The Ets transcription factor ELF5 functions as a tumor suppressor in the kidney.

Renal cell carcinoma is an important clinical disease with poorly understood etiology. ELF5 is an epithelial-specific member of the Ets family of transcription factors, characterized by the 80 amino acid Ets domain that binds the purine-rich GGAA/T Ets motif found in the promoter regions of a variety of genes. Since ELF5 is highly expressed in kidney and has been postulated to function as a tumor suppressor, at least in the context of the breast, we investigated its role in kidney cancer. In renal cell carcinoma ELF5 expression was consistently decreased in tumor samples versus normal. ELF5 mRNA was decreased in 94% of lesions tested and ELF5 protein was undetectable in 40/40 kidney-derived carcinomas. Re-expression of the ELF5 gene in 786-O renal carcinoma cells suppressed their tumorigenic capacity in vitro and in vivo. This work is the first to suggest that ELF5 has tumor suppressor activity in the kidney.

Cancer/testis antigens can be immunological targets in clonogenic CD133+ melanoma cells.

"Cancer stem cells" that resist conventional treatments may be a cause of therapeutic failure in melanoma. We report a subpopulation of clonogenic melanoma cells that are characterized by high prominin-1/CD133 expression in melanoma and melanoma cell lines. These cells have enhanced clonogenicity and self-renewal in vitro, and serve as a limited in vitro model for melanoma stem cells. In some cases clonogenic CD133(+) melanoma cells show increased expression of some cancer/testis (CT) antigens. The expression of NY-ESO-1 in an HLA-A2 expressing cell line allowed CD133(+) clonogenic melanoma cells to be targeted for killing in vitro by NY-ESO-1-specific CD8(+) T-lymphocytes. Our in vitro findings raise the hypothesis that if melanoma stem cells express CT antigens in vivo that immune targeting of these antigens may be a viable clinical strategy for the adjuvant treatment of melanoma.

ECSA/DPPA2 is an embryo-cancer antigen that is coexpressed with cancer-testis antigens in non-small cell lung cancer.

PURPOSE: Cancer cells recapitulate many behaviors of pluripotent embryonic cells such as unlimited proliferation, and the capacity to self-renew and to migrate. Embryo-cancer sequence A (ECSA), later named developmental pluripotency associated-2 (DPPA2), is an embryonic gene initially isolated from pluripotent human preimplantation embryos. We hypothesized that ECSA/DPPA2 would be quiescent in most normal tissues but expressed in cancers and may therefore be a useful target for immunotherapy. EXPERIMENTAL DESIGN: ECSA/DPPA2 expression was examined in a panel of normal and tumor tissue by reverse transcription PCR, quantitative real-time PCR, and immunohistochemistry. A panel of 110 non-small cell lung cancers (NSCLC) were further investigated for the presence of ECSA/DPPA2 transcripts and several cancer testis antigens (CTA). Sera from 104 patients were analyzed for spontaneous ECSA/DPPA2 antibody production by ELISA and Western blot. RESULTS: ECSA/DPPA2 transcripts were limited to normal testis, placenta, bone marrow, thymus, and kidney but expressed in a variety of tumors most notably in 30% of NSCLC. Enrichment for CTAs in ECSA/DPPA2-positive NSCLC was observed. Immunohistochemistry confirmed nuclear and cytoplasmic localization in subpopulations of cells with coexpression of the CTA MAGE-A3. Antibodies to recombinant ECSA/DPPA2 protein were detected in the sera of 4 of 104 patients with NSCLC but not in healthy controls. CONCLUSIONS: The restricted expression in normal tissues, expression in tumors with coexpression of CTAs, and spontaneous immunogenicity indicate that ECSA/DPPA2 is a promising target for antigen-specific immunotherapy in NSCLC.

Mycoplasma Infection Alters Cancer Stem Cell Properties in Vitro.

Cancer cell lines can be useful to model cancer stem cells. Infection with Mycoplasma species is an insidious problem in mammalian cell culture. While investigating stem-like properties in early passage melanoma cell lines, we noted poorly reproducible results from an aliquot of a cell line that was later found to be infected with Mycoplasma hyorhinis. Deliberate infection of other early passage melanoma cell lines aliquots induced variable and unpredictable effects on expression of putative cancer stem cell markers, clonogenicity, proliferation and global gene expression. Cell lines established in stem cell media (SCM) were equally susceptible. Mycoplasma status is rarely reported in publications using cultured cells to study the cancer stem cell hypothesis. Our work highlights the importance of surveillance for Mycoplasma infection while using any cultured cells to interrogate tumor heterogeneity.

Immunohistochemical and molecular analysis of human melanomas for expression of the human cancer-testis antigens NY-ESO-1 and LAGE-1.

