Konrad Klinghammer

Expression of Amphiregulin and EGFRvIII Affect Outcome of Patients with Squamous Cell Carcinoma of the Head and Neck Receiving Cetuximab–Docetaxel Treatment

Purpose: Constitutive activation of epidermal growth factor receptor (EGFR) as a result of gene amplification, mutation, or overexpression of its ligands has been associated with response to EGFR targeting strategies. The role of these molecular mechanisms for the responsiveness of squamous cell carcinoma of the head and neck (SCCHN) to cetuximab-containing regimens remains unknown. Experimental Design: Tumor biopsies from 47 patients, enrolled in a single-arm phase II multicenter study for second-line treatment of recurrent or metastatic SCCHN with cetuximab and docetaxel, were analyzed by immunohistochemistry for expression of EGFR, its deletion variant III (EGFRvIII) and its ligand amphiregulin (AREG). The relation between expression levels and disease control rate (DCR) was evaluated by logistic regression. Association between expression levels, progression-free survival (PFS), and overall survival (OS) was determined by Kaplan–Meier analysis, log-rank test, and uni- and multivariate Cox regression analysis. Results: High expression of EGFR, EGFRvIII, and AREG was detected in 73%, 17%, and 45% of SCCHN cases, respectively. Expression levels of EGFR had no impact on PFS or OS. High expression levels of EGFRvIII were significantly associated with reduced DCR and shortened PFS (HR: 3.3, P = 0.005) but not with OS. Patients with high AREG expression in tumor cells had significantly shortened OS (HR: 2.2, P = 0.002) and PFS (HR 2.2, P = 0.019) compared with patients with low expression score. Multivariate Cox analysis revealed an independent association of AREG and EGFRvIII with PFS but only AREG was an independent prognosticator of OS. Conclusions: High EGFRvIII and AREG expression levels identify SCCHN patients who are less likely to benefit from combination treatment with cetuximab and docetaxel. Clin Cancer Res; 17(15); 5197–204. ©2011 AACR.

Association of epidermal growth factor receptor polymorphism, skin toxicity, and outcome in patients with squamous cell carcinoma of the head and neck receiving cetuximab-docetaxel treatment.

Purpose: Cetuximab, a monoclonal antibody targeting epidermal growth factor receptor (EGFR), has shown clinical efficacy in squamous cell carcinoma of the head and neck with prolonged progression-free (PFS) and overall survival (OS). In this study, we analyzed whether cetuximab-induced skin rash was correlated with distinct polymorphisms within the EGFR gene known to modulate EGFR expression, ligand binding, or signaling activity. Experimental Design: Fifty-one patients enrolled in a single-arm phase II multicenter study for second-line treatment of recurrent or metastatic squamous cell carcinoma of the head and neck with cetuximab/docetaxel were genotyped for two genetic variations in the EGFR gene, a point substitution G→A in exon 13 resulting in an amino acid substitution in position 521 (EGFR-R521K) and a CA repeat (CA-SSR) polymorphism in intron 1. Association between genotypes and incidence/grade of skin rash was determined by Fishers exact test. The predictive value of genotypes for PFS and OS was determined using the log-rank test. Results: Overall, 21 patients (41%) developed skin rash with grade >1 within 6 weeks of treatment. The common EGFR-R521K genotype (G/G) was significantly associated with increased skin toxicity (P = 0.024) and showed a trend toward reduced risk of tumor progression (hazard ratio, 0.55; 95% confidence interval, 0.27-1.08; P = 0.08), whereas no correlation of the EGFR-R521K genotype with OS could be observed (P = 0.20). No significant interaction between CA-SSR polymorphism and skin toxicity, PFS, or OS could be detected. Conclusions: Our study revealed an influence of the EGFR-R521K genotype on skin toxicity and suggested its relation to clinical activity of cetuximab/docetaxel treatment. Clin Cancer Res; 16(1); 304–10

A comprehensively characterized large panel of head and neck cancer patient-derived xenografts identifies the mTOR inhibitor everolimus as potential new treatment option

Patient‐derived xenograft (PDX) models have shown to reflect original patient tumors better than any other preclinical model. We embarked in a study establishing a large panel of head and neck squamous cell carcinomas PDX for biomarker analysis and evaluation of established and novel compounds. Out of 115 transplanted specimens 52 models were established of which 29 were characterized for response to docetaxel, cetuximab, methotrexate, carboplatin, 5‐fluorouracil and everolimus. Further, tumors were subjected to sequencing analysis and gene expression profiling of selected mTOR pathway members. Most frequent response was observed for docetaxel and cetuximab. Responses to carboplatin, 5‐fluorouracil and methotrexate were moderate. Everolimus revealed activity in the majority of PDX. Mutational profiling and gene expression analysis did not reveal a predictive biomarker for everolimus even though by trend RPS6KB1 mRNA expression was associated with response. In conclusion we demonstrate a comprehensively characterized panel of head and neck cancer PDX models, which represent a valuable and renewable tissue resource for evaluation of novel compounds and associated biomarkers.

