Defining HPV-specific B cell responses in patients with head and neck cancer.

Tumours often contain B cells and plasma cells but the antigen specificity of these intratumoral B cells is not well understood1-8. Here we show that human papillomavirus (HPV)-specific B cell responses are detectable in samples from patients with HPV-positive head and neck cancers, with active production of HPV-specific IgG antibodies in situ. HPV-specific antibody secreting cells (ASCs) were present in the tumour microenvironment, with minimal bystander recruitment of influenza-specific cells, suggesting a localized and antigen-specific ASC response. HPV-specific ASC responses correlated with titres of plasma IgG and were directed against the HPV proteins E2, E6 and E7, with the most dominant response against E2. Using intratumoral B cells and plasma cells, we generated several HPV-specific human monoclonal antibodies, which exhibited a high degree of somatic hypermutation, consistent with chronic antigen exposure. Single-cell RNA sequencing analyses detected activated B cells, germinal centre B cells and ASCs within the tumour microenvironment. Compared with the tumour parenchyma, B cells and ASCs were preferentially localized in the tumour stroma, with well-formed clusters of activated B cells indicating ongoing germinal centre reactions. Overall, we show that antigen-specific activated and germinal centre B cells as well as plasma cells can be found in the tumour microenvironment. Our findings provide a better understanding of humoral immune responses in human cancer and suggest that tumour-infiltrating B cells could be harnessed for the development of therapeutic agents.

Functional HPV-specific PD-1+ stem-like CD8 T cells in head and neck cancer.

T cells are important in tumour immunity but a better understanding is needed of the differentiation of antigen-specific T cells in human cancer1,2. Here we studied CD8 T cells in patients with human papillomavirus (HPV)-positive head and neck cancer and identified several epitopes derived from HPV E2, E5 and E6 proteins that allowed us to analyse virus-specific CD8 T cells using major histocompatibility complex (MHC) class I tetramers. HPV-specific CD8 T cells expressed PD-1 and were detectable in the tumour at levels that ranged from 0.1% to 10% of tumour-infiltrating CD8 T lymphocytes (TILs) for a given epitope. Single-cell RNA-sequencing analyses of tetramer-sorted HPV-specific PD-1+ CD8 TILs revealed three transcriptionally distinct subsets. One subset expressed TCF7 and other genes associated with PD-1+ stem-like CD8 T cells that are critical for maintaining T cell responses in conditions of antigen persistence. The second subset expressed more effector molecules, representing a transitory cell population, and the third subset was characterized by a terminally differentiated gene signature. T cell receptor clonotypes were shared between the three subsets and pseudotime analysis suggested a hypothetical differentiation trajectory from stem-like to transitory to terminally differentiated cells. More notably, HPV-specific PD-1+TCF-1+ stem-like TILs proliferated and differentiated into more effector-like cells after in vitro stimulation with the cognate HPV peptide, whereas the more terminally differentiated cells did not proliferate. The presence of functional HPV-specific PD-1+TCF-1+CD45RO+ stem-like CD8 T cells with proliferative capacity shows that the cellular machinery to respond to PD-1 blockade exists in HPV-positive head and neck cancer, supporting the further investigation of PD-1 targeted therapies in this malignancy. Furthermore, HPV therapeutic vaccination efforts have focused on E6 and E7 proteins; our results suggest that E2 and E5 should also be considered for inclusion as vaccine antigens to elicit tumour-reactive CD8 T cell responses of maximal breadth.

Efficacy, long-term toxicity, and mechanistic studies of gold nanorods photothermal therapy of cancer in xenograft mice.

