Differential effects of PD-1 and CTLA-4 blockade on the melanoma-reactive CD8 T cell response.

Immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) have revolutionized the treatment of melanoma patients. Based on early studies addressing the mechanism of action, it was assumed that PD-1 blockade mostly influences T cell responses at the tumor site. However, recent work has demonstrated that PD-1 blockade can influence the T cell compartment in peripheral blood. If the activation of circulating, tumor-reactive T cells would form an important mechanism of action of PD-1 blockade, it may be predicted that such blockade would alter either the frequency and/or the breadth of the tumor-reactive CD8 T cell response. To address this question, we analyzed CD8 T cell responses toward 71 melanoma-associated epitopes in peripheral blood of 24 melanoma patients. We show that both the frequency and the breadth of the circulating melanoma-reactive CD8 T cell response was unaltered upon PD-1 blockade. In contrast, a broadening of the circulating melanoma-reactive CD8 T cell response was observed upon CTLA-4 blockade, in concordance with our prior data. Based on these results, we conclude that PD-1 and CTLA-4 blockade have distinct mechanisms of action. In addition, the data provide an argument in favor of the hypothesis that anti-PD-1 therapy may primarily act at the tumor site.

MAGE-A gene expression in peripheral blood serves as a poor prognostic marker for patients with lung cancer.

BACKGROUND: MAGE-A genes belong to the cancer/testis antigens family. The prognostic significance of MAGE-A expression in the peripheral blood of patients with lung cancer is unknown. Therefore, this study evaluated the expression and possible prognostic significance of MAGE-A in the peripheral blood of patients with lung cancer. METHODS: In this study, we detected MAGE-A gene expression in the peripheral blood of 150 patients with lung cancer and 30 healthy donors using multiplex semi-nested PCR and analyzed their correlation with clinicopathological risk factors. RESULTS: MAGE-A expression was associated with factors indicating poor prognosis. The expression of MAGE-A and each individual MAGE-A gene were also associated with low overall survival in patients with lung cancer. CONCLUSION: The expression of MAGE-A genes in peripheral blood may act as a poor prognostic marker in patients with lung cancer.

A novel electrochemical immunosensor based on ITO modified by carboxyl-ended silane agent for ultrasensitive detection of MAGE-1 in human serum.

A new, low-cost electrochemical immunosensor was developed for rapid detection of Melanoma-associated antigen 1 (MAGE-1), a cancer biomarker. The fabrication procedure of immunosensor was based on the covalent immobilization of anti-MAGE-1, biorecognition molecule, on ITO electrode by carboxyethylsilanetriol (CTES) monolayer. The biosensing MAGE-1 antigen was monitored by using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) technique. Apart from these techniques, single frequency impedance (SFI) was used for investigation of antibody-antigen interactions. Scanning electron microscopy (SEM), fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM) were utilized for characterization of the proposed biosensor. To fabricate highly sensitive, good stability immunosensor, some parameters were optimized. Under optimal conditions, the developed electrochemical immunosensor for MAGE-1 exhibited a dynamic range of 4 fg/mL and 200 fg/mL with a low detection limit of 1.30 fg/mL. It had acceptable repeatability (5.05%, n = 20) and good storage stability (3.58% loss after 10 weeks). Moreover, this electrochemical immunosensor has been successfully applied to the determination of MAGE-1 in human serum samples.

Detection of circulating tumor cells by reverse transcription­quantitative polymerase chain reaction and magnetic activated cell sorting in the peripheral blood of patients with hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies worldwide. Circulating tumor cells (CTCs) are considered a major cause of recurrence and metastasis in cancer; however, the detection of CTCs is challenging owing to their very low numbers in peripheral blood (around 10 CTCs per 1,000,000 erythrocytes). Cancer­testis antigens (CTAs) are specific tumor markers for CTCs. The present study aimed to evaluate the sensitivity and specificity of reverse transcription­quantitative polymerase chain reaction (RT­qPCR) for the detection of nine CTAs as well as placenta­specific antigen 1 (PLAC1) in peripheral blood mononuclear cell (PBMC) samples collected from 51 patients with HCC. The effectiveness of magnetic­activated cell sorting (MACS) for tumor­cell enrichment, through the depletion of CD45+ leukocytes in PBMC samples, was also assessed. Immunocytochemistry along with hematoxylin and eosin staining demonstrated that RT­qPCR achieved an overall positive detection rate for CTAs and PLAC1 of 70.6%; the highest rates were observed for melanoma­associated antigen A3 (MAGEA3), synovial sarcoma X breakpoint 1, MAGEA1, NY­ESO­1, L antigen 1 and PLAC1. MACS­detected intact CTCs in PBMCs were confirmed by H&E staining and morphological assessment; 12 out of 19 (63.2%) patients were identified as positive for CTAs. Screening for these five CTAs and PLAC1 by RT­qPCR may offer a potentially valuable prognostic tool with good sensitivity and specificity in patients with HCC that may be enhanced by MACS.

