Localization of distant metastases defines prognosis and treatment efficacy in patients with FIGO stage IV ovarian cancer.

BACKGROUND: Patients with ovarian cancer who are diagnosed with Federation of Gynecology and Obstetrics (FIGO) stage IV disease are a highly heterogeneous group with possible survival differences. The FIGO staging system was therefore updated in 2014. OBJECTIVE: To evaluate the 2014 changes to FIGO stage IV ovarian cancer on overall survival. METHODS: We identified all patients diagnosed with FIGO stage IV disease between January 2008 and December 2015 from the Netherlands Cancer Registry. We analyzed the prognostic effect of FIGO IVa versus IVb. In addition, patients with extra-abdominal lymph node involvement as the only site of distant disease were analyzed separately. Overall survival was analyzed by Kaplan-Meier curves and multivariable Cox regression models. RESULTS: We identified 2436 FIGO IV patients, of whom 35% were diagnosed with FIGO IVa disease. Five-year overall survival of FIGO IVa and IVb patients (including those with no or limited therapy) was 8.9% and 13.0%, respectively (p=0.51). Patients with only extra-abdominal lymph node involvement had a significant better overall survival than all other FIGO IV patients (5-year overall survival 25.9%, hazard ratio 0.77 [95% CI 0.62 to 0.95]). CONCLUSION: Our study shows that the FIGO IV sub-classification into FIGO IVa and IVB does not provide additional prognostic information. Patients with extra-abdominal lymph node metastases as the only site of FIGO IV disease, however, have a better prognosis than all other FIGO IV patients. These results warrant a critical appraisal of the current FIGO IV sub-classification.

Ten-year results of the PORTEC-2 trial for high-intermediate risk endometrial carcinoma: improving patient selection for adjuvant therapy.

BACKGROUND: PORTEC-2 was a randomised trial for women with high-intermediate risk (HIR) endometrial cancer, comparing pelvic external beam radiotherapy (EBRT) with vaginal brachytherapy (VBT). We evaluated long-term outcomes combined with the results of pathology review and molecular analysis. METHODS: 427 women with HIR endometrial cancer were randomised between 2002-2006 to VBT or EBRT. Primary endpoint was vaginal recurrence (VR). Pathology review was done in 97.4%, combined with molecular analysis. RESULTS: Median follow-up was 116 months; 10-year VR was 3.4% versus 2.4% for VBT vs. EBRT (p = 0.55). Ten-year pelvic recurrence (PR) was more frequent in the VBT group (6.3% vs. 0.9%, p = 0.004), mostly combined with distant metastases (DM). Ten-year isolated PR was 2.5% vs. 0.5%, p = 0.10, and DM 10.4 vs. 8.9% (p = 0.45). Overall survival for VBT vs. EBRT was 69.5% vs. 67.6% at 10 years (p = 0.72). L1CAM and p53-mutant expression and substantial lymph-vascular space invasion were risk factors for PR and DM. EBRT reduced PR in cases with these risk factors. CONCLUSION: Long-term results of the PORTEC-2 trial confirm VBT as standard adjuvant treatment for HIR endometrial cancer. Molecular risk assessment has the potential to guide adjuvant therapy. EBRT provided better pelvic control in patients with unfavourable risk factors.

Preliminary stop of the TOPical Imiquimod treatment of high-grade Cervical intraepithelial neoplasia (TOPIC) trial.

The "TOPical Imiquimod treatment of high-grade Cervical intraepithelial neoplasia" (TOPIC) trial was stopped preliminary, due to lagging inclusions. This study aimed to evaluate the treatment efficacy and clinical applicability of imiquimod 5% cream in high-grade cervical intraepithelial neoplasia (CIN). The lagging inclusions were mainly due to a strong patient preference for either of the two treatment modalities. This prompted us to initiate a new study on the same subject, with a non-randomized, open-label design: the 'TOPical Imiquimod treatment of high-grade Cervical intraepithelial neoplasia (TOPIC)-3' study. Original TOPIC-trial: Medical Ethics Committee approval number METC13231; ClinicalTrials.gov Identifier: NCT02329171, 22 December 2014. TOPIC-3 study: Medical Ethics Committee approval number METC162025; ClinicalTrials.gov Identifier: NCT02917746, 16 September 2016.

Markers of the p53 pathway further refine molecular profiling in high-risk endometrial cancer: A TransPORTEC initiative.