Gold nanorods (AuNRs)-assisted plasmonic photothermal therapy (AuNRs-PPTT) is a promising strategy for combating cancer in which AuNRs absorb near-infrared light and convert it into heat, causing cell death mainly by apoptosis and/or necrosis. Developing a valid PPTT that induces cancer cell apoptosis and avoids necrosis in vivo and exploring its molecular mechanism of action is of great importance. Furthermore, assessment of the long-term fate of the AuNRs after treatment is critical for clinical use. We first optimized the size, surface modification [rifampicin (RF) conjugation], and concentration (2.5 nM) of AuNRs and the PPTT laser power (2 W/cm2) to achieve maximal induction of apoptosis. Second, we studied the potential mechanism of action of AuNRs-PPTT using quantitative proteomic analysis in mouse tumor tissues. Several death pathways were identified, mainly involving apoptosis and cell death by releasing neutrophil extracellular traps (NETs) (NETosis), which were more obvious upon PPTT using RF-conjugated AuNRs (AuNRs@RF) than with polyethylene glycol thiol-conjugated AuNRs. Cytochrome c and p53-related apoptosis mechanisms were identified as contributing to the enhanced effect of PPTT with AuNRs@RF. Furthermore, Pin1 and IL18-related signaling contributed to the observed perturbation of the NETosis pathway by PPTT with AuNRs@RF. Third, we report a 15-month toxicity study that showed no long-term toxicity of AuNRs in vivo. Together, these data demonstrate that our AuNRs-PPTT platform is effective and safe for cancer therapy in mouse models. These findings provide a strong framework for the translation of PPTT to the clinic.

Visualization of the Cellular Uptake and Trafficking of DNA Origami Nanostructures in Cancer Cells.

DNA origami is a promising molecular delivery system for a variety of therapeutic applications including cancer therapy, given its capability to fabricate homogeneous nanostructures whose physicochemical properties (size, shape, surface chemistry) can be precisely tailored. However, the correlation between DNA-origami design and internalization efficiency in different cancer cell lines remains elusive. We investigated the cellular uptake of four DNA-origami nanostructures (DONs) with programmed sizes and shapes in multiple human cancer cell lines. The cellular uptake efficiency of DONs was influenced by size, shape, and cell line. Scavenger receptors were responsible for the internalization of DONs into cancer cells. We observed distinct stages of the internalization process of a gold nanoparticle (AuNP)-tagged rod-shape DON, using high-resolution transmission electron microscopy. This study provides detailed understanding of cellular uptake and intracellular trafficking of DONs in cancer cells, and offers new insights for future optimization of DON-based drug delivery systems for cancer treatment.

Systemic Delivery of Bc12-Targeting siRNA by DNA Nanoparticles Suppresses Cancer Cell Growth.

Short interfering RNA (siRNA) is a promising molecular tool for cancer therapy, but its clinical success is limited by the lack of robust in vivo delivery systems. Rationally designed DNA nanoparticles (DNPs) have emerged as facile delivery vehicles because their physicochemical properties can be precisely controlled. Nonetheless, few studies have used DNPs to deliver siRNAs in vivo, and none has demonstrated therapeutic efficacy. Herein, we constructed a number of DNPs of rectangular and tubular shapes with varied dimensions using the modular DNA brick method for the systemic delivery of siRNA that targets anti-apoptotic protein Bcl2. The siRNA delivered by the DNPs inhibited cell growth both in vitro and in vivo, which suppressed tumor growth in a xenograft model that specifically correlated with Bcl2 depletion. This study suggests that DNPs are effective tools for the systemic delivery of therapeutic siRNA and have great potential for further clinical translation.

Evasion of anti-growth signaling: A key step in tumorigenesis and potential target for treatment and prophylaxis by natural compounds.

The evasion of anti-growth signaling is an important characteristic of cancer cells. In order to continue to proliferate, cancer cells must somehow uncouple themselves from the many signals that exist to slow down cell growth. Here, we define the anti-growth signaling process, and review several important pathways involved in growth signaling: p53, phosphatase and tensin homolog (PTEN), retinoblastoma protein (Rb), Hippo, growth differentiation factor 15 (GDF15), AT-rich interactive domain 1A (ARID1A), Notch, insulin-like growth factor (IGF), and Krüppel-like factor 5 (KLF5) pathways. Aberrations in these processes in cancer cells involve mutations and thus the suppression of genes that prevent growth, as well as mutation and activation of genes involved in driving cell growth. Using these pathways as examples, we prioritize molecular targets that might be leveraged to promote anti-growth signaling in cancer cells. Interestingly, naturally occurring phytochemicals found in human diets (either singly or as mixtures) may promote anti-growth signaling, and do so without the potentially adverse effects associated with synthetic chemicals. We review examples of naturally occurring phytochemicals that may be applied to prevent cancer by antagonizing growth signaling, and propose one phytochemical for each pathway. These are: epigallocatechin-3-gallate (EGCG) for the Rb pathway, luteolin for p53, curcumin for PTEN, porphyrins for Hippo, genistein for GDF15, resveratrol for ARID1A, withaferin A for Notch and diguelin for the IGF1-receptor pathway. The coordination of anti-growth signaling and natural compound studies will provide insight into the future application of these compounds in the clinical setting.

Heregulin and HER3 are prognostic biomarkers in oropharyngeal squamous cell carcinoma.

BACKGROUND: Although heregulin and human epidermal growth factor receptor 3 (HER3) are frequently expressed at high levels in patients with head and neck cancer, their prognostic value remains unclear. The authors explored the prognostic significance of heregulin/HER3 expression in patients with oropharyngeal squamous cell carcinoma (OPSCC), taking into account other HER family members as well as p16 status. METHODS: Ninety-six primary tumor specimens from patients with OPSCC were retrospectively collected and analyzed for heregulin messenger RNA (mRNA) using in situ hybridization and for HER3, epidermal growth factor receptor, and human epidermal growth factor receptor 2 (HER2) using quantitative immunohistochemistry. Heregulin and HER3 mRNA levels were also examined among different tumor types using The Cancer Genome Atlas database. RESULTS: High heregulin mRNA (> the median) correlated significantly with poor overall survival (OS) (hazard ratio [HR], 8.48; 95% confidence interval [95% CI], 2.17-33.17 [P =.002]) but not disease-free survival (HR, 1.52; 95% CI, 0.64-3.65 [P =.341]) in patients with OPSCC. Heregulin mRNA correlated negatively with OS in both patients with p16-positive (P =.049) and p16-negative (P =.091) OPSCC on univariate analysis. High HER3 (> the median) also correlated with poor OS (HR, 4.68; 95% CI, 1.47-14.90 [P =.009]) on multivariate analysis. Epidermal growth factor receptor levels independently correlated with disease-free survival (P =.025) and inversely correlated with p16 status (P =.012). In addition, The Cancer Genome Atlas data demonstrated that head and neck squamous cell carcinoma exhibits higher heregulin expression compared with other solid tumor types examined. CONCLUSIONS: High heregulin mRNA and high HER3 protein levels were found to independently correlate with poor OS in patients with OPSCC. These data support targeting HER3 in patients with heregulin-high OPSCC and warrant further clinical investigation.

The prometastatic ribosomal S6 kinase 2-cAMP response element-binding protein (RSK2-CREB) signaling pathway up-regulates the actin-binding protein fascin-1 to promote tumor metastasis.

Metastasis is the leading cause of death in patients with breast, lung, and head and neck cancers. However, the molecular mechanisms underlying metastases in these cancers remain unclear. We found that the p90 ribosomal S6 kinase 2 (RSK2)-cAMP response element-binding protein (CREB) pathway is commonly activated in diverse metastatic human cancer cells, leading to up-regulation of a CREB transcription target Fascin-1. We also observed that the protein expression patterns of RSK2 and Fascin-1 correlate in primary human tumor tissue samples from head and neck squamous cell carcinoma patients. Moreover, knockdown of RSK2 disrupts filopodia formation and bundling in highly invasive cancer cells, leading to attenuated cancer cell invasion in vitro and tumor metastasis in vivo, whereas expression of Fascin-1 significantly rescues these phenotypes. Furthermore, targeting RSK2 with the small molecule RSK inhibitor FMK-MEA effectively attenuated the invasive and metastatic potential of cancer cells in vitro and in vivo, respectively. Taken together, our findings for the first time link RSK2-CREB signaling to filopodia formation and bundling through the up-regulation of Fascin-1, providing a proinvasive and prometastatic advantage to human cancers. Therefore, protein effectors of the RSK2-CREB-Fascin-1 pathway represent promising biomarkers and therapeutic targets in the clinical prognosis and treatment of metastatic human cancers.

Adaptive c-Met-PLXDC2 Signaling Axis Mediates Cancer Stem Cell Plasticity to Confer Radioresistance-associated Aggressiveness in Head and Neck Cancer.

