The effects of mutational profiles on phenotypic presentation of myeloproliferative neoplasm subtypes in Bosnia: 18 year follow-up

Keywords: MPN, myeloproliferative neoplasm, JAK2, janus kinase 2, CALR, calreticulin, MPL, myeloproliferative leukemia virus, mutant allele burden

Abstract

The identification of mutually exclusive somatic mutations shared among myeloproliferative neoplasm (MPN) subtypes has provided a powerful tool for studying disease evolution. Clinical features, gene mutations, and survival over 18 years were analyzed in MPN patients. One hundred thirty-eight MPN patients were subcategorized according to MPN subtypes: essential thrombocythemia (ET, n = 41), polycythemia vera (PV, n = 56), primary myelofibrosis (PMF, n = 10), and MPN unclassified (MPN-U, n = 31). Patient characteristics included clinical parameters, overall survival (OS), and mutational status of the Janus kinase 2 (JAK2), calreticulin (CALR), and myeloproliferative leukemia virus oncogene (MPL) genes. We compared hematologic and clinical features of JAK2V617F-ET vs. CALR-mutated ET vs. JAK2V617F-PV patients. JAK2V617F-patients had higher values of erythrocytes, hemoglobin, and hematocrit compared to CALR-mutated patients (p < 0.05). The mutant allele burden in JAK2V617F-PV and JAK2V617F-ET patients directly correlated with erythrocyte, hemoglobin, and hematocrit values, but it inversely correlated with platelet count. Thus, mutant allele burden was an indicator of the clinical phenotype in JAK2V617F-MPN patients. OS was not affected by the mutational status. In general, mutated JAK2, CALR, and MPL genes left specific hematological signatures.

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The effects of mutational profiles on phenotypic presentation of myeloproliferative neoplasm subtypes in Bosnia: 18 year follow-up
Published
2020-05-01
How to Cite
1.
Kurtovic-Kozaric A, Islamagic E, Komic H, Bilalovic N, Eminovic I, Burekovic A, Uzunovic A, Kurtovic S. The effects of mutational profiles on phenotypic presentation of myeloproliferative neoplasm subtypes in Bosnia: 18 year follow-up. Bosn J of Basic Med Sci [Internet]. 2020May1 [cited 2021Oct.21];20(2):236-47. Available from: https://www.bjbms.org/ojs/index.php/bjbms/article/view/4391
Section
Translational and Clinical Research

INTRODUCTION

Myeloproliferative neoplasms (MPNs) are a group of clonal myeloid disorders that affect normal blood cell production in the bone marrow [1-3]. According to the 2016 revision of the World Health Organization (WHO) classification of tumors of hematopoietic and lymphoid tissues, MPNs are categorized into chronic myeloid leukemia (BCR-ABL1 positive), polycythemia vera (PV), primary myelofibrosis (PMF, prefibrotic/early and overt fibrotic stage), essential thrombocythemia (ET), chronic eosinophilic leukemia (CEL), chronic neutrophilic leukemia (CNL), mastocytosis, and unclassifiable MPNs (MPN-U) [4-9]. The most common BCR-ABL1 negative MPNs include PV, ET and PMF, while the other subtypes are rare [4]. These diseases have shared clinical features and molecular basis [10]. Around 5% of patients suffer from progression to more advanced disease, including transformation to acute myeloid leukemia (AML) [11].

The WHO 2016 revision lists the presence of driver mutations as one of several major criteria in the diagnosis of these diseases [12]. BCR-ABL1 negative MPN patients carry driver mutations in the JAK2 (Janus kinase 2), CALR (calreticulin), and MPL (myeloproliferative leukemia virus oncogene) genes [13,14]. A high proportion of MPN patients (75%) carries the unique JAK2V617F mutation in exon 14; subsequently, exon 12 mutations were found in 5% of patients with PV [15]. Somatic MPL exon 10 mutations include W515L and W515K and were first described in 2006 [16]. In 2013, CALR mutations were found in nonmutated JAK2 and MPL ET and PMF patients [17]. In fact, about 80% of CALR-mutated patients harbor one of two mutually exclusive mutation variants: type 1 (52-bp deletion) or type 2 (5-bp TTGTC insertion) [18].

