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. 2022 Jul 21;17(7):e0262877. doi: 10.1371/journal.pone.0262877

High prevalence of p16 staining in malignant tumors

Noémi De Wispelaere 1,#, Sebastian Dwertmann Rico 1,#, Marcus Bauer 1, Andreas M Luebke 1, Martina Kluth 1, Franziska Büscheck 1, Claudia Hube-Magg 1, Doris Höflmayer 1, Natalia Gorbokon 1, Sören Weidemann 1, Katharina Möller 1, Christoph Fraune 1, Christian Bernreuther 1, Ronald Simon 1,*, Christian Kähler 1, Anne Menz 1, Andrea Hinsch 1, Frank Jacobsen 1, Patrick Lebok 1, Till Clauditz 1, Guido Sauter 1, Ria Uhlig 1, Waldemar Wilczak 1, Stefan Steurer 1, Eike Burandt 1, Rainer Krech 2, David Dum 1, Till Krech 1,2, Andreas Marx 1,3, Sarah Minner 1
Editor: Surinder K Batra4
PMCID: PMC9302831  PMID: 35862385

Abstract

p16 (CDKN2A) is a member of the INK4 class of cell cycle inhibitors, which is often dysregulated in cancer. However, the prevalence of p16 expression in different cancer types is controversial. 15,783 samples from 124 different tumor types and 76 different normal tissue types were analyzed by immunohistochemistry in a tissue microarray format. p16 was detectable in 5,292 (45.0%) of 11,759 interpretable tumors. Except from adenohypophysis in islets of Langerhans, p16 staining was largely absent in normal tissues. In cancer, highest positivity rates were observed in uterine cervix squamous cell carcinomas (94.4%), non-invasive papillary urothelial carcinoma, pTaG2 (100%), Merkel cell carcinoma (97.7%), and small cell carcinomas of various sites of origin (54.5%-100%). All 124 tumor categories showed at least occasional p16 immunostaining. Comparison with clinico-pathological data in 128 vulvar, 149 endometrial, 295 serous ovarian, 396 pancreatic, 1365 colorectal, 284 gastric, and 1245 urinary bladder cancers, 910 breast carcinomas, 620 clear cell renal cell carcinomas, and 414 testicular germ cell tumors revealed only few statistically significant associations. Comparison of human papilloma virus (HPV) status and p16 in 497 squamous cell carcinomas of different organs revealed HPV in 80.4% of p16 positive and in 20.6% of p16 negative cancers (p<0.0001). It is concluded, that a positive and especially strong p16 immunostaining is a feature for malignancy which may be diagnostically useful in lipomatous, urothelial and possibly other tumors. The imperfect association between p16 immunostaining and HPV infection with high variability between different sites of origin challenges the use of p16 immunohistochemistry as a surrogate for HPV positivity, except in tumors of cervix uteri and the penis.

Introduction

The p16 protein is encoded by the cyclin dependent kinase inhibitor 2A gene (CDKN2A, syn. MTS-1, INK4a or p16INK4) located at chromosome 9p21 [1]. p16 inhibits cell cycle progression from G1 to S phase [2] through binding and inactivating cyclin dependent kinases CDK4 and CDK6 [3]. In its cell cycle inhibiting function, p16 interplays with the retinoblastoma (RB1) and the p53 tumor suppressor genes. In case of an inactivation of p53 or RB1 and especially in case of inactivation of both proteins, p16 can be markedly upregulated. Accordingly, a particularly strong up-regulation is seen in human papilloma virus (HPV) infected cells, where both p53 and RB1 are inactivated by the HPV proteins E6 and E7 [4,5].

More than 3,000 studies have employed immunohistochemistry to study the role of p16 expression in normal and neoplastic tissues. Due to its strong overexpression in HPV infected cells, p16 immunohistochemistry is routinely used in diagnostic pathology as a surrogate parameter for HPV infection and a marker for HPV related anogenital and oropharyngeal neoplasia. The role of p16 expression in other cancer types is less clear. The rate of reported p16 positivity is highly variable for many tumors. For example, the fraction of p16 positive cases ranged from 43% to 100% in squamous cell carcinoma of the cervix [6,7], 9% to 98% in colorectal adenocarcinoma [8–10], 12% to 64% in hepatocellular carcinoma [11,12], 4% to 96% in malignant melanoma [13–15], 20% to 62% in mesothelioma [16–18], and 0% to 100% in liopsarcomas [19–21]. Although many studies have described a prognostic role of reduced or increased p16 expression, these results have often not been confirmed by others. In several cancer types including breast, prostate, ovarian, and colorectal cancer, both reduced expression [22–25] and overexpression [26–29] have been reported to be linked to poor prognosis. Altogether, these conflicting data are likely to be caused by the use of different antibodies, immunostaining protocols, and criteria to determine p16 positivity in these studies.

To better understand the role of p16 immunohistochemistry in different tumor types, a comprehensive study analyzing a large number of neoplastic and non-neoplastic tissues under highly standardized conditions is needed. We thus analyzed p16 expression in more than 15,000 tumor tissue samples from 124 different tumor types and subtypes as well as 76 non-neoplastic tissue types by immunohistochemistry in a tissue microarray (TMA) format.

