Investigating the validity of web-enabled mechanistic case diagramming scores to assess students' integration of foundational and clinical sciences.

As medical schools have changed their curricula to address foundational and clinical sciences in a more integrated fashion, teaching methods such as concept mapping have been incorporated in small group learning settings. Methods that can assess students' ability to apply such integrated knowledge are not as developed, however. The purpose of this project was to assess the validity of scores on a focused version of concept maps called mechanistic case diagrams (MCDs), which are hypothesized to enhance existing tools for assessing integrated knowledge that supports clinical reasoning. The data were from the medical school graduating class of 2018 (N = 136 students). In 2014-2015 we implemented a total of 16 case diagrams in case analysis groups within the Mechanisms of Health and Disease (MOHD) strand of the pre-clinical curriculum. These cases were based on topics being taught during the lectures and small group sessions for MOHD. We created an overall score across all 16 cases for each student. We then correlated these scores with performance in the preclinical curriculum [as assessed by overall performance in MOHD integrated foundational basic science courses and overall performance in the Clinical and Professional Skills (CAPS) courses], and standardized licensing exam scores [United States Medical Licensing Exam (USMLE)] Step 1 (following core clerkships) and Step 2 Clinical Knowledge (at the beginning of the fourth year of medical school). MCD scores correlated with students' overall basic science scores (r = .46, p = .0002) and their overall performance in Clinical and Professional Skills courses (r = .49, p < .0001). In addition, they correlated significantly with standardized exam measures, including USMLE Step 1 (r = .33, p ≤ .0001), and USMLE Step 2 CK (r = .39, p < .0001). These results provide preliminary validity evidence that MCDs may be useful in identifying students who have difficulty in integrating foundational and clinical sciences.

Interferon treatment of human keratinocytes harboring extrachromosomal, persistent HPV-16 plasmid genomes induces de novo viral integration.

Interferons (IFNs) have been used to treat epithelial lesions caused by human papillomavirus (HPV) persistence. Here, we exposed primary human keratinocytes (HFKs) immortalized by persistently replicating HPV-16 plasmid genomes to increasing levels of IFN-γ. While untreated HFKs retained replicating HPV-16 plasmids for up to 60-120 population doublings, IFN led to rapid HPV-16 plasmid loss. However, treated cultures eventually gave rise to outgrowth of clones harboring integrated HPV-16 genomes expressing viral E6 and E7 oncogenes from chimeric virus-cell mRNAs similar to those in cervical and head and neck cancers. Surprisingly, every HPV-16 integrant that arose after IFN exposure stemmed from an independent integration event into a different cellular gene locus, even within parallel cultures started from small cell inocula and cultured separately for ≥ 25 doublings to permit the rise and expansion of spontaneous integrants. While IFN treatment conferred a growth advantage upon preexisting integrants added to mixed control cultures, our results indicate that IFN exposure directly or indirectly induces HPV-16 integration, rather than only selects preexisting, spontaneous integrants that appear to be much less frequent. We estimate that IFN exposure increased integration rates by ≥ 100-fold. IFN-induced HPV-16 integration involved a wide range of chromosomal loci with less apparent selection for recurrent insertions near genes involved in cancer-related pathways. We conclude that IFNs and other potential treatments targeting high-risk HPV persistence that disrupt viral genome replication may promote increased high-risk HPV integration as a step in cancer progression. Therapies against high-risk HPV persistence thus need to be evaluated for their integration-inducing potential.

Human papillomavirus type 16 (HPV-16) genomes integrated in head and neck cancers and in HPV-16-immortalized human keratinocyte clones express chimeric virus-cell mRNAs similar to those found in cervical cancers.

