Prognostic factors related to add-on dendritic cell vaccines on patients with inoperable pancreatic cancer receiving chemotherapy: a multicenter analysis

Objective

Dendritic cell (DC)-based cancer vaccines may have a significant benefit to patients with advanced pancreatic cancer. However, variations among clinical studies make it difficult to compare clinical outcomes. Here, we identified factors that determined the clinical benefits by analyzing data obtained at seven Japanese institutions that employed the same DC preparation and treatment regimens.

Methods

Of 354 patients who met the inclusion criteria, 255 patients who received standard chemotherapy combined with peptide-pulsed DC vaccines were analyzed.

Results

The mean survival time from diagnosis was 16.5 months (95 % CI 14.4–18.5) and that from the first vaccination was 9.9 months (95 % CI 8.0–12.9). Known prognostic baseline factors related to advanced pancreatic cancer, namely ECOG-PS, peritoneal metastasis, liver metastasis, and the prognostic nutrition index, were also representative. Importantly, we found that erythema reaction after vaccination was an independent and treatment-related prognostic factor for better survival and that OK-432 might be a good adjuvant enhancing the antitumor immunity during DC vaccination.

Conclusions

This is the first report of a multicenter clinical study suggesting the feasibility and possible clinical benefit of an add-on DC vaccine in patients with advanced pancreatic cancer who are undergoing chemotherapy. These findings need to be addressed in well-controlled prospective randomized trials.     

The Value of Expression of M2-PK and VEGF in Patients with Advanced Gastric Cancer

Glycolytic pyruvate kinase isoenzyme type M2 (M2-PK) plays a key role in tumor metabolism and energy production. Vascular endothelial growth factor (VEGF) is critical in regulating angiogenesis which is an essential process required for tumor growth and metastasis. These two genes may function in accordance with tumor development. The purpose of this study was to investigate the relationship between the expression of M2-PK and VEGF, and their association with clinicopathological features in patients with advanced gastric cancer. Expression of M2-PK and VEGF were examined in 142 cases of paraffin-embedded tissue blocks from patients with advanced gastric cancer. M2-PK expression was found to strongly correlate with that of VEGF (r = 0.718). In addition, expression of M2-PK and VEGF correlates with tumor size (p = 0.0001, and p = 0.0017, respectively), depth of invasion (p = 0.0024, and p = 0.0261, respectively), and lymph node metastasis (p = 0.036, and p = 0.028, respectively). The high expression levels of M2-PK and VEGF may indicate poor prognosis in patients with advanced gastric cancer.     

Prognostic significance of survivin expression in renal cell cancer and its correlation with radioresistance

Survivin, an important inhibitor of apoptosis, has been found to play an important role in the initiation, progression, and chemoradioresistance of human malignancies. Previously, we have reported that upregulation of survivin in oral squamous cell carcinoma correlates with poor prognosis and chemoresistance. The aim of this study was to assess prognostic significance of survivin protein expression in RCC and analyze its correlation with radiosensitivity of RCC cells. RT-PCR and Western blot assays were performed to detect survivin mRNA and protein expression in normal human kidney epithelial cell line (HKEC) or RCC cell lines. The expression of survivin mRNA in RCC and corresponding nontumor kidney tissues was also detected by RT-PCR. Immunohistochemistry was performed to determine survivin protein expression in 75 cases of RCC tissue samples. Moreover, the association of survivin protein expression with clinicopathogical factors and prognosis of RCC patients was statistically analyzed. Small interfering RNA was used to knockdown the endogenous survivin expression in RCC cell line (ACHN) and evaluate the effects of survivin knockdown on proliferation, apoptosis, and radiosensitivity of RCC cell line. RCC cells showed sufficient expression of survivin mRNA and protein, but the expression of survivin gene was not detected in normal HKEC. Moreover, the expression level of survivin mRNA in RCC tissues was significantly higher than that in corresponding nontumor kidney tissues. The immunostaining of survivin protein was mainly located in cytoplasm of RCC tumor cells. Tumor pathological stage (P = 0.028), grade (P = 0.004), and lymph node metastasis (P = 0.017) of RCC patients were significantly correlated with survivin protein expression. In addition, patients with high survivin levels had a significantly shorter overall survival than those with low levels (P < 0.001), and the expression of survivin protein was an independent prognostic factor for RCC patients (P = 0.008). The expression of survivin gene could be reduced in RCC cell line and survivin knockdown could inhibit growth and enhance in vivo radiosensitivity of RCC cell line by inducing apoptosis enhancement. Taken together, the status of survivin protein expression may be an independent factor for predicting the prognosis of RCC patients and tumor-specific survivin knockdown combined with radiotherapy will be a potential strategy for RCC therapy.     

