Expression of p16(INK4A) has a major impact on treatment response and survival in patients with head and neck cancer treated with conventional radiotherapy.

Introduction The lack of large panels of validated antibodies, tissue handling variability, and intratumoral heterogeneity potentially hamper comprehensive study of the functional proteome in non-microdissected solid tumors. The purpose of this study was to address these concerns and to demonstrate clinical utility for the functional analysis of proteins in non-microdissected breast tumors using reverse phase protein arrays (RPPA). Methods Herein, 82 antibodies that recognize kinase and steroid signaling proteins and effectors were validated for RPPA. Intraslide and interslide coefficients of variability were <15%. Multiple sites in non-microdissected breast tumors were analyzed using RPPA after intervals of up to 24 h on the benchtop at room temperature following surgical resection. Results Twenty-one of 82 total and phosphoproteins demonstrated time-dependent instability at room temperature with most variability occurring at later time points between 6 and 24 h. However, the 82-protein functional proteomic “fingerprint” was robust in most tumors even when maintained at room temperature for 24 h before freezing. In repeat samples from each tumor, intratumoral protein levels were markedly less variable than intertumoral levels. Indeed, an independent analysis of prognostic biomarkers in tissue from multiple tumor sites accurately and reproducibly predicted patient outcomes. Significant correlations were observed between RPPA and immunohistochemistry. However, RPPA demonstrated a superior dynamic range. Classification of 128 breast cancers using RPPA identified six subgroups with markedly different patient outcomes that demonstrated a significant correlation with breast cancer subtypes identified by transcriptional profiling. Conclusion Thus, the robustness of RPPA and stability of the functional proteomic “fingerprint” facilitate the study of the functional proteome in non-microdissected breast tumors.

Hypoxia, a common feature of the microenvironment in solid tumors, is associated with resistance to radiotherapy, reduced therapeutic response, and a poorer clinical outcome. In head and neck squamous cell carcinomas (HNSCC), the negative effect of hypoxia on radiotherapy can be counteracted via addition of hypoxic modification to the radiotherapy. To predict which patients harbor hypoxic tumors and would therefore benefit from hypoxic modification, clinically applicable methods for pretherapeutic hypoxic evaluation and categorization are needed. In this study, we developed a hypoxia classifier based on gene expression. Through study of xenograft tumors from human squamous cell carcinoma cell lines, we verified the in vivo relevance of previously identified in vitro derived hypoxia-induced genes. We then evaluated a training set of 58 hypoxia-evaluated HNSCCs to generate a gene expression classifier containing 15 genes. This 15-gene hypoxia classifier was validated in 323 patients with HNSCC randomized for hypoxic modification or placebo in combination with radiotherapy. Tumors categorized as hypoxic on the basis of the classifier were associated with a significantly poorer clinical outcome than nonhypoxic tumors. This outcome was improved and equalized to the nonhypoxic tumors by addition of hypoxic modification. Thus, findings show that the classifier attained both prognostic and predictive impact, and its pretherapeutic use may provide a method to identify those patients who will benefit from hypoxic modification of radiotherapy. Cancer Res; 71(17); 5923-31. Ó2011 AACR.

This study emphasizes the role of FAZA PET/CT imaging as a suitable assay with prognostic potential for detection of hypoxia in HNSCC.

No abstract