Clinical and Hematological Relevance of JAK2V617F, CALR, and MPL Mutations in Vietnamese Patients with Essential Thrombocythemia

Background: The picture of Vietnamese patients with essential thrombocythemia (ET) remains mostly undetermined. Our study intended to determine the frequency of JAK2V617F, CALR exon 9, and MPL exon 10 mutations as well as to analyze clinical characteristics associated with different mutational status in Vietnamese ET patients. Methods: We explored mutations of JAK2V617F, MPL, and CALR from 395 patients using allele specific oligonucleotide – polymerase chain reaction and Sanger sequencing techniques; then, the clinical and hematological features were compared according to mutation patterns. Results: We found that JAK2V617F, CALR exon 9, and MPL exon 10 mutations were present in 56.2%, 27.6%, and 1% of the 395 patients with ET, respectively. Twelve different types of CALR mutation were detected in 109 patients, with the CALR type 1 mutation (c.1099_1150del; L367fs*46) was the most common, followed by CALR type 2 mutation (c.1154_1155insTTGTC; K385fs*47). The JAK2V617F-positive patients had older age, higher white blood cell counts and higher hemoglobin levels but lower platelet counts than patients with CALR mutations or patients negative for triple tests. There was no significant difference regarding sex ratio, white blood cell counts, platelet counts and hemoglobin levels among CALR mutation subtypes. Conclusion: we reported high frequency of JAK2V617F, CALR, and MPL mutations in Vietnamese patients with ET and underscored the importance of combined genetic tests for diagnosis and classification of ET into different subtypes.


Introduction
higher risk of myelofibrotic transformation, especially in cases with CALR type 1 mutation (Pietra et al., 2016).
To the best of our knowledge, the characteristics of Vietnamese patients with ET remains mostly undetermined. In this study, we investigated the profiles of JAK2V617F, MPL, and CALR mutations in Vietnamese ET patients using allele specific oligonucleotide -polymerase chain reaction (ASO-PCR) and conventional Sanger sequencing method. The clinical and hematological features were compared according to mutation patterns.

Patients and samples
This was a retrospective study of 395 patients diagnosed with ET between 2008 and 2017 at Blood Transfusion and Hematology Hospital at Ho Chi Minh City, Vietnam. The diagnosis of ET was established based on the 2008 WHO diagnostic criteria (Campo et al., 2011). In brief, patient was diagnosed with ET when he/she had thrombocytosis, megakaryocyte proliferation, and did not meet WHO criteria for other MPNs, myelodysplastic syndrome (MDS) or myeloid neoplasm. Clinical and hematological findings at diagnosis were obtained by reviewing the medical records. Written informed consents for mutation analyses were obtained from patients enrolled in this study. Genomic DNA was extracted from peripheral blood samples using the GeneJET Genomic DNA Purification Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer's instruction.

Mutation analysis
All primers used in this study were newly designed. All 395 samples were assessed for JAK2V617F status using ASO-PCR technique. Genomic DNA was amplified in a 35-cycle PCR reaction at an annealing temperature of 60 o C using three primers. The reaction contained 25 -50 ng of genomic DNA, 1X PCR Buffer, 200 µM each dNTP, 0.5 U Taq Hot Start Polymerase (Takara Bio, Shiga, Japan), 0.2 µM common forward primer, 0.1 µM each of reverse primers. The mutant allele showed two bands at 453 base pairs (bp) and 279 bp, while the wild-type allele had only one band at 453 bp. Primers were as follows: reverse wild-type -specific primer, 5'-attgctttcctttttcacaagat-3'; reverse mutant -specific primer, 5'-gttttacttactctcgtctccacaaaa-3'; and common forward primer, 5'-tcctcagaacgttgatggcag-3'.
Patients with non-mutated JAK2V617F were further evaluated for CALR exon 9 and MPL exon 10 mutations using Sanger sequencing method. The CALR exon 9 was amplified with primers CALR-F (5'-gaaaccctgtccaaagcaag -3') and CALR-R (5'-agagacattatttggcgcgg-3'); while MPL exon 10 was amplified with primers MPL-F (5'-tttgggtcaaacagacgctg-3') and MPL-R (5'-cacagagcgaaccaagaatg-3). Each reaction consists of 1X PCR Buffer, 1.5 mM MgCl2, 200 µM each dNTP, 0.5 U Taq Hot Start Polymerase (Takara Bio), 0.1 µM each forward and reverse primers, and 25 -50 ng of genomic DNA. PCR involved an initial denaturation at 98°C for 3 min followed by 40 cycles of 98°C for 10 sec, 60°C for 30 sec, and 72°C for 1 min with a final elongation of 72°C for 5 min. PCR products were checked for size and purity using 1.5% agarose gel electrophoresis. PCR products were purified enzymatically using ExoSAP IT™ PCR Product Cleanup Reagent (Thermo Scientific) for removal of excess primers and dNTPs prior to Sanger sequencing using a BigDye Terminator v3.1 Kit and ABI 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). PCR fragments were sequenced and analyzed in both directions.

Statistical analysis
The clinical and hematological findings were summarized by each of the four groups of mutational status (JAK2, CALR, MPL, and triple-negative) and were compared between each pair of these groups using two-sided Fisher's exact test for categorical variables and Mann-Whitney U test for numeric variables, where appropriate. The thrombotic-event-free survival rate was described by mutational status using Kaplan-Meier estimate. Statistical significance was defined as P-value less than 0.05. All statistical analyses were performed using the statistical software R version 3.4.4.

Baseline clinical characteristics and prevalence of mutation
Among 395 patients diagnosed with ET, the follow-up duration ranged from 1 to 13 years, with the median length of follow-up of 3 years. The baseline clinical characteristics are shown in Table 1. There were more females than males (249/146). The median age was 54 years and more than 75% of the patients were middle-aged or older. There were 34 patients (8.6%) with history of arterial thrombotic diseases. According to the IPSET-thrombosis risk score, 130 patients (32.9%) had high risk and 112 patients (28.4%) had intermediate risk of thrombosis. The laboratory data showed normal median values of red blood cell (RBC) counts, hemoglobin (HGB) concentration, and white blood cell (WBC) counts. The median platelet count was 1037 × 10 9 /L. There were also high values of megakaryocytes, lactate dehydrogenase (LDH), and serum uric acid.
Among four different types of MPL exon 10 mutations detected (S505N, W515K, W515L and W515S), S505N was reported as a founder mutation in several pedigrees with familial thrombocytosis (Ding et al., 2004). However, it was also found as an acquired somatic mutation in rare ET patients (Beer et al., 2008;Vainchenker and Kralovics, 2017). In this study, the patient carrying MPLS505N was an 80-year-old man with HGB level of 10.2 g/dL, platelet count of 1,421 x 10 9 /L, and WBC count of 5.71 x 10 9 /L at diagnosis. He had history of thrombotic event and developed secondary bone marrow fibrosis three years after diagnosis.
Among JAK2V617, CALR and triple-negative groups, we showed here that triple-negative ET patients were the youngest, quite similar to previous studies (Al Assaf et al., 2015;Ojeda et al., 2018). Also, consistent with previous reports, patients with JAK2 mutations had significantly higher HGB level, higher RBC and WBC counts, and higher thrombosis risk score, but lower platelet counts compared with patients with CALR mutations or triple-negative for mutations (Al Assaf et al., 2015;Ojeda et al., 2018;Rumi et al., 2014;Tefferi et al., 2014).
When comparing the clinical and hematological findings among CALR type 1, CALR type 2, and other CALR types, we found that three CALR mutation groups were similar in their sex ratio, IPSET-thrombosis risk score, HGB level and WBC counts. In a similar cohort study of 402 ET patients, Tefferi et al concluded that patients with CALR type 2 had significantly higher platelet count compared with CALR type 1 (Tefferi et al., 2014). In our study, patients with CALR type 2 showed a tendency of higher platelet counts; however, there was no significant difference among these three CALR mutation groups (Table 3).
Within a 3-year median time of follow-up, thrombotic events and mortality were rare, with two death cases and ten thrombotic events. Long-term follow-up is required to further explore thrombotic events and mortality rate based on mutational groups. A limitation of our study is that this was a single-center retrospective study with a limited sample size. Therefore, only four patients harbored MPL mutations detected, with two of them experiencing thrombotic events. Although the prevalence of thrombotic events in the MPL mutation group was higher than in other groups, we could not conclude that this group was associated with worse outcome due to small number of patients.
In conclusion, this study is the first comprehensive investigation of gene mutations in Vietnamese patients with ET. The combined genetic tests can clarify approximately 85% of the ET patients with JAK2V617F, CALR exon 9, and MPL exon 10 mutations, which might improve the diagnosis and classification of ET in Vietnam.