Overall, the incidence of the mutations within individual subtypes of MPN is: PV (98% of patients with JAK2 mutations), ET (60% JAK2, 22% CALR, and 3% MPL) and PMF (58% JAK2, 25% CALR, and 7% MPL) [12,13]. JAK2V617F PV patients have significantly higher mutant allele burden (MAB) in comparison to JAK2V617F ET patients [19]. Mutually exclusive somatic mutations in the JAK2, CALR, and MPL genes gave a new insight into the pathogenesis and diagnostics of MPN.

Even though the disease presentation might be different, recent work has shown that JAK2V617F ET and PV are diseases that evolve from a single neoplasm [10]. On the other hand, ET with CALR mutations is a separate disease category, both clinically and molecularly, from JAK2V617F ET. Thus, JAK2 and CALR mutations determine the evolution of the MPN subtype. The aim of this study was to evaluate disease phenotypes and evolution in regards to the detected JAK2, CALR, and MPL mutations in a population of MPN patients in Bosnia and Herzegovina. We compared hematologic and clinical phenotypes of ET patients carrying either JAK2 or CALR mutations with JAK2V617F PV patients to determine the correlation of clinical features and disease evolution.

MATERIALS AND METHODS

Study population

A cohort of 138 patients diagnosed with MPN and treated at the Department of Hematology, Clinical Center of the University of Sarajevo and the Department of Internal Medicine, Cantonal Hospital Zenica, in the period January 2000–June 2018 was included in this study. Procedures performed in the study were in accordance with the 1964 Helsinki Declaration. Diagnoses of PV, ET, PMF, and MPN-U were made according to the WHO 2008 or 2016 classification criteria for hematopoietic malignancies, according to the criteria at the time of diagnosis [4]. Briefly, 24 patients were diagnosed before 2008, 57 patients were diagnosed between 2008 and 2016, and 56 patients were diagnosed after 2016. All major and minor criteria for MPN diagnosis according to the WHO classification were collected, except for erythropoietin (EPO) levels which had not been determined routinely for all patients. Analyzed patients were subcategorized according to BCR-ABL1 negative MPN subtypes: ET (n = 56), PV (n = 41), PMF (n = 10), and MPN-U (n = 31). Patient characteristics included complete blood count (CBC), bone marrow morphology, hepato/splenomegaly, and overall survival (OS).

JAK2, MPL, and CALR mutation analysis

All molecular analyses were performed at the Clinical Center of the University of Sarajevo, Department of Clinical Pathology, Cytology and Human Genetics, Laboratory for Human Genetics. Genomic DNA was extracted from patient peripheral blood using standard protocol (Qiagen QIAamp® DNA mini kit, USA). JAK2 mutational analysis and MAB were performed by quantitative PCR (qPCR) using a quantitative allelic discrimination assay (Ipsogen MutaQuant and MutaScreen kit, Qiagen, USA). JAK2V617F positive patients were not tested for CALR and MPL mutation.

JAK2WT patients were tested for CALR mutations in exon 9. Primers for allele-specific oligonucleotide (ASO)-PCR were designed to detect mutations type 1 and 2 in one reaction, as previously described [20]: F1 5′-GCA GCA GAG AAA CAA ATG AAG G-3′, F2 5′-GCA GAG GAC AAT TGT CGG A-3′ and R 5′-AGA GTG GAG GAG GGG AAC AA-3′ (Invitrogen, Thermo Fisher Scientific, USA). Double negative patients (JAK2 and CALR) were sequenced to detect mutations, type 1 (W515L, 1544G>T) or type 2 (W515K, 1543_1544TG>AA), in MPL gene (exon 10). We used primers for the amplification of 212 bp region; F 5′-TGG GCC GAA GTC TGA CCC TTT-3′ and R 5′-ACA GAG CGA ACC AAG AAT GCC TGT-3′ (Invitrogen, Thermo Fisher Scientific, USA).

