Letters to the Editor
In the current study, JAK2 V617F has been simultaneously analyzed in granulocytes, erythroblasts, megakaryocytes and platelets from the same patient. The results found in the first five ET patients (no. 1–5) demonstrated that myelopoiesis in these patients is entirely clonal and is related to JAK2 V617F. This mutation was not found in one patient who showed eCFU-MK formation; nevertheless, this patient had monoclonal myelopoiesis (as determined by the human androgen receptor gene polymorphic marker clonality assay) (results not shown). These results support the hypothesis that some cases of true ET might be related to other molecular defects, as already has been suggested in the literature. In our experience, all ET patients with JAK2 V617F in peripheral blood granulocytes carry the mutation in the platelet-megakaryocytic lineage. This is the lineage predominantly involved in ET and indicates that in ET the mutation should be searched by more sensitive techniques and directly on platelets. In conclusion, our study describes the presence of JAK2 V617F in all myeloid cells including the megakaryocytic lineage. These results strongly support the implication of different myelopoietic cell lineages in ET and confirm the biological heterogeneity of this disease.
Acknowledgements This work was supported by the Grants FIS PI030345, C03/07 and C03/10 from the Spanish Ministry of Health.
L Florensa1,2, B Bellosillo2,3, C Besses2,4, E Puigdecanet1,2,3, B Espinet2,3, E Pe´rez-Vila1,2, R Longaro´n3, RM Vila`1, F Sole´2,3 and S Serrano1,2,3 1 Laboratori de Citologia Hematolo`gica, Departament de Patologia, Hospital del Mar, IMAS, Barcelona, Spain; 2 Unitat de Recerca en Neopla`sies Hematolo`giques-Parc Recerca Biome`dica Barcelona (URNHE-PRBB), Barcelona, Spain;
3
Laboratori de Citogene`tica i Biologia Molecular, Departament de Patologia, Hospital del Mar, IMAS, Barcelona, Spain and 4 Servei d’Hematologia Clı´nica, Hospital del Mar, IMAS, Barcelona, Spain E-mail:
[email protected]
1905
References 1 Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 2005; 365: 1054–1061. 2 Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ et al. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 2005; 7: 387–397. 3 James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C et al. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 2005; 434: 1144–1148. 4 Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005; 352: 1779–1790. 5 Lu X, Levine R, Tong W, Wernig G, Pikman Y, Zarnegar S et al. Expression of a homodimeric type I cytokine receptor is required for JAK2V617F-mediated transformation. Proc Natl Acad Sci USA 2005; 102: 18962–18967. 6 Campbell PJ, Scott LM, Buck G, Wheatley K, East CL, Marsden JT et al. Definition of subtypes of essential thrombocythaemia and relation to polycythaemia vera based on JAK2 V617F mutation status: a prospective study. Lancet 2005; 366: 1945–1953. 7 Florensa L, Besses C, Woessner S, Sole´ F, Acı´n P, Pedro C et al. Endogenous megakaryocyte and erythroid colony formation from blood in essential thrombocythaemia. Leukemia 1995; 9: 271–273. 8 Bellosillo B, Besses C, Florensa L, Sole F, Serrano S. JAK2 V617F mutation, PRV-1 overexpression and endogenous erythroid colony formation show different coexpression patterns among Phnegative chronic myeloproliferative disorders. Leukemia 2006; 102: 18962–18967.
