mtm has created a CINtec® p16 Staining Atlas for use by clinicians. This atlas can be viewed  by using the slide-menu below:

Fig. 1: Scheme of p16 immunostaining in dysplasia: gradual increase in p16 positivity with progression of dysplasia, starting from basal layer.

Upregulation of p16 has been suggested to be a consequence of the functional inactivation of the retinoblastoma protein (pRb).
In the case of cervical cancer, inactivation of pRb is mediated by binding of the E7 oncoprotein of high risk human papillomaviruses (HR-HPV) to pRb. Since expression of p16 underlies a negative feedback control through functional pRb, overexpression might represent a useful biomarker for cells with active expression of HPV oncogenes.
p16 in cervical dysplasia is unrelated to differentiation /maturation and can therefore be expressed in dysplastic, i.e. proliferating cells.

In these cells, strongly overexpressed p16 is easily detectable with immunochemical means.

	Fig. 2a Irregular infiltrates of tumor cells in connective tissue with inflammatory reaction.
	Fig. 2b Strong cytoplasmic and nuclear positivity for p16 of carcinoma cells.

	Fig. 3a: Endocervical adenocarcinoma (Grade I) Neoplastic endocervical glands are lined by a pseudostratified epithelium, consisting of neoplastic columnar cells with hyperchromatic, irregularly shaped nuclei.
	Fig. 3b: Endocervical adenocarcinoma (Grade I) Strong cytoplasmic and nuclear positivity for p16 with a diffuse distribution.

	Fig. 4a: Carcinoma in situ (CIN3) CIN 3 (Carcinoma in situ) extending into endocervical gland. Neoplastic cells with relatively uniform, hyperchromatic nuclei throughout the epithelium.
	Fig. 4b: Carcinoma in situ (CIN3) Carcinoma in situ extending into endocervical gland. Strong nuclear and cytoplasmic p16 positivity in neoplastic epithelium in a diffuse distribution.

	Fig. 5a Dysplastic cells with large, hyperchromatic nuclei almost throughout the entire epithelium.
	Fig. 5b: severe dysplasia (CIN3) Mostly strong, nuclear and cytoplasmic positivity for p16 in dysplastic epithelium with a diffuse distribution.

	Fig. 6a: CIN 2 without koilocytosis Dysplastic cells in the lower half of the epithelium.
	Fig. 6b: CIN 2 without koilocytosis In the lower half of the epithelium dysplastic cells show cytoplasmic and nuclear p16 positivity in a diffuse distribution. The cells in the superficial layers show no or only weak p16 positivity.

	Fig. 7a: CIN 2 with Koilocytosis Moderate dysplasia showing koilocytes with perinuclear cavity and mildly irregular, hyperchromatic nuclei in upper epithelial cell layers (Arrowhead: Mitosis).
	Fig. 7b: CIN 2 with koilocytosis p16 positivity only in dysplastic cells of basal and parabasal layers, koilocytes in upper layers are mostly p16 negative.

Fig. 8: Scheme of p16 immunostaining, stratified squamous epithelium

The stratified squamous epithelium of the cervix uteri proliferates and matures under estrogenic stimulation. Progesterone inhibits maturation of squamous epithelium.
Proliferation is limited to the basal and parabasal cells.
Maturation occurs in the intermediate zone and is manifested by an increase in the amount of cytoplasma with accumulation of glycogen.
The superficial layer contains terminally differentiated squamous cells that eventually exfoliate.

In normal maturation / differentiation p16 is expressed at a level that is not detectable with immunochemical means.

 

	Fig. 9a: Normal stratified squamous epithelium, superficial cell layers Superficial differentiated squamous cells with large and flat cell bodies. Pyknotic nuclei.
	Fig. 9b: Normal stratified squamous epithelium, superficial cell layers Negative for p16.

	Fig. 10a: Stratified squamous epithelium, intermediate cell layer Medium large, differentiating polygonal cells. Vesicular nuclei. Uniformly fine granular chromatin. Vacuolic cell body due to glycogen content.
	Fig. 10b: Stratified squamous epithelium, intermediate cell layer Negative for p16.

	Fig. 11a: Stratified squamous epithelium, basal and parabasal cell layer Relatively small squamous cells with round to oval shape. Round to oval vesicular nuclei. Arrowhead: Clearly delineated row of palisading basal cells. Bracket: Parabasal cell layers.
	Fig. 11b: Stratified squamous epithelium, basal and parabasal cell layer Negative for p16. Arrowhead: Clearly delineated row of palisading basal cells. Bracket: Parabasal cell laye

	Fig. 12a: Atrophic squamous epithelium Stratified squamous epithelium with little maturation. Epithelium is composed almost entirely of basal and parabasal cells.
	Fig. 12b: Atrophic squamous epithelium Negative for p16 with occasional positive cells in superficial layers (Arrow).

	Fig. 13a: Endocervical mucosa, columnar cells Mucus producing cells in a single layer lining an endocervical gland. Distinct cell borders. Round to oval, basally located nuclei of uniform size. Mucus in glandular lumen.
	Fig. 13b: Endocervical mucosa, columnar cells Overwhelming majority of cells negative for p16, interspersed single cells may occasionally show positive p16 reaction in cytoplasm and nuclei.

	Fig. 14a: Endometrium during menstruation Glands and stromal cells of endometrial tissue shrunken and partly dissociated with cellular aggregates between fresh hemorrhages.
	Fig. 14b: Endometrium during menstruation Single cells may occasionally show p16 positivity.

Fig. 15: Scheme of p16 immunostaining, transformation zone with squamous metaplasia / tissue repair

In the cervix, squamous metaplasia is the replacement of the mucin producing columnar epithelium by stratified squamous epithelium. The undifferentiated subcolumnar reserve cells proliferate and eventually differentiate into stratified squamous epithelium.

In the maturation/differentiation process in squamous metaplasia p16 is expressed in some metaplastic cells that are committed to differentiation, i.e. cells in more superficial ayers. These cells are unable to proliferate. In these cells p16 is detectable with mmunochemical means. In reserve cell hyperplasia, the earliest stage of squamous metaplasia however, the proliferating cells do not show signs of differentiation and hence do not express p16.

	Fig. 16a: Immature squamous metaplasia Reserve cells, partly covered by a single layer of endocervical columnar cells (arrowheads) with focal differentiation to metaplastic squamous cells. Round to oval nuclei with finely granular chromatin.
	Fig. 16b: Immature squamous metaplasia No positivity for p16 in subcolumnar reserve cells. Arrowhead: Endocervical columnar cells.

	Fig. 17a: Transformation zone, maturing squamous cell metaplasia Regularly maturing squamous cells bordering to endocervical columnar cells (arrowhead).
	Fig. 17b: Transformation zone, maturing squamous cell metaplasia Some of the maturing squamous cells show cytoplasmic and nuclear positivity for p16 in a sporadic distribution of the positive cells in superficial layers.

Fig. 18a: Tubal metaplasia

Ciliated columnar cells in a single cell layer lining an endocervical gland. Metaplastic cells show positivity for p16 only in part of the epithelial cells, neighboring cells are negative (arrowheads). Insert: Higher Magnification to demonstrate ciliated cells (H&E)

The CINtec® p16 Staining Atlas was compiled and written by: