Anatomic Pathology / PANCREATICOBILIARY CYTOKERATINS Cytokeratins 7, 17, and 20 Reactivity in Pancreatic and Ampulla of Vater Adenocarcinomas Percentage of Positivity and Distribution Is Affected by the Cut-Point Threshold
نویسندگان
چکیده
We studied reactivity of cytokeratins (CK) 7, 17, and 20 in 64 pancreaticobiliary adenocarcinomas to examine the effect of different cut-point thresholds on “positive” results, compare ampulla of Vater and pancreas adenocarcinomas, and provide additional experience with CK17 reactivity. Almost all neoplasms had extensive CK7 reactivity. The number of CK20positive cases decreased from 29 (45%; any stained cells) to 19 (30%; >25% staining) to 14 (22%; >50% staining) with an increasing threshold of reactive cells. Similar shifts in the distribution of CK7 and CK20 reactivity occurred when different thresholds of reactivity were used for a positive result. There were no differences in CK7 or CK20 reactivity in pancreas only, ampulla only, and neoplasms involving both sites. Of 64 adenocarcinomas, 29 (45%) had no or single-cell CK17 reactivity, and 19 (30%) had reactivity in more than 50% of neoplastic cells. Ampulla of Vater and pancreas adenocarcinomas have similar CK immunophenotypes that cannot assist in distinguishing ampullary from pancreatic neoplasms on endoscopically procured tissue. CK17 staining occurs in approximately 50% of pancreaticobiliary adenocarcinomas and is usually patchy. Single antibody staining results, especially CK7 and CK20 coordinate reactivity, are influenced by the reactivity threshold used. Pancreaticobiliary adenocarcinomas can be difficult to diagnose because the primary tumor can be occult, its signs and symptoms can mimic those produced by other adenocarcinomas, and they share morphologic features with other adenocarcinomas, especially those in the stomach. The cytokeratin (CK) immunophenotype of an adenocarcinoma can assist in the identification of its primary site; CK7 and CK20 are two of the most extensively studied. Wang et al1 were the first to use CK7 and CK20 in tandem, producing 4 CK7 and CK20 coordinate staining patterns based on positive and negative reactivity. The number of pancreaticobiliary adenocarcinomas studied in this manner is small, and the reported distribution of CK7 and CK20 coordinate staining varies to such a degree that no consistent staining pattern can be elicited.1-4 One reason for the spectrum of reported staining patterns may be methodologic. Differences in the percentage of reactive cells necessary for a positive result could substantially alter the distribution of CK7 and CK20 results. Another factor that has contributed to inconsistent CK staining results may be inclusion of ampulla of Vater adenocarcinomas with pancreatic duct and common bile duct adenocarcinomas. One group of authors reported ampullary adenocarcinomas had a CK immunophenotype similar to bowel adenocarcinomas that was distinct from pancreaticobiliary adenocarcinomas.3 Last, a preliminary study of 17 formalin-fixed paraffin embedded, pancreatic adenocarcinomas reported that CK17 was potentially useful for identifying pancreatic adenocarcinomas.5 Confirmation of these results and additional experience with CK17 reactivity is needed before it can be integrated into an antibody panel. We studied the CK immunophenotype of 64 adenocarcinomas derived from pancreaticoduodenal resection specimens Am J Clin Pathol 2001;115:695-702 695 © American Society of Clinical Pathologists Goldstein and Bassi / PANCREATICOBILIARY CYTOKERATINS to clarify their CK7-CK20 coordinate reactivity and to provide additional experience with CK17 staining of pancreatic and ampulla of Vater adenocarcinomas. We also explored whether there were differences in the CK expression between ampulla of Vater and pancreatic duct adenocarcinomas. Materials and Methods From the files of William Beaumont Hospital Surgical Pathology Department (Royal Oak, MI), we identified 93 consecutive cases of carcinoma treated by pancreaticoduodenal resection from January 1995 through December 2000. Slides were reviewed from each case, and cases were excluded if they were not primary ampullary invasive adenocarcinoma or