2012;125(11):2076-2079

It is still argued as to whether chronic myeloproliferative disorders (CMPD) and chronic lymphoproliferative diseases (CLPD) are clonally related. Recently, a recurrent mutation in the JH2 pseudokinase domain of the cytoplasmatic tyrosine kinase, Janus kinase (JAK2) gene in the hematopoietic cells of patients with CMPDs has been reported, and about 90% of patients with PV and 50%–60% of patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF) have this mutation. The mutated JAK2 leads to constitutive tyrosine phosphorylation activity that finally causes CMPD.¹ Yet the frequency of JAK2 V617F mutation is extremely rare in lymphoid malignancies. Hence, whether or not the JAK2 V617F mutation is involved in the development of CLPD is a matter of debate.

 

To obtain more information we detected genetic lesions in neutrophils and B- and T-lymphocytes by fluorescence in situ hybridization (FISH) and searched for JAK2 V617F mutation status in lymphocytes in a patient having CD5⁻ chronic lymphocytic leukemia (B-CLL) and ET.

 

 

A 94-year-old male patient was diagnosed with CLL in January 2008. There were no clinical symptoms. Clinical examination revealed an enlarged spleen which was 2 cm below the left costal margin and the right clavicle, bilateral neck, and axillary lymphadenopathies. The hemoglobin level was 105 g/L, platelets 109´10⁹/L, and white blood cells (WBC) was 12.7´10⁹/L. The circulating lymphocyte was 54% and granulocyte was 40%. Serum biochemical parameters were normal. In the bone marrow, the cellularity and lymphocyte compartments were increased. Bone marrow biopsy confirmed an infiltration by small lymphocytes in small clusters, and the reticulin pattern was normal. Flow cytometric analysis of patient's peripheral blood displayed an atypical CLL phenotype. Total lymphocyte count was 6.56´10⁹/L gated CD45⁺ cells showed CD19⁺ (85.8%), CD20⁺ (61.5%), CD23⁺ (54.6%), cCD79a⁺ (49.4%), CD2⁻ (15.5%), CD5⁻ (10.2%), CD10⁻ (0.1%), CD38⁻ (14.7%), FMC7⁻ (0), CD5⁺CD19⁺ (7.3%), and ZAP-70⁺CD5⁺CD19⁺ (0). B-lymphocyte count was 5.628´10⁹/L. The diagnosis of CLL was then made, with stage B of Binet classification. The patient was followed up for more than two years without any treatment. Blood examination showed that the WBC was between 13´10⁹/L and 18´10⁹/L, lymphocyte percentage was between 54% and 58%, hemoglobin level was between 100 g/L and 110 g/L, and platelets was between 100´10⁹/L and 200´10⁹/L.

 

In May 2010, circulating platelets was 547´10⁹/L. In August 2010, platelets increased to 1161´10⁹/L, hemoglobin level to 101 g/L, WBC to 16.8×10⁹/L with 55% lymphocytes. Circulating lymphocytes still revealed an atypical CLL phenotype (CD19⁺, CD20⁺, CD23⁺, cCD79a⁺, CD5⁻, CD10⁻, and CD38⁻). In the bone marrow, the cellularity and lymphocyte compartments were increased and the platelets were distributed in large clusters (Figure 1). Bone marrow biopsy showed a diffused infiltration by small lymphocytes, megacaryocyte compartments, and reticulin fibers were

increased (Figure 1). BCR/ABL translocation was negative by RT-PCR analysis. Cytogenic studies revealed normal 46, XY karyotype.

 

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Figure 1. At the time of CLL and ET diagnosis. A: Typical small lymphocytes, “smudge” cells, myeloid cells, and diffuse infiltration with platelets are seen in peripheral blood smear (Wright staining, original magnification ×100). B: Predominantly mature lymphocytes and aggregates of platelets are evident in bone marrow aspirate (Wright staining, original magnification ×200). C: Bone marrow biopsy reveals hypercellularity with lymphoid cells and dysmorphic megakaryocytes (HE staining, original magnification ×100). D: Reticulin stain revealing 2+ fibrosis (original magnification ×200).

 

 

Then we performed FISH and detected the JAK2 V617F mutational status of the peripheral blood nucleated cells (PBNC), neutrophils, and the B- and T-lymphocyte subpopulations in the peripheral blood.

