2.1 Clinical Characteristics of Enrolled Patients
The JAK2V617F mutation was identified in 96 of 159 cases (60.3%), the CALR mutation in 59 cases (37.1%), and the MPL mutation in 2 cases (1.3%); 2 cases (1.3%) were TN. Due to low number of MPL and TN mutated cases, they were not used in cohort study, and only input the data. Age of presentation was lower in CALR mutated patients (52.25 ± 13.79 years) than JAK2V617Fmutated patients (63.93 ± 10.95 years) (t = 5.82, p = 0.000), there were no significant difference in gender.
Compared to those in patients with JAK2 mutations, leukocyte count (9.70 ± 4.57 × 109/L versus 12.86 ± 6.91 × 109/L; t = 3.11, p = 0.002) and Hb levels (130.17 ± 12.25 g/L versus 149.22 ± 19.30 g/L, t = 6.78, p = 0.000) and RBC count (4.37 ± 0.51 × 1012/L versus 4.97 ± 0.83 × 1012/L; t = 4.97, p = 0.000) were lower in patients with CALR mutation. Plt count was higher in patients who had CALR mutations, (942.98 ± 376.10 × 109/L versus 824.01 ± 263.10 × 109/L; t=-2.31, p = 0.022). The serum level of LDH was similar (335.06 ± 154.43 U/L versus 378.07 ± 164.81 U/L; t=-2.31, p = 0.096).
Among the 14 patients with thrombotic events, three (5%) had a CALR mutation, other 11 patients with JAK2 mutation (11.5%)(p = 0.03)(Table 1).
Among the CALR frame shift mutations, 27 patients had typical type 1 deletions (c.1099-1150del52; p.L367fs*46), and 32 patients had type 2 insertions (c.1154-1155insTTGTC;p.K385fs*47). There were no significant differences regarding age, count of leukocytes, RBC, PLT, levels of Hb, the serum levels of D-dimer and LDH between patients with type 1 or type 2 mutations (P༞0.05).
Compared with patients with JAK2V617F ( Table 1 ), patients with CALR1 or CARL2 mutations were younger (t=-3.814, P = 0.000; t=-5.579, P = 0.000), and presented lower RBC count (t=-3.274, P = 0.000; t=-4.107, P = 0.000), Hb level (t=-4.397, P = 0.001; t=-5.693, P = 0.000), respectively. But lower WBC counts (t=-2.996, P = 0.003) was found in patients with CARL2 mutation. And higher PLT count was only found in patients with CALR1 mutation (t = 3.138, P = 0.002).
No statistical differences were observed for the serum levels of LDH, and D-dimer between patients with CALR1 or CARL2, and JAK2V617F mutations (P༞0.05).
Table 1
Clinical and Laboratory characteristics of 155 Patients with essential thrombocythaemia
Variable | CALR | JAK2V617F |
CALR1 | CALR2 |
Male/Female | 11/16 | 13/19 | 50/46 |
Age (years) | 54.37 ± 13.31 | 50.47 ± 14.14 | 63.93 ± 10.95 |
WBC count (× 109/L) | 10.55 ± 5.08 | 8.99 ± 4.04 | 12.86 ± 6.91 |
RBC count (× 1012/L) | 4.41 ± 0.63 | 4.34 ± 0.39 | 4.97 ± 0.83 |
HB(g/L) | 131.48 ± 15.32 | 129.06 ± 9.01 | 149.22 ± 19.30 |
PLT count (× 109/L) | 1022.52 ± 373.47 | 875.88 ± 370.81 | 824.01 ± 263.10 |
LDH(IU/L) | 369.89 ± 92.16 | 386.63 ± 208.72 | 335.06 ± 154.433 |
D-dimer | 0.31 ± 0.27 | 0.25 ± 0.10 | 0.42 ± 0.47 |
Thrombotic events | 1 | 2 | 11 |
2.2 BM Biopsy Histopathological Section
BM Biopsy of the JAK2-mutated cases with ET showed active proliferation of granulocytic, erythroid and megakaryocytic lineages, a total of 15,714 megakaryocytes were observed in the study, the largest diameter of megakaryocyte was 49.77 ± 11.22 um, median 48.39 um (range 35.34 ~ 86.48 um). BM Biopsy of the CALR mutated cases were only showed active proliferation of megakaryocytic lineages, total number of megakaryocyte were 4750, and the largest diameter of megakaryocyte was 46.65 ± 8.02um, median 47um (range 30.36 ~ 70.76 um), there were no significant differences among these two genotypes (p = 0.16) (Table 2). In addition, patients with JAK2 mutations had a higher median number of megakaryocytes (16.65 ± 5.11/HPF vs 8.05 ± 2.77 /HPF, P = 0.001) and median number of clusters of megakaryocytes (2.83 ± 0.60 vs 0.48 ± 0.64/HPF, P = 0.001)(Fig. 1,Fig. 2). There were no significant differences among fibrosis 1(10% vs 14.4%,P = 0.14).