pancreaticobiliary invasive adenocarcinoma. Undifferentiated, giant cell, acinic, and neuroendocrine carcinomas were excluded. No distinction between pancreatic duct and intrapancreatic common bile duct was made. In situ carcinoma components, either intraductal within the pancreas or ampulla of Vater adenoma, were excluded from evaluation. The 64 remaining specimens constituted the study group. Each adenocarcinoma was classified into 1 of 3 groups based on the tissue(s) infiltrated by the adenocarcinoma to determine whether there were staining differences between ampullary and pancreatic adenocarcinomas. The 3 tumor groups were: 1. Ampulla only, defined as invasive adenocarcinoma involving the ampulla and periampullary duodenum, with maximal extension no farther than the outer layer of the duodenal muscularis propria. One block of invasive ampullary adenocarcinoma was chosen for immunohistochemical staining from each case. 2. Pancreas only, defined as invasive adenocarcinoma confined to the pancreas and peripancreatic soft tissues without involvement of the duodenal muscularis propria or ampulla of Vater. One block of invasive pancreatic adenocarcinoma was chosen for immunohistochemical staining from each case. 3. Ampulla and pancreas, defined as invasive adenocarcinoma involving the ampulla, duodenum, and pancreatic parenchyma. Two blocks were chosen for immunohistochemical staining from each case. One block contained only invasive adenocarcinoma from the ampulla, and the second block contained only invasive adenocarcinoma from the pancreas parenchyma. Immunohistochemical Staining We cut 3-μm-thick consecutive sections from each block, and each section was placed on a charged slide. Sections were deparaffinized using sequential immersions into 2 xylene baths, 3 baths of decreasing alcohol concentrations, and 2 water baths, followed by a 1-minute wash in water. Slides were immersed in EDTA buffer (pH 7.0) and put into a commercial vegetable steamer at 95°C for 30 minutes. The slides were allowed to cool on the countertop, remaining immersed in the heated EDTA buffer–filled containers for 5 minutes, followed by a 2-minute rinse with water while remaining in the containers. The slides were transferred into tris(hydroxymethyl)aminomethane–filled containers (pH 7.0) and allowed to cool for an additional 10 minutes on the countertop. They then were transferred to a commercial immunohistochemistry autostainer (DAKO, Carpinteria, CA) and were first washed with buffer, followed by a hydrogen peroxide incubation. The latter was rinsed off, and the primary antibody was applied. The primary antibody was incubated over the sections for 20 minutes at room temperature. After the primary antibody was washed off, the components of the Envision-plus (DAKO) detection system were applied, including an antimouse polymer, 2 distilled-water washes, and a final diaminobenzidine incubation for 4 minutes. Sections were counterstained with hematoxylin and coverslipped. A positive control slide, containing known CK-reactive tissues was stained with each batch of simultaneously stained slides. A slide from each study block was stained with CK 19 as a positive control of adenocarcinoma cell integrity. Cytokeratin 19 strongly and diffusely stains pancreaticobiliary and ampulla of Vater adenocarcinomas.6-8 All of the adenocarcinomas in the study had strong and diffuse cytoplasmic CK19 reactivity. All of the primary antibodies were obtained from DAKO. The following antibody clones and dilutions were used: CK7 (clone OV-TL-12/30, 1:800 dilution), CK17 (clone E3, 1:200 dilution), CK19 (clone RCK108, 1:50 dilution), and CK20 (clone KS20.8, 1:500 dilution). The percentage of reactive invasive adenocarcinoma cells was quantified as 0%, less than 5%, 5% to 25%, 26% to 50%, 51% to 75%, or more than 75%. Intensity of stained cells was quantified into 1 of 3 categories: (1) weak reactivity: required high magnification to detect; (2) moderate reactivity: could be seen easily at medium magnification and after careful examination at low magnification; (3) strong reactivity: staining could be seen easily at low magnification.
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