Frequency of JAK2V617F and CALR somatic mutations in Egyptian patients with thrombocytosis: relation with clinical and hematological phenotype

Gehan Mostafa Hamed; Mariam Fathy Abdelmaksoud; Doha Osama Abdulrahman; Yasmin Nabil El Sakhawy

doi:10.4103/ejh.ejh_66_21

ORIGINAL ARTICLE

Year : 2022 | Volume : 47 | Issue : 3 | Page : 167-173

Frequency of JAK2V617F and CALR somatic mutations in Egyptian patients with thrombocytosis: relation with clinical and hematological phenotype

Gehan Mostafa Hamed, Mariam Fathy Abdelmaksoud, Doha Osama Abdulrahman, Yasmin Nabil El Sakhawy
Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt

Date of Submission	11-Nov-2021
Date of Acceptance	07-Dec-2021
Date of Web Publication	03-Jan-2023

Correspondence Address:
Gehan Mostafa Hamed
Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Sheraton Heliopolis, Cairo
Egypt

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/ejh.ejh_66_21

Abstract

Background Thrombocytosis has a multitude of potential etiologies: spurious, reactive, and clonal. Clonal thrombocytosis carries a greater risk of thrombosis than reactive causes. Therefore, careful distinction between the causes of thrombocytosis is important and challenging as it carries implications for evaluation, prognosis, and treatment strategies. Aim of the work To determine the frequency of JAK2V617F and calreticulin (CALR) somatic mutations in patients with thrombocytsis and their relation with clinical and hematological phenotype. Patients and methods A total of 50 BCR-ABL-negative patients with persistent thrombocytosis were tested for both JAK2V617F mutation by real-time polymerase-chain reaction (RT-PCR) and CALR exon-9 mutation by high-resolution melting PCR. Results JAK2V615F mutation was detected in 17 (34%), whereas CALR exon-9 mutation was detected in 10 (20%) out of the 50 studied patients with thrombocytosis. One patient with essential thrombocythemia was heterozygous for both mutations. The incidence of JAK2V615F mutation was significantly higher in males (P=0.007), with higher mean age (P=0.001), higher incidence of thrombosis (0.034), and leukocytosis (0.035) compared with CALR and dual-negative mutations. Meanwhile, anemia (P=0.001), platelets (P=0.009), and lactate dehydrogenase (P=0.009) were significantly higher in CALR-mutated patients. Conclusion Both JAK2 and CALR somatic mutations were detected in 52% of patients with thrombocytosis. CALR-mutated cases show clinical and hematological phenotype different from JAKV617F-positive ones and might be considered as a distinct disease entity with more indolent course.

Keywords: CALR, JAK2V167F, phenotype, thrombocytosis

How to cite this article:
Hamed GM, Abdelmaksoud MF, Abdulrahman DO, El Sakhawy YN. Frequency of JAK2V617F and CALR somatic mutations in Egyptian patients with thrombocytosis: relation with clinical and hematological phenotype. Egypt J Haematol 2022;47:167-73

How to cite this URL:
Hamed GM, Abdelmaksoud MF, Abdulrahman DO, El Sakhawy YN. Frequency of JAK2V617F and CALR somatic mutations in Egyptian patients with thrombocytosis: relation with clinical and hematological phenotype. Egypt J Haematol [serial online] 2022 [cited 2023 Mar 30];47:167-73. Available from: http://www.ehj.eg.net/text.asp?2022/47/3/167/366867

Introduction

Thrombocytosis is a common incidental finding in up to 2.2% of the population aged more than 40 consulting primary care [1]. The differential diagnosis for thrombocytosis is broad and the diagnostic process can be challenging [2]. Evaluation of a patient with thrombocytosis requires careful consideration of patient history, comorbid conditions, other hematologic parameters, bone marrow (BM) aspirate and trephine-biopsy morphological features, and the presence or absence of clonal genetic abnormalities [2,3]. In general, the major causes of thrombocytosis can be divided into reactive and clonal [3]. The most common secondary (or reactive) causes of thrombocytosis are infection, inflammation, iron deficiency, tissue damage, hemolysis, severe exercise, malignancy, hyposplenism, and other causes of an acute-phase response [4,5].

