15-color highly sensitive flow cytometry assay for post anti-CD19 targeted therapy (anti-CD19-CAR-T and blinatumomab) measurable residual disease assessment in B-lymphoblastic leukemia/lymphoma: Real-world applicability and challenges.

OBJECTIVES: Measurable residual disease (MRD) is the most relevant predictor of disease-free survival in B-cell acute lymphoblastic leukemia (B-ALL). We aimed to establish a highly sensitive flow cytometry (MFC)-based B-ALL-MRD (BMRD) assay for patients receiving anti-CD19 immunotherapy with an alternate gating approach and to document the prevalence and immunophenotype of recurrently occurring low-level mimics and confounding populations. METHODS: We standardized a 15-color highly-sensitive BMRD assay with an alternate CD19-free gating approach. The study included 137 MRD samples from 43 relapsed/refractory B-ALL patients considered for anti-CD19 immunotherapy. RESULTS: The 15-color BMRD assay with CD22/CD24/CD81/CD33-based gating approach was routinely applicable in 137 BM samples and could achieve a sensitivity of 0.0005%. MRD was detected in 29.9% (41/137) samples with 31.7% (13/41) of them showing <.01% MRD. Recurrently occurring low-level cells that showed immunophenotypic overlap with leukemic B-blasts included: (a) CD19+CD10+CD34+CD22+CD24+CD81+CD123+CD304+ plasmacytoid dendritic cells, (b) CD73bright/CD304bright/CD81bright mesenchymal stromal/stem cells (CD10+) and endothelial cells (CD34+CD24+), (c) CD22dim/CD34+/CD38dim/CD81dim/CD19-/CD10-/CD24- early lymphoid progenitor/precursor type-1 cells (ELP-1) and (d) CD22+/CD34+/CD10heterogeneous/CD38moderate/CD81moderate/CD19-/CD24- stage-0 B-cell precursors or ELP-2 cells. CONCLUSIONS: We standardized a highly sensitive 15-color BMRD assay with a non-CD19-based gating strategy for patients receiving anti-CD19 immunotherapy. We also described the immunophenotypes of recurrently occurring low-level populations that can be misinterpreted as MRD in real-world practice.

KIT exon 17 mutations are predictive of inferior outcome in pediatric acute myeloid leukemia with RUNX1::RUNX1T1.

BACKGROUND: Pediatric core binding factor acute myeloid leukemia (CBF-AML), although considered a favorable risk subtype, exhibits variable outcomes primarily driven by additional genetic abnormalities, such as KIT mutations. PROCEDURE: In this study, we examined the prognostic impact of KIT mutations in 130 pediatric patients with CBF-AML, treated uniformly at a single center over 4 years (2017-2021). KIT mutations were detected via next-generation sequencing using a myeloid panel comprising 52 genes for most patients. RESULTS: Our findings revealed that KIT mutations were present in 31% of CBF-AML cases. Exon 17 KIT mutation was most commonly (72%) seen with notable occurrences at the D816 and N822 residue in 48% and 39% of cases, respectively. The 3-year cumulative incidence of relapse (CIR) and overall survival (OS) for patients with exon 17 KIT mutation were 36% and 40%, respectively, and was significantly worse in comparison to other site KIT mutations (3-year CIR: 11%; OS: 64%) and without KIT mutation (3-year CIR: 13%; OS:71%). Notably, the prognostic impact of KIT mutations was prominent in patients with RUNX1::RUNX1T1, but not in those with CBFB::MYH11 fusion. Additionally, a high KIT variant-allele frequency (VAF) (>33%) predicted for a higher disease relapse; 3-year CIR of 40% for VAF greater than 33% versus 7% for VAF less than 33%. When adjusted for site of KIT mutation and end-of-induction measurable residual disease, VAF greater than 33% correlated with poor OS (hazard ratio [HR]: 4.4 [95% CI: 1.2-17.2], p = .034). CONCLUSION: Exon 17 KIT mutations serve as an important predictor of relapse in RUNX1::RUNX1T1 pediatric AML. In addition, a high KIT VAF may predict poor outcomes in these patients. These results emphasize the need to incorporate KIT mutational analysis into risk stratification for pediatric CBF-AML.

Does granulocyte transfusion help pediatric cancer patients with sepsis?

