The exploration of cytokeratin (CK) started in the 1980s, when it only belonged to the scope of histopathological studies and the interest was mainly focused on studying the basic biological functions of CK. Subsequently, the study of CK developed slowly. In recent years, new discoveries have been made about CK, and research on CK in combination with clinical studies has gradually become active, and some of them have shown promising clinical applications. In this paper, we review the expression of CK in benign and malignant migratory epithelium of the urinary tract and its clinical application value.
I. Biological properties of CK
CK is a water-soluble polymeric polypeptide with molecular weights of 40,000-68,000 Mr. It is distributed in most epithelial and mesothelial cells and is a component of the intermediate filament backbone. the structure of the cytoskeleton [1]. In humans, 20 CK species have been clearly identified, ranging from CK19 (40,000 Mr) to CK1 (68,000 Mr. In 1990, Moll et al [2] identified a CK with a molecular weight of 46,000 Mr, designated CK20. It was studied that CK20 is expressed only in migratory epithelium, digestive tract epithelium and Merkel cells [3].
Currently, it is believed that CK is a marker of epithelial cell differentiation, and in humans, except for renal podocytes and clear cells, each epithelium has more than two different combinations of CK expression [4], and in pathological tissues with different differentiation there are different types or expression of CK, some with enhanced expression, some with diminished expression or even loss of expression, and some with new combinations of expression, so it can be based on the specific CK expression type or mode of tumor tissue cells Therefore, the diagnosis of tumor can be made based on the specific CK expression type or mode of tumor cells [5,6].
The association between the type and mode of CK expression and epithelial cells suggests a very close relationship between CK and epithelial tissue structure and function, but it is difficult to state the exact relationship between CK expression and epithelial cell phenotype. There is evidence that CK8/CK18 may be important in tumor infiltration. Hembrough et al [7,8,9] found that CK8 can be expressed on the outer surface of tumor cells and can act as a fibrinogen binding factor to enhance the activity of the local fibrinogen cascade reaction, which may be associated with tumor infiltration. Non-epithelial cell lines transfected with CK8/CK18 resulted in increased infiltrative activity [10].Booth et al [11] also found that highly infiltrative bladder cancer cell lines (EJ cell lines) showed extracellular deposition of CK8/CK18. In addition, mutations in the CK gene can be present in certain congenital dermatoses and are directly associated with pathogenesis [12]. Gijbels et al [13] found that epithelial cells in VitA-deficient mice had altered expression of CK prior to the development of histological changes in the bladder and elsewhere; Harnden et al [14] suggested that CK14 could be a marker of squamous metaplastic epithelium. In the bladder migratory epithelium there is no evidence for a direct relationship between CK and bladder disease, although there are many reports suggesting an association, but CK expression may be a manifestation of poor bladder migratory epithelial differentiation rather than a direct factor in the development of disease.
II.CK expression in the urinary tract epithelium
(A) CK expression in normal urinary tract epithelium
The migratory epithelium is distributed in the renal pelvis, ureter and bladder to adapt to changes in the luminal contents volume. The urinary tract migratory epithelium seems to resemble a complex epithelium, but whether it is a stratified or pseudo-stratified epithelium is still controversial [15]. And in fact the three layers of cells of the urinary tract migratory epithelium are still more evident, namely the basal, intermediate and superficial layers of cells. With the advent of monoclonal antibodies, CK expression in each layer of cells can be easily detected. Currently, CK7, CK8, CK18, and CK19 are considered to be expressed in the whole layer; CK5 and CK17 are expressed in basal layer cells; CK13 is expressed in basal and intermediate layer cells; CK20 expression is mainly associated with superficial layer umbrella cells, and Harden et al [16,17] also found that some normal intermediate layers also have a small number of CK20-positive cells, while There is still evidence of occasional CK4 expression in the urinary epithelium, but it remains inconclusive as to which type of urinary epithelium these cells belong to [18,19,20]. homology, whereas both are not homologous with bladder epithelium expressing CK7, suggesting that the urinary tract migratory epithelium may be morphologically distinct epithelial types.
(B) CK expression in metastatic epithelial carcinoma (TCC)
CK expression associated with TCC has been the subject of numerous studies. cK8/CK18 and cK7/CK19 appear to be expressed in all TCC, while other CKs have widely differing expression patterns in the development of TCC and are associated with tumor type, malignancy, and degree of squamification [18].
