Comprehensive T-cell immunophenotyping and next-generation sequencing of human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinomas.

The success of programmed cell death 1 (PD-1) inhibition in achieving a clinical response in a subset of head and neck squamous cell carcinoma (HNSCC) patients emphasizes the need to better understand the immunobiology of HNSCC. Immunophenotyping was performed for 30 HCSCC patients [16 human papillomavirus (HPV)-positive; 14 HPV-negative] on matched tissue from the primary tumour site, locally metastatic cervical lymph nodes (LNs), uninvolved local cervical LNs, and peripheral blood. CD4+ and CD8+ T-cell lymphocytes obtained from tissue were analysed for expression levels of the inhibitory receptors PD-1, TIM-3 and CTLA-4. Next-generation sequencing of the T-cell receptor (TCR) ß chain was performed on patients (n = 9) to determine receptor repertoire diversity and for clonality analysis. HPV-negative HNSCC patients, particularly those with stage IV disease, had significantly higher proportions of CD8+ T cells expressing CTLA-4 in tumour tissue (P = 0.0013) and in peripheral blood (P = 0.0344) than HPV-positive patients, as well as higher expression levels of TIM-3+ PD-1+ CD8+ T cells (P = 0.0072) than controls. For all patients, PD-1 expression on CD8+ T cells - particularly in HPV-negative HNSCC cases - strongly correlated (r = 0.63, P = 0.013) with tumour size at the primary site. The top CD8+ TCR clones from tumour tissue significantly overlapped with circulating peripheral blood TCR clones (r = 0.946), and HPV-positive patients had frequently expanded TCR clones that were more hydrophobic - and potentially more immunogenic - than those from HPV-negative patients. Collectively, our findings demonstrate, for the first time, that high-stage HPV-negative HNSCC patients with primary tumours at different sites in the head and neck have elevated peripheral CTLA-4+ CD8+ T-cell levels, that tumour-familiar CD8+ T cells are detectable in peripheral blood from HNSCC patients, and that TCRs from HPV-positive HNSCC patients potentially recognize distinctly immunogenic cognate antigens. However, our findings are preliminary, and need to be further confirmed in a larger patient cohort; also, how these factors affect patient response to immunotherapy needs to be determined. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Peritumoral cuffing by T-cell tumor-infiltrating lymphocytes distinguishes HPV-related oropharyngeal squamous cell carcinoma from oral cavity squamous cell carcinoma.

BACKGROUND: It is unclear why human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC) has improved clinical behavior compared to HPV-negative HNSCC. We sought to better characterize the immune microenvironment of tongue cancers by examining the CD3 and CD8 TIL pattern in HPV-positive and HPV-negative tumors. METHODS: Histologic sections from 40 oral tongue and oropharyngeal cases were analyzed (n=21 HPV DNA-positive, n=19 HPV DNA-negative). CD3 and CD8 T-cell immunostaining were performed on whole-slide sections to quantify tumor-infiltrating lymphocyte (TIL) density and assess its morphology. RESULTS: A subset of cases (HPV-positive) displayed a unique TIL pattern consisting of circumferential peritumoral population T cells, which was absent in the HPV-negative cases. The presence of peritumoral cuffing was strongly predictive of improved recurrence-free survival compared to cases that lacked this morphologic pattern of immune infiltrate. Four HPV-positive cases lacked the pattern, including two cases with disease recurrence. CONCLUSIONS: For the first time, we show an architectural pattern of immune infiltrate in HNSCC is seen exclusively in HPV-positive patients with improved recurrence-free survival and suggests an organized host immunological response contributes to disease control.

The role of elective neck dissection in patients with adenoid cystic carcinoma of the head and neck.