Radiotherapy plays an essential role in the treatment of head and neck squamous cell carcinoma (HNSCC), yet radioresistance remains a major barrier to therapeutic efficacy. A better understanding of the predominant pathways determining radiotherapy response could help develop mechanism-informed therapies to improve cancer management. Here we report that radioresistant HNSCC cells exhibit increased tumor aggressiveness. Using unbiased proteome profiler antibody arrays, we identify that upregulation of c-Met phosphorylation is one of the critical mechanisms for radioresistance in HNSCC cells. We further uncover that radioresistance-associated HNSCC aggressiveness is effectively exacerbated by c-Met but is suppressed by its genetic knockdown and pharmacologic inactivation. Mechanistically, the resulting upregulation of c-Met promotes elevated expression of plexin domain containing 2 (PLXDC2) through activating ERK1/2-ELK1 signaling, which in turn modulates cancer cell plasticity by epithelial-mesenchymal transition (EMT) induction and enrichment of the cancer stem cell (CSC) subpopulation, leading to resistance of HNSCC cells to radiotherapy. Depletion of PLXDC2 overcomes c-Met-mediated radioresistance through reversing the EMT progress and blunting the self-renewal capacity of CSCs. Therapeutically, the addition of SU11274, a selective and potent c-Met inhibitor, to radiation induces tumor shrinkage and limits tumor metastasis to lymph nodes in an orthotopic mouse model. Collectively, these significant findings not only demonstrate a novel mechanism underpinning radioresistance-associated aggressiveness but also provide a possible therapeutic strategy to target radioresistance in patients with HNSCC. Significance: This work provides novel insights into c-Met-PLXDC2 signaling in radioresistance-associated aggressiveness and suggests a new mechanism-informed therapeutic strategy to overcome failure of radiotherapy in patients with HNSCC.

Downregulation of E-Cadherin enhances proliferation of head and neck cancer through transcriptional regulation of EGFR.

BACKGROUND: Epidermal growth factor receptor (EGFR) has been reported to downregulate E-cadherin (E-cad); however, whether the downregulation of E-cad has any effect on EGFR expression has not been elucidated. Our previous studies have found an inverse correlation between EGFR and E-cad expression in tissue specimens of squamous cell carcinoma of the head and neck (SCCHN). To understand the biological mechanisms underlying this clinical observation, we knocked down E-cad expression utilizing E-cad siRNA in four SCCHN cell lines. RESULTS: It was observed that downregulation of E-cad upregulated EGFR expression compared with control siRNA-transfected cells after 72 hours. Cellular membrane localization of EGFR was also increased. Consequently, downstream signaling molecules of the EGFR signaling pathway, p-AKT, and p-ERK, were increased at 72 hours after the transfection with E-cad siRNA. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed EGFR mRNA was upregulated by E-cad siRNA as early as 24 hours. In addition, RT-PCR revealed this upregulation was due to the increase of EGFR mRNA stability, but not protein stability. Sulforhodamine B (SRB) assay indicated growth of E-cad knocked down cells was enhanced up to 2-fold more than that of control siRNA-transfected cells at 72-hours post-transfection. The effect of E-cad reduction on cell proliferation was blocked by treating the E-cad siRNA-transfected cells with 1 µM of the EGFR-specific tyrosine kinase inhibitor erlotinib. CONCLUSION: Our results suggest for the first time that reduction of E-cad results in upregulation of EGFR transcriptionally. It also suggests that loss of E-cad may induce proliferation of SCCHN by activating EGFR and its downstream signaling pathways.

The Proteomic Landscape of Growth Factor Signaling Networks Associated with FAT1 Mutations in Head and Neck Cancers.

