MAGE-A4, NY-ESO-1 and SAGE expression rates and co-expression relationships in solid tumours

Background Cancer testis (CT) antigens are promising targets for cancer immunotherapies such as cancer vaccines and genetically modified adoptive T cell therapy. In this study, we evaluated the expression of three CT antigens, melanoma-associated antigen A4 (MAGE-A4), New York oesophageal squamous cell carcinoma 1 (NY-ESO-1) and sarcoma antigen gene (SAGE). Methods MAGE-A4, NY-ESO-1 and/or SAGE antigen expression in tumour samples was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Informed consent was obtained from individuals prior to study enrolment. Results In total, 585 samples in 21 tumour types were evaluated between June 2009 and March 2018. The positive expression rates of these CT antigens were as follows: MAGE-A4, 34.6% (range, 30.7-38.7); NY-ESO-1, 21.0% (range, 17.2-25.1); and SAGE, 21.8% (range, 18.5-25.4). The MAGE-A4 antigen was expressed in 54.9% of oesophageal cancers, 37.5% of head and neck cancers, 35.0% of gastric cancers and 34.2% of ovarian cancers; the NY-ESO-1 antigen was expressed in 28.6% of lung cancers, 25.3% of oesophageal cancers and 22.6% of ovarian cancers; and the SAGE antigen was expressed in 35.3% of prostate cancers, 32.9% of oesophageal cancers and 26.3% of ovarian cancers. The most common tumour type was oesophageal cancer in this study. MAGE-A4, NY-ESO-1 and SAGE antigen expression were assessed in 214 oesophageal cancer samples, among which 24 (11.2%) were triple-positive, 58 (27.1%) were positive for any two, 59 (27.6%) were positive for any one, and 73 (34.1%) were triple-negative. Conclusions Oesophageal cancer exhibited a relatively high rate of CT antigen positivity. Our data indicate that oesophageal cancer is a promising candidate tumour for CT antigen-targeting immunotherapy and immune monitoring.

Chlamydia trachomatis and mycoplasma infections in tubal pregnancy

Chlamydia trachomatis (CT) infection is an important factor for tubal pregnancy. However, whether Ureaplasma urealyticum (UU) and Mycoplasma hominis (MH) infections are also involved in tubal pregnancy remains unknown. This study is aimed to detect CT, UU, and MH in cervical secretions from patients with tubal pregnancy and control women in early pregnancy, to explore their prevalence rates and drug susceptibilities. Analysis was performed on patients with tubal pregnancy and those requiring termination of early pregnancy at <12 weeks from July 2013 to March 2014. Cervical secretions were tested for UU/MH with a UU/MH isolation and culture kit and for CT antigen by an immunochromatographic assay. Mycoplasma samples were tested for resistance to 12 antibiotics. There were no cases of CT infection detected. Mycoplasma infection rates (single or mixed) were similar in the tubal pregnancy and control groups, but the total rate of infection was higher for tubal pregnancy. All MH samples were sensitive to tetracyclines as well as josamycin and azithromycin. Josamycin and clarithromycin were effective against all UU cultures. Over 50% of the samples tested were resistant to ciprofloxacin.

IMMU-50. SYSTEMIC ADOPTIVE TRANSFER IMMUNOTHERAPY WITH TCR-TRANSDUCED T-CELLS TARGETING NY-ESO-1 FOR MENINGIOMA

INTRODUCTION The lack of immunotherapeutic antigen targets selectively expressed on meningiomas have historically hindered its immunotherapy development. However, recent literature showed that in meningiomas, NY-ESO-1 is the most frequently expressed Cancer-Testis(CT)-antigen, which is a group of proteins silent in somatic cells but reactivated in cancers. NY-ESO-1 also has higher expression in higher-grade meningiomas. Decitabine is known to upregulate CT-antigens and augment immunotherapy. To evaluate systemic adoptive transfer for meningiomas, we tested the efficacy of NY-ESO-1 T-cell-receptor(TCR)-transduced T-cells in vitro and in vivo, and the role of decitabine in meningioma immunotherapy. METHODS NY-ESO-1 TCR-Transduced T-cells were generated by double-transfection with supernatants from PG-13 retroviral packaging cell line encoding HLA-A*0201–restricted NY-ESO-1(157 – 165)-specific TCR. In vitro cytolysis was measured using the xCelligence Real-Time Cell Analyzer System. We utilized human meningioma cells SF1335(Grade I, HLA-A2.1 positive) and CH157(Grade III, HLA-A2.1 negative) in vitro and in vivo. RESULTS CH157 and SF1335 have high and low NY-ESO-1 expression, respectively. Decitabine upregulated NY-ESO-1 mRNA expression in SF1335 by 6-fold after 2 days and by 100-fold after 9 days, with similar effect on protein expression. Co-culturing CH157, CH157-HLA-A2.1(CH157 transduced with HLA-A2.1 vector), SF1335, and decitabine-pretreated-SF1335 cells individually with NY-ESO-1 TCR transduced T-cells at a ratio of 1:1 resulted in 0%, 65%, 20% and 40% cytolysis at 10hours, respectively. Systemic(intravenous) adoptive transfer of TCR-transduced T-cells significantly increased overall survival in NSG mice with intracranial xenografts of CH157-HLA-A2.1 (p=0.04) and SF1335(not treated with decitabine) (p=0.06). CONCLUSIONS NY-ESO-1 TCR-transduced T-cells induce NY-ESO-1 and HLA-A2.1-specific cytolysis in meningioma in vitro, and systemic adoptive transfer confers a statistically significant survival benefit in vivo for meningiomas with high NY-ESO-1 expression. Decitabine upregulates NY-ESO-1 expression and increases tumor cytolysis by TCR-transduced T-cells in meningiomas. Targeting NY-ESO-1 may be a clinically feasible immunotherapeutic strategy to treat aggressive meningiomas.

