The Outcome of Pediatric Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia: Experience from a Referral Center in South India.

Although improved survival in children with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-ALL) has been demonstrated in trials, the outcome appears to be inferior in low- and middle-income countries (LMIC). Methods A file review of children aged ≤ 15 years diagnosed with Ph-ALL from 2010 to 2019 was performed. Minimal residual disease (MRD) was assessed by flow-cytometry. Real-time polymerase chain reaction (qRT-PCR) was used to quantify the BCR::ABL1 transcripts during treatment. Results The mean age of the 20 patients in the study was 91 months. Of 19 patients in whom the BCR::ABL1 transcript was confirmed, 10(50%) had P210, 7(35%) had P190, and two showed dual expression. The mean dose of imatinib that was administered was 294 ± 41 mg/m2/day. qRT-PCR for BCR::ABL1 was < 0.01% in all patients who were in remission or had a late relapse and was ≥ 0.01% in patients who had an early relapse. Two patients underwent HSCT. The 3-year event-free survival (EFS) was 35.0 ± 10.7%. Patients with a good prednisolone response (GPR) and a negative end-of-induction MRD demonstrated a superior EFS to those who lacked either or both (80.0 ± 17.9% vs. 16.7 ± 15.2%, P = 0.034). Conclusion The 3-year EFS of 20 children with Ph-ALL treated with chemotherapy and TKI was < 50%. An unusually high proportion of patients with p210 transcript expression; sub-optimal TKI dosing and lesser intensity of chemotherapy, due to the concern of high treatment-related mortality in LMIC are possible reasons for the poor outcome. Conventional treatment response parameters such as GPR and MRD predict outcomes in Ph-ALL. qRT-PCR for BCR::ABL1 may have a role in predicting early relapse. Supplementary Information: The online version contains supplementary material available at 10.1007/s12288-023-01684-9.

Metformin pretreatment ameliorates busulfan-induced liver endothelial toxicity during haematopoietic stem cell transplantation.

The success of Haematopoietic cell transplantation (HCT) is often limited by regimen-related toxicity (RRT) caused by conditioning regimen drugs. Among different conditioning drugs, busulfan (Bu) and treosulfan (Treo), although widely used in HCT, exhibit different toxicity profiles, the mechanism of which is still unclear. Here we investigated the effects of Bu and Treo in endothelial cells. While both Bu and Treo induced DNA damage in endothelial cells, we observed Bu alone to induce oxidative stress and sustained activation of phospho-ERK1/2, leading to apoptosis. However, Treo-treated cells exhibited no oxidative stress/apoptosis of endothelial cells. Screening of pharmacological inhibitors of both ROS and p-ERK revealed that metformin effectively ameliorates Bu-mediated toxicity in endothelial cells. In Balb/c mice, we observed a significant reduction in bone marrow endothelial cells in Bu-treated mice compared to Treo-treated mice. Further, liver sinusoidal endothelial cells (LSEC) was damaged by Bu, which is implicated in liver vasculature and their functional capacity to uptake FITC-albumin. However, Treo-treated mice liver vasculature was morphologically and functionally normal. When mice were pretreated with metformin followed by Bu, LSECs damage was ameliorated morphologically and functionally. Bone marrow transplants done on these mice did not affect the engraftment of donor cells.

Haploidentical Natural Killer Cell Therapy as an Adjunct to Stem Cell Transplantation for Treatment of Refractory Acute Myeloid Leukemia.

