Cetuximab In Head And Neck Squamous Cell Carcinoma Research Articles

Palbociclib inhibits the progression of head and neck cancer and improves the Cetuximab response under specific condition in vitro and in vivo.

In head and neck squamous cell carcinoma (HNSCC) with human papillomavirus (HPV)-negative, deregulation of cell cycle is partly due to inactivation of p16INK4 and overexpression of cyclin D1. Here we investigated the in vitro and in vivo effects of the CDK4/6 inhibitor Palbociclib alone or combined with EGFR inhibitor Cetuximab in HNSCC. CCK-8, soft agar assay, colony formation assay, wound healing assay and transwell assay, β-galactosidase assay, western blotting, and cell-derived xenografts were used to investigated the in vitro and in vivo activity of drugs. Cell viability and colony formation decreased in a dose-dependent manner in Tu686, AMC-HN8, and Fadu cells under Palbociclib treatment. Palbociclib remarkably inhibited migration, invasion and the expression MMP-9 in HNSCC cells. Palbociclib also induced senescence. Palbociclib caused the dephosphorylation of RB but increased the cyclin D1 level in a dose-dependent manner. Moreover, combination with Cetuximab could significantly prevent the induction of cyclin D1 and activation of EGFR signals from Palbociclib treatment. Nevertheless, only within the range of certain concentrations, a synergistic inhibitory effect on cell growth was observed when combined with Palbociclib and Cetuximab. Although the synergistic effect in Palbociclib/Cetuximab combined therapy was comparable to that in traditional chemotherapeutic regimens (cisplatin/Cetuximab) in Fadu tumor xenograft, the combination therapy was less active than Cetuximab monotherapy in Tu686 tumor xenograft. In HPV-negative HNSCC, CDK4/6 inhibitor shows promising anti-tumor effects, which exhibits a synergistic effect when combined with EGFR inhibitor only in specific drug concentration and mouse model.

SOX18 meditates the resistance of Bmi1-expressing cells to cetuximab in HNSCC.

Head and neck squamous cell carcinoma (HNSCC) is the most common type of malignancy in the head and neck region worldwide. The therapeutic strategies for HNSCC remain unsatisfying and limited. Here, we found a population of resistant Bmi1-expressing cells in the presence of cetuximab treatment and reported a novel role of SRY-box transcription factor 18 (SOX18), a member of the SOX family, in promoting HNSCC resistance to cetuximab. This study aimed to investigate the regulatory mechanism of Sox18 in Bmi1-positive cells and to search for better therapeutic targets. We successfully obtained Bmi1CreER, RosatdTomato, and RosaDTA mice and identified Bmi1-expressing cells through lineage tracing. SOX18 expression in HNSCC and normal tissues was analyzed by immunohistochemistry, colocalization of Sox18, and Bmi1-expressing cells was analyzed by immunofluorescence, and SOX18 expression in SCC9 cell lines was quantified by western blotting and quantitative real-time PCR. The investigation of the mechanism of SOX18-mediated cetuximab resistance in Bmi1-positive cells was based on the analysis of single-cell RNA-seq data obtained from the Gene Expression Omnibus (GEO) database. Western blotting was performed to verify the results obtained from the single-cell RNA-seq analysis. In our study, we demonstrated that Bmi1-expressing cells were resistant to cetuximab treatment and that depletion of Bmi1-expressing cells improved cetuximab efficacy in HNSCC. We then discovered that Sox18 mediated the stem cell-like properties of Bmi1-expressing cells and promoted cellular cetuximab resistance through an oxidative phosphorylation pathway. There was a significant downregulation of key genes in the oxidative phosphorylation pathway in Sox18 knockout cell lines. Taken together, the findings of our study suggest that Sox18 mediates the resistance of Bmi1-expressing cells to cetuximab in HNSCC via the oxidative phosphorylation pathway.

C-Met Signaling as a Therapeutic Target in Head and Neck Cancer.

