Printer Friendly

Reevaluation and identification of the best immunohistochemical panel (pVHL, maspin, S100P, IMP-3) for ductal adenocarcinoma of the pancreas.

Differentiation of ductal adenocarcinoma (DAC) of the pancreas from nonneoplastic pancreatic tissues can be challenging, especially for well-differentiated DAC in small core biopsy specimens, pancreatic surgical resection margins, and fine-needle aspiration specimens on cell blocks. Many tumor-associated markers have been reported to be useful in this regard; (1-46) however, the reproducibility of these markers has not been tested and confirmed in a study using a single immunostaining system. This study investigates the utility of 26 different immunohistochemical markers (CAM 5.2, CK [cytokeratin] 7, CK20, CK17, CK19, MUC1, MUC2, MUC4, MUC5AC, MUC6, p53, DPC4/ SMAD4, CDX2, pVHL [von Hippel-Lindau tumor suppressor gene protein], S100P, IMP-3 [insulin-like growth factor 2 messenger RNA binding protein 3], maspin, mesothelin, claudin 4, claudin 18, annexin A8, fascin, PSCA [prostate stem cell antigen], MOC31, CEA [carcinoembryonic antigen], and CA19-9 [cancer antigen 19-9]) in the diagnosis of ductal adenocarcinoma of the pancreas by using 60 cases of pancreatic DAC on routine and tissue microarray (TMA) sections. The results demonstrate that pVHL, maspin, S100P, and IMP-3 constitute the most effective panel of markers in the distinction of pancreatic DAC from benign/reactive pancreatic ducts. The clinical utility of this diagnostic panel (pVHL, maspin, S100P, and IMP-3) has been further tested and confirmed in 67 cases of pancreatic DAC and benign pancreatic tissues from fineneedle aspiration biopsy (FnAB) specimens on cell blocks.

MATERIALS AND METHODS

Immunohistochemical Staining of Surgical Specimens

The study was approved by the institutional review board at Geisinger Medical Center, Danville, Pennsylvania. Sixty cases of ductal adenocarcinoma of the pancreas were retrieved from the archives of the Department of Laboratory Medicine at Geisinger Medical Center from 2000-2008, including 10 well-differentiated, 35 moderately differentiated, and 15 poorly differentiated ductal adenocarcinomas. Two TMA blocks, one containing 40 cases of pancreatic DAC and the other containing 20 cases of nonneoplastic pancreatic tissue, were constructed as previously described. (47) Immunohistochemical staining was performed for these 60 cases of pancreatic DAC (20 conventional tissue sections and 40 TMA sections) and 40 cases of normal/nonneoplastic pancreatic tissues taken from patients with pancreatic carcinoma (20 conventional tissue sections and 20 TMA sections) by using the following markers: (1) epithelial markers (CAM 5.2, CK7, CK20, CK17, CK19); (2) mucin gene products (MUC1, MUC2, MUC4, MUC5AC, MUC6); (3) tumor suppressor genes and transcription factors (p53, DPC4/SMAD4, CDX2, pVHL); and (4) tumor-associated proteins (S100P, IMP-3, maspin, mesothelin, claudin 4, claudin 18, annexin A8, fascin, PSCA, MOC31, CEA, CA19-9). The staining was done on the Dako staining system (Carpinteria, California), based on the previously published protocol. (1,6,47) Detailed information about the antibodies and staining conditions is summarized in Table 1. The staining intensity for both tumor cases and normal/nonneoplastic pancreatic tissues was graded as weak or strong. The distribution (number of tumor cells stained) wasrecordedasnegative(<5% of tumor cells stained), 1+ (5%-25%), 2+ (26%-50%), 3+ (51%-75%), or 4+ (.75%). Two surgical pathologists (F.L., H.L.) independently evaluated the immunostained slides.

