| Abstract|| |
Introduction: Somatic mutation in the exon 14 of Janus Kinase 2 gene is an established diagnostic marker in bcr-abl negative myeloproliferative neoplasms, especially primary idiopathic myelofibrosis (PIMF). Aim: Our primary aim was to find out the correlation between the JAK2V617F mutational status and the clinico-hematologic characteristics, as well as the international prognostic scoring system (IPSS) scoring of patients with PIMF. Materials and Methods: Clinical and hematologic features were reviewed for 68 patients with primary idiopathic myelofibrosis (PIMF). JAK2V617F mutation status was analyzed by amplification refractory mutation screening-polymerase chain reaction. The patients were further stratified into low, intermediate-1, intermediate-2 and high-risk groups on the basis of IPSS scoring. Results: The JAK2V617F mutation was detected in 58.8% patients. Univariate analysis of variables at presentation identified that JAK2V617F negative patients were significantly associated with more severe anemia (P = 0.045), younger age (P = 0.008), higher transfusion requirement (P = 0.017), and thrombocytopenia (P = 0.015). Patients who were homozygous for JAK2V617F mutation were associated with thrombocytosis (P = 0.014) and also had higher median total leucocyte count (P = 0.20) than the other groups. No significant correlation was detected between JAK2V617F mutational status and the presence of constitutional symptoms, spleen size, grade of bone marrow fibrosis or prognostic risk stratification of the PIMF patients. Conclusion: The variations in the prognostic implication of PIMF patients with mutation status as stated by various publications worldwide, reinstates the need for larger prospective studies using standardized JAK2V617F quantification methods as well as estimation of other newer molecular markers to develop deeper insight into various molecular alterations involving PIMF patients in India as well as worldwide.
Keywords: Janus Kinase 2 mutation, polymerase chain reaction, primary myelofibrosis
|How to cite this article:|
Singh N, Sazawal S, Upadhyay A, Chhikara S, Mahapatra M, Saxena R. Correlation of JAK2V617F mutational status in primary myelofibrosis with clinico-hematologic characteristics and international prognostic scoring system scoring: A single center experience. Indian J Pathol Microbiol 2015;58:187-91
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Singh N, Sazawal S, Upadhyay A, Chhikara S, Mahapatra M, Saxena R. Correlation of JAK2V617F mutational status in primary myelofibrosis with clinico-hematologic characteristics and international prognostic scoring system scoring: A single center experience. Indian J Pathol Microbiol [serial online] 2015 [cited 2019 Jun 20];58:187-91. Available from: http://www.ijpmonline.org/text.asp?2015/58/2/187/155311
| Introduction|| |
An acquired "gain of function" mutation in exon 14 of the Janus Kinase 2 (JAK2V617F) is found in around 60% of patients with primary idiopathic myelofibrosis (PIMF). This mutation represents a major criterion for diagnosis according to the 2008 World Health Organization (WHO) classification of myeloid neoplasms. , However, it is not specific for this disorder and is shared by other Philadelphia-negative myelo-proliferative neoplasms such as polycythemia vera (PV), essential thrombocythemia (ET), and refractory anemia with ringed sideroblasts and thrombocytosis.  JAK2V617F mutated genotype as well as the differences in their V617F allele burden, has been variably associated with unique clinico-hematologic characteristics in patients with PV or ET. However, such information in the setting of PIMF is limited to few studies. Several retrospective studies have tried to evaluate the correlation between JAK2V617F mutation and disease phenotype in PIMF subjects and more importantly, the disease outcome in terms of overall survival (OS) and leukemia-free survival (LFS) but the results of all these studies have been inconsistent. Few studies found that JAK2V617F homozygous patients had poorer survival while others proclaimed that patients with lower allele burden or unmutated status were associated with adverse prognostic outcome.
