Clinical, laboratory, and risk stratification characteristics of patients enrolled: Our final analysis included 533 children with B-ALL with a male-to-female ratio of 1.99:1 and a median age of 5 years. According to the NCI risk stratification, most patients were classified as standard risk (SR, 67.2%). However, with the final ICiCLe risk stratification, the majority (57.4%) were categorized as high risk (HiR), followed by SR (22.9%) and intermediate risk (IR, 19.7%). Nearly one-third of patients (32.2%, out of 519 assessed) were detected to harbor measurable residual disease (defined as MRD ≥ 0.01%) at the PI timepoint. Similarly, 9.8% (out of 244 assessed) were positive at the PC timepoint. A summary of clinical, laboratory, and genetic characteristics, as well as MRD responses, can be seen in Table 1.
Table 1
Demographic and Laboratory Characteristics, WHO 2016 Classification, MRD characteristics and Risk Stratification of B-ALL Patients
Demographics | |
Total number of patients accrued | 533 |
Age (years) | Range:- 1–15, Median:- 5 |
Gender | Male: Female- 1.99:1 |
Laboratory characteristics | |
TLC at presentation | |
1. ≥50 x 109/L | 81 (15.2%) |
2. <50 x 109/L | 452 (84.8%) |
WHO 2016 Genetic categories | |
1. Hyperdiploidy | 182 (34.1%) |
2. ETV6::RUNX1 | 68 (12.8%) |
3. TCF3::PBX1 | 42 (7.9%) |
4. BCR::ABL1 | 29 (5.4%) |
5. BCR::ABL1 like | 24 (4.5%) |
6. KMT2A rearranged | 19 (3.6%) |
7. Hypodiploidy | 6 (1.1%) |
8. iAMP21 | 3 (0.6%) |
9. NOS | 160 (30.0%) |
NCI Risk | |
SR | 358 (67.2%) |
HR | 175 (32.8%) |
ICiCLe Initial Risk (n-533) | |
SR | 205 (38.4%) |
IR | 148 (27.8%) |
HR | 180 (33.8%) |
ICiCLe Risk (n-519) | |
SR | 119 (22.9%) |
IR | 102 (19.7%) |
HR | 298 (57.4%) |
Post-induction MRD (n = 519) | |
Positive | 167 (32.2%), Median- 0.14, Range- 0.01-88 |
Negative | 352 (67.8%) |
Post-consolidation MRD (n = 244) | |
Positive | 24 (9.8%), Median- 0.2, Range- 0.01-86 |
Negative | 220 (90.2%) |
Characterization of Genetic Subgroups and their Correlations: Through the integration of conventional PCR, targeted RNA sequencing, cytogenetics, and immunophenotyping, we identified 13 distinct genetic subgroups within our cohort (Table 2). PAX5 alt (24, 4.5%), BCR::ABL1-like (23, 4.3%), DUX4-r (12, 2.3%), MEF2D-r (13, 2.4%), ZNF384-r (3, 0.6%) and TCF3::HLF (4, 0.7%) subgroups of B-ALL were exclusively identified using RNA-sequencing (Fig. 1). Among the BCR::ABL1-like category, almost equal division was seen among the BCR::ABL1-like: ABL-class (n-11) and JAK-STAT activated (n-13) classes. Among the BCR::ABL1-like: ABL-class, the 3’ partner were PDGFRB (EBF1; n = 5), ABL1 (n = 4; one each of ETV6, NUP214, RANBP2, FOXP1), ABL2 (RCSD1) and CSF1R (LBD1) and those in JAK-STAT activated class were CRLF2 (n = 9; P2RY8; n = 8 and one IGH), JAK2 (n = 4; comprising PAX5; n = 3 and one EBF1). For PAX5alt category, the 3’ fusion partners were AUTS2 (n = 5), succeeded by MBNL1 (n = 3), ETV6 (n = 2), IGH (n = 2), ZNF521 (n = 2) and one each with CBFA2T3, FOXP1, GSE1, ANKRD12, CA10, and ESSRB. For MEF2D-r, the fusion partners were BCL9 (n = 8), HNRNPUL (n = 3), DASAP1 (n = 1) and SF1 (n = 1). For ZNF384-r cases, the fusion partners were TCF3 (n = 2) and EP300 (n = 1).
Table 2
Age and Sex Distribution, WBC Counts, and MRD characteristics across Genetic Subgroups. *p-values are not provided for genetic subgroups with fewer than 10 cases.
