Immunohistochemistry Applications in Gastrointestinal Cancer

Mike Lacey, M.D.

Incidence
Deaths
Lifetime risk of Colorectal Cancer
Signs and Symptoms
Immunohistochemistry and Colon Cancer
Metastatic Carcinoma
[Webinar] Colorectal Cancer and Immunohistochemistry
Materials
References

Incidence

Colorectal cancer is the third most common cancer in both men and women. An estimated 136,000 cases of colorectal cancer are expected to occur in 2016. Colorectal cancer incidence rates have been decreasing for most of the past two decades (from 66.3 per 100,000 persons in 1985 to 45.5 cases in 2006). This has been attributed to increases in the use of colorectal screening tests that allow the detection and removal of colorectal polyps before they progress to cancer. In contrast to overall declines, among adults younger than 50 years, for whom screening is not recommended for those at average risk, colorectal cancer incidence rates have been increasing by about 2% per year since 1994 in both men and women¹.

Deaths

An estimated 49,140 deaths from colorectal cancer are expected in 2016 in the U.S., accounting for 9% of all cancer deaths. Mortality rates for colorectal cancer have declined in both men and women over the past two decades, with steeper declines in more recent time periods (3.9% per year from 2002 to 2006 in men and 3.4% per year from 2001 to 2006 in women). This decrease reflects declining incidence rates and improvements in early detection and treatment¹.

Lifetime risk of Colorectal Cancer

The risk of developing colorectal cancer is slightly lower in women than in men. Overall, the lifetime risk is about 1 in 21 (4.7%) for men and 1 in 23 (4.4%) for women. Other factors such as family history, genetics, obesity, and diet, can also affect your risk for developing colorectal cancer.

Signs and Symptoms

Early stage colorectal cancer does not usually have symptoms; therefore, screening is often necessary to detect colorectal cancer in its early stages. Advanced disease may cause rectal bleeding, blood in the stool, a change in bowel habits, and cramping pain in the lower abdomen¹.

Immunohistochemistry and Colon Cancer

Immunohistochemical applications surrounding colon cancer are seen at several levels such as: characterization of the tumor (endocrine or epithelial type), hereditary disposition, and for prognostic purposes (MSI testing). MSI testing has been discussed previously and thus will not be discussed, in detail, here other than to say that the Bethesda protocol is rapidly gaining acceptance regarding this testing. The more prevalent use of IHC is in the presence of possible or suspected metastatic disease in which the colon is a possible primary. The common locations for metastases from colon cancers are the liver and lung; both organs of which can produce cancer morphology essentially identical to metastases from the colon.

Metastatic Carcinoma

The most common use of immunohistochemistry in the study of liver tumors is to identify the site of origin of a metastatic tumor when the primary site has not been previously identified. The development and implementation of a panel of immunostains can help resolve almost all diagnostic problems.^2,3-6 Cytokeratin 7 and CK20 are the first step in the immunohistochemical identification of many tumors, and with additional immunostains, some relatively specific for tumors of males and of females, one is able to identify potential sites of origin. More recently, other antibodies such as cadherin-17, SATB2, CDX-2, and villin have found use in the differential diagnosis of suspected metastatic adenocarcinoma.

Stomach

Immunohistochemical studies are generally not needed for the evaluation of benign and malignant epithelial tumors of the stomach because the histopathology is generally diagnostic but are used in the study of metastatic gastric carcinoma when the site of origin is not clear or when the macroscopic/radiologic appearance of the tumor is confusing (eg, gastric carcinoma directly and massively invading the liver and histologically indistinguishable from cholangiocarcinoma).

Adenocarcinoma

Gastric adenocarcinomas will react with many antibodies directed against keratins, including AE1&AE3, CK35betaH11, CK18, CK19, CK7, and CK20. When CK7 and CK20 are used together, many gastric adenocarcinomas will stain with both CK7 and CK20.^5,7-9 Approximately 25% will be positive for one and negative for the other (eg, CK7+/CK20-, CK7-/CK20+), and a small number of cases will be negative for both. CDX-2, initially thought to be specific for colon carcinoma, will be reactive in more than 50% of cases^10,11 and may be indicative of a lesser degree of invasiveness.^12,13 Even HepPar-1, a useful marker for hepatocytes, will be positive in more than 50% of gastric cancers, including signet-ring cell carcinoma.¹⁴ The quantity and quality of mucus production by gastric carcinoma, as evaluated by immunohistochemical study of mucins, may be prognostically important; MUC2 expression is associated with poor survival.¹⁵

