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. 2021 Sep 24;480(2):433–447. doi: 10.1007/s00428-021-03204-4

Cytokeratin 5 and cytokeratin 6 expressions are unconnected in normal and cancerous tissues and have separate diagnostic implications

Cosima Völkel 1, Noémi De Wispelaere 1,2, Sören Weidemann 1, Natalia Gorbokon 1, Maximilian Lennartz 1, Andreas M Luebke 1, Claudia Hube-Magg 1, Martina Kluth 1, Christoph Fraune 1, Katharina Möller 1, Christian Bernreuther 1, Patrick Lebok 1, Till S Clauditz 1, Frank Jacobsen 1, Guido Sauter 1, Ria Uhlig 1, Waldemar Wilczak 1, Stefan Steurer 1, Sarah Minner 1, Rainer H Krech 3, David Dum 1, Till Krech 1,3, Andreas H Marx 1,4, Ronald Simon 1,✉, Eike Burandt 1, Anne Menz 1
PMCID: PMC8986736  PMID: 34559291

Abstract

Cytokeratins (CKs) 5 and 6 are functionally unrelated but often analyzed together using bispecific antibodies in diagnostic immunohistochemistry. To better understand the diagnostic utility of CK5 or CK6 alone, tissue microarrays with > 15,000 samples from 120 different tumor types as well as 608 samples of 76 different normal tissues were analyzed by immunohistochemistry. In normal tissues, both CKs occurred in the squamous epithelium; CK5 dominated in basal and CK6 in suprabasal layers. CK5 (not CK6) stained basal cells in various other organs. Within tumors, both CK5 and CK6 were seen in > 95% of squamous cell carcinomas, but other tumor entities showed different results: CK5 predominated in urothelial carcinoma and mesothelioma, but CK6 in adenocarcinomas. Joint analysis of both CK5 and CK6 obscured the discrimination of epithelioid mesothelioma (100% positive for CK5 alone and for CK5/6) from adenocarcinoma of the lung (12.8% positive for CK5 alone; 23.7% positive for CK5/6). CK5 and CK6 expressions were both linked to high grade, estrogen receptor, and progesterone receptor negativity in breast cancer (p < 0.0001 each), grade/stage progression in urothelial cancer (p < 0.0001), and RAS mutations in colorectal cancer (p < 0.01). Useful diagnostic properties which are commonly attributed to CK5/6 antibodies such as basal cell staining in the prostate, distinction of adenocarcinoma of the lung from squamous cell carcinoma and epithelioid mesothelioma, and identification of basal-type features in urothelial cancer are solely driven by CK5. At least for the purpose of distinguishing thoracic tumors, monospecific CK5 antibodies may be better suited than bispecific CK5/6 antibodies.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00428-021-03204-4.

Keywords: Cytokeratin 5, Cytokeratin 6, Tissue microarray, Immunohistochemistry, Diagnostic

Introduction

Cytokeratins 5 and 6 are basic type II cytokeratins which are not functionally related [1]. Cytokeratin 5 (CK5) forms heterodimers with cytokeratin 14, and cytokeratin 6 (CK6) forms heterodimers with cytokeratin 16 [2, 3]. However, cytokeratins 5 and 6 are often jointly examined by immunohistochemistry because common antibodies recognize both cytokeratins 5 and 6 and the use of these bispecific antibodies has clinical utility [4–6]. Cytokeratin 5/6 (CK5/CK6) antibodies are for example applied to identify basal cells or myoepithelial cells for ruling out invasive breast and prostate cancer, to detect squamous cell origin in poorly differentiated carcinomas [6], and to distinguish epithelioid mesothelioma (mostly CK5/6 positive) from lung adenocarcinoma (mostly CK5/6 negative) [5]. CK5/6 immunostaining has also been proposed to have prognostic utility in triple-negative breast cancer [7–9], urothelial carcinoma [10–12], and other tumors [13–15].

Most previous immunohistochemical studies on the diagnostic and prognostic role of CK5 and CK6 have employed antibodies directed against both proteins. Studies analyzing either CK5 or CK6 alone are limited to less than 100 but the results were still partly conflicting. For example, CK5 positivity has been described in 13.6 to 91% of bladder carcinomas [11, 16], 2.5 to 100% of breast carcinomas [17, 18], 59.5 to 100% of head and neck carcinoma [14, 19], 0 to 100% of lung carcinomas [20–25] and 74.8 to 93.8% of mesothelioma carcinomas [26–28]. Even less is known about CK6 positivity alone, which has been reported to occur in 18% of endometrial stromal sarcomas [29], 28% of gastric cancers [30], 38% of basal cell carcinomas of the skin [31], and 100% of squamous cell cancers of the head and neck [32, 33].

To better understand the clinical utility of immunohistochemical analysis of CK5 and CK6 alone, both proteins were analyzed in more than 14,000 tumor tissue samples from 120 different tumor types and subtypes as well as 76 non-neoplastic tissue categories by immunohistochemistry (IHC) in a tissue microarray (TMA) format in this study.