Sepsis-related mortality continues to be a major concern while treating pediatric cancer patients, more so with the rise in the incidence of multidrug-resistant organisms (MDRO). In this retrospective study conducted between January 2021 and December 2022 at a tertiary cancer center in India, granulocyte transfusion was offered in addition to standard antimicrobial therapy to 64 children with hematolymphoid malignancy who developed 75 episodes of severe sepsis following intensive chemotherapy. Forty-four (83%) of 53 blood culture proven sepsis was caused by MDROs. Thirty-seven (70%) patients with blood culture proven sepsis cleared the organism after granulocyte transfusion. Thirty-day mortality was 25% for the entire study cohort and 32% for patients with MDRO sepsis.

Pediatric cancer-associated thrombosis: Analysis from a tertiary care cancer center in India.

BACKGROUND AND AIMS: Thrombotic events (TEs) have been extensively studied in adult cancer patients, but data in children are limited. We prospectively analyzed pediatric cancer-associated thrombosis (PCAT) in children with malignancies. METHODS: Children below 15 years of age with confirmed malignancies, treated at a large tertiary cancer center in India from July 2015 to March 2020 developing any TE were eligible. A standardized approach for detection and management was followed. Data were collected after informed consent. RESULTS: Of 6132 eligible children, 150 (2.44%) had 152 TEs, with median age 8.5 years and male:female of 1.83:1. Most TEs occurred on chemotherapy: 111 (74.0%). The most common site was central nervous system (CNS) 59 (39.3%), followed by upper-limb venous system 37 (24.7%). Hemato-lymphoid (HL) malignancies were more prone to PCAT than solid tumors (ST) (incidence 3.23% vs. 1.58%; odds ratio [OR] = 2.06, 95% confidence interval [CI] [1.36-2.88]; p < .001). Malignancies associated with PCAT were acute lymphoblastic leukemia (ALL) 2.94%, acute myeloid leukemia (AML) 6.66%, and non-Hodgkin lymphomas 5.35%. Response imaging done in 106 (70.7%) children showed complete to partial resolution in almost 90% children. Death was attributable to TE in seven (4.66%) children. Age above 10 years (OR 2.33, 95% CI [1.59-3.41]; p < .001), AML (OR 4.62, 95% CI [1.98-10.74]; p = .0062), and non-Hodgkin lymphoma (OR 4.01, 95% CI [1.15-14.04]; p = .029) were significantly associated with TEs. In ALL, age more than 10 years (OR 1.86, 95% CI [1.06-3.24]; p < .03), T-ALL (OR 3.32, 95% CI [1.69-6.54]; p = .001), and intermediate-risk group (OR 4.97, 95% CI [1.12-22.02]; p = .035) were significantly associated with thrombosis. The 2-year event-free survival (EFS) for HL malignancies with PCAT was 55.3% versus 72.1% in those without PCAT (p = .05), overall survival (OS) being 84.6% versus 80.0% (p = .32). CONCLUSION: Incidence of PCAT was 2.4%, and occurred predominantly in older children with hematolymphoid malignancies early in treatment. Most resolved completely with low molecular weight heparin (LMWH) and mortality was low. In hematolymphoid malignancies, PCAT reduce EFS, highlighting the need for prevention.

Human Immunodeficiency Virus Associated Plasmablastic Lymphoma Involving Bones and Peritoneum in a 4-Year-old Child.

In children with underlying Human Immunodeficiency virus infection and AIDS, hematolymphoid cancers, especially non-hodgkin lymphomas are common. Plasmablastic lymphoma is one such non-hodgkin lymphomas arising from the head and neck region (especially sinonasal) but extremely rare. We describe the clinical course in a 4-year-old boy who presented with a solitary bony swelling of the right knee joint, which on diagnostic work-up turned out to be plasmablastic lymphoma. With combination chemotherapy, intrathecal chemotherapy, and early institution ofHighly active anti-retroviral therapy, the child continues to be in remission.

Impact of a pediatric oncology nutrition program: Lessons learnt over a decade.

