The characteristics of CALR mutations in myeloproliferative neoplasms: a clinical experience from a tertiary care center in Qatar and a literature review

ABSTRACT

Background

Myeloproliferative neoplasms (MPNs) are hematological disorders characterized by abnormal production of myeloid cells due to genetic mutations. Since 2013, researchers have identified somatic mutations in the Calreticulin (CALR) gene, primarily insertions or deletions, in two Philadelphia chromosome-negative MPNs; essential thrombocytosis (ET) and primary myelofibrosis (PMF), and occasionally in chronic myelomonocytic leukemia (CMML). This study aims to identify the various types of CALR mutations and their impact on CALR-positive MPN patients’ clinical manifestations and outcomes.

Methods

A single-center retrospective study was conducted. The data was collected from pre-existing records. The study was carried out on Philadelphia-negative MPN patients who were being followed up on at the NCCCR (National Center for Cancer Care and Research) to assess the clinical manifestation and outcome of disease treatment. All patients included, were followed in our center between January 1, 2008, and November 20, 2021.

Results

A total of 50 patients with CALR-positive MPN were reviewed with a median follow-up of three years (1–11). This cohort included 31 (62%) patients with ET, 10 (20%) patients with PMF, and 9 (18%) patients with prefibrotic myelofibrosis (pre-MF). The study involved 38 (76%) male and 12 (24%) female patients. There were 16 (32%) patients diagnosed before the age of 40, 24 (48%) patients diagnosed between the ages of 40 and 60; and 10 (20%) patients diagnosed after the age of 60. Molecular analysis showed 24 (48%) patients with CALR type 1, 21 (42%) patients with CALR type 2, and 5 (10%) patients with none Type 1, none Type 2 CALR mutations. Two patients have double mutations; 1(2%) with none Type 1, none Type 2 CALR and JAK2 mutations, and 1(2%) with CALR type 1 and MPL mutations. The thrombotic events were 3 (6%) venous thromboembolisms, 3 (6%) abdominal veins thromboses, 2 (4%) strokes, and 4 (8%) ischemic cardiac events. Only 4 (8%) patients progressed to Myelofibrosis and were carrying CALR 1 mutations, and 1 (2%) patient progressed to AML with CALR 2 mutation.

Conclusion

The data shows a significant rise in CALR-positive MPN diagnoses in younger people, emphasizing the need for a better assessment tool to improve disease management and reduce complications.

KEYWORDS:

• Myeloproliferative neoplasms
• CALR mutation
• thrombosis
• essential thrombocytosis
• myelofibrosis

Introduction

The term myeloproliferative disorders, now known as myeloproliferative neoplasms (MPN), was first coined in 1951 to describe a group of overlapping hematological disorders that eventually lead to inappropriate production of mature myeloid cells [1]. In 1960, the Philadelphia chromosome was first identified in two chronic myeloid leukemia (CML) patients [2]. Later, in 1973, the reciprocal translocation between chromosomes 9 and 22 constituting the Philadelphia chromosome was discovered, subsequently known to harbor the BCR-ABL mutation [3]. Since then, the classic MPN has been divided broadly into Philadelphia chromosome-positive (Chronic Myeloid Leukemia – CML) and Philadelphia chromosome-negative (Polycythemia Vera – PV, Essential Thrombocythemia – ET, and Primary Myelofibrosis – PMF). Further research into the Philadelphia chromosome-negative MPN category has led to the discovery of Janus Kinase 2 (JAK2) mutation in 2005 and Myeloproliferative Leukemia virus oncogene (MPL) mutation in 2006 [4, 5]. The latest breakthrough in this field was made in 2013 with the identification of Calreticulin (CALR) mutation in ET and PMF [6].

