Expression of tumor-specific antigen MAGE, GAGE and BAGE in ovarian cancer tissues and cell lines

<p>Abstract</p> <p>Background</p> <p>To observe mRNA expression of tumor-specific antigen MAGE, BAGE and GAGE in epithelial ovarian cancer tissues and cell lines, to explore the relationship between gene expression and diagnosis, treatment and prognosis of ovarian cancer, and to evaluate the feasibility of their gene products as markers, and an immunotherapy target for ovarian cancer.</p> <p>Methods</p> <p>mRNA expression of MAGE-1, MAGE-3, GAGE-1/2 and BAGE were determined by reverse transcription polymerase chain reaction (RT-PCR) in 14 cases of normal ovarian tissue, 20 cases of ovarian benign tumor specimens, 41 cases of ovarian cancer specimens, and ovarian cancer cell lines SKOV3, A2780, and COC1.</p> <p>Results</p> <p>MAGE, GAGE and BAGE genes were not expressed in normal ovarian tissue. In benign tumors, only the MAGE gene was expressed; the expression rate of this gene in benign tumors was 15% (3/20). In ovarian cancer tissues, MAGE-1 and MAGE-3 was highly expressed, with expression rates of 53.7% (22/41) and 36.6% (15/41), while GAGE-1/2 and BAGE had relatively low expression, with rates of 26.8% (11/41) and 14.6% (6/41). In metastatic lesions of ovarian cancer, only MAGE-1 and BAGE were expressed, with expression rates of 28.6% (2/7) and 14.3% (1/7). The positive expression rates of MAGE-1 and MAGE-3 in serous cystadenocarcinoma were significantly higher than that in other types of ovarian cancer (<it>P </it>< 0.05). Gene expression rate was not correlated with menopause or lymph node metastasis. Positive expression of MAGE-1 and MAGE-3 was positively correlated with tumor differentiation and the clinical stage of the ovarian cancer. In addition, the positive expression rate of BAGE was significantly higher in ovarian cancer patients with ascites (<it>P </it>< 0.05). The mRNA expression profiles of MAGE, GAGE and BAGE in ovarian carcinoma cell lines SKOV3, A2780 and COC1 varied, but there was at least one gene expressed in each cell line.</p> <p>Conclusion</p> <p>Tumor-specific antigen MAGE, BAGE and GAGE may play a role in the occurrence and development of ovarian cancer. These genes can be used as one of the important indicators for early diagnosis, efficacy evaluation and prognostic determination of ovarian cancer.</p

Phase III study of ACVBP versus ACVBP plus rituximab for patients with localized low-risk diffuse large B-cell lymphoma (LNH03-1B)

Background The superiority of a chemotherapy with doxorubicin, cyclophosphamide, vindesine, bleomycin and prednisone (ACVBP) in comparison with cyclophosphamide, doxorubicin, vincristin and prednisone plus radiotherapy for young patients with localized diffuse large B-cell lymphoma (DLBCL) was previously demonstrated. We report the results of a trial which evaluates the role of rituximab combined with ACVBP (R-ACVBP) in these patients. Patients and methods Untreated patients younger than 66 years with stage I or II DLBCL and no adverse prognostic factors of the age-adjusted International Prognostic Index were randomly assigned to receive three cycles of ACVBP plus sequential consolidation with or without the addition of four infusions of rituximab. Results A total of 223 patients were randomly allocated to the study, 110 in the R-ACVBP group and 113 in the ACVBP group. After a median follow-up of 43 months, our 3-year estimate of event-free survival was 93% in the R-ACVBP group and 82% in the ACVBP group (P = 0.0487). Three-year estimate of progression-free survival was increased in the R-ACVBP group (95% versus 83%, P = 0.0205). Overall survival did not differ between the two groups with a 3-year estimates of 98% and 97%, respectively (P = 0.686). Conclusion In young patients with low-risk localized DLBCL, rituximab combined with three cycles of ACVBP plus consolidation is significantly superior to ACVBP plus consolidation alon

