Absolute lymphocyte count (ALC) has been correlated with overall survival and event free survival in acute lymphoblasti leukemia (ALL). In T-cell Acute lymphoblastic leukemia (T-ALL), studies have reported the data in combination with B-ALL [1-7]; separate analysis of T-ALL is lacking. Minimal residual disease (MRD) has emerged as the most important predictor of outcome [8]. Current protocols use MRD for risk stratification and treatment [9]. The drawback of MRD is that it is not available at all centers. ALC is the most readily available prognostic factor. Hence, we studied if ALC at baseline, day 8, day 15 and at the end of induction (EOI) correlates with MRD, steroid response (SR), complete response (CR), and if it can be used as a surrogate marker for MRD in T-ALL.

We conducted a prospective study from July 2015 to June 2017. The study was approved by the institutional ethics committee of our institute, and informed consent was obtained from parents. Assent was obtained from those patients who were above 7 years of age. Newly diagnosed T-ALL cases with age ³1 to 18 years were included. Patients partially treated outside the institution and those with relapsed ALL, were excluded.

Patients were treated on Indian Childhood Collaborative Leukemia Group protocol (Unpublished- CTRI/2015/12/006434). All patients were treated with steroid prophase for seven days. Good SR was defined as day 8 blasts in peripheral smear less than 1×10⁹ /l. All patients irrespective of SR and baseline features received four doses of vincristine and eight doses of L-asparaginase. MRD and bone marrow morphology was assessed on day 35 at the EOI.

MRD assessment was done by 10-color flow cytometry using stain-lyse-wash method [10]. Briefly, bone marrow aspirate, was incubated for 15-20 minutes in the dark at room temperature with antibody panel: anti-CD20-FITC, CD123-PE, CD34-ECD, CD10-PC5, CD 19-PC7, CD58-APC, CD38-AF700, CD33-APC-AF 750, (Beckmann Coulter/Immunotech, Miami, FL, USA), CD13-BV421, CD45-BV510 (Biolegend, San Diego, USA). Samples were lysed using ammonium chloridelysis buffer, then washed twice in phosphate-buffered saline, re-suspended in 1 ml of phosphate-buffered saline and analyzed by flow cytometry. All samples were processed within 24 hours after collection. For flow cytometry analysis, at least 10,00,000 cells were acquired in all cases, and data were stored in list mode files. Data analysis was done using Kaluza software (version 1.3). The cutoff levels used for MRD positivity were ³0.01%. MRD was said to be not evaluable when two consecutive bone marrow samples were not analyzable for MRD.

The association between continuous and categorical data was examined by Mann-Whitney U-test. For analyzing cut-off of ALC with MRD Fisher’s Exact test was used. Statistical analyses were carried out using Stata ver. 14 software. The criterion for significance in all analyses was P <0.05.

A total of 52 patients presented during the study period. Nine patients were excluded (5 deaths during induction and 4 opted for no treatment); and remaining 43 were analyzed. Baseline characteristics and induction outcome are shown in Table I. Out of 43 patients, 39 (90.7%) achieved CR. MRD data were available for 28 patients. Correlation of ALC with a day 8 SR, CR and MRD is shown in Table II. At the EOI ALC was significantly higher in patients who were MRD negative, so cutoffs were analyzed. Patients having ALC ³0.7×109/l (n=16, MRD negative 15) were significantly more likely to be MRD negative than patients with ALC<0.7×109/l (n=8, MRD positive 4), P= 0.028.

TABLE I	Baseline Characteristics and Induction Outcomes in Children With T-All (N=43)

TABLE II Correlation of Absolute Lymphocyte Count With Various Outcome Measures  in Children With T-All (N=43) 

We found that ALC at any point during induction does not correlate with SR and CR. We propose following explanations for our negative results. Mutations in interleukin 7 receptor signalling pathways form the basis of steroid resistance in T ALL [11]. So, lymphocytes may have a limited role in the attainment of SR. In the achievement of CR, instead of ALC, specific lymphocyte subsets could be responsible in eradicating leukemic cells. Since lymphocyte subsets were not analyzed and correlated with CR, it might explain the lack of relationship between CR and ALC. Another possibility is that the number of patients, who failed to achieve CR, was too small to yield statistically significant results.

MRD at the EOI is a favorable factor in T-ALL [12]. We interpret that ALC ³0.7×10⁹/l is a favorable factor and ALC may act as a surrogate marker of MRD at the EOI. Rolf, et al. [13] have shown that ALC at the EOI in B-ALL has a very high correlation with CD3+T cells and dendritic cells, which are known to mediate potent antileukemic activity. Probably, lymphocytes have a role in eradicating residual leukemic cells in T-ALL at the EOI, as higher ALC correlated with MRD negative status.