Sequential Approach to Improve the Molecular Classification of Childhood Acute Lymphoblastic Leukemia

Abstract

Identification of specific leukemia subtypes is a key to successful risk-directed therapy in childhood acute lymphoblastic leukemia (ALL). Although RNA sequencing (RNA-seq) is the best approach to identify virtually all specific leukemia subtypes, the routine use of this method is too costly for patients in resource-limited countries. This study enrolled 295 patients with pediatric ALL from 2010 to 2020. Routine screening could identify major cytogenetic alterations in approximately 69% of B-cell ALL (B-ALL) cases by RT-PCR, DNA index, and multiplex ligation-dependent probe amplification. STIL-TAL1 was present in 33% of T-cell ALL (T-ALL) cases. The remaining samples were submitted for RNA-seq. More than 96% of B-ALL cases and 74% of T-ALL cases could be identified based on the current molecular classification using this sequential approach. Patients with Philadelphia chromosome-like ALL constituted only 2.4% of the entire cohort, a rate even lower than those with ZNF384-rearranged (4.8%), DUX4-rearranged (6%), and Philadelphia chromosome-positive (4.4%) ALL. Patients with ETV6-RUNX1, high hyperdiploidy, PAX5 alteration, and DUX4 rearrangement had favorable prognosis, whereas those with hypodiploid and KMT2A and MEF2D rearrangement ALL had unfavorable outcomes. With the use of multiplex ligation-dependent probe amplification, DNA index, and RT-PCR in B-ALL and RT-PCR in T-ALL followed by RNA-seq, childhood ALL can be better classified to improve clinical assessments.

Figure 1

A flowchart of patients with the genetic diagnosis of acute lymphoblastic leukemia (ALL) enrolled in this study. ALL, acute lymphoblastic leukemia; B-ALL, B-cell acute lymphoblastic leukemia; DI, DNA index; MLPA, multiplex ligation–dependent probe amplification; T-ALL, T-cell acute lymphoblastic leukemia; TPOG, Taiwan Pediatric Oncology Group.

Figure 2

Integrative acute lymphoblastic leukemia (B-ALL) subtypes. Gene expression profiling of reference samples with clear B-ALL subtype predefined and Taiwan Pediatric Oncology Group (TPOG)–B-ALL samples shown in a two-dimensional t-distributed stochastic neighbor embedding plot. Each dot represents a sample. Major subtypes of reference samples are represented as circles in different colors and TPOG–B-ALL samples are represented as light blue triangles.

Figure 3

Integrative T-cell acute lymphoblastic leukemia (T-ALL) subtypes. Gene expression profiling of reference samples with clear T-ALL subtype predefined and Taiwan Pediatric Oncology Group (TPOG)–T-ALL samples shown in a two-dimensional uniform manifold approximation and projection plot. Each dot represents a sample. Major subtypes of reference samples are represented as circles in different colors and TPOG–T-ALL samples are represented as light blue triangles.

Figure 4

The 5-year event-free survival (EFS) (A) and 5-year overall survival (OS) (B) according to the risk groups by genetic subtypes. The EFS and OS were estimated using the Kaplan-Meier method, and P values were calculated using the log-rank test.