
Essential thrombocythemia (ET) is an acquired chronic myeloproliferative neoplasm (MPN) that basically involves the megakaryocytic lineage. It is characterized by constant elevation of peripheral blood platelet count and increased numbers of large, mature megakaryocytes in the bone marrow and clinically by the tendency to thrombosis or hemorrhage [1]. To diagnose patients with ET, other reactive causes of high platelet count must be excluded such as inflammatory or infectious diseases, hemorrhage, and other MPNs. However, a major insight into the genetic lesions for the enhanced myeloproliferation and clonal dominance that characterizes the Philadelphia-negative MPNs including polycythemia vera (PV), primary myelofibrosis (PMF), and ET has been reported [2]. Mutations that disrupt the protein tyrosine kinase signaling are found to be associated with these disorders [3]. The Janus Kinase 2 gene (
This cross-sectional study was conducted following approval by the local institutional ethical committee (approval No. 59/6.2019). Informed consent was obtained from all enrolled subjects. A total of 73 patients diagnosed with ET at Hiwa Hemato-Oncology Hospital, Sulaymaniyah Province, Kurdistan Region of Iraq were included in this study between June to November 2019. The patients were identified as ET based on the 2016 WHO diagnostic criteria. Briefly, the patients were diagnosed as ET when they had unexplained thrombocytosis, megakaryocytic proliferation, and did not meet WHO criteria for other MPNs. Bone marrow aspiration and biopsy was performed for all patients through posterior iliac spine and the aspirate smears were stained with Leishman stain (Merck/Darmstadt, Germany). A Trephine biopsy specimen of 1.5 cm in length placed in 10% buffered neutral formalin and sent to histopathology laboratory. Following overnight fixation, biopsy specimens were slowly decalcificated with ethylene diamine tetra acetate (EDTA). Three micrometer thick sections were cut from the paraffin-wax blocks with the rotary microtomes and stained with H&E. Figure 1 through 4 show the characteristic megakaryocyte in patients with ET. Clinical and hematological information were collected by reviewing the medical records. Genomic DNA for the analysis of the three driver mutations was isolated from peripheral blood samples using a fully automated magnetic bead nucleic acid extraction system (Zinexts, Taiwan). The Zinexts DNA extraction kit was used for the separation of genomic DNA by using 400 μL of whole blood with a final 100 μL DNA elute according to the manufacturer’s instruction. The isolated DNA was stored at −20°C until the time of analysis by real-time PCR with LineGene9600PLUS (Bioer, China). Five different fluorescence dyes attached to the DNA probe running in four different channels were used to detect wild type alleles, mutant type alleles for the three examined genes, and the internal control in each patient. For the
Newly designed primers were used in the current study. All 73 patients were examined for
PCR program for the detection of JAK2 V617F, MPL W515L, and CALR type 1 & 2 mutations
PCR steps | Temperature (°C) | Time | Cycle |
---|---|---|---|
Pre-heating | 50 | 2 minutes | 1 |
Pre-denaturation | 95 | 10 minutes | 1 |
Denaturation | 95 | 25 seconds | 40 |
Annealing, elongation and detection | 59 for JAK2 V617F 58 for MPL W515L 60 for CALR type 1 & 2 | 25 seconds 30 seconds 20 seconds |
Sequences of oligonucleotide primers and probe for the detection of JAK2 V617F mutation
Primer | Direction | Length | Sequence | Modification |
---|---|---|---|---|
JAK2-F | Forward | 24 bp | 5′- CTTTCTTTGAAGCAGCAAGTATGA -3′ | - |
JAK2-WR | Reverse wild type allele | 30 bp | 5′ -GTAGTTTTACTTACTCTCGTCTCCACATAC -3′ | - |
JAK2-MR | Reverse mutant type allele | 30 bp | 5′ - GTAGTTTTACTTACTCTCGTCTCCACATAA -3′ | - |
JAK2 Probe | Probe | 30 bp | 5’-TGAGCAAGCTTTCTCACAAGCATTTGGTTT-3’ | Hex-BHQ |
Sequences of oligonucleotide primers and probe for the detection of MPL W515L mutation
Primer | Direction | Length | Sequence | Modification |
---|---|---|---|---|
MPL-F | Forward mutant type allele | 20 bp | 5′- TGGTGACCGCTCTGCATCTA -3′ | - |
MPL-R | Reverse mutant type allele | 16 bp | 5′ - AGTGTGCAGGAAACTG -3′ | - |
MPL Probe | Probe | 18 bp | 5’- CTGGGCCTCAGCGCCGTC -3’ | Cy5-BHQ |
Sequences of oligonucleotide primers and probe for the detection of CALR type 1 & 2 mutations
Primer | Direction | Length | Sequence | Modification |
---|---|---|---|---|
CARL-F | Forward primer | 21 bp | 5′- TGGTCCTGGTCCTGATGTCGG -3′ | - |
CARL-R | Reverse primer | 21 bp | 5′ - CCCAAATCCGAACCAGCCTGG -3′ | - |
CARL-M1 probe | Deletion mutant probe | 22 bp | 5′ - CGAGGAGCAGAGGACAAGGAGG -3′ | FAM-BHQ |
CARL-M2 probe | Insertion mutant probe | 25 bp | 5’- TTGTCGGAGGAAGATGAGGAGGAAG-3’ | TexasRed-BHQ |
Sequences of internal control (template, probe and primer)
Primer | Direction | Length | Sequence | Modification |
---|---|---|---|---|
IC-F | Forward | 21 bp | 5′- CCA AGT GTG AGG GCT GAA AAG -3′ | - |
IC-R | Reverse | 26 bp | 5′ -TGT TGT GGC TGA TGA ACT ATA AAA GG -3′ | - |
IC-Probe | Probe | 26 bp | 5′ - CCC CAG TCT CTG TCA GCA CTC CCT TC -3′ | Cy5.5-BHQ |
IC-Template | DNA Template | 80 bp | 5’-CCA AGT GTG AGG GCT GAA AAG AAT GCC CCA GTC TCT GTC AGC ACT CCC TTC TTC CCT TTT ATA GTT CAT CAG CCA CAA CA-3’ | - |
The statistical package SPSS version 25 (IBM Corp., Armonk, NY, USA) was used to analyze the data: The continuous data are summarized as the means (SD) and ranges. The Chi-Square test was applied to compare the differences in the categorical data. An independent t-test was used for differences in continuous data.
