Detection of CALR Mutations Using High Resolution Melting Curve Analysis (HRM-A); Application on a Large Cohort of Greek ET and MF Patients.

BACKGROUND AND OBJECTIVES: Somatic mutations in the calreticulin gene (CALR) are detected in approximately 70% of patients with essential thrombocythemia (ET) and primary or secondary myelofibrosis (MF), lacking the JAK2 and MPL mutations. To determine the prevalence of CALR frameshift mutations in a population of MPN patients of Greek origin, we developed a rapid low-budget PCR-based assay and screened samples from 5 tertiary Haematology units. This is a first of its kind report of the Greek patient population that also disclosed novel CALR mutants. METHODS: MPN patient samples were collected from different clinical units and screened for JAK2 and MPL mutations after informed consent was obtained. Negative samples were analyzed for the presence of CALR mutations. To this end, we developed a modified post Real Time PCR High-Resolution Melting Curve analysis (HRM-A) protocol. Samples were subsequently confirmed by Sanger sequencing. RESULTS: Using this protocol we screened 173 MPN, JAK2 and MPL mutation negative, patients of Greek origin, of whom 117 (67.63%) displayed a CALR exon nine mutation. More specifically, mutations were detected in 90 out of 130 (69.23%) essential thrombocythaemia cases (ET), in 18 out of 33 (54.55%) primary myelofibrosis patients (pMF) and in 9 out of 10 (90%) cases of myelofibrosis secondary to ET (post-ET sMF). False positive results were not detected. The limit of detection (LoD) of our protocol was 2%. Furthermore, our study revealed six rare novel mutations which are to be added in the COSMIC database. CONCLUSIONS: Overall, our method could rapidly and cost-effectively detect the mutation status in a representative cohort of Greek patients; the mutation make-up in our group was not different from what has been published for other national groups.

An adult patient with systemic mastocytosis and B-acute lymphoblastic leukemia.

Mastocytosis is a myeloproliferative neoplasm characterized by clonal expansion of abnormal mast cells, ranging from the cutaneous forms of the disease to mast cell leukemia. In a significant proportion of patients, systemic mastocytosis (SM) coexists with another hematologic malignancy, termed systemic mastocytosis with an associated hematologic nonmast cell lineage disorder (SM-AHNMD). Despite the pronounced predominance of concomitant myeloid neoplasms, the much more unusual coexistence of lymphoproliferative diseases has also been reported. Imatinib mesylate (IM) has a role in the treatment of SM in the absence of the KITD816V mutation. In the setting of SM-AHNMD, eradicating the nonmast cell malignant clone greatly affects prognosis. We report a case of an adult patient with SM associated with B-lineage acute lymphoblastic leukemia (B-ALL). Three cases of concurrent adult ALL and mastocytosis have been reported in the literature, one concerning SM and two concerning cutaneous mastocytosis (CM), as well as six cases of concomitant CM and ALL in children.