 

Patient's PBNC were isolated from freshly collected heparinized blood by differential centrifugation over a Ficoll–Paque gradient, and the contaminating red cells were removed by hypotonic lysis. Then, PBNC were stained with monoclonal fluorescent antibodies for CD15⁺, CD19⁺, and CD3⁺ cells using IOTest^Ò-CD15/CD19/CD3-PE (Beckman Coulter). CD15⁺, CD19⁺, and CD3⁺ cells were separated from the PBNC by flow cytometry. FISH analysis were performed on CD15⁺, CD19⁺, and CD3⁺ cells separately according to the methods described by Liu et al.² Five commercial probes (Gpmedical) were used: LSI 13q14.3 deletion probe, RB1 probe, the CEP12 DNA probe, locus-specific identifier (LSI) 11q22.3 (ATM) specific DNA probe, and LSI p53 (17p13.1) probe. Signal number was enumerated in 200 interphase nuclei of the patient's CD15⁺, CD19⁺, and CD3⁺ cells separately.

 

FISH analysis was performed on the PBNC collected from 20 hematologically disease-free individuals. To rule out the presence of the false positive results, at least 500 interphase nuclei were analyzed. Cut-off level was established at a frequency of mean + 3 standard deviation (SD): 13q14.3 (3.1%), RB1 (4.7%), trisomy 12 (2.7%), 11q22.3 (3.2%), and p53 (4.1%).

 

Interphase FISH analysis allowed to detect 13q14.3 deletion in 31% of B-lymphocyte nuclei and RB1 deletion in 27% of B-lymphocyte nuclei (Figure 2). Trisomy 12, 11q22.3 (ATM) deletion, and p53 (17p13.1) deletion were not observed. No genetic lesions were found in neutrophils and T-lymphocytes.

 

 

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Figure 2. Interphase FISH analysis (DAPI staining). 13q14.3 and 11q22.3 hybridization signals are seen as red spots. RB1 and p53 hybridization signals are seen as green spots. A: CD19+ lymphocytes are positive for 13q14.3 deletion. Deletion is indicated by the existence of one red spot instead of two in the blue nucleus (original magnification ×1000). B: CD19+ lymphocytes are positive for RB1 deletion. Deletion is indicated by the existence of one green spot instead of two (original magnification ×1000).

 

 

Patient's PBNC, CD15⁺, CD19⁺, and CD3⁺ cells were obtained as described above. Genomic DNA was extracted from patient's PBNC, CD15⁺, CD19⁺, and CD3⁺ cells separately by PCR analysis by the methods described by Baxter et al.¹ Briefly, 80 ng of patient's DNA was amplified in a 36-cycle PCR at an annealing temperature of 58°C. We used 1 mmol/L of common reverse primer and 0.5 mmol/L of two forward primers (reverse primer: 5′-CTG AAT AGT CCT ACA GTG TTT TCA GTT TCA-3′, forward primer (specific): 5′-AGC ATT TGG TTT TAA ATT ATG GAG TAT ATT-3′, forward primer (internal control): 5′-ATC TAT AGT CAT GCT GAA AGT AGG AGA AAG-3′). The first forward primer is specific for the mutant allele and contains an intentional mismatch at the third nucleotide from the 3' end to improve specificity (giving a 203-bp product); the second amplifies a 364-bp product from both mutant and wild type alleles and serves as an internal PCR control.

 

We identified JAK2 V617F mutation in the patient's PBNC, neutrophils, whereas no mutation signal could be detected in B- and T-lymphocyte populations (Figure 3).

 

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Figure 3. Screening JAK2 V617F mutation. Genomic DNA from the control and patients' nucleated cells, neutrophils, T-lymphocytes, and B-lymphocytes were amplified by allele-specific PCR for JAK2 wild-type fragment (364 bp) and V617F mutant fragment (203 bp). The 203-bp mutation band is present in patient's PBNC and neutrophils, but not in the T-cells and B-cells. Lane 1: 100 bp DNA ladder; Lane 2: positive control; Lane 3: patientt PBNC; Lane 4: patient CD15+ cell; Lane 5: patient CD19+ cell; Lane 6: patient CD3+ cell; Lane 7: healthy control; Lane 8: DL2000 DNA ladder.

