Risk-based classification of leukemia by cytogenetic and multiplex molecular methods: results from a multicenter validation study

Abstract

Modern management of leukemia and selection of optimal treatment approaches entails the analysis of multiple recurrent cytogenetic abnormalities with independent diagnostic or prognostic value. We report the first multicenter validation of a multiplex molecular assay for 12 relevant fusion transcripts relative to cytogenetic methods. Performance was evaluated using a set of 280 adult and pediatric acute or chronic leukemias representative of the variety of presentations and pre-analytical parameters encountered in the clinical setting. The positive, negative and overall agreements were >98.5% with high concordance at each of the four sites. Positive detection of cases with low blast count or at relapse was consistent with a method sensitivity of 1%. There was 98.7% qualitative agreement with independent reference molecular tests. Apparent false negatives corresponded to rare alternative splicing isoforms not included in the panel. We further demonstrate that clinical sensitivity can be increased by adding those rare variants and other relevant transcripts or submicroscopic abnormalities. We conclude that multiplex RT-PCR followed by liquid bead array detection is a rapid and flexible method attuned to the clinical laboratory workflow, complementing standard cytogenetic methods and generating additional information valuable for the accurate diagnosis, prognosis and subsequent molecular monitoring of leukemia.

An optimized technology platform for the rapid multiplex molecular analysis of genetic alterations associated with leukemia

“…The average negative probe signal in these experiments was 102 MFI with a maximum at 273 MFI and 95% of the signals between 0 and 177 MFI (data not shown). These results are in agreement with an independent limit of blank study showing that 95% of the signals generated in known negative samples are at least two-fold below 350 MFI for each probe (11). …”

“…By optimizing primers concentration and probes/amplicons length, every target in the original leukemia translocation panel was detected at about 1% cell line dilution or 2.E4 copies per RT reaction (Figure 1). This level of sensitivity, equivalent to the detection of one leukemia cell in a background of 100 normal cells, is superior to current clinical reference methods such as fluorescence in situ hybridization, Sanger sequencing or karyotyping (5 to 20%) and was shown to be adequate for the characterization of leukemia samples at diagnosis (11,12). Using an optimized RT system and PCR buffer (see Materials and Method) we demonstrated that the multiplex assay system can be further enhanced to reach 0.1 to 0.01% dilution or 2.E3 copies per RT reaction.…”

“…Using an insertion-specific assay design (Supplemental Figure 5, Strategy #4), NPM1 mutant transcripts were also detected at low levels even in the presence of a large excess of competing wild-type transcripts expressed in both normal and leukemia cells (20–22). Following appropriate clinical validation, this improved analytical sensitivity could extend the utility of the technology platform to other applications such as semi-quantitative monitoring of treatment response beyond cytogenetic remission or earlier qualitative detection of molecular relapse (11,21). …”

“…Multiplex reactions for twelve fusion transcripts and an endogenous control transcript (GAPDH) were performed in 96-well plates using the Signature ® LTx v2.0 Kit (for research use only, not for use in diagnostic procedures, Asuragen Inc., Austin, TX, USA) as described in Gocke et al (11). The protocol for the prototype expanded panels detecting six additional ALL splicing variants or nineteen different fusion transcripts was the same except that modified primer mixes and bead mixes were used at the PCR and hybridization steps, respectively.…”

“…We took advantage of the 96-well plate format and automation capabilities of this platform to develop a rapid multiplex assay for the qualitative detection of twelve fusion transcripts resulting from seven chromosomal abnormalities commonly found in chronic myelogenous leukemia (CML), acute lymphoblastic leukemia (ALL), or acute myeloid leukemia (AML). The assay was successfully evaluated with over 600 representative leukemia specimens in multiple laboratories highlighting its potential value as a complement to cytogenetic analyses (11,12). The goals of the present study were to expand the panel beyond the initial twelve targets, including for submicroscopic abnormalities that cannot be detected by classic cytogenetics, and to fully characterize the analytical sensitivity, specificity and precision of the assay system.…”