Expression profiling of blood samples from an SU5416 Phase III metastatic colorectal cancer clinical trial: a novel strategy for biomarker identification - PubMed (original) (raw)

Expression profiling of blood samples from an SU5416 Phase III metastatic colorectal cancer clinical trial: a novel strategy for biomarker identification

Samuel E DePrimo et al. BMC Cancer. 2003.

Abstract

Background: Microarray-based gene expression profiling is a powerful approach for the identification of molecular biomarkers of disease, particularly in human cancers. Utility of this approach to measure responses to therapy is less well established, in part due to challenges in obtaining serial biopsies. Identification of suitable surrogate tissues will help minimize limitations imposed by those challenges. This study describes an approach used to identify gene expression changes that might serve as surrogate biomarkers of drug activity.

Methods: Expression profiling using microarrays was applied to peripheral blood mononuclear cell (PBMC) samples obtained from patients with advanced colorectal cancer participating in a Phase III clinical trial. The PBMC samples were harvested pre-treatment and at the end of the first 6-week cycle from patients receiving standard of care chemotherapy or standard of care plus SU5416, a vascular endothelial growth factor (VEGF) receptor tyrosine kinase (RTK) inhibitor. Results from matched pairs of PBMC samples from 23 patients were queried for expression changes that consistently correlated with SU5416 administration.

Results: Thirteen transcripts met this selection criterion; six were further tested by quantitative RT-PCR analysis of 62 additional samples from this trial and a second SU5416 Phase III trial of similar design. This method confirmed four of these transcripts (CD24, lactoferrin, lipocalin 2, and MMP-9) as potential biomarkers of drug treatment. Discriminant analysis showed that expression profiles of these 4 transcripts could be used to classify patients by treatment arm in a predictive fashion.

Conclusions: These results establish a foundation for the further exploration of peripheral blood cells as a surrogate system for biomarker analyses in clinical oncology studies.

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Figures

Figure 1

Differential expression of candidate biomarker transcripts in patient PBMC at day 56 relative to day 1 of therapy. The diagram is a depiction of the Affymetrix Difference Calls assigned to each day 56:day 1 expression comparison among the patient sample pairs analyzed via GeneChip hybridization analysis. Letters within blocks represent the Difference Call assigned to each relative expression comparison. The abbreviations are: I = Increase, MI = Marginally Increased, NC = Not changed; MD = Marginally Decreased; D = Decreased. Cases in which an Increased or Marginally Increased call is assigned to a day 56:day 1 comparison are shaded in gray. Each column represents a different patient. Column headings in each grid represent patient response assessed at end of first treatment cycle: PR = partial response, CR = complete response, PD = progressive disease.

Figure 2

Differential expression of 6 transcripts as measured by microarray and quantitative RT-PCR. The percentage of cases in 5-FU/LV (control) and 5-FU/LV + SU5416 trial arms with increased expression (at predose day 56 relative to predose day 1) of each transcript is displayed. Panel A displays results from Affymetrix analysis and Panel B displays results from SYBR Green RT-PCR verification. For the Affymetrix data, an increase is determined by Difference Call status; for the SYBR Green data, an increase is defined here as relative expression value of 2-fold or greater. A total of 31 sample pairs were used in RT-PCR analysis; 18 were from SU5416 arm (5 PR, 1 CR, 11 PD, and 1 SD response at end of cycle 1), and 13 were from the control arm (9 PR, 3 PD, and 1 SD). The relative expression values as determined in the RT-PCR analysis for each patient are displayed (see Additional File 1).

Figure 3

Differential expression of four transcripts in a second Phase III trial as measured by quantitative RT-PCR. Percentage of cases in CPT-11/5-FU/LV (control) and CPT-11/5-FU/LV + SU5416 trial arms with increased expression (at predose day 42 relative to predose day 1) of 4 candidate biomarker transcripts in a second SU5416 Phase III clinical trial is displayed. The convention is the same as in panel B in Figure 2. A total of 36 sample pairs was included in this analysis; 18 from the SU5416 arm and 18 from the control arm (8 PR and 10 SD responses at end cycle 1 in each group). The relative expression values for each patient in this group are displayed (see Additional File 1).

Figure 4

Hierarchical clustering of relative expression ratios for four biomarker transcripts. This mosaic depicts association between patient samples and relative expression of the 4 potential biomarker transcripts. Natural log-transformed SYBR Green RT-PCR ratio data (relative expression of day 56: day 1) were used in analysis. In the color scheme, higher ratios are indicated in red, lower ones in green (scale ranges from -4 to +4). Results from individual patients are oriented as rows and transcripts are oriented as columns. Red bars on the right side of the map indicate cases from the SU5416 arm. The hierarchical clustering method is average linkage and the distance metric is Euclidean. A table containing the relative expression values that were used in the clustering analysis can be viewed (see Additional File 1).

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Expression profiling of blood samples from an SU5416 Phase III metastatic colorectal cancer clinical trial: a novel strategy for biomarker identification - PubMed (original) (raw)