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Clinical Targeted NGS Detection of Type 1 CALR Mutation in a case of Essential Thrombocythemia

Posted on July 19 2017


58-year-old female with thrombocytosis. Platelet counts have been elevated at least since January, 2014, and have reportedly been as high as 722k. Current CBC: WBC 6320, HGB 14.4 g/dL, hct 43.3 %, MCV 91.7 fL; plt 597 k; absolute neutrophils 3690, absolute lymphocytes 1990, absolute monocytes 400.



NGS confirmed the presence of a classical 52 base pair deletion in exon 9 (CALR type 1 mutation; CALR p.L367fs*46 / c.1092_1143del52) (image 2). This mutation involved 11.4% of total DNA sequence reads covering this position. NGS confirmed wild type status for the other potentially actionable loci targeted for analysis in JAK2 and MPL.


Genomic DNA was extracted from bone marrow aspirate and Next Generation Sequencing was performed on the Illumina MiSeqDx platform. Analysis was targeted to assess for pathogenic mutations involving the following loci: JAK2 (V617F and exon 12 mutations), CALR (type 1 and type 2 mutations), and MPL, p.W515. Read data was aligned to the human reference genome (build hg19) using MiSeq Reporter. Variants were identified using the somatic Variant Caller Pipeline, and variant calls were annotated and filtered in Illumina Variant Studio. Alignment settings were adjusted from the Illumina default workflow parameters and were optimized to allow for detection of large indels according to the recommendations by Gardner et al (Am J Clin Pathol. 2016 Oct;146(4):448-55).


Marrow biopsy showed normal overall cellularity for age and trilineage hematopoiesis that included increased numbers of megakaryocytes, often in abnormal compact clusters (image 1). Megakaryocytes showed often larger size and hyperlobulated nuclei, compatible with "staghorn" nuclear features (image 1). Marrow aspirate smear manual differential demonstrated a mildly increased myeloid/erythroid ratio and increased numbers of megakaryocytes with atypical cytologic features similar to those seen in the core biopsy.


Parallel flow cytometry performed on bone marrow aspirate was negative for increased myeloblasts, and no aberrant myelomonocytic antigen expression was detected.

CD34 and CD117 immunohistochemical stains were negative for myeloblast expansion (<5% of total cellularity).

Marrow karyotype demonstrated a NORMAL FEMALE KARYOTYPE, 46,XX[20].

Myeloproliferative neoplasm FISH panel evaluation was negative for BCR/ABL gene rearrangement or abnormalities involving chromosomes 8, 9, 13, 20, and 22.


CALR+ Myeloproliferative Neoplasm best classified as Essential Thrombocythemia


Pathogenic CALR mutations have been reported to be prognostically favorable in the setting of essential thrombocythemia (Rumi et al. Blood 2014 123:1544-1551).


BCR/ABL negative myeloproliferative neoplasms (MPNs) are a central class of diseases in routine clinical hematopathology practice and chiefly include Essential Thrombocythemia (ET), Primary myelofibrosis (PMF), and Polycythemia vera (PV). These disorders may show overlapping clinicopathologic features, however ET is most often manifested by elevated platelet counts, generally benign clinical course, sometimes adverse thrombotic events, and only very rarely, progression to acute leukemia. Pathogenic mutations in JAK2, CALR, or MPL are seen in ~90% of these cases overall, and are usually mutually exclusive of one another. Identification of pathogenic driver mutations involving these genes effectively excludes reactive, non-clonal etiologies (e.g. inflammatory states, infection, postsplenectomy states, etc.). In ET, JAK2, p.V617F mutations are seen in ~50-60% of cases. Indel type mutations involving CALR are the second most frequent mutation type seen in ~15-25% of cases, and mutations in MPL comprise ~5% of ET cases (image 3). CALR mutations are most often “type 1” 52 base pair deletions in exon 9 (CALR p.L367fs*46 / c.1092_1143del52), resulting in loss of normal CALR protein function and Calcium binding. The second most frequent mutation subtype is a “type 2” 5-base pair insertion, also involving exon 9 (CALR p.K385fs*47 / c.1154_1155insTTGTC). Compared to JAK2 V617F mutated ET, CALR mutated ET has been associated with relatively favorable clinical prognosis and reduced incidence of adverse vascular events or progression to acute leukemia (Rumi et al. Blood 2014 123:1544-1551). Mutation studies are typically performed on peripheral blood or marrow aspirate. Based on the relative frequencies of recurrent mutations in BCR/ABL negative MPNs, many laboratories employ reflexive cascaded testing algorithms for evaluation of these neoplasms, however this approach presents some practical challenges. Traditional reflexive algorithms are time intensive, and may results in lengthy overall turnaround times for clinical reporting and final diagnostic subclassification. Traditional cascaded approaches may also involve utilization of disparate testing methodologies, each associated with varying technical performance characteristics and diagnostic sensitivities. Our laboratory performs an NGS core mutation panel composed of JAK2 (V617F and Exon 12), CALR, and MPL, in order to simultaneously assess for pathogenic mutations involving these key driver genes in clinically suspected cases of BCR/ABL negative MPNs. Clinical targeted NGS panels confer significant practical benefits in this setting. NGS panels are able to simultaneously assess for numerous disease relevant mutations of varying type in a single analysis. Overall turnaround times for clinical reporting are reduced, and this approach avoids cascaded testing algorithms often found to be complex and cumbersome by ordering personnel. Because NGS laboratory workflow is streamlined into a single assay, training and proficiency testing requirements are also consolidated and simplified.