Many human papillomavirus (HPV)-positive high-grade lesions and cancers of the uterine cervix harbor integrated HPV genomes expressing the E6 and E7 oncogenes from chimeric virus-cell mRNAs, but less is known about HPV integration in head and neck cancer (HNC). Here we compared viral DNA status and E6-E7 mRNA sequences in HPV-16-positive HNC tumors to those in independent human keratinocyte cell clones derived from primary tonsillar or foreskin epithelia immortalized with HPV-16 genomes. Three of nine HNC tumors and epithelial clones containing unintegrated HPV-16 genomes expressed mRNAs spliced from HPV-16 SD880 to SA3358 and terminating at the viral early gene p(A) signal. In contrast, most integrated HPV genomes in six HNCs and a set of 31 keratinocyte clones expressed HPV-16 major early promoter (MEP)-initiated mRNAs spliced from viral SD880 directly to diverse cellular sequences, with a minority spliced to SA3358 followed by a cellular DNA junction. Sequence analysis of chimeric virus-cell mRNAs from HNC tumors and keratinocyte clones identified viral integration sites in a variety of chromosomes, with some located in or near growth control genes, including the c-myc protooncogene and the gene encoding FAP-1 phosphatase. Taken together, these findings support the hypothesis that HPV integration in cancers is a stochastic process resulting in clonal selection of aggressively expanding cells with altered gene expression of integrated HPV genomes and potential perturbations of cellular genes at or near viral integration sites. Furthermore, our results demonstrate that this selection also takes place and can be studied in primary human keratinocytes in culture.

Risk factors and survival by HPV-16 E6 and E7 antibody status in human papillomavirus positive head and neck cancer.

High-risk human papillomavirus types (HPV-HR) are associated with head and neck cancer (HNC) risk and better survival. Most patients with HPV-HR DNA-positive tumors develop anti-HPV E6/E7 antibodies; however, it is unclear whether those who mount an immune response have similar risk factors or clinical outcomes as those who do not. HPV-16 DNA tumor-positive HNC cases were evaluated for HPV-16 E6 and E7 antibodies using a GST capture ELISA system. Among 57 HPV-16 DNA tumor-positive HNC cases, 67% were detected with HPV-16 E6 and/or E7 antibodies. Male gender (76% vs. 42%, p = 0.02), younger age (63% vs. 16%, p = 0.001) but not tobacco or alcohol were associated with E6 and/or E7 seropositivity. Seropositivity was associated more often with late stage (76%), poor grade (65%), positive nodes (82%). and in the oropharynx (82%), Median disease-specific and recurrence-free survival were longer in E6 and/or E7 seropositive compared to E6/E7-negative cases (2.2 years vs. 1.4 years, both outcomes), although results were not statistically significant. When examined jointly with p16 expression, E6 and/or E7-positive/p16-positive cases had better disease-specific (2.1 years vs. 1.1 years, p = 0.06) and recurrence-free (2.3 years vs. 1.1 years, p = 0.03) survival compared to E6-/E7-/p16- cases. These findings suggest there are 2 distinct HNC patient groups with HPV DNA-positive tumors, distinguishable by E6 and/or E7 antibody status. Differences in antibody status are associated with distinct risk factors and clinical outcomes. This information can be available as a simple blood test at initial presentation, before the removal of tissue through biopsy or surgery.

Upstream regulatory region alterations found in human papillomavirus type 16 (HPV-16) isolates from cervical carcinomas increase transcription, ori function, and HPV immortalization capacity in culture.

Human papillomavirus (HPV) DNAs isolated from cervical and head and neck carcinomas frequently contain nucleotide sequence alterations in the viral upstream regulatory region (URR). Our study has addressed the role such sequence changes may play in the efficiency of establishing HPV persistence and altered keratinocyte growth. Genomic mapping of integrated HPV type 16 (HPV-16) genomes from 32 cervical cancers revealed that the viral E6 and E7 oncogenes, as well as the L1 region/URR, were intact in all of them. The URR sequences from integrated and unintegrated viral DNA were found to harbor distinct sets of nucleotide substitutions. A subset of the altered URRs increased the potential of HPV-16 to establish persistent, cell growth-altering viral-genome replication in the cell. This aggressive phenotype in culture was not solely due to increased viral early gene transcription, but also to augmented initial amplification of the viral genome. As revealed in a novel ori-dependent HPV-16 plasmid amplification assay, the altered motifs that led to increased viral transcription from the intact genome also greatly augmented HPV-16 ori function. The nucleotide sequence changes correlate with those previously described in the distinct geographical North American type 1 and Asian-American variants that are associated with more aggressive disease in epidemiologic studies and encompass, but are not limited to, alterations in previously characterized sites for the negative regulatory protein YY1. Our results thus provide evidence that nucleotide alterations in HPV regulatory sequences could serve as potential prognostic markers of HPV-associated carcinogenesis.