The CALR gene is located in chromosome 19p13.2 and encodes for the Calcium-binding protein Calreticulin [6]. CALR plays a vital role in protein folding and calcium homeostasis when located in the endoplasmic reticulum (ER). While it is mainly found in the ER, it has become evident that it is also present in the cytosol, cell membrane, and extracellular matrix, with a wide range of biological effects that are not fully revealed yet [7]. Since its discovery, several studies have identified multiple frameshift mutations in CALR that eventually lead to JAK/STAT signaling activation in ET and PMF patients with non-mutated JAK2 or MPL [8]. These CALR mutations have produced disease phenotypes that are clinically distinct from the well-known manifestations of MPN [9]. In our study, we aim to identify the clinical characteristics of CALR mutations in Qatar and provide a summary of the significant articles addressing this topic up to this date.

Materials and methods

This retrospective study was conducted on Philadelphia-negative MPN patients under follow-up at the National Center for Cancer Care and Research (NCCCR). The primary aim was to evaluate the disease's clinical manifestation and treatment outcomes. All participants were previously followed at our center between January 1, 2008, and November 20, 2021.

Study population

We reviewed the records of about 100 patients from the NCCCR in Qatar, from which 50 patients met our inclusion criteria and were included in the study.

Inclusion criteria

Adult patients aged 18 years or older diagnosed with Philadelphia-negative MPN (ET, PV, MF, and prefibrotic myelofibrosis – Pre-MF) based on the 2008 and 2016 WHO criteria, and tested positive for any of the CALR mutations at the NCCCR between 2008 and 2021.

Exclusion criteria

MPN patients with Philadelphia-positive or other genetic mutations.

Sequencing analysis

DNA sampling have undergone molecular analysis for mutations within exon 9 of the CALR gene using fragment analysis and DNA sequencing. We observed 36 different types of CALR mutations, all of which were somatic insertions or deletions in exon 9. The sensitivity of fragment analysis for detection of type 1 and type 2 mutations is approximately 5%. Sensitivity of detection for other mutation types may vary up to approximately 10%. Genbank accession number is NM004343.3. Mutation nomenclature is according to HGVS guidelines.

Statistical analysis

We used descriptive statistics to present the demographic data of the study cohort. We classified CALR mutations as type 1, 2, or other (none Type 1, none Type 2). Continuous variables were summarized as mean (SD) and median (IQR), while categorical variables were summarized as percentages. We compared continuous parametric variables using unpaired t-tests and ANOVA. Man-Whitney U and Kruskal – Wallis tests compared non-parametric continuous data. We used the Chi-square test to compare categorical variables.

Results

We reviewed 50 patients with CALR-positive MPN who presented to our tertiary care center in Qatar. The median follow-up duration was three years. The baseline characteristics of the study population is summarized in Table 1 and subcategorized further according to the CALR mutations in Table 2. Most of the patients were diagnosed with ET (31, 62%). The rest were having PMF (10, 20%) and pre-MF (9, 18%). Our population was predominantly males (38, 76%), Arabs (27, 54%), and relatively young (40, 80% below the age of 60). Most CALR mutations were type 1 (24, 48%). Type 2 mutations were (21, 42%) and the none Type 1, none Type 2 CALR mutations were (5, 10%). The patients were treated with hydroxyurea (22, 44%), JAK2 inhibitor (6, 12%), anagrelide (1, 2%), and antiplatelet (36, 72%). The complication rates were relatively low with ischemic cardiac events being the highest (4, 8%), followed by venous thromboembolisms (3, 6%), abdominal veins thromboses (3, 6%), strokes (2, 4%). It is also noted that the cardiac events were preferentially occurring the most in the none Type 1 none Type 2 CALR mutation with statistical significance. The progression of CALR-positive MPNs to Myelofibrosis and acute myeloid leukemia (AML) was rare (4, 8% and 1, 2% respectively). We reported one mortality case, and that happened in the CALR type 2 group. One patient with CALR type 1 mutation had a concurrent MPL mutation, and one patient with non-type 1, non-type 2 CALR mutation had a concurrent JAK2 mutation. We saw a trend towards lower levels of hemoglobin, White cell counts, and platelets in CALR type 1 compared to other mutations. We also noted the development of secondary malignancies happened only in the CALR type 1 mutation group.

