Background: We have previously described that TNBC patients whose tumors have both HER2 Y1248 phosphorylation (pHER2) “high” and phospho-EGFR Y1173 (pEGFR) “high” have increased response (pCR) to neratinib in the I-SPY2 TRIAL. We hypothesize that the paradoxical finding of a response prediction signature comprised of HER2 activation in a HER2 IHC/FISH-negative population means there must be a ligand-driven biochemical event responsible for the HER2 phosphorylation because HER2 mutations were also not found to be significant. Exploratory analysis of additional cellular signaling events and protein expression levels in pre-treatment, LCM-purified tumor epithelium by reverse phase protein microarray (RPPA) included semi-quantitative measurement of total levels of estrogen receptor alpha (ERα), which has been previously shown to be able to act as a membrane non-genomic signaling molecule through direct interaction with various tyrosine kinases including EGFR and HER2. Since ERα has been previously shown to act as a ligand and co-stimulate (activate) HER2 and EGFR when present at low levels, we investigated whether or not RPPA-measured ERα levels in the TNBC cohort analyzed to date were higher in tumors with both pHER2 “high” and pEGFR “high” levels and thus provide evidence explaining how HER2-EGFR activation is occurring in TNBC.
Methods: Using RPPA analysis, we measured 118 analytes in lysates of LCM tumor epithelium obtained from the pre-treatment biopsy samples of 86 TNBC (Allred=0) patients in the I-SPY2 TRIAL analyzed to date. Cutpoints for pEGFR and pHER2 were determined previously by ROC analysis for pCR correlation in the neratinib treated TNBC population, and used here to dichotomize the pHER2 and pEGFR data in the larger TNBC population. Wilcoxon Rank Sum testing was performed using the continuous variable total ERα data and compared the TNBC that were both pHER2 and pEGFR “high” (N=39) to the rest of the TNBC population (N=47). Total ERα values were then divided into “high” and “low” groups based on the TNBC population median value in order to determine frequency/percentages within each class. Our study is exploratory with no claims for generalizability of the data, and calculations are descriptive (e.g. p-values are measures of distance with no inferential content).
Results: Total ERα values were obtained in 84/86 TNBC tumors analyzed. Total levels of ERα were higher (p< 0.006) in TNBC tumors with pHER2 and pEGFR “high” levels. 68% (26/38) of tumors in the pHER2 and pEGFR “high” group had ERα levels above the population median compared to 35% (16/46) in the rest of the TNBC population.
Conclusion: Our exploratory analysis reveals that ERα levels are significantly higher in TNBC with pHER2 and pEGFR activation and may be behaving as a direct signaling ligand in TNBC and driving HER2-EGFR signaling. This ERα-pHER2/pEGFR association was missed by current ER and HER2 clinical laboratory testing techniques, and if validated in larger independent study sets could suggest that utilization of new protein-based techniques defining ER more quantitatively could be helpful to understand tumor biology and therapeutic response prediction, especially in the context of TNBC that are ostensibly ER negative.