1.CK8/CK18
Schaafsma et al [21] detected the expression of CK8/CK18 in superficial cells using monoclonal antibodies LE41 (reacting with superficial cells CK8) and 2C8 (reacting with superficial cells CK18) and found that in the non-infiltrating areas of low-grade TCC showed normal immunoreactivity, while 2C8 showed stronger immunoreactivity in G3TCC, and both monoclonal antibodies showed higher immunoreactivity in the infiltrative zone of TCC, especially in tumor cells close to the mesenchyme. Therefore, Schaafsma speculated that there may be functional similarities between the cytoskeletal organization of normal migratory epithelium and invasive cancer cells, with the former requiring the ability to adapt to changes in luminal volume, which in turn makes it easier for tumor cells to infiltrate into the mesenchyme.
2.CK13
Two studies have shown that CK13 expression is attenuated or even de-expressed in high-grade TCC [18,21]. Both studies showed that CK13 is largely normally expressed in G1 and G2 stage TCC, while its expression is significantly reduced in the muscle infiltrating areas of G3 stage TCC.
3.CK14.
Although CK14 is not expressed in normal metastatic epithelium, it has been shown to be expressed in TCC [18,21], however, the clinicopathological evidence for CK14 expression is insufficient. a recent study by Harden et al [14] showed that CK14 expression is associated with TCC squamification, suggesting that CK14 expression may be a pre-expression of the appearance of significant squamification.
4.CK17
In most normal bladder epithelial samples, CK17 is expressed in basal cells [22], and newly Guelstein et al [23] examined CK17 expression in a group of early TCC and found that in most TCC above G2 stage, CK17 was expressed in the whole layer; in degenerative G3 TCC its expression was attenuated, with occasional focal CK17 positivity, and these foci were distributed in In degenerative G3TCC, its expression is diminished, and occasionally focal CK17 positivity is seen, and these foci are distributed in cells containing squamous components in the basal region, suggesting that CK17 may be a marker of proliferative activity in the basal cell population, which may be associated with tumor infiltration.
5. CK20
As mentioned previously, the distribution of CK20 is highly tissue-specific and its expression is limited to superficial umbrella cells of the metastatic epithelium and very few intermediate layer cells.Moll et al [24] studied a group of TCC cases and found that all 7 TCC cases graded 1 and 2 expressed CK20 and only 1 of 17 TCC cases graded 3 and 4 did not express CK20. Harnden et al [25] studied 29 specimens of non-invasive papillary tumors graded 1-2 by immunohistochemistry and found that 19 cases were positive for CK20, with a positive rate of 65.5%; after 5 years of follow-up, 11 of them recurred and the remaining 8 did not, with a significant difference in CK expression between recurrence and non-recurrence. harnden et al [26] recently studied 51 cases of They defined superficial CK20 positivity with occasional deep cell positivity as a “normal expression pattern” in 10 cases, and marked CK20 positivity in whole cells as an “abnormal expression pattern” in 41 cases. After 5 years of follow-up, 30 of the 41 cases with abnormal expression were found to have recurrence, with a recurrence rate of 73%; none of the 10 cases with normal expression had recurrence. The above study showed that the expression of CK20 in TCC is enhanced, and the expression of CK20 may have a predictive effect on the recurrence of early non-invasive papilloma.
III. Clinical significance of CK in the diagnosis of metastatic epithelial carcinoma
TCC has a complex course, grows in an infiltrative manner, recurrence is very common (50%-70%), and although it can be controlled with local therapy, 10%-15% of superficial TCC eventually develop into muscle-invasive TCC or metastasize [27]. Its prognosis is related to the grading and staging of the tumor, and mucosal dysplasia and squamification are also poor prognostic factors [28,29]. Early diagnosis is important for the choice of treatment strategy. patients with TCC are not in early stage when they present with hematuria; urine cytology is not very sensitive and is often used for initial screening of TCC; DNA flow cytology analysis improves some sensitivity, but its high technical requirements and interobserver variability limit its wide use; cystoscopy can confirm the diagnosis of bladder tumors, but the limitations of the biopsy area reduce its sensitivity, and cystoscopy is a very painful and invasive test for patients; so many new methods for detecting TCC have emerged in recent years, such as: detection of IL-2 expression, detection of CD44 expression, detection of NMP-22 expression, microsatellite foci analysis, detection of telomerase expression, and in recent years, detection of CK (cytokeratin) expression.