Human papillomavirus (HPV) type 18 induces extended growth in primary human cervical, tonsillar, or foreskin keratinocytes more effectively than other high-risk mucosal HPVs.

Mucosal high-risk (HR) human papillomaviruses (HPVs) that cause cervical and other anogenital cancers also are found in approximately 25% of head and neck carcinomas (HNCs), especially those arising in the oropharynx and the tonsils. While many HR HPV types are common in anogenital cancer, over 90% of HPV-positive HNCs harbor HPV type 16 (HPV-16). Using a quantitative colony-forming assay, we compared the ability of full-length mucosal HPV genomes, i.e., the low-risk HPV-11 and HR HPV-16, -18, and -31, to persist in and alter the growth of primary human keratinocytes from the foreskin, cervix, and tonsils. The HR HPV types led to the formation of growing keratinocyte colonies in culture independent of the site of epithelial origin. However, HPV-18 induced colony growth in all keratinocytes >4-fold more effectively than HPV-16 or HPV-31 and >20-fold more efficiently than HPV-11 or controls. HPV-11-transfected or control colonies failed to expand beyond 32 to 36 population doublings postexplantation. In contrast, individual HR HPV-transfected clones exhibited no apparent slowdown of growth or "crisis," and many maintained HPV plasmid persistence beyond 60 population doublings. Keratinocyte clones harboring extrachromosomal HR HPV genomes had shorter population doubling times and formed dysplastic stratified epithelia in organotypic raft cultures, mirroring the pathological features of higher-grade intraepithelial lesions, yet did not exhibit chromosomal instability. We conclude that, in culture, the HR HPV type, rather than the site of epithelial origin of the cells, determines the efficacy of inducing continued growth of individual keratinocytes, with HPV-18 being the most aggressive mucosal HR HPV type tested.

Tobacco and alcohol use increases the risk of both HPV-associated and HPV-independent head and neck cancers.

Tobacco, alcohol, and human papillomavirus (HPV) are major risk factors for head and neck cancer (HNC), but it is unclear whether there are two distinct HNC risk groups, one associated with HPV and the other with tobacco/alcohol. Because HPV-positive HNC are clinically distinct from HPV-negative cases in treatment response and with more favorable prognoses, determining whether these differences result from infection alone or in association with other HNC risk factors is important for developing future therapeutic strategies. Incident cases of HNC (n = 201) and age-gender frequency-matched controls (n = 324) were recruited to assess anti-HPV VLP (virus like particles) antibodies 16, 18, 31, and 33. Multivariate logistic regression and stratified analyses were used to calculate adjusted odds ratios (OR). HPV-seronegative and seropositive/heavy tobacco users had similar increased adjusted risks of HNC (HPV-seronegative OR = 2.6, 1.4-5.0; HPV-seropositive OR = 2.3, 1.1-4.8), as did HPV-seronegative (OR = 4.3, 2.1-9.1) versus HPV-seropositive/heavy alcohol users (OR = 3.9, 1.6-9.4). Similar HPV/tobacco/alcohol risk profiles also were seen in oropharyngeal and oral cavity tumor sites. Our finding that tobacco/alcohol use increased the risk of HNC in both HPV-seropositive and HPV-seronegative individuals is consistent with the observation that HPV infection is not a sufficient cause of HNC but requires the accumulation of additional cellular changes.

Evidence for vertical transmission of HPV from mothers to infants.

