A pilot study on therapeutic drug monitoring of asparaginase in Indian children with high-risk ALL: evidence for suboptimal exposure

Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy in India, with survival rates (60%–70%) significantly lower than in high-income countries (85%–90%). Asparaginase, a cornerstone of ALL therapy, has a narrow therapeutic window with substantial interpatient pharmacokinetic variability. However, therapeutic drug monitoring is rarely performed in resource-limited settings. The prospective pilot study assessed serum asparaginase activity during induction therapy in Indian children with high-risk B-cell ALL. Of 30 enrolled patients, nine high-risk cases (aged 1–12 years) received L-asparaginase according to the modified ICiCLe protocol. Serum asparaginase activity was measured using a spectrophotometric assay at pre-dose (trough) and 24-h post-dose (peak) time points. Treatment-related toxicities were monitored using standardized criteria. Among the nine high-risk patients, five completed inductions, three died from treatment-related complications, and one was transferred. All evaluable patients (n = 5) had subtherapeutic trough asparaginase activity (<0.5 IU/mL; target ≥0.5 IU/mL). Peak levels showed marked interpatient variability (range: 0.52–1.97 IU/mL; median: 1.15 IU/mL). Higher enzyme activity correlated with biochemical toxicities: hepatotoxicity (5/5), elevated blood urea nitrogen (4/5), hypertriglyceridemia (3/5), and subclinical pancreatitis (2/5). This pilot study demonstrates consistently subtherapeutic trough asparaginase levels in a small cohort of Indian children with high-risk ALL, suggesting potential inadequate drug exposure during induction therapy. These findings highlight the potential role of therapeutic drug monitoring in optimizing dosing and minimizing toxicity in resource-limited settings. Larger, adequately powered studies are required to validate these observations and establish population-specific pharmacokinetic parameters and dosing strategies.

Acute lymphoblastic leukemia (ALL) is characterized by abnormal proliferation of immature lymphocytes and represents the most common pediatric malignancy worldwide. Despite being highly curable in high-income countries, ALL remains a leading cause of cancer-related mortality in children from resource-limited settings. In India, the annual incidence of ALL is estimated to be 1.5 per 100,000 children with an overall survival rate of 60%–70% for pediatric ALL in India, significantly lower than the 85%–90% observed in high-income countries (1). This survival disparity is multifactorial, involving differences in disease biology, treatment protocols, supportive care, drug availability, and chemotherapeutic regimen (2, 3). Asparaginase (ASP) is a critical component of ALL therapy that depletes circulating asparagine by catalyzing its hydrolysis to aspartic acid and ammonia (4). Unlike normal cells, leukemic lymphoblasts have limited capacity to synthesize asparagine de novo due to low or absent expression of asparagine synthetase (ASNS), making them dependent on exogenous asparagine sources for survival (5). This metabolic dependency underlies the therapeutic efficacy of ASP in inducing leukemic cell death.

However, resistance mechanisms have been described in preclinical models, particularly upregulation of ASNS expression in leukemic cells, demonstrated through forced expression and selection studies in vitro (6, 7). Clinical evidence for ASNS upregulation as a resistance mechanism in patients remains limited and heterogeneous, varying by leukemia subtype and genetic background (8, 9). In addition, the bone marrow microenvironment may contribute to resistance, as mesenchymal stromal cells express high levels of ASNS and can supply extracellular asparagine to leukemic lymphoblasts in coculture models (9). The proposed role of hepatocytes in supporting lymphoblast survival through asparagine supply remains speculative and lacks definitive clinical evidence (6). Maintaining adequate serum ASP activity is therefore essential to ensure sustained depletion of circulating asparagine. The therapeutic challenge lies in balancing the enzyme’s activity to maximize its efficacy while minimizing toxicity (10, 11). When ASP activity is excessively high, the resultant overproduction of aspartic acid and ammonia can exacerbate toxicity, potentially leading to systemic side effects (12). Conversely, insufficient enzyme activity can result in inadequate depletion of asparagine, allowing leukemic cells to survive and proliferate (13). This suboptimal therapeutic response not only leads to poor clinical outcomes but also results in the development of drug resistance and toxicity (14). A critical challenge in asparaginase therapy is the phenomenon of “silent inactivation,” defined as the immune-mediated neutralization and/or rapid clearance of administered asparaginase that lowers enzyme activity in the absence of clinical allergic signs (15, 16). Unlike overt hypersensitivity reactions, which manifest with clinical symptoms such as urticaria or anaphylaxis, silent inactivation occurs when anti-asparaginase antibodies develop and neutralize enzyme activity without producing observable allergic symptoms (17). This phenomenon is particularly insidious because patients appear clinically asymptomatic despite loss of therapeutic drug effect, leading to inadequate asparagine depletion and subtherapeutic exposure (15). Silent inactivation has been associated with inferior oncological outcomes in several cohorts, with studies reporting increased relapse risk and reduced event-free survival when enzyme activity is not maintained (18, 19). The detection of silent inactivation is therefore the primary clinical indication for therapeutic drug monitoring of asparaginase, as it cannot be identified through clinical observation alone and requires laboratory measurement of serum asparaginase activity (16, 17).

Therapeutic drug monitoring (TDM) of asparaginase is therefore important for two major reasons: 1) detection of silent inactivation to ensure adequate asparagine depletion and optimize oncological outcomes and 2) correlation of enzyme activity with treatment-related toxicities to guide supportive care and dose adjustments (16, 20). International expert consensus now recommends routine measurement of serum asparaginase activity to identify both clinical hypersensitivity and silent inactivation, with timely switching to alternative preparations when therapeutic activity is compromised (16). Despite this consensus, TDM is rarely implemented in resource-limited settings, including India, due to limited laboratory infrastructure, assay availability, and cost constraints. The absence of routine monitoring may contribute to suboptimal outcomes, as undetected silent inactivation can result in inadvertent omission of effective asparaginase exposure during critical phases of therapy (18, 19).

The systemic effects of ASP toxicity are varied and can impact multiple organ systems. Common toxicities include hepatotoxicity, pancreatitis, coagulopathy, and hypersensitivity reactions (21, 22). Hepatotoxicity, manifested by elevated liver enzymes and jaundice, can lead to hepatic dysfunction and necessitate discontinuation of therapy (21, 23). Pancreatitis, a severe inflammation of the pancreas, can cause abdominal pain, nausea, and potentially life-threatening complications (24, 25). Coagulopathy, due to decreased synthesis of clotting factors, increases the risk of bleeding and thrombosis (22, 23). Hypersensitivity reactions, ranging from mild allergic responses to anaphylaxis, can impede the continuation of ASP therapy (17, 26). If discontinued, the patient is at significant risk of relapse (21, 27). Therefore, monitoring of ASP activity becomes crucial.

Drug resistance in ALL arises mainly when ASP therapy fails. Leukemic cells develop resistance by upregulating asparagine synthetase, allowing autonomous asparagine synthesis. Moreover, discontinuing ASP due to side effects gives these cells a survival advantage, weakening the treatment regimen and lowering remission rates (21, 27).

Globally, ASP activity monitoring protocols are standardized, with routine measurement of serum ASP levels being a common practice in high-income countries (28, 29). The primary rationale for TDM is to detect silent inactivation, the antibody-mediated loss of asparaginase activity without allergic symptoms which occurs in 4.5%–7% of patients and is associated with higher relapse rates when undetected (16, 17). Unlike overt hypersensitivity reactions, silent inactivation can only be identified through enzyme activity measurement, making routine TDM essential for treatment efficacy (11). In the United States and Europe, guidelines recommend regular monitoring to identify silent inactivation and optimize dosing. For instance, the Children’s Oncology Group (COG) and the European Organization for Research and Treatment of Cancer (EORTC) include specific protocols for ASP activity monitoring (16, 30–33). However, in resource-limited settings, including India, routine TDM is challenging due to limited access to specialized enzyme activity assays, variability in healthcare infrastructure, and economic constraints, which prevent the detection of silent inactivation and may compromise treatment outcomes.

The therapeutic range of ASP in serum as decided by published studies in the West might not actually be applicable for Indian patients owing to geographic, environmental, nutritional, and other diverse cohort factors affecting therapeutic drug monitoring and overall chemotherapy outcome. It would also be interesting to note whether the nutritional status of the patient bears any effect on the serum ASP levels, since this is impaired in protein energy malnourishment, prevalent in almost all patients of the pediatric age group.

High-risk B-ALL is a relatively rare but clinically significant group with the highest rates of relapse, resistance, and mortality. The patients require intensive therapy and meticulous monitoring to manage systemic toxicity caused by aggressive treatment regimens and are particularly vulnerable to relapse and toxicity. Understanding correlations between ASP risk and B-ALL may improve outcomes by optimizing treatment strategies and addressing poor prognostic markers like IKZF1 deletions. Intensified asparaginase regimens reduce minimal residual disease, enhance survival, and refine toxicity management, underscoring the drug’s critical role in achieving remission and long-term survival (4, 5, 34, 35). Despite growing consensus on asparaginase TDM, there is a lack of real-world pharmacokinetic and exposure toxicity data from low- and middle-income countries, particularly India. Most evidence is derived from high-income settings and may not be generalizable due to differences in clinical and patient-related factors. Additionally, the feasibility and clinical impact of TDM in resource-limited, high-risk pediatric ALL populations remain underexplored, representing an important knowledge gap. Therefore, in this study, we aim to monitor serum ASP activity in children with high-risk ALL to evaluate its efficacy and establish an optimal dosing regimen. By measuring serum ASP activity between frequent doses, we wish to determine whether enzyme levels remain within the therapeutic range. Additionally, we assessed the relationship between enzyme activity, systemic toxicity, and biochemical parameters, indicative of toxicity and impact on treatment outcomes.

This prospective observational pilot study was conducted at Vardhaman Mahavir Medical College and Safdarjung Hospital, after approval from the Institutional Ethics Committee (Protocol No: IEC/VMMC/SJH/Project/2023-08/CC-362; approved on 11 August 2023), with written informed consent obtained from the parents/guardians. The primary objective was to estimate the population variance of L-asparaginase pharmacokinetic parameters to inform future multicenter trial sample size calculations, while the secondary objectives included assessing feasibility of real-time therapeutic drug monitoring, adherence to sparse sampling, identifying logistical barriers, and generating preliminary exposure outcome data.

Thirty treatment-naive children (1–12 years) with newly diagnosed B-cell ALL were enrolled between March and July 2024. Diagnosis was confirmed by flow cytometry, and eligibility required adequate organ function. Patients with prior chemotherapy, T-cell ALL, asparaginase hypersensitivity, or severe organ dysfunction were excluded. Risk stratification followed the modified ICiCLe protocol (36). Standard-risk patients received conventional therapy, while high-risk patients underwent sparse pharmacokinetic sampling with real-time dose adjustment.

