Is there a pressing need for improving prognostication strategies in therapy-related myelodysplastic syndromes?

Leukemia accepted article preview online, 31 July 2017. doi:10.1038/leu.2017.237.

PARP-2 sustains erythropoiesis in mice by limiting replicative stress in erythroid progenitors.

Erythropoiesis is a tightly regulated process in which multipotential hematopoietic stem cells produce mature red blood cells. Here we show that deletion of poly(ADP-ribose) polymerase-2 (PARP-2) in mice leads to chronic anemia at steady state, despite increased erythropoietin plasma levels, a phenomenon not observed in mice lacking PARP-1. Loss of PARP-2 causes shortened lifespan of erythrocytes and impaired differentiation of erythroid progenitors. In erythroblasts, PARP-2 deficiency triggers replicative stress, as indicated by the presence of micronuclei, the accumulation of γ-H2AX (phospho-histone H2AX) in S-phase cells and constitutive CHK1 and replication protein A phosphorylation. Transcriptome analyses revealed the activation of the p53-dependent DNA-damage response pathways in PARP-2-deficient cells, culminating in the upregulation of cell-cycle and cell death regulators, concomitant with G2/M arrest and apoptosis. Strikingly, while loss of the proapoptotic p53 target gene Puma restored hematocrit levels in the PARP-2-deficient mice, loss of the cell-cycle regulator and CDK inhibitor p21 leads to perinatal death by exacerbating impaired fetal liver erythropoiesis in PARP-2-deficient embryos. Although the anemia displayed by PARP-2-deficient mice is compatible with life, mice die rapidly when exposed to stress-induced enhanced hemolysis. Our results pinpoint an essential role for PARP-2 in erythropoiesis by limiting replicative stress that becomes essential in the absence of p21 and in the context of enhanced hemolysis, highlighting the potential effect that might arise from the design and use of PARP inhibitors that specifically inactivate PARP proteins.

Impact of adjunct cytogenetic abnormalities for prognostic stratification in patients with myelodysplastic syndrome and deletion 5q.

This cooperative study assessed prognostic factors for overall survival (OS) and risk of transformation to acute myeloid leukemia (AML) in 541 patients with de novo myelodysplastic syndrome (MDS) and deletion 5q. Additional chromosomal abnormalities were strongly related to different patients' characteristics. In multivariate analysis, the most important predictors of both OS and AML transformation risk were number of chromosomal abnormalities (P<0.001 for both outcomes), platelet count (P<0.001 and P=0.001, respectively) and proportion of bone marrow blasts (P<0.001 and P=0.016, respectively). The number of chromosomal abnormalities defined three risk categories for AML transformation (del(5q), del(5q)+1 and del(5q)+ ≥ 2 abnormalities) and two for OS (one group: del(5q) and del(5q)+1; and del(5q)+ ≥ 2 abnormalities, as the other one); with a median survival time of 58.0 and 6.8 months, respectively. Platelet count (P=0.001) and age (P=0.034) predicted OS in patients with '5q-syndrome'. This study demonstrates the importance of additional chromosomal abnormalities in MDS patients with deletion 5q, challenges the current '5q-syndrome' definition and constitutes a useful reference series to properly analyze the results of clinical trials in these patients.

Immunophenotype of acute myeloid leukemia with NPM mutations: prognostic impact of the leukemic compartment size.

