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This phase I trial assessed the safety and tolerability of G3139 when administered in combination with carboplatin and paclitaxel chemotherapy. The effect of G3139 treatment on Bcl-2 expression in peripheral blood mononuclear cells (PBMC) and paired tumor biopsies was also determined.
Patients with advanced solid malignancies received various doses of G3139 (continuous i.v. infusion days 1–7), carboplatin (day 4), and paclitaxel (day 4), repeated in 3-week cycles, in a standard cohort-of-three dose-escalation schema. Changes in Bcl-2/Bax transcription/expression were assessed at baseline and day 4 (pre-chemotherapy) in both PBMCs and paired tumor biopsies. The pharmacokinetic interactions between G3139 and carboplatin/paclitaxel were measured.
Forty-two patients were evaluable for safety analysis. Primary toxicities were hematological (myelosuppression and thrombocytopenia). Dose-escalation was stopped with G3139 at 7 mg/kg/day, carboplatin at AUC 6, and paclitaxel at 175 mg/m2 due to significant neutropenia seen in cycle 1, and safety concerns in further escalating chemotherapy in this phase I population. With G3139 at 7 mg/kg/day, 13 patients underwent planned tumor biopsies, of which 12 matched pairs were obtained. Quantitative increases in intratumoral G3139 with decreases in intratumoral Bcl-2 gene expression were seen. This paralleled a decrease in Bcl-2 protein expression observed in PBMCs.
Although the MTD was not reached, the observed toxicities were consistent with what one would expect from carboplatin and paclitaxel alone. In addition, we show that achievable intratumoral G3139 concentrations can result in Bcl-2 down-regulation in solid tumors and PBMCs.
Apoptosis is a program of cellular suicide that removes unnecessary cells throughout life. It reflects the balance of many pro- and anti-apoptotic regulatory signals, the sum of which determines the fate of the cell. Bcl-2 is an anti-apoptotic protein that is frequently over-expressed in cancer cells, and may play a large role in the development of many solid tumors, including prostate1,2, melanoma3, breast4, lung5, renal6, and ovarian7 cancers. In addition, up regulated Bcl-2 has been implicated as a possible mechanism for chemotherapy and radiotherapy resistance8,9.
G3139 (Genasense™, oblimersen sodium) is an 18-mer antisense phosphorothioate oligodeoxynucleotide (ODN) that binds to the first six codons of the human Bcl-2 mRNA. When administered alone or in combination with chemotherapy to human cell lines in vitro, G3139 down-regulates Bcl-2 protein expression resulting in a greater degree of apoptosis10. Antitumor activity with single-agent G3139 has been reported in non-Hodgkin's lymphoma11. Likewise, disease specific trials in melanoma12, small cell lung13, colorectal14, breast15 and prostate16 cancers have been performed with G3139, alone or in combination with chemotherapy. The dose-limiting toxicities (DLT) seen with G3139 in the Phase I non-Hodgkin's lymphoma study, which used a continuous 14-day subcutaneous infusion, were thrombocytopenia, hypotension, fever, and asthenia. However, when G3139 was given as a continuous intravenous infusion for 14–21 days in an advanced solid malignancy phase I study, the drug was well tolerated with the main treatment-related adverse events being fatigue and transaminase elevations. Steady-state plasma concentrations of G3139 were achieved approximately 10 hours from start of the infusion with a terminal plasma half-life of approximately 2 hours17.
The motivation to combine G3139 with carboplatin and paclitaxel include the wide applicability of this chemotherapy regimen to many solid tumor types, including lung, bladder, head/neck, breast, and ovarian cancers. By decreasing the tumor's resistance to apoptosis with G3139, potentially less toxic doses of carboplatin and paclitaxel could be used with equal or greater antitumor activity. In addition, there is evidence that paclitaxel induces prolonged mitotic arrest, resulting in hyperphosphorylation of Bcl-218. It is suggested that the hyperphosphorylation of Bcl-2 is one important mechanism for paclitaxel's antitumor activity19,20,21,22. If so, the potential of combining G3139 with a taxane-based regimen may be beneficial. Certainly, preclinical data supports at least potential additive benefits of combining a Bcl-2 antisense oligodeoxynucleotide with paclitaxel23.
