Purpose: Tumor-infiltrating lymphocytes (TIL), in particular CD8þ T cells and CD20 þ B cells, are strongly associated with survival in ovarian cancer and other carcinomas. Although CD8 þ TIL can mediate direct cytolytic activity against tumors, the role of CD20 þ TIL is poorly understood. Here, we investigate the possible contributions of CD20 þ TIL to humoral and cellular tumor immunity.Experimental Design: Tumor and serum specimens were obtained from patients with high-grade serous ovarian cancer. CD8þ and CD20 þ TIL were analyzed by immunohistochemistry and flow cytometry.Immunoglobulin molecules were evaluated by DNA sequencing. Serum autoantibody responses to the tumor antigens p53 and NY-ESO-1 were measured by ELISA.Results: The vast majority of CD20 þ TIL were antigen experienced, as evidenced by class-switching, somatic hypermutation, and oligoclonality, yet they failed to express the canonical memory marker CD27. CD20 þ TIL showed no correlation with serum autoantibodies to p53 or NY-ESO-1. Instead, they colocalized with activated CD8 þ TIL and expressed markers of antigen presentation, including MHC class I, MHC class II, CD40, CD80, and CD86. The presence of both CD20 þ and CD8 þ TIL correlated with increased patient survival compared with CD8 þ TIL alone. Conclusions:In high-grade serous ovarian tumors, CD20 þ TIL have an antigen-experienced but atypical CD27 À memory B-cell phenotype. They are uncoupled from serum autoantibodies, express markers of antigen-presenting cells, and colocalize with CD8 þ T cells. We propose that the association between CD20 þ TIL and patient survival may reflect a supportive role in cytolytic immune responses.

BackgroundEpithelial ovarian cancer is a significant cause of mortality both in the United States and worldwide, due largely to the high proportion of cases that present at a late stage, when survival is extremely poor. Early detection of epithelial ovarian cancer, and of the serous subtype in particular, is a promising strategy for saving lives. The low prevalence of ovarian cancer makes the development of an adequately sensitive and specific test based on blood markers very challenging. We evaluated the performance of a set of candidate blood markers and combinations of these markers in detecting serous ovarian cancer.Methods and FindingsWe selected 14 candidate blood markers of serous ovarian cancer for which assays were available to measure their levels in serum or plasma, based on our analysis of global gene expression data and on literature searches. We evaluated the performance of these candidate markers individually and in combination by measuring them in overlapping sets of serum (or plasma) samples from women with clinically detectable ovarian cancer and women without ovarian cancer. Based on sensitivity at high specificity, we determined that 4 of the 14 candidate markers-MUC16, WFDC2, MSLN and MMP7-warrant further evaluation in precious serum specimens collected months to years prior to clinical diagnosis to assess their utility in early detection. We also reported differences in the performance of these candidate blood markers across histological types of epithelial ovarian cancer.ConclusionsBy systematically analyzing the performance of candidate blood markers of ovarian cancer in distinguishing women with clinically apparent ovarian cancer from women without ovarian cancer, we identified a set of serum markers with adequate performance to warrant testing for their ability to identify ovarian cancer months to years prior to clinical diagnosis. We argued for the importance of sensitivity at high specificity and of magnitude of difference in marker levels between cases and controls as performance metrics and demonstrated the importance of stratifying analyses by histological type of ovarian cancer. Also, we discussed the limitations of studies (like this one) that use samples obtained from symptomatic women to assess potential utility in detection of disease months to years prior to clinical detection.

BackgroundPancreatic intraepithelial neoplasias (PanINs) are precursors of malignant pancreatic cancer, an ideal stage for early cancer detection. We applied quantitative proteomics to identify aberrantly elevated proteins in pancreatic juice samples derived from patients with PanIN3.ResultsTwenty proteins were found elevated in all three PanIN juices by at least two-fold. Among these proteins, anterior gradient-2 (AGR2) was found to be 2-10 fold elevated in PanIN3 juice samples analyzed by quantitative proteomics. An ELISA assay was developed to evaluate AGR2 levels in 51 pancreatic juice samples and 23 serum samples from patients with pancreatic cancer, pre-malignant lesions (including PanIN3, PanIN2, Intraductal Papillary Mucinous Neoplasms (IPMNs)) and benign disease controls (including chronic pancreatitis). AGR2 levels in the pancreatic juice samples were found significantly elevated in patients with pre-malignant conditions (PanINs and IPMNs) as well as pancreatic cancer compared to control samples (p ≤ 0.03). By ROC analysis, the AGR2 ELISA achieved 67% sensitivity at 90% specificity in predicting PanIN3 juice samples from the benign disease controls.ConclusionsThese results suggest that elevation of AGR2 levels in pancreatic juice occurs in early pancreatic cancer progression and could be further investigated as a potential candidate juice biomarker for early detection of pancreatic cancer.