Of the 30 enrolled patients, nine were classified as high risk and selected for therapeutic drug monitoring. During induction, three died due to sepsis and one was transferred, leaving five patients for final pharmacokinetic analysis. These five patients completed induction therapy with native Escherichia coli L-asparaginase, contributing a total of 42 serial samples (≈8 per patient). Participant flow and exclusions are detailed in the STROBE-compliant diagram (Figure 1).

Pharmacological formulations (ASP)

ALL patients in the study were administered conventional L-asparaginase (L-ASP) during their induction period. Two patients out of the initial nine patients taken for the study were administered PEG-ASP. The five patients who completed the induction cycle and analyzed in the study were all given conventional ASP. The patients who were administered PEG-ASP failed to complete induction or had mortality before completion of induction. Each vial of L-Asgen (Adley Formulations, India) contains 10,000 IU of ASP, accompanied by 80 mg of mannitol as a sterile lyophilized plug or powder. These vials are designed for reconstitution and subsequent intravenous or intramuscular injections.

Dosing and sample collection schedule

The chemotherapy regimen followed is mentioned in the Supplementary Materials as per the modified ICiCLe-ALL regimen. According to the ICiCLe-ALL-14 protocol (36), high-risk (HR) patients with B-ALL receive a tailored asparaginase (L-ASP) regimen based on their risk group and treatment phase. During the induction phase, HR patients receive eight doses of L-ASP. In the consolidation phase, they are administered eight doses in two hemi-blocks. In the delayed intensification phase, all risk groups, including HR, uniformly receive four doses of L-ASP. Therefore, ASP dosing as per the protocol was E. coli L-ASP administered intramuscularly every 72 h during the induction phase with the high-risk groups receiving eight doses on days 9, 12, 15, 18, 21, 24, 27, and 30, respectively. Day 1 of induction represents the start of chemotherapy, and day 9 represents the ninth day from the initiation of therapy when the first asparaginase dose is administered. Molecular subtyping, cytogenetic profiling, and flow cytometry for all patients were conducted at baseline. The treatment regimen was determined based on risk stratification according to the NCI risk category and modified ICiCLe-ALL-14, with further details provided in Supplementary Table 3. Potential confounders affecting asparaginase activity and toxicity included nutritional status, infection (e.g., febrile neutropenia, sepsis), baseline disease severity (age, WBC count, cytogenetics), and concurrent medications (e.g., corticosteroids, chemotherapy). Although routinely recorded, these variables were not formally adjusted due to the study’s small sample size and exploratory design.

Note: Peripheral blood samples for asparaginase activity monitoring were collected at multiple time points relative to the first asparaginase dose (administered on day 9 of induction). The sample identifiers denote days since therapy initiation, not days relative to the first ASP dose. Supplementary Table 8 provides a comprehensive timeline correlating induction days, asparaginase administration schedule, and sample collection points to clarify the temporal relationship between treatment and monitoring.

Treatment and ASP monitoring

L-ASP was administered intramuscularly every 3 days, starting from the ninth day of the induction phase, at a dose of 10,000 IU/m 2. The number of doses administered ranged from 4 to 8, depending on the patient’s risk level, following the modified ICICLE protocol as mentioned in Table 1. Native E. coli asparaginase was selected due to its availability and cost-effectiveness in our resource-limited clinical setting. However, alternative formulations such as PEG-asparaginase and Erwinia asparaginase are available and are known to have different pharmacokinetic profiles (37). Native E. coli asparaginase has an intermediate half-life of approximately 1.2 days, which necessitates repeated dosing every few days to maintain therapeutic enzyme activity (37).

We initially screened 30 patients diagnosed with ALL, among whom nine high-risk patients were selected for TDM. Among these nine patients, five successfully completed induction therapy and were included in the final analysis. Treatment modifications were made when required based on toxicity assessment in accordance with institutional treatment protocols.

Throughout the treatment phases, all patients underwent physical examinations, nutritional status assessments, and comprehensive laboratory testing. These tests included a complete hemogram, liver and kidney function assessments, pancreatic function tests, lipid profile, and coagulation profile. Evaluations were conducted both at the beginning and end of the induction phase or after the final ASP dose in cases of complete induction therapy.

The ASP Activity Assay Kit, Catalog Number MAK007 (Merck, Darmstadt, Germany), was employed to monitor enzyme activity in plasma derived from peripheral blood samples collected from patients following ASP treatment. The assay is based on a coupled enzymatic colorimetric reaction in which L-asparaginase catalyzes the hydrolysis of L-asparagine to L-aspartate and ammonia. The generated L-aspartate is subsequently converted through enzymatic reactions producing a measurable colorimetric signal proportional to asparaginase activity. Peripheral blood samples were collected from five patients (P1–P5) at various time points following asparaginase treatment.

Immediately after collection, peripheral blood samples were centrifuged within 30 min at 3,000 rpm for 10 min to separate plasma. The separated plasma was aliquoted and stored at −80°C until analysis. Enzyme activity was not measured immediately after blood collection; instead, samples were stored for batch analysis. The duration of storage prior to analysis ranged from approximately 2 weeks to 3 months. All samples underwent a single freeze–thaw cycle prior to analysis to minimize potential loss of enzyme activity.

Following storage, the plasma samples were subjected to the colorimetric assay using the ASP Activity Assay Kit (MAK007, Merck, Darmstadt, Germany) to determine ASP activity, within a measurable range of 0.28 to 153.9 IU/mL, ensuring accurate and consistent quantification of enzyme levels.