Generation of cytotoxic responses in mice and human individuals against hematological malignancies using survivin-RNA-transfected dendritic cells

Survivin is a member of the inhibitors of apoptosis family and is overexpressed in many types of human cancers, making it an attractive target for T cell-based immunotherapeutic strategies. Recently, HLA-A2-binding peptides derived from the survivin protein were identified as capable of inducing specific T cell responses in cancer patients. Here we demonstrate that human survivin-specific CTLs generated from PBMC by stimulation with autologous dendritic cells transfected with survivin-RNA were cytotoxic for a range of hemopoietic malignant cell lines and primary tumor cells isolated from patients with acute myeloid leukemia. We also show that vaccination of mice with survivin-RNA-transfected dendritic cells leads to long term resistance to challenge by a survivin-expressing lymphoma, demonstrating the potential of survivin as a tumor rejection Ag. Our data provide evidence for the use of survivin as a target structure for immunotherapeutic strategies against hematological neoplasms.     

Hypochlorous acid enhances immunogenicity and uptake of allogeneic ovarian tumor cells by dendritic cells to cross-prime tumor-specific T cells

Background: Ovarian cancer commonly relapses after remission and new strategies to target microscopic residual diseases are required. One approach is to activate tumor-specific cytotoxic T cells with dendritic cells loaded with tumor cells. In order to enhance their immunogenicity, ovarian tumor cells (SK-OV-3, which express two well-characterized antigens HER-2/neu and MUC-1) were killed by oxidation with hypochlorous acid (HOCl). Results: Treatment for 1 h with 60 μM HOCl was found to induce necrosis in all SK-OV-3 cells. Oxidized, but not live, SK-OV-3 was rapidly taken up by monocyte-derived dendritic cells, and induced partial dendritic cell maturation. Dendritic cells cultured from HLA-A2 healthy volunteers were loaded with oxidized SK-OV-3 (HLA-A2⁻) and co-cultured with autologous T cells. Responding T cells were tested for specificity after a further round of antigen stimulation. In ELISPOT assays, T cells produced interferon-gamma (IFN-γ) in response to the immunizing cellular antigen, and also to peptides coding for MUC-1 and HER-2/neu HLA-A2 restricted epitopes, demonstrating efficient cross-presentation of cell-associated antigens. In contrast, no responses were seen after priming with heat-killed or HCl-killed SK-OV-3, indicating that HOCl oxidation and not cell death/necrosis per se enhanced the immunogenicity of SK-OV-3. Finally, T cells stimulated with oxidized SK-OV-3 showed no cross-reaction to oxidized melanoma cells, nor vice versa, demonstrating that the response was tumor-type specific. Conclusions: Immunization with oxidized ovarian tumor cell lines may represent an improved therapeutic strategy to stimulate a polyclonal anti-tumor cellular immune response and hence extend remission in ovarian cancer.     

Immunogenicity of HLA Class I and II Double Restricted Influenza A-Derived Peptides

The aim of the present study was to identify influenza A-derived peptides which bind to both HLA class I and -II molecules and by immunization lead to both HLA class I and class II restricted immune responses. Eight influenza A-derived 9-11mer peptides with simultaneous binding to both HLA-A*02:01 and HLA-DRB1*01:01 molecules were identified by bioinformatics and biochemical technology. Immunization of transgenic HLA-A*02:01/HLA-DRB1*01:01 mice with four of these double binding peptides gave rise to both HLA class I and class II restricted responses by CD8 and CD4 T cells, respectively, whereas four of the double binding peptides did result in HLA-A*02:01 restricted responses only. According to their cytokine profile, the CD4 T cell responses were of the Th2 type. In influenza infected mice, we were unable to detect natural processing in vivo of the double restricted peptides and in line with this, peptide vaccination did not decrease virus titres in the lungs of intranasally influenza challenged mice. Our data show that HLA class I and class II double binding peptides can be identified by bioinformatics and biochemical technology. By immunization, double binding peptides can give rise to both HLA class I and class I restricted responses, a quality which might be of potential interest for peptide-based vaccine development.     