Real-World Insights on the Management of Immune-Mediated Thrombotic Thrombocytopenic Purpura (iTTP) With Caplacizumab

peer reviewe

Harmonization guidelines for HLA-peptide multimer assays derived from results of a large scale international proficiency panel of the Cancer Vaccine Consortium

PURPOSE: The Cancer Vaccine Consortium of the Cancer Research Institute (CVC-CRI) conducted a multicenter HLA-peptide multimer proficiency panel (MPP) with a group of 27 laboratories to assess the performance of the assay. EXPERIMENTAL DESIGN: Participants used commercially available HLA-peptide multimers and a well characterized common source of peripheral blood mononuclear cells (PBMC). The frequency of CD8+ T cells specific for two HLA-A2-restricted model antigens was measured by flow cytometry. The panel design allowed for participants to use their preferred staining reagents and locally established protocols for both cell labeling, data acquisition and analysis. RESULTS: We observed significant differences in both the performance characteristics of the assay and the reported frequencies of specific T cells across laboratories. These results emphasize the need to identify the critical variables important for the observed variability to allow for harmonization of the technique across institutions. CONCLUSIONS: Three key recommendations emerged that would likely reduce assay variability and thus move toward harmonizing of this assay. (1) Use of more than two colors for the staining (2) collect at least 100,000 CD8 T cells, and (3) use of a background control sample to appropriately set the analytical gates. We also provide more insight into the limitations of the assay and identified additional protocol steps that potentially impact the quality of data generated and therefore should serve as primary targets for systematic analysis in future panels. Finally, we propose initial guidelines for harmonizing assay performance which include the introduction of standard operating protocols to allow for adequate training of technical staff and auditing of test analysis procedures

Phase III study of ACVBP versus ACVBP plus rituximab for patients with localized low-risk diffuse large B-cell lymphoma (LNH03-1B).

Background The superiority of a chemotherapy with doxorubicin, cyclophosphamide, vindesine, bleomycin and prednisone (ACVBP) in comparison with cyclophosphamide, doxorubicin, vincristin and prednisone plus radiotherapy for young patients with localized diffuse large B-cell lymphoma (DLBCL) was previously demonstrated. We report the results of a trial which evaluates the role of rituximab combined with ACVBP (R-ACVBP) in these patients. Patients and methods Untreated patients younger than 66 years with stage I or II DLBCL and no adverse prognostic factors of the age-adjusted International Prognostic Index were randomly assigned to receive three cycles of ACVBP plus sequential consolidation with or without the addition of four infusions of rituximab. Results A total of 223 patients were randomly allocated to the study, 110 in the R-ACVBP group and 113 in the ACVBP group. After a median follow-up of 43 months, our 3-year estimate of event-free survival was 93% in the R-ACVBP group and 82% in the ACVBP group (P = 0.0487). Three-year estimate of progression-free survival was increased in the R-ACVBP group (95% versus 83%, P = 0.0205). Overall survival did not differ between the two groups with a 3-year estimates of 98% and 97%, respectively (P = 0.686). Conclusion In young patients with low-risk localized DLBCL, rituximab combined with three cycles of ACVBP plus consolidation is significantly superior to ACVBP plus consolidation alone

A stochastic individual-based model to explore the role of spatial interactions and antigen recognition in the immune response against solid tumours