Note: It should be noted that the MAK007 assay kit is designated for research use only (RUO), and therefore, its application in this study was limited to investigational therapeutic drug monitoring rather than routine clinical diagnostics. This represents a methodological limitation of the present study. Additionally, according to the manufacturer’s technical documentation, endogenous metabolites such as aspartate, oxaloacetate, and pyruvate present in biological samples may contribute to the background signal and interfere with accurate measurement of enzyme activity. Although appropriate controls and sample handling procedures were implemented, the possibility of background signal interference cannot be completely excluded and should be considered when interpreting the results. Also, we acknowledge that target activity thresholds remain a subject of international debate, with different protocols using values ranging from 0.05 to 0.5 IU/mL (16, 38). Despite this variability, there is a strong international consensus supporting routine TDM of L-asparaginase to detect silent inactivation, guide switching decisions, and optimize treatment outcomes (16).

Sample size justification

A target sample size of 30 patients was selected based on pilot study guidelines to support both variance estimation and feasibility assessment. For variance estimation, recommendations suggest ~15 participants per arm for a future trial detecting a medium effect size, which aligned with our expectation that ~50% of enrolled patients (n ≈ 15) will be evaluable (39). For feasibility outcomes, guidance recommends 30 to 40 participants to ensure adequate precision, making 30 appropriate and achievable given institutional recruitment rates (40). Additionally, population pharmacokinetic methods using sparse sampling allow reliable parameter estimation with small samples, supporting this size in a rare pediatric population. Consistent with pilot study principles, this study is designed to estimate key parameters and feasibility rather than test efficacy and thus does not require formal power calculations (41, 42).

Bias assessment

Potential sources of bias were considered given the exploratory nature of this pilot study. Selection bias was minimized through consecutive enrollment of patients at a single tertiary care center using predefined eligibility criteria and a standardized treatment protocol, reducing investigator-driven inclusion. However, survivor bias is a key limitation, as only five of nine high-risk patients completed induction and were included in the pharmacokinetic analysis, with attrition due to early mortality (sepsis) or transfer; thus, findings may primarily reflect patients who were clinically stable and may not be generalizable to more critically ill populations. Measurement bias was addressed through standardized sample handling, including processing within 30 min, plasma separation, and storage at −80°C with a single freeze–thaw cycle prior to batch analysis; nonetheless, the use of a research-use-only assay introduces potential limitations such as background signal interference from endogenous metabolites. Additionally, confounding bias cannot be excluded, as factors including infection, nutritional status, disease severity, and concomitant medications were not adjusted for due to the small sample size. Accordingly, the findings should be interpreted in an exploratory and hypothesis-generating manner.

Pharmacokinetics and correlation of serum asparaginase activity with systemic toxicities

For each patient, pre-treatment (baseline) serum ASP activity defined as the apparent enzyme activity measured prior to the first dose was established. Given the use of a colorimetric assay (MAK007, Merck), baseline values likely reflect background signal from endogenous metabolites or assay interference rather than true circulating enzyme activity. Baseline values were used descriptively and were not subtracted; all reported ASP activity values represent absolute measured activity. Simultaneously, laboratory parameters were assessed to explore correlations with systemic toxicity associated with excessive asparaginase enzyme activity levels. Treatment-related toxicities were evaluated using clinical and laboratory parameters monitored during induction therapy and described based on deviations from institutional laboratory reference ranges; although not formally graded, these abnormalities correspond to established Common Terminology Criteria for Adverse Events (CTCAE) defined criteria. Clinically relevant toxicities were interpreted using standard diagnostic thresholds where applicable. The various clinicopathological variables monitored to evaluate treatment-related toxicities and immune-mediated hypersensitivity reactions, based on the pharmacokinetic profile of ASP, included the following: coagulation parameters—international normalized ratio (INR), fibrinogen (FIB), and activated partial thromboplastin time (APTT); lipid profile—total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and serum triglycerides; inflammatory and cardiac markers—procalcitonin (PCT) and N-terminal pro-B-type natriuretic peptide (NT-proBNP); renal function tests—uric acid, blood urea, blood urea nitrogen (BUN), and creatinine; hematological parameters—hemoglobin (Hgb) and serum lactate dehydrogenase (LDH); liver function tests—aspartate aminotransferase (AST/SGOT), alkaline phosphatase (ALP), alanine aminotransferase (ALT/SGPT), bilirubin, serum ammonia, and albumin/total protein (TP); and pancreatic function tests—amylase and lipase. The results were plotted at the time of peak serum ASP enzymatic activity in each patient to assess potential associations between enzyme levels and biochemical toxicity markers.

Statistical analysis

Spearman’s correlation (ρ) was used to assess relationships between variables. This non-parametric method is suitable for small sample sizes (n = 5) and does not assume normality or linearity. The number of paired observations (XY), 95% confidence interval, and two-tailed p -value were calculated, with α = 0.05 as the significance threshold. Analyses were performed using GraphPad Prism version 8.0.2.

A total of 30 patients with newly diagnosed B-cell ALL were enrolled in the study. Of these, nine patients (30%) were classified as high risk according to the modified ICiCLe criteria and were selected for TDM. The median age of the high-risk cohort was 6 years (range: 1–12 years), with a male predominance (77.8%, 7/9). High-risk features included age ≥10 years, white blood cell count ≥50,000/μL, and the presence of high-risk cytogenetic abnormalities. Regarding treatment outcomes within the high-risk group, three patients died during induction therapy due to sepsis, and one patient was transferred to another center prior to completion of treatment. The remaining five patients successfully completed induction therapy and were included in the final analysis. A detailed summary of participant flow, including reasons for attrition, is provided in the STROBE-compliant flow diagram (Figure 1).