Choosing wisely – Preclinical test models in the era of precision medicine

Through the introduction of a steadily growing variety of preclinical test models drug development and biomarker research has advanced. Next to classical used 2D cell line cultures, tissue-slice cultures, 3D organoid cell cultures, genetically engineered mouse models, cell line derived mouse models and patient derived xenografts may be selected for a specific question. All models harbor advantages and disadvantages. This review focuses on the available preclinical test models, novel developments such as humanized mice and discusses for which question a particular model should be employed.

Methylation of RAD51B, XRCC3 and other homologous recombination genes is associated with expression of immune checkpoints and an inflammatory signature in squamous cell carcinoma of the head and neck, lung and cervix

Immune checkpoints are emerging treatment targets, but mechanisms underlying checkpoint expression are poorly understood. Since alterations in DNA repair genes have been connected to the efficacy of checkpoint inhibitors, we investigated associations between methylation of DNA repair genes and CTLA4 and CD274 (PD-L1) expression. A list of DNA repair genes (179 genes) was selected from the literature, methylation status and expression of inflammation-associated genes (The Cancer Genome Atlas data) was correlated in head and neck squamous cell carcinoma (HNSCC), cervical and lung squamous cell carcinoma. A significant positive correlation of the methylation status of 15, 3 and 2 genes with checkpoint expression was identified, respectively. RAD51B methylation was identified in all cancer subtypes. In HNSCC and cervical cancer, there was significant enrichment for homologous recombination genes. Methylation of the candidate genes was also associated with expression of other checkpoints, ligands, MHC- and T-cell associated genes as well as an interferon-inflammatory immune gene signature, predictive for the efficacy of PD-1 inhibition in HNSCC. Homologous recombination deficiency might therefore be mediated by DNA repair gene hypermethylation and linked to an immune-evasive phenotype in SCC. The methylation status of these genes could represent a new predictive biomarker for immune checkpoint inhibition.

PI3K/PTEN/AKT/mTOR polymorphisms: Association with clinical outcome in patients with head and neck squamous cell carcinoma receiving cetuximab-docetaxel

The purpose of this study was to determine whether single nucleotide polymorphisms (SNPs) in AKT1, AKT2, FRAP1, PIK3CA, and PTEN were associated with treatment response and clinical outcome in patients with head and neck squamous cell carcinoma (HNSCC).

Basal subtype is predictive for response to cetuximab treatment in patient derived xenografts of squamous cell head and neck cancer

Cetuximab is the single targeted therapy approved for the treatment of head and neck cancer (HNSCC). Predictive biomarkers have not been established and patient stratification based on molecular tumor profiles has not been possible. Since EGFR pathway activation is pronounced in basal subtype, we hypothesized this activation could be a predictive signature for an EGFR directed treatment. From our patient‐derived xenograft platform of HNSCC, 28 models were subjected to Affymetrix gene expression studies on HG U133+ 2.0. Based on the expression of 821 genes, the subtype of each of the 28 models was determined by integrating gene expression profiles through centroid‐clustering with previously published gene expression data by Keck et al. The models were treated in groups of 5–6 animals with docetaxel, cetuximab, everolimus, cis‐ or carboplatin and 5‐fluorouracil. Response was evaluated by comparing tumor volume at treatment initiation and after 3 weeks of treatment (RTV). Tumors distributed over the 3 signature‐defined subtypes: 5 mesenchymal/inflamed phenotype (MS), 15 basal type (BA), 8 classical type (CL). Cluster analysis revealed a strong correlation between response to cetuximab and the basal subtype. RTV MS 3.32 vs. BA 0.78 (MS vs. BA, unpaired t‐test, p 0.0002). Cetuximab responders were distributed as following: 1/5 in MS, 5/8 in CL and 13/15 in the BA group. Activity of classical chemotherapies did not differ between the subtypes. In conclusion basal subtype was associated with response to EGFR directed therapy in head and neck squamous cell cancer patient‐derived xenografts.