BACKGROUND: The morphological classification of high-risk endometrial cancer is of limited prognostic value. Recent attempts to stratify tumours according to molecular signatures have shown considerable promise. Here we attempted to further refine molecular classifications using markers of the p53 pathway. METHODS: We analysed the expression of p53 as well as three downstream markers of the p53 pathway, p21, mdm2 and phospho-p63 (pp63), by immunohistochemistry in a series of 114 endometrial cancers (86 endometrioid, 28 non-endometrioid subtype) with high-risk features (such as high tumour grade and deep myometrial invasion) and correlated results with clinical outcome. The Cancer Genome Atlas (TCGA) data were used to analyse TP63 mutations and copy-number alterations using cBioPortal. TP53 was silenced in two endometrial cancer cell lines to study its effect on p21 and p63. RESULTS: About half of the tumours showed a p53 mutant phenotype and there was a strong negative correlation with p21 expression. Being marker positive for pp63 or mdm2 was associated with a significantly increased likelihood of dying, [hazard ratios 5.93 (95% CI 2.37-7.27) and 7.48 (95% CI 3.04-9.39), respectively]. These findings were seen in both p53 wildtype and p53 mutant tumours. Only 11% of TCGA endometrial cancers had a functional TP63 alteration. Upon silencing of TP53, p21 expression was decreased in one cell line, but no effects on p63 were observed. CONCLUSION: Markers of the p53 pathway improve stratification of endometrial cancers and provide novel insights into the role of this pathway in the disease.

Compliance with adjuvant treatment guidelines in endometrial cancer: room for improvement in high risk patients.

OBJECTIVES: Compliance of physicians with guidelines has emerged as an important indicator for quality of care. We evaluated compliance of physicians with adjuvant therapy guidelines for endometrial cancer patients in the Netherlands in a population-based cohort over a period of 10years. METHODS: Data from all patients diagnosed with endometrial cancer between 2005 and 2014, without residual tumor after surgical treatment, were extracted from the Netherlands Cancer Registry (N=14,564). FIGO stage, grade, tumor type and age were used to stratify patients into risk groups. Possible changes in compliance over time and impact of compliance on survival were assessed. RESULTS: Patients were stratified into low/low-intermediate (52%), high-intermediate (21%) and high (20%) risk groups. Overall compliance with adjuvant therapy guidelines was 85%. Compliance was highest in patients with low/low-intermediate risk (98%, no adjuvant therapy indicated). The lowest compliance was determined in patients with high risk (61%, external beam radiotherapy with/without chemotherapy indicated). Within this group compliance decreased from 64% in 2005-2009 to 57% in 2010-2014. In high risk patients with FIGO stage III serous disease compliance was 55% (chemotherapy with/without radiotherapy indicated) and increased from 41% in 2005-2009 to 66% in 2010-2014. CONCLUSION: While compliance of physicians with adjuvant therapy guidelines is excellent in patients with low and low-intermediate risk, there is room for improvement in high risk endometrial cancer patients. Eagerly awaited results of ongoing randomized clinical trials may provide more definitive guidance regarding adjuvant therapy for high risk endometrial cancer patients.

The impact of centralization of services on treatment delay in ovarian cancer: A study on process quality.

OBJECTIVE: Emphasis on improving healthcare quality has led to centralization of services for patients suspected of ovarian cancer. As centralization of services may induce treatment delays, we aimed to assess compliance with health system interval guidelines in patients suspected of ovarian cancer. DESIGN: Evaluation of health system intervals, comparison between direct and indirect referrals and between 2013 and 2014. SETTING: A managed clinical network (MCN) comprising 11 hospitals in the Netherlands. PARTICIPANTS: Patients that were treated for ovarian cancer within the University Medical Center Groningen in 2013 and 2014. INTERVENTION: Introduction of an MCN to centralize services for patients suspected of ovarian cancer. MAIN OUTCOME MEASURE: Compliance with national guidelines regarding health system intervals. RESULTS: Between 2013 and 2014 a clinically relevant improvement in compliance with guidelines was demonstrated. Within this period, median treatment intervals decreased from 34 to 29 days, and the percentage of patients in which treatment interval guidelines were met increased from 63.5 to 72.2%. New regulations and increased awareness of health system intervals inspired changes in local practice leading to improved compliance with guidelines. Compliance was highest in patients that were directly referred to our academic hospital. CONCLUSION: Evaluation of health system intervals in patients suspected of ovarian cancer was feasible and may be applicable to other MCNs. Though compliance with guidelines improved within the study period, there is potential for improvement. To facilitate real-time evaluation of compliance with national guidelines establishing uniformity of electronic patient files in the MCN is deemed essential.

