Bone marrow pathology in essential thrombocythemia: interobserver reliability and utility for identifying disease subtypes

Blood ◽  
2008 ◽  
Vol 111 (1) ◽  
pp. 60-70 ◽  
Author(s):  
Bridget S. Wilkins ◽  
Wendy N. Erber ◽  
David Bareford ◽  
Georgina Buck ◽  
Keith Wheatley ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Essential Thrombocythemia ◽  
Who Classification ◽  
Interobserver Variability ◽  
Interobserver Reliability ◽  
Diagnostic Category ◽  
Jak2 V617f ◽  
World Health ◽  
Major Hemorrhage ◽  
Fibrotic Process

The role of histopathology in the diagnosis of essential thrombocythemia (ET) is controversial, and there has been little attempt to quantitate interobserver variability. Diagnostic bone marrow trephine biopsy specimens from 370 patients with ET by Polycythemia Vera Study Group (PVSG) criteria were assessed by 3 experienced hematopathologists for 16 different morphologic features and overall diagnosis according to the World Health Organization (WHO) classification. Our results show substantial interobserver variability, particularly for overall diagnosis and individual cellular characteristics such as megakaryocyte morphology. Reticulin grade was the dominant independent predictor of WHO diagnostic category for all 3 hematopathologists. Factor analysis identified 3 independent factors likely to reflect underlying biologic processes. One factor related to overall and lineage-specific cellularity and was significantly associated with JAK2 V617F status (P < .001), a second factor related to megakaryocyte clustering, and a third was associated with the fibrotic process. No differences could be discerned between patients labeled as having “prefibrotic myelofibrosis” or “true ET” in clinical and laboratory features at presentation, JAK2 status, survival, thrombosis, major hemorrhage, or myelofibrotic transformation. These results show that histologic criteria described in the WHO classification are difficult to apply reproducibly and question the validity of distinguishing true ET from prefibrotic myelofibrosis on the basis of subjective morphologic criteria. This study was registered at http://isrctn.org as #72251782 and at http://eudract.emea.europa.eu/ as #2004-000245-38.

scholarly journals Reticulin Accumulation in Essential Thrombocythemia: Prognostic Significance and Relationship to Therapy

2009 ◽  
Vol 27 (18) ◽  
pp. 2991-2999 ◽  
Author(s):  
Peter J. Campbell ◽  
David Bareford ◽  
Wendy N. Erber ◽  
Bridget S. Wilkins ◽  
Penny Wright ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Essential Thrombocythemia ◽  
Arterial Thrombosis ◽  
Interobserver Reliability ◽  
Prognostic Significance ◽  
Underlying Disease ◽  
Major Hemorrhage ◽  
Independent Prognostic Marker ◽  
Bone Marrow Histology ◽  
Duration Of Disease

PurposeEssential thrombocythemia (ET) manifests substantial interpatient heterogeneity in rates of thrombosis, hemorrhage, and disease transformation. Bone marrow histology reflects underlying disease activity in ET but many morphological features show poor reproducibility.Patients and MethodsWe evaluated the clinical significance of bone marrow reticulin, a measure previously shown to have relatively high interobserver reliability, in a large, prospectively-studied cohort of ET patients.ResultsReticulin grade positively correlated with white blood cell (P = .05) and platelet counts (P = .0001) at diagnosis. Elevated reticulin levels at presentation predicted higher rates of arterial thrombosis (hazard ratio [HR], 1.8; 95% CI, 1.1 to 2.9; P = .01), major hemorrhage (HR, 2.0; 95% CI, 1.0 to 3.9; P = .05), and myelofibrotic transformation (HR, 5.5; 95% CI, 1.7 to 18.4; P = .0007) independently of known risk factors. Higher reticulin levels at diagnosis were associated with greater subsequent falls in hemoglobin levels in patients treated with anagrelide (P < .0001), but not in those receiving hydroxyurea (P = .9). Moreover, serial trephine specimens in patients randomly assigned to anagrelide showed significantly greater increases in reticulin grade compared with those allocated to hydroxyurea (P = .0003), and four patients who developed increased bone marrow reticulin on anagrelide showed regression of fibrosis when switched to hydroxyurea. These data suggest that patients receiving anagrelide therapy should undergo surveillance bone marrow biopsy every 2 to 3 years and that those who show substantially increasing reticulin levels are at risk of myelofibrotic transformation and may benefit from changing therapy before adverse clinical features develop.ConclusionOur results demonstrate that bone marrow reticulin grade at diagnosis represents an independent prognostic marker in ET, reflecting activity and/or duration of disease, with implications for the monitoring of patients receiving anagrelide.

