Baukje-Nynke Hoogeboom

Prognostic significance of tumor-infiltrating T-lymphocytes in primary and metastatic lesions of advanced stage ovarian cancer

PurposeOvarian cancer patients with intra-tumoral CD3+ T-lymphocytes in primary tumor tissue have a better prognosis. This study aims to analyze the presence and relative influence of three important T-lymphocyte subsets, tumor-infiltrating CD8+ cytotoxic T-lymphocytes (CTL), CD45R0+ memory T-lymphocytes, and FoxP3+ regulatory T-lymphocytes (Treg), in primary tumor tissue and omental metastases of patients with ovarian cancer.Experimental designThe number of CD8+, CD45R0+, and FoxP3+ T-lymphocytes was determined by immunohistochemistry on a tissue micro array containing ovarian tumor tissue and/or omental metastases obtained at primary debulking surgery from 306 FIGO stage I–IV ovarian cancer patients. Immunohistochemistry data were correlated to clinicopathological parameters and survival data.ResultsHigh number of CD8+ CTL and a high CD8+/FoxP3+ ratio in ovarian-derived tumor tissue were associated with increased disease-specific survival and proved to be independent prognostic factors in multivariate analyses. In advanced stage patients, the presence of CD8+ CTL, CD45R0+ memory T-lymphocytes, FoxP3+ Treg or a high CD8+/FoxP3+ ratio in ovarian-derived tumor tissue was associated with an increased disease specific survival in univariate analysis, as was the presence of CD45R0+ memory T-lymphocytes and FoxP3+ Treg in omental metastases. Furthermore, in advanced stage patients CD8+ cytotoxic and FoxP3+ regulatory T-lymphocytes infiltrating ovarian-derived tumor tissue were independent predictors of increased prognosis.ConclusionsT-lymphocytes infiltrating primary and metastatic ovarian cancer sites are associated with improved prognosis. These associations are especially distinct in advanced stage patients, underlining the potential for immunotherapy as a broadly applicable therapeutic strategy.

Immunization with a P53 synthetic long peptide vaccine induces P53-specific immune responses in ovarian cancer patients, a phase II trial

The prognosis of ovarian cancer, the primary cause of death from gynecological malignancies, has only modestly improved over the last decades. Immunotherapy is one of the new treatment modalities explored for this disease. To investigate safety, tolerability, immunogenicity and obtain an impression of clinical activity of a p53 synthetic long peptide (p53‐SLP) vaccine, twenty patients with recurrent elevation of CA‐125 were included, eighteen of whom were immunized 4 times with 10 overlapping p53‐SLP in Montanide ISA51. The first 5 patients were extensively monitored for toxicity, but showed no ≥ grade 3 toxicity, thus accrual was continued. Overall, toxicity was limited to grade 1 and 2, mostly locoregional, inflammatory reactions. IFN‐γ producing p53‐specific T‐cell responses were induced in all patients who received all 4 immunizations as measured by IFN‐γ ELISPOT. An IFN‐γ secretion assay showed that vaccine‐induced p53‐specific T‐cells were CD4+, produced both Th1 and Th2 cytokines as analyzed by cytokine bead array. Notably, Th2 cytokines dominated the p53‐specific response. P53‐specific T‐cells were present in a biopsy of the last immunization site of at least 9/17 (53%) patients, reflecting the migratory capacity of p53‐specific T‐cells. As best clinical response, stable disease evaluated by CA‐125 levels and CT‐scans, was observed in 2/20 (10%) patients, but no relationship was found with vaccine‐induced immunity. This study shows that the p53‐SLP vaccine is safe, well tolerated and induces p53‐specific T‐cell responses in ovarian cancer patients. Upcoming trials will focus on improving T helper‐1 polarization and clinical efficacy.

Potentiation of a p53‐SLP vaccine by cyclophosphamide in ovarian cancer: A single‐arm phase II study

