Human Papillomavirus Negative Samples Research Articles

Clinical implications of activation of the LIMD1-VHL-HIF1α pathway during head-&-neck squamous cell carcinoma development.

Background & objectives Given the importance of the role of hypoxia induced pathway in different cancers including head-and-neck squamous cell carcinoma (HNSCC), this study delved into elucidating the molecular mechanism of hypoxia-inducible factor-1α (HIF1α) activation in HNSCC. Additionally, it analyzes the alterations of its regulatory genes [von Hippel-Lindau (VHL) and LIM domain containing 1 (LIMD1)] and target gene vascular endothelial growth factor (VEGF) in head-and-neck lesions at different clinical stages in relation with human papillomavirus (HPV) infection. Methods Global mRNA expression profiles of HIF1α, VHL, LIMD1 and VEGF were evaluated from public datasets of HNSCC, followed by validation of their expression (mRNA/protein) in an independent set of HPV+ve/-ve HNSCC samples of different clinical stages. Results A diverse expression pattern of the HIF1α pathway genes was observed, irrespective of HPV infection, in the datasets. In validation in an independent set of HNSCC samples, high mRNA expressions of HIF1α/VEGF were observed particularly in HPV positive samples. However, VHL/LIMD1 mRNA expression was low in tumours regardless of HPV infection status. In immunohistochemical analysis, high/medium (H/M) expression of HIF1α/VEGF was observed in basal/parabasal layers of normal epithelium, with significantly higher expression in tumours, especially in HPV-positive samples. Conversely, high cytoplasmic VHL expression in these layers gradually decreased with the progression of HNSCC, regardless of HPV infection. A similar trend was noted in LIMD1 expression (nuclear/cytoplasmic) during the disease development. The methylation pattern of VHL and LIMD1 promoters in the basal/parabasal layers of normal epithelium correlated with their expression, exhibiting a gradual increase with the progression of HNSCC. The H/M expression of HIF1α/VEGF proteins and reduced VHL expression was associated with poor clinical outcomes. Interpretation & conclusions The results of this study showed differential regulation of the LIMD1-VHL-HIF1α pathway in HPV positive and negative HNSCC samples, illustrating the molecular distinctiveness of these two groups.

P16INK4a and pRb expression in laryngeal squamous cell carcinoma with and without infection by EBV or different genotypes of HPV: a retrospective study

BackgroundLaryngeal squamous cell carcinoma (LSCC) represents one of the principal tumors of the head and neck. Human papillomavirus (HPV) and Epstein–Barr virus (EBV) are considered risk factors for the development and the clinical prognosis of LSCC. High levels of p16INK4a are suggested as a surrogate marker of HPV or EBV infection in some head and neck tumors but in LSCC is still controversial. Furthermore, pRb expression may be considered an additional biomarker but it has not been clearly defined. This work aimed to compare the expression of pRb and p16INK4a as possible biomarkers in tumor tissues with and without infection by EBV or different genotypes of HPV from patients with LSCC.MethodsTumor samples from 103 patients with LSCC were previously investigated for the presence and genotypes of HPV using the INNO-LiPA line probe assay and for the infection of EBV by qPCR. p16 INK4a and pRb expression was assessed by immunohistochemistry.ResultsOf the 103 tumor samples, expression of p16INK4a was positive in 55 (53.4%) and of this, 32 (56.1%) were positive for HPV whereas 11 (39.3%) were EBV positive but both without a significantly difference (p > 0.05). pRb expression was positive in 78 (75.7%) and a higher frequency of this expression was observed in HPV negative samples (87.0%) (p = 0.021) and in high-risk HPV negative samples (85.2%) (p = 0.010). No difference was observed when comparing pRb expression and EBV infection status (p > 0.05).ConclusionOur results support the suggestion that p16INK4a is not a reliable surrogate marker for identifying HPV or EBV infection in LSCC. On the other hand, most of our samples had pRb expression, which was more frequent in tumors without HPV, suggesting that pRb could indicate HPV negativity. However, more studies with a larger number of cases are required, including controls without LSCC and evaluating other molecular markers to determine the real role of p16INK4a and pRb in LSCC.

Large cell neuroendocrine carcinoma originating in the subglottic larynx.