FLIP (Long) and FLIP (Short) Expression in Bone Marrow Cells in Patients with Myelodysplastic Syndrome: Correlation with FAB and WHO Classification.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4926-4926
Author(s):  
Paula Campos ◽  
Fabiola Traina ◽  
Adriana Duarte ◽  
Bruno Benites ◽  
Marcelo Brandao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
Myelodysplastic Syndrome ◽  
Mrna Expression ◽  
Who Classification ◽  
Fas Ligand ◽  
Low Risk ◽  
World Health ◽  
Caspase 8 ◽  
Marrow Cells

Abstract The paradox of peripheral cytopenias despite of normo/hypercellular marrow in myelodysplastic syndrome (MDS) has been ascribed to excessive intramedullary hematopoietic cell apoptosis. Several apoptosis-inducing systems, including Fas/Fas ligand and TNF-related apoptosis-inducing ligand (TRAIL) and its receptors, are upregulated in MDS. FLIP (FLICE (FAS-associated death-domain-like IL-1β-converting enzyme)-inhibitory protein) was identified as a FAS and TRAIL signal inhibitor. The largest variant FLIPLong (FLIPL) was originally characterized as a molecule with inhibitory activity on caspase-8. The short splice form termed FLIPShort (FLIPS) has also been characterized as a potent (TRAIL-induced) apoptosis inhibitor. However, whereas FLIPL and FLIPS have been described as death receptor pathway inhibitors, recent data suggest that physiologically, FLIPL may have caspase-8-activating properties. This study aims to characterize the expression of FLIPL and FLIPS based on mRNA, by Real-time quantitative PCR, in marrow cells from MDS patients and to correlate the expression with French-American-British (FAB) and World Health Organization (WHO) classification. For each sample, results were first calculated as a ratio of the total transcript number of FLIPL or FLIPS and the total transcript number of the endogenous reference gene (β-actin) to obtain a normalized target value. Transcript ratios of each sample were normalized against the respective ratio of a pool of 6 normal bone marrow donors (NBM), and the ratio between the two was used as measure for the relative FLIPL or FLIPS level. We hypothesized that FLIPL and FLIPS expression differed between low and high risk of MDS. Marrow aspirates were obtained from 6 NBM and 16 patients with MDS out of treatment (7 males, 9 females; 23–78 (median 64) yo). The National Ethical Committee Board approved this study, informed-written consent was obtained from all patients and donors. According to FAB classification, patients were distributed as: 10 RA, 2 RARS and 4 RAEB. According to WHO classification: 10 RCMD, 2 RCMD-RS, 3 RAEB-1 and 1 RAEB-2. FLIPS mRNA expression were significantly higher in high risk DS according to FAB and WHO classification; RA/RARS compared with AREB (0.08 [0.0–2.3] vs 0.67 [0.36–1.54]; P = 0.03); RCMD and RCMD-RS compared with RAEB-1 and RAEB-2 (0.08 [0.0–2.3] vs 0.67 [0.36–1.54]; P = 0.03). However, FLIPL mRNA expression also tended to be higher in high risk MDS according to FAB and WHO classification, though not significantly different: RA/RARS compared with AREB (1.18 [0.06–3.43] vs 1.65 [0.51–3.63]; P = 0.46); RCMD and RCMD-RS compared with RAEB-1 and RAEB-2 (1.18 [0.06–3.43] vs 1.65 [0.51–3.63]; P = 0.46). Lower FLIPS level in low risk MDS marrows, in addition to the well described upregulation of extracellular proapoptotic signals, would explain the increased susceptibility of hematopoietic cells in low risk MDS marrow to death-inducing stimuli. The fact that FLIPL expression did not differ according to FAB and WHO classification could be related to the hypothesis that FLIPL may have caspase-8-activating properties rather than anti-apoptotic activity. Differential regulation of FLIPL and FLIPS according to risk groups in MDS patients might result in different rates of apoptosis. Further studies are needed to elucidate the mechanisms controlling and regulating FLIP expression in normal and malignant hemopoietic cells.