Neuroendocrine Carcinoma

Neuroendocrine carcinoma characteristically stains with synaptophysin, chromogranin, villin, and CD57.^16,17 In contrast to gastric adenocarcinoma, carcinomas occurring in the second part of the duodenum may be negative for both synaptophysin and chromogranin but will often react with somatostatin. The proliferation marker Ki-67 and the adhesion molecule E-cadherin have been used to assess aggressiveness of neuroendocrine carcinoma.¹⁸ A high (>60%) Ki-67 proliferation index predicts aggressive behavior, and loss of E-cadherin may predict lymph node metastasis. Only 1 study has investigated cadherin-17 expression in neuroendocrine tumors of the gastrointestinal tract. In that investigation, all 27 of the well-differentiated neuroendocrine tumors (carcinoid tumors) of the small intestine and all 10 of those originating in the appendix were reported to be cadherin-17 positive.⁵⁰

Gastrointestinal Adenocarcinoma with Neuroendocrine Differentiation

Gastric adenocarcinomas, both intestinal type and signet- ring cell type, can have neuroendocrine differentiation that may not be obvious with hematoxylin-eosin staining but will show staining with chromogranin and synaptophysin.¹⁹

Gastrointestinal Stromal Tumors

CD117 stains most cases of gastrointestinal stromal tumor, including metastases.^20-23 Although there may sometimes be variation in distribution of CD117 positivity within a given tumor, in most cases staining is diffuse. When CD117 is positive in tumors other than gastrointestinal stromal tumor, the staining is almost always patchy. CD34 staining can also be seen in gastrointestinal stromal tumor. 10 to 15% of GIST are negative for CD117. DOG1 antibody has been shown to label the majority of CD117 negative GISTs.⁴⁹

Colon

Adenomas and Adenocarcinoma

Adenomas (tubular adenoma, tubulovillous adenoma, villous adenoma) demonstrate the same immunohistochemical reactions as colonic adenocarcinoma. Almost all react with antibody directed against CK20 and a minority will also stain focally with CK7, in contrast to pancreatic adenocarcinomas, most of which are CK20 negative and CK7 positive. Differentiation of metastatic colorectal adenocarcinoma from adenocarcinoma arising at other sites can sometimes be challenging. Pulmonary adenocarcinoma can resemble colorectal adenocarcinoma. Cytokeratin 7 and CK20 can be helpful in this regard, with CK7 usually strongly positive in lung adenocarcinomas and CK20 usually negative; the reverse pattern is seen with colorectal adenocarcinoma. In addition, thyroid transcription factor 1 (TTF-1) is generally positive in lung cancers, and CDX-2 and beta-catenin are generally positive in colorectal cancers. Endometrioid-type carcinomas can also be histologically indistinguishable from colorectal carcinoma. Here, again, CK7 is positive in almost all endometrioid adenocarcinomas and only mildly reactive in colorectal adenocarcinomas.

Cadherin-17

Cadherin-17 is a member of a multigene family of calcium-dependent, transmembrane proteins that mediates cell-cell adhesion, plays important roles during embryogenesis, and is crucial for tissue morphogenesis and maintenance. Cadherin-17 is exclusively expressed in the epithelial cells of embryonic and adult small intestine and colon, and pancreatic ducts. It has also been reported to be frequently expressed in adenocarcinomas arising in the gastrointestinal tract and pancreas. Owing to its restricted expression in these groups of tumors, cadherin-17 has proven to be a useful immunohistochemical marker for assisting in distinguishing these neoplasms from other malignancies with which they may be confused.

In the intestinal epithelial cells, E-cadherin, which is one of the classic cadherins, is concentrated in the adherens junctions, whereas cadherin-17 is evenly distributed along the lateral contact areas. Cadherin-17 is comprised of 832 amino acids, has a molecular weight of B92 kDa, and is encoded by the CDH17 gene located on chromosome 8q22.1.

