Search

Oral leukoplakia is common and may, in some cases, progress to carcinoma. Proliferative leukoplakia is a progressive, often multifocal subtype with a high rate of malignant transformation compared with the more common localized leukoplakia. We hypothesized that the immune microenvironment and gene expression patterns would be distinct for proliferative leukoplakia compared with localized leukoplakia. We summarize key clinicopathologic features among proliferative leukoplakia and localized leukoplakia and compare cancer-free survival (CFS) between subgroups. We analyze immunologic gene expression profiling in proliferative leukoplakia and localized leukoplakia tissue samples (NanoString PanCancer Immune Oncology Profiling). We integrate immune cell activation and spatial distribution patterns in tissue samples using multiplexed immunofluorescence and digital image capture to further define proliferative leukoplakia and localized leukoplakia. Among N = 58 patients (proliferative leukoplakia, n = 29; localized leukoplakia, n = 29), only the clinical diagnosis of proliferative leukoplakia was associated with significantly decreased CFS (HR, 11.25; P < 0.01; 5-year CFS 46.8% and 83.6% among patients with proliferative leukoplakia and localized leukoplakia, respectively). CD8+ T cells and T regulatory (Treg) were more abundant among proliferative leukoplakia samples (P < 0.01) regardless of degree of epithelial dysplasia, and often colocalized to the dysplasia–stromal interface. Gene set analysis identified granzyme M as the most differentially expressed gene favoring the proliferative leukoplakia subgroup (log2 fold change, 1.93; Padj < 0.001). Programmed death ligand 1 (PD-L1) was comparatively overexpressed among proliferative leukoplakia samples, with higher (>5) PD-L1 scores predicting worse CFS (Padj < 0.01). Proliferative leukoplakia predicts a high rate of malignant transformation within 5 years of diagnosis. A prominent CD8+ T-cell and Treg signature along with relative PD-L1 overexpression compared with localized leukoplakia provides strong rationale for PD-1/PD-L1 axis blockade using preventative immunotherapy. Significance: This is the first in-depth profiling effort to immunologically characterize high-risk proliferative leukoplakia as compared with the more common localized leukoplakia. We observed a notable cytotoxic T-cell and Treg signature with relative overexpression of PD-L1 in high-risk proliferative leukoplakia providing a strong preclinical rationale for investigating PD-1/PD-L1 axis blockade in this disease as preventative immunotherapy.

Purpose: In a head and neck squamous cell carcinoma (HNSCC) “window of opportunity” clinical trial, we reported that trametinib reduced MEK-Erk1/2 activation and resulted in tumor responses in a subset of patients. Here, we investigated resistance to trametinib and molecular correlates in HNSCC cell lines and patient samples. Experimental Design: HNSCC cell lines were treated with trametinib to generate resistant lines. Candidate bypass pathways were assessed using immunoblotting, CRISPR knockout, and survival assays. Effectiveness of combined trametinib and verteporfin targeting was evaluated. Patient-derived xenografts (PDXs) from responder patients were treated with trametinib and resistant tumors were analyzed. Window trial clinical samples were subjected to whole-exome and RNA sequencing. Results: HNSCC cell lines developed resistance (CAL27-TR and HSC3-TR) after prolonged trametinib exposure. Downstream effectors of the Hippo pathway were activated in CAL27-TR and HSC3-TR, and combined trametinib and verteporfin treatment resulted in synergistic treatment response. We defined the Hippo pathway effector Yap1 as an induced survival pathway promoting resistance to trametinib in HSC3-TR. Yap1 was necessary for HSC3-TR trametinib resistance, and constitutively active Yap1 was sufficient to confer resistance in parental HSC3. Analysis of trametinib neoadjuvant trial patient tumors indicated canonical MEK-Erk1/2 pathway activating mutations were infrequent, and Yap1 activity increased following trametinib treatment. Trametinib treatment of a PDX from a responder patient resulted in evolution of resistance with increased Yap1 expression and activity. Conclusions: These studies identify a Yap1-dependent resistance to trametinib therapy in HNSCCs. Combined Yap1 and MEK targeting may represent a strategy to enhance HNSCC response.

Cancer is characterized by an accumulation of somatic mutations that represent a source of neoantigens for targeting by antigen‐specific T cells. Head and neck squamous cell carcinoma (HNSCC) has a relatively high mutation burden across all cancer types, and cellular immunity to neoantigens likely plays a key role in HNSCC clinical outcomes. Immune checkpoint inhibitors (CPIs) have brought new treatment options and hopes to patients with recurrent and/or metastatic HNSCC. However, many patients do not benefit from CPI therapies, highlighting the need for novel immunotherapy or combinatorial strategies. One such approach is personalized cancer vaccination targeting tumor‐associated antigens and tumor‐specific antigens, either as single agents or in combination with other therapies. Recent advances in next‐generation genomic sequencing technologies and computational algorithms have enabled efficient identification of somatic mutation‐derived neoantigens and are anticipated to facilitate the development of cancer vaccine strategies. Here, we review cancer vaccine approaches against HNSCC, including fundamental mechanisms of a cancer vaccine, considerations for selecting appropriate antigens, and combination therapies., Many patients with head and neck squamous cell carcinoma (HNSCC) do not benefit from checkpoint inhibitor therapies, which highlights the need for novel immunotherapy or combinatorial strategies. One such approach is personalized cancer vaccination targeting tumor‐associated antigens and tumor‐specific antigens, either as single agents or in combination with other therapies. Here, we review cancer vaccine approaches against HNSCC, including fundamental mechanisms of cancer vaccine, considerations for selecting appropriate antigens, and combination therapies.

Purpose: Surgery often represents the best chance for disease control in locoregionally recurrent squamous cell carcinoma of the head and neck (SCCHN). We investigated dual immune-checkpoint inhibition [anti–PD-1, nivolumab (N), and anti-KIR, lirilumab (L)] before and after salvage surgery to improve disease-free survival (DFS). Patients and Methods: In this phase II study, patients received N (240 mg) + L (240 mg) 7 to 21 days before surgery, followed by six cycles of adjuvant N + L. Primary endpoint was 1-year DFS; secondary endpoints were safety, pre-op radiologic response, and overall survival (OS). Correlatives included tumor sequencing, PD-L1 scoring, and immunoprofiling. Results: Among 28 patients, the median age was 66, 86% were smokers; primary site: 9 oral cavity, 9 oropharynx, and 10 larynx/hypopharynx; 96% had prior radiation. There were no delays to surgery. Grade 3+ adverse events: 11%. At the time of surgery, 96% had stable disease radiologically, one had progression. Pathologic response to N + L was observed in 43% (12/28): 4/28 (14%) major (tumor viability, TV ≤ 10%) and 8/28 (29%) partial (TV ≤ 50%). PD-L1 combined positive score (CPS) at surgery was similar regardless of pathologic response (P = 0.71). Thirteen (46%) recurred (loco-regional = 10, distant = 3). Five of 28 (18%) had positive margins, 4 later recurred. At median follow-up of 22.8 months, 1-year DFS was 55.2% (95% CI, 34.8–71.7) and 1-year OS was 85.7% (95% CI, 66.3–94.4). Two-year DFS and OS were 64% and 80% among pathologic responders. Conclusions: (Neo)adjuvant N + L was well tolerated, with a 43% pathologic response rate. We observed favorable DFS and excellent 2-year OS among high-risk, previously treated patients exhibiting a pathologic response. Further evaluation of this strategy is warranted.