The purpose of the current phase II single‐arm clinical trial was to evaluate whether pretreatment with low‐dose cyclophosphamide improves immunogenicity of a p53‐synthetic long peptide (SLP) vaccine in patients with recurrent ovarian cancer. Patients with ovarian cancer with elevated serum levels of CA‐125 after primary treatment were immunized four times with the p53‐SLP vaccine. Each immunization was preceded by administration of 300 mg/m2 intravenous cyclophosphamide as a means to affect regulatory T cells (Tregs). Vaccine‐induced p53‐specific interferon‐gamma (IFN‐γ)‐producing T cells evaluated by IFN‐γ ELISPOT were observed in 90% (9/10) and 87.5% (7/8) of evaluable patients after two and four immunizations, respectively. Proliferative p53‐specific T cells, observed in 80.0% (8/10) and 62.5% (5/8) of patients, produced both T‐helper 1 and T‐helper‐2 cytokines. Cyclophosphamide induced neither a quantitative reduction of Tregs determined by CD4+FoxP3+ T cell levels nor a demonstrable qualitative difference in Treg function tested in vitro. Nonetheless, the number of vaccine‐induced p53‐specific IFN‐γ‐producing T cells was higher in our study compared to a study in which a similar patient group was treated with p53‐SLP monotherapy (p ≤ 0.012). Furthermore, the strong reduction in the number of circulating p53‐specific T cells observed previously after four immunizations was currently absent. Stable disease was observed in 20.0% (2/10) of patients, and the remainder of patients (80.0%) showed clinical, biochemical and/or radiographic evidence of progressive disease. The outcome of this phase II trial warrants new studies on the use of low‐dose cyclophosphamide to potentiate the immunogenicity of the p53‐SLP vaccine or other antitumor vaccines.

Sunitinib depletes myeloid-derived suppressor cells and synergizes with a cancer vaccine to enhance antigen-specific immune responses and tumor eradication

The high efficacy of therapeutic cancer vaccines in preclinical studies has yet to be fully achieved in clinical trials. Tumor immune suppression is a critical factor that hampers the desired antitumor effect. Here, we analyzed the combined effect of a cancer vaccine and the receptor tyrosine kinase inhibitor sunitinib. Sunitinib was administered intraperitoneally, alone or in combination with intramuscular immunization using a viral vector based cancer vaccine composed of Semliki Forest virus replicon particles and encoding the oncoproteins E6 and E7 (SFVeE6,7) of human papilloma virus (HPV). We first demonstrated that treatment of tumor-bearing mice with sunitinib alone dose-dependently depleted myeloid-derived suppressor cells (MDSCs) in the tumor, spleen and in circulation. Concomitantly, the number of CD8+ T cells increased 2–fold and, on the basis of CD69 expression, their activation status was greatly enhanced. The intrinsic immunosuppressive activity of residual MDSCs after sunitinib treatment was not changed in a dose-dependent fashion. We next combined sunitinib treatment with SFVeE6,7 immunization. This combined treatment resulted in a 1.5- and 3-fold increase of E7-specific cytotoxic T lymphocytes (CTLs) present within the circulation and tumor, respectively, as compared to immunization only. The ratio of E7-specific CTLs to MDSCs in blood thereby increased 10- to 20-fold and in tumors up to 12.5-fold. As a result, the combined treatment strongly enhanced the antitumor effect of the cancer vaccine. This study demonstrates that sunitinib creates a favorable microenvironment depleted of MDSCs and acts synergistically with a cancer vaccine resulting in enhanced levels of active tumor-antigen specific CTLs, thus changing the balance in favor of antitumor immunity.

Long‐term clinical and immunological effects of p53‐SLP® vaccine in patients with ovarian cancer

Vaccine‐induced p53‐specific immune responses were previously reported to be associated with improved response to secondary chemotherapy in patients with small cell lung cancer. We investigated long‐term clinical and immunological effects of the p53‐synthetic long peptide (p53‐SLP®) vaccine in patients with recurrent ovarian cancer. Twenty patients were immunized with the p53‐SLP® vaccine between July 2006 and August 2007. Follow‐up information on patients was obtained. Clinical responses to secondary chemotherapy after p53‐SLP® immunizations were determined by computerized tomography and/or tumor marker levels (CA125). Disease‐specific survival was compared to a matched historical control group. Immune responses were analyzed by flow cytometry, proliferation assay, interferon gamma (IFN‐γ) ELISPOT and/or cytokine bead array. Lymphocytes cultured from skin biopsy were analyzed by flow cytometry and proliferation assay. Of 20 patients treated with the p53‐SLP® vaccine, 17 were subsequently treated with chemotherapy. Eight of these patients volunteered another blood sample. No differences in clinical response rates to secondary chemotherapy or disease‐specific survival were observed between immunized patients and historical controls (p = 0.925, resp. p = 0.601). p53‐specific proliferative responses were observed in 5/8 patients and IFN‐γ production in 2/7 patients. Lymphocytes cultured from a prior injection site showing inflammation during chemotherapy did not recognize p53‐SLP®. Thus, treatment with the p53‐SLP® vaccine does not affect responses to secondary chemotherapy or survival, although p53‐specific T‐cells do survive chemotherapy.