Materials and methods

Tissue Microarrays (TMAs)

To study p16 expression in normal and neoplastic human tissues, preexisting TMAs containing 15,783 primary tumors from 124 tumor types and subtypes as well as 608 samples of 76 different normal tissues were used. Detailed histopathological data on grade, pT and pN status were available for 7,598 cancers (invasive breast carcinoma of no special type, colorectal carcinoma, endometroid endometrial carcinoma, clear cell renal cell carcinoma, serous high grade ovarian carcinoma, adenocarcinoma of the pancreas, adenocarcinoma of the stomach, germ cell tumors, carcinoma of the vulva and urinary bladder carcinoma). Clinical follow up data were available for 254 patients who had undergone cystectomy for muscle invasive (pT≥2) urinary bladder cancer (median follow-up time = 14 (range 1–77) months) and 978 patients with invasive breast carcinoma of no special type (median follow-up time = 50 (range 1–88) months). The composition of both normal and cancer TMAs is described in detail in the results section. All samples were derived from the archives of the Institute of Pathology, University Hospital of Hamburg, Germany, the Institute of Pathology, Clinical Center Osnabrueck, Germany, and the Department of Pathology, Academic Hospital Fuerth, Germany. Tissues were fixed in 4% buffered formalin and then embedded in paraffin. TMA tissue spot diameter was 0.6 mm. Informed patient consent was not required for this retrospective study. The use of anonymized archived remnants of diagnostic tissues for manufacturing of TMAs and their analysis for research purposes as well as patient data analysis has been approved by local laws (HmbKHG, §12) and by the local ethics committee (Ethics commission Hamburg, WF-049/09). All work has been carried out in compliance with the Helsinki Declaration.

Immunohistochemistry

Freshly cut TMA sections were immunostained on one day and in one experiment. Slides were deparaffinized with xylol, rehydrated through a graded alcohol series and exposed to heat-induced antigen retrieval for 5 minutes in an autoclave at 121°C in pH 9 DakoTarget Retrieval Solution™ (Agilent, CA, USA; #S2367). Endogenous peroxidase activity was blocked with Dako Peroxidase Blocking Solution™ (Agilent, CA, USA; #52023) for 10 minutes. Primary antibody specific against p16 protein (rabbit recombinant clone MSVA-016R; MS Validated Antibodies GmbH, Hamburg, Germany) was applied at 37°C for 60 minutes at a dilution of 1:150. Bound antibody was then visualized using the EnVision Kit™ (Agilent, CA, USA; #K5007) according to the manufacturer’s directions. The sections were counterstained with haemalaun. For tumor tissues, the percentage of positive neoplastic cells was estimated, and the staining intensity was semiquantitatively recorded (0, 1+, 2+, 3+). For statistical analyses, the staining results were categorized into four groups. Tumors without any staining were considered as negative. Tumors with 1+ staining intensity in ≤70% of cells or 2+ intensity in ≤30% of cells were considered weakly positive. Tumors with 1+ staining intensity in >70% of cells, 2+ intensity in 31–70%, or 3+ intensity in ≤30% were considered moderately positive. Tumors with 2+ intensity in >70% or 3+ intensity in >30% of cells were considered strongly positive.

HPV polymerase chain reaction (PCR) and sequencing

HPV status was analyzed in a subset of 551 squamous cell carcinomas including 80 oral, 60 pharyngeal, 60 laryngeal, 80 cervical, 30 vaginal, 80 vulvar, 80 penile, 40 skin and 41 anal canal tumors. Detection of HPV-DNA was performed on formalin-fixed, paraffin-embedded tumor specimens. One 4μm microtome section was taken from each sample for DNA extraction using the Maxwell® RSC DNA FFPE Kit (Promega, Fitchburg, WI, USA) according to the manufacturer’s protocol. Suitability of the isolated DNA for PCR analysis was verified by amplification of a ß-globin sequence with primers generating an amplicon of 217 bp (forward 5′-GCCATCACTAAAGGCACCGAGC-3′ and reverse 5′-TGGGCATGTGGAGACAGAGAAGA-3′). Detection of HPV was performed using primers HPV-GP 6+ (5´-GAAAAATAAACTGTAAATCATATTC-3´) and HPV-GP 5+ (5´-TTTGTTACTGTGGTAGATACTAC-3´) which generate amplicons ranging between 139–145 bp. The thermocycler protocol included initial denaturation at 95°C for 10 min, followed by 34 cycles of 95°C for 90 sec, 55°C for 90 sec and 72°C for 120 sec, and a final extension step at 72°C for 7 min. PCR products were visualized by standard agarose gel electrophoresis. Samples with negative ß-globin PCR were excluded from further analysis. Samples with positive ß-globin PCR but negative HPV PCR were reported as HPV-negative. Samples with positive ß-globin PCR and positive HPV PCR were reported as HPV-positive and subjected to bidirectional Sanger sequencing employing the Genetic Analyzer 3130 xl device (Applied Biosystems, Foster City, CA, USA) using primers GP 5+ and GP 6+. Sequences were analyzed with NCBI’s Basic Local Alignment Search Tool (BLAST) [30] to determine the HPV type.

Statistics

Statistical calculations were performed with JMP14 software (SAS Institute Inc., NC, USA). Contingency tables and the chi2-test were performed to search for associations between p16 and tumor phenotype. Survival curves were calculated according to Kaplan-Meier. The Log-Rank test was applied to detect significant differences between groups. A p-value of ≤0.05 was considered as statistically significant.

Results

Technical issues

A total of 11,759 (74.5%) of 15,783 tumor samples and 405 (66.6%) of 608 normal samples were interpretable for p16 immunostaining in our TMA analysis. Non-interpretable samples (4,024; 25.5%) either lacked unequivocal tumor cells or were absent on the TMA.

p16 in normal tissue

p16 staining was strongest in islets of Langerhans of the pancreas (Fig 1A) and in a large fraction of cells in the adenohypophysis (Fig 1B). Positive staining was also found in a fraction of cells of corpuscles of Hassall‘s of thymus (Fig 1C), scattered adrenocortical cells (Fig 1D), and endothelial cells of blood vessels in a normal placenta (Fig 1E) and in an otherwise p16 negative clear cell carcinoma of the kidney (Fig 1F).