Cytoreductive Therapeutic Approach in the Essential Thrombocythemia (ET) Patients of the Registro Italiano Trombocitemia (RIT): Preliminary Data

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5248-5248
Author(s):  
Luigi Gugliotta ◽  
Alessia Tieghi ◽  
Anna Candoni ◽  
Monia Lunghi ◽  
Gianluca Gaidano ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Essential Thrombocythemia ◽  
Therapeutic Approach ◽  
Jak2 V617f ◽  
Bone Marrow Biopsies ◽  
Thrombotic Risk ◽  
Significant Difference ◽  
Cytoreductive Treatment ◽  
Wbc Count

Abstract Background: the Registro Italiano Trombocitemia, that is a GIMEMA project, has been activated to registry Italian Essential Thrombocythemia (ET) patients, to improve the diagnosis appropriateness (WHO criteria), to verify the prognostic value of the clinical and biological parameters, to evaluate the compliance to the therapeutical Italian guidelines (1), and to create a network for activation of new studies. Objective: this analysis is mainly devoted to describe the ET patients registered in the RIT and to evaluate the therapeutic approach adopted in the 102 participating hematological centers. Material and methods: two thousand and fifteen ET patients have been registered after the written informed consent was obtained, and data validation by various expert panels is in progress. This preliminary report considers 1785 patients, diagnosed mainly (1078, 60.4%) since the publication in the year 2004 of the ET therapy Italian guidelines (1). Results: the patients, 678 (38%) males and 1107 (62%) females, showed at diagnosis: age 60.3 ± 16.8 years with higher values in males than in females (61.7 ± 15.3 vs. 59.4 ± 17.7, p&lt;0.05), being the patients below 40 years 14% and those over 70 years 33% of cases; PLT count (109/L) 846 ± 309 with lower values in males than in females (813 ± 261 vs. 866 ± 334, p&lt;0.002), and with values 1001–1500 and over 1500 in 16% and 4% of cases, respectively; WBC count (109/L) 9.1 ± 2.9, without difference by sex, and with values 12–15 in 10% and over 15 in 3% of cases; Hgb (g/dL) 14.2 ± 1.6 with higher values in males than in females (14.8 ± 1.5 vs. 13.8 ± 1.5, p&lt;0.001), and with values over 16.5 in 8.5% of males and 2.7% of females, respectively; splenomegaly in 488 (27%), echo-documented in 324 cases (18%); history of hemorrhage and thrombosis in 90 (5%) and 325 (19%) of cases, respectively; disease-related symptoms in 41% and general thrombotic risk factors in 93% of cases, respectively. The WHO 2001 diagnostic criteria were reported for 33% of cases observed before the year 2004 and for 53 % of cases observed since the year 2004. Detailed data at diagnosis were reported as follows: bone marrow biopsy in 1087 cases (61%) with a frequency of 51% and 68% before and since the year 2004, respectively; bcr-abl study in 1045 cases (59%); cytogenetics in 828 cases (46%) with karyotype abnormalities in 27 patients (3%). The JAK2 V617F mutation, searched in 574 cases (32%), was observed in 320 of them (56%). The patient follow-up was 4.5 ± 4.5 years with a total of 5245 pt-yrs. During the follow-up the hemorrhagic events were 5.7% (1.3/100 pt-yrs), being the major events 1.9% (0.4/100 pt-yrs); the thrombotic complications were 14.9 % (3.3/100 pt-yrs), resulting the major arterial 9.4% (2.1/100 pt-yrs), the major venous 3.5% (0.8/100 pt-yrs) and the minor thrombosis 2% (0.4/100 pt-yrs). An antiplatelet treatment, almost always with low dose aspirin, was performed in 75% of the patients, without significant difference in the cases diagnosed before and since the 2004. A cytoreductive treatment was done with use of Hydroxyurea (HU, 64%), Interferon alpha (IFN, 16%), Anagrelide (ANA, 15%), Busulfan (BUS, 4%), and Pipobroman (PIPO, 2 %). In the ET patients diagnosed since the year 2004 respect those diagnosed before, it was observed a decrease in the use of all the cytoreductive drugs, particularly BUS (−62%), IFN ((−62%), and ANA ((−68%). The use of the cytoreductive drugs was related to the patient mean age (years): BUS (76), PIPO (72), HU (67), ANA (53), IFN (48). In the patients diagnosed since the 2004 as compared with those before 2004, the mean age of the treated patients increased for BUS (from 69 to 81 yrs, p&lt;0.001) and for HU (from 64 to 69 yrs, p&lt;0.001) while it decreased for IFN (from 49 to 46 yrs, p&lt;0.05). Conclusion: in the analyzed patients of the ET Italian registry the diagnosis appropriateness resulted improved in the cases observed since the year 2004 respect those observed before, with an increase of bone marrow biopsies from 51% to 68% of patients. Moreover, in accord with the ET therapy Italian guidelines, the use of the cytoreductive drugs was less frequent in the patients diagnosed since the year 2004 than before (particularly for BUS, IFN, and ANA) and the more safe molecules IFN and ANA were preferentially deserved to the younger patients.

