35946088

Pan-keratin Immunostaining in Human Tumors: A Tissue Microarray Study of 15,940 Tumors

International Journal of Surgical Pathology 2023, Vol. 31(6) 927-938 @ The Author(s) 2022

cc O Article reuse guidelines: sagepub.com/journals-permissions DOI: 10.1177/10668969221117243

journals.sagepub.com/home/ijs

S Sage

Anne Menz, MD1, Natalia Gorbokon, MD1, Florian Viehweger, MD1, Maximilian Lennartz, MD1, Claudia Hube-Magg, MD1, Lisa Hornsteiner, MD1, Martina Kluth, MD1, Cosima Volkel, MD1, Andreas M. Luebke, MD1, Christoph Fraune, MD1, Ria Uhlig, MD1, Sarah Minner, MD1, David Dum, MD1, Doris Höflmayer, MD1, Guido Sauter, MD1, Ronald Simon, MD1 (D, Eike Burandt, MD1 iD, Till S. Clauditz, MD1, Patrick Lebok, MD1, Frank Jacobsen, MD1, Stefan Steurer, MD1, Till Krech, MD2, Andreas H. Marx, MD3, and Christian Bernreuther, MD1

Abstract

To evaluate the efficiency of pan-keratin immunostaining, tissue microarrays of 13,501 tumor samples from 121 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types were analyzed by immunohistochem- istry. In normal tissues, strong pan-keratin immunostaining was seen in epithelial cells. Staining intensity was lower in hepa- tocytes, islets of Langerhans, and pneumocytes but markedly reduced in the adrenal cortex. Pan-keratin was positive in ≥98% of samples in 62 (83%) of 75 epithelial tumor entities, including almost all adenocarcinomas, squamous cell and uro- thelial carcinomas. Only 17 of 121 tumor entities (13%) had a pan-keratin positivity rate between 25% and 98%, including tumors with mixed differentiation, endocrine/neuroendocrine tumors, renal cell carcinomas, adrenocortical tumors, and particularly poorly differentiated carcinoma subtypes. The 15 entities with pan-keratin positivity in 0.9%-25% were mostly of mesenchymal origin. Reduced/absent pan-keratin immunostaining was associated with high UICC stage (p=0.0001), high Thoenes grade (p=0.0183), high Fuhrman grade (p=0.0049), advanced tumor stage (p<0.0001) and lymph node metas- tasis (p=0.0114) in clear cell renal cell carcinoma, advanced pT stage (p=0.0007) in papillary renal cell carcinoma, and with advanced stage (p=0.0023), high grade (p=0.0005) as well as loss of ER and PR expression (each p <0.0001) in invasive breast carcinoma of no special type (NST). In summary, pan-keratin can consistently be detected in the vast majority of epithelial tumors, although pan-keratin can be negative a fraction of renal cell, adrenocortical and neuroendocrine neo- plasms. The data also link reduced pan-keratin immunostaining to unfavorable tumor phenotype in in epithelial neoplasms.

Keywords

pan-cytokeratin, pan-keratin, tissue microarray, immunohistochemistry, cancer

Introduction

Pan-keratin antibodies are mixtures of two or several anti- bodies that detect multiple low and high molecular weight keratins. These antibody cocktails have been designed to immunohistochemically detect all epithelial cell types irrespective of their tissues of origin with one single diag- nostic tool. In surgical pathology they are typically employed to document the epithelial origin of neoplastic or non-neoplastic tissue or for detection of small metasta- ses in lymph nodes. There are, however, limitations to the concept that pan-keratin antibodies stain all epithelial tumors and that non-epithelial tissues are “keratin nega- tive”. For a large variety of different epithelial tumors,

pan-keratin negative tumors have been described 1-6 and “keratin positive” mesenchymal tumors have also been reported across various mesenchymal tumor enti- ties.7-12 The frequencies reported for pan-keratin negative

1Institute of Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

2Institute of Pathology, Clinical Center Osnabrueck, Osnabrueck, Germany

3Department of Pathology, Academic Hospital Fuerth, Fuerth Germany

Corresponding Author:

Prof Dr Ronald Simon, Institute of Pathology, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany. Email: R.Simon@uke.de

carcinomas and pan-keratin positive non-epithelial tumors varies considerably in the literature, however. For example, pan-keratin positivity has been described in 15% to 45% of hepatocellular carcinoma carcinomas,3,5 0% to 95% of adrenocortical carcinomas,2,13,14 33% to 100% of clear cell15,16% and 73% to 100% of papillary renal cell carcinomas,17,18 20% to 78% of angiosarco- mas,19,20 and 17% to 100% of leiomyosarcomas. These conflicting data may be caused by the use of differ- ent antibodies, immunostaining protocols, and criteria to determine “positivity” in these studies.

To generate a comprehensive dataset on the prevalence of pan-keratin positivity in epithelial and non-epithelial neo- plasms, a set of tissue microarray (TMAs) was analyzed in this study that contained more than 15,500 tumor tissue samples from 121 different tumor types and subtypes as well as 76 different non-neoplastic tissue types.

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 tumor TMAs contained a total of 15,940 primary tumors from 121 tumor types and subtypes. The composition of normal and tumor TMAs is described in the results section. Detailed histopathological and molecu- lar data as well as clinical-follow up data were obtained from 1157 kidney and 1475 breast cancer patients. The median follow-up time was 39 months for kidney cancer (range 1-250) and 43 months for breast cancer patients (range 1-88). All samples were retrieved 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.23,24 In brief, one tissue spot (diameter: 0.6 mm) was transmitted from a representative tumor containing donor block into an empty recipient par- affin block. The use of archived remnants of diagnostic tissues for TMA manufacturing, their analysis for research purposes, and use of patient data were according to local laws (HmbKHG, §12) and analysis had been 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 one day and in one experiment. Slides were deparaffinized and exposed to heat-induced antigen retrieval for 5 min in an autoclave at 121 ℃ in a pH 9.0 buffer. The pan-cytokeratin antibody pan- keratin (recombinant rabbit, MSVA-000R, MS Validated Antibodies, GmbH, Hamburg, Germany) was applied at

37 °℃ for 60 min 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. For tumor tissues, the percentage of positive neoplastic cells was estimated, and the staining intensity was semi- quantitatively recorded (0, 1+, 2+, 3+). For statistical analyses, the staining results were categorized into four groups. Tumors without any staining were considered neg- ative. Tumors with 1 + staining intensity in ≤70% of cells

Figure 1. Pan-keratin immunostaining of normal tissues. The panels show a strong pan-keratin positivity of epithelial cells of the stomach (A), the urothelium of the urinary bladder (B), and the squamous epithelium of the oral cavity (C). In the pancreas, acinar cells show a strong staining while cells of islets of Langerhans stain only weakly (D). In the liver pan-keratin staining is variable (weak to moderate) in hepatocytes but strong in bile ducts (E). In the adrenal gland, only a subset of cortical cells shows a weak staining (F). In the myometrium, groups of spindle-shaped pan-keratin positive cells are found (G). In lymph nodes, a delicate fibrillar staining caused by fibroblastic reticulum cells occurs mainly in the interfollicular area (H).

