Prostate-63 Cancer Diagnostic Test Data Sheet

P63 Antibody
Monoclonal Mouse
Anti-Human p63 Protein
Clone 4A4
Code No. A3375
Lot 00001797, Editin 16.06.03 (These are from Dako)

Intended use
For in vitro diagnostic use.
Monoclonal Mouse Anti-Human p63 Protein, Clone 4A4, is intended for use in immunocytochemistry. The antibody labels the nuclei of basal or progenitor cells in a variety of epithelia (1) and may be a useful tool for the differential diagnosis of benign versus malignant prostatic lesions (2) and for the identification of squamous cell carcinomas (3). Differential identification is aided by the results from a panel of antibodies. Interpretation must be made within the context of the patient.s clinical history and other diagnostic tests by a qualified pathologist.

Synonyms for antigen
KET, p51, p40, and p73L (4).

Introduction
The p63 protein is a member of the p53 family, which also includes p73. At least 6 different transcripts of p63 derives from alternative splicing events and encodes proteins with two different N termini (TA and ?N) and three different C termini (?, ? and ?). The protein isotypes TAp63?, TAp63?, and TAp63? contain the N-terminal transactivation (TA) domain, whereas the other three isotypes ?Np63?, ?Np63?, and ?Np63?, lack this domain, and when present in sufficient concentration act in a dominant-negative manner with respect to wild-type p63 and p53 protein (1, 4). The predominant localization of p63 protein is in the basal layer of stratified squamous and transitional epithelia (1). These basal cells act as the progenitors of the suprabasal cells, which undergo differentiation and cell death in regenerative epithelia (5). p63-deficient mice lack all squamous epithelia and their derivatives, including hair, whiskers, teeth, as well as mammary, lacrimal, and salivary glands. Particularly stricking are severe limb truncations with forelimbs showing a complete absence of the phalanges and carpals, and variable defects of ulnae and radiae and hindlimbs that are lacking altogether. A similar pattern is observed in patients with the EEC syndrome, an autosomal dominant disorder characterized by ectrodactyly, ectodermal dysplasia, and facial clefts, where unrelated patients are found to have dominant heterozygous p63 mutations (6).

Reagent provided
Monoclonal mouse antibody provided in liquid form as cell culture supernatant dialysed against 0.05 mol/L Tris/HCl, pH 7.2, and containing 15 mmol/L NaN3. Clone: 4A4 (1). Isotype: IgG2a, kappa. Mouse IgG concentration: 375 mg/L. Total protein concentration: 15.0 g/L.

Immunogen
Glutathione-S-transferase (GST)-p63 recombinant protein comprising amino acids 1-205 of the N-terminal portion of human ?Np63 protein (1).

Specificity
In Western blotting of lysates of primary human foreskin keratinocytes (HFK), human cervical carcinoma (ME180), and baby hamster kidney cells (BHK), expressing different p63 isotypes (TAp63?, TAp63??, ?Np63?, and ?Np63?), the antibody labels bands corresponding to the different p63 isotypes (1). In Western blotting of lysate of normal prostate basal cells (PrEC), the antibody labels a major band of ?80 kDa and a fainter band of ?60 kDa, corresponding to p63. Western blotting of lysates of normal prostate stromal cells (PrSC), and of theprostate cancer cell lines, LNCaP, PC3, and DU145 are negative (2). In immunocytochemistry the antibody labels 80% of cells in the normal prostate cell line, PrEC, whereas the prostate cancer cell lines, LNCaP, PC3, and DU145, are negative (2). As demonstrated by immunocytochemistry, the antibody cross-reacts with the p63-equivalent protein in mouse (7).

Precautions
1. For in vitro diagnostic use.
2. This product contains sodium azide (NaN3), a chemical highly toxic in pure form. At product concentrations, though not classified as hazardous, sodium azide may react with lead and copper plumbing to form highly explosive build-ups of metal azides. Upon disposal, flush with large volumes of water to prevent metal azide build-up in plumbing.