Refractory acute myeloid leukemia (AML), defined as failure of two cycles of induction therapy at diagnosis or of one cycle at relapse, represents a subgroup with poor outcomes. Haploidentical natural killer cell (NK) therapy is a strategy that is being explored in refractory malignancies. Historically, at our center, patients with refractory AML have been treated with cytoreductive therapy (fludarabine + cytosine + granulocyte colony-stimulating factor ± idarubicin or mitoxantrone + etoposide) followed by 1-week rest and then reduced-intensity transplant with fludarabine + melphalan. We used the same backbone for this trial (CTRI/2019/02/017505) with the addition of CD56-positive cells from a family donor infused 1 day after the completion of chemotherapy. CD56-positive selection was done using a CliniMACS Prodigy system (Miltenyi Biotec, Bergisch Gladbach, Germany) followed by overnight incubation in autologous plasma with 2 micromolar arsenic trioxide and 500 U/mL of interleukin-2. From February 2019, 14 patients with a median age of 29 years (interquartile range [IQR]: 16.5-38.5) were enrolled in this trial. Six were females. Six had primary refractory AML while eight had relapsed refractory AML. The median CD56-cell dose infused was 46.16 × 106/kg (IQR: 25.06-70.36). One patient withdrew consent after NK cell infusion. Of the 13 patients who proceeded to transplant, five died of immediate post-transplant complications while two did not engraft but were in morphologic leukemia-free state (both subsequently died of infective complications after the second transplant). Of the remaining six patients who engrafted and survived beyond 1 month of the transplant, two developed disease relapse and died. The remaining four patients are alive and relapse free at the last follow-up (mean follow-up duration of surviving patients is 24 months). The 2-year estimated overall survival for the cohort was 28.6% ± 12.1% while the treatment-related mortality (TRM) with this approach was 38.5% ± 13.5%. Haploidentical NK cell therapy as an adjunct to transplant is safe and needs further exploration in patients with AML. For refractory AML, post-transplant NK infusion and strategies to reduce TRM while using pre-transplant NK infusion merit exploration.

Clinical significance of end of induction measurable residual disease monitoring in B-cell acute lymphoblastic leukemia: A single center experience.

The assessment of measurable residual disease (MRD) has emerged as a powerful prognostic tool for both pediatric and adult acute lymphoblastic leukemia (ALL). This retrospective study aimed to evaluate the prognostic relevance of the end of induction MRD in B-cell acute lymphoblastic leukemia (B ALL) patients. The study included 481 patients who underwent treatment for B ALL between August 2012 and March 2019 and had their MRD at the end of induction assessed by flow cytometry. Baseline demographic characteristics were collected from the patient's clinical records. Event free survival (EFS) and relapse free survival (RFS) were calculated using Kaplan-Meier analysis and survival estimates were compared using the log-rank test. End of induction MRD and baseline karyotype were the strongest predictors of EFS and RFS on multivariate analysis. The EFS was inversely related to the MRD value and the outcomes were similar in patients without morphological remission at the end of induction and patients in remission with MRD ≥1.0%. Even within the subgroups of ALL based on age, karyotype, BCR::ABL1 translocation and the treatment protocol, end of induction MRD positive patients had poor outcomes compared to patients who were MRD negative. The study outcome would help draft end of induction MRD-based treatment guidelines for the management of B ALL patients.

What happens to children with acute lymphoblastic leukemia in low- and middle-income countries after relapse? A single-center experience from India.

Managing a child with acute lymphoblastic leukemia (ALL) after relapse is arduous in low- and middle-income countries. A file review of children aged ≤15 years diagnosed with relapsed ALL from 2010 to 2019 was performed. Classification of relapse followed the Berlin-Frankfurt-Münster (BFM) scheme. The majority of patients were treated with a modified ALL-REZ-BFM protocol. Of 764 children treated for ALL in the study period, 163 (21.3%) relapsed. The median age at relapse was 101 months (range: 8-297). The immunophenotype was B-ALL and T-ALL in 140 (86%) and 23 (14%) patients. The site of relapse was extramedullary, combined, and medullary in 46 (28%), 45 (28%), and 72 (44%) patients. Very early, early, and late relapses were observed in 57 (35%), 66 (40%), and 40 (25%) patients. The proportions of extramedullary and medullary sites were greater among patients with early and late relapses, respectively (p = 0.039). Eighty-four (52%) patients were treated with palliative intent. The 2-year event-free survival (EFS) of patients treated with curative intent was 36.3 ± 6.3%. The 2-year EFS for very early/early and late relapses were 18.2 ± 6.2% and 67.6 ± 10.4% (p < 0.001). The 2-year EFS did not differ between extramedullary, combined, and medullary relapses. Treatment-related mortality occurred in 14 (20%) patients. More than 50% of the patients with relapse were treated with the intent of palliation. Extramedullary relapses were more likely to be early and did not have a better outcome than medullary relapses. Children with late relapse had a fair chance of survival with chemotherapy.

