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PurposeNeoadjuvant targeted therapy provides a brief, preoperative window of opportunity that can be exploited to individualize cancer care based on treatment response. We investigated whether response to neoadjuvant therapy during the preoperative window confers survival benefit in patients with operable head and neck squamous cell carcinoma (HNSCC).Patients and methodsA pooled analysis of treatment-naïve patients with operable HNSCC enrolled in one of three clinical trials from 2009 to 2020 (NCT00779389, NCT01218048, NCT02473731). Neoadjuvant regimens consisted of EGFR inhibitors (n = 83) or anti-ErbB3 antibody therapy (n = 9) within 28 days of surgery. Clinical to pathologic stage migration was compared with disease-free survival (DFS) and overall survival (OS) while adjusting for confounding factors using multivariable Cox regression. Circulating tumor markers validated in other solid tumor models were analyzed.Results92 of 118 patients were analyzed; all patients underwent surgery following neoadjuvant therapy. Clinical to pathologic downstaging was more frequent in patients undergoing neoadjuvant targeted therapy compared with control cohort (P = 0.048). Patients with pathologic downstage migration had the highest OS [89.5%; 95% confidence interval (CI), 75.7-100] compared with those with no stage change (58%; 95% CI, 46.2-69.8) or upstage (40%; 95% CI, 9.6-70.4; P = 0.003). Downstage migration remained a positive prognostic factor for OS (HR, 0.22; 95% CI, 0.05-0.90) while adjusting for measured confounders. Downstage migration correlated with decreased circulating tumor markers, SOX17 and TAC1 (P = 0.0078).ConclusionsBrief neoadjuvant therapy achieved pathologic downstaging in a subset of patients and was associated with significantly better DFS and OS as well as decreased circulating methylated SOX17 and TAC1.

The epidermal growth factor receptor (EGFR) inhibitor cetuximab is the only FDA-approved oncogene-targeting therapy for head and neck squamous cell carcinoma (HNSCC). Despite variable treatment response, no biomarkers exist to stratify patients for cetuximab therapy in HNSCC. Here, we applied unbiased hierarchical clustering to reverse-phase protein array molecular profiles from patient-derived xenograft (PDX) tumors and revealed 2 PDX clusters defined by protein networks associated with EGFR inhibitor resistance. In vivo validation revealed unbiased clustering to classify PDX tumors according to cetuximab response with 88% accuracy. Next, a support vector machine classifier algorithm identified a minimalist biomarker signature consisting of 8 proteins - caveolin-1, Sox-2, AXL, STING, Brd4, claudin-7, connexin-43, and fibronectin - with expression that strongly predicted cetuximab response in PDXs using either protein or mRNA. A combination of caveolin-1 and Sox-2 protein levels was sufficient to maintain high predictive accuracy, which we validated in tumor samples from patients with HNSCC with known clinical response to cetuximab. These results support further investigation into the combined use of caveolin-1 and Sox-2 as predictive biomarkers for cetuximab response in the clinic.

Immune checkpoint blockade (ICB) therapy has revolutionized head and neck squamous cell carcinoma (HNSCC) treatment, but

Cetuximab, an anti-EGFR monoclonal antibody (mAb), is approved for advanced head and neck squamous cell carcinoma (HNSCC) but benefits a minority. An established tumor-intrinsic resistance mechanism is cross-talk between the EGFR and hepatocyte growth factor (HGF)/cMet pathways. Dual pathway inhibition may overcome cetuximab resistance. This Phase I study evaluated the combination of cetuximab and ficlatuzumab, an anti-HGF mAb, in patients with recurrent/metastatic HNSCC. The primary objective was to establish the recommended Phase II dose (RP2D). Secondary objectives included overall response rate (ORR), progression-free survival (PFS), and overall survival (OS). Mechanistic tumor-intrinsic and immune biomarkers were explored. Thirteen patients enrolled with no dose-limiting toxicities observed at any dose tier. Three evaluable patients were treated at Tier 1 and nine at Tier 2, which was determined to be the RP2D (cetuximab 500 mg/m2 and ficlatuzumab 20 mg/kg every 2 weeks). Median PFS and OS were 5.4 (90% CI = 1.9-11.4) and 8.9 (90% CI = 2.7-15.2) months, respectively, with a confirmed ORR of 2 of 12 (17%; 90% CI = 6-40%). High circulating soluble cMet levels correlated with poor survival. An increase in peripheral T cells, particularly the CD8+ subset, was associated with treatment response whereas progression was associated with expansion of a distinct myeloid population. This well-tolerated combination demonstrated promising activity in cetuximab-resistant, advanced HNSCC.