BACKGROUND: The management of malnutrition in children with cancer remains a challenge in low-middle-income countries (LMICs). We describe our pediatric oncology nutrition program and its impact over the past decade. METHODS: We evaluated the impact of our nutrition program in accordance with the International Society of Paediatric Oncology-Paediatric Oncology in Developing Countries (SIOP PODC) Nutritional Program Evaluation in the areas of service delivery (number served, increments in delivery, number of trained care providers), patients at-risk (proportion identified with malnutrition at diagnosis/follow-up), and efficiency of nutritional interventions (proportion assessed, proportion achieved healthy weight, clinicians trained). We analyzed available data for trends between 2009 and 2020, and comparisons were made using the Fisher t test. This study was approved by our institutional ethics committee. RESULTS: From 2010 to 2020, 17 749 children treated at our center were beneficiaries of the nutritional program, including assessment and intervention. During this period, trained pediatric nutritionists increased from 2 to 8; SIOP PODC level from 2 to 3-4, and nutrition budget increased 15-fold. At diagnosis (n = 5618) and six-month follow-up (n = 2674), 59.6% and 51.2% children were undernourished, 34.8% and 43% well nourished, and 4.7% and 5.7% overnourished. From 2016 onward, fewer children were undernourished at follow-up-69.5% (2016), 60% (2018), 54% (2019), and 55% (2020, P < 0.001). The program helped train over 500 clinicians in nutrition. CONCLUSIONS: Improved financial support and capacity building have helped build and sustain an effective nutrition program. Priority areas include implementation of best practices, early nutritional intervention, continued education, and locally relevant research.

A comparison of asparaginase activity in generic formulations of E.coli derived L- asparaginase: In-vitro study and retrospective analysis of asparaginase monitoring in pediatric patients with leukemia.

AIMS: L-asparaginase is an essential medicine in the treatment of pediatric acute lymphoblastic leukemia (ALL) and the quality of generic formulations is an area of concern. We compared nine generic formulations of L-asparaginase available in India with the innovator. METHODS: The quality of formulations was assessed by measuring 72-hour trough asparaginase activity in children with ALL during induction following administration of 10,000 IU/m2 of L-asparaginase. In-vitro analysis of the label claim was assessed by measuring activity of three generic formulations. Liquid chromatography-mass spectrometry (LC/MS) was used to determine the amount of host contaminant proteins (HCPs) in the formulations. RESULTS: Between March 2015 to June 2018, 240 samples from 195 patients were analyzed. The number of samples analyzed ranged from 7-66 per generic brand (median: 18) and seven of the innovator. The proportion of generic formulations that failed to achieve a predefined clinical threshold activity of 50 IU/L ranged from 16.7% (2/12) to 84.9% (28/33) in the highest activity to lowest activity generic respectively. On other hand, all innovator samples had activity greater than 50 IU/L. In-vitro asparaginase activity in the three generic formulations tested ranged from 71.4-74.6% of the label claim (10,000 IU) compared to 93.5% for the innovator. LC/MS analysis of generic 5 identified 25 HCPs with a relative peptide count of 27.1% of the total peptides. CONCLUSIONS: Generic formulations had lower asparaginase activity which raises serious clinical concerns regarding their quality. Until stringent regulatory enforcement improves the quality of these generics, dose adaptive strategies coupled with therapeutic drug monitoring need to be considered.

Folic acid, one-carbon metabolism & childhood cancer.

Folate has been studied in relation to many diseases, especially cancer. Although it has been postulated to exert a dual effect on development of cancer, its role remains to be clearly defined. Its effect on cancer is the result of gene-nutrient interaction between the genes in folate metabolic pathway and dietary folate availability; mutations in genes of folate metabolism have been shown to alter individual susceptibility to certain childhood cancers as well as response to cancer chemotherapy. Although mandatory fortification of food items with folate has been initiated in some countries, many countries are yet to adopt this due to concerns about undesired adverse effects of high folate levels on health, especially cancer. However, initial reports suggest that folate fortification has led to reduction in incidence of certain childhood cancers such as neuroblastoma, wilms tumour and leukaemias. Despite studies showing folate depletion during antifolate chemotherapy and higher toxicity of chemotherapy in folate-depleted individuals, folate supplementation during cancer chemotherapy is not routinely recommended. Studies investigating the precise effect of folate supplementation during chemotherapy on both short- and long-term outcomes of cancer are needed to arrive at a consensus guideline.

Glutathione-S-transferase polymorphism and acute lymphoblastic leukemia (ALL) in north Indian children: a case-control study and meta-analysis.

Various studies on association of glutathione S-transferase (GST) polymorphisms and childhood acute lymphoblastic leukemia (ALL) have yielded conflicting results. We examined this association among north Indian children and conducted an updated meta-analysis to overcome sample size-related limitations. GSTM1, GSTP1 and GSTT1 genotypes in 100 children with ALL and 300 healthy controls were compared. GSTT1 null mutation (odds ratio (OR) 2.54, 95% confidence interval (CI) 1.50-4.32) and GSTP1 homozygous mutation (OR 3.13, 95%CI 1.48-6.59) were found to increase the risk of childhood ALL, while GSTM1 did not alter the risk. Meta-analysis included 22, 10 and 20 studies examining the association of childhood ALL with GSTM1, GSTP1 and GSTT1 genotypes, respectively. Only GSTM1 genotype (OR 1.29, 95%CI 1.10-1.62) was associated with increased risk in the overall analysis. However, both GSTM1 (OR 1.54, 95%CI 1.12-2.10) and GSTT1 (OR 1.63, 95%CI 1.32-1.99) null genotypes were associated with increased risk in Asian subjects. The risk of developing childhood ALL was not associated with GSTP1 genotype.

Causes, outcome and prevention of abandonment in retinoblastoma in India.

BACKGROUND: The high-cure rates of 90% in retinoblastoma are not replicated in developing countries due to late presentation and poor compliance to treatment. The present study takes a closer look at causes of abandonment of therapy and effectiveness of counselling in reducing abandonment. PROCEDURE: A retrospective study of children with retinoblastoma registered at our centre from March 2008 through August 2011. RESULTS: Fifty (49.50%) of 101 children registered for treatment abandoned therapy. Abandonment rates were significantly higher in rural as compared to urban children (P = 0.02). There was no significant difference in rate of abandonment between stages or laterality of disease and other socio-demographic factors. Telephone calls were more effective than letters in tracing patients (31.2% vs. 2.4%). Major reasons cited behind abandonment were financial problems (30%) and unwillingness to enucleate (20%). Of the 12 children who returned and were retreated 6 (50%) died of progressive disease. Nineteen (73%) of those who did not return died at home. Abandonment rates steadily declined from 71.42% in 2008 to 16.66% in 2011 (P = 0.01) due to effective pre-abandonment counselling by a support team under the National Retinoblastoma Registry of India from 2009. CONCLUSIONS: Abandonment rates for children with retinoblastoma continue to be unacceptably high. Rural background, financial constraints and hesitancy to enucleate were important causes behind abandonment. Outcome of patients who abandoned treatment was uniformly dismal. Inclusion of support team and intensified initial counselling helped in improving compliance.

Treatment of Pediatric Acute Promyelocytic Leukemia with Retinoic Acid and Arsenic Trioxide along with Chemotherapy.

OBJECTIVES: Outcomes of childhood acute promyelocytic leukemia (APL) have exceeded 90% in the era of differentiating agents. In resource-limited settings, early mortality secondary to coagulopathy remains a significant challenge. Differentiation syndrome is a unique complication of APL therapy that requires a high degree of suspicion for timely initiation of therapy. METHODS: A retrospective study of children ≤15 y of age with APL diagnosed between January-2013 and June-2019 treated at a tertiary cancer centre was conducted. Patients with a total leukocyte count ≥10,000/µL were risk stratified as high-risk. Treatment included differentiating agents, all-trans retinoic acid and arsenic trioxide along with chemotherapy. Baseline demographics, clinical complications and outcomes were analysed. RESULTS: Out of 90 patients treated, 48 (53%) had high-risk APL and 25 (28%) presented with significant bleeding manifestations. Response to therapy was excellent with 96% of evaluable patients achieving molecular remission by the end of consolidation phase. Differentiation syndrome occurred in 23 (25%) patients of which two expired. Early mortality rate was 5.5% and was due to severe hemorrhage most often at the time of presentation. The 3-y overall survival of the entire cohort was 91% (95% CI: 85-97%). Two of 4 patients with relapse of disease could be salvaged with only differentiating agents followed by autologous transplantation. CONCLUSIONS: Long-term outcomes of Indian children with APL are excellent. Timely management of coagulopathy and prompt initiation of differentiating agents along with appropriate cytoreductive measures is critical. Efforts to build academic-community partnerships to ensure timely diagnosis and emergency care in order to reduce early mortality are needed.

