Uptake of HPV vaccination among boys after the introduction of gender-neutral HPV vaccination in Germany before and during the COVID-19 pandemic.

BACKGROUND: HPV vaccination has been recommended and reimbursed for girls in Germany since 2007. In June 2018 the German Standing Committee on Vaccination (STIKO) recommended the gender-neutral vaccination of adolescents aged 9 to 14 years with catch-up through age 17. Objectives of this study were to describe the uptake of vaccination in boys before and during the COVID-19 pandemic. METHODS: The study used data from a proprietary electronic medical record database and a database with information on nationally dispensed vaccine doses. The monthly number of first doses of HPV vaccinations in boys and girls aged 9-17 years in the period from 01/2018 to 12/2021 was determined. In addition, for boys the cumulative vaccination rates were calculated for initiated and completed vaccination series. RESULTS: Four months after the introduction of mandatory reimbursement for boys, the monthly numbers of first doses were comparable to that of girls. Compared to the same month in 2019, the number of first doses declined by up to 49% (girls) in 2020 and 71% (boys) in 2021. At the end of 2021, the vaccination rate for 15-year-old boys (2006 birth cohort) reached 44.4% for initiated and 26.4% for completed series. CONCLUSION: After an initial dynamic increase in HPV vaccinations in boys, the impact of COVID-19 was particularly strong in the second year of the pandemic. At the end of 2021 vaccination rates were still low. Efforts are needed to catch-up on adolescents that missed doses during the pandemic and to increase uptake.

Healthcare costs of congenital cytomegalovirus (cCMV) disease in infants during the first two years of life: a retrospective German claims database analysis.

BACKGROUND: Congenital cytomegalovirus (cCMV) infection can cause severe neurological damage, growth retardation, hearing loss, and microcephaly in infants. We aimed at assessing healthcare costs of infants with recorded cCMV diagnosis in an administrative claims database in the first 2 years of life. METHODS: We conducted a retrospective, controlled cohort study using German claims data from the Institute for Applied Health Research Berlin (InGef) database. Incremental healthcare costs during the first and second year of life were assessed by matching (1:60) infants with cCMV diagnoses ≤ 90 days after birth (cCMV90 cohort) to infants without cCMV diagnosis ("representative" controls) and infants with cCMV diagnoses ≤ 21 days after birth plus specific symptoms (cCMV21-S) to infants without cCMV and any ICD-10-GM records (besides Z00-Z99) until 4th preventive health check-up ("healthy" controls). Due to missing data, mean imputation was applied for aids and remedies costs. RESULTS: We identified 54 and 24 infants born 2014-2018 for the cCMV90 and cCMV21-S cohorts, respectively. During the first year, mean (median) healthcare costs were significantly higher in cCMV90 cases vs. "representative" controls (€22,737 (€9759) vs. €3091 (€863), p < 0.001), with 87.2% inpatient costs. Healthcare costs for cCMV21-S cases compared to "healthy" controls were €34,498 (€20,924) vs. €680 (€569), p < 0.001. Differences decreased for both comparisons in the second year but remained statistically significant. CONCLUSIONS: cCMV comprises a considerable economic burden for the German healthcare system (€19,646 to €33,818 higher mean costs for infants with recorded cCMV diagnosis in the first year of life). Attempts should be made to reduce this burden.

Healthcare resource utilization and costs in 23-25-year-old women with human papillomavirus (HPV) associated anogenital diseases in Germany - a retrospective analysis of statutory health insurance claims data.

