Lisa M. Schumaker

Background: Human papillomavirus (HPV)‐associated cancers are preventable with vaccination. Serologic assays monitor antibody responses, but their complexity and cost pose limitations when evaluating population level immunity. We developed an L1‐based assay for the detection of multiple type‐specific HPV antibodies. Methods: The Luminex‐based assay used recombinant L1 proteins from seven HPVs (HPV‐6/11/16/18/35/45/58) for the detection of type‐specific immunoglobulin (Ig)G and IgA. Cutoffs were established using plasma from infants and children. The assay was optimized and qualified using a sample set from a blinded proficiency panel developed at the Frederick National Laboratory for Cancer Research. Results: Sensitivity and specificity for anti‐HPV‐6/11/16/18/58 IgG were 100% at the Mean + 3 standard deviation [SD] and Mean + 5 SD thresholds. Sensitivity for HPV‐45 was 100% and 92.9% at the +3 SD and +5 SD thresholds, respectively. IgA detection showed moderate positivity among vaccinated individuals, with peak estimates of 96.4% (HPV‐11) and 85.7% (HPV‐58) at the +3 SD threshold. IgA was correlated with IgG (Spearman's ρ = 0.74). The L1‐assay differentiated high, intermediate, low, and negative response groups from the blinded samples for all HPV genotypes ( p < 0.0001). Conclusion: The L1‐binding assay has the potential to be a scalable option for high‐throughput detection of broad‐spectrum HPV immunoglobulins, but further standardization and validation are needed.

Radiomics has the potential to improve risk stratification in head and neck squamous cell carcinoma (HNSCC), but clinical adoption is limited by inconsistent performance across institutions. A key source of variability is how radiomic features are generated, preprocessed, and selected prior to model development. This multicenter study evaluated how radiomics parameterization and feature selection strategies affect external model performance, feature stability, and time-to-event risk stratification. We studied pre-treatment CT scans from 752 patients with primary HNSCC from three hospitals. For each scan, 1648 radiomic features were computed using 20 different preparation methods that varied in scaling, outlier removal, and gray-level bin width. We compared five feature selection methods: Graph-FS with connected components, Boruta, Lasso, RFE-RF, and mRMR. The classification models used were Random Forest, XGBoost, CatBoost, and Logistic Regression. We measured performance using external ROC-AUC, bootstrap confidence intervals, Brier score, and RobustScore. Stability of feature selection was assessed using the Kuncheva and Jaccard indices. Cox proportional hazards models confirmed time-to-event results, and consensus SHAP analysis helped explain the models. Radiomics parameterization influenced model performance, and no single configuration was optimal across all analyses. Radiomics-only models outperformed clinical-only models, while clinical–radiomics models achieved the highest overall performance. mRMR and Lasso produced the highest average external AUCs, while Graph-FS showed the greatest stability. The best classification model achieved an external AUC of 0.817. In Cox validation, the best clinical–radiomics configuration achieved an external C-index of 0.662 and separated high- and low-risk patients in the external cohort.

Background: Oropharyngeal cancers associated with oral human papillomavirus (oHPV) are becoming more common. Understanding factors associated with oHPV among African men, including those living in settings where broader contextual factors shape how HPV-associated cancers are prioritized, may help predict future cancer risk and inform screening interventions. Methods: From 2014 to 2018, a cross-sectional study of the prevalence of oHPV was conducted among men who have sex with men (MSM) in Abuja and Lagos, Nigeria. Participants completed HIV testing and behavioral questionnaires before providing oral gargle samples. Gargle samples were genotyped for oHPV and oral high-risk HPV (oHR-HPV16/18/31/33/35/39/45/51/52/56/58/59/68). Multivariable logistic regression estimated the adjusted odds ratio (aOR) and 95% confidence intervals (CIs). Results: Among 584 men, median age was 24 years (interquartile range: 22–28) and 69% were living with HIV. The prevalence of oHPV, oHR-HPV and HPV16 was 16.6% (95% CI: 13.7–19.9), 5.7% (95% CI: 3.9–7.8), and 1.2% (95% CI: 0.5–2.5), respectively. Living with HIV was positively associated with oHPV (aOR: 1.97, 95% CI: 1.14–3.41) and trended similarly for oHR-HPV (aOR: 2.30, 95% CI: 0.93–5.66). Oral HPV was associated with having insertive anal sex (aOR: 1.85, 95% CI: 1.06–3.24) as compared to both insertive and receptive. Oral HPV was associated with having sex with women over 5 years ago (aOR: 3.13, 95% CI: 1.28–7.67) but not in the past 5 years (aOR: 1.70, 95% CI: 0.91–3.18) as compared to no sex with women. Oral HR-HPV was independently associated with insertive anal sex (aOR: 2.59, 95% CI: 1.11–6.08). Conclusions: Living with HIV was significantly associated with oHPV prevalence, though oHR-HPV prevalence was lower than expected. Longitudinal studies are needed to define risk pathways for oropharyngeal cancer, particularly in settings where social and structural contexts may affect access to appropriate care.