Large cell neuroendocrine carcinoma (LCNEC) of the larynx is an exceedingly rare cancer of the head and neck that is difficult to diagnose. Few case reports of laryngeal LCNEC exist within the literature, and it was not until recently that LCNEC was recognized as a discrete subtype of neuroendocrine carcinoma. Given its recent recognition as a distinct subtype, histologic characteristics distinguishing LCNEC from other poorly differentiated carcinomas remain under investigation. Various reports have shown genetic alterations such as p53 and/or p16 overexpression, which are typically associated with infection by human papilloma virus (HPV). However, some reports have shown p53 and/or p16 overexpression in HPV negative samples. In this case, we discuss a 67-year-old patient with a history of extensive alcohol and tobacco use with a newly diagnosed T4N0M0, high grade, LCNEC of the subglottic larynx. Tumor pathology demonstrated positive staining for typical neuroendocrine (NE) markers like synaptophysin and chromogranin A; however, there was diffuse CK34βE12 and p16 expression. LCNEC is a newly classified subtype of poorly differentiated neuroendocrine (NE) tumors, and the diagnosis requires consideration of the clinical presentation, microscopic features, and immunostaining markers.

Lachnoclostridium and Immune Inflamed Gene Expression Signature Association in Head & Neck Cancers

<h3>Purpose/Objective(s)</h3> The immune microenvironment plays an essential role in the progression of head and neck squamous cell carcinoma (HNSCC). The association of the tumor microbiota and immune microenvironment is not well defined. The Cancer Genome Atlas Project (TCGA) analysis provided the diversity of microbiota in different cancer types. We analyzed the TCGA microbiome database to better understand the role of the tumor microbiome in HNSCC. <h3>Materials/Methods</h3> Human papillomavirus (HPV) positive and HPV negative HNSCC samples in the TCGA database were analyzed. Data were accessed using cBioPortal. Spearman's test was used to correlate Lachnoclostridium microbiome signature with immune-related gene expressions. <h3>Results</h3> Microbiome signatures were compared in PDL1 low and PDL1 high HNSCC. Lachnoclostridium was among the top genus enriched in the PDL1 high expression group in both HPV+ and HPV- HNSCC. Lachnoclostridium was correlated with immune inflamed gene expression profile. To evaluate the differential gene expressions in Lachnoclostridium enriched HNSCC, the gene expression profile of Lachnoclostridium high and low HNSCC samples were compared. MHC class I genes; HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, TAP1, and TAP2 were among the top differentially expressed genes. Then we evaluated the correlation of MHC-I genes with Lachnoclostridium enrichment. Lachnoclostridium signature was significantly correlated with expression of these MHC I genes, with Spearman's correlation coefficient ranging between 0.51-0.59. <h3>Conclusion</h3> Lachnoclostridium is a recently defined genus found in the human gut microbiome. Here we, for the first time, identify the enrichment of this genus in high PDL1 expressing HNSCC. The association of Lachnoclostridium signature with the immune inflamed gene expression profile and MHC-I gene signature suggests its potential role in modulating the immune microenvironment in HNSCC.

Negative Impact of Elevated DNA Fragmentation and Human Papillomavirus (HPV) Presence in Sperm on the Outcome of Intra-Uterine Insemination (IUI).

We wanted to determine the sperm DNA fragmentation index (DFI) cutoff for clinical pregnancies in women receiving intra-uterine insemination (IUI) with this sperm and to assess the contribution of Human Papillomavirus (HPV) infection on sperm DNA damage and its impact on clinical pregnancies. Prospective non-interventional multi-center study with 161 infertile couples going through 209 cycles of IUI in hospital fertility centers in Flanders, Belgium. Measurement of DFI and HPV DNA with type specific quantitative PCRs (HPV 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66 and 68) in sperm before its use in IUI. Clinical pregnancy (CP) rate was used as the outcome to analyze the impact on fertility outcome and to calculated the clinical cutoff value for DFI. A DFI criterion value of 26% was obtained by receiver operating characteristic (ROC) curve analysis. Couples with a male DFI > 26% had significantly less CPs than couples with DFI below 26% (OR 0.0326; 95% CI 0.0019 to 0.5400; p = 0.017). In sperm, HPV prevalence was 14.8%/IUI cycle. Sperm samples containing HPV had a significantly higher DFI compared to HPV negative sperm samples (29.8% vs. 20.9%; p = 0.011). When HPV-virions were present in sperm, no clinical pregnancies were observed. More than 1 in 5 of samples with normal semen parameters (17/78; 21.8%) had an elevated DFI or was HPV positive. Sperm DFI is a robust predictor of clinical pregnancies in women receiving IUI with this sperm. When DFI exceeds 26%, clinical pregnancies are less likely and in vitro fertilization techniques should be considered.