As demonstrated in Table 6, out of 73 patients with ET, 37 (50.7%) patients carried
Frequency of the three driver mutations in 73 patients with ET
Driver Mutations | No. | % |
---|---|---|
JAK2 V617F |
37 | 50.7% |
CALR type 1 & 2 | 10 | 13.7% |
MPL W515L | 7 | 9.6% |
Triple negative | 19 | 26.0% |
Total | 73 | 100.0% |
aIncluding the combined cases; JAK2 V617F+ CALR Type 1 & 2=5 cases, JAK2 V617F+ MPL W515L=4 cases, JAK2 V617F+ CALR Type 1 & 2+ MPL W515L=1 case.
Main clinical and hematological features of the 73 patients with ET
Parameters | Value |
---|---|
Gender No. (%) | |
Male | 37 (50.7) |
Female | 36 (49.3) |
Age (years) | |
Mean (SD) | 55 (17) |
Range | 12∼89 |
Leukocytes (×109/L) | |
Mean (SD) | 9.5 (3.2) |
Range | 4.0∼15.3 |
Hemoglobin (g/dL) | |
Mean (SD) | 13.2 (1.3) |
Range | 11.0∼15.6 |
Platelets (×109/L) | |
Mean (SD) | 949 (387) |
Range | 443∼1900 |
Correlation of the three mutational status with the main clinical and hematological characteristics in 73 Iraqi Kurdish patients with ET
Characteristics | JAK2 1 |
CALR 2 |
MPL 3 |
Com. Mut 4 |
Trip-ve 5 |
1 v 2 |
1 v 3 |
1 v 4 |
1 v 5 |
2 v 3 |
2 v 4 |
2 v 5 |
3 v 4 |
3 v 5 |
4 v 5 |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
No. of Pt | 27 | 10 | 7 | 10 | 19 | ||||||||||
Sex M/F | 14/13 | 7/3 | 2/5 | 7/3 | 7/12 | 0.32 | 0.27 | 0.32 | 0.31 | 0.09 | 1.00 | 0.09 | 0.09 | 0.67 | 0.08 |
Age, years(range) | 56 (27∼89) |
61 (36∼80) |
53.5 (12∼70) |
59 (40∼72) |
48.1 (18∼79) |
0.09 | 0.76 | 0.63 | 0.15 | 0.39 | 0.72 | 0.06 | 0.51 | 0.51 | 0.09 |
WBC, ×109/L(range) | 12 (8.0∼15.3) |
6.6 (4.0∼10.0) |
7.5 (5.0∼10.0) |
12 (7.0∼15.0) |
6.8 (5.0∼11.2) |
0.000 | 0.000 | 0.99 | 0.000 | 0.41 | 0.000 | 0.81 | 0.001 | 0.39 | 0.000 |
Hb, g/dL(range) | 14.2 (12.0∼15.6) |
12.8 (11.0∼15.0) |
11.7 (11.0∼13.0) |
14.07 (13.0∼15.0) |
12.1 (11.0∼14.0) |
0.007 | 0.000 | 0.75 | 0.000 | 0.09 | 0.01 | 0.13 | 0.000 | 0.28 | 0.000 |
Platelets, ×109/L(range) | 1084 (600∼1820) |
867 (550∼1271) |
696 (464∼1357) |
1049 (600∼1900) |
841 (443∼1723) |
0.11 | 0.02 | 0.81 | 0.04 | 0.23 | 0.22 | 0.86 | 0.06 | 0.40 | 0.19 |
Abbreviations: Pt, Patients; Com Mut, combined mutations; Trip –ve, triple-negative; v, versus.
This is the first study to characterize the genotype profile of Iraqi Kurdish patients with ET. Using real-time PCR with specific primers and probes for the three driver mutations, we found that 54/73 (74%) of the ET patients carried
In the current study, no statistically significant difference in age and gender was observed among different genotypes. This finding is in agreement with earlier reports [4, 5, 7, 8, 10, 16, 20]. Similar to previous reports, patients with mutated
In conclusion, our data reveal that the mutation profile in ET patients in our locality is in line with earlier reports. Further our findings demonstrate similarities with these records in concern to hematological features of patients with ET. However, we notice a rather higher frequency of
We greatly thank the Laboratory staff of Sulaymaniyah Hiwa Hemato-oncology Center for their enormous participation and cooperation. Very special thanks and appreciation to management staff of the center for their tremendous role, fruitful ideas, advices, and collaboration.
None
Saeed BM1, Researcher; Getta HA2, M.D.; Khoshnaw N1, M.D.; Abdulqader G2, M.D.; Abdulqader AMR2, M.D.; Mohammed AI2, M.D.