While the principal objective of this Phase I trial was to define the maximal tolerated dose (MTD) of G3139 in combination with carboplatin and paclitaxel, an extensive pharmacokinetic and pharmacodynamic analysis was performed to better define the activity of G3139. These studies included pharmacokinetic analysis of G3139, carboplatin, and paclitaxel in plasma, as well as intratumoral G3139 levels from paired tumor biopsies in a planned cohort of 12 subjects. Quantitative changes in Bcl-2/Bax transcription and protein expression levels from the paired tumor biopsies were assayed by reverse-transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC). Changes in Bcl-2/Bax transcription and protein expression in peripheral blood mononuclear cells were assessed by RT-PCR and flow cytometry.
Eligible patients had progressive metastatic, or unresectable, solid malignancies with no standard curative therapy. All patients had an Eastern Cooperative Oncology Performance Status ≤ 2 with a life-expectancy greater than 3 months, a total leukocyte count ≥ 3,000/mm3, absolute neutrophil count ≥ 1,500/mm3, platelet ≥ 100,000/mm3, normal total bilirubin, aspartate (AST) and alanine (ALT) aminotransferase ≤ 2.5 × upper limit of normal, and normal serum creatinine (or creatinine clearance ≥ 60 mL/min/1.73m2). No known brain metastasis, pre-existing grade ≥ 2 neuropathy, or other chemotherapy/radiotherapy/investigational therapy within 4 weeks of registration was allowed. This study was reviewed and approved by the University of Wisconsin Hospital and Clinic Institutional Review Board and other appropriate committees, and all patients gave written informed consent for clinical and research aspects of this study according to federal and institutional guidelines prior to screening.
All patients underwent placement of a central venous catheter. G3139 was administered as a continuous intravenous infusion (CIV) on days 1 through 7 through a portable continuous infusion pump, with paclitaxel administered on day 4 by intravenous (IV) infusion over 3 hours, followed immediately by carboplatin administered via rapid IV infusion over 30 minutes. Standard chemotherapy premedications including antiemetics, dexamethasone, and antihistamines (H1 and H2 blockers) were used per standard practice guidelines.
The starting dose of G3139 was 3 mg/kg/day, and was escalated successively to the initial planned maximum of 7 mg/kg/day. During the G3139 dose-escalation phase, the chemotherapy dose was fixed with paclitaxel at 150 mg/m2 and carboplatin at an area under the curve (AUC) of 5. Standard cohorts of three were used with dose-escalations proceeding if no DLT was encountered. If one patient experienced a DLT at a given dose level, a total of six patients were entered at the dose level. If two out of a maximum of six evaluable patients experience a DLT, then the maximum-tolerated dose (MTD) would be exceeded and additional patients enrolled at the dose-level below. The MTD was defined as the highest dose at which less than two of six patients experienced a DLT in cycle 1. A DLT was defined as a toxicity that was probably or definitely related to G3139 (in combination with paclitaxel and carboplatin), with either grade 4 neutropenia lasting ≥ 7 days (or febrile neutropenia), platelets ≤ 25,000/mm3 lasting ≥ 7 days (or ≤ 50,000/mm3 associated with severe bleeding), or any grade ≥ 3 non-hematologic toxicity (except nausea/vomiting and diarrhea unless this occurs despite maximal supportive care). The National Cancer Institute Common Toxicity Criteria Version 2.0 was used for grading of all toxicities. At either dose-level 5 (G3139 at 7 mg/kg) or the MTD, a planned additional cohort of 12 patients were to be enrolled for correlative assessment, including procurement of paired tumor biopsies on all subjects. Based on the results of these studies, a decision would be made to either escalate chemotherapy doses to define the MTD (if not reached) or increase G3139 to 9 mg/kg (if Bcl-2 suppression not evident). A summary of the dose-escalation schema is shown in Table 1.
G3139 (Genta, Inc) was supplied through the National Cancer Institute in 10-mL vials containing 300 mg, which was diluted to the appropriate volume and concentration (final solution concentration maintained between 10mg/ml to 30mg/ml) with 0.9% saline solution. Commercial paclitaxel and carboplatin were used in this study. A cycle of therapy was defined as 21 days. There was no limit to the number of courses that could be administered for patients benefiting and tolerating therapy.