Utility of FDG-PET scanning in lymphoma by WHO classification

Blood ◽  
2003 ◽  
Vol 101 (10) ◽  
pp. 3875-3876 ◽  
Author(s):  
Rebecca Elstrom ◽  
Liang Guan ◽  
Gary Baker ◽  
Khozaim Nakhoda ◽  
Jo-Anne Vergilio ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Fdg Pet ◽  
Who Classification ◽  
Cell Lymphoma ◽  
Bone Marrow Involvement ◽  
B Cell Lymphoma ◽  
World Health ◽  
Bone Marrow Biopsies ◽  
Positron Emission ◽  
Health Organization

Abstract We retrospectively evaluated 18fluoro-2-deoxyglucose positron emission tomography (FDG-PET) scans in 172 patients with lymphoma and correlated results with pathologic diagnosis using the World Health Organization (WHO) classification system. In total, FDG-PET detected disease in at least one site in 161 patients (94%) and failed to detect disease in 11 patients (6%). The most frequent lymphoma diagnoses were diffuse large B-cell lymphoma (LBCL; n = 51), Hodgkin lymphoma (HL; n = 47), follicular lymphoma (FL; n = 42), marginal zone lymphoma (MZL; n = 12), mantle cell lymphoma (MCL; n = 7), and peripheral T-cell lymphoma (PTCL; n = 5). FDG-PET detected disease in 100% of patients with LBCL and MCL and in 98% of patients with HL and FL. In contrast, FDG-PET detected disease in only 67% of MZL and 40% of PTCL. Comparison with bone marrow biopsies showed that FDG-PET was not reliable for detection of bone marrow involvement in any lymphoma subtype.

Essential Thrombocythemia in Patients with Platelet Counts below 600×109/L: Applicability of the 2008 World Health Organization Diagnostic Criteria Proposal

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5239-5239
Author(s):  
Mi Kwon ◽  
Santiago Osorio Prendes ◽  
Carolina Muñoz ◽  
Jose Manuel Sanchez ◽  
Monica Ballesteros ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Platelet Count ◽  
Essential Thrombocythemia ◽  
World Health ◽  
Jak2 Mutation ◽  
Who Criteria ◽  
Platelet Counts ◽  
Bone Marrow Histology ◽  
Health Organization

