Rayaz Ahmed

Improved Clinical Outcomes of High Risk β Thalassemia Major Patients Undergoing a HLA Matched Related Allogeneic Stem Cell Transplant with a Treosulfan Based Conditioning Regimen and Peripheral Blood Stem Cell Grafts

Improving clinical outcomes among high risk Class III β thalassemia major patients (Class IIIHR) receiving an allogeneic SCT remains a challenge. From October, 2009 a treosulfan based regimen (TreoFluT) was used for all consecutive Class III patients (n = 50). The clinical outcomes were compared with the historical conventional busulfan (BuCy) based regimen (n = 139). Use of TreoFluT was associated with a significantly reduced incidence of sinusoidal obstruction syndrome (SOS) among Class IIIHR cases (78% to 30%; P = 0.000) and early TRM (46% to 13%; p = 0.005). There was also a trend towards better engraftment in the Class IIIHR subset (P = 0.055). However, the use of bone marrow (BM) as source of stem cells along with the TreoFluT regimen was associated with 50% early mixed chimerism which reduced to 8.5% with the use of a peripheral blood stem cell graft (PBSC). Use of a PBSC graft was not associated with a significant increase in the incidence of acute or chronic graft versus host disease (GVHD). The overall and event free survival was significantly better among the Class IIIHR subset with the use of TreoFluT Vs. BuCy (86.6±7.3 Vs. 39.4±6.8%; P = 0.002 and 77.8±8.8 Vs. 32.4±6.5%; P = 0.003 respectively). A TreoFluT conditioning regimen with a PBSC graft can significantly improve clinical outcomes of Class IIIHR patients.

Role of minimal residual disease monitoring in acute promyelocytic leukemia treated with arsenic trioxide in frontline therapy

Data on minimal residual disease (MRD) monitoring in acute promyelocytic leukemia (APL) are available only in the context of conventional all-trans retinoic acid plus chemotherapy regimens. It is recognized that the kinetics of leukemia clearance is different with the use of arsenic trioxide (ATO) in the treatment of APL. We undertook a prospective peripheral blood RT-PCR-based MRD monitoring study on patients with APL treated with a single agent ATO regimen. A total of 151 patients were enrolled in this study. A positive RT-PCR reading at the end of induction therapy was significantly associated on a multivariate analysis with an increased risk of relapse (relative risk = 4.9; P = .034). None of the good risk patients who were RT-PCR negative at the end of induction relapsed. The majority of the relapses (91%) happened within 3 years of completion of treatment. After achievement of molecular remission, the current MRD monitoring strategy was able to predict relapse in 60% of cases with an overall sensitivity and specificity of 60% and 93.2%, respectively. High-risk group patients and those that remain RT-PCR positive at the end of induction are likely to benefit from serial MRD monitoring by RT-PCR for a period of 3 years from completion of therapy.

Cytidine deaminase genetic variants influence RNA expression and cytarabine cytotoxicity in acute myeloid leukemia

AIM Cytidine deaminase (CDA) irreversibly deaminates cytarabine (Ara-C), a key component of acute myeloid leukemia (AML) induction and consolidation therapy. CDA overexpression results in Ara-C resistance, while decreased expression is associated with toxicity. We evaluated factors influencing variation in CDA mRNA expression in adult AML patients and normal controls, and how they contributed to Ara-C cytotoxicity in AML cells. MATERIALS & METHODS CDA mRNA expression in 100 de novo AML patients and 36 normal controls were determined using quantitative reverse-transcriptase PCR. Genetic variants in the CDA gene were screened by direct sequencing. IC₅₀ of Ara-C was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. RESULTS CDA RNA expression as well as Ara-C IC₅₀ showed wide variation in AML samples and normal controls. Fourteen sequence variants were identified, three of which (-33delC, intron 2 TCAT repeat and the 3´untranslated region 816delC variants) showed significant association with RNA expression and the nonsynonymous coding variant 79A>C was associated with Ara-C cytotoxicity. CONCLUSION CDA genetic variants explain the variation in RNA expression and may be candidates for individualizing Ara-C therapy.

Standardizing minimal residual disease by flow cytometry for precursor B lineage acute lymphoblastic leukemia in a developing country

In addition to standard risk criteria at diagnosis, minimal residual disease (MRD) following initiation of therapy is a well‐recognized risk factor to predict relapse. Literature from developing countries addressing therapeutic or laboratory practices related to MRD, is largely lacking. In a first paper from India, we describe our experience in establishing a flow cytometry‐based MRD assay for precursor B lineage ALL (BCP‐ALL) with emphasis on the assay standardization and cost.

Carbonyl reductase 1 expression influences daunorubicin metabolism in acute myeloid leukemia

PurposeThe present study aimed to investigate the role of expression of daunorubicin-metabolizing enzymes carbonyl reductase 1 and 3 (CBR1 and CBR3) on the in vitro cytotoxicity of daunorubicin in primary acute myeloid leukemia (AML) cells and the effect of genetic variants in CBR1 and CBR3 on the plasma pharmacokinetics of daunorubicin and daunorubicinol (DOL) in AML patients.MethodsRNA expression of CBR1 and CBR3, intracellular daunorubicin and DOL levels, and in vitro cytotoxicity of daunorubicin were measured in bone marrow mononuclear cells of 104 adult AML patients. Plasma pharmacokinetics of daunorubicin and DOL was measured in 24 patients receiving daunorubicin-based induction chemotherapy for AML.ResultsIncreased expression of CBR1 significantly reduced the in vitro cytotoxicity of daunorubicin and also positively correlated with intracellular DOL levels. Polymorphisms in CBR1 and CBR3 did not show any association with intracellular daunorubicin or DOL levels, but there was a trend towards significant increase in plasma daunorubicin systemic exposure in patients with a variant genotype for CBR1 polymorphism rs25678.ConclusionsThis pilot study suggests that CBR1 RNA expression may be helpful in identifying AML patients at risk of developing resistance or toxicity to daunorubicin due to increased formation of DOL. Further confirmation of these findings in a larger sample pool would be required to determine the applicability of these results. Inhibition of CBR1 can be an option to improve the efficacy and prevent toxicity related to the treatment. Influence of daunorubicin and DOL plasma levels on clinical outcome, if any, remains to be evaluated.

The use of a fludarabine-based conditioning regimen in patients with severe aplastic anemia – a retrospective analysis from three Indian centers

Between 2001 and 2009, 121 patients with severe aplastic anemia (SAA) underwent hematopoietic stem cell transplantation (HSCT) using a conditioning protocol of fludarabine and cyclophosphamide at three Indian hospitals. Donors were HLA‐identical sibling or family donors. Seventy‐six patients were considered “high risk” as per criteria. The graft source included peripheral blood stem cells in 109 and G‐CSF‐stimulated bone marrow in 12. GVHD prophylaxis consisted of cyclosporine and mini‐methotrexate. Engraftment occurred in 117 (96.6%) while two had graft failure and two expired in the first two wk. Neutrophil engraftment was seen at 12.3 d (range: 9–19) while platelet engraftment occurred at 12.4 d (range: 8–32). Grade II–IV acute GVHD was seen in 26.7% and grade IV GVHD in 8.6%. Chronic GVHD occurred in 44% and was extensive in 10%. The five‐yr overall survival for the entire cohort is 75.8 ± 3.9% with a survival of 95.6 ± 3.1% in the low‐risk group (n = 45) and 64.0 ± 5.6% in the high‐risk group (n = 76). Conditioning with fludarabine and cyclophosphamide is associated with very good long‐term survival in patients undergoing HSCT for SAA.

Two novel missense mutations in iron transport protein transferrin causing hypochromic microcytic anaemia and haemosiderosis: molecular characterization and structural implications

This study was supported in part by a grant BT/PR-13968/MED/12/465/2010 from the Department of Biotechnology, Government of India to ES and by the grant SAF2012-40106 from the Spanish Secretary of Research, Development and Innovation (MINECO) and the grant CIVP16A1857 “Ayudas a proyectos de Investigacion en Ciencias de la Vida- Fundacion Ramon Areces” to M.S. M.S. held a research contract under the Ramon y Cajal program from the Spanish Ministry of Science and Innovation (RYC-2008-02352).

Clinicopathological features of hepatosplenic T cell lymphoma: a single centre experience from India

Abstract In a first series from India, we report 9 cases of hepatosplenic T cell lymphoma (HSTCL) seen in 23 months accounting for 4.2% of all mature T-non-Hodgkin lymphomas (NHLs) in our institution. All patients presented with organomegaly, cytopenias and had evidence of bone marrow involvement. The tumor cells had a blastic (55%) morphology with predominantly intrasinusoidal (33.3%) or intrasinusoidal with an additional interstitial component (33.3%). On flow cytometry, the classical phenotype (CD3+, CD7+, CD4−, CD8−, CD5−, CD56+/−) was seen only in 4 patients. Unusual variations included CD45 (overexpression), CD7 (dim expression), CD3 (overexpression, heterogeneous and dim), CD2 (overexpression), CD5 (heterogeneous), CD8 (heterogeneous or dim or overexpression) and aberrant expression of CD19. Fluoresvent in situ hybridisation (FISH) and karyotyping was abnormal in 5 out of 7 patients evaluated. All of the 5 cases showed abnormalities in chromosome 7 (ring chromosome or isochromosome 7q). Five patients died of disease and related complications in a span of 1–3 months after diagnosis whereas 4 were alive at their last follow up out of which 2 had documented a relapse. In our series, HSTCL was characterized by typical clinical and variable immunophenotypic features and a dismal clinical outcome.