NPM mutations are the most common genetic abnormalities found in non-promyelocytic AML. NPM-positive patients usually show a normal karyotype, a peculiar morphologic appearance with frequent monocytic traits and good prognosis in the absence of an associated FLT3 mutation. This report describes the immunophenotypic and genetic characteristics of a consecutive series of NPM-mutated de novo AML patients enroled in the CETLAM trial. Eighty-three patients were included in the study. Complete immunophenotype was obtained using multiparametric flow cytometry. Associated genetic lesions (FLT3, MLL, CEBPA and WT1 mutations) were studied by standardized methods. Real-time PCR was employed to assess the minimal residual status. The most common pattern was CD34-CD15+ and HLA-DR+. Small CD34 populations with immunophenotypic aberrations (CD15 and CD19 coexpression, abnormal SSC) were detected even in CD34 negative samples. Nearly all cases expressed CD33 (strong positivity), CD13 and CD117, and all were CD123+. The stem cell marker CD110 was also positive in most cases. Biologic parameters such as a high percentage of intermediate CD45+ (blast gate) (>75% nucleated cells), CD123+ and FLT3-ITD mutations were associated with a poor outcome. Quantitative PCR positivity had no prognostic impact either after induction or at the end of chemotherapy. Only PCR positivity (greater than 10 copies) detected in patients in haematological remission was associated with an increased relapse rate. Further studies are required to determine whether the degree of leukemic stem cell expansion (CD45+CD123+cells) increases the risk of acquisition of FLT3-ITD and/or provides selective advantages.

Refractory anemia with ringed sideroblasts associated with thrombocytosis: comparative analysis of marked with non-marked thrombocytosis, and relationship with JAK2 V617F mutational status.

The World Health Organization (WHO) classification of tumors of hematopoietic and lymphoid tissues (2001) defined a provisional entity named refractory anemia with ringed sideroblasts associated to marked thrombocytosis (RARS-MT). Diagnosis of RARS-MT requires more than 15% of ringed sideroblasts in bone marrow aspirate and the existence of a thrombocytosis in blood, with a platelet count above 600 x 10(9)/L. Nevertheless, controversy exists regarding this platelet count "cut-off" value and, when RARS-MT was defined, the JAK2 mutation and its importance in the study of myeloproliferative disorders was unknown. We present the results of a Spanish retrospective multicentric study, which includes 76 cases of RARS with associated thrombocytosis (platelet count above 400 x 10(9)/L) at diagnosis (RARS-T), 36 of them with a platelet count above 600 x 10(9)/L. Our aim was to analyze their clinical, analytical and morphological characteristics, and to establish correlations with the JAK2 mutational status.

Gene expression profiling distinguishes JAK2V617F-negative from JAK2V617F-positive patients in essential thrombocythemia.

To explore the gene expression signature in essential thrombocythemia (ET) patients in relation to JAK2V617F mutational status, expression profiling in circulating granulocytes was performed. Twenty ET were studied by microarray analysis and the results were confirmed by real-time quantitative RT-PCR in 40 ET patients, not receiving cytoreductive treatment. A heterogeneous molecular signature characterized by two main gene expression patterns was found: one with an upregulation of inflammatory genes related to neutrophil activation and thrombosis, and the other with significantly lower expression of these genes. Supervised clustering analysis showed 30 genes differentially expressed between JAK2V617F-negative and JAK2V617F-positive ET patients. Among the JAK2V617F-negative, a set of 14 genes (CISH, C13orf18, CCL3, PIM1, MAFF, SOCS3, ID2, GADD45B, KLF5, TNF, LAMB3, HRH4, TAGAP and TRIB1) showed an abnormal expression pattern. In this group of patients, CISH, SOCS2, SOCS3 and PIM1 genes, all involved in JAK-STAT signalling pathway, presented a lower expression. A two-gene predictor model was built comprising FOSB and CISH genes, which were the best discriminators of JAK2V617F status. In conclusion, JAK2V617F-negative ET patients present a characteristic gene expression profile, different from JAK2V617F-positive patients. Other pathways, besides JAK-STAT, might be implicated in the pathophysiology of JAK2V617F-negative ET patients.

Acute myeloid dendritic cell leukaemia with specific cutaneous involvement: a diagnostic challenge.

