An MDS-specific frailty index based on cumulative deficits adds independent prognostic information to clinical prognostic scoring.

The frailty index (FI) is based on the principle that the more deficits an individual has, the greater their risk of adverse outcomes. It is expressed as a ratio of the number of deficits present to the total number of deficits considered. We developed an MDS-specific FI using a prospective MDS registry and assessed its ability to add prognostic power to conventional prognostic scores in MDS. The 42 deficits included in this FI included measurements of physical performance, comorbidities, laboratory values, instrumental activities of daily living, quality of life and performance status. Of 644 patients, 440 were eligible for FI calculation. The median FI score was 0.25 (range 0.05-0.67), correlated with age and IPSS/IPSS-R risk scores and discriminated overall survival. With a follow-up of 20 months, survival was 27 months (95% CI 24-30.4). By multivariate analysis, age >70, FI, transfusion dependence, and IPSS were significant covariates associated with OS. The incremental discrimination improvement of the frailty index was 37%. We derived a prognostic score with five risk groups and distinct survivals ranging from 7.4 months to not yet reached. If externally validated, the MDS-FI could be used as a tool to refine the risk stratification of current clinical prognostication models.

Overexpression of Arginase 1 is linked to DNMT3A and TET2 mutations in lower-grade myelodysplastic syndromes and chronic myelomonocytic leukemia.

Immune dysregulation is a common feature of myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML), particularly in early stages. However, the genetic basis remains poorly understood. We recently reported that macrophages from mice deficient in tet methylcytosine dioxygenase 2 (Tet2), a model of MDS/CMML, are hyperinflammatory and have increased expression of arginase 1 (Arg1). In macrophages and myeloid derived suppressor cells (MDSCs) expression of Arg1 contributes to T-cell suppression and immune evasion by L-arginine depletion, in the setting of chronic inflammation and cancer. Since human MDS and CMML are driven by TET2 mutations and associated with chronic inflammation, we hypothesized that arginase enzymatic activity and ARG1 expression would be increased in human MDS/CMML bone marrow. Elevated arginase activity was observed in bone marrow mononuclear cells of MDS and CMML patients with lower-grade features. Immunohistochemical studies confirmed that myelomonocytic cells overexpress ARG1. Additionally, mutations in the epigenetic regulators TET2 and DNMT3A corresponded to high ARG1 expression and activity. These findings suggest ARG1 is a biomarker of immune dysregulation in early MDS and CMML. Recent murine findings have implicated Tet2 and Dnmt3a in regulation of innate immunity. Our study suggests similar changes may be driven by human TET2 and DNMT3A mutations.

Prophylactic RhCE and Kell antigen matching: impact on alloimmunization in transfusion-dependent patients with myelodysplastic syndromes.

BACKGROUND AND OBJECTIVES: Thirty to 80 per cent of patients with myelodysplastic syndromes (MDS) become transfusion-dependent and are at risk for red blood cell (RBC) alloimmunization. This study compared alloimmunization rates in transfusion-dependent patients with MDS at an institution with a policy of prophylactic antigen matching for RhCE and K (PAM) with those transfused at institutions without such a policy (non-PAM). MATERIALS AND METHODS: Transfusion records were retrospectively reviewed to determine total number of RBC transfusions received, whether RBC phenotyping was performed, the type and date of first alloantibody development and receipt of prophylactic antigen matching for RhCE and K. RESULTS: In 176 transfusion-dependent patients with MDS, the overall rate of new alloimmunization was 17%; the majority of patients (87%) developed at least one alloantibody to Rh or Kell antigens. The alloimmunization rate at the institution with a PAM policy was 11% compared with 23% at non-PAM institutions (P = 0·06). The rate of Rh/K alloimmunization was 7 vs. 22%, respectively (P = 0·008). No patient who received PAM developed a Rh/K alloantibody. CONCLUSION: The rate of alloimmunization was 11% at an institution with a PAM policy which was non-significantly lower than 23% at institutions without a PAM policy. However, rates of Rh/K alloimmunization were significantly lower. Such a policy should be considered in transfusion-dependent patients with MDS, although further studies on cost-effectiveness and careful consideration of resource availability in the local context are required.

Monthly blood transfusions decrease after four months of azacitidine.

