CAL2 Immunohistochemical Staining Accurately IdentifiesCALRMutations in Myeloproliferative Neoplasms

Abstract

Objectives: Mutations in CALR (calreticulin) have been discovered in 50% to 80% of JAK2 (Janus kinase 2) and MPL (myeloproliferative leukemia protein) wild-type patients with Philadelphia-negative myeloproliferative neoplasm (MPNs). We evaluate the performance of a monoclonal antibody for immunohistochemical detection of CALR mutations.Methods: A computerized archival search was performed for cases of non-chronic myeloid leukemia (CML) MPNs with available CALR and JAK2 V617F mutational analysis data. Bone marrow biopsy specimens were stained with monoclonal antibody CAL2, and the percentage of stained megakaryocytes was calculated. In select cases, double immunofluorescence staining was done with CAL2 and each of the following: CD61, myeloperoxidase, CD34, and glycophorin A.

Histological evaluation of myeloproliferative neoplasms

“…Using the molecular biology assay with Sanger sequencing, 100% specificity was confirmed. 19 Considering that several similar reports have been published, 20,21 immunostaining by CAL2 monoclonal antibodies is a promising and simple method to screen for mutations. At our institution, when BCR-ABL1-negative MPNs are suspected, in addition to trilineage hematopoietic cell markers (erythroid lineage: CD71; granulocytic lineage: myeloperoxidase, megakaryocytic lineage: CD42b; and hematopoietic stem cells: CD34), immunostaining by CAL2 monoclonal antibodies is performed ( Fig.…”

Megakaryocytes, erythropoietic and granulopoietic cells express CAL2 antibody in myeloproliferative neoplasms carrying CALR gene mutations

“…However, the lineage of similar cells could not 1 4 be clarified in the first study assessing the monoclonal CAL2 antibody [3] .The results from 1 5 subsequent publications were diverse. In one 2016 study, the nine positive cases show staining in 1 6 the majority of megakaryocytes with little or no staining in any other cell types [5] .On the other 1 7 hand, Nomani et al in the same year noted staining of small mononuclear in CALR mutant cases 1 8 [6] . By performing double immunofluorescence staining, they proposed that the small cells 1 9 appeared to be myeloid cells or blasts.…”

“…Additionally, we investigated by double staining whether expression of 1 mutated CALR can also be demonstrated on cells of the erythroid and myeloid lineage. 2 3 Material and Methods 4 Tissue Samples 5 One hundred and eighty two bone marrow biopsies from patients with myeloid neoplasms: series 6 of MPNs (n=66) and a control group of acute myeloid leukaemia (AML) (n=116) were retrieved 7 from the files of the Department of Histopathology at University College London Hospitals. The London Hospitals following the criteria of the 2008 WHO classification [1] .…”

“…There utility of the CAL2 antibody [3] and are comparable to four following studies. [5][6][7][8] 1 0 1 1…”

Clonal independence of <i>JAK2</i> and <i>CALR</i> or <i>MPL</i> mutations in comutated myeloproliferative neoplasms demonstrated by single cell DNA sequencing

“…Val617Phe, CALR exon 9 frameshift mutations and MPL exon 10 mutations collectively are detectable in 83% of ET and 92% of PMF 2 . Mutations in these genes are typically mutually exclusive, however, JAK2 Val617Phe/JAK2 exon 12 3,4 , JAK2/CALR 5,6 , JAK2/MPL 4,6,7 and very rarely CALR/MPL 8 co-mutations have been reported. Further, the presence of multiple independent JAK2 Val617Phe mutations has been demonstrated in the majority of ET patients in one case series, and is one of several lines of evidence that suggest a predisposition in some patients to the development of JAK2 (and other MPN driver) mutations, the nature of which has not been elucidated but may include, genetic predisposition, a preceding clonal mutation or a permissive microenvironment 9 .…”