FRM is funded by the Engineering and Physical Sciences Research Council (EPSRC).Spatial interactions between cancer and immune cells, as well as the recognition of tumour antigens by cells of the immune system, play a key role in the immune response against solid tumours. The existing mathematical models generally focus only on one of these key aspects. We present here a spatial stochastic individual-based model that explicitly captures antigen expression and recognition. In our model, each cancer cell is characterised by an antigen profile which can change over time due to either epimutations or mutations. The immune response against the cancer cells is initiated by the dendritic cells that recognise the tumour antigens and present them to the cytotoxic T cells. Consequently, T cells become activated against the tumour cells expressing such antigens. Moreover, the differences in movement between inactive and active immune cells are explicitly taken into account by the model. Computational simulations of our model clarify the conditions for the emergence of tumour clearance, dormancy or escape, and allow us to assess the impact of antigenic heterogeneity of cancer cells on the efficacy of immune action. Ultimately, our results highlight the complex interplay between spatial interactions and adaptive mechanisms that underpins the immune response against solid tumours, and suggest how this may be exploited to further develop cancer immunotherapies.PostprintPeer reviewe

Dendritic cell vaccination and immune monitoring

We exploited dendritic cells (DC) to vaccinate melanoma patients. We recently demonstrated a statistical significant correlation between favorable clinical outcome and the presence of vaccine-related tumor antigen-specific T cells in delayed type hypersensitivity (DTH) skin biopsies. However, favorable clinical outcome is only observed in a minority of the treated patients. Therefore, it is obvious that current DC-based protocols need to be improved. For this reason, we study in small proof of principle trials the fate, interactions and effectiveness of the injected DC

Future perspectives in melanoma research: meeting report from the "Melanoma Bridge";: Napoli, December 3rd-6th 2014.

The fourth "Melanoma Bridge Meeting" took place in Naples, December 3-6th, 2014. The four topics discussed at this meeting were: Molecular and Immunological Advances, Combination Therapies, News in Immunotherapy, and Tumor Microenvironment and Biomarkers. Until recently systemic therapy for metastatic melanoma patients was ineffective, but recent advances in tumor biology and immunology have led to the development of new targeted and immunotherapeutic agents that prolong progression-free survival (PFS) and overall survival (OS). New therapies, such as mitogen-activated protein kinase (MAPK) pathway inhibitors as well as other signaling pathway inhibitors, are being tested in patients with metastatic melanoma either as monotherapy or in combination, and all have yielded promising results. These include inhibitors of receptor tyrosine kinases (BRAF, MEK, and VEGFR), the phosphatidylinositol 3 kinase (PI3K) pathway [PI3K, AKT, mammalian target of rapamycin (mTOR)], activators of apoptotic pathway, and the cell cycle inhibitors (CDK4/6). Various locoregional interventions including radiotherapy and surgery are still valid approaches in treatment of advanced melanoma that can be integrated with novel therapies. Intrinsic, adaptive and acquired resistance occur with targeted therapy such as BRAF inhibitors, where most responses are short-lived. Given that the reactivation of the MAPK pathway through several distinct mechanisms is responsible for the majority of acquired resistance, it is logical to combine BRAF inhibitors with inhibitors of targets downstream in the MAPK pathway. For example, combination of BRAF/MEK inhibitors (e.g., dabrafenib/trametinib) have been demonstrated to improve survival compared to monotherapy. Application of novel technologies such sequencing have proven useful as a tool for identification of MAPK pathway-alternative resistance mechanism and designing other combinatorial therapies such as those between BRAF and AKT inhibitors. Improved survival rates have also been observed with immune-targeted therapy for patients with metastatic melanoma. Immune-modulating antibodies came to the forefront with anti-CTLA-4, programmed cell death-1 (PD-1) and PD-1 ligand 1 (PD-L1) pathway blocking antibodies that result in durable responses in a subset of melanoma patients. Agents targeting other immune inhibitory (e.g., Tim-3) or immune stimulating (e.g., CD137) receptors and other approaches such as adoptive cell transfer demonstrate clinical benefit in patients with melanoma as well. These agents are being studied in combination with targeted therapies in attempt to produce longer-term responses than those more typically seen with targeted therapy. Other combinations with cytotoxic chemotherapy and inhibitors of angiogenesis are changing the evolving landscape of therapeutic options and are being evaluated to prevent or delay resistance and to further improve survival rates for this patient population. This meeting's specific focus was on advances in combination of targeted therapy and immunotherapy. Both combination targeted therapy approaches and different immunotherapies were discussed. Similarly to the previous meetings, the importance of biomarkers for clinical application as markers for diagnosis, prognosis and prediction of treatment response was an integral part of the meeting. The overall emphasis on biomarkers supports novel concepts toward integrating biomarkers into contemporary clinical management of patients with melanoma across the entire spectrum of disease stage. Translation of the knowledge gained from the biology of tumor microenvironment across different tumors represents a bridge to impact on prognosis and response to therapy in melanoma