SUMMARYBackgroundPembrolizumab improved survival of patients with recurrent or metastatic head and neck squamous-cell carcinoma (HNSCC). The aims of this phase 2 trial were to determine if pembrolizumab administered to patients with resectable locally advanced, human papillomavirus (HPV)-unrelated HNSCC would be safe, result in pathologic tumor response (pTR), and lower the relapse rate.MethodsNeoadjuvant pembrolizumab (200 mg) was administered 2-3 weeks before surgery. Resection of the primary tumor and involved/at-risk nodes was performed. Post-operative (chemo) radiation was planned. Patients with high-risk pathology (positive margins and/or extranodal extension) were to receive adjuvant pembrolizumab. pTR was quantified as the proportion of the resection bed with tumor necrosis, keratinous debris, and giant cells/histiocytes: pTR-0 (NCT02296684), and is ongoing but closed to accrual.FindingsBetween June 30, 2015, and March 30, 2018, 36 patients enrolled. After neoadjuvant pembrolizumab, serious (grades 3-4) adverse events and unexpected surgical delays/complications did not occur. pTR-2 occurred in eight patients (22%), and pTR-1 in eight other patients (22%). pTR ≥10% correlated with baseline tumor PD-L1 expression, immune infiltrate, and IFN-γ pathway activity. Matched sample analysis showed compensatory upregulation of multiple immune inhibitory checkpoints in patients with pTR-0, and confirmed that clonal loss occurred in some patients. The one-year relapse rate among the eighteen patients with high-risk pathology was 16.7% (95%CI: 3.6-41.4%).ConclusionsAmong patients with locally advanced, HPV-unrelated HNSCC, neoadjuvant pembrolizumab was safe, and resulted in pTR-1 or pTR-2 in 44% of patients. The one-year relapse rate in patients with high-risk-pathology was lower than historical.FundingMerck, NCI, NIDCR, NHGRI and The V Foundation.

6053 Background: Locoregional recurrence (LRR) is a major cause of death for patients (pts) with squamous cell carcinoma of the head and neck (SCCHN). With therapy options limited by prior treatment, surgery often represents the best chance for disease control. Emerging data suggests a role for neoadjuvant immunotherapy in upfront resectable SCCHN and the importance of NK cells in the tumor microenvironment. We hypothesized that dual immune checkpoint inhibition (anti-PD-1, nivolumab [N] and anti-KIR, lirilumab [L]) before and after salvage surgery would improve 1-year disease-free survival (DFS). Methods: Pts with operable LRR of SCCHN (any HPV or smoking status) with a disease-free interval of > 8 weeks after curative intent therapy were eligible for this phase II trial. Pts received a single dose of pre-op N (240 mg) + L (240 mg) 7-21 days before surgery, followed by 6-cycles of adjuvant N+L on days 1, 15 (N alone) of a 28-day cycle (C) for C1-3; and on day 1 for C4-6. Primary endpoint was 1-year DFS; 37 DFS events among N = 54 pts provided 81% power to detect improvement in 1-year DFS from 57% to 67.5% (one-sided 10% Wald’s test). Secondary endpoints: safety, radiologic response (RECIST v1.1) to pre-op N+L, and overall survival (OS). Correlatives included tumor sequencing, PD-L1 status, and immunoprofiling. Results: Between 3/15/18 and 5/29/20, N = 29 enrolled (stopped due to expiration of drug supply). Among 28 treated pts, median age: 66, 18% (5/28) women, 83% smokers; primary site: 10 oral cavity, 8 oropharynx (5/8 HPV+), and 10 larynx/hypopharynx. 96% (27/28) had prior HN radiation; 71% (20/28) prior chemotherapy. There were no delays to surgery. Grade 3+ adverse events: 11% (3/28); no deaths from treatment. At time of surgery, 96% (27/28) had stable disease radiologically with 3 showing regression, 4% (1/28) had disease progression. Pathologic response to N+L was observed in 43% (12/28): 4/28 (14%) major (tumor viability, TV ≤10%); 8/28 (29%) partial (TV ≤50%). PD-L1 CPS at surgery was similar regardless of pathologic response (p = 0.63). 68% (19/28) completed all 6-cycles of adjuvant N+L; N = 1 came off for toxicity. Ten pts (36%) recurred (local = 8, distant = 2). 5/28 (18%) had positive margins, of which 4 (80%) recurred; 4/28 (14%) declined to start adjuvant N+L, of which 3 (75%) later recurred. At median follow-up of 20.2 months, 1-year DFS70% (95%CI, 48-84%) and 1-year OS: 85% (95%CI, 65-94%). Median tumor mutational burden was 4 (range, 1-11). TP53 was the most frequent alteration (78%, 21/27). CD39 expression by TILs and CD38 expression by circulating CD4/8+ T cells increased after N+L exposure (p < 0.05). Conclusions: Neoadjuvant and adjuvant N+L was safe and well tolerated. We observed a 43% pathologic response rate prior to salvage surgery, with a favorable 1-year DFS of 70% and 1-year OS > 80% among previously irradiated pts. Further evaluation of this strategy is warranted (NCT03341936).

6008 Background: We reported that one cycle of neoadjuvant pembrolizumab induced pathologic tumor response in >10% (pTR-any) and in >50% (pTR-2) of the resection bed in 44% and 22% of patients (pts) with surgically resectable HPV-negative, Stage III/IV HNSCC ( Clin Cancer Res 2020). We hypothesized that two cycles of neoadjuvant pembrolizumab would induce pTR-2 in 50% of pts. Increasing the pathologic response rate may favorably impact clinical outcomes. Methods: Multi-institutional phase 2 trial where pts with locally advanced, HPV-negative HNSCC received two cycles of pembrolizumab (200 mg), given 42 and 21 days prior to surgery. Resected tumor was analyzed by two independent pathologists for pTR (tumor necrosis and/or giant cell/histiocytic reaction to keratinous debris) in the resection bed (primary tumor and/or lymph nodes). Additional definitions: pTR-1 (>10-49%) and major pathologic response ( > 90%). The primary endpoint was pTR-2. A sample size of 26 pts was needed to detect a significantly higher pTR-2 rate of 50%, with 80% power using a one-sided alpha level of 0.05. Pts were followed for serious adverse events (AEs) for 30 days after surgery and for AEs of clinical interest for 90 days following the last dose of pembrolizumab. Pts underwent baseline blood collection and tumor biopsies to match with blood and surgical specimens obtained post-pembrolizumab. Planned correlatives included PD-L1 expression, immune function, and molecular signatures of activation in the pre- and post-treatment blood and tumor tissue. Results: Characteristics of 29 enrolled and treated pts were median age 62 (30-82) yrs, smoking history 62% (18 pts); clinical stage T2 (n = 6), T3 (n = 5), T4 (n = 18) and N0/1 (n = 17), N2 (n = 12). All treated patients received two cycles of neoadjuvant pembrolizumab, which was tolerated well with only one (3%) grade 3 AE (rash) and no grade 4 AEs. The primary endpoint was evaluable in 25 pts, and not evaluable in 4 pts (one pt withdrew before surgery and in three pts, pTR review was pending). pTR-2 occurred in 44% (11 of 25 pts), and 4 (16%) of these pts had a major pathologic response including 1 (4%) pathologic CR at the primary site. Conclusions: Two (vs one) cycles of neoadjuvant pembrolizumab resulted in a two-fold increase in the frequency of pTR-2 (44% vs 22%). These data imply that the frequency of pTR to neoadjuvant pembrolizumab can be improved by increasing the number of cycles and the treatment interval. Clinical trial information: NCT02296684.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have not been effective in unselected head and neck squamous cell carcinoma (HNSCC) populations. We previously reported an exceptional response to a brief course of erlotinib in a patient with advanced HNSCC whose tumor harbored a MAPK1E322K somatic mutation. MAPK1E322Kwas associated with increased p-EGFR, increased EGFR downstream signaling and increased sensitivity to erlotinib. In this study, we investigated the mechanism of MAPK1E322K-mediated EGFR activation in the context of erlotinib sensitivity. We demonstrated increased AREG secretion in HNSCC cell lines harboring endogenous or exogenous MAPK1E322K compared to wild type MAPK1. We found inhibition or knockdown of MAPK1 with siRNA resulted in reduced secretion of AREG and decreased sensitivity to erlotinib in the setting of MAPK1E322K. MAPK1E322K was associated with increased AREG secretion leading to an autocrine feedback loop involving AREG, EGFR and downstream signaling. Knockdown of AREG in HNSCC cells harboring MAPK1E322K abrogated EGFR signaling and decreased sensitivity to erlotinib in vitro and in vivo. These cumulative findings implicate increased AREG secretion and EGFR activation as contributing to increased erlotinib sensitivity in MAPK1E322K HNSCC.