PurposeErbB3 and its ligand neuregulin-1 (NRG1) are widely expressed in head and neck squamous cell carcinoma (HNSCC) and associated with tumor progression. A "window-of-opportunity" study (NCT02473731) was conducted to evaluate the pharmacodynamic effects of CDX-3379, an anti-ErbB3 mAb, in patients with HNSCC.Patients and methodsTwelve patients with newly diagnosed, operable HNSCC received two infusions of CDX-3379 (1,000 mg) at a 2-week interval prior to tumor resection. The primary study objective was to achieve ≥50% reduction in tumor ErbB3 signaling (phosphorylation of ErbB3; pErbB3) in ≥30% of patients. Other potential tumor biomarkers, pharmacokinetics, safety, and tumor measurements were also assessed.ResultspErbB3 was detectable in all tumors prior to treatment and decreased for 10 of 12 (83%) patients following CDX-3379 dosing, with ≥50% reduction in 7 of 12 (58%; P = 0.04; 95% confidence interval, 27.7%-84.8%). Target trough CDX-3379 serum levels were achieved in all patients. CDX-3379 treatment-related toxicity was grade 1-2 and included diarrhea, fatigue, and acneiform dermatitis. Five of 12 (42%) patients had shrinkage in tumor burden, including a marked clinical response in a patient with human papillomavirus-negative oral cavity HNSCC. All patients with tumor shrinkage had tumors that expressed both NRG1 and ErbB3 and demonstrated reduced pErbB3 with CDX-3379 treatment.ConclusionsThis study demonstrates that CDX-3379 can inhibit tumor ErbB3 phosphorylation in HNSCC. CDX-3379 was well tolerated and associated with measurable tumor regression. A phase II study (NCT03254927) has been initiated to evaluate CDX-3379 in combination with cetuximab for patients with advanced HNSCC.

BYL719 is a PI3K inhibitor that has demonstrated efficacy in the treatment of head and neck squamous cell carcinoma. BYL719 exerts its therapeutic effect by suppressing AKT and other proliferative signaling mechanisms. Despite PI3K inhibition and AKT suppression, residual activity of protein S6, a core marker of proliferative activation, has been observed. HER3, either via dimerization or activation by its ligand neurgeulin (NRG), is known to activate PI3K. Thus, we hypothesized that co-targeting HER3 and PI3K would lead to greater suppression of the PI3K-AKT signaling pathway and greater tumor suppression than with BYL719 alone. We investigated biochemical expression and activation of the HER3-PI3K-AKT-S6 pathway in HNSCC cell lines and patient-derived xenografts (PDXs). Antitumor effects of HER3 and PI3K inhibitors alone and in combination were evaluated in cell culture and murine models. Treatment of HNSCC cell lines with BYL719 significantly reduced AKT activation and suppressed tumor growth. However, S6 was persistently activated despite suppression of AKT. Combination treatment with KTN3379, a monoclonal antibody targeted against HER3, and BYL719 led to enhanced suppression of in vitro and in vivo cancer growth and durable suppression of AKT and S6. Therefore, inhibition of HER3 with KTN3379 enhanced the effects of PI3K inhibition in pre-clinical HNSCC models. These data support co-targeting HER3 and PI3K for the treatment of HSNCC.