Despite a dearth of activating driver mutations in head and neck squamous cell carcinoma (HNSCC), aberrant activation of the oncogenes, epidermal growth factor receptor (EGFR), and c-Met is near-universal in human papillomavirus (HPV)-negative disease. Although EGFR activation drove the successful development of the anti-EGFR monoclonal antibody cetuximab in HNSCC, no c-Met-targeting therapy has gained regulatory approval. Inhibition of the c-Met pathway may subvert oncogenesis within the tumor-intrinsic compartment, blocking tumoral proliferation, invasion, migration, and metastasis, or the tumor-extrinsic compartment, modulating the immunosuppressive tumor microenvironment. This review discusses the rationale and current drug development strategies for targeting c-Met or its exclusive ligand hepatocyte growth factor (HGF) in HNSCC.

Upregulation of secreted phosphoprotein 1 affects malignant progression, prognosis, and resistance to cetuximab via the KRAS/MEK pathway in head and neck cancer.

Acquired resistance is a barrier to cetuximab efficacy in patients with head and neck squamous cell carcinoma (HNSCC). Secreted phosphoprotein 1 (SPP1) is involved in various biological processes, including immune responses, cancer progression, and prognosis in many cancers, while little is known in HNSCC. Bioinformatics methods were used to identify candidate genes and further in vivo and in vitro experiments were performed to examine and validate the function of SPP1. We found that SPP1 was upregulated and has been found to have an oncogenic role in HNSCC. We further confirmed that overexpression of SPP1 affected proliferation, migration, invasion, and survival, and inhibited apoptosis, whereas silencing of SPP1 yielded opposite results to those of SPP1 overexpression. In addition, activation of the KRAS/MEK pathway contributed to the SPP1-induced malignant progression of HNSCC and resistance to cetuximab. Furthermore, SPP1 knockdown or an MEK inhibitor overcame this cetuximab-resistance pattern. Taken together, our findings for the first time identify the role of SPP1 in tumor promotion, prognostic prediction, and potential therapeutic targeting, as well as resistance to cetuximab in HNSCC.

Cetuximab-Containing Combinations in Locally Advanced and Recurrent or Metastatic Head and Neck Squamous Cell Carcinoma.

Cetuximab remains to date the only targeted therapy approved for the treatment of head and neck squamous cell carcinoma (HNSCC). The EGFR pathway plays a key role in the tumorigenesis and progression of this disease as well as in the resistance to radiotherapy (RT). While several anti-EGFR agents have been tested in HNSCC, cetuximab, an IgG1 subclass monoclonal antibody against EGFR, is the only drug with proven efficacy for the treatment of both locoregionally-advanced (LA) and recurrent/metastatic (R/M) disease. The addition of cetuximab to radiotherapy is a validated treatment option in LA-HNSCC. However, its use has been limited to patients who are considered unfit for standard of care chemoradiotherapy (CRT) with single agent cisplatin given the lack of direct comparison of these two regimens in randomized phase III trials and the inferiority suggested by metanalysis and phase II studies. The current use of cetuximab in HNSCC is about to change given the recent results from randomized prospective clinical trials in both the LA and R/M setting. Two phase III studies evaluating RT-cetuximab vs. CRT in Human Papillomavirus (HPV)-positive LA oropharyngeal squamous cell carcinoma (De-ESCALaTE and RTOG 1016) showed inferior overall survival and progression-free survival for RT-cetuximab combination, and therefore CRT with cisplatin remains the standard of care in this disease. In the R/M HNSCC, the EXTREME regimen has been the standard of care as first-line treatment for the past 10 years. However, the results from the KEYNOTE-048 study will likely position the anti-PD-1 agent pembrolizumab as the new first line treatment either alone or in combination with chemotherapy in this setting based on PD-L1 status. Interestingly, cetuximab-mediated immunogenicity through antibody dependent cell cytotoxicity (ADCC) has encouraged the evaluation of combined approaches with immune-checkpoint inhibitors in both LA and R/M-HNSCC settings. This article reviews the accumulated evidence on the role of cetuximab in HNSCC in the past decade, offering an overview of its current impact in the treatment of LA and R/M-HNSCC disease and its potential use in the era of immunotherapy.