OBJECTIVE: To investigate the frequency and outcomes of elective neck dissection (END) for adenoid cystic carcinoma (ACC) of the head and neck. METHODS: The National Cancer Database was queried for a cohort study of patients with ACC of the major salivary glands, nasal cavity/nasopharynx, hard/soft palate, tongue, floor of mouth, larynx, and oral cavity who underwent primary surgical resection from 2004 to 2014. Multivariable logistic regression was used to identify predictors of END and occult nodal metastasis. Overall survival (OS) was estimated using the Kaplan-Meier method and modeled with Cox proportional hazards regression. RESULTS: Among 2,807 patients with ACC treated surgically, 636 (22.7%) underwent END. Patients with ACC of the salivary glands and tongue most frequently underwent END; patients with hard/soft palate (odds ratio [OR] 0.06, P < 0.001) and nasal cavity/nasopharynx (OR 0.05, P < 0.001) ACC rarely underwent END compared to patients with major salivary gland cancer. Increasing tumor (T) stage (T4 vs. T1, OR 3.02, P < 0.001) was associated with END. Patients with advanced T3 to T4 ACC of the major salivary glands demonstrated extended OS associated with END (5-year OS 78.1% vs. 70.4%, P = 0.041) on Kaplan-Meier analysis and with END with adjuvant radiation therapy (hazard ratio 0.55, P = 0.027) using Cox proportional hazards regression. Elective neck dissection for T4 ACC of the salivary glands (21.3%) and tongue (25.5%) most consistently revealed occult nodal metastasis. CONCLUSION: Elective neck dissection for ACC of the major salivary glands or tongue is most likely to reveal occult nodal metastasis. Elective neck dissection is associated with extended OS for advanced-stage ACC of the major salivary glands. LEVEL OF EVIDENCE: NA Laryngoscope, 129:2094-2104, 2019.

Cancer Immunotherapies: Are They as Effective in the Elderly?

Almost two-thirds of all new cancer diagnoses are made in persons over the age of 65 years, yet it is unclear if age affects patient responsiveness to immunotherapy, which is increasingly becoming first-line therapy in advanced stages of different tumor types. Preclinical animal studies may be difficult to translate into humans since they frequently use young mice (2-3 months of age) equivalent to adolescent human subjects. Nevertheless, ex vivo studies from humans are concordant with mice tissue findings-older patients have an increased density of circulating regulatory immune cells and a decreased ratio of naïve-to-memory T cells. A review of different immunotherapy trials reveals that contrary to expectations, advanced age generally does not hinder safety and clinical response to different treatment modalities. A growing number of immune checkpoint inhibitor immunotherapy trials have been published with basic safety and clinical response data stratified by age. We present the clinical response data from 21 phase II/III clinical trials based on age stratification into young and old subgroups. Data from these trials indicate that these agents have an overall low toxicity profile and that they are similarly well-tolerated in young and old patient subgroups. However, drug-specific differences exist for immune checkpoint inhibition in elderly subjects when comparing overall survival and progression-free survival hazard ratios with those of young subjects. Additional work is needed to better stratify 'responders' and 'nonresponders' within the elderly age group in order to optimize immunotherapy use in a heterogeneous patient population.

Free Flap Reconstruction Monitoring Techniques and Frequency in the Era of Restricted Resident Work Hours.

Importance: Free flap reconstruction of the head and neck is routinely performed with success rates around 94% to 99% at most institutions. Despite experience and meticulous technique, there is a small but recognized risk of partial or total flap loss in the postoperative setting. Historically, most microvascular surgeons involve resident house staff in flap monitoring protocols, and programs relied heavily on in-house resident physicians to assure timely intervention for compromised flaps. In 2003, the Accreditation Council for Graduate Medical Education mandated the reduction in the hours a resident could work within a given week. At many institutions this new era of restricted resident duty hours reshaped the protocols used for flap monitoring to adapt to a system with reduced resident labor. Objectives: To characterize various techniques and frequencies of free flap monitoring by nurses and resident physicians; and to determine if adapted resident monitoring frequency is associated with flap compromise and outcome. Design, Setting, and Participants: This multi-institutional retrospective review included patients undergoing free flap reconstruction to the head and/or neck between January 2005 and January 2015. Consecutive patients were included from different academic institutions or tertiary referral centers to reflect evolving practices. Main Outcomes and Measures: Technique, frequency, and personnel for flap monitoring; flap complications; and flap success. Results: Overall, 1085 patients (343 women [32%] and 742 men [78%]) from 9 institutions were included. Most patients were placed in the intensive care unit postoperatively (n = 790 [73%]), while the remaining were placed in intermediate care (n = 201 [19%]) or in the surgical ward (n = 94 [7%]). Nurses monitored flaps every hour (q1h) for all patients. Frequency of resident monitoring varied, with 635 patients monitored every 4 hours (q4h), 146 monitored every 8 hours (q8h), and 304 monitored every 12 hours (q12h). Monitoring techniques included physical examination (n = 949 [87%]), handheld external Doppler sonography (n = 739 [68%]), implanted Doppler sonography (n = 333 [31%]), and needle stick (n = 349 [32%]); 105 patients (10%) demonstrated flap compromise, prompting return to the operating room in 96 patients. Of these 96 patients, 46 had complete flap salvage, 22 had partial loss, and 37 had complete loss. The frequency of resident flap checks did not affect the total flap loss rate (q4h, 25 patients [4%]; q8h, 8 patients [6%]; and q12h, 8 patients [3%]). Flap salvage rates for compromised flaps were not statistically different. Conclusions and Relevance: Academic centers rely primarily on q1h flap checks by intensive care unit nurses using physical examination and Doppler sonography. Reduced resident monitoring frequency did not alter flap salvage nor flap outcome. These findings suggest that institutions may successfully monitor free flaps with decreased resident burden.

