In this study, we investigated the expression of XRCC1 in HNSCC patients and its association with clinicopathologic factors and outcome. Our results show that XRCC1 protein expression is common in HNSCC and that high XRCC1 protein expression, regardless of primary tumor site, stage, and p16INK4a status confers poorer survival as compared with low XRCC1 expression. Furthermore, this association was strongest in the subgroup of patients who received CRT.
XRCC1 facilitates efficient DNA damage processing and is,therefore,especially pertinentinpatientsundergoing CRT (3
). Ionizing radiation kills cells by inducing DNA damage such as base damage, SSB, double-strand breaks, and interstrand DNA cross-links. Chemotherapy, particularly platinum-based chemotherapy, binds to DNA forming DNA adducts, which distort DNA structure, causing damage and cell death. Combining chemotherapy with radiation greatly increases the overall cytotoxic effect of radiotherapy by inducing further DNA damage and interfering with DNA repair. This is mitigated to a certain extent by nonspecific DNA repairsystemssuchasnucleotideexcisionrepair(NER) and BER multistep enzymatic complexes. Therefore, high XRCC1 expression may increase the DNA repair capacity of tumor cells leading to increased tolerance to DNA damage from CRT. Our study findings of high XRCC1 protein expression being associated with poorer survival after CRT areconsistentwiththishypothesisaswellaspreviousreports (7
). Our study also shows that the coding SNP Arg194Trp is associated with treatment outcome among CRT patients, whereas the noncoding RS2682558 in the 3′-UTR may have a regulatory role and thus affect XRCC1 protein expression levels. Other studies have shown that XRCC1 Arg194Trp was associated with treatment response to platinum-based chemotherapy and may predict for PFS (16
Despite the strong association between XRCC1 protein expression status and survival, we were not able to show any relationship between XRCC1 expression and response to primary CRT or RT. Response to CRT/RT was assessed 6 to 12 weeks after treatment completion, and categorized as either CR or persistent disease. From a clinical standpoint, this was to facilitate planning for early surgical salvage of patients with residual disease. However, assessment of response is often difficult in HNSCC in which imaging may not always be accurate, particularly in the immediate postradiation period. Furthermore, some patients take longer to respond completely to CRT/RT and may, therefore, have been inappropriately categorized as persistent disease at the 6- to 12-week posttreatment time point.
Presence of HPV has been established as a favorable prognostic factor in oropharyngeal HNSCC (19
IHC has been shown to correlate with HPV status (19
) and patients with HPV-DNA positive and p16 expressing tumors, inparticular, have a favorable prognosis(23
). Consistent with previous studies, our results showed that p16INK4a
-positive status was associated with reduced risk of death. Interestingly, XRCC1 expression status was able to further discriminate within each p16INK4a
category. Among p16INK4a
-positive patients, who were expected to have good prognosis, patients with high XRCC1 expression had poorer survival compared with patients with low XRCC1 expression. Furthermore, survival was similar between patients who were p16INK4a
negative/XRCC1 low and p16INK4a
positive/XRCC1 high, implying XRCC1 expression status has significant prognostic implications. These findings are consistent with recent reports of subsets of HPV-positive patients with differing survival outcomes, depending on factors such as Bcl2 expression, epidermal growth factor receptor expression, and smoking history (19
Nevertheless, other DNA repair enzymes may also influence treatment outcomes and require consideration. PARP-1 is involved in DNA repair and is required for the assembly and stability of XRCC1 (27
). ERCC1 is one of the key rate-limiting enzymes involved in NER and studies in HNSCC have shown that low ERCC1 levels are associated with good outcomes from CRT (29
) and induction chemotherapy (30
). Enzymes that modulate the availability of platinum compounds (such as glutathione S-transferase π) or modulate cell death and apoptosis (Bcl-2 and p53) have also been reported to affect the treatment outcomes in HNSCC (27
This study had several limitations. First, the study was retrospective and comprised a heterogeneous cohort of HNSCC patients of different stages, tumor sites, and treatments, although the baseline characteristics of patients in the high and low XRCC1 expression cohorts were similar. Second, the study population was divided into high- and low-XRCC1 expression cohorts using the population median score as the cutoff point. We believe that this use of the Allred score is clinically relevant because it defines 2 distinct patient populations with different IHC staining patterns of XRCC1 expression: (i) homogenous and intense staining throughout the epithelium; and (ii) heterogeneous (average of <67% of tumor cells stained) and/or lower intensity staining (average intensity <3). This makes XRCC1 staining evaluation a potentially useful biomarker in HNSCC, as there is little ambiguity with regard to high-versus low-XRCC1 expression status. Third, only one antibody was used for XRCC1 IHC, however, we showed its specificity for XRCC1 using Western immunoblot of patient tumors, as well as Western and immunocytochemical analysis of RNAi-mediated knockdown of XRCC1 in a head and neck cancer cell line. Our data showing loss of native XRCC1 detection after RNAi support that this antibody, indeed, is detecting specifically XRCC1. Fourth, the non-CRT cohort consisted of patients treated with 3 different treatment modalities and thus it may have been inappropriate to analyze them as a single group. Fifth, we did not have information on tumor differentiation and were unable to correlate XRCC1 expression to tumor differentiation or assess its effect on survival.
Taken together, our findings presented herein suggest that XRCC1 may be an important biomarker in HNSCC. Assessment of XRCC1 protein expression status by IHC may be useful in clinical decision making. Patients with high expression may have poorer outcomes from CRT, although still being subject to toxicity, and thus could be directed to alternative treatment modalities and/or clinical trials. Conversely, patients with low XRCC1 expression may benefit from CRT, particularly if they are also p16INK4a-positive. Future studies will be required to define the role of XRCC1, as a prognostic or predictive marker, in different tumor sites and for different treatment modalities, and also to address the interactions and roles that different repair proteins and prognostic markers may have in determining the outcome of CRT in HNSCC.