CAL2 Immunohistochemical Staining Accurately Identifies CALR Mutations in Myeloproliferative Neoplasms

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1 AJCP /ORIGINAL ARTICLE CAL2 Immunohistochemical Staining Accurately Identifies CALR Mutations in Myeloproliferative Neoplasms Laila Nomani, MD, 1 Juraj Bodo, PhD, 1 Xiaoxian Zhao, PhD, 1 Lisa Durkin, 1 Sanam Loghavi, MD, 2 and Eric D. Hsi, MD 1 From the 1 Department of Laboratory Medicine, Cleveland Clinic, Cleveland, OH, and 2 Department of Hematopathology, MD Anderson Cancer Center, Houston, TX. Key Words: Myeloproliferative neoplasm; Immunohistochemistry; CAL2 antibody; CALR; Essential thrombocythemia; Primary myelofibrosis Am J Clin Pathol October 2016;146: DOI: /AJCP/AQW135 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. Results: We studied 38 bone marrow biopsy specimens of non- CML MPNs (primary myelofibrosis, n ¼ 21; essential thrombocythemia, n ¼ 15; and n ¼ 2 post polycythemia vera myelofibrosis) from 31 patients. All eight bone marrow biopsy specimens from patients with mutant CALR showed strong cytoplasmic staining of the megakaryocytes (83.5%; range, 50%-98%; median, 87%) with the CAL2 antibody. Double immunofluorescence staining of the small mononuclear cells seen in CALR mutant cases revealed them to be myeloid blasts. Conclusions: Immunohistochemistry in routinely processed bone marrow biopsy specimens for mutated CALR is feasible and accurately identifies mutated cases, including rare cases with additional driver mutations. Mutations in one of three genes Janus kinase 2 (JAK 2), myeloproliferative leukemia protein (MPL)andcalreticulin (CALR) can be found in most patients with Philadelphia-negative myeloproliferative neoplasms (MPNs) and represent driver mutations central to the pathogenesis of these diseases. 1 CALR mutations were discovered in 50% to 80% of patients with JAK2 and MPL wild-type essential thrombocythemia (ET) and primary myelofibrosis (PMF), representing 20% to 35% of all patients with ET and PMF. This discovery was a landmark finding in understanding the pathogenesis of JAK2- andmpl-negative MPNs. 2,3 Initially reported as mutually exclusive with JAK2 and MPL, there have been rare reports of CALR with either a JAK2 or a BCR- ABL1 comutation. 4-6 Calreticulin (CALR), a highly conserved protein present in the endoplasmic reticulum, is responsible for the folding of newly synthesized glycoproteins and calcium homeostasis Mutations in CALR, all located in exon 9, may be either insertions or deletions that cause a frameshift. This leads to a unique alternative reading frame, which codes for a novel protein C-terminus consisting of approximately 36 amino acids. 2,3 The mutant CALR protein lacks the C-terminal endoplasmic reticulum retention signal (KDEL) and most likely has impaired Ca2þ binding function. They ultimately lead to hyperactivity of the JAK/STAT signaling pathway in megakaryocytic and granulocytic progenitor and precursor cells. 11 Recently, two groups worked on developing antibodies for immunohistochemical staining of bone marrow biopsy specimens to detect mutant CALR. 12,13 Currently, molecular methods are the standard for detection of these mutations. An immunohistochemical stain that consistently American Society for Clinical Pathology, All rights reserved. Downloaded For from permissions, please journals.permissions@oup.com on 04 February Am J Clin Pathol 2016;146: DOI: /ajcp/aqw135

2 Nomani et al /IMMUNOHISTOCHEMISTRY OF CALR MUTATIONS detects the mutation in formalin-fixed bone marrow biopsy specimens would be a rapid and inexpensive way to identify patients with these mutations in routine practice. We analyzed the performance of a recently published and commercially available mutant CALR specific monoclonal antibody (CAL2) suitable for immunohistochemistry in formalin-fixed, decalcified bone marrow biopsy specimens. 