A sensitive and disposable indium tin oxide based electrochemical immunosensor for label-free detection of MAGE-1.

MAGE-1 (MAGE, for melanoma antigen), was identified by virtue of its processing and cell surface expression as a tumor-specific peptide bound to major histocompatibility complexes which was reactive with autolytic T cells. 3-Glycidoxypropyltrimethoxysilane (3-GOPS) is frequently employed for the preparation of dense heterometal hybrid polymers which are used, e.g., for hard coatings of organic polymers and contact lens materials in the optical industry. In this study, we have improved a new immunological biosensor with indium tin oxide (ITO). Then, Anti-MAGE-1 antibody was covalently immobilized with 3-GOPS which formed a self-assembled monolayers (SAMs) on modified ITO electrodes. Analytical characteristics such as square wave voltammetry, linear determination range, repeatability, reproducibility and regeneration of biosensors are determined. All characterization steps are monitored by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV). The developed biosensor has wide determination range (0.5fg-15fg/mL). To investigate long shelf life of the fabricated biosensor, the immunosensors were stored at 4°C for periods ten weeks. Futhermore, binding kinetics of MAGE1 to antiMAGE-1 is monitored by single frequency technique in real time. Additionally, Kramer's-Kronig transform was used to understand whether the impedance spectra of biosensor system are affected from the variation that occurred because of external factor. Morphological characteristics of constructed biosensor were observed by scanning electron microscopy. Real human serum samples were also analyzed by the proposed biosensor, successfully. A commercial ELISA kit was also used as a reference method to validate the results obtained by the biosensor. Finally, this biosensor was tried in real blood sample and that showed it could be utilized in clinical applications. This biosensor can be preferred due to it has a wide linear range and it can be prepared easily.

Melanoma antigens are biomarkers for ipilimumab response.

BACKGROUND: Novel immunotherapy modalities significantly improve survival of patients with metastatic melanoma. However, CTLA-4-blocking monoclonal antibody ipilimumab is effective only in a small proportion of patients. Biomarkers for prediction of treatment response are indispensably needed. OBJECTIVE: To determine the utility of multimarker detection of circulating melanoma cells as prognostic and pharmacodynamic biomarker in patients with metastatic melanoma treated with ipilimumab. METHODS: Patients (n = 62) with metastatic melanoma in unresectable stage III or metastatic stage IV treated with ipilimumab were recruited prospectively. The values of four melanoma markers on circulating cells Melan-A, gp100, MAGE-3 and melanoma inhibitory antigen prior to the treatment and within the therapy were compared to the data collected at baseline - after the melanoma surgery. RESULTS: The immunotherapy pretreatment marker level was found to be prognostic of overall survival; lower levels were linked to longer survival time. Moreover, longitudinal follow-up of melanoma markers in patients treated with ipilimumab correlates with therapy response. A decline of marker levels by >30% at week 6 (in 83% of the responding subjects) to week 9 (in all responders) of ipilimumab administration was associated with response to therapy. Elevation of the tumour markers during the treatment precedes clinical progression and gives an early warning of treatment failure. CONCLUSION: Melanoma circulating cells hold potential as predictive and pharmacodynamic biomarker of immunotherapy.

A Threshold of Systemic MAGE-A Gene Expression Predicting Survival in Resected Non-Small Cell Lung Cancer.

Purpose: Quantitative measurement of minimal residual disease predicting recurrence in individual cancer patients is available only in very few indications, such as acute lymphoblastic leukemia, but is still missing in most solid tumors, including non-small cell lung cancer (NSCLC).Experimental Design: MAGE-A expression levels in blood and bone marrow determined as calibrator-normalized relative ratios by quantitative multimarker real-time RT-PCR for transcript amplification of MAGE-A1, -A2, -A3/6, -A4, -A10, and -A12 in 94 patients with completely resected NSCLC were correlated with survival in a clinical study.Results: Patients with MAGE-A expression levels ≥0.2 in at least one sample of bone marrow or blood at tumor surgery had a significantly reduced overall (P = 0.007), cancer-free (P = 0.002), and distant metastasis-free survival (P < 0.001) versus patients below 0.2 in all samples without significant difference in locoregional recurrence-free survival. The corresponding HRs (≥0.2 vs. <0.2) for death, cancer-related death, and development of distant metastasis were 2.56 [95% confidence interval (CI), 1.42-4.63], 3.32 (95% CI, 1.66-6.61), and 4.03 (95% CI, 1.77-9.18), respectively. Five-year Kaplan-Meier estimates of distant metastasis-free survival were 43% (MAGE-A ≥ 0.2) versus 87% (MAGE-A < 0.2).Conclusions: MAGE-A expression in blood or bone marrow at tumor surgery is an independent predictor of survival in resected NSCLC. The reliable prediction of distant metastasis in individual patients with a statistically proven impact on overall survival may help to refine patient selection for adjuvant therapy urgently needed, especially in the clinical management of elderly patients. Clin Cancer Res; 23(5); 1213-9. ©2016 AACR.