Fig 1. p16 immunostaining in non-neoplastic tissue.

Fig 1

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The panels show a nuclear and cytoplasmic p16 staining of a fraction of cells of pancreatic islets of Langerhans (A), a large fraction of epithelial cells in the adenohypophysis (B), a fraction of cells of corpuscles of Hassall‘s of thymus (C), and of scattered adrenocortical cells (D). A p16 positivity is also seen in endothelial cells of blood vessels in a normal placenta (E) and in an otherwise p16 negative clear cell carcinoma of the kidney (F).

p16 immunostaining was absent in endothelium and media of the aorta, the heart, striated muscle, tongue muscle, myometrium of the uterus, muscular wall of the appendix, esophagus, stomach, ileum, colon descendens, kidney pelvis, and urinary bladder, corpus spongiosum of the penis, ovarian stroma, fat, skin, hair follicle and sebaceous glands of the skin, oral mucosa of the lip, oral cavity, surface epithelium of the tonsil, transitional mucosa and skin of the anal canal, ectocervix, squamous epithelium of the esophagus, urothelium of the kidney pelvis and urinary bladder, amnion and chorion of the mature placenta, spleen, antrum and corpus of the stomach, epithelium of the gallbladder, liver, Brunner gland of the duodenum, cortex and medulla of the kidney, seminal vesicle, epididymis, testis, lung, endocervix, mucosa of the fallopian tube, decidua of the early placenta, in the cerebellum, and white and grey matter of the cerebrum.

p16 immunostaining in tumor cells

Positive p16 immunostaining was detectable in 5,292 (45.0%) of the 11,759 analyzable tumors, including 3,152 (26.8%) with weak, 683 (5.8%) with moderate, and 1,457 (12.4%) with strong immunostaining. The staining pattern was heterogenous and included cases with variable percentage of positive tumor cells as well as tumors with predominantly cytoplasmic and predominantly nuclear staining. Representative images of p16 positive tumors are shown in Fig 2. All 124 tumor categories showed a detectable p16 expression in at least one case with 71 (57.3%) tumor categories showing at least one case with strong positivity (Table 1). A comparison between p16 expression in normal tissues und the corresponding tumor types is given in S2 Table. A graphical representation of a ranking order of p16 positive and strongly positive cancers is given in Fig 3.

Fig 2. p16 immunostaining of tumors and related normal tissues.

Fig 2

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In the pancreas, a moderate p16 immunostaining is regularly seen in a subset of islet cells and only occasionally occurs in few scattered cells of excretory ducts (A), but p16 expression can be strong in cases of ductal adenocarcinoma (B) and of neuroendocrine carcinoma (C). In normal lymphatic tissues, a weak p16 staining occurs in germinal centre macrophages and in some scattered lymphocytes (D) but a strong staining is seen in neoplastic cells of some Hodgkin‘s (E) and diffuse large B-cell lymphomas (F). In the stomach, few normal epithelial cells may show p16 staining (G) while p16 staining can be strong in gastric adenocarcinoma (H). In the esophagus, few cells with weak to moderate p16 staining can be found in some samples of normal squamous epithelium (I) but p16 staining can be intense in squamous cell carcinoma (J). p16 immunostaining is usually absent in normal myometrium (K), fat (L), urothelium (M), and cervical squamous epitheium (N) while staining can be intense in tumors derived from these tissues such as leimyosarcoma of the uterus (O), liposarcoma (P), urothelial carcinoma (Q) as well as adenocarcinoma (R) and squamous cell carcinoma (S) of the uterine cervix. A similarly strong p16 staining can also be seen in other squamous cell carcinomas such as of the skin (T).

Table 1. p16 immunostaining in human tumors.