13 Using double immunofluorescence, we also further characterize immunoreactivity of CAL2 in bone marrow cells. Materials and Methods Case Selection A computerized search was performed for bone marrows of non chronic myeloid leukemia (CML) MPNs with known CALR and JAK2 V617F mutational status. For the known CALR-mutated patients, we also gathered all available bone marrow samples if serial biopsy specimens existed. Thirtyeight biopsy specimens were found from 31 patients and were re-reviewed to confirm the diagnosis on the basis of the World Health Organization (WHO) classification criteria. Three additional patients with comutations of CALR and JAK2 V617 (patient A) and CALR and BCR-ABL1 (patients B and C) were also included in our study. Patient C was previously reported. 11 The control group consisted of 35 cases of chronic myelomonocytic leukemia (CMML); 10 cases of benign bone marrow submitted for lymphoma staging, cytopenias, and other nonneoplastic processes; and 10 cases of chronic phase CML as well as a tissue microarray of 90 normal tissues from various organ sites (five different samples each from normal brain, breast, colon, heart, liver, kidney, lung, endometrium, skin, prostate, testis, ovary, stomach, spleen, small bowel, bladder, tonsil, and pancreas). Processing of Bone Marrow Biopsy Specimens The bone marrow biopsy specimens were received in zinc formalin (Poly Scientific, Bayshore, NY) and allowed to fix for 2 hours. The biopsy tissues were then placed in individual cassettes and washed with deionized water for 5 minutes. The cassettes were then placed in decalcifying solution (Thermo Scientific Richard-Allan Scientific, Kalamazoo, MI) for 30 minutes each and then washed with deionized water for another 30 minutes. After completion of these steps, the specimens were submitted for further processing and embedding. Immunohistochemistry: Mutant CALR and Wild-Type CALR Briefly, 4-lm unstained sections of each biopsy specimens (38 non-cml MPNs, five comutated MPNs, 10 normal bone marrows, 10 CMLs, and 35 CMMLs) and five clot sections each from CALR wild-type MPNs and CALRmutated MPNs were cut onto electrostatically charged glass slides. Immunohistochemistry was performed on an automated immunostainer (Leica Bond Max; Leica Biosystems, Buffalo Grove, IL). CALR antibody (clone CAL2; HistoBioTec, Miami Beach, FL) staining used a 1:25 dilution for 1 hour at room temperature, antigen retrieval with a high ph EDTA-based buffer (Epitope Retrieval 2; Leica Biosystems), and a 15-minute Background SNIPER block (Biocare, Concord, CA). Visualization was performed using Bond Polymer Refine Detection (Leica Biosystems). Five bone marrow biopsy specimens each of CALRmutated MPNs and CALR wild-type MPNs, as well as five normal bone marrow biopsy specimens, were stained with wild-type CALR antibody. CALR antibody (clone FMC 75; Enzo, Farmingdale, NY) staining used a 1:3,000 dilution for 15 minutes at room temperature, antigen retrieval with a low ph EDTA-based buffer (Epitope Retrieval 1; Leica Biosystems), and a 15-minute Background SNIPER block (Biocare). Visualization was performed using Bond Polymer Refine Detection (Leica Biosystems). Any cytoplasmic staining of the cells with CAL2 antibody was considered positive immunostaining. The number of CAL2-immunoreactive megakaryocytes over the total number of morphologically recognizable megakaryocytes was calculated by counting at least 100 megakaryocytes/slide and expressed as a percentage of total megakaryocytes. Immunofluorescence Double Staining: Myeloperoxidase, CD61, CD34, Glycophorin A, and CAL2 Deparaffinization and heat-induced epitope retrieval of bone marrow biopsy samples were performed using the Bond Max instrument (Leica Biosystems). Avidin and biotin were blocked with block A or block B reagents (ThermoFisher, Waltham, MA), respectively. Before adding antibody against mutated calreticulin (CAL2, 1:25; HistoBioTec) for 1 hour, samples were incubated in 10% normal horse serum (Vector, Burlingame, CA) and 5% bovine serum albumin/phosphatebuffered saline (Sigma-Aldrich, St Louis, MO). CAL2 was visualized with biotinylated anti mouse antibody (Vector) followed by streptavidin-conjugated QD655 (ThermoFisher). Subsequent staining with CD61 (Ready to use (RTU); Leica Biosystems), CD34 (1:50; CellMarque, Rocklin, CA), myeloperoxidase (MPO) (1:500; CellMarque), or glycophorin A (GYPA) (Abcam, Cambridge, MA) was preceded by avidin and biotin blocking and incubation with anti mouse IgG2a unconjugated antibody (blocking an excess of CAL2 antibody). Second primary antibodies were visualized with biotinylated anti mouse antibody followed by streptavidinconjugated QD565 (ThermoFisher). As a nuclear 432 Am J Clin Pathol 2016;146: American Society for Clinical Pathology 432 DOI: /ajcp/aqw135 Downloaded from