Objective Src family kinase (SFK) activation circumvents epidermal growth factor receptor (EGFR) targeting in head and neck squamous cell carcinoma (HNSCC); dual SFK-EGFR targeting could overcome cetuximab resistance. Patients and methods We conducted a Simon two-stage, phase II trial of the SFK inhibitor, dasatinib, and cetuximab in biomarker-unselected patients with cetuximab-resistant, recurrent/metastatic HNSCC. Pre- and post-treatment serum levels of interleukin-6 (IL6) were measured by ELISA. HNSCC cell lines were assessed for viability and effects of IL6 modulation following dasatinib-cetuximab treatment. Results In the first stage, 13 patients were evaluable for response: 7 had progressive and 6 had stable disease (SD). Enrollment was halted for futility, and biomarker analysis initiated. Low serum IL6 levels were associated with SD (raw p = 0.028, adjusted p = 0.14) and improved overall survival (p = 0.010). The IL6 classifier was validated in a separate trial of the same combination, but was unable to segregate survival risk in a clinical trial of cetuximab and bevacizumab suggesting serum IL6 may be specific for the dasatinib-cetuximab combination. Enhanced in vitro HNSCC cell death was observed with dasatinib-cetuximab versus single agent treatment; addition of IL6-containing media abrogated this effect. Conclusion Clinical benefit and overall survival from the dasatinib-cetuximab combination were improved among patients with low serum IL6. Preclinical studies support IL6 as a modifier of dasatinib-cetuximab response. In the setting of clinical cetuximab resistance, serum IL6 is a candidate predictive marker specific for combined dasatinib-cetuximab. The trial was modified and redesigned as a biomarker-enriched Phase II study enrolling patients with undetectable IL6.

Background The purpose of this article was to study the association of human papillomavirus (HPV) with clinical outcomes in patients with recurrent or metastatic squamous cell carcinoma of the head and neck (SCCHN). Patients and methods Archival baseline tumor specimens were obtained from patients treated on two clinical trials in recurrent or metastatic SCCHN: E1395, a phase III trial of cisplatin and paclitaxel versus cisplatin and 5-fluorouracil, and E3301, a phase II trial of irinotecan and docetaxel. HPV DNA was detected by in situ hybridization (ISH) with a wide-spectrum probe. p16 status was evaluated by immunohistochemistry. Clinical outcomes of interest were objective response, progression-free survival (PFS) and overall survival (OS). Results We analyzed 64 patients for HPV ISH and 65 for p16. Eleven tumors (17%) were HPV+, 12 (18%) were p16+, whereas 52 (80%) were both HPV- and p16-. The objective response rate was 55% for HPV-positive versus 19% for HPV-negative (P = 0.022), and 50% for p16-positive versus 19% for p16-negative (P = 0.057). The median survival was 12.9 versus 6.7 months for HPV-positive versus HPV-negative patients (P = 0.014), and 11.9 versus 6.7 months for p16-positive versus p16-negative patients (P = 0.027). After adjusting for other covariates, hazard ratio for OS was 2.69 (P = 0.048) and 2.17 (P = 0.10), favoring HPV-positive and p16-positive patients, respectively. The other unfavorable risk factor for OS was loss of ≥5% weight in previous 6 months (P = 0.0021 and 0.023 for HPV and p16 models, respectively). Conclusion HPV is a favorable prognostic factor in recurrent or metastatic SCCHN that should be considered in the design of clinical trials in this setting. Clinical Trial Identifier NCT01487733 Clinicaltrials.gov.

Objectives/Hypothesis To identify factors contributing to local tumor recurrence in oral cavity and laryngeal squamous cell carcinomas treated with surgery alone, to approximate tumor age from time to recurrence data by applying Collins' law, and to identify factors that may affect time to recurrence. Study Design Retrospective review. Methods A retrospective review of all patients treated for head and neck cancer from 1997 to 2013 at the University of Pittsburgh Medical Center was performed. Results A total of 517 patients treated with surgery alone qualified for the study, of which 84 had local recurrence, and 433 had no local recurrences. History of tobacco use (P = .017), pack-year cigarette history (P = .001), and T stage (P = .03) were associated with disease recurrence. Overall, never tobacco users, those with fewer pack-years of smoking history, and those with lower T stage were more likely to recur. Time to recurrence was significantly shorter for laryngeal tumors compared to oral cavity tumors (P = .027). Median time to recurrence for oral cavity tumors was 8.6 months and laryngeal tumors was 7.1 months. Conclusions In this study, lower pack-year smoking history, lower T stage, and history of never tobacco use were associated with local tumor recurrence. Applying the concept of Collins' law to estimate the age of tumors at diagnosis, the median tumor age at diagnosis was estimated to be 8.6 months for oral cavity tumors and 7.1 months for laryngeal tumors. Level of Evidence 4 Laryngoscope 124:2297–2304, 2014

Objectives/Hypothesis: Postoperative chemoradiation (CRT) has been shown to be more effective than postoperative radiotherapy (RT) alone in high risk head and neck squamous cell carcinoma (HNSCC) patients. Multimodality therapy is associated with more treatment related-toxicity. In this study, we assessed cervical lymph node histological characteristics to detect prognostic and predictive value differences to help guide therapeutic decision making. Study Design: Retrospective analysis of Cancer Registry data. Methods: HNSCC surgical patients who had tumor resection and neck dissection at our institution from 1980 to 2008 were identified (n=1510). Multivariable Cox proportional hazards regression models were developed to identify significant predictors of three outcomes: overall survival (OS), disease-specific survival (DSS), and neck disease recurrence (NDR). Hazard ratios were estimated for the number of cervical nodal metastases and presence of extracapsular spread (ECS) by adjuvant treatment after controlling for significant covariates. Results: Increasing number of positive nodes was significantly associated with poorer outcomes in OS, DSS, and NDR models (p