A

B

C

D

E

F

G

H

Table 1. Pan-Keratin Immunostaining in Human Tumors.

	Tumor entity	on TMA (n)	Pan-keratin immunostaining result
			analyzable (n)	neg. (%)	weak (%)	mod. (%)	strong (%)
Tumors of the skin	Basal cell carcinoma	88	82	0.0	0.0	0.0	100.0
	Benign nevus	29	25	100.0	0.0	0.0	0.0
	Squamous cell carcinoma of the skin	90	88	0.0	1.1	0.0	98.9
	Malignant melanoma	48	43	93.0	0.0	4.7	2.3
	Merkel cell carcinoma	46	43	0.0	2.3	11.6	86.0
Tumors of the head and neck	Squamous cell carcinoma of the larynx	110	104	0.0	0.0	1.9	98.1
	Squamous cell carcinoma of the pharynx	60	58	0.0	0.0	0.0	100.0
	Oral squamous cell carcinoma (floor of the mouth)	130	125	0.8	0.8	0.0	98.4
	Pleomorphic adenoma of the parotid gland	50	35	0.0	0.0	8.6	91.4
	Warthin tumor of the parotid gland	49	45	0.0	0.0	0.0	100.0
	Basal cell adenoma of the salivary gland	15	15	0.0	0.0	0.0	100.0
Tumors of the lung, pleura and thymus	Adenocarcinoma of the lung	246	176	0.0	0.0	0.0	100.0
	Squamous cell carcinoma of the lung	130	73	0.0	0.0	2.7	97.3
	Small cell carcinoma of the lung	20	15	6.7	0.0	0.0	93.3
	Mesothelioma, epitheloid	39	29	0.0	0.0	3.4	96.6
	Mesothelioma, other types	76	63	3.2	0.0	1.6	95.2
	Thymoma	29	28	0.0	0.0	3.6	96.4
Tumors of the female genital tract	Squamous cell carcinoma of the vagina	78	73	0.0	0.0	1.4	98.6
	Squamous cell carcinoma of the vulva	130	124	0.0	0.0	0.0	100.0
	Squamous cell carcinoma of the cervix	130	126	0.0	0.0	0.0	100.0
	Endometrioid endometrial carcinoma	236	220	0.9	0.5	0.9	97.7
	Endometrial serous carcinoma	82	70	1.4	0.0	2.9	95.7
	Carcinosarcoma of the uterus	48	44	15.9	2.3	2.3	79.5
	Endometrial carcinoma, high grade, G3	13	12	25.0	0.0	0.0	75.0
	Endometrial clear cell carcinoma	8	8	0.0	0.0	0.0	100.0
	Endometrioid carcinoma of the ovary	110	102	0.0	0.0	0.0	100.0
	Serous carcinoma of the ovary	559	509	0.4	0.2	0.6	98.8
	Mucinous carcinoma of the ovary	96	77	0.0	0.0	0.0	100.0
	Clear cell carcinoma of the ovary	50	45	0.0	0.0	2.2	97.8
	Carcinosarcoma of the ovary	47	39	15.4	5.1	0.0	79.5
	Brenner tumor	9	9	0.0	0.0	0.0	100.0
Tumors of the breast	Invasive breast carcinoma of no special	1391	1152	0.3	0.6	1.0	98.1
	type
	Lobular carcinoma of the breast	294	241	0.0	0.0	0.0	100.0
	Medullary carcinoma of the breast	26	26	0.0	0.0	0.0	100.0
	Tubular carcinoma of the breast	27	21	0.0	0.0	0.0	100.0
	Mucinous carcinoma of the breast	58	44	0.0	0.0	0.0	100.0
	Phyllodes tumor of the breast	50	43	0.0	0.0	0.0	100.0
Tumors of the digestive system	Adenomatous polyp, low-grade	50	50	0.0	0.0	0.0	100.0
	dysplasia
	Adenomatous polyp, high-grade	50	46	0.0	0.0	0.0	100.0
	dysplasia
	Adenocarcinoma of the colon	1932	1417	0.0	0.0	0.0	100.0
	Adenocarcinoma of the small intestine	10	7	14.3	0.0	0.0	85.7
	Gastric adenocarcinoma, diffuse type	226	158	0.0	0.0	0.6	99.4
	Gastric adenocarcinoma, intestinal type	224	162	1.2	0.0	0.0	98.8
	Gastric adenocarcinoma, mixed type	62	59	0.0	0.0	0.0	100.0
	Adenocarcinoma of the esophagus	133	77	0.0	0.0	1.3	98.7
	Squamous cell carcinoma of the	124	71	0.0	1.4	1.4	97.2
	esophagus
	Squamous cell carcinoma of the anal	91	87	0.0	0.0	0.0	100.0
	canal

(continued)

Table 1. (continued).

	Tumor entity	on TMA	Pan-keratin immunostaining result
			analyzable	neg.	weak	mod.	strong
		(n)	(n)	(%)	(%)	(%)	(%)
	Cholangiocarcinoma	114	106	0.0	0.0	0.0	100.0
	Hepatocellular carcinoma	50	50	0.0	10.0	12.0	78.0
	Ductal adenocarcinoma of the pancreas	662	551	0.2	0.0	0.4	99.5
	Pancreatic/Ampullary adenocarcinoma	119	84	0.0	0.0	0.0	100.0
	Acinar cell carcinoma of the pancreas	15	14	0.0	0.0	0.0	100.0
	Gastrointestinal stromal tumor (GIST)	50	47	95.7	0.0	2.1	2.1
Tumors of the urinary system Tumors of the male genital organs Tumors of endocrine organs	Non-invasive papillary urothelial	177	147	0.0	0.0	0.0	100.0
	carcinoma, pTa G2 low grade
	Non-invasive papillary urothelial	141	122	0.0	0.0	0.0	100.0
	carcinoma, pTa G2 high grade
	Non-invasive papillary urothelial	187	118	0.0	0.0	2.5	97.5
	carcinoma, pTa G3
	Urothelial carcinoma, pT2-4 G3	1207	862	0.9	0.3	0.9	97.8
	Small cell neuroendocrine carcinoma of	18	18	27.8	16.7	0.0	55.6
	the bladder
	Sarcomatoid urothelial carcinoma	25	21	23.8	4.8	4.8	66.7
	Clear cell renal cell carcinoma	858	791	8.0	18.6	19.5	54.0
	Papillary renal cell carcinoma	255	236	0.8	1.3	1.7	96.2
	Clear cell (tubulo) papillary renal cell	21	21	0.0	4.8	4.8	90.5
	carcinoma
	Chromophobe renal cell carcinoma	131	126	0.8	0.0	0.0	99.2
	Oncocytoma	177	168	1.2	0.6	1.2	97.0
	Adenocarcinoma of the prostate,	83	83	0.0	0.0	0.0	100.0
	Gleason 3 + 3
	Adenocarcinoma of the prostate,	80	78	0.0	0.0	0.0	100.0
	Gleason 4 + 4
	Adenocarcinoma of the prostate,	85	85	0.0	0.0	0.0	100.0
	Gleason 5 + 5
	Adenocarcinoma of the prostate	261	252	0.0	0.0	2.0	98.0
	(recurrence)
	Small cell neuroendocrine carcinoma of	17	15	6.7	0.0	0.0	93.3
	the prostate
	Seminoma	621	584	77.1	17.1	4.5	1.4
	Embryonal carcinoma of the testis	50	46	0.0	0.0	4.3	95.7
	Yolk sac tumor	50	42	0.0	0.0	4.8	95.2
	Teratoma	50	24	0.0	0.0	0.0	100.0
	Squamous cell carcinoma of the penis	80	78	0.0	0.0	0.0	100.0
	Adenoma of the thyroid gland	114	106	0.0	0.0	0.0	100.0
	Papillary thyroid carcinoma	392	366	0.0	0.3	0.0	99.7
	Follicular thyroid carcinoma	158	152	0.0	0.0	0.0	100.0
	Medullary thyroid carcinoma	107	103	0.0	0.0	0.0	100.0
	Anaplastic thyroid carcinoma	45	36	19.4	13.9	13.9	52.8
	Adrenal cortical adenoma	50	46	60.9	13.0	15.2	10.9
	Adrenal cortical carcinoma	26	25	52.0	16.0	4.0	28.0
	Phaeochromocytoma	50	47	100.0	0.0	0.0	0.0
	Appendix, neuroendocrine tumor	22	16	0.0	0.0	6.3	93.8
	Colorectal, neuroendocrine tumor	11	11	0.0	0.0	0.0	100.0
	Ileum, neuroendocrine tumor	49	46	0.0	0.0	0.0	100.0
	Lung, neuroendocrine tumor	19	18	5.6	0.0	0.0	94.4
	Pancreas, neuroendocrine tumor	98	91	0.0	1.1	4.4	94.5
	Colorectal, neuroendocrine carcinoma	12	10	20.0	0.0	0.0	80.0
	Gallbladder, neuroendocrine carcinoma	4	4	50.0	0.0	0.0	50.0
	Pancreas, neuroendocrine carcinoma	14	14	0.0	7.1	0.0	92.9
Tumors of	Hodgkin Lymphoma	103	103	99.0	1.0	0.0	0.0

(continued)

Table 1. (continued).

	Tumor entity	on TMA (n)	Pan-keratin immunostaining result
			analyzable (n)	neg. (%)	weak (%)	mod. (%)	strong (%)
haemotopoetic and	Non-Hodgkin Lymphoma	62	61	100.0	0.0	0.0	0.0
lymphoid tissues	Small lymphocytic lymphoma, B-cell type	50	50	100.0	0.0	0.0	0.0
	Diffuse large B cell lymphoma	114	114	100.0	0.0	0.0	0.0
	Follicular lymphoma	88	88	100.0	0.0	0.0	0.0
	T-cell Non Hodgkin lymphoma	24	24	100.0	0.0	0.0	0.0
	Mantle cell lymphoma	18	18	100.0	0.0	0.0	0.0
	Marginal zone lymphoma	16	16	100.0	0.0	0.0	0.0
	Diffuse large B-cell lymphoma in the testis	16	16	100.0	0.0	0.0	0.0
	Burkitt lymphoma	5	3	100.0	0.0	0.0	0.0
Tumors of soft tissue	Tenosynovial giant cell tumor	45	45	100.0	0.0	0.0	0.0
and bone	Granular cell tumor	53	46	100.0	0.0	0.0	0.0
	Leiomyoma	50	44	86.4	13.6	0.0	0.0
	Leiomyosarcoma	87	85	81.2	12.9	4.7	1.2
	Liposarcoma	132	116	94.8	3.4	1.7	0.0
	Malignant peripheral nerve sheath tumor	13	13	100.0	0.0	0.0	0.0
	Myofibrosarcoma	26	26	100.0	0.0	0.0	0.0
	Angiosarcoma	73	65	75.4	9.2	3.1	12.3
	Angiomyolipoma	91	86	88.4	10.5	1.2	0.0
	Dermatofibrosarcoma protuberans	21	17	94.1	5.9	0.0	0.0
	Ganglioneuroma	14	12	100.0	0.0	0.0	0.0
	Kaposi sarcoma	8	5	100.0	0.0	0.0	0.0
	Neurofibroma	117	107	99.1	0.9	0.0	0.0
	Sarcoma, not otherwise specified (NOS)	75	73	90.4	4.1	1.4	4.1
	Paraganglioma	41	41	100.0	0.0	0.0	0.0
	Ewing Sarcoma	23	15	100.0	0.0	0.0	0.0
	Rhabdomyosarcoma	7	7	71.4	28.6	0.0	0.0
	Schwannoma	121	117	96.6	3.4	0.0	0.0
	Synovial sarcoma	12	11	81.8	18.2	0.0	0.0
	Osteosarcoma	43	33	87.9	0.0	9.1	3.0
	Chondrosarcoma	38	18	100.0	0.0	0.0	0.0

and 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 werde 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 pan-keratin and tumor phe- notype. Survival curves were calculated according to Kaplan-Meier. The Log-Rank test was applied to detect significant differences between groups.