Melanoma-associated antigen genes: a new trend to predict the prognosis of breast cancer patients.

MAGE-A are normally expressed in testis and placenta. Among MAGEs, the MAGE-A subtype has been the most characterized in cancers. Our study was conducted to assess the expression of (MAGE-A1-MAGE-A6) m-RNA using MMRPs and MAGE-A12 m-RNA in blood for evaluating their clinical implications in breast cancer patients. RT-PCR was carried out to detect the expression of (MAGE-A1-MAGE-A6) m-RNA using MMRPs and MAGE-A12 m-RNA in blood. The study included 100 breast cancer cases aged 41-62 years and 100 controls aged 36-53 years. MAGE m-RNA expression was not detected in healthy donors. In breast cancer patients, the positivity of (MAGE-A1-MAGE-A6) m-RNA was 44 % (44 cases), while MAGE-A12 m-RNA was expressed in 13 % (13 cases). The gene expressions of MAGE-A1-A6 and MAGE-A12 were significantly associated with advanced TNM stages (P = 0.001 and 0.034, respectively). Simultaneous estimation of the gene expressions of MAGE-A1-A6 and MAGE-A12 can detect occult hematogenous dissemination of tumor cells and may help to monitor the effectiveness of the therapy and the development of effective immunotherapeutic strategies in breast cancer.

Circulating cell-free cancer-testis MAGE-A RNA, BORIS RNA, let-7b and miR-202 in the blood of patients with breast cancer and benign breast diseases.

BACKGROUND: MAGE-A (melanoma-associated antigen-A) are promising targets for specific immunotherapy and their expression may be induced by the epigenetic factor BORIS. METHODS: To determine their relevance for breast cancer, we quantified the levels of MAGE-A1, -A2, -A3, -A12 and BORIS mRNA, as well as microRNAs let-7b and miR-202 in pre- and postoperative serum of 102 and 34 breast cancer patients, respectively, and in serum of 26 patients with benign breast diseases and 37 healthy women by real-time PCR. The mean follow-up time of the cancer patients was 6.2 years. RESULTS: The serum levels of MAGE-A and BORIS mRNA, as well as let-7b were significantly higher in patients with invasive carcinomas than in patients with benign breast diseases or healthy women (P<0.001), whereas the levels of miR-202 were elevated in both patient cohorts (P<0.001). In uni- and multivariate analyses, high levels of miR-202 significantly correlated with poor overall survival (P=0.0001). Transfection of breast cancer cells with synthetic microRNAs and their inhibitors showed that let-7b and miR-202 did not affect the protein expression of MAGE-A1. CONCLUSIONS: Based on their cancer-specific increase in breast cancer patients, circulating MAGE-A and BORIS mRNAs may be further explored for early detection of breast cancer and monitoring of MAGE-directed immunotherapies. Moreover, serum miR-202 is associated with prognosis.

Multiple MAGE-A genes as surveillance marker for the detection of circulating tumor cells in patients with ovarian cancer.

Ovarian cancer is a leading cause of death among gynecologic malignancies. In this study, we reported the expression of melanoma-associated antigens A (MAGE-A) genes in peripheral blood from 80 patients with ovarian cancer and 30 healthy donors. MAGE-As expression was associated with the factors indicating poor prognosis. The expressions of MAGE-As and each individual MAGE-A genes were also associated with low overall survival of patients with ovarian cancer. Our results suggested MAGE-A genes may have the potential to be surveillance markers for the detection of circulating tumor cells and represent a poor prognosis for patients with ovarian cancer.

Augmentation of anti-HLA-E antibodies with concomitant HLA-Ia reactivity in IFNγ-treated autologous melanoma cell vaccine recipients.