Whole-exome sequencing in cervical adenocarcinoma in mainland Chinese patients.

BackgroundCervical cancer is the most common gynecological malignancy worldwide. Adenocarcinoma is an important pathological type of cervical cancer. In recent years, the incidence of adenocarcinoma is rising in some countries and the prognosis of it remains poor. A precise description of the mutational landscape in cervical adenocarcinoma may provide insights into a better selection of treatments and improve prognosis.MethodsIn this study, we conducted whole-exome sequencing (WES) for cervical adenocarcinomas and matched blood samples from a cohort of 24 mainland Chinese patients. Additionally, the Human-Papilloma virus (HPV) infection statuses of these tumor samples were detected, and the genes that were enriched in both HPV positive and negative samples were also analyzed.ResultsThe results of WES revealed the gene expression profile of cervical adenocarcinoma of women in mainland China and identified multiple genes/pathways, which are frequently mutated in these tumors, including the PI3K-AKT (KRAS, PIK3CA and PTEN), estrogen signaling (KRAS, PIK3CA and GNAS) and NK cell-mediated antibody-dependent cellular cytotoxicity pathways. Besides, seven patients had HPV infection, and the mutated genes in HPV-positive tumor tissues were relatively consistent, while the mutation profiles of HPV-negative tumor tissues were relatively scattered.ConclusionsTaken together, these findings provide novel insights into the pathogenesis of cervical adenocarcinomas. They suggest the potential for individualized treatment of cervical adenocarcinoma according to genomic information.

Prevalence of human papillomavirus in oral epithelial dysplasia: Systematic review and meta-analysis.

The purpose of this systematic review and meta-analysis was to estimate the overall and type-specific prevalence of human papillomavirus (HPV) DNA in oral epithelial dysplasia and assess p16INK4a overexpression in relation to HPV-status. A systematic literature search identified 31 eligible studies (832 cases) evaluating the presence of HPV DNA in oral epithelial dysplasia cases by PCR. Of these, six studies evaluated p16INK4a overexpression in relation to HPV-status. The overall pooled prevalence of HPV DNA in oral epithelial dysplasia was 27.2% (95% CI: 17.6-38.1). We observed substantial interstudy heterogeneity, which could not be explained by differences in continent, tissue type, or severity of epithelial dysplasia. HPV16 was the predominant genotype detected. Moreover, 62.2% of HPV positive and 17.8% of HPV negative oral epithelial dysplasia samples stained intensively positive for p16INK4a . This meta-analysis found that 27% of oral epithelial dysplasia harbor HPV DNA. Whether this represents a transient infection or has a carcinogenic role is unknown.

Rapid and efficient screening of human papillomavirus by Raman spectroscopy based on GA-SVM

In this study, a rapid, noninvasive and efficient screening method for human papillomavirus (HPV) was proposed by combining cervical secretion Raman spectral data with a pattern recognition algorithm. We measured the cervical secretion Raman spectra of 58 HPV positive and 196 HPV negative samples. The preliminary assignment of the Raman peaks in the measured cervical secretion spectra indicated changes in specific biomolecules between the groups, and we analyzed them according to their differences.The specificity of the airPLS-PLS-GA-SVM model was 100 %, the sensitivity was 94.4 %, and the total accuracy was 98.6 %. The results show that the combination of cervical secretion Raman spectroscopy and multivariate statistical analysis has great potential in the early screening of HPV and the samples of low-level lesions and high-level lesions were preliminarily classified effectively, providing a basis for the design of computer-aided diagnosis systems in the future.

HPV infection and bacterial microbiota in breast milk and infant oral mucosa.