Table 2
Number and histological features in patients with essential thrombocythemia according to genotype
Variable | CALR | JAK2V617F | P Value |
Largest diameter of megakaryocyte | 46.65 ± 8.02 | 49.77 ± 11.22 | 0.16 |
Median number of megakaryocytes per HPF in BM | 8.05 ± 2.77 | 16.65 ± 5.11 | 0.001 |
Median number of clusters of megakaryocytes per HPF in BM | 0.48 ± 0.64 | 2.83 ± 0.60 | 0.001 |
Fibrosis M1 | 4 | 11 | 0.14 |
3.Discussion
It is well known that the type of gene mutation can identify different sub-types of ET.
Previous study [3, 8, 9] showed that the age of presentation was the highest in JAK2-mutated patients, it was same that the majority of patients are older than 60 in JAK2 mutated patients in this study. It was reported that the patients with ET who had CALR mutation showed a preference for males, the highest Plt count, the lowest leukocytosis, Hb level, LDH level, the lowest number of thrombotic events and cardiovascular events, low risk of transformation to myelofibrosis and leukemia, and long-term survival rates and better prognosis[3, 10].
In our study, the CALR mutated patients was younger than the JAK2 mutated patients. The ET patients with CALR mutation were also associated with normal WBC count and RBC count, but higher Plt count.
In addition, patients with CALR1 mutation or CALR2 mutation with lower leukocyte counts and hemoglobin levels than those with JAK2 mutation, in accordance with previous reports [10]. The patients with CALR1 and CALR2 mutations displayed the similar leukocyte counts, hemoglobin levels, but patients with CALR1 mutation revealed higher platelet counts than those with JAK2 mutation. These findings were not consistent with the report by Guo et al [10], it was reported patients with CALR2 mutation exhibited lower WBC counts and higher platelet counts than patients with JAK2 mutation. These observations indicate that CALR mutation and JAK2 mutation exert different effect on leukocyte and erythroid cell. Patients with CALR1 mutation displayed higher platelet counts than those with JAK2 mutation. These findings indicate that it is worth further bone marrow pathology to see the count and distribution of megakaryocytes.
The most overt abnormalities in BM biopsy was increased quantity and volume of megakaryocytes (more than 13/HPF), JAK2V617F- mutated had high BM cellularity, the highest number of dysmorphic megakaryocytes, and few “staghorn” megakaryocytes [11]. CALR-mutated ET showed a reduced BM cellularity, many clusters of large megakaryocytes and only a few dysmorphic megakaryocytes[12];
Megakaryocytes exhibited specific morphological features in the various categories of MPNs. ET exhibited giant megakaryocytes, abundant mature large cytoplasm, staghorn, hyperlobated nuclei in dense clusters[13, 14].In this study, the most prominent feature of megakaryocytes in bone marrow biopsy is the hyperproliferation of megakaryocytes, which is characterized by a large number of large cell bodies with abundant cytoplasm, multi-lobulated nuclei and different sizes. The majority of megakaryocytes are mature megakaryocytes, and megakaryocytes are pleomorphic. in accordance with previous reports [13, 14]. No statistical differences were observed for diameter range of megakaryocytes between ET patients with CALR and JAK2 mutations. CALR mutations group presented loose and rare clusters of megakaryocytes, JAK2 mutations group showed dense clusters of megakaryocytes, and the median number of megakaryocytes and median number of clusters of megakaryocytes per HPF in JAK2-mutated ET were significantly higher than that CALR-mutated ET. and no statistical differences were observed for diameter range, the median number of megakaryocytes and median number of clusters of megakaryocytes per HPF of megakaryocytes between ET patients with CALR1 and CALR2 mutations. However, these findings were not consistent with the report by Achille Pich [12], this difference in genetic background is probably associated with the different population, these should be certainly verified in large clinical study.
Patients with ET who had CALR mutations had slightly higher ratios of progression to post -ET myelofibrosis than did patients with JAK2 mutations, but this difference was not statistically significant [4, 15]. In this study, none of the patients had fibrosis or leukemia transformation during follow-up. In our data, no correlation analysis was performed because of the small number of patients with MPL-mutated and TN ET.
The limitations of this study are the retrospective nature of the study, but the differential diagnosis of this disease can be confirmed by observing and analyzing morphological characteristics and distribution of the megakaryocytes in Bone morrow (BM)biopsy.