Challenging conventional karyotyping by next-generation karyotyping in 281 intensively treated patients with AML

Abstract

Although copy number alterations (CNAs) and translocations constitute the backbone of the diagnosis and prognostication of acute myeloid leukemia (AML), techniques used for their assessment in routine diagnostics have not been reconsidered for decades. We used a combination of 2 next-generation sequencing–based techniques to challenge the currently recommended conventional cytogenetic analysis (CCA), comparing the approaches in a series of 281 intensively treated patients with AML. Shallow whole-genome sequencing (sWGS) outperformed CCA in detecting European Leukemia Net (ELN)–defining CNAs and showed that CCA overestimated monosomies and suboptimally reported karyotype complexity. Still, the concordance between CCA and sWGS for all ELN CNA–related criteria was 94%. Moreover, using in silico dilution, we showed that 1 million reads per patient would be enough to accurately assess ELN-defining CNAs. Total genomic loss, defined as a total loss ≥200 Mb by sWGS, was found to be a better marker for genetic complexity and poor prognosis compared with the CCA-based definition of complex karyotype. For fusion detection, the concordance between CCA and whole-transcriptome sequencing (WTS) was 99%. WTS had better sensitivity in identifying inv(16) and KMT2A rearrangements while showing limitations in detecting lowly expressed PML-RARA fusions. Ligation-dependent reverse transcription polymerase chain reaction was used for validation and was shown to be a fast and reliable method for fusion detection. We conclude that a next-generation sequencing–based approach can replace conventional CCA for karyotyping, provided that efforts are made to cover lowly expressed fusion transcripts.

CBFB Break-Apart FISH Testing: An Analysis of 1629 AML Cases with a Focus on Atypical Findings and Their Implications in Clinical Diagnosis and Management

“…To date, the next-generation sequencing (NGS)-based methods such as whole genome sequencing (WGS) [34,35], whole transcriptome sequencing (WTS) [36][37][38], and targeted RNA sequencing (RNA-Seq) [39][40][41], with the enormous power of precise detection of all known and even novel translocations and/or fusions simultaneously, have been implemented as a diagnostic tool for hematologic malignancies including inv(16)/t(16;16) AML.…”

“…Interestingly, FISH assays including CBFB FISH have been applied either as a tool for the confirmation of novel fusions and/or copy number variants (CNVs) or solving the discrepancies between these new methods and karyotype analysis in these reports [34,35,37]. Although implementation of these NGS-based methods in clinical diagnosis of myeloid neoplasia is still at a stage of development and validation in our institute and none of the cases in this cohort was tested with any of these new methods yet, however, based on the biology of these methods and the parameters used in the published reports, we can postulate the results, as summarized in Table 5, of these methods were applied to all 271 cases with CBFB rearrangement in our study.…”

What Is Abnormal in Normal Karyotype Acute Myeloid Leukemia in Children? Analysis of the Mutational Landscape and Prognosis of the TARGET-AML Cohort

“…Notably, six gene fusions not reported in the cytogenetic data were detected by WGS in NK-AML. This highlights the relevance of genomic sequencing to be implemented as clinical cytogenomic analyses as recently suggested [ 27 , 28 ]. Certain cryptic cytogenetic events of prognostic importance are difficult to capture in routine cytogenetic examinations, including, for instance, inv(16)(p13q24) ( CBFA2T3 - GLIS2 ) [ 29 ] and rearrangements involving 11p15 ( NUP98 fusions) [ 10 ].…”

“…Certain cryptic cytogenetic events of prognostic importance are difficult to capture in routine cytogenetic examinations, including, for instance, inv(16)(p13q24) ( CBFA2T3 - GLIS2 ) [ 29 ] and rearrangements involving 11p15 ( NUP98 fusions) [ 10 ]. For these cryptic events, WGS or transcriptome (RNA) sequencing may have a higher diagnostic yield detecting gene fusions or their expressed fusion transcripts, respectively [ 27 , 28 , 30 , 31 ].…”

A Study Protocol for Validation and Implementation of Whole-Genome and -Transcriptome Sequencing as a Comprehensive Precision Diagnostic Test in Acute Leukemias

“…Furthermore, a head-to-head comparison of WGS (tumor-only, 60×) analysis to conventional cytogenetics and targeted sequencing not only confirmed accurate genomic profiling by WGS but also showed that WGS identified additional clinically relevant events in 17% (40/235) of patients and reclassified risk group assignments in 16% (19/117) of patients ( 13 ). Shallow WGS and WTS also demonstrated greater sensitivity in CNA detection and fusion detection in AML patients, respectively, compared with conventional cytogenetic analysis ( 19 ); concordance was high between cytogenetic analysis and shallow WGS (approximately 96%) or WTS (approximately 99%). Likewise, clinical value of WGS/WGTS in routine care of patients with solid tumors has been shown or is being investigated; e.g., the Hartwig Medical Foundation ( 20 ), the MASTER ( 21 ), and the WIDE ( 22 ) studies.…”