3 AJCP /ORIGINAL ARTICLE Table 1 Non-CML MPN Cases With JAK2 and CALR Mutational Status and CAL2 IHC Diagnosis Patient No. Sample No. CAL2 IHC % Megakaryocyte Staining CALR Mutation JAK2 V617F Biopsy PMF 1 a N 0 NA N Diagnostic b P 94 Type 2 N 312 mo 2 a P 50 NA N Diagnostic b P 80 NA N 105 mo c P 90 Type 1 N 120 mo 3 P 96 Type 1 N Diagnostic 4 N 0 N P Diagnostic 5 N 0 N N Diagnostic 6 N 0 N N Diagnostic 7 N 0 N P Diagnostic 8 N 0 N P Diagnostic 9 N 0 N N Diagnostic 10 N 0 N N 5 mo 11 N 0 N P 12 mo 12 N 0 N N Diagnostic 13 N 0 N P Diagnostic 14 N 0 N P 24 mo 15 N 0 N N 36 mo 16 N 0 N N 36 mo 17 N 0 N N Diagnostic 18 N 0 N P Diagnostic ET 19 a P 72 NA N Diagnostic b P 87 Type 1 N 15 mo c P 87 NA N 21 mo 20 a P 80 NA N Diagnostic b P 85 NA N 26 mo c P 60 Type 2 N 51 mo 21 P 95 Type 2 N Diagnostic 22 P 95 Type 1 N Diagnostic 23 P 98 Type 2 N Diagnostic 24 N 0 N N Diagnostic 25 N 0 N P Diagnostic 26 N 0 N P Diagnostic 27 N 0 N P Diagnostic 28 N 0 N P 36 mo 29 N 0 N P Diagnostic Post-PV MF 30 N 0 N P Diagnostic 31 N 0 N P Diagnostic CALR, calreticulin; JAK2, Janus kinase 2; CML, chronic myeloid leukemia; ET, essential thrombocythemia; IHC, immunohistochemistry; MF, myelofibrosis; MPN, myeloproliferative neoplasm; N, negative; NA, not available; P, positive; PMF, primary myelofibrosis; PV, polycythemia vera. counterstain, Hoechst (ThermoFisher) was used. Imaging was done using Nuance multispectral imaging system (CRi, Woburn, MA). Results Immunostaining for Mutated CALR in Nonneoplastic Bone Marrows, CMML, CML, and Normal Tissues Ten nonneoplastic bone marrows showed no immunostaining of the megakaryocytic, erythroid, myeloid, or other bone marrow stromal cells with the CAL2 monoclonal antibody. All 35 cases of CMML (which might be considered in the differential diagnosis of MPNs) were negative for staining with CAL2. Similarly, there was no CAL2 immunoreactivity seen in the 10 bone marrow biopsy specimens from CML cases. A Basic Local Alignment Search Tool (BLAST) search revealed high homology of the peptide immunogen to ubiquitin ligase (TMEM129), which is reported to be highly expressed by normal liver and testis. We thus also performed CAL2 immunostaining on a normal tissue microarray of 90 normal tissues (see Materials and Methods) representing multiple examples of major tissue types, including liver and testis, which showed no reactivity. CAL2 Immunostaining in CALR-Mutated and CALR Wild-Type Bone Marrow Biopsy Specimens From Non-CML MPNs We studied 38 bone marrow biopsy specimens of non- CML MPNs (PMF, n ¼ 21; ET, n ¼ 15; and n ¼ 2post polycythemia vera myelofibrosis) from 31 patients Table 1. American Society for Clinical Pathology Am J Clin Pathol 2016;146: Downloaded 433 from DOI: /ajcp/aqw135

4 Nomani et al /IMMUNOHISTOCHEMISTRY OF CALR MUTATIONS A B C Image 1 CAL2 immunohistochemical staining (all 400) showing strong cytoplasmic staining of megakaryocytes (A, B): A,Calreticulin(CALR) mutated/janus kinase 2 (JAK2)V617 wild-type essential thrombocythemia (ET). B, CALR-mutated/ JAK2 V617 wild-type primary myelofibrosis. C, No CAL2immunostaining seen in megakaryocytes (arrows) from a CALR wildtype/jak2 V617 mutated ET. Twenty-five biopsies were done for diagnostic purposes in patients with pancytopenia, unclassified thrombocytosis, or splenomegaly of unknown origin. Thirteen biopsies were done for patients with known MPN with worsening cytopenias and for clinical trial enrollment and follow-up. Fourteen patients had JAK2 V617F mutation, and eight harbored a CALR mutation. Type 1 mutation was seen in four patients and type 2 in four patients. All of the CALR-mutated cases had wild-type MPL. All 23 bone marrow biopsy