In the preclinical setting, phosphorylation and subsequent proteosomal degradation of the proapoptotic protein BIM confers resistance to paclitaxel in solid tumors with RAS/RAF/MAPK pathway activation. Concurrent administration of the proteasome inhibitor bortezomib enables paclitaxel-induced BIM accumulation, restoring cancer cell apoptosis in vitro and producing tumor regression in mice in vivo. A phase I study was conducted to determine the maximum tolerated dose (MTD) of paclitaxel and bortezomib combinatorial treatment. Sixteen patients with refractory solid tumors commonly exhibiting mitogen-activated protein kinase (MAPK) pathway activation were treated weekly with paclitaxel and bortezomib. Starting doses were 40 mg/m2 for paclitaxel and 0.7 mg/m2 for bortezomib. A modified continual reassessment method adapted for 2-drug escalation was used for MTD determination with 3-patient cohorts treated at each dose level. MTD was reached at 60 mg/m2 paclitaxel and 1.0 mg/m2 bortezomib, the recommended phase II dose. Therapy was overall well tolerated. Most frequently observed toxicities included anemia (in 43.75% of patients, one grade 3 event), fatigue (in 43.75% of patients, one grade 3 event beyond cycle 1), and neuropathy (in 31.25% of patients, one grade 3 event after cycle 1). Of 15 evaluable patients, one non–small-cell lung carcinoma (NSCLC) patient with paclitaxel exposure at the adjuvant setting had a partial response and five patients had stable disease (SD); median disease stabilization was 143.5 days; three NSCLC patients had SD lasting 165 days or longer. Thus, rationally designed weekly treatment with paclitaxel and bortezomib in solid tumors with MAPK pathway activation, including previously taxane-treated malignancies, is a tolerable regimen with preliminary signals of antitumor activity worthy of further investigation. Mol Cancer Ther; 10(8); 1509–19. ©2011 AACR.
Paclitaxel, a microtubule-stabilizing agent causing late G2–M cell-cycle arrest followed by apoptotic cell death, is commonly used for solid malignancy treatment. Similar to other chemotherapeutic agents, cancer cell resistance to paclitaxel occurs frequently (1), many times because of acquired apoptosis resistance, which provides malignant cells with a survival advantage, thus, compromising cancer therapy (2, 3). Understanding the mechanisms of paclitaxel-induced programmed cell death and by which tumors evade this process is critical for pharmacologic reactivation of cancer cell apoptosis and clinical benefit.
Apoptosis is controlled by antiapoptotic BCL-2 family proteins and proapoptotic BAX, BAK, and BH3-only proteins. Highly stressful stimuli initiate apoptosis through BH3-only proteins, such as BIM, by inhibiting BCL-2–like proteins or activating BAX and BAK, resulting in cell death by mitochondrial outer membrane permeabilization (4). Upregulation of antiapoptotic and/or downregulation of proapoptotic proteins influences tumorigenesis and treatment response; thus, deciphering involved mechanisms may guide rational therapy design.
Earlier in vitro and in vivo animal studies indicated that epithelial tumor resistance to paclitaxel was conferred by RAS/RAF/mitogen-activated protein kinase (MAPK) pathway activation causing phosphorylation, proteosomal degradation, and thus inactivation of the BH3-only proapoptotic protein BIM, which is a major determinant of cell sensitivity to paclitaxel (5). In a baby mouse kidney epithelial cell model (5), paclitaxel induced selective BIM accumulation, in turn promoting cancer cell apoptosis in vitro and in allograft tumors in mice in vivo. Responsiveness to paclitaxel specifically depended on BIM and not other proapoptotic proteins as shown by BIM deficient, but not wild-type or other BH3-only-deficient, cell resistance to paclitaxel. Constitutive RAS/RAF/MAPK pathway activation suppressed BIM induction by phosphorylating BIM and targeting it to proteasomes for degradation, thus causing cancer cell resistance paclitaxel-induced apoptosis. In cancer cells and tumors with RAS/RAF/MAPK pathway activation treated with paclitaxel and a proteasome inhibitor combination, BIM degradation was blocked and paclitaxel-dependent BIM accumulation and apoptosis were restored, as manifested by cancer cell death in vitro and tumor regression in animal studies.