Our next generation sequencing (NGS)-based human papillomavirus (HPV) genotyping assay showed a high degree of concordance with the Roche Linear Array (LA) with as little as 1.25 ng formalin-fixed paraffin-embedded-derived genomic DNA in head and neck and cervical cancer samples. This sensitive genotyping assay uses barcoded HPV PCR broad-spectrum general primers 5+/6+ (BSGP)5+/6+ applicable to population studies, but it’s diagnostic performance has not been tested in cases with multiple concurrent HPV infections. We conducted a cross-sectional study to compare the positive and negative predictive value (PPV and NPV), sensitivity and specificity of the NGS assay to detect HPV genotype infections as compared to the LA. DNA was previously extracted from ten anal swab samples from men who have sex with men in Nigeria enrolled on the TRUST/RV368 cohort study. Two-sample tests of proportions were used to examine differences in the diagnostic performance of the NGS assay to detect high vs. low-risk HPV type-specific infections. In total there were 94 type-specific infections detected in 10 samples with a median of 9.5, range (9 to 10) per sample. Using the LA as the gold standard, 84.4% (95% CI: 75.2–91.2) of the same anal type-specific infections detected on the NGS assay had been detected by LA. The PPV and sensitivity differed significantly for high risk (PPV: 90%, 95% CI: 79.5–96.2; sensitivity: 93.1%, 95% CI: 83.3–98.1) as compared to low risk HPV (PPV: 73%, 95% CI: 54.1–87.7; sensitivity: 61.1, 95% CI: 43.5–76.9) (all p < 0.05). The NPV for all types was 92.5% (95% CI: 88.4–95.4). The NPV and specificity were similar for high and low risk HPVs (all p > 0.05). The NGS assay detected 10 HPV genotypes that were not among the 37 genotypes found on LA (30, 32, 43, 44, 74, 86, 87, 90, 91, 114). The NGS assay accurately detects multiple HPV infections in individual clinical specimens with limited sample volume and has extended coverage compared to LA.

While we previously reported a striking racial difference in the prevalence of human papilloma virus (HPV)–positive squamous cell carcinoma of the oropharynx (OPSCC), less is known about differences in outcomes and trends over time in OPSCC by HPV status and race. We conducted a retrospective analysis of 467 patients with OPSCC treated at the University of Maryland Greenebaum Cancer Center (Baltimore, MD) between 1992 and 2007, of which 200 had tissue available for HPV16 testing. HPV16-positive patients were significantly more likely to be white, with 45.5% of whites and 15.5% of blacks testing positive for HPV16. There was a significant increase in HPV16-positive OPSCC for all patients over time from 15.6% in 1992 to 1995 to 43.3% in 2004 to 2007 (P = 0.01). From 1992 to 1995, 33% of white patients were HPV16-positive, with no black patients positive. From 2004 to 2007, 17.7% of black patients and 54% of white patients were HPV16-positive. White and black patients with HPV16-positive tumors had an identical and favorable overall survival (OS; median, 8.1 and 8.1 years, respectively). However, among HPV16-negative patients, whites had an improved OS compared with blacks (median, 2.3 vs. 0.9 years, respectively; P = 0.02), including when analyzed in a multivariable Cox regression model. From 1992 to 2007, the percentage of HPV16-positive OPSCC increased for white patients and was seen for the first time in black patients. While survival for HPV-positive black and white patients was similar and favorable, outcomes for HPV-negative patients were poor, with blacks having worse survival even after controlling for baseline characteristics.Cancer Prev Res; 8(1); 12–19. ©2014 AACR. See related article by E. Cohen and C. Fakhry, p. 9