Table 1. Baseline characteristics of patients with CALR mutation in myeloproliferative neoplasm

Characteristic	CALR Mutated MPN Patients (N = 50), n (%)
Male Gender	38 (76%)
Age At Diagnosis
<40 years	16 (32%)
40–60 years	24 (48%)
>60 years	10 (20%)
Weight (kg, mean)	77.5
Height (cm, mean)	169.9
Ethnicity
Asian	15 (30%)
Black/African	6 (12%)
White	2 (4%)
Arabs	27 (54%)
Comorbid Medical Conditions
Diabetes mellitus	9 (18%)
Hypertension	18 (36%)
Obesity	11 (22%)
Cigarette Smoking	5 (10%)
Baseline Blood Works Results
Hemoglobin (Median, IQR)	13.05 (11.7-14.28)
WBCs (Median, IQR)	8.20 (5.98-10.30)
Platelets (Median, IQR)	850.00 (525.25-1120.25)
LDH (Median, IQR)	214.00 (177.00-317.00)
Molecular Tests
CALR type 1	24 (48%)
CALR type 2	21 (42%)
Other CALR	5 (10%)
JAK2 (with other CALR)	1 (2%)
MPL (with CALR type 1)	1 (2%)
Diagnosis
preMF	9 (18%)
ET	31 (62%)
PMF	10 (20%)
Treatment
Hydroxyurea	22 (44%)
JAK2 inhibitors	6 (12%)
Anagrelide	1 (2%)
Antiplatelet (Aspirin or Clopidogrel)	36 (72%)
Progression
MF (carrying CALR 1 mutations)	4 (8%)
AML (carrying CALR 2 mutations)	1 (2%)
ALL	0
CML	0
Complications
VTE	3 (6%)
Stroke	2 (4%)
Cardiac ischemic events	4 (8%)
Abdominal veins thrombosis	3 (6%)
Hemorrhage	1 (2%)
Secondary malignancy (carrying CALR type 1 mutations)	3 (6%)
Mortality (carrying CALR type 2 mutation)	1 (2%)

CALR: Calreticulin; MPN: Myeloproliferative Neoplasm; kg: Kilogram; cm: Centimeter; IQR: Interquartile Range; WBC: White Blood Cell; LDH: Lactate Dehydrogenase; JAK2: Janus kinase 2; MPL: Myeloproliferative Leukemia virus oncogene; pre-MF: Prefibrotic Myelofibrosis; ET: Essential Thrombocytopenia; P/MF: Primary/Myelofibrosis; AML: Acute Myeloid Leukemia; ALL: Acute Lymphocytic Leukemia; CML: Chronic Myeloid Leukemia; VTE: Venous Thromboembolism

Table 2. Association between Different CALR Mutations and Baseline Characteristics of Patients.

Characteristics	CALR Mutations			P Value
	Type 1 (n = 24), n (%)	Type 2 (n = 21), n (%)	Other (n = 5), n (%)
Male Gender	19 (79.2)	14 (66.7)	4 (80.0)	0.602
Age Group
Below 40	5 (20.8)	9 (42.8)	2 (40.0)	0.396
40–60	10 (41.6)	12 (50.0)	2 (40.0)	0.884
Above 60	7 (29.2)	2 (9.5)	1 (20.0)	0.259
Arab Ethnicity	12 (50.0)	14 (66.7)	1 (20.0)	0.082
Comorbid Medical Conditions
Diabetes mellitus	3 (12.5)	5 (23.8)	1 (20.0)	0.611
Hypertension	11 (45.8)	7 (33.3)	0 (0)	0.143
Obesity	2 (8.3)	7 (33.3)	2 (40.0)	0.077
Cigarette Smoking	4 (16.7)	1 (4.8)	0 (0)	0.304
Baseline Blood Works Results
Hemoglobin (Median, IQR)	11.8 (2.7)	13.4 (3.2)	13.95 (1.6)	0.323
WBCs (Median, IQR)	6.45 (2.8)	8.3 (5.2)	7.0 (3.6)	0.557
Platelets (Median, IQR)	713.0 (363.0)	874.0 (673.0)	1062.5 (738)	0.673
LDH (Median, IQR)	207.5 (281.0)	221.0 (80.0)	180.5 (232.0)	0.464
Molecular Tests
JAK2	0 (0)	0 (0)	1 (20.0)	0.010
MPL	1 (4.2)	0 (0)	0 (0)	0.575
Diagnosis
preMF	3 (12.5)	4 (19.1)	2 (40.0)	0.501
ET	16 (66.7)	12 (57.1)	3 (60.0)	0.928
PMF	5 (20.8)	5 (23.8)	0 (0)	0.484
Treatment
Hydroxyurea	8 (33.3)	11 (52.4)	3 (60.0)	0.329
JAK2 inhibitors	3 (12.5)	3 (14.3)	0 (0)	0.673
Anagrelide	1 (4.2)	0 (0)	0 (0)	0.575
Aspirin	14 (58.3)	15 (71.4)	3 (60.0)	0.844
Clopidogrel	2 (8.3)	0 (0)	2 (40.0)	0.012
Progression
MF	4 (16.7)	0 (0)	0 (0)	0.095
AML	0 (0)	1 (4.8)	0 (0)	0.494
ALL	-	-	-	N/A
CML	-	-	-	N/A
Complications
VTE	1 (4.2)	2 (9.5)	0 (0)	0.630
Stroke	0 (0)	2 (9.5)	0 (0)	0.237
Cardiac ischemic events	1 (4.2)	1 (4.8)	2 (40.0)	0.021
Abdominal veins thrombosis	2 (8.4)	0 (0)	1 (20.0)	0.078
Hemorrhage	1 (4.2)	0 (0)	0 (0)	0.575
Secondary malignancy	3 (12.5)	0 (0)	0 (0)	0.750
Mortality	0 (0)	1 (4.8)	0 (0)	0.494

Chi-square analysis and Kruskal-Wallis test were applied at alpha = 0.05. CALR: Calreticulin; IQR: Interquartile Range; WBC: White Blood Cell; LDH: Lactate Dehydrogenase; JAK2: Janus kinase 2; MPL: Myeloproliferative Leukemia virus oncogene; pre-MF: Prefibrotic Myelofibrosis; ET: Essential Thrombocytopenia; P/MF: Primary/Myelofibrosis; AML: Acute Myeloid Leukemia; ALL: Acute Lymphocytic Leukemia; CML: Chronic Myeloid Leukemia; VTE: Venous Thromboembolism

Discussion and conclusion

CALR mutations have been identified in two of the three classic Philadelphia chromosome-negative (BCR-ABL-negative) MPN, namely ET and PMF [6]. Several mutations were detected in the CALR gene, but more than 80% of them are attributed to two mutations: 52-bp deletion (known as type 1) and 5-bp insertion (known as type 2) [9]. The clinical implications of these CALR mutations are yet to be explored, and it is an emerging field that can have therapeutic implications in the future. In a recent animal-based study, CARL was investigated as being a novel target of monoclonal antibody (4D7) used to treat PMF [10].

The research and knowledge of the CALR gene and its mutations is rapidly growing. Since 2013, several published articles addressed CALR mutations and their impact on the course of BCR-ABL-negative MPNs. It is evident from the current literature that CALR-mutated patients tend to be younger and have male predominance compared to JAK2-mutated counterparts [11–18]. It is well-established that CALR mutations are associated with higher platelet and lower hemoglobin and white cell counts in ET than JAK2 mutations [6, 11–17, 19]. Despite the increase in platelet production, CALR-mutated patients have a lower risk of thrombosis and at least equal, or even better, overall survival than any other molecular subtypes [6, 11–17, 19–21]. On the other hand, most studies reported an increased risk of myelofibrotic transformation in CALR-mutated ET patients. In contrast, others showed them to have an equivalent risk to JAK2 mutations [6, 11–17, 19–22]. We summarized the key elements of the main studies that looked into the CALR-mutated MPNs in Table 3.

Table 3. Summary of Major CALR Studies.

