Five-Color Multiplex Real-Time PCR Technology to Detect Over 75 Recurrent Chromosomal Abnormalities in Acute Myeloid Leukemia; Benefits for Minimal Residual Disease Detection
Authors: Plachy Radek , Zejskova Lenka , Cmejla Radek , Hardekopf David Warren , Jancuskova Tereza , Krutilkova Lucie , Karas Michal , Koza Vladimir , Vydra Jan , Zak Pavel , Smolej Lukas , Pekova Sona 
Published: October 2019

Abstract
Abstract 2526
Many studies on childhood and adult leukemias have demonstrated that thorough molecular analysis at the time of diagnosis and minimal residual disease (MRD) follow-up are significantly related to the prognosis, and overall- and event-free survival, of patients with acute leukemias. In acute myeloid leukemia (AML), such complex molecular diagnostics and subsequent MRD evaluations are important factors for proper stratification, disease prognosis, assessment of the treatment response, optimal dosage and duration of chemotherapy, and estimation of the optimal timing for hematopoietic stem cell transplantation.
Upon diagnosis, the bone marrow samples of AML patients are routinely screened for an array of recurrent chromosomal abnormalities, including chromosomal translocations (e.g. PML/RARa, AML1/ETO, CBFb/MYH11, MLL fusions, BCR/ABL) or leukemia-associated genetic mutations (e.g. mutations in genes NPM1, WT1, FLT3, MLL or CEBPa). Given the fact that laboratories often deal with a limited amount of material sampled for molecular investigations, and the number of possible genomic aberrations and molecular targets is high, it is desirable to implement in routine practice a flexible tool that allows testing for as many genetic abnormalities as possible, while reducing the amount of biological material required for analyses to a minimum.
In our laboratory, we have developed a multiplex Real-Time PCR technique allowing us to examine over 75 recurrent chromosomal aberrations in only 10 multiplex PCR reactions (Table 1). The methodology makes use of a set of fluorescently labeled TaqMan hybridization probes, labeled by 5 different fluorophores. Using this methodology we are able to assess the presence of both rare as well as recurrent chromosomal translocations/aberrations in one setting, from a limited amount of starting material. This approach is not only extremely beneficial for the leukemic patient - which is always the primary goal - but also for the overall budgeting of routine molecular screening of diagnostic AML samples.Table 1:List of the AML-associated chromosomal abnormalities included in the 5-Color Multiplex Real-Time PCR system. The multiplexing of individual molecular targets is indicated by the individual PCR tubes (A to J) used in the diagnostic screening of AML in our laboratory.
Importantly, a clone-specific chromosomal abnormality found at the time of diagnosis using our 5-Color Multiplex Real-Time PCR system allows us to molecularly follow-up the MRD level with a high sensitivity of 10e-4 to 10e-6, as assessed by serial dilutions of cloned standards harboring the individual aberrant genetic targets. This complex molecular approach considerably helps hematooncologists in clinical decision making and the adjustment and modulation of the treatment of AML patients in respect to their individual needs and their individual disease course.
Since 2005 we have molecularly investigated 398 adult AML cases. Using the 5-Color Multiplex Real-Time PCR technique and mutational screening we were able to identify a clone-specific abnormality in 45.2% of cases. The clone-specific genetic markers were then used as specific molecular targets for a clone-specific MRD follow-up.
Although in approximately 50% of AML patients we are still not able to identify any clone-specific abnormality to be used for either stratification (recurrent abnormalities) or molecular MRD follow-up (both recurrent and unique/rare abnormalities) of these leukemic patients, our 5-Color Multiplex Real-Time PCR system, being an open platform, enables us to flexibly implement any newly identified chromosomal aberrations to the diagnostic portfolio, thus increase the probability of finding a clone-specific molecular marker with all the positive consequences in respect to the management of patients with acute leukemia.

Disclosures:
Smolej: GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Travel Grants; Roche: Honoraria, Travel Grants; Genzyme: Honoraria, Travel Grants.