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ORIGINAL ARTICLE
Year : 2021 | Volume
: 46
| Issue : 3 | Page : 175-180
Calreticulin mutational analysis in patients with myelofibrosis
May AlMoshary MD 1, Eman Al Mussaed1, Ahmad A Raza2
1 Department of Basic Science (Hematology), College of Medicine, Princess Nourah Bint Abdulrahman University, Riyadh, Saudi Arabia
2 Department of Hematology, Lady Reading Hospital, Peshawar, Pakistan
Correspondence Address:
May AlMoshary
Department of Basic Science (Haematology), Princess Nourah Bint Abdulrehman University, Riyadh, Zip Code 11564, Saudi Arabia
Saudi Arabia
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/ejh.ejh_42_20
Objective Our study aimed to determine the occurrence of CALR and JAK-2 mutation in patients with myelofibrosis.
Patients and methods This cross-sectional study was conducted in the Department of Hematology, Lady Reading Hospital, Peshawar, and Princess Nourah Bint AbdulRehman University, Saudi Arabia. Blood samples and data were collected from patients in the Hematology Department of Lady Reading Hospital, Peshawar, Pakistan. Nonprobability convenience sampling technique was used. All patients of JAK-2 V617F-positive and JAK-2 V617F-negative primary or secondary myelofibrosis due to polycythemia vera or essential thrombocythemia were included. After taking blood and bone marrow sampling, DNA extraction was done manually, and these samples were analyzed for CALR mutations by Sanger sequencing technique. Data were recorded and analyzed in SPSS, version 20.
Results Of 118 patients of myelofibrosis, we have found two types of genetic variations. One is single nucleotide polymorphism (SNP) in the 3’ UTR variant and the other is a novel indel frameshift mutation in the form of p.Leu 367 Thr Fx 63. Among these 118 patients, 14.40% patients had the indel frameshift mutation, whereas 46.61% patients had SNPs. The remaining patients did not harbor any significant changes.
Conclusion Our study concluded that most patients had a SNP in the 3’ UTR variant and a novel frameshift mutation. Further large-scale studies should be organized to determine the co-occurrence of calreticulin mutations in the JAK-2-positive patients of myelofibrosis. The influence of this coexistence on the phenotype and clinical course of myelofibrosis should also be studied to better understand the diagnosis and prognosis of the patients with myelofibrosis.
Keywords: calreticulin, JAK-2, mutation, myelofibrosis
Myelofibrosis is a stem cell disorder classified under the category of Philadelphia chromosome-negative ‘myeloproliferative neoplasms’ (MPNs) and includes ‘essential thrombocythemia’ (ET) and ‘polycythemia vera’ (PV) [1]. The disease is characterized by hepatosplenomegaly, variable degrees of cytopenia, thrombosis, bone marrow fibrosis, and constitutional symptoms [2]. The annual incidence is 0.5–1.5 cases per 100 000 individuals, making it the least common of MPNs [3],[4]. MPN generally affects older individuals with a median age around 65 years at the time of diagnosis, although it can affect any age group [5]. The median length of survival is ∼69 months with a worse prognosis compared with PV and ET [3]. The main causes of death are infections, bleeding, thromboembolism, and transformation into acute leukemia [6].
Most patients with myelofibrosis (50–60%) carry a JAK-2 V617F mutation in exon 14 of JAK-2 gene, which is located on chromosome 9p24 [7],[8]. In JAK-2-negative cases of primary myelofibrosis (PMF) (35%), somatic mutations are identified over the CALR gene located on chromosome 19p13.2 [9]. These insertion and deletion (indel) mutations in the exon 9 of calreticulin gene are present in nearly 85% of the non-JAK-2 mutated cases of PMF [10],[11]. However, a few recent studies have reported the mutual existence of CALR and JAK-2 mutations in MPNs [12]. Calreticulin is an ‘endoplasmic reticulum’ (ER) protein that is responsible for the proper folding of the newly synthesized glycoproteins and calcium homeostasis within ER [13],[14]. Outside ER, it plays a key role in proliferation, apoptosis, and immune-mediated cell death [15],[16],[17],[18]. N, P, and C are the three structural and functional domains of calreticulin protein, of which ‘C’ is a negatively charged acidic domain with multiple calcium-binding sites [19]. CALR mutation results in the substitution of this negatively charged C-terminal amino acids by positively charged polypeptides impairing the Ca++-binding capacity of calreticulin protein [9]. Because calreticulin mutations are the second most common in myelofibrosis, its screening should be included in the diagnostic workup of non-JAK-2 mutated myelofibrosis [20]. Overall survival, using multivariate regression analysis, showed to be better in CALR mutations compared with JAK-2/MPL mutations. CALR mutations are found in 15.6–35% of patients with unselected myelofibrosis, corresponding to 56–88% of patients with JAK-2/MPL-negative myelofibrosis [21].
The rationale of conducting this study was to determine the frequency of co-occurrence of CALR/JAK-2 double mutations in patients with myelofibrosis. Patients with MF with calreticulin mutation have a better survival as compared with JAK-2-positive patients, so CALR mutation screening should be included in the diagnostic workup of patients with myelofibrosis. This study gave us local magnitude of the CALR mutations. The results of this study will put us one step further in designing future research recommendations and guidelines in the diagnostic and therapeutic workup of patients with myelofibrosis. This will ultimately help us in improving better survival of these patients.
Patients and methodsThis descriptive cross-sectional study was carried out in the Department of Hematology, Lady Reading Hospital, Peshawar, from January 2017 to January 2019. Samples of blood and other information were gathered from patients with myelofibrosis admitted to the hospital for treatment. Selection of participants for the study was done using the nonprobability convenient sampling method.
All patients of JAK-2 V617F-positive and negative PMF or secondary MF owing to PV and ET were included. Patients with secondary MF owing to other conditions like tuberculosis, fungal infections, Hodgkin or non-Hodgkin lymphoma, and other variants of MPNs (PV, ET, and chronic myelogenous leukemia) were excluded.
The study was initiated after endorsement acquired from Advanced Studies and Research Board (ASARB) and Ethical Board of Lady Reading Hospital, Peshawar. An informed consent was obtained from all the participants in writing. Data were collected through a specially designed proforma, which consisted of two parts: the first part covered the demographic aspects of the patients, whereas the second part included the clinical presentation and investigation profile consisting of peripheral smear, bone marrow biopsy, and ultrasound abdomen findings.
Blood samples were gathered in plastic Whole Blood Tube coated with K2EDTA (lavender top; Vacutainer; Vacutainer®, Becton Dickinson, Plymouth, UK) by trained personnel at different diagnostic centers and hospitals, applying standard techniques. Automated complete blood counts for all the samples were performed on Cell dyne, Sysmex (Sysmex Corporation, Kobe, Japan), hematology analyzer.
A blood spot measuring 4 mm in diameter was placed on a glass slide about 5 mm from the end. A second glass slide (spreader) was positioned at 45° and backed into the blood spot to spread it by capillary action. The spreader slide was then smoothly pushed so that it joined end to end with the sample slide, resulting in smear formation. The slide was left to be dried in air after which it was fixed using methanol solution of 100% concentration before staining. After fixation, the slides were dipped for 20 min in Giemsa stain diluted to 1 : 20 in ddH2O, flushed with faucet water, and then left in air to be dried. Once the preparation of slides was done, they were examined using light microscopy to look for findings suggestive of MPNs. The blood samples were stored at 2–8°C until further used, such as for DNA extraction and mutation analysis.
Competent pathologists analyzed the peripheral smears to determine indications for aspiration of bone marrow. Once the indications were established, aspiration of the bone marrow was done using spinal needles of gauge 16 and 2% lignocaine for local anesthesia, ensuring the conditions were sterile throughout the procedure. The site of bone marrow aspiration was posterior superior iliac spine in all cases. Using a 20-ml syringe, ∼0.5 ml of bone marrow was aspirated and placed on a clean slide. Smear was prepared without any delay.
Bone marrow slides and reports were taken from all patients for reconfirmation and grading of myelofibrosis. For this purpose, staining with reticulin stain was carried out. The stained slides were then re-examined for morphological diagnosis and re-confirmed by a competent hematologist after blinding.
Nanodrop 2000 spectrophotometer (Thermo Fisher, Waltham, Massachusetts, United States) was used to ensure the extracted DNA was of desired quality and quantity. These DNA primers were used for the detection of JAK-2 V617F mutations by conventional PCR technology. Sanger sequencing technology was employed to detect presence or absence of CALR mutations in both JAK-2 positive and negative patients.
IBM SPSS (New York USA) (v.20) computer application was used for statistical analyses. Mean and SD were calculated for numerical variables (e.g. age). Frequencies and percentages were calculated for categorical variables, including sex and CALR mutations. Age and sex-wise stratification was done. Results were presented as tables and graphs.
ResultsThis cross-sectional descriptive study was conducted at the Department of Hematology, Lady Reading Hospital, Peshawar, in which 118 patients with myelofibrosis were studied. All the participants were analyzed based on age distribution; their age ranged from 20 to 80 years. A total of 32 (27.11%) patients were in the age range 20–40 years (group I), 62 (52.54%) patients were in age group 41–60 years (group II), and 24 (20.33%) patients were in the age range 61–80 years (group III). Mean age of our study group was 51.63±12.081 years. Mean age of male patients was 52.23±13.74, whereas that of female patients was 45.72±11.89. Overall, 67.79% (80) patients were male and 32.21% (38) patients were female.
The commonest presenting clinical features in our study patients were fever, pallor, and splenomegaly. Fever was observed in 81 (68.64%) patients, pallor in 69 (58.47%) patients, and splenomegaly 110 (93.22%). Easy bruising and hepatomegaly were observed in 18 (15.25%) and 10 (8.4%) patients, respectively.
Hematological parameters are shown in [Table 1].