Determination of minimum and peak ASP activity from the patient’s blood plasma

After analysis of plasma from five patients, baseline levels for ASP activity in each patient were determined. The activity levels varied among the patients, ranging from 0.43 to 0.48 IU/mL. The maximum ASP activity levels were also seen to vary significantly among the patients, ranging from 0.52 to 1.97 IU/mL. Patient 1 exhibited a peak activity level of 1.05 IU/mL on day 6 after the first ASP treatment. Patient 2 showed a maximum activity level of 0.52 IU/mL on day 1, while patient 3 demonstrated the highest recorded activity level of 1.97 IU/mL on day 21 after ASP treatment. Patient 4 reached a maximum level of 0.65 IU/mL on day 4, and patient 5 showed a peak activity of 0.53 IU/mL on day 13. The minimum trough ASP activity levels recorded ranged from 0.43 to 0.47 IU/mL (median: 0.44 IU/mL). These values are below our institutional target of ≥0.5 IU/mL, indicating subtherapeutic exposure. However, all measured trough levels remained above the commonly cited international minimum threshold of ≥0.1 IU/mL (≥100 IU/L) (16, 43; Figure 2, Table 2), which should be interpreted with caution when assessing adequacy of drug exposure.

Correlations between ASP activity and hepatic dysfunction

Spearman’s rank correlation analysis identified two statistically significant associations between peak ASP activity and laboratory parameters. Hematocrit and aspartate aminotransferase (AST/SGOT) both showed a strong correlation (both p = 0.0167, respectively). All the other parameters, including total cholesterol, albumin/total protein, LDH, alanine aminotransferase (ALT), alkaline phosphatase (ALP), BUN, and creatinine, were not statistically significant (p > 0.05), as detailed in Supplementary Table 1. Although some variables showed moderate to strong correlations (e.g., albumin/TP, LDH, ALT, ALP), these did not reach statistical significance and should be interpreted cautiously given the small sample size.

Note: For this, no blank correction was applied (i.e., subtraction of pre-dose ASP value from post-dose ASP value), as all samples were processed uniformly according to manufacturer instructions, ensuring internal consistency across time points. Serum ASP activity was then sequentially plotted by day of administration to generate individual pharmacokinetic activity time curves.

Excessive enzymatic activity and metabolic alterations

The laboratory parameters suggesting excessive enzymatic activity in all patients were studied at baseline and compared during febrile neutropenia and/or when the patient was admitted for sepsis. The values have been shown patient-wise in Supplementary Table 1. Liver dysfunction was evident by elevated LDH levels in all patients (100%, n = 5), with AST and ALT levels increased in 80% (n = 4) of the cases. BUN levels were elevated in 80% (n = 4) of patients, likely reflecting increased urea production from corticosteroid-induced catabolism, chemotherapy-related protein turnover, and ammonia generation from L-asparaginase, rather than renal dysfunction, as serum creatinine and eGFR remained normal. Tumor lysis syndrome was excluded clinically and biochemically. Metabolic abnormalities included hyperuricemia (40%), dyslipidemia (60% elevated cholesterol/LDL; 100% hypertriglyceridemia), and universal hypoalbuminemia/hypoproteinemia, suggesting treatment-related hepatic effects. Notably, subclinical pancreatitis was observed in 50% of patients, with elevated amylase and lipase in 40% each despite the absence of symptoms. Four patients died of sepsis, with underlying pancreatitis potentially contributing as a confounding factor associated with asparaginase toxicity. Total cholesterol and LDL levels were elevated in 60% (n = 3) of the patients, and triglyceride levels were elevated in all patients (100%, n = 5), suggesting metabolic disturbances possibly linked to chemotherapy side effects as shown in Supplementary Table 2. Albumin/total protein levels were low in all patients (100%, n = 5), suggesting potential subclinical hepatotoxicity by chemotherapy.

The most significant finding observed in our pilot study was the subclinical evidence of pancreatitis noted in half of the patients. Pancreatic enzyme level assessment revealed elevated amylase levels in 40% (n = 2) of the patients, while 20% (n = 1) had low/normal amylase levels. Similarly, elevated serum lipase levels were found in 40% (n = 2) of the patients, and 20% (n = 1) showed low/normal lipase levels. No patients exhibited symptoms of pancreatitis, a known systemic toxicity associated with asparaginase therapy. Out of these five patients, four died due to sepsis; however, underlying pancreatitis may have been a confounding factor, suggesting allergic reactions to therapeutic doses of ASP.

Patient mortality and enzyme activity

All the patients in our study had serum ASP level peaks at variable intervals. However, the mortality data correlation needs individual assessment owing to the pilot study’s small sample size. Given the very small evaluable sample size (n = 5), these findings should be interpreted as exploratory and hypothesis-generating. The study is underpowered for robust statistical inference, and the observed correlations may be influenced by individual variability rather than true biological associations. The first patient initially exhibited moderate ASP activity levels (0.47 IU/mL), within the established therapeutic window range of 0.1 to 0.5 IU/mL. Activity levels peaked significantly on day 6, reaching 1.05 IU/mL, which was sufficiently above the minimum therapeutic threshold before stabilizing. This increase in activity likely indicated an effective therapeutic response, although the patient later developed complications. The second patient demonstrated an initially high ASP activity level (0.46 IU/mL), which increased to 0.52 IU/mL on day 2 but fluctuated thereafter, remaining below 0.5 IU/mL, still within the therapeutic threshold but close to the upper limit. Despite this, the second patient, who was categorized as high risk, passed away a week after completing induction therapy due to sepsis. The third patient’s ASP activity remained consistently high, staying near the therapeutic threshold of 0.5 IU/mL until day 19. By day 21, activity had surged to 1.97 IU/mL, likely as a result of aggressive treatment. This increase in activity contributed to the patient’s adverse outcome, as they succumbed to sepsis during induction therapy. The fourth patient exhibited a gradual increase in ASP activity, peaking on day 4 at 0.65 IU/mL, well above the therapeutic threshold. Despite maintaining elevated levels throughout treatment, this high-risk patient also died due to sepsis. The fifth patient initially showed lower ASP activity levels but experienced a moderate rise (0.52 IU/mL on day 13), eventually requiring intensive care in the Pediatric Intensive Care Unit (PICU) due to severe toxicity and complications. This patient, also classified as high risk, experienced significant toxicity and eventually succumbed to sepsis (Figure 2, Table 2).