Results

Technical issues. A total of 13,501 (85%) of 15,940 tumor samples were interpretable. The remaining 2439 (15%)

samples were not analyzable due to the lack of unequivocal tumor cells or missing tissue spots on the TMA. For the normal tissue TMA, a sufficient number of samples was always interpretable per tissue to determine pan-keratin immunostaining.

Pan-keratin in normal tissues. A positive pan-keratin immunostaining was seen in virtually all normal epithelial and mesothelial cells. The most significant exception was the adrenal cortex, where only a fraction of cells, typically arranged in groups, fascicles or sheets, stained weakly to moderately positive. A somewhat lower, but still moder- ate to strong staining intensity than in most other epithe- lial cells was seen in hepatocytes, Langerhans islets in the pancreas, and pneumocytes of the lung. In lymph nodes, tonsil, spleen, and the thymus, a delicate fibrillar staining caused by fibroblastic reticulum cells was regularly seen, mainly in the interfollicular area. Occasional pan-keratin positive spindle shaped myofibroblasts occurred in

multiple organs, especially in case of degenerative or chronic inflammatory conditions. They were, for example, seen in the media of the aorta, muscular wall of the gallbladder, placental stroma, or in the ovary in the vicinity of a corpus luteum. Groups of spindle-shaped pan-keratin positive cells were also found in the myome- trium. Pan-keratin immunostaining was absent in the testis, endothelial cells, the heart, striated muscle, muscu- lar wall of the appendix, esophagus, stomach, ileum, colon, renal pelvis and urinary bladder, corpus spongio- sum of the penis, ovarian stroma, fat, testis, neurohy- pophysis, cerebellum and cerebrum. Representative

Figure 2. Pan-keratin immunostaining in tumors. The panels show a cytoplasmatic pan-keratin immunostaining of variable intensity in samples from a colorectal adenoma (A: strong staining), two clear cell renal cell carcinomas (B: strong; C: weak), a sarcoma NOS (D: strong), an angiosarcoma (E: strong), a gastrointestinal stromal tumor (GIST) (F: moderate), and a seminoma (G: weak). Pan-keratin immunostaining is lacking in an adrenocortical carcinoma (H).

A

B

C

D

E

F

G

H

images of pan-keratin positive normal tissues are shown in Figure 1.

Pan-keratin in tumors. A membranous and cytoplas- mic pan-keratin immunostaining was observed in 11,323 (84%) of 13,501 analyzable tumors, including 79% with strong, 2.1% with moderate, and 2.6% with weak staining intensity. At least an occasional weak pan-keratin positiv- ity could be detected in 101 of 121 (84%) different tumor types and tumor subtypes (Table 1). Among 75 epithelial tumor entities, the pan-keratin positivity rate was 100% in 50 (67%) and 98 - 99.9% in 12 categories (16%). These tumors included almost all adenocarcinomas,

Figure 3. Ranking order of pan-keratin immunostaining in tumors. Both the frequency of positive tumors (blue cross) and the frequency of strongly positive tumors (orange rhombus) are shown.

Frequency of Pan-keratin staining (%)

8

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

Basal cell carcinoma

Squamous cell carcinoma of the pharynx

Warthin tumor of the parotid gland

Basal cell adenoma of the salivary gland

Adenocarcinoma of the lung

Squamous cell carcinoma of the vulva

Squamous cell carcinoma of the cervix

Endometrial clear cell carcinoma

Endometrioid carcinoma of the ovary

Mucinous carcinoma of the ovary

Brenner tumor

Lobular carcinoma of the breast

Medullary carcinoma of the breast

Tubular carcinoma of the breast

Mucinous carcinoma of the breast

Phyllodes tumor of the breast

Adenomatous polyp, low-grade dysplasia

Adenomatous polyp, high-grade dysplasia

Adenocarcinoma of the colon

Gastric adenocarcinoma, mixed type

Squamous cell carcinoma of the anal canal

Cholangiocarcinoma

Pancreatic/Ampullary adenocarcinoma

Acinar cell carcinoma of the pancreas

Non-invasive papillary urothelial carcinoma, pTa G2 low grade

Non-invasive papillary urothelial carcinoma, pTa G2 high grade

Adenocarcinoma of the prostate, Gleason 3+3

Adenocarcinoma of the prostate, Gleason 4+4

Adenocarcinoma of the prostate, Gleason 5+5

Teratoma

Squamous cell carcinoma of the penis

Adenoma of the thyroid gland

Follicular thyroid carcinoma

Medullary thyroid carcinoma

Colorectal, neuroendocrine tumor (NET)

Ileum, neuroendocrine tumor (NET)

Papillary thyroid carcinoma

Gastric adenocarcinoma, diffuse type

Squamous cell carcinoma of the skin

Adenocarcinoma of the esophagus

Squamous cell carcinoma of the vagina

Adenocarcinoma of the prostate (recurrence)

Clear cell carcinoma of the ovary

Non-invasive papillary urothelial carcinoma, pTa G3

Squamous cell carcinoma of the lung

Squamous cell carcinoma of the esophagus

Mesothelioma, epitheloid

Thymoma

Embryonal carcinoma of the testis

Appendix, neuroendocrine tumor (NET)

Pancreas, neuroendocrine carcinoma (NEC)

Pleomorphic adenoma of the parotid gland

Clear cell (tubulo) papillary renal cell carcinoma

Merkel cell carcinoma

Hepatocellular carcinoma

Squamous cell carcinoma of the larynx

Yolk sack tumor

Pancreas, neuroendocrine tumor (NET)

Ductal adenocarcinoma of the pancreas

Invasive breast carcinoma of no special type

Serous carcinoma of the ovary

Chromophobe renal cell carcinoma

Oral squamous cell carcinoma (floor of the mouth)

Papillary renal cell carcinoma

Endometrioid endometrial carcinoma

Urothelial carcinoma, pT2-4 G3

Oncocytoma

Gastric adenocarcinoma, intestinal type

Endometrial serous carcinoma

Mesothelioma, other types

Lung, neuroendocrine tumor (NET)

Small cell carcinoma of the lung

Small cell neuroendocrine carcinoma of the prostate

Clear cell renal cell carcinoma

Adenocarcinoma of the small intestine

Carcinosarcoma of the ovary

Carcinosarcoma of the uterus

Anaplastic thyroid carcinoma

Colorectal, neuroendocrine carcinoma (NEC)