Statistical analysis

Numerical variables were presented by their range and median, and categorical variables by count and relative frequency (%) of each category (Tables S1 and 1). Comparisons of quantitative variables between groups of patients were carried out by the nonparametric Wilcoxon rank-sum test. Correlation between numerical variables was tested by the nonparametric Spearman’s ρ (ρ) coefficient.

TABLE S1: Clinical characteristics and mutational status of 138 MPN patients at diagnosis in Bosnia and Herzegovina
TABLE 1: Demographic, hematologic, and clinical features at diagnosis of patients with PV, ET, and PMF, subdivided according to JAK2, CALR, or MPL mutational status

OS was estimated using the Kaplan–Meier method, and survival curves were compared by the log-rank test. Survival probabilities were estimated with the Kaplan–Meier method and compared using the log-rank test. Data were analyzed using IBM SPSS Statistics for Windows, Version 21.0 (IBM Corp., Armonk, NY, USA). Statistical significance was determined at the level p < 0.05 (2-tailed).

RESULTS

Clinical characteristics

We included 138 MPN patients in this study, diagnosed with PV (n = 41), ET (n = 56), PMF (n = 10), and MPN-U (n = 31). Patient characteristics are shown in Table S1, which reports clinical parameters and mutational status of each MPN subtype at diagnosis. Median follow-up was 33 months (60, 45.5, 69, and 20 months for PV, ET, PMF, and MPN-U subtypes, respectively). Age at diagnosis among different MPN subtypes did not show significant differences (p > 0.05). Regarding mutational analysis, three genes were analyzed: JAK2, CALR, and MPL. JAK2V617F mutation was found in 71% of all MPN patients (Table S1); CALR mutations, type 1 and 2, were detected in 13% of all MPN patients; MPL mutations, type 1 (W515L) and type 2 (W515K), were found in 4% of all MPN patients. Table 1 shows clinical parameters of each MPN subtype at diagnosis according to the presence of JAK2V617F, CALR, and MPL mutations.

Mutational status vs. CBC in PV patients

Hematologic parameters in PV patients compared to mutational status (JAK2V617F and JAK2WT) are presented in Figure 1. Among PV patients, the presence of JAK2V617F mutation was associated with higher platelet count (p < 0.05). However, no effect was found on clinical parameters such as white blood cell (WBC), red blood cell (RBC), hemoglobin (Hb), and hematocrit (Hct). WBC and RBC values were higher in JAK2V617F PV patients, although the difference was not significant.

FIGURE 1: Hematologic parameters in JAK2V617F and JAK2WT PV patients. Data are presented in a box and whisker plot showing the upper and lower values (highest and lowest horizontal line, respectively) and upper and lower quartile (box) with median value. Patients with JAK2V617F PV had markedly higher platelet count values (p < 0.05) than patients without JAK2V617F mutation. WBC and RBC levels were higher in JAK2V617F PV. JAK2: Janus kinase 2; PV: Polycythemia vera; WBC: White blood cell; RBC: Red blood cell.

Mutational status vs. CBC in ET patients

Hematologic parameters in ET patients compared to mutational status (JAK2V617F, JAK2WT, CALR-mutated, CALRWT, MPL-mutated, and MPLWT) are presented in Figure 2. RBC levels were higher in JAK2V617F compared to JAK2WT ET patients. Between JAK2V617F vs. CALR-mutated ET patients, JAK2V617F mutation was present in patients with lower platelet count (p < 0.05). We also categorized ET patients based on the presence or absence of driver mutations (triple negative vs. patients with driver mutations) and found no significant differences in hematologic parameters.