BACKGROUND AND OBJECTIVES: Azacitidine (AZA) improves overall survival and transfusion independence in patients with myelodysplastic syndrome (MDS). We aimed to quantify the reduction in red blood cell (RBC) transfusions and to determine when this reduction occurs, in MDS patients treated with AZA. MATERIALS AND METHODS: We performed a retrospective audit of changes in RBC transfusion burden in 51 patients with predominantly higher risk MDS (26.5% high risk, 51.0% intermediate-2) who received AZA. Transfusion requirements were audited 6 months prior to and up to 18 months after therapy initiation, and data were analysed using a generalized linear mixed model. RESULTS: At baseline, 30 patients (58.8%) were transfusion dependent (TD). Seventeen patients (56.7%) achieved transfusion independence (TI) by 18 months, and 8 of these patients (47.1%) achieved this response by 4 months on therapy. Achievement of TI was not consistently durable in these 17 patients, as 11 patients reverted to TD while on therapy. Meanwhile, 6 of 21 patients who were TI at baseline became TD on therapy. The monthly average of RBC units transfused decreased significantly beginning at 4 months, with a reduction from 2.50 units per month at baseline to 1.00 units per month at month 4. This 60% reduction was significant (P = 0.002) and sustained beyond 12 months. CONCLUSION: These results bolster the notion that AZA significantly reduces transfusion burden and resource utilization and illustrate the limitations of the current WHO erythroid response criteria which do not account for differing durability and fluctuations of response.

Optimizing outcomes with azacitidine: recommendations from Canadian centres of excellence.

Myelodysplastic syndromes (mdss) constitute a heterogeneous group of malignant hematologic disorders characterized by marrow dysplasia, ineffective hematopoiesis, peripheral blood cytopenias, and pronounced risk of progression to acute myeloid leukemia. Azacitidine has emerged as an important treatment option and is recommended by the Canadian Consortium on Evidence-Based Care in mds as a first-line therapy for intermediate-2 and high-risk patients not eligible for allogeneic stem cell transplant; however, practical guidance on how to manage patients through treatment is limited. This best practice guideline provides recommendations by a panel of experts from Canadian centres of excellence on the selection and clinical management of mds patients with azacitidine. Familiarity with the referral process, treatment protocols, dose scheduling, treatment expectations, response monitoring, management of treatment breaks and adverse events, and multidisciplinary strategies for patient support will improve the opportunity for optimizing treatment outcomes with azacitidine.

Identification of a role for the nuclear receptor EAR-2 in the maintenance of clonogenic status within the leukemia cell hierarchy.

Identification of genes that regulate clonogenicity of acute myelogenous leukemia (AML) cells is hindered by the difficulty of isolating pure populations of cells with defined proliferative abilities. By analyzing the growth of clonal siblings in low passage cultures of the cell line OCI/AML4 we resolved this heterogeneous population into strata of distinct clonogenic potential, permitting analysis of the transcriptional signature of single cells with defined proliferative abilities. By microarray analysis we showed that the expression of the orphan nuclear receptor EAR-2 (NR2F6) is greater in leukemia cells with extensive proliferative capacity than in those that have lost proliferative ability. EAR-2 is expressed highly in long-term hematopoietic stem cells, relative to short-term hematopoietic stem and progenitor cells, and is downregulated in AML cells after induction of differentiation. Exogenous expression of EAR-2 increased the growth of U937 cells and prevented the proliferative arrest associated with terminal differentiation, and blocked differentiation of U937 and 32Dcl3 cells. Conversely, silencing of EAR-2 by short-hairpin RNA initiated terminal differentiation of these cell lines. These data identify EAR-2 as an important factor in the regulation of clonogenicity and differentiation, and establish that analysis of clonal siblings allows the elucidation of differences in gene expression within the AML hierarchy.

Validation of a scoring system to establish the probability of myelodysplastic syndrome in patients with unexplained cytopenias or macrocytosis.

Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders primarily seen in the elderly that are characterized by ineffective hematopoiesis and a propensity to develop AML. Clinicians may be hesitant to refer older patients with unexplained cytopenias and/or macrocytosis for a bone marrow biopsy (BM), and consequently undiagnosed patients may be deprived access to effective treatments. Previously, we described factors which were independently predictive of a diagnosis of MDS at time of bone marrow: age ≥65, mean cell volume (MCV), red cell distribution width (RDW), and lactate dehydrogenase (LDH), and suggested a scoring system to calculate the post-test probability of MDS [1]. In this study we validate this scoring system in a cohort of 313 individuals who underwent bone marrow examinations for the investigation of unexplained cytopenias and or macrocytosis over a 3 year period at our institution (2006-2008). Thirty-two percent of all patients were diagnosed with MDS and 9% had suspected MDS. The post-test likelihood of a diagnosis of MDS increased from 12% when none of the four identified factors were present to 48% when 3 or more factors were present. This scoring system can be used to guide the diagnostic testing of patients presenting with unexplained cytopenias or macrocytosis.

Evidence of functional interaction between NuMA-RARalpha and RXRalpha in an in vivo model of acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is characterized by reciprocal balanced chromosomal translocations involving retinoic acid receptor-alpha (RARalpha). RARalpha heterodimerizes with the retinoid X receptor-alpha (RXRalpha) and transcriptionally regulates myeloid differentiation in response to ATRA (all-trans retinoic acid). Several lines of evidence suggest that APL fusion proteins interact with RXRalpha. To elucidate the role of RXRalpha in APL, we conditionally knocked out RXRalpha in the hCG-NuMA-RARalpha APL mouse model. Phenotype analysis of NuMA-RARalpha+ mice demonstrated that these mice developed a myeloproliferative disease-like myeloid leukemia within 4 months of birth. While hemizygous and homozygous RXRalpha conditional knockout mice were phenotypically normal as late as 12 months of age, we observed that the leukemic phenotype in NuMA-RARalpha+ mice was dependent on the presence of functional RXRalpha. Bone marrow promyelocyte counts were significantly reduced in NuMA-RARalpha+ mice with RXRalpha knocked down. Significant differences in the accumulations of Gr-1+ and Mac-1+ cells were also seen. We further observed that genes previously identified to be cooperating events in APL were also regulated in an RXRalpha-dependent manner. We therefore propose that the APL fusion protein NuMA-RARalpha cooperates with RXRalpha in the development of leukemia in hCG-NuMA-RARalpha transgenic mice and suggest a novel role for RXRalpha in the pathogenesis of APL.

Assessment of the downstream portion of the mitochondrial pathway of caspase activation in patients with acute myeloid leukemia.

Most chemotherapeutic agents used in the treatment of acute myeloid leukemia (AML) induce apoptosis by triggering the mitochondrial pathway of caspase activation. To investigate the downstream portion of the mitochondrial pathway of caspase activation in patients with AML, cytosolic lysates were stimulated with cytochrome c and dATP and hydrolysis of Ac-DEVD-AFC by effector caspases was measured. Defects in the distal mitochondrial pathway were more common in samples from patients with AML that relapsed rapidly after induction chemotherapy compared to samples from treatment naïve patients. The incidence of blocked pathways did not differ based on response to induction chemotherapy, as even nonresponders generally had an intact pathway. When the distal mitochondrial pathway was blocked, defects were usually at the level of the effector caspases. Thus, functional defects in the distal portion of the mitochondrial pathway of caspase activation may help explain the nature of response and relapse after treatment.

Treatment of elderly patients with acute lymphoblastic leukemia--evidence for a benefit of imatinib in BCR-ABL positive patients.

All patients with acute lymphoblastic leukemia (ALL) over age 60 years seen at Princess Margaret Hospital over an 11-year period were analysed retrospectively. Of 53 patients, 45 received multiagent induction chemotherapy using a variety of regimens. There were 13 BCR-ABL positive patients, 9 of who received imatinib mesylate, either during induction or post-remission therapy. The overall complete remission (CR) rate of all 45-induction patients was 56%, with a 27% induction-related mortality rate. The CR rate was not influenced by induction regimen, age, initial WBC, LDH or BCR-ABL status. The median overall survival of the induction patients was 9 months, while the median progression-free survival (PFS) of the patients achieving CR was 10 months. The estimated overall survival (OS) at 3 years was 18.4% (95% CI: 9.8-34.3%). Age and initial WBC did not significantly predict for OS when evaluating the entire group of induction patients. However, there was a strong trend for BCR-ABL status to favorably predict for PFS, and for OS when only patients treated after July 2000 (when imatinib became available) were evaluated. The results indicate that ALL remains a poor prognosis disease in elderly patients, and that aggressive induction regimens designed for younger patients are very toxic for these patients. These data suggest that BCR-ABL+ ALL is becoming a relatively more favorable prognosis disease in the elderly, likely due to the influence of imatinib therapy. Further regimens should explore the use of less aggressive regimens in elderly patients and should evaluate the optimal way of combining imatinib with conventional agents in BCR-ABL+ patients.

