Abstract
BackgroundThe role of basic fibroblast growth factor (bFGF) in chemoresistance is controversial; some studies showed a relationship between higher bFGF level and chemoresistance while other studies showed the opposite finding. The goal of the present study was to quantify bFGF levels in archived tumor tissues, and to determine its relationship with chemosensitivity.MethodsWe established an image analysis-based method to quantify and convert the immunostaining intensity of intra-tumor bFGF to concentrations; this was accomplished by generating standard curves using human xenograft tumors as the renewable tissue source for simultaneous image analysis and ELISA. The relationships between bFGF concentrations and tumor chemosensitivity of patient tumors (n = 87) to paclitaxel were evaluated using linear regression analysis.ResultsThe image analysis results were compared to our previous results obtained using a conventional, semi-quantitative visual scoring method. While both analyses indicated an inverse relationship between bFGF level and tumor sensitivity to paclitaxel, the image analysis method, by providing bFGF levels in individual tumors and therefore more data points (87 numerical values as opposed to four groups of staining intensities), further enabled the quantitative analysis of the relationship in subgroups of tumors with different pathobiological properties. The results show significant correlation between bFGF level and tumor sensitivity to the antiproliferation effect, but not the apoptotic effect, of paclitaxel. We further found stronger correlations of bFGF level and paclitaxel sensitivity in four tumor subgroups (high stage, positive p53 staining, negative aFGF staining, containing higher-than-median bFGF level), compared to all other groups. These findings suggest that the relationship between intra-tumoral bFGF level and paclitaxel sensitivity was context-dependent, which may explain the previous contradictory findings on the merit of using plasma or urine bFGF level as a prognostic indicator.ConclusionThe present study established a quantitative image analysis method that enabled the measurement of intratumoral bFGF level in archived tissues. The ability to quantify a potential biomarker provided the opportunity to study the relationship between the biomarker and chemosensitivity in tumor subgroups and thereby enabled hypothesis generation for additional translational research.
Highlights
The role of basic fibroblast growth factor in chemoresistance is controversial; some studies showed a relationship between higher bFGF level and chemoresistance while other studies showed the opposite finding
Development of image analysis method The image analysis-based measurement was accomplished in 4 steps: (a) Identified the parameters for image analysis and developed a macro that automated the procedures. (b) Identified the human xenografts tumors that yielded a 100-fold range of bFGF levels as measured by ELISA. (c) Comparison of bFGF levels in xenograft tumors measured by image analysis and ELISA, to obtain standard curves. (d) Using the standard curve to convert the image analysis readings to protein levels
The threshold value for the intensity parameter was set by incrementally changing the range such that the final value found in 20 randomly selected slides (4 from each tumor type) accurately identified the visually bFGFstained areas while excluding the visually non-stained areas
Summary
The role of basic fibroblast growth factor (bFGF) in chemoresistance is controversial; some studies showed a relationship between higher bFGF level and chemoresistance while other studies showed the opposite finding. The clinical development was done without the knowledge whether the intended targets were present or important for the patients enrolled in clinical trials. While gefitinib, an inhibitor of epidermal growth factor receptor (EGFR) typrosine kianse, improved the objective response rate in non-small cell lung cancer patients, it did not produce survival benefits. A subsequent study identified several qualitative (EGFR mutation status) and quantitative markers (number of EGFR gene copies, EGFR protein level) as potentially important prognostic indicators for response rate and survival [5]. Taken together, these examples illustrate that successful translation of molecular discoveries to useful clinical interventions is possible. The gefitinib example further highlights the potential importance of quantifying the levels of molecular markers
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