Abstract WHO criteria defines platelet counts above 600×109/L as the threshold for essential thrombocythemia (ET) diagnosis. It has been argued that such threshold excludes a number of patients with ET with platelet counts below 600×109/L. Recently, a proposal for revision of the World Health Organization (WHO) diagnostic criteria for ET has been published, which includes the combination of histological bone marrow study and testing of JAK2 mutation. Design and methods: Retrospective analysis of 92 patients with ET diagnosis between 1989 and February 2008, isolating the subgroup of patients with platelet counts below 600×109/L. The aim of this study was to analyze the applicability of the 2008 WHO criteria in this subgroup. Results: Of the 92 patients, 30 patients did not fulfill the WHO criteria due to platelet counts &lt;600×109/L and in some cases also due to the coexistence of alternative causes of thrombocytosis. There were no significant differences between the entire group and the borderline platelet count subgroup in demographics, clinical and laboratory parameters (Table 1). The median age of the borderline platelet count group was 51 years (range 19–83 years) and 20 were female (70%). At diagnosis their median platelet count was 527×109/L (range 424–597). Fifteen patients (50%) showed the presence of JAK2 mutation. Remarkably, 74% of the patients presented as high-intermediate risk at diagnosis. From the 30 patients who did not fulfill the WHO criteria due to low platelet counts, 26 (87%) satisfied the modified criteria allowing ET diagnosis. Among them, 1 patient showed an alternative cause of thrombocytosis, however JAK2 mutation was positive confirming the primary cause of the disorder. Four patients remained not fulfilling the new criteria due to insufficient bone marrow sample or incompatible histology, however one of these patients showed JAK2 mutation confirming ET. The median follow-up was 2.54 years (range 0.07–18.7). During this period, none of the 30 patients had a spontaneous decrease of platelet count to within the normal range. Furthermore, transformation from ET to IMF was observed in 2 cases supporting the diagnosis of ET. During follow-up, 27 out of 30 patients were treated with antiaggregating drugs, 3 with antithrombotic therapy, and 20 with myelosuppressive therapy. The 11 patients who did not receive myelosuppressive therapy remained with platelet counts above 400×109/L. Conclusions: In our study, patients with platelet counts below 600×109/L did not show significant differences compared with the whole ET patients group. This subgroup can be diagnosed as having ET following the 2008 WHO criteria. The detection of JAK2 mutation in this setting enables the accurate diagnosis not only in cases with borderline thrombocytosis but more importantly in cases with alternative potential causes and also in cases where bone marrow sample is not available or incompatible. This observation raises de question of the role of bone marrow histology as a subjective diagnostic tool in ET diagnosis as opposed to JAK2 mutation detection. In JAK2 negative patients, subsequent follow-up of untreated patients confirmed the diagnosis since platelet counts remained high. The modified criteria facilitates the clinician to make an early diagnosis of ET in this subgroup of patients. Furthermore, a high proportion of these patients may be at risk of vascular complications, who may beneficiate from being correctly treated. TABLE 1 Total of patients Platelet count &lt;600×10e9/L Number 92 30 Female (number, %) 59 (64%) 20 (70%) Age (median, range) 51 (19–84) 51 (19–83) Risk Low 26 (28%) 8 (26%) Intermediate 21(22%) 6 (19%) High 45 (48%) 16 (53%) Platelets ×10e9/L 693 (424–2,777) 527 (424–597) Hb g/dL 14.5 (11–18) 14.3 (11–16.8) Leucocytes ×10e9/L 8,5 (3,6–24,2) 8,5 (5,2–13,8) LDH UI/L 380 (39–1413) 337 (39–938) Splenomegaly 16 (17%) 5 (17%) JAK2 mutation 47 (51%) 15 (50%) Bone Marrow histology Celullarity &gt;3.5 30/88 (34%) 8/28 (29%) Fibrosis grade I 2/90 (2%) 0 Compatible histology 79/89 (86%) 21/28 (75%) Abnormal Cytogenetics 2/26 (8%) 0 Symptoms 13 (14%) 4 (13%) Thrombotic event 16 (18%) 5 (17%) Haemorrhagic event 3 (4%) 0

Acute Panmyelosis with Myelofibrosis: Clinicopathological and Molecular Features of a Rare Malignant Bone Marrow Disease.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3101-3101
Author(s):  
Silke Kapp-Schwoerer ◽  
Renate Looser ◽  
Franz Schaub ◽  
Michael Hummel ◽  
Manfred Olschewski ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Who Classification ◽  
Laboratory Data ◽  
Myeloproliferative Neoplasm ◽  
Jak2 V617f ◽  
Genetic Alterations ◽  
Control Group ◽  
Stromal Compartment ◽  
Molecular Features ◽  
Mutational Status

