Circulating metabolic signatures as a superior alternative to Absolute Blast Count (ABC) to assess the prognosis of pediatric Acute Lymphoblastic Leukaemia (ALL)

Aims:

a) To identify and validate the metabolomic markers that serve as prognostic markers of pediatric ALL. b) To identify and validate the metabolomic signatures that serve as predictive markers of steroid resistance in pediatric ALL. Objectives: a) To identify circulating baseline metabolomic signatures in pediatric ALL. b) Identify metabolomic biomarkers as an objective and superior alternative to ABC for predicting the prognosis of pediatric ALL. c) Contrast the differences in the circulating metabolomic baseline signatures in steroid sensitive and steroid resistant pediatric ALL.

Justification: Acute Lymphoblastic Leukaemia (ALL) is the commonest leukaemia in paediatric population. Glucocorticoids (GCs) hold a central role in the multi-drug regimen used to treat ALL and high dose GCs are administered from Day 1 to Day 7 (induction phase) of the treatment regimen (Inaba & Pui, 2021). Response to GCs is monitored by an absolute blast count (ABC) on Day 8 of initiation of therapy, with an ABC of >1000/L reflective of a poor prognosis and possible GC resistance. Unfortunately, ABC is a subjective assessment and inter-assessor variability is high (Hodes, et al., 2019), necessitating the need for developing an objective marker for predicting the prognosis. A metabolomic profile takes into consideration both the genomic and epigenomic aspects of an individual. Hence, the metabolomic signatures reflect changes resulting from expression of genes to epigenetic factors, providing an excellent indicator of the disease spectrum in an individual. Moreover, being an objective marker, it can be used reliably to predict treatment response and prognosis than ABC. Steroid resistance is one of the most important factors predicting a poor treatment outcome. Since steroid receptors are omnipresent, steroid resistance would result in a perturbation of circulating metabolites, quantification of which should help in developing a marker more objective than ABC.

INCLUSION CRITERIA (Test group): a) Children (either gender) below the age of 12 years diagnosed with acute lymphoblastic leukemia for the first time. b) No treatment initiated. EXCLUSION CRITERIA (Test group): a) Children (either gender) above the age of 12 years b) Already diagnosed and treatment initiated children with ALL from any hospital/clinic. INCLUSION CRITERIA (Control group): a) Children (either gender) below the age of 12 years with normal developmental milestones and no active infections/diagnosis of malignancies. EXCLUSION CRITERIA (Control group): a) Children above the age of 12 years. b) Any child with an active infection.

Samplesize: To the best of our knowledge, no studies have been done in identification of metabolomic markers for predicting the prognosis and paeditaric ALL or as a marker of steroid resistance. As such, we would like to do a pilot study recruiting a total of 30 patients with ALL and 30 age matched controls

Procedure:

The samples will be collected from newly diagnosed cases of pediatric ALL on Day 0 (baseline) and Day 7 (last day of treatment with steroids). The sequence of events from the first day is as follows: Day 0: Patient counselling followed by collection of informed consent. This will be followd by the collection of blood sample. From vacutainer that is sent to Biochemistry department, left over serum would be collected (after the billed tests are processed) in 1.5 mL Eppendorf tubes. After labelling, these tubes will be stored at -80 degree Celcius. Blood samples sent to the Biochemistry laboratory from age-matched children (sent from Department of Pediatrics) would be taken as controls. The investigator would liason with a faculty member from Department of Pediatrics to ensure that the samples sent are appropriate to be taken for the study. A faculty from Department of Biochemistry would then anonymize the samples before handing them over to the PI. ; Day 7 (last day of treatment with steroids): From vacutainer that is sent to Biochemistry department, left over serum would be collected (after the billed tests are processed) in 1.5 mL Eppendorf tubes. After labelling, these tubes will be stored at -80 degree Celcius. Once all the samples (cases and controls) are collected, the samples will be analysed using a Gas Chromatograph – Mass Spectrometer housed in the Department of Biochemistry, Kasturba Medical College, Manipal. The following parameters will be quantified: Organic acids; Amino Acids; Fatty Acids. Once the samples are processed, the data from cases will be compared with the data from controls. Data analysis will be done using the apt statistical tool. Then ,the data will be compared to Absolute Blast Count (ABC) and a potential metabolomic marker would be identified that can be used as an objective and superior marker.

**Outcome measures:**i) Establishment of reliable and superior circulating metabolomic markers consistent with the prognostic outcomes of pediatric ALL compared to ABC. ii)  Establishment of metabolomic markers predictive of glucocorticoid resistance.