In view of this, our primary aim was to determine the correlations if any, between the JAK2V617F mutation status or the V617F allele burden and the prognostic risk stratification of the PIMF patients as per the International Prognostic Scoring System (IPSS). We also tried to determine whether there was some association between the JAK2V617F mutation status and the specific clinical and laboratory parameters in our subset of Indian patients, as no such study has been published from India till date.
| Materials and Methods|| |
This retrospective study involved both the newly diagnosed as well as the follow-up patients of PIMF, satisfying the 2008 WHO criteria, who presented in the Hematology OPD during the period of 2012-2013. Patients were eligible for the study if their blood samples for JAK2V617F genotyping had been collected at the time of presentation, before the institution of any therapy. Written informed consent was taken from all the patients, and ethical clearance was obtained before the conduct of this study.
Cases of secondary myelofibrosis developing in the setting of prior history of PV or ET and other causes of myelofibrosis such as hematologic neoplasms and auto-immune diseases were excluded from the study. These patients were further stratified into low, intermediate-1, intermediate-2 and high-risk groups on the basis of the IPSS, which includes age >65 years, total leucocyte count (TLC) >25 10 3 /L, hemoglobin <10 g/dl, blast percentage ≥1, and presence of constitutional symptoms. Dynamic IPSS Plus scoring could not be applied in our patients due to nonavailability of the results of conventional cytogenetics in all patients. Reverse transcriptase-polymerase chain reaction (PCR) for bcr-abl was performed in all these patients to exclude the possibility of chronic myeloid leukemia. Information was also obtained regarding the grade of bone marrow fibrosis by reticulin staining on bone biopsies, presence or absence of constitutional symptoms, transfusion dependence, spleen size, and baseline laboratory parameters like hemoglobin, total leukocyte count and platelet count etc., at the time of presentation. JAK2V617F mutation status, whether negative, heterozygous or homozygous was also compiled.
The presence of JAK2V617F mutation was assessed by DNA tetra-primer based amplification refractory mutation screening-polymerase chain reaction (ARMS-PCR) exactly as described by Jones et al.  Granulocytes were separated from peripheral blood (10 ml), and genomic DNA was extracted using conventional gradient centrifugation technique. ARMS-PCR was performed using 2 primer pairs to amplify the normal and mutant sequences and a positive control band in a single reaction. Each 25 ul reaction contained approximately 25 ng DNA template, 2.5 ul 10 buffer (final concentration of Mgcl2: 1.5 mM) 1.0 U Taq polymerase (Roche diagnostics, GmbH Germany) and 200 uM deoxynucleoside triphosphates (dNTPs; Roche diagnostics, GmbH Germany). The final concentration of outer primers was 1 uM, and the mutant/wild type-specific inner primer was 0.5 uM. PCR cycling parameters were: One cycle of 94°C for 3 min; 30 cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 1 min; followed by one cycle of 72°C for 10 min. The amplification of PCR product was confirmed by electrophoresis on an ethidium bromide-impregnated 2% agarose gel [Figure 1].
|Figure 1: Agarose gel analysis for the detection of JAK2V617F mutation in genomic DNA by amplification refractory mutation screening-polymerase chain reaction assay. Lane 1, 4, 5, 6 and 8 show a normal genotype, Lane 2 and 3 show a single mutant band (279 bp) and were scored as homozygous for the mutation; Lane 7 showed mutant band of similar intensity to the normal band and was therefore scored as heterozygous; the Lane 9 is negative control and M is 100 bp ladder. Arrows indicate the product of amplification|
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Data were analyzed using Stata 12 [Stata Corp 4905, Texas, USA] and presented in mean (standard deviation), median (inter-quartile-range) and frequency (percentage). Categorical variables were analyzed using Chi-square or Fisher exact test as applicable. Continuous data were compared among groups by one-way ANOVA, Kruskal-Wallis equality-of-population rank test or Wilcoxon rank-sum test, as applicable. P < 0.05 was considered statistically significant.
| Results|| |
Sixty-eight patients of bcr-abl negative PIMF were enrolled in our study, out of which 28/68 (41.2%) were JAK2-negative while the rest 40/68 (58.8%) were JAK2-positive. Information regarding the mutational status of JAK2V617F positive cases was available in only 27/40 patients. Of these 27 patients, twelve were heterozygous (44.4%), and 15 were homozygous (55.6%). Their demographic and baseline characteristics have been shown in [Table 1].