      p16 immunostaining
  tumor entity on TMA (n) analyzable (n) negative (%) weak (%) moderate (%) strong (%) positive (%)
Tumors of the skin Pilomatrixoma 35 28 32.1 64.3 3.6 0.0 67.9
Basal cell carcinoma 88 67 9.0 68.7 20.9 1.5 91.0
Benign nevus 29 25 4.0 60.0 20.0 16.0 96.0
Squamous cell carcinoma of the skin 90 85 64.7 20.0 5.9 9.4 35.3
Malignant melanoma 48 41 53.7 19.5 19.5 7.3 46.3
Merkel cell carcinoma 46 44 2.3 2.3 9.1 86.4 97.7
Tumors of the head and neck Squamous cell carcinoma of the larynx 110 90 76.7 13.3 4.4 5.6 23.3
Squamous cell carcinoma of the pharynx 60 52 51.9 7.7 7.7 32.7 48.1
Oral squamous cell carcinoma (floor of the mouth) 130 114 76.3 7.9 3.5 12.3 23.7
Pleomorphic adenoma of the parotid gland 50 33 6.1 81.8 12.1 0.0 93.9
Warthin tumor of the parotid gland 49 41 2.4 87.8 9.8 0.0 97.6
Basal cell adenoma of the salivary gland 15 13 0.0 100.0 0.0 0.0 100.0
Tumors of the lung, pleura and thymus Squamous cell carcinoma of the lung 77 40 82.5 5.0 10.0 2.5 17.5
Adenocarcinoma of the lung 200 107 58.9 27.1 10.3 3.7 41.1
Small cell carcinoma of the lung 20 16 18.8 12.5 6.3 62.5 81.3
Mesothelioma, epitheloid 39 30 76.7 23.3 0.0 0.0 23.3
Mesothelioma, other types 76 63 63.5 12.7 0.0 42.9 55.6
Thymoma 29 25 36.0 60.0 4.0 0.0 64.0
Tumors of the female genital tract Squamous cell carcinoma of the vagina 78 73 37.0 11.0 9.6 42.5 63.0
Squamous cell carcinoma of the vulva 130 116 60.3 12.1 8.6 19.0 39.7
Squamous cell carcinoma of the cervix 130 125 5.6 4.0 11.2 79.2 94.4
Adenocarcinoma of the cervix uteri 50 48 10.4 52.1 22.9 14.6 89.6
Endometrioid endometrial carcinoma 236 195 19.0 63.6 12.3 5.1 81.0
Endometrial serous carcinoma 82 69 13.0 26.1 20.3 40.6 87.0
Carcinosarcoma of the uterus 48 39 10.3 20.5 38.5 30.8 89.7
Endometrial carcinoma, high grade, G3 13 8 12.5 62.5 12.5 12.5 87.5
Endometrial clear cell carcinoma 8 5 0.0 80.0 20.0 0.0 100.0
Endometrial stromal sarcoma 12 12 75.0 16.7 0.0 8.3 25.0
Endometrioid carcinoma of the ovary 115 89 18.0 47.2 19.1 15.7 82.0
Serous carcinoma of the ovary 567 446 11.7 24.0 16.4 48.0 88.3
Mucinous carcinoma of the ovary 97 66 78.8 18.2 3.0 0.0 21.2
Clear cell carcinoma of the ovary 54 38 36.8 47.4 15.8 0.0 63.2
Carcinosarcoma of the ovary 47 36 22.2 30.6 19.4 27.8 77.8
Brenner tumor 9 9 22.2 66.7 11.1 0.0 77.8
Tumors of the breast Invasive breast carcinoma of no special type 1387 960 61.1 29.5 3.6 5.7 38.9
Lobular carcinoma of the breast 294 168 67.3 30.4 1.2 1.2 32.7
Medullary carcinoma of the breast 26 22 22.7 13.6 13.6 50.0 77.3
Tubular carcinoma of the breast 27 14 28.6 71.4 0.0 0.0 71.4
Mucinous carcinoma of the breast 58 30 53.3 40.0 6.7 0.0 46.7
Phyllodes tumor of the breast 50 42 7.1 71.4 14.3 7.1 92.9
Tumors of the digestive system Adenomatous polyp, low-grade dysplasia 50 49 53.1 44.9 2.0 0.0 46.9
Adenomatous polyp, high-grade dysplasie 50 49 26.5 63.3 10.2 0.0 73.5
Adenocarcinoma of the colon 1882 1434 48.2 48.6 2.6 0.6 51.8
Adenocarcinoma of the small intestine 10 10 80.0 0.0 10.0 10.0 20.0
Gastric adenocarcinoma, diffuse type 176 101 54.5 28.7 10.9 5.9 45.5
Gastric adenocarcinoma, intestinal type 174 127 59.1 23.6 7.1 10.2 40.9
Gastric adenocarcinoma, mixed type 62 45 62.2 28.9 0.0 8.9 37.8
Adenocarcinoma of the esophagus 133 57 87.7 1.8 1.8 8.8 12.3
Squamous cell carcinoma of the esophagus 124 44 81.8 0.0 4.5 13.6 18.2
Squamous cell carcinoma of the anal canal 91 87 24.1 4.6 10.3 60.9 75.9
Cholangiocarcinoma 130 102 73.5 20.6 3.9 2.0 26.5
Hepatocellular carcinoma 50 50 94.0 6.0 0.0 0.0 6.0
Ductal adenocarcinoma of the pancreas 612 410 81.2 13.2 3.2 2.4 18.8
Pancreatic/Ampullary adenocarcinoma 89 52 69.2 21.2 5.8 3.8 30.8
Acinar cell carcinoma of the pancreas 13 12 75.0 8.3 8.3 8.3 25.0
Gastrointestinal stromal tumor (GIST) 50 45 35.6 40.0 8.9 15.6 64.4
Tumors of the urinary system Non-invasive papillary urothelial carcinoma, pTa G2 low grade 177 116 0.0 0.0 2.6 97.4 100.0
Non-invasive papillary urothelial carcinoma, pTa G2 high grade 141 106 0.0 0.9 8.5 90.6 100.0