Double Immunostaining Using a Mouse Monoclonal Anti-maspin Antibody and a Rabbit Polyclonal Anti-pVHL Antibody on Surgical Specimens

Double immunostaining with maspin and pVHL was performed on the Ventana BenchMark ULTRA instrument (Ventana Medical Systems, a member of the Roche Group, Tucson, Arizona). Briefly, the TMA slides containing 40 cases of adenocarcinoma of the pancreas were deparaffinized on the ULTRA at 72[degrees]C with a proprietary detergent solution, followed by blocking for endogenous peroxidase. Automated antigen retrieval was performed for 36 minutes at 95[degrees]C with a proprietary solution at pH 8.0. Anti-maspin antibody (1:200) was applied to the tissue sections, and slides were incubated at 37[degrees]C for 40 minutes. Slides were subsequently rinsed in a proprietary buffer, and visualization with the ultraView DAB Detection Kit was achieved. Antibody denaturation was performed by heating the slides to 90[degrees]C for 8 minutes. After denaturation, 12 minutes of enzymatic epitope retrieval was completed. Anti-pVHL antibody (1:100) was applied, and the tissue sections were incubated for 40 minutes at 37[degrees]C. Slides were subsequently rinsed in a proprietary buffer, and visualization with the ultraView Red Detection Kit was achieved. The signal was amplified with Ventana's Amplification Kit. Gill II hematoxylin was used as a counterstain.

Immunohistochemical Staining of FNAB Specimens on Cell Blocks

From the above study results, the panel of antibodies containing pVHL, maspin, S100P, and IMP-3 was identified as the most effective diagnostic panel for distinction of DAC of the pancreas from benign/reactive pancreatic ducts. Immunohistochemical evaluation of the expression of maspin, S100P, IMP-3, and pVHL was further performed for 67 cases of FNAB specimens of the pancreas on cell block sections. The preparation of the cell block was as previously described. (48) In brief, the RPMI sample containing tumor cells was placed in a 50-ml Falcon tube and spun in a Beckman Coulter Allegra 6 Centrifuge for 10 minutes at 2000 rpm (Beckman Coulter, Brea, California). The supernatant was poured off, which left the small pellet/ button in the bottom of the tube. Approximately 5 drops of plasma were added to the cell button to resuspend it. Then, approximately 5 drops of bovine thrombin (item No. 23-306291, Fisher Scientific, Pittsburgh, Pennsylvania) were added, and the mixture was allowed to stand for 10 minutes. The cell pellet was fixed in 10% formalin for 10 minutes. The cell block material was transferred to a biopsy bag by pouring the contents of the Falcon tube into a biopsy bag over a funnel and beaker. The cell block/ biopsy bag was placed into a histology cassette. The cassette was processed as a routine surgical specimen. The immunostaining protocol was as described for surgical specimens. (1,6,47)

The 67 cases were divided into 3 groups: group 1 (44 cases of pancreatic DAC with adequate cellularity); group 2 (13 cases with a suspicious or indeterminate diagnosis); and group 3 (10 benign cases). In group 1, thirty cases were well-differentiated or moderately differentiated adenocarcinoma; the remaining 14 cases were moderately differentiated or moderately to poorly differentiated adenocarcinoma. Only adenocarcinoma cases with an adequate cellularity in the cell block preparation were included in this study. The adequate cellularity in the cell block was tentatively defined as at least 2 groups of atypical epithelial cells (more than 10 cells in each group) and single atypical cells. Most cell blocks contained more than 5 groups of atypical epithelial cells. The staining intensity (weak or strong) and distribution (negative [<5% of tumor cells stained], 1+ [5%-25%], 2+ [26%-50%], 3+ [51%-75%], or 4+ [>75%]) were recorded. For S100P and maspin, nuclear staining only or nuclear and cytoplasmic staining was regarded as positive. Two surgical pathologists (F.L. and V.A.) independently evaluated the immunostained slides. Follow-up surgical or clinical data confirmed the diagnosis of pancreatic DAC for all cases in group 1. Surgical or clinical follow-up data were available in 9 cases in group 2, and all 9 were pancreatic carcinomas.

RESULTS

Overall Staining Results for Adenocarcinoma and Normal Pancreatic Tissue in Surgical Specimens

The staining results for both DAC and normal pancreatic tissues (ducts, acini, and islets) are summarized in Table 2. The results demonstrated that (1) more than 90% of cases were positive for maspin, S100P, and IMP-3; (2) nearly all adenocarcinomas were negative for pVHL, whereas nonneoplastic ducts and acini were strongly positive for pVHL in all cases; (3) normal/reactive pancreatic ducts were positive for CAM 5.2, CK7, CK19, MUC1, MUC6, CA19-9, MOC31, PSCA, mesothelin, an nexin A8, claudin 4, and claudin 18; (4) normal pancreatic ducts were usually negative for IMP-3, maspin, S100P, CK17, MUC2, MUC4, and MUC5AC; (5) 60% of adenocar cinoma cases showed loss of expression of DPC4/SMAD4; and (6) strong background staining was frequently seen with fascin (including staining for endothelial cells and stromal cells), PSCA, and annexin A8.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