Background We conducted a phase I dose escalation study to evaluate the safety and immunologic response to peptide immunomodulatory vaccines GL-0810 (HPV16) and GL-0817 (MAGE-A3) in HPV16 and MAGE-A3-positive RM–SCCHN patients, respectively. Methods Three dose levels (500, 1,000, and 1,500 µg) of GL-0810 or GL-0817 with adjuvants Montanide (1.2 ml) and GM-CSF (100 µg/m2) were administered subcutaneously q2 weeks for a total of four vaccinations in HPV16 and MAGE-A3-positive RM–SCCHN patients, respectively. Results Nine and seven patients were enrolled in the HPV16 and MAGE-A3 cohorts, respectively. No dose-limiting toxicities were observed, and toxicity was predominantly local and grade 1 (erythema, pain, and itching at the injection site). In those patients who received all four vaccinations, 80 % (4/5) of the HPV16 cohort and 67 % (4/6) of the MAGE-A3 cohort developed antigen-specific T cell and antibody responses to the vaccine. Significant concordance between T cell and antibody responses was observed for both groups. No clear dose–response correlation was seen. All patients progressed by RECIST at first repeat imaging, except for one patient in the MAGE-A3 500 µg cohort who had stable disease for 10.5 months. The median PFS and OS for the MAGE-A3 cohorts were 79 and 183 days, respectively, and for the HPV16 cohort 80 and 196 days, respectively. Conclusions GL-0810 and GL-0817 were well tolerated in patients with RM–SCCHN with T cell and antibody responses observed in the majority of patients who received all four vaccinations.

Background: Racial outcome disparities have been observed in head and neck squamous cell carcinoma (HNSCC) with diminished survival for black patients compared with white patients.Methods: We retrospectively analyzed 1318 patients with primary HNSCC treated at the University of Maryland Greenebaum Cancer Center (UMGCC) from 2000 to 2010.Results: Of all the patients, 65.9% were white, 30.7% were black, and 3.3% were of other races. Black patients were less likely to present with oral cavity cancer, and more likely to present with laryngeal or hypopharyngeal cancers. White patients were more likely to have early stage disease, especially in the oral cavity. Black race was independently associated with worse overall survival (OS) in the entire cohort. Black patients had a significantly worse OS among oral cavity and oropharyngeal cancers, with the largest disparity in oropharyngeal cancer. However, in multivariate analysis, race was only still significant in oropharyngeal cancer.Conclusion: We observed differences by race in distribution of disease site, stage, and OS. Survival disparity in the entire cohort was driven mostly by differences among oropharyngeal cancer. © 2015 Wiley Periodicals, Inc. Head Neck 38: 564–572, 2016

HPV associated HNSCC has emerged as a rapidly growing virus associated cancer in recent years. Studies show that the burden of HPV positive HNSCC is lower in African Americans compared to White Americans and the increase in incidence over time has been statistically significant only for White Americans. Differences in prevalence of oral sex behaviors have been blamed for this. There has been no previous study of the secular trend in HPV associated HNSCC in African population. We used QIAamp DNA FFPE Tissue Kit to extract DNA from Formalin fixed, Paraffin-Embedded (FFPE) HNC cancers blocks from 3 tertiary hospitals in Nigeria. We identified HPV types using Roche LINEAR ARRAY® HPV Genotyping Test. We examined 115 Head and Neck Cancers collected over 22 years (1990–2011); 94 (82%) were eligible for DNA core analysis, DNA purification was successful in 84 (73%). PCR amplification was successful in 49 (43%), Linear Array amplification was successful in 17 of these cases resulting in an overall success rate of 15%. These were from the larynx (6), cervical lymph nodes (3), nasal cavity (2), parotid (1), palate (1), maxillary sinus (1) and mandible (1). Two were non-specific head and neck masses and none from the oropharynx. Forty-one percent (7) were squamous cell carcinomas (larynx 6, maxillary sinus 1). None were found to be positive for HPV of any type. HPV associated HNSCC seems low in this population, this may be a result of a birth cohort effect and also because oral sex rates are believed to be low in this population. Public health education on the role of oral sex with multiple partners in the etiology of HPV associated HNSCC may mitigate future rise in incidence of HNSCC in this population. Study limitations are the small sample size and the low success rate for PCR and Linear Array amplification due to poor sample quality. Future studies with large sample size and improved sampling methods are required to aid improved understanding of the epidemiology of HPV associated HNSCC and of disparities in incidence and outcome among different populations.

