Metabolic adaptation drives arsenic trioxide resistance in acute promyelocytic leukemia.

Acquired genetic mutations can confer resistance to arsenic trioxide (ATO) in the treatment of acute promyelocytic leukemia (APL). However, such resistance-conferring mutations are rare and do not explain most disease recurrence seen in the clinic. We have generated stable ATO-resistant promyelocytic cell lines that are less sensitive to all-trans retinoic acid (ATRA) and the combination of ATO and ATRA compared with the sensitive cell line. Characterization of these resistant cell lines that were generated in-house showed significant differences in immunophenotype, drug transporter expression, anti-apoptotic protein dependence, and promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA) mutation. Gene expression profiling revealed prominent dysregulation of the cellular metabolic pathways in these ATO-resistant APL cell lines. Glycolytic inhibition by 2-deoxyglucose (2-DG) was sufficient and comparable to the standard of care (ATO) in targeting the sensitive APL cell line. 2-DG was also effective in the in vivo transplantable APL mouse model; however, it did not affect the ATO-resistant cell lines. In contrast, the resistant cell lines were significantly affected by compounds targeting mitochondrial respiration when combined with ATO, irrespective of the ATO resistance-conferring genetic mutations or the pattern of their anti-apoptotic protein dependency. Our data demonstrate that combining mitocans with ATO can overcome ATO resistance. We also show that this combination has potential for treating non-M3 acute myeloid leukemia (AML) and relapsed APL. The translation of this approach in the clinic needs to be explored further.

A phase II study evaluating the role of bortezomib in the management of relapsed acute promyelocytic leukemia treated upfront with arsenic trioxide.

The standard-of-care for patients with acute promyelocytic leukemia (APL) relapsing after upfront arsenic trioxide (ATO) therapy is not defined. The present study was undertaken to evaluate the safety of addition of bortezomib to ATO in the treatment of relapsed APL based on our previously reported preclinical data demonstrating synergy between these agents. This was an open label, nonrandomized, phase II, single-center study. We enrolled 22 consecutive patients with relapsed APL. The median age was 26.5 years (interquartile range 17.5 to 41.5). The median time from initial diagnosis to relapse was 23.1 months (interquartile range 15.6 to 43.8). All patients achieved hematological remission at a median time of 45 days (range 40-63). Nineteen patients were in molecular remission at the end of induction. Grade 3 adverse events occurred in eight instances with one patient requiring discontinuation of therapy for grade 3 neuropathy. Twelve (54.5%) patients underwent autologous transplantation (auto-SCT) in molecular remission while the rest opted for maintenance therapy. The median follow-up was 48 months (range 28-56.3). Of the patients undergoing auto-SCT, all except one was alive and relapse free at last follow-up. Of the patients who opted for maintenance therapy, three developed a second relapse. For treatment of APL relapsing after upfront ATO therapy, addition of bortezomib to a standard ATO-based salvage regimen is safe and effective. This trial was registered at www.clinicaltrials.gov as NCT01950611.

Stromal cells downregulate miR-23a-5p to activate protective autophagy in acute myeloid leukemia.

Complex molecular cross talk between stromal cells and the leukemic cells in bone marrow is known to contribute significantly towards drug-resistance. Here, we have identified the molecular events that lead to stromal cells mediated therapy-resistance in acute myeloid leukemia (AML). Our work demonstrates that stromal cells downregulate miR-23a-5p levels in leukemic cells to protect them from the chemotherapy induced apoptosis. Downregulation of miR-23a-5p in leukemic cells leads to upregulation of protective autophagy by targeting TLR2 expression. Further, autophagy inhibitors when used as adjuvants along with conventional drugs can improve drug sensitivity in vitro as well in vivo in a mouse model of leukemia. Our work also demonstrates that this mechanism of bone marrow stromal cell mediated regulation of miR-23a-5p levels and subsequent molecular events are relevant predominantly in myeloid leukemia. Our results illustrate the critical and dynamic role of the bone marrow microenvironment in modulating miRNA expression in leukemic cells which could contribute significantly to drug resistance and subsequent relapse, possibly through persistence of minimal residual disease in this environment.

Prevalence of FVIII inhibitors in severe haemophilia A patients: Effect of treatment and genetic factors in an Indian population.