Few large studies have evaluated concordance based on a broad spectrum of human papillomavirus (HPV) types in oral and genital specimens of mothers and their recently born infants. This information is important in determining whether HPV vaccines administered prior to pregnancy may be useful for preventing vertical transmission. HPV DNA was positive in 30% of mothers and 1.5% of newborns. Maternal/newborn concordance (HPV+/+ or HPV-/-) was 71%. Among HPV DNA+ mothers, only 3% of their infants were DNA+ and only 1 pair had the same HPV type. Among HPV- women, 0.8% of infants were HPV+. HPV DNA detected in hospitalized newborns reflects current infection transmitted to infants during pregnancy or delivery. None of the mother/baby HPV DNA+ concordance pairs detected viral types found in HPV vaccines suggesting that vaccination prior to pregnancy is unlikely to be efficacious in preventing vertical transmission.

Interferon-beta treatment increases human papillomavirus early gene transcription and viral plasmid genome replication by activating interferon regulatory factor (IRF)-1.

Interferons (IFNs) have been used to treat mucosal lesions caused by human papillomavirus (HPV) infection, such as intraepithelial precursor lesions to cancer of the uterine cervix, genital warts or recurrent respiratory papillomatosis, to potentially reduce or eliminate replicating HPV plasmid genomes. Mucosal HPVs have evolved mechanisms that impede IFN-beta synthesis and downregulate genes induced by IFN. Here we show that these HPV types directly subvert a cellular transcriptional response to IFN-beta as a potential boost in infection. Treatment with low levels of human IFN-beta induced initial amplification of HPV-16 and HPV-11 plasmid genomes and increased HPV-16 or HPV-31 DNA copy numbers up to 6-fold in HPV-immortalized keratinocytes. IFN treatment also increased early gene transcription from the major early gene promoters in HPV-16, HPV-31 and HPV-11. Furthermore, mutagenesis of the viral genomes and ectopic interferon regulatory factor (IRF) expression in transfection experiments using IRF-1(-/-), IRF-2(-/-) and dual knockout cell lines determined that these responses are due to the activation of IRF-1 interaction with a conserved interferon response element demonstrated in several mucosal HPV early gene promoters. Our results provide a molecular explanation for the varying clinical outcomes of IFN therapy of papillomatoses and define an assay for the modulation of the HPV gene program by IFNs as well as other cytokines and signaling molecules in infection and therapy.

Cellular factor YY1 downregulates the human papillomavirus 16 E6/E7 promoter, P97, in vivo and in vitro from a negative element overlapping the transcription-initiation site.

Cellular factors that bind to cis sequences in the human papillomavirus 16 (HPV-16) upstream regulatory region (URR) positively and negatively regulate the viral E6 and E7 oncogene promoter, P97. DNase I footprinting has revealed the binding of cellular proteins to two previously undetected cis elements overlapping and 3' of the transcription-initiation site of the P97 promoter. Mutations within homologous motifs found in both of these cis elements abolished their negative function in vivo and the binding of the same cellular complex in vitro. This factor was identified as YY1 by complex mobility and binding specificity in comparison with vaccinia virus-expressed, purified recombinant YY1 protein and by antigenic reactivity with YY1 antisera. Cis mutations in the 'initiator' YY1 site activated the P97 promoter in vivo and in vitro. P97 was also activated threefold in vitro by depletion of endogenous YY1 with wild-type, but not mutant, YY1 oligonucleotides from the IgH kappa E3' enhancer. Furthermore, increasing concentrations of exogenous, purified recombinant YY1 repressed wild-type P97 transcript levels by up to threefold, but did not influence the P97 promoter mutated in the 'initiator' YY1 site. Thus, the promoter-proximal YY1 site was not necessary for correct transcription initiation at the P97 promoter, but was found to be required for downregulation of P97 transcription in vivo and in vitro. In contrast to other viral and cellular promoters, where YY1 is thought to function as a positive transcription-'initiator' factor, HPV-16 P97 transcription is downregulated by YY1 from a critical motif overlapping the transcription start site.

Functional mapping of the human papillomavirus type 16 E1 cistron.