PIK3CA is the most commonly altered oncogene in head and neck squamous cell carcinoma (HNSCC). We evaluated the impact of nonsteroidal anti-inflammatory drugs (NSAIDs) on survival in a PIK3CA-characterized cohort of 266 HNSCC patients and explored the mechanism in relevant preclinical models including patient-derived xenografts. Among subjects with PIK3CA mutations or amplification, regular NSAID use (≥6 mo) conferred markedly prolonged disease-specific survival (DSS; hazard ratio 0.23, P = 0.0032, 95% CI 0.09-0.62) and overall survival (OS; hazard ratio 0.31, P = 0.0043, 95% CI 0.14-0.69) compared with nonregular NSAID users. For PIK3CA-altered HNSCC, predicted 5-yr DSS was 72% for NSAID users and 25% for nonusers; predicted 5-yr OS was 78% for regular NSAID users and 45% for nonregular users. PIK3CA mutation predicted sensitivity to NSAIDs in preclinical models in association with increased systemic PGE2 production. These findings uncover a biologically plausible rationale to implement NSAID therapy in PIK3CA-altered HNSCC.

BackgroundCetuximab combined with radiation therapy (RT) is an evidence-based treatment for locally advanced head and neck squamous cell carcinoma (HNSCC); however, locoregional failure remains the primary cause of cancer-related death in this disease. Intratumoral injection of epidermal growth factor receptor (EGFR)-antisense plasmid DNA (EGFR-AS) is safe and has been associated with promising lesional responses in patients who have recurrent/metastatic HNSCC. For the current study, the authors investigated the antitumor effects of cetuximab and EGFR-AS in preclinical HNSCC models and reported their phase 1 experience adding intratumoral EGFR-AS to cetuximab RT.MethodsAntitumor mechanisms were investigated in cell line and xenograft models. Phase 1 trial eligibility required stage IVA through IVC HNSCC and a measurable lesion accessible for repeat injections. Patients received standard cetuximab was for 9 weeks. EGFR-AS was injected weekly until they achieved a lesional complete response. RT was delivered by conventional fractionation for 7 weeks, starting at week 3. Research biopsies were obtained at baseline and week 2.ResultsWhen added to cetuximab, EGFR-AS decreased cell viability and xenograft growth compared with EGFR-sense control, partially mediated by reduced EGFR expression. Six patients were enrolled in the phase 1 cohort. No grade 2 or greater EGFR-AS-related adverse events occurred. The best lesional response was a complete response (4 patients), and 1 patient each had a partial response and disease progression. EGFR expression decreased in 4 patients who had available paired specimens.ConclusionsIn preclinical models, dual EGFR inhibition with cetuximab and EGFR-AS enhanced antitumor effects. In a phase 1 cohort, intratumoral EGFR-AS injections, cetuximab, and RT were well tolerated. A phase 2 trial is needed to conduct an extended evaluation of safety and to establish efficacy.

Human papillomavirus (HPV) type 16 is implicated in approximately 75% of head and neck squamous cell carcinomas (HNSCC) that arise in the oropharynx, where viral expression of the E6 and E7 oncoproteins promote cellular transformation, tumor growth, and maintenance. An important oncogenic signaling pathway activated by E6 and E7 is the PI3K pathway, a key driver of carcinogenesis. The PI3K pathway is also activated by mutation or amplification of PIK3CA in over half of HPV(+) HNSCC. In this study, we investigated the efficacy of PI3K-targeted therapies in HPV(+) HNSCC preclinical models and report that HPV(+) cell line- and patient-derived xenografts are resistant to PI3K inhibitors due to feedback signaling emanating from E6 and E7. Receptor tyrosine kinase profiling indicated that PI3K inhibition led to elevated expression of the HER3 receptor, which in turn increased the abundance of E6 and E7 to promote PI3K inhibitor resistance. Targeting HER3 with siRNA or the mAb CDX-3379 reduced E6 and E7 abundance and enhanced the efficacy of PI3K-targeted therapies. Together, these findings suggest that cross-talk between HER3 and HPV oncoproteins promotes resistance to PI3K inhibitors and that cotargeting HER3 and PI3K may be an effective therapeutic strategy in HPV(+) tumors.Significance: These findings suggest a new therapeutic combination that may improve outcomes in HPV(+) head and neck cancer patients. Cancer Res; 78(9); 2383-95. ©2018 AACR.