FAT1 is frequently mutated in head and neck squamous cell carcinoma (HNSCC), but the biological and clinical effects of FAT1 mutations in HNSCC remain to be fully elucidated. We investigated the landscape of altered protein and gene expression associated with FAT1 mutations and clinical outcomes of patients with HNSCC. FAT1 mutation was stratified with clinical information from The Cancer Genome Atlas HNSCC databases with more than 200 proteins or phosphorylated sites. FAT1 mutation was significantly more prevalent among HPV(-), female, and older patients and was enriched in oral, larynx, and hypopharynx primary tumors. FAT1 mutation was also significantly associated with lower FAT1 gene expression and increased protein expression of HER3_pY1289, IRS1, and CAVEOLIN1. From an independent International Cancer Genome Consortium dataset, FAT1 mutation in oral cancer co-occurred with top mutated genes TP53 and CASP8. Poorer overall survival or progression-free survival was observed in patients with FAT1 mutation or altered HER3_pY1289, IRS1, or CAVEOLIN1. Pathway analysis revealed dominant ERBB/neuregulin pathways linked to FAT1 mutations in HNSCC, and protein signature panels uncovered the heterogeneity of patient subgroups. Decreased pEGFR, pHER2, and pERK and upregulated pHER3 and HER3 proteins were observed in two FAT1 knockout HNSCC cell lines, supporting that FAT1 alterations lead to altered EGFR/ERBB signaling. In squamous cancers of the lung and cervix, a strong association of FAT1 and EGFR gene expressions was identified. Collectively, these results suggest that alteration of FAT1 appears to involve mostly HPV(-) HNSCC and may contribute to resistance to EGFR-targeted therapy. SIGNIFICANCE: Integrative bioinformatics and statistical analyses reveal a panel of genes and proteins associated with FAT1 mutation in HNSCC, providing important insights into prospective clinical investigations with targeted therapies.

Tipifarnib enhances anti-EGFR activity of cetuximab in non-HRas mutated head and neck squamous cell carcinoma cancer (HNSCC).

OBJECTIVE: To test the potential ability of tipifarnib to impair proliferation and to enhance the activity of the EGFR inhibitor cetuximab in wild-type H-Ras HNSCC, which accounts for the majority of HNSCC. MATERIALS AND METHODS: Cell growth, apoptosis and signaling changes in HNSCC cells following tipifarnib exposure in vitro were assessed by SRB, colony formation assay, annexin V staining and Western blot, respectively. A patient-derived xenograft (PDX) animal model was adopted to evaluate the efficacy of tipifarnib in vivo with and without cetuximab. RESULTS: Treatment of wild-type H-Ras HNSCC cell lines in vitro with tipifarnib reduced cell growth and increased levels of defarnesylated H-Ras in a dose-dependent manner. In a PDX mouse model, treatment with single-agent tipifarnib led to only near-significant growth inhibition. The addition of cetuximab resulted in increased anti-proliferative effect both in culture and in PDX models, which was also mirrored by Western blot and apoptosis assay results. CONCLUSION: Tipifarnib has only a moderate ability to slow tumor growth as a single agent in HNSCC with wild type H-Ras, despite specifically inhibiting the farnesyltransferase upon which the function of H-Ras depends. The combination of cetuximab and tipifarnib appears to enhance the anti-proliferative effect of single-agent tipifarnib and marginally enhance that of single agent cetuximab. These findings deserve further evaluation.

Co-expression of fibroblast growth factor receptor 3 with mutant p53, and its association with worse outcome in oropharyngeal squamous cell carcinoma.

Fibroblast growth factor receptor 3 (FGFR3) is expressed in squamous cell carcinoma of the head and neck (SCCHN) including oropharyngeal squamous cell carcinoma (OPSCC) and is a potential therapeutic target. However, information on its correlation with other relevant cancer related proteins stratified by p16 status and its prognostic significance in OPSCC is limited. We examined FGFR3 expression and its correlation with clinical characteristics, p16 status, and mutant p53 (mp53) among 220 retrospectively collected OPSCC cases and 40 prospectively collected SCCHN cases, including a majority of OPSCC. Correlations of FGFR3 Weighted Index (WI) with p16 status and mp53 WI as well as its association with disease-free survival (DFS) and overall survival (OS) were evaluated. FGFR3 expression was detected in 61% and 70% of cases in cohorts 1 and 2, respectively. FGFR3 level was significantly higher in p16-negative tumors in both cohorts (p<0.001 and 0.006). FGFR3 expression was highly correlated with mp53 expression in both p16 + and p16- OPSCC (p<0.0001 and p = 0.0006, respectively). In cohort 1, univariate analysis showed that FGFR3 was associated with DFS but not OS. Kaplan-Meier analysis showed that higher FGFR3 and mp53 level correlated with worse DFS (p = 0.025) and OS (p = 0.009). As expected, p16 positive status was associated with improved OS and DFS (p<0.001 for both). Our results suggest that high FGFR3 expression is associated with p16 negative status and mp53 expression in OPSCC and correlates with a worse clinical outcome. The biological relationship between FGFR3 and mp53 in OPSCC deserves further investigation.