Replication of the double-stranded, circular human papillomavirus (HPV) genomes requires the viral DNA replicase E1. Here, we report an initial characterization of the E1 cistron of HPV type 16 (HPV-16), the most common oncogenic mucosal HPV type found in cervical and some head and neck cancers. The first step in HPV DNA replication is an initial burst of plasmid viral DNA amplification. Complementation assays between HPV-16 genomes carrying mutations in the early genes confirmed that the expression of E1 was necessary for initial HPV-16 plasmid synthesis. The major early HPV-16 promoter, P97, was dispensable for E1 production in the initial amplification because cis mutations inactivating P97 did not affect the trans complementation of E1- mutants. In contrast, E1 expression was abolished by cis mutations in the splice donor site at nucleotide (nt) 226, the splice acceptor site at nt 409, or a TATAA box at nt 7890. The mapping of 5' mRNA ends using rapid amplification of cDNA ends defined a promoter with a transcription start site at HPV-16 nt 14, P14. P14-initiated mRNA levels were low and required intact TATAA (7890). E1 expression required the HPV-16 keratinocyte-dependent enhancer, since cis mutations in its AP-2 and TEF-1 motifs abolished the ability of the mutant genomes to complement E1- genomes, and it was further modulated by origin-proximal and -distal binding sites for the viral E2 gene products. We conclude that P14-initiated E1 expression is critical for and limiting in the initial amplification of the HPV-16 genome.

The E8--E2 gene product of human papillomavirus type 16 represses early transcription and replication but is dispensable for viral plasmid persistence in keratinocytes.

A conserved E8(wedge)E2 spliced mRNA is detected in keratinocytes transfected with human papillomavirus type 16 (HPV-16) plasmid DNA. Expression of HPV-16 E8--E2 (16-E8--E2) is independent of the major early promoter, P97, and is modulated by both specific splicing events and conserved cis elements in the upstream regulatory region in a manner that differs from transcriptional regulation of other early viral genes. Mutations that disrupt the predicted 16-E8--E2 message also increase initial HPV-16 plasmid amplification 8- to 15-fold and major early gene (P97) transcription 4- to 5-fold over those of the wild type (wt). Expressing the 16-E8--E2 gene product from the cytomegalovirus (CMV) promoter represses HPV-16 early gene transcription from P97 in a dose-dependent manner, as detected by RNase protection assays. When expressed from the CMV promoter, 16-E8--E2 also inhibits the amplification of an HPV-16 plasmid and a heterologous simian virus 40 (SV40) ori plasmid that contains E2 binding sites in cis. In contrast, cotransfections with HPV-16 wt genomes that express physiologic levels of 16-E8--E2 are sufficient to repress HPV-16 plasmid amplification but are limiting and insufficient for the repression of SV40 amplification. 16-E8--E2-dependent repression of HPV-16 E1 expression is sufficient to account for this observed inhibition of initial HPV-16 plasmid amplification. Unlike with other papillomaviruses, primary human keratinocytes immortalized by the HPV-16 E8 mutant genome contain more than eightfold-higher levels of unintegrated plasmid than the wt, demonstrating that 16-E8(wedge)E2 limits the viral copy number but is not required for plasmid persistence and maintenance.

Fundamental differences in cell cycle deregulation in human papillomavirus-positive and human papillomavirus-negative head/neck and cervical cancers.

Human papillomaviruses (HPV) are associated with nearly all cervical cancers, 20% to 30% of head and neck cancers (HNC), and other cancers. Because HNCs also arise in HPV-negative patients, this type of cancer provides unique opportunities to define similarities and differences of HPV-positive versus HPV-negative cancers arising in the same tissue. Here, we describe genome-wide expression profiling of 84 HNCs, cervical cancers, and site-matched normal epithelial samples in which we used laser capture microdissection to enrich samples for tumor-derived versus normal epithelial cells. This analysis revealed that HPV(+) HNCs and cervical cancers differed in their patterns of gene expression yet shared many changes compared with HPV(-) HNCs. Some of these shared changes were predicted, but many others were not. Notably, HPV(+) HNCs and cervical cancers were found to be up-regulated in their expression of a distinct and larger subset of cell cycle genes than that observed in HPV(-) HNC. Moreover, HPV(+) cancers overexpressed testis-specific genes that are normally expressed only in meiotic cells. Many, although not all, of the hallmark differences between HPV(+) HNC and HPV(-) HNC were a direct consequence of HPV and in particular the viral E6 and E7 oncogenes. This included a novel association of HPV oncogenes with testis-specific gene expression. These findings in primary human tumors provide novel biomarkers for early detection of HPV(+) and HPV(-) cancers, and emphasize the potential value of targeting E6 and E7 function, alone or combined with radiation and/or traditional chemotherapy, in the treatment of HPV(+) cancers.

