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Cytokeratin 7 and Cytokeratin 20 in Primary Urinary Bladder Carcinoma and Matched Lymph Node Metastasis.

The cytokeratin family consists of at least 20 types of cytoplasmic intermediate filaments found in epithelial cells.[1] Expression of these cytoskeletal proteins is determined by cell type, stage of development, and differentiation.[2,3] Abnormal expression of cytokeratin has been found in various forms of neoplasia and other diseases.[4]

Cytokeratin 20 (CK20), a low-molecular-weight cytokeratin, is specifically expressed in the superficial and in some of the intermediate cells of the normal urothelium.[5] Cytokeratin 20 is detected in some urothelial carcinomas and is associated with recurrence in the bladder.[6] Cytokeratin 7 (CK7) is another intermediate filament that is found in urothelial neoplasia of the urinary bladder. Immunohistochemical staining of CK7 and CK20 has been shown to be a helpful diagnostic aid in differentiating the origin of carcinomas, including urothelial carcinoma of urinary bladder.[7,8] It is anticipated that the pattern of CK7 and CK20 expression in metastatic urothelial carcinoma is similar to its primary counterpart. As a result, immunohistochemical staining of metastatic tumor of the lymph node may help in determining the origin of the primary tumor. However, limited information is available about the pattern of CK7 and CK20 expression in lymph node metastases of urothelial carcinoma. In the present study, we sought to determine the expression of these 2 cytokeratins in primary bladder carcinomas and their co-existing lymph node metastases from 26 patients who underwent radical cystectomy and bilateral lymphadenectomy for urothelial carcinoma.

MATERIALS AND METHODS

We studied 26 lymph node-positive patients who underwent radical cystectomy with bilateral lymphadenectomy between 1989 and 1998 at Indiana University Hospital, Indianapolis, In. All histologic slides of the radical cystectomy and lymphadenectomy specimens were reviewed. The 1997 TNM (tumor, lymph node, metastasis) system was used for pathologic staging.[9] In the new classification, T3 is defined as tumor extending beyond the muscularis propria into adjacent perivesical adipose tissue. Tumor invading the deep muscle (formerly stage T3a) is classified as stage T2b. Grading was performed according to the 1998 World Health Organization/International Society of Urologic Pathology classification.[10]

Immunohistochemical staining for CK7 and CK20 was performed on both the primary tumor and lymph node metastases from all of the cases. Briefly, formalin-fixed 5-[micro]m tissue sections were deparaffinized and rehydrated. Endogenous peroxidase activity was blocked with 0.3% (vol/vol) hydrogen peroxide. The primary antibodies for CK7 (1:50 dilution; Dako Corporation, Carpinteria, Calif) and CK20 (1:50 dilution; Dako) were incubated overnight at 4 [degrees] C, followed by biotinylated secondary antibody and peroxidase-labeled streptavidin. Three-aminoethylcarbazole was used as chromogen in the presence of hydrogen peroxide. Positive and negative controls were run in parallel with each batch, and appropriate results were obtained. The percentage of positive tumor cells was recorded by counting 1000 cells per slide.

RESULTS

The patients ranged in age from 36 to 86 years (median = 62 years). The male-female ratio was 3:1. All of the primary tumors were high grade (grade 3). The pathologic stage was T2a in 5 patients, T2b in 7, T3a in 13, and T4a in 1. The corresponding lymph node metastases were also poorly differentiated. The number of lymph nodes containing metastases ranged from 1 to 9 per case. Immunoreactivity for CK20 was confined to the cytoplasm of the tumor cells (Figure). The occasional remaining benign urothelium showed focal staining in the surface epithelium. The percentage of CK20-positive cells varied in each case (Table). None had squamous or glandular differentiation in the sections evaluated. The results of CK20 immunoreactivity in the primary bladder tumor and the corresponding lymph node metastases are shown in the Table. Positive immunoreactivity for CK20 was found in 12 (46%) of the primary tumors (range of positive tumor cells, 5% to 95%; mean, 72%). All of the corresponding lymph node metastases also showed CK20 reactivity (range, 5% to 100%; mean, 70%). Fourteen cases (54%) showed no CK20 staining in both the primary and metastatic tumors.

[ILLUSTRATION OMITTED]
CK 20 and CK 7 Immunostaining in Bladder Urothelial
Carcinomas and Lymph Node Metastases(*)

 CK 20 CK 7
 Positivity, % Positivity, %
 Case
Number Tumor Grade T Stage Primary Node Primary Node

 1 High grade T2a 90 5 95 95
 2 High grade T2a 0 0 95 95
 3 High grade T2a 0 0 95 95
 4 High grade T2a 0 0 95 95
 5 High grade T2a 0 0 95 95
 6 High grade T2b 90 90 95 95
 7 High grade T2b 0 0 80 95
 8 High grade T2b 95 95 95 95
 9 High grade T2b 0 0 95 95
 10 High grade T2b 70 80 95 95
 11 High grade T2b 95 95 95 95
 12 High grade T2b 95 95 90 90
 13 High grade T3a 0 0 95 95
 14 High grade T3a 90 90 95 95
 15 High grade T3a 0 0 95 95
 16 High grade T3a 80 80 95 95
 17 High grade T3a 0 0 95 95
 18 High grade T3a 95 95 95 95
 19 High grade T3a 0 0 95 95
 20 High grade T3a 0 0 95 95
 21 High grade T3a 10 5 95 95
 22 High grade T3a 0 0 95 95
 23 High grade T3a 50 100 95 95
 24 High grade T3a 0 0 95 95
 25 High grade T3a 5 5 95 95
 26 High grade T4a 0 0 80 95

(*) CK indicates cytokeratin.


Cytokeratin 7 expression was also confined to the cytoplasm of the tumor cells. Positive immunostaining for CK7 was observed in from 80% to 95% of the tumor cells in both the primary and metastatic tumors in all of the cases (Table).

COMMENT

In the present study, we examined the expression of CK7 and CK20 in primary urothelial carcinomas and their co-existing metastases. We found that CK20 was diffusely expressed in almost half of the primary tumors (46%). All of the lymph node metastases from these tumors also showed immunoreactivity for CK20. Correspondingly, all of the lymph node metastases from CK20-negative primary tumors were also CK20 negative. Cytokeratin 7 staining was positive in both the primary and metastatic tumors of all the cases studied.

Utilization of expression of CK20 as a diagnostic aid in urothelial carcinomas has been explored by several investigators. The percentage of positive CK20 in urothelial carcinoma ranges from 15% to 97% in different studies. Wang et al[7] found that 89% (17 out of 19) of urothelial carcinomas were positive for CK20. Chu et al[8] reported recently that only 7 of 24 urothelial carcinomas (29%) were positive for CK20. Moll et al[11] found that 97% of the urothelial carcinomas in a series of 24 cases were CK20 positive. However, the histologic grade and pathologic staging information were not addressed in these 3 studies. In a study of 46 cases of high-grade urothelial carcinoma, CK20 immunoreactivity was found in only 15% of cases.[12] In the study conducted by Bassily et al,[13] expression of CK20 was reported in two thirds of 28 bladder urothelial carcinomas. Five (83%) of 6 of low-grade tumors were CK20 positive, whereas only 9 of the 17 high-grade (grade 3 or 4) carcinomas (52%) showed CK20 immunoreactivity. These findings are similar to ours, in that 46% of high-grade primary bladder tumors expressed CK20. Whether CK20 immunoreactivity is less frequent in high-grade urothelial carcinomas than in lower-grade tumors needs further investigation. It is unlikely that those tumors that did not stain for CK20 can be attributed to false-negative reactions from tissue processing, because diffuse CK7 immunoreactivity was present in all the cases.

The combination of CK7 and CK20 staining has been shown to be helpful in differentiating adenocarcinomas of different origins, especially in metastatic tumors, where it is difficult to distinguish cell origin by morphology alone. For example, Loy and Calaluce[14] reported that the CK7-positive/ CK20-negative immunophenotype was found in 86% of pulmonary adenocarcinomas and in 0% of colonic adenocarcinomas. On the other hand, a CK7-negative/ CK20-positive immunophenotype was seen in 77% of colonic adenocarcinomas and in 0% of pulmonary adenocarcinomas. Rullier et al[15] found that the combined detection of CK7 and CK20 is useful for differential diagnosis between cholangiocarcinoma and colorectal carcinoma metastatic to the liver. In the present study, we showed the combined CK7 and CK20 positivity in 46% of urinary bladder urothelial carcinomas. On the other hand, prostatic cancer has been reported to be negative for both CK7 and CK20.[7,8] Hence, immunohistochemical staining for CK7 and CK20 can be useful in differentiating prostatic cancer and urothelial carcinoma of the bladder invading into the prostate.

