J Clin Pathol 1998;51:679–684 679 The limited diVerence between keratin patterns of J Clin Pathol: first published as 10.1136/jcp.51.9.679 on 1 September 1998. Downloaded from squamous cell carcinomas and adenocarcinomas is explicable by both cell lineage and state of diVerentiation of tumour cells EBLvanDorst,GNPvanMuijen, S V Litvinov, G J Fleuren Abstract For normal epithelia, distribution patterns of Aim—To study the diVerentiation of epi- specific keratins are well established. The thelial tissues within their histological markers used in this study are generally context, and to identify hypothetically, on distributed in the following way: keratins 8 and the basis of keratin pattern, the putative 18 are always present in columnar epithelia, tissue origin of a (metastatic) carcinoma. while keratins 19 and 7 may also be found11; Methods—Using well characterised non-keratinising stratified squamous epithelia monoclonal antibodies against individual contain keratins 4 and 13 suprabasally, and keratins 7, 8, 18, and 19, which are keratins 14 and 19 in the basal compart- predominantly found in columnar epithe- ments12 13; keratinising stratified squamous epi- lia, and keratins 4, 10, 13, and 14, predomi- thelia express keratin 14 in basal compart- nantly expressed in (non)-keratinising ments up to the stratum corneum and keratin squamous epithelia, the keratin patterns 10 suprabasally as a marker for phenotypic for a series of 45 squamous cell carcino- keratinisation14 15; and the myoepithelial cells of mas and 44 adenocarcinomas originating bronchial epithelium16 and mammary epithe- from various epithelial tissues were char- lium also contain keratin 14.17 18 acterised. The value of monoclonal antibodies to keratin Results—The predominant keratins in all 18 for identifying adenocarcinomas has been adenocarcinomas proved to be 8, 18, and described in several reports,19–21 though this does 19. In addition, these keratins were also not apply to keratin 18 positive focal areas of less abundantly present in squamous cell car- diVerentiated cells in squamous cell carcinomas cinomas of the lung, cervix, and rectum of the uterine cervix, the epiglottis, and and, to a lesser extent, of the larynx, lung.5 22–25 It has been reported that keratin 7 oesophagus, and tongue, but not in those of distinguishes between colon carcinomas (nega- the vulva and skin. Keratins 4, 10, 13, and tive) and other adenocarcinomas.5 21 26–30 Re- 14 were present in almost all squamous cell cently, keratin 20 was found to distinguish http://jcp.bmj.com/ carcinomas, but also focally in some of the colonic adenocarcinomas, mucinous ovarian adenocarcinomas studied. tumours, and transitional cell and Merkel cell Conclusions—There is a limited diVeren- carcinomas (positive) from squamous cell carci- tial expression of distinctive keratin nomas, adenocarcinomas of breast, lung, and filaments between squamous cell carcino- endometrium, non-mucinous ovarian tumours, mas and adenocarcinomas. Apparently, and small cell lung carcinoma (negative).31 32 squamous cell carcinomas that originate We performed a detailed systematic study to from columnar epithelium by squamous define the keratin expression patterns in epithe- on September 29, 2021 by guest. Protected copyright. metaplasia gain the keratins of squamous lial neoplasms with respect to cell lineage and cells but retain the keratins of columnar diVerentiation, and the diVerential diagnostic epithelial cells. However, the simultaneous questions for which keratin typing may be Department of expression of two of three squamous kerat- informative. Special attention was paid to the Pathology, University ins (4, 10, and 13) identifies a squamous cell diagnostic value of keratin patterns in defining of Leiden, Leiden, The carcinoma, and thus might be useful in squamous cell carcinoma and adenocarcinoma. Netherlands solving diVerential diagnostic problems. In clinical practice, for example, the diVerential EBLvanDorst (J Clin Pathol 1998;51:679–684) S V Litvinov diagnosis for squamous cell carcinoma of the G J Fleuren Keywords: keratin; carcinoma; diVerentiation cervix uteri from adenocarcinoma is highly important, since the prognosis and treatment of Department of The main structural proteins of the intermediate the two conditions are quite diVerent. The Pathology, University filament based cytoskeleton of epithelial cells are results show that the patterns of keratin expres- Hospital, Nijmegen, the keratins.1 The composition of these fila- sion in adenocarcinomas and squamous cell The Netherlands GNPvanMuijen ments is highly heterogeneous. Approximately carcinomas are dependent on both cell lineage 20 polypeptides of this multigene family have and state of tumour cell diVerentiation. 