Alessandra Perego

Wide-transcriptome analysis and cellularity of bone marrow CD34+/lin- cells of patients with chronic-phase chronic myeloid leukemia at diagnosis vs. 12 months of first-line nilotinib treatment

BACKGROUND Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder with heterogeneous biological and clinical features. The biomolecular mechanisms of CML response to tyrosine-kinase inhibitors are not fully defined. OBJECTIVE We undertook a gene expression profiling (GEP) study of selected bone marrow (BM) CD34+/lin- cells of chronic-phase CML patients at diagnosis and after 12 months of TKI nilotinib to investigate molecular signatures characterizing both conditions.

Nilotinib induced bone marrow CD34+/lin-Ph+ cells early clearance in newly diagnosed CP-chronic myeloid leukemia

To the Editor: Chronic myeloid leukemia (CML) is a clonal disorder characterized by Philadelphia (Ph) chromosome and/or BCR/ABL tyrosine-kinase (TK) presence. Target therapy with imatinib has greatly improved its outcome while deeper and faster responses are reported with the secondgeneration TKIs dasatinib and nilotinib. Sustained responses may enable TKI discontinuation. However, a fraction of treatment-free remission patients, including those in long-term complete molecular response (MR), may experience disease recurrence possibly due to persistence of quiescent leukemic precursor cells [ie, leukemic progenitor cells (LPCs) CD341/CD381/lin– and leukemic stem cells (LSCs) CD341/CD38–/lin–]. Time-course and mechanisms of LPCs/LSCs clearance during TKI treatment have not been clearly established yet, although in vitro data suggests that quiescent LSCs are indeed not sensitive to BCR/ABL inhibition. In vivo preliminary data suggest that residual CD341 Ph1 cells are very rarely detected in nilotinib-treated patients in complete cytogenetic response (CCyR), compared to imatinib-treated ones. Moreover, in five patients in CCyR at 3 months of nilotinib-treatment, undetectable CD341 Ph1 cells were reported. Despite the very limited number of patients reported, these in vivo data suggest that the rapid inhibitory activity of nilotinib on CML burden may affect stem cells as well. The PhilosoPhi34 (EudraCT: 2012–005062-34) is a multi-center, prospective single-arm study conducted on behalf of the Rete Ematologica Lombarda (REL). Its primary endpoint was to evaluate the rate of residual CD341/ lin–Ph1 cells in the bone marrow (BM) of CCyR patients after 6 months of nilotinib-treatment by cell selection system and fluorescence in situ hybridization (FISH) analysis. Secondary endpoints evaluated were: clearance of CD341/ lin–Ph1 cells in the BM of CCyR patients at 3 and 12 months of treatment; CCyR rate and the MRs in peripheral blood (PB) at 3, 6, and 12 months of treatment, respectively. The protocol design also included an exploratory study aimed at evaluating the Gene Expression Profiling (GEP) of selected CD341/ lin– cells at diagnosis and at 12 months of treatment using Affymetrix GeneChip Instruments and Software Systems, and Affymetrix GeneChip Human Genome HTA 2.0. This exploratory study was planned to initially include 30 consecutive patients. A total of 87 patients were enrolled and their median time onstudy was 26 months at data analysis. Of the enrolled patients, two discontinued nilotinib by 3 months of treatment while five additional patients discontinued treatment between 6 and 8 months (see Supporting Information Table 2). Of the 84 6-month evaluable samples, 79 showed CCyR and 78 were adequate for FISH analysis; 7 negative tests were excluded since less than 200 nuclei were analyzed. Only 5/71 (7%; CI 95%: 2.3%15.7%) evaluable FISH tested positive. Of the 84 3-month evaluable samples, 76 showed CCyR and 75 were adequate for FISH analysis (10 negative tests excluded); only 8/65 (12.3%; CI95%: 5.5%-22.8%) evaluable FISH tested positive. Of the 80 12-month evaluable samples, 79 showed CCyR and 78 were adequate for FISH analysis (9 negative tests excluded); none of the 69 evaluable FISH tested positive (0.0%; CI95%: 0.0%-5%) (Figure 1A). The 3-month MR of the FISH-positive patients (at 3 and 6 months) tested 0.175% international scale (IS). Evaluation of Response to Treatment. Five of 87 patients failed treatment; none of them progressed to an accelerated or blastic phase; 2 harbored a mutation. Intention to treat (ITT) cytogenetic analysis was as follows: CCyR 77/87 patients (88.5%) at 3 months; CCyR 79/87 patients (90.8%) at 6 months; CCyR 77/87 patients (88.5%) at 12 months. Figure 1B summarizes the ITT and per protocol (PP) rate of MRs at the different time points. Gene Expression Profiling of CD341/LIN– Cells. Gene expression profiling (GEP) experiments, for details see Ref. 5, were conducted on the first 30 evaluable patients enrolled. Since the number of selected CD341/lin– cells collected at diagnosis and at 3, 6, and 12 months showed two opposite trends, the first GEP experiments was performed on the CD341/lin– cells stored at diagnosis. Experiment 1. Patients were grouped according to CD341/lin– cell trends: class 1 (n524) showed highly reduced levels of CD341/ lin– cells while class 2 (n56) demonstrated increasing levels of CD341/lin– cells at both 3 and 6 months. Bioinformatics analysis was performed: 56 transcripts could be selected which differed between the two classes (Supporting Information Table 4). Of note, a large part of the transcripts identified have not been characterized yet; 31/56 transcripts were located on chromosome 15. Among the transcripts with known function, we focused on the NFKBIA gene for its biological relevance in the regulation of apoptotic pathways and cell proliferation. Sample analysis showed an over expression of NFKBIA in class 1 patients, ie those showing decreasing number of C341/lin– cells over time.