 

 

 

The CD5 surface marker was considered to be a pan-T-cell marker, a pan-thymocyte marker that is also expressed in the B1 subset of the human B-lymphocytes. Although CLL usually express B-phenotype and the CD5 antigen, among B-CLL, 7%–20% do not express CD5 or express low levels of this antigen. The survival of CD5^−/+ patients was on the borderline of being significantly shorter than that of CD5⁺ patients.³

 

B-CLL typical genetic lesions such as trisomy 12, deletion of 13q14.3, 11q22.3, p53 (17p13), and 6q21 are indispensable and do fully explain the natural history of the disease. The expansion of the leukemic clone in B-CLL has been related to signals from the micro-environment that influence the balance between anti-apoptotic and pro-apoptotic signals. Patients with CLL are predisposed to the development of a second malignancy due to impaired immune system or chemotherapy. The second malignancy in the majority of the cases is non-hematologic and occurs several years after the diagnosis of CLL.

 

Thus, the second hematologic malignancy like CMPDs developed in patients with CLL is exceptional. This case describes a rare occurrence of sequential CD5⁻ B-CLL and ET in a patient.

 

As we can see in Table 1, only two instances had ET after the diagnosis of CLL, and all the patients reported were CD5 positive CLL. To our knowledge, therefore, this is the first such case in the literature. Yet, the underlying pathogenesis remains unknown. Results of interphase FISH analysis did provide some evidence that the deletion of 13q14.3 and RB1 was restricted to B-lymphocytes in this patient. It was likely that these genetic aberrants of CLL did not lead to ET.

 

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Table 1. Reports of patients with B-CLL and ET

 

 

Does CLL and ET originate at the level of the pluripotent hematopoietic stem cell? And was it caused by the V617F JAK2 mutation? Until now, there have been only three instances reporting the results of V617F JAK2 mutation in the lymphoid compartment with CD5⁺ B-CLL and ET coexisting patients. However, the consequence was controversial. In our patient, analysis of JAK2 V617F mutation seems to testify that the CD5⁻ B lymphatic leukemic clone was derived from a V617F-negative multipotent stem cell. As originally defined, a common lymphoid progenitor cell could give rise to B-, T- and natural killer cells but not to myeloid cells, whereas a common myeloid progenitor cell could generate myeloid but not lymphoid cells. A coincidence of both disorders is possible, given the interval from the diagnosis of CLL to the development of ET, and different mutagenic events would independently induce the lymphoid and myeloid malignant proliferation.

 

Nevertheless, Hou et al⁴ have identified a human B-cell/myeloid common progenitor cell capable of producing B-cells and differentiating into myeloid lineages. There was one case that reported JAK2 V617F mutation status in stem cells, myeloid cell lineages, and lymphoid cells in patients both diseases simultaneously occurred. It is possible that a common pluripotent (myeloid and lymphoid) cell can give rise to coexisting CLL and ET. In most cases reported in literature (Table 1), both the disorders developed simultaneously^5-9 or ET preceded the development of CLL.^8,10-12 According to the results of the present case and the report of Musolino et al,¹² it seems that there was no relationship between the two diseases that developed sequentially.

 

On the other hand, the mutagenic role of hydroxyurea remains controversial, but no increased frequency of CLPD has been reported following hydroxyurea treatment. As seen in Table 1, the patients who had simultaneous occurrence of CLL and ET did not previously use hydroxyurea. And neither of the patients who had an onset ET after CLL was treated with hydroxyurea or alkylation agents after the diagnosis of CLL.¹³ Moreover, in the group of CLL after the diagnosis of ET, only half were exposed to hydroxyurea. There seems, therefore, to be no therapeutic interference between the two diseases.

 

As a result, it was concluded that at least in this patient the CD5^– B-CLL and JAK2 V617F positive ET represents two separate neoplasms of the myeloid and lymphoid lineage. Our report provides a new case of CD5⁻ B-CLL co-existing with JAK2 V617F positive ET, which may give more data to clarify the mechanism of coincidence of CMPD and CLPD. The further studies involving analysis of genetic profiles of CD5⁻ B-CLL cells and JAK2 V617F positive neutrophils from this patient compared with those from patients having either only CD5^– B-CLL or JAK2 V617F positive ET may provide information needed for the molecular confirmation of our results.

 

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