Representative photomicrographs for maspin, S100P, IMP-3, CK17, MUC5AC, and pVHL are shown in Figure 1, A through F. Positive staining for pVHL in normal pancreatic ducts is shown in Figure 2, A and a rare pancreatic DAC case with focal (1+) staining for pVHL is shown in Figure 2, B. Double staining for maspin and pVHL in DAC demonstrated the same result: positive staining for maspin and concurrent negative staining for pVHL. A representative case of pancreatic DAC with double staining for maspin (brown) and pVHL (purple) is shown in Figure 3, A and B. Figure 4 demonstrates expression of p53 and Figure 5, loss of expression of DPC4/SMAD4 in pancreatic DAC.

Expression of pVHL, Maspin, S100P, and IMP-3 in Pancreatic DAC in Surgical Specimens

The positive staining for both maspin and S100P was generally strong and diffuse (3+ to 4+). The staining intensity for IMP-3 tended to be intermediate to weak when compared to that for maspin and S100P. The 3 S100P-negative cases were positive for both maspin and IMP-3. Similarly, all 6 IMP-3-negative cases were positive for both maspin and S100P. The results are summarized in Table 3. Only 2 cases showed very focal (1+) positivity for pVHL, as shown in Figure 2, B.

Expression of Immunostaining Markers in Normal Pancreatic Ducts, Acini, and Islets

CAM 5.2 and CK7 expression was diffusely (4+) and strongly positive in benign/reactive ducts and acini in all cases. Normal/reactive ducts were only focally and weakly positive for CK19 and frequently negative for CK17, CEA, MUC2, MUC4, and MUC5AC. Benign ducts can show focal cytoplasmic positivity for S100P, but nuclear staining was absent. Maspin staining was usually negative in benign ducts and acini. Focal (1+ to 2+) positivity for IMP-3 was observed in 3 cases involving pancreatic ducts. Both benign ducts and acini were diffusely (3+ to 4+) and strongly positive for pVHL, with a membranous staining pattern. Pancreatic islet cells were positive for pVHL, IMP-3 (KOC), claudin4, claudin 18, and PSCA, and negative for both CK7 and CK20. Benign ducts were frequently positive for CA19-9, mesothelin, MUC1, MUC6, MOC31, PSCA, claudin 4, and claudin 18, which limits the application of these markers in distinguishing DAC from nonneoplastic pancreatic ducts. Strong background staining was frequently seen with annexin A8, PSCA, and fascin; in addition, many stromal cells and endothelial cells were strongly positive for fascin. Approximately 10% of cases of pancreatic ducts and acini were focally positive for CDX2.

Expression of pVHL, Maspin, S100P, and IMP-3 in FNAB Specimens

Strong and diffuse staining (3+ or 4+) for maspin, S100P, and IMP-3 was seen in 33 (79%), 35 (80%), and 19 (48%) cases, respectively, in group 1; and 9 (70%), 7 (54%), and 5 (39%) cases, respectively, in group 2. The 3 pVHL-negative cases in group 3 (benign group) had very low cellularity. The maspin-positive and S100P-positive cells in the benign group were gastric contaminants. The results are summarized in Table 4. Representative photomicrographs of the expression of these 4 markers and of the cell block section with hematoxylin-eosin stain are shown in Figure 6, A through E.

COMMENT

Our previous studies (1,6) showed overexpression of S100P but no expression of pVHL in pancreatic DAC and intraductal pancreatic neoplasia and an inverse expression pattern of these 2 markers in normal/reactive pancreatic ducts (positive for pVHL and negative for S100P). The present study further demonstrates that pVHL, maspin, S100P, and IMP-3 constitute the best panel of markers in the distinction of pancreatic DAC from normal/reactive pancreatic ducts. (41) Applications and pitfalls of each of these 4 markers will be discussed in detail, including a brief discussion of the utility of these 4 markers in FNAB specimens, followed by discussion of other tested markers in both carcinomas and normal pancreatic tissues.

