Coexisting JAK2V617F and CALR Exon 9 Mutations in Myeloproliferative Neoplasms-Do They Designate a New Subtype?

Myeloproliferative neoplasms (MPNs) are a heterogeneous group of chronic myeloid neoplasms with the potential to progress to acute leukemia. All MPNs are clonal disorders with an initial hit in the HSCs resulting in an excessive production of blood cells because of hypersensitivity or independence from normal cytokine regulation. According to WHO classification system 2008, MPNs comes under eight clinicopathological entities: chronic myelogenous leukemia (CML), polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF), chronic neutrophilic leukemia, chronic eosinophilic leukemia-not otherwise specified, mastocytosis and MPN-unclassifiable (Vardiman et al., 2009). MPNs involve the three main myeloid lineages but predominate in one of them: the erythroid lineage for PV, the megakaryocyte (MK)/platelet lineage for ET, and the MK/granulocytic lineages for PMF (Vainchenker et al., 2011). Majority of clinical features of MPNs is shared to a


Introduction
Myeloproliferative neoplasms (MPNs) are a heterogeneous group of chronic myeloid neoplasms with the potential to progress to acute leukemia. All MPNs are clonal disorders with an initial hit in the HSCs resulting in an excessive production of blood cells because of hypersensitivity or independence from normal cytokine regulation.