P53-specific T cell responses in patients with malignant and benign ovarian tumors : Implications for p53 based immunotherapy

Despite intensive treatment, 70% of the ovarian cancer patients will develop recurrent disease, emphasizing the need for new approaches such as immunotherapy. A promising antigenic target for immunotherapy in ovarian cancer is the frequently overexpressed p53 protein. The aim of the study was to evaluate the nature and magnitude of the baseline anti‐p53 immune response in ovarian cancer patients. P53‐specific T cell responses were detected in both half of the ovarian cancer patients as in the group of control subjects, consisting of women with benign ovarian tumors and healthy controls. Importantly, while in the control group p53‐specific immunity was detected among the CD45RA+ naïve subset of T cells only, the p53‐specific T‐cell responses in ovarian cancer patients were also present in the CD45RO+ memory T‐cell subset, suggesting that in the cancer patients sufficient amounts of cancer‐derived p53 was presented to induce the formation of a p53‐specific memory T‐cell response. Further characterization of the p53‐specific memory T‐cell responses revealed that in addition to the type 1 cytokine IFN‐γ also the type 2 cytokines IL‐4 and IL‐5, as well as the immunosuppressive cytokine IL‐10 were produced. Notably, p53‐specific T cells were not only detected in the peripheral blood, but also among tumor infiltrating lymphocytes and in tumor‐draining lymph nodes. In conclusion, the existence of a weak mixed T‐helper type 1 and 2 p53‐specific T‐cell repertoire supports the rationale of using p53 long peptides in vaccination strategies aiming at the induction of p53‐specific Th1/CTL immunity.

Therapeutic immunization and local low-dose tumor irradiation, a reinforcing combination

Therapeutic cancer vaccines show promise in preclinical studies, yet their clinical efficacy is limited. Increased recruitment of immune cells into tumors and suppression of the immune suppressive tumor environment are critical components toward effective cancer immunotherapies. Here, we report how local low‐dose irradiation, alone or with a therapeutic immunization based on Semliki Forest virus (SFV) against human papillomavirus (HPV)‐related cancer, influences these immune mechanisms. We first demonstrated that immunization with SFVeE6,7 or SFVeOVA, replicon particles expressing either HPV16 E6/E7 or ovalbumin, resulted in an antigen‐specific migration of CD8+ T cells into HPV‐ and OVA‐specific tumors. Local low‐dose tumor irradiation alone resulted in a 2‐fold increase of intratumoral CD8+ T cells. When 14 Gy irradiation was combined with immunization, intratumoral numbers of CD8+ T cells increased 10‐fold and the number of CD8+ T cells specific for the E7‐ epitope increased more than 20‐fold. Irradiation alone however also increased the number of intratumoral myeloid‐derived suppressor cells (MDSCs) 3.5‐fold. Importantly, this number did not further increase when combined with immunization. As a result, the ratio of antigen‐specific CD8+ T cells and MDSCs in tumors increased up to 85‐fold compared to the control. We furthermore demonstrated that following irradiation CCR2 and CCL2, CXCR6 and CCL16, chemokines and ligands involved in tumor homing of immune cells, were significantly up regulated. This study demonstrates that local low‐dose tumor irradiation influences the intratumoral immune population induced by SFVeE6,7 immunization by a strong increase in the ratio of antitumoral to immune suppressive cells, thus changing the intratumoral immune balance in favor of antitumor activity.

Enhancement of human papilloma virus type 16 E7 specific T cell responses by local invasive procedures in patients with (pre)malignant cervical neoplasia

