Evaluation of the primitive fraction by functional in vitro assays at the RNA and DNA level represents a novel tool for complementing molecular monitoring in chronic myeloid leukemia

// Maria Sol Ruiz 1 , Maria Belen Sanchez 1, 2 , Leandro Gutierrez 3 , Daniel Koile 4 , Patricio Yankilevich 4 , Celeste Mosqueira 2 , Santiago Cranco 5 , Maria del Rosario Custidiano 5 , Josefina Freitas 6 , Cecilia Foncuberta 5 , Beatriz Moiraghi 7 , Carolina Pavlovsky 8 , Mariel Ana Perez 9 , Veronica Ventriglia 6 , Julio Sanchez Avalos 5 , Jose Mordoh 1 , Irene Larripa 3 and Michele Bianchini 1 1 Centro de Investigaciones Oncologicas-Fundacion Cancer (CIO-FUCA), Ciudad Autonoma de Buenos Aires, Argentina 2 Argenomics, Ciudad Autonoma de Buenos Aires, Argentina 3 Instituto de Medicina Experimental, CONICET/Academia Nacional de Medicina, Ciudad Autonoma de Buenos Aires, Argentina 4 Instituto de Investigacion en Biomedicina de Buenos Aires (IBioBA), CONICET, Partner Institute of the Max Planck Society, Ciudad Autonoma de Buenos Aires, Argentina 5 Instituto Alexander Fleming, Ciudad Autonoma de Buenos Aires, Argentina 6 Hospital Nacional Posadas, El Palomar, Buenos Aires, Argentina 7 Hospital J. M. Ramos Mejia, Ciudad Autonoma de Buenos Aires, Argentina 8 Fundaleu, Ciudad Autonoma de Buenos Aires, Argentina 9 Hospital Interzonal General de Agudos, Prof. Dr. R. Rossi, La Plata, Buenos Aires, Argentina Correspondence to: Michele Bianchini, email: mbianchini@conicet.gov.ar Keywords: leukemic stem cells; chronic myeloid leukemia; tyrosine-kinase inhibitors; therapy discontinuation; persistence Received: September 01, 2017     Accepted: March 06, 2018     Published: April 17, 2018 ABSTRACT Quantification of BCR-ABL1 mRNA levels in peripheral blood of chronic myeloid leukemia patients is a strong indicator of response to tyrosine-kinase inhibitors (TKI) treatment. However, additional prognostic markers are needed in order to better classify patients. The hypothesis of leukemic stem cells (LSCs) heterogeneity and persistence, suggests that their functional evaluation could be of clinical interest. In this work, we assessed the primitive and progenitor fractions in patients at diagnosis and during TKI treatment using functional in vitro assays, defining a “functional leukemic burden” (FLB). We observed that the FLB was reduced in vivo in both fractions upon treatment. However, different FLB levels were observed among patients according to their response to treatment, suggesting that quantification of the FLB could complement early molecular monitoring. Given that FLB assessment is limited by BCR-ABL1 mRNA expression levels, we developed a novel detection method of primitive cells at the DNA level, using patient-specific primers and direct nested PCR in colonies obtained from functional in vitro assays. We believe that this method could be useful in the context of discontinuation trials, given that it is unknown whether the persistent leukemic clone represents LSCs, able to resume the leukemia upon TKI removal.