Genetic category | N (%) | Median age in years (range), p-value* | Male n (%), p-value* | Median WBC (range) at diagnosis (X 109/L), p-value* | Percentage of PI MRD positive cases (median, range), p-value* |
All categories | 533 | 5 (1–15) | 355 (66.5) | 6.37 (0.23–692.3) | 32.2 (0.14, 0.01- 88) |
High hyperdiploidy | 182 (34.1) | 4 (1–13), < 0.001 | 125 (67.2), 1.000 | 4.63 (0.61 − 253.0), < 0.001 | 30.3 (0.01, 0.01- 11), 0.38 |
ETV6::RUNX1 | 68 (12.8) | 4 (1–15), 0.003 | 44 (66.7), 1.000 | 4.85 (0.23 − 90.1), 0.064 | 24.2 (0.02, 0.01–5.8), 0.10 |
TCF3::PBX1 | 42 (7.9) | 6(2–14), 0.154 | 24 (57.1), 0.177 | 14.185 (1.22 − 281.0), 0.003 | 26.2 (0.03, 0.01–3.60), 0.55 |
BCR::ABL1 | 29 (5.4) | 10 (2–14), < 0.001 | 22 (75.9), 0.318 | 61.71 (1.11 − 559.4), <0.01 | 48.3 (0.48, 0.01–23.50), 0.01 |
BCR::ABL1 like | 24 (4.5) | 11.5 (3–15), 0.001 | 17 (70.8), 0.825 | 33.525 (0.93 − 295.1), 0.005 | 70.8 (1.6, 0.01-62), 0.000033 |
PAX alt | 24 (4.5) | 4 (1–14), 0.354 | 16 (69.6), 0.825 | 8.15 (0.59 − 208.5), 0.637 | 23.8 (0.04, 0.01–1.1), 0.46 |
KMT2A-r | 19 (3.6) | 3 (0–14), 0.001 | 12 (63.2), 0.806 | 19.54 (1.37 − 424.71), 0.055 | 26.3 (0.26, 0.01-50), 0.76 |
MEF2D-r | 13 (2.4) | 9 (3–14), 0.004 | 5 (38.5), 0.038 | 11.15 (1.31 − 119.11), 0.252 | 30.8 (0.05, 0.01–0.24), 1.00 |
DUX4-r | 12 (2.3) | 6 (3–14), 0.167 | 9 (75.0), 0.759 | 5.14 (1.56 − 27.73), 0.358 | 33.3 (0.03, 0.01–3.5), 1.00 |
Hypodiploidy | 6 (1.1) | 11 (4–13) | 4 (66.7) | 3.27 (1.34 − 155.1) | 33.3 (0.01, 0.01–4.3) |
PAX5 P80R | 4 (0.7) | 12 (8–14) | 4 (100) | 55.87 (1.99–86.8) | 0 |
TCF3::HLF | 4 (0.7) | 8 (4–14) | 3 (75) | 6.335 (1.32 − 9.74) | 75.0 (1.25, 0.01–6.5) |
ZNF384-r | 3 (0.6) | 5 (4–16) | 1 (33.3) | 14.33 (4.37 − 22.32) | 66.7 (6.20, 0.32–12.09) |
iAMP21 | 3 (0.6) | 6 (2–10) | 2 (66.7) | 3.04 (2.94–5.4) | 0, 0.56 |
NOS | 100 (18.8) | 7 (1–14), < 0.001 | 70 (70.0), 0.481 | 8.07 (0.29–692.3), 0.147 | 33.0 (0.05, 0.01–71.7), 1.00 |
Targeted DNA sequencing further helped identify additional PAX5alt (n = 4) and the PAX5 P80R group (n = 4). Using this approach, we could identify a genomic abnormality in 81.2% of all cases when compared to legacy approaches (61.7%)
Patients harboring BCR::ABL1 (median age- 10 years; p < 0.001), BCR::ABL1-like (median age- 11.5 years, p = 0.001), and MEF2D-r (median age- 9 years; p = 0.004) were significantly older compared to the entire cohort (median age- 5 years; range- 1 to 15 years). BCR::ABL1 (median WBC- 61.7 x 109/L; p = 0.003) and BCR::ABL1 like (median WBC- 33.5 x 109/L; p = 0.005) showed higher WBC counts as compared to the entire cohort (median WBC- 6.4 x 109/L; range- 0.2 to 692.3 x 109/L). Additionally, the BCR::ABL1 (48.3% MRD-positive; median MRD: 0.48; p = 0.02) and BCR::ABL1-like (70.8% MRD-positive; median MRD: 1.6; p < 0.0001) groups exhibited higher PI MRD positivity as compared to the rest (32.3% PI MRD-positive; median PI MRD: 0.14%). The MRD characteristics of each sub-group are detailed in supplementary data and highlighted in Supplementary Fig. 3.