Sarcomatoid urothelial carcinoma

Endometrial carcinoma, high grade, G3

Small cell neuroendocrine carcinoma of the bladder

Gallbladder, neuroendocrine carcinoma (NEC)

Adrenal cortical carcinoma

Adrenal cortical adenoma

Rhabdomyosarcoma

Angiosarcoma

Seminoma

Leiomyosarcoma

Synovial sarcoma

Leiomyoma

Osteosarcoma

Angiomyolipoma

Sarcoma, not otherwise specified (NOS)

Malignant melanoma

any pan-keratin positivity

Dermatofibrosarcoma protuberans

Liposarcoma

Gastrointestinal stromal tumor (GIST)

strong pan-keratin positivity

Schwannoma

Hodgkin Lymphoma

Neurofibroma

Table 2. Pan-Keratin Immunostaining and Tumor Phenotype in Breast and Clear Cell Renal and Papillary Renal Cell Cancer.

			n	Pan-keratin IHC result
				negative	weak	moderate	strong
				(%)	(%)	(%)	(%)	P
Breast carcinoma of no special type	Primary Tumor	PT1	547	0.5	0.5	0.0	98.9	0.0023
		PT2	400	0.0	1.0	1.5	97.5
		PT3-4	89	0.0	0.0	3.4	96.6
	Grade	G1	171	0.6	0.6	0.0	98.8	0.0005
		G2	538	0.2	0.4	0.0	99.4
		G3	366	0.3	1.1	2.7	95.9
	Regional Lymph	pN0	444	0.0	0.7	0.9	98.4	0.7401
	Nodes
		PN1	217	0.0	0.5	0.9	98.6
		PN2	72	0.0	0.0	0.0	100.0
		PN3	50	0.0	0.0	0.0	100.0
	HER2 status	negative	840	0.4	0.6	1.1	98.0	0.5544
		positive	114	0.0	0.0	0.9	99.1
	ER status	negative	198	0.5	1.5	4.5	93.4	<0.0001
		positive	713	0.1	0.3	0.1	99.4
	PR status	negative	381	0.3	0.8	2.6	96.3	<0.0001
		positive	564	0.2	0.4	0.0	99.5
	Triple negative	no	749	0.1	0.3	0.3	99.3	<0.0001
		yes	133	0.8	2.3	6.0	91.0
Clear cell renal cell carcinoma
	ISUP grade	1	236	7.6	17.8	21.2	53.4	0.1907
		2	248	7.7	17.3	21.0	54.0
		3	206	7.8	20.9	19.4	51.9
		4	42	21.4	26.2	9.5	42.9
	Fuhrmann grade	1	38	7.9	5.3	15.8	71.1	0.0049
		2	443	7.4	18.1	21.2	53.3
		3	210	7.6	21.0	21.0	50.5
		4	50	20.0	26.0	6.0	48.0
	Thoenes grade	1	268	6.3	17.2	20.1	56.3	0.0183
		2	406	8.6	18.0	21.4	52.0
		3	67	14.9	29.9	9.0	46.3
	UICC stage	1	340	3.5	15.0	17.9	63.5	0.001
		2	35	8.6	8.6	22.9	60.0
		3	91	14.3	24.2	17.6	44.0
		4	74	13.5	14.9	21.6	50.0
	Primary Tumor	1	446	4.9	17.0	17.9	60.1	<0.0001
		2	76	10.5	19.7	26.3	43.4
		3-4	214	15.0	22.4	21.0	41.6
	Regional Lymph	0	127	9.4	22.0	22.0	46.5	0.0114
	Nodes
		≥1	18	16.7	0.0	11.1	72.2
	Distant Metastasis	0	113	6.2	15.9	22.1	55.8	0.192
		≥1	76	15.8	14.5	22.4	47.4
Papillary renal cell carcinoma		1	40			2.5
	ISUP grade			0.0	0.0		97.5	0.933
		2	92	1.1	2.2	1.1	95.7
		3	58	1.7	1.7	3.4	93.1
		4	1	0.0	0.0	0.0	100.0
	Fuhrmann grade	1	2	0.0	0.0	0.0	100.0	0.9965
		2	130	0.8	1.5	1.5	96.2
		3	56	1.8	1.8	3.6	92.9
		4	3	0.0	0.0	0.0	100.0

(continued)

Table 2. (continued).

		n	Pan-keratin IHC result				P
			negative	weak	moderate	strong
			(%)	(%)	(%)	(%)
Thoenes grade	1	49	0.0	0.0	2.0	98.0	0.664
	2	133	1.5	2.3	2.3	94.0
	3	9	0.0	0.0	0.0	100.0
UICC stage	1	104	0.0	1.0	0.0	99.0	0.0062
	2	18	0.0	0.0	5.6	94.4
	3	5	20.0	0.0	40.0	40.0
	4	12	0.0	0.0	0.0	100.0
Primary Tumor	1	133	0.0	0.8	0.0	99.2	0.0007
	2	37	2.7	2.7	2.7	91.9
	3-4	15	6.7	6.7	20.0	66.7
Regional Lymph	0	19	0.0	0.0	5.3	94.7	0.423
Nodes
	≥1	7	0.0	0.0	0.0	100.0
Distant Metastasis	0	27	0.0	0.0	3.7	96.3	0.4935
	≥1	7	0.0	0.0	0.0	100.0

squamous cell and urothelial carcinomas. Only 17 of 121 tumor entities (13%) had a pan-keratin positivity rate between 25% and 98%. This group mainly included tumors with mixed differentiation, endocrine/neuroendo- crine tumors, renal cell carcinomas, adrenocortical tumors, and particularly poorly differentiated carcinoma subtypes (anaplastic, small cell, sarcomatoid). The tumors with a pan-keratin positivity in the range of 0.9%-25% included 15 tumor entities. Most of these were of mesenchymal origin and often showed a weaker staining than epithelial neoplasms. The 20 (17%) tumor entities that were always pan-keratin negative were all of mesenchymal and hemato-lymphatic origin. Representative images of pan-keratin positive tumors are shown in Figure 2. A graphical representation of a ranking order of pan-keratin positive tumors and the observed staining intensities in these tumors is given in Figure 3. These data also show, that most tumor entities with 90%-100% positive tumors showed a strong pan-keratin immunostaining while the staining intensity decreased in tumors entities with lower positivity rates. That significant pan-keratin staining can occur in sarcomas was also confirmed by using a second independent pan-keratin antibody (Supplementary Figure 1).

