REL-Protocol PhilosoPhi34: An Open Label, Single Arm, Phase II Study of Nilotinib 300 Mg BID in Newly Diagnosed Chronic Phase Chronic Myeloid Leukaemia Patients, to Study the Disappearance of CD34+/Lin-Ph+ Cells from Bone Marrow during Treatment. Preliminary Data

Background. Chronic Myeloid Leukaemia (CML) can be effectively treated with the first generation Tyrosine Kinase Inhibitor (TKI) Imatinib, and more effectively with the second generation TKI, like Nilotinib. However, despite the deeper and faster responses induced by nilotinib in a large proportion of patients, the possible eradication of the pathological stem cells is not yet clearly elucidated. In fact, in vitro data suggest that quiescent stem cells are not sensitive to Bcr/Abl inhibition (Corbin AS, et al 2011; Hamilton A, et al 2012). A preliminary in-vivo study (Defina M, et al 2012) shows that in patients in CCyR even after short-term of nilotinib therapy, residual leukemic progenitors are rarely detected.

Methods. On behalf of Rete Ematologica Lombarda (REL), Italy, we designed a single arm prospectic study, PhilosoPhi34 (EudraCT: 2012-005062-34), with the aim to investigate the efficacy of nilotinib 300 mg BID in obtaining the disappearance of Bone Marrow (BM) leukemic stem cells (CD34+/lin-Ph+) in newly diagnosed CP-CML.

Primary objective of the study: to enumerate the BM CD34+/lin-Ph+ cells at the end of 6 months of treatment.

Secondary objectives: to enumerate the BM CD34+/lin-Ph+ cells at 3 and 12 months; to assess the percentage of patients showing MR ≤10% IS at 3 months and MR ≤1% IS at 6 months and the MMR IS and MR4.5 IS by 3, 6 and 12 months of treatment.

BM blood  samples (range of 5-20 ml) were collected at diagnosis and after 3, 6 and 12 months of nilotinib treatment. BM mononuclear cells were purified by density gradient centrifugation and then CD34+/lin- cells were isolated using Diamond CD34 Isolation Kit (Miltenyi Biotec). The purity of CD34+/lin- cells was about 97% as determined by flow cytometry. BM CD34+/lin- cells were counted and a range of 100,000-800,000 has been noted at diagnosis.

After the treatment we observed that the number of CD34+/lin- cells dramatically decreased after 3 (1,000-600,000), 6 (1,000-260,000) and 12 months (100-130,000). In particular, CD34+/lin- cells were even less than 1000 at 12 month of treatment.

In order to verify the disappearance of leukemic stem cells, isolated CD34+/lin- cells were tested by standard FISH (i.e. to categorize a sample as negative at least 200 nucleus were examined).

From April 2013 and June 2015 we enrolled 87 pts, as for protocol. We report here the preliminary results.

Results. Of 56 patients in CCyR after 6 months of treatment, FISH performed on BM CD34+/lin- cells nuclei was evaluable in 51 cases (5 negative cases were excluded because of less than 200 nucleus were analysed). In 4 out of 51 patients (7.8%), Ph+ nuclei were detected. The Sokal score of these 4 patients was 1 low, 2 intermediate and 1 high risk with a ratio (positive nuclei/total nuclei) of 295/300, 1/200, 2/92, 3/300, respectively. Among 58 patients tested at 3 months and 44 tested at 12 months of treatment, the number of evaluable patients was 48 and 37, respectively; 8/48 (16.6%) and 0/37 (0%)patients showed Ph+ nuclei. Only 2 out of 8 positive patients had a high Sokal score.

Regarding efficacy of treatment, Table 1 summarizes the MRs IS observed after 3, 6 and 12 months of treatment in 71, 57 and 41 patients, respectively.

Conclusion. Data of this prospective study confirms that nilotinib 300 mg BID, rapidly and progressively induces the clearance of BM CD34+/lin-Ph+ cells in CP-CML patients. In particular, on CD34+/lin- cells, after 6 months of treatment, only 7.8% of patients showed positive nuclei. On 37 patients after 12 months of treatment, no positive nuclei were detected. So far, the kinetic of reduction of such cells seems not influenced by Sokal score.

According to international studies, PhilosoPhi34 shows a very high efficacy of Nilotinib to induce MRs in CP-CML patients, at the standard time points.

Table 1. Molecular Response (MR) in CP-CML patients treated with Nilotinib 300mg BID from diagnosis.

MR IS	3 months	6 months	12 months
≤10%	67/71    94%	57/57         100%	40/41       97.50%
≤1%	57/71    80%	55/57       96.50%	40/41       97.50%
≤0.1%	17/71    24%	41/57           72%	35/41           85%
≤0.01%	3/71        4%	19/57            33%	20/41       48.70%
MR4.5(UD)	2/71      2.80%	10 (6)/57  17.5% (10.5%)	15 (9)/41 36.5% (22%)

UD: undetectable

We acknowledge all REL Colleagues for their  collaboration and Novartis SpA for the partial financial support to the study.