Richard B. S. Roden

Comparison of candidate serologic markers for type I and type II ovarian cancer

OBJECTIVEnTo examine the value of individual and combinations of ovarian cancer associated blood biomarkers for the discrimination between plasma of patients with type I or II ovarian cancer and disease-free volunteers.nnnMETHODSnLevels of 14 currently promising ovarian cancer-related biomarkers, including CA125, macrophage inhibitory factor-1 (MIF-1), leptin, prolactin, osteopontin (OPN), insulin-like growth factor-II (IGF-II), autoantibodies (AAbs) to eight proteins: p53, NY-ESO-1, p16, ALPP, CTSD, B23, GRP78, and SSX, were measured in the plasma of 151 ovarian cancer patients, 23 with borderline ovarian tumors, 55 with benign tumors and 75 healthy controls.nnnRESULTSnWhen examined individually, seven candidate biomarkers (MIF, Prolactin, CA-125, OPN, Leptin, IGF-II and p53 AAbs) had significantly different plasma levels between type II ovarian cancer patients and healthy controls. Based on the receiver operating characteristic (ROC) curves constructed and area under the curve (AUC) calculated, CA125 exhibited the greatest power to discriminate the plasma samples of type II cancer patients from normal volunteers (AUC 0.9310), followed by IGF-II (AUC 0.8514), OPN (AUC 0.7888), leptin (AUC 0.7571), prolactin (AUC 0.7247), p53 AAbs (AUC 0.7033), and MIF (AUC 0.6992). p53 AAbs levels exhibited the lowest correlation with CA125 levels among the six markers, suggesting the potential of p53 AAbs as a biomarker independent of CA125. Indeed, p53 AAbs increased the AUC of ROC curve to the greatest extent when combining CA125 with one of the other markers. At a fixed specificity of 100%, the addition of p53 AAbs to CA125 increased sensitivity from 73.8% to 85.7% to discriminate type II cancer patients from normal controls. Notably, seropositivity of p53 AAbs is comparable in type II ovarian cancer patients with negative and positive CA125, but has no value for type I ovarian cancer patients.nnnCONCLUSIONSnp53 AAbs might be a useful blood-based biomarker for the detection of type II ovarian cancer, especially when combined with CA125 levels.

p53 autoantibodies, cytokine levels and ovarian carcinogenesis

OBJECTIVEnTo address the hypothesis that type II ovarian carcinoma, mutation of p53 and plasma levels of particular cytokines are associated with the generation of p53-specific serum autoantibody (AAb) responses in patients.nnnMETHODSnLevels of CA125, 17 cytokines and AAbs to tumor-associated antigens including p53 were measured in plasma of 130 gynecologic tumor patients and 84 healthy controls. TP53 exons 4-9 were sequenced in tumor specimens.nnnRESULTSnp53 AAbs are associated with high grade, but not low grade ovarian carcinoma. Seropositivity for p53 AAb occurred only in those ovarian carcinoma patients whose tumors contained mutated TP53, regardless of the exon targeted. Higher p53 AAb levels were detected in ovarian carcinoma patients who had higher stage disease, but p53 AAb levels were not correlated with CA125 levels. Among high-grade carcinoma patients, there was no relationship between p53 AAb seropositivity and seropositivity to other tumor-associated antigens tested, CA125 level or survival outcome. Both high and low grade ovarian carcinoma patients exhibited elevated levels of IL6, IL8 and IL10 as compared to healthy volunteers, although increased levels of IL5, MCP1, MIP1 and TNFalpha were associated only with high grade and advanced disease. Higher levels of p53AAb responses were correlated with elevated circulating IL4 and IL12, but reduced IL8 levels.nnnCONCLUSIONnType II, but not type I, ovarian carcinoma patients had elevated serum levels of p53 AAb. P53 AAb is associated with mutation of TP53, higher plasma IL4 and IL12 but lower plasma IL8 levels and no survival advantage.

Human Papillomavirus Type-16 Virus-Like Particles Activate Complementary Defense Responses in Key Dendritic Cell Subpopulations

Human papillomavirus type-16 (HPV16) L1 virus-like particles (VLPs) activate dendritic cells (DCs) and induce protective immunity. In this study, we demonstrate, using global gene expression analysis, that HPV16 VLPs produce quite distinct innate responses in murine splenic DC subpopulations. While HPV16 VLPs increase transcription of IFN-γ and numerous Th1-related cytokines and chemokines in CD8α+CD11c+ DCs, CD4+CD11c+ DCs up-regulate only type I IFN and a different set of Th2-associated cytokines and chemokines. Type I IFN, but not IFN-γ, potentiates humoral immunity, notably production of VLP-specific IgG2a. However, HPV16 VLP-stimulated IL-12 production by CD8α+CD11c+ DCs is augmented by autocrine IFN-γ signaling. Thus, before adaptive immunity, HPV16 VLPs signal complementary defense responses in key DC subpopulations, indicating specialized DC lineages with predetermined polarization.

