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Assessing proliferation, cell-cycle arrest and apoptotic end points in human buccal punch biopsies for use as
D R Camidge1, M N Pemberton, J W Growcott
1Edinburgh Cancer Centre, Western General Hospital, Edinburgh EH4 2XU, UK.
Abstract:
Easily accessible normal tissues expressing the same molecular site(s) of drug action as malignant tissue offer an enhanced potential for early proof of anticancer drug mechanism and estimation of the biologically effective dose. Studies were undertaken in healthy male volunteers to assess the tolerability of single and multiple (four in 24 h) 3 mm punch biopsies of the buccal mucosa, and to determine the feasibility of detecting and quantifying a range of proliferation, cell-cycle arrest and apoptosis markers by immunohistochemistry (IHC) for use as potential pharmacodynamic (PD) end points. The biopsy procedure was well tolerated with 100% of volunteers stating that they would undergo single (n = 10) and multiple (n = 12) biopsies again. Total retinoblastoma protein (pRb), phosphorylated pRb (phospho-pRb), total p27, phosphorylated p27 (phospho-p27), phosphorylated-histone H3 (phospho-HH3), p21, p53, Cyclin A, Cyclin E, Ki67 all produced good signal detection, but M30, cleaved caspase 3 and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling did not. Total pRb, phospho-pRb, total p27 and phospho-p27 were quantified further in a multiple biopsy study to allow components of variability to be addressed to inform future sizing decisions on intervention studies. Neither site of biopsy within the oral cavity, nor the nominal time of biopsy had any significant impact on any of the four markers expression levels. Inter- and intrasubject coefficients of variation (CVs) that could be used to size future intervention studies for pRb, phospho-pRb, total p27 and phospho-p27 were 14, 19, 18 and 16%; and 18, 29, 25 and 19%, respectively. In conclusion, quantitation of such markers in 3 mm buccal punch biopsies would be suitable to explore as PD end points within intervention studies of drugs acting on these pathways.
Insights
Buccal mucosa biopsies are well-tolerated and suitable for measuring anticancer drug effects. This method allows for early proof of drug mechanism and dose estimation using pharmacodynamic (PD) markers.
Area of Science:
- Pharmacology
- Oncology
- Translational Medicine
Background:
- Accessible normal tissues mirroring drug targets in tumors can aid early anticancer drug evaluation.
- Pharmacodynamic (PD) endpoints are crucial for assessing drug efficacy and determining optimal dosing.
- Buccal mucosa offers a readily accessible site for biopsy to evaluate drug effects.
Purpose of the Study:
- To assess the tolerability of single and multiple buccal mucosa biopsies.
- To determine the feasibility of quantifying proliferation, cell-cycle arrest, and apoptosis markers via immunohistochemistry (IHC).
- To establish these markers as potential pharmacodynamic (PD) endpoints for anticancer drug studies.
Main Methods:
- Healthy male volunteers underwent single or multiple (four within 24 hours) 3 mm punch biopsies of the buccal mucosa.
- Immunohistochemistry (IHC) was used to detect and quantify various protein markers including retinoblastoma protein (pRb), p27, p21, p53, Cyclins, and Ki67.
- Statistical analysis was performed to assess marker variability and inform future study designs.
Main Results:
- The biopsy procedure was well-tolerated, with 100% of volunteers willing to undergo repeat procedures.
- Good signal detection was achieved for markers like total pRb, phospho-pRb, total p27, phospho-p27, phospho-histone H3, p21, p53, Cyclin A, Cyclin E, and Ki67.
- Neither biopsy site nor time significantly impacted marker expression levels, and inter/intrasubject coefficients of variation were established.
Conclusions:
- Quantification of selected markers in 3 mm buccal punch biopsies is a feasible and suitable approach for pharmacodynamic (PD) endpoint assessment.
- This methodology can support early-phase intervention studies evaluating anticancer drugs targeting specific cellular pathways.
- Buccal mucosa biopsies provide a valuable tool for translational research in oncology, bridging preclinical findings to clinical application.
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