Association between p53 and human papillomavirus in head and neck cancer survival.

BACKGROUND: High-risk human papillomavirus (HPV-HR) is a significant risk factor for head and neck cancer (HNC), abrogating normal p53 function. In addition, HPV and p53 have been associated with prognosis of these tumors but the findings have been inconsistent. We examined p53 expression and HPV-HR individually and jointly for differences in predicting HNC survival. METHODS: HNC patients (n = 294) were evaluated for p53 by immunohistochemical staining. HPV was detected by PCR/dot blot hybridization and sequencing. RESULTS: HNC tumors showed 48% with p53 overexpression and 27% with HPV-HR. Multivariate analyses showed that p53 positivity was significantly associated with higher risk of disease-specific [hazard ratio (HR); 2.0; 95% confidence interval (95% CI), 1.1-3.7] and recurrence-free mortality (HR, 2.8; 95% CI, 1.4-5.3). HPV- cases had significantly worse disease-specific survival (HR, 2.8; 95% CI, 1.3-6.3) compared with HPV-HR cases. When analyzed jointly, with p53(-)/HPV-HR tumors as the reference group, p53(+)/HPV(-) patients had the worst disease-specific (HR, 5.3; 58% versus 15%, P = 0.006) and recurrence-free survival rates (HR, 9.5; 17% versus 89%, P = 0.001), in contrast to the p53(-)/HPV(-) and p53(+)/HPV-HR groups, which had less elevated and different risks for disease-specific survival (HR, 2.5 and 1.7, respectively) and recurrence-free survival (HR, 4.2 and 7.2, respectively). CONCLUSION: Joint assessment of p53/HPV status provides different HRs for each clinical outcome in the four biomarker groups that are distinct from the individual biomarkers. These findings suggest that joint assessment of p53/HPV provides a better indicator of prognosis and potentially different types of treatments.

Does pretreatment seropositivity to human papillomavirus have prognostic significance for head and neck cancers?

BACKGROUND: Human papillomavirus (HPV) is a risk factor for head and neck cancers (HNC), yet HPV-associated tumors have better prognosis than HPV-negative tumors. METHODS: We evaluated whether pretreatment presence of antibodies to HPV capsids [virus-like particles (VLP)] or to HPV-16 oncoproteins E6 and E7 was a predictor of HPV-positive HNC and clinical outcomes. Sera from 156 HNC patients were tested for antibodies to HPV-16-derived antigens using ELISA. HPV-16 in tumors was evaluated by PCR and DNA sequencing. RESULTS: HPV-16 antibodies were found in 33% with HPV-16 VLP, 21% with HPV-16 E6, and 21% with E7. HPV-16 was detected in 26% of tumors. There was a strong correlation between detection of HPV-16 tumor DNA and antibodies to HPV-16 E6 or E7 (kappa = 0.7) but not to HPV-16 VLP (kappa = 0.4). Multivariate analyses showed significantly better disease-specific survival in seropositive HPV-16 VLP [hazard ratio (HR), 0.4; 95% confidence interval (95% CI), 0.1-0.9], HPV-16 E6 (HR, 0.1; 95% CI, 0.02-0.5), and HPV-16 E7 (HR, 0.3; 95% CI, 0.1-0.9) cases. Less disease recurrence occurred among those with antibodies to both E6 and E7 compared with those negative to both (P = 0.003). There was better disease-specific survival in patients who were E6 positive at baseline and remained positive at follow-up compared with individuals who were E6 negative at both time points (P = 0.03; kappa = 0.9). CONCLUSIONS: The presence of antibodies to HPV-16 E6 and E7 is associated with HPV in tumor cells and with better clinical outcomes. These findings suggest that the presence of E6/E7 antibodies before treatment is predictive of better clinical outcomes and that they may serve as biomarkers for selecting targeted therapeutic modalities developed for HPV-associated tumors.

