Introduction

Myeloproliferative neoplasms are chronic myeloid cancers divided in Philadelphia positive (Ph+) or negative causing in this case: polycythemia vera (PV) essential thrombocythemia (ET), and primary myelofibrosis (PMF). Most Ph- cases have an activating JAK2 or MPL mutation. Recently, somatic mutations in the calreticulin gene (CALR) have been detected in 56–88% of JAK2/MPL-negative patients affected by ET or PMF.

Technical features

We developed a novel detection assay in order to identify the two most common mutations: type-1 and 2 CALR mutations by PNA directed PCR clamping. The assay resulted to be more sensitive, specific and cheaper than Sanger sequencing and it could be applied even in laboratories lacking a basic Sanger sequencing service, a scenario quite common in small laboratories especially those located in developing countries. The method displays a quite high sensitivity, allowing to detect an amount of mutated template as low as 10% for CALR type-1 and 0,1% for CALR type-2 mutations, which cannot be identified by Sanger sequencing. Our diagnostic test shows a sensitivity of 100% (CI 79.41% to 100.00%) and a specificity of 98,5% (CI 91.96% to 99.96%) with an AUC corresponding to 0.99.

Possible Applications

• Diagnostic tool for Philadelphia chromosome negative myeloproliferative neoplasms.

Advantages

• Discriminates 3 mutated forms of the CALR gene from its wild type form;
• Faster and cheaper;
• High sensitivity;
• High specificity.