As mentioned earlier, in histopathology CK can be used as a marker of epithelium to identify the tissue origin of primary and secondary tumors, and also the expression of each CK subtype in bladder cancer can be used to determine the biological function of the tumor, such as the degree of differentiation, tendency to infiltrate, proliferative activity, and the presence of dysplasia or squamification, etc. The very exciting study by Harnden et al. is that the different expression patterns of CK20 based on predict whether the tumor recurs and can be used to identify benign migratory cell papilloma from TCC. they also found that abnormal CK20 expression patterns may also be an objective marker of mucosal dysplasia [28].
Burchill et al [30] used RT-PCR to detect the presence of CK transcripts in the peripheral circulation, suggesting potential tumor cell metastasis in the peripheral blood, and they found that the expression of CK20 mRNA in peripheral blood could be helpful in colon cell tumors or TCC.Fujii et al [31] studied the expression of CK20 in the peripheral blood of a group of TCC patients and found that 13 cases of superficial tumors were negative, 4 of 21 locally infiltrating tumors were positive (19%), and 5 of 6 metastatic tumors were positive (83%), suggesting that detection of peripheral blood CK20 expression is not very helpful for early diagnosis.
The expression of CK20 in urine exfoliated cells has been detected by RT-PCR. Buchumensky et al [32] examined 192 urine specimens by RT-PCR, among which 131 of 144 patients with confirmed bladder cancer were positive for CK20 (sensitivity 91%), none of the 21 healthy volunteers expressed CK20, and 7 of 27 patients with cystoscopically confirmed benign tumors were positive for CK20. 7 of the 27 patients with cystoscopy confirmed benign were positive for CK20, and these 7 cases were chronic inflammation (2 cases), atypical hyperplasia (3 cases), chemosis (1 case), and normal but had a clear history of TCC (1 case), suggesting that CK20 expression is not only related to TCC but also to the precancerous state of TCC, but there was no evidence of a relationship with the grading of the tumor, and this experiment has yet found that detection of Rotem et al [33] used the same method to detect 122 urine specimens and found that the sensitivity was 86.9% and the specificity was 96.7%, and found that the higher the grade of TCC the higher the positive rate of CK20. 9 of 11 patients with cystoscopically confirmed benign CK20 were positive, and the 6-month follow-up of these 9 patients revealed that there were Four of these nine patients were found to have recurrence at 6 months of follow-up, suggesting a relationship between the expression of CK20 and recurrence. The detection of CK20 expression in urine exfoliated cells by RT-PCR has great advantages: 1) easy sample acquisition; 2) extremely sensitive, with Klein suggesting that 1 cancer cell per 1,000,000 urine exfoliated cells can be detected [34]; 3) non-invasive; and 4) can be useful for early diagnosis and recurrence of TCC. The sensitivity and specificity reported by the above two groups of studies were high, but their work was questioned by Southgate et al [35,36], since normal human metastatic epithelial superficial cells are known to express CK20. therefore, a large number of studies are needed to further justify the significance of detecting CK20 expression in urine exfoliated cells by RT-PCR.
Immunohistochemical methods have also been used to detect urinary exfoliated cell CK20 expression in patients with bladder cancer, with a sensitivity of 81.6% and specificity of 77%, disappointingly with the possibility of false negatives in highly graded tumors [37]. Also more recently studied is the detection of CYFRA21-1 in urine as a tumor marker for bladder cancer, a fragment of CK19, which has been studied as a tool to monitor tumor progression, recurrence and reoccurrence [38].
IV. Outlook
CK has been increasingly studied and has shown its clinical application in many aspects. the study of CK20 is perhaps the most important progress, and multiple evidences suggest that CK20 can be used as a clinical indicator for early diagnosis of migratory cell carcinoma and that CK20 can have a predictive effect on the recurrence of TCC. The detection of CK expression in urine exfoliated cells by RT-PCR is even more non-invasive and extremely sensitive. In the near future, CK may play an important role in the diagnosis of migratory cell carcinoma and in the selection of postoperative treatment strategies.