Lymphotoxin-β Interacts with Methylated EGFR to Mediate Acquired Resistance to Cetuximab in Head and Neck Cancer.

Purpose: In head and neck squamous cell carcinoma (HNSCC), the incidence of RAS mutation, which is the major cause of cetuximab resistance, is relatively rare compared with the other types of cancers, and the mechanism mediating acquired resistance is unclear compared with the driver gene mutation-mediated de novo resistance. Here, we investigated the driver gene mutation-independent mechanism for cetuximab resistance in HNSCC.Experimental Design: We used the in vitro-selected and in vivo-selected cetuximab-resistant sublines of HNSCC cell lines for investigating the mechanism of acquired resistance to cetuximab. Zebrafish model was applied for evaluating the synergistic effect of combinatory drugs for overcoming cetuximab resistance.Results: The cetuximab-resistant HNSCC cells undergo a Snail-induced epithelial-mesenchymal transition. Mechanistically, Snail induces the expression of lymphotoxin-β (LTβ), a TNF superfamily protein that activates NF-κB, and protein arginine methyltransferase 1 (PRMT1), an arginine methyltransferase that methylates EGFR. LTβ interacts with methylated EGFR to promote its ligand-binding ability and dimerization. Furthermore, LTβ activates the NF-κB pathway through a LTβ receptor-independent mechanism. Combination of an EGFR tyrosine kinase inhibitor and a NF-κB inhibitor effectively suppressed cetuximab-resistant HNSCC and interfering with the EGFR-LTβ interaction reverses resistance.Conclusions: Our findings elucidate the mechanism of driver gene mutations-independent mechanism of acquired resistance to cetuximab in HNSCC and also provide potential strategies for combating cetuximab resistance. Clin Cancer Res; 23(15); 4388-401. ©2017 AACR.

Abstract 4113: Acquired resistance to cetuximab is mediated by HER3 activation in head and neck squamous cell carcinoma

Abstract Cetuximab, an EGFR monocloanal antibody, is commonly known to be an effective treatment agent in head and neck squamous cell carcinoma (HNSCC). However, despite the clinical efficacy of cetuximab, a majority of patients with good initial response still suffer from side effects as the acquired resistance to cetuximab. To understand the mechanisms of acquired resistance to cetuximab, we developed a model by exposing a head and neck cancer cell line Cal27 to increasing concentrations of cetuximab and established cetuximab-resistant clones (CETr) derived from the cetuximab sensitive (CETp). We examined how the acquired resistance in the CETr influenced the signaling pathway compared to the CETp. We investigated the effect of lapatinib, a tyrosine kinase inhibitor inhibiting HER3, with or without cetuximab combination on CETr clones and in a xenograft mouse model. Cetuximab-resistant(CETr) clones showed robust overexpression on the HER family receptors HER3. CETr clones also expressed upregulated EGFR, HER2, and HER3 activation resulting in activation of PI3K/ATK and ERK signaling. We also showed that CETr clones exhibited increased EGFR/HER3 dimerization. Treatment of cetuximab and siHER3 RNA together reduced EGFR activation in CETr clones to re-sensitize cells against cetuximab and robustly decreased cell proliferation. Combined treatment of CETr clones with cetuximab and lapatinib led to potent anti-proliferative effects. Co-treatment with cetuximab and lapatinib blocked EGFR, HER2 and HER3 activities and inhibited downstream signaling pathways. Co-treatment resulted in suppression of cell growth more effectively than each drug alone and induced apoptotic cell death through mitochondrial ROS. Furthermore, Co-treatment with cetuximab and lapatinib also led to suppression of tumor growth in orthotopic xenograft mouse model of oral tongue cancer. Our results suggested the upregulation of HER3 as a mechanism underlying resistance to cetuximab in HNSCC, supporting further clinical treatment strategy for tumors displaying acquired resistance to cetuximab. Citation Format: Yeon Ju Yang, Min Hee Cho, Yoo Jung Oh, Da Hee Kim, Jung Min Kim, Hyung Kwon Byeon, Myung Jin Ban, Ji Hoon Kim, Jae Wook Kim, Min Hee Ku, Jae Moon Yang, Eun Chang Choi, Yoon Woo Koh, Jeong Yeon Lee. Acquired resistance to cetuximab is mediated by HER3 activation in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4113. doi:10.1158/1538-7445.AM2017-4113