Vaccination with p53-peptide–pulsed dendritic cells,of patients with advanced breast cancer: report from a phase I study

Peptides derived from over-expressed p53 protein are presented by class I MHC molecules and may act as tumour-associated epitopes. Due to the diversity of p53 mutations, immunogenic peptides representing wild-type sequences are preferable as a basis for a broad-spectrum p53-targeting cancer vaccine. Our preclinical studies have shown that wild-type p53-derived HLA-A2–binding peptides are able to activate human T cells and that the generated effector T cells are cytotoxic to human HLA-A2⁺, p53⁺ tumour cells. In this phase I pilot study, the toxicity and efficacy of autologous dendritic cells (DCs) loaded with a cocktail of three wild-type and three modified p53 peptides are being analysed in six HLA-A2⁺ patients with progressive advanced breast cancer. Vaccinations were well tolerated and no toxicity was observed. Disease stabilisation was seen in two of six patients, one patient had a transient regression of a single lymph node and one had a mixed response. ELISpot analyses showed that the p53-peptide–loaded DCs were able to induce specific T-cell responses against modified and unmodified p53 peptides in three patients, including two of the patients with a possible clinical benefit from the treatment. In conclusion, the strategy for p53-DC vaccination seems safe and without toxicity. Furthermore, indications of both immunologic and clinical effect were found in heavily pretreated patients with advanced breast cancer. An independent clinical effect of repeated administration of DCs and IL-2 can not of course be excluded; further studies are necessary to answer these questions.     

Dendritic cell-based vaccination of patients with advanced pancreatic carcinoma: results of a pilot study

Background and aims

Dendritic cell (DC)-based vaccination can induce antitumor T cell responses in vivo. This clinical pilot study examined feasibility and outcome of DC-based tumor vaccination for patients with advanced pancreatic adenocarcinoma.

Methods

Tumor lysate of patients with pancreatic carcinoma was generated by repeated freeze?Cthaw cycles of surgically obtained tissue specimens. Patients were eligible for DC vaccination after recurrence of pancreatic carcinoma or in a primarily palliative situation. DC were generated from peripheral blood mononuclear cells (PBMC), loaded with autologous tumor lysate, stimulated with TNF-?? and PGE₂ and injected intradermally. All patients received concomitant chemotherapy with gemcitabine. Disease response was the primary endpoint. Individual immunological responses to DC vaccination were analyzed by T cell-based immunoassays using pre- and post-vaccination samples of non-adherent PBMC.

Results

Twelve patients received DC vaccination and concomitant chemotherapy. One patient developed a partial remission, and two patients remained in stable disease. Median survival was 10.5?months. No severe side effects were observed. Tumor-reactive T cells could be detected prior to vaccination. DC vaccination increased the frequency of tumor-reactive cells in all patients tested; however, the degree of this increase varied. To quantify the presence of tumor-reactive T cells, stimulatory indices (SI) were calculated as the ratio of proliferation-inducing capacity of lysate-loaded versus -unloaded DC. The patient with longest overall survival of 56?months had a high SI of 6.49, indicating that the presence of a pre-vaccination antitumor T cell response might be associated with prolonged survival. Five patients survived 1?year or more.

Conclusion

DC-based vaccination can stimulate an antitumoral T cell response in patients with advanced or recurrent pancreatic carcinoma receiving concomitant gemcitabine treatment.     

Survivin-specific T-cell reactivity correlates with tumor response and patient survival: a phase-II peptide vaccination trial in metastatic melanoma

Background

Therapeutic vaccination directed to induce an anti-tumoral T-cell response is a field of extensive investigation in the treatment of melanoma. However, many vaccination trials in melanoma failed to demonstrate a correlation between the vaccine-specific immune response and therapy outcome. This has been mainly attributed to immune escape by antigen loss, rendering us in the need of new vaccination targets.