Combinatorial approaches targeting the EGFR family and c-Met in SCCHN.

OBJECTIVE: We aimed to develop novel combinations of inhibitors targeting EGFR family members and c-Met for the treatment of recurrent SCCHN. MATERIALS AND METHODS: Three different c-Met inhibitors in combination with a pan-HER inhibitor (crizotinib/afatinib, tivantinib/afatinib and cabozantinib/afatinib) were investigated for their anti-tumor effects on SCCHN cell lines in vitro. In vivo activity of the combinations was tested in SCCHN cell line xenografts and patient-derived xenograft (PDX) animal models generated from patients with recurrent SCCHN. RESULTS: Western blot assay indicated that activation of EGFR, HER2, HER3, and c-Met was blocked by all three combinations and the downstream PI3K/AKT and ERK signaling pathways were inhibited. Sulforhodamine B colorimetric assay revealed SCCHN cell growth was more effectively inhibited by the combinations than by single agents, particularly in cell lines with high c-Met expression. Furthermore, the combinations were more potent in inducing apoptosis than each of the single agents. In the PDX models, the combination treatments exhibited significantly better efficacy in tumor growth inhibition compared to the respective single agents. CONCLUSION: In conclusion, we demonstrated that the simultaneous targeting of EGFR, HER2, and c-Met is more effective than the individual inhibition of these targets in vitro and in SCCHN cell line xenograft and PDX models. Our findings pave the way for further clinical investigation of such combinations in SCCHN.

A Historic Perspective and Overview of H-Ras Structure, Oncogenicity, and Targeting.

H-Ras is a unique isoform of the Ras GTPase family, one of the most prominently mutated oncogene families across the cancer landscape. Relative to other isoforms, though, mutations of H-Ras account for the smallest proportion of mutant Ras cancers. Yet, in recent years, there have been renewed efforts to study this isoform, especially as certain H-Ras-driven cancers, like those of the head and neck, have become more prominent. Important advances have therefore been made not only in the understanding of H-Ras structural biology but also in approaches designed to inhibit and impair its signaling activity. In this review, we outline historic and present initiatives to elucidate the mechanisms of H-Ras-dependent tumorigenesis as well as highlight ongoing developments in the quest to target this critical oncogene.

Tumor Membrane Vesicle Vaccine Augments the Efficacy of Anti-PD1 Antibody in Immune Checkpoint Inhibitor-Resistant Squamous Cell Carcinoma Models of Head and Neck Cancer.

Immune checkpoint inhibitor (ICI) immunotherapy improved the survival of head and neck squamous cell carcinoma (HNSCC) patients. However, more than 80% of the patients are still resistant to this therapy. To test whether the efficacy of ICI therapy can be improved by vaccine-induced immunity, we investigated the efficacy of a tumor membrane-based vaccine immunotherapy in murine models of HNSCC. The tumors, grown subcutaneously, are used to prepare tumor membrane vesicles (TMVs). TMVs are then incorporated with glycolipid-anchored immunostimulatory molecules GPI-B7-1 and GPI-IL-12 by protein transfer to generate the TMV vaccine. This TMV vaccine inhibited tumor growth and improved the survival of mice challenged with SCCVII tumor cells. The tumor-free mice survived for several months, remained tumor-free, and were protected following a secondary tumor cell challenge, suggesting that the TMV vaccine induced an anti-tumor immune memory response. However, no synergy with anti-PD1 mAb was observed in this model. In contrast, the TMV vaccine was effective in inhibiting MOC1 and MOC2 murine oral cancer models and synergized with anti-PD1 mAb in extending the survival of tumor-bearing mice. These observations suggest that tumor tissue based TMV vaccines can be harnessed to develop an effective personalized immunotherapy for HNSCC that can enhance the efficacy of immune checkpoint inhibitors.