A Web-based database for pathology faculty effort reporting.

To ensure appropriate mission-based budgeting and equitable distribution of funds for faculty salaries, our compensation committee developed a pathology-specific effort reporting database. Principles included the following: (1) measurement should be done by web-based databases; (2) most entry should be done by departmental administration or be relational to other databases; (3) data entry categories should be aligned with funding streams; and (4) units of effort should be equal across categories of effort (service, teaching, research). MySQL was used for all data transactions (http://dev.mysql.com/downloads), and scripts were constructed using PERL (http://www.perl.org). Data are accessed with forms that correspond to fields in the database. The committee's work resulted in a novel database using pathology value units (PVUs) as a standard quantitative measure of effort for activities in an academic pathology department. The most common calculation was to estimate the number of hours required for a specific task, divide by 2080 hours (a Medicare year) and then multiply by 100. Other methods included assigning a baseline PVU for program, laboratory, or course directorship with an increment for each student or staff in that unit. With these methods, a faculty member should acquire approximately 100 PVUs. Some outcomes include (1) plotting PVUs versus salary to identify outliers for salary correction, (2) quantifying effort in activities outside the department, (3) documenting salary expenditure for unfunded research, (4) evaluating salary equity by plotting PVUs versus salary by sex, and (5) aggregating data by category of effort for mission-based budgeting and long-term planning.

P16INK4a expression, human papillomavirus, and survival in head and neck cancer.

Development of head and neck cancer (HNC) is associated with human papillomavirus high-risk (HPV-HR) types. The HPV E7 oncoprotein inactivates the pRB protein increasing expression of p16(INK4a). P16 expression and HPV status have been associated with differences in clinical outcomes for HNC. This study examined whether HNC prognosis was different when these biomarkers were examined as individual or joint molecular effects. Tumor tissue from 301 HNC patients were analyzed and sequenced for HPV types. P16 was evaluated by immunohistochemical staining. p16 was expressed in 35% and HPV-HR was detected in 27% of HNC patients. After adjustment for age, tobacco, and alcohol, p16+ tumors were statistically significantly associated with HPV-HR (OR=13.3, 7.1-24.9), histology, stage, grade, tumor site, and node involvement. Compared to p16+ HNC cases, those who did not express p16 had significantly worse disease-specific (DS) survival (Hazards Ratio, adj.HR=2.0. 1.0-3.9) and recurrence (adj.HR=3.6, 1.6-8.2); and HPV- cases had worse DS survival (adj.HR=2.8, 1.1-7.1) and recurrence (adj.HR=2.0, 0.8-4.8) compared to HPV-HR patients. Each of the p16/HPV groups had different survival outcomes: p16+/HPV-HR cases (referent) had the best and p16-/HPV- cases had the worst DS survival (adj.HR=3.6; 53% versus 13%, p=0.004) whereas p16+/HPV- and p16-/HPV-HR had similar DS survival (adj.HR=2.7/2.8). p16-/HPV-HR cases had a worse recurrence rate (adj.HR=7.0; 60% versus 0%, referent, p=0.08) than p16-/HPV- (adj.HR=4.5) or p16+/HPV- (adj.HR=1.8) cases. The combined p16/HPV biomarker data are associated with different survival outcomes of HNC compared to each marker evaluated separately, indicating that the two molecular mechanisms evaluated together may provide a more accurate prediction of clinical outcomes.

Web-Enabled Mechanistic Case Diagramming: A Novel Tool for Assessing Students' Ability to Integrate Foundational and Clinical Sciences.