Patients and methods

This phase-II trial investigated a peptide vaccination against survivin, an oncogenic inhibitor-of-apoptosis protein crucial for the survival of tumor cells, in HLA-A1/-A2/-B35-positive patients with treatment-refractory stage-IV metastatic melanoma. The study endpoints were survivin-specific T-cell reactivity (SSTR), safety, response, and survival (OS).

Results

Sixty-one patients (ITT) received vaccination therapy using three different regimens. 55 patients (PP) were evaluable for response and survival, and 41/55 for SSTR. Patients achieving progression arrest (CR?+?PR?+?SD) more often showed SSTRs than patients with disease progression (p?=?0.0008). Patients presenting SSTRs revealed a prolonged OS (median 19.6 vs. 8.6?months; p?=?0.0077); multivariate analysis demonstrated SSTR as an independent predictor of survival (p?=?0.013). The induction of SSTRs was associated with gender (female vs. male; p?=?0.014) and disease stage (M1a/b vs. M1c; p?=?0.010), but not with patient age, HLA type, performance status, or vaccination regimen.

Conclusion

Survivin-specific T-cell reactivities strongly correlate with tumor response and patient survival, indicating that vaccination with survivin-derived peptides is a promising treatment strategy in melanoma.     

Modified vaccinia Ankara expressing survivin combined with gemcitabine generates specific antitumor effects in a murine pancreatic carcinoma model

Survivin is overexpressed by 70–80% of pancreatic cancers, and is associated with resistance to chemotherapy and a poor prognosis. Gemcitabine has been a standard treatment for patients with advanced pancreatic cancer for a decade. Recent reports have demonstrated that gemcitabine treatment attenuates the tumor-suppressive environment by eliminating CD11b⁺/Gr-1⁺ myeloid-derived suppressor cells (MDSCs). We hypothesize that a cancer vaccine targeting survivin can achieve enhanced efficacy when combined with gemcitabine. In this study, we tested this hypothesis using modified vaccinia Ankara (MVA) expressing full-length murine survivin. The poorly immunogenic mouse pancreas adenocarcinoma cell line, Pan02, which expresses murine survivin and is syngeneic to C57BL/6, was used for this study. Immunization with MVA-survivin resulted in a modest therapeutic antitumor effect on established Pan02 tumors. When administered with gemcitabine, MVA-survivin immunization resulted in significant tumor regression and prolonged survival. The enhanced vaccine efficacy was associated with decreased CD11b⁺/Gr-1⁺ MDSCs. To analyze the survivin-specific immune response to MVA-survivin immunization, we utilized a peptide library of 15mers with 11 residues overlapping from full-length murine survivin. Splenocytes from mice immunized with MVA-survivin produced intracellular γ-interferon in response to in vitro stimulation with the overlapping peptide library. Increased survivin-specific CD8⁺ T cells that specifically recognized the Pan02 tumor line were seen in mice treated with MVA-survivin and gemcitabine. These data suggest that vaccination with MVA-survivin in combination with gemcitabine represents an attractive strategy to overcome tumor-induced peripheral immune tolerance, and this effect has potential for clinical benefit in pancreatic cancer.     

STEAP, a prostate tumor antigen, is a target of human CD8+ T cells

STEAP is a recently identified protein shown to be particularly overexpressed in prostate cancer and also present in numerous human cancer cell lines from prostate, pancreas, colon, breast, testicular, cervical, bladder and ovarian carcinoma, acute lymphocytic leukemia and Ewing sarcoma. This expression profile renders STEAP an appealing candidate for broad cancer immunotherapy. In order to investigate if STEAP is a tumor antigen that can be targeted by specific CD8⁺ T cells, we identified two high affinity HLA-A*0201 restricted peptides (STEAP_86–94 and STEAP_262–270). These peptides were immunogenic in vivo in HLA-A*0201 transgenic HHD mice. Peptide specific murine CD8 T cells recognized COS-7 cells co-transfected with HHD (HLA-A*0201) and STEAP cDNA constructs and also HLA-A*0201⁺ STEAP⁺ human tumor cells. Furthermore, STEAP_86–94 and STEAP_262–270 stimulated specific CD8⁺ T cells from HLA-A*0201⁺ healthy donors, and these peptide specific CD8⁺ T cells recognized STEAP positive human tumor cells in an HLA-A*0201-restricted manner. Importantly, STEAP_86–94-specific T cells were detected and reactive in the peripheral blood mononuclear cells in NSCLC and prostate cancer patients ex vivo. These results show that STEAP can be a target of anti-tumor CD8⁺ T cells and that STEAP peptides can be used for a broad-spectrum-tumor immunotherapy.     