High ZAP-70 expression correlates with worse clinical outcome in mantle cell lymphoma Leukemia (2006) 20, 1905–1908. doi:10.1038/sj.leu.2404362; published online 17 August 2006
Zeta-associated protein 70 (ZAP-70) is a z-chain, CD3-receptorassociated protein tyrosine kinase (PTK) that is critical for initiating T-cell signaling. Relatively little is known with regard to its function in B cells, although there is evidence that ZAP-70 may enhance signal transduction via the B-cell receptor complex. In chronic lymphocytic leukemia (CLL), the presence of 420% of ZAP-70-positive CLL cells detectable by flow cytometry was found to correlate with rapid disease progression and shorter overall survival in stage Binet A patients.1 High level of ZAP-70 expression also has been associated with a relative lack of somatic mutations in the variable regions of the immunoglobulin heavy chain gene (VH), which is an established prognostic marker for CLL.2 Several recent studies have confirmed that ZAP-70 is expressed in a subset of mantle cell lymphoma (MCL).3–5 In this study, we aimed to determine if ZAP-70 carries any clinical significance in newly diagnosed MCL patients.
Immunohistochemistry was employed to assess ZAP-70 expression in 64 MCL tumors. The clinical characteristics of these patients are summarized in Table 1. All cases were newly diagnosed and collected between 1994 and 2003 at the Department of Laboratory Medicine and Pathology, Cross Cancer Institute. Treatment for each MCL patient was determined during our weekly lymphoma conference based on our institutional treatment protocol. For first-line treatment, 32 of the 64 patients received CHOP-based (cyclophosphamide, doxorubicin, vincristine, prednisone) chemotherapy, 15 had chlorambucil-based chemotherapy, and five received other treatments such as proteasome inhibitor PS341 and flavopirodol. The diagnosis of all MCL cases was based on the criteria described in the World Health Organization Classification Scheme; all cases were positive for CD5 and/or CD43 and cyclin D1, and negative for CD23. All tissues were routinely processed, formalin-fixed and paraffin-embedded. All but four cases had the small cell morphology. Three of the remaining four cases were blastoid MCL. The morphology of the other case was composed of diffuse areas of small lymphoma cells admixed with Leukemia
Letters to the Editor
1906 Table 1
Summary of clinical data in ZAP-70high and ZAP-70low MCL patients Overall (N ¼ 64)
ZAP-70low (N ¼ 27)
ZAP-70high (N ¼ 37)
67 65.6
63 65.1
69 65.9
53 11
22 (81%) 5 (19%)
31 (84%) 6 (16%)
Ann Arbor stage N ¼ 62 I–III IV
21 (34%) 41 (66%)
7 (27%) 19 (73%)
14 (39%) 22 (61%)
Lactate dehydrogenase N ¼ 43 Normal Elevated
34 (79%) 9 (21%)
18 (90%) 2 (10%)
16 (70%) 7 (30%)
Performance status N ¼ 57 0–1 2–4
46 (81%) 11 (19%)
20 (83%) 4 (17%)
26 (79%) 7 (21%)
International prognostic index N ¼ 49 0–2 3–5
37 (76%) 12 (24%)
17 (81%) 4 (19%)
20 (71%) 8 (29%)
76
27
Age N ¼ 64 Median (years) Mean (years) Gender N ¼ 64 Male Female
ZAP-70low versus ZAP-70high (P-values) 0.041
1.00
0.38
0.14
0.75
0.52
Median overall survival (months)
0.03
Abbreviation: MCL, mantle cell lymphoma; ZAP-70, Zeta-associated protein 70. P-values were provided for comparison of each clinical parameter between the ZAP-70low group and the ZAP-70high group. The Fisher’s exact test was used for all comparisons except for age of patient and median survival, for which Mann–Whitney and Cox proportional hazards model were used, respectively.