Complete medical history, physical examination, and routine laboratory studies were performed at baseline and prior to each new cycle of therapy. Routine laboratories included: complete blood count, albumin, alkaline phosphatase, total bilirubin, electrolytes, creatinine, phosphate, total protein, AST, ALT, and lactate dehydrogenase (LDH). A baseline electrocardiogram was required, as well as pretreatment radiographs of all known sites of measurable and evaluable metastasis. Repeat radiographic evaluations for response were performed after every two cycles of therapy. Response Evaluation Criteria in Solid Tumors (RECIST) guidelines were used for determination of tumor response24.
Plasma concentrations of G3139 were determined by high performance liquid chromatography (HPLC)25 and tumor concentrations were evaluated by enzyme-linked immunosorbent assay (ELISA) as previously described26. A total of three blood samples were obtained for analysis of G3139 plasma concentrations. The first was obtained immediately before initiation of the G3139 infusion, the second on day 4, prior to paclitaxel and carboplatin administration (96 hours after initiating G3139), and the third, on day 5 (24 hours after carboplatin and paclitaxel administration and 120 hours after initiating the G3139 continuous infusion). Biopsies were obtained (dose level 5 only) at baseline and day 4 (prior to carboplatin and paclitaxel infusion). The sample used for pharmacokinetics was flash frozen and stored at −70C until analysis.
To analyze G3139, 100 μl of plasma was placed in a microfuge tube with 300 μl of IPEGAL/(phosphate buffered saline) (1:1) added and vortexed. Subsequently, 400 μl acetonitrile was added and vortexed for 30 sec. The sample was then centrifuged at 9000 × g for 7 min. The supernatant was placed into an autosampler vial and analyzed by UV detection at 267 nm. HPLC was performed with a gradient on a Dionex DNA Pac PA-100, 2mm × 10 mm, 13 μm, alkyl quaternary amine column.
The standard curve was linear from 0.234 to 30 ug/ml (r2 =0 .994). Intraday variability was 3.96 % for high (10 ug/ml) standard (n=3) and 0.56% for low (0.039 ug/ml) standard (n = 3). Interday variability was 0.46% for high standard (n = 4) and 4.34% for the low standard (n = 4) over 7 days. The lower limit of quantitation was 0.234 ug/ml and the absolute recovery from plasma compared to water was greater than 95%.
G3139 in solid tumor tissue was measured by a 96-well plate quantitative sandwich immunoassay with data normalized to protein values by standard methods. This assay involves hybridization of G3139 to the 5'-end overhang of a 3'-biotinylated capture oligonucleotide, ligation to a digoxigenin-labeled probe, and detection by an anti–digoxigenin-alkaline phosphatase system (AttoPhos AP Fluorescent Substrate System, Promega Corp, Madison, WI). The plate was read at 430 nm (excitation) and 570 nm (emission) using a Molecular Devices SpectraMax Gemini plate reader.
The standard curve was linear from 0.18 to 10,000 ng/mL G3139 (r2 =0 .992). Intraday variability was less than 14% for high (10,000 ng/ml) standard (n=3) and 0.56% for low (0.18 ng/ml) standard (n = 3). Interday variability was 12% for high standard (n = 4) and 11.15% for the low standard (n = 4) over 7 days. The lower limit of quantitation was 0.18 ng/mL and the absolute recovery from plasma compared to water was greater than 95%.
Carboplatin concentrations were evaluated with a Varian SpectrAA 10/20 with GTA-96 atomic absorption spectrophotometer adjusted to detect platinum (265.8–266) as previously described27,28. Samples were obtained immediately prior to the carboplatin infusion, 40 minutes into the infusion, at the end of the infusion, 30 minutes, and 1, 2, 4, 6, 8 12, 24, and 48 hours after the end of the infusion. Blood samples were collected in heparinized tubes, and plasma was separated from cells by centrifugation (1,200 × g, 4°C, 10 minutes) within 15 minutes of blood collection. Free drug was then separated from the protein bound drug by centrifugation (1,200 × g, 4°C, 15 minutes) through an ultrafiltration membrane (Centrifree YM-30, Amicon Bioseparations, Millipore Corporation, Bedford, MA). Ultrafiltrate samples were stored at −70°C until analysis. Fifty microliters of each standard or sample to be analyzed were placed in a plastic atomic absorption vial, to which was then added 0.95 mL of 0.1% HNO3. The three calibration samples of 0.05, 0.1 and 0.2 μg/ml were used to calibrate the atomic absorption spectrophotometer, and the calibration was checked by running the calibration samples after the sample runs.