The preclinical data described above suggested that paclitaxel and proteasome inhibitor combination may be therapeutically beneficial against malignancies with activated RAS/RAF/MAPK pathway, such as pancreas, colon, lung, ovarian, thyroid, breast, and prostate cancers (6–8), as well as malignancies traditionally treated with paclitaxel and those exhibiting paclitaxel resistance (9, 10). Independent preclinical studies confirmed the importance of BIM accumulation for drug-induced apoptosis in different tumor types (11–14). We, thus, decided to clinically investigate combining paclitaxel with the proteasome inhibitor bortezomib for restoration of BIM levels in tumors with activated RAS/RAF/MAPK pathway (5) and potentiation of paclitaxel-induced cancer cell apoptosis to enhance tumor regression and improve clinical response.
Paclitaxel and bortezomib combination was previously explored in several small studies. In a National Cancer Institute-sponsored, multicenter phase I clinical trial, bortezomib was escalated in combination with paclitaxel at 100 mg/m2. Paclitaxel given on days 1 and 8 in combination with bortezomib at 1.8 mg/m2 on days 2 and 9 showed acceptable toxicity and disease stabilization in 7/44 (16%) patients, including 3 patients with advanced pancreatic cancer (15). An earlier study compared 2 different administration schedules with schedule A patients receiving paclitaxel and carboplatin on day 1 followed by bortezomib on days 2, 5, and 8, and schedule B patients receiving bortezomib on days 1, 4, and 8 with paclitaxel and carboplatin combination administered on day 2. Toxicities were primarily hematologic, and schedule B patients showed improved responses. A phase II dose of bortezomib at 1.3 mg/m2, carboplatin at area under the concentration-time curve (AUC) 6, and paclitaxel at 135 mg/m2 was recommended (16); however, a subsequent phase II trial of the same drug combination and schedule B, but using a higher paclitaxel dose, was terminated because of insufficient clinical activity and significant associated toxicity (17). A third study escalated both bortezomib (on days 1, 4, 8, and 11 of a 3-week cycle, starting at 0.7 mg/m2) and paclitaxel (on days 1 and 8, starting at 80 mg/m2) and resulted in a 30% partial response (PR) rate, which was considered similar to that of single agent paclitaxel and was accompanied by significant peripheral neuropathy (76% of patients; grades 3 and 4 in 9%) that precluded further clinical development (18).
We now report the findings of a rationally designed, single center phase I study of weekly paclitaxel and bortezomib combination in patients with advanced and refractory solid tumors commonly exhibiting MAPK pathway activation using an adaptive dose-finding approach. In our trial, the 2 drugs were given on the same day weekly, in a schedule different than those used in earlier studies and expected to more actively induce BIM-mediated tumor cell apoptosis, as our in vitro and in vivo preclinical data (5) argued for concurrent, rather than sequential, drug administration. Despite earlier reports of antagonistic effects and decreased prostate cancer cell apoptosis upon simultaneous exposure to bortezomib and paclitaxel (19), concurrently given drugs clearly showed killing synergy in baby mouse kidney cancer cell lines and allograft tumors with RAS/RAF/MAPK pathway activation (5). Therefore, a phase I clinical trial of paclitaxel and bortezomib combination for the treatment of tumors commonly exhibiting RAS/RAF/MAPK pathway activation was rationally designed to identify the maximum tolerated dose (MTD) of weekly treatment using a modified continual reassessment method (MCRM; refs. 20–23) adapted for 2-drug escalation. Correlative studies determined BIM protein level changes in peripheral blood mononuclear cells (PBMC) during treatment as a surrogate marker for intratumoral treatment-induced BIM expression alterations. Cancer cell RAS/RAF/MAPK pathway activation was evaluated by phospho-p44/42 MAPK immunohistochemistry (IHC) on archived tumor biopsies. Finally, paclitaxel plasma concentrations in bortezomib presence were compared with historical pharmacokinetic (PK) data for this drug.