Materials and methods

Tissue microarrays (TMAs)

Our normal tissue TMA was composed of 8 samples from 8 different donors for each of 76 different normal tissue types (608 samples on one slide). The cancer TMAs contained a total of 15,966 primary tumors from 120 tumor types and subtypes. Histopathological data on pathological tumor stage (pT), histological grade, and pathological lymph node status (pN) were available from up to 2,075 breast, 1,663 bladder, 327 gastric, 598 pancreatic, 2,351 colorectal, 524 ovarian, and 259 endometrial cancers. Clinical follow-up data were available from 1,183 breast cancer and 254 urinary bladder cancer patients with a median follow-up time of 49/14 months (range 1–88/1–77). Molecular data on HER2, estrogen receptor (ER), and progesterone receptor (PR) status, microsatellite instability (MSI), and RAS mutations were available from previous studies [34, 35]. The composition of both normal and cancer TMAs is described in the results section. All samples were from the archives of the Institutes of Pathology, University Hospital of Hamburg, Germany, the Institute of Pathology, Clinical Center Osnabrueck, Germany, and Department of Pathology, Academic Hospital Fuerth, Germany. Tissues were fixed in 4% buffered formalin and then embedded in paraffin. The TMA manufacturing process was described earlier in detail [36, 37]. In brief, one tissue spot (diameter: 0.6 mm) was transmitted from a representative cancer-containing donor block in an empty recipient paraffin block. The use of archived remnants of diagnostic tissues for manufacturing of TMAs and their analysis for research purposes as well as patient data analysis were according to local laws (HmbKHG, §12) and approved 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 1 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 min 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; #52,023) for 10 min. Primary antibodies specific for CK5 protein (mouse monoclonal, clone MSVA-605 M) and CK6 protein (rabbit recombinant, clone MSVA-606R), both from MS Validated Antibodies, Hamburg, Germany were applied at a dilution of 1:150 each at 37 °C for 60 min. Bound antibody was then visualized using the EnVision Kit™ (Agilent, CA, USA; #K5007) according to the manufacturer’s directions. 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 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 or 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.

Statistics

Statistical calculations were performed with JMP 14 software (SAS Institute Inc., NC, USA). Contingency tables and the chi2-test were performed to search for associations between CK5 or CK6 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 12,525 (78.5%) of 15,966 tumor samples were interpretable for CK5 and 12,898 (80.8%) of 15,966 tumor samples were interpretable for CK6 in this TMA analysis. The remaining samples were not analyzable due to the lack of unequivocal tumor cells or loss of the tissue spot during the technical procedures. In the normal tissue TMA, a sufficient number of samples was always interpretable per tissue type to determine CK5 and CK6 expressions.

CK5 in normal tissues

Strong CK5 immunostaining was seen in all keratinizing and non-keratinizing squamous epithelia (Fig. 1A) with a predominance of the staining in the basal cells of the epidermis, hair follicles, sebaceous glands, all epithelial cells of tonsil crypts, and of the thymus (Fig. 1B). In the urothelium, only the basal cell layers stained CK5 positive (Fig. 1C). CK5 immunostaining was also seen in myoepithelial cells and basal cells of excretion ducts of salivary and bronchial glands, basal cells of the prostate (Fig. 1D), seminal vesicle, respiratory epithelium, endocervix (not all glands), columnar cells (not all), and basal cells of the epididymis, myoepithelial cells of the breast. Amnion and chorion cells of the placenta also showed strong CK5 staining. CK5 immunostaining was absent in the lung, liver, pancreas, testis, kidney, gastrointestinal epithelial cells, Brunner glands, fallopian tube, adrenal gland, parathyroid gland, brain, adeno- and neurohypophysis, spleen, lymph node, all hematopoetic cell types, and all mesenchymal tissues.

Fig. 1.

Fig. 1

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CK5 and CK6 immunostaining of normal tissues. The panels show for CK5 a strong staining of all cell layers of squamous epithelium of the uterine cervix (A), virtually all thymic epithelial cells (B), the basal cell layer of the urothelium (C), and prostate gland basal cells (D). Although CK6 immunostaining did often involve similar tissues as seen for CK5, CK6 staining was largely lacking in the basal cell layer of the squamous epithelium of the uterine cervix (E), thymic epithelial cells except corpuscles of Hassal’s (F), the urothelium (G), and basal cells of the prostate (H). The images A–D and E–H were taken from consecutive tissue sections

CK6 in normal tissues

A preferential immunostaining in squamous epithelial cells was also seen for CK6 but the staining was most intense in suprabasal cell layers and basal cells were negative or only weakly stained (Fig. 1E). A strong CK6 positivity was also seen in hair follicles, sebaceous glands, a fraction of the squamous cells in tonsil crypts, corpuscles of Hassall’s (but not in other epithelial cells) of the thymus (Fig. 1F), intercalated ducts of salivary and bronchial glands, basal cells and sometimes also ciliated cells (but not ciliae) of respiratory epithelium, few scattered endometrial cells (only in few samples), and in amnion/chorion cells (but not trophoblastic cells) of the placenta. CK6 immunostaining was absent in the urothelium (Fig. 1G), lung, liver, pancreas, prostate (Fig. 1H), seminal vesicle, epididymis, testis, kidney, gastrointestinal epithelial cells, Brunner glands, fallopian tube, adrenal gland, thyroid, parathyroid gland, brain, adeno- and neurohypophysis, spleen, lymph node, all hematopoietic cell types, and all mesenchymal tissues.

CK5 and CK6 in tumors

Our tumor analysis revealed CK5 positivity in 21.1% (4.4% weak, 2.7% moderate, 14.0% strong) and CK6 positivity in 22.8% (8.4% weak, 4.1% moderate, 10.4% strong) of tumors. Of 2,920 CK5 and/or CK6 positive tumors, 1,921 (66%) showed staining for both CK5 and CK6, 461 (16%) stained positive only for CK5, and 538 (18%) showed positivity for CK6 alone. A detailed description of the results for CK5 and CK6 is given in Table 1. Representative images are shown in Fig. 2. Both antibodies showed positive staining in > 95% of squamous cell carcinomas from various origins. For most other tumor entities, CK5 and CK6 showed different positivity rates. CK5 was the predominant staining in mesothelioma, basal cell carcinoma of the skin, urothelial carcinoma, thymoma, and salivary gland tumors, while CK6 predominated in various adenocarcinomas. It was noticeable for all tumor entities that either CK5 or CK6 was the predominant staining and that adding the second cytokeratin did not relevantly increase the fraction of positive cases (Supplementary Fig. 1). A separate analysis of thoracic tumors (mesothelioma vs. squamous cell carcinoma of the lung vs. adenocarcinoma of the lung) revealed that the combined use of both CK5 and CK6 immunostaining hindered the discrimination of these tumors because the positivity rate of lung adenocarcinomas increased from 12.8% (CK5 alone) to 23.7% (CK5 and/or CK6 positive) while both squamous cell carcinomas and epithelioid mesotheliomas were positive in 100% irrespective of whether CK5 alone or CK5 and CK6 were jointly applied (Fig. 3).