Paclitaxel concentrations were evaluated with a Spectra Physics P2000 HPLC as previously described29. Samples were obtained immediately prior to the paclitaxel infusion, 90 minutes into the infusion, at the end of the infusion, 30 minutes, and 1, 2, 4, 6, 8, 12, 24, and 48 hours after the end of the infusion. Blood samples were collected in heparinized tubes, and plasma was separated from cells by centrifugation (1,200 × g, 4°C, 10 minutes) within 15 minutes of blood collection. Samples were stored at −70°C until analysis.
The plasma standard curve was linear from 0.05 to 5.0 μg/ml (r2 = 0.999). Intraday variability was low; under 4% for 0.05 ug/ml and 2% for the high standard 5 μg/ml (n = 3). Interday variability was under 9% for the low standard and approximately 14% for the high standard (n = 6) over 6 months, and the lower limit of quantitation (LLOQ) was determined to be 0.1 μg/ml.
Pharmacokinetic analysis was performed by noncompartmental methods using the WinNonlin program, version 5.2 (Pharsight, Cary, NC). The maximum plasma concentration (Cmax) and the corresponding time of the maximum concentration were identified from the measured samples and recorded. Plasma concentration versus time data were plotted on a semilogarithmic scale, and the terminal log-linear phase was identified by best fit. The elimination rate constant (λ) was determined as the slope of the linear regression for the terminal log-linear portion of the plasma concentration versus time curve. A terminal half-life value was calculated as ln(2)/λ. AUC was calculated by the trapezoidal method using extrapolation to infinity.
Peripheral blood specimens were collected on day −4, day 1 (pre-G3139), and day 4 (pre-paclitaxel/carboplatin). At each time point, a PAXgene (Qiagen, Valencia, CA) tube was obtained and RNA isolated by standard methods (Ultraspec II RNA Isolation Systems, Houston, TX)30. RNA was quantified spectrophotometrically, and transcribed into cDNA by standard methods. A total of 250ng of cDNA was used in each 25 μl PCR reaction. For tumor samples, laser capture microdissection of 1000 cells was performed as previously described31, RNA was extracted and transcribed into cDNA by standard methods, and the total volume was used in the PCR reaction. Sample with mRNA concentrations above the standard curve were diluted and retested.
Gene expression was performed using the MyIQ real-time thermocycler (Bio-Rad, Hercules, CA) with the iCycler parameters as follow, for 95°C/2 min × 1 cycle, 95°C/30 sec, 61.7°C/1 min, 72°C/30 sec for 50 cycles, 60°C/7 sec + 0.5°C × 70 cycles and analysis by the Eragen MultiCode RTx Analysis Software version 1.0.14 (Eragen, Madison, WI). The following primers were used: Bcl-2 forward: 5'-GCT CTT CAG GGA CGG G-3', Bcl-2 reverse(probe): 5'-/56-FAM/Me-isod C/GC TCT CCA CAC ACA TGA CC-3 Bax forward(probe): 5'-/56-FAM/Me-isod C/CG GAC CCG GCG AGA-3', Bax reverse: 5'-CGC CTC TGG GCT GCT-3' (IDT, Coralville, IA)
The Bcl-2 assay was validated using cDNA obtained from the DU145 prostate cancer cell line. Total cDNA concentration was determined spectrophotometrically and the standard curve was generated by amplifying in 39ng–2500ng total DNA, on 4 days over a period of 4 weeks. The standard curve was linear from 39–2500 ng, with an intraday variability of 3.5%. Interday variability over 4 weeks was 3.3%. The Bax assay similarly was validated using cDNA obtained from the DU145 prostate cancer cell line. Total cDNA concentration was determined spectrophotometrically and the standard curve was generated by amplifying in 6.25ng–100ng total DNA, on 6 days over a period of 6 weeks. The standard curve was linear from 6.25–100ng, with an intraday variability of 14.1%. Interday variability over 6 weeks was 2.9%.