FoxM1 regulates mammary luminal cell fate.

Elevated expression of FoxM1 in breast cancer correlates with an undifferentiated tumor phenotype and a negative clinical outcome. However, a role for FoxM1 in regulating mammary differentiation was not known. Here, we identify another function of FoxM1, the ability to act as a transcriptional repressor, which plays an important role in regulating the differentiation of luminal epithelial progenitors. Regeneration of mammary glands with elevated levels of FoxM1 leads to aberrant ductal morphology and expansion of the luminal progenitor pool. Conversely, knockdown of FoxM1 results in a shift toward the differentiated state. FoxM1 mediates these effects by repressing the key regulator of luminal differentiation, GATA-3. Through association with DNMT3b, FoxM1 promotes methylation of the GATA-3 promoter in an Rb-dependent manner. This study identifies FoxM1 as a critical regulator of mammary differentiation with significant implications for the development of aggressive breast cancers.

Using LASSO regression to detect predictive aggregate effects in genetic studies.

We use least absolute shrinkage and selection operator (LASSO) regression to select genetic markers and phenotypic features that are most informative with respect to a trait of interest. We compare several strategies for applying LASSO methods in risk prediction models, using the Genetic Analysis Workshop 17 exome simulation data consisting of 697 individuals with information on genotypic and phenotypic features (smoking, age, sex) in 5-fold cross-validated fashion. The cross-validated averages of the area under the receiver operating curve range from 0.45 to 0.63 for different strategies using only genotypic markers. The same values are improved to 0.69-0.87 when both genotypic and phenotypic information are used. The ability of the LASSO method to find true causal markers is limited, but the method was able to discover several common variants (e.g., FLT1) under certain conditions.

An evolutionary optimization strategy using graphics processing units to efficiently investigate gene-gene interactions in genetic association studies.

The analysis of gene-gene interactions related to common complex human diseases is complicated by the increasing scale of genetic association analysis. Concurrent with the advances in genetic technology that led to these large data sets, improvements have been made in parallel computing with graphics processing units (GPUs). The data-intensive nature of genetic association analysis makes this problem particularly suitable for improved computation with the powerful computing resources available in GPUs. In this study, we present a GPU-accelerated discrete optimization strategy to improve the computational efficiency of multi-locus association analysis. We implemented an adaptive evolutionary algorithm that takes advantage of linkage disequilibrium to reduce the need for exhaustive search for combinations of genetic markers. The proposed GPU algorithm was shown to have improved efficiency and equivalent power relative to the CPU version.

Localization of brainstem auditory evoked potentials in primates: a comparison of localization techniques applied to deep brain sources.

The objective of this study was to evaluate the performance of source localization techniques through localization of deep brain sources. To accomplish this, two replications of a brainstem auditory evoked potential (BAEP, left ear 60 dB nHL clicks) were recorded from five normal rhesus monkeys. We analyzed waves III and IV, as this portion of the BAEP corresponds to the deepest signal. Data were analyzed using five different source localization techniques: Moving Dipoles, Fixed Dipoles, MUSIC (Multiple Signal Classification) dipole scan, LORETA (Low Resolution Tomography), and LCMV (Linearly Constrained Minimum Variance) spatial filtering. The moving dipole, fixed dipole and MUSIC solutions were found to be, on average, 25.1 mm from the brainstem generators. LORETA detected sources within the brainstem 65% of the time. However, 90% of these localization results also included false detections defined as regions of the brain that were more than 2 cm away from the auditory pathway. LCMV included the brainstem in 90% of the trials and false detections in 40% of the cases. These findings indicate that evoked electrical activity from deep brain sources can be localized with cm accuracy. The dipole methods performed better than LORETA and LCMV. Given the depth and amplitude of the sources analyzed in this study, these results can be interpreted as an upper bound on the accuracy of each technique.