Table 1.

CK5, CK6, and CK5/CK5 immunostaining in human tumors (*n analyzable for CK5 and CK6; w, weak; m, moderate; s, strong; neg, negative; pos positive)

Tumor entity CK5 CK6 CK5/CK6
on TMA (n) n* w (%) m (%) s (%) w (%) m (%) s (%) CK5 neg / CK6 pos CK5 pos / CK6 neg CK5 pos / CK6 pos
Tumors of the skin
  Pilomatrixoma 35 22 8.7 26.1 8.7 9.7 12.9 0.0 0.0 18.2 22.7
  Basal cell carcinoma 88 37 0.0 0.0 100.0 41.8 44.8 7.5 0.0 10.8 89.2
  Benign nevus 29 25 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Squamous cell carcinoma of the skin 90 86 0.0 1.1 97.7 1.2 0.0 96.5 0.0 1.2 97.7
  Malignant melanoma 48 43 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Merkel cell carcinoma 46 31 6.5 0.0 0.0 0.0 0.0 0.0 0.0 6.5 0.0
Tumors of the head and neck
  Squamous cell carcinoma of the larynx 110 95 1.0 2.0 96.0 3.1 2.1 92.8 1.1 2.1 96.8
  Squamous cell carcinoma of the pharynx 60 51 1.8 0.0 96.4 3.6 12.5 83.9 2.0 0.0 98.0
  Oral squamous cell carcinoma (floor of the mouth) 130 120 1.6 0.8 96.0 4.8 5.6 88.0 0.8 0.8 97.5
  Pleomorphic adenoma of the parotid gland 50 20 6.1 6.1 72.7 25.8 25.8 0.0 5.0 45.0 35.0
  Warthin tumor of the parotid gland 49 39 0.0 52.1 47.9 56.3 33.3 6.3 0.0 5.1 94.9
  Basal cell adenoma of the salivary gland 15 10 6.7 0.0 93.3 26.7 33.3 20.0 0.0 30.0 70.0
Tumors of the lung, pleura, and thymus
  Adenocarcinoma of the lung 246 156 9.1 3.6 2.4 16.9 5.6 6.8 10.9 0.0 12.8
  Squamous cell carcinoma of the lung 130 68 0.0 1.4 97.3 2.8 4.2 87.5 0.0 1.5 97.1
  Small cell carcinoma of the lung 20 14 7.1 7.1 0.0 0.0 14.3 0.0 0.0 0.0 14.3
  Mesothelioma, epithelioid 39 17 5.3 10.5 84.2 20.0 10.0 20.0 0.0 52.9 47.1
  Mesothelioma, other types 76 35 5.3 7.9 52.6 39.1 0.0 0.0 2.9 22.9 40.0
  Thymoma 29 21 0.0 3.7 92.6 15.4 19.2 3.8 0.0 76.2 23.8
Tumors of the female genital tract
  Squamous cell carcinoma of the vagina 78 66 1.4 0.0 98.6 5.8 4.3 88.4 0.0 0.0 100.0
  Squamous cell carcinoma of the vulva 130 116 0.0 0.8 99.2 0.8 2.5 96.6 0.0 0.0 100.0
  Squamous cell carcinoma of the cervix 130 111 0.0 2.5 95.0 1.7 8.3 86.8 0.0 0.0 97.3
  Endometrioid endometrial carcinoma 236 183 23.8 11.6 4.2 15.6 14.2 2.2 3.8 14.2 24.6
  Endometrial serous carcinoma 82 55 16.9 6.8 0.0 14.7 8.8 1.5 7.3 7.3 18.2
  Carcinosarcoma of the uterus 48 37 21.1 10.5 13.2 13.0 15.2 2.2 0.0 16.2 27.0
  Endometrial carcinoma, high grade, G3 13 11 8.3 8.3 8.3 8.3 8.3 8.3 0.0 9.1 18.2
  Endometrial clear cell carcinoma 8 4 25.0 25.0 0.0 50.0 12.5 0.0 0.0 0.0 50.0
  Endometrioid carcinoma of the ovary 110 72 25.3 8.9 8.9 20.3 10.1 2.5 5.6 18.1 25.0
  Serous carcinoma of the ovary 559 355 23.6 6.2 8.3 32.6 5.8 1.1 12.1 13.2 25.1
  Mucinous carcinoma of the ovary 96 69 2.8 1.4 2.8 7.0 1.4 1.4 4.3 1.4 4.3
  Clear cell carcinoma of the ovary 50 37 5.3 2.6 0.0 17.9 0.0 0.0 16.2 5.4 2.7
  Carcinosarcoma of the ovary 47 29 3.2 9.7 9.7 7.0 4.7 7.0 3.4 10.3 10.3
  Brenner tumor 9 8 12.5 37.5 25.0 0.0 0.0 12.5 0.0 62.5 12.5
Tumors of the breast
  Invasive breast carcinoma of no special type 1391 930 3.9 1.7 4.4 7.8 6.1 3.4 8.1 1.7 8.0
  Lobular carcinoma of the breast 294 171 1.6 0.0 1.6 3.7 1.6 1.1 2.3 0.6 2.3
  Medullary carcinoma of the breast 26 18 20.0 0.0 20.0 0.0 9.1 4.5 0.0 33.3 5.6
  Tubular carcinoma of the breast 27 13 13.3 0.0 0.0 0.0 0.0 0.0 0.0 15.4 0.0
  Mucinous carcinoma of the breast 58 37 2.6 0.0 0.0 2.6 0.0 0.0 0.0 0.0 2.7
  Phyllodes tumor of the breast 50 41 23.3 11.6 9.3 26.1 6.5 4.3 9.8 17.1 26.8
Tumors of the digestive system
  Adenomatous polyp, low-grade dysplasia 50 38 0.0 0.0 0.0 10.5 2.6 0.0 13.2 0.0 0.0
  Adenomatous polyp, high-grade dysplasia 50 47 0.0 0.0 0.0 6.4 0.0 0.0 6.4 0.0 0.0
  Adenocarcinoma of the colon 1932 1520 2.9 1.1 0.4 13.4 3.6 1.9 12.2 0.3 3.8
  Gastric adenocarcinoma, diffuse type 226 120 0.0 0.0 0.7 4.5 1.5 2.3 8.3 0.0 0.8
  Gastric adenocarcinoma, intestinal type 224 142 7.3 4.6 4.6 12.6 7.9 7.9 12.0 1.4 14.1
  Gastric adenocarcinoma, mixed type 62 39 2.3 2.3 0.0 11.4 9.1 4.5 20.5 0.0 2.6
  Adenocarcinoma of the esophagus 133 75 5.0 5.0 1.3 11.5 3.8 5.1 8.0 0.0 10.7
  Squamous cell carcinoma of the esophagus 124 58 1.5 1.5 94.1 4.5 10.6 81.8 0.0 0.0 98.3
  Squamous cell carcinoma of the anal canal 91 68 0.0 1.4 97.3 5.5 4.1 90.4 0.0 0.0 100.0
  Cholangiocarcinoma 114 96 7.8 3.9 6.8 7.7 4.8 9.6 4.2 1.0 14.6
  Hepatocellular carcinoma 50 47 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Ductal adenocarcinoma of the pancreas 662 447 12.5 5.7 14.5 27.4 13.7 16.3 20.4 0.2 31.3
  Pancreatic/Ampullary adenocarcinoma 119 77 17.9 5.1 5.1 24.4 8.5 4.9 13.0 5.2 22.1
  Acinar cell carcinoma of the pancreas 16 15 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Gastrointestinal stromal tumor (GIST) 50 47 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Tumors of the urinary system
  Non-invasive papillary urothelial ca., pTa G2 low grade 177 115 31.1 23.8 32.8 6.7 4.5 2.2 0.0 75.7 12.2
  Non-invasive papillary urothelial ca., pTa G2 high grade 141 85 27.8 25.6 18.9 14.4 7.7 2.9 0.0 48.2 22.4
  Non-invasive papillary urothelial ca., pTa G3 187 85 17.4 17.4 14.1 14.7 3.9 2.0 1.2 29.4 18.8
  Urothelial carcinoma, pT2-4 G3 1207 635 7.9 7.3 43.8 13.6 5.5 27.9 1.7 12.9 45.5
  Small cell neuroendocrine carcinoma of the bladder 18 13 0.0 0.0 0.0 0.0 6.3 0.0 0.0 0.0 0.0