The role of allogeneic bone marrow transplantation in adult patients below the age of 55 years with acute lymphoblastic leukemia in first complete remission: a donor vs no donor comparison.

The role of allogeneic bone marrow transplantation (alloBMT) in adults with acute lymphoblastic leukemia (ALL) in first complete remission (CR1) remains controversial. At our institution, the policy is to offer alloBMT to ALL patients in CR1 up to the age of 55 years if a related donor is available. In addition, unrelated donor transplants are offered to patients with Philadelphia (Ph+) ALL. We report the results on 92 patients with ALL treated according to this policy from September 1992 to October 2001. Of the 87 patients achieving CR1, the comparison of patients with (n=48) or without donors (n=39) was done using an intention-to-treat approach. Of the 48 patients with donors (39 related and nine unrelated), 35 (73%) received alloBMT in CR1. No significant difference in 3-year event-free survival (EFS) (40 vs 39%, P=0.74) or overall survival (OS) (46 vs 58%, P=0.41) was seen in 'donor' vs 'no-donor' groups. For Ph+ patients, 3-year EFS and OS in 'donor' group were 46 and 57%, respectively, none of the patients in 'no-donor' group survived beyond 3 years. With our treatment strategy, 3-year OS of Ph+ patients was equivalent to Ph-negative (Ph-) patients (51 vs 52%, P=0.77). In conclusion, our data show that the policy of performing alloBMT if a sibling donor is available has not resulted in better outcome in Ph- patients.

Deregulation of NPM and PLZF in a variant t(5;17) case of acute promyelocytic leukemia.

Greater than 95% of acute promyelocytic leukemia (APL) cases are associated with the expression of PML-RARalpha. This chimeric protein has been strongly implicated in APL pathogenesis because of its interactions with growth suppressors (PML), retinoid signaling molecules (RXRalpha), and nuclear hormone transcriptional co-repressors (N-CoR and SMRT). A small number of variant APL translocations have also been shown to involve rearrangements that fuse RARalpha to partner genes other than PML, namely PLZF, NPM, and NuMA. We describe the molecular characterization of a t(5;17)(q35;q21) variant translocation involving the NPM gene, identified in a pediatric case of APL. RT-PCR, cloning, and sequence studies identified NPM as the RARalpha partner on chromosome 5, and both NPM-RARalpha and RARalpha-NPM fusion mRNAs were expressed in this patient. We further explored the effects of the NPM-RARalpha chimeric protein on the subcellular localization of PML, RXRalpha, NPM, and PLZF using immunofluorescent confocal microscopy. While PML remained localized to its normal 10-20 nuclear bodies, NPM nucleolar localization was disrupted and PLZF expression was upregulated in a microspeckled pattern in patient leukemic bone marrow cells. We also observed nuclear co-localization of NPM, RXRalpha, and NPM-RARalpha in these cells. Our data support the hypothesis that while deregulation of both the retinoid signaling pathway and RARalpha partner proteins are molecular consequences of APL translocations, APL pathogenesis is not dependent on disruption of PML nuclear bodies.

Fusion of retinoic acid receptor alpha to NuMA, the nuclear mitotic apparatus protein, by a variant translocation in acute promyelocytic leukaemia.

Acute promyelocytic leukaemia (APL) is uniquely associated with chromosomal translocations that disrupt the gene encoding the retinoic acid receptor, RARA. In more than 99% of cases, this disruption results in the formation of a PML-RARA gene fusion. Two rare variants of APL have been described, in which RARA is fused to one of two other genes, PLZF and NPM. Although RARA dysregulation is evidently important in APL, the role of the various fusion partners remains unclear. We have characterized a fourth APL gene fusion, which links exons encoding the retinoic acid and DNA-binding domains of RARA to 5' exons of NuMA, a gene that encodes the nuclear mitotic apparatus protein. The NuMA-RARA fusion protein exists in sheet-like nuclear aggregates with which normal NuMA partly co-localizes. In contrast to t(15;17) APL, the intracellular distribution of PML is normal in these cells. Our results suggest that interference with retinoid signalling, and not disruption of PML organization, is essential to the APL phenotype and implicates for the first time an element of the mitotic apparatus in the molecular pathogenesis of human malignancy.