IKZF1 deletion was identified in 69 (15.1%) of the 458 patients tested and showed a high prevalence in the BCR::ABL1 (56.5%) and BCR::ABL1-like (55.6%) genetic subgroups. Additionally, IKZF1 deletions were detected across various other subgroups, including NOS, high hyperdiploidy, PAX5alt, DUX4-r, KMT2A-r, hypodiploidy, ETV6::RUNX1, and iAMP21 (Supplementary Fig. 4).
Re-stratification from the WHO 2016 to WHO 2022 classification: There was no change in the classification of 373 patients (ETV6::RUNX1, High hyperdiploidy, BCR::ABL1, TCF3::PBX1, BCR::ABL1 like KMT2A-r, hypodiploidy, iAMP21) when they were classified as per the WHO 2022 compared to the WHO 2016. Among WHO 2016-defined NOS subgroup (n = 160, 30% of the entire cohort), 60 (37.5%) children were reclassified into newly recognized subtypes such as PAX5alt (n = 24, 15%), DUX4-r (n = 12, 7.5%), MEF2D-r (n = 13, 8.1%), PAX5 P80R (n = 4, 2.5%), TCF3::HLF (n = 4, 2.5%), and ZNF384-r (n = 3, 1.9%) as seen in Fig. 1A.
Patient Outcomes
The 2-year EFS and OS of the entire cohort were 75.8% and 85.1%, respectively (median EFS and OS not reached). The median follow-up of the cohort was 26.8 months. The presence of PI MRD predicted a significantly inferior EFS [HR- 2.0; 95% CI- 1.42 to 2.89; (p < 0.0001)] and OS [HR- 1.7; 95% CI- 1.08 to 2.64; (p = 0.01)]. Similarly, PC MRD positivity was associated with markedly worse EFS [HR- 22.0; 95% CI- 4.37 to 110.93; (p < 0.0001)] and OS [HR- 2.4; 95% CI- 0.78 to 7.70; (p = 0.02)] (Supplementary Fig. 5). The impact of ICiCLe risk stratification on patient outcomes is detailed in the supplementary data (Supplementary Fig. 6).
Outcomes for individual genetic subgroups: On evaluating the impact of genetic subgroups, the following were associated with inferior EFS: PAX5alt (HR- 2.8; 95% CI- 1.18 to 6.68; p = 0.0001), BCR::ABL1 (HR- 2.5; 95% CI- 1.17 to 5.51; p = 0.0002), MEF2D-r (HR- 2.3; 95% CI- 0.75 to 7.20; p = 0.024), and BCR::ABL1-like with ABL-class fusion (HR-2.3; 95% CI- 0.68 to 7.55; p = 0.043) (Table 3). Subgroups associated with inferior OS were PAX5alt (HR-4.3; 95% CI- 1.46 to 12.96; p < 0.0001) and BCR::ABL1 (HR-2.4; 95% CI- 0.98 to 6.11; p = 0.004). Similarly, IKZF1 deletion was associated with inferior EFS (HR-3.0; 95% CI- 1.72 to 5.07; p < 0.0001) and OS (HR-1.8; 95% CI- 0.95 to 3.51; p = 0.022). TCF3::HLF subgroup also exhibited poor EFS, with three out of four affected children having refractory disease and one experiencing an early relapse; however, statistical analysis was not performed due to the small sample size.
Table 3
Event-free and overall survival rates for genetic categories of B-ALL, as classified by WHO 2022, including hazard ratios and p-values. The genetic categories with fewer than 10 cases are not included in the table.