HLA-E expressed on the surface of melanoma cells and shed into circulation are known to inhibit killing of tumor cells by binding to CD94/NKGA2 receptors on cytotoxic T- and NKT cells. Interferon (IFN)-γ is known to promote HLA-E over-expression on the cell surface and shedding. The shed HLA-E heavy chain may expose cryptic epitopes to elicit antibodies (Abs). The anti-HLA-E Abs may bind to shed HLA-E or to the tumor cell surface to block its interaction with CTL/NKT cells. This is the basis for a melanoma cell vaccine that will generate anti-HLA-E Abs. The objective of this study was to characterize the antibody response and characterize the cross-reactivity of the antibodies produced in melanoma patients immunized with autologous melanoma cells treated with IFNγ. Anti-HLA-E murine mAbs and serum anti-HLA-E Abs in healthy individuals were known to react with HLA-Ia alleles, which is attributed to the presence of peptide sequences shared between HLA-E and HLA-Ia. Therefore, pre- and post-immune (weeks 4 and 24) serum Abs reacting to both HLA-E and HLA-Ia alleles were measured by multiplex Luminex®-based immunoassay. To ascertain whether the reactivity of the serum Abs to HLA-Ia was due to anti-HLA-E Abs, the shared-peptides were used to inhibit anti-HLA-E and HLA-Ia reactivities. The level of anti-HLA-E IgG in sera has increased post-immunization from its pre-immune level. Concomitantly, the HLA-Ia reactivity of the sera was also augmented. The reactivity of both anti-HLA-E Abs and HLA-Ia were inhibited by the shared-peptides. The HLA-Ia reactivity of the anti-HLA-E Abs in patients' sera is similar to the HLA-Ia reactivity of the anti-HLA-E mAbs and anti-HLA-E Abs in normal sera. The results establish the immunogenicity of HLA-E and also ascertain that the HLA-Ia reactivity of the anti-HLA-E Abs is due to shared-peptide epitopes.

Novel myeloma-associated antigens revealed in the context of syngeneic hematopoietic stem cell transplantation.

Targets of curative donor-derived graft-versus-myeloma (GVM) responses after allogeneic hematopoietic stem cell transplantation (HSCT) remain poorly defined, partly because immunity against minor histocompatibility Ags (mHAgs) complicates the elucidation of multiple myeloma (MM)-specific targets. We hypothesized that syngeneic HSCT would facilitate the identification of GVM-associated Ags because donor immune responses in this setting should exclusively target unique tumor Ags in the absence of donor-host genetic disparities. Therefore, in the present study, we investigated the development of tumor immunity in an HLA-A0201(+) MM patient who achieved durable remission after myeloablative syngeneic HSCT. Using high-density protein microarrays to screen post-HSCT plasma, we identified 6 Ags that elicited high-titer (1:5000-1:10 000) Abs that correlated with clinical tumor regression. Two Ags (DAPK2 and PIM1) had enriched expression in primary MM tissues. Both elicited Ab responses in other MM patients after chemotherapy or HSCT (11 and 6 of 32 patients for DAPK2 and PIM1, respectively). The index patient also developed specific CD8(+) T-cell responses to HLA-A2-restricted peptides derived from DAPK2 and PIM1. Peptide-specific T cells recognized HLA-A2(+) MM-derived cell lines and primary MM tumor cells. Coordinated T- and B-cell immunity develops against MM-associated Ags after syngeneic HSCT. DAPK1 and PIM1 are promising target Ags for MM-directed immunotherapy.

Detection of melanoma-derived cancer-testis antigen CT16 in patient sera by a novel immunoassay.

Cancer-testis antigens (CTAs) are expressed mainly in various cancer tissues and in testis or placenta. Because of their restricted expression pattern, the CTAs can be potentially used for vaccine development and diagnostic applications. CTA CT16 has been found to be expressed in lung and renal cancers as well as in melanomas. Detection of CT16 protein directly from patient serum could facilitate monitoring of tumor growth and response to therapy in CT16-positive patients. A highly sensitive time-resolved fluorescence-based immunoassay measuring CTA CT16 in serum was developed. Generally, CTAs have not been measured directly from body fluids. CT16 level was detectable in 14 of 23 (61%) patients with metastatic melanoma, whereas none of the nine healthy volunteers collected by us had measurable CT16 level. For an unknown reason, 1 of 20 commercial control serum samples gave a positive result. The Wilcoxon-Mann-Whitney exact test showed statistically significant difference when patients with metastatic melanoma were compared to our control group (p = 0.006) or to the commercial set (p < 0.001). Four melanoma patients had exceptionally high serum CT16 level. CT16 did not correlate either with S100B, a recognized marker of progressing melanoma, or with unspecific serum marker lactate dehydrogenase. Elevation of CT16 titers preceded or followed the clinical diagnosis of disease progression in four patients with metastatic melanoma. As a conclusion, our results show that CT16 protein can be measured directly from patient serum, and the developed assay has a potential for clinical use.