Wilm's tumour gene, WT1

The WT1 protein is encoded by the WT1 gene located on chromosome 11p13¹⁵ and has four protein isoforms²⁷. It encodes a zinc finger protein that binds to the same DNA sequences as does ERG-1¹¹. While ERG-1 activates transcription, WT1 suppresses transcription. The suppressor function is lost in mutations that affect the zinc finger region¹¹. Candidate target genes for WT-1 include e-cadherin and bcl-2, and there is interaction with p53²¹.

Germline deletion of one WT-1 allele, along with the neighbouring PAX6 gene, occur in the WAGR syndrome (Wilms tumour, aniridia, genitourinary abnormalities and mental retardation)¹⁹. In the Denys-Drash syndrome, there is a missense mutation effecting zinc finger 3 or a or truncating mutation, resulting in Wilms tumour, nephropathy and genital malformation, but with partial penetrance¹⁹. A homozygous knockout mouse model fails to develop kidneys or gonads and has small heart and lungs with a incomplete diaphragm¹⁹.

Alternative RNA splicing results at least twenty four isoforms²² of WT1 with variation in the carboxy-end zinc finger region. Of two major isoforms, the -KTS form appears to mediate transcriptional activation of genes, while the +KTS form may be involved in mRNA processing²³. This variety of isoforms might theoretically influence immunoreactivity of the polyclonal antibody C-19, which is directed at the carboxyl end. The monoclonal 6F-H2 is directed at the amino terminus¹³. The 6F-H2 antibody is reported to produce stronger homogeneous reactivity than the C-19 clone in serous ovarian tumours¹³, but to produce no immunoreactivity in desmoplastic small round cell tumours¹.

Although WT-1 is expressed in Wilms tumour, only 10% of sporadic cases show WT-1 mutations¹⁹. There is commonly loss of heterozygocity in ovarian tumours²¹.

Immunohistochemical expression

The informative immunoreactivity is nuclear.

• the mesodermally derived tissue of the developing urogenital tract: fetal and adult kidney and developing ovary and testis show positivity¹⁹. The epithelia of the Fallopian tube and the ovarian surface are immunoreactive¹⁶, while endometrium and cervical epithelia are not¹⁴.

• breast epithelium and myoepithelium are positive but many carcinomas are negative, using antibody C-19²⁰.

• fetal spleen

• non-germ cells of the gonads²⁸

• fetal and adult²⁸ mesothelium, and mesothelioma. Staining is reportedly weaker in post mortem specimens that in the corresponding ante mortem mesothelioma specimens².

• The ovarian surface epithelium and ovarian inclusion cysts stain²⁹. Ovarian adenocarcinoma is positive, most other adenocarcinomas are negative:

	Ovary	Assorted	25/30 (8 primary serous carcinomas, 5 primary endometrioid adenocarcinomas, and 17 metastatic serous carcinomas to the lung [6] or to the peritoneum [11])6
		serous	16/1710, 38/41 (moderate or strong but patchy in 26 cases, weak in 12 cases.)15, 29/30 (The 6F-H2 antibody produced stronger homogeneous reactivity than did the C-19 clone)13, 25/25 In all cases, at least 5% of nuclei stained.)16, 9/917, 36/3818, 14/16 (borderline tumours)18, positive (results given as average number of nuclei staining, 42±34% cross 27 cases)14, 24/28 (median H-score 45, mean 67 ± 12)21
		mixed serous/endometrioid	2/210, 2/2 (serous component positive)17
		endometrioid	1/110, 0/1516, 1/517, 0/3818, negative (results given as average number of nuclei staining, 2 ± 3% cross 11 cases)14, 0/1121, 2/1824
		mucinous	0/1215, 0/1516, 0/117, ? (results given as average number of nuclei staining, 24 ± 29% cross 10 cases)14, 0/1121
		Brenner tumour, benign	4/1717
		Brenner tumour, malignant	14/1717
		clear cell	0/210, 0/1516, 1/417, ? (results given as average number of nuclei staining, 10 ± 17% across 21 cases)14, 4/18 (median H-score 0)21, 0/1124
		small cell carcinoma of hypercalcaemia type	14/1530
	Fallopian tube	primary serous carcinoma	13/13 (In all cases, staining was diffuse and strong. 11 cases showed metastases, which were also positive.)16, 12/12 (median H-score 180)21
		primary endometrioid carcinoma	0/221
	Peritoneum, primary serous carcinoma		3/316, 6/618, 19/20 (median H-score 108)21
	Breast		2/29 (metastases from breast)10, 0/256, 6/21 (using antibody C-19: positivity is more common in high-grade ER-negative / p53-positive tumours)20
	Uterine	serous	0/1813, 0/516, 5/25 (3+ staining in only two cases)18, 10/16 (median H-score 7.5, mean 23 ± 8)21
		endometrial	0/210, 0/718, 0/3521
		clear cell	0/1821
	Transitional cell carcinoma of bladder		0/1517
	Lung		0/3310, 0/406
	Oesophagus		0/210
	Gallbladder		0/110
	Stomach		0/310
	Colon		0/106
	Pancreaticobiliary		0/210, 0/6415
	Kidney		0/106
	Prostate		0/210, 0/106
	Thyroid		0/106