Peripheral blood specimens were collected on day −4, day 1 (pre-G3139), and day 4 (pre-paclitaxel/carboplatin). One hundred microliters of blood was added to tubes and lysed with 3 ml of FACSLyse (BD Biosciences, San Jose, CA). Samples were incubated for 10 minutes at room temperature, centrifuged at 400 × g for 10 minutes and washed with Dulbecco's Phosphate Buffered Saline (DPBS-Media Tech, Herndon, VA). Cell pellets were resuspended in permeabilizing buffer [0.1% saponin and 0.1% bovine serum albumin (both from Sigma, St. Louis, MO) in DPBS] and incubated for 10 minutes at room temperature and then centrifuged at 400 × g for 10 minutes. Antibodies or isotype controls were added to the cell pellets in the volumes given and incubated for 45 minutes at room temperature. Samples were washed with 3 ml of permeabilizing buffer and centrifuged again at 400 g for 10 minutes. The following unconjugated or phycoerythrin (PE)-conjugated antibodies were added to the resulting cell pellets: anti-Bcl-2-PE, hamster IgG, and goat anti-mouse IgG from BD Biosciences, San Jose, CA; anti-Bax was purchased from Beckman-Coulter, Hialeah, Florida. Three ml of DPBS were used to wash the samples that were centrifuged at 400 g for 10 minutes. The resulting cell pellets were resuspended in 300 μl of DPBS with 1% fetal calf serum and analyzed on a LSRII cytometer (BD Biosciences, San Jose, CA) with FACSDiVa software. Raw data were acquired until 50,000 lymphocyte events were counted, identified by light scatter. At the same instrument settings data was acquired on Quantibrite Phycoerythrin standard beads (BD Biosciences, San Jose, CA). Data was analyzed with FloJo analysis software (Treestar, Ashland, OR). The fluorescence of the Bcl-2 was expressed as molecules of Phycoerythrin. The ratio of Bax to Bcl-2 was been calculated by creating a ratio of the FITC (Bax) to the PE (Bcl-2). All values were normalized to the isotype control.
Immunohistochemistry was performed on tissue sections of formalin fixed, paraffin embedded tumor biopsies using an automated immunostainer (Ventana Medical Systems (VMS), Tucson, AZ). Tissue sections underwent antigen retrieval prior to staining. The antigen retrieval for Bcl-2 staining was done on-line using mild heat induced retrieval with Cell Conditioning Solution #1 (CC1, a proprietary VMS-pH buffer). Bax antigen retrieval was performed by incubating slides in an EDTA buffer (pH 8.0), in an electric pressure cooker (Decloaking Chamber, Biocare Medical, Walnut Creek, CA) for 2 minutes at approximately 20 PSI. The slides were incubated with the prediluted primary antibody Bcl-2 (Clone 124 from VMS) or a 1:50 dilution in Zymed antibody diluent of anti-Bax (Clone 2D2, Zymed Laboratories, South San Francisco, CA for 32 minutes. Slides receiving the Bcl-2 were subsequently put through an amplification step using VMS amplification kit. After incubation with a universal secondary, target detection was by an indirect biotin avidin system (VMS) including diaminobenzidine. Endogenous peroxidase quenching and biotin blocking were done on-instrument with kit reagents (VMS). Immunostained slides were counterstained with hematoxylin on the instrument, and subsequently dehydrated through a series of graded alcohols and coverslipped on a Tissue-Tek automated coverslipper (Sakura, Torrance, CA). The immunohistochemical staining pattern of each case was assessed by the same pathologist and scored according to staining intensity and proportion of positive cells.
A total of 46 patients were enrolled on the dose-escalation and expanded correlative parts of this study. Four of these patients were deemed unevaluable. Three patients at dose level 1 were unevaluable for toxicity (two due G3139 infusion pump malfunctioning and one secondary to the development of spinal cord compression on day 15 requiring emergent radiotherapy). One additional patient at dose level 5 was unevaluable secondary to failure to complete cycle 1 of treatment because of the development of a recto-vaginal fistula attributed to her cancer and recent surgery. Table 2 summarizes relevant patient characteristics.