Materials and Methods
Patients were eligible if older than 18, with Eastern Cooperative Oncology Group (ECOG) performance status 0–2 and histologically confirmed malignancy that was metastatic or unresectable and for which no standard curative or palliative measures existed. Entry was restricted to patients with solid tumors commonly exhibiting RAS/RAF/MAPK pathway activation, that is, pancreas, non–small-cell lung carcinoma (NSCLC), melanoma, colon, breast, prostate, ovary, and papillary thyroid cancer (6, 7). Unlimited number of prior chemotherapies and past paclitaxel or bortezomib treatment were also allowed. Adequate organ function was required: white blood cell ≥ 3,500/μL and absolute neutrophil count ≥ 1,500/μL, platelets ≥ 100,000/μL, creatinine ≤ 2× the upper limit of normal (ULN), total bilirubin ≤ 1.5× ULN, aspartate, and alanine aminotrasnferases ≤ 2.5× ULN (or ≤ 5× ULN, if tumor-involved liver). Patients with untreated or uncontrolled brain metastases, active infections, significant comorbid cardiac disease, neuropathy common terminology criteria for adverse events grade 1 or above with pain within 14 days, or having received other anticancer treatment within 4 weeks before enrollment were excluded. Prophylactic use of antiemetics, antidiarrheals, and other supportive care measures was allowed; growth factor support was allowed after cycle 1 at treating physician's discretion. The Cancer Institute of New Jersey (CINJ) Institutional Review Board approved the study protocol, and all patients signed informed consent before treatment initiation.
Study design and treatment
This was a phase I study of paclitaxel and bortezomib given on days 1, 8, and 15 of a 21-day cycle with primary objective to identify this combination's MTD. Starting paclitaxel dose (40 mg/m2) was half of the maximum Food and Drug Administration-approved dose for weekly paclitaxel treatment of ovarian and metastatic breast cancer. Starting bortezomib dose (0.7 mg/m2) was shown to effectively inhibit proteasome function (24) and was the lowest dose used in bortezomib monotherapy when peripheral neuropathy symptoms necessitated dose reduction (25). Dose limiting toxicities (DLT) were defined as any grade 3 or 4 treatment-related nonhematologic toxicity (except nausea and vomiting occurring in the absence of antiemetic regimen and elevated alanine and/or aspartate that decreased to grade 2 or lower within 1 week) or grade 4 hematologic toxicity during treatment or within 1 week of treatment completion and (i) lasting more than 7 days or (ii) resulting in omission or delay of 2 or more drug doses in a cycle because of toxicity or (iii) resulting in toxicity-associated cycle initiation delay beyond 2 weeks.
MTD was determined by an MCRM (20–23) adapted for 2-drug escalation and taking into account all treatment-related toxicities grade 2 and above (grade 1 with pain and above for neurotoxicity) that could be intolerable if sustained, rather than solely the DLTs. 40 to 80 mg/m2 and 0.7 to 1.3 mg/m2 were considered the useful paclitaxel and bortezomib ranges, respectively, when both drugs were given weekly. Dose toxicity relationships in the paclitaxel and bortezomib ranges were assumed similar and a standardized effective dose (SED) approach was used. Actual paclitaxel and bortezomib dose ranges were standardized to a 10 to 100 range, that is, linear transformations were used so that SED of 10 represented paclitaxel 40 mg/m2 or bortezomib 0.7 mg/m2 and SED of 100 represented paclitaxel 80 mg/m2 or bortezomib 1.3 mg/m2. The paclitaxel and bortezomib combination SED was the sum of paclitaxel and bortezomib SEDs. With starting doses of bortezomib at 0.7 mg/m2 and paclitaxel at 40 mg/m2 (i.e., combination SED of 20), cohorts of 3 patients were treated at each dose level. Subsequent bortezomib and paclitaxel combination SED levels were determined by the CINJ Biometrics Department using MCRM with a 2-parameter logistic model adaptation. The 2 drugs were assumed equally important, hence both were modified by the same dose change percentage to determine subsequent SED doses, which