2. Clinical Applications of RPPA
RPPA is increasingly being used to determine deregulated signaling networks in cancer tissues. For example, RPPA was one of the methods to determine if multi-omic molecular profiling based treatment improves the clinical course of patients with metastatic breast cancer measured by growth of modulation index (GMI). GMI was calculated as the ratio of length of time between treatment and further growth of the primary tumor or metastases. Multi-omic molecular profiling-based therapy differed in all 25 patients from the treatment selected by the treatment selection committee. It could be shown that the molecular profiling-rationalized treatment recommendation can improve the progression free time period, indicating RPPA as a suitable tool to select an appropriate anti-cancer therapy [22]. Besides profiling signaling pathways or entire networks in human cancer tissues, one of the three most common methods to validate mass spectrometry discovered biomarkers is RPPA [23]. If a validated antibody is available for a potential new biomarker, RPPA can be used to validate independent patient-derived sample sets, separate from those initially used for discovery [24].
RPPA analyses have not only focused on protein detection for biomarker discovery and quantification but also to analyze protein expression profile changes during the pre-analytical phase. In several publications, it has been shown that tissue is still alive after resection and gene expression as well as protein levels (especially amounts of phosphoproteins) can change during cold ischemia [25,26,27], i.e., the time period from resection until sample stabilization. One of the more recent publications investigated multiple tissue samples from the same specimen in different patients using targeted and non-targeted technologies [28]. Besides mass spectrometry, RPPA was used to determine potential fluctuations of protein levels. The data allowed the classification of protein and phosphoprotein levels during the pre-analytical phase in three groups: (1) predictable stable; (2) predictable unstable; (3) unpredictable. As most phosphoproteins belonged to the latter group, the authors recommend tissue fixation or stabilization after specimen collection without delay. Thus, tissue procurement guidelines should be aligned [29]. 
RPPA has also been used to analyze heterogeneity of protein levels within a tumor and in primary tumor and lymph node metastases of the same patient [30,31,32,33]. All of these studies revealed a significant heterogeneity of a subset of proteins within a tumor and between primary tumor and metastases, suggesting that for molecular diagnosis the use of multiple tumor samples from distinct locations rather than the analysis of one single sample should be envisaged.