TOPical Imiquimod treatment of high-grade Cervical intraepithelial neoplasia (TOPIC trial): study protocol for a randomized controlled trial.

BACKGROUND: Cervical intraepithelial neoplasia (CIN) is the premalignant condition of cervical cancer. Whereas not all high grade CIN lesions progress to cervical cancer, the natural history and risk of progression of individual lesions remain unpredictable. Therefore, high-grade CIN is currently treated by surgical excision: large loop excision of the transformation zone (LLETZ). This procedure has potential complications, such as acute haemorrhage, prolonged bleeding, infection and preterm birth in subsequent pregnancies. These complications could be prevented by development of a non-invasive treatment modality, such as topical imiquimod treatment. The primary study objective is to investigate the efficacy of topical imiquimod 5% cream for the treatment of high-grade CIN and to develop a biomarker profile to predict clinical response to imiquimod treatment. Secondary study objectives are to assess treatment side-effects, disease recurrence and quality of life during and after different treatment modalities. METHODS/DESIGN: The study design is a randomized controlled trial. One hundred forty women with a histological diagnosis of high-grade CIN (CIN 2-3) will be randomized into two arms: imiquimod treatment during 16 weeks (experimental arm) or immediate LLETZ (standard care arm). Treatment efficacy will be evaluated by colposcopy with diagnostic biopsies at 20 weeks for the experimental arm. Successful imiquimod treatment is defined as regression to CIN 1 or less, successful LLETZ treatment is defined as PAP 1 after 6 months. Disease recurrence will be evaluated by cytology at 6, 12 and 24 months after treatment. Side-effects will be evaluated using a standardized report form. Quality of life will be evaluated using validated questionnaires at baseline, 20 weeks and 1 year after treatment. Biomarkers, reflecting both host and viral factors in the pathophysiology of CIN, will be tested at baseline with the aim of developing a predictive biomarker profile for the clinical response to imiquimod treatment. DISCUSSION: Treatment of high-grade CIN lesions with imiquimod in a selected patient population may diminish complications as a result of surgical intervention. More knowledge on treatment efficacy, side effects and long-term recurrence rates after treatment is necessary. TRIAL REGISTRATION: EU Clinical Trials Register EU-CTR2013-001260-34 . Registered 18 March 2013. Medical Ethical Committee approval number: NL44336.068.13 (Medical Ethical Committee Maastricht University Hospital, University of Maastricht). Affiliation: Maastricht University Hospital. Registration number ClinicalTrials.gov: NCT02329171.

Improved outcomes due to changes in organization of care for patients with ovarian cancer in the Netherlands.

OBJECTIVES: Objectives of this study were to evaluate the effect of changes in patterns of care, for example centralization and treatment sequence, on surgical outcome and survival in patients with epithelial ovarian cancer (EOC). METHODS: Patients diagnosed with FIGO stage IIB-IV EOC (2004-2013) were selected from the Netherlands Cancer Registry. Primary outcomes were surgical outcome (extent of macroscopic residual tumor after surgery) and overall survival. Changes in treatment sequence (primary debulking surgery and adjuvant chemotherapy (PDS+ACT) or neo-adjuvant chemotherapy and interval debulking surgery (NACT+IDS)), hospital type and annual hospital volume were also evaluated. RESULTS: Patient and tumor characteristics of 7987 patients were retrieved. Most patients were diagnosed with stage III-IV EOC. The average annual case-load per hospital increased from 8 to 28. More patients received an optimal cytoreduction (tumor residue≤1cm) in 2013 (87%) compared to 2004 (55%, p<0.001). Complete cytoreduction (no macroscopic residual tumor), registered since 2010, increased from 42% to 52% (2010 and 2013, respectively, p<0.001). Optimal/complete cytoreduction was achieved in 85% in high volume (≥20 cytoreductive surgeries annually), 80% in medium (10-19 surgeries) and 71% in small hospitals (<10 surgeries, p<0.001). Within a selection of patients with advanced stage disease that underwent surgery the proportion of patients undergoing NACT+IDS increased from 28% (2004) to 71% (2013). Between 2004 and 2013 a 3% annual reduction in risk of death was observed (HR 0.97, p<0.001). CONCLUSION: Changes in pattern of care for patients with EOC in the Netherlands have led to improvement in surgical outcome and survival.

