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Cutaneous immunohistochemical staining pattern of p53β isoforms
  1. Christine J Ko1,
  2. Peggy Myung1,
  3. David J Leffell1,
  4. Jean-Christophe Bourdon2
  1. 1 Department of Dermatology, Yale University School of Medicine, New Haven, Connecticut, USA
  2. 2 Department of Dermatology, School of Medicine, University of Dundee, Dundee, UK
  1. Correspondence to Christine J Ko, Yale University School of Medicine, New Haven 06511, Connecticut, USA; christine.ko{at}yale.edu

Abstract

p53 is considered the guardian of the genome and as such has numerous functions. The TP53 gene is the most commonly mutated gene in cancer, and yet the exact biological significance of such mutations remains unclear. There are at least 12 different isoforms of p53, and the complexity of the p53 pathway may be in part related to these isoforms. Prior research has often not teased out what isoforms of p53 are being studied, and there is evidence in the literature that p53 isoforms are expressed differently. In this paper, we document the staining pattern of p53β isoforms in the skin and correlate it with mutational status in a subgroup of squamous proliferations of the skin. p53β isoforms are present in the cytoplasm of the differentiated layer of the epidermis and hair follicles (granular layer, infundibular and isthmus–catagen). p53β isoforms are diffusely expressed within the cytoplasm of well-differentiated squamous tumours with tetramerisation (C-terminal) domain mutations in TP53. Our results lend support to p53β isoforms being a marker of differentiation in keratinocytes.

  • P53
  • staining
  • skin

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Introduction

p53β, a C-terminal truncated form of p53 (figure 1) that has been linked to differentiation and senescence in fibroblasts and T lymphocytes,1 2 has not been well studied in the skin. Expression of p53β isoforms correlates with a better prognosis in breast cancer and colon cancer.3 4 Expression of p53β isoforms in the skin has not been well studied, and we hypothesised that these isoforms might be expressed differently in squamous proliferations with known mutations in the TP53 gene.

Figure 1

p53α and isoforms of p53β. Full-length p53α has a carboxy-terminal tetramerisation domain that is absent in p53β isoforms. Three of the four p53β isoforms have different amino-terminal ends compared with full-length p53α. The epitope (amino acids 20–25: SDLWKL) for p53 (DO-1) antibody, a commonly used and commercially available p53 antibody, is present in the amino-terminal end of p53α, p53β and p53γ (the latter is not shown). The epitope (amino acids 332–341: DQTSFQKENC) for KJC8 antibody against p53β isoforms is present in the carboxy-terminal end of all β isoforms.

Materials and methods

Thirty atypical squamous proliferations (ASPs) on the legs of women, as previously described,5 were stained with KJC8 antibody that specifically detects the expression of p53β isoforms, with examination of tumour as well as flanking epidermis. As previously described, 16 of the leg lesions were keratoacanthoma-like (KA-like) and lacked significant atypia at the tumour–stroma interface (but were treated precluding assessment of spontaneous regression), and 14 had significant atypia of the keratinocytes adjacent to stroma and were classified by that criterion as squamous cell carcinoma (SCC).5 Ten of these lesions had detectable mutations in the TP53 gene.5 One of the leg tumours did not have enough of the tumour remaining on the slide for valid assessment of staining. Due to the staining pattern that was detected in the epidermis, five isthmus–catagen cysts and one sebaceous adenoma were also stained with KJC8 antibody.

The peptide-affinity purified p53β isoforms (KJC8) antibody was raised in rabbit. The specificity of the KJC8 antibody was rigorously validated on endogenously expressed p53β protein isoforms extracted from different wild-type and mutant TP53 cell lines transfected with siRNA targeting specifically the p53β mRNA variants.6 7 KJC8 detects the epitope (DQTSFQKENC) present in p53β, Δ40p53β, Δ133p53β and Δ160p53β. A 20 µg/mL dilution of KJC8 was used on formalin-fixed, paraffin-embedded samples. Samples were processed in xylene, then absolute ethanol. Endogenous peroxidase was quenched with 3% H202/methanol for 10 min, with slides washed in phosphate buffered saline (PBS). Slides were treated in microwave using citrate at pH 6.0 for 25 min at 95°C. Slides were then cooled, transferred to PBS, blocked with 10% bovine serum albumin/PBS for 1 hour at 37°C. Excess was blotted off with incubation of primary antibody using Van Gogh diluent (BioCare) overnight at 4°C. Slides were washed with PBS/Tween 0.05% for 4 min. A secondary antibody (Envision Dual link; Dako, Carpinteria, California, USA) was applied for 30 min. Subsequent washing was with PBS-T and PBS before application of diaminobenzidine until colour developed with washing in H20 before counterstaining with Mayer’s haematoxylin. Staining of <10% of the tumour was considered negative. Staining of >50% of the tumour was considered positive. All tumours had a positive internal control–staining of the granular layer of the epidermis (figure 2A).