Abstract Abstract 3101 Poster Board III-38 Background Acute panmyelosis with myelofibrosis (APMF) is listed among the category of “AML, not otherwise specified” in the actual WHO classification of hematopoietic tumors. Since specific genetic alterations have not yet been defined, this myeloid neoplasm (MN) remains clinicopathologically assigned. The WHO classification addresses the difficulties concerning the differential diagnosis between APMF, myelodysplastic syndromes (MDS) with excess of blasts and concomitant fibrosis (RAEB-F) and acute megakaryoblastic leukaemia (AMGL). Methods Our files were searched for patients with rapid-onset MN without a prior history of a myeloproliferative neoplasm or a MDS fulfilling the 2008 WHO criteria of APMF. An age- and gender-matched control group of RAEB-F cases with multilineage dysplasia was retrieved for comparison. Our approach included: i) the careful review of morphological, clinical and laboratory data with special regard to the proportion of immature CD34 + precursors/blasts in bone marrow (BM) biopsies; ii) the grading of BM fibrosis according to the European consensus guidelines; iii) the analysis of the JAK2-V617F, the MPL W515L and the GATA1 mutational status, the cytoplasmic localization of nucleophosphamin1 (NPM1) in BM blasts, and the expression the inhibitor of differentiation protein-2 (Id2) in hematopoietic and stroma cells; iv) the evaluation of the prognostic relevance of these parameters. Results Fifty-four patients (32 males, 22 females) with a median age of 66, 9 years from our institutions were diagnosed APMF. Sufficient DNA for allele specific PCR for JAK2 genotyping was obtained from paraffin-embedded EDTA-decalcified BMB specimens from 22 APMF and 14 RAEB-F patients. In addition, an analysis of MPL W515L and GATA1 mutations was performed on 15 APMF cases. The relevant clinicopathological and molecular data are summarized in the table. Semiquantitative evaluation revealed a decreased expression level of Id2 in erythropoiesis of the APMF samples, while Id2 was increased in the stromal compartment. Within the total APMF cohort, patients aged ≤60 years had a longer OS (p=0,013). A comparison between the Kaplan-Meier curves of the APMF patients who underwent hematopietic cell transplantation (HCT) with those treated by conventional chemotherapy clearly showed that HCT significantly improved OS (median survival 6,1 vs. 11,1 months, p=0,005). Within the non-transplanted group, a bone marrow blast count ≤25% was associated with a longer OS (p=0,006). Conclusions Our results point to significant differences between APMF and RAEB-F with regard to OS, MCV, LDH, BM fibrosis and BM blast cells. We detected a higher frequency of mutated JAK2 V616F in the MB samples than generally reported in the literature for other categories of AML or MDS. However, the JAK2 V617F mutation status or an aberrant cytoplasmic NPM1 localization were both not predictive of outcome. In contrast to data published for AMGL, no association between a MPL W515L mutation and APMF was observed. Survival was significantly worse in APMF than in RAEB-F. A rapid diagnosis based on a multiparameter approach and a proper management including the option of HCT even in elderly patients seems mandatory for APMF patients. Targeting the fibrotic BM microenvironment which is characterized by an increased Id2 expression may be considered for future therapeutic strategies. Disclosures Skoda: Genentech Inc.: Consultancy.

JAK2 V617F-Negative and MPL W515K/L-Negative Essential Thrombocythemia: A High Resolution SNP Array Study

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5258-5258
Author(s):  
Carla AL Assaf ◽  
Els Lierman ◽  
Timothy Devos ◽  
Carlos Graux ◽  
Johan Billiet ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Essential Thrombocythemia ◽  
Copy Number ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Snp Array ◽  
Jak2 V617f ◽  
Common Mutation ◽  
Nicotinamide Phosphoribosyltransferase ◽  
Array Data

