Phospho-T356RB1 predicts survival in HPV-negative squamous cell carcinoma of the head and neck

Abstract

Locally advanced squamous cell carcinoma of the head and neck (SCCHN) that is not associated with human papillomavirus (HPV) has a poor prognosis in contrast to HPV-positive disease. To better understand the importance of RB1 activity in HPV-negative SCCHN, we investigated the prognostic value of inhibitory CDK4/6 phosphorylation of RB1 on threonine 356 (T356) in archival HPV-negative tumor specimens from patients who underwent surgical resection and adjuvant radiation. We benchmarked pT356RB1 to total RB1, Ki67, pT202/Y204ERK1/2, and TP53, as quantified by automatic quantitative analysis (AQUA), and correlated protein expression with tumor stage and grade. High expression of pT356RB1 but not total RB1 predicted reduced overall survival (OS; P = 0.0295), indicating the potential relevance of post-translational phosphorylation. Paired analysis of The Cancer Genome Atlas (TCGA) data for regulators of this RB1 phosphorylation identified loss or truncating mutation of negative regulator CDKN2A (p16) and elevated expression of the CDK4/6 activator CCND1 (cyclin D) as also predicting poor survival. Given that CDK4/6 inhibitors have been most effective in the context of functional RB1 and low expression or deletion of p16 in other tumor types, these data suggest such agents may merit evaluation in HPV-negative SCCHN, specifically in cases associated with high pT356RB1.

Keywords: CDK4/6; E2F; RB1; biomarkers; head and neck cancer.

Figure 1. Validation of antibodies and immunofluorescence microscopy

A. Western blots for the relevant protein markers in the presence or absence of siRNA, B. representative high and low staining immunofluorescent microscopy images for each marker. LC = loading control (β-actin), DAPI = nuclear stain, CK = cytokeratin (epithelial tumor stain).

Figure 2. Kaplan-Meier survival analysis for high and low expression levels of TP53, RB1, p^T356RB1, p^T202/Y204ERK1/2, and Ki67

Patients treated with surgery only and patients treated with surgery and radiation therapy were included. OS = overall survival; HR = hazard ratio; CI = confidence interval. See Supplementary Table S1 for additional details regarding the HR.

Figure 3. Expression correlation between markers and between markers and tumor stage and grade

A. Statistically significant correlations between marker expression levels, increasing saturation of blue indicates higher correlation, correlations with P > 0.05 are suppressed, B. correlations between marker expression levels and tumor grade, T-stage and N-stage, blue squares indicate low marker expression levels and red squares indicate high maker expression levels, C. hematoxylin and eosin (H&E) stained samples for high and low grade tumors with the corresponding p^T356RB1 staining. (**) P < 0.05.

Figure 4. Genomic and transcriptomic analysis of RB1, CDK4, CDK6, CDKN2A (p16) and CCND1

A. Genomic alterations of RB1, CDKN2A (p16), CDK4, CDK6 in 243 HPV negative SSCHN TCGA specimens (only tumors with changes affecting at least one of these genes are shown), B. Kaplan-Meier survival analysis of CDKN2A with deletions (Del.) compared to WT, C. Kaplan-Meier survival analysis for cases of simultaneous CCND1 amplification (Amp.) and CDKN2A homozygous deletion (Del.), D. Kaplan-Meier survival analysis of high versus low RNA expression for CDKN2A (p16) and E. CCND1. F. Summary of RB1-pathway alterations and the survival impact thereof. See Supplementary Figure S4 for additional data. Shallow Deletion = LOH = loss of heterozygosity; Deep Deletion = homozygous deletion; Mut. = mutation; WT = wild type; green arrow = increase; red arrow = decrease; horizontal line = no change; co-occur. = trend towards co-occurrence.