| Overall survival | Event free survival |
| 2 years OS | P-value | Hazard ratio (95% Confidence Interval) | 2 years EFS | P-value | Hazard ratio (95% Confidence Interval) |
IKZF1 del | 77.5 | 0.022 | 1.8 (0.95 to 3.51) | 46.2 | < 0.0001 | 3.0 (1.72 to 5.07) |
PAX5alt | 46.6 | < 0.0001 | 4.3 (1.46 to 12.96) | 39.1 | 0.0001 | 2.8 (1.18 to 6.68) |
BCR::ABL1 | 67.6 | 0.004 | 2.4 (0.98 to 6.11) | 50.6 | 0.0002 | 2.5 (1.17 to 5.51) |
MEF2D-r | 66.1 | 0.114 | 2.2 (0.51 to 9.33) | 53.8 | 0.024 | 2.3 (0.75 to 7.20) |
BCR::ABL1 like, ABL-Class | 90.9 | 0.821 | 0.9 (0.23 to 3.11) | 60.6 | 0.043 | 2.3 (0.68 to 7.55) |
NOS | 84.2 | 0.714 | 1.1 (0.67 to 1.79) | 65.9 | 0.015 | 1.6 (1.03 to 2.33) |
TCF3::PBX1 | 77.3 | 0.133 | 1.6 (0.77 to 3.26) | 67.4 | 0.253 | 1.3 (0.76 to 2.36) |
Hypodiploidy | 83.3 | 0.961 | 1.1 (0.14 to 7.90) | 66.7 | 0.684 | 1.3 (0.27 to 6.64) |
KMT2A-r | 72.0 | 0.214 | 1.8 (0.5469 to 5.6249) | 67.5 | 0.645 | 1.2 (0.50 to 2.95) |
BCR::ABL1 like, JAK-STAT | 84.6 | 0.730 | 1.2 (0.35 to 4.33) | 63.5 | 0.98 | 1.0 (0.37 to 2.65) |
DUX4-r | 82.5 | 0.791 | 0.8 (0.23 to 2.97) | 73.3 | 0.58 | 0.7 (0.27 to 1.94) |
ETV6::RUNX1 | 100 | 0.027 | 0.4 (0.23 to 0.72) | 98.0 | 0.006 | 0.4 (0.27 to 0.65) |
High Hyperdiploidy | 92.1 | 0.0006 | 0.4 (0.29 to 0.65) | 88.9 | < 0.0001 | 0.4 (0.26 to 0.49) |
Conversely, high hyperdiploidy was associated with superior EFS (HR-0.4; 95% CI- 0.26 to 0.49; p < 0.0001) and OS (HR-0.4; 95% CI- 0.29 to 0.65; p = 0.0006). ETV6::RUNX1 also predicted superior EFS (HR-0.4; 95% CI- 0.27 to 0.65; p = 0.006) and OS (HR- 0.4; 95% CI- 0.23 to 0.72; p = 0.027) as seen in Table 3.
Genetic risk assignment: We devised a 3-tier genetic risk model (SGR, IGR, and HGR) incorporating variables found to be significantly predictive of EFS on univariate analysis in the present study. Furthermore, if the number of available cases was < 10 in our study, available literature for those categories was considered (for, e.g., TCF3::HLF). Genetic categories with a hazard ratio (HR) < 0.75 were classified as SGR, those with an HR between 0.75 and 2 as IGR, and those with an HR > 2 as HGR. The details of this classification can be seen in Fig. 3. The 2-year EFS for SGR, IGR, and HGR was 92.6%, 71.2%, and 50.3%, respectively (p < 0.0001), and the 2-year OS for SGR, IGR, and HGR were 94.3%, 81.9%, and 71.6%, respectively (p < 0.0001; Table 4).
Table 4
Genetic risk-based survival outcomes and the impact of post-induction measurable residual disease positivity.
Impact of Genetic Risk on Patient Outcomes (n-533) |
| Event free survival | Overall survival |
| Hazard ratio (95% confidence interval) | 2 year EFS | p-value | Hazard ratio (95% confidence interval) | 2 year EFS | p-value |
Standard genomic risk (SGR) | 1 | 92.6% | < 0.0001 | 1 | 94.3% | < 0.0001 |
Intermediate genomic risk (IGR) | 2.77 (1.91 to 4.02) | 71.2% | 2.37 (1.48 to 3.79) | 81.9% |
High genomic risk (IGR) | 5.34 (3.61 to 7.89) | 50.3% | 3.81 (2.34 to 6.19) | 71.6% |
Survival analyses in genetic risk categories in ICiCLe initial SR/IR risk categories (n-352) |
| Event free survival | Overall survival |
| Hazard ratio (95% confidence interval) | 2 year EFS | p-value | Hazard ratio (95% confidence interval) | 2 year EFS | p-value |
ICiCLe SR/IR & SGR | 1 | 93.8% | < 0.0001 | 1 | 94.2% | < 0.0001 |
ICiCLe SR/IR & IGR | 2.81 (1.76 to 4.48) | 74.0% | 2.34 (1.34 to 4.08) | 81.4% |
ICiCLe SR/IR & HGR | 5.70 (3.18 to 10.20) | 50.0% | 4.38 (2.21 to 8.68) | 70.6% |
Survival analyses in genetic risk categories in ICiCLe initial HiR risk categories (n-181) |
| Event free survival | Overall survival |