Once a reactive thrombocytosis is excluded and thrombocytosis is persistent, the diagnostic evaluation should turn to distinguishing between the various causes of clonal thrombocytosis. Clonal thrombocytosis is often due to one of the Philadelphia-negative myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) [6]. Three-quarters of these patients carry the unique JAK2 (V617F) mutation [7,8], which is present in about 95% of patients with PV and in about 60% of those with ET or PMF [9]. Mutations of MPL exon 10 are present in about 5% of those with ET or PMF [10]. Most patients with ET or PMF with nonmutated JAK2 and MPL carry a somatic mutation of the gene encoding calreticulin (CALR) [11].

The identification of somatic mutation in CALR that is mutually exclusive with JAK2 and MPL mutations has provided a new powerful tool for studying MPNs, and according to the 2016 World Health Organization (WHO) classification of MPNs, it is considered one of the major criteria for diagnosis of ET, pre-PMF, and overt PMF. This study aimed to detect the frequency of JAK2 and CALR somatic mutations in patients with persistent thrombocytosis to distinguish reactive from clonal causes, and to detect the clinical and hematological phenotype of patients harboring one of these mutations. To our knowledge, this is the first study to detect the prevalence of both CALR and JAK2 mutations in Egyptian patients with thrombocytosis in relation to clinical and hematological phenotype.

Patients and methods

Study group

This study was conducted on 50 Egyptian patients with thrombocytosis who were attending the Hematology and Oncology Unit of Ain-Shams University Hospitals. An informed consent was obtained from each participant before enrolment in the study. The study was approved by the ethical committee of Ain Shams University. Inclusion criteria of the studied patients included sustained elevation in platelet count ≥450 × 10⁹/l for a minimum of 3 months and BCR-ABL negativity. Patients who were receiving chemotherapy were excluded. Consistent with the WHO classification of MPNs (2016) [12], the studied patients were divided into two groups: Primary thrombocytosis (group I) that included 30 patients [26 patients (86.7%) had ET and 4 patients (13.3%) had prefibrotic/pre-PMF]. Reactive thrombocytosis (group II) that included 20 patients with thrombocytosis secondary to autoimmune diseases (35%), iron-deficiency anemia and chronic gastrointestinal tract (GIT) bleeding (35%), infection (15%), and cancer (15%) [hepatocellular carcinoma (10%), colon cancer (5%)].

Blood sampling and laboratory investigations

Blood and BM-aspiration samples were collected under complete aseptic conditions on ethylenediaminetetraacetic acid, potassium salt (K₂-EDTA) (1.2 mg/ml) for complete blood count (CBC), and molecular analysis of genetic mutations. In all, 2–3 cm of BM trephine-biopsy samples were obtained under complete aseptic conditions and were kept in proper fixative after making imprint slides.

All studied patients were subjected to detailed history and thorough clinical examination, laying stress on organomegaly, thromboembolic complications, and bleeding. Laboratory workup included CBC using Coulter LH 750 (Beckman Coulter, Inc., Fullerton, CA), BM aspiration and trephine biopsy with examination of Leishman-stained peripheral blood and BM smears, genetic analysis for JAK2 V617F mutation by real-time polymerase-chain reaction (RT-PCR) using Slan 96 P Real Time PCR System (Sansure Biotech, Inc., China) using Ipsogen JAK2 Muta Screen Kit, and genetic analysis for CALR exon-9 mutation by high-resolution melting PCR (HRM-PCR) using Rotor-Gene Q MDx 5plex HRM (Qiagen, MD, USA). The DNA was extracted from fresh BM or peripheral blood samples using QIAamp®DNA Blood Mini Kit (Qiagen, MD, USA). The extracted DNA was stored at −20°C.

