In this Phase I study, we report the safety, feasibility, and bioactivity of a vaccine comprised of autologous DC pulsed with autologous tumor lysate as an adjuvant following surgical resection with standard chemo-radiotherapy. Unlike our previous reported DC vaccination strategy (8
) and those reported by other groups (6
), we included “booster” vaccinations with the innate immune response modifiers, 5% imiquimod (Aldara™) or poly-ICLC (Hiltonol™) based on our pre-clinical studies suggesting that pro-inflammatory innate immune signals could enhance DC activation, trafficking to lymph nodes, and the priming of anti-tumor antigen-specific T lymphocytes (15
). There were no dose-limiting toxicities and no detectable differences in safety or efficacy among the three DC dose levels tested. Of note, there was a significant difference in the average age of patients in the 10 million DC cohort compared to the other dose cohorts, which could influence the difference in overall survival. However, another possible hypothesis is that this trend in the data was a reflection of a dilutional decrease in antigens available for presentation by DC at the highest DC dose cohort (10×106
cells), given that the quantity of lysate was fixed (at 100 µg per dose) despite the increased DC cell dose.
The concomitant administration of 5% imiquimod or poly ICLC with DC vaccination was also found to be safe and did not result in any additional toxicity or adverse events. To our knowledge, this is the first report of the use of TLR agonists in conjunction with DC vaccination strategies in brain tumor patients. Because TLR agonists were used only in patients in the booster phase, it is unclear whether or to what extent the addition of the TLR agonists contributed to the potential efficacy and overall survival of these patients. Furthermore, imiquimod and poly-ICLC are two different biological agents, targeting different TLRs. Imiquimod activates TLR-7, while poly-ICLC activates TLR-3, but both induce pro-inflammatory cytokine secretion. These complexities make it somewhat difficult to determine how these innate immune modifiers actually contributed to our study endpoints. Nevertheless, this current study establishes the safety of these TLR agonists in conjunction with glioma lysate-loaded DC, and further Phase II studies directly comparing these TLR agonists at the time of initial vaccination (not only in the booster phase) are currently underway.
While the number of glioblastoma patients entered in this Phase I clinical trial was not powered to measure efficacy, the clinical results of this trial are still noteworthy. The median OS from the time of initial surgical diagnosis was 31.4 months for all glioblastoma patients (n=23) treated in this study, including both those enrolled as newly diagnosed and recurrent tumor patients. For those treated in the newly diagnosed setting, the OS was 35.9 months; and the OS was 17.9 months for those who received vaccination at recurrence. In addition, we have had three patients survive over six years to date. Such statistics are compelling in the face of the expected median survival for this disease, which is currently still reported as about 14 months for newly diagnosed patients that receive standard surgery, radiation and temozolomide chemotherapy (4
). This compares favorably even when compared with published data for the best clinically defined prognostic group of glioblastoma patients (Recursive Partitioning Analysis, RPA class III: age < 50 years and KPS ≥ 90), whose 2-year survivals were 40% and 29% for RPA III and IV patients, respectively, following treatment with standard radiation and temozolomide (31
). Such data is also favorable compared to other recent brain tumor DC-based vaccine trials without booster injections and TLR adjuvants, where the OS was reported as 21.4 months (mean, 11 newly diagnosed and 23 recurrent glioblastoma patients) (10
) and 9.6 months (median) in a recurrent glioblastoma population (6
Glioblastomas are primarily identified by histologic features assigned to cytologically malignant, mitotically active, necrosis-prone tumors established by the World Health Organization (WHO) (32
). Such histologic features are generally associated with patient survival, together with performance status, extent of surgical resection, and age. Yet, histologically identical tumors can behave in different ways; a situation that may underlie the biology of this heterogeneous disease. More recently, extensive genetic profiling of these tumors has been able to identify molecularly classifiable subgroups of glioblastoma (i.e., proneural, proliferative, and mesenchymal subtypes),(20
) which can better predict survival than conventional histopathologic analysis. Such new classification techniques are of interest so that patients can be more appropriately stratified for new treatment strategies (20
The mesenchymal subgroup of glioblastomas typically have a poorer prognosis than the more common proneural subgroup (21
). However, in our study, patients with the mesenchymal
gene expression signatures had significantly extended survival compared with a large, multi-institutional cohort (n=82) of glioblastoma samples of the same molecular subgroup treated with various other therapies. No such survival difference was observed in patients from this clinical trial with proneural
signatures, compared to other control glioblastoma subjects of the proneural subgroup (n=60). Admittedly, such comparisons with concurrent and historical controls are not meant to imply efficacy, since this Phase I trial did not have a prospectively matched, placebo-controlled arm. Although some prognostic factors, such as age and Karnofsky performance status, were relatively matched in our comparison groups, the extent of surgical resection was not directly compared between the patients in this trial and our concurrent/historical controls. Since we need adequate amounts of tumor (>2 grams) to generate the autologous vaccines, tumor resectability was taken into account in the eligibility criteria. Therefore, it is possible that the extent of surgical resection may have been greater in our DC vaccinated patients compared to concurrent/historical controls, which could have influenced our survival results. Nevertheless, the median OS (31.4 mo.) of our DC-vaccinated patients is still noteworthy, when compared to large series of glioblastoma patients who underwent gross total tumor resections and were treated with concomitant chemo-radiotherapy, where the median survival was reported to be 18.6 months (31
It is unclear whether the extended survival of our patients with mesenchymal gene expression signatures is a direct result of the vaccine effects, or good responses to follow-up therapies after failing the vaccine. Since mesenchymal signatures represent glioblastoma subgroups that are more resistant to conventional therapy, it can be speculated that DC vaccination somehow makes these tumors more susceptible to subsequent treatments (39
). Because adjuvant temozolomide treatment was coordinated into the schedule of the DC booster vaccinations, this is a difficult distinction to make from our study design. Nevertheless, our results do suggest that mesenchymal
gene expression signatures express elevated inflammatory gene transcripts and possess an increased density of tumor-infiltrating CD3+
lymphocytes compared with glioblastomas expressing other genetic signatures. As such, we hypothesize that the expression of inflammatory genes (e.g., IL-1R, TNF-α signaling factors, and chemokines) may facilitate the priming and trafficking of tumor-specific T cells into the tumor parenchyma, which might be enhanced by DC vaccination and innate immune response modifiers. Hence, the mesenchymal gene expression signature may have a direct impact on the bioactivity of the vaccine itself, irrespective of post-vaccine therapy. Prospectively designed, randomized, multi-center Phase III clinical trials will be required to validate such hypotheses and proof of clinical benefit remains to be established.
Overall, the results reported here may provide novel insights for prospective patient selection in future immunotherapy studies and lend additional credence for the ability of genetic expression signatures to impart relevant data for personalized cancer treatment. Based on the results of this Phase I trial, we will continue developing more advanced clinical trials with this particular approach. We currently are planning a randomized, multi-center Phase II/III clinical trial of DC vaccination for newly diagnosed glioblastoma (DCVax-Brain™), which will hopefully help to further define which subgroups of patients may respond to tumor vaccination strategies. This in turn may lead to further optimization and refinements of related trials of DC-based vaccines for patients with glioblastoma, with the ultimate goal of developing novel immunotherapeutic strategies for brain cancer patients.
Statement of Translational Relevance
The selective identification of patients who will respond to a particular therapy is of paramount importance, especially for patients diagnosed with malignant glioma. Patients diagnosed with glioblastoma (WHO grade IV) have an expected 5 year survival of less 3.3%. In this study, we report the results of a phase I clinical trial in which glioblastoma patients were treated with a personalized immunotherapy approach, comprised of autologous tumor lysate-pulsed dendritic cell vaccination. In addition, we utilized gene expression profiling to identify a group of patients with a particular gene expression signature (mesenchymal glioblastoma) that had longer survival following DC vaccination, compared to contemporary/historical control patients of the same gene expression subgroup, who did not receive vaccination. This signature was associated with inflammatory transcripts and enhanced tumor infiltrating T lymphocytes. Thus, these results suggest that gene expression signatures may be able to identify an immunogenic subgroup of glioblastoma that could be more responsive to immune-based therapies.