Preclinical Evaluation of the Association of the Cyclin-Dependent Kinase 4/6 Inhibitor, Ribociclib, and Cetuximab in Squamous Cell Carcinoma of the Head and Neck.

Epidermal growth factor receptor (EGFR) overexpression is observed in 90% of human papillomavirus (HPV)-negative squamous cell carcinomas of the head and neck (SCCHN). Cell cycle pathway impairments resulting in cyclin-dependent kinase (CDK) 4 and 6 activation, are frequently observed in SCCHN. We investigated the efficacy of ribociclib, a CDK4/6 inhibitor, in combination with cetuximab, a monoclonal antibody targeting the EGFR, in HPV-negative SCCHN patient-derived tumor xenograft (PDTX) models. The combination of cetuximab and ribociclib was not significantly more active than cetuximab monotherapy in all models investigated. In addition, the combination of cetuximab and ribociclib was less active than ribociclib monotherapy in the cetuximab-resistant PDTX models. In these models, a significant downregulation of the retinoblastoma (Rb) protein was observed in cetuximab-treated mice. We also observed Rb downregulation in the SCCHN cell lines chronically exposed and resistant to cetuximab. In addition, Rb downregulation induced interleukin 6 (Il-6) secretion and the Janus kinase family member/signal transducer and activator of transcription (JAK/STAT) pathway activation that might be implicated in the cetuximab resistance of these cell lines. To conclude, cetuximab is not an appropriate partner for ribociclib in cetuximab-resistant SCCHN models. Our work has significant clinical implications since the combination of anti-EGFR therapy with CDK4/6 inhibitors is currently being investigated in clinical trials.

Liquid biopsy for mutational profiling of locoregional recurrent and/or metastatic head and neck squamous cell carcinoma.

OBJECTIVES: The molecular landscape of head and neck squamous cell carcinoma (HNSCC) harbors potentially actionable genomic alterations. We aimed to study the utility of liquid biopsy to (i) characterize the mutational landscape of recurrent/metastatic HNSCC using a comprehensive gene panel and (ii) estimate the concordance between DNA mutations identified from circulating tumor DNA (ctDNA) and matched tumor tissues. MATERIALS AND METHODS: Targeted next-generation sequencing (NGS) was performed on cell-free DNA (cfDNA) of 39 patients with locoregional recurrent (n = 19) and/or metastatic (n = 20) HNSCC. Tumor biopsy (n = 18) was sequenced using the same technique. RESULTS: ctDNA was detected in 51% of patients (20/39) with a higher probability of detection in metastatic than locoregional recurrent disease (70% versus 30%, p = 0.025). 81% and 58% of the tissue tumor variants were not detected in plasma when considering all patients and only metastatic patients with detectable ctDNA, respectively. In a multivariate analysis, the likelihood of detecting the tissue tumor variant in plasma was related to metastatic status (p = 0.012), tumor variant allele frequency (p < 0.001) and ctDNA quantity (p < 0.001). 26% of the variants were detected only in liquid and not in the solid biopsy. Three patients without an available tumor sample had plasma containing three different potentially actionable PIK3CA mutations. CONCLUSION: CtDNA detection and characterization using targeted NGS is feasible in metastatic HNSCC. Liquid biopsies do not reflect the complete mutation profile of the tumor but have the potential to identify actionable mutations when tumor biopsies are not available as well as variants not found in matched tumor tissue.

Preclinical Activity of Ribociclib in Squamous Cell Carcinoma of the Head and Neck.

Cell-cycle pathway impairments resulting in CDK4 and 6 activation are frequently observed in human papillomavirus (HPV)-negative squamous cell carcinoma of the head and neck (SCCHN). We investigated the activity of ribociclib, a CDK4/6 inhibitor, in SCCHN models with the aim of identifying predictive biomarkers of response. HPV-negative or HPV-positive SCCHN cell lines (n = 8) and patient-derived tumor xenograft (PDTX) models (n = 6) were used. The models were classified according to their sensitivity to ribociclib to investigate potential predictive biomarkers. Ribociclib had a cytostatic effect in some HPV-negative SCCHN models but had no effect in HPV-positive models. In SCCHN cell lines and PDTXs, the retinoblastoma (Rb) protein expression level correlated with ribociclib activity. Rb knockdown was, however, not sufficient to block G0-G1 arrest induced by ribociclib in Detroit-562 where p107, p130, and Forkhead BOX M1 (FOXM1) were also implicated in ribociclib activity. Cell lines harboring epithelial-to-mesenchymal transition (EMT) features were less sensitive to ribociclib than those with an epithelial phenotype. Rb downregulation induced EMT in our Rb-expressing SCCHN cell lines. However, ribociclib still had significant activity in one PDTX model with high Rb and vimentin expression, suggesting that the presence of vimentin alone is not enough to induce ribociclib resistance. These findings suggest that CDK4/6 inhibitors should be investigated in patients with HPV-negative SCCHN with high Rb expression and an epithelial phenotype. Although these biomarkers are not predictive in all cases, they may enrich the population that could benefit from CDK4/6 inhibitors.