ANO1 is a calcium-activated chloride channel that is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC) and other cancers. While ANO1 expression negatively correlates with survival in several cancers, its epigenetic regulation is poorly understood. We analyzed HNSCC samples from TCGA and a separate dataset of HPV+ oropharyngeal squamous cell carcinoma (OPSCC) samples to identify differentially methylated regions. E6 and E7 transfected normal oral keratinocytes (NOK) were used to induce hypermethylation of the ANO1 promoter. We found three CpG islands that correlated with ANO1 expression, including two positively correlated with expression. Using two HNSCC datasets with differential expression of ANO1, we showed hypermethylation of positively correlated CpG islands potentiates ANO1 expression. E7 but not E6 transfection of NOK cells led to hypermethylation of a positively correlated CpG island without a change in ANO1 expression. ANO1 promoter methylation was also correlated with patient survival. Our results are the first to show the contribution of positively correlated CpG's for regulating gene expression in HNSCC. Hypermethylation of the ANO1 promoter was strongly correlated with but not sufficient to increase ANO1 expression, suggesting methylation of positively correlated CpG's likely serves as an adjunct to other mechanisms of ANO1 activation.

Purpose: Human papillomavirus (HPV) 16 plays an etiologic role in a growing subset of head and neck squamous cell carcinomas (HNSCC), where viral expression of the E6 and E7 oncoproteins is necessary for tumor growth and maintenance. Although patients with HPV+ tumors have a more favorable prognosis, there are currently no HPV-selective therapies. Recent studies identified differential receptor tyrosine kinase (RTK) profiles in HPV+ versus HPV- tumors. One such RTK, HER3, is overexpressed and interacts with phosphoinositide-3-kinase (PI3K) in HPV+ tumors. Therefore, we investigated the role of HPV oncoproteins in regulating HER3-mediated signaling and determined whether HER3 could be a molecular target in HPV+ HNSCC.Experimental Design: HER3 was investigated as a molecular target in HPV+ HNSCC using established cell lines, patient-derived xenografts (PDX), and human tumor specimens. A mechanistic link between HPV and HER3 was examined by augmenting E6 and E7 expression levels in HNSCC cell lines. The dependency of HPV+ and HPV- HNSCC models on HER3 was evaluated with anti-HER3 siRNAs and the clinical stage anti-HER3 monoclonal antibody KTN3379.Results: HER3 was overexpressed in HPV+ HNSCC, where it was associated with worse overall survival in patients with pharyngeal cancer. Further investigation indicated that E6 and E7 regulated HER3 protein expression and downstream PI3K pathway signaling. Targeting HER3 with siRNAs or KTN3379 significantly inhibited the growth of HPV+ cell lines and PDXs.Conclusions: This study uncovers a direct relationship between HPV infection and HER3 in HNSCC and provides a rationale for the clinical evaluation of targeted HER3 therapy for the treatment of HPV+ patients. Clin Cancer Res; 23(12); 3072-83. ©2016 AACR.

TMEM16A, a Ca2+ -activated Cl- channel, contributes to tumor growth in breast cancer and head and neck squamous cell carcinoma (HNSCC). Here, we investigated whether TMEM16A influences the response to EGFR/HER family-targeting biological therapies. Inhibition of TMEM16A Cl- channel activity in breast cancer cells with HER2 amplification induced a loss of viability. Cells resistant to trastuzumab, a monoclonal antibody targeting HER2, showed an increase in TMEM16A expression and heightened sensitivity to Cl- channel inhibition. Treatment of HNSCC cells with cetuximab, a monoclonal antibody targeting EGFR, and simultaneous TMEM16A suppression led to a pronounced loss of viability. Biochemical analyses of cells subjected to TMEM16A inhibitors or expressing chloride-deficient forms of TMEM16A provide further evidence that TMEM16A channel function may play a role in regulating EGFR/HER2 signaling. These data demonstrate that TMEM16A regulates EGFR and HER2 in growth and survival pathways. Furthermore, in the absence of TMEM16A cotargeting, tumor cells may acquire resistance to EGFR/HER inhibitors. Finally, targeting TMEM16A improves response to biological therapies targeting EGFR/HER family members.

