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
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María Sol Ruiz1, María Belén Sanchez1,2, Leandro Gutiérrez3, Daniel Koile4, Patricio Yankilevich4, Celeste Mosqueira2, Santiago Cranco5, María del Rosario Custidiano5, Josefina Freitas6, Cecilia Foncuberta5, Beatriz Moiraghi7, Carolina Pavlovsky8, Mariel Ana Pérez9, Verónica Ventriglia6, Julio Sanchez Ávalos5, José Mordoh1, Irene Larripa3 and Michele Bianchini1
1Centro de Investigaciones Oncológicas-Fundación Cáncer (CIO-FUCA), Ciudad Autónoma de Buenos Aires, Argentina
2Argenomics, Ciudad Autónoma de Buenos Aires, Argentina
3Instituto de Medicina Experimental, CONICET/Academia Nacional de Medicina, Ciudad Autónoma de Buenos Aires, Argentina
4Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA), CONICET, Partner Institute of the Max Planck Society, Ciudad Autónoma de Buenos Aires, Argentina
5Instituto Alexander Fleming, Ciudad Autónoma de Buenos Aires, Argentina
6Hospital Nacional Posadas, El Palomar, Buenos Aires, Argentina
7Hospital J. M. Ramos Mejía, Ciudad Autónoma de Buenos Aires, Argentina
8Fundaleu, Ciudad Autónoma de Buenos Aires, Argentina
9Hospital Interzonal General de Agudos, Prof. Dr. R. Rossi, La Plata, Buenos Aires, Argentina
Michele Bianchini, email: [email protected]
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
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.
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