BACKGROUND: New treatment strategies for locally advanced head and neck squamous cell carcinoma combine induction chemotherapy and chemoradiation. Identifying the predictors of outcome in sequentially treated patients is critical for focusing therapeutic research. In this analysis, the authors evaluated human papillomavirus type 16 (HPV‐16) status and the expression levels of a defined set of biomarkers to identify predictors of response to this treatment modality.METHODS: In total, 114 patients with oropharyngeal cancer (OPC) who were treated on the TAX 324 trial (cisplatin and fluorouracil with or without docetaxel in patients with locally advanced head and neck squamous cell carcinoma) had pretreatment biopsy specimens that were evaluable for HPV‐16 DNA and immunohistochemical expression of the following biomarkers: beta‐tubulin II (βT‐II), glutathione S‐transferase (GST‐π), p53, and B‐cell lymphoma 2 (Bcl‐2). Patients were categorized into risk groups based on their HPV status and biomarker expression levels.RESULTS: Patients with high‐risk OPC were defined by HPV‐negative status and either elevated expression of βT‐II or levels of at least 2 of the other 3 adverse markers (elevated GST‐π, elevated p53, or low Bcl‐2). All other HPV‐negative patients were categorized as moderate risk. In total, 55 patients were HPV‐positive, and 59 patients were HPV‐negative, with 34 were categorized as high risk and 25 categorized as moderate risk. The median survival for HPV‐positive patients was not reached. The median survival was 44.2 months for moderate‐risk patients (95% confidence interval, 20.9 months to not reached) and 12.1 months for high‐risk patients (95% confidence interval, 7.5‐19.7 months). The 24‐month survival rate was 89% for HPV‐positive patients, 67% for moderate‐risk patients, and 29% for high‐risk patients (P < .0001).CONCLUSIONS: The molecular data set in this study readily differentiated between 2 distinct groups of patients with locally advanced, HPV‐negative OPC. This risk‐stratification strategy may serve as a guide for treatment selection. Cancer 2012;. © 2011 American Cancer Society.

Background: The association between human papillomavirus (HPV) and overall survival (OS) in oropharynx cancer (OPC) was retrospectively examined in TAX 324, a phase III trial of sequential therapy for locally advanced head and neck cancer. Methods: Accrual for TAX 324 was completed in 2003 and data updated through 2008. Pretherapy tumor biopsies were studied by PCR for human papillomavirus type 16 and linked to OS, progression-free survival (PFS) and demographics. Results: Of 264 patients with OPC, 111 (42%) had evaluable biopsies; 56 (50%) were HPV+ and 55 (50%) were HPV−. HPV+ patients were significantly younger (54 versus 58 years, P = 0.02), had T1/T2 primary cancers (49% versus 20%, P = 0.001), and had a performance status of zero (77% versus 49%, P = 0.003). OS and PFS were better for HPV+ patients (OS, hazard ratio = 0.20, P < 0.0001). Local–regional failure was less in HPV+ patients (13% versus 42%, P = 0.0006); at 5 years, 82% of HPV+ patients were alive compared with 35% of HPV− patients (P < 0.0001). Conclusions: HPV+ OPC has a different biology compared with HPV− OPC; 5-year OS, PFS, and local–regional control are unprecedented. These results support the possibility of selectively reducing therapy and long-term morbidity in HPV+ OPC while preserving survival and approaching HPV− disease with more aggressive treatment.