BACKGROUND: Human papilloma virus (HPV) causes multiple anogenital diseases including cervical cancer and is the most common sexually transmitted infection. Healthcare resource utilization (HRU) associated with HPV-related anogenital diseases includes diagnostic and disease specific treatment regimens. A recent study showed disease burden of young women aged 23-25 years, who were the first populations eligible to receive HPV vaccination after its introduction in Germany. Cost for the German statutory health insurance (SHI) due to HPV­related anogenital diseases in this population are unknown. This study aimed at assessing HRU and costs related to HPV-associated anogenital diseases for the Germany SHI. METHODS: We used a retrospective, matched cohort design to leverage the prior identified cohort of 23-25-year-old women born between 1989-1992 diagnosed with HPV-related anogenital disease from the Institute for Applied Health Research Berlin (InGef) Research Database. German SHI claims data from 2012-2017 were analyzed. The prior identified cases were matched (direct, without replacement) to women without anogenital diseases (1:10 ratio). HRU and costs for inpatient care, outpatient care, and pharmaceutical during a 3-year observation period were determined for both cases and controls and increments between the groups were assessed. RESULTS: 2,972 women diagnosed with anogenital diseases (cases) who were matched to 29,720 women without anogenital diseases (controls). Cases had more outpatient visits (52.4 visits vs. 39.2 visits) and more cases (45.2% vs. 31.7%) were hospitalized at least once in the 3­year observation period. Most common outpatient procedures performed in cases were conization of the cervix uteri (4.4% cases; n < 5 controls), followed by other excision and destruction of diseased tissue of the cervix uteri (3.1% in cases; 0.0% in controls). Median difference in total healthcare costs of €684 (mean difference: €1,089, 95%CI: €752-1,426) suggest that HPV-related anogenital diseases were responsible for approximately €3.2 Million more healthcare costs for the identified cases in the four birth cohorts within the 3­year observation period in the InGef Research Database. Costs were mainly driven by outpatient care (41.6% of total costs). CONCLUSION: In Germany, HPV-related anogenital diseases among young women are associated with considerable HRU and financial expenditures, mostly driven by outpatient care.

Burden of CIN2+ diagnoses and conizations in women aged 18-45 years-a retrospective secondary data analysis of German statutory health insurance claims data.

PURPOSE: High grade cervical intraepithelial neoplasia (CIN2+) may progress to cervical cancer. They may be detected by screening and are usually treated by conization. This study aimed at assessing annual proportions of screening, prevalent and incident CIN2+ diagnoses, as well as proportions of (re-)conizations during 24 months follow-up after conization in Germany. METHODS: A descriptive retrospective claims data analysis of the years 2013-2018 was conducted using the InGef Research Database. Women aged 18-45 years with CIN2+ diagnoses were identified by ICD-10-GM codes (N87.1, N87.2, D06.-, and C53.-). Cervical conizations were identified by OPS codes (5-671.0* or 5-671.1*). Screening participation was identified by EBM codes (01730, 01733, 32819 or 32820). Annual proportions were calculated as women with the respective documented codes divided by all women in the respective age group per calendar year. RESULTS: Overall annual proportions of screened women spanned from 60.01 to 61.33% between 2013 and 2018. The overall annual prevalence of CIN2+ diagnoses (regardless of screening participation) ranged from 0.72 to 0.84% between 2013 and 2018, with highest proportions observed in women aged 27-45 years. Also, CIN2+ incidence was highest in women 27-45 years. Annual proportion of women undergoing conization was 0.24% in 2013 and 0.21% in 2018. During a 24-month follow-up period after conization, 2.91% of women underwent a re-conization 3 months or later after the initial conization. CONCLUSION: This analysis demonstrates a considerable burden of CIN2+, conizations and re-conizations in Germany, especially in women aged 27-45 years. This highlights the need for intensified prevention efforts such as expanding human papillomavirus (HPV) vaccination.

Burden of HPV related anogenital diseases in young women in Germany - an analysis of German statutory health insurance claims data from 2012 to 2017.

BACKGROUND: Most individuals are infected with human papillomavirus (HPV) at least once in their lifetime. Infections with low-risk types can cause genital warts, whereas high-risk types can cause malignant tumors. The aim of this study was to determine the burden of anogenital diseases potentially related to HPV in young women based on German statutory health insurance claims data. METHODS: We conducted a retrospective claims data analysis using the "Institute for Applied Health Research Berlin" (InGef) Research Database, containing claims data from approximately 4 million individuals. In the period from 2012 to 2017 all women born in1989-1992, who were continuously insured between the age of 23-25 years were identified. Using ICD-10-GM codes (verified diagnosis in the outpatient sector or primary or secondary diagnosis in the inpatient sector) the administrative prevalence (95% confidence interval) of genital warts (A63.0), anogenital diseases grade I (K62.8, N87.0, N89.0, N90.0), grade II (N87.1, N89.1, N90.1) and grade III (D01.3, D06.-, D06.0, D07.1, D07.2, N87.2, N89.2, N90.2) was calculated (women with diagnosis divided by all women). RESULTS: From 2012 to 2017, a total of 15,358 (birth cohort 1989), 16,027 (birth cohort 1990), 14,748 (birth cohort 1991) and 14,862 (birth cohort 1992) women at the age of 23-25 were identified. A decrease of the administrative prevalence was observed in genital warts (1.30% (1.12-1.49) birth cohort 1989 vs. 0.94% (0.79-1.10) birth cohort 1992) and anogenital diseases grade III (1.09% (0.93-1.26) birth cohort 1989 vs. 0.71% (0.58-0.86) birth cohort 1992). In anogenital diseases grade III, this trend was especially observed for severe cervical dysplasia (N87.2) (0.91% (0.76-1.07) birth cohort 1989 vs. 0.60% (0.48-0.74) birth cohort 1992). In contrast, anogenital diseases grade I (1.41% (1.23-1.61) birth cohort 1989 vs. 1.31% (1.14-1.51) birth cohort 1992) and grade II (0.61% (0.49-0.75) birth cohort 1989 vs. 0.52% (0.42-0.65) birth cohort 1992) remained stable. CONCLUSIONS: A decrease of the burden of anogenital disease potentially related to HPV was observed in the younger birth cohorts. This was observed especially for genital warts and anogenital diseases grade III. Further research to investigate this trend for the upcoming years in light of varying HPV vaccination coverage for newer birth cohorts is necessary.