Figure 2

(A, B) Non-tumoural epidermis and hair follicles. KJC8 staining against p53β isoforms present in the granular layer of the epidermis and the infundibulum of the hair follicle (arrows). (C, D) Squamous proliferation of the leg, classified as squamous cell carcinoma by microscopy. The criterion used to classify this atypical squamous proliferation of the leg as a squamous cell carcinoma was the predominance of atypical keratinocytes at the tumour–stroma interface. An obvious granular layer was not present in this tumour, with keratinocytes with abundant cytoplasm merging into dense keratin towards the tumour surface. This tumour had a tetramerisation domain frameshift mutation at amino acid 341 (p.Phe341fs). There is KJC8 cytoplasmic staining of keratinocytes with abundant cytoplasm (arrows) in the upper half of the tumour.

Results

Non-tumoural epidermis and hair follicles

The granular layer of the uninvolved epidermis invariably showed weak nuclear and cytoplasmic staining for the p53β isoforms (p53β, Δ40p53β, Δ133p53β and Δ160p53β) as detected with KJC8 (figure 2, red arrows). Several follicles were represented in the cases examined, and there was staining of keratinocytes within the granular layer of the infundibulum with KJC8; the stem and bulb did not stain (figure 2B).

As the follicular isthmus and catagen follicles were not represented in the original examined slides, five isthmus–catagen cysts (pilar cysts) as well as one sebaceous adenoma were stained with KJC8 in order to document staining of the follicular isthmus. Four of five pilar cysts and the sebaceous adenoma showed weak cytoplasmic staining of the subcorneal keratinocytes adjacent to the dense keratin within the cysts and in the isthmic area of the sebaceous adenoma.

Atypical squamous proliferations of the leg

Twenty-nine of 30 ASPs had enough tissue for staining. While these tumours were classified on histopathological grounds (see Materials and methods section) as SCC or KA-like, our clinical experience is that these tumours have the same biologic behaviour with conservative treatment (ie, shave removal). Many tumours had foci of a well-differentiated granular layer towards the surface of the tumour; foci without a granular layer had keratinocytes with abundant, eosinophilic cytoplasm merging directly with somewhat dense keratin, reminiscent of trichilemmal keratinisation. The two tumours as previously described5 with mutations in the tetramerisation domain of TP53 showed cytoplasmic staining of enlarged keratinocytes in >50% of the tumour (figure 2C,D). Twenty-seven tumours had KJC8 staining that was essentially negative (0 to <10% of tumour cells staining).

Discussion

This study documents the staining pattern of KJC8 antibody, which detects p53β isoforms, in human epidermis, the hair follicle and ASPs on the legs of women. KJC8 stains the granular layer of the epidermis (figure 2), a pattern of in vivo staining which has been previously described for some, but not all, p53 antibodies, depending on the epitope.8 DO-1, a commonly used p53 antibody that is commercially available (see figure 1), does not stain the normal epidermis in vivo.8 Cytoplasmic KJC8 staining of enlarged keratinocytes in the upper stratum spinosum with KJC8 was diffusely positive only in two tumours previously described5 with TP53 truncating and frameshift mutations in the tetramerisation domain (p.R342X and p.Phe341fs) (figure 2C,D). Staining was relatively weak, and further study using other methods like RNA in situ hybridisation may more accurately reflect levels of transcription. As 27/29 tumours had essentially negative staining, p53β isoforms cannot be used as a positive biomarker of these ASPs on the legs of women.

The exact signals required for epidermal and follicular differentiation are not fully elucidated, but p53 does play a complex role.9 10 It is therefore compelling that KJC8 stains both the differentiated granular layer (figure 2A,B), which is typical of epidermal and follicular infundibular differentiation, and enlarged/eosinophilic keratinocytes that merge into denser keratin, which is reminiscent of trichilemmal-type differentiation (figure 2C,D). Trichilemmal-type differentiation is normally present only in the follicular isthmus and catagen hair follicles, and such keratinisation at the epidermal surface is abnormal. Further study of the eosinophilic, enlarged keratinocytes within these lesions with different markers of follicular differentiation may aid in classifying their keratinisation pattern as follicular, isthmus–catagen type.

Indolent squamous proliferations include KA, a tumour that mimics SCC but spontaneously regresses,11 and ASPs on the legs of women above the age of 75, many of which are KA-like but are not known to spontaneously regress.12 13 The absence of significant expression of p53 isoforms in 27/29 ASPs may reflect true lack of expression or poor detection of the isoforms due to masking of the epitope. The positive staining in two squamous tumours previously described5 with tetramerisation domain mutations in TP53, microscopically classified as SCC, is suggestive of a functional correlate of the genotype. Further study is necessary to elucidate the biological significance of increased p53β in squamous tumours with tetramerisation domain mutations.

Take home messages

  • Different splice isoforms of p53 have not been well-studied in the skin.

  • p53β isoforms are expressed in the differentiated layers of the epidermis and hair follicle.

  • Squamous proliferations with tetramerization domain mutations in TP53 have increased p53β isoforms expression.

References

Footnotes

  • Handling editor Cheok Soon Lee.

  • Contributors CJK wrote the paper, designed the study and analysed the data. PM analysed the data and edited the paper. DL edited the paper. J-CB provided the p53β antibody and edited the paper.

  • Funding This study was funded by Women’s Health Research at Yale.

  • Competing interests None declared.

  • Patient consent Not required.

  • Ethics approval Yale University Institutional Review Board.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data sharing statement No unpublished data will be available.