Study	Population Size	CALR Patients Demographics	CALR Prevalence	Clinical Phenotype	Thrombotic Complications	Hematologic Transformation	Survival and Follow-up Duration
Klampfl et al, 2013 [6]	311 ET & 203 MF	-	25% of ET and 35% of MF had CALR mutation. Among CALR mutations, type 1 and type 2 percentages were 53% and 31.7%, respectively.	ET with CALR mutation had ↓ Hgb, ↓ WBC, &↑ Plt at diagnosis than mutated JAK2. MF with CALR mutation had ↓ WBC, &↑ Plt then mutated JAK2.	ET with CALR mutation had a lower risk of thrombosis than mutated JAK2.	-	CALR mutation had a more prolonged overall survival than mutated JAK2.
Nangalia et al, 2013 [19]	151 ET & MF	-	CALR mutations were found in 70-84% of MPNs.	MPN with CALR mutation had ↑ Plt & ↓ Hgb than mutated JAK2.	-	ET with CALR mutation had a significantly higher incidence of transformation to MF than mutated JAK2.	There were no significant differences in rates of survival.
Rumi et al, 2013 [11]	717 ET	ET with CALR mutation patients were significantly younger and had more males compared to those with JAK2-mutated ET. CALR-1 patients were younger than CALR-2.	24.5% of ET had CALR mutation. Among CALR mutations, type 1, type2, and others percentages were 46%, 38%, and 16%, respectively.	ET with CALR mutation had ↓ Hgb, ↓ WBC, ↑ Plt, &↑ serum EPO, then mutated JAK2. CALR-1, compared to CALR-2, had ↓ Hgb, ↓ WBC, ↓ frequency of splenomegaly, & ↓ LDH, ↑Plt, ↑ serum EPO, & ↑ CD34 count.	ET with CALR mutation had a lower risk of thrombosis at diagnosis than mutated JAK2.	No significant difference in myelofibrotic transformation was noted, and no polycythemic transformation was observed in CALR-mutated. Patients.	CALR-mutated PMF, but not ET, had better overall survival than JAK2. The median follow-up period was 5.2 years (range 0–32 years)
Andrikovics et al, 2014 [9]	289 ET & 99 PMF	CALR-mutated patients were younger compared to those with JAK2-mutation	50% and 32% of MPN carried type 1 and type 2 CALR, respectively. 33% of ET and 25% of MF had CALR mutation.	CALR mutation had ↑ Plt than mutated JAK2.	ET with CALR mutation had a lower risk of venous thrombosis than mutated JAK2.	ET with CALR mutation had a higher risk of progression than mutated JAK2.	CALR-mutated PMF, but not ET, had better overall survival than JAK2.
Li et al., 2014 [13]	357 Chinese patients with PMF	CALR-mutated patients were younger compared to those with JAK2-mutation	21% of PMF had CALR mutation. 32% and 64% of CALR mutations were type-1 and type-2, respectively.	PMF with CALR type 2 mutation had ↓ Hgb, ↓ WBC, abnormal platelet levels (<100 × 109/L, or >450 x 109/L), & no splenomegaly compared to mutated JAK2.	-	-	Type 2 CALR-mutated, but not type 1, PMF had significantly shorter survival compared with JAK2 mutations. The median follow-up period was 28 months (range 1-385).
Rotunno et al, 2014 [14]	576 ET	CALR-mutated patients were preferentially males compared with JAK2 mutation.	15.5% of ET had CALR mutation	ET with CALR mutation had ↓ Hgb, ↓ WBC, & ↑ Plt than mutated JAK2.	ET with CALR mutation had a lower risk of thrombosis than mutated JAK2.	CALR mutation had no impact on transformation to post-ET Myelofibrosis	CALR mutation had no impact on survival. Median follow-up was 71.9 months (range, 2–257 months)
Gisslinger et al, 2014 [20]	115 ET & 85 pre-MF	-	18% of ET and 39% of pre-MF had CALR mutations	-	-	-	CALR-mutated pre-MF, but not ET, had better overall survival than JAK2. Such a striking difference was not seen at the time of transformation into overt Myelofibrosis.
Chen et al, 2014 [15]	147 ET	CALR-mutated patients were younger compared to those with JAK2-mutation	22.5% of ET had CALR mutations. 54.5%, 27.3%, and 6.1% of CALR mutations were type-1, type-2, and type-3, respectively.	ET with CALR mutation had ↓ WBC, & ↑ Plt then mutated JAK2.	ET with CALR mutation had a lower risk of thrombosis than mutated JAK2.	CALR-mutated patients had 3.3% post-ET MF Transformation.	CALR-mutated ET had a favorable impact on thrombosis-free survival (TFS) than JAK2.
Al Assaf et al, 2015 [16]	160 ET	CALR-mutated patients were younger compared to those with JAK2-mutation. CALR type 1 is strongly associated with male gender compared to the JAK2.	63.7% of double-negative and 56% of JAK-negative ET were CALR positive. 60.3% and 31% of double-negative ET CALR were type 1 and type 2, respectively. 34%, 17%, and 5% of JAK2-negative ET CALR were type-1, type-2, and other types, respectively.	ET with CALR mutation had ↓ Hgb, ↓ WBC, & ↑ Plt than mutated JAK2.	CALR mutation had no impact on thrombotic complications.	ET with CALR mutation had a higher risk of progression to MF than mutated JAK2.	CALR-mutated ET had better overall survival than JAK2, in particular patients of age 60 years or younger.
Pietra et al, 2016 [17]	908 ET & 374 PMF	CALR-mutated ET was younger compared to those with JAK2-mutation.	24% of ET and 26% of PMF had CALR mutation. 65%, 32%, and 3% of CALR mutations were type-1 like, type-2 like, and others, respectively.	CALR type 1-like mutations were mainly associated with a Myelofibrosis phenotype, while CALR type 2 was preferentially associated with the ET phenotype.	Despite higher platelet count, CALR type 2 mutation in ET had a lower risk of thrombosis than mutated JAK2 or type 1-like CALR mutation.	ET with type 1-like CALR mutations were mainly associated with a significantly higher risk of myelofibrotic transformation but no impact on leukemic transformation.	Type 1-like CALR-mutated PMF, but not ET, had better overall survival than JAK2. The median follow-up period was 4.7 years (range 0–31 years).
Kong et al, 2016 [22]	3,141 ET and 1,605 PMF (Meta-analysis)	-	19% of ET and 22% of PMF had CALR mutation.	-	-	ET with CALR mutations had a slight trend toward fibrotic transformation but no impact on leukemic transformation.	-
Tefferi et al, 2017 [18]	709 PMF	CALR type 1 is associated with male gender. Type 2 is associated with younger age.	16% and 3.4% of PMF carried CALR type 1-like and CALR type 2-like mutations, respectively.	PMF with CALR type 2-like mutation had ↓ Hgb, ↑ WBC, ↑ Plt, & ↑ circulating blasts than mutated JAK2.	-	CALR mutation did not impact the progression rate to Myelofibrosis or the risk of leukemic transformation. CALR mutations had the lowest red cell transfusion need.	CALR-mutated PMF, but not ET, had better overall survival than JAK2. The median follow-up period was 3.5 years.
Kim et al, 2022 [21]	510 ET & MF	-	6.2% of ET and 3.3% of PMF had CALR mutation. 49% and 51% of CALR mutations were type 1-like and type 2-like, respectively.	CALR type 1-like mutations were associated with a Myelofibrosis phenotype (↓ Hgb & ↓ Plt), while CALR type 2-like was associated with ET phenotype (↑ Plt & ↑ ANC).	Despite higher platelet counts, CALR type 2 mutation in ET had no thrombotic events.	-	-