23 Correspondence to: been characterised. Findings revealing the dif- Professor G J Fleuren, ferentiation and cell type specificity for expres- Department of Pathology, 2 Leiden University, Building sion of individual combinations of keratins Methods 1, L1-Q, PO Box 9600, 2300 raised the question of whether the analysis of the Frozen tissue specimens of 44 primary adeno- RC Leiden, The individual keratins present in fetal and normal carcinomas and 45 primary squamous cell car- Netherlands. epithelia, carcinomas, and their metastases cinomas originating from various epithelial tis- Accepted for publication could serve as a basis for a histologically mean- sues were obtained from our frozen tissue 11 March 1998 ingful classification of neoplasms.4–10 collection (tables 1–3). Keratin expression was 680 van Dorst, van Muijen, Litvinov, et al Table 1 Keratin expression in 44 adenocarcinomas J Clin Pathol: first published as 10.1136/jcp.51.9.679 on 1 September 1998. Downloaded from Stomach (n= 5) Colon (n = 9) Pancreas (n = 5) Lung (n = 6) Kidney (n = 5) Keratin 7 8 18 19 7 8 18 19 7 8 18 19 7 8 18 19 7 8 18 19 ++++ – 4 5 5 – 9 9 9 2 5 5 5 1 5 5 6 – 5 4 2 +++–1–– –––– 2––– 11–– 1––1 ++–––– –––– 1––– 1–1– ––11 + 3––– –––– –––– –––– –––– – 2––– 9––– –––– 3––– 4––1 Keratin 4 10 13 14 4 10 13 14 4 10 13 14 4 10 13 14 4 10 13 14 ++++ – – – – – – – – – – – – – – – – – – – – +++–––– –––– –––– –––– –––– ++–––– –––– –––– –––– –––– + 2––2 –––– 1–21 2––1 –––– – 3553 9999 4534 4665 5555 studied immunohistochemically using chain Results specific mouse monoclonal antikeratin anti- KERATIN PATTERNS IN ADENOCARCINOMAS bodies in serial slides of a tissue specimen The predominant keratins of the adenocarci- (table 4). The reacted monoclonal antibodies noma tissues were represented by keratins 8, were detected using antimouse IgG peroxidase 18, and 19, that were present abundantly and conjugate.40 Normal tissues from the uterine homogeneously. Only in renal cell carcinomas cervix that contained epithelial cells (both was keratin 19 not abundantly present, and it squamous and columnar) expressing all the was even found to be completely absent in one keratins studied were used as a positive control. case. The staining patterns of M9 and RGE 53, As a negative control, we used isotype matched both antikeratin 18 antibodies, were identical. irrelevant mouse monoclonal antibodies. The Keratin 7 expression was variable among the number of positive cells was determined semi- types of adenocarcinoma tested. All pancreatic, quantitatively: −, no positive tumour cells, +, oesophageal, and breast carcinomas contained 0–25%, ++, 25–50%, +++, 50–75%, and variable numbers of positive cells. Three of six ++++, 75–100% positive tumour cells. All fro- adenocarcinomas of the lung and two of five zen tissue samples were reviewed in paraYn cervical adenocarcinomas were negative for keratin 7. Only a few groups of tumour cells embedded sections and found representative. expressed keratin 7 in three of the stomach In addition, we compared the staining carcinomas (fig 1A), and only one renal cell patterns for two antikeratin 18 antibodies— carcinoma expressed keratin 7, though this was RGE53 and M9—and two anti-keratin 14 present in approximately 75% of the tumour antibodies—CKB1 and LL002—in serial dilu- cells. No colon carcinomas were found to be http://jcp.bmj.com/ tions in a selected series of squamous cell car- positive for keratin 7. cinomas and adenocarcinomas. Keratins 4, 13, and 14 were sporadically The specificity of RGE53 and M9 were veri- detected in a limited number of cells of 30% of fied by immunoblotting, using lysates of the adenocarcinomas studied, except for the cultured carcinoma cells. For the preparation colonic and renal cell carcinomas. These kerat- of keratin extracts, we used MCF-7, a cell line ins were not confined to a particular type of derived from a breast carcinoma, and SCC-15, tumour cell, for instance cells with histological derived from squamous cell carcinoma of the signs of squamous diVerentiation. The two on September 29, 2021 by guest. Protected copyright. tongue, in accordance with a published antikeratin 13 antibodies used did not show a 12 protocol. The cells were cultured under diVerence in staining pattern. The selected standard conditions and harvested at conflu- series of 12 adenocarcinomas stained with two ency. According to Laemmli,41 10% polyacryla- antikeratin 14 antibodies in several dilutions mide gels were used for one dimensional gel showednodiVerences in staining patterns. electrophoresis. Keratin 10 was not observed in any of the Table 2 Keratin expression in 44 squamous cell carcinomas Skin (n= 8) Vulva* (n = 5) Larynx† (n = 7) Lung (n = 10) Cervix (n = 5) Keratin 7 8 18 19 7 8 18 19 7 8 18 19 7 8 18 19 7 8 18 19 ++++ – – – – – – – – 2 – 3 – 5 – 9 – – – – 5 +++–––– –––– 11–– 1331 143– ++–––– –––– ––1– ––3– 112– + –––– –––– 1242 123– 2––– – 8888 5555 5222 8–1– 1––– Keratin 4 10 13 14 4 10 13 14 4 10 13 14 4 10 13 14 4 10 13 14 ++++ – – – 5 – – – 1 – – – 2 – – – 1 – – – 2 +++–3–2 –1–1 –––3 1––1 1––2 ++–2–1 –––– –––2 211– 2121 + –2–– 241– 565– 4442 132– – 818– 3–4– 212– 3551 111– *Two tissues stained for keratin 14; †five tissues stained for keratin 14. Keratin patterns of squamous cell carcinomas and adenocarcinomas 681 Table 1 continued Table 3 Comparison of RGE53 and M9 reactivity with J Clin Pathol: first published as 10.1136/jcp.51.9.679 on 1 September 1998.