ObjectiveWe investigated the association between bacterial microbiota in breast milk and the infant mouth. The influence of human papilloma virus (HPV) infection on infant oral microbiota was also assessed.Material and methodsAltogether 35 breast milk and 35 infant oral samples with known HPV status were selected from the Finnish Family HPV Study cohort. In total, there were 31 mother-infant pairs. The microbiota composition was characterized by 16S rRNA gene sequencing (V3-V4 region).ResultsHPV DNA was present in 8.6% (3/35) of the breast milk and 40% (14/35) of the infant oral samples. Eight shared genera between breast milk and infant oral were found; these included Streptococcus, Staphylococcus, Unclassified Gemellaceae, Rothia, Veillonella, Haemophilus, Propionibacterium and Corynebacterium. HPV status was not associated with either microbiota richness or diversity in the infant mouth. However, the infant oral microbiota clustered in different groups according to HPV status. We detected higher abundance of Veillonella dispar (p = 0.048) at species level in HPV negative infant oral samples. We did not detect differences in the breast milk microbiota composition related to HPV infection due to only three HPV positive milk samples.ConclusionsHPV infection is associated with distinct oral bacterial microbiota composition in infants. The direction of causality underlying the phenomenon remains unclear.

Abstract A26: Network integration of epigenomic data in HPV-associated head and neck cancer

Abstract The majority of published studies investigating driver genes have focused primarily on genomic mutations which have led to novel study designs (basket trials) where patients with a rare mutation, regardless of tumor histology, are matched to a drug expected to work through the mutated pathway. This dominant focus on genomic mutations has overshadowed consideration of inclusion of epigenetic information. Epigenetic silencing of driver genes leads to various genomic alterations, including mismatch repair deficiency, altered DNA repair and loss of chromosomal stability. In human papilloma virus (HPV) positive head and neck squamous cell carcinoma (HNSCC), recent studies are beginning to establish a mechanistic role for DNA methylation with potential to impact improved survival outcomes. The purpose of this study was to illustrate network integration of epigenomic data in head and neck cancer to prioritize elements that drive biological states. Causal Networks are small hierarchical networks of regulators whose activity can be modulated by the expression of downstream target genes to enhance understanding of the effect of upstream master regulators on disease or function. To further establish the driver potential of 11 genes: C14orf162/ CCDC177, CDH8, CRMP1, ELMO1, HTR1E, MEI1, MSX2, PCDH10, PCDHB11, PITX2, SYN2 previously reported as significantly differentially methylated between HPV positive and HPV negative HNSCC tumor samples, and subsequently validated by our group in two independent sample sets, their master regulatory networks were identified utilizing Causal Network Analysis (CNA) software from Ingenuity Pathway Analysis. To reflect expected gene expression direction implied by methylation changes, the inverse of the methylation ratio from HPV positive vs. HPV negative HNSCC was used for CNA. CNA identified 23 top hierarchical networks (significant z score of absolute 2) associated with HNSCC and their corresponding master regulatory molecules characterized as transcription regulators and kinase inhibitors among others. Of the 11 target genes, 7 had representation in multiple networks; CRMP1 in 21/23, MSX2 and SYN2 in 20/23, CDH8 and PITX2 in 17/23, PCDH10 in 10/23, and ELMO1 in 5/23 networks. Of the 23 networks, 12 indicated activation and 11 inhibition by at least 4 or more of the 7 target genes. Master regulators were within 2 or 3 hops (intermediate regulators) to the target genes. CNA raised the profile of CDH8, CRMP1, ELMO1, MSX2, PCDH10, SYN2, and PITX2 (7/11 target genes) for further consideration as epigenetic drivers of HPV-associated HNSCC. Support: Komen Foundation: KG110218 Citation Format: Maria J. Worsham, Kang Mei Chen, Indrani Datta, Josena K. Stephen, Dhananjay Chitale, Tamer Ghanem, Lamont Jones, Laura Garcia-Rodriquez, George Divine. Network integration of epigenomic data in HPV-associated head and neck cancer [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr A26.

