Polymeric structure and host Toll-like receptor 4 dictate immunogenicity of NY-ESO-1 antigen in vivo.

In search of intrinsic factors that contribute to the distinctively strong immunogenicity of a non-mutated cancer/testis antigen, we found that NY-ESO-1 forms polymeric structures through disulfide bonds. NY-ESO-1 binding to immature dendritic cells was dependent on its polymeric structure and involved Toll-like receptor-4 (TLR4) on the surface of immature dendritic cells in mouse and human. Gene gun-delivered plasmid encoding the wild-type NY-ESO-1 readily induced T cell-dependent antibody (Ab) responses in wild-type C57BL/10 mice but not TLR4-knock-out C57BL/10ScNJ mice. Disrupting polymeric structures of NY-ESO-1 by cysteine-to-serine (Cys-to-Ser) substitutions lead to diminished immunogenicity and altered TLR4-dependence in the induced Ab response. To demonstrate its adjuvant effect, NY-ESO-1 was fused with a major mugwort pollen allergen Art v 1 and a tumor-associated antigen, carbonic anhydrase 9. Plasmid DNA vaccines encoding the fusion genes generated robust immune responses against otherwise non-immunogenic targets in mice. Polymeric structure and TLR4 may play important roles in rendering NY-ESO-1 immunogenic and thus serve as a potent molecular adjuvant. NY-ESO-1 thus represents the first example of a cancer/testis antigen that is a also damage-associated molecular pattern.

Carotid intima-media thickness and plaque assessment by trained medical residents: validation and preliminary testing of a training protocol.

BACKGROUND: Training of nonsonographer physicians or staff members is needed to implement carotid intima-media thickness (CIMT) and plaque screening by ultrasound for the assessment of subclinical atherosclerosis. The purpose of this study was to determine the effect of formal training on CIMT assessment and plaque detection by medical residents. METHODS: A medical resident (R1) was trained using an abbreviated American Society of Echocardiography CIMT protocol. CIMT and plaque assessment by R1 were compared against an expert scanner on 60 subjects using a portable US system. A second medical resident (R2) was then trained on the CIMT protocol focusing on plaque visualization after the results of the first phase of the study were analyzed, and the results were compared against an expert on an additional 10 subjects. RESULTS: In the first phase of the study, a total of 106 images (94% interpretable) were available for CIMT and plaque assessment by both R1 and the expert. CIMT measurements were bioequivalent within the limits of ultrasound resolution, with 88% agreement. Variability on plaque presence was high, with only 53% agreement. R2 and the expert each scanned 10 new subjects twice, from whom 40 images were available for interpretation. R2 demonstrated CIMT agreement (93%) comparable with that observed in phase 1 but with greatly improved plaque agreement (100%). Intraobserver variability during phase 2 for both R2 and the expert was extremely low. CONCLUSIONS: Medical residents can undergo rapid training for CIMT measurement and plaque visualization to detect subclinical atherosclerosis compared with an expert.

Dendritic cell surface calreticulin is a receptor for NY-ESO-1: direct interactions between tumor-associated antigen and the innate immune system.

How the immune system recognizes endogenously arising tumors and elicits adaptive immune responses against nonmutated tumor-associated Ags is poorly understood. In search of intrinsic factors contributing to the immunogenicity of the tumor-associated Ag NY-ESO-1, we found that the NY-ESO-1 protein binds to the surface of immature dendritic cells (DC), macrophages, and monocytes, but not to that of B cells or T cells. Using immunoprecipitation coupled with tandem mass spectrometry, we isolated DC surface calreticulin as the receptor for NY-ESO-1. Calreticulin Abs blocked NY-ESO-1 binding on immature DC and its cross-presentation to CD8+ T cells in vitro. Calreticulin/NY-ESO-1 interactions provide a direct link between NY-ESO-1, the innate immune system, and, potentially, the adaptive immune response against NY-ESO-1.

Dominant B cell epitope from NY-ESO-1 recognized by sera from a wide spectrum of cancer patients: implications as a potential biomarker.