Background staining for S100P was sometimes present. The staining may occur in background stromal cells, inflammatory cells, and endothelial cells, with both nuclear and cytoplasmic staining. We have also noticed that tissues that have been processed in a microwave processor usually show very little background staining. In the instance of background staining, S100A6 can be a good substitute, although weak nuclear and cytoplasmic staining for S100A6 can be seen in normal/reactive pancreatic ducts. (1) In contrast, normal/reactive pancreatic ducts were either negative or had only cytoplasmic positivity for S100P.

[FIGURE 6 OMITTED]

The cytoplasmic staining signal for IMP-3 was generally weaker and less diffuse than that for maspin and S100P. Approximately 10% of DACs were negative for IMP-3 in this study. The negative rate may be even higher when the immunostaining is applied on a limited sample, such as core biopsy specimens and cell blocks from FNAB samples, because of the focal/patchy nature of the staining.

In contrast to S100P and IMP-3, maspin appears to be the best positive marker for identifying pancreatic DAC. Like S100P, maspin demonstrates both nuclear and cytoplasmic staining when expression is positive. Pure cytoplasmic staining should be regarded as negative expression. Importantly, more than 80% of maspinpositive cases in this study were strongly and diffusely (3+ or 4+) positive. In addition, no background staining was observed in this study.

pVHL is a negative marker for pancreatic DAC. This was observed in nearly all cases in this study and our previous studies. (1,41) A detailed discussion of pVHL can be reviewed in our previous studies. (1,6) The positive staining for pVHL in normal pancreatic ducts and acini in our study was always associated with strong membranous staining. Two adenocarcinoma cases showed very focal pVHL positivity (less than 10% of tumor cells stained). A small caveat is that protease digestion, which is used as the antigen retrieval method, may lead to overdigestion, especially in a small tissue sample such as a core biopsy sample or cytologic specimens on cell block preparation. As a result of overdigestion, the tissue may be distorted and lose some morphologic details, and weak nonspecific cytoplasmic staining may occur. In this instance, caution should be taken to avoid any overinterpretation of a positive staining. Other biliary tract carcinomas, including extrahepatic bile duct, gallbladder, and most ampullary adenocarcinomas, have also been reported to be negative for pVHL. (1,7,49) Our preliminary study (50) demonstrated that most intrahepatic cholangiocarcinomas were positive for pVHL. If this finding can be confirmed in a large series of cases, then pVHL can be potentially used as a marker to differentiate intrahepatic cholangiocarcinoma from other pancreatic/extrahepatic bile duct/gallbladder carcinomas.

S100P, maspin, and IMP-3 are not entirely specific for pancreatic DAC. They can be expressed in other malignancies from various organs. Our unpublished data demonstrated that (1) maspin was expressed in 100% (N = 30) of esophageal adenocarcinomas, (51) 90% (N = 38) of colorectal adenocarcinomas, and 65% (N = 40) of urothelial carcinomas; (51) (2) IMP-3 was also expressed in nearly all germ cell tumors (including seminoma, yolk sac tumor, and embryonal carcinoma), but it usually yielded a negative result in breast carcinoma of both ductal and lobular types; (51) (3) S100P was positively expressed in 74% (N = 23) of gallbladder adenocarcinomas and 65% (N = 40) of invasive urothelial carcinomas; (51) and (4) pVHL was a useful marker for confirming a diagnosis of renal cell carcinoma, as well as clear cell carcinoma of the ovary and uterus. (6) More recently, we have also demonstrated that pVHL can be positively expressed in 17% (N = 18) of hepatocellular carcinomas and 13% (N = 30) of esophageal adenocarcinomas. (51)

In addition to surgical specimens, this panel of 4 markers (pVHL, maspin, S100P, and IMP-3) has been tested in 44 cases of ductal adenocarcinoma of the pancreas from FNAB specimens on cell blocks. In this study, the expression of maspin, S100P, and IMP-3 was observed in 100%, 100%, and 93% of cases, respectively, whereas the expression of pVHL was negative in all cases with a diagnosis of malignancy. (42) Strong and diffuse staining (3+ or 4+) for maspin, S100P, and IMP-3 was observed in most cases, which is important to ensure a high diagnostic sensitivity of FNAB specimens, especially when a case with low cellularity is encountered.