samples from patients lacking a CALR mutation were negative by CAL2 immunohistochemistry (IHC) Image 1C. As can be seen in Table 1, we had the opportunity to study serial biopsy specimens from the CALR-mutated patients, although concomitant CALR mutational testing from each sample was not available in some of these serial samples. From the eight CALR-mutated patients, there were 15 biopsy samples. All eight samples with concomitant CALR mutational testing were CAL2 IHC positive. Six of the seven remaining serial samples were also positive. All samples showed moderate to strong cytoplasmic staining of the megakaryocytes with the CAL2 antibody Image 1A and Image 1B. The mean percentage of immunostained megakaryocytes in CAL2-positive samples was 83.5% (range, 50%-98%; median, 87%). No difference in the pattern of staining was seen based on whether type 1 or type 2 CALR mutation was present. The final serial sample from the CALR-mutated patients, sample 1a, lacked CAL2 immunoreactivity and was a historic sample taken 312 months prior to the sample in which CALR mutation was demonstrated. It had been diagnosed as an unclassifiable thrombocytosis. There was no material available from this biopsy specimen to conduct molecular tests for CALR mutation. Given the apparent excellent performance characteristics of the CAL2 antibody, we suggest that the CALR mutation was 434 Am J Clin Pathol 2016;146: American Society for Clinical Pathology 434 DOI: /ajcp/aqw135 Downloaded from

5 AJCP /ORIGINAL ARTICLE Table 2 Comutated Cases With Serial Biopsy Specimens Patient No. Sample No. CAL2 IHC Megakaryocyte Staining, % CALR Mutation JAK2 V617 A 1 P 20 Type 1 P B 1 P 97 Type 1 N 2 (44 mo) P 67 NA NA 3 (54 mo) P 30 Type 1 NA C 1 P 87 Type 1 N CALR, calreticulin; JAK2, Janus kinase 2; IHC, immunohistochemistry; N, negative; NA, not available; P, positive. not present, present at such a low level as not to be detectable by IHC, acquired during clonal evolution, or there was loss of antigenicity of the mutated CALR protein, given that this was an archived sample more than 25 years old. Unfortunately, we cannot definitively resolve this case. We also stained clot sections from five CALR-mutated and five CALR wild-type non-cml MPNs with CAL2 antibody. A similar staining pattern was seen as in the biopsy specimens. Thus, this antibody is suitable for both clot sections and decalcified bone marrow biopsy specimens. In addition to megakaryocytes, occasional CAL2 cytoplasmic staining of small mononuclear was noted in CALR mutant cases. To determine the lineage of these cells, we performed double immunofluorescence staining of bone marrow biopsy specimens with mutated CALR and each of the following markers: CD61, CD34, MPO, and GYPA Image 2. These studies showed that the small mononuclear cells seen in CALR mutant cases variably coexpressed mutated CALR and CD34 and MPO with only rare coexpression of mutated CALR and GYPA. Thus, these CALRmutated mononuclear cells appear to be myeloid cells or blasts rather than micro-megakaryocytes. Wild-Type CALR Immunostaining in CALR-Mutated and CALR Wild-Type Bone Marrows From Non-CML MPNs Given the unusual distribution of mutant CALR protein, largely limited to megakaryocytes and rare mononuclear cells, we wished to understand the expression of wild-type CALR in normal bone marrow and non-cml MPN bone marrows. Biopsy specimens from five CALR wild-type non- CML MPNs and five CALR-mutated non-cml MPNs were stained for wild-type CALR. The wild-type CALR antibody was titrated in normal bone marrow such that a range of reactivity was seen, allowing comparison to MPN samples. In normal bone marrow, megakaryocytes and some granulocytes and myeloid precursors expressed CALR. Very few erythroid precursors stained for wild-type CALR. The staining pattern in megakaryocytes was cytoplasmic. The Mann- Whitney U test showed a significant difference in the staining with a higher proportion of megakaryocytes staining in the CALR wild-type non-cml MPNs compared with CALRmutated non-cml MPNs (P ¼.034). Mutated CALR Immunostaining Pattern in Patients Harboring Comutations in