High E6 Gene Expression Predicts for Distant Metastasis and Cancer-Specific Survival in Patients With HPV-Positive Oropharyngeal Squamous Cell Carcinoma

Abstract

Purpose/Objective(s)Human papillomavirus–positive (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) patients have an excellent prognosis. However, 10% to 15% of patients will develop distant recurrence despite standard of care treatment. Here, we aim to identify a biomarker to risk stratify HPV+ OPSCC patients.Materials/MethodsHPV status was determined by quantitative real-time PCR (qPCR) using RNA extracted from formalin-fixed paraffin-embedded primary tumors. Two HPV+ OPSCC cohorts were utilized. The initial group comprised 39 patients with stage III/IV disease treated definitively with chemoradiation (20%) or with postoperative radiation with or without chemo (80%). Nineteen patients in this cohort had metastatic disease (DM+) and 20 had no recurrence after a median follow-up of 5 years (DM-). RNA extracted from DM+ and DM- primaries were prepared for gene expression studies using the established RNA sequencing technique. The second group served as a validation cohort, consisting of 93 HPV+ OPSCC patients with stage III/IV disease treated from 2000 to 2011 with definitive chemoradiation (15%) or with postoperative radiation with or without chemo (76%). E6 gene expression was assayed in the validation cohort using qPCR. ROC analysis was employed to establish an E6 cutoff that predicts distant recurrence. Patients with E6 expression levels above the ROC cutoff were denoted as E6 high and those below the cutoff as E6 low.ResultsGene expression profiling of the initial cohort revealed that DM+ had a 2-fold higher E6 expression level compared to DM- (P = .02). This observation was confirmed in the validation cohort, in which after a median follow-up of 3 years, 15 distant recurrences (16%) and 13 cancer-related deaths (14%) were noted. Mean normalized E6 expression level in the 15 recurring primary specimens was 13±2 compared to 8 ± 1 in the remaining 78 nonrecurring primaries (P = .001). ROC analysis of the validation cohort established an E6 expression level of 7.3 as a cutoff for worse recurrence-free survival (RFS). The resulting E6-high cohort (n = 51, 55%) had more cancer-related deaths (23% vs 2%, P < .001) and distant recurrences (26% vs 5%, P < .001) compared to the E6-low cohort (n = 42, 45%). Kaplan-Meier survival analyses revealed that the E6-high group had worse 3-year actuarial RFS (95% vs 69%, P = .004) and cancer-specific survival (CSS) (97% vs 79%, P = .007). E6-high status was significant for both RFS and CSS on multivariate COX regression balancing surgery, chemo, nodal stage, and smoking.ConclusionHigh E6 expression level identifies a subset of HPV+ OPSCC patients with 5-fold greater risk of distant recurrence and worse cancer-specific survival. We propose that E6 expression level can be used in future prospective studies to stratify HPV+ OPSCC patients with higher recurrence risk. Purpose/Objective(s)Human papillomavirus–positive (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) patients have an excellent prognosis. However, 10% to 15% of patients will develop distant recurrence despite standard of care treatment. Here, we aim to identify a biomarker to risk stratify HPV+ OPSCC patients. Human papillomavirus–positive (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) patients have an excellent prognosis. However, 10% to 15% of patients will develop distant recurrence despite standard of care treatment. Here, we aim to identify a biomarker to risk stratify HPV+ OPSCC patients. Materials/MethodsHPV status was determined by quantitative real-time PCR (qPCR) using RNA extracted from formalin-fixed paraffin-embedded primary tumors. Two HPV+ OPSCC cohorts were utilized. The initial group comprised 39 patients with stage III/IV disease treated definitively with chemoradiation (20%) or with postoperative radiation with or without chemo (80%). Nineteen patients in this cohort had metastatic disease (DM+) and 20 had no recurrence after a median follow-up of 5 years (DM-). RNA extracted from DM+ and DM- primaries were prepared for gene expression studies using the established RNA sequencing technique. The second group served as a validation cohort, consisting of 93 HPV+ OPSCC