Although the function of cadherin-17 is not completely understood, it is believed that it is involved in the morphologic organization of the liver and intestine, and also that it may function as a peptide transporter. Cadherin-17 expression is thought to be regulated by CDX-2, an intestine-specific caudal-related homeobox transcription factor that plays an important role in the regulation of the development and homeostasis of intestinal epithelial cells and in the maintenance of the intestinal phenotype.⁵⁰

In 3 immunohistochemical studies^54-56 in which a large number of normal tissues were investigated for cadherin-17 expression, strong positivity was reported in the surface epithelium of the duodenum (but not in the Brunner glands), ileum, appendix, and colorectum. Cadherin-17 expression was not found in the normal gastric mucosa of the body of the stomach or the antrum, but it was seen in areas with intestinal metaplasia. In the liver, cadherin-17 expression was not found in hepatocytes, but on occasion, weak staining was seen in intrahepatic bile duct epithelial cells. In the normal pancreas, cadherin-17 expression was found in the pancreatic ducts, but absent in pancreatic acinar and islet cells.⁵⁰

Of those adenocarcinomas arising in the gastrointestinal tract, cadherin-17 expression has been reported in the vast majority of primary (96% to 100%) and metastatic (100%) colorectal adenocarcinomas. It has also been reported in esophageal (67% to 82%) and gastric (23% to 90%) adenocarcinomas. In a combined review of 6 large published series on the expression of this marker in primary gastrointestinal adenocarcinomas, 329 (99%) of 333 of the colon, 247 (44%) of 565 of the stomach, and 51(75%) of 68 of the esophagus were cadherin-17 positive.⁵⁰

When cadherin-17 expression in adenocarcinomas of the gastrointestinal tract is compared with that of CDX-2, a gastrointestinal-associated transcription factor that is at present commonly used as an immunohistochemical marker for assisting in the diagnosis of adenocarcinomas of the gastrointestinal tract and pancreas, it appears that cadherin-17 is more sensitive than CDX-2 for those tumors arising in the colon (99% vs. 89%) and esophagus (75% vs. 69%), but less sensitive for those originating in the stomach (44% vs. 62.5%).⁵⁰

Villin

Villin is another marker that has been recommended as being useful in the diagnosis of adenocarcinomas of the gastrointestinal tract that, when compared with cadherin-17, is less sensitive for colorectal adenocarcinomas (93% vs. 99%), but more sensitive for gastric adenocarcinomas (49% vs. 44%).⁵⁰

SATB2

SATB2 is a recently recognized immunohistochemical marker that, because its expression is highly restricted to colorectal adenocarcinoma, has found utility as an immunohistochemical marker in the diagnosis of these tumors. Magnusson et al⁵³ show that SATB2 is a sensitive and highly specific marker for colorectal carcinoma (CRC) with distinct positivity in 85% of all CRCs, and that SATB2 and/or CK20 are positive in 97% of CRCs. The specific expression of SATB2 in a large majority of CRCs suggests that SATB2 can be used as an important complementary tool for the differential diagnosis of carcinoma of unknown primary origin. When cadherin-17 is compared with SATB2, it is more sensitive for colorectal adenocarcinomas (99% vs. 87%), but less specific.⁵⁰

Appendiceal Adenocarcinoma

Appendiceal adenocarcinoma will typically show staining for MUC5AC, in contrast to colonic adenocarcinoma in which this antibody is rarely reactive.^24-25 This is particularly useful in studying mucinous adenocarcinomas that have metastasized in the abdomen. Beta-Catenin is another differentiating antibody, positive in almost all colonic adenocarcinomas and negative in appendiceal adenocarcinomas. In women with abdominal mucinous carcinomatosis, distinction of colonic and appendiceal adenocarcinoma from ovarian adenocarcinoma is important. In colonic tumors, both villin and beta-catenin are often expressed; in appendiceal metastases villin is often expressed, but beta-catenin is unusual and in ovarian mucinous adenocarcinomas neither villin nor beta-catenin are seen.²⁶ Similar to appendiceal lesions, ovarian carcinomas express MUC5AC and similar to colorectal adenocarcinoma, ovarian mucinous adenocarcinomas express CDX-2. Useful supplements to the basic panels for unknown primaries in which appendiceal or ovarian mucinous tumors are suspected are MUC5AC and beta-catenin. Villin can also be helpful because it typically has a ‘‘brush-border’’ pattern of staining in both colonic and appendiceal adenocarcinoma and is typically cytoplasmic in ovarian and pancreatic lesions.²⁷

