Novel biomarker panel predicts prognosis in human papillomavirus-negative oropharyngeal cancer: an analysis of the TAX 324 trial.

BACKGROUND: New treatment strategies for locally advanced head and neck squamous cell carcinoma combine induction chemotherapy and chemoradiation. Identifying the predictors of outcome in sequentially treated patients is critical for focusing therapeutic research. In this analysis, the authors evaluated human papillomavirus type 16 (HPV-16) status and the expression levels of a defined set of biomarkers to identify predictors of response to this treatment modality. METHODS: In total, 114 patients with oropharyngeal cancer (OPC) who were treated on the TAX 324 trial (cisplatin and fluorouracil with or without docetaxel in patients with locally advanced head and neck squamous cell carcinoma) had pretreatment biopsy specimens that were evaluable for HPV-16 DNA and immunohistochemical expression of the following biomarkers: beta-tubulin II (ßT-II), glutathione S-transferase (GST-π), p53, and B-cell lymphoma 2 (Bcl-2). Patients were categorized into risk groups based on their HPV status and biomarker expression levels. RESULTS: Patients with high-risk OPC were defined by HPV-negative status and either elevated expression of ßT-II or levels of at least 2 of the other 3 adverse markers (elevated GST-π, elevated p53, or low Bcl-2). All other HPV-negative patients were categorized as moderate risk. In total, 55 patients were HPV-positive, and 59 patients were HPV-negative, with 34 were categorized as high risk and 25 categorized as moderate risk. The median survival for HPV-positive patients was not reached. The median survival was 44.2 months for moderate-risk patients (95% confidence interval, 20.9 months to not reached) and 12.1 months for high-risk patients (95% confidence interval, 7.5-19.7 months). The 24-month survival rate was 89% for HPV-positive patients, 67% for moderate-risk patients, and 29% for high-risk patients (P < .0001). CONCLUSIONS: The molecular data set in this study readily differentiated between 2 distinct groups of patients with locally advanced, HPV-negative OPC. This risk-stratification strategy may serve as a guide for treatment selection.

Plasma microRNAs as potential biomarkers for non-small-cell lung cancer.

Non-small-cell lung cancer (NSCLC) is the leading cause of cancer-related death. Developing minimally invasive techniques that can diagnose NSCLC, particularly at an early stage, may improve its outcome. Using microarray platforms, we previously identified 12 microRNAs (miRNAs) the aberrant expressions of which in primary lung tumors are associated with early-stage NSCLC. Here, we extend our previous research by investigating whether the miRNAs could be used as potential plasma biomarkers for NSCLC. We initially validated expressions of the miRNAs in paired lung tumor tissues and plasma specimens from 28 stage I NSCLC patients by real-time quantitative reverse transcription PCR, and then evaluated diagnostic value of the plasma miRNAs in a cohort of 58 NSCLC patients and 29 healthy individuals. The altered miRNA expressions were reproducibly confirmed in the tumor tissues. The miRNAs were stably present and reliably measurable in plasma. Of the 12 miRNAs, five displayed significant concordance of the expression levels in plasma and the corresponding tumor tissues (all r>0.850, all P<0.05). A logistic regression model with the best prediction was defined on the basis of the four genes (miRNA-21, -126, -210, and 486-5p), yielding 86.22% sensitivity and 96.55% specificity in distinguishing NSCLC patients from the healthy controls. Furthermore, the panel of miRNAs produced 73.33% sensitivity and 96.55% specificity in identifying stage I NSCLC patients. In addition, the genes have higher sensitivity (91.67%) in diagnosis of lung adenocarcinomas compared with squamous cell carcinomas (82.35%) (P<0.05). Altered expressions of the miRNAs in plasma would provide potential blood-based biomarkers for NSCLC.

Comparative antigen-induced gene expression profiles unveil novel aspects of susceptibility/resistance to adjuvant arthritis in rats.