Several clinical and laboratory parameters were compared among the JAK2 negative, heterozygous, and homozygous groups. Univariate analysis of variables at presentation identified that JAK2 negative patients were significantly associated with more severe anemia (P = 0.045), younger age (P = 0.008), higher transfusion requirement (P = 0.017), and thrombocytopenia (P = 0.015). Patients who were homozygous for JAK2 mutation were associated with thrombocytosis, which was statistically significant (P = 0.014) and also had higher median TLC (P = 0.20) than the other groups [Table 2]. No significant correlation was detected between JAK2V617F mutational status and the presence of constitutional symptoms, spleen size or grade of bone marrow fibrosis in PIMF patients. Most JAK2-negative patients belonged to intermediate to high-risk categories while homozygosity was more commonly found to be associated with low to intermediate-1 risk groups but we could not find any statistically significant association in this regard (P = 0.64; χ2 = 0.90). None of these patients had episodes of thrombosis, and only one patient progressed to acute myeloid leukemia. Majority of our patients were continued on cytoreductive therapy with hydroxyurea apart from dexamethasone and lenalidomide combinations in few patients, due to financial constraints. Two patients underwent splenectomy while two others were taken for up-front bone marrow transplantation. The follow-up duration in all these patients ranged from 6 months to 7 years.
|Table 2: Comparison of the clinico-hematologic characteristics and prognostic risk stratification of the three broad groups|
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| Discussion|| |
Primary myelofibrosis is associated with a worse prognosis in comparison with other classic BCR/ABL-negative chronic myeloproliferative neoplasms. , However, survival may range from a few months to an excess of a decade, especially in younger patients. Therefore, identification of variables associated with prognosis is of considerable importance for driving therapeutic decisions, particularly in the case of hematopoietic stem cell transplantation, which is the only potentially curative therapeutic approach. , The discovery of the JAK2V617F mutation and its association with distinct hematologic and clinical characteristics has prompted further research into assessing the predictive value of its mutational status or allele burden on prognostic outcome or progression free survival in myeloproliferative neoplasms in general and PIMF in particular. Overall prevalence of JAK2V617F mutation in PIMF patients in our subset of Indian population was found to be 60%, similar to the reported data worldwide. Moreover, homozygosity was commoner among our JAK2V617F positive patients (55.6%), which correlates with a previous publication from our institution, reinstating the fact that ours being a developing country, patients may be presenting later in the course of disease. 
In numerous studies, the JAK2V617F allele has been variably associated with higher indices of erythropoiesis, unchanged or decreased platelet count, increased bone marrow fibrosis, older age, and longer duration of disease. ,,, We found no significant correlation of leukocytosis, higher blast percentage, spleen size, presence of constitutional symptoms, and grade of bone marrow fibrosis with JAK2V617F mutational status. However, there was statistically significant association between homozygosity and thrombocytosis (P = 0.014) and also of JAK2V617F negative clone with more severe anemia, younger age group and increasing transfusion dependence (P = 0.017) in PIMF patients. Our results were partially similar to the Korean study by Ha et al.  who found no statistically significant correlation of mutational status with any of the clinical and laboratory parameters discussed above. The study by Tefferi et al.  also showed some similarities with our study; they found older age group, normal to high platelet count and blast percentage <3% to be associated with positive JAK2V617F mutational status. Another study by Guglielmelli et al.  was also in line with our observations. They proclaimed that anemia was more common in patients with wild-type JAK2V617F allele and that the JAK2V617F mutated group showed a significant correlation with erythrocytosis, leucocytosis, and thrombocytosis. They also found that there was no statistically significant impact of presence or absence of mutation on spleen size, blast percentage, grade of bone marrow fibrosis, and constitutional symptoms. There were clear-cut differences in our observations from that of Barosi et al. 2007  who stated that the JAK2V617F heterozygous state favored a higher platelet count whereas the homozygous state was more commonly linked to hyperproliferative profile substantiated by larger splenomegaly, and greater need of cytoreductive therapy. There is a possibility for some differences in the observations of the various studies due to ethnic or geographic distribution, which needs to be elucidated with further clinical trials. We did not perform the quantitative estimation of the JAK2 V617F allele burden and so were unable to make any comment on the correlation of lower or higher allele burden with any of the clinico-hematologic parameters or more so, the prognostic outcome of patients, although some clue in this context can be achieved by the fact that homozygosity corresponds to >50% of JAK2V617F mutant allele burden.
