JAK2 V617F mutation is uncommon in patients with the 3q21q26 syndrome

Human Pathology (2010) 41, 758–762

www.elsevier.com/locate/humpath

Original contribution

JAK2 V617F mutation is uncommon in patients with the 3q21q26 syndrome Pei Lin MD ⁎, Rajyalakshmi Luthra PhD, Roberto H. Nussenzveig PhD, L. Jeffrey Medeiros MD Department of Hematopathology, The University of Texas M. D. Anderson Cancer Center, Box 72, Houston, TX 77030, USA Received 23 April 2009; revised 2 November 2009; accepted 4 November 2009

Keywords: 3q21q26 syndrome; JAK2 V617F; EVI1

Summary The 3q21q26 syndrome is recognized as a distinct clinicopathologic entity. Patients have a myeloid neoplasm associated with 3q21q26 cytogenetic abnormalities and present with anemia, leukopenia, and either thrombocytosis or a normal platelet count associated with dysplasia. To determine if JAK2 V617F mutation is implicated in the abnormal thrombopoiesis of the 3q21q26 syndrome, we analyzed bone marrow samples of 12 patients, including 10 patients with acute myeloid leukemia and 2 patients with a myelodysplastic syndrome, associated with either inv(3)(q21;q26) or t(3;3)(q21;q26). The platelet count ranged from 142 to 597 × 103/μL. Using polymerase chain reaction and pyrosequencing assays, no evidence of JAK2 V617F was identified in 11 of 12 cases. A JAK2 V617F mutation was identified in one patient who had acute myeloid leukemia with concurrent mast cell disease. Separate DNA analysis of myeloblasts and mast cells after laser capture microdissection confirmed that JAK2 V617F was present in both components. We conclude that JAK2 V617F mutation is uncommon in the 3q21q26 syndrome and that its presence may indicate an unusual coexistence of a myeloproliferative neoplasm. © 2010 Elsevier Inc. All rights reserved.

1. Introduction Patients who have a myeloid neoplasm associated with 3q21q26 cytogenetic abnormalities often present with the so-called 3q21q26 syndrome characterized by anemia, leukopenia, and either thrombocytosis or a normal platelet count [1]. The 3q21q26 syndrome usually occurs in the setting of acute myeloid leukemia (AML) or a high-grade myelodysplastic syndrome (MDS) and is most commonly associated with either inv(3)(q21q26) or t(3;3)(q21;q26). The characteristic morphologic findings in bone marrow aspirate smears and biopsy specimens are megakaryocytic ⁎ Corresponding author. Tel.: +1 713 794 1746; fax: +1 713 794 1800. E-mail address: [email protected] (P. Lin). 0046-8177/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2009.11.004

hyperplasia and dysplasia with increased micromegakaryocytes. As 3q21q26 abnormalities are not entirely specific for this entity, the 3q21q26 syndrome is currently defined by a constellation of clinical and morphologic findings associated with 3q21q26 cytogenetic abnormalities. The disease is clinically aggressive and tends to be refractory to therapy. Thrombocytosis and megakaryocytic hyperplasia are typically features of myeloproliferative neoplasm (MPN). In this regard, the 3q21q26 syndrome resembles 5q− syndrome in that both are primarily myelodysplastic syndrome with certain myeloproliferative features. Studies on the 5q− syndrome and other diseases that display overlapping MDS and MPN features, such as refractory anemia with ringed sideroblasts and thrombocytosis, have

JAK2 V617F in 3q21q26 syndrome implicated JAK2 V617F mutation in a subset of cases [2-9]. In a recent report, histone deacetylase inhibitors were shown to selectively target cells with JAK2 V617F [10]. Knowledge of JAK2 status in patients with the 3q21q26 syndrome may be of value in designing rational therapy. For this reason, we collected bone marrow samples of 12 patients with the 3q21q26 syndrome and analyzed for JAK2 V617F.