42 CD56-positive lymphocyte infiltration in correlation with outcome and human papilloma virus association of oropharyngeal squamous cell carcinoma

Tumor infiltrating lymphocytes and immune factors within the tumor microenvironment are critically related to cancer patient’s prognosis. Human papillomavirus (HPV)-related oropharyngeal tumors are clinical and biological distinct from their HPV unrelated counterparts and patients with HPV relates oropharyngeal tumors display improved prognosis. We investigated possible immune cell infiltrations associated with this tumor phenotype. We randomly selected a cohort of 140 squamous cell carcinoma of the oropharynx (OSCC) and analyzed the presence of immune cells and HPV prevalence by immunohistochemistry and PCR followed by bead-based hybridization (Luminex technology). Cytotoxic activity of immune cells was determined by immunofluorescence double staining for representative HPV associated and HPV negative samples (n = 20). We have found significantly higher numbers of CD56+ cells in tumor (t-test: p = 0.004) and surrounding microenvironment (t-test: p = 0.002) in HPV associated compared to HPV negative OSCC. The prognostic value of presence of CD56+ cells was measured by Kaplan–Meier and Cox regression model analyses and correlated with a better outcome in HPV negative OSCC (Kaplan–Meier: p = 0.05) and within the entire cohort independent from HPV (Kaplan–Meier: p = 0.002; Cox regression: p = 0.046). Immunofluorescence localization of granular GZMB within CD56+ cells and coexpression of CD16 and CD56 suggests that the detected CD56+ cells mainly represent cytotoxic NK cells. The differential presence and distribution of cytotoxic NK-cells might be in part responsible for the improved prognosis of patients with oropharyngeal tumors and this effect is pronounced in HPV related OSCC.

MBL2 polymorphisms in women with atypical squamous cells of undetermined significance.

Infection with high risk Human papillomavirus (HPV) is the main known cause of cervical cancer. HPV induces different grades of lesions: among them, Atypical squamous cells of undetermined significance are abnormal lesions that could evolve in pre-cancer lesions or spontaneously regress. The mannose binding lectin (MBL) is an innate immunity serum protein also found in cervico-vaginal mucosa, whose expression is known to be affected by polymorphisms in exon 1 and promoter of the MBL2 gene. In the present study the possible association between MBL2 functional polymorphisms and susceptibility to develop atypical squamous cells of undetermined significance was investigated in a group of women from North-East of Italy, stratified for HPV infection status. The MBL2 D and O alleles and the deficient producer combined genotypes, responsible for low MBL production, were more represented among atypical squamous cells of undetermined significance positive women than healthy controls and the results were confirmed when only HPV negative samples were considered. These results suggest a possible involvement of MBL2 functional polymorphisms in atypical squamous cells of undetermined significance susceptibility.

Human papillomavirus, lichen sclerosus and penile cancer: A study in Belgium

PurposeThe prevalence of penile cancer varies between 1.5 (industrialized countries) and 4.5 per 100,000 men (non-industrialized countries). Predominant histological subtype is squamous cell carcinoma (SCC). Human papillomavirus (HPV) is found in 40–46% of cases: penile cancer is considered to behave as vulvar cancer. Non HPV related risk factors are lack of circumcision, phimosis, chronic inflammation, and smoking. The role of lichen sclerosus (LS) is unclear. Clinical diagnosis is difficult and treatment often mutilating. Preventive measures can be taken since the risk factors are known: the use of the prophylactic HPV vaccines may contribute. We measured the prevalence of HPV and LS in penile cancer in Belgium. Materials and methodsWe found 76 samples of penile lesions in the archives of the departments of Histology of four university hospitals in Belgium. Real-time PCR of type-specific HPV DNA was performed targeting 18 HPV types. Principal resultsPatients with penile intraepithelial neoplasia (PeIN) were 56.1 years of age: patients with invasive penile cancer (IPC) 68.5 (p=0.009). Fifty-five samples (55/76) were adequate for HPV targeting. Overall HPV DNA was 70.9%: 89.5% in samples of PeIN (n=19) and 61.1% in samples of IPC (n=36). Invasive penile cancer samples were less likely to be HPV infected (p=0.028). HPV 16 was most prevalent: 48.3%: 20% PeIN, and 28.3% IPC. HPV DNA of the types, included in the prophylactic vaccines, was found in 33% of PeIN and 31.7% of IPC samples. Thrice, low risk HPV (lrHPV) types 6 (1 IPC) and 11 (1 PeIN, 1 IPC) were solely present. There was no difference in the presence of LS between HPV positive and HPV negative samples (p=0.944). ConclusionsPrevalence of HPV DNA in penile lesions in Belgium is high. However, the prophylactic vaccines may contribute to primary prevention of only a subset of cases. The role of LS remains unclear.