The question of a possible impact of JAK2V617F mutation on risk stratification or OS of the patients with PIMF has not been clearly answered till date as different publications provide conflicting results. In our study, the mutational status of JAK2V617F was not found to significantly correlate with the IPSS score or progression-free survival of the patients, although there was a slight predilection of unmutated JAK2V617F status for intermediate-2 to high-risk categories. Furthermore, we found no significant difference between homozygous and heterozygous patients in terms of prognostic scoring. Our results were similar to few other published studies  involving larger subset of patients, who showed that the presence of JAK2V617F mutation was not associated with either the prognostic score or LFS. Similarly, in the study by Guglielmelli et al.,  they found that the number of JAK2V617F mutated patients in the low-risk categories was significantly higher than in the unmutated group. Few others like Tefferi et al.,  have found correlation between a lower allele burden with reduced OS, although they also supported one of our inferences that mere presence or absence of this mutation does not carry any prognostic relevance. They inferred that a lower allele burden or unmutated status may possibly be overwhelmed by a more dominant JAK2V617F negative clone with higher propensity to undergo clonal evolution.
We differed remarkably from the results of Barosi et al.  who proclaimed a more aggressive clinical phenotype with homozygosity for JAK2V617F mutation. They postulated that dysregulation of homologous recombination leading to loss of heterozygosity, over-expression of oncogenes such as LMO2 due to phosphorylation of histone proteins and MDM2 mediated suppression of p53 proto-oncogene may contribute to severity of disease in homozygotes.
On this basis, it is clear that there is marked variation in the results of different studies conducted worldwide, for reasons still unknown. Recent research has shown that the clinical heterogeneity of myelofibrosis patients is closely linked to their genetic complexity, , because in addition to JAK2V617F, many other genetic and epigenetic abnormalities seem to play a crucial role in the biology of this myeloproliferative disease like MPL, CALR, TET2, ASXL1, EZH2, IDH, RUNX1 etc. This brings us to the impression that the study of just one gene is not enough to develop a complete insight into the pathogenesis and prognostication of PIMF patients.
The major limitations of our study were the smaller sample size and relatively shorter duration of clinical follow-up which precluded any strong impression about the OS and LFS of our patients. Furthermore, correlation with IPSS scoring could not identify the patients with extremely poor prognosis, including key biologic characteristics, such as karyotypic and molecular aberrations.
| Conclusions|| |
The current knowledge indicates that JAK2 mutational status or the V617F allele burden has a role in clinical phenotype and disease evolution. These correlations have been consistently observed in clinical practice, but it would be premature to consider them as surrogate for prognostication in PIMF patients. The variations in the prognostic implication of PIMF patients with mutation status as stated by various publications worldwide call for the need for larger prospective studies. Study of well-defined population of patients using standardized JAK2V617F quantification methods as well as estimation of other newer molecular markers is required to develop deeper insight into various molecular alterations involving PIMF patients and their prognostic significance in India as well as worldwide.
| Author Contributions|| |
NS collected clinical details, analyzed the data, and drafted the manuscript. SS coordinated the study and participated in its design. AU performed statistical analysis. SC performed experimental studies. MM and RS provided valuable clinical inputs, and reviewed the paper. All authors read and approved the final manuscript.
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Dr. Renu Saxena
Department of Hematology, All India Institute of Medical Sciences, New Delhi - 110 029
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2]