Essential Thrombocythemia with High Hemoglobin Levels According to the Revised WHO Classification

Letters to the Editor 2092 6 Jaso JM, Yin CC, Lu VW, Zhao M, Abruzzo LV, You MJ et al. B acute lymphoblastic 9 Stirewalt DL, Radich JP. The role of FLT3 in haematopoietic malignancies. Nat Rev leukemia with t(14;19)(q32;p13.1) involving IGH/EPOR: a clinically aggressive Cancer 2003; 3: 650–665. subset of disease. Mod Pathol 2013. 10 Vu HA, Xinh PT, Kano Y, Tokunaga K, Sato Y. The juxtamembrane domain in ETV6/ 7 Matthews W, Jordan CT, Wiegand GW, Pardoll D, Lemischka lR. A receptor tyrosine FLT3 is critical for PIM-1 up-regulation and cell proliferation. Biochem Biophys Res kinase speciﬁc to hematopoietic stem and progenitor cell-enriched populations. Commun. 2009; 383: 308–313. Cell 1991; 65: 1143–1152. 11 Chonabayashi K, Hishizawa M, Kawamata S, Nagai Y, Ohno T, Ishikawa T et al. 8 Rosnet O, Mattei M-G, Marchetto S, Birnbaum D. Isolation and chromosomal Direct binding of Grb2 has an important role in the development of localization of a novel FMS-like tyrosine kinase gene. Genomics 1991; 9: myeloproliferative disease induced by ETV6/FLT3. Leukemia 2013; 27: 380–385. 1433–1436.

Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)

Essential thrombocythemia with high hemoglobin levels according to the revised WHO classiﬁcation

Leukemia (2014) 28, 2092–2094; doi:10.1038/leu.2014.175 In a recently published study, JAK2V617F ET patients were reported to transform to PV with a cumulative risk of 29% at Differentiation between essential thrombocythemia (ET) and 15 years, whereas no transformation to PV was observed in CALR- mutated ET patients.11 According to the investigators, these polycythemia vera (PV) persists to be a matter of controversy, 10 particularly in patients who present with increased hemoglobin findings were in keeping with the one-single disease hypothesis. Regarding the design of this study, but especially recruitment of (HB) levels accompanied by very high platelet counts. Although 1 cases partly dating before 2002, it can be assumed that a considered as a ‘diagnostic gold standard’, the impact and considerable fraction of the total cohort of 745 ET patients would practicability of red cell mass (RCM) measurements in patients have been diagnosed according to the PVSG guidelines.1 with the clinical differential diagnosis of PV versus ET has to be Moreover, 142 patients of this cohort11 displayed evidence of discussed.2,3 In this context the diagnostic accuracy of different HB erythrocytosis or leukocytosis about 22 months before the clinical or hematocrit (HCT) threshold values and bone marrow (BM) diagnosis of PV, and in these patients the HB values, white morphology as surrogate markers for RCM have been blood cell counts, serum erythropoietin levels and degree of considered.4,5 To capture PV patients with a borderline increase splenomegaly were above the usual range for ET according to the 8 in HB ( 18.5 g/dl in men and 16.5 g/dl in women), in addition WHO definition. Contrasting the significantly elevated rate of p p 11 to JAK2V617F or JAK2 exon 12 mutations and characteristic BM transformation into PV in this trial, other authors found biopsy features,6 a lowering of the HB and HCT values was independent of mutation status in strictly WHO-defined large postulated for the prospective revision of the 2008 WHO series of ET patients, a transformation rate to PV of only 0.9% 12 13 classification.7 On the basis of the previous studies regarding after 10 years or 5% after 12 years. Concerning outcomes in so-called masked PV, threshold values included a HB level of our first cohort after a median follow-up of 6.2 years, clinical 16.5 g/dl in men and 16 g/dl for women or a HCT level of 49% in events were very few but their frequencies appeared to be similar men and 48% in women to be the optimal cutoff levels for to ET with lower HB values (major arterial thrombosis in 47 cases distinguishing JAK2V617 ET from masked PV.5 On the other hand, presenting with high HB level ET 9% vs 12% in the 844 cases with no knowledge exists on how many patients with World Health low HB level ET, death rate 11% vs 10%, respectively). Similar Organization (WHO)-confirmed ET including consistent BM results could be assessed in the validation sample with only 2% of features present with HB levels exceeding these postulated cases with overall PV transformation after 5 years of observation thresholds, that is, clinically suggesting (early/masked) PV.3,5 in the high HB level category of ET. A possible explanation Our experience regarding this critical diagnostic pitfall was for this disturbing discrepancy concerning incidences of PV based on 891 patients with ET derived from an international transformation should include the inadvertent inclusion of 5,13 study8 (cohort 1) and diagnosed according to the classification so-called masked/early PV. It is well established that, proposed by the WHO.9 Moreover, a validation sample (cohort 2) although displaying characteristic BM features of PV, a number was considered including 248 patients selected from the Frankfurt of these cases may clinically present as ET at onset before a later 14,15 database again with strictly WHO-defined ET diagnosis. manifestation as overt PV. Examination of the HB values at diagnosis in our first cohort In conclusion, a small fraction of ET patients (ranging between disclosed increased levels in about 5% without significant 3 and 5%) diagnosed according to the WHO criteria may difference to the validation cohort (3%). Specifically, in 27 of 370 presentwithhigherHBvalues(X16.5 g/dl and X16.0 g/dl in males (7.3%) in WHO-confirmed ET, a high level HB (X16.5 g/dl) males and females, respectively) and therefore clinically could was observed, and in the 521 female ET patients, 20 cases (3.8%) mimic PV. However, about 20–30% of these cases do not have showed a HB exceeding 16.0 g/dl (Figure 1). Of note was that, in JAK2V617F or JAK2 exon 12 mutations and consequently cannot both cohorts these higher HB thresholds were associated with be regarded as PV. An accurate differentiation already at the JAK2 positivity ranging between 70 and 80% (Table 1). It is well beginning between ET with high HB and PV has certainly a known that patients with JAK2-mutated ET diagnosed according significant impact on therapeutic modalities. On the other to the Polycythemia Vera Study Group (PVSG) criteria1 may share a hand, all of these patients revealed characteristic BM features number of molecular genetic and clinical features with PV, and consistent with ET. This finding would indicate that BM therefore it has been postulated that both may belong to a morphology is a valid tool to distinguish these two MPN entities biological continuum representing different phenotypes of a in mutation-positive or questionable cases and therefore may single myeloproliferative neoplasm (MPN).10,11 replace RCM measurements.

Accepted article preview online 3 June 2014; advance online publication, 24 June 2014

Leukemia (2014) 2090 – 2115 & 2014 Macmillan Publishers Limited Letters to the Editor 2093 20.0 a 17.5

15.0

12.5

% 10.0

7.5

5.0 Hb ≥ 16 g/dL # 20 females 2.5 (3.8%)

0 7 8 91011 12 13 14 15 16 17 18 19 20 hb

17.5 b

15.0

12.5

10.0 % 7.5

5.0 Hb ≥ 16.5 g/dL # 27 males 2.5 (7.3%)

0 9 9.5 10 11 12 13 14 15 16 17 18 19 20 10.5 11.5 12.5 13.5 14.5 15.5 16.5 17.5 18.5 19.5 hb Figure 1. Distribution of hemoglobin at diagnosis in females (panel A) and males (panel B) in 891 WHO-ET patients (cohort 1).

Table 1. Association between JAK2 mutation status and different subtypes of WHO-deﬁned ET according to hemoglobin levels (HB: males X16.5 and females X16.0 g/dl) in the two cohorts

JAK2 wild type JAK2V617F Total

Cohort 1 (n ¼ 690a) Low HB level,b n (%) 261 (40%) 392 (60%) 653 (100%) High HB level,c n (%) 7 (19%) 30 (81%) 37 (100%) Total 268 (39%) 422 (61%) 690 (100%)

Cohort 2 (n ¼ 248) Low HB level,b n (%) 119 (49%) 122 (51%) 241 (100%) High HB level,c n (%) 2 (29%) 5 (71%) 7 (100%) Total 121 (49%) 127 (51%) 248 (100%) Abbreviations: ET, essential thrombocythemia; HB, hemoglobin; WHO, World Health Organization. aJAK2 mutational status available on 690 out of 891 patients; bLow HB level (females HBo16 g/dl; males HBo16.5 g/dl); cHigh HB level (females HBX16 g/dl; males HBX16.5 g/dl).

& 2014 Macmillan Publishers Limited Leukemia (2014) 2090 – 2115 Letters to the Editor 2094 CONFLICT OF INTEREST 5 Barbui T, Thiele J, Carobbio A, Guglielmelli P, Rambaldi A, Vannucchi A et al. The authors declare no conflict of interest. Discriminating between essential thrombocythemia and masked polycythemia vera in JAK2 mutated patients. Am J Hematol 2014; 89: 588–590. 6 Thiele J, Kvasnicka HM. Diagnostic impact of bone marrow histopathology in ACKNOWLEDGEMENTS polycythemia vera (PV). Histol Histopathol 2005; 20: 317–328. TB and AMV were supported by a grant from Associazione Italiana per la Ricerca sul 7 Tefferi A, Thiele J, Vannucchi AM, Barbui T. An overview on CALR and Cancro (AIRC, Milano) ‘Special Program Molecular Clinical Oncology 5 Â 1000’ to CSF3R mutations and a proposal for revision of WHO diagnostic criteria for AGIMM (AIRC-Gruppo Italiano Malattie Mieloproliferative). myeloproliferative neoplasms. Leukemia 2014; 28: 1407–1413. 8 Barbui T, Thiele J, Passamonti F, Rumi E, Boveri E, Ruggeri M et al. Survival and 1,6 2,6 3,6 1,6 disease progression in essential thrombocythemia are significantly influenced by T Barbui , J Thiele , HM Kvasnicka , A Carobbio , accurate morphologic diagnosis: an international study. J Clin Oncol 2011; 29: 4,6 5,6 AM Vannucchi and A Tefferi 3179–3184. 1 Research Foundation, Papa Giovanni XXIII Hospital, Bergamo, Italy; 9 Swerdlow S, Campo E, Harris N, Jaffe E, Pileri S, Stein H et al. (eds) WHO 2Institute of Pathology, University of Cologne, Cologne, Germany; Classification of Tumours of Haematopoietic and Lympoid Tissues. IARC Press: Lyon, 3Senckenberg Institute of Pathology, University of Frankfurt, France, 2008. Frankfurt, Germany; 10 Campbell PJ, Scott LM, Buck G, Wheatley K, East CL, Marsden JT et al. Definition of 4Department of Experimental and Clinical Medicine, subtypes of essential thrombocythaemia and relation to polycythaemia vera University of Florence, Florence, Italy and based on JAK2 V617F mutation status: a prospective study. Lancet 2005; 366: 5Hematology Division, Mayo Clinic, Rochester, MN, USA 1945–1953. 11 Rumi E, Pietra D, Ferretti V, Klampfl T, Harutyunyan AS, Milosevic JD et al. E-mail: [email protected] 6 JAK2 or CALR mutation status defines subtypes of essential thrombocythemia All authors contributed equally to this work. with substantially different clinical course and outcomes. Blood 2014; 123: 1544–1551. 12 Rotunno G, Mannarelli C, Guglielmelli P, Pacilli A, Pancrazzi A, Pieri L et al. REFERENCES Impact of calreticulin mutations on clinical and hematological phenotype and 1 Murphy S. Diagnostic criteria and prognosis in polycythemia vera and essential outcome in essential thrombocythemia. Blood 2014; 123: 1552–1555. thrombocythemia. Semin Hematol 1999; 36(1 Suppl 2): 9–13. 13 Tefferi A, Wassie EA, Lasho TL, Finke C, Belachew AA, Ketterling RP et al. 2 Tefferi A. The rise and fall of red cell mass measurement in polycythemia vera. Calreticulin mutations and long-term survival in essential thrombocythemia. Curr Hematol Rep 2005; 4: 213–217. Leukemia 2014, e-pub ahead of print 5 May 2014; doi:1038/leu.2014.148. 3 Silver RT, Chow W, Orazi A, Arles SP, Goldsmith SJ. Evaluation of WHO criteria 14 Thiele J, Kvasnicka HM, Diehl V. Initial (latent) polycythemia vera with for diagnosis of polycythemia vera: a prospective analysis. Blood 2013; 122: thrombocytosis mimicking essential thrombocythemia. Acta Haematol 2005; 113: 1881–1886. 213–219. 4 McMullin MF, Bareford D, Campbell P, Green AR, Harrison C, Hunt B et al. 15 Gianelli U, Vener C, Bossi A, Cortinovis I, Iurlo A, Fracchiolla NS et al. The European Guidelines for the diagnosis, investigation and management of polycythaemia/ Consensus on grading of bone marrow fibrosis allows a better prognostication of erythrocytosis. Br J Haematol 2005; 130: 174–195. patients with primary myelofibrosis. Mod Pathol 2012; 25: 1193–1202.

Bone marrow stromal cell-fueled multiple myeloma growth and osteoclastogenesis are sustained by protein kinase CK2

Leukemia (2014) 28, 2094–2097; doi:10.1038/leu.2014.178 reticulum stress/unfolded protein response and the sensitivity to classic and novel chemotherapeutics.7–9 CK2 can stimulate NF-kB by direct phosphorylation of Ser529 on p65/RelA.10 CK2 activation Multiple myeloma (MM) is an incurable B-cell malignancy character- of STAT3 signaling can result from triggering of upstream JAK ized by uncontrolled growth of clonal plasma cells.1 MM cells’ kinases and/or direct phosphorylation of Ser727.11 Remarkably, the dwelling in the bone marrow (BM) niche is associated with oral adenosine 50-triphosphate-competitive small CK2 inhibitor alterations of the surrounding microenvironment, which confers CX-4945 has entered phase I clinical trials in the USA in solid resistance to cytotoxic noxae. These changes are partly sustained by tumors and MM12 and displayed activity also against other B-cell delivery of growth signals from BM stromal cells (BMSCs) and tumors.13 Although CK2 was demonstrated to protect malignant perturbations affecting bone-remodeling mechanisms.2 Secretion of plasma cells from apoptosis-inducing noxae, its role in the several growth factors/cytokines (that is, IL-6 and TNFa) promotes MM–BMSC–OC cross-talk was never investigated, nor was its the evolution of the malignant clone(s). The NF-kB and JAK/STAT3 function in osteoclastogenesis/osteoblasts. are among the most active signaling cascades triggered in malignant Here, we addressed the consequences of CK2 inhibition on plasma cells.3,4 Homing of MM cells to the BM niche are largely (1) the survival of MM cells and growth signals delivered in the BM dependent on BMSC-secreted chemokine SDF1a/CXCL12 and its microenvironment, (2) the expression and function of the homing/ receptor CXCR4 on MM. Disruption of this circuit causes mobilization trafﬁcking receptor CXCR4 and (3) OC and osteoblast formation of MM cells outside the BM, loss of the protective niche and and viability. increased sensitivity to chemotherapy.5 Moreover, about 70% of MM To test whether CK2 inhibition could cause MM cell death even patients present the MM-associated bone disease (MMABD) caused in the presence of BMSC, we cultured INA-6 MM cells (IL-6- or by derangements between bone resorption (osteoclast (OC) activity) BMSC-dependent) or plasma cells from patients with the BMSC and bone deposition (osteoblast activity) in the MM BM milieu.6 line HS-5 or MM patients’ BMSC, and treated the co-cultures with We and others have previously shown that protein kinase CK2 is CX-4945 (Figure 1a). The inhibitor caused a signiﬁcant amount of an essential regulator of malignant plasma cell survival by apoptosis of CD45 þ INA-6 cells although they had been cultured impinging on NF-kB and STAT3 signaling, the endoplasmic with HS-5 or MM patient BMSC (Figure 1b, n ¼ 6, Po0.05). On the Accepted article preview online 4 June 2014; advance online publication, 27 June 2014

Leukemia (2014) 2090 – 2115 & 2014 Macmillan Publishers Limited