Detection of Jak2 V617f Mutation, Secondary to the Presence of Bcr-Abl1 Translocation in a Patient with Chronic Myeloid Leukemia: Report of a Case and Review of the Literature

The Myeloproliferative Neoplasms (MPN) are classified as four major diseases: Chronic Myeloid Leukemia (CML), Polycythemia Vera (PV), Primary Myelofibrosis (PMF) and Essential Thrombocythemia (ET) [1]. The translocation t(9;22)(q34;q11) produces the Philadelphia chromosome and causes the BCR-ABL1 transcript; this alteration is commonly found in CML [1]. In 2008 the World Health Organization (WHO) reported that MPN patients with the BCR-ABL1 transcript (Philadelphia positive) should be classified as chronic myeloid leukemia, and those who are Philadelphia negative (BCR-ABL1 negative) should be classified as Polycythemia Vera (PV), Essential Thrombocytosis (ET ) or myelofibrosis (PMF) [1]. JAK2 is a tyrosine kinase gene that has a role in the signaling pathways of hematopoietic factors. The JAK2 V617F mutation is found in 95% of PV patients, and 50% of ET and PMF patients [2,3]. Previously, it was thought that the JAK2 V617F mutation and BCR-ABL1 translocation were mutually exclusive. However a few rare cases have been reported that are positive for both alterations [4-17]. We report the first case in Central American with the presence of JAK2 V617F mutation, in a patient with the diagnosis of Philadelphia positive chronic myeloid leukemia.


Introduction
The Myeloproliferative Neoplasms (MPN) are classified as four major diseases: Chronic Myeloid Leukemia (CML), Polycythemia Vera (PV), Primary Myelofibrosis (PMF) and Essential Thrombocythemia (ET) [1]. The translocation t(9;22)(q34;q11) produces the Philadelphia chromosome and causes the BCR-ABL1 transcript; this alteration is commonly found in CML [1]. In 2008 the World Health Organization (WHO) reported that MPN patients with the BCR-ABL1 transcript (Philadelphia positive) should be classified as chronic myeloid leukemia, and those who are Philadelphia negative (BCR-ABL1 negative) should be classified as Polycythemia Vera (PV), Essential Thrombocytosis (ET ) or myelofibrosis (PMF) [1]. JAK2 is a tyrosine kinase gene that has a role in the signaling pathways of hematopoietic factors. The JAK2 V617F mutation is found in 95% of PV patients, and 50% of ET and PMF patients [2,3]. Previously, it was thought that the JAK2 V617F mutation and BCR-ABL1 translocation were mutually exclusive. However a few rare cases have been reported that are positive for both alterations [4][5][6][7][8][9][10][11][12][13][14][15][16][17]. We report the first case in Central American with the presence of JAK2 V617F mutation, in a patient with the diagnosis of Philadelphia positive chronic myeloid leukemia.

Patients' samples
We collected blood samples from patients with a diagnosis of myeloproliferative neoplasm. The samples were collected from patients referred from different national hospitals. Every patient signed the informed consent form approved by the local ethics committee. The technique used for extracting leukocytes from blood was via ammonium lysis. Cells obtained by this technique were stored at -20ºC.

Case report information
All the information analyzed in this case report was given by the patient and the participant hospitals. The diagnosis of the BCR-ABL1 transcript was made by FISH by other national laboratory.
RNA AND DNA EXTRACTION: The RNA was extracted by the trizol (Life technologies, NY, USA) method according to the manufacture's procedures. The DNA was extracted using the Purelink Genomic DNA Minikit (Invitrogen, NY, USA). The RNA and DNA quantitation was made by fluorometry, using the quantifluor equipment and method (Promega, WI, USA).

JAK2 V617F Allele specific-PCR
The reaction was standardized according to the thermal cycler used. The primers used were described by Jones et al. and Chen, et al. [18,19]. The PCR conditions included 10 minutes at 94°C, 30 cycles each of 30 seconds at 94°C, 45 seconds at 54°C, 1 minute at 72°C, and a final extension of 5 minutes at 72°C.

BCR-ABL1 real time PCR quantitation
The real time PCR was made according the Molecular MD One- Step qRT-PCR BCR-ABL kit procedures. (Molecular MD, Portland OR, USA).