DNA fusion-gene vaccination in patients with prostate cancer induces high-frequency CD8+ T-cell responses and increases PSA doubling time

We report on the immunogenicity and clinical effects in a phase I/II dose escalation trial of a DNA fusion vaccine in patients with prostate cancer. The vaccine encodes a domain (DOM) from fragment C of tetanus toxin linked to an HLA-A2-binding epitope from prostate-specific membrane antigen (PSMA), PSMA_27–35. We evaluated the effect of intramuscular vaccination without or with electroporation (EP) on vaccine potency. Thirty-two HLA-A2⁺ patients were vaccinated and monitored for immune and clinical responses for a follow-up period of 72 weeks. At week 24, cross-over to the immunologically more effective delivery modality was permitted; this was shown to be with EP based on early antibody data, and subsequently, 13/15 patients crossed to the +EP arm. Thirty-two HLA-A2^? control patients were assessed for time to next treatment and overall survival. Vaccination was safe and well tolerated. The vaccine induced DOM-specific CD4⁺ and PSMA₂₇-specific CD8⁺ T cells, which were detectable at significant levels above baseline at the end of the study (p = 0.0223 and p = 0.00248, respectively). Of 30 patients, 29 had a measurable CD4⁺ T-cell response and PSMA₂₇-specific CD8⁺ T cells were detected in 16/30 patients, with or without EP. At week 24, before cross-over, both delivery methods led to increased CD4⁺ and CD8⁺ vaccine-specific T cells with a trend to a greater effect with EP. PSA doubling time increased significantly from 11.97 months pre-treatment to 16.82 months over the 72-week follow-up (p = 0.0417), with no clear differential effect of EP. The high frequency of immunological responses to DOM-PSMA₂₇ vaccination and the clinical effects are sufficiently promising to warrant further, randomized testing.     

Phase I trial of a multi-epitope-pulsed dendritic cell vaccine for patients with newly diagnosed glioblastoma

Background

This study evaluated the safety and immune responses to an autologous dendritic cell vaccine pulsed with class I peptides from tumor-associated antigens (TAA) expressed on gliomas and overexpressed in their cancer stem cell population (ICT-107).

Methods

TAA epitopes included HER2, TRP-2, gp100, MAGE-1, IL13Rα2, and AIM-2. HLA-A1- and/or HLA-A2-positive patients with glioblastoma (GBM) were eligible. Mononuclear cells from leukapheresis were differentiated into dendritic cells, pulsed with TAA peptides, and administered intradermally three times at two-week intervals.

Results

Twenty-one patients were enrolled with 17 newly diagnosed (ND-GBM) and three recurrent GBM patients and one brainstem glioma. Immune response data on 15 newly diagnosed patients showed 33 % responders. TAA expression by qRT-PCR from fresh-frozen tumor samples showed all patient tumors expressed at least three TAA, with 75 % expressing all six. Correlations of increased PFS and OS with quantitative expression of MAGE1 and AIM-2 were observed, and a trend for longer survival was observed with gp100 and HER2 antigens. Target antigens gp100, HER1, and IL13Rα2 were downregulated in recurrent tumors from 4 HLA-A2+ patients. A decrease in or absence of CD133 expression was seen in five patients who underwent a second resection. At a median follow-up of 40.1 months, six of 16 ND-GBM patients showed no evidence of tumor recurrence. Median PFS in newly diagnosed patients was 16.9 months, and median OS was 38.4 months.

Conclusions

Expression of four ICT-107 targeted antigens in the pre-vaccine tumors correlated with prolonged overall survival and PFS in ND-GBM patients. The goal of targeting tumor antigens highly expressed on glioblastoma cancer stem cells is supported by the observation of decreased or absent CD133 expression in the recurrent areas of gadolinium-enhanced tumors.     

Immunogenicity of the carcinoembryonic antigen derived peptide 694 in HLA-A2 healthy donors and colorectal carcinoma patients

Carcinoembryonic antigen (CEACAM5) is commonly overexpressed in human colon cancer. Several antigenic peptides recognized by cytolytic CD8+ T-cells have been identified and used in colon cancer phase-I vaccination clinical trials. The HLA-A*0201-binding CEA_694–702 peptide was recently isolated from acid eluted MHC-I associated peptides from a human colon tumor cell line. However, the immunogenicity of this peptide in humans remains unknown. We found that the peptide CEA_694–702 binds weakly to HLA-A*0201 molecules and is ineffective at inducing specific CD8+ T-cell responses in healthy donors. Immunogenic-altered peptide ligands with increased affinity for HLA-A*0201 were identified. Importantly, the elicited cytolytic T lymphocyte (CTL) lines and clones cross-reacted with the wild-type CEA_694–702 peptide. Tumor cells expressing CEA were recognized in a peptide and HLA-A*0201 restricted fashion, but high-CEA expression levels appear to be required for CTL recognition. Finally, CEA-specific T-cell precursors could be readily expanded by in vitro stimulation of peripheral blood mononuclear cell (PBMC) from colon cancer patients with altered CEA peptide. However, the CEA-specific CD8+ T-cell clones derived from cancer patients revealed low-functional avidity and impaired tumor-cell recognition. Together, using T-cells to demonstrate the processing and presentation of the peptide CEA694-702, we were able to corroborate its presentation by tumor cells. However, the low avidity of the specific CTLs generated from cancer patients as well as the high-antigen expression levels required for CTL recognition pose serious concerns for the use of CEA694-702 in cancer immunotherapy.     

Immunogenicity Evaluation of a Rationally Designed Polytope Construct Encoding HLA-A*0201 Restricted Epitopes Derived from Leishmania major Related Proteins in HLA-A2/DR1 Transgenic Mice: Steps toward Polytope Vaccine

Background

There are several reports demonstrating the role of CD8 T cells against Leishmania species. Therefore peptide vaccine might represent an effective approach to control the infection. We developed a rational polytope-DNA construct encoding immunogenic HLA-A2 restricted peptides and validated the processing and presentation of encoded epitopes in a preclinical mouse model humanized for the MHC-class-I and II.

Methods and Findings

HLA-A*0201 restricted epitopes from LPG-3, LmSTI-1, CPB and CPC along with H-2Kd restricted peptides, were lined-up together as a polytope string in a DNA construct. Polytope string was rationally designed by harnessing advantages of ubiquitin, spacers and HLA-DR restricted Th1 epitope. Endotoxin free pcDNA plasmid expressing the polytope was inoculated into humanized HLA-DRB1*0101/HLA-A*0201 transgenic mice intramuscularly 4 days after Cardiotoxin priming followed by 2 boosters at one week interval. Mice were sacrificed 10 days after the last booster, and splenocytes were subjected to ex-vivo and in-vitro evaluation of specific IFN-γ production and in-vitro cytotoxicity against individual peptides by ELISpot and standard chromium-51(⁵¹Cr) release assay respectively. 4 H-2Kd and 5 HLA-A*0201 restricted peptides were able to induce specific CD8 T cell responses in BALB/C and HLA-A2/DR1 mice respectively. IFN-γ and cytolytic activity together discriminated LPG-3-P1 as dominant, LmSTI-1-P3 and LmSTI-1-P6 as subdominant with both cytolytic activity and IFN-γ production, LmSTI-1-P4 and LPG-3-P5 as subdominant with only IFN-γ production potential.

Conclusions

Here we described a new DNA-polytope construct for Leishmania vaccination encompassing immunogenic HLA-A2 restricted peptides. Immunogenicity evaluation in HLA-transgenic model confirmed CD8 T cell induction with expected affinities and avidities showing almost efficient processing and presentation of the peptides in relevant preclinical model. Further evaluation will determine the efficacy of this polytope construct protecting against infectious challenge of Leishmania. Fortunately HLA transgenic mice are promising preclinical models helping to speed up immunogenicity analysis in a human related mouse model.     