Pan-keratin immunostaining, tumor phenotype, and prognosis. The relationship between pan-keratin immunostaining and clinico-pathological data could be analyzed in breast and kidney cancer (Table 2). Reduced or absent pan-keratin immunostaining was associated with high UICC stage (p=0.001), high Thoenes grade (p=0.0183), high Fuhrman grade (p= 0.0049), advanced tumor stage (p<0.0001) as well as lymph node metastasis (p=0.0114) in clear cell renal

cell carcinomas, with high UICC stage (p=0.0062) and advanced pT stage (p=0.0007) in papillary renal cell carcinoma, and with advanced stage (p=0.0023), high grade (p = 0.0005) as well as loss of estrogen recep- tor (ER) and progesteron receptor (PR) expression and a triple-negative status (p<0.0001 each) in invasive breast carcinoma of no special type (NST). Despite a tendency towards shortened recurrence free (Figure 4a; p=0.1890) and overall survival (Figure 4b, p= 0.1234) for pan-keratin negative clear cell renal cell car- cinomas, this difference did not reach statistical signifi- cance. Reduced pan-keratin immunostaining was not statistically linked to poor outcome in NST (Figure 4c, p= 0.2863) but there were only 6 patients with reduced pan- keratin staining for which clinical follow-up data were available.

Discussion

The standardized analysis of 13,501 tumors provided a comprehensive overview on pan-keratin immunostaining in different tumor types. That the graphical representation of the frequencies of pan-keratin immunostaining among 121 different tumor entities resulted in an S-shaped curve reflects the fact that intense pan-keratin immunostaining is common in epithelial neoplasms while non-epithelial tumors are usually pan-keratin negative.

Among epithelial tumor entities, 50 of 75 (67%) showed Pan-keratin positivity in 100% of tumors and 12 (16%) were positive in ≥98% of tumors. These entities include virtually all important types of adenocarcinomas and squamous cell carcinomas. We assume that a fraction of the few negative tumors in these cancer types may be

Figure 4. Pan-keratin immunostaining and recurrence-free survival (A) and overall survival (B) in clear cell renal cancer. Pan-keratin immunostaining in NST breast cancer and overall survival (C).

A

1.0

Recurrence-free survival

0.8

pan-keratin positive (n=421)

0.6

0.4

pan-keratin negative (n=30)

0.2

Clear cell renal cell carcinoma

p=0.1890

0.0

0

50

100

150

200

250

Months

B

1.0

pan-keratin positive (n=456)

0.8

Overall survival

0.6

pan-keratin negative (n=32)

0.4

0.2

Clear cell renal cell carcinoma

p=0.1234

0.0

0

50

100

150

200

250

Months

C

1.0

pan-keratin negative/weak (n=6)

0.8

Overall survival

0.6

pan-keratin moderate/strong (n=581)

0.4

0.2

NST breast carcinoma

p=0.2863

0.0

0

10

20

30

40

50

60

70

80

90

Months

caused by technical issues. Some unexpected negative immunostaining results always occur in TMAs because not all tissues are properly fixed in all areas.25 Unequal

immunostaining in tissues can results in an immunostain- ing gradient across a tissue block and can thus cause false negative immunostaining, if TMA cores are taken from areas with poor reactivity.26 It is likely that - in at least a fraction of these tumors - some immunoreactive areas will be found if larger tissue samples, perhaps derived from different blocks, are analyzed.

The group of tumor entities with a pan-keratin positivity in 25% to 98% of analyzed tumors made up for only 17 (13%) of analyzed entities. Most of these tumors could be categorized into the following 5 groups: tumors with mixed differentiation, endocrine/neuroendocrine tumors, kidney tumors, adrenocortical tumors, and particularly poorly differentiated carcinoma subtypes. The group of tumors with mixed epithelial-mesenchymal differentiation includes carcinosarcoma of the uterus and the ovary, phyl- lodes tumor of the breast, teratoma of the testis, and malig- nant mesothelioma. In these tumors, epithelial but not mesenchymal tumor areas are pan-keratin positive. The pan-keratin TMA result therefore depends on whether epi- thelial components are present in the TMA spot or not. The rather low positivity rate in kidney cancers reflects the fact that these tumors have low cytokeratin levels and tend to express vimentin instead.27,2

For adrenocortical, neuroendocrine and endocrine tumors, the intermediate positivity rate appears to mirror the rather low pan-keratin immunostaining in correspond- ing normal cells. Hepatocellular carcinoma, another tumor for which reduced cytokeratin has often been reported 5,29,30 was always pan-keratin positive in this study, although staining was only weak or moderate in 22% of tumors.

Very poorly differentiated cancers, such as small cell and sarcomatoid cancers as well as anaplastic cancers of the thyroid, often showed lower pan-keratin immunostain- ing rates as compared to corresponding normal tissues and more differentiated tumors from these organs. This may reflect that a reduced expressions of keratins is a feature of tumors dedifferentiation that can occur during cancer progression. This interpretation is also in line with our find- ings in kidney and breast carcinomas showing significant associations between reduced pan-keratin immunostaining and several unfavorable phenotypic tumor features. Other investigators had previously also described significant cor- relations between reduced expression of specific keratins 31-34 or reduced pan-keratin immunostaining 35 and poor patient prognosis or unfavorable tumor phenotype in various tumor types. A reduced expression of keratins in tumors derived from keratin positive progenitor cells is likely to represent a feature of cellular dedifferentiation which regularly goes along with cancer progression. Our data also emphasize that pan-keratin immunostaining is not uncommon in mesenchymal tumors although the expression levels are usually lower in these neoplasms as compared to carcinomas. In this study, pan-keratin

immunostaining was observed in 13 mesenchymal tumor entities with highest rates in rhabdomyosarcomas (29%) and angiosarcomas (25%). These findings are in line with earlier studies describing keratin expression in 20%, 36%, 100% of rhabdomyosarcomas 36-38 and in 20%, 33%, and 88% of angiosarcomas.19,20,39 Given the signifi- cant pan-keratin staining in the majority of cells is several sarcomas of different types, cytokeratin positivity - even if strong - should not automatically lead to a diagnosis of “sarcomatoid carcinoma”.