  Clear cell renal cell carcinoma 1226 794 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.1 0.0
  Papillary renal cell carcinoma 320 220 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Clear cell (tubulo) papillary renal cell carcinoma 28 18 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Chromophobe renal cell carcinoma 151 102 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Oncocytoma 199 145 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Tumors of the male genital organs
  Adenocarcinoma of the prostate, Gleason 3 + 3 83 83 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Adenocarcinoma of the prostate, Gleason 4 + 4 80 80 0.0 0.0 0.0 1.3 0.0 0.0 1.3 0.0 0.0
  Adenocarcinoma of the prostate, Gleason 5 + 5 85 84 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Adenocarcinoma of the prostate (recurrence) 261 244 0.0 0.0 0.4 0.0 1.2 0.0 0.8 0.0 0.0
  Small cell neuroendocrine carcinoma of the prostate 17 12 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Seminoma 621 594 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Embryonal carcinoma of the testis 50 21 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Yolk sac tumor 50 32 0.0 0.0 0.0 3.0 0.0 0.0 3.1 0.0 0.0
  Teratoma 50 20 3.7 7.4 25.9 7.4 11.1 11.1 0.0 15.0 15.0
  Squamous cell carcinoma of the penis 80 77 0.0 0.0 100.0 3.8 1.3 94.9 0.0 0.0 100.0
Tumors of endocrine organs
  Adenoma of the thyroid gland 114 108 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Papillary thyroid carcinoma 392 365 0.0 0.0 0.0 1.1 0.5 0.0 1.1 0.0 0.0
  Follicular thyroid carcinoma 158 144 0.0 0.0 0.0 0.0 0.0 0.7 0.0 0.0 0.0
  Medullary thyroid carcinoma 107 95 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Anaplastic thyroid carcinoma 45 40 2.4 0.0 17.1 0.0 0.0 17.1 0.0 2.5 17.5
  Adrenal cortical adenoma 50 27 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Adrenal cortical carcinoma 26 23 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Phaeochromocytoma 50 43 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Appendix, neuroendocrine tumor (NET) 22 11 8.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Colorectal, neuroendocrine tumor (NET) 11 11 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Ileum, neuroendocrine tumor (NET) 49 41 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Lung, neuroendocrine tumor (NET) 19 18 5.6 0.0 0.0 5.6 0.0 0.0 5.6 5.6 0.0
  Pancreas, neuroendocrine tumor (NET) 98 79 0.0 0.0 1.2 1.3 3.8 0.0 3.8 1.3 0.0
  Colorectal, neuroendocrine carcinoma (NEC) 12 7 0.0 0.0 10.0 0.0 0.0 0.0 0.0 14.3 0.0
  Gallbladder, neuroendocrine carcinoma (NEC) 4 4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Pancreas, neuroendocrine carcinoma (NEC) 14 10 0.0 0.0 0.0 9.1 0.0 0.0 10.0 0.0 0.0
Tumors of hematopoietic and lymphoid tissues
  Hodgkin Lymphoma 103 92 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Non-Hodgkin Lymphoma 62 58 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Small lymphocytic lymphoma, B-cell type (B-SLL/B-CLL) 50 47 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Diffuse large B-cell lymphoma (DLBCL) 114 109 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Follicular lymphoma 88 86 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  T-cell non-Hodgkin lymphoma 24 22 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Mantle cell lymphoma 18 18 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Marginal zone lymphoma 16 14 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Diffuse large B-cell lymphoma (DLBCL) in the testis 16 16 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Burkitt lymphoma 5 3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Tumors of soft tissue and bone
  Tenosynovial giant cell tumor 45 37 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Granular cell tumor 53 36 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Leiomyoma 50 45 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Leiomyosarcoma 87 80 1.3 0.0 0.0 0.0 0.0 0.0 0.0 1.3 0.0
  Liposarcoma 132 99 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Malignant peripheral nerve sheath tumor (MPNST) 13 12 8.3 0.0 0.0 0.0 0.0 0.0 0.0 8.3 0.0
  Myofibrosarcoma 26 23 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Angiosarcoma 73 49 0.0 0.0 0.0 1.9 0.0 0.0 0.0 0.0 0.0
  Angiomyolipoma 91 68 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Dermatofibrosarcoma protuberans 21 13 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Ganglioneuroma 14 12 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Kaposi sarcoma 8 3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Neurofibroma 117 107 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Sarcoma, not otherwise specified (NOS) 75 63 1.5 0.0 0.0 0.0 0.0 0.0 0.0 1.6 0.0
  Paraganglioma 41 40 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Ewing sarcoma 23 9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Rhabdomyosarcoma 7 5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Schwannoma 121 118 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Synovial sarcoma 12 8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Osteosarcoma 43 27 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  Chondrosarcoma 38 9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
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Fig. 2.