Metabolic Imaging Using Hyperpolarized Pyruvate-Lactate Exchange Assesses Response or Resistance to the EGFR Inhibitor Cetuximab in Patient-Derived HNSCC Xenografts.

PURPOSE: Optimal head and neck squamous cell carcinoma (HNSCC) patient selection for anti-EGFR-based therapy remains an unmet need since only a minority of patients derive long-term benefit from cetuximab treatment. We assessed the ability of state-of-the-art noninvasive in vivo metabolic imaging to probe metabolic shift in cetuximab-sensitive and -resistant HNSCC patient-derived tumor xenografts (PDTXs). EXPERIMENTAL DESIGN: Three models selected based on their known sensitivity to cetuximab in patients (cetuximab-sensitive or acquired-resistant HNC007 PDTXs, cetuximab-naïve UCLHN4 PDTXs, and cetuximab-resistant HNC010 PDTXs) were inoculated in athymic nude mice. RESULTS: Cetuximab induced tumor size stabilization in mice for 4 weeks in cetuximab-sensitive and -naïve models treated with weekly injections (30 mg/kg) of cetuximab. Hyperpolarized 13C-pyruvate-13C-lactate exchange was significantly decreased in vivo in cetuximab-sensitive xenograft models 8 days after treatment initiation, whereas it was not modified in cetuximab-resistant xenografts. Ex vivo analysis of sensitive tumors resected at day 8 after treatment highlighted specific metabolic changes, likely to participate in the decrease in the lactate to pyruvate ratio in vivo. Diffusion MRI showed a decrease in tumor cellularity in the HNC007-sensitive tumors, but failed to show sensitivity to cetuximab in the UCLHN4 model. CONCLUSIONS: This study constitutes the first in vivo demonstration of cetuximab-induced metabolic changes in cetuximab-sensitive HNSCC PDTXs that were not present in resistant tumors. Using metabolic imaging, we were able to identify hyperpolarized 13C-pyruvate as a potential marker for response and resistance to the EGFR inhibitor in HNSCC.

2'-deoxy-2'-[18F] fluoro-D-glucose positron emission tomography, diffusion-weighted magnetic resonance imaging, and choline spectroscopy to predict the activity of cetuximab in tumor xenografts derived from patients with squamous cell carcinoma of the head and neck.

We investigated changes on 2'-deoxy-2'-[18F]fluoro-D-glucose positron emission tomography (18FDG-PET), diffusion-weighted magnetic resonance imaging (DW-MRI), and choline spectroscopy as early markers of cetuximab activity in squamous cell carcinoma of the head and neck (SCCHN). SCCHN patient-derived tumor xenografts models were selected based on their cetuximab sensitivity. Three models were resistant to cetuximab and two were sensitive (one was highly sensitive and the other one was moderately sensitive). Cetuximab was infused on day 0 and 7. Maximal standardized uptake values (SUVmax), apparent diffusion coefficient (ADC), and total choline pool were measured at baseline and at day 8. To investigate the possible clinical relevance of our pre-clinical findings, we also studied the SUVmax and ADC modifications induced by cetuximab in five patients. Cetuximab induced a significant decrease in SUVmax and an increase in ADC at day 8 compared to baseline in the most cetuximab-sensitive model but not in the other models. At day 8, in one resistant model, SUVmax was decreased compared to baseline and was significantly lower than the controls. Choline spectroscopy was not able to predict cetuximab activity. The five patients treated with cetuximab had a 18FDG-PET partial response. One patient had a partial response according to RECISTv1.1. Interestingly, this last had also an increase in ADC value above 25%. Our preclinical data support the use of PDTX to investigate imaging techniques to detect early treatment response. Our pre-clinical and clinical data suggest that DW-MRI and 18FDG-PET should be further investigated to predict cetuximab activity.