Diagnostic accuracy of p16INK4a immunohistochemistry in oropharyngeal squamous cell carcinomas: A systematic review and meta-analysis.

The accurate diagnosis of human papillomavirus (HPV) causality in oropharyngeal squamous cell carcinomas (OPSCC) is likely to influence therapeutic decisions in affected patients in the near future. We conducted a systematic review and meta-analysis to determine the diagnostic accuracy of p16INK4a immunohistochemistry (IHC) to identify HPV-induced OPSCC. We identified all studies that performed p16INK4a IHC (index test) and HPV E6/E7 mRNA detection using an amplification-based method (gold standard to indicate a transforming relevance of HPV) in OPSCC. Testing with one or more comparator tests (HPV DNA PCR, HPV DNA in situ hybridization (ISH) and p16INK4a IHC/HPV DNA PCR combined testing) was an optional criterion for inclusion. Among 1,636 retrieved studies 24 fulfilled the inclusion criteria. The pooled sensitivity of p16INK4a IHC, HPV DNA PCR, HPV DNA ISH and p16INK4a IHC/HPV DNA PCR combined testing was 94% (95%-confidence interval (CI) 91-97%), 98% (CI 94-100%), 85% (CI 76-92%) and 93% (CI 87-97%), respectively. The pooled specificity was 83% (CI 78-88%), 84% (CI 74-92%), 88% (CI 78-96%) and 96% (CI 89-100%), respectively. p16INK4a IHC/HPV DNA PCR combined testing was as sensitive as either p16INK4a IHC or HPV DNA PCR alone but significantly more specific than either separate test. In conclusion, p16INK4a IHC is highly sensitive but moderately specific to diagnose HPV-transformed OPSCC when used as a single test. Combined p16INK4a IHC and HPV DNA PCR testing significantly enhances specificity while maintaining high sensitivity. This diagnostic test combination thus represents an attractive testing strategy for the reliable diagnosis of HPV-induced OPSCC in the clinical setting and may constitute an inclusion criterion for future therapeutic trials.

Human papillomavirus association is the most important predictor for surgically treated patients with oropharyngeal cancer.

BACKGROUND: Upfront surgery is a valuable treatment option for oropharyngeal squamous cell carcinoma (OPSCC) and risk stratification is emerging for treatment de-escalation in human papillomavirus (HPV)-related OPSCC. Available prognostic models are either based on selected, mainly non-surgically treated cohorts. Therefore, we investigated unselected OPSCC treated with predominantly upfront surgery. METHODS: All patients diagnosed with OPSCC and treated with curative intent between 2000 and 2009 (n=359) were included. HPV association was determined by HPV-DNA detection and p16INK4a immunohistochemistry. Predictors with significant impact on overall survival (OS) in univariate analysis were included in recursive partitioning analysis. RESULTS: Risk models generated from non-surgically treated patients showed low discrimination in our cohort. A new model developed for unselected patients predominantly treated with upfront surgery separates low-, intermediate- and high-risk patients with significant differences in 5-year OS (86%, 53% and 19%, P<0.001, respectively). HPV status is the most important parameter followed by T-stage in HPV-related and performance status in HPV-negative OPSCC. HPV status and ECOG remained important parameters in risk models for patients treated with or without surgery. CONCLUSIONS: Regardless of treatment strategies, HPV status is the strongest predictor of survival in unselected OPSCC patients. The proposed risk models are suitable to discriminate risk groups in unselected OPSCC patients treated with upfront surgery, which has substantial impact for design and interpretation of de-escalation trials.