Decentralized manufacturing of anti CD19 CAR-T cells using CliniMACS Prodigy®: real-world experience and cost analysis in India.

Chimeric Antigen Receptor (CAR) T cell therapy is an accepted standard of care for relapsed/refractory B cell malignancies. However, the high cost of existing industry-driven centralized production makes this therapy unaffordable in low and middle-income countries. Decentralized or point of care manufacturing has the potential to overcome some of these challenges. Here we demonstrate a decentralized manufacturing process for anti-CD19-CAR-T cells using a fully automated closed system (Miltenyi CliniMACS Prodigy®) is feasible in a developing country setting. Validation run data, as part of a pre-clinical trial safety evaluation, demonstrates the successful and robust manufacturing of anti-CD19 CAR-T cells with T cell expansion of 25 to 47-fold. The median transduction efficiency was 48.8%, with a median viability of 98% and fulfillment of all standard release criteria assays for clinical application. Evaluation of production costs in an academic, not for profit setting in India provide a benchmark for low and middle-income pricing which could greatly increase access to this therapy. Based on our analysis, the cost per product would be approximately $35,107 US dollars. Our data highlights the safety, efficacy, and reproducibility of the process for use in planned future clinical trials.

Generation of good manufacturing practice grade virus-specific T cells for the management of post-transplant CMV infections.

Adoptive transfer of antigen-specific T cells has recently emerged as a successful strategy to treat viral infections following hematopoietic cell transplantation (HCT). Ex-vivo expanded donor-derived virus-specific T cells (VSTs) can be safe and effective, devoid of all the drug-related adverse effects. The study aimed to manufacture cGMP grade VSTs from healthy donors, characterize the VST product and demonstrate its safety and efficacy. Peripheral blood mononuclear cells (PBMCs) collected from six healthy donors were stimulated with pepmix that mimics the pp65 antigenic epitope of CMV and cultured for 14 days in G-Rex culture tubes. Post pepmix exposure and expansion the median CD3% was 98.8% (range:95.5% to 99.9%) while the median CD4% and CD8% were 49.1% (range:21.3% to 86.6%) and 43.9% (range:12.7% to 75.5%) respectively. The percentage of IFNγ+ cells was much higher among the CD8+ T cells (median - 18.47%; range 6.50% - 45.82%) when compared to CD4+ T cells (median - 2.74%; range 0.47% - 18.58%) and there was a switch from the CD45RA+ naive phenotype to CD45RA- effector memory phenotype in the 4 samples that achieved a >5 fold expansion. The VSTs were cytotoxic to the pepmix pulsed lymphoblasts (efficacy) while they did not induce cytolysis in the lymphoblasts that were not exposed to the pepmix (safety). This feasibility exercise helped us optimize the starting cell dose for the culture and clinical grade culture strategies, subset characterization and cytotoxicity assays. The approach could be applied to the clinical practice where virus-specific T cell infusions could be given for post-transplant viral infections.

Mutation Profile in BCR-ABL1-Negative Myeloproliferative Neoplasms: A Single-Center Experience From India.

OBJECTIVE/BACKGROUND: Recurrent somatic mutations in the JAK2, calreticulin (CALR), and the MPL genes are described as drivers of BCR-ABL1-negative myeloproliferative neoplasms (MPN) that includes polycythemia vera (PV), essential thrombocytosis (ET), primary myelofibrosis (PMF), and MPN unclassified (MPN-U). METHODS: We describe the mutation profile and clinical features of MPN cases diagnosed at a tertiary care center. JAK2V617F and MPL (S505/W515) mutations were screened by allele-specific polymerase chain reaction, while CALR exon 9 and JAK2 exon 12 mutations were screened by fragment analysis/Sanger sequencing. Among the 1,570 patients tested for these mutations during the study period, 407 were classified as MPN with a diagnosis of PV, ET, PMF, and MPN-U seen in 30%, 17%, 36%, and 17%, respectively, screened. RESULTS: Similar to previous reports from Asian countries, the incidence of PMF was the highest among the classic MPN. JAK2V617F mutation was detected in 90% of PV, 38% of ET, 48% of PMF, and 65% of MPN-U. JAK2 exon 12 mutations were seen in 5.7% of PV and 1.4% of PMF. CALR exon 9 mutations were seen in 33% of ET, 33% of PMF, and 12% of MPN-U. MPL mutations were detected in 2.8%, 2.7%, and 2.9% of ET, PMF, and MPN-U, respectively. Fifteen % of PMF, 26% of ET, and 22% of MPN-U were triple negative. CONCLUSION: There was a significantly higher incidence of CALR mutation in PMF and ET cases. Our study highlights the challenges in the diagnosis of JAK2-negative PV and the need for harmonization of criteria for the same.