Nutritional status and complications in children with diabetic ketoacidosis.

OBJECTIVE: Diabetic ketoacidosis in children continues to be an important cause of morbidity and mortality, especially in developing economies as a result of malnutrition, a high rate of infections, and delay in seeking timely medical care. Malnutrition also increases the risk of diabetic ketoacidosis-related complications. The objective of this study was to assess the nutritional status of patients presenting with diabetic ketoacidosis and correlate it with the incidence of complications at presentation and those encountered during the course of illness. DESIGN: Prospective study. SETTING: Pediatric emergency and intensive care units, Advanced Pediatrics Centre, PGIMER, Chandigarh, India. PATIENTS: Thirty-three children between 1 month and 12 yrs of age presenting with diabetic ketoacidosis between July 2008 and June 2009 were enrolled consecutively and assessed for nutritional status by anthropometric parameters (body weight, crown-heel length/height, mid-upper arm circumference, triceps and subscapular skin fold thicknesses), biochemical parameters (serum albumin, zinc, magnesium, vitamin A levels), and preillness dietary history (by pretested Food Frequency Questionnaire). Patients were classified as malnourished or normally nourished based on the weight for age criteria matched for Indian standards. The incidence of complications (electrolyte imbalances, hypoglycemia, sepsis, cerebral edema, etc.) and outcome in terms of survival or death in both the groups were compared with Student's t-test for parametric data, Mann-Whitney U test for nonparametric data, and chi-square test for categorical variables. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Anthropometric assessment showed that 11 of 33 (33.3%) were malnourished. Preillness dietary history revealed that 16 (48.5%) were calorie- and protein-deficient (known diabetic n = 7; new onset n = 9), whereas 11 (33.3%) were only calorie-deficient (known diabetic n = 2). Hypoalbuminemia was seen in 21 (63.6%), hypovitaminosis A in eight (24.2%), and low zinc levels in three (9%). The malnourished and normally nourished groups were similar with respect to demographics, precipitating factors, severity of diabetic ketoacidosis, treatment received, and outcome. However, the incidence and severity of therapy-related hypokalemia (100% vs. 72.7%; p = .05) and hypoglycemia (63.6 vs. 13.6%; p = .004) were significantly higher in the former as compared with the latter. The mean ± SD admission serum potassium levels were similar in both the groups (3.4 ± 0.8 mEq/L in the malnourished vs. 3.5 ± 0.7 mEq/L in the normally nourished) with the malnourished group showing a significant fall at 6 hrs after start the of diabetic ketoacidosis protocol (2.8 ± 0.8 mEq/L vs. 3.6 ± 0.7 mEq/L; p = .033), although the mean rate and dose of insulin infusion were similar. The fall in blood glucose (mean ± SD mg/dL) at 12, 24, and 36 hrs after onset of the diabetic ketoacidosis protocol was also significantly greater in the malnourished group as compared with the normally nourished diabetic ketoacidosis (195 ± 69.1 and 272.61 ± 96.3, p = .02; 171 ± 58.5 and 257 ± 96.3, p = .05; and 153.75 ± 49.6 and 241.71 ± 76.3, p = .04, respectively). The incidence of hypophosphatemia, hypomagnesemia, cerebral edema, renal failure, sepsis, and septic shock was similar in both the groups. There were two deaths, both resulting from complicating cerebral edema and renal failure and unrelated to the nutritional status of the patients. CONCLUSIONS: The incidence and severity of therapy-related hypokalemia and hypoglycemia were significantly higher in the malnourished as compared to the normally nourished diabetic ketoacidosis. Other diabetic ketoacidosis-related complications and outcome were similar in both the groups.

COVID-19 in Children with Cancer and Continuation of Cancer-Directed Therapy During the Infection.