Antigen design enhances the immunogenicity of Semliki Forest virus-based therapeutic human papillomavirus vaccines.

Cellular immunity against cancer can be achieved with viral vector- and DNA-based immunizations. In preclinical studies, cancer vaccines are very potent, but in clinical trials these potencies are not achieved yet. Thus, a rational approach to improve cancer vaccines is warranted. We previously demonstrated that the relatively low intrinsic immunogenicity of DNA vaccines could be enhanced by inclusion of endoplasmic reticulum (ER) targeting and universal helper epitopes within the vaccine. We now evaluated whether an optimal antigen format, as defined in DNA vaccines, can further enhance the effectiveness of recombinant Semliki Forest virus (rSFV) vaccines. To this purpose, we generated, characterized and evaluated the efficacy of rSFV replicon particles expressing human papillomavirus E6 and/or E7 proteins fused to several helper T-cell epitopes and an ER targeting signal. Here, we show that inclusion of a helper cassette and an ER targeting signal enhanced protein stability and markedly augmented the frequencies of human papillomavirus-specific T cells. Even at an immunization dose of as low as 10(5) replicon particles, this novel vaccine achieved tumor regression and protection. Thus, even highly effective viral vector vaccines can benefit from an improved antigen format, based on the inclusion of defined helper epitopes and ER targeting.

Prediction model for regional or distant recurrence in endometrial cancer based on classical pathological and immunological parameters.

BACKGROUND: Adjuvant therapy increases disease-free survival in endometrial cancer (EC), but has no impact on overall survival and negatively influences the quality of life. We investigated the discriminatory power of classical and immunological predictors of recurrence in a cohort of EC patients and confirmed the findings in an independent validation cohort. METHODS: We reanalysed the data from 355 EC patients and tested our findings in an independent validation cohort of 72 patients with EC. Predictors were selected and Harrell's C-index for concordance was used to determine discriminatory power for disease-free survival in the total group and stratified for histological subtype. RESULTS: Predictors for recurrence were FIGO stage, lymphovascular space invasion and numbers of cytotoxic and memory T-cells. For high risk cancer, cytotoxic or memory T-cells predicted recurrence as well as a combination of FIGO stage and lymphovascular space invasion (C-index 0.67 and 0.71 vs 0.70). Recurrence was best predicted when FIGO stage, lymphovascular space invasion and numbers of cytotoxic cells were used in combination (C-index 0.82). Findings were confirmed in the validation cohort. CONCLUSIONS: In high-risk EC, clinicopathological or immunological variables can predict regional or distant recurrence with equal accuracy, but the use of these variables in combination is more powerful.

A phase I trial combining carboplatin/doxorubicin with tocilizumab, an anti-IL-6R monoclonal antibody, and interferon-α2b in patients with recurrent epithelial ovarian cancer.

BACKGROUND: The immune system is important in epithelial ovarian cancer (EOC). Interleukin-6 is associated with chemoresistance and an immune-suppressive tumor microenvironment. We investigated whether a combination of chemotherapeutics, blockade of interleukin 6 (IL-6) receptor (IL-6R; tocilizumab), and immune enhancer interferon-α (Peg-Intron) is feasible, safe, and able to enhance immunity in patients with recurrent EOC. PATIENTS AND METHODS: In this dose-escalation study, patients received tocilizumab 1, 2, 4, or 8 mg/kg i.v., q4 weeks during the first three cycles of carboplatin (AUC5) plus doxorubicin [pegylated liposomal doxorubicin (PLD) 30 mg/m(2) or doxorubicin 50 mg/m(2) i.v., day 1, q4 weeks, for six cycles]. At the highest tocilizumab dose (8 mg/kg), Peg-Intron (1 µg/kg s.c.) was added. Peripheral blood mononuclear cells were collected for immunomonitoring at baseline, after three and six cycles. Dose-limiting toxicity (DLT), CA-125, and radiologic response were evaluated. RESULTS: In the 23 patients enrolled, no DLT was established. The most frequent grade 3/4 adverse events (CTCAE v4.03) were neutropenia (23%), febrile neutropenia (19%), and ileus (19%). No treatment-related deaths occurred. Using CT evaluation, 11 of 21 assessable patients responded, 6 had stable disease and 3 progressive disease. Patients receiving highest dose tocilizumab showed a functional blockade of IL-6R with increased levels of serum IL-6 (P = 0.02) and soluble IL-6R (P = 0.008). Consequently, immune cells displayed decreased levels of pSTAT3, myeloid cells produced more IL-12 and IL-1ß while T cells were more activated and secreted higher amounts of effector cytokines interferon-γ and tumor necrosis factor-α. An increase in sIL-6R was potentially associated with a survival benefit (P = 0.03). CONCLUSIONS: Functional IL-6R blocking is feasible and safe in EOC patients treated with carboplatin/(pegylated liposomal)doxorubicin, using 8 mg/kg tocilizumab. This combination is recommended for phase II evaluation based on immune parameters. CLINICAL TRIAL REGISTER: NCT01637532.

Elevated serum CXCL16 is an independent predictor of poor survival in ovarian cancer and may reflect pro-metastatic ADAM protease activity.

BACKGROUND: In certain cancers, expression of CXCL16 and its receptor CXCR6 associate with lymphocyte infiltration, possibly aiding anti-tumour immune response. In other cancers, CXCL16 and CXCR6 associate with pro-metastatic activity. In the current study, we aimed to characterise the role of CXCL16, sCXCL16, and CXCR6 in ovarian cancer (OC). METHODS: CXCL16/CXCR6 expression was analysed on tissue microarray containing 306 OC patient samples. Pre-treatment serum sCXCL16 was determined in 118 patients using ELISA. In vitro, (primary) OC cells were treated with an ADAM-10/ADAM-17 inhibitor (TAPI-2) and an ADAM-10-specific inhibitor (GI254023x), whereupon CXCL16 levels were evaluated on the cell membrane (immunofluorescent analysis, western blots) and in culture supernatants (ELISA). In addition, cell migration was assessed using scratch assays. RESULTS: sCXCL16 independently predicted for poor survival (hazard ratio=2.28, 95% confidence interval=1.29-4.02, P=0.005), whereas neither CXCL16 nor CXCR6 expression correlated with survival. Further, CXCL16/CXCR6 expression and serum sCXCL16 levels did not associate with lymphocyte infiltration. In vitro inhibition of both ADAM-17 and ADAM-10, but especially the latter, decreased CXCL16 membrane shedding and strongly reduced cell migration of A2780 and cultured primary OC-derived malignant cells. CONCLUSIONS: High serum sCXCL16 is a prognostic marker for poor survival of OC patients, possibly reflecting ADAM-10 and ADAM-17 pro-metastatic activity. Therefore, serum sCXCL16 levels may be a pseudomarker that identifies patients with highly metastatic tumours.

Status of cellular immunity lacks prognostic significance in vulvar squamous carcinoma.

OBJECTIVE: It is generally recognized that the immune system has an important role in regulating cancer development. Evidence indicating a prognostic role of the immune system in vulvar carcinoma is scarce. This study investigated the presence and prognostic significance of several aspects of the immune system in vulvar squamous carcinoma. METHODS: The number of intratumoral CD8(+) and Foxp3(+) T-lymphocytes, next to HLA class I (HLA-A, HLA-B/C and ß(2)-m) and indoleamine 2,3-dioxygenase (IDO) expression was determined by immunohistochemistry in a consecutively selected cohort of 286 vulvar squamous carcinoma patients, all treated in the University Medical Center Groningen, the Netherlands. Associations between immunohistochemistry expression and the influence on survival were determined. RESULTS: The number of tumor-infiltrating CD8(+) T-lymphocytes was significantly lower in tumors with loss of HLA-A (p=0.004), HLA-B/C (p=0.024) or ß(2)-m (p=0.025) expression compared with tumors with expression of HLA class I. No association was found between the number of intratumoral CD8(+) T-lymphocytes and Foxp3(+) T-lymphocytes, HLA class I and IDO expression and survival of vulvar squamous carcinoma patients. CONCLUSION: Our results indicate that the immune system does not seem to have a major influence on prognosis of patients with vulvar squamous carcinoma.

The prognostic influence of tumour-infiltrating lymphocytes in cancer: a systematic review with meta-analysis.