CALR and JAK2 V617F or BCR-ABL1 We identified samples from three additional patients with comutations, one with CALR and JAK2 V617 (patient A) and the other two with mutated CALR and BCR-ABL1 (patients B and C) Table 2. Patient A reportedly had a history of an MPN (biopsy specimen not available) and was subsequently diagnosed as having PMF on his initial biopsy specimen (A) at our institution. Molecular analysis revealed JAK2 V617 mutation and concomitant CALR mutation. Immunohistochemistry showed fewer (20%) immunoreactive megakaryocytes compared with typical specimens from patients with only the CALR mutation Image 3A. Patient B with the BCR-ABL1 mutation was initially diagnosed as having PMF in 2011 (B1) with wild-type JAK2 mutational status. Subsequently, a second bone marrow biopsy (B2) was done in 2014 and BCR-ABL1 transcripts were detected, but morphologically, the biopsy specimen was consistent with PMF. Ten months later, after the patient had been treated with tyrosine kinase inhibitors, his BCR-ABL1 transcripts were undetectable, and a third biopsy (B3) was done that showed persisting PMF. Molecular analysis revealed that this patient harbors a CALR mutation, and CAL2 antibody staining was also positive at this time. A large number of smaller cells in this biopsy specimen also showed CAL2 staining Image 3B. To determine their lineage, we performed double immunofluorescence staining of these cells. We found that the smaller cells were CD61 and CAL2 positive Image 3C. This led us to conclude that these cells were micro-megakaryocytes. We attempted to perform molecular studies for CALR mutation on the DNA from the first and second biopsy specimens. One biopsy specimen (B1) had the CALR mutation (type 1), while testing was unsuccessful for the other biopsy specimen (B2). Immunohistochemistry using the CAL2 antibody on both these biopsy specimens showed immunoreactive megakaryocytes. It could not be determined with certainty whether these findings represented one neoplasm (ie, PMF with American Society for Clinical Pathology Am J Clin Pathol 2016;146: Downloaded 435 from DOI: /ajcp/aqw135

6 Nomani et al /IMMUNOHISTOCHEMISTRY OF CALR MUTATIONS Image 2 Double immunofluorescence staining of a Calreticulin (CALR) mutated essential thrombocythemia (red, QD655; green, QD565; blue, Hoechst 33342; 630). Top row: megakaryocytes expressing mutant CALR as seen by strong staining with CAL2 (red), CD61 (green), and merged CAL2/CD61. Middle row: lineage of small CAL2 immunohistochemistry-positive cells was revealed to be myeloid as elucidated by double staining with CAL2 (red), myeloperoxidase (MPO) (green), and merged CAL2/MPO. Bottom row: a rare myeloid blast with dual expression of CAL2 (red), CD34 (green), and merged CAL2/CD34. secondary acquisition of a BCR-ABL1 translocation) or two concurrent neoplasms (ie, PMF plus CML), but we favor the former in keeping with a prior report of similar CALR and BCR-ABL1 comutated cases demonstrating a common clonal origin. 6 Patient C also had a BCR-ABL1 and CALR comutation and has been previously reported. 11 IHC with the CAL2 antibody showed staining in 87% of the megakaryocytes. From these studies, we can conclude that the presence of JAK2 V617F or BCR-ABL1 with the CALR mutation does not interfere with detection of mutant CALR. Discussion We confirmed the utility of the commercially available CAL2 monoclonal antibody in successfully identifying bone marrow biopsy specimens with mutant CALR. There was 100% concordance in biopsy specimens with the concomitant molecular results. The antibody showed no cross-reactivity with other cell types as well as no staining in normal bone marrows or in CALR wild-type MPN cases. The staining was predominantly seen in megakaryocytes, but some smaller mononuclear cells were also immunoreactive as initially reported. Immunofluorescence double staining revealed them to be predominantly myeloid (MPOþ) with rare CD34- positive cells and a very rare cell with faint coexpression of CAL2 and GYPA. This is consistent with the work of Nanglia et al, 2 who suggested that CALR mutations occur in a multipotent progenitor capable of generating both erythroid and myeloid progeny. Wild-type CALR is highly expressed in many cells. However, as reported by others and shown in the present work, mutated CALR is preferentially expressed in 436 Am J Clin Pathol 2016;146: American Society for Clinical Pathology 436 DOI: /ajcp/aqw135 Downloaded from

