In Vitro Assessment of the Expression and T Cell Immunogenicity of the Tumor-Associated Antigens BORIS, MUC1, hTERT, MAGE-A3 and Sp17 in Uterine Cancer.

BACKGROUND: While immunotherapy moved to the forefront of treatment of various cancers, it remains underexplored for uterine cancer. This might be due to the small patient population with advanced endometrial carcinoma and uterine sarcoma. Data about immunotherapeutic targets are scarce in endometrial carcinoma and lacking in uterine sarcoma. METHODS: Expression of five tumor-associated antigens (TAA) (BORIS, MUC1, hTERT, MAGE-A3 and Sp17) was validated in uterine tumor samples by immunohistochemistry (IHC) and/or quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). TAA immunogenicity was analyzed by determining spontaneous T cell responses towards overlapping peptide pools covering the whole TAA in patient blood. RESULTS: At mRNA level, MAGE-A3 and Sp17 were overexpressed in a minority of patients and BORIS was moderately overexpressed (26% in endometrial carcinoma and 62% in uterine sarcoma). hTERT was overexpressed in the vast majority of tumors. On protein level, MUC1 was upregulated in primary, recurrent and metastatic EMCAR and in metastatic US tumors. hTERT protein was highly expressed in both normal and malignant tissue. Spontaneous TAA-specific T cell responses were detected in a minority of patients, except for hTERT to which T cell responses occurred more frequently. CONCLUSIONS: These data point to MUC1 and hTERT as most suitable targets based on expression levels and T cell immunogenicity for use in immunotherapeutic regimens.

External validation of non-imaging models for predicting distant metastasis in patients with endometrial cancer.

OBJECTIVE: To evaluate two non-imaging models designed to predict distant metastasis (stages IIIC-IV) in endometrial carcinoma (EC). Both used preoperative histological and biological findings. One used primary tumoral size, the other did not. METHODS: 374 patients operated on for EC by hysterectomy and at least bilateral pelvic lymphadenectomy were included. Patient's characteristics, preoperative histological, biological findings and primary tumoral size were used to calculate for each patient two scores (one for each model) for distant metastasis. The accuracy of the models was evaluated in terms of areas under the receiver operating characteristic curves (AUCs), rates of false negatives, and number of patients in the group at low risk to predict stages IIIC-IV. RESULTS: 309 and 65 patients had FIGO stages IA-IIIB and IIIC-IV respectively. Thrombocytosis and leukocytosis were not significantly different between patients who had distant metastasis and those who did not. CA125 serum level was significantly higher in patients who had distant metastasis (71.2 vs 32.0U/mL, p<0.001). High-risk preoperative histology and primary tumor diameter >3cm were more frequently observed in patients who had distant metastasis (55.4% and 39.9%, p=0.02 and 21.3% and 8.5%, p=0.003). The AUC were 0.65 [0.63-0.67] and 0.68 [0.63-0.67] with 54% and 93.4% sensitivity, 64% 19.1% specificity. Two hundred and twenty nine patients (61.2%) and 62 (17.0%) were classified as low risk; among them, 30 patients (13.2%) and 4 (6.4%) had final stage IIIC or IV. CONCLUSION: Both models turned out to have a low discrimination power in our population. However, the score using primary tumoral size permits to identify a subgroup of patients in whom metastatic probability is low and lymphadenectomy unnecessary. Preoperative CA125 level, histological findings and primary tumoral size remain prognostic factors of stages IIIC-IV and should be included in predictive models.

Evaluation of models to predict lymph node metastasis in endometrial cancer: A multicentre study.

BACKGROUND: Several models (preoperative and postoperative) have been developed to predict lymph node metastasis (LNM) in patients with endometrial cancer. The purpose of our investigation was to compare available models in a multicentre study. METHODS: In a cohort of 519 patients with endometrial cancer who had undergone primary hysterectomy and at least a pelvic lymphadenectomy, we compared the areas under the receiver-operating characteristic curves (AUCs), calibrations, rates of false negatives (FN), and the number of patients at low-risk for LNM using ten different models (three preoperative and seven postoperative). RESULTS: In all, 17.5% of patients among the study population (91 in 519) had LNM. Only one of the three preoperative models and three of the seven postoperative models had an AUC >0.75. Six models were well calibrated. Eight models yielded an FN rate of <5%. Six models could assign more than a third of patients to the low-risk group. One postoperative (a French nomogram) and one preoperative (the Korean Gynecologic Oncology Group [KGOG]) model had an AUC >0.75, to yield an FN rate of <5%, and could assign more than a third of patients to the low-risk group. CONCLUSIONS: This study supports the use of the KGOG model to decide upon lymphadenectomy preoperatively in patients with endometrial cancer. For patients who did not have lymphadenectomy, a French nomogram could be applied using pathological characteristics to decide on a secondary lymphadenectomy.

In Vitro Validation of Survivin as Target Tumor-associated Antigen for Immunotherapy in Uterine Cancer.

Survivin is an antiapoptotic protein, not expressed in terminally differentiated adult tissues, yet overexpressed in several tumors. Therefore, it is an interesting target for immunotherapeutic strategies. In addition to specific overexpression in tumors, tumor survival is mediated by survivin and hence, tumor survival can be tackled by targeting survivin. Survivin expression in uterine cancer was validated by quantitative real-time polymerase chain reaction and immunohistochemistry. In addition, we evaluated survivin immunogenicity by analyzing spontaneous B-cell and T-cell responses in patients. Survivin as a protein was expressed in only a minority of normal tissues, whereas it was being expressed in all of the currently analyzed uterine cancers, both endometrial carcinoma (n = 52) and uterine sarcoma (n = 52). Survivin RNA transcripts were overexpressed in more aggressive tumors and survivin protein was overexpressed in recurrent endometrial tumors compared with primary tumors. Spontaneous T-cell responses were seen in 10/39 endometrial cancer patients and 3/16 uterine sarcoma patients. In normal controls, T-cell responses were found only in 1 donor (n = 21). Although increased antibody titers were found in more aggressive and far-advanced tumors, no differences in B-cell responses were seen. Overall, when compared with normal controls, a B-cell response was only measured in 1/41 uterine sarcoma patients. In conclusion, we currently validated the presence of survivin in uterine cancer. In addition, spontaneous T-cell responses were found in 23.6% of the total patient population. These data indicate that a survivin-specific immune response may be induced spontaneously in patients, further fortifying the eligibility of survivin as an immunotherapeutic target.

Dendritic cell immunotherapy in uterine cancer.

Uterine cancer is the most common pelvic gynecological malignancy. Uterine sarcomas and relapsed uterine carcinomas have limited treatment options. The search for new therapies is urgent. Dendritic cell (DC) immunotherapy holds much promise, though has been poorly explored in uterine cancer. This commentary gives an insight in existing DC immunotherapy studies in uterine cancer and summarizes the possibilities and the importance of the loading of tumor antigens onto DC and their subsequent maturation. However, the sole application of DC immunotherapy to target uterine cancer will be insufficient because of tumor-induced immunosuppression, which will hamper the establishment of an effective anti-tumor immune response. The authors give an overview on the limited existing immunosuppressive data and propose a novel approach on DC immunotherapy in uterine cancer.

Variability in CRP, regulatory T cells and effector T cells over time in gynaecological cancer patients: a study of potential oscillatory behaviour and correlations.

BACKGROUND: The inflammatory marker, C reactive protein has been proposed to also be a biomarker for adaptive immune responses in cancer patients with a possible application in time based chemotherapy. Fluxes in serum CRP levels were suggested to be indicative of a cyclical process in which, immune activation is followed by auto-regulating immune suppression. The applicability of CRP as a biomarker for regulatory or effector T cells was therefore investigated in a cohort of patients with gynaecological malignancies. METHODS: Peripheral blood samples were obtained from a cohort of patients at 7 time points over a period of 12 days. Serum and mononuclear cells were isolated and CRP levels in serum were detected using ELISA while regulatory and effector T cell frequencies were assessed using flow cytometry. To test periodicity, periodogram analysis of data was employed while Pearson correlation and the Wilcoxon signed rank test were used to determine correlations. RESULTS: The statistical analysis used showed no evidence of periodic oscillation in either serum CRP concentrations or Teff and Treg frequencies. Furthermore, there was no apparent correlation between serum CRP concentrations and the corresponding frequencies of Tregs or Teffs. Relative to healthy individuals, the disease state in the patients neither significantly affected the mean frequency of Tregs nor the mean coefficient of variation within the Treg population over time. However, both Teff mean frequency and mean coefficient of variation were significantly reduced in patients. CONCLUSION: Using our methods we were unable to detect CRP oscillations that could be used as a consistent serial biomarker for time based chemotherapy.

Mapping the immunosuppressive environment in uterine tumors: implications for immunotherapy.

The major hurdle for cancer vaccines to be effective is posed by tumor immune evasion. Several common immune mechanisms and mediators are exploited by tumors to avoid immune destruction. In an attempt to shed more light on the immunosuppressive environment in uterine tumors, we analyzed the presence of PD-L1, PDL2, B7-H4, indoleamine 2,3-dioxygenase (IDO), galectin- 1, galectin-3, arginase-1 activity and myeloid-derived suppressor cell (MDSC) infiltration. IDO, PD-L1, PD-L2 and B7-H4 were analyzed by immunohistochemistry. PDL2 was mostly expressed at low levels in these tumors. We found high IDO expression in 21 % of endometrial carcinoma samples and in 14 % of uterine sarcoma samples. For PD-L1 and B7-H4, we found high expression in 92 and 90 % of endometrial cancers, respectively, and in 100 and 92 % of the sarcomas. Galectin-1 and 3 were analyzed in tissue lysates by ELISA, but we did not find an increase in both molecules in tumor lysates compared with benign tissues. We detected expression of galectin-3 by fibroblasts, immune cells and tumor cells in single-cell tumor suspensions. In addition, we noted a highly significant increase in arginase-1 activity in endometrial carcinomas compared with normal endometria, which was not the case for uterine sarcomas. Finally, we could demonstrate MDSC infiltration in fresh tumor suspensions from uterine tumors. These results indicate that the PD-1/PD-L1 interaction and B7-H4 could be possible targets for immune intervention in uterine cancer patients as well as mediation of MDSC function. These observations are another step toward the implementation of inhibitors of immunosuppression in the treatment of uterine cancer patients.

Wilms' Tumor Gene 1 (WT1)--loaded dendritic cell immunotherapy in patients with uterine tumors: a phase I/II clinical trial.

AIM: Treatment options are limited in uterine cancer, leading to a poor prognosis. Overexpression of Wilms' tumor gene 1 (WT1), the highest ranked tumor antigen, is attractive for immunotherapy. PATIENTS AND METHODS: Six pre-treated patients with uterine cancer received four weekly vaccines of autologous dendritic cells (DCs) electroporated with WT1 mRNA. Safety, feasibility and immunogenicity were assessed. In cases of response, patients received monthly booster vaccines. RESULTS: The technique was feasible. One patient had a local allergic reaction. Three out of four Human Leucocyte Antigen-A2 (HLA-A2)-positive patients showed an oncological response; an enrichment of WT1-specific T-cells was observed in two of them. Two HLA-A2-negative patients did not show an oncological or an immunological response. CONCLUSION: A first series of six patients with uterine cancer treated with WT1 mRNA-electroporated DCs is presented herein. Oncological and immunological responses were observed and are supportive for further research.

Immunological response after WT1 mRNA-loaded dendritic cell immunotherapy in ovarian carcinoma and carcinosarcoma.

BACKGROUND: Dendritic cell (DC)-based immunotherapy is an emerging new treatment option in ovarian cancer, an important cause of cancer-related mortality. PATIENTS AND METHODS: One patient with ovarian carcinosarcoma (OCS) and one with serous ovarian cancer (SOC) received four weekly vaccinations of autologous DCs electroporated with mRNA coding for the Wilms' tumor gene 1 (WT1). Safety, feasibility and immunogenicity were assessed. RESULTS: Vaccination was feasible without toxicity. In an ex vivo antigen re-stimulation assay of peripheral blood mononuclear cells, both patients showed increasing cluster of differentiation 137 (CD137+) antigen-specific T-cells and interleukin 10 (IL-10) production post-vaccination. Moreover, interleukin-2 (IL-2) production increased (OCS) as well as interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) (SOC). Disease in patients progressed after four vaccines and patients continued with conventional therapies. After cessation of immunotherapy, they had an extended survival of 19 (OCS) and 12 (SOC) months. CONCLUSION: To our knowledge, we report for the first time the feasibility and T-cell immunogenicity of WT1 mRNA-loaded DC immunotherapy in ovarian cancer.