Prognostic Markers

In recent years, a number of immunohistochemical and molecular markers have been developed to predict outcome and also to help select therapies. Colon adenocarcinoma, the most frequent of malignant epithelial tumors affecting the gastrointestinal system, has been particularly evaluated in this way. Currently useful markers that can be studied with immunohistochemical techniques include evaluation of p27, p53, thymidylate synthase,²⁸ and EGFR,²⁹ although the usefulness of these markers, with the exception of EGFR, is still in question. The absence of p27 has been suggested to be a strong negative prognostic marker, particularly in stage II colon cancer, and may help select patients who will benefit from adjuvant therapy. p53 nuclear expression is also associated with shortened survival and might be useful as an independent predictor in patients in whom colorectal carcinoma is metastatic to regional lymph nodes. Overexpression of thymidylate synthase has also been associated with poor prognosis and resistance to 5-flourouracil chemotherapy. EGFR expression is also an indicator of increased likelihood of metastases and decreased survival, but immunoreactivity is also associated with clinical response to cetuximab (Erbitux) therapy.²⁹ Loss of expression of SMAD4 (DPC4), a gene found on chromosome 4, is also associated with poor prognosis in colorectal carcinoma.³⁰ This immunostain is not diagnostically useful because it is also expressed in other carcinomas, including pancreas.^{31, 32}

Microsatellite instability can also be tested with immunohistochemical reactions that evaluate the proteins MLH1, MSH2, MSH6, and PMS2.^33,34 Mutations in one or several DNA mismatch repair genes lead to the development of microsatellite instability (MSI) in hereditary nonpolyposis colorectal cancer and in 15% to 20% of patients with sporadic colorectal adenocarcinoma. MSI is associated with improved survival in both sporadic and hereditary tumors. Mutations in mismatch repair genes result in absence of expression of one or more of the proteins; this is seen as negative immunostaining and is strongly associated with MSI. The presence of immunostaining is evidence of normal expression of the protein and shows that the mutation has not occurred. In this regard, immunohistochemistry is a sensitive (>90%) and extremely specific (nearly 100%) method for screening for DNA mismatch repair defects. The predictive value for the absence of MSI with normal immunohistochemistry results is 97%, and the predictive value for the presence of MSI with abnormal immunohistochemistry results is 100%.^33,34 High SATB2 expression is an independent marker of good prognosis in colon cancer and may modulate sensitivity to chemotherapy and radiation.⁵¹

Pancreas

Invasive Ductal Adenocarcinoma

Adenocarcinoma of the pancreas usually derives from precursor stages of pancreatic ductal dysplasia. The immunostaining pattern of high-grade pancreatic intraepithelial neoplasia is the same as that of invasive pancreatic adenocarcinoma and cannot be used to differentiate between them. Pancreatic ductal adenocarcinomas resemble adenocarcinomas of the bile ducts and gallbladder in their light microscopy appearances and also in their immunophenotypical presentations. Pancreatic adenocarcinomas react with a variety of keratin antibodies, including CK8, CK17, CK18, CK19, CAM 5.2, and AE1&AE3.^5,35 Pancreatic adenocarcinoma is generally both CK7 and CK20 positive. Pancreatic adenocarcinoma can also be faintly CDX-2 positive.^4,10 Almost all pancreaticobiliary adenocarcinomas are CEA positive and CA-125 positive. They may also have a minor component of neuroendocrine cells, which will react with somatostatin, synaptophysin, chromogranin, or other neuroendocrine markers.³⁶ Mucin evaluation may be useful in evaluating prognosis.^{37, 38} Therapy outcomes can also be predicted with positive vascular endothelial growth factor and negative SMAD4 (DPC4) immunostaining.³² Loss of expression of SMAD4 has also been shown in bile duct epithelium in cases of chronic gallstone disease.³⁹ Well-differentiated metastatic pancreatic carcinoma to the liver may be difficult to distinguish from benign bile duct lesions in biopsy material. Unlike the benign lesions, however, they typically express p53, cytoplasmic mCEA, and other markers including CA-125.⁴⁰

In addition to adenocarcinoma of the gastrointestinal tract, a relatively large percentage of pancreatic adenocarcinomas express cadherin-17. Because of the rather restricted expression of this marker in this type of tumor, cadherin-17 immunostaining, when it is used in conjunction with other immunohistochemical markers, can assist in the differential diagnosis of pancreatic adenocarcinomas.⁵⁰