CALR: Calreticulin; MPN: Myeloproliferative Neoplasm; ET: Essential Thrombocytopenia; P/MF: Primary Myelofibrosis; Hgb: Hemoglobin; WBC: White Blood Cell; Plt: Platelet; JAK2: Janus kinase 2; EPO: erythropoietin; pre-MF: Prefibrotic Myelofibrosis; ANC: Absolute Neutrophilic Count

In this retrospective study, we evaluated 50 patients diagnosed with MPN who tested positive for CALR mutations with a median follow-up period of three years. Whether CALR mutations are more common in ET or PMF is a question that is not fully answered yet, as shown in Table 3. More than half of our study population were ET patients; the rest were either PMF or Pre-MF (62%, 20%, and 18%, respectively). CALR type 1 mutation was more common than type 2 (48% vs. 42%), which is consistent with most of the available literature [6, 11, 12, 15–18]. The incidence of thrombotic events recorded in our study is quite low, which supports the known overall low risk of thrombosis in CALR-mutated MPNs. We did not find statistically significant differences in myelofibrotic transformation between the different CALR mutations. However, the number of events leaned towards a higher risk with CALR type 1 rather than the others. This important finding can be examined in detail with more extensive studies in the future.

In our study, we had five patients (10%) who carried CALR mutations that were none Type 1 or Type 2. Such mutations are scarce, and little is known about their clinical significance. It is worth mentioning that despite the few patients who carry such a mutation type in our sample, we had a statistically significant increase in cardiac events. This raises concerns about their potential higher cardiac thrombotic risk and fatal outcomes. An initial presentation of myocardial infarction has been reported in the literature in this particular population [23]. It is interesting to note that one of those five patients had a concurrent JAK2 mutation, and diagnosed with pre-MF. The patient was a young female who developed portal vein thrombosis. Based on our review, we could not find a similar reported case of pre-MF in the literature. Given the rarity of CALR mutations other than Type 1 and Type 2, this area remains a topic with many unmet needs including its co-existence with other mutations.

To date, we do not have a treatment that targets CALR mutations. MPNs are treated the same, whether they carry CALR mutations or not, with cytoreductive agents [24–26]. This is also true in our study, as both groups were treated with Hydroxyurea, JAK2 inhibitor, and antiplatelet agents, regardless of the mutation status. While such interventions might provide some clinical response, developing a targeted therapy is obviously the standard of care and will be an active field for research in the coming years.

It is unknown whether CALR mutations have malignant transformation roles. CALR is a chaperon protein that is largely present in the endoplasmic reticulum (ER). ER activity is increased in proliferating malignant cells leading to tumor progression and preventing cell death signaling. This process involves the utilization of ER chaperon proteins to promote the protein folding capacity and maintain ER homeostasis [27]. In our study population, secondary malignancy was diagnosed in three patients carrying CALR type 1 mutation, which can open the door for more research to investigate this finding further.

Our study is unique since it has the largest sample size for research conducted in this geographic region, showing a descriptive analysis of CALR mutation. In addition, It involves a relatively young population (80% below the age of 60), and more than half of them are Arab (54%). Furthermore, the differential occurrence of MF transformation and secondary malignancy raises questions about the possible involvement of CALR type 1 mutations in these processes. We believe that the small sample size is the major limitation of our study, as it is reflected in the statistical significance of most of our results. Conducting prospective or randomized-controlled trials can help to provide answers regarding some of the interesting points raised in our paper. In addition, this study did not investigate the response to the treatment modalities used in managing CALR-mutated patients.

Authorship contributions

Conception and design of the study: Dr. M. Najim, Dr. M. Abu-Tineh, Dr. M. Yassin.

Methodology: Dr. A. Alshurafa, Dr. M.Ibrahim.

Validation: Dr. M. Najim, Dr. M. Abu-Tineh, Dr. M. Yassin

Acquisition of data: Dr. S. Ansari, Dr. H. Faraj, Dr. S. Alateeg. Dr. S. Akiki.

Drafting the manuscript: Dr. M. Najim, Dr. M. Abu-Tineh, Dr. S. Akiki.

Revising the manuscript for intellectual content: Dr. M. Najim, Dr. M. Abu-Tineh,

Approval of the version of the manuscript to be published: All authors have read and agreed to the published version of the manuscript

Supervision: Dr. M. Yassin

Statement of ethics

The study was approved by Hamad Medical Corporation Medical Research center.

Acknowledgment

We acknowledge the contributions of cytogenetic and molecular lab for their significant Contribution.

Data availability statement

All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

Open Access funding provided by the Qatar National Library.