We prospectively examined pretreatment biopsies from patients with advanced SCCOP enrolled on a clinical trial in which all patients were treated with IC; responders were given CRT, and nonresponders underwent surgery.43
This allowed us to determine how molecular factors and patient characteristics affected response to chemotherapy, CRT, and survival in a group of homogeneously treated patients.
In our cohort, EGFR, HPV16,1
and p16 each independently predicted survival and, as combined markers, identified the patients with the best and worst survival. If a patient's tumor had high EGFR expression and contained HPV or expressed high p16, the survival probability was improved compared with those without HPV or p16. HPV titer and p16 expression were inversely related to EGFR expression. Kim et al45
reported an association between HPV and p16 expression and an inverse relationship between HPV and EGFR expression but did not report any association with outcome in a series of patients with tonsillar cancer (73% HPV positive). In patients with oropharyngeal cancer (28% HPV positive), Reimers et al46
found only a trend for EGFR to predict outcome, but the combination of p16 and EGFR expression stratified patients with better and worse OS and DSS. They also found a tendency for lower EGFR expression in p16-positive tumors. Prior reports in patients with laryngeal cancer47
and with laryngeal papillomatosis48
suggested that the HPV-E5 protein increased EGFR expression. However, because 15 of 25 HPV-positive patient cases in our study expressed low EGFR, it is unlikely that high EGFR expression is secondary to HPV in most instances. This is the first report that has combined HPV titer, expression of EGFR, and p16 in pretreatment SCCOP biopsies and has analyzed these markers in relation to smoking, sex, response to therapy, and outcome.
To understand why women in our study had worse outcomes than men, we examined differences in biomarker expression and clinical factors. As a group, the women were older than the men, were either current or past smokers, and were less likely to be HPV positive. Similarly, there was a strong trend for higher EGFR expression among the tumors from women. Thus, for each category, the women had less favorable statuses. We and others2,49,50
found an inverse correlation between smoking and HPV. We also observed that EGFR expression was significantly higher in current smokers than in past smokers, who in turn had higher EGFR levels than those who never smoked. This suggests that smoking may contribute to increased EGFR expression, possibly through increased hypoxia in the tumor tissue of smokers.22
When patients were stratified according to smoking status and EGFR expression, those who were current smokers with high tumor EGFR expression had poorer DSS than patients in all other groups.
In this cohort, p53 and Bcl-xL together are markers of outcome and survival that identify tumors with good and poor outcome, independent of HPV. This observation is consistent with in vitro studies, in which we observed that HNSCC tumor cells with low (wild-type) p53 and low Bcl-xL undergo apoptosis in response to cisplatin and that those with low (wild-type) p53 and high Bcl-xL are cisplatin resistant.41
Interestingly, in our current study, the cisplatin-sensitive phenotype translated into improved DSS and OS. Of the patients whose tumors had low p53 and low Bcl-xL expression, eight of nine were alive at last follow-up, whereas five of seven with low p53 and high Bcl-xL had died as a result of their cancers. The predictive value of these markers may be explained by high expression of the apoptosis-blocking protein, Bcl-xL, which inhibits apoptosis in tumors with wild-type p53
. Our in vitro studies showed that cells with this phenotype arrest, induce DNA repair, and begin growing again within 24 to 48 hours after cisplatin treatment (Bauer et al, submitted for publication). This behavior would be consistent with poor outcome in patients. In contrast, the favorable outcome of patients whose tumors have low p53 and low Bcl-xL is likely a result of p53-mediated apoptosis secondary to treatment-induced DNA damage, as we have observed in vitro.41
We recently observed a similar association of the low p53 and low Bcl-xL phenotype with larynx preservation and with a trend for improved survival in patients on the chemotherapy arm of the VA larynx cancer trial.51,52
Intermediate survival results were obtained with the tumors that overexpressed p53 irrespective of Bcl-xL expression. In our in vitro studies, tumor cells that overexpressed mutant p53 generally were more sensitive to cisplatin than those with wild-type p53
and high Bcl-xL,53
presumably because cells with mutant p53
lack p53-mediated arrest and repair. In our cohort, only a subset of patients with SCCOP that overexpressed p53 harbored p53
mutations. Many of the tumors that overexpressed p53 were positive for HPV, which, in itself, influences outcome and response to therapy. Others also observed p53 overexpression in HPV-positive tumors.54,55
The mechanism responsible for overexpression of wild-type p53 in the context of HPV is not known and is the subject of continuing study in our lab.
Smoking cessation strategies are warranted in patients with SCCOP, because current smokers benefited far less than past smokers from this successful therapeutic approach. Pretreatment biopsies to identify those nonsmoking patients with high HPV loads and low EGFR expression for the least aggressive therapy also may be indicated to minimize treatment morbidity. Targeted use of EGFR inhibitors in patients with high tumor EGFR expression, either as single agents or in combination with current therapy, could improve the outcome for the group of patients with the poorest survival. Likewise, for patients who have the low p53 and high Bcl-x combination, inhibitors of cell survival proteins have promise as targeted agents.56-58
Our results showed highly significant associations between biomarkers and outcome and identified patients that would or would not benefit from current treatments. However, ours is a relatively small sample size; these findings must be validated in a larger cohort of patients so that individualized targeted treatments can be designed to improve survival in the patients at greatest risk.