HPV-independent Differentiated Vulvar Intraepithelial Neoplasia (dVIN) is Associated With an Aggressive Clinical Course.

Differentiated vulvar intrapeithelial neoplasia (dVIN) is an human papillomavirus (HPV)-independent precursor of squamous cell carcinoma (SCC), and the aim of this study was to better characterize its natural history. Cases of dVIN were identified from the pathology archives. Outcomes of patients with dVIN only, without associated invasive SCC, were compared with a cohort of patients with high-grade squamous intraepithelial lesion [HSIL(VIN3)]. Eighteen patients diagnosed with dVIN with adjacent invasive SCC (SCC/dVIN) and 7 patients with dVIN only, without invasive carcinoma, were identified. Mean age in both cohorts was 75 yr. All lesions but 1 were unifocal. In 35% of SCC/dVIN cases the surgical resection margins were positive for SCC, with 75% and 60% having margins positive for dVIN in the SCC/dVIN and dVIN-only cohorts, respectively. In total, 23/25 women with dVIN only or dVIN/SCC, for whom there was follow-up information, experienced either progression to or recurrence of invasive SCC, respectively, at a median of 1.1 yr, including all but 1 case of dVIN only, where the median time of progression to invasive SCC was 1.9 yr. A total of 22/25 women died of disease with a median overall survival of 3.4 yr. The outcome (i.e. progression to invasive carcinoma) of patients with dVIN only was significantly worse than that of a comparison group of 18 patients with HSIL(VIN3) (progression-free survival log-rank, P<0.001; disease-specific survival, P=0.04; overall survival, P=0.01). Six of 7 patients with dVIN only developed invasive carcinoma on follow-up, compared with 0 of 18 patients with HSIL(VIN3). The diagnosis of dVIN indicates the presence of a high-risk human papillomavirus-negative precursor of invasive SCC. These patients are likely to progress to invasive carcinoma over a relatively short period, at which point their prognosis is guarded.

CD56-positive lymphocyte infiltration in relation to human papillomavirus association and prognostic significance in oropharyngeal squamous cell carcinoma.

Human papillomavirus (HPV)-related squamous cell carcinoma of the oropharynx (OSCC) are clinical and biological distinct from their HPV-unrelated counterparts. Patients with HPV-related OSCC display improved prognosis and therefore we investigated possible immune cell infiltrations associated with this tumor phenotype. We retrospectively analyzed a randomly selected cohort of 140 OSCC for presence of immune cells and HPV by immunohistochemistry and PCR followed by bead-based hybridization (Luminex technology). HPV prevalence was 24.3% as determined by positive staining of p16INK4a and detection of high risk HPV-DNA. We found significantly higher numbers of CD56 positive (CD56+) cells in tumor and surrounding microenvironment in HPV-associated compared to HPV-negative OSCC (t-test: p = 0.004 and p = 0.002). For the entire cohort presence of CD56+ cells was associated with increased overall survival independent from HPV (Kaplan-Meier: p = 0.002; Cox regression: p = 0.042). Presence of CD56+ cells also correlated with a better outcome in HPV-negative and especially in HPV-negative OSCC with alcohol consumption ≤ 2 standard drinks per day (Kaplan-Meier: p = 0.05 and p = 0.003). Immunofluorescence localization of granular Granzyme B (GZMB) within CD56+ cells and coexpression of CD16 and CD56 suggests that detected CD56+ cells mainly represent cytotoxic Natural Killer (NK) cells. The fraction of potentially cytotoxic NK cells was significantly higher in HPV-associated compared to HPV-negative OSCC (Mann-Whitney-U-Test: p = 0.011). The elevated abundance and activity of cytotoxic NK cells in OSCC with HPV driven carcinogenesis might contribute to favorable outcome in HPV-related OSCC.

A phase 1/2a study to test the safety and immunogenicity of a p16(INK4a) peptide vaccine in patients with advanced human papillomavirus-associated cancers.