BACKGROUND: Tumour-infiltrating lymphocytes (TILs) are often found in tumours, presumably reflecting an immune response against the tumour. We carried out a systematic review and meta-analysis, aiming to establish pooled estimates for survival outcomes based on the presence of TILs in cancer. METHODS: A Pubmed and Embase literature search was designed. Studies were included, in which the prognostic significance of intratumoural CD3+, CD4+, CD8+, and FoxP3+ lymphocytes, as well as ratios between these subsets, were determined in solid tumours. RESULTS: In pooled analysis, CD3+ TILs had a positive effect on survival with a hazard ratio (HR) of 0.58 (95% confidence interval (CI) 0.43-0.78) for death, as did CD8+ TILs with a HR of 0.71 (95% CI 0.62-0.82). FoxP3+ regulatory TILs were not linked to overall survival, with a HR of 1.19 (95% CI 0.84-1.67). The CD8/FoxP3 ratio produced a more impressive HR (risk of death: HR 0.48, 95% CI 0.34-0.68), but was used in relatively few studies. Sample size and follow-up time seemed to influence study outcomes. CONCLUSION: Any future studies should be carefully designed, to prevent overestimating the effect of TILs on prognosis. In this context, ratios between TIL subsets may be more informative.

Immunological and clinical effects of vaccines targeting p53-overexpressing malignancies.

Approximately 50% of human malignancies carry p53 mutations, which makes it a potential antigenic target for cancer immunotherapy. Adoptive transfer with p53-specific cytotoxic T-lymphocytes (CTL) and CD4(+) T-helper cells eradicates p53-overexpressing tumors in mice. Furthermore, p53 antibodies and p53-specific CTLs can be detected in cancer patients, indicating that p53 is immunogenic. Based on these results, clinical trials were initiated. In this paper, we review immunological and clinical responses observed in cancer patients vaccinated with p53 targeting vaccines. In most trials, p53-specific vaccine-induced immunological responses were observed. Unfortunately, no clinical responses with significant reduction of tumor-burden have occurred. We will elaborate on possible explanations for this lack of clinical effectiveness. In the second part of this paper, we summarize several immunopotentiating combination strategies suitable for clinical use. In our opinion, future p53-vaccine studies should focus on addition of these immunopotentiating regimens to achieve clinically effective therapeutic vaccination strategies for cancer patients.

Prognostic image-based quantification of CD8CD103 T cell subsets in high-grade serous ovarian cancer patients.

CD103-positive tissue resident memory-like CD8+ T cells (CD8CD103 TRM) are associated with improved prognosis across malignancies, including high-grade serous ovarian cancer (HGSOC). However, whether quantification of CD8, CD103 or both is required to improve existing survival prediction and whether all HGSOC patients or only specific subgroups of patients benefit from infiltration, remains unclear. To address this question, we applied image-based quantification of CD8 and CD103 multiplex immunohistochemistry in the intratumoral and stromal compartments of 268 advanced-stage HGSOC patients from two independent clinical institutions. Infiltration of CD8CD103 immune cell subsets was independent of clinicopathological factors. Our results suggest CD8CD103 TRM quantification as a superior method for prognostication compared to single CD8 or CD103 quantification. A survival benefit of CD8CD103 TRM was observed only in patients treated with primary cytoreductive surgery. Moreover, survival benefit in this group was limited to patients with no macroscopic tumor lesions after surgery. This approach provides novel insights into prognostic stratification of HGSOC patients and may contribute to personalized treatment strategies in the future.

B lymphocytes secrete antigen-presenting vesicles.

Antigen-presenting cells contain a specialized late endocytic compartment, MIIC (major histocompatibility complex [MHC] class II-enriched compartment), that harbors newly synthesized MHC class II molecules in transit to the plasma membrane. MIICs have a limiting membrane enclosing characteristic internal membrane vesicles. Both the limiting membrane and the internal vesicles contain MHC class II. In this study on B lymphoblastoid cells, we demonstrate by immunoelectron microscopy that the limiting membrane of MIICs can fuse directly with the plasma membrane, resulting in release from the cells of internal MHC class II-containing vesicles. These secreted vesicles, named exosomes, were isolated from the cell culture media by differential centrifugation followed by flotation on sucrose density gradients. The overall surface protein composition of exosomes differed significantly from that of the plasma membrane. Exosome-bound MHC class II was in a compact, peptide-bound conformation. Metabolically labeled MHC class II was released into the extracellular medium with relatively slow kinetics, 10 +/- 4% in 24 h, indicating that direct fusion of MIICs with the plasma membrane is not the major pathway by which MHC class II reaches the plasma membrane. Exosomes derived from both human and murine B lymphocytes induced antigen-specific MHC class II-restricted T cell responses. These data suggest a role for exosomes in antigen presentation in vivo.

