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CDX2 Immunostaining in Primary and Secondary Ovarian Carcinomas
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11 CDX2 Immunostaining in Primary and Secondary Ovarian Carcinomas Gad Singer and Luigi Tornillo
Introduction Histologic subtypes of ovarian carcinoma (OC) include serous papillary, endometrioid, clear cell, and mucinous carcinomas (Lee et al., 2003). Earlier studies concluded that mucinous carcinomas represent 10–12% of all OCs. Newer studies indicate that primary mucinous ovarian adenocarcinomas are rare, occurring in only about 2% of all OCs (Seidman et al., 2003). Most mucinous carcinomas of the ovary are probably metastatic, with the majority of intestinal provenance. In a 2003 study of 124 consecutive ovarian mucinous carcinomas, only 24% were primary and nearly 60% were derived from the intestine or pancreas (Seidman et al., 2003). On morphologic examination, many metastases to the ovary may also closely mimic other primary OCs (Young and Hart, 1992), many showing a so-called “pseudoendometrioid” pattern. The most challenging task is therefore the distinction between primary and metastatic OCs (Lee et al., 2003). This distinction is crucial because of different implications for therapy and prognosis. Immunohistochemistry (IHC) plays a fundamental role in the differential diagnosis of adenocarcinomas of unknown origin. In regard to OC, mainly the combination of cytokeratin 7 (CK7) and CK20 is in Handbook of Immunohistochemistry and in situ Hybridization of Human Carcinomas, Volume 4: Molecular Genetics, Gastrointestinal Carcinoma, and Ovarian Carcinoma
wide use (Berezowski et al., 1996; Dionigi et al., 2000; Lagendijk et al., 1998). CDX2 protein, the product of the homebox cdx2 gene, is involved in the development of the gastrointestinal (GI) tract (Suh et al., 1994; Suh and Traber, 1996) and has been shown to be a specific marker of intestinal-type differentiation (Moskaluk et al., 2003). The reported prevalence of CDX2 expression in primary OCs is inconsistent (Barbareschi et al., 2003; Fraggetta et al., 2003; Moskaluk et al., 2003; Werling et al., 2003). We therefore decided to further investigate the role of CDX2 in primary OC and to include metastases from carcinomas to the ovary (Tornillo et al., 2004), to specifically assess the utility of CDX2 as a marker for the distinction between primary OCs and metastases to the ovary.
METHODS A monoclonal antibody to CDX2 protein (clone CDX2-88 BioGenex, Norris Canyon Road, San Ramon, CA) was used, dilution 1/100. For detection commercial kits (DAB [diaminobenzidine] detection kit, amplification kit, endogenous
393
Copyright © 2006 by Elsevier (USA). All rights reserved.
III Ovarian Carcinoma
394 biotin blocking kit [Ventana Medical Systems, Tucson, AZ]) were used.
Solutions 1. Phosphate buffer saline (PBS), 0.01 M (pH 7.4). 2. Citrate buffer (10 mM, pH 6.0) (Quartett Immunodiagnostika GmbH, Berlin, Germany). 3. 0.3% H2O2 in methanol.
Antibody
25. Wash in PBS buffer at 37°C. 26. Add copper sulphate at 37°C for 4 min. 27. Add haematoxylin for 2 min. 28. Wash in PBS buffer. 29. Add 0.1 M lithium carbonate in 0.5 M sodium carbonate and treat for 2 min. 30. Wash in PBS buffer at 37°C. 31. Rinse in 80% alcohol for 3 min. 32. Rinse in 95% alcohol for 3 min. 33. Rinse in 100% alcohol. 34. Rinse in xylene 2× for 3 min each. 35. Mount on slide.
Monoclonal antibody to CDX2 protein, clone CDX2-88 (BioGenex), dilution 1/100.
RESULTS AND DISCUSSION Tissue Fix tissue in 10% buffered formalin and embed in paraffin. Cut 5 µm thick sections and place them on sialinized slides.
Procedure 1. Dry overnight at 37°C. 2. Rinse in xylene 2× for 5 min each. 3. Rinse in absolute alcohol twice for 3 min each. 4. Rinse in 95% alcohol for 3 min. 5. Rinse in 80% alcohol for 3 min. 6. Rinse in running water for 5 min. 7. Pretreat in microwave oven at 98°C for 30 min in citrate buffer. 8. Wash in PBS buffer at 37°C. 9. Add 0.3% H2O2 in methanol at 37°C for 4 min. 10. Wash in PBS buffer at 37°C. 11. Add CDX2 antibody 37°C for 32 min. 12. Wash in PBS buffer at 37°C. 13. Add prediluted rabbit anti-mouse immunoglobulin G (IgG) heavy and light chains in PBS buffer at 37°C for 8 min. 14. Wash in PBS buffer at 37°C. 15. Add prediluted rabbit anti-mouse IgG heavy chains in PBS buffer at 37°C for 8 min. 16. Wash in PBS buffer at 37°C. 17. Add 2.5 mg egg-white avidin in 0.1 M PBS at 37°C for 4 min. 18. Wash in PBS buffer at 37°C. 19. Add 2.5 mg egg-free biotin in 0.1 M PBS at 37°C for 4 min. 20. Add anti-rabbit biotinylated antibody. 21. Wash in PBS buffer at 37°C (2×). 22. Add avidin horseradish peroxidase at 37°C for 8 min. 23. Wash in PBS buffer at 37°C. 24. Add chromogen (DAB) at 37°C for 8 min.
The CDX2 protein is a homebox transcription factor, which derives its name from the Drosophila homeotic caudal (Cad) gene (Hinoi et al., 2003). Cad plays an important role in segmentation and formation of posterior structures in Drosophila, such as the posterior midgut and hindgut (Mlodzik and Gehring, 1987). A number of Cad-related genes have been identified in mammals, including at least two homologs in humans, termed cdx1 and cdx2. Although CDX2 protein is rather broadly expressed in embryogenesis and in adult tissues of mice and humans, its expression appears to be essentially restricted to epithelial cells in the small intestine and colon (Beck et al., 1995). CDX2 protein is localized in the more differentiated epithelial cells of the villus or the crypt tip (James et al., 1994), being involved in the control of differentiation (Suh et al., 1994; Suh and Traber, 1996). Expression of CDX2 protein is very high in the small intestine and the cecum and decreases in the distal colon (Chawengsaksophak et al., l997; Freund et al., l998; Jin and Drucker, 1996). Its homeotic function is showed by experiments in mice: cdx2–/– animals die during development and cdx2+/– ones develop hamartomatous polyps and adenomas in the GI tract, with characteristic heteroplasias underlining the role of the protein in the determination of position along the anterior–posterior axis of the gut (Silberg et al., 2002). Cdx2 probably acts as a tumor-suppressor gene because forced expression of CDX2 in various intestinal epithelial cell lines increases cell differentiation and apoptosis and inhibits proliferation (Lorentz et al., 1997; Mallo et al., 1998; Suh and Traber, 1996), while its level is lowered in human colorectal cancer cells (Ee et al., 1995; Hinoi et al., 2003; Kaimaktchiev et al., 2004; Mallo et al., 1997). The detailed distribution of CDX2 immunohistochemical expression in normal tissues and in tumors has been shown in large series (Moskaluk et al., 2003). CDX2 protein has been reported to be expressed in 85–100% of colon
11 CDX2 Immunostaining in Primary and Secondary Ovarian Carcinomas adenocarcinomas (Kaimaktchiev et al., 2004), 20– 70% of oesophageal and gastric adenocarcinomas (Kaimaktchiev et al., 2004), and 10–60% of pancreas and gallbladder adenocarcinomas (Werling et al., 2003). Moreover, CDX2 immunoreactvity has been reported in well-differentiated neuroendocrine tumors of the GI tract (Barbareschi et al., 2004; La Rosa et al., 2004) and so-called goblet-cell–type mucinous carcinomas of the lung (Rossi et al., 2004). These studies, together with the reported positive immunoexpression of CDX2 in Barret’s esophagus (Groisman et al., 2004; Moons et al., 2004), underline its specificity as a marker of intestinal differentiation. The most important differential diagnosis of OCs is metastatic disease that may present clinically as a primary ovarian tumor (Lee and Young, 2003). Most of metastases to the ovary arise in carcinomas of the intestines, pancreas, bilary tract, or uterine cervix (Seidman et al., 2003). Several morphologic patterns might be suggestive for metastatic disease. Metastatic tumors tend to occur bilaterally, usually measure below 10 cm, and show superficial involvement and vascular invasion (Lee et al., 2003). Immunohistochemical markers that are in use for the distinction between primary and metastatic OCs include CK7, CK20, Dpc4, and beta-catenin (Chou et al., 2003; Ji et al., 2002), but no individual marker is entirely specific. Therefore, primary OCs and metastatic carcinomas to the ovary should be identifiable by a distinct pattern of markers or by more reliable markers. To investigate the role of CDX2 for the differential diagnosis between primary and metastatic OCs, we have studied the expression of CDX2 protein in 237 primary ovarian tumors and 20 metastases to the ovary (Tornillo et al., 2004). Only a clearcut nuclear staining was considered as positive. Serous (n = 129), endometrioid (n = 68), and clear cell (n = 24) OCs were negative for CDX2. Among primary mucinous tumors only one showed a focal weak nuclear positivity (<25% of tumor cells). Metastases from gastric, pancreatic, and cervical adenocarcinomas were negative for CDX2. On the contrary, 14 (all moderately differentiated adenocarcinomas) out of 16 (2 additional poorly differentiated adenocarcinomas) metastatic tumors to the ovary of intestinal origin as well as their primaries showed strong immunoreactivity for CDX2 in more than 75% (3+) of the tumor cells (Tornillo et al., 2004) (Figure 64). In the literature, two studies found a lack of immunostaining for CDX2 (Barbareschi et al., 2003; Werling et al., 2003) in “nonmucinous” and serous OCs, whereas one study found CDX2 immunoreactivity in 20% of endometrioid carcinomas (Moskaluk et al., 2003). In ovarian mucinous carcinomas, CDX2 expression was detected in 20–100% of the neoplasms
395
A
B
C Figure 64. Nuclear staining of CDX-2. A: Normal colon mucosa. B: Primary carcinoma of the colon. C: Ovarian metastasis of colonic carcinoma.
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396 (Barbareschi et al., 2003; Werling et al., 2003). Fraggetta et al. (2003) reported an investigation of CDX2 immunoexpression in a series of 62 mucinous tumors of the ovary, including 16 carcinomas. They found at least a focal CDX2 reactivity in 15 of the 16 carcinomas. In contrast, a 2004 fine-needle aspiration study (Saad et al., 2004) found CDX2 expression only in metastatic carcinomas. Some contrasting reports of CDX2 immunopositivity in OCs might be partially explained by a misclassification of metastases as ovarian primaries. Conceivably, focal positivity of CDX2 (Fraggetta et al., 2003) might be related to a partial “intestinal” differentiation. In summary, CDX2 IHC is a useful and largely specific marker in the differential diagnosis between primary and metastatic tumors of the ovary. Although CDX2 appears to be an excellent marker for metastases to the ovaries from intestinal carcinomas, it should be noted that poorly differentiated intestinal carcinomas may lose their CDX2 expression and that rare primary ovarian tumors may be focally positive for CDX2. Ideally, it should be used in a panel with other markers such as CK7 or CK20.
References Barbareschi, M., Murer, B., Colby, T.V., Chilosi, M., Macri, E., Loda, M., and Doglioni, C. 2003. CDX-2 homeobox gene expression is a reliable marker of colorectal adenocarcinoma metastases to the lungs. Am. J. Surg. Pathol. 27:141–149. Barbareschi, M., Roldo, C., Zamboni, G., Capelli, P., Cavazza, A., Macri, E., Cangi, M.G., Chilosi, M., and Doglioni, C. 2004. CDX-2 homeobox gene product expression in neuroendocrine tumors: its role as a marker of intestinal neuroendocrine tumors. Am. J. Surg. Pathol. 28:1169–1176. Beck, F., Erler, T., Russell, A., and James, R. 1995. Expression of Cdx-2 in the mouse embryo and placenta: possible role in patterning of the extra-embryonic membranes. Dev. Dyn. 204:219–227. Berezowski, K., Grimes, M.M., Gal, A., and Kornstein, M.J. 1996. CD5 immunoreactivity of epithelial cells in thymic carcinoma and CASTLE using paraffin-embedded tissue. Am. J. Clin. Pathol. 106:483–486. Chawengsaksophak, K., James, R., Hammond, V.E., Kontgen, F., and Beck, F. 1997. Homeosis and intestinal tumours in Cdx2 mutant mice. Nature 386:84–87. Chou, Y.Y., Jeng, Y.M., Kao, H.L., Chen, T., Mao, T.L., and Lin, M.C. 2003. Differentiation of ovarian mucinous carcinoma and metastatic colorectal adenocarcinoma by immunostaining with beta-catenin. Histopathology 43:151–156. Dionigi, A., Facco, C., Tibiletti, M.G., Bernasconi, B., Riva, C., and Capella, C. 2000. Ovarian metastases from colorectal carcinoma: clinicopathologic profile, immunophenotype, and karyotype analysis. Am. J. Clin. Pathol. 114:111–122. Ee, H.C., Erler, T., Bhathal, P.S., Young, G.P., and James, R.J. 1995. Cdx-2 homeodomain protein expression in human and rat colorectal adenoma and carcinoma. Am. J. Pathol. 147:586–592. Fraggetta, F., Pelosi, G., Cafici, A., Scollo, P., Nuciforo, P., and Viale, G. 2003. CDX2 immunoreactivity in primary and metastatic ovarian mucinous tumors. Virchows Arch. 443:782–786.
Freund, J.N., Domon-Dell, C., Kedinger, M., and Duluc, I. 1998. The Cdx-1 and Cdx-2 homeobox genes in the intestine. Biochem. Cell. Biol. 76:957–969. Groisman, G.M., Amar, M., and Meir, A. 2004. Expression of the intestinal marker Cdx2 in the columnar-lined esophagus with and without intestinal (Barrett’s) metaplasia. Mod. Pathol. 17:1282–1288. Hinoi, T., Loda, M., and Fearon, E.R. 2003. Silencing of CDX2 expression in colon cancer via a dominant repression pathway. J. Biol. Chem. 278:44608–44616. James, R., Erler, T., and Kazenwadel, J. 1994. Structure of the murine homeobox gene cdx-2. Expression in embryonic and adult intestinal epithelium. J. Biol. Chem. 269:15229–15237. Ji, H., Isacson, C., Seidman, J.D., Kurman, R.J., and Ronnett, B.M. 2002. Cytokeratins 7 and 20, Dpc4, and MUC5AC in the distinction of metastatic mucinous carcinomas in the ovary from primary ovarian mucinous tumors: dpc4 assists in identifying metastatic pancreatic carcinomas. Int. J. Gynecol. Pathol. 21: 391–400. Jin, T., and Drucker, D.J. 1996. Activation of proglucagon gene transcription through a novel promoter element by the caudalrelated homeodomain protein cdx-2/3. Mol. Cell. Biol. 16:19–28. Kaimaktchiev, V., Terracciano, L., Tornillo, L., Spichtin, H., Stoios, D., Bundi, M., Korcheva, V., Mirlacher, M., Loda, M., Sauter, G., and Corless, C.L. 2004. The homeobox intestinal differentiation factor CDX2 is selectively expressed in gastrointestinal adenocarcinomas. Mod. Pathol. 17:1392–1399. La Rosa, S., Rigoli, E., Uccella, S., Chiaravalli, A.M., and Capella, C. 2004. CDX2 as a marker of intestinal EC-cells and related well-differentiated endocrine tumors. Virchows Arch. 445: 248–254. Lagendijk, J.H., Mullink, H., Van Diest, P.J., Meijer, G.A., and Meijer, C.J. 1998. Tracing the origin of adenocarcinomas with unknown primary using immunohistochemistiy: differential diagnosis between colonic and ovarian carcinomas as primary sites. Hum. Pathol. 29:491–497. Lee, K.R., Tavassoli, F.A., Prat, J., Dietel, M., Gersell, D.J., Karseladze, A.I., Hauptmann, S., Rutgers, J., Russell, P., Buckley, C.H., Pisani, P., Schwartz, P., Goldgar, D.E., Silva, E., Caduff, R., and Kubik-Huch, R.A., (eds). 2003. Surface Epithelial-Stromal Tumors. World Health Classification of Tumors. Lyon: IARC Press. Lee, K.R., and Young, R.H. 2003. The distinction between primary and metastatic mucinous carcinomas of the ovary: gross and histologic findings in 50 cases. Am. J. Surg. Pathol. 27:281–292. Lorentz, O., Duluc, I., Arcangelis, A.D., Simon-Assmann, P., Kedinger, M., and Freund, J.N. 1997. Key role of the Cdx2 homeobox gene in extracellular matrix-mediated intestinal cell differentiation. J. Cell Biol. 139:1553–l565. Mallo, G.V., Rechreche, H., Frigerio, J.M., Rocha, D., Zweibaum, A., Lacasa, M., Jordan, B.R., Dusetti, N.J., Dagorn, J.C., and Iovanna, J.L. 1997. Molecular cloning, sequencing and expression of the mRNA encoding human Cdx1 and Cdx2 homeobox: down-regulation of Cdx1 and Cdx2 mRNA expression during colorectal carcinogenesis. Int. J. Cancer 74:35–44. Mallo, G.V., Soubeyran, P., Lissitzky, J.C., Andre, F., Farnarier, C., Marvaldi, J., Dagorn, J.C., and Iovanna, J.L. 1998. Expression of the Cdx1 and Cdx2 homeotic genes leads to reduced malignancy in colon cancer-derived cells. J. Biol. Chem. 273: 14030–14036. Mlodzik, M., and Gehring, W.J. 1987. Expression of the caudal gene in the germ line of Drosophila: formation of an RNA and protein gradient during early embryogenesis. Cell 48:465–478. Moons, L.M., Bax, D.A., Kuipers, E.J., Van Dekken, H., Haringsma, J., Van Vliet, A.H., Siersema, P.D., and Kusters, J.G. 2004.
11 CDX2 Immunostaining in Primary and Secondary Ovarian Carcinomas The homeodomain protein CDX2 is an early marker of Barrett’s oesophagus. J. Clin. Pathol. 57:1063–1068. Moskaluk, C.A., Zhang, H., Powell, S.M., Cerilli, L.A., Hampton, G.M., and Frierson, H.F., Jr. 2003. Cdx2 protein expression in normal and malignant human tissues: an immunohistochemical survey using tissue microarrays. Mod. Pathol. 16:913–919. Rossi, G., Murer, B., Cavazza, A., Losi, L., Natali, P., Marchioni, A., Migaldi, M., Capitanio, G., and Brambilla, E. 2004. Primary mucinous (so-called colloid) carcinomas of the lung: a clinicopathologic and immunohistochemical study with special reference to CDX-2 homeobox gene and MUC2 expression. Am. J. Surg. Pathol. 28:442–452. Saad, R.S., Essig, D.L., Silverman, J.F., and Liu, Y. 2004. Diagnostic utility of CDX-2 expression in separating metastatic gastrointestinal adenocarcinoma from other metastatic adenocarcinoma in fine-needle aspiration cytology using cell blocks. Cancer 102:168–173. Seidman, J.D., Kunnan, R.J., and Ronnett, B.M. 2003. Primary and metastatic mucinous adenocarcinomas in the ovaries: incidence in routine practice with a new approach to improve intraoperative diagnosis. Am. J. Surg. Pathol. 27:985–993.
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Silberg, D.G., Sullivan, J., Kang, E., Swain, G.P., Moffett, J., Sund, N.J., Sackett, S.D., and Kaestner, K.H. 2002. Cdx2 ectopic expression induces gastric intestinal metaplasia in transgenic mice. Gastroenterology 122:689–696. Suh, E., Chen, L., Taylor, J., and Traber, P.G. 1994. A homeodomain protein related to caudal regulates intestine-specific gene transcription. Mol. Cell. Biol. 14:7340–7351. Suh, E., and Traber, P.G. 1996. An intestine-specific homeobox gene regulates proliferation and differentiation. Mol. Cell Biol. 16:619–625. Tornillo, L., Moch, H., Diener, P.A., Lugli, A., and Singer, G. 2004. CDX-2 immunostaining in primary and secondary ovarian carcinomas. J. Clin. Pathol. 57:641–643. Werling, R.W., Yazji, H., Bacchi, C.E., and Gown, A.M. 2003. CDX2, a highly sensitive and specific marker of adenocarcinomas of intestinal origin: an immunohistochemical survey of 476 primary and metastatic carcinomas. Am. J. Surg. Pathol. 27:303–320. Young, R.H., and Hart, W.R. 1992. Renal cell carcinoma metastatic to the ovary: a report of three cases emphasizing possible confusion with ovarian clear cell adenocarcinoma. Int. J. Gynecol. Pathol. 11:96–104.
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11 CDX2 Immunostaining in Primary and Secondary Ovarian Carcinomas Gad Singer and Luigi Tornillo
Introduction Histologic subtypes of ovarian carcinoma (OC) include serous papillary, endometrioid, clear cell, and mucinous carcinomas (Lee et al., 2003). Earlier studies concluded that mucinous carcinomas represent 10–12% of all OCs. Newer studies indicate that primary mucinous ovarian adenocarcinomas are rare, occurring in only about 2% of all OCs (Seidman et al., 2003). Most mucinous carcinomas of the ovary are probably metastatic, with the majority of intestinal provenance. In a 2003 study of 124 consecutive ovarian mucinous carcinomas, only 24% were primary and nearly 60% were derived from the intestine or pancreas (Seidman et al., 2003). On morphologic examination, many metastases to the ovary may also closely mimic other primary OCs (Young and Hart, 1992), many showing a so-called “pseudoendometrioid” pattern. The most challenging task is therefore the distinction between primary and metastatic OCs (Lee et al., 2003). This distinction is crucial because of different implications for therapy and prognosis. Immunohistochemistry (IHC) plays a fundamental role in the differential diagnosis of adenocarcinomas of unknown origin. In regard to OC, mainly the combination of cytokeratin 7 (CK7) and CK20 is in Handbook of Immunohistochemistry and in situ Hybridization of Human Carcinomas, Volume 4: Molecular Genetics, Gastrointestinal Carcinoma, and Ovarian Carcinoma
wide use (Berezowski et al., 1996; Dionigi et al., 2000; Lagendijk et al., 1998). CDX2 protein, the product of the homebox cdx2 gene, is involved in the development of the gastrointestinal (GI) tract (Suh et al., 1994; Suh and Traber, 1996) and has been shown to be a specific marker of intestinal-type differentiation (Moskaluk et al., 2003). The reported prevalence of CDX2 expression in primary OCs is inconsistent (Barbareschi et al., 2003; Fraggetta et al., 2003; Moskaluk et al., 2003; Werling et al., 2003). We therefore decided to further investigate the role of CDX2 in primary OC and to include metastases from carcinomas to the ovary (Tornillo et al., 2004), to specifically assess the utility of CDX2 as a marker for the distinction between primary OCs and metastases to the ovary.
METHODS A monoclonal antibody to CDX2 protein (clone CDX2-88 BioGenex, Norris Canyon Road, San Ramon, CA) was used, dilution 1/100. For detection commercial kits (DAB [diaminobenzidine] detection kit, amplification kit, endogenous
393
Copyright © 2006 by Elsevier (USA). All rights reserved.
III Ovarian Carcinoma
394 biotin blocking kit [Ventana Medical Systems, Tucson, AZ]) were used.
Solutions 1. Phosphate buffer saline (PBS), 0.01 M (pH 7.4). 2. Citrate buffer (10 mM, pH 6.0) (Quartett Immunodiagnostika GmbH, Berlin, Germany). 3. 0.3% H2O2 in methanol.
Antibody
25. Wash in PBS buffer at 37°C. 26. Add copper sulphate at 37°C for 4 min. 27. Add haematoxylin for 2 min. 28. Wash in PBS buffer. 29. Add 0.1 M lithium carbonate in 0.5 M sodium carbonate and treat for 2 min. 30. Wash in PBS buffer at 37°C. 31. Rinse in 80% alcohol for 3 min. 32. Rinse in 95% alcohol for 3 min. 33. Rinse in 100% alcohol. 34. Rinse in xylene 2× for 3 min each. 35. Mount on slide.
Monoclonal antibody to CDX2 protein, clone CDX2-88 (BioGenex), dilution 1/100.
RESULTS AND DISCUSSION Tissue Fix tissue in 10% buffered formalin and embed in paraffin. Cut 5 µm thick sections and place them on sialinized slides.
Procedure 1. Dry overnight at 37°C. 2. Rinse in xylene 2× for 5 min each. 3. Rinse in absolute alcohol twice for 3 min each. 4. Rinse in 95% alcohol for 3 min. 5. Rinse in 80% alcohol for 3 min. 6. Rinse in running water for 5 min. 7. Pretreat in microwave oven at 98°C for 30 min in citrate buffer. 8. Wash in PBS buffer at 37°C. 9. Add 0.3% H2O2 in methanol at 37°C for 4 min. 10. Wash in PBS buffer at 37°C. 11. Add CDX2 antibody 37°C for 32 min. 12. Wash in PBS buffer at 37°C. 13. Add prediluted rabbit anti-mouse immunoglobulin G (IgG) heavy and light chains in PBS buffer at 37°C for 8 min. 14. Wash in PBS buffer at 37°C. 15. Add prediluted rabbit anti-mouse IgG heavy chains in PBS buffer at 37°C for 8 min. 16. Wash in PBS buffer at 37°C. 17. Add 2.5 mg egg-white avidin in 0.1 M PBS at 37°C for 4 min. 18. Wash in PBS buffer at 37°C. 19. Add 2.5 mg egg-free biotin in 0.1 M PBS at 37°C for 4 min. 20. Add anti-rabbit biotinylated antibody. 21. Wash in PBS buffer at 37°C (2×). 22. Add avidin horseradish peroxidase at 37°C for 8 min. 23. Wash in PBS buffer at 37°C. 24. Add chromogen (DAB) at 37°C for 8 min.
The CDX2 protein is a homebox transcription factor, which derives its name from the Drosophila homeotic caudal (Cad) gene (Hinoi et al., 2003). Cad plays an important role in segmentation and formation of posterior structures in Drosophila, such as the posterior midgut and hindgut (Mlodzik and Gehring, 1987). A number of Cad-related genes have been identified in mammals, including at least two homologs in humans, termed cdx1 and cdx2. Although CDX2 protein is rather broadly expressed in embryogenesis and in adult tissues of mice and humans, its expression appears to be essentially restricted to epithelial cells in the small intestine and colon (Beck et al., 1995). CDX2 protein is localized in the more differentiated epithelial cells of the villus or the crypt tip (James et al., 1994), being involved in the control of differentiation (Suh et al., 1994; Suh and Traber, 1996). Expression of CDX2 protein is very high in the small intestine and the cecum and decreases in the distal colon (Chawengsaksophak et al., l997; Freund et al., l998; Jin and Drucker, 1996). Its homeotic function is showed by experiments in mice: cdx2–/– animals die during development and cdx2+/– ones develop hamartomatous polyps and adenomas in the GI tract, with characteristic heteroplasias underlining the role of the protein in the determination of position along the anterior–posterior axis of the gut (Silberg et al., 2002). Cdx2 probably acts as a tumor-suppressor gene because forced expression of CDX2 in various intestinal epithelial cell lines increases cell differentiation and apoptosis and inhibits proliferation (Lorentz et al., 1997; Mallo et al., 1998; Suh and Traber, 1996), while its level is lowered in human colorectal cancer cells (Ee et al., 1995; Hinoi et al., 2003; Kaimaktchiev et al., 2004; Mallo et al., 1997). The detailed distribution of CDX2 immunohistochemical expression in normal tissues and in tumors has been shown in large series (Moskaluk et al., 2003). CDX2 protein has been reported to be expressed in 85–100% of colon