Our findings suggest that CK20 expression is limited to a subset of urothelial carcinomas. Even in the same tumor, CK20 expression differs in different cell populations; the percentage of CK20-positive cells varied from case to case. The matched lymph node metastases maintained the same pattern of expression as their primary tumors. Cytokeratin 20 and CK7 staining might have diagnostic utility in detecting small metastatic foci in lymph nodes. However, it should be kept in mind that negative immunostains for CK20 alone in metastatic tumors do not rule out the possibility of bladder origin, because a small metastatic focus may not be detected by CK20 staining.

In summary, CK20 immunoreactivity is reliably observed in metastases from bladder carcinoma when the primary tumor expresses CK20 (about 50% of all cases). Cytokeratin 7 is expressed in both primary and metastatic bladder carcinoma in all, or nearly all, cases.

References

[1.] Southgate J, Harnden P, Trejdosiewicz LK. Cytokeratin expression patterns in normal and malignant urothelium: a review of the biological and diagnostic implications. Histol Histopathol. 1999;24:657-664.

[2.] Moll R, Franke WW, Schiller DL, et al. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell. 1982; 31:11-24.

[3.] Cooper D, Schermer A, Sun TT. Biology of human epithelia and their neoplasms using monoclonal antibodies to keratins: strategies, applications, and limitations. Lab Invest. 1985;52:243-256.

[4.] Lane EB, Alexander CM. Use of keratin antibodies in tumor diagnosis. Semin Cancer Biol. 1990;1:165-179.

[5.] Moll R, Schiller DL, Franke WW. Identification of protein IT of the intestinal cytoskeleton as a novel type I cytokeratin with unusual properties and expression patterns. J Cell Biol. 1990;111:567-580.

[6.] Harnden P, Mahmood N, Southgate J. Expression of cytokeratin 20 redefines urothelial papillomas of the bladder. Lancet. 1999;353:974-977.

[7.] Wang NP, Zee S, Zarbo RJ, et al. Coordinate expression of cytokeratin 7 and 20 defines unique subsets of carcinomas. Appl Immunohistochem. 1995;51: 219-223.

[8.] Chu P, Wu E, Weiss LM. Cytokeratin 7 and cytokeratin 20 expression in epithelial neoplasms: a survey of 435 cases. Mod Pathol. 2000;13:962-972.

[9.] Fleming ID, Cooper JS, Henson DE, et al. American Joint Committee on Cancer: Cancer Staging Manual. Philadelphia, Pa: Raven & Lippincott; 1997.

[10.] Epstein JI, Amin MB, Reuter VR, et al. The World Health Organization/ International Society of Urologic Pathology consensus classification of urothelial (transitional cell) neoplasms of the urinary bladder. Am J Surg Pathol. 1998;22: 1435-1448.

[11.] Moll R, Lowe A, Laufer J, Franke WW. Cytokeratin 20 in human carcinomas: a new histodiagnostic marker detected by monoclonal antibodies. Am J Pathol. 1992;140:427-447.

[12.] Hutchinson B, Reuter VE, Gaudin PB. Immunophenotype of intermediate and high grade prostatic and urothelial carcinoma. Mod Pathol. 1998;12:98A.

[13.] Bassily NH, Vallorosi CJ, Akdas G, et al. Coordinate expression of cytokeratin 7 and 20 in prostate adenocarcinoma and bladder urothelial carcinoma. Am J Clin Pathol. 2000;113:383-388.

[14.] Loy TS, Calaluce RD. Utility of cytokeratin immunostaining in separating pulmonary adenocarcinomas from colonic adenocarcinomas. Am J Clin Pathol. 1994;102:764-767.

[15.] Rullier A, Bail BL, Fawaz F, et al. Cytokeratin 7 and 20 expression in cholangiocarcinomas varies along the biliary tract but still differs from that in colorectal carcinomas metastasis. Am J Pathol. 2000;24:870-876.

Accepted for publication February 20, 2001.

From the Departments of Pathology (Drs Jiang, Ulbright, Younger, Sanchez, Eble, and Cheng) and Urology (Drs Koch and Cheng), Indiana University School of Medicine, Indianapolis, Ind; and the Bostwick Laboratories, Richmond, Va (Dr Bostwick).

Reprints: Liang Cheng, MD, Department of Pathology, University Hospital 3465, Indiana University School of Medicine, 550 North University Blvd, Indianapolis, IN 46202.
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Article Details
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Author:Jiang, Jiazhong; Ulbright, Thomas M.; Younger, Cheryl; Sanchez, Katya; Bostwick, David G.; Koch, Mic
Publication:Archives of Pathology & Laboratory Medicine
Geographic Code:1USA
Date:Jul 1, 2001
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