PURPOSE: NY-ESO-1 and LAGE-1 are homologous cancer-testis antigens, which are expressed in many different cancers. It is essential to type tumors accurately to assess patient suitability for clinical trials which target these. This study evaluates typing strategies used to distinguish these two homologous but distinct antigens and to characterize and quantitate expression of each in clinical samples. EXPERIMENTAL DESIGN: We typed 120 malignant melanomas for the expression of NY-ESO-1 and LAGE-1 with immunohistochemistry, reverse transcription-PCR (RT-PCR), and quantitative real-time (qRT-PCR), which was also used to explore the relationship between NY-ESO-1 and LAGE expression. RESULTS: The two monoclonal antibodies ES121 and E978 had very similar immunohistochemistry reactivities. Both were specific for NY-ESO-1 because neither bound to homologous LAGE-1 peptides despite 84% overall amino acid homology. Of 120 melanomas tested by immunohistochemistry, NY-ESO-1 was expressed in >50% of cells in 23 melanomas (19%), between 11 and 50% cells in 15 (12.5%), <11% cells in 16 (13.5%), and negative in 66 (55%). Although specific for both antigens, the PCR methods did not provide this information about microheterogeneity. Polymorphisms in the LAGE-1 gene resulted in false negative LAGE-1 typing by qRT-PCR by inhibiting binding of oligonucleotide primers, thereby showing the exquisite specificity of qRT-PCR as a typing method. CONCLUSIONS: For NY-ESO-1 typing, immunohistochemistry compared favorably with the RT-PCR, with the added advantage of being able to characterize heterogeneity of antigen expression. Because neither mAb bound LAGE and because there was no coordinate expression LAGE and NY-ESO-1, separate typing for each is required.

Rapid detection of Clostridium difficile toxins from stool samples using real-time multiplex PCR.

In this study, a total of 650 stool samples were tested to show that our method is capable of detecting four Clostridium difficile genes; tcdA, tcdB, encoding toxin A (TcdA) and toxin B (TcdB), and the binary toxin C. difficile transferase genes (cdtA and/or cdtB) encoding CDT toxin. Besides detecting the targeted C. difficile genes, our method can be used to detect the presence of any inhibitory components in the PCR. This assay, combined with a selective culture medium, such as the chromID™ C. difficile, can be applied directly for screening C. difficile-associated disease. The PCR-based assay developed here is rapid (4 h per 21 stool samples) and accurate in diagnosing C. difficile infection, 100 % assay sensitivity and negative predictive value (NPV) were obtained. However, the assay specificity of 99.1 % and positive predictive value (PPV) of 94.9 % were slightly lower than the optimal value of 100 %. The assay protocol outlined here can be used as a rapid screening tool to assist infection control units and in managing infected patients by reducing the number of patients requiring isolation and extended hospitalization. Rapid detection can prevent unnecessary antibiotic therapy and potentially reduce the spread of infection by emerging hypervirulent C. difficile strains.

Human perforin mutations and susceptibility to multiple primary cancers.

Loss-of-function mutations in the gene coding for perforin (PRF1) markedly reduce the ability of cytotoxic T lymphocytes and natural killer cells to kill target cells, causing immunosuppression and impairing immune regulation. In humans, nearly half of the cases of type 2 familial hemophagocytic lymphohistiocytosis are due to bi-allelic PRF1 mutations. The partial inactivation of PRF1 due to mutations that promote protein misfolding or the common hypomorphic allele coding for the A91V substitution have been associated with lymphoid malignancies in childhood and adolescence. To investigate whether PRF1 mutations also predispose adults to cancer, we genotyped 566 individuals diagnosed with melanoma (101), lymphoma (65), colorectal carcinoma (30) or ovarian cancer (370). The frequency of PRF1 genotypes was similar in all disease groups and 424 matched controls, indicating that the PRF1 status is not associated with an increased susceptibility to these malignancies. However, four out of 15 additional individuals diagnosed with melanoma and B-cell lymphoma during their lifetime expressed either PRF1A91V or the rare pathogenic PRF1R28C variant (p = 0.04), and developed melanoma relatively early in life. Both PRF1A91V- and PRF1R28C-expressing lymphocytes exhibited severely impaired but measurable cytotoxic function. Our results suggest that defects in human PRF1 predispose individuals to develop both melanoma and lymphoma. However, these findings require validation in larger patient cohorts.

Influenza antiviral resistance in the Asia-Pacific region during 2011.