11 CDX2 Immunostaining in Primary and Secondary Ovarian Carcinomas adenocarcinomas (Kaimaktchiev et al., 2004), 20– 70% of oesophageal and gastric adenocarcinomas (Kaimaktchiev et al., 2004), and 10–60% of pancreas and gallbladder adenocarcinomas (Werling et al., 2003). Moreover, CDX2 immunoreactvity has been reported in well-differentiated neuroendocrine tumors of the GI tract (Barbareschi et al., 2004; La Rosa et al., 2004) and so-called goblet-cell–type mucinous carcinomas of the lung (Rossi et al., 2004). These studies, together with the reported positive immunoexpression of CDX2 in Barret’s esophagus (Groisman et al., 2004; Moons et al., 2004), underline its specificity as a marker of intestinal differentiation. The most important differential diagnosis of OCs is metastatic disease that may present clinically as a primary ovarian tumor (Lee and Young, 2003). Most of metastases to the ovary arise in carcinomas of the intestines, pancreas, bilary tract, or uterine cervix (Seidman et al., 2003). Several morphologic patterns might be suggestive for metastatic disease. Metastatic tumors tend to occur bilaterally, usually measure below 10 cm, and show superficial involvement and vascular invasion (Lee et al., 2003). Immunohistochemical markers that are in use for the distinction between primary and metastatic OCs include CK7, CK20, Dpc4, and beta-catenin (Chou et al., 2003; Ji et al., 2002), but no individual marker is entirely specific. Therefore, primary OCs and metastatic carcinomas to the ovary should be identifiable by a distinct pattern of markers or by more reliable markers. To investigate the role of CDX2 for the differential diagnosis between primary and metastatic OCs, we have studied the expression of CDX2 protein in 237 primary ovarian tumors and 20 metastases to the ovary (Tornillo et al., 2004). Only a clearcut nuclear staining was considered as positive. Serous (n = 129), endometrioid (n = 68), and clear cell (n = 24) OCs were negative for CDX2. Among primary mucinous tumors only one showed a focal weak nuclear positivity (<25% of tumor cells). Metastases from gastric, pancreatic, and cervical adenocarcinomas were negative for CDX2. On the contrary, 14 (all moderately differentiated adenocarcinomas) out of 16 (2 additional poorly differentiated adenocarcinomas) metastatic tumors to the ovary of intestinal origin as well as their primaries showed strong immunoreactivity for CDX2 in more than 75% (3+) of the tumor cells (Tornillo et al., 2004) (Figure 64). In the literature, two studies found a lack of immunostaining for CDX2 (Barbareschi et al., 2003; Werling et al., 2003) in “nonmucinous” and serous OCs, whereas one study found CDX2 immunoreactivity in 20% of endometrioid carcinomas (Moskaluk et al., 2003). In ovarian mucinous carcinomas, CDX2 expression was detected in 20–100% of the neoplasms
395
A
B
C Figure 64. Nuclear staining of CDX-2. A: Normal colon mucosa. B: Primary carcinoma of the colon. C: Ovarian metastasis of colonic carcinoma.
III Ovarian Carcinoma
396 (Barbareschi et al., 2003; Werling et al., 2003). Fraggetta et al. (2003) reported an investigation of CDX2 immunoexpression in a series of 62 mucinous tumors of the ovary, including 16 carcinomas. They found at least a focal CDX2 reactivity in 15 of the 16 carcinomas. In contrast, a 2004 fine-needle aspiration study (Saad et al., 2004) found CDX2 expression only in metastatic carcinomas. Some contrasting reports of CDX2 immunopositivity in OCs might be partially explained by a misclassification of metastases as ovarian primaries. Conceivably, focal positivity of CDX2 (Fraggetta et al., 2003) might be related to a partial “intestinal” differentiation. In summary, CDX2 IHC is a useful and largely specific marker in the differential diagnosis between primary and metastatic tumors of the ovary. Although CDX2 appears to be an excellent marker for metastases to the ovaries from intestinal carcinomas, it should be noted that poorly differentiated intestinal carcinomas may lose their CDX2 expression and that rare primary ovarian tumors may be focally positive for CDX2. Ideally, it should be used in a panel with other markers such as CK7 or CK20.
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