BACKGROUND: The cyclin-dependent kinase inhibitor p16(INK4a) is strongly and consistently overexpressed in all human papillomavirus (HPV)-associated cancers. Therefore, the authors hypothesized that p16(INK4a) may be a vaccine target antigen for HPV-associated cancers. To test this hypothesis, the authors performed a phase 1/2a first-in-human trial to evaluate the safety and immunogenicity of a p16(INK4a) -based peptide vaccine. METHODS: A total of 26 patients with different, advanced, p16(INK4a) -overexpressing, HPV DNA-positive cancers were included after the completion of standard treatment. According to protocol, 12 subcutaneous injections of a p16(INK4) peptide (P16_37-63) mixed in a water-in-oil emulsion with immunoadjuvant activity (Montanide ISA-51 VG) were administered over a 6-month period. RESULTS: A total of 20 patients received at least 4 injections and were evaluable for immune responses against P16_37-63. Clusters of differentiation (CD) 4 T cells were detected in 14 of 20 patients (3 of whom had preexisting CD4 T cells before vaccination), CD8 T cells were detected in 5 of 20 patients, and antibodies were detected in 14 of 20 patients (1 of whom had preexisting antibodies). No suspected unexpected serious adverse reaction or serious adverse drug reaction was documented. All reported serious adverse events were expected and not considered to be related to study therapy. None of the patients discontinued trial participation due to unacceptable toxicities and no dose-limiting toxicities occurred. Tumor response could be assessed in 14 patients. Of these, 9 patients (64%) had stable disease as their best overall response and 5 patients (36%) developed progressive disease. CONCLUSIONS: Vaccination with the p16(INK4a) -derived peptide P16_37-63 appears to induce cellular and humoral immune responses and does not cause severe toxicities. The results of the current study pave the way for the further clinical development of p16(INK4a) -based cancer immunotherapeutics. Cancer 2016;122:1425-1433. © 2016 American Cancer Society.

Oncogenic human papillomaviruses activate the tumor-associated lens epithelial-derived growth factor (LEDGF) gene.

The expression of the human papillomavirus (HPV) E6/E7 oncogenes is crucial for HPV-induced malignant cell transformation. The identification of cellular targets attacked by the HPV oncogenes is critical for our understanding of the molecular mechanisms of HPV-associated carcinogenesis and may open novel therapeutic opportunities. Here, we identify the Lens Epithelial-Derived Growth Factor (LEDGF) gene as a novel cellular target gene for the HPV oncogenes. Elevated LEDGF expression has been recently linked to human carcinogenesis and can protect tumor cells towards different forms of cellular stress. We show that intracellular LEDGF mRNA and protein levels in HPV-positive cancer cells are critically dependent on the maintenance of viral oncogene expression. Ectopic E6/E7 expression stimulates LEDGF transcription in primary keratinocytes, at least in part via activation of the LEDGF promoter. Repression of endogenous LEDGF expression by RNA interference results in an increased sensitivity of HPV-positive cancer cells towards genotoxic agents. Immunohistochemical analyses of cervical tissue specimens reveal a highly significant increase of LEDGF protein levels in HPV-positive lesions compared to histologically normal cervical epithelium. Taken together, these results indicate that the E6/E7-dependent maintenance of intracellular LEDGF expression is critical for protecting HPV-positive cancer cells against various forms of cellular stress, including DNA damage. This could support tumor cell survival and contribute to the therapeutic resistance of cervical cancers towards genotoxic treatment strategies in the clinic.

The clinical impact of using p16(INK4a) immunochemistry in cervical histopathology and cytology: an update of recent developments.

Cervical cancer screening test performance has been hampered by either lack of sensitivity of Pap cytology or lack of specificity of Human Papillomavirus (HPV) testing. This uncertainty can lead to unnecessary referral and treatment, which is disturbing for patients and increases costs for health care providers. The identification of p16(INK4a) as a marker for neoplastic transformation of cervical squamous epithelial cells by HPVs allows the identification of HPV-transformed cells in histopathology or cytopathology specimens. Diagnostic studies have demonstrated that the use of p16(INK4a) immunohistochemistry substantially improves the reproducibility and diagnostic accuracy of histopathologic diagnoses. p16(INK4a) cytology has substantially higher sensitivity for detection of cervical precancer in comparison to conventional Pap tests. Compared to HPV DNA tests, immunochemical detection of p16(INK4a) -stained cells demonstrates a significantly improved specificity with remarkably good sensitivity. About 15 years after the initial observation that p16(INK4a) is overexpressed in HPV-transformed cells we review the accumulated clinical evidence suggesting that p16(INK4a) can serve as a useful biomarker in the routine diagnostic work up of patients with HPV infections and associated lesions of the female anogenital tract.