Maspin is also positively expressed in both normal gastric antral and fundic mucosa as well as duodenal mucosa. Additionally S100P expression is frequently positive in gastric mucosa, with both nuclear and cytoplasmic staining. Therefore, caution should be taken when using this panel of markers in endoscopic ultrasound-guided FNAB samples of the pancreas, since gastrointestinal contaminants are frequently seen in this type of sample.

Normal pancreatic ducts and acini are usually positive for MOC31, PSCA, claudin 4, and claudin 18, which limits the application of these markers in the distinction between pancreatic DAC and reactive ducts. Strong background staining is frequently seen with annexin A8 and fascin; in addition, many stromal cells and endothelial cells are frequently positive for fascin.

Among the group of cytokeratins being tested (CK7, CK20, CK17, CK19, and CAM 5.2), CK17 appears to be the only promising marker in differentiating pancreatic DAC from normal/reactive ducts, since it is usually not expressed in nonneoplastic pancreatic ducts.

Loss of DPC4/SMAD4 expression has been reported in approximately 60% of pancreatic DACs--both in the literature (52) and in our current study--which can be useful in differentiating pancreatic DAC from nonneoplastic pancreatic ducts. Caution should be taken, since the staining for DPC4/SMAD4 in normal pancreatic ducts tends to be weak. Additionally, loss of DPC4/SMAD4 expression is not absolutely specific for pancreatic DAC, since it has been reported in other tumors, including metastatic colonic adenocarcinomas. (53)

In summary, our data demonstrate that pVHL, maspin, S100P, and IMP-3 constitute the best diagnostic panel of immunomarkers for confirming the diagnosis of ductal adenocarcinoma of the pancreas in both surgical specimens and FNAB specimens on cell blocks.

This study is partly supported by Geisinger Medical Center Targeted Funding TRA-040--Evaluation of S100P as a Biomarker for Pancreatic Cancer (F.L.).

References

(1.) Lin F, Shi J, Liu H, et al. Diagnostic utility of S100P and von Hippel-Lindau gene product (pVHL) in pancreatic adenocarcinoma-with implication of their roles in early tumorigenesis. Am J Surg Pathol. 2008; 32(1):78-91.

(2.) Deng H, Shi J, Wilkerson M, Meschter S, Dupree W, Lin F. Usefulness of S100P in diagnosis of adenocarcinoma of pancreas on fine-needle aspiration biopsy specimens. Am J Clin Pathol. 2008; 129(1):81-88.

(3.) Dowen SE, Crnogorac-Jurcevic T, Gangeswaran R, et al. Expression of S100P and its novel binding partner S100PBPR in early pancreatic cancer. Am J Pathol. 2005; 166(1):81-92.

(4.) Crnogorac-Jurcevic T, Missiaglia E, Blaveri E, et al. Molecular alterations in pancreatic carcinoma: expression profiling shows that dysregulated expression of S100 genes is highly prevalent. J Pathol. 2003; 201(1):63-74.

(5.) Sato N, Fukushima N, Matsubayashi H, Goggins M. Identification of maspin and S100P as novel hypomethylation targets in pancreatic cancer using global gene expression profiling. Oncogene. 2004; 23(8):1531-1538.

(6.) Lin F, Shi J, Liu H, et al. Immunohistochemical detection of the von HippelLindau gene product (pVHL) in human tissues and tumors: a diagnostic marker for metastatic renal cell carcinoma and clear cell carcinoma of the ovary and uterus. Am J Clin Pathol. 2008; 129:592-605.

(7.) Levy M, Lin F, Xu H, Dhall D, Spaulding BO, Wang HL. S100P, von HippelLindau gene product, and IMP3 serve as a useful immunohistochemical panel in the diagnosis of adenocarcinoma on endoscopic bile duct biopsy. Hum Pathol. 2010; 41(9):1210-1219.

(8.) Yantiss RK, Woda BA, Fanger GR, et al. KOC (K homology domain containing protein overexpressed in cancer): a novel molecular marker that distinguishes between benign and malignant lesions of the pancreas. Am J Surg Pathol. 2005; 29(2):188-195.

(9.) Zhao H, Mandich D, Cartun RW, Ligato S. Expression of K homology domain containing protein overexpressed in cancer in pancreatic FNA for diagnosing adenocarcinoma of pancreas. Diagn Cytopathol. 2007; 35(11):700 704.