In summary, our data show that pan-keratin can consis- tently be detected in the vast majority of epithelial tumors but also identify renal cell, hepatocellular, adrenocortical and neuroendocrine neoplasms as tumors lacking pan-keratin immunostaining in a fraction of tumors. Moreover, pan- keratin immunostaining - usually at lower levels - can also occur in a broad range of mesenchymal tumors.

Acknowledgements

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

Contributiors

AM, RS, GS, CB: contributed to conception, design, data collection, data analysis and manuscript writing. NG, FV, ML, AML, EB, DH, CF, PL, RU, FJ, SM, TSC, DD: participated in pathology data anal- ysis and data interpretation. TK, AHM collection of samples

AM, CB: immunohistochemistry analysis. RS, LH, MK, CHM: data analysis. AM, RS, GS, CB: study supervision. All authors agree to be accountable for the content of the work.

Data Availability Statement

All data generated or analyzed during this study are included in this published article

Declaration of Conflicting Interests

The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: The Pan-keratin antibody clone MSVA-000R was received from MS Validated Antibodies GmbH (owned by a family member of GS).

Ethical Approval

The use of archived remnants of diagnostic tissues for TMA man- ufacturing, their analysis for research purposes, and use of patient data were according to local laws (HmbKHG, §12) and analysis had been approved by the local ethics committee (Ethics commis- sion Hamburg, WF-049/09). All work has been carried out in compliance with the Helsinki Declaration.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Informed Consent

Not applicable, because this article does not contain any studies with human or animal subjects.

Trial Registration

Not applicable, because this article does not contain any clinical trials.

ORCID iDs

Ronald Simon ID https://orcid.org/0000-0003-0158-4258 Eike Burandt İD https://orcid.org/0000-0002-5705-9084

Supplemental Material

Supplemental material for this article is available online.

References

1. Badzio A, Czapiewski P, Gorczynski A, et al. Prognostic value of broad-spectrum keratin clones AE1/AE3 and CAM5.2 in small cell lung cancer patients undergoing pulmo- nary resection. Acta Biochim Pol. Feb 22 2019;66(1):111-114. doi:10.18388/abp.2018_2773

2. Sangoi AR, Fujiwara M, West RB, et al. Immunohistochemical distinction of primary adrenal cortical lesions from metastatic clear cell renal cell carcinoma: a study of 248 cases. Am J Surg Pathol. May 2011;35(5):678-686. doi:10.1097/PAS. 0b013e3182152629

3. Lau SK, Prakash S, Geller SA, Alsabeh R. Comparative immunohistochemical profile of hepatocellular carcinoma, cholangiocarcinoma, and metastatic adenocarcinoma. Hum Pathol. Dec 2002;33(12):1175-1181. doi:10.1053/hupa. 2002.130104

4. Cote RJ, Cordon-Cardo C, Reuter VE, Rosen PP. Immunopathology of adrenal and renal cortical tumors. Coordinated change in antigen expression is associated with neoplastic conversion in the adrenal cortex. Am J Pathol. May 1990;136(5):1077-1084.

5. Christensen WN, Boitnott JK, Kuhajda FP. Immunoperoxidase staining as a diagnostic aid for hepatocellular carcinoma. Mod Pathol. Jan 1989;2(1):8-12.

6. Wick MR, Cherwitz DL, McGlennen RC, Dehner LP. Adrenocortical carcinoma. An immunohistochemical com- parison with renal cell carcinoma. Am J Pathol. Feb 1986;122(2):343-352.

7. Lin XY, Liu Y, Zhang Y, Yu JH, Wang EH. The co-expression of cytokeratin and p63 in epithelioid angiosar- coma of the parotid gland: a diagnostic pitfall. Diagn Pathol. Sep 3 2012;7(1):118. doi:10.1186/1746-1596-7-118

8. Miettinen M, Fetsch JF. Distribution of keratins in normal endothelial cells and a spectrum of vascular tumors: implica- tions in tumor diagnosis. Hum Pathol. Sep 2000;31(9):1062- 1067. doi:10.1053/hupa.2000.9843

9. Gu M, Antonescu CR, Guiter G, Huvos AG, Ladanyi M, Zakowski MF. Cytokeratin immunoreactivity in Ewing’s sarcoma: prevalence in 50 cases confirmed by molecular diagnostic studies. Am J Surg Pathol. Mar 2000;24(3):410- 416. doi:10.1097/00000478-200003000-00010

10. Iwata J, Fletcher CD. Immunohistochemical detection of cytokeratin and epithelial membrane antigen in leiomyosar- coma: a systematic study of 100 cases. Pathol Int. Jan 2000;50(1):7-14. doi:10.1046/j.1440-1827.2000.01001.x

11. Meis-Kindblom JM, Kindblom LG. Angiosarcoma of soft tissue: a study of 80 cases. Am J Surg Pathol. Jun 1998;22(6):683-697. doi:10.1097/00000478-199806000- 00005

12. Miettinen M. Immunoreactivity for cytokeratin and epithelial membrane antigen in leiomyosarcoma. Arch Pathol Lab Med. Jun 1988;112(6):637-640.

13. Lapinski JE, Chen L, Zhou M. Distinguishing clear cell renal cell carcinoma, retroperitoneal paraganglioma, and adrenal cortical lesions on limited biopsy material: utility of immu- nohistochemical markers. Appl Immunohistochem Mol Morphol. Oct 2010;18(5):414-421. doi:10.1097/PAI.0b013 e3181ddf7b9

14. Pinkus GS, Etheridge CL, O’Connor EM. Are keratin pro- teins a better tumor marker than epithelial membrane antigen? A comparative immunohistochemical study of various paraffin-embedded neoplasms using monoclonal and polyclonal antibodies. Am J Clin Pathol. Mar 1986;85(3):269-277. doi:10.1093/ajcp/85.3.269

15. He H, Trpkov K, Martinek P, et al. “High-grade oncocytic renal tumor”: morphologic, immunohistochemical, and molecular genetic study of 14 cases. Virchows Arch. Dec 2018;473(6):725-738. doi:10.1007/s00428-018-2456-4

16. Hayes M, Peckova K, Martinek P, et al. Molecular-genetic analysis is essential for accurate classification of renal carci- noma resembling Xp11.2 translocation carcinoma. Virchows Arch. Mar 2015;466(3):313-322. doi:10.1007/s00428-014- 1702-7

