Diagnostic Role of Calreticulin Mutation in Patients with Ph/JAK2/MPL Negative Myeloproliferative Neoplasm Not Identified at Disease Presentation

BACKGROUND: Calreticulin (CALR) somatic mutation is present in the majority of patients with Ph-negative MPNs without either JAK2 or MPL mutation . Since the discovery of CALR mutation its molecular, pathogenic and clinical role has been thoroughly investigated. Based on current knowledge CALR mutation has recently been included in revised World Health Organization (WHO) diagnostic criteria for myeloproliferative neoplasms (MPN). In our study, we retrospectively evaluated diagnostic role of CALR mutation in patient cohort without CALR testing at the time of initial clinical suspicion for MPN. Some patients not having proper work-up by a hematologist could have been inadequately investigated and consequently misdiagnosed.

METHODS: Based on clinical or laboratory findings suspicious for MPN patients' blood samples were sent for molecular analysis by general practitioners or hematologists in community hospitals in the period from 2011 to 2016. Prior bone marrow examination was not performed and Ph chromosome, JAK2 V617F and MPL mutations were excluded. To retrospectively detect the presence of CALR mutation we used High Resolution Melting curve (HRM) method which detected two most common mutations, 52bp deletion and 5bp insertion. Sanger sequencing was used to detect other mutations in the CALR gene. The presence of all mutations was confirmed by gel electrophoresis. Newly identified patients had their medical files reviewed for clinical follow-up.

RESULTS: We analyzed blood samples from 332 patients. CALR mutation was detected in 21 (6.3%) patients. The most common laboratory finding in CALR positive patients was thrombocytosis which was present in 18 (85.7%) patients, one (4.8%) patient had polycythemia and two (9.5%) patients had no laboratory abnormalities. One (4.8%) patient suffered from portal vein thrombosis. Nineteen (90.5%) patients had a full diagnostic work-up and were diagnosed with Ph-negative MPN according to 2008 WHO diagnostic criteria. Only one (4.8%) patient did not reach the 2008 WHO diagnostic criteria for MPN and was misdiagnosed as MPN negative. One (4.8%) patient was diagnosed with splenic marginal cell lymphoma after performing bone marrow biopsy while two patients had two concomitant hematologic diseases (MPN and lymphoma) based on bone marrow examination. On follow-up, four (19.0%) patients developed thrombotic and hemorrhagic complications, one patient presented with a retinal hemorrhage, one patient with DVT, one with portal venous thrombosis while one patient presented with ischemic stroke. One (4.8%) patient diagnosed with essential thrombocytosis progressed to secondary myelofibrosis.

CONCLUSION: Retrospective CALR analysis on a large cohort of Ph/JAK2/MPL negative patients with clinical or laboratory suspicion of MPN identified a small number of patients with true MPN. Most were adequately diagnosed and treated based on other criteria, mainly bone marrow biopsy. In rare cases CALR mutation detection could add diagnostic value in patient management. In our patient group 9.2% of newly identified patients with CALR mutation could have benefited with routine CALR mutation analysis at the time of MPN suspicion, probably in the early stage of the disease or with concomitant hematologic disease obscuring MPN clinical picture.