Xenograft assessment of predictive biomarkers for standard head and neck cancer therapies

Abstract

Head and neck squamous cell carcinoma (HNSCC) remains a challenging cancer to treat with overall 5-year survival on the order of 50–60%. Therefore, predictive biomarkers for this disease would be valuable to provide more effective and individualized therapeutic approaches for these patients. While prognostic biomarkers such as p16 expression correlate with outcome; to date, no predictive biomarkers have been clinically validated for HNSCC. We generated xenografts in immunocompromised mice from six established HNSCC cell lines and evaluated response to cisplatin, cetuximab, and radiation. Tissue microarrays were constructed from pre- and posttreatment tumor samples derived from each xenograft experiment. Quantitative immunohistochemistry was performed using a semiautomated imaging and analysis platform to determine the relative expression of five potential predictive biomarkers: epidermal growth factor receptor (EGFR), phospho-EGFR, phospho-Akt, phospho-ERK, and excision repair cross-complementation group 1 (ERCC1). Biomarker levels were compared between xenografts that were sensitive versus resistant to a specific therapy utilizing a two-sample t-test with equal standard deviations. Indeed the xenografts displayed heterogeneous responses to each treatment, and we linked a number of baseline biomarker levels to response. This included low ERCC1 being associated with cisplatin sensitivity, low phospho-Akt correlated with cetuximab sensitivity, and high total EGFR was related to radiation resistance. Overall, we developed a systematic approach to identifying predictive biomarkers and demonstrated several connections between biomarker levels and treatment response. Despite these promising initial results, this work requires additional preclinical validation, likely involving the use of patient-derived xenografts, prior to moving into the clinical realm for confirmation among patients with HNSCC.

Defining the boundaries and expanding the utility of head and neck cancer patient derived xenografts

“…Current investigations have been underway to identify predictive biomarkers for standard HNSCC therapies including cisplatin, cetuximab and radiation. Lower levels of the nucleotide excision repair pathway member ERCC1 are correlated with cisplatin sensitivity[14, 26, 27]. Investigators have studied total EGFR levels in relation to cetuximab response[28, 29], while others evaluated the association between total EGFR and ERCC1 with respect to radiation sensitivity.…”

“…Investigators have studied total EGFR levels in relation to cetuximab response[28, 29], while others evaluated the association between total EGFR and ERCC1 with respect to radiation sensitivity. [30, 31] Our work with cell line xenografts extended these analyses to include related EGFR-signaling members including pEGFR, pAkt and pERK in relation to both cetuximab and radiation response[14]. …”

“…Moreover, we revealed that putative predictive biomarkers maintain stability across passaged PDXs. This is important information as we attempt to utilize predictive biomarker identification in the PDXs and translate this information into therapeutic pre-clinical and clinical studies[32]. …”

“…The TMAs were sectioned (5 μm) and H&E stains were carried out on the thirtieth section. Remaining sections were stained by immunohistochemistry (IHC) for the expression of EGFR, pEGFR, pAkt, pERK and ERCC1 by standard IHC techniques[14], and detected with DAB. Additional details available in supplemental methods.…”

“…We followed a standardized approach to quantitatively evaluate the expression of the TMA cores for each biomarker. [14] The inForm Software outputs the DAB mean optical density (MOD) as the measure of expression of each marker as a continuous value from 0 to 1. To evaluate the stability of protein expression, the expression of each biomarker in the first and last passaged PDXs were compared.…”