Targeted Therapy of Head and Neck Cancer

Head and neck squamous cell carcinoma (HNSCC) is one of the most common solid cancers worldwide. It is mainly caused by exposure to tobacco smoke and alcohol as well as infection with the human papilloma virus (HPV). The prognosis is poor, especially once it recurs or metastasizes. Current therapeutic options include surgery, radio- and chemotherapy. Epidermal growth factor receptor (EGFR) inhibitors are so far the only targeted agents that have been approved in head and neck cancer. Primary or secondary resistance is frequent or will eventually develop. Several driver mutations and other genomic aberrations have been described in HNSCC including EGFR overexpression and amplification. Yet, no predictive biomarkers for the application of EGFR inhibitors have been identified. Further targeted agents are in development for HNSCC, of which inhibitors of the PI3K pathway are the closest to clinical application. In recent years, the incidence of HPV-driven HNSCC has risen in Western countries. HPV-positive and -negative HNSCC are distinct molecular tumor entities, and consequences for targeted therapies have been discussed. This review looks at approved and investigational targeted treatment strategies as well as potential predictive biomarkers such as the HPV status to guide treatment.

A phosphoarray platform is capable of personalizing kinase inhibitor therapy in head and neck cancers

Tyrosine kinase inhibitors are effective treatments for cancers. Knowing the specific kinase mutants that drive the underlying cancers predict therapeutic response to these inhibitors. Thus, the current protocol for personalized cancer therapy involves genotyping tumors in search of various driver mutations and subsequently individualizing the tyrosine kinase inhibitor to the patients whose tumors express the corresponding driver mutant. While this approach works when known driver mutations are found, its limitation is the dependence on driver mutations as predictors for response. To complement the genotype approach, we hypothesize that a phosphoarray platform is equally capable of personalizing kinase inhibitor therapy. We selected head and neck squamous cell carcinoma as the cancer model to test our hypothesis. Using the receptor tyrosine kinase phosphoarray, we identified the phosphorylation profiles of 49 different tyrosine kinase receptors in five different head and neck cancer cell lines. Based on these results, we tested the cell line response to the corresponding kinase inhibitor therapy. We found that this phosphoarray accurately informed the kinase inhibitor response profile of the cell lines. Next, we determined the phosphorylation profiles of 39 head and neck cancer patient derived xenografts. We found that absent phosphorylated EGFR signal predicted primary resistance to cetuximab treatment in the xenografts without phosphorylated ErbB2. Meanwhile, absent ErbB2 signaling in the xenografts with phosphorylated EGFR is associated with a higher likelihood of response to cetuximab. In summary, the phosphoarray technology has the potential to become a new diagnostic platform for personalized cancer therapy.

pN status predicts outcomes in surgically treated pT1–pT2 patients of various disease stages with squamous cell carcinoma of the head and neck: a 17-year retrospective single center cohort study

ObjectivesThe optimal treatment for a substantial proportion of patients with pT1–pT2 squamous cell carcinomas of the head and neck (SCCHN) remains to be refined. The extent of surgery, role and potential benefit of adjuvant treatment are to be balanced against therapy-induced side effects. We compared the outcomes of surgery with or without adjuvant radiotherapy (RT) or chemotherapy (CRT) and investigated the prognostic value of established clinicopathological parameters.MethodsData were retrospectively collected for 227 patients who were treated by surgery alone (n = 31), RT (n = 87) and CRT (n = 109) in a single center.ResultsPatients with stage I/II disease who had received adjuvant RT showed a better disease-free survival (DFS) (P = 0.04) than those who had received adjuvant CRT treatment. Conversely, patients with stage III/IV disease who had received CRT showed a better overall survival (OS) (P = 0.003) and DFS (P = 0.03) than those who had received surgery alone or adjuvant RT without chemotherapy. Survival analysis demonstrated that patients with pN0 to pN1 had better OS (P = 0.02), disease-specific survival (DSS) (P = 0.003), DFS (P = 0.02) and metastases free survival (MFS) (P = 0.002) compared to patients with pN2 to pN3. Multivariate analysis showed that the pN status was an independent factor for OS (P = 0.03), DSS (P = 0.04), relapse-free survival (P = 0.03), DFS (P = 0.03).ConclusionThe pN status is the most important prognostic factor for pT1 to pT2 SCCHN. Adjuvant CRT was associated with significantly better survival outcomes in patients with pN1 and pN2-3 or more advanced stage, while adjuvant RT showed significantly better outcomes in patients with pN0.