Monitoring serum ASP activity is essential in ALL to ensure insufficient and excessive activity that leads to suboptimal outcomes or toxicity, respectively (44). Certain studies, like the study conducted by Burke et al., have highlighted the clinical consequences of hypersensitivity reactions to ASP, emphasizing the need for accurate monitoring. They demonstrated that immune-mediated hypersensitivity can significantly impact treatment efficacy and patient safety. In this context, the pilot study provides the first systematic assessment of serum ASP activity during induction therapy in Indian children with high-risk B-cell ALL, revealing uniformly subtherapeutic trough levels despite standard dosing under the modified ICiCLe protocol. These findings suggest inadequate drug exposure and emphasize the need for treatment optimization in settings lacking routine therapeutic drug monitoring.

The analysis of plasma samples from five patients revealed variability in ASP activity levels, both at baseline and during treatment. According to studies by Pieters, Appel et al., Salzer, Bostrom et al., Lenicek Krleza, and Katusic Bojanac et al., the outcome of patients with serum ASP levels consistently above 0.5 IU/mL at day 3 had an overall good prognosis when compared to patients having serum ASP levels less than 0.5 IU/mL at day 3 of the first dose of ASP (3, 34–36, 44, 45). In our pilot study, baseline levels ranged from 0.43 to 0.48 IU/mL, comparable to published values (≤0.5 IU/mL) (11). Maximum activity levels in our pilot fluctuated significantly, with patients showing peaks ranging from 0.52 to 1.97 IU/mL at different times (between days 1 and 21), similar to those published by Ahuja et al. Despite these variations, three of the five patients (60%) exhibited ASP levels below 0.5 IU/mL by day 3, indicating suboptimal dosage for achieving therapeutic efficacy, and these three patients succumbed to complications related to the disease eventually after completion of induction therapy (Figure 2).

The universal finding of subtherapeutic trough asparaginase activity in our cohort raises concerns regarding inadequate drug exposure during induction therapy. Although the exact cause cannot be determined, potential mechanisms include silent inactivation due to antibody-mediated neutralization without clinical hypersensitivity, inadequate dosing, or interpatient pharmacokinetic variability (16, 18). Silent inactivation is particularly problematic because it leads to insufficient systemic asparagine depletion, which may compromise treatment efficacy and increase relapse risk (15, 19). Since this phenomenon cannot be detected through clinical symptoms alone, patients may appear clinically stable despite receiving ineffective therapy. These findings emphasize the importance of TDM of serum asparaginase activity to identify silent inactivation, guide timely switching to alternative preparations, and maintain therapeutic enzyme levels during treatment (16, 20).

These findings should be interpreted in the context of the ongoing international debate regarding optimal asparaginase activity thresholds. Although our institutional protocol used a trough target of ≥0.5 IU/mL, reported thresholds across protocols range from 0.05 to 0.5 IU/mL (16, 38), with ≥0.1 IU/mL most commonly cited in contemporary literature and adopted in several prospective therapeutic drug monitoring studies (16, 43). This variation reflects differences in assay methodology, sampling timing, asparaginase formulation, and local validation. Nevertheless, expert consensus supports routine therapeutic drug monitoring to detect silent inactivation and ensure adequate drug exposure (16). In our cohort, all patients had trough levels <0.5 IU/mL, indicating subtherapeutic exposure and highlighting the need for systematic monitoring (16, 43).

An important consideration for TDM is that different asparaginase formulations possess distinct pharmacokinetic properties, necessitating formulation-specific monitoring strategies. Native E. coli asparaginase, which was used in this study, has an intermediate half-life of approximately 1.2 days and therefore requires dosing every few days to maintain therapeutic activity (37). In contrast, PEG-asparaginase has a substantially longer half-life of approximately 5–7 days, allowing less frequent administration while maintaining sustained enzymatic activity (37, 46). Conversely, Erwinia chrysanthemi -derived asparaginase exhibits the shortest half-life (approximately 0.6–0.8 days), requiring more frequent dosing schedules to achieve adequate asparagine depletion (37, 46, 47). These pharmacokinetic differences directly influence therapeutic drug monitoring strategies, including the optimal timing of sample collection, frequency of monitoring, and interpretation of therapeutic activity thresholds (16, 46). Additionally, the development of anti-asparaginase antibodies may accelerate enzymatic clearance, thereby reducing effective drug exposure and necessitating closer pharmacokinetic monitoring (38, 46). In resource-limited healthcare settings such as India, native E. coli preparations remain more widely available and economically accessible compared with PEGylated or Erwinia -derived formulations (23). Therefore, our findings further emphasize the importance of implementing therapeutic drug monitoring strategies tailored to the pharmacokinetic characteristics of the locally available formulation to ensure adequate therapeutic exposure.