Lewis (LEW) and Wistar Kyoto (WKY) rats of the same major histocompatibility complex (MHC) haplotype (RT.1(l)) display differential susceptibility to adjuvant-induced arthritis (AIA). LEW are susceptible while WKY are resistant to AIA. To gain insights into the mechanistic basis of these disparate outcomes, we compared the gene expression profiles of the draining lymph node cells (LNC) of these two rat strains early (day 7) following a potentially arthritogenic challenge. LNC were tested both ex vivo and after restimulation with the disease-related antigen, mycobacterial heat-shock protein 65. Biotin-labeled fragment cRNA was generated from RNA of LNC and then hybridized with an oligonucleotide-based DNA microarray chip. The differentially expressed genes (DEG) were compared by limiting the false discovery rate to <5% and fold change ≥2.0, and their association with quantitative trait loci (QTL) was analyzed. This analysis revealed overall a more active immune response in WKY than LEW rats. Important differences were observed in the association of DEG with QTL in LEW vs. WKY rats. Both the number of upregulated DEG associated with rat arthritis-QTL and their level of expression were relatively higher in LEW when compared to WKY rat; however, the number of downregulated DEG-associated with rat arthritis-QTL as well as AIA-QTL were found to be higher in WKY than in LEW rats. In conclusion, distinct gene expression profiles define arthritis-susceptible versus resistant phenotype of MHC-compatible inbred rats. These results would advance our understanding of the pathogenesis of autoimmune arthritis and might also offer potential novel targets for therapeutic purposes.

Induction of MAGE-A3 and HPV-16 immunity by Trojan vaccines in patients with head and neck carcinoma.

BACKGROUND: We performed a pilot study using Trojan vaccines in patients with advanced squamous cell carcinoma of the head and neck (SCCHN). These vaccines are composed of HLA-I and HLA-II restricted melanoma antigen E (MAGE)-A3 or human papillomavirus (HPV)-16 derived peptides, joined by furin-cleavable linkers, and linked to a "penetrin" peptide sequence derived from HIV-TAT. Thirty-one patients with SCCHN were screened for the trial and 5 were enrolled. METHODS: Enrolled patients were treated with 300 µg of Trojan peptide supplemented with Montanide and granulocyte-macrophage colony-stimulating factor (GM-CSF) at 4-week intervals for up to 4 injections. RESULTS: Following vaccination, peripheral blood mononuclear cells (PBMCs) from 4 of 5 patients recognized both the full Trojan constructs and constituent HLA-II peptides, whereas responses to HLA-I restricted peptides were less pronounced. CONCLUSION: This treatment regimen seems to have acceptable toxicity and elicits measurable systemic immune responses against HLA-II restricted epitopes in a subset of patients with advanced SCCHN.

Fully recombinant IgG2a Fc multimers (stradomers) effectively treat collagen-induced arthritis and prevent idiopathic thrombocytopenic purpura in mice.

INTRODUCTION: Soluble immune aggregates bearing intact Fc fragments are effective treatment for a variety of autoimmune disorders in mice. The better to understand the mechanisms by which Fc-bearing immune complexes suppress autoimmunity, and to develop a platform for clinical translation, we created a series of fully recombinant forms of polyvalent IgG2a Fc, termed stradomers, and tested their efficacy in a therapeutic model of collagen-induced arthritis (CIA) and preventive models of both idiopathic thrombocytopenic purpura (ITP) and graft-versus-host disease (GVHD). METHODS: Stradomers were created by engineering either the human IgG2 hinge sequence (IgG2H) or the isoleucine zipper (ILZ) onto either the carboxy or amino termini of murine IgG2a Fc. Multimerization and binding to the canonical Fc receptors and the C-type lectin SIGN-RI were evaluated by using sodium dodecylsulfate-polymerase chain reaction (SDS-PAGE) and Biacore/Octet assays. The efficacy of stradomers in alleviating CIA and preventing ITP and GVHD was compared with "gold standard" therapies, including prednisolone and intravenous immune globulin (IVIG). RESULTS: Stradomers exist as both homodimeric and highly ordered sequential multimers. Higher-order multimers demonstrate increasingly stable associations with the canonic Fcγ receptors (FcγRs), and SIGN-R1, and are more effective than Fc homodimers in treating CIA. Furthermore, stradomers confer partial protection against platelet loss in a murine model ITP, but do not prevent GVHD. CONCLUSION: These data suggest that fully human stradomers might serve as valuable tools for the treatment of selected autoimmune disorders and as reagents to study the function of Fc:FcR interactions in vivo.