Myeloid or type 1 dendritic cell leukaemia is an exceedingly rare haematopoietic neoplasm characterized by a specific immunophenotypic profile close to plasmacytoid dendritic cell and acute myelogenous leukaemia. A 77-year-old man presenting specific cutaneous infiltration by myeloid dendritic cell leukaemia is reported. The clinical features as well as the cutaneous histopathological and immunohistochemical features led to the initial diagnosis of CD4+/CD56+ haematodermic neoplasm. However, extensive immunophenotypic studies performed from peripheral blood blasts disclosed that leukaemic cells expressed myeloid dendritic cell markers, confirming the diagnosis. The diagnostic difficulties of specific cutaneous involvement by myeloid dendritic cell leukaemia on the basis of routine histopathological and immunohistochemical features are highlighted.

Prognostic value of minimal residual disease (MRD) in acute myeloid leukemia (AML) with favorable cytogenetics [t(8;21) and inv(16)].

Most patients with acute myeloid leukemia (AML) and t(8;21) or inv(16) have a good prognosis with current anthracycline- and cytarabine-based protocols. Tandem analysis with flow cytometry (FC) and real-time RT-PCR (RQ-PCR) was applied to 55 patients, 28 harboring a t(8;21) and 27 an inv(16), including one case with a novel CBFbeta/MYH11 transcript. A total of 31% (n=17) of CR patients relapsed: seven with t(8;21) and 10 with inv(16). The mean amount of minimal residual disease (MRD) detected by FC in relapsed and nonrelapsed patients was markedly different: 0.3 vs 0.08% (P=0.002) at the end of treatment. The mean number of fusion transcript copies/ ABL x 10(4) also differed between relapsed and non-relapsed patients: 2385 vs 122 (P=0.001) after induction, 56 vs 7.6 after intensification (P=0.0001) and 75 vs 3.3 (P=0.0001) at the end of chemotherapy. Relapses were more common in patients with FC MRD level >0.1% at the end of treatment than in patients with < or = 0.1%: cumulative incidence of relapse (CIR) was 67 and 21% (P=0.03), respectively. Likewise, using RQ-PCR, a cutoff level of >10 copies at the end of treatment correlated with a high risk of relapse: CIR was 75% for patients with RQ-PCR >10 compared to 21% for patients with RQ-PCR levels < or = 10 (P=0.04). Combined use of FC and RQ-PCR may improve MRD detection, and provide useful clinical information on relapse kinetics in AML patients.

Erythrokeratoderma variabilis-like ichthyosis in Chanarin-Dorfman syndrome.

Neutral lipid storage disease (Chanarin-Dorfman syndrome) is an autosomal recessive metabolic disorder associated with congenital ichthyosis and a multisystemic accumulation of neutral lipids (lipid droplets) in various types of cells. The clinical presentation has been reported to correspond to that of nonbullous congenital ichthyosiform erythroderma. We report a 4-year-old boy presenting a generalized ichthyosiform disorder manifested by migrating scaly plaques alternating with areas of normal-looking skin, showing erythematous borders with sharp margins, clinically suggestive of erythrokeratoderma variabilis (EKV). A peripheral blood smear revealed cytoplasmic vacuoles in most granulocytes. Genetic studies from the patient and his parents revealed that the patient carried two different and novel mutations of the ABHD5 gene: a nonsense mutation in exon 6 (transmitted by the father) and an insertion/deletion in exon 4 (transmitted by the mother). Our observation demonstrates the clinical heterogeneity of the ichthyosiform dermatoses observed in Chanarin-Dorfman syndrome and widens the clinical range of conditions presenting migrating scaly plaques mimicking EKV.

Adverse prognostic impact of CD36 and CD2 expression in adult de novo acute myeloid leukemia patients.