Background Combined inhibition of epidermal growth factor receptor (EGFR) and Src family kinases (SFK) may lead to improved therapeutic effects. We evaluated the combination of dasatinib, an inhibitor of SFK and other kinases, and cetuximab, an anti-EGFR monoclonal antibody. Patients and methods Patients with advanced solid malignancies received cetuximab intravenously on a standard weekly schedule and dasatinib orally, once daily at 3 dose levels: (1) 100 mg, (2) 150 mg, (3) 200 mg. Pharmacokinetic and pharmacodynamic studies of dasatinib were performed prior to starting cetuximab and following 14 days of treatment. Results Twenty-five patients (3 dose level 1; 19 dose level 2; 3 dose level 3) were initially treated. Three patients developed dose-limiting toxicities: 1 at dose level 2 (headache) and 2 at dose level 3 (headache, nausea). Grade 3–4 toxicities in more than 2 patients included: dyspnea (4), vomiting (4), nausea (3), hypersensitivity reactions (3), headache (3) and anemia (3). Twenty-one patients developed headache (8 grade 1; 10 grade 2), which occurred after the loading of cetuximab and lasted 1–3 days. Six additional patients were treated with dasatinib starting 3 days after the loading dose of cetuximab; none developed headache after dasatinib. Dasatinib pharmacokinetics and a transient decrease in SFK PY416 levels in peripheral blood mononuclear cells were not altered by cetuximab. Patients with higher plasma TGF-alpha levels had worse progression-free survival. Conclusions Dasatinib 150 mg once daily plus weekly cetuximab is recommended for phase II studies. Early-onset headache was ameliorated by starting dasatinib after cetuximab.

Purpose Squamous cell carcinoma of the head and neck (SCCHN) is characterized by upregulation of the epidermal growth factor receptor (EGFR). We developed a novel strategy to target EGFR by using a therapeutic gene that consisted of an EGFR antisense (AS) gene sequence under U6 promoter control. A phase I clinical trial was conducted to evaluate the safety and biologic effects of EGFR AS. Patients and Methods Patients with advanced SCCHN who were refractory to standard therapies and who had at least one assessable and accessible lesion were enrolled. The EGFR AS dose was escalated in successive cohorts (six dose levels; 60 to 1,920 μg/injection). Patients received four weekly intratumoral EGFR AS injections. Tumor biopsies were performed before and after completion of therapy. Treatment response was assessed by tumor volume measurements (positron emission tomography/computed tomography), and levels of target proteins were assessed by immunohistochemistry. Results Seventeen assessable patients were treated. No grades 3 to 4 or dose-limiting toxicities were noted, and a maximum-tolerated dose was not reached. Five patients (29%) achieved a clinical response, which included two complete responses (CRs) and three partial responses (PRs); two additional patients had stable disease (SD) as the best response. Patients with disease control (CR + PR + SD) had tumors with higher EGFR and lower STAT3 expression at baseline compared with patients who had progressive disease (P = .0312 and P = .095, respectively). Conclusion Intratumoral EGFR AS was safe and resulted in antitumor activity in patients with advanced SCCHN. Baseline levels of high EGFR and low STAT3 may be associated with antitumor effects.

Epidermal growth factor receptor (EGFR), a member of the ErbB family of receptor tyrosine kinases (RTKs), is highly expressed in head and neck squamous cell carcinoma (HNSCC) where increased EGFR expression levels in tumors are associated with decreased survival. HNSCC patient responses to EGFR-targeted monotherapies in clinical trials, though significant, have been limited. Tumor signaling pathway components that work in cooperation with EGFR or provide compensation for the loss of EGFR-initiated signaling will be ideal targets for therapies to be used in combination with EGFR-targeted agents. Based on the current understanding of molecular signaling pathways and available agents, ErbB family-targeted and Src family-targeted agents represent strategies for further exploration. Here, we discuss agents targeting ErbB and Src family kinases in clinical development, provide an overview of completed and ongoing clinical trials, and outline a molecular rationale for combining ErbB- and Src-targeted therapeutics.

Importance Randomized clinical trials demonstrate no benefit for epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in unselected patients with head and neck squamous cell carcinoma (HNSCC). However, a patient with stage IVA HNSCC received 13 days of neoadjuvant erlotinib and experienced a near-complete histologic response. Objective To determine a mechanism of exceptional response to erlotinib therapy in HNSCC. Design, Setting, and Participants Single patient with locally advanced HNSCC who received erlotinib monotherapy in a window-of-opportunity clinical trial (patients scheduled to undergo primary cancer surgery are treated briefly with an investigational agent). Whole-exome sequencing of pretreatment tumor and germline patient samples was performed at a quaternary care academic medical center, and a candidate somatic variant was experimentally investigated for mediating erlotinib response. Intervention A brief course of erlotinib monotherapy followed by surgical resection. Main Outcomes and Measures Identification of pretreatment tumor somatic alterations that may contribute to the exceptional response to erlotinib. Hypotheses were formulated regarding enhanced erlotinib response in preclinical models harboring the patient tumor somatic variant MAPK1 E322K following the identification of tumor somatic variants. Results No EGFR alterations were observed in the pretreatment tumor DNA. Paradoxically, the tumor harbored an activating MAPK1 E322K mutation (allelic fraction 0.13), which predicts ERK activation and erlotinib resistance in EGFR -mutant lung cancer. The HNSCC cells with MAPK1 E322K exhibited enhanced EGFR phosphorylation and erlotinib sensitivity compared with wild-type MAPK1 cells. Conclusions and Relevance Selective erlotinib use in HNSCC may be informed by precision oncology approaches.

© 2014 Elsevier Ireland Ltd. EGFR polymorphisms have not been thoroughly evaluated for association with head and neck squamous cell carcinoma (HNSCC) risk. We genotyped 578 HNSCC patients and 588 cancer-free controls for 60 EGFR single nucleotide polymorphisms (SNPs) and tested associations with HNSCC risk.EGFR intronic SNPs rs12535536, rs2075110, rs1253871, rs845561 and rs6970262 and synonymous SNP rs2072454 were associated with HNSCC risk among all subjects (p < 0.05). SNPs rs12538371, rs845561, and rs6970262 were significantly associated with HNSCC risk (p < 0.05) among never tobacco users. We identified EGFR variants that likely modify risk for HNSCC including three variants that contribute to tobacco-independent risk.

The primary aims of this study were to establish a high-throughput assay and analytic procedures that would allow us to assess with accuracy and reproducibility the presence of anti-cyclin B1 antibodies in a large cohort of subjects at-risk for development of lung cancer. The overall goal is to identify factors significantly associated with immune responses against this lung tumor antigen and to evaluate the relationship between anticyclin B1 antibodies and lung cancer risk in a prospective cohort. Successful cancer treatment relies on early detection. The immune system could respond to tumor antigens arising from altered host factors and provide an early readout for cancer presence. This study examines immune responses to the tumor antigen cyclin B1. Lung cancer patients have antibodies against cyclin B1. Unscheduled overexpression of this cell cycle regulator occurs early in tumorigenesis and might be detected by the immune system long before clinical diagnosis of cancer. Antibodies recognizing cyclin B1 were measured using semiautomated ELISAs in sera samples from subjects without detectable lung cancer enrolled in the Pittsburgh Lung Screening Study (PLuSS), a longitudinal study of long-term smokers over age 50. Factors significantly associated with antibody presence were identified using linear regression analysis. We have established a highly reproducible, semiautomated ELISA-based assay for the high-throughput assessment of serum antibody titers. Using this technology and a small subset of PluSS subjects, cyclin B1 antibody levels were found to be high in a small proportion of subjects tested. Regression analysis identified gender as well as age in women smokers to be significant determinants of cyclin B1 antibody levels, association not seen in male subjects. This work represents an important first step toward defining the importance of the presence of anticyclin B1 antibodies in an at-risk population and assessing the predictive value of serum cyclin B1 antibody as relates to lung cancer risk.