were then reviewed and, if necessary, adjusted by the principal investigator on the basis of clinical and safety factors. SEDs of 10 and 200 were assumed to have DLT rates of 20% and 98%, respectively, and the target toxicity level (probability of treatment-related DLT at the MTD) was set at 25%. With these assumptions, the DLT rate at the starting dose level (SED of 20) was 25%. Starting at the presumed MTD is one of the reasons that MCRM is more efficient than the traditional 3 + 3 design in reaching actual MTD. The above assumptions initiated the MCRM fitting process and were modified or discarded as more outcome data became available. For subsequent SED level calculations, ordinal values of 0, 0, 0.2, 1, and 1 were used to represent grades 0, 1, 2, 3, and 4 drug-related toxicities, respectively, except for neuropathy, which was given greater weight (values of 0.5 and 1 were used for grade 1 with pain or grade 2 toxicities and grade 2 with pain or grades 3 or 4 toxicities, respectively). If no grade 3 hematologic toxicity or a DLT was observed, the maximum dose escalation was no more than 75% of the previous SED level. Upon grade 3 hematologic toxicity or a DLT, the maximum dose escalation was no more than 50% of the previous SED level. Process continued until 25 patients entered the study or changes in the next recommended SED were no more than 10% for 2 consecutive cohorts, whichever came first.
Toxicities were evaluated and graded using National Cancer Institute Common Terminology Criteria version 3.0. A history, physical examination, chest X-ray, electrocardiogram, and laboratory tests, including a complete blood count with differential, chemistry, and urinalysis, were obtained at baseline. A complete blood count was drawn weekly during cycle 1. In subsequent cycles, the complete blood count and chemistry were obtained before cycle initiation. Baseline and restaging imaging studies were done within 4 weeks before study enrollment and every 2 cycles thereafter, respectively. Response was assessed using Response Evaluation Criteria in Solid Tumors (26).
PK sampling, assay, and analysis
Blood specimens (5 mL) were collected in heparinized tubes immediately before and at 1, 2, 3, 4, 5, 6, 7, 8, 27, 51, and 75 hours after the start of paclitaxel infusion on day 1 of cycle 1; centrifuged at 1,000 × g for 10 minutes at 4°C; plasma was separated and stored at −80°C until further analysis. Paclitaxel concentration in plasma was determined by high-performance liquid chromatography (HPLC) by using modifications of a previously described method (27). Calibration plasma standards and patient samples (0.5 mL each) were spiked with Baccatin III (100 ng), extracted in acetonitrile (5 mL), and evaporated to dryness. Extracts were reconstituted in acetonitrile/water (80:20, 1 mL), transferred to microfuge tubes, and dried under nitrogen. Residues were reconstituted in water/acetonitrile (1:1, 400 μL), centrifuged at 18,000 × g for 12 minutes at 4°C. Clear supernatant (250 μL) was injected into an HPLC system consisting of Hitachi L-7000 series ultraviolet detector, autosampler, and quaternary gradient solvent delivery pump. Analytes were isolated by using Luna PFP column (4.6 × 250 mm, 5 μL; Phenomenex), maintained at 37°C, and analyzed in a mobile phase gradient of water and acetonitrile at the following ratios and times: 61:39, 0–12 minutes; 61:39 to 53:47, 12–30 minutes; 53:47 to 61:39, 30–45 minutes, at a flow rate of 0.8 mL/min, detector set at 229 nm. Paclitaxel concentrations in standards and samples were calculated using least-square linear regression analysis of paclitaxel and internal standard peak height ratios over nominal concentrations. The PK parameters, AUC, total body clearance (CL), peak plasma concentration (Cmax), time to maximum concentration (Tmax), half-life (T½), and volume of distribution (Vz), were determined using a noncompartmental model with WinNonlin 2.1 software (Pharsight Corp.). AUC was estimated by the log-linear trapezoidal method up to the last measurable concentration (Clast). CL was calculated by dividing dose by AUC.
Pharmacodynamic studies: treatment-induced BIM expression changes in PBMCs
Blood samples (8 mL) before and at 27 and 51 hours after paclitaxel infusion start on day 1 of cycle 1 were collected in cell preparation tubes tubes (BD-Vacutainer Cell Preparation Tubes), and mononuclear cells were prepared according to the manufacturer's instructions. Cells were washed with phosphate-buffered saline and stored at −80°C. Upon request, cells were thawed, lysed in 0.15 mol/L NaCl, 0.01 mol/L Tris-HCl, pH 7.4, (with pepstatin, leupeptin, DTT, protease inhibitors, and phosphatase inhibitor cocktails 1 and 2), mixed with Laemmli sample buffer, boiled at 95°C for 5 minutes, and loaded on 12% SDS-PAGE followed by transfer to nitrocellulose membranes. As a positive control, MCF-7 breast cancer cells were prepared and lysed similarly to PBMCs. BIM immunoblotting was carried out by using a rabbit monoclonal antibody (C34C5, Cell Signaling Technology; refs. 11, 28).
Tumor evaluation for RAS/RAF/MAPK pathway activation
RAS/RAF/MAPK pathway activation was evaluated in paraffin-embedded archived tumors by phospho-p44/42 MAPK IHC using a rabbit polyclonal antibody (#4377; Cell Signaling Technology) as previously described (29). Protein expression was manually and independently evaluated by 2 study investigators, Drs. E. White and V. Karantza, and was considered positive when more than 5% of tumor specimen exhibited at least low (1+) to moderate (2+) staining intensity.
Between April 2007 and March 2008, 16 patients, more commonly with NSCLC and colon cancer, were enrolled in study (Table 1). In total, 44 treatment cycles were administered with a median number of 3.3 cycles per patient. Seven patients had been previously treated with taxane-containing regimen (6 with paclitaxel; 1 with docetaxel). No patient had prior bortezomib exposure. Patient study participation ended because of disease progression (15) or consent withdrawal (1).
Treatment-related toxicities and MTD
Table 2 summarizes drug doses and toxicities observed at all dose levels during cycle 1. The initial cohort was expanded to include 4 patients, as 1 patient died of disease progression during cycle 1 and was thus nonevaluable for tumor response; however, toxicity data were included in MCRM. At the first dose level, treatment-related grade 2 anemia (2 events) and grade 2 fatigue (1 event) were reported, but no DLTs, and combination SED was increased by 10% to paclitaxel 45 mg/m2 and bortezomib 0.75 mg/m2. At the second and third dose levels, no treatment-related grade 2 or above toxicities were observed, hence combination SED was increased by 14% and 12%, respectively, to paclitaxel 60 mg/m2 and bortezomib 1.0 mg/m2. At the fourth dose level, treatment-related grade 3 myalgias lasting for 7 days before resolution (1 event) and grade 2 anemia (1 event) were reported and a combination SED decrement by 4% was recommended. However, paclitaxel and bortezomib doses were kept unchanged, as a 4% dose decrease was considered clinically nonsignificant for both drugs. Finally, at the fifth dose level, treatment-related grades 2 and 3 anemia (1 event each) occurred and a combination SED decrease by 6.3% was recommended. Because the recommended change in combination SED was less than 10% at 2 consecutive (fourth and fifth) dose levels, MCRM was terminated and MTD was reached at the fifth dose level (paclitaxel 60 mg/m2 and bortezomib 1.0 mg/m2). In summary, using MCRM, paclitaxel was escalated from 40 to 45, 51, and 60 mg/m2, whereas bortezomib was escalated from 0.70 to 0.75, 0.86, and finally 1.0 mg/m2.
Weekly paclitaxel and bortezomib combination was overall well tolerated. The most serious treatment-related toxicities during cycle 1 included fatigue (1 grade 2 event), myalgias (1 grade 3 event), and anemia (2 grade 2 and 1 grade 3 events), but no event satisfied DLT criteria. Thus, MTD was reached with only 5 dose levels and no DLTs, indicating that our initial MCRM assumptions were near target and quite efficiently used.