Neuroendocrine and Endocrine Cell Tumors, Low Grade and High Grade

Low- and high-grade neuroendocrine tumors tend to show similar immunophenotypic expressions, but, in general, the intensity of staining is less with high-grade tumors. They can be grouped by the predominant secreted hormone (eg, somatostatin, gastrin) but usually also stain with synaptophysin and chromogranin, as well as with various keratins, including CK8, CK18, and CK35betaH11. CK7 and CK20 are generally negative.^41-45 CD56 and CD57 tend to stain more intensely in high-grade neuroendocrine tumors than in low grade, in a membranous pattern; CD56 will also be positive in a variety of other tumors.^41,46 Serotonin can also be demonstrated.⁴⁷ High-grade neuroendocrine tumors can also stain with calcitonin, and metastatic lesions can be misinterpreted as having arisen in the thyroid.⁴⁵ Epithelial cytoplasmic expression of CD10, in contrast to membrane staining, is more commonly seen in malignant than in benign pancreatic endocrine tumors.⁴⁸

[Webinar] Colorectal Cancer and Immunohistochemistry

References

1. American Cancer Society.

2. Lau SK, Prakash S, Geller SA, Alsabeh R. 2002. Comparative immunohistochemical profile of hepatocellular carcinoma, cholangiocarcinoma, and metastatic adenocarcinoma. Human Pathology. 33(12):1175-1181. https://doi.org/10.1053/hupa.2002.130104

3. 1999. Tot T. Adenocarcinomas metastatic to the liver: the value of cytokeratins 20 and 7 in the search for unknown primary tumors. Cancer. ;85:..171–177.

4. 2004. Tot T. Identifying colorectal metastases in liver biopsies: the novel CDX2 antibody is less specific than the cytokeratin 20_/7- phenotype. Med Sci Monit. ;10:BR139–BR143..

5. Wang NP, Zee S, Zarbo RJ, et al. . 1995. Coordinate expression of cytokeratins 7 and 20 defines unique subsets of carcinomas. Appl Immunohistochem..(3):99–107..

6. Rullier A, Le Bail B, Fawaz R, Blanc JF, Saric J, Bioulac-Sage P. 2000. Cytokeratin 7 and 20 Expression in Cholangiocarcinomas Varies Along the Biliary Tract But Still Differs From That in Colorectal Carcinoma Metastasis. The American Journal of Surgical Pathology. 24(6):870-876. https://doi.org/10.1097/00000478-200006000-00014

7. Kende AI, Carr NJ, Sobin LH. 2003. Expression of cytokeratins 7 and 20 in carcinomas of the gastrointestinal tract. Histopathology. 42(2):137-140. https://doi.org/10.1046/j.1365-2559.2003.01545.x

8. Park SY, Kim HS, Hong EK, Kim WH. 2002. Expression of cytokeratins 7 and 20 in primary carcinomas of the stomach and colorectum and their value in the differential diagnosis of metastatic carcinomas to the ovary. Human Pathology. 33(11):1078-1085. https://doi.org/10.1053/hupa.2002.129422

9. Chu P, Wu E, Weiss LM. 2000. Cytokeratin 7 and Cytokeratin 20 Expression in Epithelial Neoplasms: A Survey of 435 Cases. Mod Pathol. 13(9):962-972. https://doi.org/10.1038/modpathol.3880175

10. Werling RW, Yaziji H, Bacchi CE, Gown AM. 2003. CDX2, a Highly Sensitive and Specific Marker of Adenocarcinomas of Intestinal Origin. The American Journal of Surgical Pathology. 27(3):303-310. https://doi.org/10.1097/00000478-200303000-00003

11. Moskaluk CA, Zhang H, Powell SM, Cerilli LA, Hampton GM, Frierson HF. 2003. Cdx2 Protein Expression in Normal and Malignant Human Tissues: An Immunohistochemical Survey Using Tissue Microarrays. Mod Pathol. 16(9):913-919. https://doi.org/10.1097/01.mp.0000086073.92773.55

12. Roessler K. 2005. Co-expression of CDX2 and MUC2 in gastric carcinomas: Correlations with clinico-pathological parameters and prognosis. WJG. 11(21):3182. https://doi.org/10.3748/wjg.v11.i21.3182