Le mode de vaccination des patients avec un antigène tumoral influence la différenciation des lymphocytes T CD8 anti-vaccinaux

The identification in 1991 of antigens recognized on human tumor cells by cytolytic T lymphocytes (CTL) opened new prospects for cancer immunotherapy. Several small scale clinical trials of vaccination with MAGE antigens have been conducted in metastatic melanoma patients with measurable disease. Tumor regressions have been observed in 15-20% of the patients, and a third of these regressions corresponded to 'partial responses' or better. These clinical results are similar whatever the vaccine modality (peptide, protein, recombinant virus, or dendritic cells). Vaccination with peptide MAGE-A3(168-176) under different modalities induced low level anti-vaccine CTL responses in the blood, reached between 10-5 and 10-3 of the CD8+ T cells. These responses were detectable after in vitro restimulation in limiting dilution condition, followed by labeling the responder cells with the HLA-A1/MAGE-A3(168-176) tetramer. The anti-vaccine CTL responses were correlated with the tumor regressions, suggesting a causative link. It is however surprising that so few anti-vaccine CTL could by themselves eliminate one or several metastases, leading in some cases to complete remission. Several hypotheses could explain this paradox, and this work consisted in exploring two of them. The low level anti-vaccine CTL responses could be associated to other anti-vaccine CTL responses, possibly of high magnitude, but not detectable by our methodology. Indeed, labeling the microcultures with the HLA-A1/MAGE-A3(168-176) tetramer, we were not able to detect T lymphocytes recognizing peptide MAGE-A3(168-176) presented by other HLA molecules than HLA-A1. I used two approaches to reasonably exclude this possibility: a screening of the microcultures with a cytokine secretion assay instead of a tetramer, and an in vitro assessment of the binding of peptide MAGE-A3(168-176) to other recombinant class I HLA molecules. I obtained negative results, strengthening the hypothesis that the low level anti-MAGE-A3(168-176)/HLA-A1 CTL responses detected in some patients played a crucial role in the tumor regressions. Anti-vaccine CTL could have functional properties that could explain their anti-tumoral effect, even in low numbers. As the low blood frequencies of anti-vaccine T cells prevented robust ex vivo functional analyses, I analyzed a representative set of 15 anti-MAGE-A3(168-176)/HLA-A1 CTL clones derived from 8 melanoma patients who displayed tumor regression. Using gene expression profiling, I observed that the expression levels of about 20 genes distinguished the anti-MAGE-A3(168-176)/HLA-A1 CTL clones derived from patients vaccinated with peptide alone or with a recombinant poxvirus containing MAGE minigenes, and the anti-MAGE-A3(168-176)/HLA-A1 CTL clones derived from patients vaccinated with peptide-pulsed dendritic cells. These results indicated that the vaccination modality with a MAGE tumor-specific antigen influences the differentiation of anti-vaccine CTL. This might impact on their capacity to trigger tumor regression. In addition they suggest that it might be important to carry out the immunological monitoring of vaccinated cancer patients with methods that do not only evaluate quantitative aspects of the response, but that can also compare the functional properties of the detected anti-vaccine T lymphocytes.La découverte d’antigènes tumoraux, reconnus à la surface des cellules tumorales par des lymphocytes T cytolytiques, a ouvert en 1991 de nouvelles perspectives pour l’immunothérapie