Subtherapeutic asparaginase activity in our cohort raises the possibility of biological resistance and microenvironmental influences. Upregulation of ASNS has been shown in vitro to confer resistance to asparaginase, while its inhibition restores drug sensitivity (6, 7). However, clinical studies indicate that ASNS expression varies across leukemia subtypes and does not consistently predict treatment response in pediatric ALL (8, 9). The bone marrow microenvironment may also contribute, as mesenchymal stromal cells express higher ASNS levels and can secrete asparagine, partially protecting leukemic cells from asparaginase-induced cytotoxicity (30). Although hepatocytes have been proposed as another potential source of asparagine, clinical evidence for their protective role remains limited (6). In our study, the consistently low trough asparaginase levels suggest that pharmacokinetic factors such as inadequate dosing, rapid clearance, or antibody-mediated inactivation are more likely responsible for subtherapeutic exposure, though cellular resistance cannot be excluded without molecular analysis (16, 37).

Furthermore, similar to our study, Egler et al. also observed substantial variability in individual responses to treatment (4). Their study reported that baseline ASP activity typically ranges between 0.05 and 0.5 IU/mL, providing valuable insights into patient-specific pharmacokinetics, which were largely in accordance with our study values of baseline activity. This is particularly important because Indian patients, who have diverse geographic and socioeconomic backgrounds, as well as various nutritional factors like protein intake, micronutrient deficiencies (like vitamins and minerals), dietary fat balance, nutrient-rich vs. calorie-dense food consumption, gut health, hydration and electrolyte balance, B-vitamin levels, socioeconomic access to nutrition, and regional dietary practices, all of which can influence drug metabolism, immune function, and overall treatment outcomes, might significantly affect ASP treatment in chemotherapy for ALL.

Studies for therapeutic drug monitoring for serum ASP levels in patients undergoing chemotherapy for leukemia from India are limited due to very few groups working on therapeutic drug monitoring in leukemia in Indian patients. However, the study conducted by Sidhu et al. highlights significant variations in the baseline enzymatic activity of E. coli ASP monitoring and serum therapeutic levels compared to the standard reference curve (48). This study suggests that the results could have suboptimal clinical efficacy, potentially compromising treatment outcomes for patients with ALL in India, and it is very important to highlight here that three of our five patients could have suboptimal activity based on our pilot results. Also, in another study conducted by Gupta et al., depicting the critical role of L-ASP in ALL treatment, high-risk patients who did not receive all the prescribed doses of ASP had worse disease-free survival (DFS) compared to those who received all the prescribed doses of PEG-ASP (45). Discontinuation of ASP doses is associated with inferior DFS in higher-risk patients; similar to our study, three of all high-risk patients succumbed to various causes at the end of induction.

The observed correlation between AST and asparaginase activity can likely reflect complex factors such as timing of drug exposure, pharmacokinetic variability, immune-mediated inactivation, and patient susceptibility rather than a direct causal relationship. Asparaginase is known to cause hepatotoxicity through lipid redistribution, steatosis, and impaired protein synthesis, leading to elevated AST, but enzyme activity levels may not align temporally with liver injury. Low measured activity can still follow earlier high exposure or antibody inactivation, while host factors (e.g., obesity, age) further influence toxicity. Clinically, this highlights the need to interpret enzyme activity and liver function tests together with dosing timing, supported by therapeutic drug monitoring and serial lab results.

Hence, based on the patient data and findings, a recommended treatment strategy for L-ASP therapy in high-risk ALL patients involves careful dosing, TDM, and individualized adjustments to optimize outcomes while minimizing toxicity. Treatment should begin with intramuscular administration of native L-ASP every 3 days during induction, at a dose of 10,000 IU/m², as outlined in the modified ICiCLe protocol. TDM is critical, with serum asparaginase activity monitored at baseline and multiple time points during treatment, such as on days 3, 7, 13, and 21, to maintain enzyme activity in the therapeutic range between 0.1 and 0.5 IU/mL (11, 31). For patients with suboptimal activity levels below 0.1 IU/mL, the activity measurements should be repeated. Conversely, patients with activity levels exceeding the therapeutic threshold require metabolic and toxicity screening, focusing on liver enzymes (AST, ALT, LDH), renal function (BUN), and lipid profiles to detect and manage side effects.

The pilot study underscores the importance of TDM in leukemia patients, particularly given multifactorial variability in treatment response and the observation that standard protocols may not yield comparable outcomes across populations, including Indian cohorts. Our findings demonstrate notable interpatient variability in post-treatment ASP activity among five patients, highlighting the individualized nature of response. L-asparaginase is an exogenously administered enzyme; thus, measurable pre-treatment activity is minimal and primarily reflects assay background rather than physiological variability (4). Differences in ASP activity are therefore attributable to pharmacokinetic and pharmacodynamic variability following administration. Patient-related factors such as genetic variability, age, nutritional status, comorbidities, and immune response may influence drug metabolism, clearance, and therapeutic effect. Additionally, treatment-related factors including ASP formulation, dosing schedule, route of administration, and concomitant medications contribute to variability. Consistent with the literature, substantial interindividual variability supports TDM to ensure adequate activity (≥0.1 IU/mL), detect suboptimal exposure, and guide individualized dose optimization (10).