NUDT15 c.415C>T Polymorphism Predicts 6-MP Induced Early Myelotoxicity in Patients with Acute Lymphoblastic Leukemia Undergoing Maintenance Therapy.

PURPOSE: Severe myelosuppression in patients with acute lymphoblastic leukemia (ALL) undergoing 6-MP-based maintenance therapy is attributed to TPMT gene polymorphisms, which is rare in Asian populations. This study aims to evaluate the role of selected polymorphisms in NUDT15, ITPA, and MRP4 genes in addition to TPMT in predicting 6-MP intolerance during ALL maintenance therapy. PATIENTS AND METHODS: We screened for the presence of NUDT15*3 (c.415 C>T, rs116855232); MRP4 c.2269 C>T (rs3765534), ITPA c.94 C>A (rs1127354) polymorphisms in addition to TPMT *2 (rs1800462), *3A (*3B and *3C; rs1800460 and rs1142345) in ALL patients with documented severe neutropenia (cohort-1; n=42). These polymorphisms were then screened in a prospective cohort of ALL patients (cohort-2; n=133) and compared with 6-MP dose reduction, early/late myelotoxicity. RESULTS: Nineteen (45%) patients in cohort-1 and 18 (14%) in cohort-2 had NUDT15 c.415 C>T variant while 4 (3%) patients in cohort-2 had TPMT*3C variant. Five (12%) in cohort-1 and 30 (24%) in cohort-2 had ITPA c.94 C>A variant while 9 (22%) and 15 (12%) had MRP4 c.2269 C>T variant in cohorts-1 and 2, respectively. All in cohort-1 and 36 (27%) in cohort-2 had severe myelotoxicity. Twenty-eight patients (66.6%) in cohort-1 and 40 (30%) patients in cohort-2 had significant 6-MP dose reduction. NUDT15 c.415 C>T variant explained severe myelotoxicity in 63% and 33% in cohort 1 and 2. TPMT*3C and ITPA c.94 C>A variants also explained myelotoxicity in cohort-2 (Median ANC: 376 vs 1014 mm3; p=0.04 and 776 vs 1023 mm3; p=0.04 respectively). NUDT15 c.415 C>T polymorphism explained significant myelotoxicity (507 vs 1298 mm3; p<0.0001) in the multivariate analysis as well (ß=-0.314, p<0.0001). CONCLUSION: NUDT15 c.415 C>T (15*3), TPMT*3C, as well as ITPA c.94 C>A and MRP4 c.2269 C>T polymorphisms explain hematotoxicities. Preemptive genotype-based (NUDT15*3, TPMT, ITPA c.94 C>A) 6-MP dosing could improve the outcome after maintenance therapy.

Clinical and Molecular Findings in Mendelian Susceptibility to Mycobacterial Diseases: Experience From India.