nodules of large blastoid cells. None of the cases demonstrated a predominantly (i.e., 485%) mantle zone pattern. A monoclonal antibody reactive with ZAP-70 (clone 2F3.2, Upstate Biotechnology, Lake Placid, NY, USA) was employed. The immunohistochemical method used in this study has been described elsewhere.6 Evaluation of the ZAP-70 staining was performed independently by three different observers (DH, HMA, RL) without the knowledge of the clinical outcome. Expression of ZAP-70 staining, based on definitive cytoplasmic staining, was scored as negative, weak (1 þ ), moderate (2 þ ) and strong (3 þ ). Reactive T cells, which had 3 þ ZAP-70 staining, were used as internal positive controls. Cases with X40% of neoplastic cells expressing 1 þ or 2 þ ZAP-70 were labeled ZAP-70high and the rest was labeled ZAP-70low. Concordance among the three observers was 485%; most of the discrepancies, which were mostly between 1 þ and 2 þ , did not result in any change in the classification of the ZAP-70 expression status. As illustrated in Figure 1a and b, T cells in the paracortical areas and those in the germinal centers were strongly positive (3 þ ) for ZAP-70. In the reactive lymphoid follicles, the mantle zones were negative, whereas the germinal center B cells were weakly positive (1 þ ). On high magnification (Figure 1b), ZAP70 staining was predominantly cytoplasmic. Figure 1c and d illustrate the ZAP-70 expression in MCL tumors. In all cases, uniform and strong ZAP-70 positivity (3 þ ) was identified in the infiltrating T cells (positive internal controls). ZAP-70 expression was heterogeneous between tumors and within the same tumor. Thirty-seven (58%) cases were categorized as ZAP-70high. None of the MCL tumors showed a ZAP-70 staining intensity level in the neoplastic cells as high as that of reactive T cells. In one case in which nodules of blastoid cells were identified, weak Leukemia
positivity (1 þ ) was found in the small cell component, whereas moderate positivity (2 þ ) was found in the blastoid cell nodules. As summarized in Table 1, 37 of 64 (58%) cases were scored as ZAP-70high. In this group, there were 31 men and six women (M:F ¼ 5.2), with a median age of 69 years. Twenty-five (68%) patients died during the follow-up, and the median survival was 27 months. Twenty-seven (42%) was labeled ZAP-70low. In this group, there were 22 men and five women (M:F ¼ 4.4), with a median age of 63 years. Twelve (44%) died during the followup, and the median survival was 76 months. ZAP-70 expression significantly correlated with the age of patient, with the ZAP-70high group being older (0.041, Mann– Whitney). Nevertheless, when the statistical analysis was performed by treating the patient age as a categorical parameter (o60 or X60 years), no significant correlation was found (P ¼ 1.00, Fisher exact test). ZAP-70 expression did not correlate with other clinical prognostic factors including clinical stage, performance status, lactate dehydrogenase and the international prognostic index (Table 1). Similar analysis of ZAP-70 status with other parameters such as B-symptoms, splenomegaly, blastoid cytology was also insignificant. A high ZAP-70 expression level correlated with worse clinical outcome in this study. The 3-year overall survival for patients with ZAP-70high tumors was 39% compared with 69% for patients with ZAP-70low tumors (hazard ratio ¼ 2.2, P ¼ 0.03, Cox proportional hazards model) (Figure 2). Univariate analysis showed that patients with a performance status of 0–1 survived longer than those with a performance status of 2–4 (hazard ratio 3.5, P ¼ 0.007, Cox proportional hazards model). An elevated lactate dehydrogenase also conferred a poor prognosis (hazard ratio 3.7, P ¼ 0.006, Cox proportional hazards model). Clinical stage and international prognostic index were not significant in our cohort. Multivariate
Letters to the Editor
1907
Figure 1 ZAP-70 expression in a reactive lymph node (a and b) and MCL tumors (c and d) detected by immunohistochemistry. (a) Strong (3 þ ) ZAP-70 positivity was detectable in the paracortical T-cell areas (thin white arrow) as well as a small subset of T cells within the germinal centers (thin white arrow). The germinal center cells were weakly (1 þ ) positive for ZAP-70 (thick white arrow), whereas the mantle zone cells were negative (open white arrow). (b) On high magnification, mantle zone cells were negative for ZAP-70 (open black arrow) and the germinal center cells were weakly positive for ZAP-70 (solid white arrow). The insert demonstrated weak (1 þ ) ZAP-70 positivity in the germinal center cells, and the staining was predominantly granular. T cells were strongly ZAP-70 positive. (c) A case of mantle cell lymphoma, small cell type, expressed no detectable ZAP-70 expression (also see insert). Infiltrating T cells with strong staining of ZAP-70 provided internal positive controls. (d) ZAP-70 expression in a case of mantle cell lymphoma showing moderately intense (2 þ ) ZAP-70 immunostaining. T cells with strong ZAP-70 staining provided internal positive controls.