During all treatment cycles, 5 patients experienced a total of 9 serious adverse events that resulted in patient hospitalization, but only 1 event was treatment related (nonneutropenic fever accompanied by mucositis, observed beyond cycle 1). Two NSCLC patients died of progressive disease (PD) during cycle 1; in both cases, death was associated with new brain metastases diagnosed shortly after study initiation.
During the entire study, treatment-related adverse events were mostly grades 1 and 2 (Table 3). For all adverse events, predominant hematologic toxicity was anemia (observed in 7 patients or 43.75%; 1 grade 3 event), but did not result in any delayed, reduced, or missed drug doses. Six patients were given erythropoietin while on trial, but 3 of them had required this support even before study participation. The most commonly observed nonhematologic toxicities included neuropathy (in 31.25% of patients; 1 grade 3 event after cycle 1) and fatigue (in 43.75% of patients; 1 grade 3 event, also beyond cycle 1). Grade 3 dyspnea (1 event after cycle 1) was noted in a patient with NSCLC and chronic obstructive pulmonary disease (COPD) in the setting of nonneutropenic fever and was initially considered possibly treatment related; however, symptoms resolved with antibiotics, oxygen, and supportive care within 7 days, thus, most likely representing COPD exacerbation.
Paclitaxel PK parameters were determined in 16 patients on paclitaxel and bortezomib combination (Table 4). Cmax increased from dose level 1 to 4 reflecting paclitaxel dose escalation and was comparable with previously reported values for weekly low-dose paclitaxel protocols (30, 31). Average AUC at 40, 45, 51, and 60 mg/m2 paclitaxel showed a dose proportional linear increase (r2 = 0.995; Fig. 1A). Paclitaxel CL ranged from 17 to 48 L/h. Mean CLs at 40 and 45 mg/m2 paclitaxel were almost the same. Mean CLs at 51 and 60 mg/m2 paclitaxel were 31.9 and 27.2 L/h, respectively, also similar to each other and not significantly different from those at other dose levels (P > 0.3; refs. 32, 33). In all cases, Tmax of paclitaxel was 1 hour, corresponding to infusion end. Paclitaxel T½ ranged from 0.55 to 3.4 hours with mean values at different dose levels ranging from 0.69 to 1.96 hours. Paclitaxel PK parameter variation was likely because of the small patient number at each dose level.
Among 15 evaluable patients (1 patient withdrew consent because of grade 3 myalgias in cycle 1 and was, thus, evaluable for toxicity, but nonevaluable for response), no complete responses were seen (Table 5). One NSCLC patient previously treated with paclitaxel during definitive chemoradiation had a confirmed PR (Fig. 2) and remained on study for 227 days. Two other NSCLC patients showed disease stabilization as compared with PD before study initiation, each remaining on study for 165 and 180 days. Stable disease (SD) as best response was also seen in 3 additional patients, 1 each with colon, ovarian, and pancreatic tumors. Mean duration of disease stabilization was 143.5 days. Two out of 5 patients with SD had been treated with a paclitaxel-containing regimen in the past. A second NSCLC patient had received paclitaxel during definitive chemoradiation, and a patient with ovarian cancer had been treated with paclitaxel in the adjuvant setting, at first recurrence, and as the most recent line of therapy before study participation. Despite recent disease progression on paclitaxel, tumor markers, ascitic fluid accumulation and peritoneal implants stabilized, and patient remained on study for 4 cycles. Of the 9 patients with disease progression (60% of evaluable patients), 3 (2 with lung and 1 with colon cancer) showed PD during cycle 1, making it unlikely that their disease trajectory was affected in any significant way by study drugs, whereas 6 other patients (2 with melanoma, 2 with colon, and 1 with breast cancer) completed 2 treatment cycles and were found to have PD at first restaging.