Distinct Patterns of Stromal and Tumor Expression of ROR1 and ROR2 in Histological Subtypes of Epithelial Ovarian Cancer

OBJECTIVE: The ROR1 and ROR2 receptor tyrosine kinases have both been implicated in ovarian cancer progression and have been shown to drive migration and invasion. There is an increasing importance of the role of stroma in ovarian cancer metastasis; however, neither ROR1 nor ROR2 expression in tumor or stromal cells has been analyzed in the same clinical cohort. AIM: To determine ROR1 and ROR2 expression in ovarian cancer and surrounding microenvironment and examine associations with clinicopathological characteristics. METHODS: Immunohistochemistry for ROR1 and ROR2 was used to assess receptor expression in a cohort of epithelial ovarian cancer patients (n = 178). Results were analyzed in relation to clinical and histopathological characteristics and survival. Matched patient sample case studies of normal, primary, and metastatic lesions were used to examine ROR expression in relation to ovarian cancer progression. RESULTS: ROR1 and ROR2 are abnormally expressed in malignant ovarian epithelium and stroma. Higher ROR2 tumor expression was found in early-stage, low-grade endometrioid carcinomas. ROR2 stromal expression was highest in the serous subtype. In matched patient case studies, metastatic samples had higher expression of ROR2 in the stroma, and a recurrent sample had the highest expression of ROR2 in both tumor and stroma. CONCLUSION: ROR1 and ROR2 are expressed in tumor-associated stroma in all histological subtypes of ovarian cancer and hold potential as therapeutic targets which may disrupt tumor and stroma interactions.     

Vaccination with autologous dendritic cells pulsed with multiple tumor antigens for treatment of patients with malignant melanoma: results from a phase I/II trial

Background aimsDendritic cells are regarded as the most effective antigen presenting cells and coordinators of the immune response and therefore suitable as vaccine basis. Here we present results from a clinical study in which patients with malignant melanoma (MM) with verified progressive disease received vaccination with autologous monocyte-derived mature dendritic cells (DC) pulsed with p53, survivin and telomerase-derived peptides (HLA-A2⁺ patients) or with autologous/allogeneic tumor lysate (HLA-A2^? patients) in combination with low-dose interleukin (IL)-2 and interferon (IFN)-α2b.ResultsOf 46 patients who initiated treatment, 10 stopped treatment within 1–4 weeks because of rapid disease progression and deterioration. After 8 weeks, 36 patients were evaluable: no patient had an objective response, 11 patients had stable disease (SD); six had continued SD after 4 months, and three patients had prolonged SD for more than 6 months. The mean overall survival time was 9 months, with a significantly longer survival (18.4 months) of patients who attained SD compared with patients with progressive disease (PD) (5 months). Induction of antigen-specific T-cell responses was analyzed by multidimensional encoding of T cells using HLA-A2 major histocompatibility complex (MHC) multimers. Immune responses against five high-affinity vaccine peptides were detectable in the peripheral blood of six out of 10 analyzed HLA-A2⁺ patients. There was no observed correlation between the induction of immune responses and disease stabilization. A significant lower blood level of regulatory T cells (CD25^high CD4 T cells) was demonstrable after six vaccinations in patients with SD compared with PD.ConclusionsVaccination was feasible and safe. Treatment-associated SD was observed in 24% of the patients. SD correlated with prolonged survival suggesting a clinical benefit. Differences in the level of regulatory T cells among SD and PD patients could indicate a significant role of these immune suppressive cells.     

