Dose selection trial of metronomic oral vinorelbine monotherapy in patients with metastatic cancer: a hellenic cooperative oncology group clinical translational study

This was a multi-institutional randomized open-label phase IB trial conducted in 6 medical centers. Eligible patients were randomly assigned to receive oral vinorelbine tartrate (Navelbine® softgel capsules) at one of three predefined flat dose levels (30 or 40 or 50 mg) taken orally 3 times a week before lunch. Treatment continued until disease progression or occurrence of treatment related toxicity grade 3 or higher or patient’s decision or maximum 24 months treatment. The study was conducted in accordance with the Declaration of Helsinki and Scientific Committees of involved institutions approved the protocol.

Eligibility criteria were similar to those of the phase IA part of the trial [22]. Eligible patients had to sign an informed consent before participation. Eligibility criteria were as follows: age 16–75 years; performance status 0 to 2 according to the WHO scale; minimum life expectancy of 16 weeks; adequate bone marrow, hepatic and renal functions; absence of brain metastasis; metastatic and locally advanced hormonal refractory prostate, or previously treated metastatic breast cancer or non-small cell lung cancer previously treated with no more than two chemotherapeutic regimens; no other concurrent anticancer chemotherapy; serum creatinine within normal limits; hemoglobin of at least 10 g/L, white blood cell counts ≥3.5x10⁹/L; absolute neutrophil count (ANC) ≥1.5x10⁹/L; platelets ≥150x10^9/L; total serum bilirubin ≤1.5× upper normal limit (UNL); transaminases ≤ 2.0 × upper normal limit (UNL) unless attributed to liver metastases. Concurrent limited field radiation therapy (RT) and any previous RT was allowed. Exclusion criteria were the following: major active infection; more than two prior chemotherapy regimens for metastatic disease; chemotherapy administered within 28 days prior to start of metronomic vinorelbine; extensive liver metastases occupying more than half the liver; ongoing anti-coagulation therapy; pregnancy or breastfeeding and any of the following if occurred within 12 months prior to randomization: myocardial infarction, severe/unstable angina, coronary/peripheral artery bypass graft, congestive heart failure, cerebrovascular accident or transient ischemic attack, pulmonary embolism, cardiac dysrhythmias of grade >/= 2, atrial fibrillation of any grade or heart rate corrected QT interval (QTc) > 450 msec for males or > 470 msec for females, uncontrolled hypertension (> 150/100 mmHg despite optimal medical therapy). Patients with severe acute or chronic medical or psychiatric condition that in the judgment of the investigator would make the patient inappropriate for entry into the trial would also be excluded. Drop-off reasons included serious adverse events possibly related to the study drug, conditions requiring therapeutic intervention not permitted by the protocol and patients personal preference. The consort diagram of the study is shown in Figure 1.

Patients attended outpatient clinics every two weeks during he first two months on treatment and monthly thereafter for clinical assessment and blood sampling. For pharmacokinetic analysis, 5 ml whole blood samples were collected into EDTA tubes (Sarstedt, Germany) before treatment initiation and at follow-up visits before taking oral vinorelbine. Samples were stored at −20°C until analysis. Additional blood plasma and blood RNA were obtained prior to treatment, 4 weeks later and on a monthly schedule in consented patients in order to study circulating angiogenesis biomarkers. Both plasma and PAXgene RNA™ tubes (Qiagen, Thessaloniki, Greece) were stored at −80°C until analysis.

The primary clinical endpoint was time to treatment failure (TTF). TTF rates per treatment arm would be compared at 4 and 6 months. Secondary endpoints were progression-free survival (PFS), time-to-progression, toxicity, correlation of baseline blood concentrations of angiogenesis-associated surrogate markers with treatment efficacy measures, and pharmacokinetics.

Toxicity was evaluated according to the National Cancer Institute Common Toxicity Criteria V3. Acute toxicity was considered any adverse event that occurred during the first 8 weeks of treatment, while chronic toxicity was characterized any side effect that was recorded four months after the initiation of treatment. Toxicity that occurred between 8 weeks to 4 months of treatment was characterized sub-acute.