anti-cancéreuse. Plusieurs études cliniques de vaccination avec des antigènes codés par les gènes MAGE ont été conduites chez des patients atteints de mélanome. Des régressions tumorales ont été observées dans 15-20% des cas. Un tiers d’entre elles constituent des réponses cliniques au minimum partielles. Ces résultats cliniques sont similaires quelle que soit la modalité vaccinale (peptides, protéines, virus recombinants, cellules dendritiques). La vaccination avec le peptide MAGE-3(168-176), administré sous différentes formes, induit de faibles réponses CTL anti-MAGE-A3(168-176)/HLA-A1 qui atteignent des fréquences entre 10-5 et 10-3 des lymphocytes T CD8+ du sang. Ces réponses ne sont donc généralement détectables qu’après restimulation in vitro des CTL circulants en condition de dilution limite, suivie par l’analyse des microcultures avec le tétramère HLA-A1/MAGE-A3(168-176). Ces réponses CTL anti-vaccinales sont corrélées aux régressions tumorales. Nous en déduisons qu’elles ont probablement joué un rôle dans les réponses cliniques survenant après vaccination. Il est surprenant que des lymphocytes T cytolytiques aussi rares puissent déclencher un processus de régression tumorale, menant parfois à une rémission complète. Ce paradoxe apparent pourrait s’expliquer de plusieurs manières. Mon travail a consisté à évaluer deux d’entre elles. Ces réponses immunologiques de faible amplitude pourraient être fréquemment associées à d’autres réponses lymphocytaires T cytolytiques contre le vaccin, potentiellement de forte amplitude, mais qui ne soient pas identifiables par notre méthodologie. En effet, par le marquage des microcultures avec le tétramère HLA-A1/MAGE-A3(168-176), nous ne pouvons pas détecter des lymphocytes T qui reconnaîtraient le peptide vaccinal MAGE-A3(168-176) présenté par un autre HLA qu’HLA-A1. Afin d’exclure raisonnablement cette hypothèse, j’ai utilisé deux méthodes indépendantes : analyser les microcultures par un test de sécrétion de cytokine à la place du tétramère et tester directement la liaison du peptide MAGE-A3(168-176) à certains HLA recombinants de classe 1. Les résultats négatifs obtenus confortent l’hypothèse que les faibles réponses CTL anti-MAGE-A3(168-176)/HLA-A1 détectées chez certains patients ont joué un rôle crucial dans les régressions tumorales observées. Les lymphocytes T cytolytiques anti-vaccinaux pourraient exprimer des propriétés particulières qui expliqueraient leur rôle dans les régressions tumorales, m��me en faible nombre. Après avoir éliminé raisonnablement ma première hypothèse, j’ai cherché à mieux caractériser ces réponses lymphocytaires T identifiées. Leur faible amplitude m’a empêché de réaliser une étude fonctionnelle ex vivo. C’est pourquoi, j’ai réalisé mes expériences sur une batterie de 15 clones CTL anti-MAGEA3(168-176)/HLA-A1 dérivés de 8 patients qui ont présenté une régression tumorale suite à la vaccination. Le niveau d’expression d’environ 20 gènes fut fortement différent entre les clones CTL dérivés de patients vaccinés avec le peptide antigénique seul ou avec ALVACminiMAGE-1/3 et les clones CTL dérivés de patients vaccinés avec des cellules dendritiques incubées avec le peptide vaccinal. Ces résultats indiquent que la modalité de vaccination avec un antigène spécifique de tumeur influence la voie de différenciation des lymphocytes T CD8 anti-vaccinaux. Cela pourrait avoir un impact sur leur capacité à initier un rejet tumoral. De plus, cette observation suggère qu’une analyse immunologique uniquement quantitative telle que pratiquée en vaccinologie humorale est probablement insuffisante en vaccinologie anti-tumorale.(SBIM 3) -- UCL, 200