Discrimination of ‘Driver’ and ‘Passenger’ HPV in Tonsillar Carcinomas by the Polymerase Chain Reaction, Chromogenic In Situ Hybridization, and p16INK4a Immunohistochemistry

Human papillomavirus (HPV) positive tonsillar squamous cell carcinoma (TSCC) is associated with a favorable clinical outcome. However, the HPV detected in a given tumor may be causal (driver HPV) or an incidental bystander (passenger HPV). There is a need to discriminate these forms of HPV in TSCCs to understand their impact on HPV as a biomarker for use in TSCC patient management. This study has compared the polymerase chain reaction (PCR), chromogenic in situ hybridization (CISH), and p16(INK4a) immunohistochemistry in the assessment of HPV status in TSCC. Archival specimens of TSCC from thirty patients were investigated. HPV was detected by PCR in 25/30 (83.3%) tumors; HPV16 (70.0%) and HPV52 (6.7%) were the most common types. HPV was corroborated by CISH in 22/25 (88.0%) specimens; integrated HPV was implicated by the presence of punctate signals in each of these cases. p16(INK4a) staining was found in 20/22 (90.9%) HPV PCR positive samples; two PCR/CISH HPV positive cases were p16(INK4a) negative and two HPV negative samples were p16(INK4a) positive. These data suggest that a minority of HPV positive TSCCs are positive for passenger HPV and that two or more assays may be required for diagnosing driver HPV status. Further studies are required to exam whether oropharyngeal tumors positive for passenger HPV have a less favorable prognosis than tumors that are driver HPV positive. The clinical significance of TSCCs that test HPV negative/p16(INK4a) positive, PCR and CISH HPV positive/p16 (INK4a) negative, or PCR HPV positive/p16 (INK4a) and CISH negative, also requires further investigation.

Abstract LB-376: Prediction of clinical outcome using NFkB target gene expression based classifier in head and neck patients with different HPV status

Abstract Purpose: Mutations and polymorphisms in various genes, tobacco/alcohol abuse as well as Human Papillomavirus (HPV) are shown to be associated with pathogenesis of Squamous Cell Carcinomas of the Head and Neck (SCCHN). NFkB is one of the common significant players in both HPV and tobacco/alcohol related carcinogenesis. Our objective is to develop a classifier based on the NFkB signaling pathway gene expression profiles that predicts the clinical outcome to chemo-radiation therapy of SCCHN patients with different HPV status. Methods: Expression levels of HPV16, p16 and 84 NFkB signaling pathway genes were determined using real time RT PCR in 29 SCCHN patient samples and 4 SCCHN cell lines that were either resistant or sensitive to radiation and chemotherapy. A gene expression pattern that correlated to chemo-radiation response was identified in the cell lines. The cell line model was blindly tested in untreated SCCHN patients to predict their clinical outcome to chemo-radiation based upon the individual tumor gene expression. Results: The gene-based predicted clinical outcome compared with patients' actual observed clinical outcome correctly identified the clinical response in 73% (21 of 29) of the cases (95% Confidence Interval: 55%, 84%). Several important genes were identified that correlated with the clinical outcome. Further, it was observed that 8 out of 11 recurrent patients (73%) were HPV16 negative and 13 out of 18 responders (72%) were HPV16 positive thus confirming that patients with HPV positive tumors respond better than HPV negative tumors to the treatment (p-value=0.01). Further, mean gene expression levels for HPV positive and negative samples for each of the 84 genes in the NFkB signaling pathway were compared by independent two-sample t-test to identify the significantly differentially expressed gene list. We identified a group of genes that were differentially expressed in HPV16 positive and negative tumors. Most of the 35 up-regulated genes in HPV positive recurrent patients had pro-survival function compared to only one gene that was up-regulated in HPV positive responder patients. These results suggest that up-regulation of NFkB target genes with pro-survival function leads to recurrence. Conclusions: We demonstrated that therapeutic response to chemo-radiation can be predicted based on NFkB signaling pathway gene expression profiles. Ongoing analyses will identify the pro-survival/pro-apoptotic genes that were significantly up-regulated in tumor samples from recurrent patients by correlating them to the patients HPV status. These genes can then be used as markers of treatment failure or success. 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 LB-376. doi:10.1158/1538-7445.AM2011-LB-376