PROBLEM: As medical schools move from discipline-based courses to more integrated approaches, identifying assessment tools that parallel this change is an important goal. APPROACH: The authors describe the use of test item statistics to assess the reliability and validity of web-enabled mechanistic case diagrams (MCDs) as a potential tool to assess students' ability to integrate basic science and clinical information. Students review a narrative clinical case and construct an MCD using items provided by the case author. Students identify the relationships among underlying risk factors, etiology, pathogenesis and pathophysiology, and the patients' signs and symptoms. They receive one point for each correctly identified link. OUTCOMES: In 2014-2015 and 2015-2016, case diagrams were implemented in consecutive classes of 150 medical students. The alpha reliability coefficient for the overall score, constructed using each student's mean proportion correct across all cases, was 0.82. Discrimination indices for each of the case scores with the overall score ranged from 0.23 to 0.51. In a G study using those students with complete data (n = 251) on all 16 cases, 10% of the variance was true score variance, and systematic case variance was large. Using 16 cases generated a G coefficient (relative score reliability) equal to 0.72 and a Phi equal to 0.65. NEXT STEPS: The next phase of the project will involve deploying MCDs in higher-stakes settings to determine whether similar results can be achieved. Further analyses will determine whether these assessments correlate with other measures of higher-order thinking skills.

Prevalence of human papillomavirus in the oral cavity/oropharynx in a large population of children and adolescents.

OBJECTIVE: Human papillomavirus (HPV) in the oral cavity or oropharynx is associated with an increased risk of laryngeal papillomatosis, head and neck cancer, and cervical and other genital cancers. We evaluated the prevalence of HPV DNA in the oral cavity/oropharynx in a cross section of children aged 2 weeks to 20 years. METHODS: A risk factor questionnaire and oral exfoliated cells were collected from children (N = 1235). HPV DNA was detected using PCR, dot blot hybridization, and DNA sequencing. RESULTS: The HPV prevalence was 1.9% in the oral cavity/oropharynx of children. A bimodal age distribution was observed with the highest HPV prevalence in the youngest and oldest groups: 2.5% aged <1 year, 0.8% aged 1 to 4 years, 1.2% aged 5 to 11 years, 1.5% in aged 12 to 15 years, and 3.3% in aged 16 to 20 years. The prevalence of the HPV quadrivalent vaccine types (HPV-6, 11, 16, 18) reached 0.9% in the 16- to 20-year age group. In this age group, female gender [odds ratio (OR): 6.9, P = 0.04], genital warts (OR: 19.3, P < 0.01), and current smoker (OR: 6.5, P = 0.01) were associated with a higher risk of being detected with an oral HPV infection. No risk factors in parents were identified with transmission of HPV to infants. CONCLUSIONS: The age-specific prevalence rates of HPV in this large cross section of children and adolescents demonstrate that HPV infection is acquired gradually in childhood. These data support a target age for HPV vaccination before puberty to prevent serious HPV-related genital and oral diseases.

Head and neck cancer associated with herpes simplex virus 1 and 2 and other risk factors.

We investigated whether herpes simplex viruses, HSV-1 and HSV-2, are cofactors of head and neck cancer (HNC) in association with tobacco, alcohol, or HPV-16 infection. The study included 164 HNC cases and 295 controls. Serologic tests were used to distinguish HSV-1 and HSV-2. Antibodies to anti-VLP HPV-16 and HPV-16 E6 and E7 were evaluated by ELISA. After adjusting for age, tobacco, alcohol use, and number of sexual partners, risk of cancer was not significantly increased in those with HSV-1 [adjusted odds ratio (OR)=0.7] or HSV-2 (OR=0.8) compared to HSV-negative patients. Although heavy use of tobacco, alcohol and HPV-16 infection was associated with an increased risk of HNC, the adjusted risk among those infected with HSV-1 or HSV-2 lowered the odds compared to those who were not infected. Heavy smokers (OR=1.7) and heavy drinkers infected with HSV-1 (OR=4.2) or HSV-2 (smokers: OR=1.6; drinkers: OR=3.2) had lower odds compared to seronegative HSV-1 heavy users (smokers: OR=2.5; drinkers: OR=5.5) or HSV-2 (smokers: OR=1.9; drinkers: OR=6.2). Those seropositive to HPV-16 E6 and/or E7 but not HSV-1 (OR=27.4) or HSV-2 (OR=18.0) had higher risk of HNC compared to those infected with HSV-1 (OR=16.7) or HSV-2 (not estimable). These findings suggest that seropositivity to HSV-1 and HSV-2, although not independent risk factors for HNC, may modify the risk of HNC associated with exposure to tobacco, alcohol, or HPV-HR.