Hypoxia Mediates Differential Response to Anti-EGFR Therapy in HNSCC Cells

Despite advances in the head and neck squamous cell carcinoma (HNSCC) treatment modalities, drug resistance and cancer recurrence are often reported. Hypoxia signaling through hypoxia-inducible factor 1 (HIF-1) promotes angiogenesis and metastasis by inducing epithelial-mesenchymal-transition (EMT). The aim of this study was to evaluate the impact of hypoxia on response to therapy as well as EMT and expression of stem cell markers in HNSCC cells. Five HNSCC cell lines (UT-SCC-2, UT-SCC-14, LK0412, LK0827, and LK0923) were selected for this study. The treatment sensitivity for radiation, cisplatin, cetuximab, and dasatinib was assessed by crystal violet assay. Gene expression of EMT and cancer stem cell (CSC) markers as well as protein level of EGFR signaling molecules were analyzed by qPCR and western blotting, respectively. Unlike UT-SCC-14 and LK0827, the LK0412 cell line became significantly more sensitive to cetuximab in hypoxic conditions. This cetuximab sensitivity was efficiently reversed after suppression of HIF-1α with siRNA. Additionally, hypoxia-induced EMT and expression of stem cell markers in HNSCC cells was partially revoked by treatment with cetuximab or knockdown of HIF-1α. In summary, our study shows that hypoxia might have a positive influence on the anti-EGFR therapy effectiveness in HNSCC. However, due to heterogeneity of HNSCC lesions, targeting HIF-1α may not be sufficient to mediate such a response. Further studies identifying a trait of hypoxia-specific response to cetuximab in HNSCC are advisable.

Abstract 1414: Treatment outcome under cetuximab-based therapy and individual ADCC capability in head and neck cancer

Abstract Introduction Cetuximab is a IgG1 monoclonal antibody against epidermal growth factor receptor (EGFR) used for head and neck squamous cell carcinoma (HNSCC) treatment. In addition to EGFR targeting, cetuximab is able to develop an antibody-dependent cell-mediated cytotoxicity (ADCC), triggered by Fc receptors (Fcγ-R) on Natural Killer cells, macrophages and polymorphonucleated leukocytes. This prospective study examined the possible prognostic value of cetuximab-mediated ADCC response in a cohort of HNSCC patients. Patients and Methods There were 63 patients treated with curative intent with chemo-radiotherapy (CT-RT), associated (N = 39) or not (N = 24) with cetuximab. Peripheral blood samples were collected at start of therapy. Intrinsic ADCC was evaluated from ex vivo NK-dependent activity measuring LDH release through the Cytotox 96® non radioactive cytotoxicity assay, previously standardized in our laboratory (Crit Rev Oncol Hematol, 2015). EGFR tumoral expression was analyzed by immunohistochemistry. Results Median ADCC response at treatment start for all patients was 56.1% (range 5-99%). Analysing the whole population, patients with ADCC above the median value (high) showed a better OS as compared to patients with ADCC below the median (low), HR = 0.3827 (95% CI, 0.1464 to 1.000), p = 0.05 (Long-rank Mantel-Cox Test). A sub-group analysis confirmed a possible link between high ADCC and a better OS (CT-RT group, p = 0.223 and CT-RT+cetuximab group, p = 0.244). Importantly, considering CT-RT+cetuximab treatment and EGFR tumoral expression (N = 21), patients with EGFR 3+ status and high ADCC showed an improved OS as compared to patients with ADCC below median value, HR = 0.09031 (95% CI, 0.009983 to 0.8170), p = 0.032 (Long-rank Mantel-Cox Test). Median OS values for patients with high EGFR expression were 34.8 months (range 9.1-53.7) and 13.1 months (range 2.1-39.5) for patients with respectively high ADCC and low ADCC. Conclusion ADCC is shown to be a strong driver of cetuximab-based therapy outcome in HNSCC. High tumoral EGFR expression and high ADCC confer a particularly long overall survival. These data suggest an ADCC-based and EGFR expression-based precision therapy under cetuximab in HNSCC and open perspectives for NK boosting. Citation Format: Cristiana Lo Nigro, Laura Lattanzio, Daniela Vivenza, Martino Monteverde, Federica Tonissi, Giuliana Strola, Marco Merlano, Gerard Milano. Treatment outcome under cetuximab-based therapy and individual ADCC capability in head and neck cancer. [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 1414.