Methods

Detection of CALR mutation

The test primers were prepared by Applied Biosystems. The forward primer was 5′-TAACA AAGGTGAGGCCTGGT-3′ and the reverse primer was 5′-GGGACATCTTCCTCCTCATCT-3′. The reaction mixture consisted of 5 ul of DNA extract, 1 ul of forward primer, 1 ul of reverse primer, 5.5 ul of nuclease-free water, and 12.5 ul of HRM-PCR master mix (ready to use) formed of hot-start Thermus Aquaticus (Taq) plus DNA polymerase, Type-it HRM-PCR buffer EvaGreen, Q solution, and deoxyribonucleotide triphosphates.

The reaction protocol started by preincubation cycle at 95°C for 10 min, 45 amplification cycles included denaturation by heating at 95°C for 10 s, annealing/extension by heating at 64°C for 10 s, and elongation by heating at 74°C. Finally, one cycle of HRM composed of denaturation by heating at 95°C for 60 s, annealing by heating at 45°C for 60 s, and melting by heating at 65–95°C for 5 s. Data analysis was done using Rotor-Gene Q Series Software 2.3.1 (Build 49). Data were given in the form of normalized and difference plots. The melting curves were normalized and temperature shifted, creating the normalized plot for direct comparison of samples. The normalized plot is the graph in which the amount of fluorescence (due to the intercalating dye remaining at any temperature point) is expressed as a fraction of the amount prior to data acquisition. Difference plots were generated by selecting a negative control as the baseline and the fluorescence of all other samples was plotted relative to this sample. Significant differences in fluorescence were indicative of mutations. Each mutant allele had its own distinctive melting curve when compared with the wild-type allele. The distinct melting curves of the mutant became more apparent when data were represented in a difference-plot format than in a normalized plot.

Detection of JAK2V617F mutation

This was done by RT-PCR using Slang 96 p real-time PCR system (Sansure Biotech, Inc.) using Ipsogen JAK2 MutaScreen kit, in which 2 TaqMan probes were used labeled with a fluorescent dye at its 5-end such as fluorescein amidites (FAM) or victoria green fluorescent protein (VIC), generation of fluorescent signal only with FAM dye indicating homozygous mutation, whereas generation of fluorescent signal from FAM and VIC indicates heterozygous mutation V617F. Positive control (PC), negative control (NC), and cutoff samples (COS) were used.

The FAM/VIC ratios for all the samples, PC, COS, and NC were calculated. The normalized ratio (N ratio) for the COS and for all the samples was calculated as the following: N ratio sample=ratio sample/ratio NC. The gray zone around the normalized ratio of the COS was calculated as the following: (N ratio COS)=[(N ratio COS×0.94): (N ratio COS×1.06)].

Statistical analysis

Analysis of data was done by IBM computer using Statistical Program for Social Science version 15 (SPSS Inc., Chicago, IL); quantitative data were described in the form of a mean, SD, median, and range, whereas qualitative data were described as number and percent. To compare quantitative parametric data between two groups, Student’s t-test was applied. Quantitative nonparametric data between two groups were compared using Mann–Whitney, whereas Kruskal–Wallis was used for comparisons between more than two groups. Analysis of variance (ANOVA) test was used for comparison between means of more than two groups. The χ² test was used to compare qualitative data. Probability or P value of <0.05 was considered statistically significant in all analyses.

Results

Comparison between patients with primary (group I) and reactive (group II) thrombocytosis regarding their studied data

As shown in [Table 1], the incidence of organomegaly and thrombotic complications was significantly higher in group I (P=0.001). Laboratory data showed significantly lower hemoglobin in group II (P=0.01). The incidence of JAK2 and CALR somatic mutations was significantly higher among patients with primary thrombocytosis (P<0.001 and P=0.018, respectively). No significant difference regarding other studied clinical and laboratory data (P>0.05).

Table 1: Comparison between patients with primary (group I) and reactive (group II) thrombocytosis regarding studied variables

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According to WHO criteria of MPNs (2016), patients with primary thrombocytosis (group I) were diagnosed as ET that included 26 (86.7%) patients and pre-PMF that included 4 (13.3%) patients.

Comparison between patients with ET and pre-PMF

As shown in [Table 2], no significant difference was found between patients with ET and pre-PMF regarding the studied epidemiological, clinical, and laboratory data. It is noteworthy that patients with pre-PMF showed higher incidence of organomegaly, higher total leukocyte count (TLC), and lactate dehydrogenase (LDH) level, whereas Hb and platelets were lower than patients with ET, however, not reaching a significant difference (P>0.05), this could be probably due to the small number of patients presenting with pre-PMF. Moreover, BM cellularity was higher in ET (66.4 ± 13.8%) compared with pre-PMF (63.8 ± 15%), however, not reaching a significant difference (P=0.730) ([Table 2]).

Table 2: Comparison between ET and pre-PMF regarding studied variables

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Frequency of JAK2V615F and CALR mutations

The result of the present study revealed that both JAK2 and CALR mutations were detected in 52% out of the 50 studied patients with persistent thrombocytosis; JAK2V615F mutation was detected in 17 (34%), whereas CALR exon-9 mutation was detected in 10 (20%) patients.

Primary thrombocytosis (group I): JAK2V615F mutation was detected in 17 out of 30 patients (56.7%), 16 (53.3%) patients were diagnosed as ET, and 1 (3.3%) patient was diagnosed as pre-PMF, whereas CALR exon-9 mutation was detected in 9 out of 30 patients (30%), 7 (23.3%) patients were diagnosed as ET, and 2 (6.7%) patients were diagnosed as PMF. Of note, one patient with ET was heterozygous for both mutations. Diagnosis of 1ry thrombocytosis was confirmed by demonstration of clonality in 83.3% of patients by detection of JAK2 and/or CALR mutations, whereas 5 (16.7%) of the patients were double-negative for both mutations.

The group of reactive thrombocytosis (group II) showed dual negativity for JAK2V615F and CALR mutations, except for one patient with metastatic colon cancer who was positive for CALR exon-9 mutation.

Comparison between patients of 1ry thrombocytosis according to JAK2V615F and CALR exon-9 mutations

According to the results of the present study, the group of 1ry thrombocytosis (group I) was subdivided into 8 (26.7%) CALR positive, 16 (53.3%) JAK2V615F positive, 1 (3.3%) double-positive for JAK2V617F/CALR, and 5 (16.7%) dual-negative for both JAK2V617F and CALR mutations.

As regards the studied clinical and epidemiological data, mean age was significantly higher in JAK2V615F-positive than CALR-mutated and the dual-negative subgroups (P=0.001). A significant difference regarding sex was noted; the incidence of JAK2V615F was significantly higher in males compared with CALR-mutated and the dual-negative subgroups (P=0.007). Clinically, the incidence of thrombosis was significantly higher in JAK2V615F positive (P=0.034). The presence of organomegaly showed no significant difference between the three subgroups (P>0.5) ([Table 3]).

Table 3: Comparison between different subgroups of patients with 1ry thrombocytopenia

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Regarding laboratory investigation, TLC and granulocytes were significantly higher in JAK2V617F-positive subgroup (P<0.001). Hemoglobin was significantly lower in CALR-mutated subgroup (P=0.007). Platelets and LDH were significantly higher in CALR-mutated patients (P=0.001 and P=0.009, respectively). Of note, MPV was significantly low in CALR-positive patients (P=0.009).

Examination of BM smears revealed significant higher cellularity in JAK2V617F positive (P=0.028). Megakaryocytes were increased in the three subgroups (based on a 5-hpf count containing the highest number of megakaryocytes); however, clusters of megakaryocytes (≥3 megakaryocytes in direct contact without intervening hematopoiesis) were significantly associated with CALR-mutated cases (88.9%), which was significantly higher than in JAK2-mutated and double-negative subgroups (P=0.004) ([Table 3]).

As shown in [Table 3], stratification of patients into ET and pre-PMF according to WHO (2016) criteria, revealed no significant difference between the three studied subgroups (P=0.422).