BACKGROUND. EGFR and Src family kinases are upregulated in head and neck squamous cell carcinoma (HNSCC). EGFR interacts with Src to activate STAT3 signaling, and dual EGFR-Src targeting is synergistic in HNSCC preclinical models. pSrc overexpression predicted resistance to the EGFR inhibitor, erlotinib, in a prior window trial. We conducted a 4-arm window trial to identify biomarkers associated with response to EGFR and/or Src inhibition. METHODS. Patients with operable stage II-IVa HNSCC were randomized to 7-21 days of neoadjuvant erlotinib, the Src inhibitor dasatinib, the combination of both, or placebo. Paired tumor specimens were collected before and after treatment. Pharmacodynamic expression of EGFR and Src pathway components was evaluated by IHC of tissue microarrays and reverse-phase protein array of tissue lysates. Candidate biomarkers were assessed for correlation with change in tumor size. RESULTS. From April 2009 to December 2012, 58 patients were randomized and 55 were treated. There was a significant decrease in tumor size in both erlotinib arms (P = 0.0014); however, no effect was seen with dasatinib alone (P = 0.24). High baseline pMAPK expression was associated with response to erlotinib (P = 0.03). High baseline pSTAT3 was associated with resistance to dasatinib (P = 0.099). CONCLUSIONS. Brief exposure to erlotinib significantly decreased tumor size in operable HNSCC, with no additive effect from dasatinib. Baseline pMAPK expression warrants further study as a response biomarker for anti-EGFR therapy. Basal expression of pSTAT3 may be independent of Src, explain therapeutic resistance, and preclude development of dasatinib in biomarker-unselected cohorts. TRIAL REGISTRATION. NCT00779389. FUNDING. National Cancer Institute, American Cancer Society, Pennsylvania Department of Health, V Foundation for Cancer Research, Bristol-Myers Squibb, and Astellas Pharma.

UnlabelledDespite advances in treatment approaches for head and neck squamous cell carcinoma (HNSCC), survival rates have remained stagnant due to the paucity of preclinical models that accurately reflect the human tumor. Patient-derived xenografts (PDX) are an emerging model system where patient tumors are implanted directly into mice. Increased understanding of the application and limitations of PDXs will facilitate their rational use. Studies to date have not reported protein profiles of PDXs. Therefore, we developed a large cohort of HNSCC PDXs and found that tumor take rate was not influenced by the clinical, pathologic, or processing features. Protein expression profiles, from a subset of the PDXs, were characterized by reverse-phase protein array and the data was compared with The Cancer Genome Atlas HNSCC data. Cluster analysis revealed that HNSCC PDXs were more similar to primary HNSCC than to any other tumor type. Interestingly, while a significant fraction of proteins were expressed similarly in both primary HNSCC and PDXs, a subset of proteins/phosphoproteins were expressed at higher (or lower) levels in PDXs compared with primary HNSCC. These findings indicate that the proteome is generally conserved in PDXs, but mechanisms for both positive and negative model selection and/or differences in the stromal components exist.ImplicationsProteomic characterization of HNSCC PDXs demonstrates potential drivers for model selection and provides a framework for improved utilization of this expanding model system.

ImportanceRandomized 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.ObjectiveTo determine a mechanism of exceptional response to erlotinib therapy in HNSCC.Design, setting, and participantsSingle 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.InterventionA brief course of erlotinib monotherapy followed by surgical resection.Main outcomes and measuresIdentification 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.ResultsNo 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 relevanceSelective erlotinib use in HNSCC may be informed by precision oncology approaches.