2. Materials and methods Patients with inv(3)(q21;q26) and t(3;3)(q21;q26) were identified from the files of our hospital. Because these cytogenetic aberrations may occur in accelerated or blast phase of chronic myelogenous leukemia, cases involved by an antecedent MPN were excluded. Only patients with a normal or elevated platelet count were included in the study. Bone marrow aspirate specimens were cultured for conventional cytogenetic analysis by established methods [11]. Chromosome aberrations were described according to the guidelines of the International System for Human Cytogenetic Nomenclature [12]. Wright-Giemsa–stained peripheral blood and bone marrow aspirate smears and hematoxylin-eosin–stained aspirate clot and biopsy sections were reviewed. Reticulin stains were also assessed on bone marrow biopsy specimens. Clinical data were obtained from the medical records. JAK2 V617F was detected by a pyrosequencing assay developed in M. D. Anderson Cancer Center's clinical molecular diagnostic laboratory. Briefly, DNA was isolated from bone marrow aspirate and/or core biopsy samples and amplified using the following primers: 5′-TCT TTC TTT GAA GCA GCA AGT ATG AT-3′ (forward) and biotinlabeled reverse primer 5′ACA AAA ACA GAT GCT CTG

Table 1

759 AGA AAG G-3′. Single-stranded biotinylated template strand was prepared for sequencing using the Pyrosequencing Vacuum Prep Tool (Biotage, Uppsala, Sweden). Pyrosequencing reactions were performed on a PSQ HS 96 instrument using a JAK2 sequencing primer: 5′-ATT ATG GAG TAT GT- 3′. All primers were obtained from Invitrogen (Carlsbad, CA), and analyses were performed using appropriate negative and positive controls. The sensitivity of detection is approximately 10% mutationcontaining cells. In one case, we also performed laser microdissection of myeloid blasts and mast cells using methods reported by Cho-Vega and colleagues [13]. For comparison, we also assessed for JAK2 V617F in cases of systemic mastocytosis (SM) involving bone marrow, either with or without an associated clonal hematolgoic non–mast cell hematologic disease, as defined using World Health Organization (WHO) criteria. These specimens were analyzed for JAK2 V617F as part of the clinical workup at our institution during the period of January 2004 to October 2009. The criterion for inclusion in this study was that the mast cell infiltrate replaced at least 10% of the bone marrow medullary space. Most of these SM cases were analyzed for KITD816V mutation using methods described previously [14].

3. Results We identified 40 patients who were diagnosed with AML or MDS with cytogenetic evidence of t(3;3)(q21;q26) or inv (3)(q21;q26) from April 2002 to April 2007. Among this group, 12 patients had a normal or elevated platelet count and other clinical features characteristic of the 3q21q26 syndrome. Ten patients had AML, and 2 patients had refractory anemia with excess blasts-type 2. The cases of leukemia

Karyotype of 12 cases of the 3q21q26 syndrome Diagnosis

Karyotype

Platelet count

JAK2 V617F

46,XX,inv(3)(q21q26)[2],45,XX,inv(3)(q21q26),−7[18]

597

Yes

2

AML-M2 + mast cell proliferation AML-M0

496

No

3 4 5

AML-M2 AML-M2 AML-M2

296 224 331

No No No

6 7 8

AML-M4 AML-M5 AML-M7

177 729 376

No No No

9 10 11 12

AML-M7 AML-M7 RAEB-2 RAEB-2

46,XX,inv(3)(q21q26),del(5)(q13),add(14)(q24)[12] 46,XX,inv(3)(q21q26),t(9;20)(q22;q13.3),del(12)(p13)[8] 46,XY,inv(3)(q21q26),del(7)(q22)[20] 46,XX,inv(3)(q21q26),del(7)(q22q34)[20] 46,XY,inv(3)(q21q26.2),del(7)(q22q34),add(20)(q12)[2] 46,XY[18] 45,XX,t(3;3)(q21;q26),−7[20] 46,XX,inv(3)(q21q26)[20] 46,XY,inv(2)(p13q21),inv(3)(q21q26)[7] 46,XY,inv(2)(p13q21),inv(3)(q21q26),del(5)(q15q33)[13] 46,XY,inv(3)(q21q26)[21] 46,XX,in(3)(q21;q26)[20] 46,XY,inv(3)(q21q26)[7],46,XY[6] 46,XY,inv(3)(q21q26),del(5)(q13)[3],46,XY[11]

316 692 243 203

No No No No

1

760 were further classified as AML with minimal differentiation (n = 1), AML with maturation (n = 4, including 1 with concurrent mast cell disease), acute myelomonocytic leuke-

P. Lin et al. Table 2

Summary of 22 cases of SM

Extent of BM involvement

c-KIT/D816V

JAK2 V617F

Diagnosis

80% 80% 60% 50% 50% 30% 30% 20% 15% 20% 20% 20% 15% 15% 15% 10% 10% 10% 10% 20% 60% 60%

Pos Pos Pos Pos Pos Pos Pos Pos Pos Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg ND Pos Neg

Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg

SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM-CMML SM-CMML

Abbreviations: BM, bone marrow; ND, not done; Pos, positive; Neg, negative.

Fig. 1 A case of AML with concurrent mast cell proliferation that is positive for inv(3)(q21q26) and JAK2 V617F. A, Bone marrow biopsy specimen shows hypercellular bone marrow with megakaryocytic hyperplasia and dysplasia. No significant fibrosis (×400, hematoxylin and eosin). B, Increased immature cells in the background and increased spindle-shaped mast cells forming sheets (×400, hematoxylin and eosin). C, Bone marrow aspirate smear shows numerous blasts and dysplastic megakaryocytes in the center surrounded by clumps of platelets (×1000, Wright-Giemsa).

mia (n = 1), acute monoblastic leukemia (n = 1), or acute megakaryoblastic leukemia (n = 3) according to the criteria of the WHO classification. Four patients had thrombocytosis with a platelet count ranging from 496 to 729 × 103/μL (reference range, 140-440 × 10 3 /μL). The remaining 8 patients had a normal platelet count. Flow cytometry immunophenotypic analysis demonstrated increased myeloblasts in each case positive for CD34, CD117, CD33, CD13, and myeloperoxidase. Conventional cytogenetic analysis identified inv(3)(q21q26) in 11 cases and t(3;3)(q21;q26) in 1 case. The 3q26 abnormality was an isolated cytogenetic finding in 4 patients and was accompanied by additional abnormalities in 8 patients. The karyotype of each neoplasm is listed in the Table 1. No evidence of JAK2 V617F was identified in 11 of 12 cases. A JAK2 V617F mutation was identified in one case of AML with maturation associated with mast cell disease. The mutant allele represented 81% of the polymerase chain reaction products. The patient was a woman who presented with infection of the left lower extremity unresponsive to antibiotic treatment. The complete blood count showed leukocytosis of 14.4 × 103/μL (reference range, 4-11 × 103/ μL), hemoglobin of 15 g/dL (reference range, 12-16 g/dL), hematocrit of 43.8% (reference range, 37%-47%), and a platelet count of 597 × 103/μL. Examination of a peripheral blood smear showed 38% of blasts. The bone marrow aspirate smears were cellular with 67% blasts in a

JAK2 V617F in 3q21q26 syndrome background of trilineage dysplasia and scattered spindleshaped mast cells. The bone marrow core biopsy displayed 100% cellularity with marked megakaryocytic hyperplasia and dysplasia and left-shifted myeloid maturation with increased blasts. There were also multiple clusters of mast cells (Fig. 1A-C). The karyotype of this case was: 46,XX,inv (3)(q21q26)[2],45,XX,inv(3)(q21q26),−7[18]. We microdissected areas that contained mast cell clusters and other areas composed predominantly of leukemic blasts. Separate analysis of DNA isolated from these different areas identified JAK2 V617F in both mast cells and the blast cell population. Despite intensive chemotherapy, the patient died 11 months after initial diagnosis. The patient did not have a history of mast cell disease. We also assessed 22 cases of SM for JAK2 V617F, and the results are summarized in Table 2. Twenty cases were classified as SM with bone marrow involvement ranging from 10% to 80%, and 2 cases were classified as SM associated with chronic myelomonocytic leukemia (SM-CMML). All 22 cases were negative for JAK2 V617F. Ten of 21 SM cases analyzed for c-KIT/D816V mutation were found to be positive, including 1 case of SM-CMML.