Fig. 2

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CK5 and CK6 immunostaining in cancer. For CK5, the panels show a strong staining of all epithelial cells of a malignant mesothelioma (A), a lack of staining in an adenocarcinoma of the lung (B), a “basal cell type” staining pattern in a non-invasive papillary (grade 2) urothelial carcinoma (C), and a diffuse positivity of all cells of an invasive urothelial carcinoma (D). The panels E–H show CK6 staining of consecutive tissue sections of the samples A–D. They show a lack of CK6 staining in a malignant mesothelioma (E), a focal positivity in an adenocarcinoma of the lung (F), absence of staining in a non-invasive papillary urothelial carcinoma (G), and a diffuse positivity of all cells of an invasive urothelial carcinoma (H)

Fig. 3.

Fig. 3

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Obstructive role of CK6 for differential diagnosis of lung adenocarcinomas and lung squamous cell cancers or mesotheliomas

A comparison with histopathological features of cancer aggressiveness and/or clinical follow-up data in 120 different tumor entities revealed only few statistical associations (Table 2). Both CK5 and CK6 expressions were linked to high grade, estrogen and progesterone receptor negativity, and “triple negativity” in breast cancer (p < 0.0001 each), grade/stage progression in urothelial cancer (p < 0.0001), and RAS mutations in colorectal cancer (p < 0.01). CK5 expression was also associated with nodal metastasis in urothelial cancer (p = 0.0007) while CK6 expression was linked to nodal metastasis in gastric and ovarian cancer (p < 0.05). The CK5 and CK6 expression status was unrelated to overall patient survival in breast cancer (Supplementary Fig. 2a–c) and recurrence-free survival in patients treated by cystectomy for their urothelial carcinomas (Supplementary Fig. 2e–g).

Table 2.

