Of the EGFR molecular characteristics evaluated in paraffin-embedded formalin-fixed tumors, we found that elevated tumor levels of EGFR by IHC were significantly associated with PFS in a patient cohort treated with surgery with curative intent without an EGFR-targeted therapy. Though our IHC staining method did not detect nuclear EGFR, which has been previously reported to be associated with increased local HNSCC recurrence rates (34
), our results are similar to a recent study finding high predominately plasma membrane and cytoplasmic tumor EGFR levels to be associated with reduced survival (40
). We found a similar decrease in PFS with elevated tumor EGFR by IHC for patients treated with chemoradiotherapy and EGFR-targeted agent cetuximab. Our two cohorts differed in treatment type and HPV-positive tumor representation, suggesting that EGFR IHC levels may be prognostic for both HPV-positive and negative tumor types.
Though we found no significant association between EGFR gene amplification and PFS in either cohort, we did note a trend towards reduced PFS with increased EGFR gene copy number in the EDRN surgical cohort that was similar to the association reported by Chung et al. (15
). Our finding of lack of significant association between increased EGFR gene copy number and PFS was probably not due to a minor difference defining high EGFR copy number where we included 4 or more EGFR copies in at least 50% of cells while Chung et al. delineated 40% as the cut-point (15
). Though our follow-up time was longer than the Chung et al. study, our cohort was smaller, which may decrease power in our study to detect a difference in PFS by copy number status. Acknowledging these caveats, our data demonstrated that tumor EGFR gene amplification status, which is currently assessed using an expensive and challenging assay, did not function as a stand-alone prognostic marker nor did it improve prognostic information provided by EGFR IHC, which is a relatively inexpensive assay performed routinely. Our finding that tumor EGFR levels by IHC had prognostic value while EGFR gene copy number did not is consistent with two recent studies in HNSCC that report similar findings (40
Even though tumor EGFR levels by IHC were associated with PFS, EGFR levels by RPPA were not. We speculate that the differences are largely technical in nature. Recent reports have demonstrated that EGFR protein levels assessed by IHC versus immunoblotting methods can differ somewhat (33
), and that the magnitude of these differences varies depending upon the antibody used (33
). We employed the anti-EGFR antibody clone H11 for our IHC studies. This clone H11 antibody was recently shown to have only a modest correlation between EGFR protein levels by automated quantitative analysis (AQUA) of IHC stained cell lines and EGFR protein levels quantified from immunoblots of lysates from these same cell lines (33
). Of four anti-EGFR antibodies tested in a study of 642 breast cancer tumors in this same report by Anagnostou et al., EGFR levels by AQUA analysis of clone H11-stained tumors did not correlate with EGFR levels by AQUA of IHC results obtained using the other anti-EGFR antibodies tested (33
). Because we employed the clone H11 antibody for IHC and a different anti-EGFR antibody for RPPA, the differences between IHC and RPPA results likely reflect inherent antibody and assay performance differences. Of interest, of the anti-EGFR antibodies tested for prognostic value in breast cancer by Anagnostou et al., only the clone H11 antibody demonstrated prognostic value (p<0.05), with shorter survival observed for patients whose tumors had high or intermediate tumor levels of EGFR as assessed by AQUA and IHC. Because of assay validation specifications, we used a different anti-EGFR antibody for our RPPA studies. Therefore, a portion of difference between IHC and RPPA results is likely attributed to differences in the antibodies performance.
In addition to inherent assay performance differences such as antibody differences and possible epitope availability differences in arrayed fresh-frozen tissue lysates compared to formalin-fixed tissues, pathologist-interpreted EGFR protein levels by IHC were confined to the cytoplasm and plasma membrane of tumor cells because nuclear EGFR was not detected using the clone H11 antibody. Subcellular architecture was lost when making lysates for RPPA. Therefore, it is possible that EGFR tumor protein in the cytoplasm, membrane and nucleus may contribute differentially to signaling.
