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.

Radiosensitization and nanoparticles.

Nanomaterials have been shown to have physical and chemical properties that have opened new avenues for cancer diagnosis and therapy. Nanoconstructs that enhance existing treatments for cancer, such as radiation therapy, are being explored in several different ways. Two general paths toward nanomaterial-enabled radiosensitization have been explored: (1) improving the effectiveness of ionizing radiation and (2) modulating cellular pathways leading to a disturbance of cellular homeostasis, thus rendering the cells more susceptible to radiation-induced damage. A variety of different agents that work via one of these two approaches have been explored, many of which modulate direct and indirect DNA damage (gold), radiosensitivity through hyperthermia (Fe), and different cellular pathways. There have been many in vitro successes with the use of nanomaterials for radiosensitization, but in vivo testing has been less efficacious, predominantly because of difficulty in targeting the nanoparticles. As improved methods for tumor targeting become available, it is anticipated that nanomaterials can become clinically useful radiosensitizers for radiation therapy.

Elemental Zn and its Binding Protein Zinc-α2-Glycoprotein are Elevated in HPV-Positive Oropharyngeal Squamous Cell Carcinoma.

Human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) is biologically distinct from HPV-negative HNSCC. Outside of HPV-status, few tumor-intrinsic variables have been identified that correlate to improved survival. As part of exploratory analysis into the trace elemental composition of oropharyngeal squamous cell carcinoma (OPSCC), we performed elemental quanitification by X-ray fluorescence microscopy (XFM) on a small cohort (n = 32) of patients with HPV-positive and -negative OPSCC and identified in HPV-positive cases increased zinc (Zn) concentrations in tumor tissue relative to normal tissue. Subsequent immunohistochemistry of six Zn-binding proteins-zinc-α2-glycoprotein (AZGP1), Lipocalin-1, Albumin, S100A7, S100A8 and S100A9-revealed that only AZGP1 expression significantly correlated to HPV-status (p < 0.001) and was also increased in tumor relative to normal tissue from HPV-positive OPSCC tumor samples. AZGP1 protein expression in our cohort significantly correlated to a prolonged recurrence-free survival (p = 0.029), similar to HNSCC cases from the TCGA (n = 499), where highest AZGP1 mRNA levels correlated to improved overall survival (p = 0.023). By showing for the first time that HPV-positive OPSCC patients have increased intratumoral Zn levels and AZGP1 expression, we identify possible positive prognostic biomarkers in HNSCC as well as possible mechanisms of increased sensitivity to chemoradiation in HPV-positive OPSCC.

Intracellular in situ labeling of TiO2 nanoparticles for fluorescence microscopy detection.

Titanium dioxide (TiO2) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. Herein, we describe two in situ post-treatment labeling approaches to stain TiO2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO2 nanoparticles with alkyne-conjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Therefore, future experiments with TiO2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here.