Pharmacodynamic and other correlative laboratory studies
PBMC pre- and posttreatment BIM expression was determined for all (16) study patients (Table 5, Fig. 1B). Archived tumors, available for 10 patients, were evaluated for RAS/RAF/MAPK pathway activation by phospho-p44/42 MAPK IHC, and the majority (8/10) showed evidence of MAPK activation (Table 5, Fig. 2B). For 4 patients (25%), BIM was neither expressed at baseline, nor was it induced by treatment in PMBCs. From 12 patients with detectable PBMC BIM before treatment, 6 (50%) exhibited BIM upregulation, 3 (25%) showed stable BIM levels, and 3 (25%) had decreased BIM expression during threatment. Of the 6 patients with objective disease stabilization or regression, 4 had detectable BIM in PBMCs at baseline; 3 of them (2 patients with SD for longer than 5 months and 1 patient with PR for 7.5 months, all with NSCLC) or 75% showed PBMC BIM upregulation at 27 and 51 hours posttreatment. For these 3 patients, archived tumors were available and all showed intratumoral MAPK pathway activation. Of the 9 patients with PD, 7 had detectable BIM in PBMCs before treatment, but only 2 of them or 28% exhibited treatment-induced increase in PBMC BIM expression.
Thus, treatment-induced BIM upregulation in PBMCs seemed to correlate with disease stabilization or regression in patients with cancers harboring MAPK pathway activation, indicating that changes in PBMC BIM expression during treatment may be a pharmacodynamic indicator of clinical benefit that warrants further investigation.
In this phase I clinical trial, drug combination, dosing schedule, and correlative PK and PD studies were rationally designed based on preclinical data, indicating that RAS/RAF/MAPK pathway activation confers cancer cell resistance to paclitaxel because of functional BIM inactivation. Although paclitaxel generally induces BIM accumulation and BIM-dependent apoptosis in vitro and in tumors in mice in vivo, the RAS/RAF/MAPK pathway suppresses BIM induction by phosphorylating BIM and targeting it to the proteasome for degradation. Bortezomib, a proteasome inhibitor, restores BIM induction in vitro, abrogates RAS-dependent cancer cell resistance to paclitaxel, and promotes BIM-dependent tumor regression (5). We hypothesized that concurrent paclitaxel and bortezomib administration would increase intratumoral BIM levels in malignancies with MAPK pathway activation and, thus, result in enhanced cancer cell death.
Given that weekly paclitaxel and bortezomib combination was a novel treatment scheme and that an earlier trial using these agents together, but on a different schedule, showed significant toxicity (18), the primary endpoint of our study was to determine the paclitaxel and bortezomib combination MTD, when both drugs were given on the same day weekly, using a MCRM adapted for 2-drug escalation. As shown in this study, MCRM can be more efficient in determining MTD than a classic 3 + 3 study design.
Paclitaxel and bortezomib combination was quite well tolerated. Most commonly observed adverse events included neuropathy (in 31.25% of patients), fatigue (in 43.75% of patients), and anemia (in 43.75% of patients), which were mostly grade 1 or 2 in nature and treatment related only in a minority of cases. A treatment-related grade 3 neuropathy event (observed beyond cycle 1) resulted in a 2-week dose delay and subsequent dose reduction, whereas an episode of treatment-related grade 3 fatigue resulted in dose delay, but resolved with supportive care and did not require dose reduction. Treatment-related grade 3 myalgias (1 event) and grade 2 (6 events) and grade 3 (1 event) anemia were also observed. These side effects were narrower in spectrum and overall milder than those observed in previously published studies using this drug combination on different schedules. In a study combining paclitaxel on days 1 and 8 and bortezomib on days 1, 4, 8, and 11, the recommended phase II dose was 100 and 1.3 mg/m2, respectively (18). One third of the patients (9/31) had PRs, but at the expense of significant toxicities, including cumulative peripheral neuropathy (in 76% of patients; grades 3–4 in 9%) that necessitated treatment discontinuation in 6 patients, followed by diarrhea (55%) and fatigue (41%). Other earlier clinical trials also showed significant hematologic toxicities (16, 17). The improved safety profile observed in our study was likely because of an MCRM-determined MTD that was lower than previously defined by other approaches (18). Paclitaxel PK parameters were comparable with earlier published values (30–33), indicating no clinically significant PK interaction between paclitaxel and bortezomib.
