Bridget S. Wilkins

Definition of subtypes of essential thrombocythaemia and relation to polycythaemia vera based on JAK2 V617F mutation status: a prospective study

BACKGROUNDnAn acquired V617F mutation in JAK2 occurs in most patients with polycythaemia vera, but is seen in only half those with essential thrombocythaemia and idiopathic myelofibrosis. We aimed to assess whether patients with the mutation are biologically distinct from those without, and why the same mutation is associated with different disease phenotypes.nnnMETHODSnTwo sensitive PCR-based methods were used to assess the JAK2 mutation status of 806 patients with essential thrombocythaemia, including 776 from the Medical Research Councils Primary Thrombocythaemia trial (MRC PT-1) and two other prospective studies. Laboratory and clinical features, response to treatment, and clinical events were compared for V617F-positive and V617F-negative patients with essential thrombocythaemia.nnnFINDINGSnMutation-positive patients had multiple features resembling polycythaemia vera, with significantly increased haemoglobin (mean increase 9.6 g/L, 95% CI 7.6-11.6 g/L; p<0.0001), neutrophil counts (1.1x10(9)/L, 0.7-1.5x10(9)/L; p<0.0001), bone marrow erythropoiesis and granulopoiesis, more venous thromboses, and a higher rate of polycythaemic transformation than those without the mutation. Mutation-positive patients had lower serum erythropoietin (mean decrease 13.8 U/L; 95% CI, 10.8-16.9 U/L; p<0.0001) and ferritin (n=182; median 58 vs 91 mug/L; p=0.01) concentrations than did mutation-negative patients. Mutation-negative patients did, nonetheless, show many clinical and laboratory features that were characteristic of a myeloproliferative disorder. V617F-positive individuals were more sensitive to therapy with hydroxyurea, but not anagrelide, than those without the JAK2 mutation.nnnINTERPRETATIONnOur results suggest that JAK2 V617F-positive essential thrombocythaemia and polycythaemia vera form a biological continuum, with the degree of erythrocytosis determined by physiological or genetic modifiers.

Guidelines for the diagnosis, investigation and management of polycythaemia/erythrocytosis.

Traditionally, polycythaemia has been used to identify a group of varied disorders with an increase in circulating red cells that are typified by a persistently raised haematocrit (Hct). Since only the red cell lineage is involved, the term erythrocytosis has more validity and will be used throughout this article. Polycythaemia will be retained in relation to the clonal disorder, polycythaemia vera (PV), in which three cell lineages are involved.

The V617F mutation in Jak2 is not found in childhood acute lymphoblastic leukaemia

Members of the Jak kinase family, including Tyk2, Jak1, Jak2 and Jak3, play an important role in mediating cellular signals between cytokine/hormone receptors and downstream effector proteins by virtue of their tyrosine phosphorylation activities. They share structural homology and have two kinase domains, JH1 and JH2. While only the JH1 domain has kinase functionality, JH2 plays an important negative-regulatory role on JH1 activity. Recently a somatic, gain of function, mutation has been identified in JAK2 in a range of Philadelphia chromosomenegative myeloproliferative diseases including polycythaemia vera, essential thrombocythaemia and chronic idiopathic myelofibrosis (Baxter et al, 2005; Kralovics et al, 2005; Levine et al, 2005). A pointmutation in exon 12 of the JAK2 gene results in substitution of valine for phenylalanine at amino acid position 617 in the JH2 domain, leading to constitutive tyrosine phosphorylation activity and cytokine hypersensitivity. Neoplastic cells can be heterozygous for themutation or hemizygous if they are associated with loss of heterozygosity (LOH) of 9p, where JAK2 is situated. The explanation for V617F yielding different chronic myeloproliferative disease phenotypes is not clear but may relate to the target cell for transformation or the presence of other co-operating genetic events. Jak2 activation is also implicated in the pathology of childhood acute lymphoblastic leukaemia (ALL). Two studies showed constitutive activation of Jak2 in both primary samples and leukaemic cell lines, with inhibition blocking leukaemic cell growth selectively, in vitro and in vivo, by inducing programmed cell death (Meydan et al, 1996). In addition, LOH of 9p is a relatively common event, with JAK2 invariably being present in the minimally deleted region. One known mechanism of JAK2 activation in ALL is chromosomal translocation with the t(9;12)(p24;p13) yielding a chimaeric Tel–Jak2 fusion protein that has deregulated Jak2 kinase activity, confers cytokine-independent proliferation and results in the formation of ALL in a mouse model system (Lacronique et al, 1997; Peeters et al, 1997; Carron et al, 2000). However, this is a rare occurrence. Taken together, these observations provide a rationale for investigating the presence of V617F in childhood ALL. To detect the V617 mutation, we performed allele-specific polymerase chain reaction using genomic DNA isolated from bone marrow samples from children with ALL treated at our institution (Baxter et al, 2005). Eighty-six samples were collected during diagnosis and 42 samples during relapse. All samples were collected with appropriate consent. Amplicons were size fractioned by standard agarose gel electrophoresis. DNA from HEL92.1.7, an erythroleukaemic cell line, served as a positive control. None of the 128 ALL samples examined were positive for the 203-bp product, indicative of V617F mutation, but all were positive for the internal control (representative gel shown in Fig 1). Using HEL92.1.7 cells spiked into normal peripheral blood, the sensitivity of the assay was determined to be between 1% and 5%. Thus the V617F mutation is absent in childhood ALL and this finding concords with a mutational screening study of the JH2 domain in a small cohort of ALL patients (Cools et al, 1999). Given the importance of the Jak2 activation in primary ALL, a comprehensive mutational screen of all of its coding exons is warranted.