Storage
Store at 2-8 °C. Do not use after expiration date stamped on vial. If reagents are stored under any conditions other than those specified, the user must verify the conditions. There are no obvious signs to indicate instability of this product. Therefore, positive and negative controls should be run simultaneously with patient specimens. If unexpected staining is observed which cannot be explained by variations in laboratory procedures and a problem with the antibody is suspected, contact our Technical Services. 101081-003 / 25-06-2003 M 7247/CE/16.06.03 p. 2/2

Specimen preparation
Paraffin sections: The antibody can be used for labelling paraffin-embedded tissue sections fixed in formalin or B5-fixative (3). Pre-treatment of tissues with heat-induced epitope retrieval is required. For tissues fixed in formalin, optimal results are obtained with DakoCytomation Target Retrieval Solution, code No. S 1700, DakoCytomation Target Retrieval Solution, High pH, code No. S 3308, 10 mmol/L citrate buffer, pH 6.0, or 10 mmol/L Tris buffer, 1 mmol/L EDTA, pH 9.0. Pre-treatment of tissues with proteinase K was found inefficient. The tissue sections should not dry out during the treatment or during the following immunocytochemical staining procedure.

Staining procedure
Dilution: Monoclonal Mouse Anti-Human p63 Protein, code No. M 7247, may be used at a dilution range of 1:25-1:50 when applied on formalin-fixed, paraffin-embedded sections of human tonsil or prostate and using 20 minutes heat-induced epitope retrieval in 10 mmol/L Tris buffer, 1 mmol/L EDTA, pH 9.0, and 30 minutes incubation at room temperature with the primary antibody. Optimal conditions may vary depending on specimen and preparation method, and should be determined by each individual laboratory. The recommended negative control is DakoCytomation Mouse IgG2a, code No. X 0943, diluted to the same mouse IgG concentration as the primary antibody. Visualization: DAKO LSAB.+/HRP kit, code No. K 0679, and DAKO EnVision.+/HRP kits, code Nos. K 4004 and K 4006, are recommended. For frozen sections and cell preparations, the DakoCytomation APAAP kit, code No. K 0670, is a good alternative if endogenous peroxidase staining is a concern. Follow the procedure enclosed with the selected visualization kit. Automation: The antibody is well-suited for immunocytochemical staining using automated platforms, such as the DakoCytomation Autostainer.

Performance characteristics
Cells labelled by the antibody display a nuclear staining pattern. Normal tissues: The antibody strongly labels proliferating basal cells of epithelial layers in the epidermis, foreskin, uterine cervix, vagina, urothelium, and prostate (1), whereas secretory cells and neuroendocrine cells in the prostate are negative (2). In the lung, the antibody labels basal and suprabasal cells in the bronchial mucosa, and myoepithelial cells of seromucous glands. Type I and II pneumocytes and Clara cells are negative (3). Abnormal tissues: In all of 48 prostate cancer specimens with intraepithelial neoplasia, dysplastic cells were negative with the antibody, but a rim of residual p63-positive basal cells could be identified. 126/130 of invasive prostate cancers were negative for p63 when using the antibody, whereas in 4 cases less than 1% of cells were positive (2). In stage l non-small-cell lung carcinoma, the antibody labelled 109/118 cases of squamous cell carcinoma, 15/95 cases of adenocarcinoma, 4/6 cases of large-cell carcinoma, and 2/2 cases of adenosquamous carcinoma. In stage l to lV neuroendocrine tumours, the antibody labelled 9/20 poorly differentiated tumours, and 1/37 typical and atypical carcinoid tumours (2).

References
1. Yang A, Kaghad M, Wang Y, Gillett E, Fleming MD, Dötsch V, et al. p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. Mol Cell 1998;2:305-16.
2. Signoretti S, Waltregny D, Dilks J, Isaac B, Lin D, Garraway L, et al. p63 is a prostate basal cell marker and is required for prostate development. Am J Pathol 2000;157:1769-75.
3. Pelosi G, Pasini F, Stenholm CO, Pastorino U, Maisonneuve P, Sonzogni A, et al. p63 immunoreactivity in lung cancer: yet another player in the development of squamous cell carcinomas? J Pathol 2002;198:100- 9.
4. Marin MC, Kaelin WG. p63 and p73: old members of a new family [minireview]. Biochim Biophys Acta 2000;1470:M93-M100.
5. Jetten AM, Harvat BL. Epidermal differentiation and squamous metaplasia: from stem cell to cell death. J Dermatol 1997;24:711-25.
6. Celli J, Duijf P, Hamel BCJ, Bamshad M, Kramer B, Smits APT, et al. Heterozygous germline mutations in the p53 homolog p63 are the cause of EEC syndrome. Cell 1999;99:143-53.
7. Yang A, Schweitzer R, Sun D, Kaghad M, Walker N, Bronson RT, et al. p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 1999;398:714-8.