We have demonstrated the feasibility of detecting and quantifying six cell-cycle-related nuclear markers (Ki67, pRb, p27, phosphop27 (phosphorylated p27), phospho-pRb (phosphorylated pRb), phospho-HH3 (phosphorylated histone H3)) in plucked human scalp and eyebrow hair. Estimates of the proportion of plucked hairs that are lost or damaged during processing plus the intra-and intersubject variability of each nuclear marker with these techniques are provided to inform sizing decisions for intervention studies with drugs potentially impacting on these markers in the future. (Moll, 1996;Gho et al, 2004); they are therefore potentially attractive as an easily accessible tissue in which to assess the pharmacodynamic (PD) effects of drugs that interfere with cell proliferation. Skin biopsies have already been used to similar ends during the development of a number of agents targeted against the epidermal growth factor receptor (Albanell et al, 2002;Malik et al, 2003;Salazar et al, 2004;Vanhoefer et al, 2004). Plucked hairs would offer certain advantages over skin biopsies for these purposes including improved tolerability, a greater potential for multiple time-point analyses and the possibility of a higher signal within hair follicles for certain key biomarkers compared to the cells of the epidermis (Albanell et al, 2002). We have developed a technique for quantifying cell-cycle-related proteins within the sheath cells of plucked human hair. MATERIALS AND METHODS Trial designIn all, 12 healthy Caucasian males, within the age range 18 -45 years, and with scalp hair greater than or equal to 5 mm in length, were recruited. Following thorough combing to remove any loose hairs, five suitable hairs with visible bulbs were plucked from each of the left eyebrow, right eyebrow and four different scalp sites using a pair of blunt-nosed forceps. Each set of five hairs was trimmed with scissors to a workable length (approximately 1 cm), retaining the bulb end of each hair, and then immersed in prelabelled 1.5 ml microcentrifuge tubes containing 1 ml 100% acetone at 41C. Plucked hairs without visible bulbs were discarded and not processed. Immunohistochemistry (IHC) and signal quantificationHair processing Following acetone fixation for 10 min, each hair was air-dried for 10 min and then placed, in batches of five from each sampling site, in 1 ml of 0.5 M, pH 7.6, Tris-buffered saline.Hair IHC In our hands, it was not possible to paraffin embed and then section the hairs for later staining without producing significant loss or damage of the fragile material; we therefore developed techniques based on staining intact hairs followed by rigid epoxy resin embedding and then sectioning.Briefly, a peroxidase block (5 min at room temperature), followed by a nonspecific protein block (30 min at room temperature), was employed on the intact hairs. A primary antibody, or buffer for negative controls, was then applied (antiKi67 at 1 : 100 for 1 h at room temperature, DAKO
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.
There are potential advantages to detecting pharmacodynamic (PD) and pharmacokinetic (PK) endpoints in a tissue-based compartment such as the skin during the development of molecularly targeted drugs. We explored regional differences between inner arm, inner thigh, lower back and buttocks in 12 healthy male Caucasian volunteers in the tolerability of skin biopsy procedures; the Ki67 proliferation index; the frequency of detecting hair follicles and sweat glands; and the percentage of melanocytes. We also explored the amounts of tissue and protein obtained, and two separate methods of splitting biopsies for processing in mutually exclusive media. Biopsies from all body sites were well tolerated. The subjective ranking order was inner arm > buttocks = back > thigh. There were no statistically significant differences in the Ki67 labelling index (P > 0.05). The frequency of detecting sweat glands was the same in all body sites, but the frequency of detecting hair follicles was higher in back and buttock, compared to arm and thigh. The percentage of melanocytes was significantly lower in the buttocks compared to the back and thigh (P < 0.05), but not compared to the arm (P = 0.07). A 4-mm punch biopsy yielded a mean of 16.8 mg of tissue (range: 9-28 mg) and 160 microg of protein (range: 80-270 microg). In vivo sample splitting, by following a 2-mm punch with a 4-mm overpunch, had a shorter time from devascularisation to immersion into processing medium than ex vivo dissection of a 4-mm sample, which may be of importance to the assessment of labile endpoints. We conclude that multiple punch biopsies of the skin are feasible, with the buttocks representing the studied body site with the optimal balance between tolerability, hair follicle density and melanocyte density for obtaining tissue in which to assess PD and PK endpoints during drug development studies.
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