Post cardiac transplantation lymphoproliferative disorder presenting as t(8;14) Burkitt leukaemia/lymphoma treated with low intensity chemotherapy and rituximab

Post‐transplant lymphoproliferative disorder (PTLD) occasionally presents as Burkitt lymphoma/L3 leukaemia (BLL).

Mismatch repair defects in childhood acute lymphoblastic leukaemia at diagnosis and relapse

Although both MRD and karyotype are powerful determinants ofnoutcome in childhood ALL, few studies have examined the kinetics ofnMRD clearance by cytogenetics. In ALL2003, patients are stratifiednby NCI criteria to a three or four drug induction. MRD is assessed atnday 29 and week 11 using a standardised and quality controlled RQPCRnof 21patient specific immunoglobulin or T-cell receptornrearrangements. MRD risk groups were defined as: (1) High risknMRD410-4 at day 29 (HR); (2) Low risk MRD negative or o10-4 atnday 29 and negative at week 11 (LR); or (3) MRD indeterminate risk.nAmong 1000 patients entered into the trial, 98% were eligible fornthese analyses, 94% had a successful cytogenetics and 57% werenassigned to a clinically relevant MRD groups. Among these latter 555npatients, 54% were MRD-HR whereas 45% were MRD-LR. Collectively,npatients with high-risk cytogenetics ‘t(9;22), o40 chromosomes,n11q23/MLL, t(17;19) and iAMP21’ were more likely to be MRDHRn‘83% vs 52%, P50.003’. Patients with ETV6-RUNX1 fusion werenless likely to be MRD-HR ‘28% vs 63%, Po0.001’ whereas highnhyperdiploid patients were more likely ‘64% vs 49%, P50.002’.nHowever, excluding ETV6-RUNX1 patients from the latter analysisnrevealed that high hyperdiploid patients were as likely to be MRDHRnas other ETV6-RUNX1 negative patients. T-ALL patients werenalso more likely to be MRD-HR compared to BCP-ALL patients ‘70%nvs 52%, P50.022’. In particular, 9/10 patients with t(5;14)/TLX3-nBCL11B fusion and 6/6 patients with SIL-TAL1 fusion were MRD-HR.nIn conclusion, we have clearly demonstrated that MRD status variesnby cytogenetic subgroup with ETV6-RUNX1 patients having thenfastest MRD clearance rate. Despite the good prognosis associatednwith high hyperdiploidy, these patients were as likely to be MRD-HRnas other standard risk patients. Longer follow-up is required tondetermine the clinical significance of this finding.Genetic alterations play a key role in the leukemogenesis of childhood ALL. Inactivation of CDKN2A (p16), a tumour suppressor gene located at 9p21, can occur by deletion, methylation or mutation. Published reports are inconsistent in terms of incidence and mode of inactivation. We report a comprehensive analysis of CDKN2A inactivation in 1230 diagnostic and 101 relapse samples, including 46 matched diagnostic and relapse pairs, from 1285 children with ALL. Using data from cytogenetics (CC) (n=1088), FISH (n=1209), SNP arrays (SNPA) (n=106), CGH arrays (aCGH) (n=106), dHPLC (n=48) and methylation specific-PCR (MSP) (n=96) we have assessed the mode and frequency of CDKN2A inactivation. Mutation or methylation of CDKN2A was rare occurring in 1 patient each (2% and 1% respectively). In contrast, CDKN2A deletion was highly prevalent. The frequencies of deletion detected by the different methodologies were: CC 166 (15%), FISH 335 (28%), SNPA 17 (16%) and aCGH 35 (33%). The proportion of biallelic deletions also varied: CC 15 (9%), FISH 174 (52%) and aCGH 15 (65%). This variation was directly related to the resolution of each technique with a high degree of concordance across samples investigated by >1 method. Analysis of 50 deletions by aCGH showed that the size of the deletion ranged from 0.03Mb to 39.1Mb with a mean of 14.8Mb. Furthermore, analysis of 15 biallelic deletions demonstrated that they comprised one large deletion (mean size 23.3Mb) and a second much smaller deletion (mean size 1.4Mb). In addition, SNPA revealed copy number neutral LOH in 8 (8%) cases, but only once in association with a CDKN2A mutation. At diagnosis CDKN2A inactivation by any method was noted in 329 (27%) patients which was not different from that observed at relapse [25 (25%)]. However, the frequencies of CDKN2A inactivation and biallelic deletion were significantly greater in T-ALL compared with B cell precursor (BCP) ALL: 135/269 (50%) v 190/918 (21%) (p1 Prognostic impact of minimal residual disease in childhood acute lymphoblastic leukaemia depends on immunological subtype: results of trial AIEOP-BFM ALL 2000 M Schrappe , C Rizzari, G Mannz, A Moericke , MG Valsecchi, M Zimmermann, CR Bartram, R PanzerGrümayerz, A Schrauder , G Cazzaniga, T Flohr , R Parasole, A Reiter , WD Ludwig, L Lo Nigrozz, G Basso, G de Rossi, E Barisone, F Niggli , H Gadnerz and V Conter Department of Paediatrics and University Medical Center Schleswig-Holstein, Kiel, Germany, Paediatric Clinic, Ospedale Nuovo S Gerardo, Monza, Italy, zDepartment of Paediatric Haemato-Oncology, St Anna Children’s Hospital, Vienna, Austria, Department of Paediatric Haemato-Oncology, Medical School Hannover, Germany, Institute of Human Genetics, Ruprecht-Karls University, Heidelberg, Germany, Department of Paediatric Haemato-Oncology, Pausilipon Hospital, Napoli, Italy, Department of Paediatric Haemato-Oncology, University Hospital, Giessen, Germany, Department of Haemato-Oncology, Robert-Roessle-Klinik at the HELIOS Klinikum Charité, Berlin, Germany, zzDepartment of Paediatric Haematology/Oncology, University Hospital, Catania, Italy, Department of Paediatrics, University Hospital, Padova, Italy, Department of Paediatric Haematology/Oncology, Hospital Bambino Gesù, Rome, Italy, Ospedale Infantile Regina Margherita, Torino, Italy, Department of Paediatric Haematology/Oncology, University Hospital, Zurich, Switzerland