(10.) Cao D, Zhang Q, Wu LS, et al. Prognostic significance of maspin in pancreatic ductal adenocarcinoma: tissue microarray analysis of 223 surgically resected cases. Mod Pathol. 2007; 20(5):570-578.

(11.) Kashima K, Ohike N, Mukai S, Sato M, Takahashi M, Morohoshi T. Expression of the tumor suppressor gene maspin and its significance in intraductal papillary mucinous neoplasms of the pancreas. Hepatobiliary Pancreat Dis Int. 2008; 7(1):86-90.

(12.) Ohike N, Maass N, Mundhenke C, et al. Clinicopathological significance and molecular regulation of maspin expression in ductal adenocarcinoma of the pancreas. Cancer Lett. 2003; 199(2):193-200.

(13.) Agarwal B, Ludwig OJ, Collins BT, Cortese C. Immunostaining as an adjunct to cytology for diagnosis of pancreatic adenocarcinoma. Clin Gastro enterol Hepatol. 2008; 6(12):1425-1431.

(14.) McCarthy DM, Maitra A, Argani P, et al. Novel markers of pancreatic adenocarcinoma in fine-needle aspiration: mesothelin and prostate stem cell antigen labeling increases accuracy in cytologically borderline cases. Appl Immunohistochem Mol Morphol. 2003; 11(3):238-243.

(15.) Ordonez NG. Application of mesothelin immunostaining in tumor diagnosis. Am J SurgPathol. 2003; 27(11):1418-1428.

(16.) Hassan R, Laszik ZG, Lerner M, Raffeld M, Postier R, Brackett D. Mesothelin is overexpressed in pancreaticobiliary adenocarcinomas but not in normal pancreas and chronic pancreatitis. Am J Clin Pathol. 2005; 124(6):838 845.

(17.) Frierson HF Jr, Moskaluk CA, Powell SM, et al. Large-scale molecular and tissue microarray analysis of mesothelin expression in common human carcinomas. Hum Pathol. 2003; 34(6):605-609.

(18.) Wente MN, Jain A, Kono E, et al. Prostate stem cell antigen is a putative target for immunotherapy in pancreatic cancer. Pancreas. 2005; 31(2):119-125.

(19.) Argani P, Rosty C, Reiter RE, et al. Discovery of new markers of cancer through serial analysis of gene expression: prostate stem cell antigen is overexpressed in pancreatic adenocarcinoma. Cancer Res. 2001; 61(11):4320 4324.

(20.) Karanjawala ZE, Illei PB, Ashfaq R, et al. New markers of pancreatic cancer identified through differential gene expression analyses: claudin 18 and annexin A8. Am J SurgPathol. 2008; 32(2):188-196.

(21.) Hewitt KJ, Agarwal R, Morin PJ. The claudin gene family: expression in normal and neoplastic tissues. BMC Cancer. 2006; 6:186.

(22.) Werling RW, Yaziji H, Bacchi CE, Gown AM. 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. 2003; 27(3):303-310.

(23.) Moskaluk CA, Zhang H, Powell SM, Cerilli LA, Hampton GM, Frierson HF Jr. Cdx2 protein expression in normal and malignant human tissues: an immunohistochemical survey using tissue microarrays. Mod Pathol. 2003; 16(9): 913-919.

(24.) De Lott LB, Morrison C, Suster S, Cohn DE, FrankelWL. CDX2 is a useful marker of intestinal-type differentiation: a tissue microarray-based study of 629 tumors from various sites. Arch Pathol Lab Med. 2005; 129(9):1100-1105.

(25.) Goldstein NS, Bassi D. Cytokeratins 7, 17, and 20 reactivity in pancreatic and ampulla of vater adenocarcinomas: percentage of positivity and distribution is affected by the cut-pointthreshold. Am J Clin Pathol. 2001; 115(5):695-702.

(26.) Hornick JL, Lauwers GY, Odze RD. Immunohistochemistry can help distinguish metastatic pancreatic adenocarcinomas from bile ductadenomas and hamartomas of the liver. Am J Surg Pathol. 2005; 29(3):381-389.

(27.) Chu P, Wu E, Weiss LM. Cytokeratin 7 and cytokeratin 20 expression in epithelial neoplasms: a survey of 435 cases. Mod Pathol. 2000; 13(9):962 972.

