RESUMO
BACKGROUND: Gene rearrangements affecting KMT2A are frequent in acute myeloid leukemia (AML) and are often associated with a poor prognosis. KMT2A gene fusions are often detected by chromosome banding analysis and confirmed by fluorescence in situ hybridization. However, small intragenic insertions, termed KMT2A partial tandem duplication (KMT2A-PTD), are particularly challenging to detect using standard molecular and cytogenetic approaches. METHODS: We have validated the use of a custom hybrid-capture-based next-generation sequencing (NGS) panel for comprehensive profiling of AML patients seen at our institution. This NGS panel targets the entire consensus coding DNA sequence of KMT2A. To deduce the presence of a KMT2A-PTD, we used the relative ratio of KMT2A exons coverage. We sought to corroborate the KMT2A-PTD NGS results using (1) multiplex-ligation probe amplification (MLPA) and (2) optical genome mapping (OGM). RESULTS: We analyzed 932 AML cases and identified 41 individuals harboring a KMT2A-PTD. MLPA, NGS, and OGM confirmed the presence of a KMT2A-PTD in 22 of the cases analyzed where orthogonal testing was possible. The two false-positive KMT2A-PTD calls by NGS could be explained by the presence of cryptic structural variants impacting KMT2A and interfering with KMT2A-PTD analysis. OGM revealed the nature of these previously undetected gene rearrangements in KMT2A, while MLPA yielded inconclusive results. MLPA analysis for KMT2A-PTD is limited to exon 4, whereas NGS and OGM resolved KMT2A-PTD sizes and copy number levels. CONCLUSIONS: KMT2A-PTDs are complex gene rearrangements that cannot be fully ascertained using a single genomic platform. MLPA, NGS panels, and OGM are complementary technologies applied in standard-of-care testing for AML patients. MLPA and NGS panels are designed for targeted copy number analysis; however, our results showed that integration of concurrent genomic alterations is needed for accurate KMT2A-PTD identification. Unbalanced chromosomal rearrangements overlapping with KMT2A can interfere with the diagnostic sensitivity and specificity of copy-number-based KMT2A-PTD detection methodologies.
RESUMO
In patients with chronic myeloid leukemia, the use of real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for measuring BCR-ABL1 transcripts has become standard methodology for the diagnosis and monitoring of minimal residual disease. In 2004 and 2005, 38 different laboratories from North America participated in three separate sample exchanges using real-time qRT-PCR to measure RNA transcript levels in unknown diluents of a BCR-ABL1-positive cell line, K562. In this study we compared results of quantitative testing for BCR-ABL1 from laboratories using different platforms, internal controls, reagents, and calculation methods. Our data showed that there can be considerable variability of results from laboratory to laboratory, with log reduction calculations varying from 1.6 to 3 log between laboratories at the same dilution. We found that none of the variables tested had a significant impact on the results reported, except for the use of ABL1 as the internal control (P < 0.001). Laboratories that used ABL1 consistently underreported their log reduction values. Regardless of the specific methodology and platform used for real-time qRT-PCR testing, it is important for laboratories to participate in proficiency testing to ensure consistent and acceptable test accuracy and sensitivity. Our study emphasizes the need for optimization of real-time qRT-PCR before offering clinical testing and the need for widely available universal standards that can be used for test calibration.