Though the stromal compartments of tumors are appreciated as important contributors to HNSCC development and progression, it is possible that stromal tissue present in RPPA samples contributed to the lack of correlation between IHC and RPPA analyses, though we were not able to retrospectively assess this. EGFR tumor levels by IHC were confined to the tumor portion of the specimen by the evaluating pathologist. EGFR levels by RPPA would represent tumor and stromal tissue levels. Our fresh-frozen tumor tissues contained at least 70% tumor; it is possible that up to 30% of our RPPA analyzed tumor tissues were stromal tissues. Our lack of concordance between tumor EGFR levels by IHC and EGFR in protein lysates was similar to previous reports comparing tumor EGFR levels by IHC and by enzyme-linked immunosorbant assays (43
We did find that high tumor levels of EGFR PY1068 but not EGFR PY992 in fresh-frozen tumor lysates contributed prognostic information alone and in addition to tumor EGFR levels by IHC, suggesting that EGFR PY1068 evaluation was not substantially hindered by subcellular localization or stromal contamination issues. Reports evaluating associations between clinical parameters and tyrosine-phosphorylated EGFR that is not site-specific have yielded mixed results (3
). Our findings suggest that there are biologically relevant differences between phosphorylation at specific sites that could impact patient survival. Our data further suggest that STAT3 signaling downstream of EGFR PY1068 may be important. We evaluated only EGFR PY992 and PY1068 because the quality of these antibodies was sufficient for RPPA. It is possible that evaluation of additional EGFR phosphorylation sites will yield more information. EGFR PY1068 had prognostic value in our predominately HPV-negative cohort. It will be important to assess its prognostic value in independent cohorts, including those with higher HPV-positive tumor representation.
The assessment of phosphoproteins in tissues is generally recognized as requiring special care because of the labile nature of the phoshorylation. We collected and processed tissues under a protocol that involved coordination of personnel and immediate transport of collected tissues to the pathology lab for examination, cataloging and freezing in order to minimize time to freezing. Beginning in 2003, our tissue bank recorded time from surgical resection to freezing, and typical banking time was within 40 minutes of resection. For evaluations of EGFR PY1068 tumor levels, time to freezing logistics will need to be considered before general use.
EGFR transcription/translational regulatory mechanisms in HNSCC are likely complex and only just beginning to be defined. Our observation of association between EGFR gene amplification and elevated EGFR protein by IHC but lack of association of EGFR mRNA levels with either characteristic suggests complex regulation and contradicts somewhat a previously published report of elevated EGFR mRNA in EGFR FISH positive lung cancer (45
). Similar to a previous report by Sok et al., we found EGFRvIII expression to be associated with EGFR wild-type expression (38
). EGFRvIII expression, which has been associated with resistance to EGFR-targeted therapies (38
), was not prognostic in our surgical cohort that was not treated with an EGFR-targeted therapy. However, our detection of EGFRvIII in approximately 20% of tumors that were EGFR-targeting agent naive suggests that EGFRvIII expression may have relevance for HNSCC de novo
resistance to EGFR-targeted therapies.
Strengths of our study include the prospective enrollment of our subjects and limited heterogeneity of treatments within each of the two cohorts evaluated. For the EDRN cohort, evaluations extended beyond paraffin-embedded tumor tissues to include fresh-frozen tissues. This study presents a multifaceted evaluation of HNSCC that includes the assessment of molecular characteristics that currently require fresh-frozen specimens. A limitation of this study was our inability to assess all patient tumors for all parameters. This limitation reflects the challenges of obtaining sufficient fresh-frozen material for study. Importantly, there are few specimen selection biases, and the evidence indicates that studied specimens are representative of the general cohort.
In conclusion, we report EGFR tumor levels by IHC assessment, which is performed routinely, are informative regarding patient prognosis. Because independent cohorts treated with and without an EGFR targeting agent had similar survival profiles by tumor EGFR IHC status, EGFR IHC likely provides prognostic rather than predictive value. We noted a trend towards reduced survival with high EGFR gene copy number; however, EGFR gene copy status did not provide prognostic information alone or in combination with tumor EGFR IHC data. We do not anticipate that EGFR FISH analysis will provide prognostic information above that acquired by tumor EGFR IHC analysis in the general HNSCC patient population. Of interest, we report that EGFR site-specific phosphorylation at PY1068 provided prognostic information that was independent of tumor EGFR levels by IHC. These data suggest a further exploration of EGFR site-specific phosphorylation events and EGFR PY1068-specific downstream signaling could provide additional insights into HNSCC progression.