Mendelian Susceptibility to Mycobacterial diseases (MSMD) are a group of innate immune defects with more than 17 genes and 32 clinical phenotypes identified. Defects in the IFN-γ mediated immunity lead to an increased susceptibility to intracellular pathogens like mycobacteria including attenuated Mycobacterium bovis-Bacillus Calmette-Guérin (BCG) vaccine strains and non-tuberculous environmental mycobacteria (NTM), Salmonella, fungi, parasites like Leishmania and some viruses, in otherwise healthy individuals. Mutations in the IL12RB1 gene are the commonest genetic defects identified. This retrospective study reports the clinical, immunological, and molecular characteristics of a cohort of 55 MSMD patients from 10 centers across India. Mycobacterial infection was confirmed by GeneXpert, Histopathology, and acid fast bacilli staining. Immunological workup included lymphocyte subset analysis, Nitro blue tetrazolium (NBT) test, immunoglobulin levels, and flow-cytometric evaluation of the IFN-γ mediated immunity. Genetic analysis was done by next generation sequencing (NGS). Disseminated BCG-osis was the commonest presenting manifestation (82%) with a median age of presentation of 6 months due to the practice of BCG vaccination at birth. This was followed by infection with Salmonella and non-typhi Salmonella (13%), Cytomegalovirus (CMV) (11%), Candida (7%), NTM (4%), and Histoplasma (2%). Thirty-six percent of patients in cohort were infected by more than one organism. This study is the largest cohort of MSMD patients reported from India to the best of our knowledge and we highlight the importance of work up for IL-12/IL-23/ISG15/IFN-γ circuit in all patients with BCG-osis and suspected MSMD irrespective of age.

Primary Immunodeficiencies in India: Molecular Diagnosis and the Role of Next-Generation Sequencing.

Primary immunodeficiency diseases (PIDs) are a group of clinically and genetically heterogeneous disorders showing ethnic and geographic diversities. Next-generation sequencing (NGS) is a comprehensive tool to diagnose PID. Although PID is common in India, data on the genetic spectrum of PIDs are limited due to financial restrictions. The study aims to characterize the clinical and genetic spectrum of PID patients in India and highlight the importance of a cost-effective targeted gene panel sequencing approach for PID in a resource-limited setting. The study includes 229 patients with clinical and laboratory features suggestive of PIDs. Mutation analysis was done by Sanger sequencing and NGS targeting a customized panel of genes. Pathogenic variants were identified in 97 patients involving 42 different genes with BTK and IL12RB1 being the most common mutated genes. Autosomal recessive and X-linked recessive inheritance were seen in 51.6% and 23.7% of patients. Mendelian susceptibility to mycobacterial diseases (MSMD) and IL12RB1 mutations was more common in our population compared to the Western world and the Middle East. Two patients with hypomorphic RAG1 mutations and one female with skewed CYBB mutation were also identified. Another 40 patients had variants classified as variants of uncertain significance (VUS). The study shows that targeted NGS is an effective diagnostic strategy for PIDs in countries with limited diagnostic resources. Molecular diagnosis of PID helps in genetic counseling and to make therapeutic decisions including the need for a stem cell transplantation.

Prognostic value of MRD monitoring based on BCR-ABL1 copy numbers in Philadelphia chromosome positive acute lymphoblastic leukemia.

Assessment of measurable residual disease (MRD) has emerged as a powerful prognostic tool in pediatric and adult acute lymphoblastic leukemia (ALL). In this single-centre retrospective study, we evaluated the prognostic relevance of MRD based on BCR-ABL1 copy numbers in Ph + ALL patients between 2006 and 2018. Molecular responses were evaluated at 3, 6, 9 and 12 months after the initiation of treatment. Patients who had their MRD assessed at three or more time points were categorized into MRD good risk or poor risk based on BCR-ABL1/ABL1 copy number ratio. MRD positive patients consistently showed a trend toward poor survival and on multivariate analysis, MRD poor risk patients had adverse outcomes when compared to MRD good risk patients in terms of overall (OS; p = .031) and event-free (EFS; p < .001) survival. In conclusion, molecular MRD based on BCR-ABL1 copy number ratio is an ideal prognostic indicator in Ph + ALL patients undergoing treatment.

Molecular Characterization of G6PD Deficiency: Report of Three Novel G6PD Variants.