Capsid display of a conserved human papillomavirus L2 peptide in the adenovirus 5 hexon protein: a candidate prophylactic hpv vaccine approach.

BackgroundInfection by any one of 15 high risk human papillomavirus (hrHPV) types causes most invasive cervical cancers. Their oncogenic genome is encapsidated by L1 (major) and L2 (minor) coat proteins. Current HPV prophylactic vaccines are composed of L1 virus-like particles (VLP) that elicit type restricted immunity. An N-terminal region of L2 protein identified by neutralizing monoclonal antibodies comprises a protective epitope conserved among HPV types, but it is weakly immunogenic compared to L1 VLP. The major antigenic capsid protein of adenovirus type 5 (Ad5) is hexon which contains 9 hypervariable regions (HVRs) that form the immunodominant neutralizing epitopes. Insertion of weakly antigenic foreign B cell epitopes into these HVRs has shown promise in eliciting robust neutralizing antibody responses. Thus here we sought to generate a broadly protective prophylactic HPV vaccine candidate by inserting a conserved protective L2 epitope into the Ad5 hexon protein for VLP-like display.MethodsFour recombinant adenoviruses were generated without significant compromise of viral replication by introduction of HPV16 amino acids L2 12–41 into Ad5 hexon, either by insertion into, or substitution of, either hexon HVR1 or HVR5.ResultsVaccination of mice three times with each of these L2-recombinant adenoviruses induced similarly robust adenovirus-specific serum antibody but weak titers against L2. These L2-specific responses were enhanced by vaccination in the presence of alum and monophoryl lipid A adjuvant. Sera obtained after the third immunization exhibited low neutralizing antibody titers against HPV16 and HPV73. L2-recombinant adenovirus vaccination without adjuvant provided partial protection of mice against HPV16 challenge to either the vagina or skin. In contrast, vaccination with each L2-recombinant adenovirus formulated in adjuvant provided robust protection against vaginal challenge with HPV16, but not against HPV56.ConclusionWe conclude that introduction of HPV16 L2 12–41 epitope into Ad5 hexon HVR1 or HVR5 is a feasible method of generating a protective HPV vaccine, but further optimization is required to strengthen the L2-specific response and broaden protection to the more diverse hrHPV.

Immune Responses in Macaques to a Prototype Recombinant Adenovirus Live Oral Human Papillomavirus 16 Vaccine

ABSTRACT Immunization with human papillomavirus (HPV) L1 virus-like particles (VLPs) prevents infection with HPV. However, the expense and logistical demands of current VLP vaccines will limit their widespread use in resource-limited settings, where most HPV-induced cervical cancer occurs. Live oral adenovirus vaccines have properties that are well-suited for use in such settings. We have described a live recombinant adenovirus vaccine prototype that produces abundant HPV16 L1 protein from the adenovirus major late transcriptional unit and directs the assembly of HPV16 VLPs in tissue culture. Recombinant-derived VLPs potently elicit neutralizing antibodies in mice. Here, we characterize the immune response to the recombinant after dual oral and intranasal immunization of pigtail macaques, in which the virus replicates as it would in immunized humans. The immunization of macaques induced vigorous humoral responses to adenovirus capsid and nonstructural proteins, although, surprisingly, not against HPV L1. In contrast, immunization elicited strong T-cell responses to HPV VLPs as well as adenovirus virions. T-cell responses arose immediately after the primary immunization and were boosted by a second immunization with recombinant virus. T-cell immunity contributes to protection against a wide variety of pathogens, including many viruses. The induction of a strong cellular response by the recombinant indicates that live adenovirus recombinants have potential as vaccines for those agents. These studies encourage and will inform the continued development of viable recombinant adenovirus vaccines.

Prevention and Treatment of Cervical Cancer by Vaccination

Causality requires a judgment based on scientific evidence from human and experimental studies, as strict causality studies are often not appropriate in humans. Evidence linking certain human papillomavirus (HPV) genotypes to cervical carcinoma is extensive and compelling. More than two decades of research has led to the fulfillment of criteria, as proposed by Hill, to establish a causal link between high risk HPV infection and cervical cancer (Table 1). HPV DNA was first isolated from biopsies of cervical cancer more than 30 years ago (1, 2). HPV DNA is detected in 99.7% of cervical carcinomas worldwide. The evidence overwhelmingly demonstrates that persistent high risk HPV infection is a necessary but not sufficient cause of this cancer (3).

