One Step Quantitative Molecular Approach for Detection of BCR/ABL Translocation and for Monitoring and Follow up of the Minimal Residual Disease in CML Patients Under Tyrosine Kines Inhibitors Treatment
Authors: Daraio Filomena , Barberis Davide , Crasto Francesca , Rege-Cambrin Giovanna , Olivo Cristina , Renzulli Matteo , Volpi Rosy , Lorenzatti Roberta , Fava Carmen , Gottardi Enrico , Saglio Giuseppe 
Published: October 2019

Abstract
Abstract 4435
In patients with chronic myeloid leukemia (CML) a correct and careful monitoring of the response to treatment with tyrosine kinase inhibitors (TKI) is essential in order to properly manage the disease.
The use of standardised rapid and highly sensitive molecular methods has become a requirement for the interpretation of the patient response to the standard therapy and to promptly switch to alternative treatments in case of resistance.
International guidelines suggested the possibility to use the absolute copy number of the control gene ABL in order to define the sensitivity of the analytical method and hence the molecular remission level achieved. A minimum of 10000 copy of ABL gene is required for the validation of the final diagnosis and a minimum of 32000 copies of ABL is desirable to define the molecular remission as MR4.5.
We are currently testing a new assay, “BCR-ABL ELITe MGB® kit” (Nanogen Advanced Diagnostics S.p.A) which allows to perform in one step the retrotranscription and the amplification of the extracted sample, thus increasing sensitivity in detection of both genes.
In a pilot study, we compared the sensitivity of “BCR-ABL ELITe MGB® kit” one step assay with our current routine assay, “Philadelphia P210 Q-PCR Alert” (Nanogen Advanced diagnostics). Peripheral whole blood samples from CML patients (n=45) at different stages of the disease and from normal individuals (n=30) were analysed. For the analysis, 500 ng of RNA were directly added into the amplification reaction. Amplification was performed on the 7500Fast Dx instrument (LifeTech, Applied Biosystem). Final results were expressed as ratio BCR-ABL/ABL % in international scale.
In the analysed group, the average value of ABL detected by “BCR-ABL ELITe MGB® kit” was 48500 copies. In CML patients ABL ranged between 19000 and 106525 copies, with a median value of about 59000 copies. In the analysis with the routine assay ABL values ranged between 6600 and 105000 copies, with a median value of about 33000 copies. High reproducibility of results on sample duplicates was observed. and the final results with the “BCR-ABL ELITe MGB® kit” complete overlapped the routine assay outcome.
In conclusion, “BCR-ABL ELITe MGB® kit” assay is rapid, accurate, sensitive. It eliminates the time consuming step of separate retrotranscription and reduces the possibility of errors during the analysis and gives more reproducibile results. BCR-ABL ELITe MGB® kit” offers an innovative approach to CML molecular monitoring and a step further in the standardisation molecular results in CML follow-up. Further analysis of PCR results is ongoing in order to complete the validation of this system.

Disclosures:
Barberis: Nanogen Advanced Diagnsotics S.p.A: Employment. Olivo:Nanogen Advanced Diagnostics S.p. A: Employment. Renzulli:Nanogen Advanced Diagnostics S.p.A.: Employment. Volpi:Nanogen Advanced Diagnostics S.p.A.: Employment.