Abstract Background: Checkpoint blockade therapies like PD-1 antibodies elicit durable long-lasting immunity in a subset of patients of certain tumor types. However, many patients across a range of tumor types are resistant to checkpoint blockade. Mechanisms that confer local response or resistance to checkpoint blockade are not well understood. Mounting evidence points to local suppression of T cell function as one of the most substantial barriers to effective antitumor immunity. Methods: The study involved multiparametric flow cytometry and genomic characterization of a subset of T cells in the tumors and lymphoid organs of syngeneic tumors, genetically engineered mouse model of cancers and human tumors. Results: A significant fraction (10-40%) of α/β TCR+ CD3+ T cells do not express CD4, CD8 or NKp46 in the tumors. The presence of double-negative T-cell subset (DN T cells) is restricted to tumor microenvironment and is relatively scarce in tumor draining lymph nodes and in spleen. To identify if DN T cells are antigen-specific, we performed AH-1 tetramer staining. While a substantial fraction of CD8 T cells are AH-1 tetramer positive, majority of DN T cells are AH-1 tetramer negative in CT26 tumors. Recently, it has been shown that higher frequency of CD39-negative bystander T cells correlated to lack of response to PD-1 therapy in non-small cell lung cancer patients. Mounting evidence also reveals presence of a specific subset of CD8 T cells with features of stem cell memory in tumors as defined by CXCR5 and TCF1 expression. Efforts are under way to understand if DN T cells exhibit bystander cell and/or memory stem-like phenotype. Majority of the DN T cells are proliferating (Ki67+) and express very low levels of PD-1 (in contrast to CD8 T cells) and Granzyme B. Treatment of mice bearing syngeneic tumors with PD-1 checkpoint blockade therapy does not impact the frequency of DN T-cell subset consistent with lower levels of PD-1 expression. Moreover, a substantial frequency (30-40%) of DN T cells in both syngeneic and KPC pancreatic GEMM tumors express CD103, a tissue resident marker. Furthermore, DN T cells are CD44+ CD127+ KLRG1- and may represent a memory precursor cell population. Upon ex vivo stimulation, these DN T cell subsets proliferate and express modest levels of PD-1. Preliminary analysis of single-cell RNA sequencing data from FACS sorted murine DN T cells reveals lack of CD4, CD8 expression and retention of CD2, CD44, CD69, CCR7 and CXCR5 expression. Flow cytometry analysis of human CD2+ TILs reveals presence of DN T cells in human tumors. Single-cell transcriptome analysis of CD2+ T cells isolated from human colorectal tumors revealed presence of DN T cells. Current efforts are focused on further characterization of this subset in both mouse and human tumors. Conclusions: In this study, we describe a tumor-resident CD4 and CD8 double-negative T-cell subset that may represent a bystander cell population with features of memory precursors and potential implications in modulation of response to checkpoint blockade therapy in cancer. Citation Format: Murali Gururajan, Ravi Kandasamy, Jessica Wong, Kalpit Shah, Shuoguo Wang, Adam Bata, Amy Truong, Sunil Kuppasani, Ching-Ping Ho, Robert Graziano, Michael Quigley, Anwar Murtaza, Jinqi Liu. High-dimensional analysis of tumor-resident CD4 and CD8 double-negative T-cell subset in multiple tumor types [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B61.
Sera from patients with ovarian cancer contain autoantibodies (AAb) to tumor-derived proteins that are potential biomarkers for early detection. To detect AAb, we probed high-density programmable protein microarrays (NAPPA) expressing 5177 candidate tumor antigens with sera from patients with serous ovarian cancer (n = 34 cases/30 controls) and measured bound IgG. Of these, 741 antigens were selected and probed with an independent set of ovarian cancer sera (n = 60 cases/60 controls). Twelve potential autoantigens were identified with sensitivities ranging from 13 to 22% at >93% specificity. These were retested using a Luminex bead array using 60 cases and 60 controls, with sensitivities ranging from 0 to 31.7% at 95% specificity. Three AAb (p53, PTPRA, and PTGFR) had area under the curve (AUC) levels >60% (p < 0.01), with the partial AUC (SPAUC) over 5 times greater than for a nondiscriminating test (p < 0.01). Using a panel of the top three AAb (p53, PTPRA, and PTGFR), if at least two AAb were positive, then the sensitivity was 23.3% at 98.3% specificity. AAb to at least one of these top three antigens were also detected in 7/20 sera (35%) of patients with low CA 125 levels and 0/15 controls. AAb to p53, PTPRA, and PTGFR are potential biomarkers for the early detection of ovarian cancer.