An integrated proteomic and glycoproteomic approach uncovers differences in glycosylation occupancy from benign and malignant epithelial ovarian tumors

BackgroundEpithelial ovarian carcinomas encompass a heterogeneous group of diseases with a poor 5-year survival rate. Serous carcinoma is the most common type. Most FDA-approved serum tumor markers are glycoproteins. These glycoproteins on cell surface or shed into the bloodstream could serve as therapeutic targets as well as surrogates of tumor. In addition to glycoprotein expressions, the analysis of protein glycosylation occupancy could be important for the understanding of cancer biology as well as the identification of potential glycoprotein changes in cancer. In this study, we used an integrated proteomics and glycoproteomics approach to analyze global glycoprotein abundance and glycosylation occupancy for proteins from high-grade ovarian serous carcinoma (HGSC) and serous cystadenoma, a benign epithelial ovarian tumor, by using LC–MS/MS-based technique.MethodsFresh-frozen ovarian HGSC tissues and benign serous cystadenoma cases were quantitatively analyzed using isobaric tags for relative and absolute quantitation for both global and glycoproteomic analyses by two dimensional fractionation followed by LC–MS/MS analysis using a Orbitrap Velos mass spectrometer.ResultsProteins and N-linked glycosite-containing peptides were identified and quantified using the integrated global proteomic and glycoproteomic approach. Among the identified N-linked glycosite-containing peptides, the relative abundances of glycosite-containing peptide and the glycoprotein levels were compared using glycoproteomic and proteomic data. The glycosite-containing peptides with unique changes in glycosylation occupancies rather than the protein expression levels were identified.ConclusionIn this study, we presented an integrated proteomics and glycoproteomics approach to identify changes of glycoproteins in protein expression and glycosylation occupancy in HGSC and serous cystadenoma and determined the changes of glycosylation occupancy that are associated with malignant and benign tumor tissues. Specific changes in glycoprotein expression or glycosylation occupancy have the potential to be used in the discrimination between benign and malignant epithelial ovarian tumors and to improve our understanding of ovarian cancer biology.

Abstract 1320: PD-L1: the hand brake of immune responses in cervical intraepithelial neoplasia and cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PAnnBackground: Prospective cohort studies have reported spontaneous regression rates of high-grade cervical intraepithelial Neoplasia (CIN2/3) close to 35%, over a period of 4-6 months. These regressions are presumably immunologically mediated. It is now well recognized that PD-L1 is an immune checkpoint used by neoplastic cells to evade the immune system, particularly T cells that are specific for tumor antigens. PD-L1 and several other immune checkpoints consist of inhibitory pathways that tightly regulate the immune system to control the duration and amplitude of immune responses in peripheral tissues in order to minimize collateral tissue damage.nnDesign: Immunohistochemistry with anti-TBX21 and anti-PD-L1 antibodies was performed in formalin-fixed paraffin-embedded tissue sections from a tissue microarray consisting of invasive cervical carcinomas (ICCs) (n = 22), CIN2/3 (n = 4) and benign cervical tissues (n = 4). Expression of TBX21 and PD-L1 was reviewed and scored by a trained pathologist blinded to the clinical outcome of each case. Statistical correlations between these markers’ score and available demographic and clinicopathologic data from each case were performed, as well as disease-free survival curves. Moreover, PD-L1 staining was performed in three patients with CIN2/3 that received peripheral (IM) therapeutic HPV vaccination. PD-L1 expression differences in pre and post-vaccination tissues were compared.nnResults: Increased expression of TBX21+ T-cells and PD-L1 was observed from benign to CIN2/3 to ICCs. Association analysis (fisher exact test) showed a significant correlation between increased intraepithelial and stromal infiltration of TBX21+ T-cells (score 2,3) and higher PD-L1 membrane expression (p = 0.04 and p = 0.005, respectively). No significant associations were found between PD-L1 expression and demographic and clinicopathologic information, likely due to small number of samples. In our vaccine cohort, we observed a stronger membrane PD-L1 expression in the epithelium and stroma of the post-vaccination tissue compared to the pre-vaccination biopsy.nnConclusion: Increased membrane expression of PD-L1 is tightly correlated with an increased epithelial and stromal infiltration of TBX21+ T cells in CIN2/3 and ICCs. This correlation was also observed in our vaccination cohort when compared to our previous published findings of immune activation after HPV therapeutic vaccination. This association could represent an immune response control mechanism induced presumably by interferons released by TBX21+ T cells. We are currently evaluating the expression of PD-L1 and B7-H4, a recently discovered inhibitory ligand, in a larger cohort of benign, CIN2/3 and ICCs. Finally, we will further explore, quantitate and compare the expression changes of these immune checkpoints in the remaining CIN2/3 cases of our vaccination cohort (n = 12) in pre and post-vaccination tissues.nnCitation Format: Leonel F. Maldonado Gonzalez, Christopher VandenBussche, Richard Roden, T.C. Wu, Benjamin Tycko, Cornelia L. Trimble. PD-L1: the hand brake of immune responses in cervical intraepithelial neoplasia and cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1320. doi:10.1158/1538-7445.AM2015-1320