We used a quantitative, pathway-specific approach to profile genes involved in important cellular pathways that are crucial in UC development. The choice for the final predictive panel was determined on the basis of the hypothesis that the most biologically relevant genes should be able to predict both recurrence and survival. In this study, the four-gene panel (JUN, MAP2K6, STAT3, and ICAM1) was a highly significant predictor of these outcomes, independent of standard prognostic criteria (ie, stage and lymph-node density). In addition, this panel identified high-risk patients; nearly all patients with favorable expressions of ≤ two of four genes experienced recurrence and died. The prognostic potential of this panel was additionally supported by an external data set that profiled genes using a completely different methodology, thereby demonstrating the robustness of this four-gene panel in predicting clinical outcome.
Gene expression profiles are usually generated using microarrays. These studies may involve inconsistencies in results and lack of reproducibility across platforms.24,25
Furthermore, the output often contains more than 20 to 100 genes, which can dilute biologic and clinical relevance while increasing noise and opportunities for random chance. Although our hypothesis-driven exploration limited potential for discovery, the rational choice allowed identification of key genes and associated pathways of prognostic value.
The univariate analysis identified six genes associated with recurrence, and 10 associated with overall survival. The protein products of IGF1
, and FOSL1
have been associated with poor prognosis in several cancers, including bladder cancer.8,22,26–31
High expressions of these genes were associated with worse prognosis, consistent with their biologic roles as oncogenes. Our study also demonstrated constitutive activation of the mitogen-activated protein kinase pathway in UC5
; low MAPK12
expression was associated with higher probability of overall survival. STAT3
overexpression corresponded with poorer prognosis, consistent with observations that signal transducer and activator of transcription 3 increases the invasiveness of UC cell lines.23
expression corresponded with decreased probability of recurrence, consistent with findings in acute myelogenous leukemia and lymphoproliferative syndromes.32
Although tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), the protein product of TNFSF10
, induces apoptosis, patients with increased TNFSF10
expression had poorer overall survival. This patient subset was probably insensitive to TRAIL-mediated apoptosis, consistent with findings demonstrating that different UC cell lines have varying degrees of susceptibility to TRAIL.33
The RP analysis also selected BMP6
as joint determinants of recurrence. Bone morphogenetic protein 6 promotes tumor angiogenesis, and elevated ICAM1
expression is associated with increased metastatic potential in UC.8,34
Patients with low BMP6
and high SOD1
expressions had the lowest recurrence rates, whereas those with normal or high BMP6
and high ICAM1
expressions had the highest. GSTM3
was also associated with overall survival in RP analysis. GSMT3
polymorphisms are linked to carcinogenesis, and GSTM3
mutations are associated with increasing risk for UC.35
In patients with low MAPK12
expression, those with low GSTM3
expression as well had the highest survival probability, whereas those with normal or high GSTM3
expression had lower survival probability.
When the interrelationships between proteins transcribed from these genes were examined, nine direct and more than 150 indirect interactions were discovered (Data Supplement, online only), which highlights the importance of their crosstalk. This led us to focus on genes that could predict both recurrence and survival. Obtaining a concise prognostic marker list is crucial in such studies, because clinical applications of such panels are more cost effective and practical. Although such prognostic panels have been previously identified, they have usually featured markers from a single cellular pathway.36,37
The four-gene panel obtained after profiling genes across multiple pathways robustly predicted clinical outcome. Additionally, the ability of this panel to accurately predict recurrence independent of stage is likely to be a useful supplement to routine staging. Furthermore, MAP2K6
were also previously identified by our group to predict nodal metastasis in UC.8
Validation of the four- and 11-gene panels on the external data set was consistent with AIC observations that although the 11-gene panel could expectedly better predict survival, its performance was not substantially superior to that of the four-gene panel. Moreover, the validation highlighted the robustness of the four-gene panel, independent of the platform used for profiling the genes.
In conclusion, using a multiplexed, biologically driven approach, we have identified a panel comprising JUN, MAP2K6, STAT3, and ICAM1 that can predict clinical outcome in UC independent of conventional prognostic criteria and identify patients with operable UC who will experience recurrence despite undergoing definitive surgery alone. These patients would clearly benefit from additional therapy. Increasing numbers of alterations in these genes predict poorer prognosis. Additional study of this panel is warranted to better characterize its ability to identify patients at higher risk. Although limited transcripts were analyzed, this does suggest that these genes and their associated pathways may serve as promising outcome predictors and potential therapeutic targets in UC.