Recommending combination paclitaxel and bortezomib doses that are lower than those commonly used in monotherapy raises the concern that drug activity may be compromised at the gain of reduced toxicity. However, the proposed doses produce plasma paclitaxel and bortezomib concentrations known to be sufficient for microtubule and proteasome modulation, respectively. Furthermore, weekly paclitaxel at 60 mg/m2 has been used for disease control as a single agent (34) and in novel combinatorial therapy trials (35–39). Concern for significant neurologic toxicities, as both paclitaxel and bortezomib can cause neuropathy, also mandated lower initial drug doses. We are pleased to report clinical benefit, mostly as disease stabilization along with 1 PR case, in a third of all evaluable—generally heavily pretreated—patients. Interestingly, 3 out of 5 NSCLC patients showed durable responses lasting 165 days or longer, including a confirmed PR. Antitumor activity was also observed in a recent phase I trial of neoadjuvant carboplatin, paclitaxel, and bortezomib in combination with concurrent radiotherapy in stage III lung cancer, where complete pathologic complete responses were noted, but trial was terminated because of delayed, unpredictable, and severe toxicities (40). Of note, 2 of the 3 NSCLC patients that showed clinical benefit on our trial had already been treated with paclitaxel in the adjuvant setting. Quite importantly, all 3 patients had tumors exhibiting MAPK pathway activation and showed treatment-induced BIM upregulation in PBMCs as a surrogate marker for intratumoral BIM activity, that is, perfectly fitted the patient profile with an expected response, as predicted by our earlier preclinical studies (5). Although our sample size is very small, these results clearly indicate that weekly paclitaxel and bortezomib combination is worthy of further investigation and may show clinical efficacy in carefully designed phase II trials.
Determination of treatment-dependent PBMC BIM level changes as a pharmacodynamic parameter is another novelty of our trial. Baseline BIM levels and treatment-induced BIM expression alterations in PBMCs cannot provide quantitative information on intratumoral BIM status and its modifications, if any, during treatment, because somatic oncogenic mutations should only affect BIM expression in tumors. However, BIM monitoring in easily accessible PBMCs may be a qualitative indicator of how study drugs affect BIM expression in all tissues, including tumor site(s), as indicated by the correlation between treatment-induced PBMC BIM upregulation and clinical benefit in the small patient number treated in our study. A phase II study for patients with documented intratumoral RAS/RAF/MAPK activation is, thus, recommended to further explore treatment-dependent PBMC BIM expression changes as a clinical response predictor. Baseline intratumoral BIM levels will also be determined and readily accessible tumor sites, such as metastatic skin lesions, will be serially biopsied for assessment of cancer cell BIM expression changes and apoptosis induction during treatment.
In conclusion, MTD for weekly paclitaxel and bortezomib combination was determined to be 60 and 1.0 mg/m2, respectively, using MCRM adapted for 2-drug escalation. Treatment was generally well tolerated and produced disease stabilization and/or regression in one third of evaluable patients, several of whom had received prior taxane exposure. Paclitaxel PK parameters were similar to those determined in previous studies, indicating no significant PK interactions between paclitaxel and bortezomib. A phase II trial combining paclitaxel and bortezomib as second line treatment for NSCLC patients is under development to more rigorously test the antitumor activity of this regimen and explore whether PBMC BIM expression changes during treatment may serve as a pharmacodynamic marker and a clinical response predictor.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
This work was supported by ECOG Paul Carbonne, MD Fellowship Award and Americal Society of Clinical Oncology Young Investigator Award (V. Karantza); NIH grant R37CA53370 (E. White)
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Millennium Pharmaceuticals, the Takeda Oncology Company, for generously providing bortezomib for this trial; the CINJ Office of Human Research Services, Pharmacy, Tissue Analytical Services, and phase I Clinic APNs Stephanie Beers, Phaedra Kirin, and Cecilia Thomas for their hard work and commitment to making clinical trials happen; Dr. William N. Hait (Johnson & Jonhson) for his support and mentoring; and, above all, the patients who participated in the study.
- Received October 12, 2010.
- Revision received April 22, 2011.
- Accepted June 1, 2011.
- ©2011 American Association for Cancer Research.