Real-life assessment of Volumetric Modulated Arc Therapy (VMAT) toxicity in Head and Neck Squamous Cell Carcinoma (HNSCC) treatment

Conclusion: The present study demonstrates the feasibility of VMAT in association with platin or cetuximab in HNSCC and reports VMAT-related acute and late toxicities for the first time. Objectives: New radiotherapy techniques, such as Volumetric Modulated Arc Therapy (VMAT) were developed to lower RT-related toxicity. The aim of the present study was to investigate acute and late toxicities of head and neck squamous cell carcinoma (HNSCC) patients treated using VMAT. Methods: This study investigated retrospectively all patients with HNSCC who received VMAT in curative intent. Results: From 2010–2013, 150 patients were treated. Seventy-five patients (50%) received concurrent chemotherapy with VMAT, 51 patients (34%) received VMAT alone and 24 patients (16%) received concurrent cetuximab with VMAT. Mean delivered dose to planning target volume tumor (PTV T), high risk nodes (PTV HNR), low risk nodes (PTV LNR) and prophylactic nodes (PTV PN) were: 65.2 Gy, 62.9 Gy, 55.4 Gy, and 51.5 Gy, respectively. PTV mean coverages were higher than 96.5%. Most common grade 3/4 acute infield toxicities were mucosis (n = 28, 19%), dysphagia (n = 24, 16%), and dermatitis (n = 24, 16%). With a median follow-up of 16.0 months, most common late toxicities were dysphagia (n = 30, 20%), xerostomia (n = 28, 19%), larynx stiff (n = 17, 11%), and skin fibrosis (n = 14, 9%).

Combination of anti-HER3 antibody MM-121/SAR256212 and cetuximab inhibits tumor growth in preclinical models of head and neck squamous cell carcinoma.

The EGFR monoclonal antibody cetuximab is the only approved targeted agent for treating head and neck squamous cell carcinoma (HNSCC). Yet resistance to cetuximab has hindered its activity in this disease. Intrinsic or compensatory HER3 signaling may contribute to cetuximab resistance. To investigate the therapeutic benefit of combining MM-121/SAR256212, an anti-HER3 monoclonal antibody, with cetuximab in HNSCC, we initially screened 12 HNSCC cell lines for total and phosphorylated levels of the four HER receptors. We also investigated the combination of MM-121 with cetuximab in preclinical models of HNSCC. Our results revealed that HER3 is widely expressed and activated in HNSCC cell lines. MM-121 strongly inhibited phosphorylation of HER3 and AKT. When combined with cetuximab, MM-121 exerted a more potent antitumor activity through simultaneously inhibiting the activation of HER3 and EGFR and consequently the downstream PI3K/AKT and ERK pathways in vitro. Both high and low doses of MM-121 in combination with cetuximab significantly suppressed tumor growth in xenograft models and inhibited activations of HER3, EGFR, AKT, and ERK in vivo. Our work is the first report on this new combination in HNSCC and supports the concept that HER3 inhibition may play an important role in future therapy of HNSCC. Our results open the door for further mechanistic studies to better understand the role of HER3 in resistance to EGFR inhibitors in HNSCC.

Predictive biomarkers in a phase II trial of weekly carboplatin (CBDCA), paclitaxel (P), and cetuximab (C) induction and chemoradiation (CRT) in patients (pts) with resectable stage III/IVa,b head and neck squamous cell carcinoma (HNSCC): Eastern Cooperative Oncology Group E2303.

6081 Background: We studied the addition of C to a sequential regimen of weekly CBDCA and P followed by CBDCA-P-radiation in pts with locally advanced resectable HNSCC. Tissue-based biomarkers may aid in pt selection for such approaches. Methods: Sixty-three eligible pts withoperablestage III/IV HNSCC participated in E2303, an Eastern Cooperative Oncology Group (ECOG) phase II trial of induction chemotherapy with weekly C, P and CBDCA x 6 followed by CRT with concurrent weekly C, paclitaxel, carboplatin. A tissue microarray was constructed and b-catenin, E-cadherin, Epidermal Growth Factor Receptor Variant III (EGFRVIII), insulin-like growth factor-1 receptor (IGF1R), NF-kappa b, p53, PI3Kp85, PI3Kp110a, PTEN, ΝRAS, and pRb protein expression levels were assessed using automated quantitative protein analysis (AQUA). For each marker, time-to-event distributions (OS, PFS, and EFS) were estimated by Kaplan-Meier estimates and compared using log-rank tests. Multivariable Cox proportional hazards models were used to estimate hazard ratios and test for significance, with primary site (oropharynx vs. non-oropharynx), disease stage (III vs. IV), and other important markers adjusted in the model. All p-values are two-sided. A level of p < 0.05 is considered statistically significant. Results: Based on the continuous scale, pRb tended to association with EFS (p=0.05). On multivariable analysis, low pRb level was a significant predictor for improved EFS (p=0.048). Our pRb data analysis was based on 32 pts with marker data available. Conclusions: pRb level is a potential predictive biomarker for response to cetuximab. HPV E7 oncoprotein binds and degrades pRb; therefore, low pRb protein level might be a surrogate marker for HPV association.Large prospective studies will be required to determine the association between pRb, HPV status and response to cetuximab in HNSCC.

EGFRv3 and PTEN as prognostic markers in head and neck squamous cell carcinoma patients treated with cetuximab.

e17032 Background: Cetuximab (CTx) is used in treatment of locally advanced (LAD) head and neck squamous cell carcinoma (HNSCC) in combination with radiotherapy (RT) or in metastatic disease (MD). There is no comparison between CTx plus RT and cisplatin plus RT. Patients treated with CTx in daily clinical practice are frequently different from the selected population treated in clinical trials. There are no biomarkers for efficacy of CTx in HNSCC. EGFR variant 3 mutation (EGFRv3), an extracellular domain mutation of EGFR, has been reported in different frequencies in HNSCC in the last years and its association with prognosis and CTx efficacy is still unknown. Methods: Retrospective review of data from patients with HNSCC treated with CTx at a single institution from 2007 to 2010. We evaluated CTx efficacy and expression of EGFRv3, EGFR, PTEN, CD44 and CD44v6 and their impact in objective response rate (ORR), progression free survival (PFS) and overall survival (OS). Biomarkers were analyzed by immunohistochemistry in tissue microarray. Results: For a median follow-up time of 13 months, 61 patients with LAD treated with RT plus CTx had a median OS of 22.7 months, and a median PFS of 8.0 months. Age adjusted Charlson Comorbidity Index (AA-CCI) and ECOG performance status were the most important predictors of poor prognosis in this population. For a median follow-up time of 10.9 months, 44 patients with MD had a median OS of 13.0 months and a median PFS of 7.0 months, for an ORR of 53.7%. EGFRv3 was expressed in 27.1% of tumor samples and was not associated with any clinical outcome. EGFR positivity was associated to higher ORR in LAD and PTEN negativity was associated with shorter OS in the MD setting. Conclusions: In a non selected population with LAD treatment results with CTx in combination with radiotherapy were worse than expected by the phase III study, median OS 22.7 months vs 49.0 months. This difference may be attributed to different population characteristics with higher ECOG and AA-CCI in our study and warrants an adequate proof of efficacy of CTx in this population. EGFRv3 is present in HNSCC but does not impact prognosis. PTEN and EGFR expression emerged as potential biomarkers in HNSCC patients treated with CTx.