A new variant translocation in acute promyelocytic leukaemia: molecular characterization and clinical correlation.

Translocation t(15;17)(q22;q21) is an acquired clonal cytogenetic change present in almost all cases of acute promelocytic leukemia (APL). The molecular genetic basis of the translocation supports its integral role in pathogenesis. We describe a patient with APL in whom the leukaemic clone was characterized by a true variant of the classical t(15;17). The patient whose disease had numerous atypical clinical features, had t(11;17)(q13;121). The chromosome 17 breakpoint was localized to intron 2 of RARA by Southern blotting, and there was no evidence at the molecular level for rearrangement at PML locus. These data, along with previous reports of rare variant translocations in APL, indicate that while dysregulation of RARA by gene fusion may be essential for the APL phenotype, the particular fusion partner may determine clinicopathological aspects, including presentation, response to treatment with all-trans retinoic acid (ATRA), and prognosis. This heterogeneity suggests that the variant fusion partners of RARA in APL encode factors with properties both common to and distinct from those of PML. Investigation of these factors promises to shed light on the complex development pathways involved in the regulation of haematopoiesis.

Chromosomal localization and regulation of the longevity determinant genes in a selected strain of Drosophila melanogaster.

A controlled chromosome substitution experiment was performed on a strain (NDC-L) selected for long life to determine if the genes responsible for the extended-longevity phenotype could be localized to any particular chromosome(s). All 27 different possible combinations of the three major chromosomes of Drosophila melanogaster were constructed and longevities were determined on 3875 individual animals of both sexes and analysed. The results are statistically significant and demonstrate that mean longevity is specified primarily by recessive genes on the third chromosome (c3). The extended longevity phenotype (ELP) is only expressed in those lines which are homozygous for the NDC-L type c3. Loci on the first (c1) and second (c2) chromosomes interact, both positively (c1) and negatively (c2), respectively, such that c1 represses c2 which in turn represses c3. The ELP is fully expressed in the mutual presence and mutual absence of c1 and c2. The significance of these results is discussed in the context of broader categories of molecular genetic mechanisms suggested previously to be involved in the modulation of longevity in Drosophila.

A family of cation ATPase-like molecules from Plasmodium falciparum.

We report the nucleotide and derived amino acid sequence of the ATPase 1 gene from Plasmodium falciparum. The amino acid sequence shares homology with the family of "P"-type cation translocating ATPases in conserved regions important for nucleotide binding, conformational change, or phosphorylation. The gene, which is present on chromosome 5, has a product longer than any other reported for a P-type ATPase. Interstrain analysis from 12 parasite isolates by the polymerase chain reaction reveals that a 330-bp nucleotide sequence encoding three cytoplasmic regions conserved in cation ATPases (regions a-c) is of constant length. By contrast, another 360-bp sequence which is one of four regions we refer to as "inserts" contains arrays of tandem repeats which show length variation between different parasite isolates. Polymorphism results from differences in the number and types of repeat motif contained in this insert. Inserts are divergent in sequence from other P-type ATPases and share features in common with many malarial antigens. Studies using RNA from the erythrocytic stages of the malarial life cycle suggest that ATPase 1 (including the sequence which encodes tandem repeats) is expressed at the large ring stage of development. Immunolocalization has identified ATPase 1 to be in the region of the parasite plasma membrane and pigment body. These findings suggest a possible model for the genesis of malarial antigens.

Multiple variants in subtelomeric regions of normal karyotypes.

We describe a human genomic cosmid clone, 56.1.1, that contains subtelomeric sequences present on multiple human chromosomes. In particular, using fluorescence in situ hybridization, we have identified 16 sites of hybridization on 12 chromosomes. In a sample of 8 unrelated individuals, 10 of these sites showed interindividual variation. Co-hybridization with other polymorphic probes allowed us to demonstrate cytologically heterozygosity at three sites in six individuals. The chromosomal distribution of hybridization sites in a family strongly suggests that these variants are inherited in a Mendelian fashion. These data show that subtelomeric repeats are a rich source of genetic variability. Possible mechanisms of generation of such variants are discussed.