Targeted-Capture Sequencing Is a Useful Method for MRD Markers Screening in KMT2A (MLL) Rearranged Leukemia

Immunoglobulin (Ig)/T-cell receptor (TCR) gene rearrangement-based molecular minimum residual disease (MRD) monitoring has become one of the most powerful prognostic indicators for patients with acute lymphoblastic leukemia (ALL). Infant ALL has a poor prognosis and requires therapeutic development based on accurate MRD assessment. However, there are some difficulties associated with the highly sensitive detection of MRD based on Ig/TCR gene rearrangements in infant ALL cases, compared to other B-ALL cases, because of the immature Ig/TCR gene rearrangement patterns and their oligoclonality. Since KMT2A(MLL) gene rearrangements are identified in infant ALL at a high frequency, the use of KMT2A rearranged sequences is an alternative method for MRD detection in KMT2A rearranged ALL cases. However, identifying the genomic breakpoints in KMT2A rearrangements by conventional methods is laborious and difficult in routine practice. To detect molecular MRD markers in infant ALL cases, we designed capture probes covering the coding and recognition signal sequences of the V, D, and J genes of the Ig/TCR loci and common genomic breakpoints in KMT2A, AFF1 (AF4), MLLT3 (AF9), MLLT1 (ENL), MLLT10 (AF10), and ELL. 88 infant ALL cases from the JPLSG MLL-10 study, including 73 cases with KMT2A rearrangements, were analyzed by high-throughput target-capture sequencing on the Illumina platform. The sequenced data were analyzed with the Ig/TCR recombination analysis tool Vidjil and the Genomon pipeline for detection of KMT2A rearrangements.

Although 40% of cases in this study were marker-negative in the conventional marker screening analysis, clonal Ig/TCR targets for MRD measurements were identified in more than 80% of the cases by target-capture sequencing. Ig / TCR rearrangements detected in infant ALL were unique and related to age at diagnosis and type of KMT2A rearragement. KMT2A-rearranged cases showed a significantly higher frequency of incomplete rearrangements of IGH and TCRD compared to KMT2A-negative infant ALL cases.　The target-capture method also showed an increased sensitivity of Ig/TCR-based MRD measurements even in cases where MRD measurement was possible.　Genomic breakpoints of KMT2A rearrangements were detected in 80% of KMT2A-rearranged cases, in which KMT2A was most frequently rearranged with AFF1, followed by MLLT1, MLLT3, and MLLT10. Other rare fusion partners of the KMT2A gene were also captured by probes for KMT2A sequences. The determined patient-specific genomic breakpoint sequences could be used for monitoring the MRD of KMT2A-rearranged cases. Thus, target-capture sequencing is an easy and useful method for screening MRD markers in infant leukemia cases.