Case presentation
In November, 2010 a Guatemalan man of 68 years presented with a problem of thrombosis in one finger. Initial laboratory tests showed that the white cell count was 22.7×10 3 /ml with 86.8% neutrophils, 6.9% lymphocytes, 2.2% monocytes 3.7% eosinophils and 0.4% basophils. The red blood count was 7.12×10 6 ml, the hemoglobin was 18 mg/ml, the HCT was 55.50% and the platelets count was 6.97×10 5 mL. Bone marrow analysis was compatible with a chronic myeloproliferative process. FISH analysis revealed the chromosome translocation t(9;22) (q34;q11). From this, the patient was diagnosed as chronic myeloid leukemia BCR-ABL1-positive. The patient was treated with 400 mg.
Imatinib, and then the dose was increased to 800 mg since there was not a complete hematologic response. The patient reached hematologic remission, and remained there, until January 2013, at which time the patient had a hematological relapse.
Molecular monitoring of BCR-ABL1 copy numbers, began in November 2011 (one year after diagnosis) and was at 0.06% IS (international system), which means a major molecular response (MMR) to therapy; unfortunately there were no monitoring of BCR-ABL1 prior to this date. Four additional BCR-ABL1 monitoring were conducted periodically, and all showed a complete molecular response. The last monitoring was performed in April 2013 (Figure 1). Note that the patient has no detectable copies BCR-ABL1, yet still has clinically  We concluded that the alteration that caused the loss of complete hematologic response was the JAK2 V617F mutation, secondary to the presence of BCR-ABL1 transcript in a patient with chronic myeloid leukemia.
In Table 1, is shown a summary of the similar reported cases. There is a wide clinical variety of the co-existence of BCR-ABL1 and JAK2 mutations. Some patients present the both alterations at diagnosis and some present one first and then the other. Only four cases reported the initial diagnosis of chronic myeloid leukemia BCR-ABL1 positive, have been initially treated with Imatinib, and after years of treatment have relapsed and subsequently shown the secondary JAK2 mutation. This is the fifth case with these features. Presence of JAK2 at diagnosis, and subsequent development of the BCR-ABL1 translocation -----------19.7-20.0 7.5 Table 1: Summary of Cases Reported [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19].

PRESENCE OF BCR-ABL1 AND
relapsed. In June 2013, the laboratory tests showed that the white cell count was 17.2×10 3 ml, while the differential blood count showed 82.8% neutrophils, 9.41% lymphocytes, 4.01% monocytes 3.49% eosinophils and 0.30% basophils. The red blood count was 6.23×10 6 / ml, the hemoglobin 13.2 mg/ml, the HCT 43.43% and a platelets count was 7.39×10 5 /ml. Because of the absence of a hematologic response to therapy, and the complete molecular response of BCR/ABL1 presented by the patient, it was thought that another genetic alteration might coexist.
Because of the results of the last hematology studies in which both the platelet and white blood cell counts were increased, it was believed that this patient had a myeloproliferative neoplasm which was BCR-ABL1 positive in combination with a mutation in the JAK2 gene. Therefore, we performed a test of the JAK2 V617F mutation by allelespecific PCR, and a positive heterozygous result was obtained for this mutation. The JAK2 mutation appeared after three years of having the BCR-ABL1 translocation, when the patient relapsed ( Figure 2).

Discussion
The JAK2 gene and the ABL1 gene regulate hematopoietic function. The JAK2 mutations leads to uncontrolled cell proliferation and produces clinical characteristic compatible with MPN. The BCR-ABL1 translocation produces an imbalance in cell division, differentiation and apoptosis, this produce a CML. The two alterations are present in this patient. The high platelet counts show the presence of a MPN syndrome, secondary to the CML presentation.
As mentioned in the introduction, the BCR-ABL1 transcript and JAK2 mutation were previously believed to be mutually exclusive (according to the WHO criteria, 2008); and the presence of either one was used to classify the MPNs into two main groups: Philadelphia positive chronic myelogenous leukemia and Philadelphia negative MPNs, which are PV, ET, PMF. However a few cases have been reported in which there was a co-existence of two genetic alterations. In some of these the BCR-ABL1 and JAK2 mutations were found to co-exist [3][4][5][6][7][8][9][10][11].
This case is the first reported from Central American and one of the few reported internationally, where the patient was initially diagnosed as having Philadelphia positive chronic myeloid leukemia, and in the following years developed the presence of the JAK2 V617F mutation. This is the first such case among 168 Guatemalan patients with Philadelphia positive chronic myeloid leukemia that we have studied.
As can be observed, our patient had a complete hematologic response only with a dose of 800 mg of Imatinib, although the recommended dose is half of that. After the patient remained well for three years, he relapsed. We discarded Imatinib resistance as an explanation of the relapse since the monitoring of BCR-ABL1 quantification indicated a continuing complete molecular response. Therefore, it was thought that