7 AJCP /ORIGINAL ARTICLE A B C Image 3 Bone marrow biopsy specimens from patients with comutations. A, Patient A with the Janus kinase 2 (JAK2)/ Calreticulin (CALR) comutation showing strong CAL2 immunostaining of megakaryocytes expressing mutant CALR (400). B, CAL2 immunostaining of smaller cells (arrows) and megakaryocytes (arrowhead) in patient B with the CALR/BCR-ABL1 comutation (400). C, Double immunofluorescence staining of CALR/BCR-ABL1 comutated case from patient B (red, QD655; green, QD565; blue, Hoechst 33342; 630). The small cells expressing mutant CALR were micromegakaryocytes as shown by the double immunofluorescence staining with CAL2 (red), CD61 (green), and merged CAL2/CD61. megakaryocytes. 12,13 The reason for this has been elucidated. 14 Mutant CALR has been reported to deregulate JAK/ STAT signaling pathways by interacting with the thrombopoietin receptor (MPL), and the extracellular domain of MPL is crucial for activation by CALR mutants Indeed, it has been recently reported that MPL binds the mutant CALR protein. The tertiary structure of the mutant protein and the net positive charge of the C-terminus were critical for binding to MPL and signaling through the JAK/STAT axis downstream of MPL. 14 Interestingly, we found that wild-type CALR protein may be expressed at lower levels in the bone marrow of CALR patients compared with wild type. Whether this has functional consequences is unknown. We also report CAL2 staining patterns in samples with CALR and other MPN driver mutations. The comutated case (JAK2 V617 and CALR) stained much more weakly (approximately 20% of the megakaryocytes) compared with cases with the CALR-only mutation. This could point toward the existence of two clones in the same patient, one with CALR mutation and the other with JAK2 V617 mutation. Although initially reported as being mutually exclusive, various reports of comutated cases have been reported in literature with low frequency The patient with the BCR-ABL1 mutation had three serial biopsy specimens available with the initial being CALR mutated/bcr- ABL1 negative/jak2 wild type. Subsequently, the patient acquired the BCR-ABL1 translocation along with the existing CALR mutation. A year later, after receiving therapy for CML, the BCR-ABL1 transcripts had been lost, but the CALR mutation was still present. The CAL2 IHC in this case was positive in all three biopsy specimens consistently. Cabagnols et al 6 and Loghavi et al 11 reported similar cases with mutation in CALR and the BCR-ABL1 fusion gene in the same dominant clone. They showed that the American Society for Clinical Pathology Am J Clin Pathol 2016;146: Downloaded 437 from DOI: /ajcp/aqw135

8 Nomani et al /IMMUNOHISTOCHEMISTRY OF CALR MUTATIONS CALR mutation was an early genetic event and BCR-ABL1 was a secondary event leading to a clinically unusual MPN. We conclude that the coexistence of other driver mutations does not interfere with detection of mutant CALR and that CAL2 IHC can be useful in following mutational status in serial biopsy specimens. CALR mutation testing is expanding rapidly as it has shown to have prognostic and diagnostic implications in PMF and ET. 22,23 However, only frameshift mutations are capable of generating the novel C-terminus in the mutant protein, and with the development of the novel antibodies, these can now be detected by IHC. It is important to note that in-frame mutations of CALR do not generate the novel C-terminus, and currently their clinical significance is unknown. 