Selective activity over a constitutively active RET-variant of the oral multikinase inhibitor dovitinib: results of the CNIO-BR002 phase I-trial

Abstract

Background: Given our preclinical data showing synergy between dovitinib and paclitaxel in preclinical models we conducted this phase I trial aiming to define the recommended phase II-dose (RP2D) on the basis of toxicity and pharmacodynamic criteria while searching for genetic variants that could sensitize patients to the regimen under study.

Patients and methods: A 3+3 escalation schedule was adopted. Seriated FGF23 and dovitinib and paclitaxel pharmacokinetic profiles were determined along a single-agent dovitinib "priming-phase" followed by a dovitinib + paclitaxel combination phase. RECIST 1.1 criteria and NCI CTCAE V.4.0 were used. In fresh pre-treatment tumor biopsy samples, FGFR1, 2 and 3 amplifications were revealed by FISH probes; 32 missense variants were genotyped in tumors and peripheral blood mononuclear cells with Taqman genotyping assays (FGFR1-3 and RET). Constructs encoding for wild-type and variant genes associated with clinical benefit were transfected into HEK-293 cells for preclinical experiments checking constitutive activation and dovitinib sensitivity of the variants.

Results: twelve patients were recruited in three dose-levels. At level 1B (200 mg dovitinib 5-days-on/2-days-off plus 60 mg/m 2-week of paclitaxel) more than 50% FGF23 upregulation was observed and no dose-limiting-toxicities (DLTs) occurred. The most frequent toxicities were asthenia, neutropenia, nausea/vomiting and transaminitis. Two patients with progressive disease prior to trial inclusion achieved prolonged disease stabilization. Both had the germline variant G2071A in the RET gene, which led to constitutive activation of the protein product and Y-905 phosphorylation, both in transfectants and in patients with the alteration. This variant was sensitive to dovitinib; in addition both patients experienced progression upon medication withdrawal.

Conclusions: Level 1B was the RP2D as it provided adequate pharmacodynamic exposure to dovitinib. The G2071A germline variant act as a genetic modifier that renders different tumors sensitive to dovitinib.

Keywords: Antiangiogenic; Clinical trial; Dovitinib; Phase I; RET genetic variant; Sensitizing genetic variant.

Figure 1

Treatment schedule. The screening included conventional assessments plus an image‐guided tumor biopsy (within 7 days before to the first dovitinib dose) and LVEF determination. Dovitinib was then administered orally for a week (“priming phase”); a pharmacokinetic profile was obtained on an inpatient basis on day 1. Blood samples for pharmacodynamic determinations were obtained at 0 and +24 h. After a 1 week “wash‐out” period, the treatment phase started with concurrent treatment of dovitinib + paclitaxel. A second pharmacokinetic profile (so that the paclitaxel‐induced modifications in dovitinib pharmacokinetics could be studied) including as well paclitaxel determinations, in order to compare the pharmacokinetics in combination with dovitinib with historical data of weekly paclitaxel (Fennelly et al., 1997), and two additional samples for pharmacodynamic determinations were obtained. The patients were discharged after 24 h and the treatment was continued with dovitinib administered orally on a schedule of 5‐days‐on/2‐days‐off in combination with weekly paclitaxel, in 28‐days cycles. Patients were visited and evaluated for toxicity weekly beginning with the first dose of dovitinib and until the end of the first cycle, bi‐weekly during the second and third cycles, and subsequently on a monthly basis. RECIST evaluations were performed every 2 cycles.

Figure 2

Pharmacokinetics and pharmacodynamics. (A) Dovitinib plasma concentration‐time profiles in the presence (solid‐line with squars) and absence (dotted‐line with triangles) of paclitaxel. (B) Average plasmatic FGF23 levels per timepoint (baseline, 24 h after the first dovitinib dose, before the first dovitinib dose of the combination phase and 24 h later) and dose levels. Error bars: standard error of the mean.

Figure 3

Activating RET variant sensitive to dovitinib. (A) Electropherogram showing the wild‐type sequence (G, left panel) and the variant sequence (A, right panel) in the 2071 position. (B) HEK‐293 cells were transfected with a wild‐type or a mutant‐RET‐encoding plasmid and blotted for anti‐905‐Y‐RET, in the presence and in the absence of dovitinib. It can be appreciated how the baseline levels of RET phosphorylation are highly increased in the mutant variant, despite similar total RET levels, whereas it is completely sensitive to dovitinib exposure. Virtually no residual phosphorylation of RET remains after incubation with 250 nM of dovitinib (2 h). (C) Western blot of anti‐905‐Y‐RET performed with lysates from tumor biopsy sample (only 5 biopsy samples yielded sufficient tissue after performing the FISH and mutational analysis). The two patients showing therapeutic benefit are those with the RET genetic variant, showing baseline increased phosphorylation. The numbers above the bands indicate how long (in months) the disease was controlled.