Description

SPECIFIC AIMS: Approximately 150 million Americans have been infected with human papillomavirus (HPV), and another 6.2 million people become newly infected each year. In cervical cancer, progression from infection of high-risk (HR) HPV subtypes to carcinoma is infrequent and can occur over decades, suggesting that HPV infection is necessary but not sufficient for carcinogenesis. HPV16 has recently been associated with the majority of oropharyngeal cancers (OPC)7, 8, which has profound implications both for the risk of oral transmission, as well as HPV vaccination strategies. The natural history of oral HPV infection is unknown as there are no standard assays for monitoring either pathologic changes (i.e. Pap smear), viral load or immunity and no known precancerous lesions. A biomarker panel for early detection of at-risk populations and for predicting recurrence would have a clear impact on the management of OPC. HPV infection induces both systemic and local humoral immune responses with IgG and IgA antibodies (Abs) to HPV-derived proteins. Serum Abs to L1 capsid protein are induced in 50-70% of infected patients months after HPV infection and are detectable for years after clearance of the infection; these antibodies represent lifetime HPV exposure. Serum Abs to HPV16 E6 and E7 proteins are detected in a subset (50-70%) of patients with invasive HPV+ cervical cancer and OPC, and are associated with improved prognosis independent of HPV status. Immunity to HPV16E6 can be detected up to 10 years prior to clinical diagnosis of OPC 5. Considering the rising incidence of HPV-related OPC, we hypothesize measuring Ab immunity to the entire HPV16 proteome will yield biomarkers for screening at-risk populations. We have developed novel protein arrays for high-throughput serologic screening of antibodies to the HPV16 proteome. This approach uses mammalian in vitro expression of cDNAs encoding protein antigens, capture of antigens using anti-tag Abs, and then binding and detection of Abs from patient sera. Because this approach is based on cDNA and expression of structural epitopes, it allows for greater flexibility and economy in antigen selection and epitope mapping. We have identified antibodies to E1, E2, E4, E6, and E7 antigens in patients with OPC, but not partners or healthy controls. We have also detected antibodies primarily to E6 and E7 in patients with invasive cervical cancer but not CIN II/III, obtained from the CDC, demonstrating these are biomarkers of cancer, not HPV infection. The heterogeneity of immune responses in these patient populations suggests that there are fundamental biologic differences in host/viral biology within patients with OPC, and supports the use of a multiplexed assay for increased sensitivity of detection of patients with OPC. Our hypothesis is that antibodies to HPV-derived proteins are biomarkers of for the early detection of oropharyngeal cancer. This research proposal aims to determine which antigens within high-risk (HR) HPVs, in particular HPV16, generate measurable B cell immunity in patients with OPC. Based on our preliminary data, we predict that: 1) HPV16 early antigen (EA)-specific antibodies (Abs) will be detected in cases at the time of diagnosis and prior to diagnosis 2) Other HR HPV-specific Abs will improve the detection of cases of non-HPV16+ OPC and 3) A multiplexed assay of multiple HPV-specific Abs will be superior to HPV16 E6 serology alone. To answer these questions, we will leverage our expertise in protein arrays and immunoassay development funded by the parent U01 grant, and two well-annotated biorepositories of sera from patients with OPC and controls. We will measure HR HPV Ab levels and correlate them with detection of OPC. We predict that these HR HPV serum Ab biomarkers will have utility for early detection, monitoring therapy and identifying early recurrence of HPV+ OPC. Specific Aim 1. Develop a NAPPA HPV protein microarray Specific Aim 2. Determine the sensitivity and specificity of serum HPV-specific antibodies in patients with newly diagnosed oropharyngeal cancers. Specific Aim 3. Determine the sensitivity and specificity of HPV16 antibodies for detection of oropharyngeal