An HLA-A3-binding prostate acid phosphatase-derived peptide can induce CTLs restricted to HLA-A2 and -A24 alleles

We previously reported peptide vaccine candidates for HLA-A3 supertype (-A3, -A11, -A31, -A33)-positive cancer patients. In the present study, we examined whether those peptides can also induce cytotoxic T lymphocyte (CTL) activity restricted to HLA-A2, HLA-A24, and HLA-A26 alleles. Fourteen peptides were screened for their binding activity to HLA-A*0201, -A*0206, -A*0207, -A*2402, and -A*2601 molecules and then tested for their ability to induce CTL activity in peripheral blood mononuclear cells (PBMCs) from prostate cancer patients. Among these peptides, one from the prostate acid phosphatase protein exhibited binding activity to HLA-A*0201, -A*0206, and -A*2402 molecules. In addition, PBMCs stimulated with this peptide showed that HLA-A2 or HLA-A24 restricted CTL activity. Their cytotoxicity toward cancer cells was ascribed to peptide-specific and CD8⁺ T cells. These results suggest that this peptide could be widely applicable as a peptide vaccine for HLA-A3 supertype-, HLA-A2-, and -A24-positive cancer patients.     

Identification of Special AT-Rich Sequence Binding Protein 1 as a Novel Tumor Antigen Recognized by CD8+ T Cells: Implication for Cancer Immunotherapy

Background

A large number of human tumor-associated antigens that are recognized by CD8⁺ T cells in a human leukocyte antigen class I (HLA-I)-restricted fashion have been identified. Special AT-rich sequence binding protein 1 (SATB1) is highly expressed in many types of human cancers as part of their neoplastic phenotype, and up-regulation of SATB1 expression is essential for tumor survival and metastasis, thus this protein may serve as a rational target for cancer vaccines.

Methodology/Principal Findings

Twelve SATB1-derived peptides were predicted by an immuno-informatics approach based on the HLA-A*02 binding motif. These peptides were examined for their ability to induce peptide-specific T cell responses in peripheral blood mononuclear cells (PBMCs) obtained from HLA-A*02⁺ healthy donors and/or HLA-A*02⁺ cancer patients. The recognition of HLA-A*02⁺ SATB1-expressing cancer cells was also tested. Among the twelve SATB1-derived peptides, SATB1_565–574 frequently induced peptide-specific T cell responses in PBMCs from both healthy donors and cancer patients. Importantly, SATB1_565–574-specific T cells recognized and killed HLA-A*02⁺ SATB1⁺ cancer cells in an HLA-I-restricted manner.

Conclusions/Significance

We have identified a novel HLA-A*02-restricted SATB1-derived peptide epitope recognized by CD8⁺ T cells, which, in turn, recognizes and kills HLA-A*02⁺ SATB1⁺ tumor cells. The SATB1-derived epitope identified may be used as a diagnostic marker as well as an immune target for development of cancer vaccines.     

Phase II clinical trial of ex vivo-expanded cytokine-induced killer cells therapy in advanced pancreatic cancer

Second-line chemotherapy in patients with gemcitabine-refractory advanced pancreatic cancer has shown disappointing survival outcomes due to rapid disease progression and performance deterioration. The aim of this phase II trial was to evaluate the efficacy and safety of adoptive immunotherapy using ex vivo-expanded, cytokine-induced killer (CIK) cells in gemcitabine-refractory advanced pancreatic cancer. Patients with advanced pancreatic cancer who showed disease progression during gemcitabine-based chemotherapy were enrolled in this study. For generation of CIK cells, peripheral blood samples were collected from each patient and cultured with anti-CD3 monoclonal antibody and IL-2. Patients received CIK cells intravenously 10 times, every week for 5 weeks and then every other week for 10 weeks. Twenty patients were enrolled between November 2009 and September 2010. The disease control rate was 25 % (4/16 patients). The median progression-free survival (PFS) was 11.0 weeks (95 % CI 8.8–13.2), and the median overall survival (OS) was 26.6 weeks (95 % CI 8.6–44.6). Grade 3 toxicities included general weakness in two patients and thrombocytopenia in one patient. Grade 4 hematologic or non-hematologic toxicity was not observed. Patients showed improvement in pancreatic pain, gastrointestinal distress, jaundice, body image alterations, altered bowel habits, health satisfaction, and sexuality when assessing quality of life (QoL). Adoptive immunotherapy using CIK cells showed comparable PFS and OS to survival data of previous trials that assessed conventional chemotherapies while maintaining tolerability and showing encouraging results in terms of patient QoL in gemcitabine-refractory advanced pancreatic cancer (clinicalTrials.gov number NCT00965718).