FIGURE 2: Hematologic parameters in JAK2V617F, JAK2WT, CALR-mutated, CALRWT, MPL-mutated, MPLWT triple-negative ET patients, and ET patients with driver mutation. Data are presented in a box and whisker plot showing the upper and lower values (highest and lowest horizontal line, respectively) and upper and lower quartile (box) with median value. JAK2V617F ET patients had markedly higher RBC values (p < 0.05) than JAK2WT patients and CALR-mutated patients had significantly higher platelet count compared to JAK2V617F ET patients. JAK2: Janus kinase 2; CALR: Calreticulin; MPL: Myeloproliferative leukemia virus oncogene; ET: Essential thrombocythemia; RBC: Red blood cell.

Since PV and ET patients share JAK2V617F mutation, we compared the clinical parameters among JAK2V617F ET vs. JAK2V617F PV patients; we found that RBC, Hb, and Hct values were higher in PV patients, but platelet count values were lower; however, the frequency of splenomegaly was higher (p < 0.001). Interestingly, we found the same results when we compared JAK2V617F PV patients and CALR-mutated ET patients (p < 0.05).

Mutational status vs. CBC in PMF patients

Hematologic parameters in PMF patients compared to mutational status are shown in Figure S1. The presence of either JAK2V617F or CALR mutation did not have an impact on the analyzed clinical parameters (p > 0.05). When we compared JAK2V617F PMF, ET, and PV patients, we found that JAK2V617F PV patients had higher RBC (p < 0.001), Hb, and Hct (p < 0.05) values; JAK2V617F PMF patients had higher WBC values compared to JAK2V617F PV and ET patients. A comparison between CALR-mutated PMF vs. CALR-mutated ET patients showed that CALR-mutated PMF patients had lower Hb (p < 0.05) and platelet (Plt) values (p < 0.001).

FIGURE S1: Hematologic parameters in JAK2V617F, JAK2WT, CALR-mutated, CALRWT, MPL-mutated, MPLWT, triple-negative PMF patients, and PMF patients with driver mutation. Data are presented in a box and whisker plot showing the upper and lower values (highest and lowest horizontal line, respectively) and upper and lower quartile (box) with median value. JAK2V617F PMF patients had higher RBC, WBC, Hb values, and platelet count than JAK2WT patients, and CALR-mutated PMF patients, even though the difference was not statistically different (p > 0.05). JAK2: Janus kinase 2; CALR: Calreticulin; MPL: Myeloproliferative leukemia virus oncogene; PMF: Primary myelofibrosis; RBC: Red blood cell; WBC: White blood cell; Hb: Hemoglobin.

Mutational status vs. CBC in MPN-U patients

The values of hematologic parameters in MPN-U patients compared to mutational status are presented in Figure S2. Platelet count was statistically different between JAK2V617F and JAK2WT MPN-U patients. A comparison among JAK2V617F MPN-U, PV, ET, and PMF patients showed that JAK2V617F MPN-U patients had higher RBC and Hb values (p < 0.05) and lower WBC values (p < 0.05) than JAK2V617F PMF patients, and lower Hb values than JAK2V617F PV patients (p < 0.05). In JAK2V617F MPN-U patients, platelet count was lower than in JAK2V617F ET patients (p < 0.05).

FIGURE S2: Hematologic parameters in JAK2V617F, JAK2WT, CALR-mutated, CALRWT, triple-negative MPN-U patients, and MPN-U patients with driver mutation. Data are presented in a box and whisker plot showing the upper and lower values (highest and lowest horizontal line, respectively) and upper and lower quartile (box) with median value. JAK2V617F MPN-U patients had higher RBC and platelet count than JAK2WT MPN-U patients, and CALR-mutated MPN-U patients, even though the difference was not statistically different (p > 0.05). JAK2: Janus kinase 2; CALR: Calreticulin; MPN-U: Unclassifiable MPNs; RBC: Red blood cell.

