JAK2V617F mutation and a previously unreported mutation in exon 14 (L611S) and we are now determining if these two separate JAK2 mutations are in cis or trans.
In summary, we developed an HRM assay suitable for detection JAK2 exons 12 and exon14 mutations in archival material. This method is suitable for routine laboratory detection of these mutations as well as screening of archived biological material for additional mutations. We report one case of JAK2V617F-positive PV presenting with an additional JAK2L611S.
This work has been supported by the MPD-RC consortium (J.P.) and a “UICC International Cancer Technology Transfer Fellowship“ awarded to Dr. Burjanivova. TB, RN and MES contributed equally to this work.

Disclosures:
No relevant conflicts of interest to declare.