Integrated Whole Genome and Transcriptome Sequencing as a Framework for Pediatric and Adolescent AML Diagnosis and Risk Assessment

Abstract

Pediatric acute myeloid leukemia (AML) exhibits distinct genetic characteristics, including unique driver alterations and mutations with prognostic and therapeutic significance. Emerging rare, recurrent genetic abnormalities and their associations with outcomes emphasize the need for high-throughput molecular diagnostic tools. Whole genome sequencing (WGS) reliably detects key AML biomarkers such as structural variants, mutations, and copy number alterations. Whole transcriptome sequencing (WTS) complements WGS by uncovering oncogene expression patterns, allele-specific expression, and gene expression signatures. In this study, we describe an integrated WGS and WTS clinical workflow for routine pediatric AML diagnosis and present a systematic evaluation of its application compared to conventional cytogenetics and standard molecular diagnostic methods. Our findings demonstrate that the integrated WGS and WTS (iWGS-WTS) approach improves the identification of clinically relevant genetic alterations, enhancing precise disease classification and risk assessment. Moreover, with advancements in workflow and bioinformatics pipelines, the testing turnaround time can be optimized to meet the demands of clinical decision-making, positioning iWGS-WTS as a practical and superior alternative to traditional diagnostic methods in pediatric AML management.

Keywords: AML; Whole genome sequencing; Whole transcriptome sequencing.

Figure 1. Detection of SNVs/indels and focal CNVs.

A) Comparison of WGS and WES in the detection of SNVs and Indels. Variant allele frequency (VAF) for Whole Genome Sequencing (WGS) and Whole Exome Sequencing (WES) is shown on the x and y axes, respectively. Variants with less than 5% VAF are shown in green for WES and orange for WGS. High concordance in VAF estimates among the variants considered reportable by both WGS and WES observed: correlation coefficient (r) = 0.9 (slope = 0.88). B) Comparison of WGS and targeted panel NGS in the detection of SNV and Indel. VAF is shown for WGS and targeted Next Generation Sequencing (NGS) on the x and y axes, respectively. Variants with less than 5% VAF are shown in green (WES) and orange (WGS). Correlation of VAF estimates showed a correlation coefficient (r) of 0.83 (slope = 0.86). C) Focal CNVs detected by WGS are presented according to size (in kb) on the x-axis. Gains are red squares, deletions blue dots and partial tandem deletions (PTDs) green triangles.

Figure 2. Comparison of WGS and WTS in the detection of AML-associated oncogenic gene fusions/chromosomal translocations.

Outer ring: Oncogenic/likely oncogenic fusions detected by WGS. Enhancer hijacking fusions are marked. The remaining events are predicted to form chimeric fusion transcripts. Inner ring: Fusion detection by WTS. Events missed by WTS are indicated in gray, those that did not show diagnostic evidence of a fusion transcript by WTS but were confirmed by other supportive evidence are shown in beige.

Figure 3. Detection of chromosomal abnormalities and comparison to conventional cytogenetics.

A) Comparison of AML-associated fusion detection between conventional G-banded Karyotyping (with complementary FISH test when available) on the y-axis and integrated WGS and WTS (iWGS-WTS) on the x-axis. *Known cytogenetically cryptic gene fusions. Note: FISH (Fluorescence in situ hybridization) analysis for KMT2A rearrangement, CBFB rearrangement and RUNX1::RUNX1T1fusion were available in a subset of cases (See Supplementary Table S1). B) Overview of the comparison of WGS and conventional cytogenetics in the detection of large-scale CNVs. Groups of events detected by cytogenetics or WGS alone are listed to each side. C) Comparison of the detection of AML-MR defining cytogenetic alterations by conventional cytogenetics (y-axis) and WGS (x-axis).

Figure 4. Molecular classification of cases in the study cohort.

Comparison between different tumor profiling approaches: WGS, WTS, and integrated WGS and WTS (iWGS-WTS), for the molecular classification of AML. Sankey diagram shows detection of AML genetic drivers by WGS only on the left, WTS only on the right and the iWGS-WTS in the middle.

Figure 5. Genomic landscape of cases in the study cohort.

A) Pathogenic/likely pathogenic genomic alterations identified in 154 AML cases by the integrated WGS and WTS approach (Note: SJ032376 sequenced by WES and WTS). Patients’ sex, treatment-related AML (tAML) and relapsed cases are shown on the top. Only alterations seen in two or more cases in this study cohort are presented for additional findings, total count is shown on the right. Molecular subtypes are indicated along the bottom. B) The donut chart breaks down the 154 AML cases into subtypes according to genetic drivers/potential drivers determined by the integrated WGS and WTS approach. Absolute count is listed as numbers on the outer ring, and the percentage of the total cohort is shown towards the inner ring. Selected additional genetic alterations, either with impact on risk assessment and/or being directly targetable/therapy-informing, were shown and specified by different symbols along with the driver alteration. AML-MR, acute myeloid leukemia - myelodysplasia related; APL, acute promyelocytic leukemia; CEBPA-ASE, CEBPA-allele specific expression; DS-AML, Down Syndrome-associated AML; LS-CNV, large-scale copy cumber variant; LOF, loss of function; CN-LOH, copy neutral loss of heterozygosity; N/A, not available; PTD, partial tandem duplication; tAML, therapy-related AML; TD, tandem duplication.

Figure 6. Advanced clinical workflow for the integrated WGS and WTS testing.

A phased data processing and reporting approach for WGS and WTS is shown with timeline indicated below. Segments in different colors indicate varying steps of the process from sample acquisition/processing to data processing and analysis. Reports can be released in three phases, as indicated above: rapid tumor report by day 7, selected integrated iWGS-WTS tumor report by day 14, and a comprehensive paired tumor-normal genomic profiling report by day 21. EMR, electronic medical record; P/LP, pathogenic/likely pathogenic; SNV, single nucleotide variant; SV, structural variant; QC, quality control.