24 The upcoming revision of WHO criteria for ET and PMF will most probably include testing for the CALR mutation. Immunohistochemical staining provides a rapid and inexpensive method of detecting these mutations in bone marrow biopsy specimens. The use of IHC would allow for initial detection of the CALR mutation in suspicious cases in smaller laboratories without access to molecular methods. Corresponding author: Eric D. Hsi, MD, Dept of Laboratory Medicine, Cleveland Clinic, 9500 Euclid Ave, Cleveland, OH 44195; hsie@ccf.org. References 1. Skoda RC, Duek A, Grisouard J. Pathogenesis of myeloproliferative neoplasms. Exp Hematol. 2015;43: Nangalia J, Massie CE, Baxter EJ, et al. Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2. N Engl J Med. 2013;369: Klampfl T, Gisslinger H, Harutyunyan AS, et al. Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med. 2013;369: Bonzheim I, Mankel B, Klapthor P, et al. CALR-mutated essential thrombocythemia evolving to chronic myeloid leukemia with coexistent CALR mutation and BCR-ABL1 translocation. Blood. 2015;125: Pagoni M, Garofalaki M, Tziotziou I, et al. Concurrent or sequential BCR-ABL1 translocation and CALR gene or JAK2V617F mutation. Blood. 2014;124: Cabagnols X, Cayuela JM, Vainchenker WA. CALR mutation preceding BCR-ABL1 in an atypical myeloproliferative neoplasm. N Engl J Med. 2015;372: Michalak M, Groenendyk J, Szabo E, et al. Calreticulin, a multi-process calcium-buffering chaperone of the endoplasmic reticulum. Biochem J. 2009;417: Wang WA, Groenendyk J, Michalak M. Calreticulin signaling in health and disease. Int J Biochem Cell Biol. 2012;44: Chao MP, Majeti R, Weissman IL. Programmed cell removal: a new obstacle in the road to developing cancer. Nat Rev Cancer. 2012;12: Gold LI, Eggleton P, Sweetwyne MT, et al. Calreticulin: non-endoplasmic reticulum functions in physiology and disease. FASEB J. 2010;24: Loghavi S, Pemmaraju N, Kanagal-Shamanna R, et al. Insights from response to tyrosine kinase inhibitor therapy in a rare myeloproliferative neoplasm with CALR mutation and BCR-ABL1. Blood. 2015;125: Vannucchi AM, Rotunno G, Bartalucci N, et al. Calreticulin mutation-specific immunostaining in myeloproliferative neoplasms: pathogenetic insight and diagnostic value. Leukemia. 2014;28: Stein H, Bob R, Dürkop H, et al. A new monoclonal antibody (CAL2) detects CALRETICULIN mutations in formalin-fixed and paraffin-embedded bone marrow biopsies. Leukemia. 2016;30: Araki M, Yang Y, Masubuchi N, et al. Activation of the thrombopoietin receptor by mutant calreticulin in CALRmutant myeloproliferative neoplasms. Blood. 2016;127: Rampal R, Al-Shahrour F, Abdel-Wahab O, et al. Integrated genomic analysis illustrates the central role of JAK-STAT pathway activation in myeloproliferative neoplasm pathogenesis. Blood. 2014;123: Abdelfattah N, Chen E, Perales-Paton J, et al. Physical Interaction between mutant calreticulin and the thrombopoietin receptor is required for hematopoietic transformation. Blood. 2015;126:LBA-LB Lavi N. Calreticulin mutations in myeloproliferative neoplasms. Rambam Maimonides Med J. 2014;5:e McGaffin G, Harper K, Stirling D, et al. JAK2 V617F and CALR mutations are not mutually exclusive: findings from retrospective analysis of a small patient cohort. Br J Haematol. 2014;167: Tefferi A, Lasho TL, Finke CM, et al. CALR vs JAK2 vs MPL-mutated or triple-negative myelofibrosis: clinical, cytogenetic and molecular comparisons. Leukemia. 2014;28: Lundberg P, Karow A, Nienhold R, et al. Clonal evolution and clinical correlates of somatic mutations in myeloproliferative neoplasms. Blood. 2014;123: Fu R, Xuan M, Zhou Y, et al. Analysis of calreticulin mutations in Chinese patients with essential thrombocythemia: clinical implications in diagnosis, prognosis and treatment. Leukemia. 2014;28: Guglielmelli P, Rotunno G, Fanelli T, et al. Validation of the differential prognostic impact of type 1/type 1-like versus type 2/type 2-like CALR mutations in myelofibrosis. Blood Cancer J. 2015;5:e Kourie HR, Ameye L, Paesmans M, et al. Improved survival of calreticulin-mutated patients compared with Janus kinase 2 in primary myelofibrosis: a meta-analysis. Clin Lymphoma Myeloma Leuk. 2016;16: He R, Hanson CA, Chen D, et al. Not all CALR mutations are created equal. Leuk Lymphoma. 2015;56: Am J Clin Pathol 2016;146: American Society for Clinical Pathology 438 DOI: /ajcp/aqw135 Downloaded from