p16(INK4a) /Ki-67 co-expression specifically identifies transformed cells in the head and neck region.

p16(INK4a) immunohistochemical overexpression is an overall reliable surrogate marker of human papillomavirus (HPV)-associated head and neck squamous cell carcinomas (HNSCC). However, cases of ambiguous p16(INK4a) overexpression are regularly detected in the head and neck: p16(INK4a) expression can be observed in non-malignant tissue, such as tonsillar crypt epithelium and a proportion of branchial cleft cysts. Additionally, diverse patterns of p16(INK4) expression can complicate interpretation of "p16(INK4a) -positivity". These aspects impede the unrestricted application of p16(INK4a) as a diagnostic marker in the head and neck. We hypothesized that combined detection of p16(INK4a) and the proliferation marker Ki-67 could support clarification of ambiguous p16(INK4a) expression in the head and neck by specifically indicating p16(INK4a) -expressing cells with proliferative activity. p16(INK4a) /Ki-67 co-expression in a combined staining procedure was correlated to distinct p16(INK4a) expression patterns and HPV status (HPV DNA followed by E6*I oncogene mRNA detection) in 147 HNSCC and 50 non-malignant head and neck samples. p16(INK4a) /Ki-67 co-expression only occurred in transformed cells of the head and neck. Co-expression was never detected in non-transformed cells. Combined p16(INK4a) /Ki-67 expression was stringently associated with a diffuse p16(INK4a) expression pattern. All HPV oncogene-expressing HNSCC showed p16(INK4a) /Ki-67 co-expression. We demonstrate that p16(INK4a) /Ki-67 co-expression occurs exclusively in transformed cells of the head and neck. Our findings indicate a substantial impact of combined p16(INK4a) /Ki-67 expression in the assessment of ambiguous p16(INK4a) expression in the head and neck by specifically identifying p16(INK4a) -expressing cells with proliferative activity. This property will be of considerable significance for head and neck histo- and cytopathology.

Methylation status of HPV16 E2-binding sites classifies subtypes of HPV-associated oropharyngeal cancers.

BACKGROUND: The human papillomavirus (HPV) E2 protein is a transcriptional repressor of the oncogenes E6/E7 and loss of E2 function is considered a key step in carcinogenesis. Integration of HPV into the host genome may disrupt the E2 gene. Furthermore, methylation of CpG dinucleotides in E2-binding sites (E2BSs) in the HPV upstream regulatory region may interfere with transcriptional repression of E6 and E7 by E2. The authors hypothesized that the CpG methylation status of E2BS identifies subtypes of HPV type 16 (HPV16)-associated oropharyngeal squamous cell cancers (OPSCC) in association with E2 gene integrity and viral integration. METHODS: Methylation of 10 CpG dinucleotides within the upstream regulatory region, encompassing E2BSs 1, 2, 3, and 4, was quantitatively analyzed by bisulfite pyrosequencing in 57 HPV16-associated OPSCC cases. E2 status was analyzed by gene amplification and quantitative real-time reverse transcriptase-polymerase chain reaction. Viral integration was determined by integration-specific polymerase chain reaction methods. RESULTS: Three subgroups with differential methylation at E2BS3 and E2BS 4 were identified: 1) complete methylation (>80%) associated with the presence of integrated HPV genomes with an intact E2 gene; 2) intermediate methylation levels (20%-80%) with predominantly episomal HPV genomes with intact E2; and 3) no methylation (<20%) with a disrupted E2 gene. Patients with high methylation levels tended to have a worse 5-year overall survival compared with patients with intermediate methylation (hazard ratio, 3.23; 95% confidence interval, 1.13-9.24 [P = .06]). CONCLUSIONS: Methylation of E2BS3 and E2BS4 in OPSCC is associated with E2 integrity and viral physical status. It might explain deregulated viral oncogene expression in the presence of E2. The prognostic significance of E2BS methylation for patients with HPV-associated OPSCC needs to be analyzed further.

Association of high CD4-positive T cell infiltration with mutations in HLA class II-regulatory genes in microsatellite-unstable colorectal cancer.