INTRODUCTION: Factor replacement therapy in treatment of haemophilia A is complicated by the production of neutralising antibodies known as inhibitors. The formation of inhibitors is multifactorial being associated with both genetic and environmental factors. AIM: To document the prevalence of inhibitors in severe haemophilia in the community where most patients receive only infrequent episodic replacement therapy and evaluate the factors which could be contributing to it. METHODS: Community based camps were conducted in different parts of the country. Patients were assessed through a structured questionnaire and blood samples were obtained for laboratory evaluation of inhibitors and defined immunological parameters. RESULTS: Inhibitors were present in 87/447 (19.5%) of the evaluated patients. High-titre inhibitor (>5 Bethesda Units [BU]) was identified in 31 (35.6%) patients. HLA DRB1-13-positive cases (RR = 2.04; 95% CI 1.06-3.911; P = 0.033) had an increased risk of inhibitor formation which was retained in the high-titre subset. A decreased risk of inhibitor formation was noted with heterozygous IL4-590 C/T allele (RR = 0.22; 95% CI 0.108-0.442: P = 0.000). There were no significant correlations between any of the evaluated environmental factors and the development of inhibitors in this study. CONCLUSION: The overall prevalence of inhibitors in patients with severe haemophilia A is similar to that reported among patients receiving regular replacement therapy. The data from this study, limited by its retrospective and cross-sectional study design, would suggest that genetic rather than environmental are more likely to impact the development of inhibitors.

Arsenic Trioxide Enhances the NK Cell Cytotoxicity Against Acute Promyelocytic Leukemia While Simultaneously Inhibiting Its Bio-Genesis.

Natural killer cells (NK) contribute significantly to eradication of cancer cells, and there is increased interest in strategies to enhance it's efficacy. Therapeutic agents used in the treatment of cancer can impact the immune system in a quantitative and qualitative manner. In this study, we evaluated the impact of arsenic trioxide (ATO) used in the management of acute promyelocytic leukemia (APL) on NK cell reconstitution and function. In patients with APL treated with single agent ATO, there was a significant delay in the reconstitution of circulating NK cells to reach median normal levels from the time of diagnosis (655 days for NK cells vs 145 and 265 days for T cells and B cells, respectively). In vitro experiments demonstrated that ATO significantly reduced the CD34 hematopoietic stem cell (HSC) differentiation to NK cells. Additional experimental data demonstrate that CD34+ sorted cells when exposed to ATO lead to a significant decrease in the expression of IKZF2, ETS1, and TOX transcription factors involved in NK cell differentiation and maturation. In contrast, exposure of NK cells and leukemic cells to low doses of ATO modulates NK cell receptors and malignant cell ligand profile in a direction that enhances NK cell mediated cytolytic activity. We have demonstrated that NK cytolytic activity toward NB4 cell line when exposed to ATO was significantly higher when compared with controls. We also validated this beneficial effect in a mouse model of APL were the median survival with ATO alone and ATO + NK was 44 days (range: 33-46) vs 54 days (range: 52-75). In conclusion, ATO has a differential quantitative and qualitative effect on NK cell activity. This information can potentially be exploited in the management of leukemia.

Leukocyte Adhesion Deficiency-I: Clinical and Molecular Characterization in an Indian Population.

OBJECTIVE: To describe clinical and flow cytometric immunophenotyping details of 26 patients of Leukocyte adhesion deficiency-I (LAD-I) along with molecular characterization of 7 patients. METHODS: Diagnosis of LAD-I was suspected on the basis of clinical features, white blood cell count and absolute neutrophil counts and flow cytometric assessment of expression of CD18 and CD11(a, b, c) on leukocytes. Mutation analysis was performed using DNA PCR and conformation sensitive gel electrophoresis (CSGE) technique followed by sequencing. RESULTS: All the patients were symptomatic by the age of 6 mo, with history of recurrent bacterial infections involving skin, mucosa or umbilical cord (omphalitis) being the most frequent presenting symptoms. White blood cells (WBC) and absolute neutrophil counts (ANC) were markedly elevated, without any specific morphological findings. On flow cytometry, CD11a and CD11c showed moderate correlation with CD18 expression. Mutation analysis was performed in 7 patients and six different mutations (4 missense, 2 nonsense and 1 splice site) were identified, all of which were homozygous in nature. CONCLUSIONS: A presentation of repeated bacterial infections during infancy, especially omphalitis, with markedly elevated absolute neutrophil counts should trigger investigations for LAD-I including flow cytometric analysis of CD11/CD18 expression.

