Study Question: Does myelosuppression secondary to Temozolomide (TMZ) potentiate or limit EGFRvIII peptide vaccine induced anti-tumor immunotherapy in glioblastoma (GBM) patients?
Methods: Adults with newly diagnosed GBM were eligible for the clinical trial if they met the following criteria: 1) gross total resection of their tumor followed by standard conformal beam radiotherapy with concurrent TMZ, 2) EGFRvIII expression via immunohistochemistry (IHC), 3) Karnofsky Performance Scale (KPS) of >70, and 4) no radiographic evidence of recurrence after irradiation. Patients were vaccinated with a peptide that spans the EGFRvIII mutation (LEEKKGNYVVTDHC) conjugated to keyhole limpet hemocyanin (PEPvIII-KLH; BioSynCorporation) along with granulocyte-macrophage colony-stimulating factor (GM-CSF), starting within 6 weeks of completing radiation. Patients were sequentially assigned to receive TMZ at a standard (STD) targeted dose of 200 mg/m2 for the first 5 days of a 28-day cycle (n = 12) or at a dose-intensified (DI) targeted dose of 100 mg/m2 for the first 21 days of a 28-day cycle (n = 10).
Delayed-type hypersensitivity (DTH) reactions were assessed within 48-72 h by a positive skin test defined as 0.5 mm induration. Humoral responses were compared based on maximum titer obtained as measured by enzyme-linked immunosorbent assay (ELISA) testing from the serum. Immunostaining for EGFRvIII was performed on paraffin-embedded tissue specimens before enrollment and at progression. Absolute and subset leukocyte counts were quantified by flow cytometry using a direct immunofluorescence.
Results: Toxicity was minimal and limited to possible allergic reactions in 4 of 22 patients. There were no autoimmune reactions or T2 changes on the MRI. One subject was removed because of the allergic reaction. Patients receiving the STD 5-day schedule exhibited at least transient grade 2 lymphopenia by the 4th cycle of TMZ. Grade 3 lymphopenia was observed in only two patients with this regimen at any time point. Sustained grade 2 lymphopenia was induced in all patients receiving the DI schedule by the 4th cycle of TMZ, and by the 6th cycle of TMZ, all patients exhibited a sustained grade 3 lymphopenia. B-cell counts increased in the STD cohort, but they were significantly reduced in the DI cohort (P = 0.004; paired t-test). TMZ treatment did not lead to a significant reduction in the proportion of Treg in the STD cohort (P = 0.134; paired t-test), but there was an increase in the proportion of Treg at vaccine 6 within the DI cohort (P = 0.008). No patient had evidence of EGFRvIII cellular or humoral immunity prior to vaccination. All patients exhibited EGFRvIII-specific humoral responses by vaccine 8 (P < 0.0001). The antibody titers were unexpectedly higher in the DI cohort when compared to the STD cohort despite worse lymphopenia (1:634,982 compared with 1:186,521; P = 0.037). When evaluated at vaccine 8, none of the STD cohort developed a DTH reaction, but 7 of 8 in the DI cohort did. Histologic samples from 12 of 17 recurrent tumors were available for EGFRvIII expression IHC analysis. Eleven of these samples had lost expression of EGFRvIII (P < 0.0001; binomial proportions). There was no difference in progression-free survival (PFS) or overall survival (OS) between the DI and STD cohort, but the study was not powered to detect such differences. The median PFS of all patients was 15.2 months (CI95: 11.0–18.5 months), the median PFS from the time of vaccination was 11.8 months (CI95: 8.1–15.6 months), the OS from the time of diagnosis was 23.6 months (CI95: 18.5–33.1 months) and the median OS from the time of vaccination was 19.3 months (CI95: 15.6–30.7 months). After adjustment for age and KPS, the risk of death of the vaccinated patients was significantly lower than observed in the TMZ-treated historical control group.
Conclusions: Despite profound lymphopenia and increase in Treg cells, both humoral and cellular vaccine-induced immune responses are enhanced by the DI TMZ regimen. Furthermore, vaccination of these patients resulted in elimination of expression of the EGFRvIII antigen and was significantly associated with increase in PFS and OS.
Perspectives: Postoperative radiation with concurrent chemotherapy followed by maintenance chemotherapy is the standard of care for GBM patients. The current study gives evidence that TMZ-induced lymphopenia does not inhibit EGFRvIII peptide vaccine-induced immune responses in GBM patients. Instead, the DI TMZ regimen which leads to increase in lymphopenia, decrease in B cells and increase in Tregs seems to potentiate the vaccine-induced immune response. These results suggest that concurrent use of myelosuppressive chemotherapy can be given effectively with vaccine therapy and this does not inhibit the induction of the desired immune response. Furthermore, these results suggest a possible survival benefit for GBM patients treated with the EGFRvIII peptide vaccine. A randomized, controlled clinical trial is currently being planned to further study this question.
Summary written by: Gordon Li, M.D.