EWSR1-FLI1 Activation of the Cancer/Testis Antigen FATE1 Promotes Ewing Sarcoma Survival

ABSTRACT Ewing sarcoma is characterized by a pathognomonic chromosomal translocation that generates the EWSR1-FLI1 chimeric transcription factor. The transcriptional targets of EWSR1-FLI1 that are essential for tumorigenicity are incompletely defined. Here, we found that EWSR1-FLI1 modulates the expression of cancer/testis (CT) antigen genes, whose expression is biased to the testes but is also activated in cancer. Among these CT antigens, fetal and adult testis expressed 1 (FATE1) is most robustly induced. EWSR1-FLI1 associates with the GGAA repeats in the proximal promoter of FATE1, which exhibits accessible chromatin exclusively in mesenchymal progenitor cells (MPCs) and Ewing sarcoma cells. Expression of EWSR1-FLI1 in non-Ewing sarcoma cells and in MPCs enhances FATE1 mRNA and protein expression. Conversely, depletion of EWSR1-FLI1 in Ewing sarcoma cells leads to a loss of FATE1 expression. Importantly, we found that FATE1 is required for survival and anchorage-independent growth in Ewing sarcoma cells via attenuating the accumulation of BNIP3L, a BH3-only protein that is toxic when stabilized. This action appears to be mediated by the E3 ligase RNF183. We propose that engaging FATE1 function can permit the bypass of cell death mechanisms that would otherwise inhibit tumor progression.

Comparison of three serological assays to measure antibody response to Chlamydia antigen Pgp3 in adolescent and young adults with pelvic inflammatory disease

Antibody-based epidemiologic surveillance to determine population-level exposure to sexually transmitted infections could help inform public health fertility preservation strategies. We compared the performance of three platforms to detect antibodies against the Chlamydia trachomatis (CT) antigen Pgp3 – multiplex bead array (MBA), enzyme-linked immunosorbent assay (ELISA), and lateral flow assay (LFA) – on sera from adolescents and young adults (AYAs) with pelvic inflammatory disease (PID). Ninety-five of 118 AYAs diagnosed with PID (80.5%) had positive antibody response to Pgp3 antigen by at least one test, and 78 (66.1%) tested positive by all three tests. Among 27 individuals with infection detected using nucleic acid amplification testing, 92.6% were positive by MBA (25/27), 77.8% (21/27) were positive by ELISA, and 74.1% (20/27) were positive by LFA. These data suggest that the MBA was the most sensitive of the three tests and could be useful in seroepidemiologic studies designed to assess population-level exposure to CT.

Expression of sheep pathogen Babesia sp. Xinjiang rhoptry-associated protein 1 and evaluation of its diagnostic potential by enzyme-linked immunosorbent assay

SUMMARYOvine babesiosis is one of the most important tick-borne haemoparasitic diseases of small ruminants. The ovine parasite Babesia sp. Xinjiang is widespread in China. In this study, recombinant full-length XJrRAP-1aα2 (rhoptry-associated protein 1aα2) and C-terminal XJrRAP-1aα2 CT of Babesia sp. Xinjiang were expressed and used to evaluate their diagnostic potential for Babesia sp. Xinjiang infections by indirect enzyme-linked immunosorbent assay (ELISA). Purified XJrRAP-1aα2 was tested for reactivity with sera from animals experimentally infected with Babesia sp. Xinjiang and other haemoparasites using Western blotting and ELISA. The results showed no cross-reactivities between XJrRAP-1aα2 CT and sera from animals infected by other pathogens. High level of antibodies against RAP-1a usually lasted 10 weeks post-infection (wpi). A total of 3690 serum samples from small ruminants in 23 provinces located in 59 different regions of China were tested by ELISA. The results indicated that the average positive rate was 30·43%, and the infections were found in all of the investigated provinces. This is the first report on the expression and potential use of a recombinant XJrRAP-1aα2 CT antigen for the development of serological assays for the diagnosis of ovine babesiosis, caused by Babesia sp. Xinjiang.