Treatment response was evaluated in patients that had completed at least 6 weeks of treatment and had at least one follow-up tumor assessment. Baseline tumor assessment was performed within 4 weeks prior to treatment initiation and thereafter every 2 months until documentation of response. Documented response should be confirmed after 4 weeks and should be regularly assessed every 4 months thereafter. Chest X-rays, computerized tomographic (CT) scans, ultrasound imaging studies and clinical measurements were used as appropriate. Response was documented using the RECIST response criteria for solid tumors [27] and Bubley Criteria for prostate cancer [28].

Plasma concentrations of basic fibroblast growth factor (FGF2), vascular endothelial growth factor-A (VEGFA), interleukin-8 (IL8) and thrombospondin-1 (TSP1) were determined by using commercially available quantitative sandwich enzyme immunoassays. In particular, Quantikine kits (R&D Systems, Inc., Minneapolis, USA) were used for FGF2, VEGF, VEGFR2 and IL8, and the ChemiKine Human TSP1 EIA Kit for TSP1 (Chemicon International, Inc., Temecula, CA, USA). Protocols, procedures, and equipment were used according to the manufacturer’s instructions. The lower detection limits for FGF2, VEGF, VEGFR2, IL8 and TSP1 were respectively 10, 31.2, 78.1, 31.2 and 9.8 pg/mL and the means for intra- and inter-assay coefficients of variation were 3.6 to 7.8 and 6.5 to 10.0, respectively. Optical densities were determined using a microfilter plate reader (DAS-A3, Roma, Italy) with filters for 450 nm (IL8, VEGF, VEGFR-2) and 490 nm (FGF2, TSP1). All analyses were carried out in duplicate. Samples available for each biomarker are shown in the REMARK diagram of Figure 2[29].

Peripheral blood was drawn in PAXgene tubes and kept frozen at −80°C. Total RNA was extracted with the PAXgene blood RNA kit, according to the manufacturer’s instructions (PreAnalytiX/Qiagen), within 6 months from collection. The additive contained in the PAXgene tubes reduces in vitro RNA degradation and minimizes in vitro gene induction [30]. RNA was extracted from 4 patients who either had an objective response (three patients) or TTF >6 months (1 patient) and 4 patients who relapsed early (within 2 months of the initiation of treatment). The analysis was done in mirrored samples: before treatment initiation and 4 weeks after treatment initiation. RNA integrity was checked by both conventional RNA electrophoresis and Agilent bioanalizer 2100. High quality RNA was reverse transcribed and analyzed on a PCR array platform (PAHS-024 F, SABiosciences) and the expression levels of 84 genes involved in angiogenesis were determined by using real time PCR.

Only RNAs with RNA integrity number (RIN) >7 were used for reverse transcription and further processing. For reverse transcription the SABiosciences RT² First Strand kit was used. In all cases RNA of 0.5 μg was reversed transcribed. Simultaneous quantification of 84 key genes involved in angiogenesis was done by using the angiogenesis RT² profiler PCR Array (PAHS-024 F, SABiosciences). Relative expression was determined with the Light Cycler 480 instrument (Roche) and the ΔΔCt method [31].

Whole blood samples were shipped on dry ice to the Institute de Recherché Pierre Fabre, Castres, France, for analysis. Concentrations of vinorelbine (VRL) and its main metabolite, 4-O-deacetylvinorelbine (DVRL) were quantified using a sensitive LC/MS/MS method previously reported [32].

Analysis was performed on an intent-to-treat basis. Regarding the definition of endpoints reported, time to treatment failure (TTF) was calculated as the time from random assignment to disease progression or death from any cause or early treatment discontinuation. Time to progression (TTP) was calculated as the time from random assignment to disease progression. Progression-free survival (PFS) was calculated as the time from random assignment to disease progression or death from any cause. Survival was calculated as the time from random assignment to death from any cause. Event-free patients at last contact were censored. Time-to-event distributions were estimated using Kaplan-Meier curves while the log-rank test was used for comparisons.