BACKGROUND AND OBJECTIVES: A consecutive series of acute myeloid leukemias (AML) patients was analyzed in conditions which reduce the inter-assay variations (the same flow cytometer, the same observers and the same panel of monoclonal antibodies) in order to investigate the prognostic information provided by flow cytometry. DESIGN AND METHODS: Two hundred and sixty-six bone marrow (BM) samples from 326 patients enrolled in the LMA-99 protocol from the CETLAM group were studied by multiparametric flow cytometry. Immunophenotyping studies were performed on erythrocyte-lysed BM samples. Antigen expression of leukemic cells was analyzed using triple stainings with fluorochrome-conjugated combinations of monoclonal antibodies. RESULTS: CD2 was positive in 21 cases (8%); an associated inv(16) was detected in eight CD2+ cases (38%). Two-year overall survival (OS) rate for CD2+/inv(16)+ patients was 75%, whereas it was 0% for CD2+/inv(16)- patients and 47% for CD2- patients (p=0.0001). CD36 was expressed in 37% of patients (n=98). Two-year leukemia-free survival (LFS) rate was 34% for CD36+ patients and 55% for CD36- patients (p=0.001). In the multivariate analysis, CD2+ (RR=8.4; p=0.0001) and adverse karyotype (RR=10.2; p=0.0001) were associated with a lower CR rate, CD36+ (RR=1.5; p=0.03), CD2+ (RR=2; p=0.04) and adverse karyotype (RR=4; p=0.0001) were associated with a lower OS and CD36+ (RR=2; p=0.002) and adverse karyotype (RR=3.5; p=0.005) predicted a lower LFS. CONCLUSIONS: CD2+ patients had a very poor OS when CD2/inv(16)+ cases were excluded. CD36 and CD2 expression at diagnosis can provide prognostically important information in adult de novo AML.

Relationship among densitometry, bone histomorphometry, and histologic stage in idiopathic myelofibrosis.

Idiopathic myelofibrosis (IMF) induces dramatic changes in bone. Bone remodeling and densitometric alterations in a series of nine patients with IMF and their relationship with the histologic stage of the disease were assessed. Patients were included at diagnosis and a bone marrow biopsy, dual-energy X-ray absorptiometry, and transiliac bone biopsy for histomorphometric analysis were performed. Five cases were classified as IMF histologic stage 1, one as stage 2, and three as stage 3. Compared with 40 age- and sex-matched controls, the following histomorphometric parameters were significantly higher in our patients: bone volume (BV/TV), osteoblast surface (Ob.S/BS), eroded surface (ES/BS), osteoclast surface (Oc.S/BS), osteoclast number (N.Oc/TA), mineralizing surface (MS/BS), reversal period (Rv.P), and remodeling period (Rm.P). Mineral apposition rate (MAR) and erosion depth (E.Depth) were significantly decreased (P < 0.05 for all comparisons). Bone mineral density (BMD) measurements showed high values for patient age and sex both at femur neck (Z score range +0.19 to +7) and total femur (Z score range -0.09 to +6.48). When densitometric values were analyzed according to IMF histologic stage, patients in stages 1 and 2 had significantly lower BMD values than to those in stage 3 (P = 0.024). In conclusion, patients with IMF present a characteristic bone histomorphometric pattern with increased bone volume and bone cells but low apposition and decreased erosion depth, suggesting a positive balance in bone remodeling units. This balance would produce the increase in bone mass observed in this disease. Given the increase in BMD observed with more advanced stages of IMF, this noninvasive method could be useful tool for assessing IMF progression.

Congenital dyserthropoietic anaemia other than type I to III with a peculiar erythroblastic morphology.

Here, we report the case of a child who, since birth, showed persistent macrocytosis and elevated mean corpuscular volume of the erythrocytes. Bone marrow biopsy revealed gross disorganisation of the erythroblastic series both at the light and electron microscopic examination, with complete absence of dysplastic features in the granulocytic and megakaryocytic series. Common causes of macrocytosis were excluded. The spectrum of morphological findings were not consistent with any of the classical types of congenital dyserythropoietic anaemias (CDAs) and serological findings of CDA type II were absent. The most outstanding feature was a marked irregularity of the nuclear outline of the late erythroblasts that presented thick-ending finger-like projections. The combination of macrocytosis without anaemia and these morphologic erythroblastic changes have not been previously reported in the setting of classical and variant forms of CDAs.