Antigen capture and major histocompatibility class II compartments of freshly isolated and cultured human blood dendritic cells.

Dendritic cells (DC) represent potent antigen-presenting cells for the induction of T cell-dependent immune responses. Previous work on antigen uptake and presentation by human DC is based largely on studies of blood DC that have been cultured for various periods of time before analysis. These cultured cells may therefore have undergone a maturation process from precursors that have different capacities for antigen capture and presentation. We have now used immunoelectron microscopy and antigen presentation assays to compare freshly isolated DC (f-DC) and cultured DC (c-DC). f-DC display a round appearance, whereas c-DC display characteristic long processes. c-DC express much more cell surface major histocompatibility complex (MHC) class II than f-DC. The uptake of colloidal gold-labeled bovine serum albumin (BSA), however, is greater in f-DC, as is the presentation of 65-kD heat shock protein to T cell clones. The most striking discovery is that the majority of MHC class II molecules in both f-DC and c-DC occur in intracellular vacuoles with a complex shape (multivesicular and multilaminar). These MHC class II enriched compartments (MIIC) represent the site to which BSA is transported within 30 min. Although MIIC appear as more dense structures with less MHC class II molecules in f-DC than c-DC, the marker characteristics are very similar. The MIIC in both types of DC are acidic, contain invariant chain, and express the recently described HLA-DM molecule that can contribute to antigen presentation. CD19+ peripheral blood B cells have fewer MIIC and surface MHC class II expression than DCs, while monocytes had low levels of MIIC and surface MHC class II. These results demonstrate in dendritic cells the elaborate development of MIIC expressing several of the components that are required for efficient antigen presentation.

Tumor eradication by wild-type p53-specific cytotoxic T lymphocytes.

The tumor suppressor protein p53 is overexpressed in close to 50% of all human malignancies. The p53 protein is therefore an attractive target for immunotherapy. Cytotoxic T lymphocytes (CTLs) recognizing a murine wild-type p53 peptide, presented by the major histocompatibility complex class I molecule H-2Kb, were generated by immunizing p53 gene deficient (p53 -/-) C57BL/6 mice with syngeneic p53-overexpressing tumor cells. Adoptive transfer of these CTLs into tumor-bearing p53 +/+ nude mice caused complete and permanent tumor eradication. Importantly, this occurred in the absence of any demonstrable damage to normal tissue. When transferred into p53 +/+ immunocompetent C57BL/6 mice, the CTLs persisted for weeks in the absence of immunopathology and were capable of preventing tumor outgrowth. Wild-type p53-specific CTLs can apparently discriminate between p53-overexpressing tumor cells and normal tissue, indicating that widely expressed autologous molecules such as p53 can serve as a target for CTL-mediated immunotherapy of tumors.

Identification of genes and pathways associated with cytotoxic T lymphocyte infiltration of serous ovarian cancer.

BACKGROUND: Tumour-infiltrating lymphocytes (TILs) are predictors of disease-specific survival (DSS) in ovarian cancer. It is largely unknown what factors contribute to lymphocyte recruitment. Our aim was to evaluate genes and pathways contributing to infiltration of cytotoxic T lymphocytes (CTLs) in advanced-stage serous ovarian cancer. METHODS: For this study global gene expression was compared between low TIL (n=25) and high TIL tumours (n=24). The differences in gene expression were evaluated using parametric T-testing. Selectively enriched biological pathways were identified with gene set enrichment analysis. Prognostic influence was validated in 157 late-stage serous ovarian cancer patients. Using immunohistochemistry, association of selected genes from identified pathways with CTL was validated. RESULTS: The presence of CTL was associated with 320 genes and 23 pathways (P<0.05). In addition, 54 genes and 8 pathways were also associated with DSS in our validation cohort. Immunohistochemical evaluation showed strong correlations between MHC class I and II membrane expression, parts of the antigen processing and presentation pathway, and CTL recruitment. CONCLUSION: Gene expression profiling and pathway analyses are valuable tools to obtain more understanding of tumour characteristics influencing lymphocyte recruitment in advanced-stage serous ovarian cancer. Identified genes and pathways need to be further investigated for suitability as therapeutic targets.