Where there is both peritoneal serous carcinoma and serous carcinoma within an endometrial polyp, they are concordantly negative for WT-1, suggesting that the peritoneal tumour is metastatic from the endometrial primary²⁷:

	Ovarian serous carcinoma	11/1227
	Primary peritoneal serous carcinoma	8/1027
	Primary uterine serous carcinoma	1/927
	Peritoneal serous carcinoma with serous carcinoma in a uterine polyp	1/9 (there is concordance of the peritoneal and endometrial components)27

• Wilm's tumour

• spleen and some leukaemias¹⁹

• desmoplastic small round cell tumour (DSRCT)¹⁶. The chimeric EWS-WT1 protein in DSRCT acts as a transcription factor¹.

It is NOT expressed by:

• Ewing's sarcoma / Primitive neuroectodermal tumour¹.

• neuroblastoma

• rhabdomyosarcoma

Diagnostic utility

• differentiation of epithelial mesothelioma from adenocarcinoma

	adenocarcinoma	mesothelioma
Amin 19953 (monoclonal antibody, non-commercial)	0/26	20/21
Kumar-Singh 19974 (monoclonal antibody, non-commercial)	3/14 (various sites of origin; the positive cases were 2 ovarian and 1 renal)	39/41
Oates 20005 (polyclonal antibody, Santa Cruz)	8/40 (primary lung adenocarcinomas)	18/42
Ordonez 20006 (polyclonal antibody, Santa Cruz)	25/115 (various sites of origin; all positive cases were ovarian)	36/50
Foster 20017 (polyclonal antibody, Santa Cruz)	0/51 (primary lung adenocarcinomas: 44 cases showed cytoplasmic staining of varying degrees)	50/67 (8 cases showed predominantly cytoplasmic staining)
Miettinen 20018 (polyclonal antibody, Santa Cruz)	not studied	12/21 (12/17 epithelioid, 0/4 sarcomatoid: staining was nuclear)
Ordonez 20039 (monoclonal antibody, Dako 6F-H2)	0/50 (primary lung adenocarcinomas)	56/60 (all epithelioid mesotheliomas: 27 cases >75% of cells, 16 cases 50-75% of cells, 9 cases 25-50% of cells, 4 cases 1-25% of cells)
Pan 200325 (polyclonal Santa Cruz)	0/1425	7/1225
Chu 200526 (monoclonal antibody, Dako 6F-H2)	29/91 (2 of 31 primary pulmonary adenocarcinomas, 4 of 34 renal, 23/26 ovarian serous)26	29/49 (19 of 33 epithelioid, 10 of 16 biphasic; also 0/4 sarcomatous)26
Overall	4% (15/348, excluding ovarian tumours)	76% (238/314)

A systematic review of eight studies (consisting of 264 epithelioid mesotheliomas and 213 pulmonary adenocarcinomas) reported sensitivities and specificities of WT-1 for epithelioid mesothelioma of 77% and 96%³¹.

Further studies are needed to see whether the high rate of positivity in epithelioid mesothelioma obtained by Ordonez⁹ using a newly available monoclonal antibody can be reproduced. Note that WT1 is commonly expressed by serous carcinomas of the ovary⁶.