CT Antigen-Specific Immune Response From DAC/DC Vaccine Correlates With Clinical Outcome In Patients With Relapsed Neuroblastoma

Neuroblastoma is the most common solid tumor in children in the first year of life. Despite high-dose chemotherapy, irradiation and autologous stem cell transplantation, nearly half of these patients relapse, a group for whom there are limited treatment options. The cancer-testis (CT) antigens MAGE-A1, MAGE-A3 and NY-ESO-1 are expressed on neuroblastoma cells in low levels and we have previously shown that the demethylating chemotherapy drug decitabine (DAC) can upregulate the expression of CT antigens in neuroblastoma. We developed a clinical study combining DAC to upregulate CT antigens followed by a dendritic cell (DC) vaccine targeting CT antigens MAGE-A1, MAGE-A3 and NY-ESO-1. Here we report the effects of DAC/DC vaccine in generating antigen-specific immune response and evaluate if there exists a correlation between development of antigen-specific immune responses and clinical responses. The treatment regimen includes 4 cycles of therapy, each consisting of DAC 10mg/m2/day for 5 days, followed by 2 weekly vaccinations consisting of autologous DC pulsed with overlapping peptide mixes derived from full length MAGE-A1, MAGE-A3 and NY-ESO-1. The number of DC administered in the vaccine was based on patient weight, and ranged from 3 to 10 x106 cells. The topical TLR agonist imiquimod was used at the site of vaccination to facilitate immune responses to the vaccine. Peripheral blood was collected weekly to assess antigen-specific immune response. Peripheral blood mononuclear cells were archived at various time points, stimulated for 24 h with MAGE-A1, MAGE-A3 and NY-ESO-1 peptide mixes and studied for the presence of CD137+ antigen-specific cells by flow cytometry. The regimen was well tolerated and highly feasible. We were able to culture DC for 10/10 neuroblastoma patients enrolled on the study. Development of an antibody or a T cell response to the vaccine was defined as either new onset or a two fold increase in the level of antibodies or number of MAGE-A1, MAGE-A3 and NY-ESO-1 specific, CD137+ T cells over baseline levels. The clinical and immunological outcomes of seven neuroblastoma patients treated so far with the DAC/CT antigen vaccine is summarized in table 1. Two patients are in complete remission, one of whom is two years from completing therapy, and another patient is 9 months from therapy. Both these patients demonstrated an increase in the number of circulating CD3+CD8+CD137+ and CD3+CD4+CD137+ T cells against one of the CT antigens in the vaccine. Of the five patients who had disease progression, one had a partial response to his chemotherapy and radiation resistant tumor 2 months post-vaccine. This patient had an antibody response to these antigens post-vaccination but no CD8+ or CD4+ T cell response. Another patient who had no evidence of disease for 8 months following the last vaccine prior to disease recurrence had an antigen-specific CD8+ T cell response against MAGE-A1, MAGE-A3 and NY-ESO-1 antigens but no CD4+ T cell response. These data indicate that DAC/DC vaccine targeting MAGE-A1, MAGE-A3 and NY-ESO-1 are efficient in generating an antigen-specific immune response in four of seven patients studied and there exist a correlation between the presence of immune response and positive clinical outcome. Disclosures: No relevant conflicts of interest to declare.

A Pilot Study of Anti-CTLA4 Antibody Ipilimumab in Patients with Synovial Sarcoma

Background. Patients with recurrent synovial sarcomas have few options for systemic therapy. Since they express large amounts of endogenous CT (cancer testis) antigens such as NY-ESO-1, we investigated the clinical activity of single agent anti-CTLA4 antibody ipilimumab in patients with advanced or metastatic synovial sarcoma.Methods. A Simon two-stage phase II design was used to determine if there was sufficient activity to pursue further. The primary endpoint was tumor response rate by RECIST 1.0. Patients were treated with ipilimumab 3 mg/kg intravenously every 3 weeks for three cycles and then restaged. Retreatment was possible for patients receiving an extra three-week break from therapy. Sera and peripheral blood mononuclear cells were collected before and during therapy to assess NY-ESO-1-specific immunity.Results. Six patients were enrolled and received 1–3 cycles of ipilimumab. All patients showed clinical or radiological evidence of disease progression after no more than three cycles of therapy, for a RECIST response rate of 0%. The study was stopped for slow accrual, lack of activity, and lack of immune response. There was no evidence of clinically significant either serologic or delayed type hypersensitivity responses to NY-ESO-1 before or after therapy.Conclusion. Despite high expression of CT antigens by synovial sarcomas of patients treated in this study, there was neither clinical benefit nor evidence of anti-CT antigen serological responses. Assessment of the ability of synovial sarcoma cell lines to present cancer-germ cell antigens may be useful in determining the reason for the observed lack of immunological or clinical activity.