Novel Tools to Improve the Challenging Diagnosis of Ph-like ALL. Comparison between Optical Genome Mapping and Digital-MLPA
Authors: Lapietra Gianfranco , Cardinali Deborah , Cisneros Adela , Beldinanzi Marco , Hernández Sánchez Alberto , Kaiser Francesca , Ribera Jordi , Navas Acosta Josgrey Del Valle Del Valle , Hernandez Rivas Jesus Maria , Torrent Anna , Granada Isabel , Navarro Jose Tomas , Foà Robin , Martelli Maurizio , Orfao Alberto , Ribera Josep-Maria , Chiaretti Sabina 
Published: December 2024

Introduction. Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL) is a heterogeneous group of B-lineage ALL (B-ALL), accounting for 20-30% of adult cases and characterized by a gene expression profile similar to that of Ph+ ALL, lacking however the typical t(9;22)(q34;q11.2)/BCR::ABL1. The recurrent aberrations involve dysregulations of the JAK-STAT pathway and of ABL-class fusions, usually associated with deletions affecting EBF1, IKZF1, CDKN2A/B, PAX5, BTG1, TCF3 and VPREB1. Overall, this group still suffers from a worse prognosis compared to the other B-ALL subtypes. Thus, the precise identification of Ph-like ALL at baseline is paramount for a proper risk-stratification and a tailored treatment strategy. Its diagnostic work-up is, however, still challenging and novel tools are being investigated. Here, we compared optical genome mapping (OGM) and digital multiplex ligation-dependent probe amplification (dMLPA) to evaluate their ability and complementarity to identify Ph-like cases within a cohort of newly diagnosed (ND) adult B-ALL patients (pts). OGM is a genome-wide technology based on fluorescent labelling and imaging of ultra-high molecular weight (UHMW) DNA to detect both structural (SVs) and copy number variations (CNVs); dMLPA is a novel technique, developed on the basis of the conventional MLPA method and Illumina next generation sequencing technology for amplicon quantification, enabling the detection of CNVs and intrachromosomal gene fusions.

Methods. For the purpose of the study, we selected a cohort of ND adult B-ALL pts with sufficient leftover material after cytogenetic routine testing (RT) (i.e. chromosomal banding analysis, fluorescent in-situ hybridization and genomic microarray). OGM was performed (in Spain) following the manufacturer's instructions (BionanoGenomics, San Diego, US). UHMW-DNA was extracted, labelled with DLE-1 enzyme, loaded onto a chip and run using the Saphyr instrument (BionanoGenomics). Data analysis was carried out through rare variant analysis algorithm using the BionanoSolve software v.3.7, and the GRCh37/h19 as genome reference. SVs and CNVs were considered if overlapping with regions included in open-access BED files for B-ALL or if ≥100 kb. The dMLPA experiments were performed (in Italy), in accordance with the manufacturer's instructions, using the D007 ALL probemix (MRC Holland, Amsterdam, NL). The sample-specific products from several reactions were pooled and loaded on an Illumina MiSeq V3 flow-cell. The data were analyzed using the bioinformatics software by MRC Holland.

Results. Twenty-two cases were collected between May 2023 and July 2024. Median age was 46.5 years (range 18-70). The male/female ratio was 1:1. Based on RT, 5 Ph-like pts (23%) were identified: three with a CRLF2-rearrangement (-r) (interstitial deletion, 2 cases; translocation to another chromosome, 1 case), 1 with a JAK2-r and 1 with a ABL1-r. OGM confirmed the JAK2-r and ABL1-r, proving they were due to inv(9)(p24.1q33.3)/STRBP::JAK2 and del(9)(q34)/ABL1::NUP214, respectively, but missed the CRLF2-r. Furthermore, it detected a t(8;22)(p11;q11)/FGFR1::BCR in a pt previously classified as B-other and a t(14;19)(q32;p13.1)/IGH::EPOR in a pt previously classified with a PAX5 alteration. Overall, 6 Ph-like cases (27%) were identified by OGM. dMLPA confirmed the ABL1-r and the 2 cases with CRLF2-r due to an interstitial deletion, showing del(X)(p22p22)/P2RY8::CRLF2. The other cases, caused by balanced aberrations, were missed. Thus, 3 Ph-like cases (14%) were detected by dMLPA. Overall, after OGM and dMLPA analysis, 7 cases (32%) were scored as Ph-like. A manual revision of CNVs was performed for EBF1, IKZF1, CDKN2A/B, PAX5, BTG1, TCF3 and VPREB1. Both techniques detected at least one deletion in one of the above-mentioned genes in all 7 cases. Indeed, dMLPA identified a delIKZF1 in 5 cases (71%): 2 in association with delCDKN2A/B (IKZF1plus), and OGM in 3 cases (43%) (1 with IKZF1plus).

Conclusions. OGM outperforms RT in the identification of SVs, unless they affect regions with repeated sequences. dMLPA is more reliable for the detection of CNVs. The genetic diagnostic work-up of Ph-like cases remains nonetheless challenging. The integration with predictive models, namely the BCR::ABL1-like predictor, is planned for the refined classification of this difficult-to-manage B-ALL subgroup.