Purpose: Treatment with cisplatin or cetuximab combined with radiotherapy each yield superior survival in locally advanced squamous cell head and neck cancer (LA-SCCHN) compared with radiotherapy alone. Eastern Cooperative Oncology Group Trial E3303 evaluated the triple combination. Experimental Design: Patients with stage IV unresectable LA-SCCHN received a loading dose of cetuximab (400 mg/m2) followed by 250 mg/m2/week and cisplatin 75 mg/m2 q 3 weeks ×3 cycles concurrent with standard fractionated radiotherapy. In the absence of disease progression or unacceptable toxicity, patients continued maintenance cetuximab for 6 to 12 months. Primary endpoint was 2-year progression-free survival (PFS). Patient tumor and blood correlates, including tumor human papillomavirus (HPV) status, were evaluated for association with survival. Results: A total of 69 patients were enrolled; 60 proved eligible and received protocol treatment. Oropharyngeal primaries constituted the majority (66.7%), stage T4 48.3% and N2-3 91.7%. Median radiotherapy dose delivered was 70 Gy, 71.6% received all three cycles of cisplatin, and 74.6% received maintenance cetuximab. Median PFS was 19.4 months, 2-year PFS 47% [95% confidence interval (CI), 33%–61%]. Two-year overall survival (OS) was 66% (95% CI, 53%–77%); median OS was not reached. Response rate was 66.7%. Most common grade ≥3 toxicities included mucositis (55%), dysphagia (46%), and neutropenia (26%); one attributable grade 5 toxicity occurred. Only tumor HPV status was significantly associated with survival. HPV was evaluable in 29 tumors; 10 (all oropharyngeal) were HPV positive. HPV+ patients had significantly longer OS and PFS (P = 0.004 and P = 0.036, respectively). Conclusions: Concurrent cetuximab, cisplatin, and radiotherapy were well tolerated and yielded promising 2-year PFS and OS in LA-SCCHN with improved survival for patients with HPV+ tumors. Clin Cancer Res; 20(19); 5041–51. ©2014 AACR.

Objective: Deconditioning and frailty may contribute to dysphagia and aspiration. Early identification of patients at risk of aspiration is important. Aspiration prevention would lead to reduced morbidity and health care costs. We therefore wondered whether objective measurements of frailty could help identify patients at risk for dysphagia and aspiration. Methods: Consecutive patients (n = 183) were enrolled. Patient characteristics and objective measures of frailty were recorded prospectively. Variables tested included age, body mass index, grip strength, and 5 meter walk pace. Statistical analysis tested for association between these parameters and dysphagia or aspiration, diagnosed by instrumental swallowing examination. Results: Of variables tested for association with grip strength, only age category ( P = .003) and ambulatory status ( P < .001) were significantly associated with grip strength in linear regression models. Whereas walk speed was not associated with dysphagia or aspiration, ambulatory status was significantly associated with dysphagia and aspiration in multivariable model building. Conclusion: Nonambulatory status is a predictor of aspiration and should be included in risk assessments for dysphagia. The relationship between frailty and dysphagia deserves further investigation. Frailty assessments may help identify those at risk for complications of dysphagia.

Preclinical data indicate that activation of Src family kinases (SFKs) provides a mechanism for circumventing epidermal growth factor receptor (EGFR) targeting in head and neck squamous cell carcinoma (HNSCC). We performed a Simon two-stage, phase II trial combining dasatinib and cetuximab in patients with recurrent/metastatic HNSCC and analyzed candidate blood and tumor biomarkers for association with response. Significant biomarkers were evaluated in additional cetuximab-treated cohorts to evaluate specificity of association with response. Patients received 150 mg daily dasatinib three days after the loading dose of cetuximab; daily dasatinib and weekly cetuximab were continued until progressive disease (PD). Responses were assessed after 6 weeks and every 12 weeks thereafter until PD. Before and after treatment serum levels of interleukin-6 (IL6), transforming growth factor-alpha (TGF-α), hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) were measured by ELISA. Phospho-SFK levels were evaluated in pretreatment archived tumor specimens. Cell line models of HNSCC were assessed for in vitro cell viability following cetuximab-dasatinib treatment and the effects of IL6 modulation on treatment response. Fourteen patients were enrolled and treated during the first stage. The median treatment duration was 47 days. Thirteen patients were evaluable for response: 6 had stable disease (SD) and 7 had PD. No partial or complete response was observed; enrollment was halted according to the pre-specified futility rule. Low baseline serum IL6 levels ( Citation Format: Laura P. Stabile, Ann Marie Egloff, Pei Zhou, William E. Gooding, Jennifer R. Grandis, Julie E. Bauman. Phase II study of dasatinib in combination with cetuximab in recurrent/metastatic head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT119.

To report the prevalence of anatomic variants on computed tomography (CT) in congenital aural atresia (CAA) and external auditory canal stenosis (EACS). Anatomic variants included inferiorly displaced/obstructing tegmen mastoideum, malleus-incus complex (MIC) directly lateral to stapes, facial nerve obstruction of oval window (OW) or middle ear, and incudostapedial joint (ISJ) angle.Cross-sectional study.Tertiary care children's hospital.An anatomic analysis of 130 CT scans (98 children, 32 bilateral) of CAA/EACS, performed by a blinded neuroradiologist. Both Jahrsdoerfer's and new/modified anatomic considerations were graded in 32 atresiaplasty and 66 nonsurgical patients. Surgical data were analyzed for anatomic correlations related to surgical findings.Prevalence of anatomic variants was as follows: 13% of the ears had mild inferior displacement of tegmen, 4% had a significantly obstructing tegmen, and 24% had MIC directly lateral to stapes. The facial nerve obstructed access to OW in 41% and middle ear in 21%. Six atresiaplasty patients were reported to have a large MIC obstructing stapes access with increased intraoperative difficulty in viewing and assessing the integrity and mobility of the ISJ and stapes. Five of these 6 (83%) were noted on CT scan. The mean ISJ angle was 101° (range, 51°-155°).A large obstructing MIC increases difficulty of atresiaplasty. Awareness of the presence of these anatomic variants is an aid in teaching temporal bone anatomy and may possibly influence the decision regarding atresiaplasty.

Expression of gastrin-releasing peptide receptor (GRPR) is elevated in mucosa adjacent to head and neck squamous cell carcinoma (HNSCC) compared with mucosa from cancer-free controls, suggesting elevated GRPR expression may indicate presence of HNSCC.We measured GRPR mRNA levels in histologically normal buccal mucosa from 65 surgical patients with HNSCC and 75 cancer-free control subjects using quantitative polymerase chain reaction (PCR). We tested for association between GRPR expression and HNSCC and evaluated differences in patient progression-free survival (PFS).Buccal GRPR expression was higher in cases but not controls who were active smokers (p = .04). High GRPR expression was associated with HNSCC (odds ratio [OR] = 3.55; 95% confidence interval [CI] = 1.15-10.93), even after adjustment for age, sex, tobacco use, and sample storage time. PFS did not differ between patients with HNSCC with high versus low GRPR expression (p = .22).Elevated buccal GRPR expression was significantly associated with HNSCC independent of known risk factors but was not an indicator of disease prognosis.

