Tumor epidermal growth factor receptor and EGFR PY1068 are independent prognostic indicators for head and neck squamous cell carcinoma

Abstract

Purpose: To assess the prognostic value of epidermal growth factor receptor (EGFR) molecular characteristics of head and neck squamous cell carcinoma (HNSCC).

Patients and methods: HNSCC tumors from patients prospectively enrolled in either an Early Detection Research Network (EDRN) study and treated with surgery without an EGFR-targeted agent (N = 154) or enrolled in a chemoradiation trial involving the EGFR-targeted antibody cetuximab (N = 39) were evaluated for EGFR gene amplification by FISH and EGFR protein by immunohistochemical staining. Fresh-frozen tumors (EDRN) were also evaluated for EGFR protein and site-specific phosphorylation at Y992 and Y1068 using reverse-phase protein array (n = 67). Tumor (n = 50) EGFR and EGFRvIII mRNA levels were quantified using real-time PCR.

Results: EGFR expression by immunohistochemistry (IHC) was significantly higher in the EDRN tumors with EGFR gene amplification (P < 0.001), and a similar trend was noted in the cetuximab-treated cohort. In the EDRN and cetuximab-treated cohorts elevated EGFR by IHC was associated with reduced survival (P = 0.019 and P = 0.06, respectively). Elevated expression of total EGFR and EGFR PY1068 were independently significantly associated with reduced progression-free survival in the EDRN cohort [HR = 2.75; 95% confidence interval (CI) = 1.26-6.00 and HR = 3.29; 95% CI = 1.34-8.14, respectively].

Conclusions: In two independent HNSCC cohorts treated with or without cetuximab, tumor EGFR levels were indicative of survival. Tumor EGFR PY1068 levels provided prognostic information independent of total EGFR.

Figure 1. EGFR protein levels by IHC were higher in EDRN tumors with EGFR gene amplification

(A) Representative EGFR (red) and chromosome 7 (green) FISH for tumors with EGFR gene amplification (left panel, n=13/57), hyperploidy without EGFR gene amplification (middle panel, n=18/57) and neither EGFR gene amplification nor hyperploidy (right panel, n=26/57). (B) EGFR protein levels determined by IHC staining categorized by tumor EGFR gene amplification and ploidy status. (C) EGFR protein levels determined by reverse phase protein array (RPPA) categorized by EGFR gene amplification and ploidy status. (D) Tumor EGFR mRNA level by EGFR gene amplification and ploidy status. Medians (black horizontal bars), overall tertiles (red horizontal bars) and p values (Kruskal-Wallis tests) are provided.

Figure 2. High EDRN tumor EGFR protein by IHC was associated with reduced progression-free survival

Kaplan-Meier progression-free survival plots by (A) tumor EGFR level by IHC Score tertile, (B) EGFR gene amplification status (presence versus absence), (C) EGFR protein level by RPPA by tertile and (D) EGFR mRNA level by tertile. Log rank tests with associated p values compare presence versus absence (B) or trend across low, intermediate and high tertiles (A, C & D).

Figure 3. High tumor EGFR protein by IHC tended to be associated with reduced progression-free survival in the cetuximab-treated cohort

(A) EGFR protein levels determined by IHC staining categorized by tumor EGFR gene amplification and ploidy status. Kruskal Wallis p value provided. (B) Kaplan-Meier progression-free survival plots by low versus high tumor EGFR protein by IHC level as defined by the median. (C) Kaplan-Meier plot of progression-free survival by tumor EGFR gene amplification status (presence versus absence). Log rank test-associated p values are provided for B and C.

Figure 4. Phosphorylation at EGFR PY1068 was associated with reduced progression-free survival in the EDRN cohort

Kaplan-Meier progression-free survival (PFS) plots by tertiles (low, intermediate and high) of tumor levels of (A) EGFR PY1068 and (B) EGFR PY992 as assessed by RPPA. Differences in PFS phosphorylation site-specific pEGFR levels across tertiles were tested using the log rank trend test. (C) EGFR intermediate/high protein levels by IHC combined with EGFR PY1068 intermediate/high levels versus lowest tertile reference. Log rank trend test-associated p value comparing across ordered categories of (1) low in EGFR and PY1068, (2) intermediate/high EGFR or PY1068 and (3) intermediate/high in both EGFR and PY1068 is provided.