17. Al-Obaidy KI, Eble JN, Cheng L, et al. Papillary renal neo- plasm with reverse polarity: a morphologic, immunohisto- chemical, and molecular study. Am J Surg Pathol. Aug 2019;43(8):1099-1111. doi:10.1097/PAS.00000000000012 88

18. Hes O, Brunelli M, Michal M, et al. Oncocytic papillary renal cell carcinoma: a clinicopathologic, immunohistochemical, ultrastructural, and interphase cytogenetic study of 12 cases. Ann Diagn Pathol. Jun 2006;10(3):133-139. doi:10. 1016/j.anndiagpath.2005.12.002

19. Nakashima Y, Inamura K, Ninomiya H, et al. Frequent expression of conventional endothelial markers in pleural mesothelioma: usefulness of claudin-5 as well as combined traditional markers to distinguish mesothelioma from angio- sarcoma. Lung Cancer. Oct 2020;148:20-27. doi:10.1016/j. lungcan.2020.07.029

20. Allison KH, Yoder BJ, Bronner MP, Goldblum JR, Rubin BP. Angiosarcoma involving the gastrointestinal tract: a series of primary and metastatic cases. Am J Surg Pathol. Mar 2004;28(3):298-307. doi:10.1097/00000478-2004030 00-00002

21. Abeler VM, Nenodovic M. Diagnostic immunohistochemis- try in uterine sarcomas: a study of 397 cases. Int J Gynecol Pathol. May 2011;30(3):236-243. doi:10.1097/PGP. 0b013e318200caff

22. Yang J, Eddy JA, Pan Y, et al. Integrated proteomics and genomics analysis reveals a novel mesenchymal to epithelial reverting transition in leiomyosarcoma through regulation of

slug. Mol Cell Proteomics. Nov 2010;9(11):2405-2413. doi:10.1074/mcp.M110.000240

23. Dancau AM, Simon R, Mirlacher M, Sauter G. Tissue micro- arrays. Methods Mol Biol. 2016;1381:53-65. doi:10.1007/ 978-1-4939-3204-7_3

24. Kononen J, Bubendorf L, Kallioniemi A, et al. Tissue micro- arrays for high-throughput molecular profiling of tumor spec- imens. Nat Med. Jul 1998;4(7):844-847. doi:10.1038/ nm0798-844

25. Tapia C, Schraml P, Simon R, et al. HER2 Analysis in breast cancer: reduced immunoreactivity in FISH non-informative cancer biopsies. Int J Oncol. Dec 2004;25(6):1551-1557.

26. Fraune C, Simon R, Hube-Magg C, et al. MMR deficiency in urothelial carcinoma of the bladder presents with temporal and spatial homogeneity throughout the tumor mass. Urol Oncol. May 2020;38(5):488-495. doi:10.1016/j.urolonc. 2019.12.012

27. Skinnider BF, Folpe AL, Hennigar RA, et al. Distribution of cytokeratins and vimentin in adult renal neoplasms and normal renal tissue: potential utility of a cytokeratin antibody panel in the differential diagnosis of renal tumors. Am J Surg Pathol. Jun 2005;29(6):747-754. doi:10.1097/01.pas. 0000163362.78475.63

28. Alexa A, Baderca F, Lighezan R, Izvernariu D, Raica M. The diagnostic value of cytokeratins expression in the renal parenchyma tumors. Rom J Morphol Embryol. 2010;51(1):27-35.

29. Van Eyken P, Sciot R, Desmet VJ. Immunocytochemistry of cytokeratins in primary human liver tumors. APMIS Suppl. 1991;23:77-85.

30. Van Eyken P, Sciot R, Paterson A, Callea F, Kew MC, Desmet VJ. Cytokeratin expression in hepatocellular carci- noma: an immunohistochemical study. Hum Pathol. May 1988;19(5):562-568. doi:10.1016/s0046-8177(88)80205-3

31. Pandey S, Soland TM, Bjerkli IH, et al. Combined loss of expression of involucrin and cytokeratin 13 is associated with poor prognosis in squamous cell carcinoma of mobile tongue. Head Neck. Nov 2021;43(11):3374-3385. doi:10. 1002/hed.26826

32. Menz A, Bauer R, Kluth M, et al. Diagnostic and prognostic impact of cytokeratin 19 expression analysis in human tumors: a tissue microarray study of 13,172 tumors. Hum Pathol. Sep 2021;115:19-36. doi:10.1016/j.humpath.2021. 05.012

33. Menz A, Weitbrecht T, Gorbokon N, et al. Diagnostic and prognostic impact of cytokeratin 18 expression in human tumors: a tissue microarray study on 11,952 tumors. Mol Med. Feb 15 2021;27(1):16. doi:10.1186/s10020-021- 00274-7

34. Vora HH, Patel NA, Rajvik KN, et al. Cytokeratin and vimentin expression in breast cancer. Int J Biol Markers. Jan-Mar 2009;24(1):38-46. doi:10.5301/jbm.2009.4965

35. Rajkovic N, Li X, Plataniotis KN, Kanjer K, Radulovic M, Milosevic NT. The pan-cytokeratin staining intensity and fractal computational analysis of breast tumor malignant growth patterns prognosticate the occurrence of distant metastasis. Front Oncol. 2018;8:348. doi:10.3389/fonc. 2018.00348

36. Le Loarer F, Cleven AHG, Bouvier C, et al. A subset of epi- thelioid and spindle cell rhabdomyosarcomas is associated

with TFCP2 fusions and common ALK upregulation. Mod Pathol. Mar 2020;33(3):404-419. doi:10.1038/s41379-019- 0323-8

37. Thompson LDR, Jo VY, Agaimy A, et al. Sinonasal tract alveolar rhabdomyosarcoma in adults: a clinicopathologic and immunophenotypic study of fifty-two cases with empha- sis on epithelial immunoreactivity. Head Neck Pathol. Jun 2018;12(2):181-192. doi:10.1007/s12105-017-0851-9

38. Stock N, Chibon F, Binh MB, et al. Adult-type rhabdomyo- sarcoma: analysis of 57 cases with clinicopathologic descrip- tion, identification of 3 morphologic patterns and prognosis. Am J Surg Pathol. Dec 2009;33(12):1850-1859. doi:10. 1097/PAS.0b013e3181be6209

39. Matoso A, Epstein JI. Epithelioid angiosarcoma of the bladder: a series of 9 cases. Am J Surg Pathol. Oct 2015;39(10):1377- 1382. doi:10.1097/PAS.0000000000000444