The predominant toxicity observed was myelosuppression and thrombocytopenia. One patient at dose level 1 developed grade 4 thrombocytopenia and one patient at dose level 5.1 had grade 4 neutropenia. These two events were the only toxicities observed that met our stringent criteria for DLTs. Although other grade 3 and 4 events were seen in cycle 1, these events did not meet criteria for a DLT (see Table 3). In all, the combination of G3139 with carboplatin and paclitaxel was found to be well tolerated, with no unusual toxicities observed that were felt likely related to G3139 and not attributable to the expected toxicities of the chemotherapy agents. The median number of cycles received per patient was 3 (range 1–14).
In total, 6 partial responses (4 confirmed, 2 unconfirmed) were observed. Two confirmed responses were in dose level 5; the first patient being an individual with esophageal cancer previously treated with two prior cytotoxic chemotherapy regimens, and the second a patient with bladder cancer treated with one prior chemotherapy regimen. At dose level 5.1, two partial responses (1 confirmed, 1 unconfirmed) were observed. Both patients had urothelial cell cancer of the bladder. One patient at dose level 5.2 also experienced a confirmed partial response. This individual had head-neck carcinoma which was previously treated with chemotherapy and an epidermal growth factor receptor tyrosine kinase inhibitor. Lastly, one patient with papillary urothelial cell carcinoma of the bladder had an unconfirmed partial response at dose level 5.2. This patient was removed from study after two cycles of treatment by physician discretion as it was felt that the patient poorly tolerated the chemotherapy despite maximal dose-reduction during cycle two. Of note, this patient had significant difficulties tolerating a prior cytotoxic chemotherapy regimen as well.
G3139 plasma concentrations were evaluated at baseline, on day 4 of continuous infusion of G3139 (prior to carboplatin and paclitaxel chemotherapy), and on day 5 of continuous infusion of G3139 (after carboplatin and paclitaxel chemotherapy) in 11 subjects enrolled onto level 5 using HPLC. All subjects had measurable G3139 concentrations on Day 4 and 5 after administration of G3139 with the mean G3139 concentration being 4.28 ± 1.40 μg/mL on Day 4 and 3.86 ± 1.36 μg/mL on Day 5. G3139 plasma levels were similar on day 4 and day 5, suggesting that G3139 pharmacokinetics are unaffected by coadministration of carboplatin and paclitaxel. G3139 concentrations in tumor were evaluated in eleven paired biopsies obtained prior to G3139 administration and on day 4. G3139 was detectable in ten of eleven tumors following treatment, and the mean tumor concentrations of G3139 was 27.9 ± 43.1 ng/per μg of tumor protein indicating the G3139 is able to penetrate tumors (see Table 4).
Pharmacokinetic studies of paclitaxel and carboplatin were conducted in eleven subjects enrolled onto level 5 using HPLC. The pharmacokinetic parameters are described in Table 4 and are similar to reported values, suggesting that administration of G3139 did not alter the pharmacokinetics of paclitaxel or carboplatin. Both carboplatin and paclitaxel exhibited wide inter-patient variability and patients on average achieved approximately 80% of the planned carboplatin AUC. Calculation of carboplatin doses used the Calvert formula with the GFR estimated from the creatinine clearance, which may have accounted for the inaccuracy in dosing32.
Bcl-2 is the target of G3139, therefore, we expected Bcl-2 gene expression in PBMCs to decrease after administration of G3139. As expected, Bcl-2 gene expression had a statistically significant decline from baseline to 4 days of G3139 infusion (p<0.001, two sided Wilcoxan signed rank test), indicating that Bcl-2 gene expression is inhibited by G3139 in PBMCs. There was no change in detectable bax expression in PBMCs after G3139 administration (Figure 1).
There were a total of thirteen Day 0 biopsy samples and ten Day 4 biopsy specimens amenable for this analysis (one patient did not undergo second biopsy; two patients did not have enough tumor tissue for laser capture microdissection and subsequent gene transcription assessment). Following laser capture microdissection of 1000 cells, Bcl-2 and bax gene expression were also evaluated in biopsy samples. Bcl-2 gene expression declined in the majority of paired samples by day 4 of G3139 treatment, with a median ratio of Day 4/Pre of 0.65, although this did not reach statistical significance. There was no change in bax expression in biopsy specimens after G3139 administration (Figure 2).
