In Frame Calr Exon 9 Mutations: Often Ignored but Potentially Significant

Philadelphia chromosome negative myeloproliferative neoplasms (MPNs) are characterized by the overproduction of mature blood cells and variable bone marrow fibrosis. MPNs attributed to dysregulation of the Janus kinase 2 (JAK2) pathway include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Somatic mutations in JAK2, thrombopoietin receptor (MPL), and calreticulin (CALR) have been identified as driver mutations with direct or upstream upregulation of JAK2. CALR mutations are the most recently described, with the two most common mutations being a 52-base pair (bp) deletion (type 1) or 5 bp insertion (type2) in exon 9. Studies have shown a prognostic advantage to type1/type 1 like CALR driven MPNs over JAK2, MPL, and type2 CALR driven MPNs. Rarer CALR exon 9 mutations have also been identified in presumed MPN patients negative for JAK2 and MPL mutations. In these cases variable predicted changes to the CALR protein have resulted in speculative interpretations as to their relevance in the diagnosis of a suspected MPN.

Here we report a patient with a longstanding history of myelofibrosis, thrombocytosis, and anemia, eventually determined to have an in-frame presumed germline (due to variant fraction and identification in the patient's child) CALR mutation downstream of a somatic type 1 CALR mutation. The overall compounded alterations generate a type 1 like mutation previously not described (to the best of our knowledge) in the literature. The patient is a 70-year old female noted to be persistently anemic all her life. While a bone marrow assessment was recommended early in life, the patient declined workup until a marrow biopsy was eventually performed at age 50. The biopsy reportedly showed mild marrow fibrosis and the patient was trailed on erythropoietin for her anemia with little benefit. At age 59 the patient was noted with thrombocytosis (478 X 10⁹/L) and mild splenomegaly. Repeat marrow biopsy showed hypercellular marrow, marked fibrosis (WHO grade 3/3), and megakaryocyte clustering. JAK2 was noted to not be mutated. Over the next decade the patient developed symptomatic splenomegly and continued to be anemic with eventual intermittent transfusion requirement at age 65, pushing her risk to DIPPS-plus intermediate-2. During this period successive treatments included Revlimid, trial sonic hedgehog (shh) inhibitor, and JAK2 inhibitors with intervening multiyear long spans without treatment. Marrow fibrosis over this time was predominantly unchanged on the various therapies but symptomatic splenomegaly decreased on shh and JAK2 inhibitors. At present the patient requires intermittent transfusions and is on a JAK2 inhibitor.

Recent NGS testing of marrow aspirate identified an in-frame presumed germline CALR mutation downstream of a compound somatic type 1 CALR mutation. A high molecular risk ASXL1 mutation was also identified. The in-frame CALR mutation results in a 9bp in frame deletion (c.1191_1199del, p.398_400delGluGluAsp), which has been reported at least 10 times in the literature. The various reports have one event mentioned as being presumably germline and non-pathogenic, while the other reports are equivocal to presumed pathogenic in light of negative JAK2 and MPL mutations in patients with clinical suspicion for a MPN. At our institution we have identified 4 instances of this 9bp deletion, 3 show allele fractions suggestive of being germline and 1 case with an allele fraction consistent with being a somatic mutation. In one germline case the patient also had a JAK2 V617F mutation and a diagnosis of a MPN. The other presumed germline case was found in an offspring of the patient described in this report and currently shows no signs of a MPN. The presumed somatic 9bp deletion was seen in patient with a hypocellular marrow myelodysplastic syndrome, found to also have a TET2 mutation and normal karyotype.

While in-frame CALR exon 9 mutations are rare and predominantly considered germline non-pathogenic polymorphisms, there may be value in reporting such events in the context of patients with myeloid neoplasms as to not miss possible disease modifying mutations which may become apparent when aggregating multi-institution data sets. The patient highlighted in this report exhibits a strikingly long and relatively indolent disease course, notably despite an adverse prognostic risk category and high molecular risk mutation.