| Hazard ratio (95% confidence interval) | 2 year EFS | p-value | Hazard ratio (95% confidence interval) | 2 year EFS | p-value |
ICiCLe HiR & SGR | 1 | 89.0% | < 0.0001 | 1 | 94.5% | 0.026 |
ICiCLe HiR & IGR | 2.67 (1.39 to 5.10) | 64.4% | 2.35 (0.96 to 5.74) | 83.8% |
ICiCLe HiR & HGR | 4.36 (2.53 to 7.51) | 51.1% | 3.28 (1.55 to 6.93) | 73.1% |
Impact of PI MRD in standard genomic risk (n-248) |
| Event free survival | Overall survival |
| Hazard ratio (95% confidence interval) | 2 year EFS | p-value | Hazard ratio (95% confidence interval) | 2 year EFS | p-value |
SGR with PI MRD Negative | 1 | 93.9% | 0.47 | 1 | 95.9% | 0.96 |
SGR with PI MRD positive | 0.75 (0.36 to 2.80) | 90.6% | 1.02 (0.42 to 2.48) | 91.7% |
Impact of PI MRD in intermediate genomic risk (n-130) |
| Event free survival | Overall survival |
| Hazard ratio (95% confidence interval) | 2 year EFS | p-value | Hazard ratio (95% confidence interval) | 2 year EFS | p-value |
IGR with PI MRD Negative | 1 | 77.4% | 0.017 | 1 | 82.8% | 0.239 |
IGR with PI MRD positive | 2.03 (1.03 to 4.00) | 58.7% | 1.56 (0.69 to 3.50) | 79.9% |
Impact of PI MRD in high genomic risk (n-141) |
| Event free survival | Overall survival |
| Hazard ratio (95% confidence interval) | 2 year EFS | p-value | Hazard ratio (95% confidence interval) | 2 year EFS | p-value |
HGR with PI MRD Negative | 1 | 67.4% | < 0.0001 | 1 | 79.6% | 0.054 |
HGR with PI MRD positive | 2.71 (1.65 to 4.43) | 34.2% | 1.86 (0.97 to 3.55) | 68.3% |
Genetic Risk Identifies Heterogeneous Outcomes within ICiCLe Risk Groups
Since patients in this cohort were treated with risk-adapted protocol, we further evaluated the effect of genetic risk stratification in risk groups treated with similar intensities. The ICiCLe initial SR and IR were combined for this analysis, while the HiR group was analyzed separately. Genetic risk stratification retained its prognostic significance within both ICiCLe initial SR/IR and HiR groups. The 2-year EFS of SGR, IGR, and HGR within ICiCLe initial SR/IR were 93.8%, 74.0%, and 50.0%, respectively (p < 0.0001), and that within ICiCLe initial HiR was 89.2%, 64.4% and 51.1% respectively (p < 0.0001) (Fig. 4). Similarly, the 2-year OS of SGR, IGR, and HGR within ICiCLe initial SR/IR were 94.2%, 81.4%, and 70.6%, respectively (p < 0.0001), and that within ICiCLe initial HiR was 94.5%, 83.8% and 73.1% respectively (p = 0.026) (Table 4).
Genetic Risk Identifies Heterogeneity in Standard Risk B-ALL Irrespective of MRD Status
Patients in the SGR group exhibited superior outcomes regardless of PI MRD status, with 2-year EFS rates of 93.9% for MRD-positive and 90.6% for MRD-negative patients (p = 0.469), and 2-year OS rates of 95.9% and 93.5%, respectively (p = 0.954). In the IGR group, OS was similar between MRD-negative (84.2%) and MRD-positive (79.9%) patients (p = 0.239). In the HGR group, OS was 79.6% for MRD-negative and 68.3% for MRD-positive patients (p = 0.054). However, MRD status remained a significant predictor of EFS in the IGR and HGR groups, with 2-year EFS rates of 58.7% for MRD-positive versus 77.4% for MRD-negative in the IGR group (p = 0.017), and 34.2% for MRD-positive versus 67.4% for MRD-negative in the HGR group (p < 0.0001) (Table 4, Fig. 4).
Multivariate analysis
On multivariate analysis, genetic risk stratification (HR-2.0; 95% CI- 1.62 to 2.45; p < 0.0001), initial ICiCLe risk (HR-1.3; 95% CI- 1.05 to 1.57; p = 0.015), and PI MRD (HR-2.0; 95% CI- 1.42 to 2.74; p < 0.0001) independently predicted EFS, with genetic risk stratification (HR-1.7; 95% CI- 1.29 to 2.17; p = 0.0001) and PI MRD positivity (HR-1.6; 95% CI- 1.03 to 2.38; p < 0.0001) also independently impacting OS (Supplementary Table 2).