This pilot study has several inherent limitations that warrant careful consideration when interpreting the findings. The most significant limitation is the small sample size, which restricted statistical power and limited the ability to establish robust correlations between ASP activity and biochemical toxicity parameters. The lack of structured, continuous monitoring further constrained the ability to capture dynamic changes in enzyme activity and associated toxicity markers over the treatment period. These factors likely contributed to the absence of strong correlations with parameters such as creatinine, AST, ALT, bilirubin, and LDH, which may otherwise have shown significant associations in a larger, more rigorously monitored cohort, as reported in Western studies. The potential influence of unmeasured confounders, including nutritional status, infection/sepsis, disease severity, and concurrent medications (e.g., corticosteroids), may also affect both asparaginase pharmacokinetics and biochemical parameters. These factors can independently alter laboratory markers, complicating attribution of toxicity solely to asparaginase. Due to the small sample size and pilot design, adjustment was not feasible, and the findings should be interpreted cautiously. Importantly, survivor bias must be acknowledged, as pharmacokinetic analyses were limited to patients who completed induction therapy, while those with early mortality or transfer were excluded; this may skew the findings toward clinically stable patients and limit the generalizability of the results, particularly in more critically ill high-risk populations. Also, as a single-center pilot study with only five patients, the generalizability of our findings is limited. Results are specific to high-risk pediatric B-cell ALL patients treated at a tertiary center and may not apply to other settings, particularly resource-limited environments or patients with early treatment failure. Institutional protocols may also influence observed outcomes. Additionally, the single geographic cohort may not reflect broader genetic and socioeconomic diversity, and evolving treatment practices may further limit applicability. Despite these constraints, the study offers important preliminary insights into potential relationships between ASP activity and hepatic function markers. The observed strong correlations with AST are consistent with the findings reported in the existing literature, where elevations in transaminases are commonly described as markers of hepatic injury during therapy (49–53). However, these findings should still be interpreted with caution given the very small sample size and require validation in larger cohorts with paired, time-resolved measurements. These findings underscore the clinical importance of comprehensive monitoring during ASP therapy, including hematocrit, liver function tests, and coagulation parameters, to detect early signs of toxicity and guide individualized treatment decisions. Moreover, this study demonstrates the feasibility of pharmacokinetic toxicity correlation studies in an Indian pediatric population with B-cell ALL, paving the way for larger, multicentric investigations aimed at optimizing asparaginase-based therapy and improving treatment safety in resource-limited settings.

The study further highlighted considerable differences in peak ASP activity levels, which can be influenced by a variety of factors like the formulations used, the route of administration affecting absorption rates, dosage frequency, patient-specific variability such as immune response, and the drug’s pharmacokinetics. Optimizing these variables is essential for effective asparagine depletion in ALL treatments and to achieve successful removal of all blasts from circulation (10).

In our pilot study of five patients, each patient achieves peak activity at different time points, reflecting the individualized kinetics of ASP metabolism. The observed variability in both baseline and peak ASP activity levels underscores the complexity of ASP metabolism and aligns with several key studies. For example, Müller et al. analyzed the pharmacokinetics of native E. coli ASP in children with ALL during reinduction therapy, showing significant variability in peak and trough ASP levels, with peaks occurring at different time points, similar to the range of peak activity times in our study (days 1 to 21) (54). This reinforces the need for TDM to ensure optimal ASP activity. The study conducted by Avramis et al. also demonstrated that peak ASP levels vary depending on the formulation used and individual patient response, with peak activity observed as early as 4 h post-treatment in some patients, but later in others, consistent with the wide range of enzymatic responses seen in our study (55). Additionally, Angiolillo et al. explored the pharmacokinetics and pharmacodynamics of calaspargase pegol; they found that while most patients maintained ASP activity within a therapeutic range, there were significant differences in how long it took for patients to reach peak activity levels (56), and this was concurrent with our pilot study findings. Some patients metabolized ASP rapidly, while others exhibited prolonged enzyme activity. This is reflected in our findings, particularly in patient 3, who demonstrated a sustained enzymatic response with the highest peak activity on day 21, further emphasizing the importance of individualized treatment plans based on real-time monitoring. These prior studies validate the variability in peak ASP activity and metabolism observed. The individualized kinetics of ASP metabolism seen in our patients are supported by these studies, emphasizing the necessity of tailored therapeutic strategies to achieve optimal outcomes in ASP therapy.

Highlighting the clinical parameters, patients exhibited significant abnormalities in their liver and renal function tests, indicating toward possible kidney dysfunction, severe liver damage, and deranged lipid profile. These findings highlighted the need for further systemic monitoring to address potential liver and kidney dysfunctions along with the standard chemotherapeutic regimen...

In conclusion, this pilot study underscores the importance of monitoring serum ASP activity in high-risk patients undergoing treatment for acute lymphoblastic leukemia. Marked variability in both baseline and peak enzyme activity was observed, highlighting the individualized nature of ASP pharmacokinetics and treatment response. TDM plays a key role in optimizing treatment efficacy by enabling the identification of suboptimal enzyme activity and early detection of hypersensitivity or treatment-related toxicities associated with asparaginase therapy. This is particularly relevant in the Indian context, where limited data are available. Our findings support the need for integrated monitoring of hematological and hepatic parameters during ASP therapy, along with careful consideration of sampling conditions and timing relative to drug administration. Importantly, this study demonstrates the feasibility of conducting pharmacokinetic and toxicity-related investigations in an Indian pediatric B-cell ALL population. However, The study’s small sample size, single-center design, and attrition mean the findings are exploratory. It primarily demonstrates feasibility and identifies recruitment and retention challenges. These findings should be interpreted with consideration of confounders (e.g., infection, nutrition, concomitant therapies), and larger adjusted studies are needed to establish causality. These preliminary observations provide a foundation for larger, multicentric studies with standardized protocols, which are essential to refine asparaginase-based treatment strategies and improve safety outcomes in resource-limited settings.