CK5 and CK6 immunostaining and tumor phenotype

CK5 CK6
n Neg. (%) Weak (%) Mod. (%) Strong (%) p n Neg. (%) Weak (%) Mod. (%) Strong (%) p
Breast cancer of no special type Tumor stage pT1 454 92.3 3.1 1.3 3.3 0.2082 479 84.1 7.3 5.8 2.7 0.5290
pT2 351 90.0 2.8 1.4 5.7 370 81.6 8.1 6.2 4.1
pT3-4 77 85.7 9.1 1.3 3.9 82 89.0 3.7 6.1 1.2
Grade G1 141 98.6 1.4 0.0 0.0  < 0.0001 151 91.4 5.3 3.3 0.0  < 0.0001
G2 442 95.7 1.6 1.4 1.4 467 85.7 6.0 6.4 1.9
G3 325 80.0 7.1 2.5 10.5 344 75.9 10.8 6.7 6.7
Nodal stage pN0 364 90.7 2.2 1.9 5.2 0.5639 379 83.6 8.2 4.5 3.7 0.9611
pN1 185 89.7 4.3 1.1 4.9 199 82.4 9.0 5.0 3.5
pN2 57 94.7 1.8 1.8 1.8 64 84.4 6.3 7.8 1.6
pN3 45 86.7 6.7 0.0 6.7 46 82.6 10.9 4.3 2.2
HER2 status Negative 736 90.1 3.7 1.8 4.5 0.5951 779 82.4 6.9 6.9 3.7 0.1808
Positive 101 94.1 2.0 1.0 3.0 110 88.2 7.3 2.7 1.8
ER status Negative 167 54.5 16.8 7.8 21.0  < 0.0001 185 54.1 18.4 11.9 15.7  < 0.0001
Positive 633 99.7 0.3 0.0 0.0 662 90.2 4.2 5.1 0.5
PR status Negative 327 76.8 8.3 4.0 11.0  < 0.0001 349 72.8 11.2 7.7 8.3  < 0.0001
Positive 503 99.0 0.6 0.2 0.2 528 89.2 4.7 5.5 0.6
Triple negative No 664 98.6 0.6 0.2 0.6  < 0.0001 699 89.0 5.0 5.3 0.7  < 0.0001
Yes 115 41.7 21.7 10.4 26.1 126 44.4 20.6 14.3 20.6
Urinary bladder cancer Tumor stage pTa G2 low 122 12.3 31.1 23.8 32.8  < 0.0001 134 86.6 6.7 4.5 2.2  < 0.0001
pTa G2 high 90 27.8 27.8 25.6 18.9 104 75.0 14.4 7.7 2.9
pTa G3 92 51.1 17.4 17.4 14.1 102 79.4 14.7 3.9 2.0
pT2 125 40.8 7.2 7.2 44.8 0.2338 138 51.4 15.2 5.8 27.5 0.6770
pT3 218 37.2 8.3 4.6 50.0 239 47.7 15.5 5.4 31.4
pT4 104 47.1 9.6 8.7 34.6 112 57.1 16.1 3.6 23.2
Nodal stage pN0 268 32.5 9.3 7.1 51.1 0.0007 295 48.5 15.9 5.4 30.2 0.5191
pN +  170 51.8 6.6 7.2 34.3 170 54.2 16.2 5.6 24.0
Endometroid endometrial carcinoma Tumor stage pT1 93 59.1 28.0 9.7 3.2 0.2312 114 67.5 14.9 14.9 2.6 0.4008
pT2 20 50.0 15.0 30.0 5.0 24 50.0 20.8 29.2 0.0
pT3-4 28 67.9 17.9 7.1 7.1 35 62.9 20.0 17.1 0.0
Nodal stage pN0 46 60.9 21.7 15.2 2.2 0.9774 50 66.0 12.0 22.0 0.0 0.6323
pN +  20 65.0 25.0 0.0 10.0 30 60.0 20.0 20.0 0.0
CK5 CK6
n Neg. (%) Weak (%) Mod. (%) Strong (%) p n Neg. (%) Weak (%) Mod. (%) Strong (%) p
Endometrioid ovarian cancer Tumor stage pT1 21 52.4 28.6 14.3 4.8 0.4956 22 63.6 22.7 13.6 0.0 0.2097
pT2 6 66.7 16.7 16.7 0.0 5 60.0 0.0 20.0 20.0
pT3 3 66.7 0.0 0.0 33.3 4 50.0 25.0 0.0 25.0
Nodal stage pN0 21 57.1 28.6 9.5 4.8 0.7389 21 57.1 28.6 14.3 0.0 0.0281
pN1 6 50.0 16.7 16.7 16.7 6 50.0 0.0 16.7 33.3
Serous ovarian cancer Tumor stage pT1 25 56.0 20.0 12.0 12.0 0.6695 28 60.7 32.1 7.1 0.0 0.4721
pT2 30 66.7 26.7 3.3 3.3 40 47.5 50.0 2.5 0.0
pT3 193 63.2 23.3 4.7 8.8 223 61.4 33.2 4.5 0.9
Nodal stage pN0 61 72.1 19.7 1.6 6.6 0.2458 73 61.6 38.4 0.0 0.0 0.0244
pN1 125 59.2 24.0 5.6 11.2 143 58.0 34.3 6.3 1.4
Stomach cancer Tumor stage pT1-2 49 100.0 0.0 0.0 0.0 0.0663 48 85.4 12.5 0.0 2.1 0.2234
pT3 90 87.8 5.6 4.4 2.2 89 77.5 11.2 5.6 5.6
pT4 87 92.0 3.4 1.1 3.4 87 80.5 6.9 6.9 5.7
Nodal stage pN0 58 94.8 3.4 1.7 0.0 0.3701 54 88.9 9.3 1.9 0.0 0.0380
pN +  167 91.0 3.6 2.4 3.0 168 78.0 9.5 6.0 6.5
Pancreatic adenocarcinomas Tumor stage pT1 15 66.7 20 0 13.3 0.075 13 46.2 30.8 23.1 0 0.5751
pT2 62 59.7 11.3 16.1 12.9 67 37.3 29.9 13.4 19.4
pT3 340 67.4 13.5 3.2 15.9 369 44.2 25.7 13.8 16.3
pT4 25 60 16 8 16 28 42.9 21.4 21.4 14.3
Grade 1 15 86.7 6.7 0 6.7 0.4984 16 68.8 18.8 6.3 6.3 0.2057
2 313 65.2 14.4 5.1 15.3 334 44 26 13.5 16.5
3 95 61.1 13.7 6.3 18.9 106 34.9 27.4 18.9 18.9
Nodal stage pN0 99 65.7 13.1 5.1 16.2 0.9962 102 41.2 30.4 12.7 15.7 0.7194
pN +  342 66.1 13.5 5.3 15.2 374 43.9 24.9 15 16.3
Colon adenocarcinoma Tumor stage pT1 56 100.0 0.0 0.0 0.0 0.3002 58 87.9 8.6 1.7 1.7 0.2232
pT2 304 97.0 1.6 1.0 0.3 313 78.9 17.6 2.6 1.0
pT3 833 95.2 3.5 1.0 0.4 881 81.6 12.5 4.1 1.8
pT4 303 95.0 2.3 2.0 0.7 314 82.8 11.1 3.5 2.5
Nodal stage pN0 786 95.4 3.1 1.1 0.4 0.9083 817 84.0 11.4 3.1 1.6 0.0675
pN +  698 96.0 2.4 1.1 0.4 737 78.7 15.1 4.2 2.0
Vessel invasion V0 1083 95.3 3.2 1.1 0.4 0.3112 1145 81.3 13.1 3.8 1.8 0.9808
V +  390 96.9 1.5 1.0 0.5 400 82.3 12.5 3.5 1.8
Lymph invasion L0 579 95.7 2.9 0.5 0.9 0.0559 596 81.9 12.8 3.4 2.0 0.8876
L1 877 95.8 2.7 1.4 0.1 930 81.4 13.1 3.9 1.6
Tumor localization Left colon 1110 96.4 2.0 1.2 0.5 0.0646 1159 83.2 11.7 3.5 1.6 0.0670
Right colon 389 94.1 4.6 1.0 0.3 413 77.2 16.5 4.1 2.2
MMR status Defective 83 100.0 0.0 0.0 0.0 0.0449 80 86.3 12.5 1.3 0.0 0.1937
Proficient 1057 95.1 3.2 1.3 0.4 1106 80.7 13.9 3.7 1.7
RAS mutation status Mutated 329 93.9 3.6 1.5 0.9 0.0054 343 75.2 17.2 4.7 2.9 0.0042
Wildtype 434 98.2 0.9 0.9 0.0 446 85.2 9.9 3.6 1.3
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Discussion