OBJECTIVE: To report the experience with COVID-19 in children with cancer at the largest tertiary-cancer care and referral center in India. METHODS: This study is a single tertiary center experience on COVID-19 in children with cancer and continuation of cancer-directed therapy in them. Children ≤ 15 y on active cancer treatment detected with COVID-19 until September 15th, 2020 were prospectively followed up in the study. Patients were managed in accordance with well-laid guidelines. Treatment was continued for children with COVID-19 who were clinically stable and on intensive treatment for various childhood cancers. RESULTS: One hundred twenty-two children (median age 8 y; range 1-15 y, male:female 1.7:1) with cancer were diagnosed with COVID-19. Of 118 children, 99 (83.9%), 60 (50.8%), 43 (36.4%), 26 (22.0%), and 6 (5.1%) had RT-PCR positivity at 14, 21, 28, 35, and 60 d from diagnosis of COVID-19, respectively. Scheduled risk-directed intravenous chemotherapy was delivered in 70 (90.9%) of 77 children on active systemic treatment with a median delay of 14 d (range 0-48 d) and no increased toxicities. All-cause mortality rate was 7.4% (n = 9) and COVID-19 related mortality rate was 4.9% (n = 6). One hundred-fifteen (94.2%) children with COVID-19 did not require any form of respiratory support during the course of infection. CONCLUSIONS: COVID-19 was not a major deterrent for the continuation of active cancer treatment despite persistent RT-PCR positivity. The long-term assessment of treatment adaptations requires further prospective follow-up and real-time addressal.

Outcomes of COVID-19 and risk factors in patients with cancer.

Patients with cancer are at higher risk for adverse coronavirus disease 2019 (COVID-19) outcomes. Here, we studied 1,253 patients with cancer, who were diagnosed with severe acute respiratory syndrome coronavirus 2 at a tertiary referral cancer center in India. Most patients had mild disease; in our settings, recent cancer therapies did not impact COVID-19 outcomes. Advancing age, smoking history, concurrent comorbidities and palliative intent of treatment were independently associated with severe COVID-19 or death. Thus, our study provides useful insights into cancer management during the COVID-19 pandemic.

Mutational landscape of Juvenile Myelomonocytic Leukemia (JMML)-A real-world context.

INTRODUCTION: Juvenile myelomonocytic leukemia (JMML) is a rare childhood neoplasm (<5% cases), which has been categorized under myelodysplastic/myeloproliferative neoplasms (MDS/MPN) in the recent classification by the World Health Organization. METHODS: We developed a 51-gene (151.5kB) low-cost targeted myeloid panel based on single-molecule molecular inversion probes to comprehensively evaluate the genomic profile of Juvenile myelomonocytic leukemia (JMML). RESULTS: A total of 50 children with clinical and pathological features of JMML were sequenced at high coverage. Among the 50 patients, 44(88%) harbored mutations in one of the RAS/MAPK-pathway genes, most frequently in NRAS (32%), followed by PTPN11 (28%) and NF1 (22%). One-fifth of children had more than one mutation, with 5 cases harboring two RAS pathway mutations. Monosomy 7 was detected in 32% (16) patients, and five of these did not harbor any RAS pathway mutations. Children with monosomy 7 showed shorter overall survival compared with their wild-type counterparts (P = .02). CONCLUSION: Our study highlights that comprehensive genomic profiling identifies at least one mutation in almost 90% of JMML patients. Performing genomic analysis at baseline might help in triaging children with JMML for allogenic stem cell transplant in resource-constrained settings.

Importance of conventional cytogenetics in the identification of ins(19;X)(q13.1;p11.2q28) and t(1;11)(q10;p10), both, novel cytogenetic abnormalities in a pediatric AML case.

Acute Myeloid Leukemia (AML) is a heterogeneous disease with respect to morphology, immunophenotype, chromosomal abnormalities and genetic lesions. While a majority of AML cases harbour recurrent chromosomal abnormalities, several rare, apparently unique or novel aberrations may be identified by conventional cytogenetics. In fact, with the prognostic relevance of chromosomal abnormalities, and with the advent of new-age, target-specific therapy, identifying such aberrations becomes vital. In this study, we present a case of pediatric AML with ins(19;X)(q13.1;p11.2q28) and t(1;11)(q10;p10), both, novel, previously unreported chromosomal abnormalities in AML. Post induction, both these clonal cytogenetic abnormalities persisted. The documentation of this case will help determine the significance of these cytogenetic abnormalities. Also, this case exemplifies the importance of cytogenetics in the complete characterization and risk stratification of AML patients.