• Differentiation of desmoplastic small round cell tumour (DSRCT) which stains positively for WT1 protein from Ewing sarcoma / primitive neuroectodermal tumour

DSRCT	EWS/PNET
13/13 positive, using C-191	0/11 positive1

• Identification of the primary site of adenocarcinomas, being commonly positive in ovarian but negative in breast carcinoma¹⁰.

• Differentiation of ovarian and peritoneal from uterine serous carcinomas. One paper reports positivity in uterine as well as ovarian serous tumours; although staining is significantly stronger in ovarian tumours, overlap was thought to limit the diagnostic value²¹. Adjuvant therapies differ, uterine serous tumours being less responsive to cisplatin-based chemotherapy than are ovarian serous tumours¹³.

References

¹ Hill AD et al. WT1 staining reliably differentiates desmoplastic small round cell tumor from Ewing sarcoma/primitive neuroectodermal tumor. An immunohistochemical and molecular diagnostic study. Am J Clin Pathol 2000;114:345-353.

² Roberts, F., McCall, A. E., Burnett, R. A. Malignant mesothelioma: a comparison of biopsy and postmortem material by light microscopy and immunohistochemistry. J Clin Pathol 2001;54:766-70.

³ Amin, K. M., L. A. Litzky, et al. (1995). "Wilms' tumor 1 susceptibility (WT1) gene products are selectively expressed in malignant mesothelioma." Am J Pathol 146(2): 344-56.

⁴ Kumar-Singh, S., K. Segers, et al. (1997). "WT1 mutation in malignant mesothelioma and WT1 immunoreactivity in relation to p53 and growth factor receptor expression, cell-type transition, and prognosis." J Pathol 181(1): 67-74.

⁵ Oates, J. and C. Edwards (2000). "HBME-1, MOC-31, WT1 and calretinin: an assessment of recently described markers for mesothelioma and adenocarcinoma." Histopathology 36(4): 341-7.

⁶ Ordonez, N. G. (2000). "Value of thyroid transcription factor-1, E-cadherin, BG8, WT1, and CD44S immunostaining in distinguishing epithelial pleural mesothelioma from pulmonary and nonpulmonary adenocarcinoma." Am J Surg Pathol 24(4): 598-606.

⁷ Foster, M. R., J. E. Johnson, et al. (2001). "Immunohistochemical analysis of nuclear versus cytoplasmic staining of WT1 in malignant mesotheliomas and primary pulmonary adenocarcinomas." Arch Pathol Lab Med 125(10): 1316-20.

⁸ Miettinen, M., J. Limon, et al. (2001). "Calretinin and other mesothelioma markers in synovial sarcoma: analysis of antigenic similarities and differences with malignant mesothelioma." Am J Surg Pathol 25(5): 610-7.

⁹ Ordonez, N. G. (2003). "The immunohistochemical diagnosis of mesothelioma: a comparative study of epithelioid mesothelioma and lung adenocarcinoma." Am J Surg Pathol 27(8): 1031-51.

¹⁰ Lee, B. H., J. L. Hecht, et al. (2002). "WT1, estrogen receptor, and progesterone receptor as markers for breast or ovarian primary sites in metastatic adenocarcinoma to body fluids." Am J Clin Pathol 117(5): 745-50.

¹¹ Rauscher, F. J., 3rd, J. F. Morris, et al. (1990). "Binding of the Wilms' tumor locus zinc finger protein to the EGR-1 consensus sequence." Science 250(4985): 1259-62.

¹² Lae, M. E., P. C. Roche, et al. (2002). "Desmoplastic small round cell tumor: a clinicopathologic, immunohistochemical, and molecular study of 32 tumors." Am J Surg Pathol 26(7): 823-35.

¹³ Goldstein, N. S. and A. Uzieblo (2002). "WT1 immunoreactivity in uterine papillary serous carcinomas is different from ovarian serous carcinomas." Am J Clin Pathol 117(4): 541-5.

¹⁴ Shimizu, M., T. Toki, et al. (2000). "Immunohistochemical detection of the Wilms' tumor gene (WT1) in epithelial ovarian tumors." Int J Gynecol Pathol 19(2): 158-63.