Acute myeloid leukaemia: challenges and real world data from India.

The management of acute myeloid leukaemia (AML) in India remains a challenge. In a two-year prospective study at our centre there were 380 newly diagnosed AML (excluding acute promyelocytic leukaemia, AML-M3) patients. The median age of newly diagnosed patients was 40 years (range: 1-79; 12.3% were ≤ 15 years, 16.3% were ≥ 60 years old) and there were 244 (64.2%) males. The median duration of symptoms prior to first presentation at our hospital was 4 weeks (range: 1-52). The median distance from home to hospital was 580 km (range: 6-3200 km). 109 (29%) opted for standard of care and were admitted for induction chemotherapy. Of the 271 that did not take treatment the major reason was lack of financial resources in 219 (81%). There were 27 (24.7%) inductions deaths and of these, 12 (44.5%) were due to multidrug-resistant gram-negative bacilli and 12 (44.5%) showed evidence of a fungal infection. The overall survival at 1 year was 70.4% ± 10.7%, 55.6% ± 6.8% and 42.4% ± 15.6% in patients aged ≤ 15 years, 15 - 60 years and ≥ 60 years, respectively. In conclusion, the biggest constraint is the cost of treatment and the absence of a health security net to treat all patients with this diagnosis.

Comparison of newly diagnosed and relapsed patients with acute promyelocytic leukemia treated with arsenic trioxide: insight into mechanisms of resistance.

There is limited data on the clinical, cellular and molecular changes in relapsed acute promyeloytic leukemia (RAPL) in comparison with newly diagnosed cases (NAPL). We undertook a prospective study to compare NAPL and RAPL patients treated with arsenic trioxide (ATO) based regimens. 98 NAPL and 28 RAPL were enrolled in this study. RAPL patients had a significantly lower WBC count and higher platelet count at diagnosis. IC bleeds was significantly lower in RAPL cases (P=0.022). The ability of malignant promyelocytes to concentrate ATO intracellularly and their in-vitro IC50 to ATO was not significantly different between the two groups. Targeted NGS revealed PML B2 domain mutations in 4 (15.38%) of the RAPL subset and none were associated with secondary resistance to ATO. A microarray GEP revealed 1744 genes were 2 fold and above differentially expressed between the two groups. The most prominent differentially regulated pathways were cell adhesion (n=92), cell survival (n=50), immune regulation (n=74) and stem cell regulation (n=51). Consistent with the GEP data, immunophenotyping revealed significantly increased CD34 expression (P=0.001) in RAPL cases and there was in-vitro evidence of significant microenvironment mediated innate resistance (EM-DR) to ATO. Resistance and relapse following treatment with ATO is probably multi-factorial, mutations in PML B2 domain while seen only in RAPL may not be the major clinically relevant cause of subsequent relapses. In RAPL additional factors such as expansion of the leukemia initiating compartment along with EM-DR may contribute significantly to relapse following treatment with ATO based regimens.

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

BACKGROUND: 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. METHODS: Normal templates for B cell development were established in 10 control patients using CD45, CD11a, CD38, CD20, CD10, CD19, CD58, CD34, CD123, and CD22. BCP-ALL samples (n = 42) were characterized at diagnosis to identify a suitable marker for follow-up during mid (D+21) and end of induction (D+33). Both, multiparametric immunophenotyping and single marker detection of LAIP were used for data analysis. RESULTS: In 95.2% of BCP-ALL at least two informative markers could be obtained when a minimum of four cocktail combinations were used. The combination CD20, CD10, CD45, and CD19 was the most useful (71.4%) followed by combinations containing CD38 (66.7%), CD22 (57.1%), CD11a (52.4%), and CD58 (33.3%). Using our approach, 60 and 47% of patients had detectable MRD at mid and end induction time points, respectively. CONCLUSION: We have described a relatively cost effective MRD panel which is applicable to over 90% of patients. We hope that this data would encourage more centers in India and other resource constrained health delivery systems to develop MRD assays.

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.

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

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.