Peripheral blood mononuclear cells were collected from patients at baseline and on day 4 after starting G3139 (pre-carboplatin and paclitaxel chemotherapy). Bcl-2 and Bax protein expression were assessed by intracellular flow cytometric analysis. Bcl-2 expression had a statistically significant decline from baseline after 4 days of G3139 infusion (p=0.008, paired t-test), suggesting that Bcl-2 expression is reduced by G3139 treatment in PBMCs. There was no significant change in Bax expression in PBMCs after G3139 administration.
A total of twelve paired core needle tumor biopsy samples were obtained, of which eleven paired samples were adequate for IHC analysis. One patient with melanoma had biopsies that were unable to be assessed due to extensive background melanin, making immunohistochemical interpretation difficult. In general, faint staining with Bax was seen in the background with focal regions of intense staining noted in areas where cells were presumably undergoing apoptosis (close to necrotic areas as seen on the H&E). Bcl-2 staining appeared similar between pre and post treatment samples for most subjects but many tumors had negative pre and post treatment Bcl-2 staining. Few Bcl-2 positive small lymphocytes (tumor infiltrating lymphocytes) were present within some tumors.
Overexpression of Bcl-2 in tumors has been reported to be associated with not only a more advanced stage at diagnosis, but also treatment resistance and a shortened survival. By targeting critical proteins such as Bcl-2, it may be possible to restore normal regulatory pathways and enhance the effectiveness of current chemotherapeutics33. Here we report the results of a phase I trial assessing the safety and feasibility of combining G3139 with carboplatin and paclitaxel, as well as an extensive evaluation of the pharmacodynamic effect of G3139 alone in PBMCs and in the tumor.
As patients are typically heavily pretreated in a phase I study, we had concerns that significant myelosuppression and thrombocytopenia would be seen secondary to the carboplatin and paclitaxel chemotherapy alone. Given the potential for additional hematological toxicity, as noted with other phosphorothiolate olignucleotides34,35,36, we chose to fix the chemotherapy dose (carboplatin AUC 5, paclitaxel 150 mg/m2) and dose-escalate the G3139 alone. Since some degree of hematological toxicity was expected, we used an aggressive definition of a hematological DLT to allow us to not only assess the safety of this combination combination, but also escalate the G3139 to levels that were felt necessary for appropriate Bcl-2 suppression. It was soon realized that G3139 could be safely combined with carboplatin and paclitaxel, with no appreciable increase in toxicity than what was consistent with carboplatin and paclitaxel alone. As a result, the protocol was modified to subsequently dose-escalate the chemotherapy in order to define the MTD. During cohort 5.2, it was obvious that all three patients had grade 4 myelosuppression. Although the neutropenia did not meet the 7 day duration criteria for a DLT, it was decided after discussion with the Cancer Therapy Evaluation Program (CTEP) at the National Cancer Institute (NCI) to stop the dose escalations, as the next planned dose level involved a chemotherapy dose that is typically administered only in the first-line setting, and not in heavily pretreated patients commonly enrolled in phase I studies. Therefore, although the MTD was not defined, we feel confident reporting that G3139 can be safely combined with standard doses of carboplatin and paclitaxel. This is consistent with the conclusion derived in multiple other solid tumor trials combining G3139 with full doses of cytotoxic chemotherapy37,38,39.
The planned expanded cohort of twelve patients with paired tumor biopsies was completed and extensive correlative analyses of G3139 pharmacokinetic and pharmacodynamic effects were performed. What is evident is that by day 4, Bcl-2 RNA was significantly decreased in the PBMCs with a parallel decrease seen in the tumor samples. Although the decrease in the tumor Bcl-2 RNA did not meet statistical significance, given detectable intratumoral G3139 in 10/11 samples available for this evaluation, and the fact that a relative decrease in Bcl-2 gene expression over baseline was observed in the majority of samples, this was felt to be highly indicative of G3139 activity.
