A New Sensitive and Fast Molecular Analysis Method for the Identification of 30 Different Markers in Acute Myeloid and Lymphoid Leukemias at Diagnosis Time

Background: Medical advances in the treatment of Acute and Chronic Leukemia in the last decade were exciting, thanks to the discovery of new drugs and transplantation of hematopoietic stem cells; but so that these weapons can be used in an optimal way, it is necessary that the leukemia is diagnosed as quickly as possible and accurately to limit the spread of leukemia cells in the patient. The ability to detect fusion genes characteristics of these diseases has become essential but laborious, time consuming and generally comprises karyotype analysis, followed by FISH (Fluorescent in situ hybridization) and molecular analysis (RT-PCR). New molecular diagnostic technologies are being developed that can identify disease markers with extreme accuracy in a few hours (2-3 hours). The Leukemia Fusion Genes (Q30) Screening Kit (QuanDx, San Jose, CA 95131 USA; Distr. Resnova S.r.l.) is a real-time multiplex RT-qPCR based assay for detection of leukemia associated fusion gene transcripts in total RNA from bone marrow or whole blood samples, it is a qualitative test for the simultaneous detection of 30 characteristic fusion genes of acute leukemias. For Acute Myeloid Leukemia (AML) transcripts we can expect tested 18 fusion genes with 72 different breakpoints: BCR-ABL1, MLL-AF9, CBFbeta-MYH11, PML-RARalpha, AML-MDS1, NPM-MLF1, AML-ETO, NPM-RARalpha, PLZF-RARalpha, DEK-CAN, MLL-ELL, AML1-EAP, MLL-AF10, ST-CAN, TEL-ABL1, TLS-ERG, FIP1L1-PDGFR alpha, TEL-PDGFRbeta. For Acute Lymphoid Leukemia (ALL) transcripts we will detected 15 fusion genes with 71 different breakpoints: MLL-AF4, TEL-AML1, MLL-ENL, E2APBX1, BCR/ABL1, SIL-TAL1, E2A-HLF, MLL-AF6, CALM-AF10, HOX11, HOX11L2, MLL-AF10, HOX11, HOX11L2, MLL-AF10, SET-CAN, TEL-ABL1, TLS-ERG.

Aim: The aim of this study is to evaluate and to standardize a new commercial kit, the Leukemia Fusion Genes (Q30) Screening Kit, that is reliable, reproducible and able to detect simultaneously, in one real-time PCR reaction and in a short time different rearrangement involved in acute leukemia at diagnosis.

Methods: 60 samples will be analyzed as follows: 51 AML and 9 ALL.All samples have been previously tested with classical PCR qualitative methods (Leukemia 1999). 55 RNAs were extracted using the Maxwell 16 Semi-automatic Extractor (Simply RNA Blood kit) from isolated cells from Ficoll or buffycoat of bone marrow or peripheral blood. 5 RNAs were extracted with manual phenol-chloroform method. Concentration was determined by spectrophotometric reading (from 20ng/µL to 200ng/µL). cDNA was added to 8 qPCR reaction tubes, which contain specific PCR primers and probes for detection of fusion genes and an internal control gene (GUSB). The qPCR is performed in a real-time thermal cycler, the kit is compatible with the ABI7500 system.

Results: 21 out of 51 AML patients (pts), tested with routine approach, had no markers, the remaining 30 AML had rearrangements and mutations as follows:1 pt CBFβ-MYH11, 2 pts PML-RARalpha, 2 pts AML1-ETO, 1 pt DEK-CAN, 4 pts FLT3 ITD, 6 pts NPM, 8 pts FLT3/NPM, 1 pt DEK-CAN/FLT3, 3 pts PML-RARα/FLT3 ITD, 1 pt IDH mut, 1 pt MLL ITD. 5/9 ALL samples had no markers, the remaining presented with t (4;11) (2 pts), t (9;22) (2 pts).All the tested samples ( 60 samples) confirmed the molecular rearrangement where it was present (14 samples) using primer sets for molecular rearrangements by RT-PCR technique. 100% concordance, 14 samples out of 14.Significantly 5 out of 60 patients showed new (previously unidentified) rearrangements with the primer sets used by the laboratory routinely: 2 pts with MLL-ELL/t(11;19), 1 pt with MLL-AF10/t(10;11) and 2 pts with AML1-MTG16/t(16;21). The kit revealed a percentage of rearranged patients equal to 32% compared to 23% analyzed with the primer sets used routinely, using a time of two/three hours against 72 hours, considering the totality of the rearrangements.

Conclusion: The Q-Fusion system provides a sensitive instrument specific, reliable and cost effective, easy to perform for routine screening in leukemias at diagnosis.The kit can detect 30 common fusion genes, it can only report the types of fusion genes, but cannot differentiate the splice variants. It is a Real-Time multiplex RT-qPCR and the Cq values cannot be used for quantification of the fusion transcript level.