Human Papillomavirus DNA and E6/E7 mRNA Status in Relation to Survival of Patients Treated for Cervical Squamous Cell Carcinoma

The prognostic significance of human papillomavirus (HPV) DNA and E6/E7 mRNA, the presence of specific types, and the physical state of HPV DNA, were studied in 202 cervical squamous cell carcinomas. Absence or non-detectable levels of high-risk (types 16, 18, 31, 33, 35, 45, 52 and 58) E6/E7 mRNA, using the real-time nucleic acid sequence based amplification (NASBA) assay, and absence of HPV high-risk/HPV 6, 26, 66, 69, 73 (all methods collectively) were associated with poor overall survival in univariate analysis (P = 0.04 and P = 0.03, respectively) and had independent prognostic value in multivariate analysis (P = 0.01 and P = 0.03, respectively) including FIGO stage and age. Based on the individual results of type-specific PCR and in situ hybridization (ISH), the presence of HPV DNA was not found to be a prognostic factor. Likewise, concerning the presence of specific HPV types and the HPV integration status (determined by ISH), no prognostic significance was found. Mutation analyses of the TP53 gene revealed mutations in 3 of the 6 HPV negative samples (50%).

Automation of a Multiplexed Competitive Luminex Immunoassay for Measuring Antibodies to Human Papillomavirus Types 6, 11, 16, and 18

Serum specimen pipetting is a labor-intensive process that can be a source of laboratory error, ergonomic stress, and a potential source of accidental exposure to infectious pathogens. Automated solutions for sample preparation reduce time spent on sample handling, minimize exposure to infectious pathogens, reduce laboratory error, reduce ergonomic stress, and save money. A fully automated method was developed for a custom-designed competitive Luminex immunoassay (cLIA) on a TECAN Genesis Workstation that measures antibodies to human papillomavirus (HPV) virus-like particles (VLPs). The automated program generated statistically similar data to assays performed manually. Overall, HPV 6, 11, 16, and 18 antibody titers obtained from samples prepared with the automated program were 3.2%, 6.6%, 3.6%, and 2.0% higher, respectively, than antibody titers from samples prepared manually. The agreement rates between methods for HPV positive and negative samples for HPV 6, 11, 16, and 18 were 100%, 96.8%, 98.4%, and 97.3%, respectively. An overview of the basic workflow of the automated HPV cLIA used to support our HPV vaccine Phase III clinical trials is described.

Reliable high risk HPV DNA testing by polymerase chain reaction: an intermethod and intramethod comparison.

BACKGROUND: The development of a reproducible, sensitive, and standardised human papillomavirus (HPV) polymerase chain reaction (PCR) test is required to implement HPV testing in cervical cancer screening programmes and for...

Role of human papillomavirus in determining the HLA associated risk of cervical carcinogenesis.

To investigate the role of human papillomavirus (HPV) in the association between HLA DQw3 and squamous cell cancer of the cervix (SCCC). Tissue from 194 cervical samples, ranging from normal, through cervical intraepithelial neoplasia, to SCCC, were typed for HPV by amplification of the L1 gene using degenerate consensus primers, followed by oligonucleotide probing. HLA DQw3 typing was undertaken in the same samples using a new PCR amplification system using primers common to all DQ loci, followed by restriction digestion with Mlu 1 to differentiate HLA DQw3 types--null, heterozygous, and homozygous. The data were analysed using chi 2 analysis and by calculating relative risks with the 95% confidence interval. Samples (n = 188) were successfully typed for HPV and 177 were typed for HLA DQw3. There was a nonsignificant rise in the prevalence of HLA DQw3 in SCCC (64.3%) compared with the group with normal histology (53.2%). Analysis of the prevalence of HLA DQw3 on the basis of HPV infection rather than histology showed that 63 of 95 (66.3%) of the HPV positive samples contained HLA DQw3 alleles, compared with 39 of 78 (50.0%) of the HPV negative samples (chi 2 4.06; p < 0.05). There was a significant association between HLA DQw3 and cervical HPV infection. This may be because people with HLA DQw3 are less able to mount an effective immune response to HPV, which predisposes them to the development of SCCC.