13. Fan Z, van de Rijn M, Montgomery K, Rouse RV. 2003. Hep Par 1 Antibody Stain for the Differential Diagnosis of Hepatocellular Carcinoma: 676 Tumors Tested Using Tissue Microarrays and Conventional Tissue Sections. Mod Pathol. 16(2):137-144. https://doi.org/10.1097/01.mp.0000052103.13730.20

14. Leteurtre E. 2006. Relationships between mucinous gastric carcinoma, MUC2 expression and survival. WJG. 12(21):3324. https://doi.org/10.3748/wjg.v12.i21.3324

15. Burke AP, Thomas RM, Elsayed AM, et al.. 1997. Carcinoids of the jejunum and ileum: an immunohistochemical and clinicopathologic study of 167 cases. Cancer. ;79:1086–1093..

16. Park J, Choe GY, Helman LJ, Gazdar AF, Yang H, Kim J, Park SH, Kim YI. 1992. Chromogranin-a expression in gastric and colon cancer tissues. Int. J. Cancer. 51(2):189-194. https://doi.org/10.1002/ijc.2910510205

17. Boo Y, Park S, Kim J, Mok Y, Kim S, Kim C. 2007. Gastric neuroendocrine carcinoma: Clinicopathologic review and immunohistochemical study of E-cadherin and Ki-67 as prognostic markers. J. Surg. Oncol.. 95(2):110-117. https://doi.org/10.1002/jso.20616

18. Blumenfeld W, Chandhoke DK, Sagerman P, et al. . 1996. Neuroendocrine differentiation in gastric adenocarcinomas: an immunohistochemical study. Arch Pathol Lab Med. ;120:478–481..

19. Wong NACS, Young R, Malcomson RDG, Nayar AG, Jamieson LA, Save VE, Carey FA, Brewster DH, Han C, Al-Nafussi A. 2003. Prognostic indicators for gastrointestinal stromal tumours: a clinicopathological and immunohistochemical study of 108 resected cases of the stomach. Histopathology. 43(2):118-126. https://doi.org/10.1046/j.1365-2559.2003.01665.x

20. Greenson JK. 2003. Gastrointestinal Stromal Tumors and Other Mesenchymal Lesions of the Gut. Mod Pathol. 16(4):366-375. https://doi.org/10.1097/01.mp.0000062860.60390.c7

21. Goldblum JR. 2002. Gastrointestinal Stromal Tumors. 117(suppl_1):S49-S61. https://doi.org/10.1309/7ewe-pf04-vwjr-g4wq

22. de Silva MC, Reid R. 2003. Gastrointestinal stromal tumors (GIST): C-kit mutations, CD117 expression, differential diagnosis and targeted cancer therapy with imatinib. Pathol. Oncol. Res.. 9(1):13-19. https://doi.org/10.1007/bf03033708

23. Lee MJ, Lee HS, Kim WH, Choi Y, Yang M. 2003. Expression of Mucins and Cytokeratins in Primary Carcinomas of the Digestive System. Mod Pathol. 16(5):403-410. https://doi.org/10.1097/01.mp.0000067683.84284.66

24. ALBARRACIN C, JAFRI J, MONTAG A, HART J, WAN S. 2000. Differential expression of MUC2 and MUC5AC mutin genes in primary ovarian and metastatic colonic carcinoma. Human Pathology. 31(6):672-677. https://doi.org/10.1053/hupa.2000.6799

25. Chou Y, Jeng Y, Kao H, Chen TJ, Mao T, Lin M. 2003. Differentiation of ovarian mucinous carcinoma and metastatic colorectal adenocarcinoma by immunostaining with beta-catenin. Histopathology. 43(2):151-156. https://doi.org/10.1046/j.1365-2559.2003.01687.x

26. Nishizuka S, Chen ST, Gwadry FG, et al.. 2003. Diagnostic markers than distinguish colon and ovarian adenocarcinomas: identification by genomic, proteomic, and tissue array profiling. Cancer Res. ;63:5243–5250..

27. Compton C, Fenoglio-Presider C, Pettigrew N, Fielding L. . 2000. American Joint Committee on Cancer prognostic factors consensus conference. Cancer. ; 88:1739–1757..