Non-invasive papillary urothelial carcinoma, pTa G3 187 132 9.8 6.1 15.2 68.9 90.2
Urothelial carcinoma, pT2-4 G3 1214 732 50.7 20.4 10.1 18.9 49.3
Small cell neuroendocrine carcinoma of the bladder 18 18 0.0 0.0 0.0 100.0 100.0
Sarcomatoid urothelial carcinoma 25 24 54.2 12.5 0.0 33.3 45.8
Clear cell renal cell carcinoma 858 648 98.2 1.7 0.2 0.0 1.8
Papillary renal cell carcinoma 255 129 67.2 31.3 1.6 0.0 32.8
Clear cell (tubulo) papillary renal cell carcinoma 21 13 92.9 7.1 0.0 0.0 7.1
Chromophobe renal cell carcinoma 131 80 77.7 22.3 0.0 0.0 22.3
Oncocytoma 177 128 95.5 4.5 0.0 0.0 4.5
Tumors of the male genital organs Adenocarcinoma of the prostate, Gleason 3+3 83 63 92.1 6.3 1.6 0.0 7.9
Adenocarcinoma of the prostate, Gleason 4+4 80 64 67.2 31.3 1.6 0.0 32.8
Adenocarcinoma of the prostate, Gleason 5+5 85 61 63.9 36.1 0.0 0.0 36.1
Adenocarcinoma of the prostate (recurrence) 330 284 35.6 57.0 2.5 4.9 64.4
Small cell neuroendocrine carcinoma of the prostate 17 16 6.3 18.8 12.5 62.5 93.8
Seminoma 620 454 90.3 9.3 0.2 0.2 9.7
Embryonal carcinoma of the testis 50 41 82.9 14.6 2.4 0.0 17.1
Yolk sack tumor 50 36 80.6 19.4 0.0 0.0 19.4
Teratoma 50 36 88.9 5.6 2.8 2.8 11.1
Squamous cell carcinoma of the penis 80 75 49.3 8.0 4.0 38.7 50.7
Tumors of endocrine organs Adenoma of the thyroid gland 50 47 83.0 17.0 0.0 0.0 17.0
Papillary thyroid carcinoma 114 96 90.6 9.4 0.0 0.0 9.4
Follicular thyroid carcinoma 392 333 70.9 26.4 2.4 0.3 29.1
Medullary thyroid carcinoma 158 130 87.7 11.5 0.8 0.0 12.3
Anaplastic thyroid carcinoma 107 80 76.3 22.5 1.3 0.0 23.8
Adrenal cortical adenoma 45 42 66.7 2.4 7.1 23.8 33.3
Adrenal cortical carcinoma 26 26 26.9 23.1 19.2 30.8 73.1
Phaeochromocytoma 50 50 58.0 38.0 4.0 0.0 42.0
Appendix, neuroendocrine tumor (NET) 22 13 38.5 53.8 7.7 0.0 61.5
Colorectal, neuroendocrine tumor (NET) 10 10 70.0 30.0 0.0 0.0 30.0
Ileum, neuroendocrine tumor (NET) 49 47 76.6 23.4 0.0 0.0 23.4
Lung, neuroendocrine tumor (NET) 19 17 82.4 17.6 0.0 0.0 17.6
Pancreas, neuroendocrine tumor (NET) 102 96 52.1 40.6 5.2 2.1 47.9
Colorectal, neuroendocrine carcinoma (NEC) 11 11 45.5 0.0 27.3 27.3 54.5
Gallbladder, neuroendocrine carcinoma (NEC) 4 4 25.0 0.0 75.0 0.0 75.0
Pancreas, neuroendocrine carcinoma (NEC) 13 11 27.3 36.4 18.2 18.2 72.7
Tumors of haemotopoetic Hodgkin Lymphoma 103 77 64.9 23.4 10.4 1.3 35.1
and lymphoid tissues Non-Hodgkin Lymphoma 62 52 53.8 44.2 1.9 0.0 46.2
Small lymphocytic lymphoma, B-cell type (B-SLL/B-CLL) 50 48 54.2 45.8 0.0 0.0 45.8
Diffuse large B cell lymphoma (DLBCL) 114 110 63.6 29.1 5.5 1.8 36.4
Follicular lymphoma 88 84 58.3 41.7 0.0 0.0 41.7
T-cell Non Hodgkin lymphoma 24 24 58.3 33.3 8.3 0.0 41.7
Mantle cell lymphoma 18 18 83.3 16.7 0.0 0.0 16.7
Marginal zone lymphoma 16 15 80.0 20.0 0.0 0.0 20.0
Diffuse large B-cell lymphoma (DLBCL) in the testis 16 15 80.0 13.3 6.7 0.0 20.0
Burkitt lymphoma 5 4 75.0 25.0 0.0 0.0 25.0
Tumors of soft tissue and bone Tenosynovial giant cell tumor 45 43 9.3 88.4 2.3 0.0 90.7
Angiomyolipoma 91 84 95.2 4.8 0.0 0.0 4.8
Angiosarcoma 73 51 47.1 41.2 9.8 2.0 52.9
Dermatofibrosarcoma protuberans 21 16 25.0 68.8 6.3 0.0 75.0
Ganglioneuroma 14 10 90.0 0.0 0.0 10.0 10.0
Granular cell tumor 23 21 38.1 57.1 4.8 0.0 61.9
Kaposi sarcoma 8 6 66.7 33.3 0.0 0.0 33.3
Leiomyoma 50 40 57.5 42.5 0.0 0.0 42.5
Leiomyosarcoma 87 77 26.0 31.2 22.1 20.8 74.0
Liposarcoma 132 99 11.1 22.2 10.1 56.6 88.9
Malignant peripheral nerve sheat tumor (MPNST) 13 12 83.3 8.3 8.3 0.0 16.7
Myofibrosarcoma 26 25 52.0 8.0 4.0 36.0 48.0
Neurofibroma 117 77 64.9 24.7 7.8 2.6 35.1
Sarcoma, not otherwise specified (NOS) 75 68 38.2 25.0 8.8 27.9 61.8
Paraganglioma 41 36 77.8 22.2 0.0 0.0 22.2
Primitive neuroectodermal tumor (PNET) 23 20 35.0 50.0 10.0 5.0 65.0
Rhabdomyosarcoma 7 7 28.6 0.0 0.0 71.4 71.4
Schwannoma 121 97 12.4 47.4 23.7 16.5 87.6
Synovial sarcoma 12 11 45.5 36.4 18.2 0.0 54.5
Osteosarcoma 39 16 87.5 12.5 0.0 0.0 12.5
  Chondrosarcoma 43 29 41.4 6.9 10.3 41.4 58.6
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Fig 3. Ranking order of p16 immunostaining in human tumors.