Despite greater than 99% of influenza A viruses circulating in the Asia-Pacific region being resistant to the adamantane antiviral drugs in 2011, the large majority of influenza A (>97%) and B strains (∼99%) remained susceptible to the neuraminidase inhibitors oseltamivir and zanamivir. However, compared to the first year of the 2009 pandemic, cases of oseltamivir-resistant A(H1N1)pdm09 viruses with the H275Y neuraminidase mutation increased in 2011, primarily due to an outbreak of oseltamivir-resistant viruses that occurred in Newcastle, as reported in Hurt et al. (2011c, 2012a), where the majority of the resistant viruses were from community patients not being treated with oseltamivir. A small number of influenza B viruses with reduced oseltamivir or zanamivir susceptibility were also detected. The increased detection of neuraminidase inhibitor resistant strains circulating in the community and the detection of novel variants with reduced susceptibility are reminders that monitoring of influenza viruses is important to ensure that antiviral treatment guidelines remain appropriate.

Cancer-testis antigen expression in primary cutaneous melanoma has independent prognostic value comparable to that of Breslow thickness, ulceration and mitotic rate.

To determine the effect of Cancer-Testis Antigen (CTAg) expression on the natural history of primary cutaneous melanoma we compared its impact on prognosis with that of known prognostic factors and its relationship with other clinicopathologic characteristics. The immunohistochemical expression of three CTAgs (MAGE-A1, MAGE-A4 and NY-ESO-1) in 348 cases of stage I and stage II primary cutaneous melanoma was analysed and correlated with clinicopathologic characteristics, relapse free survival (RFS) and overall survival (OS). A Cox proportional hazards regression model was used to analyse factors which independently predicted RFS. All three CTAgs were significantly co-expressed with each other (p < 0.001). The median RFS for patients with CTAg-negative tumours and CTAg-positive tumours was 72 months and 45 months, respectively, (P = 0.008). Univariate analysis demonstrated that the impact of CTAg expression on RFS was comparable in magnitude to that of Breslow thickness, ulceration and tumour mitotic rate. Multivariate Cox regression analysis indicated that CTAg expression was a powerful independent predictor of RFS (risk ratio (RR) = 1.715, 95% confidence interval (CI) = 0.430-0.902, P = 0.010). In contrast, CTAg expression was demonstrated to have no prognostic impact on overall survival. This study demonstrates that CTAg expression in primary cutaneous melanoma is a strong independent predictor of RFS and it is comparable to other known important prognostic factors. CTAg expression has no relationship with overall survival, suggesting anti-melanoma immunity directed towards CTAg expression may contribute to the natural history of the disease. In view of these results, further investigation of the function of CTAgs and their potential use in therapeutic targeting is warranted.

Frequent MAGE mutations in human melanoma.

BACKGROUND: Cancer/testis (CT) genes are expressed only in the germ line and certain tumors and are most frequently located on the X-chromosome (the CT-X genes). Amongst the best studied CT-X genes are those encoding several MAGE protein families. The function of MAGE proteins is not well understood, but several have been shown to potentially influence the tumorigenic phenotype. METHODOLOGY/PRINCIPAL FINDINGS: We undertook a mutational analysis of coding regions of four CT-X MAGE genes, MAGEA1, MAGEA4, MAGEC1, MAGEC2 and the ubiquitously expressed MAGEE1 in human melanoma samples. We first examined cell lines established from tumors and matching blood samples from 27 melanoma patients. We found that melanoma cell lines from 37% of patients contained at least one mutated MAGE gene. The frequency of mutations in the coding regions of individual MAGE genes varied from 3.7% for MAGEA1 and MAGEA4 to 14.8% for MAGEC2. We also examined 111 fresh melanoma samples collected from 86 patients. In this case, samples from 32% of the patients exhibited mutations in one or more MAGE genes with the frequency of mutations in individual MAGE genes ranging from 6% in MAGEA1 to 16% in MAGEC1. SIGNIFICANCE: These results demonstrate for the first time that the MAGE gene family is frequently mutated in melanoma.

Activin-A: a novel dendritic cell-derived cytokine that potently attenuates CD40 ligand-specific cytokine and chemokine production.

Activin-A is a transforming growth factor-beta (TGF-beta) superfamily member that plays a pivotal role in many developmental and reproductive processes. It is also involved in neuroprotection, apoptosis of tumor and some immune cells, wound healing, and cancer. Its role as an immune-regulating protein has not previously been described. Here we demonstrate for the first time that activin-A has potent autocrine effects on the capacity of human dendritic cells (DCs) to stimulate immune responses. Human monocyte-derived DCs (MoDCs) and the CD1c(+) and CD123(+) peripheral blood DC populations express both activin-A and the type I and II activin receptors. Furthermore, MoDCs and CD1c(+) myeloid DCs rapidly secrete high levels of activin-A after exposure to bacteria, specific toll-like receptor (TLR) ligands, or CD40 ligand (CD40L). Blocking autocrine activin-A signaling in DCs using its antagonist, follistatin, enhanced DC cytokine (IL-6, IL-10, IL-12p70, and tumor necrosis factor-alpha [TNF-alpha]) and chemokine (IL-8, IP-10, RANTES, and MCP-1) production during CD40L stimulation, but not TLR-4 ligation. Moreover, antagonizing DC-derived activin-A resulted in significantly enhanced expansion of viral antigen-specific effector CD8(+) T cells. These findings establish an immune-regulatory role for activin-A in DCs, highlighting the potential of antagonizing activin-A signaling in vivo to enhance vaccine immunogenicity.