¹⁵ Goldstein, N. S., D. Bassi, et al. (2001). "WT1 is an integral component of an antibody panel to distinguish pancreaticobiliary and some ovarian epithelial neoplasms." Am J Clin Pathol 116(2): 246-52.

¹⁶ Hashi, A., T. Yuminamochi, et al. (2003). "Wilms tumor gene immunoreactivity in primary serous carcinomas of the fallopian tube, ovary, endometrium, and peritoneum." Int J Gynecol Pathol 22(4): 374-7.

¹⁷ Logani, S., E. Oliva, et al. (2003). "Immunoprofile of ovarian tumors with putative transitional cell (urothelial) differentiation using novel urothelial markers: histogenetic and diagnostic implications." Am J Surg Pathol 27(11): 1434-41.

¹⁸ Al-Hussaini, M., A. Stockman, et al. (2004). "WT-1 assists in distinguishing ovarian from uterine serous carcinoma and in distinguishing between serous and endometrioid ovarian carcinoma." Histopathology 44(2): 109-15.

¹⁹ Pritchard-Jones, K. and L. King-Underwood (1997). "The Wilms tumour gene WT1 in leukaemia." Leuk Lymphoma 27(3-4): 207-20.

²⁰ Silberstein, G. B., K. Van Horn, et al. (1997). "Altered expression of the WT1 wilms tumor suppressor gene in human breast cancer." Proc Natl Acad Sci U S A 94(15): 8132-7.

²¹ Acs, G., T. Pasha, et al. (2004). "WT1 is differentially expressed in serous, endometrioid, clear cell, and mucinous carcinomas of the peritoneum, fallopian tube, ovary, and endometrium." Int J Gynecol Pathol 23(2): 110-8.

²² Scharnhorst, V., A. J. van der Eb, et al. (2001). "WT1 proteins: functions in growth and differentiation." Gene 273(2): 141-61.

²³ Lee, S. B. and D. A. Haber (2001). "Wilms tumor and the WT1 gene." Exp Cell Res 264(1): 74-99. This study used clone 6F-H2.

²⁴ Ramalingam, P., A. Malpica, et al. (2004). "The use of cytokeratin 7 and EMA in differentiating ovarian yolk sac tumors from endometrioid and clear cell carcinomas." Am J Surg Pathol 28(11): 1499-1505.

²⁵ Pan, C. C., P. C. Chen, et al. (2003). "Expression of calretinin and other mesothelioma-related markers in thymic carcinoma and thymoma." Hum Pathol 34(11): 1155-62.

²⁶ Chu AY, Litzky LA, Pasha TL, Acs G,Zhang PJ Utility of D2-40, a novel mesothelial marker, in the diagnosis of malignant mesothelioma. Mod Pathol 2005; 18:105-10

²⁷ Euscher ED, Malpica A, Deavers MT, et al. Differential expression of WT-1 in serous carcinomas in the peritoneum with or without associated serous carcinoma in endometrial polyps. Am J Surg Pathol 2005; 29:1074-8

²⁸ Gulyas M,Hjerpe A Proteoglycans and WT1 as markers for distinguishing adenocarcinoma, epithelioid mesothelioma, and benign mesothelium. J Pathol 2003; 199:479-87

²⁹ O'Neill CJ, Deavers MT, Malpica A, et al. An immunohistochemical comparison between low-grade and high-grade ovarian serous carcinomas: significantly higher expression of p53, MIB1, BCL2, HER-2/neu, and C-KIT in high-grade neoplasms. Am J Surg Pathol 2005; 29:1034-41

³⁰ McCluggage WG, Oliva E, Connolly LE, et al. An immunohistochemical analysis of ovarian small cell carcinoma of hypercalcemic type. Int J Gynecol Pathol 2004; 23:330-6

³¹ King JE, Thatcher N, Pickering CA, et al. Sensitivity and specificity of immunohistochemical markers used in the diagnosis of epithelioid mesothelioma: a detailed systematic analysis using published data. Histopathology 2006; 48:223-32

This page last revised 16.2.2006.

©SMUHT/PW Bishop