Molecular basis of Wiskott–Aldrich syndrome in patients from India

To the Editor: Wiskott–Aldrich syndrome (WAS) [OMIM: 301000] is an X-linked immunodeficiency disease characterized by thrombocytopenia and small platelets, eczema, recurrent infections, with an increased risk for autoimmunity and malignancy (1, 2). The gene responsible for this syndrome, WAS, comprises 12 exons and approximately 1.8 kb in length (3). WAS encodes a 502-amino-acid protein (WASp) that is expressed selectively in hematopoietic stem cell– derived lineages and is involved in cell signaling and cytoskeleton reorganization (4). A milder allelic variant caused by WAS gene mutation leads to X-linked thrombocytopenia (XLT), a congenital disorder characterized by thrombocytopenia and small platelets but, in general, without the other complications of WAS (5). So far, ~369mutations have been reported in WAS gene (http://www.hgmd.cf.ac.uk/ ac/gene.php?gene=WAS). Detection of additional mutations in this gene is important for the precise genetic diagnosis in families affected by this disorder as well as for studying the molecular basis of this disease (6). We report here for the first time the WAS gene mutations identified in patients with WAS/XLT from India and their genotype–phenotype correlations. Ten patients from eight families were evaluated at the Department of Haematology, Christian Medical College, Vellore, with clinical features suggestive of WAS on written, informed consent. All patients underwent hematological and biochemical evaluation (Table 1). Blood was collected in citrated buffer and in ethylenediamine tetra-acetic acid (EDTA) from the probands and, whenever available, from family members. Similar samples were also collected from healthy controls. Blood smears were stained using modified Giemsa–Wright stain (Beckman, Duarte, CA, USA) and evaluated for morphology. Platelet count and the mean platelet volume (MPV) were estimated in a cell counter (Coulter LH 755; Beckman Coulter). A mononuclear cell suspension in phosphate-buffered saline (PBS) was isolated from heparinized blood by Ficoll gradient centrifugation. The cells were fixed (Fix and Perm Medium; Invitrogen, Carlsbad, CA, USA) and permeabilized (Fix and Perm medium B; Invitrogen) for intracellular staining and incubated with phycoerythrin (PE)-conjugated antibody against WASP DI (SantaCruz Biotechnology, SantaCruz, CA, USA) and WAS B9 (Santa Cruz Biotechnology) with appropriate IgG1 and IgG2a controls (BD Pharmingen, San Jose, CA, USA). After processing, cells were analyzed for intracellular bound florescence in a flow cytometer (BD FACS Calibur, Manifield, MA, USA). The patient WASP expression was compared to normal controls processed at the same time, and the data are expressed as percentage of normal WASP-positive cells (7). Genomic DNA from EDTA anti-coagulated blood was isolated by standard phenol–chloroform method. The human WAS gene exonic and flanking intronic regions were amplified by twelve pairs of primers as described previously (8). Nucleotide changes in the amplified fragments were screened by a conformation-sensitive gel electrophoresis (CSGE) and DNA sequencing strategy (9). Samples displaying abnormal CSGE patterns were sequenced by the Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Warrington, UK) on an ABI 3130 genetic analyzer (PE Applied Biosystems, Foster City, CA, USA). All the novel mutations identified were confirmed as unique to the patients by screening 100 normal control alleles. The normal controls were recruited based on a written informed consent. Mutations at or near the splice junction consensus sequences were analyzed by ‘Splice Site Prediction program’ (http://www.fruitfly.org/seq_tools/splice.html) to predict changes in RNA splicing. The clinical features, and hematological and molecular genetic data of all the 10 patients are detailed in Table 1. Diagnosis was based on low platelet count (9000–95 000/ mm), presence of small platelets (mean platelet volume – 6.2 ± 1.5) and reduced levels of WASp by flow cytometry. Of these, two were sibling pairs (WAS-9/WAS-10; WAS15/WAS-35). The median age at first clinical symptoms of these patients was 2.5 yr (range 1–12 yr). Six of them had a family history of bleeding. To differentiate between classical WAS and XLT in these patients, the disease was scored from 1 to 5 based on the presence of thrombocytopenia, small platelets, eczema, immunodeficiency, infections, autoimmunity or malignancy and congenital neutropenia as described previously (10). In patients who were lost to follow-up, we could not comprehensively analyze the biochemical or genetic data, and this was a limitation to our study. Genotypic analysis revealed mutations in eight of ten patients (Table 1). Among them, three had nonsense mutations, two splice site variations, two deletions, and one missense mutation. Two of these eight mutations were novel. These included a single ‘T-nucleotide’ deletion (c.108delT),

Evaluating New Markers for Minimal Residual Disease Analysis by Flow Cytometry in Precursor B Lymphoblastic Leukemia

Minimal residual disease is currently the most powerful prognostic indicator in Precursor B lymphoblastic leukemia. Multiparameter flow cytometry is the most commonly used modality. Seventy three B ALL cases and 15 normal marrows were evaluated for expression patterns of leukemia markers (CD38, CD58, CD73) in all 73 cases and CD66c, CD86 and CD123 in 23 cases. CD73 was aberrantly expressed in 90.41% cases and CD86 in 60.87% B ALL cases. Thus addition of these markers in MRD panels can increase the sensitivity of the assay.