In our study, of 118 patients, most [95 (80.50%)] were diagnosed as having PMF, followed by post-PV [15 (12.71%)], and post-essential thrombocythemia (ET) [eight (6.77%)]. Among the 118 patients of myelofibrosis under study, 104 (88.13%) were harboring JAK-2 mutations, whereas it was not present in only 14 (11.86%) patients.
Although we have not detected any CALR mutations in the 118 patients of myelofibrosis under study, we came across certain genetic sequencing. One is in the form of single nucleotide polymorphism (SNP) in the 3’ UTR and the other is a novel indel frameshift mutation in the form of p.Leu 367 Thr Fx 63.
Among these 118 patients, 17 (14.40%) patients had the indel frameshift mutation, whereas 55 (46.61%) patients had SNP in the 3’ UTR variant. Rest of the patients did not harbor any significant variation, as shown in [Figure 1],[Figure 2],[Figure 3].
Figure 2 Chromatograph showing the G/T change in the SNP. SNP, single nucleotide polymorphism. Discussion As a diverse group of diseases, MPNs have kept hematologists and hematopathologists confused, owing to their multifaceted clinical presentations and various morphologies. The idea for classifying MPNs based on molecular alterations has recently been gaining popularity because ample evidence has been gathered which indicates there is a better correlation between molecular or chromosomal alterations and clinical presentation, response to therapies, and prognosis, than conventional classification based on morphology. A significant number of gene mutations have been identified in MPNs, of which JAK-2 and MPL are the major ones. A significant gap is still present because many cases of MPNs do not harbor any of these hallmark mutations. This gap can be filled by the recent discovery of calreticulin (CALR) mutation in MPNs without JAK-2 mutation [9].
Our study concludes that diagnosis of myelofibrosis is made relatively late in life with male predominance. These demographic findings of our study are supported by Tefferi [22], in which they have studied 1000 patients of myelofibrosis with male predominance (male : female=3 : 2), and most of the patient were diagnosed later in life (median age of 60 years).
All the 118 patients were studied for JAK-2 V617F and CALR mutations after DNA extraction. Only 14 (11.86%) patients were JAK-2 nonmutated, whereas rest of the 104 (88.13%) had JAK-2 mutations. The discovery of JAKinase-2 pathway mutations provided us with new opportunities to understand pathophysiology of MPNs and develop new treatment strategies. JAK-2 gene is located on chromosome 9p24 and is a nonreceptor intracytoplasmic tyrosine kinase of Janus family [23]. The frequency of JAK-2 V617F mutation ranges between 43 and 59% in MF, and it results from a somatic G to T (G/T) point mutation, leading to the substitution of valine to phenylalanine [24]. This mutation renders JAK-STAT signaling pathway to be active constitutively which results in unchecked proliferation of hematopoietic stem cells. Mutations in the JAK-2 gene appear to account for hyperresponsiveness of the hematopoietic progenitor cells to various cellular growth factors and other cytokines [25]. However, the presence of this mutation alone cannot explain all the other findings associated with myelofibrosis, including cytopenia and the tendency to transform into acute myeloid leukemia.
Co-occurrence of the JAK-2 and CALR mutations has been reported in only a few cases of MPN (ET and MF) across different ethnic groups with frequency below 1% [26]. There are still not many reports on the phenotype and clinical course of the patients who are positive for these double mutations. The first data pertaining to phenotype and clinical manifestations in CALR and JAK-2 positive patients were provided by Xu et al. [27]. We have organized this study to see JAK-2/CALR double mutation in primary and secondary (post-PV and post-ET) myelofibrosis in our part of the world as no previous study is available in this regard. As far CALR mutation is concerned, we were unable to detect any mutations in the calreticulin gene performing Sanger sequencing technique; however, we came across certain genetic variations. These variations are in the form of a novel indel frameshift mutation and a SNP. In this study, 14.40% of patients had the indel frameshift mutation, whereas 46.61% of patients had SNPs. The novel mutation is a frameshift indel mutation in which CTT is substituted by AC (CTT>AC) at position no. 1172 of the cDNA. This substitution resulted in leucine replacement by threonine at position number 367 (p: Leu 367 Thr Fx 63). This is a novel mutation as it is not described in any previous medical literature. Future studies should be designed for the determination of this mutation and the resultant phenotypic effects of this mutation on patients with myelofibrosis.
ConclusionOur study concluded that most patients had SNP in the 3’ UTR variant in which most of the patients were in age group II and found a novel frameshift mutation. Further large-scale studies should be organized to determine the coexistence of CALR mutations in JAK-2-positive patients of myelofibrosis and study the influence of this coexistence on the phenotype and clinical course to better understand the diagnosis and prognosis in patients with myelofibrosis.
AcknowledgementsAuthors Contribution: M.A.M., E.A.M., conceived, designed, and did statistical analysis and editing of manuscript. A.A.R. did data collection and manuscript writing. A.A.R., M.A.M., and E.A.M. did review and final approval of the manuscript.
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Conflicts of interest
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