Objective: Report incidence of anatomic variants in temporal bone CT scans with congenital aural atresia (CAA) or external auditory canal stenosis (EACS). Anatomic variants include: inferiorly displaced tegmen, malleus/incus complex (MIC) directly lateral to stapes, facial nerve obstruction at oval window (OW) and middle ear, middle ear area, incudostapedial (IS) joint angle.Method: Prospective anatomic study of 134 CT scans (102 children, 32 bilateral) of CAA/EACS, graded by a blinded neuroradiologist. Both Jahrsdoerfer and absence or presence of new/refined anatomic considerations were graded. There were 32 atresiaplasty and 70 nonsurgical patients. Surgical and audiological data were reviewed retrospectively for anatomic correlations related to outcomes.Results: Prevalence of anatomical variants: inferiorly displaced tegmen X (X%), MIC directly lateral to stapes Y (Y%), facial nerve obstructed access to oval window Z (Z%), and middle ear B (B%). Large MIC obstructing stapes access (5/7 patients) with i...

Purpose: A high frequency of head and neck squamous cell cancers (HNSCC) contain constitutively activated signal transducer and activator of transcription 3 (STAT3). To further elucidate the prognostic role of STAT3 in HNSCC, the expression pattern of STAT3 was correlated with outcome in two independent data sets. Experimental Design: STAT3 protein expression analysis was done on a test cohort of 102 patients with HNSCC recruited between 1992 and 2005. Automated quantitative analysis was used to assess STAT3 protein expression. We evaluated associations with clinicopathologic parameters and survival prognosis. Associations were validated in a second, independent cohort of 58 patients with confirmed HNSCC enrolled in the Early Detection Research Network–sponsored study who underwent surgical resection with curative intent at the University of Pittsburgh Medical Center between 2000 and 2004. Results: STAT3 displayed mixed nuclear and cytoplasmic staining. Survival analysis showed that high nuclear STAT3 expression (top tertile versus the rest) was associated with longer progression-free survival (n = 70, mean survival of 88.9 versus 46.7 months, P = 0.012 for the first cohort; n = 37, mean survival of 60.3 versus 33.0 months, P = 0.009 for the second cohort). After best model selection in the multivariable analysis context, only STAT3 was significant, revealing a lower risk of progression and death for patients with high nuclear STAT3-expressing tumors (hazard ratio, 0.28; 95% confidence interval, 0.10-0.82; P = 0.019; and hazard ratio, 0.23; 95% confidence interval, 0.07-0.76; P = 0.016, respectively). Conclusions: Our results indicate that high nuclear STAT3 expression levels by automated quantitative analysis are associated with favorable outcome in HNSCC. Clin Cancer Res; 16(8); 2427–34. ©2010 AACR.

The epidermal growth factor receptor- (EGFR-) directed antibody, cetuximab, was FDA-approved for the treatment of squamous cell carcinoma of the head and neck (SCCHN) in 2006. Additional EGFR-targeting agents in clinical development for SCCHN include other EGFR-directed antibodies, tyrosine kinase inhibitors and antisense DNA. Although the majority of SCCHN overexpress EGFR, SCCHN clinical responses to EGFR-targeting agents have been modest. Molecular predictors for SCCHN response to EGFR-targeted therapies have not been identified. However, molecular correlate studies in lung cancer and colon cancer, which have EGFR-targeted therapeutics FDA-approved for treatment, may provide insights. We describe candidate predictive markers for SCCHN response to EGFR-targeted therapies and their prevalence in SCCHN. Clinical response will likely be improved by targeted therapy combination treatments. Src family kinases mediate EGFR-dependent and -independent tumor progression pathways in many cancers including SCCHN. Several Src-targeting agents are in clinical development for solid malignancies. Molecular correlate studies for Src-targeting therapies are few and biomarkers correlated with patient response are limited. Identifying SCCHN patients who will respond to combined EGFR- and Src-targeting will require further characterization of molecular correlates. We discuss rationale for EGFR and Src co-targeting for SCCHN treatment and describe recent clinical trials implementing combined Src- and EGFR-targeted therapeutics.

Background: Although cetuximab has demonstrated only modest response rates in head and neck squamous cell carcinoma (HNSCC) patients in clinical studies, most HNSCC cell line xenograft models have demonstrated sensitivity to cetuximab. We sought to characterize molecular and phenotypic changes associated with the acquisition of cetuximab resistance by evaluating patient-derived xenograft (PDX) models with and without cetuximab selection in vivo. Methods: A single PDX was established from a patient tongue HNSCC and a separate PDX from an independent patient lymph node by subcutaneous propagation in nod scid gamma (NSG) immunodeficient mice. Established PDXs were implanted into 2 flanks of NSG mice, and mice were either untreated (parental) or treated with cetuximab using modulated dosing dependent upon tumor growth (0.04-0.2 mg 1-2 times weekly) with the goal of PDX growth at the maximum dose. Tumor histology and short tandem repeat (STR) profiling were evaluated for HNSCC tumors and paired PDX models. Cell lines were derived from PDX tumors grown in NSG hypoxanthine-guanine phosphoribosyltransferase (Hprt)-deficient mice, allowing negative selection of mouse fibroblasts in vitro. Cell lines were retrovirally transduced to express either green fluorescent protein (GFP) or telomerase (HTERT). Cell lines were evaluated for presence of human cells using a PCR-based assay to detect human amelogenin alleles (sex id assay). Candidate gene mutations and protein/phosphoprotein levels will be evaluated in parental and cetuximab-selected PDXs using Ion Torrent and immunoblotting technologies, respectively. Results: Patient HNSCC tumors and paired parental PDXs displayed similar histologies and consistent STR profiles. Four of 14 PDXs derived from the tongue HNSCC displayed growth in the presence of cetuximab at the maximum dose, and of 18 PDXs derived from lymph node, 3 grew in the presence of cetuximab at the maximum dose. Average tumor volume at treatment initiation was 134 mm3 and average duration of treatment was 8 months. For tumors that eventually grew in presence of maximum dose cetuximab, initial tumor volume and duration of treatment were 156.5 mm3 and 9 months, respectively. Cetuximab-resistant tumors were heterogeneous regarding their resistance to cetuximab in a subsequent passage. Of the two cetuximab-resistant tumors STR evaluated, 1 was consistent with the patient and parental PDX and 1 could not be genotyped. In vitro cell cultures were established from parental PDX tumor cells transduced with HTERT and GFP, and these cells were confirmed to be of human origin by the sex id assay. Molecular profiling and establishment of cell lines derived from cetuximab-resistant, genotype confirmed PDXs are currently ongoing. Conclusions: It was possible to obtain PDX tumors grown under conditions of chronic treatment with cetuximab. However, dose modulation was required and propagation of the trait of cetuximab-resistant growth upon PDX passage was not guaranteed. The acquisition of cetuximab-resistant PDX tumors was a lengthy process that required care regarding dosing modulation and genotype validation. Once established, these models will provide valuable insights regarding molecular and phenotypic changes associated with acquired cetuximab resistance. Citation Format: Pei Zhou, Hua Li, Sarah Wheeler, Jennifer R. Grandis, Laura A. Stabile, Ann Marie Egloff. Generation of head and neck cancer patient-derived xenografts with in vivo acquired cetuximab resistance. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B39.

