Challenging Conventional Diagnostic Methods by Comprehensive Molecular Diagnostics: A Nationwide Prospective Comparison in Children With ALL

PURPOSE Treatment stratification in ALL includes diverse (cyto)genetic aberrations, requiring diverse tests to yield conclusive data. We optimized the diagnostic workflow to detect all relevant aberrations with a limited number of tests in a clinically relevant time frame. METHODS In 467 consecutive patients with ALL (0-20 years), we compared RNA sequencing (RNAseq), fluorescence in situ hybridization (FISH), reverse transcriptase polymerase chain reaction (RT-PCR), karyotyping, single-nucleotide polymorphism (SNP) array, and multiplex ligation-dependent probe amplification (MLPA) for technical success, concordance of results, and turnaround time. RESULTS To detect stratifying fusions (ETV6::RUNX1, BCR::ABL1, ABL-class, KMT2Ar, TCF3::HLF, IGH::MYC), RNAseq and FISH were conclusive for 97% and 96% of patients, respectively, with 99% concordance. RNAseq performed well in samples with a low leukemic cell percentage or low RNA quality. RT-PCR for six specific fusions was conclusive for >99% but false-negative for six patients with alternatively fused exons. RNAseq also detected gene fusions not yet used for stratification in 14% of B-cell precursor-ALL and 33% of T-ALL. For aneuploidies and intrachromosomal amplification of chromosome 21, SNP array gave a conclusive result in 99%, thereby outperforming karyotyping, which was conclusive for 64%. To identify deletions in eight stratifying genes/regions, SNP array was conclusive in 99% and MLPA in 95% of patients, with 98% concordance. The median turnaround times were 10 days for RNAseq, 9 days for FISH, 10 days for SNP array, and <7 days for MLPA and RT-PCR in this real-world prospective study. CONCLUSION Combining RNAseq and SNP array outperformed current diagnostic tools to detect all stratifying genetic aberrations in ALL. The turnaround time is <15 days matching major treatment decision time points. Moreover, combining RNAseq and SNP array has the advantage of detecting new lesions for studies on prognosis and pathobiology.