The successful analysis of a broad range of normal tissues and of more than 10,000 cancers for CK5 and CK6 by immunohistochemistry demonstrates important differences in the expression patterns of these cytokeratins. The data collected in this study suggest a superior diagnostic utility of monospecific CK5 or CK6 antibodies for immunohistochemical analysis as compared to bispecific CK5/6 antibodies. Additionally, literature data from various tumor types on CK5 expression (Supplementary Fig. 3) or CK6 expression (Supplementary Fig. 4) clearly demonstrate that such information cannot be easily obtained from the literature due to highly discrepant data across many studies.

That cytokeratins 5 and 6 have very different expression patterns is particularly demonstrated by the results of our extensive normal tissue analysis. Although both proteins were found in the majority of squamous epithelia, their staining patterns differed considerably with CK5 preferentially staining basal cells and CK6 preferably occurring in suprabasal layers. Given the complementary staining patterns of CK5 (basal) and CK6 (suprabasal), one might expect that the combined use of both antibodies could improve the positivity rate in squamous cell carcinomas of different sites of origin. That the combined analysis of CK5 and CK6 only increased the fraction of positive squamous cell carcinomas by 0–1% (average 0.1%) if compared to CK5 analysis alone and by 0–2% (average 0.5%) if compared to CK6 analysis alone does not provide strong evidence for superiority of using a combined CK5/6 antibody for the identification of squamous cell carcinomas, however.

The analysis of 109 non-squamous cell cancer tumor entities also did not suggest a particular rationale for combining anti-CK5 and anti-CK6 antibodies. The separate analysis of these antibodies showed for the vast majority of analyzed cancers that their CK5/6 positivity rate was largely driven by either CK5 or CK6 and that the addition of the other cytokeratin only minimally increased the positivity rate. Moreover, the positivity rate of both CK5 and CK6 was generally so low that the combination of both cytokeratins did still not result in a diagnostically useful information. Whether a tumor entity such as endometrioid carcinoma of the ovary is CK5 positive in 43% or CK5/6 positive in 49% does not impact the diagnostic information obtained by analyzing CK5 and/or CK6.

Especially in the case of thoracic tumors, the isolated CK5 analysis appears to be advantageous as compared to CK5/6 immunohistochemistry. For example, CK5 alone was positive in 100% of epithelioid mesotheliomas but in only 12.8% of lung adenocarcinoma and can thus be used in panels designed to distinguish these entities. Adding CK6 increases the positivity rate in lung adenocarcinoma to 23.7% and thus reduces the diagnostic potential for distinguishing mesothelioma from adenocarcinomas. CK6-positive adenocarcinomas also limit the utility of CK5/6 in the distinction of squamous cell carcinoma vs. adenocarcinoma of the lung which may be difficult and often requires the use of IHC panels. In another major application of CK5/6 IHC—the detection of basal cells in the prostate—CK5 is solely responsible for the beneficial effects, while CK6 is not staining any basal cells.

The availability of clinical follow-up data or histopathological data related to cancer aggressiveness enabled us to investigate the potential role of aberrant CK5 and CK6 expressions in 8 different cancer entities. The findings in bladder cancer further challenged the utility of combined CK5/6 analysis. In normal urothelium, CK5 is expressed in the basal cell layers. Because this basal layer staining is retained in the vast majority of non-invasive urothelial carcinomas, especially if they are of low grade, most pTa grade 1/2 tumors were scored as “weak” or “moderately” CK5 positive in our scoring system. In pTa grade 3 and especially in muscle-invasive urothelial carcinomas, the tumors often either completely lose CK5 expression or show CK5 expression in all cells. As a result, the fraction of CK5 negative and of strongly positive cases increased markedly with bladder cancer grade and stage. A link between CK5 expression and bladder cancer progression was earlier reported by several authors [38–41]. Most of these studies have employed antibodies against CK5/6 and it has been assumed that CK5/6 positivity reflects a “basal-type” molecular subgroup of urothelial carcinoma [42, 43]. Our separate analysis of CK6 revealed, however, that CK6 is unrelated to urothelial basal cells. CK6 upregulation has been considered a feature of squamous cell differentiation in urothelial cancer by others [44, 45].