Description

The overall goal of this project is to identify autoantibody biomarkers in sera that can be readily used for the early detection of cancer. Antibodies are induced by tumor-specific alterations in protein expression, mutation, degradation, or localization, with high specificity. We have developed a novel programmable protein microarray technology (NAPPA), which uses printed cDNA encoding tumor antigens that are translated in vitro. We have been funded for the past four years as an EDRN Biomarker Development Laboratory to develop NAPPA as a clinical research tool and to employ it for the detection of autoantibody biomarkers for breast cancer, and have screened over 700 sera and identified 32 novel breast and 23 ovarian biomarkers that have undergone blinded validation studies. Here, we propose to increase the feature density of NAPPA arrays in order to accommodate the increasing size of our cDNA collection, which now approaches 10,000 unique genes (Aim 1) and to develop a method for producing glycoproteome arrays (Aim 2). Both of these methods will enhance autoantibody biomarker discovery by increasing the number and quality of the antigens screened. Aim 3 will use the results of Aims 1 and 2 to expand the top breast cancer biomarkers that have been identified to focus on particular subtypes of breast cancer that are more difficult to detect including Her2+ breast cancers, triple-negative (ER-PR-Her2-) breast cancers, and cancers that occur in the setting of high breast density. Association of antibody detection and tumor antigen expression will be explored. Aim 4 will assess the performance characteristics of the combined set of top breast cancer biomarkers in Phase II studies using sera collected in multicenter clinical studies. Aim 5 will focus on the utility of these biomarkers to detect disease prior to clinical diagnosis. At the end of these experiments, our goal is to identify autoantibody serum signatures that can be used by EDRN CEVCs in further Phase III independent validation studies for the early detection of breast cancer according to the EDRN biomarker discovery guideline.Q