Teaching Cases from the Royal Marsden and St Mary's Hospitals. Case 23: An Unexpected Finding During Investigation of Thrombocytopenia

A 69-year-old retired Ashkenazi-Jewish man presented to our Chest and Allergy clinic with a history of rhinitis. Apart from hypertension (for which he was taking bendrofluazide and losartan) he was in good health. There was no family history of note. FBC showed a WBC 7.8 £ 10/l, Hb 15.7 g dl, MCV 83 fl and platelet count of 29 £ 10 l; which led to a haematology referral. The blood film showed anisopoikilocytosis with occasional acanthocytes and irregularly contracted cells, platelet anisocytosis with some giant hypogranular forms, neutrophils with borderline hypogranularity and increased nuclear projections and an occasional blast cell (Fig. 1). There was no history of easy bruising or bleeding and he had undergone an adenoidectomy as a child without incident. Physical examination was unremarkable with no lymphadenopathy or hepatosplenomegaly; abdominal ultrasound confirmed the lack of organomegaly. Further blood tests, which included a coagulation screen, liver function tests, assays of serum vitamin B12, red cell folate and serum ferritin, and an autoimmune profile were all normal. A bone marrow aspiration and trephine biopsy were performed. The aspirate was normocellular and normoblastic with some hypolobated megakaryocytes (Fig. 2), hypogranular neutrophil precursors, dysplastic neutrophils (as in the peripheral blood) and large abnormal macrophages (Fig. 2). Iron stores were increased but there were no ring sideroblasts. The trephine biopsy showed large focal aggregates of abnormal macrophages (Fig. 3) and small hypolobated megakaryocytes. Further