Besides being expressed on professional antigen-presenting cells, HLA class II antigens are expressed on various tumors of non-lymphoid origin, including a subset of colorectal cancers (CRC). Information about the regulation of HLA class II antigen expression is important for a better understanding of their role in the interactions between tumor and immune cells. Whether lack of HLA class II antigen expression in tumors reflects the selective immune destruction of HLA class II antigen-expressing tumor cells is unknown. To address this question, we tested whether lack of HLA class II antigen expression in CRC was associated with immune cell infiltration. We selected microsatellite-unstable (MSI-H) CRC, because they show pronounced tumor antigen-specific immune responses and, in a subset of tumors, lack of HLA class II antigen expression due to mutations inactivating HLA class II-regulatory genes. We examined HLA class II antigen expression, mutations in regulatory genes, and CD4-positive T cell infiltration in 69 MSI-H CRC lesions. Mutations in RFX5, CIITA, and RFXAP were found in 13 (28.9%), 3 (6.7%), and 1 (2.2%) out of 45 HLA class II antigen-negative tumors. CD4-positive tumor-infiltrating lymphocyte counts were significantly higher in HLA class II antigen-negative tumors harboring mutations in HLA class II-regulatory genes (107.4 T cells per 0.25 mm(2)) compared to tumors without mutations (55.5 T cells per 0.25 mm(2), p = 0.008). Our results suggest that the outgrowth of tumor cells lacking HLA class II antigen expression due to mutations of regulatory genes is favored in an environment of dense CD4-positive T cell infiltration.

Targeted deep sequencing of mucinous ovarian tumors reveals multiple overlapping RAS-pathway activating mutations in borderline and cancerous neoplasms.

BACKGROUND: Mucinous ovarian tumors represent a distinct histotype of epithelial ovarian cancer. The rarest (2-4 % of ovarian carcinomas) of the five major histotypes, their genomic landscape remains poorly described. We undertook hotspot sequencing of 50 genes commonly mutated in human cancer across 69 mucinous ovarian tumors. Our goals were to establish the overall frequency of cancer-hotspot mutations across a large cohort, especially those tumors previously thought to be "RAS-pathway alteration negative", using highly-sensitive next-generation sequencing as well as further explore a small number of cases with apparent heterogeneity in RAS-pathway activating alterations. METHODS: Using the Ion Torrent PGM platform, we performed next generation sequencing analysis using the v2 Cancer Hotspot Panel. Regions of disparate ERBB2-amplification status were sequenced independently for two mucinous carcinoma (MC) cases, previously established as showing ERBB2 amplification/overexpression heterogeneity, to assess the hypothesis of subclonal populations containing either KRAS mutation or ERBB2 amplification independently or simultaneously. RESULTS: We detected mutations in KRAS, TP53, CDKN2A, PIK3CA, PTEN, BRAF, FGFR2, STK11, CTNNB1, SRC, SMAD4, GNA11 and ERBB2. KRAS mutations remain the most frequently observed alteration among MC (64.9 %) and mucinous borderline tumors (MBOT) (92.3 %). TP53 mutation occurred more frequently in carcinomas than borderline tumors (56.8 % and 11.5 %, respectively), and combined IHC and mutation data suggest alterations occur in approximately 68 % of MC and as many as 20 % of MBOT. Proven and potential RAS-pathway activating changes were observed in all but one MC. Concurrent ERBB2 amplification and KRAS mutation were observed in a substantial number of cases (7/63 total), as was co-occurrence of KRAS and BRAF mutations (one case). Microdissection of ERBB2-amplified regions of tumors harboring KRAS mutation suggests these alterations are occurring in the same cell populations, while consistency of KRAS allelic frequency in both ERBB2 amplified and non-amplified regions suggests this mutation occurred in advance of the amplification event. CONCLUSIONS: Overall, the prevalence of RAS-alteration and striking co-occurrence of pathway "double-hits" supports a critical role for tumor progression in this ovarian malignancy. Given the spectrum of RAS-activating mutations, it is clear that targeting this pathway may be a viable therapeutic option for patients with recurrent or advanced stage mucinous ovarian carcinoma, however caution should be exercised in selecting one or more personalized therapeutics given the frequency of non-redundant RAS-activating alterations.

A multiplex method for the detection of serum antibodies against in silico-predicted tumor antigens.

