Glioblastoma (GBM) is a poor prognosis malignant WHO grade 4 glioma whose standard therapy, after surgical resection, consists of radio-therapy (RT) and chemotherapy (CT) with temozolomide (TMZ) according to Stupp et al. However, the prognosis remains poor with a 5% five-year survival (1). In recent years, there has been renewed interest in immunotherapy of cancer due to new drugs and effective therapies, such as immune checkpoints inhibitors (ICIs), adoptive T-cell approaches, dendritic cell-based vaccines, or combinations of these. Although studies with active immunotherapy have used different types of molecules, their results have not been consistent enough to be approved by the FDA (2). GBM has an immunosuppressive microenvironment due to tumor-associated factors: overexpression of inhibitory cytokines or checkpoint molecules, low levels of HLA expression on tumor cells, and an abundance of infiltrating regulatory T cells (Treg) (3). Thus, it remains an aggressive cancer with limited therapeutic options due to the interactions between tumor cells and the microenvironment, which require more targeted agents against both components (4). Dendritic cells (DCs) are the most potent professional antigen-presenting cells due to their linking function between innate and adaptive immune responses, becoming a promising way to generate a specific immune response against cancer (5). Regarding the use of dendritic cell vaccination in HGGs (High-grade gliomas), numerous studies have been published and are underway to evaluate the safety and efficacy of DC-based vaccines in GBM patients (6, 7). Moreover, in 2014 two meta-analyses were published, indicating improved survival (OS) and progression-free survival (PFS) with a DC vaccination in HGG patients. In 2023 Oster et al. identified the main phase III clinical trials for adult GBM and among these, he cited Kong because his study investigated cytokine-induced killer (CIK) cells combined with standard radio-chemotherapy which prolonged PFS (8.1 months) (8, 9). Moreover, a two most recent meta-analysis were relevant to demonstrate a different outcome in GBM patients treated with DC vaccination in terms of OS and PFS. The authors agreed on the safety, in fact they didn’t report severe adverse events (AEs), regardless of the number of cycles, dosages and the administration route (10, 11). Finally, a phase III trial by Liau et al. found that OS was longer in the arm treated with their autologous DC vaccine compared to the standard therapy (12).They randomized the 331 patients to receive DCVax-L plus temozolomide vs placebo plus temozolomide; in case of disease progression/relapse during treatment, crossover was allowed. Considering this, our study is monocentric and all patients after surgery had received the same treatment because the placebo was not expected.

The study is divided into two parts: a pre-screening phase and a main study. Pre-screening and patient enrollment occur after surgery. As surgery is a clinical activity, it is not part of the study. During the pre-screening phase, patients undergo procedures necessary to obtain the biological material needed for DCvax manufacturing (leukapheresis) and are treated with standard RT-CT (according to the Stupp regimen).

After the pre-screening phase, patients are enrolled based on the following

Inclusion Criteria:

Then the main exclusion criteria are:

The experimental treatment consists of an induction phase with four-weekly doses of the DCvax (1.0 × 10⁷ cells), administered intradermally (weeks one to four), followed by a maintenance phase consisting of 28-day cycles with vaccine administration at the start of week seven, and TMZ (150-200mg/m2/day) assumed orally from day one to five q28 (starting week five). The combined maintenance treatment continues until disease progression, unacceptable toxicity, the patient withdrawing consent, or until the maximum of one-year treatment time ( Figure 1A ). To evaluate response and progression, we used the international Response Evaluation Criteria from the Brain Tumors Committee (Response Assessment in Neuro-Oncology guidelines, RANO), associated with Perfusion MRI by DSC (dynamic susceptibility contrast). Our objective was to distinguish between the real progression of GBM from radionecrosis and pseudo-progression by evaluating the five ROIs (Region Of Interest) of the perfusion study and correlating them with RANO imaging criteria. In particular, we wanted to demonstrate that the presence in at least three ROIs of rCBV (Relative Cerebral Blood Volume) values> 2.5 identifies real disease progression according to the RANO imaging criteria.

For this study, patients are reevaluated for response at the end of the induction phase and every two cycles during the maintenance phase (16). (Protocol in Supplementary Documents )

In December 2022, nine patients were evaluated for safety and efficacy, allowing the pre-planned analysis to be performed. Screening failure occurred in 13 patients: for 8 patients, we did not collect enough tumor tissue to ensure that we could perform at least 75% of the induction treatment (3 out of 4 planned vaccines), 3 patients withdrew consent, 2 patients had a rapid worsening of their clinical condition ( Figure 1B ).

All the patients (5 males and 4 females) with a median age of 58 years (range: 58-69) underwent radical surgery and leukapheresis before starting RT-CT. Four out of 9 patients, due to a shortage of vaccine vials and their clinical stability, continued with TMZ alone for the maintenance phase. However, all patients completed the induction phase, a necessary condition for them to be considered evaluable by protocol and after which the first re-evaluation was planned.

One week after completing RT-CT, they began the vaccine induction phase. Table 1 describes patients characteristics. The median vaccine cycle was six (range: 3-12).At baseline DTH skin test was negative in all nine patients, but after the induction phase it turned out positive on KLH test alone in four patients and on both KLH and tumor Homogenate in one patient. In contrast, we did not observe any positivity on tumor homogenate test alone. Finally the DTH test result correlated neither with MGMT status nor with residual disease post surgery.

All 9 patients were evaluable for AE analysis (patients who had at least 30 days of observation after first vaccine administration). Table 2 summarizes the targeted AEs reported by AE type and maximum grade. All G3-4 toxicities observed were related to TMZ and in line with the drug’s expected ones. As shown in Table 3 two patients progressed within three months after leukapheresis. At the cut-off date, all patients were alive except one and were being treated with TMZ. However, the patients who had shown disease progression according to study criteria but had clinical benefit and had not a clinical significant progression in MRI continued the TMZ treatment outside the study. None of these patients had yet to start a second line treatment. At the start of the induction phase, the median amount of dexamethasone consumed by patients, during all the entire treatment, was 2 milligrams (prednisolone equivalent dose 13 mg), which gradually reduced during the four weeks of treatment (compatible with the patient’s symptoms). Seven out of nine patients had a functional perfusion evaluation done because of a pathologic enhancement, whereas two patients had insufficient residual disease for at least three consecutive controls, making the functional perfusion study unnecessary. Of the latter, one patient underwent 10 DCvax cycles and the other underwent 12 DCvax cycles, achieving long-term disease control. At the end of the study, a correlation will be performed between the functional study, immunoRANOs (18), RANOs, and clinical outcomes to meet the exploratory objectives. As of June 2023, the median follow-up after surgery was 13.8 months (range: 13.3-24.7 months). The median PFS from leukapheresis was 11.3 months (95%CI: 3.2-Not estimable) and 12.2 months from surgery (95%CI: 4.7-Not estimable). The median OS from leukapheresis was 23.1 months and 24.7 from surgery (95%:13.0-Not estimable) ( Table 3 ).

GBM has a poor prognosis with a median survival of approximately 12-14 months and less than a 5% five-year survival, even when patients receive the Stupp regimen treatment post-surgery. Nowadays, the only prognostic factor related to the outcome remains the methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) gene promoter. The cut off value of methylation cited in literature is higher than 9% and may be a predictive marker of sensitivity to alkylating agents (19). In January 2023, Liau et al. published a phase III trial. They compared OS between the Stupp regimen (concomitant RT and TMZ followed by TMZ), and the same treatment associated with DC vaccine. Three hundred thirty-one patients were enrolled. The trial raised a lot of criticism regarding its methodology, not only because it removed PFS from the primary endpoint but because of its post hoc introduction of “control patients” by pooled external control data (20). Despite these limitations, the trial outcome showed evidence of a statistically significant longer OS for patients receiving the combination therapy. In addition to their primary endpoint of OS, they conducted an exploratory analysis on biomarkers and immunogenicity that may correlate with OS and responses to the DC vaccine. Similar results were reported in the Lepski et al. study, which investigated the difference between a combination treatment and an allogeneic DC vaccine after a standard RT-CT regimen. In recurrent patients, the vaccination leads to an OS of 27.6 months (21). These recent trials and our first-step results emphasize the potential impact of immunotherapy, particularly DC cell-based approaches concerning patients with a poor prognosis. Our previous data (on melanoma patients) demonstrated that TMZ could selectively reduce the circulating T regs, and DCvax may favor objective responses to subsequent therapies (14, 22). In melanoma patients we also demonstrated that patients who developed in vivo immune responses (DTH skin test positivity after treatment) gained a survival advantage (13). Based on the data, we suggest combining the vaccine with standard therapies to increase drugs’ synergistic effect. To understand the full impact of the vaccine, we added some exploratory objectives, including a functional radiological study of perfusion to correlate with iRANOs, immunological responses, and clinical outcomes. A complete analysis of all objectives will be performed upon completion of the study. Out of the first nine patients, we observed that two patients receiving treatment for one year, with 10 and 12 DCvax cycles, respectively, had minimal residual disease, which didn’t require any further ROI evaluation. Furthermore, they had three consecutive negative radiological controls, making the functional perfusion study unnecessary. The study has very restrictive radiological criteria to determine progression, resulting in the exit of patients from the study. To prioritize patient safety and maintain conservative approach, continuation of treatment beyond progression is not planned by study. However, 8 out of 9 patients, due to clinical benefit and to marginal radiological disease progression confirmed during multidisciplinary discussion, continued TMZ treatment outside the study. Only 1 patient out of 9 proceeded immediately to second-line therapy. The DCvax treatment was able to induce in vivo immune response in 5 patients, which we saw was associated with improved outcome in melanoma patients. The main limitation of our study lies in the vaccine manufacture that has to comply with Good Manufacturing Practice (GMP) rules. Furthermore, our vaccine is entirely autologous, and the main reason for the screening failure was the limited quantity of tumor tissue that was available, which prevented us from producing enough vaccine vials. On the other hand, the excellent toxicity profile, the capacity to elicit immunological responses, and the easy route of administration are strengths. The advantage of using autologous tumor homogenate (as DCVax-L by Liau), instead of allogeneic antigens (DC vaccine by Lepski) could be to target the individual patient’s antigens repertoire, while addressing the extreme heterogeneity of glioblastoma. Our vaccine’s favorable toxicity profile and mechanisms of action make it an ideal candidate for future combination therapies trials.

When used with standard RT-CT treatments, the here proposed autologous DCVax is safe and has met the first step primary endpoint, allowing enrollment to continue.