Abstract Background JAK2 V617F is the most common mutation in essential thrombocythemia (ET), occurring in approximately 50 % of cases. Second to JAK2 V617F is MPL W515K/L, accounting for about 10 % of cases. The molecular cause of the remaining ET cases is still largely unknown. Aims We sought to investigate JAK2 V617F-negative and MPL W515K/L-negative ET for regions of copy number variations (CNV) and loss of heterozygosity (LOH). Methods We studied blood or bone marrow samples from a series of 64 JAK2 V617F-negative and MPL W515K/L-negative ET cases. They were subjected to 2.7M SNP array by Affymetrix, which has 2,761,979 copy number markers including 400,103 SNP markers. The array data were analyzed for recurrent CNVs with Array Studio (OmicSoft), and for individual CNVs or recurrent LOHs (≥3 Mbs) with the Chromosome analysis suite (ChAS, Affymetrix). Results Only 8 recurrent gains were identified, in 5/64 patients. Interestingly, the most common gain, occurring in 5 cases was a gain of chr7 q22.3, including the gene encoding Nicotinamide phosphoribosyltransferase (NAMPT). NAMPT is known to be overexpressed in several cancers such as multiple myeloma. It catalyzes the rate-limiting step of the nicotinamide adenine dinucleotide (NAD+) biosynthesis pathway. It is also required for cell growth and survival. We checked in the 5 patients for NAMPT amplification by quantitative PCR (qPCR) on genomic DNA in comparison to controls and by normalizing to ALB and RPPH1. We were able to validate the gain in 2/5 patients. The gain in these 2 patients was demonstrated to be acquired by qPCR of NAMPT in buccal swab DNA. Other recurrent gains involved regions of chromosomes 2, 5, 7, 12, 13, and 22. These gains included, amongst others, LCP1 on chr13 q14.3 and CYTIP on chr2 q24.1, occurring in 2/64 and in 3/64 respectively. We also checked for non-recurrent gains and losses in our cohort. This analysis generated a total of 8 CNVs in 6 different patients, comprising 5 regional gains in chromosomes 2, 8, 12, and 15 and 3 regional losses in chromosomes 5, 8 and 11. The array data were also analyzed for recurrent LOHs on ChAS, yielding 17 recurrent copy neutral LOHs (CN-LOH) in 35 patients (circos plot). The most common CN-LOH region was on chromosome 3 appearing in 8 patients. Other CN-LOH regions involved chromosomes 1, 2, 3, 4, 5, 6, 7, 10, 12, 15, and 17 and they occurred in 2-5/64 patients. However, as small regions of CN-LOH can be constitutional, we suspect that most of these CN-LOH regions are not acquired. The largest region of CN-LOH observed was 12 Mbs in size. Conclusions Previous studies in unselected series of BCR-ABL1-negative myeloproliferative neoplasms have shown that copy number alterations are rare in ET as well as in polycythemia vera. In this series of 64 JAK2 V617F-negative and MPL W515K/L-negative ET patients we found recurrent gains not reported previously in the database of genomic variants in only 8% of patients, and small areas of CN-LOH in ∼55% of cases. However, most of the latter probably are constitutional. Our SNP array study provides further evidence that gains, losses or CN-LOH of small genomic regions do not play an essential role in the pathogenesis of the majority of JAK2 V617F-negative and MPL W515K/L-negative ET. However, the low frequency of megakaryocytes and unknown level of clonal involvement of the myeloid compartment in JAK2 V617F-negative and MPL W515K/L-negative ET bone marrow remain a caveat. Next generation sequencing technology is expected to bring new insights on the molecular pathogenesis of this elusive ET subset. Circos plot showing the recurrent CN-LOHs Left half represents a total of 35 patients carrying recurrent CN-LOHs and the right half represents the chromosomes and their associated properties. Right outermost layer depicts 1+log-gene density (min, 1; max 42) where cancer, OMIM and other genes are colored in red, blue and green respectively. Right middle and innermost layers designates SNP density (blue, values < 0,02; gray values, <0,06; red values >0,06; max scale 0,013) and absolute SNP numbers (min, 1; max, 12074) per windows of 50kb. Each 5Mb distance is marked with a tick underneath the innermost layer. Links from each chromosome are colored differently. Regions that are more confined on the same chromosome are least transparent and regions that are shared by more patients are drawn on top of lesser links. Disclosures: No relevant conflicts of interest to declare.

Comparison of Clinical Outcomes Between Pediatric Aplastic Anemia and Refractory Cytopenia of Childhood

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1498-1498
Author(s):  
Asahito Hama ◽  
Atsushi Manabe ◽  
Daisuke Hasegawa ◽  
Kazue Nozawa ◽  
Atsushi Narita ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Aplastic Anemia ◽  
Clinical Outcomes ◽  
Who Classification ◽  
Chromosomal Abnormalities ◽  
Response Rates ◽  
World Health ◽  
Trisomy 8 ◽  
Monosomy 7 ◽  
Cell Lineages

