Abstract P1-07-06: Effect of biospecimen variables on proteomic biomarker assessment in breast cancer

Abstract

Background: PI3K/Akt/mTOR signaling is being actively pursued as a therapeutic target. We sought to determine how tumor heterogeneity and biospecimen variables affect assessment of PI3K/Akt/mTOR pathway activation. Methods: Intraoperative image-guided core-needle biopsies (CNB) of primary breast tumors were prospectively collected in 53 patients with invasive breast cancer. After surgery, specimens were collected from the center and periphery of the excised tumor. CNB, central and peripheral surgical specimens were assessed with reverse phase proteomic arrays (RPPA), H&E and immunohistochemistry (IHC). Results: The expression of standard of care markers ER, PR, and HER2 by RPPA correlated well between biospecimen types. Overall, there was a significant correlation between the expression of 132 (86%) of 154 different markers in the center and periphery; the correlation was significantly higher for smaller tumors, and with shorter cold ischemia time. Expression of many investigational prognostic markers and druggable targets on CNB correlated with expression in the surgical specimen (average of center and periphery), while others, such as EGFR and c-MET, had a weak correlation. Of 154 RPPA markers, 132 (86%) were not statistically different between the center and periphery, and 97 (67%) were not different between the CNB and the surgical specimen. On analysis of the PI3K/AKT/mTOR pathway, pAkt S473 and PTEN had a significant correlation between central and peripheral specimens, and between CNB and surgical specimens. However, pAkt S473, pS6 S235/236 and pS6 240/244 levels were higher in CNB than the central specimens both by RPPA and by IHC. When patients were classified by RPPA PI3K pathway activation score, there was a moderate agreement between classification on the CNB and central specimens (Cohen's Kappa 0.539). However 9 of 20 tumors classified as having PI3K activation on CNB were classified as not having pathway activation on central specimens. Conclusions: There is remarkable homogeneity in expression of biomarkers within a tumor. However, proteomic markers are differentially expressed by biospecimen type and other preanalytic variables. PI3K pathway activation is greater in CNB compared to surgical samples. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-06.

PI3K Pathway Activation in High-Grade Ductal CarcinomaIn Situ—Implications for Progression to Invasive Breast Carcinoma

“…The vast majority of cases (69% of pure DCIS and 75% of DCIS adjacent to IBC) harboring PTEN or INPP4B loss of expression, or PIK3CA or AKT1 mutations, displayed pAKT expression, whereas pS6 expression was found in 6% and 21% of pure DCIS and DCIS adjacent to IBC harboring these molecular aberrations. The lack of pAKT and pS6 expression in cases with alterations in these genes may stem from the fact that we have i) assessed pAKT and pS6 expression in surgical specimens and that previous analyses have shown that their immunohistochemical assessment is affected by pre-analytical variables, and its expression levels are significantly lower in surgical specimens than in core biopsies(48, 49); and ii) employed antibodies that recognize only a few phosphorylation sites of AKT (i.e. Ser473) and S6 (i.e.…”

“…Third, given the evidence to suggest that low-and high-grade DCIS and IBCs are likely to evolve through distinct pathways (reviewed in(1)), we have focused only on the subset of high-grade lesions; hence, our conclusions should be considered relevant only to high-grade disease. Fourth, the surrogate markers employed to determine activation of the PI3K pathway, pAKT and pS6 immunohistochemistry, have been shown to be affected by pre-analytical parameters, including delayed fixation and the type of specimen(48, 49). Although the frequency of PI3K pathway activation may have been underestimated in this study due to the use of surgical specimens, given that all tissues were collected during the same time frame at a single institution, the reduction in pAKT and pS6 expression driven by pre-analytical parameters should be equally prevalent among all groups.…”