N-GAL Expression Correlates with Disease Status in Chronic Myelogenous Leukemia (CML).
Authors: Villalva Claire , Sorel Nathalie , Bonnet Marie-Laure , Guilhot Francois , Chomel Jean-Claude , Turhan Ali G. 
Published: April 2020

Abstract
N-GAL (neutrophil gelatinase associated lipocalin) is a secreted protein constitutively expressed in the granulocytic lineage and induced in epithelial cells during inflammatory and neoplastic processes. In the hematopoietic lineage N-GAL has been shown to induce a negative regulation of erythropoiesis and associated with the stimulation of differentiation of the granulocytic lineage. Its expression has been shown to be increased by BCR-ABL in murine models (Lin et al, 2005) and in a TET-inducible BCR-ABL-expressing cell line (Flamant et al, 2005). In order to determine the relevance and significance of N-GAL expression in primary CML cells, we have evaluated its expression by real time quantitative PCR (RTQ-PCR) in CML patients at diagnosis before any treatment (n=28) and in patients with complete molecular response (n= 30) as compared to normal marrow donors used as controls (n=10). We performed the same analysis in patients with accelerated phase of their disease (n= 17) and exhibiting an imatinib-mesylate resistant disease (n= 27). The expression of N-GAL measured by RTQ-PCR, was normalized as compared to ABL expression in each patient and this value was normalized with regard to a N-GAL / ABL value obtained from 10 normal controls, allowing to calculate a N-GAL patient / N-GAL control (P/C) ratio for each sample. Some patients have also been studied using sequential analyses. N-GAL Patient / Control ratio was found to be highly increased in all CML patients at diagnosis with a mean value of 20 (Range 1 – 43). This major increase did not correlate with leukocyte counts. Similarly, the N-GAL expression did not correlate with BCR-ABL expression levels nor with hemoglobin values and platelet counts. In patients with molecular remission, the N-GAL Patient / Control ratio was found to be highly reduced as compared to the levels observed at diagnosis with a mean value of 0.28 (Range 0.07 – 1.47 n = 30). In 17 patients with accelerated phase of their disease, the N-GAL P/C ratio was increased to a mean value of 2.4 (0.01– 10) and it was found to be further increased in patients with an imatinib-mesylate resistant disease (mean N-GAL P/C ratio = 9.25; Range 0.03 – 44 n = 27). In 13 of these 27 patients, an ABL-kinase domain mutation was present including M351T (3 patients), E255K (2 patients), M244V (4 patients), F359V (1 patient), G250E (1 patient), V379I (1 patient) and T315I (1 patient). N-GAL P/C ratio was also measured sequentially in 4 patients, allowing the study of the effects of imatinib mesylate-induced remissions on the expression of N-GAL over a period of 1–3 years. In three patients, Imatinib-induced remissions were accompanied by the reduction of N-GAL P/C ratios by 2–3 logs, whereas in the 4th patient, who exhibited imatinib-resistance this ratio remained high and slightly increased despite a remission induced by dasatinib. In conclusion, N-GAL expression could represent a surrogate marker of CML disease status and its role in the pathophysiology of CML phenotype as well as its possible role in progression warrants further study. Current experiments will correlate the N-GAL P/C ratio with disease parameters in a larger series of patients to determine the significance of high levels of N-GAL expression in CML at diagnosis, the expression of N-GAL in purifed cell populations and its role as a progression / IM-resistance marker.