Abstract In the diagnosis of childhood bone marrow failures (BMFs), differentiating aplastic anemia (AA) from hypoplastic myelodysplastic syndrome (MDS) is challenging. The 2008 World Health Organization (WHO) classification has proposed a provisional entity, "refractory cytopenia of childhood (RCC)". The spectrum of patients with RCC is wide, ranging from patients with severe hypocellular bone marrow (BM) and mild dysplasia to those with normocellular BM and distinct dysplasia meeting the criteria for refractory cytopenia with multilineage dysplasia (RCMD) defined for adults with MDS. Currently, it is recommended that children who meet the criteria for RCMD should be classified as RCC in the WHO classification until the number of lineages involved has been fully evaluated with regard to their relative importance as prognostic factors. Until now, few studies have addressed the question whether the current WHO classification reflects clinical outcomes of childhood BMFs. To determine the clinical differences among AA, RCC, and RCMD, we compared clinical outcomes for patients with AA, RCC, and RCMD in Japan. From February 2009 to December 2013, 252 patients were registered to the central morphology review system of the Japanese Society of Hematology and Oncology and were diagnosed with BMFs. Peripheral blood (PB) and BM smears were reviewed by two pediatric hematologists, and BM trephine biopsies were reviewed by a hematopathologist. RCC is defined as persistent cytopenia with <2% and <5% blasts in PB and BM, respectively. BM aspirate smears show dysplastic changes in >2 cell lineages or >10% within one cell lineage. On the other hand, the criteria of RCMD is defined as persistent cytopenia with <1% and <5% blasts in PB and BM, respectively. BM smears show >10% dysplastic changes in >2 cell lineages. Patients with inherited BMFs were excluded by family history and physical examination. Further, Fanconi anemia was excluded by chromosome fragility test　and Dyskeratosis congenita was screened by measuring the telomere length of the peripheral lymphocytes by flowcytometry. Out of 252 patients, 63 were classified as AA, 131 as RCC, and 58 as RCMD. Median ages in AA, RCC, and RCMD groups were 10, 8, and 7 years, respectively (p=0.07). The median of leukocyte, neutrophil, reticulocyte, and platelet count, and mean corpuscular volume were significantly lower in AA than in RCC and RCMD groups (p<0.01). Chromosomal abnormalities were detected in 1 patient with AA (trisomy 8), 3 patients with RCC (trisomy 8, n=2; other, n=1), and 9 patients with RCMD (trisomy 8, n=5; monosomy 7, n=1; other, n=3) at the time of diagnosis (p<0.01). Out of 252 patients, 82 (AA, n=3; RCC, n=46; RCMD, n=33) were observed without any treatments (watch and wait, WW). 5-year overall survival (OS)/failure free survival (FFS) rates in WW group were 67%/67% in AA, 98%/54% in RCC, and 100%/69% in RCMD patients (p<0.01/p=0.97). Immunosuppressive therapy (IST) with rabbit antithymocyte globulin and cyclosporine was performed in 110 (AA, n=39; RCC, n=57; RCMD, n=14) patients. Six months after the IST, the response rates to the IST were not significantly different among AA (40%), RCC (63%), and RCMD (64%) (p=0.08). The development of additional chromosomal aberrations was found in 2 patients with RCC, and 1 with RCMD. The 5-year OS/FFS rates in IST group were 89%/36% in AA, 94%/38% in RCC, and 93%/23% in RCMD patients (p=0.64/p=0.86). Stem cell transplantation (SCT) as a first line therapy was performed in 19 patients with AA, 10 with RCC, and 5 with RCMD. The rejection was found in 2 patients with RCC and 3 with RCMD. Although 5-year OS rates in patients who underwent SCT were not different among 3 groups (p=0.26), FFS rate (30%) in patients with RCMD was significantly lower than in those with AA (100%) and RCC (78%) (p<0.01). In conclusion, we could not find any clinical relevance of separating RCC from AA because response rates to IST and the development of clonal evolution did not significantly differ between AA and RCC. The entity of RCMD should be adopted to childhood MDS classification because children with RCMD exhibited a distinct characteristic of morphology and a frequent chromosomal aberration at the time of diagnosis. The optimal treatment strategy including preconditioning regimen of SCT should be established for children with acquired BMFs based on the BM cellularity and morphological classification. Disclosures Kojima: SANOFI: Honoraria, Research Funding.

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