The Fisher’s exact test and the Jonckheere-Terpstra exact test were used to examine differences in toxicity rates between treatment groups. Association between biomarkers at baseline and response/activity (patients without objective response and/or progressing within 4 months vs those with objective response or PFS > 4 months vs healthy controls) was assessed with the Kruskall-Wallis test. Correlations among biomarkers were calculated using the Pearson’s correlation test. Significance was determined at the level of 5% (two-sided).

The sample size for this 3-arm randomized study was estimated under the assumption that the expected 4 and 6 month TTF rate for the 30 mg, 40 mg and 50 mg groups would be 10%, 30% and 50% and 5%, 20% and 40% respectively. Using a global chi-square test at the 2.5% level of significance (Bonferroni adjusted) the study had 80% power to reject the null hypothesis that all the arms had the same TTF rate [33].

Antitumor efficacy was seen at all dose arms. Confirmed partial remissions were documented in four cases of patients. Those were two NSCLC patients who received 30 mg (response duration 4 weeks) and 50 mg (response duration 100 weeks) one breast cancer patient treated at 40 mg (response duration 18 weeks) and one prostate cancer patient treated at 50 mg (response duration 30 weeks) (Figure 3). Eleven patients achieved a TTF longer than 6 months. No arm was found superior at the primary endpoint (Table 2, Figure 4). The median time to treatment failure was eight weeks in all three arms, while the 4 month TTF rate point estimates and 95% Confidence Intervals (CIs) for the 30 mg, 40 mg and 50 mg vinorelbine groups were 25.9% (11.1%-46.2%), 33.3% (15.6%-55.3%) and 18.2% (5.2%-40.3%) respectively. The p-value of the Fisher’s exact test for equality of the rates is 0.56. Similarly, the 6 month TTF rate point estimates and 95% CIs were respectively 22.2% (8.6%-57.7%), 8.3% (1.0%-27.0%) and 13.6% (2.91%-34.9%) for the 30 mg, 40 mg and 50 mg groups respectively. The p-value of the Fisher’s exact test is 0.39. In both cases the TTF rate did not vary statistically significant among the treatment arms. No differences were noticed among the three arms with regard to PFS and overall survival.

The side effects observed were generally mild and negligible. No differences in grade 3 and 4 acute toxicities were seen among the three dose levels. The only statistically significant difference observed was lymphopenia (worse in lower dose levels) and gastrointestinal (worse at dose level 40) (Table 3). Chronic toxicities did not occur.

Trough levels of vinorelbine were measured in 237 blood samples drawn from 44 consented patients (61% of treated patients) over a time that spanned duration of therapy from 2 to 36 weeks. Steady state concentrations were similar to those recorded in the phase IA trial with no evidence of accumulation over time (Figure 5). Mean values and standard deviation (SD) for the 30 mg dose was 1.8 ng/ml (SD 1.10), 2.2 ng/ml (SD 1.87) for the 40 mg dose and 2.6 ng/ml (SD 0.69) for the 50 mg dose. A dose proportional increase of steady state concentrations was noted but did not reach statistical significance (ANOVA p value = 0.5).

Of the investigated angiogenesis modulating proteins, baseline pre-treatment levels of FGF2 and IL8 were found to have a power to predict favorable response to metronomic vinorelbine (p value = 0.02 and 0.006 respectively). In particular, patients with baseline levels approximating those of healthy individuals had a chance to have a favorable outcome to therapy. VEGF and VEGFR2 baseline levels did not differ between patients with favorable versus non-favorable therapy outcome and TSP-1 data were inconclusive at a marginal p-value (Table 4).

Data analysis of circulating gene transcripts by using the ΔΔCt method revealed that TEK gene transcript was significantly up-regulated before treatment in non responders, while gene transcripts of SPHK1, CCL2, KDR, CCL1, FGF1, FGFR3, FIGF, HAND2, IFNA1, IGF1, MDK, MMP2, PLG were down-regulated about two fold and the CXCL9 gene was down-regulated 2.92 fold. FGF2 transcript post treatment initiation was found to be up-regulated in non responders and IL8, PGF, FIGF, IGF1 gene transcripts were down-regulated more than 2 folds (Figure 6).