survival analysis was performed using the Cox proportional hazard model. ZAP-70 expression remained to be a significant prognostic factor independent of performance status. As the number of patients for whom the lactate dehydrogenase levels were available was relatively small (i.e., 43), we did not include this parameter in the multivariate analysis. The IgVH status was assessed in 33 of 64 MCL patients, and the protocol was adapted from that described by Timm et al.7 Five to 12 clones were subjected to DNA sequencing using the BigDye Terminators (ABI3100, San Jose, CA, USA). The DNA sequence from each clone was aligned to the immunoglobulin gene sequence database (www.ncbi.nlm.nih.gov/igblast). The germline IgVH region possessing the highest homology to the DNA sequence being analyzed was regarded as the IgVH region utilized, and each individual clone was given a degree of homology compared to its most closely related specific germline IgVH region, which was expressed as a percentage. An IgVH homology of o98% was defined as mutated, while a homology of X98% was defined as unmutated. The IgVH mutation status was available in 33 cases. Of the 18 ZAP-70high cases, seven
had the mutated IgVH genotype. Among the 15 ZAP-70low MCL cases, 12 had the mutated IgVH genotype. High ZAP-70 expression was significantly associated with the unmutated IgVH genotype (0.041, Fisher’s exact test). Cox proportional hazards survival analysis using the IgVH genotype was not significant (P ¼ 0.12), although the sample size is relatively small. In summary, we found 58% of the MCL to express a relatively high level of ZAP-70, and patients with ZAP-70High tumors tend to have shorter overall survival. We also found that relatively high ZAP-70 expression in MCL significantly correlated with the unmutated IgVH genotype. In contrast with our findings, Crespo et al.,2 did not detect any ZAP-70 expression in six MCL samples by immunohistochemistry. This discrepancy is probably related to the use of different anti-ZAP-70 antibodies. In fact, two other recent studies using the same commercially available anti-ZAP70 antibody as ours revealed positivity in MCL tumors, ranging from 8 to 19%.3–5 We believe that there are two significant differences in the methodology that may explain our higher frequency of ZAP-70 positivity in MCL. First, while the three Leukemia
Letters to the Editor
1908 possibility that ZAP-70 expression may be upregulated during disease progression.
Acknowledgements Research grant support was received from the National Cancer Institute of Canada and Canadian Cancer Society.
D Hui1, L Dabbagh2, J Hanson3, HM Amin4 and R Lai2,5 Department of Medicine, University of Alberta, Edmonton, Alberta, Canada; 2 Department of Laboratory Medicine and Pathology, Cross Cancer Institute, Edmonton, Alberta, Canada; 3 Department of Epidemiology, Cross Cancer Institute, Edmonton, Alberta, Canada; 4 Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA and 5 Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada E-mail:
[email protected]
1
References Figure 2 Correlation of ZAP-70 status and overall survival (Kaplan–Meier analysis). ZAP-70low MCL patients survived significantly longer than ZAP-70high MCL patients (P ¼ 0.03).