It has been suggested that local invasive procedures may alter the natural course of (pre)malignant cervical disease. This could be due to partial excision of the lesions, or via induction of cellular immunity against human papillomavirus (HPV) by the local invasive procedures. We studied the influence of local invasive procedures on HPV‐16 E7 specific immune responses in patients with different grades of cervical intra‐epithelial neoplasia (CIN) and different stages of cervical cancer. Blood was obtained at intake and after invasive procedures from patients with CIN or cervical cancer. Antigen specific T‐cell responses were measured by IFN‐γ ELISPOT analysis, after stimulation with recombinant HPV‐16 E7 protein. As expected, HPV‐16 E7 specific IFN‐γ T cell responses were more frequent in HPV‐16 DNA positive patients compared with that in HPV‐16 DNA negative patients (39/50 vs. 16/36, (p = 0.006, χ2 test). After invasive procedures, a small number of HPV‐16 DNA positive CIN patients, but a considerable proportion of HPV‐16 DNA positive cervical cancer patients, showed an enhancement of T cell responses against HPV‐16 E7. Induction of T cell reactivity was most pronounced in cervical cancer patients who had undergone previous invasive procedures. Both CD4+ and CD8+ T cells showed E7 specific IFN‐γ production upon in‐vitro stimulation. Our study shows that invasive procedures may enhance HPV‐specific cell‐mediated immunity in a considerable number of patients with cervical cancer, but in only a minority of CIN patients. Our data indicate that invasive procedures should be considered as possible confounding factors when analyzing the effectiveness of therapeutic immunization studies, especially, when induction of HPV‐specific immune responses is used as intermediate end‐point.

Heterologous Prime-Boost Immunizations with a Virosomal and an Alphavirus Replicon Vaccine

Heterologous prime-boost immunization strategies in general establish higher frequencies of antigen-specific T lymphocytes than homologous prime-boost protocols or single immunizations. We developed virosomes and recombinant Semliki Forest virus (rSFV) as antigen delivery systems, each capable of inducing strong CTL responses in homologous prime-boost protocols. Here, we demonstrate that a heterologous prime-boost with recombinant Semliki Forest virus (rSFV) encoding a fusion protein of E6 and E7 of human papillomavirus (HPV) type 16 and virosomes containing the HPV16 E7 protein resulted in higher numbers of antigen-specific CTL in mice than homologous protocols. Evasion of vector-specific immunity appeared to play a role in establishing these high frequencies, as coinduction of vector-specific responses during the prime immunization reduced the frequency of antigen-specific CTL after a heterologous booster. However, the high numbers of CTL initially primed by the heterologous protocols did not correlate with enhanced responsiveness to in vitro antigenic stimulation, nor in improved cytolytic activity or antitumor responses in vivo compared to a homologous protocol with rSFV. This lack of correlation could not be explained by changes in numbers of regulatory T cells. However, we observed differences in the frequencies of T cell subsets within the E7-specific CD8(+) T cell population, e.g. higher frequencies of central memory T cells upon homologous immunizations compared to heterologous immunizations. The induction of central memory T cells is crucial for a cancer vaccine as these cells are known to rapidly expand upon recall stimulation. This study demonstrates that the strongly increased number of antigen-specific CTL as induced by heterologous prime-boost immunizations, often used as a proof for the enhanced efficacy of such regimes, does not necessarily equal superior functional antitumor responses.

A rationally designed combined treatment with an alphavirus-based cancer vaccine, sunitinib and low-dose tumor irradiation completely blocks tumor development.

The clinical efficacy of therapeutic cancer vaccines remains limited. For effective immunotherapeutic responses in cancer patients, multimodal approaches capable of both inducing antitumor immune responses and bypassing tumor-mediated immune escape seem essential. Here, we report on a combination therapy comprising sunitinib (40 mg/kg), single low-dose (14 Gy) tumor irradiation and immunization with a therapeutic cancer vaccine based on a Semliki Forest virus vector encoding the oncoproteins E6 and E7 of human papillomavirus (SFVeE6,7). We previously demonstrated that either low–dose irradiation or sunitinib in single combination with SFVeE6,7 immunizations enhanced the intratumoral ratio of antitumor effector cells to myeloid-derived suppressor cells (MDSCs). On the basis of these results we designed a triple treatment combinatorial regimen. The trimodal sunitinib, low–dose irradiation and SFVeE6,7 immunization therapy resulted in stronger intratumoral MDSC depletion than sunitinib alone. Concomitantly, the highest levels of intratumoral E7-specific CD8+ T cells were attained after triple treatment. Approximately 75% of these cells were positive for the early activation marker CD69. The combination of sunitinib, low-dose tumor irradiation and SFVeE6,7 immunization dramatically changed the intratumoral immune compartment. Whereas control tumors contained 0.02 E7-specific CD8+ T cells per MDSC, triple treatment tumors contained more than 200 E7-specific CD8+ T cells per MDSC, a 10,000-fold increased ratio. As a result, the triple treatment strongly enhanced the immunotherapeutic antitumor effect, blocking tumor development altogether and leading to 100% tumor-free survival of tumor-bearing mice. This study demonstrates that this multimodal approach elicits superior antitumor effects and should be considered for clinical applications.