patients with stage III/IV disease treated from 2000 to 2011 with definitive chemoradiation (15%) or with postoperative radiation with or without chemo (76%). E6 gene expression was assayed in the validation cohort using qPCR. ROC analysis was employed to establish an E6 cutoff that predicts distant recurrence. Patients with E6 expression levels above the ROC cutoff were denoted as E6 high and those below the cutoff as E6 low. HPV status was determined by quantitative real-time PCR (qPCR) using RNA extracted from formalin-fixed paraffin-embedded primary tumors. Two HPV+ OPSCC cohorts were utilized. The initial group comprised 39 patients with stage III/IV disease treated definitively with chemoradiation (20%) or with postoperative radiation with or without chemo (80%). Nineteen patients in this cohort had metastatic disease (DM+) and 20 had no recurrence after a median follow-up of 5 years (DM-). RNA extracted from DM+ and DM- primaries were prepared for gene expression studies using the established RNA sequencing technique. The second group served as a validation cohort, consisting of 93 HPV+ OPSCC patients with stage III/IV disease treated from 2000 to 2011 with definitive chemoradiation (15%) or with postoperative radiation with or without chemo (76%). E6 gene expression was assayed in the validation cohort using qPCR. ROC analysis was employed to establish an E6 cutoff that predicts distant recurrence. Patients with E6 expression levels above the ROC cutoff were denoted as E6 high and those below the cutoff as E6 low. ResultsGene expression profiling of the initial cohort revealed that DM+ had a 2-fold higher E6 expression level compared to DM- (P = .02). This observation was confirmed in the validation cohort, in which after a median follow-up of 3 years, 15 distant recurrences (16%) and 13 cancer-related deaths (14%) were noted. Mean normalized E6 expression level in the 15 recurring primary specimens was 13±2 compared to 8 ± 1 in the remaining 78 nonrecurring primaries (P = .001). ROC analysis of the validation cohort established an E6 expression level of 7.3 as a cutoff for worse recurrence-free survival (RFS). The resulting E6-high cohort (n = 51, 55%) had more cancer-related deaths (23% vs 2%, P < .001) and distant recurrences (26% vs 5%, P < .001) compared to the E6-low cohort (n = 42, 45%). Kaplan-Meier survival analyses revealed that the E6-high group had worse 3-year actuarial RFS (95% vs 69%, P = .004) and cancer-specific survival (CSS) (97% vs 79%, P = .007). E6-high status was significant for both RFS and CSS on multivariate COX regression balancing surgery, chemo, nodal stage, and smoking. Gene expression profiling of the initial cohort revealed that DM+ had a 2-fold higher E6 expression level compared to DM- (P = .02). This observation was confirmed in the validation cohort, in which after a median follow-up of 3 years, 15 distant recurrences (16%) and 13 cancer-related deaths (14%) were noted. Mean normalized E6 expression level in the 15 recurring primary specimens was 13±2 compared to 8 ± 1 in the remaining 78 nonrecurring primaries (P = .001). ROC analysis of the validation cohort established an E6 expression level of 7.3 as a cutoff for worse recurrence-free survival (RFS). The resulting E6-high cohort (n = 51, 55%) had more cancer-related deaths (23% vs 2%, P < .001) and distant recurrences (26% vs 5%, P < .001) compared to the E6-low cohort (n = 42, 45%). Kaplan-Meier survival analyses revealed that the E6-high group had worse 3-year actuarial RFS (95% vs 69%, P = .004) and cancer-specific survival (CSS) (97% vs 79%, P = .007). E6-high status was significant for both RFS and CSS on multivariate COX regression balancing surgery, chemo, nodal stage, and smoking. ConclusionHigh E6 expression level identifies a subset of HPV+ OPSCC patients with 5-fold greater risk of distant recurrence and worse cancer-specific survival. We propose that E6 expression level can be used in future prospective studies to stratify HPV+ OPSCC patients with higher recurrence risk. High E6 expression level identifies a subset of HPV+ OPSCC patients with 5-fold greater risk of distant recurrence and worse cancer-specific survival. We propose that E6 expression level can be used in future prospective studies to stratify HPV+ OPSCC patients with higher recurrence risk.