The regulatory T cell-associated transcription factor FoxP3 is expressed by tumor cells.

FoxP3 is a member of the forkhead family of transcription factors critically involved in the development and function of CD25(+) regulatory T cells (Treg). Until recently, FoxP3 expression was thought to be restricted to the T-cell lineage. However, using immunohistochemistry and flow cytometric analysis of human melanoma tissue, we detected FoxP3 expression not only in the tumor infiltrating Treg but also in the melanoma cells themselves. FoxP3 is also widely expressed by established human melanoma cell lines (as determined by flow cytometry, PCR, and Western blot), as well as cell lines derived from other solid tumors. Normal B cells do not express FoxP3; however, expression could be induced after transformation with EBV in vitro and in vivo, suggesting that malignant transformation of healthy cells can induce FoxP3. In addition, a FOXP3 mRNA variant lacking exons 3 and 4 was identified in tumor cell lines but was absent from Treg. Interestingly, this alternative splicing event introduces a translation frame-shift that is predicted to encode a novel protein. Together, our results show that FoxP3, a key regulator of immune suppression, is not only expressed by Treg but also by melanoma cells, EBV-transformed B cells, and a wide variety of tumor cell lines.

Striking immunodominance hierarchy of naturally occurring CD8+ and CD4+ T cell responses to tumor antigen NY-ESO-1.

Immunodominance has been well-demonstrated in many antiviral and antibacterial systems, but much less so in the setting of immune responses against cancer. Tumor Ag-specific CD8+ T cells keep cancer cells in check via immunosurveillance and shape tumor development through immunoediting. Because most tumor Ags are self Ags, the breadth and depth of antitumor immune responses have not been well-appreciated. To design and develop antitumor vaccines, it is important to understand the immunodominance hierarchy and its underlying mechanisms, and to identify the most immunodominant tumor Ag-specific T cells. We have comprehensively analyzed spontaneous cellular immune responses of one individual and show that multiple tumor Ags are targeted by the patient's immune system, especially the "cancer-testis" tumor Ag NY-ESO-1. The pattern of anti-NY-ESO-1 T cell responses in this patient closely resembles the classical broad yet hierarchical antiviral immunity and was confirmed in a second subject.

Blood dendritic cells generated with Flt3 ligand and CD40 ligand prime CD8+ T cells efficiently in cancer patients.

Flt3 ligand mobilizes dendritic cells (DCs) into blood, allowing generation in vivo of large numbers of DCs for immunotherapy. These immature DCs can be rapidly activated by soluble CD40 ligand (CD40L). We developed a novel overnight method using these cytokines to produce DCs for cancer immunotherapy. Flt3 ligand-mobilized DCs (FLDCs) were isolated, activated with CD40L, loaded with antigenic peptides from influenza matrix protein, hepatitis B core antigen, NY-ESO-1, MAGE-A4, and MAGE-A10, and injected into patients with resected melanoma. Three injections were given at 4-week intervals. Study end points included antigen-specific immune responses (skin reactions to peptides alone or peptide-pulsed FLDCs; circulating T-cell responses), safety, and toxicity. No patient had a measurable tumor. Six patients were entered. FLDCs were obtained, enriched, and cultured under Good Manufacturing Practice grade conditions. Overnight culture with soluble CD40L caused marked up-regulation of activation markers (CD83 and HLA-DR). These FLDCs were functional and able to stimulate antigen-specific T cells in vitro. No significant adverse events were attributable to FLDCs. Peptide-pulsed FLDCs caused strong local skin reactions up to 60 mm diameter with intense perivascular infiltration of T cells, exceeding those seen in our previous peptide-based protocols. Antigen-specific blood T-cell responses were induced, including responses to an antigen for which the patients were naive (hepatitis B core antigen) and MAGE-A10. MAGE-A10-specific T cells with a skewed T-cell receptor repertoire were detected in 1 patient in blood ex vivo and from tumor biopsies. Vaccination with FLDCs pulsed with peptides is safe and primes immune responses to cancer antigens.