Assessing proliferation, cell-cycle arrest and apoptotic end points in human buccal punch biopsies for use as pharmacodynamic biomarkers in drug development

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 ¼ 10) and multiple (n ¼ 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.

Skin biopsies for the measurement of clinical pharmacodynamic biomarkers

“…More recently, Camidge and colleagues [17 ] have evaluated the tolerability of multiple 3 mm buccal punch biopsies (skin samples taken from the inside of the cheek) and the feasibility of using these to measure PD effects of some anticancer compounds. Tolerability is an important factor that affects the difficulty of recruiting subjects to undergo biopsy procedures in a clinical trial.…”

Pathological Aspects of Oral Precancer

“…Punch biopsies have been reported to cause less morbidity than scalpel biopsies and can be closed with a single suture or left to heal by primary intention. In a volunteer study to assess tolerance of 3 mm buccal punch biopsies, all subjects stated that they would be willing to undergo a subsequent biopsy procedure [2].…”

Aurora kinase inhibitors: identification and preclinical validation of their biomarkers

“…Several studies were performed in order to evaluate the optimal body site for skin biopsies and to assess PD and PK end points [89] , or to assess the feasibility of detecting signals for a range of different immunohistochemistry markers within the buccal mucosa [90] and plucked human hair [91] .…”