(28.) Chu PG, Schwarz RE, Lau SK, Yen Y, Weiss LM. Immunohistochemical staining in the diagnosis of pancreatobiliary and ampulla of Vater adenocarcinoma: application of CDX2, CK17, MUC1, and MUC2. Am J Surg Pathol. 2005; 29(3):359-367.

(29.) Lau SK, Prakash S, Geller SA, Alsabeh R. Comparative immunohistochemical profile of hepatocellular carcinoma, cholangiocarcinoma, and metastatic adenocarcinoma. Hum Pathol. 2002; 33(12):1175-1181.

(30.) Nagata K, Horinouchi M, Saitou M, et al. Mucin expression profile in pancreatic cancer and the precursor lesions. J Hepatobiliary Pancreat Surg. 2007; 14(3):243-254.

(31.) Bhardwaj A, Marsh WL Jr, Nash JW, Barbacioru CC, Jones S, Frankel WL. Double immunohistochemical staining with MUC4/p53 is useful in the

distinction of pancreatic adenocarcinoma from chronic pancreatitis: a tissue microarray-based study. Arch Pathol Lab Med. 2007; 131(4):556-562.

(32.) Apple SK, Hecht JR, Lewin DN, Jahromi SA, Grody WW, Nieberg RK.

Immunohistochemical evaluation of K-ras, p53, and HER-2/neu expression in hyperplastic, dysplastic, and carcinomatous lesions of the pancreas: evidence for multistep carcinogenesis. Hum Pathol. 1999; 30(2):123-129.

(33.) DiGiuseppe JA, Hruban RH, Goodman SN, et al. Overexpression of p53 protein in adenocarcinoma of the pancreas. Am J Clin Pathol. 1994; 101(6):684-688.

(34.) Swierczynski SL, Maitra A, Abraham SC, et al. Analysis of novel tumor markers in pancreatic and biliary carcinomas using tissue microarrays. Hum Pathol. 2004; 35(3):357-366.

(35.) Jhala N, Jhala D, Vickers SM, et al. Biomarkers in diagnosis of pancreatic carcinoma in fine-needle aspirates. Am J Clin Pathol. 2006; 126(4):572-579.

(36.) Cao D, Maitra A, Saavedra JA, Klimstra DS, Adsay NV, Hruban RH. Expression of novel markers of pancreatic ductal adenocarcinoma in pancreatic nonductal neoplasms: additional evidence of different genetic pathways. Mod Pathol. 2005; 18(6):752-761.

(37.) Chhieng DC, Benson E, Eltoum I, et al. MUC1 and MUC2 expression in pancreatic ductal carcinoma obtained by fine-needle aspiration. Cancer. 2003; 99(6):365-371.

(38.) Giorgadze TA, Peterman H, Baloch ZW, et al. Diagnostic utility of mucin profile in fine-needle aspiration specimens of the pancreas: an immunohistochemical study with surgical pathology correlation. Cancer. 2006; 108(3):186 197.

(39.) Yamaguchi H, Inoue T, Eguchi T, et al. Fascin overexpression in intraductal papillary mucinous neoplasms (adenomas, borderline neoplasms, and carcinomas) of the pancreas, correlated with increased histological grade. Mod Pathol. 2007; 20(5):552-561.

(40.) van Heek T, Rader AE, Of ferhaus GJ, et al. K-ras, p53, and DPC4 (MAD4) alterations in fine-needle aspirates of the pancreas: amolecula rpanel correlates with and supplements cytologic diagnosis. Am J Clin Pathol. 2002; 117(5):755-765.

(41.) Lin F, Shi J, Wang HL, Liu J. Reevaluation and identification of the best immunohistochemical panel for adenocarcinoma of the pancreas [abstract 1613]. Mod Pathol. 2010; 23(suppl 1s):363A.

(42.) Anandan V, Shi J, Liu H, Meschter S, Lin F. Identification of an effective antibody panel in the diagnosis of pancreatic ductal adenocarcinoma on fine needle aspiration biopsy specimens [abstract 381]. Mod Pathol. 2010; 23 (suppl 1s):87A.

