Effects of cold ischemia and inflammatory tumor microenvironment on detection of PI3K/AKT and MAPK pathway activation patterns in clinical cancer samples

Abstract

The accuracy of common markers for PI3K/AKT and MAPK pathway activation in preclinical and clinical cancer biomarker studies depends on phosphoepitope stability and changes of phosphorylation under ischemia. Herein, we define conditions under which phosphoepitope-specific duplex immunohistochemistry (IHC) on formalin-fixed, paraffin-embedded tumor tissues reflects pathway activation in situ as accurately as possible, and identify activation patterns linked to mutational status, pathway dependency and tumor microenvironment in clinical tumor samples, cell culture and xenograft tissues. Systematically assessing robustness of pAKT, pERK1/2, pMEK1/2 and pmTOR detection and related markers in xenograft tissues exposed to ischemia, we show that control of preprocessing and ischemia times allows accurate interpretation of staining results. Phosphorylation patterns were then analyzed in 33 xenograft models and in 58 cases with breast cancer, including 21 paired samples of core-needle biopsies with corresponding mastectomy specimens, and 37 mastectomy samples obtained under rigorously controlled conditions minimizing ischemia time. Patterns of pAKT and pERK1/2 staining (predominant PI3K/AKT, predominant MAPK and concomitant activation) were associated with sensitivity to pathway inhibition and partially with the mutational status in cell lines and corresponding xenograft tumors. In contrast, no clear correlation between mutational status and staining patterns was observed in clinical breast cancer samples, suggesting that interaction with the human tumor microenvironment may interfere with the use of phosphoepitope-specific IHC as potential markers for pathway dependency. In contrast to core needle biopsies, surgically resected breast cancer samples showed evidence of severe signal changes comparable to those effects observed in xenograft tumors exposed to controlled ischemia.Robust assessment of kinase phosphorylation in tumor biopsy samples is necessary to understand how mutations translate into aberrant oncogenic signaling, and it has been successfully used as pharmacodynamic marker to monitor the effect of therapeutic inhibition of cancer-related pathways.

ERK Signal Suppression and Sensitivity to CH5183284/Debio 1347, a Selective FGFR Inhibitor

“…The loss of immunoreactive phospho-AKT and phospho-ERK during sample procurement has been reported (41). Therefore, we must consider multiple preanalytical factors that influence the stability of phosphorylated proteins during procurement and preservation of clinical samples (42). Noninvasive samples such as circulating tumor cells and circulating tumor DNA obtained from a liquid biopsy could be the next-generation source of pharmacodynamic markers (43).…”

Genome-Wide Analysis of miRNA Signature Differentially Expressed in Doxorubicin-Resistant and Parental Human Hepatocellular Carcinoma Cell Lines

“…Among the analyzed miRNAs, some have common target genes in the MAPK signaling pathway that combined to a systematic network (Figure 3). In HCC, MAPK signaling pathways are up-regulated and generally considered to promote tumor growth [26], [40]–[43]. It has been addressed to participate in the drug-induced tumor cell proliferation and apoptosis in other studies [44]–[46].…”

Quantification of the Heterogeneity of Prognostic Cellular Biomarkers in Ewing Sarcoma Using Automated Image and Random Survival Forest Analysis

“…Moreover, such observations undermine any analysis of separately collected and stored tissues under variable pre-analytic processing conditions, as combining these data sets are likely to compound either biased high content or even standard histological scoring systems using immuno-histochemistry. For future prospective validation, improved tissue ischaemic time and optimal preservation, such as with combined paraffin coating and nitrogen storage, will likely be mandatory [44], [62], [63].…”