Predictors for response to cetuximab in a prospective clinical trial (E2303) of patients with head and neck squamous cell carcinoma (HNSCC).

5576 Background: Theidentification of resistance mechanisms to Epidermal Growth Factor Receptor (EGFR) inhibitors remains critical lack in the management of HNSCC. We sought to determine predictors for response to cetuximab in a phase II clinical trial. Methods: 63 patients (pts) with operable stage III/IV HNSCC participated in E2303, an Eastern Cooperative Oncology Group (ECOG) phase II trial of induction chemotherapy with weekly cetuximab, paclitaxel and carboplatin x 6 followed by chemoradiotherapy with the same regimen. A tissue microarray was constructed and EGFR, ERK1/2, Met, pAkt and STAT protein expression levels were assessed using AQUA. The objectives of analysis were to determine association of biomarkers with E2303 efficacy outcomes (best objective response (OR), overall survival (OS), progression-free survival (PFS), and event-free survival (EFS)). The logistic regression model was used to examine relationship between marker measurements (on a continuous scale) and OR. The univariate and multivariate Cox proportional hazards models were used to evaluate the relationship between markers and event-time distributions. Fisher’s exact test was used to evaluate differences in response rate between groups (high vs. low AQUA scores). Event-time distributions were estimated by the Kaplan-Meier method and compared by the log-rank test. Results: Cytoplasmic ERK1/2 levels weresignificantly associated with PFS and OS (p=0.03 and 0.01, respectively). Nuclear ERK1/2 levels were significantly associated with OS (p=0.02) and tended towards significance for PFS (p=0.09). The multivariate Cox regression analysis shows that cytoplasmic and nuclear ERK1/2 are significantly associated with OS and PFS after controlling for primary site and disease stage, respectively There was no significant association between cytoplasmic or nuclear ERK1/2 status and OR (p-values 0.98 and 0.41, respectively).No association was found between expression of any of other biomarkers and outcome measures. Our data analysis was based on 35 pts with marker data available. Conclusions: Ras/MAPK/ERKpathway may be associated with resistance to cetuximab in HNSCC.

Autocrine epidermal growth factor receptor ligand production and cetuximab response in head and neck squamous cell carcinoma cell lines

Predictive strategies for the treatment efficacy of cetuximab are currently not available for head and neck cancer. We investigated the correlation between the expression of epidermal growth factor receptor (EGFR) ligands and EGFR expression, and the growth inhibitory activity of cetuximab in a panel of head and neck squamous cell carcinoma (HNSCC) cell lines. The growth inhibiting effect of cetuximab was measured for eight HNSCC cell lines and correlated with the autocrine production of five EGFR ligands as measured by ELISA, and the mRNA expression of two ligands, as measured by quantitative RT-PCR. EGFR expression was assessed by western blot analysis. There was a good correlation between the expression of four of the EGFR ligands (TGF-α, amphiregulin, epiregulin and epigen) and the growth inhibiting effect of cetuximab. TGF-α had the highest predictive potential but had to be combined with epigen for full prediction. EGFR expression also correlated with cetuximab sensitivity but less clearly. The results indicate that the expression of several EGFR ligands has to be used to predict sensitivity to cetuximab in HNSCC. This has to be further evaluated in clinical samples.