We observed that Bcl-2 protein expression in PBMCs was also significantly decreased on day 4, although the change in intratumoral Bcl-2 expression was not appreciably different. This raises many questions regarding the utility of using PBMCs as a surrogate marker for changes within the tumor itself. One confounding factor that we retrospectively identified is a potential change in peripheral lymphocyte populations due to effects from dexamethasone, which is routinely administered prior to paclitaxel administration. Since glucocorticoids are well known to induce lymphocyte apoptosis, as well as lymphocyte demargination, the relative subpopulations of lymphocytes measured in peripheral blood may be altered, affecting Bcl-2 determination. At present, a separate cohort of 6 subjects is being enrolled to evaluate specifically PBMC changes due to G3139 alone (without glucocorticoids). Complete data with regards to the immunologic correlative of this study and ongoing evaluation will be published separately once complete.
The lack of significant change in tumor Bcl-2 protein expression is also complex. This could be due to differences in the tumor types being assessed, the inconsistent nature of tumor biopsies (tumors are by nature heterogeneous with focal areas of necrosis and inflammation and thus specific site of biopsy can greatly affect results), as well as the fact that a repeat biopsy on day 4 biopsy might not provide adequate time to see changes in Bcl-2 protein expression. Given that the optimal Bcl-2 RNA suppression is likely not achieved until day 3 of G3139, and the reportedly long Bcl-2 half-life of approximately 14 hours40, the measurement of Bcl-2 protein expression on day 4 was probably too early to detect Bcl-2 protein changes. No significant change in Bax gene expression was noted in either PBMCs or tumor samples.
This study represents the most complete assessment of G3139 activity in solid tumors to date, and includes the highest number of paired tumor biopsies obtained. What our data suggests is that a reduction in PBMC Bcl-2 RNA appears to correlate with reductions in Bcl-2 RNA within the tumors. Whether these changes will result in a significant decrease in tumor Bcl-2 protein expression, leading to clinically meaningful anti-tumor activity, is of course still open for debate and can only be addressed in the context of a randomized trial. Given issues related to tumor heterogeneity, reproducibility of paired needle biopsies, and increased risk for our patients, further assessment of anti-Bcl-2 activity using paired tumor biopsies is not recommended. Future assessments of new anti-Bcl-2 agents might include novel functional imaging modalities evaluating apoptosis, such as Annexin-V positron emission tomography (PET) scans41, as it could allow for non-invasive assessments at multiple time points. By assessing changes over time, we may be better able to define drug effect and the optimum biological dose and schedule of these and other novel agents.
Lastly, oligodeoxynucleotides, like G3139, contain CpG dinucleotides within specific-sequence contexts (CpG motifs), which have been shown to activate rodent and primate immune cells via toll-like receptor 9 (TLR9)42. Thus, G3139 might function as an immune stimulator in addition to its effects on Bcl-2. Future analysis of the effects of G3139 on T-cell functioning and signaling may be warranted.
Here we report a pharmacodymanic trial assessing the reduction of Bcl-2 RNA and change in protein expression levels in peripheral blood mononuclear cells and paired tumor biopsy samples after exposure to G3139. The reduction of Bcl-2 RNA in the tumors and peripheral blood mononuclear cells was observed in conjunction with achievable intratumoral concentrations of G3139. In summary, G3139 remains an interesting agent with a variety of effects that still need exploring. Future plans for this particular regimen are uncertain, although one possible disease interest may be in urothelial cell cancers given the prognostic significance of Bcl-2 over-expression. Evaluation of G3139 in combination with other agents remains under active investigation, especially in diseases such as melanoma and chronic lymphocytic leukemia. Likewise, there may be potential to combine G3139 with tumor vaccines, increasing immunologic activity by not only its anti-Bcl-2 effects, but also potentially by effects mediated by its CpG motif.
Special thanks to Heidi Bakken, Maria Kruse, Jessica Weiss, Molly Houston, Marcia Pomplun, Amy Dresen, and Barbara Woodhouse for their assistance in the conduct of this study.
Grant support: U01 CA062491, Early Clinical Trials of Anti-Cancer Agents With Phase I Emphasis, NCI; CTEP Translational Research Initiative Contract 22XS096; and M01 RR03186, General Clinical Research Center Program of the National Center for Research Resources, NIH