Driver gene MPN patients

We hypothesized that the presence of specific mutations in JAK2, CALR, or MPL gene is the driver of MPN pathogenesis. Thus, we combined data for all PV/ET/PMF/MPN-U patients that carried JAK2V617F mutation. Accordingly, we organized data for CALR and MPL positive MPN patients (Figure 3). We found that JAK2V617F patients had higher values for RBC, Hb, and Hct compared to CALR-mutated patients (p < 0.05). MPL-mutated patients had higher values for Plt compared to JAK2V617F patients (p < 0.05).

FIGURE 3: PV/ET/PMF/MPN-U patients are categorized according to detected driver gene mutation (JAK2, CALR, and MPL). Presence of driver mutation in JAK2 gene had an impact on higher RBC and Hb values (*) compared to CALR-mutated patients and MPL mutation had an impact on higher Plt values (**) compared to JAK2-mutated patients. PV: Polycythemia vera; ET: Essential thrombocythemia; PMF: Primary myelofibrosis; MPN: Myeloproliferative neoplasm; MPN-U: Unclassifiable MPNs; JAK2: Janus kinase 2; CALR: Calreticulin; MPL: Myeloproliferative leukemia virus oncogene; RBC: Red blood cell; Hb: Hemoglobin; Plt: Platelet.

Allele burden

We assessed the MAB in 45 JAK2V617F MPN patients. The median JAK2V617F MAB at diagnosis was significantly lower in JAK2V617F ET than in PV patients (21% vs. 40%, p < 0.05). In other words, in 12% of JAK2V617F ET patients, the MAB was higher than 50%, compared to 47% of PV patients.

A correlation between hematologic parameters (RBC, Hb, Hct, and Plt) and MAB was calculated for JAK2V617F PV vs. JAK2V617F ET patients, as illustrated in Figure 4. A direct correlation was found between the MAB and RBC; however, Hb and Hct were directly correlated and Plt count was inversely correlated with MAB, but without statistical significance. These findings suggest that the MAB is an indicator of the phenotypic presentation of JAK2V617F MPN.

FIGURE 4: Relationship between JAK2V617F allele burden and hematologic parameters in ET and PV patients. The mutant allele burden was directly correlated with RBC values (p = 0.362, p < 0.05), Hb level (p = 0.140, p > 0.05), and hematocrit (p = 0.206, p > 0.05), and inversely correlated with Plt count (p = -0.12, p > 0.05). JAK2: Janus kinase 2; ET: Essential thrombocythemia; PV: Polycythemia vera; RBC: Red blood cell; Hb: Hemoglobin; Plt: Platelet.

Overall survival

Kaplan–Meier estimates were performed to generate and analyze survival-time data. We compared survival rates among different MPN subtypes (PV, ET, PMF, and MPN-U). At 120 months, OS for PV, ET, PMF, and MPN-U patients was 80%, 90%, 100%, and 77%, respectively. Also, all MPN patients were categorized according to the presence of driver mutation (JAK2V617F, CALR-mutated, and MPL-mutated). Interestingly, JAK2V617F patients had worse survival compared to CALR-mutated and MPL-mutated patients, even though significant differences were not found (p > 0.05). The presence of JAK2V617F mutation in PV patients did not confer better survival. No significant differences in OS were found among ET patients who were triple negative or carried a driver mutation (Figure S3).

FIGURE S3: Kaplan–Meier estimates of overall survival rate in (A) MPN subtypes; (B) PV, ET, PMF and MPN-U patients categorized according to presence of driver mutation (JAK2V617F, CALR-mutated, and MPL-mutated); (C) PV patients with and without JAK2 mutation; and (D) triple-negative and ET patients with detected driver mutation (JAK2V617F, CALR-mutated, and MPL-mutated). Statistically significant values regarding survival rate were found only for comparison among different MPN subtypes (p < 0.05). MPN: Myeloproliferative neoplasm; PV: Polycythemia vera; ET: Essential thrombocythemia; PMF: Primary myelofibrosis; MPN-U: Unclassifiable MPNs; JAK2: Janus kinase 2; CALR: Calreticulin; MPL: Myeloproliferative leukemia virus oncogene.