<div>Abstract<p><b>Purpose:</b> Tumor-infiltrating lymphocytes (TIL), in particular CD8⁺ T cells and CD20⁺ B cells, are strongly associated with survival in ovarian cancer and other carcinomas. Although CD8⁺ TIL can mediate direct cytolytic activity against tumors, the role of CD20⁺ TIL is poorly understood. Here, we investigate the possible contributions of CD20⁺ TIL to humoral and cellular tumor immunity.</p><p><b>Experimental Design:</b> Tumor and serum specimens were obtained from patients with high-grade serous ovarian cancer. CD8⁺ and CD20⁺ TIL were analyzed by immunohistochemistry and flow cytometry. Immunoglobulin molecules were evaluated by DNA sequencing. Serum autoantibody responses to the tumor antigens p53 and NY-ESO-1 were measured by ELISA.</p><p><b>Results:</b> The vast majority of CD20⁺ TIL were antigen experienced, as evidenced by class-switching, somatic hypermutation, and oligoclonality, yet they failed to express the canonical memory marker CD27. CD20⁺ TIL showed no correlation with serum autoantibodies to p53 or NY-ESO-1. Instead, they colocalized with activated CD8⁺ TIL and expressed markers of antigen presentation, including MHC class I, MHC class II, CD40, CD80, and CD86. The presence of both CD20⁺ and CD8⁺ TIL correlated with increased patient survival compared with CD8⁺ TIL alone.</p><p><b>Conclusions:</b> In high-grade serous ovarian tumors, CD20⁺ TIL have an antigen–experienced but atypical CD27⁻ memory B-cell phenotype. They are uncoupled from serum autoantibodies, express markers of antigen-presenting cells, and colocalize with CD8⁺ T cells. We propose that the association between CD20⁺ TIL and patient survival may reflect a supportive role in cytolytic immune responses. <i>Clin Cancer Res; 18(12); 3281–92. ©2012 AACR</i>.</p></div>

<p>PDF file, 54KB, CA125 levels and NY-ESO-1 autoantibody titres before (Pre) and after (Post) primary surgery and chemotherapy for the 6 HGSC patients shown in Figure 4C. Titres represent mean values from three independent experiments.</p>

<div>Abstract<p><b>Purpose:</b> Tumor-infiltrating lymphocytes (TIL), in particular CD8⁺ T cells and CD20⁺ B cells, are strongly associated with survival in ovarian cancer and other carcinomas. Although CD8⁺ TIL can mediate direct cytolytic activity against tumors, the role of CD20⁺ TIL is poorly understood. Here, we investigate the possible contributions of CD20⁺ TIL to humoral and cellular tumor immunity.</p><p><b>Experimental Design:</b> Tumor and serum specimens were obtained from patients with high-grade serous ovarian cancer. CD8⁺ and CD20⁺ TIL were analyzed by immunohistochemistry and flow cytometry. Immunoglobulin molecules were evaluated by DNA sequencing. Serum autoantibody responses to the tumor antigens p53 and NY-ESO-1 were measured by ELISA.</p><p><b>Results:</b> The vast majority of CD20⁺ TIL were antigen experienced, as evidenced by class-switching, somatic hypermutation, and oligoclonality, yet they failed to express the canonical memory marker CD27. CD20⁺ TIL showed no correlation with serum autoantibodies to p53 or NY-ESO-1. Instead, they colocalized with activated CD8⁺ TIL and expressed markers of antigen presentation, including MHC class I, MHC class II, CD40, CD80, and CD86. The presence of both CD20⁺ and CD8⁺ TIL correlated with increased patient survival compared with CD8⁺ TIL alone.</p><p><b>Conclusions:</b> In high-grade serous ovarian tumors, CD20⁺ TIL have an antigen–experienced but atypical CD27⁻ memory B-cell phenotype. They are uncoupled from serum autoantibodies, express markers of antigen-presenting cells, and colocalize with CD8⁺ T cells. We propose that the association between CD20⁺ TIL and patient survival may reflect a supportive role in cytolytic immune responses. <i>Clin Cancer Res; 18(12); 3281–92. ©2012 AACR</i>.</p></div>

<p>PDF file, 54KB, CA125 levels and NY-ESO-1 autoantibody titres before (Pre) and after (Post) primary surgery and chemotherapy for the 6 HGSC patients shown in Figure 4C. Titres represent mean values from three independent experiments.</p>

<p>PDF file, 56KB, B cells in HGSC tumors exhibit oligoclonal and somatically hypermutated immunoglobulin sequence.</p>