Fig 3

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Both the frequency of positive cases (blue dots) and the frequency of strongly positive cases (orange dots) is shown.

p16 immunostaining, tumor phenotype and prognosis

A comparison of p16 expression with pT, pN, histologic grade, and patient prognosis in 128 analyzable vulvar carcinomas, 149 endometrioid endometrial carcinomas, 295 serous high grade ovarian carcinomas, 910 invasive breast carcinomas of no special type, 1245 urinary bladder carcinomas, 620 clear cell renal cell carcinomas, 414 germ cell tumors of the testis, 284 gastric adenocarcinomas, 396 pancreatic adenocarcinomas and 1365 colorectal adenocarcinomas revealed only few statistically significant associations (Table 2). Positive p16 immunostaining was associated with high pT category in urinary bladder carcinoma (p<0.0001) and gastric adenocarcinoma (p = 0.0212), and with high grade in invasive breast carcinoma of no special type (p<.0001). In colorectal adenocarcinoma, a significant association was found between p16 positivity (at least weak immunostaining) and MMR (mismatch repair) status, with a higher percentage of tumors showing positive p16 staining in the MMR-proficient group (56%) than in the MMR-deficient group (27%, p<0.0001). In cohorts of 502 invasive breast cancers and 151 urothelial carcinomas with clinical follow-up data, p16 immunostaining was unrelated to overall survival (Fig 4).

Table 2. p16 immunostaining and tumor phenotype.

    analyzable n* p16 immunostaining p value
    negative (%) weak (%) moderate (%) strong (%)
Vulvar carcinoma all cancers 128 57.0 6.3 12.5 24.2  
pT1 43 51.2 2.3 18.6 27.9 0.0880
pT2 67 58.2 7.5 11.9 22.4
pT3-4 14 78.6 14.3 0.0 7.1
G1 16 68.8 12.5 18.8 0.0 0.1567
G2 62 54.8 6.5 11.3 27.4
G3 30 56.7 6.7 13.3 23.3
pN0 68 50.0 4.4 16.2 29.4 0.1448
pN+ 37 64.9 10.8 8.1 16.2
endometrioid endometrial carcinoma all cancers 149 22.1 66.4 10.1 1.3  
pT1 94 21.3 67.0 9.6 2.1 0.9198
pT2 23 21.7 65.2 13.0 0.0
pT3-4 29 20.7 69.0 10.3 0.0
pN0 7 15.6 75.6 6.7 2.2 0.4994
pN+ 5 23.8 57.1 14.3 4.8
serous high grade ovarian carcinoma all cancers 295 15.2 12.9 49.2 22.7
pT1 20 5.0 0.0 70.0 25.0 0.1266
pT2 38 18.4 13.2 42.1 26.3
pT3 208 15.9 14.4 47.1 22.6
pN0 65 15.4 9.2 53.9 21.5 0.1883
pN1 138 10.9 18.8 44.2 26.1
Invasive breast carcinoma of no special type all cancers 910 61.1 30.2 3.0 5.7  
pT1 452 61.5 32.7 1.8 4.0 0.0738
pT2 358 62.8 25.7 3.6 7.8
pT3-4 70 61.4 28.6 2.9 7.1
G1 127.0 76.4 22.8 0.8 0.0 <0.0001
G2 433.0 63.5 33.7 1.6 1.2
G3 349.0 52.7 28.4 5.4 13.5
pN0 468.0 65.5 27.4 3.1 4.1 0.8944
pN+ 343.0 60.7 30.6 2.2 6.6
Urinary bladder carcinoma all cancers 1245 45.7 32.5 8.4 13.4  
pTa G2 low 145 19.3 80.0 0.7 0.0 <0.0001
pTa G2 high 121 51.2 43.0 2.5 3.3
pTaG3 138 26.8 48.6 13.8 10.9
pT≥2 G3 780 51.3 20.3 10.0 18.5
normal urothelium 24 91.7 8.3 0.0 0.0 0.0465
dysplasia 12 53.3 33.3 8.3 0.0
Clear cell renal cell carcinoma all cancers 620 98.9 1.0 0.1 0.0  
pT1 365 98.9 1.1 0.0 0.0 0.4494
pT2 63 100.0 0.0 0.0 0.0
pT3-4 187 98.4 1.1 0.5 0.0
ISUP 1 192 99.5 0.5 0.0 0.0 0.7094
ISUP 2 204 99.0 1.0 0.0 0.0
ISUP 3 177 98.3 1.1 0.6 0.0
ISUP 4 38 97.4 2.6 0.0 0.0
pN0 98 99.0 1.0 0.0 0.0 0.5927
pN≥1 15 100.0 0.0 0.0 0.0
Germ cell tumors of the testis all cancers 414 91.0 8.7 0.0 0.2
pT1 266 92.9 6.8 0.0 0.4 0.5059
pT2 101 89.1 10.9 0.0 0.0
pT3 40 87.5 12.5 0.0 0.0
Gastric carcinoma all cancers 284 64.8 23.9 5.6 5.6  
pT1-2 45 62.2 33.3 2.2 2.2 0.0212
pT3 92 75.0 14.1 3.3 7.6
pT4 94 55.3 28.7 8.5 7.5
pN0 54 51.9 29.6 7.4 11.1 0.1350
pN+ 178 68.5 21.9 4.5 5.1
Adenocarcinoma of the pancreas all cancers 396 79.8 14.6 2.8 2.8  
pT1 14 85.7 0.0 0.0 14.3 0.0486
pT2 65 70.8 20.0 3.1 6.2
pT3 290 81.4 13.8 3.1 1.7
pT4 26 80.8 19.2 0.0 0.0
G1 13 69.2 23.1 0.0 7.7 0.8606
G2 271 79.3 14.8 3.3 2.6
G3 87 80.5 14.9 2.3 2.3
pN0 84 82.1 10.7 3.6 3.6 0.6210
pN+ 310 79.0 15.8 2.6 2.6
R0 216 74.5 16.7 4.6 4.2 0.0007
R1 155 87.1 11.6 0.0 1.3
Colorectal adenocarcinoma all cancers 1365 48.2 48.6 2.6 0.6  
pT1 57 47.4 52.6 0.0 0.0 0.4898
pT2 271 45.8 50.9 3.0 0.4
pT3 745 48.2 48.2 3.1 0.5
pT4 280 50.0 47.5 1.4 1.1
pN0 698 48.3 48.6 2.6 0.6 0.9965
pN+ 639 47.7 49.0 2.7 0.6
MMR proficient 71 73.2 25.4 0.0 1.4 <0.0001
MMR deficient 983 44.2 52.6 2.7 0.5
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Abbreviation: pT: pathological tumor stage, pN: pathological lymph node status, G: grade, ISUP: International Society of Urological Pathology, R: resection margin, MMR: mismatch repair.