28. Cunningham D, Humblet Y, Siena S, Khayat D, Bleiberg H, Santoro A, Bets D, Mueser M, Harstrick A, Verslype C, et al. 2004. Cetuximab Monotherapy and Cetuximab plus Irinotecan in Irinotecan-Refractory Metastatic Colorectal Cancer. N Engl J Med. 351(4):337-345. https://doi.org/10.1056/nejmoa033025

29. Isaksson-Mettavainio M, Palmqvist R, Forssell J, Stenling R, Oberg A. . 2006. SMAD4/DPC4 expression and prognosis in human colorectal cancer. Anticancer Res. ;26:507–510..

30. Sheehan GM, Kallakury BV, Sheehan CE, Fisher HA, Kaufman RP, Ross JS. 2005. Smad4 protein expression correlates with grade, stage, and DNA ploidy in prostatic adenocarcinomas. Human Pathology. 36(11):1204-1209. https://doi.org/10.1016/j.humpath.2005.08.015

31. KHORANA A, HU Y, RYAN C, KOMOROWSKI R, HOSTETTER G, AHRENDT S. 2005. Vascular Endothelial Growth Factor and Predict Adjuvant Therapy Outcomes in Resected Pancreatic Cancer. Journal of Gastrointestinal Surgery. 9(7):903-911. https://doi.org/10.1016/j.gassur.2005.06.021

32. Lindor NM, Burgart LJ, Leontovich O, Goldberg RM, Cunningham JM, Sargent DJ, Walsh-Vockley C, Petersen GM, Walsh MD, Leggett BA, et al. 2002. Immunohistochemistry Versus Microsatellite Instability Testing in Phenotyping Colorectal Tumors. JCO. 20(4):1043-1048. https://doi.org/10.1200/jco.2002.20.4.1043

33. Jass JR. 2006. Re: Wardet al. Routine testing for mismatch repair deficiency in sporadic colorectal cancer is justified.J Pathol 2005;207:377?384. J. Pathol.. 208(4):590-591. https://doi.org/10.1002/path.1933

34. Yamaguchi K, Enjoji M, Tsuneyoshi M. 1991. Pancreatoduodenal carcinoma: A clinicopathologic study of 304 patients and immunohistochemical observation for CEA and CA19-9. J. Surg. Oncol.. 47(3):148-154. https://doi.org/10.1002/jso.2930470303

35. Tamiolakis D, Simopoulos C, Venizelos J, Lambropoulou M, Nikolaidou S, Tsikouras P, Koutsougeras G, Alexiadis G, Menegaki M, Papadopoulos N. 2005. Distribution of somatostatin in pancreatic ductal adenocarcinoma remodels the normal pattern of the protein during foetal pancreatic development: an immunohistochemical analysis. Clin. Exper.Med.. 5(3):106-111. https://doi.org/10.1007/s10238-005-0073-4

36. Kigure S.. 2006. Immunohistochemical study of the association between the progression of pancreatic ductal lesions and the expression of MUC1, MUC2, MUC5AC, and E-cdherin. Rinsho Byori. ;54:447–452..

37. Saitou M. 2005. MUC4 expression is a novel prognostic factor in patients with invasive ductal carcinoma of the pancreas. Journal of Clinical Pathology. 58(8):845-852. https://doi.org/10.1136/jcp.2004.023572

38. Lee K, Chang W, Wang S, Chuang S, Chai C, Hu S. 2006. Expression of DPC4/Smad4 gene in stone-containing intrahepatic bile duct. J. Surg. Oncol.. 94(4):338-343. https://doi.org/10.1002/jso.20517

39. Hornick JL, Lauwers GY, Odze RD. 2005. Immunohistochemistry Can Help Distinguish Metastatic Pancreatic Adenocarcinomas From Bile Duct Adenomas and Hamartomas of the Liver. The American Journal of Surgical Pathology. 29(3):381-389. https://doi.org/10.1097/01.pas.0000149710.01559.fe

40. Mertz H, Vyberg M, Paulsen SM, Teglbj??rg PS. 1998. Immunohistochemical Detection of Neuroendocrine Markers in Tumors of the Lungs and Gastrointestinal Tract. Applied Immunohistochemistry. 6(4):175-180. https://doi.org/10.1097/00022744-199812000-00001

41. Bordi C, Yu JY, Baggi MT, et al.. 1991. Gastric carcinoids and their precursor lesions: a histologic and immunohistochemical study of 23 cases. Cancer. ; 67:663–672..

