Tineke E Buffart1,2, Rosanne A H M van den Oord1,3, Adriënne van den Berg4, Riet Hilhorst4, Niek Bastiaensen4, Hans F M Pruijt3, Adriaan van den Brule5, Peet Nooijen6, Mariette Labots1, Richard R de Goeij-de Haas1, Henk Dekker1, Sander R Piersma1, Thang V Pham1, Theo van der Leij4, Rik de Wijn4, Rob Ruijtenbeek4,7, Connie R Jiménez1, Henk M W Verheul8,9. 1. Department of Medical Oncology, Amsterdam UMC, VU University Medical Center, Amsterdam, The Netherlands. 2. Department of Gastrointestinal Oncology, Netherlands Cancer Institute, Amsterdam, The Netherlands. 3. Department of Medical Oncology, Jeroen Bosch Hospital, 'S-Hertogenbosch, The Netherlands. 4. PamGene International BV, 'S-Hertogenbosch, The Netherlands. 5. Laboratory of Molecular Diagnostics, Jeroen Bosch Hospital, 'S-Hertogenbosch, The Netherlands. 6. Department of Pathology, Jeroen Bosch Hospital, 'S-Hertogenbosch, The Netherlands. 7. GenMab BV, Utrecht, The Netherlands. 8. Department of Medical Oncology, Amsterdam UMC, VU University Medical Center, Amsterdam, The Netherlands. Henk.verheul@radboudumc.nl. 9. Department of Medical Oncology, Radboud University Medical Center, Geert Grooteplein 10, 6525GA, Nijmegen, The Netherlands. Henk.verheul@radboudumc.nl.
Abstract
BACKGROUND: Based on their potential to analyze aberrant cellular signaling in relation to biological function, kinase activity profiling in tumor biopsies by peptide microarrays and mass spectrometry-based phosphoproteomics may guide selection of protein kinase inhibitors in patients with cancer. Variable tissue handling procedures in clinical practice may influence protein phosphorylation status and kinase activity and therewith may hamper biomarker discovery. Here, the effect of cold ischemia time (CIT) on the stability of kinase activity and protein phosphorylation status in fresh-frozen clinical tissue samples was studied using peptide microarrays and mass spectrometry-based phosphoproteomics. METHODS: Biopsies of colorectal cancer resection specimens from five patients were collected and snap frozen immediately after surgery and at 6 additional time points between 0 and 180 min of CIT. Kinase activity profiling was performed for all samples using a peptide microarray. MS-based global phosphoproteomics was performed in tumors from 3 patients at 4 time points. Statistical and cluster analyses were performed to analyze changes in kinase activity and phosphoproteome resulting from CIT. RESULTS: Unsupervised cluster analysis of kinase activity and phosphoproteome data revealed that samples from the same patients cluster together. Continuous ANOVA analysis of all 7 time points for 5 patient samples resulted in 4 peptides out of 210 (2%) with significantly (p < 0.01 and fold change > 2) altered signal intensity in time. In 4 out of 5 patients tumor kinase activity was stable with CIT. MS-based phosphoproteomics resulted in the detection of 10,488 different phosphopeptides with on average 6044 phosphopeptides per tumor sample. 2715 phosphopeptides were detected in all samples at time point 0, of which 90 (3.3%) phosphopeptides showed significant changes in intensity with CIT (p < 0.01). Only two phosphopeptides were significantly changed in all time points, including one peptide (PKP3) with a fold change > 2. CONCLUSIONS: The vast majority of the phosphoproteome as well as the activity of protein kinases in colorectal cancer resection tissue is stable up to 180 min of CIT and reflects tumor characteristics. However, specific changes in kinase activity with increasing CIT were observed. Therefore, stringent tissue collection procedures are advised to minimize changes in kinase activity during CIT.
BACKGROUND: Based on their potential to analyze aberrant cellular signaling in relation to biological function, kinase activity profiling in tumor biopsies by peptide microarrays and mass spectrometry-based phosphoproteomics may guide selection of protein kinase inhibitors in patients with cancer. Variable tissue handling procedures in clinical practice may influence protein phosphorylation status and kinase activity and therewith may hamper biomarker discovery. Here, the effect of cold ischemia time (CIT) on the stability of kinase activity and protein phosphorylation status in fresh-frozen clinical tissue samples was studied using peptide microarrays and mass spectrometry-based phosphoproteomics. METHODS: Biopsies of colorectal cancer resection specimens from five patients were collected and snap frozen immediately after surgery and at 6 additional time points between 0 and 180 min of CIT. Kinase activity profiling was performed for all samples using a peptide microarray. MS-based global phosphoproteomics was performed in tumors from 3 patients at 4 time points. Statistical and cluster analyses were performed to analyze changes in kinase activity and phosphoproteome resulting from CIT. RESULTS: Unsupervised cluster analysis of kinase activity and phosphoproteome data revealed that samples from the same patients cluster together. Continuous ANOVA analysis of all 7 time points for 5 patient samples resulted in 4 peptides out of 210 (2%) with significantly (p < 0.01 and fold change > 2) altered signal intensity in time. In 4 out of 5 patientstumor kinase activity was stable with CIT. MS-based phosphoproteomics resulted in the detection of 10,488 different phosphopeptides with on average 6044 phosphopeptides per tumor sample. 2715 phosphopeptides were detected in all samples at time point 0, of which 90 (3.3%) phosphopeptides showed significant changes in intensity with CIT (p < 0.01). Only two phosphopeptides were significantly changed in all time points, including one peptide (PKP3) with a fold change > 2. CONCLUSIONS: The vast majority of the phosphoproteome as well as the activity of protein kinases in colorectal cancer resection tissue is stable up to 180 min of CIT and reflects tumor characteristics. However, specific changes in kinase activity with increasing CIT were observed. Therefore, stringent tissue collection procedures are advised to minimize changes in kinase activity during CIT.
Entities:
Keywords:
Cancer; Cold ischemia; Mass spectrometry; Peptide microarray; Phosphoproteomics; Protein kinase
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