Description

This section describes the research plan for Carlos A. Morales-Betanzos funded by the requested supplement to my EDRN Biomarker Development grant: Biomarker Detection Using NAPPA Tumor Antigen Arrays. I. Parent Grant Abstract. The overall goal of the parent grant is to identify autoantibody biomarkers in sera that can be readily used for the early detection of cancer. Antibodies are induced by tumor-specific alterations in protein expression, mutation, degradation, or localization, with high specificity. We have developed a novel programmable protein microarray technology (NAPPA), which uses printed cDNA encoding tumor antigens that are translated in vitro. We have been funded for the past four years as an EDRN Biomarker Development Laboratory to develop NAPPA as a clinical research tool and to employ it for the detection of autoantibody biomarkers for breast cancer. We have screened over 700 sera and identified 32 novel breast and 23 ovarian biomarkers that have undergone blinded validation studies. In our approach, we propose to increase the feature density of NAPPA arrays in order to accommodate the increasing size of our cDNA collection, which now approaches 10,000 unique genes (Aim 1) and to develop a method for producing glycoproteome arrays (Aim 2). Both of these methods will enhance autoantibody biomarker discovery by increasing the number and quality of the antigens screened. Aim 3 will use the results of Aims 1 and 2 to expand the top breast cancer biomarkers that have been identified to focus on particular subtypes of breast cancer that are more difficult to detect including Her2+ breast cancers, triple-negative (ER-PR-Her2-) breast cancers, and cancers that occur in the setting of high breast density. Association of antibody detection and tumor antigen expression will be explored. Aim 4 will assess the performance characteristics of the combined set of top breast cancer biomarkers in Phase II studies using sera collected in multicenter clinical studies. Aim 5 will focus on the utility of these biomarkers to detect disease prior to clinical diagnosis. At the end of these experiments, our goal is to identify autoantibody serum signatures that can be used by EDRN CEVCs in further Phase III independent validation studies for the early detection of breast cancer according to the EDRN biomarker discovery guideline. II. Candidate Research. Carlos A. Morales-Betanzos is a graduate student who recently joined my lab, the Virginia G. Piper Center for Personalized Diagnostics at Arizona State University (CPD). He will be under my supervision while he carries out the project: Characterization of the phosphorylation events mediated by ShcC, a protein overexpressed in high risk/poor outcome neuroblastoma, as markers of prognosis. Project description.Neuroblastoma (NB) is a type of cancer that primarily affects young children in the first 5 years of age. It affects either the adrenal gland or the sympathetic nerve chain located in the neck, chest or abdomen. According to the National Cancer Institute NB is the third most common childhood tumor, following leukemia and brain cancer, and accounted for 7.1% of the total childhood tumors in 2010 in the United States. Neuroblastoma is characterized by its significant heterogenic behavior: 90% of NB patients with low and medium risk (LR and MR) cases will recover, but only a dismal 20-30% of patients with high risk (HR) cases will survive after 5 years of receiving the treatment. The intrinsic mechanisms responsible for the differences in behavior among the various types of NB are still not clear1,2,3,4. Approach. In our approach we propose to expand the current knowledge of neuroblastoma tumorigenicity and attempt to identify novel prognostic biomarkers for high risk neuroblastoma. We plan to accomplish this by studying ShcC, a scaffold proteinover expressed in high risk (HR) neuroblastoma cases but not in low risk cases (LR). We will identify the specific phosphorylation events that ShcC mediates in neuroblastoma. Phosphorylation is a post-translational modification (PTM) that represents a crucial factor in mediation of intracellular signaling. It is responsible for the signal transduction of the most relevant cellular processes: growth, development, differentiation, apoptosis and many others. Because cancer cells present abnormalities in one or more of these pathways, concomitant phosphorylation changes have been detected and are currently being used as an attractive source of novel biomarkers for hormonal response, drug resistance and prognosis.5,6,7,8Proteins modifying phosphorylations arealso an attractive target for current drug development and arecurrently considered to be one of the three most important cancer drug targets9,10. Proteins associated with poor prognosis in neuroblastoma. NMYC is the most important neuroblastoma prognosis biomarker known today. The NMYC gene encodes for a 60-63 KDa transcription factor that has been used to stratify neuroblastoma from local to invasive types. The role of the NMYC protein in cell proliferation, differentiation, transformation and apoptosis has been identified in cells and patients. However, the action mechanism of NMYC remains mostly unknown. The clinical significance of NMYC expression in children with neuroblastoma is also controversial in multivariable settings. This is in part because of the disparities observed in different patient populations and the contradictory results observed in retrospective analysis of patients with advanced stages of neuroblastoma and normal NMYC count numbers11. 4 Program Director/Principal Investigator (Last, First, Middle): LaBaer, Joshua PHS 398/2590 (Rev. 06/09) Page Continuation Format Page It is therefore likely that in multivariate neuroblastoma settings, NMYC alone is not sufficient to distinguish the patients who are likely to survive from those destined to fail the treatment. Therefore,we propose that a more precise discrimination between risk stages may be accomplished by supplementing NMYC with additional markers.12 ShcC is a scaffold protein normally involved in cell proliferation and development of normal neurons. In 2005, Terui et al. analyzed 52 neuroblastoma tumors from patients using semi-quantitative reverse transcription PCR and found that patients with high levels of ShcC mRNA had poor prognosis compared with patients with lower levels13. In 2008, Miyake et al. also correlated the presence of ShcC with poor prognosis using western blot14. The latter study evaluated the effect of ShcC knockdown in neuroblastoma differentiation and xenograft tumors in mice using RNA interference (RNAi) and showed that neuroblastoma cells treated with ShcC RNAi differentiated and were less tumorigenic when injected into nude mice. This study suggests that ShcC inhibits neuroblastoma cell differentiation and neurite development and therefore may represent a novel prognostic biomarker for neuroblastoma.
StatusFinished
Effective start/end date8/1/106/30/16

Funding

  • HHS-NIH: National Cancer Institute (NCI): $4,043,332.00

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Neoplasm Antigens
Neuroblastoma
Oropharyngeal Neoplasms
Biomarkers
Antibodies
Breast Neoplasms
Autoantibodies
Serum
Protein Array Analysis
Papillomavirus Infections
Antigens
Tumor Biomarkers
Early Detection of Cancer
Complementary DNA
Validation Studies
Neoplasms
Phosphorylation
Proteins
Organized Financing
Immunity