That both CK5 and CK6 expressions were statistically linked to high grade in breast cancer is consistent with data from various earlier studies [46–48]. Accordingly, CK5/6 expression is an established feature of basal-type breast cancer which is well known for its poor clinical outcome [49]. These findings may reflect a general phenomenon. Multiple studies have described a tendency towards a poor prognosis and/or unfavorable tumor phenotype in cancers that show de novo expression of a cytokeratin which is not expressed in its normal cell type of origin [50–55]. An altered expression pattern of intermediate filaments appears to represent a common feature of cancer cell dedifferentiation that occurs during cancer progression and will thus be linked to unfavorable tumor features [56]. These significant associations with relevant histological tumor aspects were not found in endometrial, ovarian, stomach, pancreatic, and colon cancers, and argue against a major role of CK5 and CK6 expressions for tumor progression. The relationship between CK5 and CK6 expression and KRAS remains unclear as direct or indirect interactions between these proteins are not known and also not expected based on the individual functions of these two proteins.

In summary, our data show that important properties which are commonly attributed to CK5/6 antibodies such as basal cell staining in the prostate, distinction of mesothelioma and squamous cell carcinoma from adenocarcinoma of the lung, and identification of basal-type features in urothelial cancer are solely driven by CK5. At least for the purpose of distinguishing thoracic tumors, monospecific CK5 antibodies may be better suited than bispecific CK5/6 antibodies in diagnostic immunohistochemistry.

Supplementary Information

Below is the link to the electronic supplementary material.

428_2021_3204_MOESM1_ESM.pdf (28.3KB, pdf)

Supplementary file1 Relative importance of CK5 and CK6 in different tumor types. a) Tumor types with a predominant role of CK6. b) Tumor types with a predominant role of CK5. Squamous cell carcinomas are shown in both diagrams. (PDF 28 KB)

428_2021_3204_MOESM2_ESM.pdf (38.1KB, pdf)

Supplementary file2 Prognostic relevance of CK5, CK6 or the joint analysis of CK5 and CK6 in a-c) breast cancers of no special type and d-f) muscle invasive urinary bladder cancers. (PDF 38 KB)

428_2021_3204_MOESM3_ESM.pdf (27.7KB, pdf)

Supplementary file3 Graphical representation of CK5 data from this study (marked by an “x”) in comparison with the previous literature. Red dots are used for studies involving 2–10 cases, orange dots are used for studies involving 11–25 cases, and green dots are used for studies involving >25 cases. (PDF 28 KB)

428_2021_3204_MOESM4_ESM.pdf (23.4KB, pdf)

Supplementary file4 Graphical representation of CK6 data from this study (marked by an “x”) in comparison with the previous literature. Red dots are used for studies involving 5 cases, green dots are used for studies involving >25 cases. Literature data [30] do not disclose the histological subtype of gastric cancers. (PDF 23 KB)

Acknowledgements

We are grateful to Melanie Witt, Inge Brandt, Maren Eisenberg, and Sünje Seekamp for excellent technical assistance.

Author contribution

CV, RS, GS, and AM contributed to the conception, design, data collection, data analysis, and manuscript writing.

NDW, SW, ML, AML, EB, TSC, WW, CB, PL, TK, and SS participated in pathology data analysis and data interpretation.

DD, KM, CF, RU, NG, FJ, and SM contributed to the immunohistochemistry analysis.

RHK, AHM, and TK contributed to the conception and design, and collection of samples.

CHM, MK, and RS performed statistical analysis.

CV, AM, RS, GS contributed to the study supervision.

All authors agree to be accountable for the content of the work.

Funding

Open Access funding enabled and organized by Projekt DEAL.

Availability of data and material

Raw data are available upon reasonable request. All data relevant to the study are included in the article.

Declarations

The usage of archived diagnostic left-over tissues for manufacturing of TMAs and their analysis for research purposes as well as patient data analysis has been approved by local laws (HmbKHG, §12,1) and by the local ethics committee (Ethics Commission Hamburg, WF-049/09). All work has been carried out in compliance with the Helsinki declaration.

Conflict of interest

The CK5 antibody clone MSVA-605 M and the CK6 antibody clone MSVA-606R were provided from MS Validated Antibodies GmbH (owned by a family member of GS).

Footnotes

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Change history

2/23/2022

The Funding information has been added.

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Associated Data

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

Supplementary Materials

428_2021_3204_MOESM1_ESM.pdf (28.3KB, pdf)

Supplementary file1 Relative importance of CK5 and CK6 in different tumor types. a) Tumor types with a predominant role of CK6. b) Tumor types with a predominant role of CK5. Squamous cell carcinomas are shown in both diagrams. (PDF 28 KB)

428_2021_3204_MOESM2_ESM.pdf (38.1KB, pdf)

Supplementary file2 Prognostic relevance of CK5, CK6 or the joint analysis of CK5 and CK6 in a-c) breast cancers of no special type and d-f) muscle invasive urinary bladder cancers. (PDF 38 KB)

428_2021_3204_MOESM3_ESM.pdf (27.7KB, pdf)

Supplementary file3 Graphical representation of CK5 data from this study (marked by an “x”) in comparison with the previous literature. Red dots are used for studies involving 2–10 cases, orange dots are used for studies involving 11–25 cases, and green dots are used for studies involving >25 cases. (PDF 28 KB)

428_2021_3204_MOESM4_ESM.pdf (23.4KB, pdf)

Supplementary file4 Graphical representation of CK6 data from this study (marked by an “x”) in comparison with the previous literature. Red dots are used for studies involving 5 cases, green dots are used for studies involving >25 cases. Literature data [30] do not disclose the histological subtype of gastric cancers. (PDF 23 KB)

Data Availability Statement

Raw data are available upon reasonable request. All data relevant to the study are included in the article.

Articles from Virchows Archiv are provided here courtesy of Springer

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