*Numbers do not always add up to the total number in the different categories because of cases with missing data.

Fig 4. p16 immunostaining and overall survival in patients with invasive breast cancer of no special type and urothelial carcinoma (pT2-4; G3).

Fig 4

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*The numbers do not add to the total number of tumors with clinical follow-up data, since only cases with evaluable p16 staining are included.

p16 immunostaining and HPV-status

HPV analysis of 535 squamous cell carcinomas of different sites of origin revealed 244 HPV positive cases (45.6%). Among these, 98.0% were high risk type (HPV type 16, 18, 33, 35, 45, 58), 1.6% intermediate risk type (HPV type 56, 67, 73) and 0.4% low risk type (HPV type 6). The comparison of HPV status and p16 staining revealed a strong but not perfect association between these parameters (Table 3). HPV was detected in 80.4% of 163 tumors with strong, 62.3% of 42 tumors with moderate, 25.9% of 42 tumors with weak p16 positivity, but also in 20.6% of 250 p16 negative cancers (p<0.0001). The association between p16 expression and HPV status varied between the organs of tumor origin and was particularly strong in squamous cell carcinomas of the cervix, squamous cell carcinoma of the penis and squamous cell carcinoma of the pharynx (p<0.0001 each). The statistical association between p16 expression and HPV status was particularly weak in squamous cell carcinomas of the larynx and of the vulva. It is of note, however, that both HPV negative cases with strong p16 positivity, and HPV positive cases with negative p16 staining were found in almost all tumor entities. Only cervical and skin cancer lacked HPV positive but p16 negative cases.

Table 3. Association between p16 immunostaining and HPV status.

  p16 status n HPV status  
  negative positive  
All cancers negative 253 79.4 20.6 <0.0001
weak 54 74.1 25.9
moderate 53 37.7 62.3
strong 163 19.6 80.4
Oral squamous cell carcinoma negative 55 89.1 10.9 0.0017
weak 3 100.0 0.0
moderate 4 100.0 0.0
strong 9 33.3 66.7
Squamous cell carcinoma of the pharynx negative 25 72.0 28.0 <0.0001
weak 4 25.0 75.0
moderate 4 25.0 75.0
strong 17 5.9 94.1
Squamous cell carcinoma of the larynx negative 47 83.0 17.0 0.3388
weak 2 100.0 0.0
moderate 2 50.0 50.0
strong 4 100.0 0.0
Squamous cell carcinoma of the cervix negative 4 100.0 0.0 <0.0001
weak 2 0.0 100.0
moderate 12 0.0 100.0
strong 57 7.0 93.0
Squamous cell carcinoma of the vagina negative 16 68.8 31.3 0.0077
weak 2 100.0 0.0
moderate 4 0.0 100.0
strong 7 28.6 71.4
Squamous cell carcinoma of the vulva negative 44 72.7 27.3 0.0571
weak 12 83.3 16.7
moderate 6 33.3 66.7
strong 13 46.2 53.8
Squamous cell carcinoma of penis negative 34 67.6 32.4 <0.0001
weak 6 16.7 83.3
moderate 3 0.0 100.0
strong 28 14.3 85.7
Squamous cell carcinoma of the skin negative 19 100.0 0.0 0.0951
weak 9 100.0 0.0
moderate 2 50.0 50.0
strong 6 100.0 0.0
Squamous cell carcinoma of the anal canal negative 6 50.0 50.0 0.0723
weak 2 0.0 100.0
moderate 5 0.0 100.0
  strong 22 9.1 90.9
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Discussion

The successful analysis of 11,759 cancers and 76 normal tissue types revealed that–as compared to normal tissues—p16 is often upregulated in cancers. While the normal tissue analysis demonstrated a moderate to strong p16 immunostaining in only few tissues, a strong p16 positivity was found in many tumors. Although p16 is a known tumor suppressor gene, upregulation can occur directly as a consequence of an altered state of the interaction partner pRb [31] or indirectly through pathway crosstalk with p53 (reviewed in [32]). Considering that p53 is the most frequently mutated tumor suppressor gene in cancer [33], that p53 inactivation can also occur in the absence of p53 gene mutations (reviewed in [34]), and that alterations of Rb (reviewed in [35]) and other p16 interaction partners such as CDK4 (cyclin dependent kinase 4) [36] are also common in cancer, the high rate of p16 upregulation is not a surprise. Our data also revealed a correlation between p16 staining and microsatellite instability in colorectal adenocarcinoma. In the microsatellite-stable group a higher percentage of tumors showed positive p16 staining than in the microsatellite-instable group. This may be explained by the known inverse relationship of p53 alterations (a well-established cause for p16 upreagulation) and MSI in colorectal cancer. Moreover, it has been shown that microsatellite instability leads to increased methylation of the p16 gene which may lead to reduced p16 expression or at least hinder p16 upregulation [37]. The results of our study do not exclude that p16 expression can also be reduced in some cancers as described in previous studies [15,38–41]. However, an entirely different experimental approach than the one selected for this study, with a much higher antibody concentration and a more sensitive staining protocol would be required to demonstrate reduced expression.

