ERCC1 is a prognostic biomarker in locally advanced head and neck cancer: results from a randomised, phase II trial

Abstract

Background: Cisplatin-radiotherapy is a preferred standard for locally advanced, head and neck squamous cell carcinoma (HNSCC). However, the cisplatin-attributable survival benefit is small and toxicity substantial. A biomarker of cisplatin resistance could guide treatment selection and spare morbidity. The ERCC1-XPF nuclease is critical to DNA repair pathways resolving cisplatin-induced lesions.

Methods: In a phase II trial, patients with untreated Stage III-IVb HNSCC were randomised to cisplatin-radiotherapy with/without erlotinib. Archived primary tumours were available from 90 of 204 patients for this planned substudy. Semi-quantitative ERCC1 protein expression (H-score) was determined using the FL297, 4F9, and 8F1 antibodies. The primary analysis evaluated the relationship between continuous ERCC1 protein expression and progression-free survival (PFS). Secondary analyses included two pre-specified ERCC1 cutpoints and performance in HPV-associated disease.

Results: Higher ERCC1 expression was associated with inferior PFS, as measured by the specific antibodies FL297 (HR=2.5, 95% CI=1.1-5.9, P=0.03) and 4F9 (HR=3.0, 95% CI=1.2-7.8, P=0.02). Patients with increased vs decreased/normal ERCC1 expression experienced inferior PFS (HR=4.8 for FL297, P=0.003; HR=5.5 for 4F9, P=0.007). This threshold remained prognostic in HPV-associated disease.

Conclusion: ERCC1-XPF protein expression by the specific FL297 and 4F9 antibodies is prognostic in patients undergoing definitive cisplatin-radiotherapy for HNSCC, irrespective of HPV status.

Trial registration: ClinicalTrials.gov NCT00410826.

Figures

Figure 1

Representative 3+ ERCC1 Staining for FL297, 4F9, and 8F1. Representative ERCC1 stains are presented for consecutive sections of a p16-negative hypopharynx tumour. Note that staining intensity cannot be compared among antibodies as it is referenced to an internal control designated 2+ (arrow in box C). (A) H&E stained invasive squamous cell carcinoma at × 20 magnification. (B) Negative control; tissue shows lack of non-specific ERCC1 staining. (C) 3+ ERCC1 staining for 4F9. (D) 3+ ERCC1 staining for 8F1. (E) 3+ ERCC1 staining for FL297.

Figure 2

The 4F9 antibody is specific for ERCC1. Specificity of 4F9 is assessed in human skin fibroblasts isolated from either a normal individual (WT) or an individual with a mutation in XPF causing near-undetectable ERCC1 (XP2YO). (A) 4F9 is specific by western blot. Only a trace amount of ERCC1 is detected in XP2YO cells with either 4F9 or the specific anti-ERCC1 antibody D-10. In contrast, the non-specific 8F1 antibody recognises an additional band migrating slightly slower than ERCC1, present both in WT and ERCC1 deficient cells (arrows). Tubulin (loading control). (B) 4F9 is specific by immunofluorescence. Only background nuclear signal is observed in ERCC1-deficient cells either with 4F9 (white asterisks) or with the antibody D-10 while nuclear staining is readily observable in WT cells. In contrast, the nuclear signal persists in ERCC1 deficient cells when 8F1 is used, confirming the lack of specificity of this antibody. ERCC1 antibodies (red); DNA stain DAPI (blue). (C) Quantitation of average nuclear fluorescence intensity represented by boxplot; p (paired t-test); * indicates statistical significance. (D) 4F9 is specific by immunohistochemistry performed on formalin-fixed paraffin-embedded cells. Only background staining is observed in ERCC1-deficient cells (black asterisks). 4F9 (brown); haematoxylin counterstain (blue).

Figure 3

Bland–Altman plots comparing H-scores for three ERCC1 assays. Mean differences were centered around zero for all assays (solid lines), but 95% limits of agreement (dashed lines) were more narrow for the FL297 and 4F9 antibodies.

Figure 4

(A–C) Kaplan–Meier curves showing PFS as a function of categorical ERCC1. For the primary analysis, PFS was treated as a continuous variable. For purposes of graphical display, PFS is presented in Figure 4 for each antibody, according to pre-defined categories of ERCC1 expression: increased (H-score ⩾2.5), normal (1.5< H-score <2.5), or decreased (H-score ⩽1.5). Hazard ratios are presented for the exploratory cutpoint, ‘increased' vs ‘normal/decreased' ERCC1 expression as detected by the two specific antibodies. * Indicates statistical significance.

Figure 5

Distribution of ERCC1 by p16 expression. Boxplots present ERCC1 expression by p16 status, for the specific antibodies FL297 and 4F9. Plotting characters identify tumour site category (oropharyngeal vs non-oropharyngeal). * Indicates statistical significance.