Monitoring the spontaneous antibody (Ab) response against a panel of relevant tumor-associated antigens (TAA) in cancer patients may provide useful information regarding the clinical status of cancer. However, current Ab detection approaches require the purification of recombinant proteins, which is often difficult to achieve. In order to bypass the purification of recombinant proteins, we identified a dominant B-cell epitope from a shared tumor antigen NY-ESO-1. A synthetic peptide of the epitope, ESO:1-40, was as sensitive as the recombinant protein for detecting Ab against NY-ESO-1 in most patients. NY-ESO-1 specific Ab present in the sera of patients with melanoma, prostate cancer, nonsmall cell lung cancer, esophageal cancer, gastric cancer and hepatocellular carcinoma reacted with the dominant peptide at a similar frequency as the recombinant protein. To our knowledge, ESO:1-40 is the first peptide epitope recognized by sera from a wide spectrum of cancer patients but not healthy donors. This simple and straightforward approach may allow the investigation of the clinical significance of spontaneous Ab responses against multiple TAA and their correlation with the clinical course of malignant diseases in the future.

Clinicopathologic and molecular correlations of necrosis in the primary tumor of patients with renal cell carcinoma.

BACKGROUND: The presence of histologic necrosis in the primary tumor of patients with renal cell carcinoma (RCC) has been suggested to be an important predictor of survival. The authors investigated the relation of tumor necrosis to other clinicopathologic factors known to be important prognostic indicators for patients with RCC. METHODS: The records of 311 patients undergoing treatment for RCC were evaluated for basic clinicopathologic information including TNM classification, nuclear grade, Eastern Cooperative Oncology Group (ECOG) performance status (PS), disease recurrence, and survival. The presence and extent of histologic necrosis of the primary tumors was recorded and correlated with clinicopathologic factors, carbonic anhydrase IX and Ki-67 expression, disease recurrence, and survival. RESULTS: The presence of necrosis in the primary tumor of patients with RCC compared with patients with RCC without necrosis was associated with higher T classification (P < 0.0001), the presence of lymph node disease (P = 0.009), the presence of metastases (P < 0.0001), higher grade (P < 0.0001), greater mean tumor size (P < 0.0001), an ECOG PS score > or = 1 (P = 0.007), higher University of California-Los Angeles Integrated Staging System (UISS) category (P < 0.0001), and higher Ki-67 expression (P < 0.0001). The extent of necrosis in the primary tumor was associated with the presence of lymph node disease (P = 0.009) and the presence of metastases (P < 0.0001), and correlated with higher T classification (sigma = 0.31, P < 0.0001), poorer ECOG PS (sigma = 0.18, P = 0.002), higher grade (sigma = 0.33, P < 0.0001), greater tumor size (sigma = 0.40, P < 0.0001), higher UISS category (sigma = 0.37, P < 0.0001), and higher Ki-67 staining (sigma = 0.32, P < 0.0001). Patients with the presence of necrosis in the primary tumor demonstrated a lower 5-year disease-specific survival compared with patients without necrosis in the primary tumor (36% vs. 75%; P < 0.0001). Multivariate analysis demonstrated that T classification (P < 0.0001), distant metastases (P < 0.0001), and ECOG PS (P < 0.0001) were independent predictors of DSS, whereas the presence of necrosis was not (P = 0.1100). Substratification into localized and metastatic disease demonstrated that the presence of necrosis was an independent predictor of survival in patients with localized (P = 0.025), but not metastatic (P = 0.44), disease. The extent of necrosis was not an independent predictor of survival (P > 0.05). Patients with the presence of necrosis in the primary tumor had a lower 5-year disease recurrence-free rate compared with patients without the presence of necrosis (62% vs. 92%, P < 0.0001). CONCLUSIONS: The presence of necrosis in the primary tumor was associated with adverse prognostic factors such as high T classification, presence of lymph node disease and metastases, high grade, large tumor size, and poor ECOG PS. The extent of necrosis was found to be associated with the presence of lymph node disease and metastases and correlated with higher T classification, higher grade, greater tumor size, poorer ECOG PS, and higher UISS category. The presence of this histologic variant was an independent predictor of poor survival in patients with localized, but not metastatic, disease. In addition, Ki-67 expression served as a valuable surrogate marker for the presence of histologic tumor necrosis.