4. Discussion The 3q21q26 syndrome typically occurs in patients with AML or high-grade MDS associated with inv(3)(q21q26) or t(3;3)(q21;q26) and is characterized by normal or elevated platelet counts, megakaryocytic hyperplasia, multilineage dysplasia, poor response to currently available therapies, and a poor prognosis [1,15]. The 3q21q26 chromosomal aberrations result in aberrant expression of the protooncogene ecotropic virus integration site–1 (EVI1) located at 3q26.2. Through mechanisms not completely understood, myeloid and erythroid differentiation is profoundly suppressed; and the megakaryocytic lineage shows marked hyperplasia. As shown in this study, despite overt leukemia and an association with other adverse cytogenetic aberrations, such as −7, 7q−, or −5, patients typically have a normal or elevated platelet count. Previous studies have shown that the thrombopoietin (TPO) gene, located on chromosome 3q26, is not responsible for thrombocytosis [16,17]. Patients with the 3q21q26 syndrome share features in common with the 5q− syndrome. In both diseases, a primarily myelodysplastic process is associated with thrombocytosis. Based on the reports that JAK2 V617F is implicated in a subset of patients with 5q− syndrome [2-9], and that therapy targeting cells with JAK2 V617F is available, we were interested in assessing the JAK2 status in patients with the 3q21q26 syndrome. In the 12 cases we studied, most were classified as AML and were associated with inv(3)(q21;q26). JAK2 V617F was identified in only 1 case, and the bone marrow of this patient displayed an abnormal proliferation of

761 mast cells along with AML. Microdissection and separate analysis of DNA samples identified JAK2 V617F in both the mast cells and leukemic blasts. Although SM is included in the overall category of MPN in the recently updated WHO classification, JAK2 V617F is typically absent in this entity. In a large study of 679 cases of various types of MPN (previously referred to as chronic myeloproliferative disease), all 28 cases of SM assessed had no evidence of JAK2 V617F [18]. Similarly, all 22 cases of SM analyzed as part of this study were negative for JAK2 V617F. There are 2 studies in the literature, reporting a total of 5 cases of SM, that found JAK2 V617F; all were associated with concurrent primary myelofibrosis [8,19]. In one study that used microdissection techniques, Sotlar and colleagues [18] identified JAK2 V617F in both mast cells and non–mast cell components in 4 of 5 cases with SM and coexistent primary myelofibrosis. One can argue that the case we report of AML associated with mast cell disease represents an example of SM with associated clonal hematologic non–mast cell lineage disorder. The finding of JAK2 V617F in this case suggests that AML associated with mast cell disease, similar to the cases of primary myelofibrosis and SM reported previously, is distinct from other types of AML and SM and that JAK2 V617F may play a role in pathogenesis. An important question is the sequence of events. Although inv(3)(q21q26) had been previously described in cases of chronic myelogenous leukemia in accelerated or blast phase and one case of primary myelofibrosis in blast phase, inv(3)(q21q26) has not been described in SM to our knowledge. Given that inv(3) (q21q26) is typically associated with AML and MDS, and the clinical presentation of the patient we studied was characterized by an acute onset and rapid downhill course without a preceding history of mast cell disease or myelofibrosis, it seems reasonable to hypothesize that the initial disease in this patient was AML with inv(3)(q21; q26). JAK2 mutation may have occurred either as an independent early event that cooperated in pathogenesis or a second hit. Alternatively, the onset of AML may have been preceded by a previously unrecognized MPN other than SM that carried the JAK2 V617F. A definitive conclusion, however, cannot be established on the basis of this single case. In summary, this is the first study analyzing for JAK2 V617F in patients with the 3q21q26 syndrome. The current findings support the concept that JAK2 V617F is usually not present and therefore cannot be the explanation for the thrombocytosis and megakaryocytic hyperplasia seen in affected patients. The presence of JAK2 V617F in one patient with the 3q21q26 syndrome in this study was unique in that this patient had both AML and mast cell disease, providing a possible explanation for detecting JAK2 V617F in this patient. Myeloid diseases associated with mast cell proliferation may be biologically distinctive and JAK2 inhibitors may have a role in the therapy of these

762 patients. Additional study, however, is needed to address this possibility.

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