The gene expression profile of preclinical autoimmune arthritis and its modulation by a tolerogenic disease-protective antigenic challenge.

INTRODUCTION: Autoimmune inflammation is a characteristic feature of rheumatoid arthritis (RA) and other autoimmune diseases. In the natural course of human autoimmune diseases, it is rather difficult to pinpoint the precise timing of the initial event that triggers the cascade of pathogenic events that later culminate into clinically overt disease. Therefore, it is a challenge to examine the early preclinical events in these disorders. Animal models are an invaluable resource in this regard. Furthermore, considering the complex nature of the pathogenic immune events in arthritis, microarray analysis offers a versatile tool to define the dynamic patterns of gene expression during the disease course. METHODS: In this study, we defined the profiles of gene expression at different phases of adjuvant arthritis (AA) in Lewis rats and compared them with those of antigen mycobacterial heat shock protein 65 (Bhsp65)-tolerized syngeneic rats. Purified total RNA (100 ng) extracted from the draining lymph node cells was used to generate biotin-labeled fragment cRNA, which was then hybridized with an oligonucleotide-based DNA microarray chip. Significance analysis of microarrays was used to compare gene expression levels between the two different groups by limiting the false discovery rate to < 5%. Some of the data were further analyzed using a fold change ≥2.0 as the cutoff. The gene expression of select genes was validated by quantitative real-time PCR. RESULTS: Intriguingly, the most dramatic changes in gene expression in the draining lymphoid tissue ex vivo were observed at the preclinical (incubation) phase of the disease. The affected genes represented many of the known proteins that participate in the cellular immune response. Interestingly, the preclinical gene expression profile was significantly altered by a disease-modulating, antigen-based tolerogenic regimen. The changes mostly included upregulation of several genes, suggesting that immune tolerance suppressed disease by activating disease-regulating pathways. We identified a molecular signature comprising at least 12 arthritis-related genes altered by Bhsp65-induced tolerance. CONCLUSIONS: This is the first report of microarray analysis in the rat AA model. The results of this study not only advance our understanding of the early phase events in autoimmune arthritis but also help in identifying potential targets for the immunomodulation of RA.

Impact of age, gender, and race on circulating γδ T cells.

A major subset of human peripheral blood γδ T cells expresses the Vγ2Vδ2 T cell receptor and responds to malignant or infectious diseases. We noted significant differences in the numbers of Vγ2Vδ2 T cells in blood samples from healthy Caucasian CA or African American (AA) donors. On average, CA donors had 3.71% ± 4.37% Vδ2 cells (as a percentage of total lymphocytes) compared with 1.18% ± 2.14% Vδ2 cells for AA donors (p < 0.0001). Age and race had the greatest impact on Vδ2 cell levels; the effect of age was similar for both racial groups. The Vδ2 cell population was dominated, for both donor groups, by cells expressing the Vγ2-Jγ1.2 Vδ2 T cell receptor, an apparent result of strong positive selection and there was substantial overlap in the public Vγ2 clonotypes from both racial groups. Mechanisms for selection and amplification of Vδ2 cells are nearly identical for both groups, despite the significant difference in baseline levels. These data show that appropriate controls, matched for age and race, may be required for clinical studies of Vγ2Vδ2 T cells in infectious disease or cancer and raise important questions about the mechanisms regulating the levels of circulating Vδ2 cells.