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.