Radioimmunohistochemical quantitation of EGFR and HER2 expression in preinvasive compared to invasive breast cancer and the relationship of HER2 expression in primary invasive breast cancer to outcome

Background: Ductal carcinoma in situ (DCIS) of the breast is being diagnosed with increasing frequency, partly due to the increased use of mammography in mass screening programs. The precise natural history of DCIS is not clear but is thought to represent a stage in the progression to frank malignancy. This group has previously described a radioimmunohistochemical technique which allows quantitation of the EGFR and HER2 in all cases of tumour examined (1;2). Application of this quantitative radioimmunohistochemistry to pure DCIS, DCIS in association with invasive disease and primary invasive breast cancers may clarify the role, if any, of EGFR and HER2 in the pathogenesis of invasive breast cancer. If EGFR downregulation or HER2 overexpression are required for progression to invasion we might expect to see more lesions with normal levels of expression in pure DCIS, and an invasive or intermediate pattern in DCIS adjacent to invasive areas. In this way, the quantitative information available from radio-immunohistochemistry may allow us to predict the biological role of EGFR and HER2 in breast carcinogenesis and progression. HER2 gene amplification or protein overexpression identifies 20-30% of breast cancer patients with a poor outlook. Current methods of HER2 analysis reveal nothing about the remaining 70 to 80% of patients. This may compromise the prognostic or predictive value of HER2. Methods: In frozen sections of pure DCIS tumours (n=37 for EGFR, n=36 for HER2) and in DCIS lesions in association with invasive cancers, (n=50 for EGFR, n=47 for HER2) receptor levels were assayed quantitatively using a radiolabelled antibody method. EGFR was quantified in 193 and HER2 in 177 frozen primary breast tumours using radioimmunohistochemistry. The results of HER2 expression were related to patient survival and major pathological variables. Results: EGFR and HER2 expression each varied by a factor of several thousand. Levels of EGFR and HER2 expression in pure DCIS, in DCIS associated with invasion and in a larger group of invasive tumours (n=193 for EGFR, n=177 for HER2) were compared. The frequency distributions for expression of both factors were comparable in pure DCIS and in DCIS associated with invasive tumours (Mann-Whitney U test, EGFR p=0.17, HER2 p=0.16). Similarly, frequency distributions for expression of both factors were comparable in DCIS associated with invasive tumours and in purely invasive tumours (Mann-Whitney U test, EGFR p=0.16, HER2 p=0.91). Comparing pure DCIS with invasive tumours , there was a trend to significance difference in the frequency distributions of these 2 growth factor receptors. Within each tumour that had both DCIS and invasion, there was no significant difference in expression of EGFR or HER2 in the DCIS and invasive components (Wilcoxon Signed Rank Test, EGFR p=0.39, HER2 p=0.087). However, on reviewing individual cases, those tumours that exhibit the largest change in expression of growth factor receptor between the DCIS and invasive components appeared to have very aggressive invasive tumours. Fifteen percent of primary invasive cancers expressed less HER2 than normal breast parenchyma, 62% had between 1 and 15 times normal and 23% between 15 and 400 fold overexpression. We have shown previously that this last group corresponds to those with HER2 gene amplification. Survival analysis indicated that prognosis was best for tumours with intermediate levels of HER2, becoming worse for both lower and higher expressing cases. The best outcome was associated with a 5.7 times the normal HER2 level. The relative hazard was 3.5 times higher for tumours with 0.1x normal expression, and 4 times higher for tumours with 100x normal expression. HER2 remained a significant predictor of survival when the pathological variables were introduced into the analysis. Conclusions: These data suggest that alterations in type I growth factor receptors occur before progression of in-situ disease to invasive cancer. High levels of HER2 overexpression in both in-situ and invasive areas suggest that the HER2 product is a potential therapeutic target for the treatment of breast cancer at an early stage. In addition, these results indicate that the role of HER2 in breast cancer is complex, as tumours with down regulation as well as those with gene amplification exhibit more aggressive behaviour. These findings are of clinical importance given the developing role of HER2 as a prognostic and predictive factor, and as a target for therapy.