Abstract Purpose: Human papillomavirus (HPV) type 16 is associated with oropharyngeal carcinomas (OPC). Antibodies (Abs) to HPV16 E6 and E7 oncoproteins have been detected in patient sera, however Abs to other early HPV-derived proteins have not been well explored. Experimental Design: Abs to the HPV16 proteome were quantified using a novel multiplexed bead assay, using C-terminal GST fusion proteins captured onto Luminex beads. Sera were obtained from two cohorts of untreated OPC patients with known Abs to HPV16 Virus-Like Particles (VLP) (N=10), HPV18 VLP (N=10), healthy controls (N=20), and from a validation cohort of untreated patients with OPC (N=30), partners of patients with HPV16+ OPC (N=11), and healthy controls (N=30). Results: OPC patients in the known VLP+ cohort had elevated serum Abs to HPV16 E1, E2, E4, E6, and E7, and L1 antibody levels but not E5. The ratios of specific median fluorescent intensity (MFI) to p21-GST compared to controls were: E1 50.7 vs. 2.1; E4 14.6 vs. 1.3; E6 11.3 vs. 2.4; E7 43.1 vs. 2.6; and L1 10.3 vs. 2.6 (each p<0.01). No cross-reactivity was detected between HPV16 E7 and HPV18 E7 Abs. In the validation cohort, HPV16 E1, E2, and E7 antibody levels were most significantly elevated compared to healthy control samples (p<0.0001) and partners of OPC patients (p<0.01). Conclusion: A novel multiplexed bead assay for the detection of HPV16-specific Abs demonstrates that patients with HPV16+ OPC have detectable Abs to multiple early genes, in particular E1, E2 and E7. These Abs are potential biomarkers for HPV-associated OPC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 902. doi:10.1158/1538-7445.AM2011-902
Cancer patients spontaneously generate autoantibodies (AAb) to tumor-derived proteins. To detect AAb, we have probed novel high-density custom protein microarrays (NAPPA) expressing 4988 candidate tumor antigens with sera from patients with early stage breast cancer (IBC), and bound IgG was measured. We used a three-phase serial screening approach. First, a prescreen was performed to eliminate uninformative antigens. Sera from stage I-III IBC (n = 53) and healthy women (n = 53) were screened for AAb to all 4988 protein antigens. Antigens were selected if the 95th percentile of signal of cases and controls were significantly different (p < 0.05) and if the number of cases with signals above the 95th percentile of controls was significant (p < 0.05). These 761 antigens were screened using an independent set of IBC sera (n = 51) and sera from women with benign breast disease (BBD) (n = 39). From these, 119 antigens had a partial area under the ROC curve (p < 0.05), with sensitivities ranging from 9-40% at >91% specificity. Twenty-eight of these antigens were confirmed using an independent serum cohort (n = 51 cases/38 controls, p < 0.05). Using all 28 AAb, a classifier was identified with a sensitivity of 80.8% and a specificity of 61.6% (AUC = 0.756). These are potential biomarkers for the early detection of breast cancer.
There is strong preclinical evidence that cancer, including breast cancer, undergoes immune surveillance. This continual monitoring, by both the innate and the adaptive immune systems, recognizes changes in protein expression, mutation, folding, glycosylation, and degradation. Local immune responses to tumor antigens are amplified in draining lymph nodes, and then enter the systemic circulation. The antibody response to tumor antigens, such as p53 protein, are robust, stable, and easily detected in serum; may exist in greater concentrations than their cognate antigens; and are potential highly specific biomarkers for cancer. However, antibodies have limited sensitivities as single analytes, and differences in protein purification and assay characteristics have limited their clinical application. For example, p53 autoantibodies in the sera are highly specific for cancer patients, but are only detected in the sera of 10-20% of patients with breast cancer. Detection of p53 autoantibodies is dependent on tumor burden, p53 mutation, rapidly decreases with effective therapy, but is relatively independent of breast cancer subtype. Although antibodies to hundreds of other tumor antigens have been identified in the sera of breast cancer patients, very little is known about the specificity and clinical impact of the antibody immune repertoire to breast cancer. Recent advances in proteomic technologies have the potential for rapid identification of immune response signatures for breast cancer diagnosis and monitoring. We have adapted programmable protein microarrays for the specific detection of autoantibodies in breast cancer. Here, we present the first demonstration of the application of programmable protein microarray ELISAs for the rapid identification of breast cancer autoantibodies.
This study examined the value of serum p53 autoantibodies (p53-AAb) as detection and prognostic biomarkers in ovarian cancer.p53-AAb were detected by ELISA in sera obtained preoperatively from women undergoing surgery for a pelvic mass. This group included women subsequently diagnosed with invasive serous ovarian cancer (n = 60), nonserous ovarian cancers (n = 30), and women with benign disease (n = 30). Age-matched controls were selected from the general population (n = 120). Receiver operating characteristic curves were constructed to compare the values of p53-AAb, CA 125, and HE4 as a screening biomarker. Kaplan-Meier curves and Cox proportional hazards modeling were used to assess its prognostic value on survival.p53-AAb were detected in 25 of 60 (41.7%) of serous cases, 4 of 30 (13.3%) nonserous cases, 3 of 30 (10%) benign disease cases, and 10 of 120 (8.3%) controls (combined P = 0.0002). p53-AAb did not significantly improve the detection of cases [area under the curve (AUC), 0.69] or the discrimination of benign versus malignant disease (AUC, 0.64) compared with CA 125 (AUC, 0.99) or HE4 (AUC, 0.98). In multivariate analysis among cases, p53-AAb correlated only with a family history of breast cancer (P = 0.01). Detectable p53 antibodies in pretreatment sera were correlated with improved overall survival (P = 0.04; hazard ratio, 0.57; 95% confidence interval, 0.33-0.97) in serous ovarian cancer.Antibodies to p53 are detected in the sera of 42% of patients with advanced serous ovarian cancer.Although their utility as a preoperative diagnostic biomarker, beyond CA 125 and HE4, is limited, p53-AAb are prognostic for improved overall survival.