Several molecular-targeted therapeutics have been tested in clinical trials for the treatment of head and neck squamous cell carcinoma (HNSCC). Of these, therapeutics targeting the epidermal growth factor receptor (EGFR) have been studied most extensively and some agents have demonstrated measurable clinical effectiveness. However, molecular studies designed to define HNSCC patient subcohorts of likely responders to EGFR-targeted therapy have not identified molecular signatures that correlate with clinical response. Here, the authors summarise the relevant clinical findings and highlight reported molecular correlative studies for EGFR-targeted therapeutics for HNSCC. The authors focus especially on molecular markers evaluated for association with clinical response and include data from EGFR-targeted clinical studies in other cancer sites that they anticipate will be of interest to the head and neck cancer research and treatment communities.

Head and neck squamous cell carcinoma (HNSCC) is frequently characterized by aggressive clinical behavior with a propensity for metastasis and recurrence. Current understanding of HNSCC pathogenesis remains limited, and there are few biomarkers that reliably predict patient outcomes such as survival or response to therapy. Here we report a comprehensive analysis of the molecular and clinical features of HNSCC that define patient survival. Building on data and infrastructure established by The Cancer Genome Atlas (TCGA), we develop an approach that integrates molecular events across multiple tiers of ‘omics data to progressively factor patients into subgroups with the greatest survival differences. We show that in HPV negative patients, the detrimental impact of TP53 mutation occurs only in combination with loss of chromosome 3p, leading to a marked decrease in median survival from >5 years for TP53 mutation only to 1.7 years for both events. Conditioned on TP53/3p, patients are stratified by a hierarchy of events incorporating MUC5B mutation, mir-548k expression, and mutation and expression of Abnormal Spindle Protein (ASPM), while in the absence of the TP53/3p event RAS signaling is an important driver. We also show in HPV positive patients that HPV-mediated inactivation of TP53 acts in synergy with 3p deletion. The major findings are replicated in an independent HNSCC cohort as well as a pan-cancer TCGA cohort of over 3000 patients. Together, the identified markers enable a new multi-tiered molecular classification of HNSCC. Citation Format: Andrew M. Gross, Ryan K. Orosco, John P. Shen, Ann Marie Egloff, Hannah Carter, Matan Hoffree, Michel Choueiri, Charles S. Coffey, Scott M. Lippman, David Neil Hayes, Ezra E. Cohen, Jennifer R. Grandis, Quyen T. Nguyen, Trey Ideker. A prognostic model of head and neck cancer ties TP53 mutation to 3p loss. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3821. doi:10.1158/1538-7445.AM2014-3821

6036 Background: Almost all survivors of FA, an inherited DNA repair deficiency syndrome, will develop H&N Ca. Preclinical data in H&N Ca cells harboring FA mut show increased sensitivity to chemo ...

6006 Background: We studied the association of human papillomavirus (HPV) and excision repair cross-complementation group 1 (ERCC1) tumor expression with clinical outcomes in patients (pts) with R/M SCCHN. Methods: Archival baseline specimens were obtained from pts on ECOG trials: E1395, phase III trial of cisplatin/paclitaxel (CP) vs. cisplatin/5-FU (CF), and E3301, phase II trial of docetaxel/irinotecan. HPV DNA was detected by in situ hybridization (ISH) with a wide spectrum HPV probe. Tumor p16 status was defined as positive if immunohistochemical staining for p16 was strong and present in >80% of tumor cells. ERCC1 expression was measured (HistoRx PM-2000) and data analyzed using AQUA algorithms, after tissue was stained with ERCC1 ab (1:5000 HPA0297731, Sigma), and a wide-spectrum cytokeratin ab (Dako Z0622) for tumor mask. A prior determined cut point for nuclear staining was utilized. Fisher's exact test and log-rank test were used to compare categorical variables and survival. Stratified logistic regression and Cox regression model were used to estimate odds ratio (OR) and hazard ratio (HR), respectively, adjusting for potential confounding factors. p-values were two-sided. Results: Tissue was evaluable from 65 tumors (T) for HPV, 66 for p16 (E1395 and E3301), and 43 for ERCC1 expression (E1395). 11 T were HPV+ (12 p16+), and 54 were HPV-/p16-. HPV+ tumors were similarly represented in all treatment groups (p=0.58). Objective response rates (RR): 67% for HPV+, 22% for HPV- (p=0.013); 60% p16+, 22% p16- (p=0.05). RR rates were calculated excluding cases with unevaluable/unknown responses. HR for OS was 2.66 (HPV, p=0.02) and 2.27 (p16, p=0.04), favoring HPV+/p16+ pts. 18 T were ERCC1 high (H) and 25 ERCC1 low (L). HR for OS (H vs. L) was 1.96 (p=.11) RR: CF, 58% (L), 29% (H); CP, 33% (L), 56% (H). A test of ERCC1 by treatment interaction (p=0.12) suggested the effect of ERCC1 may be different for taxane vs. non-taxane regimens. Conclusions: This is the first study to show that HPV+/p16+ status is associated with a significant improvement in RR and OS among pts treated for R/M SCCHN. ERCC1 L was associated with a trend towards a better OS.

Objective To determine predictive factors for residual disease and hearing outcomes of surgery for congenital cholesteatoma (CC). Design Retrospective record review of surgery for CC from January 1, 1998, through December 31, 2010. The initial extent of CC was staged using the system as defined by Potsic et al. Setting Tertiary care children's hospital. Patients Eighty-one children (82 ears) underwent a total of 230 operations for CC. The mean (SD) age was 5.3 (2.9) years, and the mean follow-up was 4.3 years. Intervention Initial and subsequent operations for CC and audiologic evaluations. Main Outcome Measures Statistical analyses were performed to determine factors associated with increased residual disease for CC and poorer hearing outcomes. Results Higher initial stage of disease, erosion of ossicles, and removal of ossicles were significantly associated with increased likelihood of residual CC (46%, 50%, and 51%, respectively; P Conclusions More extensive initial disease, ossicular erosion, and the need for ossicular removal were associated with residual disease. On the basis of our data, the best chance for completely removing CC at initial surgery involves removing involved ossicles if they are eroded, if the CC is abutting or enveloping the incus or stapes, if the CC is medial to the malleus or incus, or if the matrix of the CC is violated. These results may help guide surgeons to achieve the best results for their patients.