Dasatinib blocks cetuximab- and radiation-induced nuclear translocation of the epidermal growth factor receptor in head and neck squamous cell carcinoma

Background and purpose The aberrant expression of epidermal growth factor receptor (EGFR) has been linked to the etiology of head and neck squamous cell carcinoma (HNSCC). The first major phase III trial combining cetuximab with radiation confirmed a strong survival advantage. However, both cetuximab and radiation can promote EGFR translocation to the nucleus where it enhances resistance to both of these modalities. In this report we sought to determine how to block cetuximab- and radiation-induced translocation of EGFR to the nucleus in HNSCC cell lines. Material and methods We utilized three established HNSCC cell lines, SCC1, SCC6 and SCC1483 and measured nuclear translocation of EGFR after treatment with cetuximab or radiation. We then utilized dasatinib (BMS-354825), a potent, orally bioavailable inhibitor of several tyrosine kinases, including the Src family kinases, to determine if SFKs blockade could abrogate cetuximab- and radiation-induced nuclear EGFR translocation. Results Cetuximab and radiation treatment of all three HNSCC lines lead to translocation of the EGFR to the nucleus. Blockade of SFKs abrogated cetuximab- and radiation-induced EGFR translocation to the nucleus. Conclusions The data presented in this report suggest that both cetuximab and radiation can promote EGFR translocation to the nucleus and dasatinib can inhibit this process. Collectively these findings may suggest that dasatinib can limit EGFR translocation to the nucleus and may enhance radiotherapy plus cetuximab in HNSCC.

Abstract 338: Enhanced cetuximab and radiosensitization with src inhibition in head and neck cancer

Abstract The epidermal growth factor receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase (RTK) that initiates a spectrum of signaling pathways that promote cell proliferation, differentiation, migration, motility, and metastasis. The EGFR has been strongly linked to the etiology of head and neck squamous cell carcinoma (HNSCC). The first major phase III clinical trial combining cetuximab with radiation in HNSCC confirmed a strong survival advantage for patients on the cetuximab arm. It has recently been reported, however, that cetuximab and radiation can induce EGFR to the nucleus where it can enhance resistance to both of these therapeutic modalities. In this report we sought to determine how to block cetuximab and radiation induced translocation of EGFR to the nucleus to increase the efficacy of this therapeutic regimen in HNSCC. We utilized three established HNSCC lines, SCC1, SCC6 and SCC1483 and measured nuclear translocation of the EGFR after treatment with cetuximab, radiation or the combination. Both cetuximab and radiation treatment could induce nuclear translocation of the EGFR in all SCC lines tested. Strikingly, time course analysis indicated that cetuximab could stimulate movement of the EGFR to the nucleus within one hour where it maintained its present for greater than 96 hours. Conversely, radiation could stimulate EGFR translocation to the nucleus within 0.5 hours, but returned to baseline levels within four hours. Both cetuximab and radiation treatment of SCC lines led to the phosphorylation of tyrosine 845 (Y845) of the EGFR. Y845 is phosphorylated specifically by the non-receptor tyrosine kinase Src and/or its family of kinases (SFKs). Blockade of SFKs by dasatinib could abrogate cetuximab and radiation induced EGFR translocation to the nucleus indicating that SFKs are crucial for the movement of the EGFR to the nucleus. In addition, pre-treatment of HNSCC cells with dasatinib, followed by cetuximab and radiation enhanced the suppression of ERK activity in all three SCC cell lines. Furthermore, combining dasatinib with cetuximab/radiation treatment exhibited additive inhibitory effects on cell growth or clonogenic formation. These data suggest that both cetuximab and radiation can induce EGFR to the nucleus and blockade of SFKs using dasatinib can abrogate this translocation. Collectively these findings may suggest that dasatinib can limit EGFR translocation to the nucleus and may enhance radiotherapy plus cetuximab in HNSCC. 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 338.