previous studies employed either heating by a steamer or pressure cooking for antigen retrieval, we used a combination of pressure cooking and microwave heating. Second, we used a higher concentration of ZAP-70 antibody for immunohistochemistry; while we used 1:50, Sup et al., employed 1:200 and Carreras et al., employed 1:400 dilution. We speculate that these two factors contributed to a higher sensitivity of detection. The fact that we were able to detect a relatively low level of ZAP-70 expression in the germinal center B cells (other than the T cells), which were reported to be negative in the studies by Admirand et al., and Sup et al., supports this concept. While we have provided evidence that ZAP-70 expression correlates with the overall survival and the IgVH mutational status in MCL, the sample size used in this pilot study is relatively small. Thus, larger studies are needed to confirm these findings. Our finding of upregulation of ZAP-70 expression in a case of MCL with foci of transformation also raises the
1 Durig J, Nuckel H, Cremer M, Fuhrer A, Halfmeyer K, Fandrey J et al. ZAP-70 expression is a prognostic factor in chronic lymphocytic leukemia. Leukemia 2003; 17: 2426–2434. 2 Crespo M, Bosch F, Villamor N, Bellosillo B, Colomer D, Rozman M et al. ZAP-70 expression as a surrogate for immunoglobulinvariable-region mutations in chronic lymphocytic leukemia. N Engl J Med 2003; 348: 1764–1775. 3 Admirand J, Rassidakis GW, Abruzzo LV, Valbuena JR, Jones D, Medeiros LJ. Immunohistochemical detection of ZAP-70 in 341 cases of non-Hodgkin and Hodgkin lymphoma. Mod Pathol 2004; 17: 954–961. 4 Sup SJ, Domiati-Saad R, Kelly TW, Steinle R, Zhao X, His ED. ZAP70 expression in B-cell hematologic malignancy is not limited to CLL/SLL. Am J Clin Pathol 2004; 122: 582–587. 5 Carreras J, Villamor N, Colomo L, Moreno C, Ramon y Cajal S, Crespo M et al. Immunohistochemical analysis of ZAP-70 expression in B-cell lymphoid neoplasms. J Pathol 2005; 205: 507–513. 6 Hui D, Reiman T, Hanson J, Linford R, Wong W, Belch A et al. Immunohistochemical detection of cdc2 is useful in predicting survival in patients with mantle cell lymphoma. Mod Pathol 2005; 18: 1223–1231. 7 Timms JM, Bell A, Flavell JR, Murray PG, Rickinson AB, TraverseGlehen A et al. Target cells of Epstein-Barr-virus (EBV)-positive posttransplant lymphoproliferative disease: similarities to EBV-positive Hodgkin’s lymphoma. Lancet 2003; 361: 217–223.
Most immature T-ALLs express Ra-IL3 (CD123): possible target for DT-IL3 therapy
Leukemia (2006) 20, 1908–1910. doi:10.1038/sj.leu.2404349; published online 10 August 2006
Receptor interleukin 3 (IL3) alpha-chain (Ra-IL3 or CD123) expression is described in approximately 80% of acute myeloid leukemias (AML) and may contribute to blast cell proliferative advantage, but the few T-acute lymphoblastic leukemias (T-ALL) reported were CD123 negative (reviewed in Testa et al.1). T-ALLs represent expansions of cells arrested at various stages of lymphoid development. Several recent studies, using different approaches, suggested the existence of an immature (IM) T-ALL subtype.2–5 We defined this IM subtype by a surface T-cell Leukemia
receptor (TCR)/cytoplasmic TCRb negative phenotype. They represent 30% of adult and 15% of pediatric cases, are CD4/ 8DN, CD1a, CD34 þ , with frequent expression of ‘myeloid’ markers (CD13, CD33, CD117) and distinct oncogenic lesions, predominantly CALM-AF10 and MLL.5 They can be further classified on the basis of their TCRd, g and b gene configurations: IM0 demonstrates a germline configuration at all three loci; IMd demonstrates TCRd rearrangement; IMg T-ALLs, TCRd and TCRg rearrangements but no TCRb V(D)J and IMb T-ALLs have undergone TCRb VDJ. More mature T-ALLs can be identified by a sTCR-/cTCRb þ phenotype (defined as pre-ab) or a surface TCR. We demonstrated that in adult T-ALLs, o50% of IM cases enter complete remission (CR) after a single