G6PD deficiency is a monogenic, X-linked genetic defect with a worldwide prevalence of around 400 million people and an overall prevalence of 8.5% in India. Hemolytic anemia is encountered in only a small proportion of patients with G6PD variants and is usually triggered by some exogenous agent. Although G6PD deficiency was reported in India more than 50 years ago, there are very few studies on molecular characterization and phenotypic correlation in G6PD deficient patients. We aimed to study the epidemiology and correlate the phenotypic expression with molecular genotypes in symptomatic G6PD deficient patients. All symptomatic hemolytic anaemia patients with a possible etiology of G6PD deficiency based on the clinical, hematological and biochemical parameters and reduced G6PD enzyme levels were included in this study. Molecular analysis of the G6PD gene was done by direct Sanger sequencing. From a total of 38 patients with hemolytic anemia suspected for G6PD deficiency, 24 patients had reduced G6PD enzyme levels and were included for the molecular analysis and mutations in the G6PD gene were identified in 21 of them (83.3%). The different mutations identified in our study include 6 patients with c.131C > G (G6PD Orissa), 3 patients with c.563C > T (G6PD Mediterranean), two patients with c.825G > T (G6PD Bangkok), one patient each with c.208T > C (G6PD Namouru), c.487G > A (G6PD Mahidol), c.949G > A (G6PD Kerala-Kalyan), c.100 G > A (G6PD Chatham), c.1178C > G (G6PD Nashville), c.1361 G > A (G6PD Andalus) and 4 patients with novel mutations (2 patients with c.1186C > T and 1 patient each with c.1288-2A > T and c.1372C > T. No disease causing genetic variants were identified in the other three cases. Co-inheritance of other red cell and hemoglobin disorders can modify the clinical phenotype of G6PD patients and the diagnostic accuracy can be improved by molecular characterization of the variant.

JAK2 exon 12 mutations in cases with JAK2V617F-negative polycythemia vera and primary myelofibrosis.

Molecular detection of JAK2 mutation (V617F or exon 12) is included as a major diagnostic criterion for polycythemia vera (PV) by the WHO 2016 guidelines. JAK2 exon 12 mutations are seen in about 2-5% of JAK2V617F-negative cases of PV. Mutations in JAK2 cause constitutive activation of JAK-STAT pathway which results in variable phenotypes. PV patients with exon 12 mutations in JAK2 present characteristically with erythrocytosis. There are limited reports describing the spectrum of JAK2 exon12 mutations in myeloproliferative neoplasms (MPNs). Here, we describe the characteristics of a series of MPN patients with mutations in exon 12 of JAK2 of which two were novel variants associated with polycythemia. Interestingly, we noted two patients presenting as myelofibrosis having JAK2 exon 12 mutations.

Screening of genetic variants in ELANE mutation negative congenital neutropenia by next generation sequencing.

AIMS: Congenital neutropenia (CN) is a rare inherited disease that results in recurrent, life-threatening bacterial infections due to a deficiency of mature neutrophils. They are usually caused by heterozygous ELANE mutations although mutations in other genes like HAX-1, G6PC3 and GFI1 have also been reported. Identifying the causative mutation aids in the establishment of diagnosis and rules out other secondary causes of neutropenia like autoimmune cytopenia and evolving aplasia. We aimed to identify the molecular defects in CN patients who had no mutations in ELANE gene, by next generation sequencing (NGS) targeting a customised panel of genes. METHODS: DNA samples were sequenced with an Illumina NextSeq sequencer using an in-house customised panel of genes at ≥100× depth. Bioinformatics analysis was carried out and the pathogenic variants were identified using a stepwise filtering and analysis strategy. Specific mutations identified were subsequently validated by Sanger sequencing. RESULTS: The pathogenic variants identified in the study includes previously reported variants in SBDS (compound heterozygous c.258+2T>C and c.1A>T), GATA2 (heterozygous c.1186C>T) and novel variants in WAS (hemizygous c.812T>C), JAGN1 (homozygous c.70G>A) and RTEL1 (heterozygous c.2893G>C) genes. CONCLUSION: This study highlights that the absence of ELANE mutations does not rule out the diagnosis of CN and this NGS based approach with a customised panel will help in diagnostic confirmation in such patients. The early onset of the disease, clinical severity and associated high risk of malignant transformation in CN strongly suggests the need for early diagnosis and therapeutic intervention.