Jak-2 and Nfkbia Gene Expression Play a Strategic Role in Chronic Myeloid Leukemia (CML) Molecular Response during Early Nilotinib Treatment: The PhilosoPhi34 Data
Authors: Pungolino Ester , D'adda Mariella , Trojani Alessandra , Perego Alessandra , Elena Chiara , Iurlo Alessandra , Malato Simona , Turrini Mauro , De Canal Gabriella , Borin Lorenza , Lodola Milena , Caramella Marianna , Artale Salvatore , Spina Francesco , Latargia Maria Luisa , Anghilieri Michela , Spedini Pierangelo , Carraro Mariacristina , Di Camillo Barbara , Gramegna Doriana , Pioltelli Maria Luisa , Rossi Giuseppe , Morra Enrica , Cairoli Roberto 
Published: October 2019

Abstract
Background Targeted therapy with Tyrosine-Kinase-Inhibitors (TKIs) totally modified the course of treatment of Chronic Myeloid Leukemia (CML). The objectives and the needs of treatment have been modified during the last years and the discontinuation of therapy is now a feasible aim. However, a lot of biological data acquired in the last twenty years, showed that degree and mechanisms of Leukemic Stem Cells (LSCs) clearance during TKI treatment are not clearly established as well as the predictive criteria for a stable and prolonged Treatment Free Remission (TFR). The multicentre, prospective, single-arm PhilosoPhi34 study (EudraCT: 2012-005062-34) was designed by the Rete Ematologica Lombarda (REL), to verify the in-vivo activity and time-course of first-line Nilotinib (NIL) therapy on Bone Marrow (BM) CD34+/lin-Ph+ cells clearance. An exploratory Gene Expression Profiling (GEP) study of CD34+/lin- cells at diagnosis and at 12 months (mos) of treatment, for the first 30 evaluable pts, was included. Preliminary GEP data suggested a correlation between different NFKBIA expression at diagnosis and at 12 mos and the achievement of a deeper Molecular Response (MR) (Pungolino et al, AJH 2018).
We report here some results of GEP analysis on all enrolled evaluable pts and their possible correlation with clinical data.
Methods BM cells were collected and stored at diagnosis and at 12 mos of treatment. CD34+/lin- cells were purified with a Diamond CD34 Isolation Kit Miltenyi (97% of purity). For GEP analysis we used Affymetrix HG-U133 Plus 2.0 microarray and Genechip platform (Affymetrix) and the Affymetrix GeneChip Scanner 3000. Data was pre-processed and normalized using the Robust Multi-array Average (RMA) algorithm. The Significant Analysis of Microarrays (SAM) was used to identify genes with statistically significant changes in expression. P-values were corrected for multiple testing using false discovery rate, for differentially expressed genes confirmation. We chose to analyse different expression of NFKBIA (the inhibitor of NF-kB onco-gene) in order to confirm the preliminary data reported on the first 30 analysed pts. Pts were monitored according to ELN-recommendation. Biological data were correlated with MR at 3, 12 and 36 mos of therapy. We use Fishers test to compare unbalanced group.
Results Out of the 87 enrolled pts, 80 completed the first 12 mos of treatment and 78 (1 failure and 77 CCyR) were evaluable for GEP analysis. We observed 2726 genes symbol differentially expressed of which 1868 are coding genes. Among these, JAK-2 showed a down regulation at 12 mos (p: .024). JAK-2 expression ranged from 2.62 to 4.95 at diagnosis and from 1.48 to 5.58 at 12 mos. Only 26/78 pts increased JAK-2 expression that was > 4 in 1/26 pts, at diagnosis; 2/26 (7.69%) pts showed a H Sokal. Other 52/78 pts decreased JAK-2 expression that was ≥ 4 in 21/52 pts, at diagnosis; 10/52 (19.23%) pts and 6/21 (28,57%) pts showed a H Sokal. Similarly, when we compared low JAK-2 expression (< 3.5) vs vary high expression (≥ 4) 2/21 vs 6/22 pts had H Sokal (9.52% vs 27.27%; p: .0057).
Considering the role of JAK-2 and NFKBIA in cell regulation and survival, we evaluated how the combination of their different expression impact on MR (i.e. NFKBIA increased expression/JAK-2 decreased expression vs NFKBIA decreased expression/JAK-2 increased expression). Data are reported in Table 1 and 2.
Conclusion GEP analysis showed a down regulation of JAK-2 expression after 12 mos of first line NIL treatment, in 78 early chronic phase CML pts. Data suggest that high expression of JAK-2, at diagnosis, correlate with H Sokal score. However, H Sokal pts with a JAK-2 down regulation, obtain during treatment similar MR compared to L Sokal pts. Additionally, the study confirms our preliminary observation on 30 pts , concerning the role of NKBIA up - regulation in increasing percentage of earlier and deeper MR . The better condition of NFKBIA and JAK-2 expression (up regulation of NFKBIA and down regulation of JAK-2) is associated with a higher percentage of early MR3 and optimal responses over time, despite the higher number of H Sokal pts in this group. A study with NIL as first line treatment combined with low dose of JAK-2 inhibitor and a natural inhibitor of NF-kB (such as curcuma), during the first year of treatment, to increase the deeper MR rate and the probability of TFR is warrented.

Disclosures
Rossi: Sandoz: Honoraria; Jazz: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria; Teva: Membership on an entity's Board of Directors or advisory committees; Mundipharma: Honoraria; Novartis: Honoraria; Pfizer: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees.