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We aimed to assess the clinical efficacy of glutaraldehyde-fixed human umbilical vein endothelial cell (HUVEC) vaccine for the treatment of patients with recurrent glioblastoma. Patients of a HUVEC vaccine group received intradermal injections of 5 × 107 HUVEC weekly during the first month, and every 2 weeks from the second month, until progression of the disease was observed. Salvage treatment consisted of multimodal chemotherapy, radiation, including gamma-knife therapy, and/or repeated surgery, when feasible. Hazard ratios for death were calculated using a Cox model. A total of 17 patients with recurrent glioblastoma were enrolled in this study. All the patients received the initial treatment consisting of maximal safe surgical resection, followed by radiotherapy of 50–80 Gy or more, with concomitant and adjuvant chemotherapy consisting of temozolomide or nimustine (ACNU). A total of 352 vaccinations were performed for the patients of the HUVEC vaccine group (median number of vaccination = 11 doses; range 3–122 doses). The median progression-free survival and overall survival were 5.5 and 11.4 months, respectively. The median overall survival from the diagnosis was 24.3 months. The HUVEC vaccine therapy significantly prolonged the tumor doubling time and contributed to reducing the tumor growth rate. Hematological adverse reactions due to chemotherapy were recognized: one patient experienced grade III leukocytopenia and one showed grade II lymphocytopenia. Associated with the HUVEC vaccine therapy, a delayed-type hypersensitivity-like skin reaction developed at the injection site. The HUVEC vaccine therapy effectively controlled disease progression, without evident adverse effects, except for a delayed-type hypersensitivity-like skin reaction at the injection site.
Glioblastoma (GBM) is one of the most devastating human tumors. Even with optimal surgical resection and standard chemoradiotherapy, GBM always recurs, and no specific treatment exists for recurrent GBM. Unfortunately, the median survival following recurrence is 5–7 months. GBM is a highly vascular tumor with high expression of vascular endothelial growth factor (VEGF).[2, 3] Bevacizumab (BEV), a humanized monoclonal antibody to VEGF, which inhibits tumor angiogenesis, consequently decreasing the intratumoral blood flow,[3-7] had been expected to reduce the volume of recurrent tumors at the time the drug was approved for clinical use and clinical studies were started. However, in patients with recurrent GBM, BEV has only limited clinical benefit. Although BEV causes a strong decrease of contrast enhancement on magnetic resonance images, vascular remodeling induced by BEV, which makes tumors more hypoxic and glycolytic,[8, 9] might result in increased invasiveness of tumor cells into the normal brain tissue. Enhanced tumor cell infiltration after anti-angiogenic treatment has been reported in other tumor models.[4, 9-11]
Human endothelial cells in culture share some properties with the angiogenic endothelium, such as the high expression of CD51 and CD105. We have been focusing on human umbilical vein endothelium cell (HUVEC), which, under culture with VEGF and basic-fibroblast growth factor, has specific properties of angiogeneic endothelium, such as the high expression of the platelet endothelial cell adhesion molecule (CD31), integrin alpha-V precursor (CD51) and endoglin (CD105), as confirmed by flow-cytometry.[12, 13] We have also confirmed the expression of these surface markers on the vascular endothelium of GBM and colorectal cancer. Based on these results, we tested and confirmed the effectiveness of glutaraldehyde-fixed allogeneic endothelial cells as a vaccine against solid tumors (in an animal model). In addition, we commenced a clinical trial of allogeneic HUVEC as a vaccine for the treatment of angiogenic solid tumors (malignant brain tumors and colorectal cancer) in humans. Patients with recurrent malignant brain tumors had better clinical response than those with metastatic colorectal cancer. This might be mainly dependent on the smaller size of the targeted tumor lesions of the patients with malignant brain tumors. In the present study, we aim to assess the clinical efficacy of glutaraldehyde-fixed HUVEC vaccine for the treatment of patients with recurrent GBM.
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The HUVEC vaccine therapy was feasible for the patients with recurrent GBM and a low KPS score. In the present study, the radiological response rate was much lower (5.9%) than that for BEV (more than 50%), but the patients had relatively long survival time: the median PFS and OS were 5.5 and 11.4 months, respectively. The median OS from the diagnosis was 24.3 months. The median OS compares favorably with that reported for other salvage therapies. The median OS for patients with GBM treated with temozolomide at first relapse was approximately 8 months. Similarly, a phase II trial of BEV-alone or the BEV-plus-irinotecan in patients with recurrent GBM demonstrated that the median OS was 9.2 and 8.7 months, respectively.[5-7]
According to the report by Carson, prognostic factors for recurrent GBM include age, KPS, corticosteroid use, and shorter time from original diagnosis to recurrence. In this study, age was the only prognostic factor, but the patients over age 50 survived longer than those under age 50. It could depend on the bias of patients' background: 10 patients (59%) had poor neurological function (KPS of 50-60%), and four patients (24%) had used corticosteroids during the study.
Because patients with recurrent GBM were clinically deteriorated, and previously treated with multimodality therapy consisting of surgery and chemoradiotherapy, treatment options were limited to palliative surgery when feasible, radiotherapy, including stereotactic radiosurgery, and chemotherapy. Because immunotherapy is associated with low risk of toxicity, it is a promising treatment strategy for recurrent GBM. Wilms tumor 1 peptide vaccine therapy for patients with recurrent GBM showed promising results: the 6-month PFS rate was 33.3% and the median OS was 9.2 months. This treatment, however, is limited to patients with the HLA-A *2402 phenotype.
We established the HUVEC vaccine therapy for patients with progressive malignancy. The HUVEC vaccine therapy was designed to target antigens specifically or preferentially on human tumor endothelium, such as CD31, integrin alpha v beta 3 and CD105: CD31 can be isolated from glioblastoma specimens; integrin alpha v beta 3 plays a key role in endothelial cell survival and migration during angiogenesis; and CD105 is considered an appropriate marker of tumor-related angiogenesis and neovascularization. Therefore, different from single-target therapies, such as “VEGF-targeted therapy” or peptide-based immunotherapy, HUVEC vaccine therapy is a multi-targeted immunotherapy. Compared with single-targeted therapies, multi-targeted immunotherapy has the advantage of reducing the risk of resistance to therapy, as well as the in vivo selection of the ideal antigen to be exposed to the immune system by antigen-presenting cells. The most important limiting factor of single peptide-based immunotherapy is the use of peptides that are effectively presented only by specific human leukocyte antigen (HLA) subtypes, eliciting HLA-restricted CTL responses. Thus, it cannot be applied to every patient, but to a limited population with a specific HLA subtype. In comparison, the HUVEC vaccine, which consists of whole cells expressing various kinds of angiogenic antigens (multi-target), may allow the different antigen presenting cells expressing different HLA to “select” the best antigenic determinant from the antigen “repertoire” to be presented and to generate the anti-angiogenic CTL. As evidence to support this theory, for at least 6 months after enrollment, the HUVEC vaccine therapy resulted in significant elongation of the tumor doubling time and delay of tumor progression in patients selected without consideration of their HLA subtypes (Fig. 2). Specific antibodies and cellular immunity reactive with HUVEC's membrane antigens were detected in monthly samples of the patients. At 1 month after enrollment, specific antibodies such as CD31, CD51, CD105 and CD146 were also detected. IFN-γ secretion by patients' peripheral blood mononuclear cells was measured in the presence of HUVEC by enzyme-linked immunospot. The evident reduction of the tumor growth rate, however, could be observed from the third month after the start of the HUVEC vaccine therapy.
In this study, hematological adverse reactions of grades 2–3 were recognized, but they were associated with the administration of chemotherapy. Although we continued the vaccination protocol for long periods of time with an expectation of inducing a long-lasting immune response, no adverse effect caused by the HUVEC vaccine therapy was observed, except for a DTH-like skin reaction at the injection site. In contrast, patients with recurrent GBM receiving BEV alone or in combination with irinotecan experienced grade 3 adverse events in 46.4 and 65.8% of cases, respectively, including hypertension, proteinuria, convulsion and neutropenia. Intracranial hemorrhage was also reported in 2 patients (2.4%) of a BEV-alone group (grade 1) and in three patients (3.8%) of a BEV plus irinotecan group (grades 1, 2 and 4, respectively). In addition, BEV has been linked to increased invasiveness. Thus, the HUVEC vaccine seemed to be superior to BEV in terms of adverse reactions. The main difference between BEV and the HUVEC vaccine is that BEV targets one angiogenic factor, namely VEGF, whereas the HUVEC vaccine targets the angiogenic vascular endothelium itself, inducing humoral and cellular immune responses against it.
Cancer cells may acquire the ability to produce angiogenic factors other than VEGF, such as acidic or basic fibroblast growth factor, interleukin-8, and epidermal growth factor, among others, overcoming the effect of BEV. Different from cancer cells, the HUVEC or angiogenic endothelial cells are normal cells, which rarely develop mutations. Because the HUVEC provide several targets, such as CD31 and CD105, different from the BEV, in which the only target is the VEGF, the HUVEC vaccine therapy might not increase the degree of invasiveness.
Once the immunity against tumor angiogenesis is stimulated, theoretically, it can be permanently reactivated by periodic vaccinations, and might be useful for the control of not only brain tumors, but also other solid tumors that also depend on angiogenesis for their growth and metastasis. Unfortunately, in the case of fast-growing and large tumors, the vaccine is not successful in controlling the growth of tumor mass (Fig. 1). The HUVEC vaccine therapy against recurrent GBM, therefore, should be used in combination with other treatment modalities. In a pilot study including metastatic colorectal cancer as well as other malignant brain tumors, such as recurrent anaplastic oligodendroglioma and recurrent pineoblastoma, specific antibodies and cellular effectors against HUVEC membrane antigens were detected in all the patients. However, patients with malignant brain tumors had better clinical response than those with metastatic colorectal cancer, which might be mainly because of smaller lesions targeted. Different from patients with GBM, colorectal cancer patients were enrolled in the HUVEC vaccine protocol only after the relapse of all available treatment modalities. Thus, a similar trial could not be conducted for colorectal cancer. Patients with other tumor types have also been included, but only a few cases have been evaluated, without conclusive findings. Because the safety of this treatment modality could be proved, similar trials should be conducted to analyze the other solid tumor types that might benefit from its application.
In conclusion, the HUVEC vaccine therapy is a promising new treatment modality, without important adverse effects. Acquisition of resistance to multi-target immunotherapy, namely the HUVEC vaccine therapy, seems to be less frequent. As a matter of course, a large-scale prospective study will help confirm our present results.