In this study we have described a novel, automated system for the ex vivo pharmacodynamic analysis of human melanoma samples. We have termed the results of such analyses functional signaling profiles. Our preliminary studies with melanoma cell lines confirmed that functional signaling profiles from this ex vivo system accurately predicted BRAF inhibitor sensitivity in virtually all cell lines, based on their genotype, known biology, and IC50 values. These studies were extended to a clinically-relevant model system, using fine needle aspiration biopsy samples of human melanoma xenografts, which resemble human FNAs of metastatic melanoma in their cell composition and procurement method 
. These xenograft studies confirmed that functional signaling profiles can be generated from such samples and that these profiles resemble those found in the preliminary cell line work. Finally, we generated functional signaling profiles from a set of samples derived from patients, including FNAs of subcutaneous melanoma metastases, as well as FNAs of surgically-excised metastatic melanoma lesions. These profiles were consistent with the known melanoma genotype, therapeutic regimen, and clinical status on that regimen.
Due to the prevalence of BRAF mutations in melanoma, the MAPK pathway has emerged as a prime target of molecularly targeted agents for this disease, including an approved inhibitor of BRAF (vemurafenib) and additional emerging inhibitors of BRAF and MEK 
. Despite success in extending overall survival in patients with BRAF V600E mutations, a spectrum of responses, ranging from no response to complete response, occurs even in this highly selected population. In addition, virtually all patients develop resistance to BRAF therapy, often through re-activation of the MAPK pathway. This suggests that many additional factors are important in the modulation and pharmacodynamics of MAPK pathway activity in these patients.
In our cell line work, the functional signaling profiles of the MAPK pathway identified ex vivo pathway blockade in every sensitive line, identified the known paradoxical pathway activation in an NRAS mutant line, and identified MAPK pathway reactivation in the known BRAF inhibitor resistant lines, with one interesting exception. The A2058 cell line contains a V600E mutation, displays ex vivo MAPK inhibition by PLX-4720, but is intrinsically resistant to BRAF inhibitors. While the exact mechanism of this resistance, despite pathway inhibition, is still under investigation, one study has suggested that it could be due to bypass pathway activation via PTEN loss and RB loss 
. These findings are clinically relevant, since they suggest that in such patients, combination therapy should include continued use of the BRAF inhibitor, with addition of a PI3K pathway inhibitor, for example. Conversely, patients with ex vivo MAPK reactivation in the presence of a BRAF inhibitor will likely require a different MAPK inhibitor, such as a MEK inhibitor, and possibly a PI3K pathway inhibitor as has been previously proposed 
The functional signaling profiles generated in this study stratify human samples into three groups that display the following key features in the ex vivo presence of BRAF inhibition: MAPK suppression; MAPK reactivation; and MAPK stimulation. Because of the enrollment criteria and the practice patterns at the referral center where the study was conducted, most of the patients in the study were either BRAF wild type or BRAF V600E patients who were clinically progressing on a MAPK inhibitor regimen (BRAF or BRAF+MEK inhibitors). Most of these patients displayed a MAPK reactivation profile, confirming the previous evidence that MAPK reactivation plays a major role in BRAF inhibitor resistance. Interestingly, several patients displayed a MAPK activation profile, suggesting the acquisition of a previously described MAPK activation mechanism 
, or a novel pathway activation mechanism. Finally, in this cohort there were two patients who displayed a MAPK suppression profile. Interestingly, these were the only patients who possessed a BRAF mutation, were receiving a BRAF inhibitor, and showed clinical signs of response.
In summary, in this proof-of-concept study we have successfully generated pharmacodynamic data from human melanoma samples using a novel, automated ex vivo platform. This study focused on the MAPK pathway in metastatic melanoma, but this system is applicable to any solid tumor and multiple other molecularly targeted agents and signaling pathways. This system has potential applications in the efficient performance of preclinical drug studies in murine xenograft or human tumorgraft models. Also, it represents a novel tool for early clinical drug development in the assessment of pharmacodynamic responses to investigational drugs. Finally, it also has the potential to serve as a powerful predictive test to stratify patients in late stage clinical trials, and ultimately to guide targeted therapy.