Phase Ib Study of Chemoprevention with Green Tea Polyphenon E and Erlotinib in Patients with Advanced Premalignant Lesions (APL) of the Head and Neck.

PURPOSE: On the basis of synergistic effects between green tea polyphenon E (PPE) and EGFR-tyrosine kinase inhibitor in preclinical studies, we conducted a phase Ib study of the PPE and erlotinib combination in patients with advanced premalignant lesions (APL) of the oral cavity and larynx. PATIENTS AND METHODS: Patients were treated with a fixed dose of PPE (200 mg three times a day) and dose escalation of erlotinib (50, 75, 100 mg daily) for 6 months with tissue biopsy at baseline and 6 months. Primary endpoints were safety and toxicity; secondary endpoints were evaluation of pathologic response, cancer-free survival (CFS), overall survival (OS), and biomarker modulation. RESULTS: Among 21 enrolled patients, 19 began treatment and 17 completed 6 months of treatment with PPE and erlotinib. Main characteristics of treated patients: 15 severe dysplasia or carcinoma in situ and 17 oral cavity. Only skin rash was associated with dose-limiting toxicity and MTD. Recommended doses for phase II studies are PPE 600 mg daily plus erlotinib 100 mg daily for 6 months. Pathologic responses in 17 evaluable patients: pathologic complete response (47%) and pathologic partial response (18%). The 5-year CFS and OS were 66.3% and 93%, respectively. Among tested biomarkers, only phosphorylated ERK was correlated with response to treatment. CONCLUSIONS: Treatment with PPE and erlotinib combination was well tolerated in patients with APLs of the head and neck, and showed a high rate of pathologic response with excellent CFS. This combination deserves further investigation for the chemoprevention and/or prevention of second primary tumors in early-stage head and neck cancer.

A continuous-flow acoustofluidic cytometer for single-cell mechanotyping.

The biophysical properties of cells such as their compressibility have been found to be closely related to disease progression such as cancer development and metastasis. As cancer cells are heterogeneous, rapid and high-throughput evaluation of cell biophysical properties at single-cell resolution is needed to assess their potential as biomarkers for cancer staging and prognosis. Acoustofluidics has shown promise as a contactless method for accurately measuring cell biophysical properties; however, previously reported methods had relatively low throughput due to their requirement of no-flow conditions. This work presents a high-throughput continuous flow-based acoustofluidic cell mechanotyping method at single-cell resolution that retains the advantage of simplicity and low-cost.

Interleukin-6/STAT3 Signaling is Prominent and Associated with Reduced Overall Survival in p16 Negative Oropharyngeal Squamous Cell Carcinoma.

This study addresses the hypothesis that IL-6/STAT3 signaling is of clinical relevance in oropharyngeal squamous cell carcinoma (OPSCC). We evaluated relationships between key components of this pathway in tumors from a unique cohort of n = 59 fully annotated, treatment-naïve patients with OPSCC. The multiplex Opal platform was utilized for immunofluorescence (IF) analysis of tissues to detect IL-6 and phosphorylated STAT3 (pSTAT3), taking into consideration its nuclear versus cytoplasmic localization. Abundant staining for both IL-6 and pSTAT3 was evident in tumor-rich regions of each specimen. IL-6 correlated with cytoplasmic pSTAT3 but not nuclear or total pSTAT3 in this cohort of OPSCC tumors, regardless of p16 status (r = 0.682, p < 0.0001). There was a significant association between increased total pSTAT3, nuclear pSTAT3, cytoplasmic pSTAT3 and IL-6 in p16 negative tumors. Our data indicate STAT3 phosphorylation was a key feature in p16-negative OPSCC tumors. When IL-6 data was stratified by median expression in tumors, there was no association with overall survival. In contrast, both total and nuclear pSTAT3 were significant predictors of poor overall and disease free survival. This strong inverse relationship with overall survival was present in p16 negative tumors for both total and nuclear pSTAT3, but not in p16 positive OPSCC tumors. Together these data indicate that activation of the STAT3 signaling pathway is a marker of p16 negative tumors and relevant to OPSCC prognosis and a potential target for treatment of this more aggressive OPSCC sub-population.