Humoral immune responses against tumor antigens are studied as indirect markers of antigen exposure and in cancer vaccine studies. An increasing number of tumor antigens potentially translated from mutant genes is identified by advances in genomic sequencing. They represent an interesting source for yet unknown immunogenic epitopes. We here describe a multiplex method using the Luminex technology allowing for the detection of antibodies against multiple in silico-predicted linear neo-antigens in large sets of sera. The approach included 32 synthetic biotinylated peptides comprising a predicted set of frameshift mutation-induced neo-antigens. The antigens were fused to a FLAG epitope to ensure monitoring antigen binding to avidin-linked microspheres in the absence of monoclonal antibodies. Analytical specificity of measured serum antibody reactivity was proven by the detection of immune responses in immunized rabbits and a colorectal cancer patient vaccinated with peptides included in the assay. The measured antibody responses were comparable to peptide ELISA, and inter-assay reproducibility of the multiplex approach was excellent (R (2) > 0.98) for 20 sera tested against all antigens. Our methodic approach represents a valuable platform to monitor antibody responses against predicted antigens. It may be used in individualized cancer vaccine studies, thereby extending the relevance beyond the model system in the presented approach.

The APTIMA HPV assay versus the Hybrid Capture 2 test in triage of women with ASC-US or LSIL cervical cytology: a meta-analysis of the diagnostic accuracy.

Testing for DNA of 13 high-risk HPV types with the Hybrid Capture 2 (HC2) test has consistently been shown to perform better in triage of women with cervical cytology results showing atypical squamous cells of undetermined significance (ASC-US) but often not in triage of low-grade squamous intraepithelial lesions (LSIL) detected in cervical cancer screening. In a meta-analysis, we compared the accuracy of the APTIMA HPV test, which identifies RNA of 14 high-risk HPV types, to HC2 for the triage of women with ASC-US or LSIL. Literature search-targeted studies where the accuracy of APTIMA HPV and HC2 for detection of underlying CIN2/3+ was assessed concomitantly including verification of all cases of ASC-US and LSIL. HSROC (Hierarchical Summary ROC) curve regression was used to compute the pooled absolute and relative sensitivity and specificity. Eight studies, comprising 1,839 ASC-US and 1,887 LSIL cases, were retrieved. The pooled sensitivity and specificity of APTIMA to triage ASC-US to detect underlying CIN3 or worse was 96.2% (95% CI = 91.7-98.3%) and 54.9% (95% CI = 43.5-65.9%), respectively. APTIMA and HC2 showed similar pooled sensitivity; however, the specificity of the former was significantly higher (ratio: 1.19; 95% CI = 1.08-1.31 for CIN2+). The pooled sensitivity and specificity of APTIMA to triage LSIL were 96.7% (95% CI = 91.4-98.9%) and 38.7% (95% CI = 30.5-47.6%) for CIN3+. APTIMA was as sensitive as HC2 but more specific (ratio: 1.35; 95% CI = 1.11-1.66). Results were similar for detection of CIN2 or worse. In both triage of ASC-US and LSIL, APTIMA is as sensitive but more specific than HC2 for detecting cervical precancer.

Differential methylation of E2 binding sites in episomal and integrated HPV 16 genomes in preinvasive and invasive cervical lesions.

Enhanced expression of the HPV 16 E6-E7 oncogenes may trigger neoplastic transformation of the squamous epithelial cells at the uterine cervix. The HPV E2 protein is a key transcriptional regulator of the E6-E7 genes. It binds to four E2 binding sites (E2BSs 1-4) in the viral upstream regulatory region (URR). Modification of E2 functions, for example, by methylation of E2BSs is hypothesized to trigger enhanced expression of the viral E6-E7 oncogenes. In the majority of HPV-transformed premalignant lesions and about half of cervical carcinomas HPV genomes persist in an extra-chromosomal, episomal state, whereas they are integrated into host cells chromosomes in the remaining lesions. Here we compared the methylation profile of E2BSs 1-4 of the HPV 16 URR in a series of 18 HPV16-positive premalignant lesions and 33 invasive cervical cancers. CpGs within the E2BSs 1, 3, and 4 were higher methylated in all lesions with only episomal HPV16 genomes compared with lesions displaying single integrated copies. Samples with multiple HPV16 integrated copies displayed high methylation levels for all CpGs suggesting that the majority of multiple copies were silenced by extensive methylation. These data support the hypothesis that differential methylation of the E2BSs 1, 3 and 4 is related to the activation of viral oncogene expression in cervical lesions as long as the viral genome remains in the episomal state. Once the virus becomes integrated into host cell chromosomes these methylation patterns may be substantially altered due to complex epigenetic changes of integrated HPV genomes.