That a minimum of one case with at least a moderate p16 positivity was found in 100 of our 124 (80.6%) analyzed cancer types demonstrates that p16 immunostaining offers only limited support for defining a tumor’s site of origin. According to our data, there are only few occasions, where p16 immunostaining can assist in diagnosing the right tumor type. This applies for example—as previously suggested [42]—in the differentiation of high grade endometrial from serous carcinoma of the endometrium. In our study 40.6% of serous endometrial carcinoma showed strong p16 immunostaining in comparison to 5.1% strong p16 positivity in endometroid endometrial carcinoma.

In normal tissues, moderate to high p16 immunostaining was only consistently seen in islets of Langerhans of the pancreas and in the pituitary gland. p16 immunostaining was largely absent in tissues which are prone to develop cancer such as urothelium and squamous cell epithelium of various sites. Given the high rate of p16 overexpression in various cancers developing from p16 negative cells, p16 immunostaining may serve as a parameter that might indicate malignancy in some organs. Based on our findings that might for example apply for liposarcoma (56.6% strong positive, vs negative in normal fat), leiomyosarcoma (21% strong positive, vs 0% strong positive cases in leiomyoma) or urothelial dysplasia (46.7% positive, vs negative in normal urothelium). The high utility of immunohistochemical p16 analysis in assessing cervical biopsies is based on the fact that almost all neoplasias in this location are due to HPV infection [43]. The imperfect correlation of p16 expression and HPV infection found in our analysis of 497 squamous cell carcinomas of different origins suggest low reliability for using p16 immunostaining as a surrogate for HPV infection in other cancer types, however. Of note, in our study HPV could be detected in 20.6% of cases with absent p16 immunostaining. On the other hand, it is not surprising, that moderate to strong p16 positivity was found in 25–100% (average 30.7%) of HPV negative extra-genital squamous cell carcinomas, given the interaction of p16 with several important pathways. That aberrant p16 expression was not only seen in endothelial cells of a few cancers, but also in rare instances in endothelial cells in non-neoplastic tissue and in fibroblasts of the tumor stroma demonstrates, that substantial p16 upregulation can occasionally also occur in non-neoplastic tissue proliferation.

Our highly standardized analysis of 11,759 tumors from 124 different tumor entities enabled us to clarify the relative importance of p16 expression across tumor entities and to generate a ranking list according to the p16 positivity rate (Fig 3). It is of note, that many of the top ranked p16 positive tumor entities such as small cell neuroendocrine carcinoma of the bladder, Merkel cell carcinoma, small cell carcinoma of the lung and small cell neuroendocrine carcinoma of the prostate exhibit neuroendocrine differentiation. This observation is in line with data from an earlier study showing, that loss of Rb function, which can cause overexpression of p16, leads to neuroendocrine hypercellularity in the lung [44]. Although we are unaware of a specific role of p16 in neuroendocrine cellular functions, it is conspicuous that our normal tissue analysis had also identified the highest expression in neuroendocrine/endocrine cells of islets of Langerhans in the pancreas, and the adenohypophysis. Moreover, it is possible that the scattered p16 positive cells in the gastrointestinal tract also represent endocrine cells. Several other studies have also shown p16 overexpression in various neuroendocrine neoplasms of different origin [45–47].

More than 3000 studies have previously analyzed p16 expression in tumors by immunohistochemistry. The summary of the results of 448 of these studies in Fig 5 demonstrates, that highly discrepant data on the prevalence of p16 positivity exist for many tumor entities. This wide range of published p16 positivity rates makes it difficult to assess the potential significance of p16 immunohistochemistry in individual tumor entities and may also be responsible for conflicting data on the potential prognostic and diagnostic relevance of p16 expression in such tumor entities. That our own analyses of associations between clinico-pathological parameters of cancer aggressiveness and p16 expression mostly revealed only weak or even no associations seems to suggest, that p16 overexpression is not a feature that is dramatically linked to lethal cancer cell properties.

Fig 5. Graphical representation of p16 data from this study (marked with a cross) in comparison with data from existing literature (marked with dots).

Fig 5

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In order to simplify the figure the percentage of weak, moderate and strong staining was merged. Red dots are used for previous studies involving 1–9 cases, yellow dots for studies involving 10–50 cases and green dots for studies involving >50 cases. All studies are quoted in a list of references in S1 Table.

In summary, these results provide a comprehensive overview on p16 expression in human normal tissues and cancers. The absence of a significant p16 expression in most normal tissues in combination with a high frequency of p16 overexpression in cancers of all types demonstrates a significant role of p16 in cancer biology and suggest a general utility of p16 immunohistochemistry as a potential aid to diagnose malignancy. The lack of striking associations of p16 immunostaining with clinico-pathological parameters for cancer aggressiveness in most analyzed cancer types argues against a major prognostic impact of p16 protein expression, however.

Supporting information

S1 Table. List of studies used to generate Fig 5.

(XLSX)

Click here for additional data file. (29.5KB, xlsx)
S2 Table. p16 positive and p16 negative normal tissues and associated tumor types.

(XLSX)

Click here for additional data file. (15KB, xlsx)

Acknowledgments

We are grateful to Melanie Witt, Inge Brandt, Sünje Seekamp, Maren Eisenberg, Gabriele Rieck, Sina Dietrich, Jana Hagemann and Tessa-Ann Saggau

for excellent technical support.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

The author(s) received no specific funding for this work.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. List of studies used to generate Fig 5.

(XLSX)

Click here for additional data file. (29.5KB, xlsx)
S2 Table. p16 positive and p16 negative normal tissues and associated tumor types.

(XLSX)

Click here for additional data file. (15KB, xlsx)

Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

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