Discussion

Thrombocytosis is usually discovered incidentally, but the differential diagnosis is important. In cases of reactive, or secondary, thrombocytosis, the underlying disease may require treatment. Clonal thrombocytosis, in contrast, is associated with both thrombotic and bleeding complications, and cytoreductive treatment may be necessary [13].

Clonal thrombocytosis is typically due to a chronic MPN, particularly ET and PMF [14]. According to the WHO classification of MPNs (2016), CALR mutation is considered one of the major criteria for diagnosis of ET, pre-PMF, and overt PMF [15], whereas JAK2V617F is present in about 60% of those with ET or PMF [9]. Therefore, we focused on detecting the frequency of JAK2 and CALR somatic mutations in 50 adult patients with persistent thrombocytosis, as well as the clinical and hematological features associated with each mutation. According to the etiology of thrombocytosis, patients were divided into two groups: 1ry and 2ry thrombocytosis.

The results of the present study revealed that CALR mutation was detected in 30% of cases with 1ry thrombocytosis, whereas JAK2 mutation was detected in 56.7% of cases, the remaining 16.7% of cases were dual-negative for both mutations. Jeong etal. [16] revealed in their study a mutational frequency of 61.1% for JAK2 V617F, 29.6% for CALR, and 14.8% for dual-negative mutations. Overall, these mutational frequencies accord well with the findings of other previous studies [11,17],[18],[19],[20].

Although JAK2 and CALR mutations are considered mutually exclusive in patients with ET and PMF, in the current work, coexpression of both mutations was found in one patient with ET. This finding adds to the exceptions to the rule of mutual exclusiveness of JAK2 and CALR mutations, which was also found in few previous studies [16,21],[22],[23],[24]. Owing to the infrequency and diversity of available reports on the phenotype and clinical course of patients positive for both mutations, the comparison is limited. Our patient was heterozygous for JAK2 and CALR somatic mutations. Clinically, there was no history of thrombosis, cerebrovascular disease, or organomegaly. Laboratory data showed leukocytosis (mainly neutrophilia), with erythrocytosis and elevated hemoglobin level. BM biopsy was mildly hypercellular with increased megakaryocytes tending to form clusters.

As regards the impact of CALR and JAK2 mutations on the clinical phenotype, our result revealed that most of JAK2-mutated cases were males, their mean ages, white-blood cell, and granulocytes were significantly higher than CALR-mutated and dual-negative cases. On the other hand, anemia and platelets were significantly higher among CALR-mutated patients compared with JAK2-mutated and dual-negative. CALR-mutated cases were younger, with lower hemoglobin level and higher platelet count than JAK2-mutated cases. These findings were described also by Chen etal. [21] and Bilbao-Sieyro etal. [24].

As for the higher platelet count in CALR mutation, the link concerning CALR mutations and megakaryocytopoiesis has been explained in some studies by the discovery of physical interaction between CALR-mutated protein and MPL receptor, thus activating JAK-STAT pathway [25]. However, the increase of megakaryocyte-commitment regulator as megakaryocyte-activating factor [26] coupled to the decrease of megakaryocyte-differentiation inhibitors, such as interferon alpha- and beta-receptor subunit 1, phosphatase and tensin homolog, and suppressor of cytokine signaling 6, could favor the switch from granulocyte to megakaryocyte commitment [27,28]. Moreover, the increased level of polo-like kinase 3 that was already described as having a pivotal role in megakaryocyte polyploidization and differentiation might justify the higher number of platelets in CALR-mutated patients compared with JAK2V617F-positive patients [29].

Our result revealed a significant higher platelet count in CALR-mutated patients, even though the incidence of thrombosis was significantly lower than those with JAK2 mutation. This could be contributed to the relatively younger age of CALR-mutated patients compared with JAK2-mutated ones. Zini etal. [29] uncovered the downmodulation of several proteins involved in thrombin and RhoA signaling in CALR mutation. In addition, several genes involved in platelet activation, aggregation, and degranulation are decreased in CALR-mutated progenitors, whereas the antithrombotic factor thrombomodulin is upregulated. Likewise, Torregrosa etal. [30] showed reduced platelet activation in CALR-mutated ET patients compared with JAK2V617F-positive ET patients. Furthermore, a study in 891 patients with WHO-defined ET clearly showed that JAK2 (V617F) represents a strong independent risk factor for thrombosis [31]. Overall, these observations could explain the low thrombotic risk in CALR-mutated patients, even though they have a higher platelet count as compared with JAK2V617F-positive patients.

Concerning the lower mean hemoglobin level detected in our studied CALR-mutated compared with JAK2-mutated patients, Zini etal. [29] unveiled a direct correlation between hemoglobin level and the expression of some erythroid-positive regulators, which are decreased in CALR-mutated patients. Adding to that, CALR frameshift mutation occurs in a small proportion of endogenous erythroid colonies, suggesting that the expression of mutant CALR does not confer erythropoietin hypersensitivity to developing red-blood cells and also, the cytokine-independent proliferation of cells carrying CALR mutations occurs only in the presence of MPL, a receptor that is not expressed by maturing red-blood cells [23,25].

Moreover, our study noted significantly higher LDH levels in CALR-mutated cases compared with JAK2-mutated patients that may reflect a higher myelofibrotic transformation potential as LDH might be a biologically accurate measure of cell turnover and a sensitive marker of occult pre-PMF [32]. Marty etal. [33] conducted a study on two murine models that developed megakaryocyte hyperplasia and isolated thrombocytosis after receiving BM cells expressing a mutated CALR protein, and accordingly, the majority of recipients developed myelofibrosis 6 months after transplantation, with anemia and thrombocytopenia in the peripheral blood, hypocellular marrow, and splenomegaly. Marked osteosclerosis and increased reticulin-fiber depositions were also observed.

In the present work, examination of BM biopsies revealed increased megakaryocytes in all cases of clonal thrombocytosis, with a tendency of cluster formation in CALR-mutated cases. Similarly, Loghavi etal. [34] stated that CALR-mutated cases were more likely to show megakaryocytic clustering and abnormal morphology, compared with their CALR wild-type counterparts. This was explained by loss of CALR function as a result of mutation, leading to Ca²⁺ export from endoplasmic reticulum in hematopoietic progenitor cells, thus decreasing the activity of the calcineurin-nuclear factor of activated T-cell signaling pathway, which in turn favors myeloid/megakaryocyte-lineage commitment and not erythroid-lineage proliferation.

Of note, in the present study, almost all studied patients with reactive thrombocytosis did not reveal to harbor JAK2V615F or CALR mutations, which accords with the findings reported by previous studies on patients with persistent thrombocytosis [24,35]. Surprisingly, we discovered CALR mutation in one patient with metastatic colon cancer, which is an uncommon finding, as most of recurrent mutations in the CALR gene occur in MPNs, despite a small number of mutations reported in different solid tumors: ovary, pancreas, and breast [36],[37],[38]. Vougas etal. [39] reported high CALR expression in colon cancer compared with the matched mirror-biopsy tissues, especially in highly malignant and poorly differentiated tumors. However, the specificity of CALR genotyping for confirming the diagnosis of MPNs lies in detecting the types of the mutation in MPNs, by sequencing techniques, which are completely different from those occurring in other tumors and mainly include type-1 mutation (52-bp deletion) and type-2 mutation (5-bp insertion), which also have different clinical features and outcome [40]. Further follow-up for evolving MPN is also important.

In conclusion, detection of JAK2 and CALR somatic mutations in patients with persistent thrombocytosis confirms the diagnosis of 1ry and 2ry thrombocytosis and might uncover the presence of ongoing clonal disorder. JAK2 and CALR mutations define subtypes of ET with different clinical and hematological phenotype; JAK2-mutated ET affects relatively older males, with higher hemoglobin, higher white-blood cells, and higher incidence of thrombosis; meanwhile, CALR mutations are relatively younger, more anemic with higher platelets, yet with lower incidence of thrombosis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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