MINI-REVIEW
Coexisting JAK2V617F and CALR Exon 9 Mutations in Myeloproliferative Neoplasms -Do They Designate a New Subtype?
Rifat Zubair Ahmed 1 *, Munazza Rashid 1 , Nuzhat Ahmed 1 , Muhammad Nadeem 2 , Tahir Sultan Shamsi 1,2 variable degree among the three MPN entities (Kralovics, 2008). Routine diagnostic tests for MPNs include red cell mass determination, bone marrow aspirate and trephine biopsy, arterial oxygen saturation and carboxyhaemoglobin level, neutrophil alkaline phosphatase level, vitamin B12 and serum urate. Although these tests strongly support the diagnosis of MPNs, the disease could still not be well discriminated from the reactive hyperplasia. Moreover, misdiagnosis could occur when the symptoms of disease are atypical (Wu et al., 2014).
In addition to morphologic evaluation, the genetic abnormality is a major diagnostic criterion for MPNs and considerable attention has been devoted in the past to explaining the genetic basis of clonal hematopoiesis in MPNs. Discovery of the Philadelphia chromosome in 1960 and the subsequent identification of the BCR-ABL fusion protein in CML was the landmark (Kralovics, 2008). The year 2005 was the time when four seminal studies published defined the molecular pathogenesis of classic"BCR-ABL1-negative" chronic MPNs PV, ET and PMF by reporting the existence of a recurrent V617F point mutation in exon 14 of Janus kinase 2 (JAK2) gene in these three disorders . The JAK2V617F mutation is found in the vast majority (~95%) of PV patients and 50-60% of those with ET or PMF (ET~55%, PMF~65%) (Klampfl et al., 2013;Nangalia et al., 2013;Guglielmelli et al., 2014;Lin et al., 2015). In most of the remaining JAK2V617F-negative PV cases, JAK2 exon 12 mutations were first described in 2007. These JAK2 exon 12 mutations, such as insertions or deletions, are relatively specific to JAK2V617F-negative PV and not found in ET and PMF. Amongst the JAK2negative ET or PMF patients 5-10% of patients carry mutations in exon 10 at codon 515 of the gene MPL encoding the thrombopoietin receptor. Both JAK2 and MPL are gain of function mutations and promote the over activation of STAT signaling largely mediated by abnormal and sustained phosphorylation of JAK2 (Tefferi et al., 2010;Vainchenker et al., 2011;Klampfl et al., 2013;Nangalia et al., 2013;Guglielmelli et al., 2014;Lin et al., 2015). The close association of JAK2V617F mutation with MPNs led to a revision of the WHO diagnostic criteria in 2008, where JAK2V617F, JAK2 exon 12 and MPL515 mutations were elected to represent new major diagnostic criteria for the three classic MPN . Prior to 2013, there was no specific molecular marker described in the remaining 30-40% of ET and PMF patients that has now largely been resolved by the discovery of mutations in the gene CALReticulin (CALR) (Klampfl et al., 2013;Nangalia et al., 2013;Guglielmelli et al., 2014;Lin et al., 2015). In December 2013 two research groups independently reported mutations in the gene (CALR found specifically in ET (67-71%) and PMF (56-88%) but not in PV (Klampfl et al., 2013;Nangalia et al., 2013). Many studies have reported important diagnostic and prognostic significance of CALR mutations in ET and PMF patients and CALR mutation screening has been proposed to be incorporated into WHO diagnostic criteria of MPNs (Tefferi et al., 2014c).
It was found initially that CALR mutations were mutually exclusive with JAK2 or MPL (Klampfl et al., 2013;Nangalia et al., 2013). However, co-occurrence of both the mutations has been reported recently in few MPN cases (Table 1)  In an ET patient it was first reported by Lundberg et al., in 2014 whereas in PV it was also reported first in 2014 by Xu et al., Though the frequency of this co-occurrence was usually below 1%, Lim et al., (2015) reported it with a higher 6.8% frequency. Majority applied Sanger sequencing to detect CALR exon 9 mutations. Lim et al., (2015) applied a sensitive HRMA method to detect CALR mutations that enabled them to detect even low allele burden mutants which might be the reason of detecting higher frequency. According to Zmora et al., (2015) the low percentage of JAK2V617F and CALR positive patients could be underestimated as the majority of groups only test CALR mutations in JAK2 V617F negative patients.
The phenotypic manifestations of CALR and JAK2V617F differ in ET and PMF. It has been observed in many studies that patients with CALR mutation often had a low risk of thrombosis, with lower hemoglobin and leukocyte counts, higher platelet count and longer survival in comparison to those who had JAK2V617F mutation (Klampfl et al., 2013;Nangalia et al., 2013;Fu et al., 2014;Rotunno et al., 2014;Tafferi and Pardanani, 2014;Tefferi et al., 2014a;2014b;2014c). Lundberg et al. (2014) analyzed a cohort of MPN patients to detect somatic mutations by targeted NGS of 104 genes and found that patients with two or more somatic mutations formed a high-risk category, with increased risk of transformation into AML and reduced survival. Though CALR and JAK2V617F co-existence has been reported in few MPN cases across different ethnic groups but unfortunately there are not many reports on the phenotype and clinical course of those patients. Patient reported in the study of McGaffin et al., (2014) was a 79 year old female who was well and had no history of bleeding, thrombosis, cerebrovascular disease or any other occlusive symptoms. Xu et al., (2015) provided the first data in detail regarding the phenotype and clinical outcomes in CALR and JAK2V617F positive patients and they have more patients with both mutations under observation. Their ET patient was a 63 year old female who was well and without history of bleeding, thrombosis, cerebrovascular disease, or any other occlusive symptoms and was in complete hematologic remission at the time of publication whereas the other one was a 65 year old male PV patient who was still in complete remission without cytoreductive therapy. Both patients also reported a positive response to interferon alfa therapy. In the study of Zmora et al., (2015) the double positive patients was 86 year old male PMF patient who died of acute myocardial infarction. Lim et al., (2015) reported the high frequency (6.8%) until now of patients with both mutations. They studied 92 ET patients and found 13 patients with both mutations. CALR and JAK2-mutated ET patients in their study were found to be associated with oldest age, higher thrombotic events and higher major arterial thrombotic events after diagnosis and more patients were in the high-risk group for thrombohemorrhagic complications. Rashid et al., (2015) reported 55 year old female ET patient who was in complete remission without cytoreductive therapy at the time of publication.
It is suggestive in diagnostic workup of MPN that CALR mutations should not be studied in those MPN patients who are already known to carry JAK2 or MPL mutations (Tafferi and Pardanani, 2014). Either it is the difference of methods to detect CALR mutations in JAK2V617F positive samples or a different mutation spectrum in Caucasian and Asian population or the fact that majority of groups follow the suggestive diagnostic workup for MPN but these double positive patients might represent a new subtype in MPNs. As suggested by Lim et al. (2015) JAK2V617F and CALR positive patients make a specific sub group of patients and require a careful followup and management. McGaffin et al. (2014) and Zmora et al. (2015) proposed that double-mutant patients might have a different phenotype and clinical course, distinct from the JAK2-positive or CALR-positive subgroups and identification of the true frequency of these patients may be an important factor for defining the prognosis, risk factors and outcomes for MPN patients when mutation status is the criterion to allocate subgroups. In current scenario when inclusion of CALR is expected in revision of the WHO diagnostic criteria it is still to decide what should be the workup for MPN with respect to CALR and JAK2V617F.

Conclusion
Many studies have suggested that CALR mutations have important diagnostic and prognostic significance in ET and PMF patients and should be incorporated into WHO diagnostic criteria of MPN. Identification of the true frequency of patients with JAK2V617F and CALR mutation and findings of their clinical course and phenotype is necessary to define the prognosis, risk factor, and outcomes for those MPN patients carrying both mutations.