DISCUSSION

MPN driver mutations in the JAK2, CALR, and MPL genes upregulate the JAK-STAT signaling pathway [21]. Ectopic expression of JAK2V617F stimulates proliferation of erythroid progenitor in EPO-dependent manner, delays final erythropoiesis due to low expression of erythroid-related genes, and activates abnormal STAT signaling through the activation of signal transducer and activator of transcription 1 (Stat1) protein [22]. JAK2, MPL, and CALR mutations in BCR-ABL1 negative MPN patients are usually mutually exclusive [23]. The discovery of these genes in MPN pathogenesis has led to the introduction of targeted therapy [23]. Therefore, it has become crucial to test all BCR-ABL1–negative MPN patients for these mutations for management and prognosis of the disease [24-27].

When we analyzed blood parameters such as RBC, WBC, Hb, Hct and Plt of all MPN patients regardless of the disease subtype, we found the following: RBC count was higher in JAK2V617F PV patients vs. CALR-mutated ET patients, suggesting that the presence of JAK2V617F helps RBC production. Furthermore, when we compared JAK2V617F PV vs. JAK2V617F ET vs. JAK2V617F PMF patients, we found that the highest RBC values were in JAK2V617F PV patients. Similarly, higher JAK2V617F expression (allele burden) was associated with increased RBC values in JAK2V617F PV vs. JAK2V617F ET patients, supporting the hypothesis that higher JAK2V617F expression leads to increased RBC values.

Higher platelet counts were associated with the presence of CALR mutation, similar to other published studies [23,28,29]. When we compared CALR-mutated ET patients vs. JAK2V617F ET patients, we found that CALR mutation is associated with increased platelet counts. Similarly, our results showed that CALR-mutated ET patients had higher platelet counts than CALR-mutated PMF patients, suggesting that there may be an additional factor that increases platelet count in ET patients.

Previous studies have hypothesized that JAK2V617F, CALR, and MPL are the drivers of MPN pathogenesis [10,30-33]. Since JAK2V617F is present in both PV and ET patients, we compared their blood parameters. Regarding platelet count, JAK2V617F ET patients vs. JAK2V617F PV patients had higher platelet values, which suggests that even though there is no CALR mutation, there might be an additional factor that increases platelet values in ET patients, differentiating them from PV patients [34]. Similar to our results, Rumi et al. [10] showed that JAK2V617F MPN patients had lower platelet counts than CALR-mutated patients. Also, the same study found that hematologic parameters of JAK2V617F ET and JAK2V617F PV patients were associated with the MAB, leading them to conclude that JAK2V617F PV and ET present distinct phenotypes of a single MPN (JAK2V617F MPN), whereas CALR-mutated ET is another disease category.

We found that within JAK2V617F disease WBC was higher in PMF than PV patients, which could imply that there is an additional factor in JAK2V617F PMF patients that increases WBC count.

Regarding OS, we found that CALR-mutated patients had higher survival rates compared to JAK2V617F patients, which is similar to the results published by Tefferi et al. and Kourie et al. [23,35]; however, statistical significance was not reached. ET patients had better survival than PV patients and MPN-U patients (p < 0.05). The presence of JAK2V617F in PV patients did not confer better survival compared to JAK2WT PV patients. Similarly, triple negative ET patients did not have worse survival compared to mutation-positive ET patients (p > 0.05). In another study performed by Tefferi et al., triple negative status in PMF patients did not show additional prognostic information for OS [36].

Acknowledgements

ACKNOWLEDGMENTS

We would like to thank Esad Kurtovic for support. We would also like to thank Adis Muhic, Zana Halilovic, and Eldina Hodzic for technical assistance. We would like to thank Sabina Halilovic for reading the manuscript.

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Conflict of interest statement: The authors declare no conflict of interests