TMEM16A, a calcium-activated chloride channel, was recently isolated from oral cavity cancer cells. TMEM16A is amplified in head and neck cancers and overexpressed in gastrointestinal stromal tumors (GIST's); however, the role that TMEM16A plays in promoting tumor growth remains unclear. The goal of this research was to establish the biologic function of TMEM16A in the context of epithelial malignancies. We hypothesized that TMEM16A contributes to tumor cell survival and may be a useful prognostic factor for patients with squamous cell carcinoma of the head and neck (SCCHN). Knockdown and overexpressing SCCHN and T24 cell lines were transduced with lentiviral particles encoding RNAi and engineered to stably overexpress TMEM16A. For functional verification, electrophysiologic assays were performed to establish that the altered TMEM16A expression was modulating the chloride current. To elucidate a mechanism of TMEM16A's proliferative effects, immunoblotting and pharmacologic inhibition of potential downstream targets were performed. Our results demonstrate that the levels of TMEM16A expression modulates proliferation in vitro and in vivo, and that stable overexpression of TMEM16A increases resistance to cisplatin therapy as compared to cancer cell lines expressing endogenous TMEM16A. Specificity of these phenotypic results were confirmed using genetic rescue experiments. Next, we identified that the MAPK/Cyclin-D1 axis was a critical signaling pathway in TMEM16A-induced proliferation, and that pMAPK/MAPK and Cyclin-D1 levels paralleled expression of TMEM16A. Finally, using pharmacologic agents niflumic acid or NPPB to inhibit the TMEM16A chloride channel activity in combination with a MAPK inhibitor (U0126) demonstrated additive effects significantly decreasing cancer cell proliferation in vitro. In summary, our results provide novel insights into the biologic function of TMEM16A and its role in tumor cell proliferation. This research has enhanced our understanding about the pathogenesis of SCCHN tumors, and can potentially be translated to many other cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-220. doi:10.1158/1538-7445.AM2011-LB-220

Methods: HNSCC surgical patients who had tumor resection and neck dissection at our institution from 1980 through 2007 were included in this retrospective study (n=1534). Cases were categorized according to number of positive cervical nodes (no positive nodes (n=719), 1-2 positive nodes (n=459) or 3+ positive nodes (n=351)) and by ECS status as negative (n=1048) or positive (n=415). Differences in disease-free survival (DFS) and overall survival (OS) were assessed using KaplanMeier and log-rank tests.

TPS206 Background: Esophageal carcinoma is highly lethal and the incidence is increasing, especially the subtype of EAC. Our group demonstrated that overexpression of the epidermal growth factor re...

5537 Background: The molecular characteristics of SCCHN associated with response to C225 alone or in combination with RT and DDP are unknown. We evaluated tumor HPV status, EGFR gene amplification,...

Purpose: Evaluate EGFR gene amplification status and EGFR mRNA and protein/phosphoprotein levels in tumors from patients with head and neck squamous cell carcinoma (HNSCC) surgically treated with curative intent from 2000-4 to define relationships between and assess the prognostic values of these biomarkers. Background: EGFR has been reported to be overexpressed in HNSCC, and high levels of EGFR protein and EGFR gene amplification have been independently reported to be associated with poorer prognosis. To our knowledge, tumor levels of the known site-specific phosphorylated EGFR (Y992, Y1068) have not been reported as prognostic indicators for HNSCC. Methods: 58 tumor specimens and 50 paired adjacent mucosal tissues from patients surgically treated for HNSCC were collected through an Early Detection Research Network (EDRN) study. Tissue microarrays (TMAs) were constructed using these paired patient samples and 30 oral mucosal specimens from cancer-free controls. Three cores from each specimen were arrayed on two TMAs and evaluated for EGFR gene amplification by dual-color FISH and for EGFR by IHC (EGFR levels in these IHC specimens have been previously reported). Corresponding fresh-frozen tumor specimens were evaluated by reverse-phase protein array (RPPA) for total EGFR, EGFR P-Y992 and P-Y1068 levels and by quantitative RT-PCR for EGFR mRNA levels. Progression-free survival (PFS) was calculated using the University of Pittsburgh Head and Neck Cancer Registry data as months from date of surgery to date of first cancer recurrence, new primary, or death. Hazard Ratios were determined using Cox proportional hazards models. Results: Tumors with high level EGFR gene amplification expressed significantly higher levels of EGFR protein as assessed by IHC than tumors without EGFR gene amplification (P Conclusions: Similar to previous findings, we find that patients with tumor EGFR gene amplification tend to have shorter PFS than do patients without EGFR gene amplification. We find that tumors with EGFR gene amplification have significantly higher EGFR protein levels as assessed by IHC. Though increased EGFR protein levels in tumors are not always associated with gene amplification, this may be an important mechanism for increased EGFR expression. Differential levels of site-specific EGFR phosphorylation at Y992 and Y1068 indicate that signaling pathways associated with these sites may play different roles in disease progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4646.

The epidermal growth factor receptor (EGFR), c-Met and G-protein-coupled receptor signaling pathways contribute to head and neck squamous cell carcinoma (HNSCC) growth. To assess these pathways in HNSCC patients, we measured plasma levels of EGFR, the EGFR ligands transforming growth factor alpha (TGF-α) and amphiregulin (AR), the c-Met ligand hepatocyte growth factor (HGF) and COX-2, a component of prostaglandin production, in HNSCC patients (n=22) and cancer-free controls (n=32) enrolled through an Early Detection Research Network- (EDRN-) sponsored effort. Analytes were measured using a multiplex enzyme-linked immunosorbent assay. Differences in analyte levels between cases and controls and the prognostic potential of each analyte were evaluated. EGFR plasma levels were significantly decreased (P=0.04, rank sum test (RST)) and HGF plasma levels significantly increased (P Plasma EGFR and HGF levels may provide indications of HNSCC cancer status, and EGFR plasma levels may be a valuable prognostic indicator for patients with HNSCC. Decreased plasma EGFR levels in HNSCC patients were likely not due to general effects related to smoking and instead may reflect tissue characteristics specific to tobacco-related cancers. We found EGFR plasma levels in HNSCC patients were not positively correlated with EGFR tumor levels. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4661.

6034 Background: Src is implicated in the resistance to epidermal growth factor receptor (EGFR) inhibitors. We studied biomarkers in the context of a phase I clinical trial of D, a small molecule inhibitor of several kinases, including Src, and C, a chimeric IgG1 monoclonal anti-EGFR antibody (Esteve et al. ASCO 2008; A14668). Methods: Plasma levels of EGFR, amphiregulin (AR), transforming growth factor-α (TGF-α), COX-2 and hepatocyte growth factor (HGF) were measured using a multiplex enzyme-linked immunosorbant assay (Searchlight TM) at day 0 and day 15. Blood mononuclear cell pSrc/Src levels were measured by Odyssey western blotting at day 0 (no C) and day 15 (with C) prior to and 2, 4, 8 and 24 hours after D. Clinical results were tested for association with biomarkers. The same biomarkers were also measured in plasma collected through an Early Detection Research Network- (EDRN-) sponsored effort from 22 squamous cell carcinoma of the head and neck (SCCHN) pts and 32 cancer-free matched controls. Results: 25 pts had baseline levels, 16 with paired samples. At day 15, EGFR plasma levels significantly decreased (p < 0.001), while AR (p < 0.001) and TGF-α (p < 0.001) significantly increased compared to pretreatment levels. HGF and COX 2 plasma levels did not change with treatment. Pre-daily D pSrc levels on day 0 and day 15 did not differ. pSrc levels decreased from pre-daily D levels by 8 hours post-D at day 0 (p = 0.036, n = 21) and day 15 (p = 0.027, n = 13) and returned to pre-D levels by 24 hours. In 17 pts evaluable for response, only low baseline levels of TGF-α were associated with an increased likelihood of disease control (p = 0.020). Elevated HGF at baseline was seen in treatment pts and SCCHN control pts compared to cancer-free controls (p = 0.006 and p = 0.001, respectively). Otherwise, there was no significant difference in plasma biomarker levels compared to the matched EDRN. Conclusions: D and C significantly decreased plasma levels of EGFR and increased EGFR ligands (AR and TGF-α). Similar data has been shown in vitro, suggesting that C binding to EGFR leads to fewer available receptors for ligand binding. pSrc transiently decreased after D dosing. We will evaluate C plus D and validate these biomarkers in a planned phase II trial in SCCHN. [Table: see text]