42. Lam K, Lo C. 1997. Pancreatic endocrine tumour: a 22-year clinico-pathological experience with morphological, immunohistochemical observation and a review of the literature. European Journal of Surgical Oncology (EJSO). 23(1):36-42. https://doi.org/10.1016/s0748-7983(97)80140-0

43. Rindi G, Paolotti D, LaRosa S, et al. . 1998. The tumours of the endocrine pancreas. Eur J Histochem. ;42:63–66..

44. Chetty R, Asa SL. 2004. Pancreatic Endocrine Tumors. Advances in Anatomic Pathology. 11(4):202-210. https://doi.org/10.1097/01.pap.0000131828.00189.29

45. Chetty R, Asa SL. 2004. Pancreatic Endocrine Tumors. Advances in Anatomic Pathology. 11(4):202-210. https://doi.org/10.1097/01.pap.0000131828.00189.29

46. Shipley WR, Hammer RD, Lennington WJ, Macon WR. 1997. Paraffin Immunohistochemical Detection of CD56, a Useful Marker for Neural Cell Adhesion Molecule (NCAM), in Normal and Neoplastic Fixed Tissues. Applied Immunohistochemistry. 5(2):87-93. https://doi.org/10.1097/00022744-199706000-00003

47. Soga J. 2005. Carcinoids of the pancreas. Cancer. 104(6):1180-1187. https://doi.org/10.1002/cncr.21291

48. Deschamps L, Handra-Luca A, O'Toole D, Sauvanet A, Ruszniewski P, Belghiti J, Bedossa P, Couvelard A. 2006. CD10 expression in pancreatic endocrine tumors: correlation with prognostic factors and survival. Human Pathology. 37(7):802-808. https://doi.org/10.1016/j.humpath.2006.02.024

49. West RB, Corless CL, Chen X, Rubin BP, Subramanian S, Montgomery K, Zhu S, Ball CA, Nielsen TO, Patel R, et al. 2004. The Novel Marker, DOG1, Is Expressed Ubiquitously in Gastrointestinal Stromal Tumors Irrespective of KIT or PDGFRA Mutation Status. The American Journal of Pathology. 165(1):107-113. https://doi.org/10.1016/s0002-9440(10)63279-8

50. Ordóñez NG. 2014. Cadherin 17 is a Novel Diagnostic Marker for Adenocarcinomas of the Digestive System. Advances In Anatomic Pathology. 21(2):131-137. https://doi.org/10.1097/pap.0000000000000008

51. Eberhard J, Gaber A, Wangefjord S, Nodin B, Uhlén M, Ericson Lindquist K, Jirström K. 2012. A cohort study of the prognostic and treatment predictive value of SATB2 expression in colorectal cancer. Br J Cancer. 106(5):931-938. https://doi.org/10.1038/bjc.2012.34

52. Wang S, Zhou J, Wang X, Hao J, Chen J, Zhang X, Jin H, Liu L, Zhang Y, Liu J, et al. 2009. Down-regulated expression of SATB2 is associated with metastasis and poor prognosis in colorectal cancer. J. Pathol.. 219(1):114-122. https://doi.org/10.1002/path.2575

53. Magnusson K, de Wit M, Brennan DJ, Johnson LB, McGee SF, Lundberg E, Naicker K, Klinger R, Kampf C, Asplund A, et al. 2011. SATB2 in Combination With Cytokeratin 20 Identifies Over 95% of all Colorectal Carcinomas. The American Journal of Surgical Pathology. 35(7):937-948. https://doi.org/10.1097/pas.0b013e31821c3dae

54. Takamura M, Sakamoto M, Ino Y, Shimamura T, Ichida T, Asakura H, Hirohashi S. 2003. Expression of liver-intestine cadherin and its possible interaction with galectin-3 in ductal adenocarcinoma of the pancreas. Cancer Science. 94(5):425-430. https://doi.org/10.1111/j.1349-7006.2003.tb01459.x

55. Su M, Yuan R, Lin C, Jeng Y. 2008. Cadherin-17 is a useful diagnostic marker for adenocarcinomas of the digestive system. Mod Pathol. 21(11):1379-1386. https://doi.org/10.1038/modpathol.2008.107

56. Lin F, Shi J, Zhu S, Chen Z, Li A, Chen T, Wang HL, Liu H. 2014. Cadherin-17 and SATB2 Are Sensitive and Specific Immunomarkers for Medullary Carcinoma of the Large Intestine. 138(8):1015-1026. https://doi.org/10.5858/arpa.2013-0452-oa