(43.) Kosarac O, Takei H, Zhai QJ, Schwartz MR, Mody DR. S100P and XIAP expression in pancreatic ductal adenocarcinoma: potential novel biomarkers as a diagnostic adjunct to fine needle aspiration cytology. Acta Cytol. 2011; 55(2): 142-148.

(44.) Nakata K, Nagai E, Ohuchida K, et al. S100P is a novel marker to identify intraductal papillary mucinous neoplasms. Hum Pathol. 2010; 41(6):824-831.

(45.) Wachter DL, Schlabrakowski A, Hoegel J, Kristiansen G, Hartmann A, Riener MO. Diagnostic value of immunohistochemical IMP3 expression in core needle biopsies of pancreatic ductal adenocarcinoma. Am J Surg Pathol. 2011; 35(6):873-877.

(46.) Dim DC, JiangF, Qiu Q, et al. Theusefulness of S100P, mesothelin, fascin, prostate stem cell antigen, and 14-3-3 sigma in diagnosing pancreatic adenocarcinoma in cytological specimens obtained by endoscopic ultrasound guided fine-needle aspiration [published online ahead of print April 28, 2011]. Diagn Cytopathol. doi:10.1002/dc.21684.

(47.) Lin F, ZhangPL, Yang XJ, et al. Human kidneyinjury molecule-1 (hKIM-1): a useful immunohistochemical marker for diagnosing renal cell carcinoma and ovarian clear cell carcinoma. Am J Surg Pathol. 2007; 31(3):371-381.

(48.) Liu H, Shi J, Wilkerson M, et al. Immunohistochemical detection of p16INKa in liquid-based cytology specimens on cell block sections. Cancer. 2007; 111(2):74-82.

(49.) Lin F, Shi J, Wang HL, Liu H. Diagnostic utility of von Hipple-Lindau gene product (pVHL), maspin, KOC, and S100Pin adenocarcinoma of the gallbladder [abstract 1612]. Mod Pathol. 2010; 23(suppl 1s):363A.

(50.) Lin F, Shi J, Liu H, Prichard JW, WangHL. CK17, MUC5AC, pVHL, andS100P are the effective antibody panel in differentiating intrahepatic cholangiocarcinoma from pancreatic adenocarcinoma [abstract 1556]. Mod Pathol. 2011; 24(suppl 1s): 367A.

(51.) Lin F, Shi J, WangHL, Wilkerson M, Liu H. UtilityofpVHL, maspin, S100P and IMP3 in diagnosis of carcinomas from various organs adenocarcinoma [abstract 1557]. Mod Pathol. 2011; 24(suppl 1s):367A.

(52.) Wilentz RE, Su GH, Dai JL, et al. Immunohistochemicallabeling for dpc4 mirrors genetic status in pancreatic adenocarcinomas: a new marker of DPC4 inactivation. Am J Pathol. 2000; 156(1):37-43.

(53.) Ji H, Isacson C, Seidman JD, Kurman RJ, Ronnett BM. Cytokeratins 7 and 20, Dpc4, and MUC5AC in the distinction of metastatic mucinouscarcinomas in the ovary from primary ovarian mucinous tumors: Dpc4 assists in identifying metastatic pancreatic carcinomas. Int J Gynecol Pathol. 2002; 21(4):391-400.

Haiyan Liu, MD; Jianhui Shi, MD, PhD; Vasuki Anandan, MD; Hanlin L. Wang, MD, PhD; David Diehl, MD; Joseph Blansfield, MD; Glenn Gerhard, MD; Fan Lin, MD, PhD

Accepted for publication September 8, 2011.

From the Departments of Laboratory Medicine (Drs Liu, Shi, Anandan, and Lin), Internal Medicine (Dr Diehl), Surgery (Dr Blansfield), and the Weis Center for Research (Dr Gerhard), Geisinger Medical Center, Danville, Pennsylvania; and the Department of Pathology, UCLA, Los Angeles, California (Dr Wang).

The authors have no relevant financial interest in the products or companies described in this article.

Presented in part at the United States and Canadian Academy of Pathology annual meetings, Washington, District of Columbia, March 2010, and San Antonio, Texas, March 2011.

Reprints: Fan Lin, MD, PhD, Department of Laboratory Medicine, MC 01-31, Geisinger Medical Center, 100 N Academy Ave, Danville, PA 17822 (e-mail: Flin1@geisinger.edu).
Table 1. Summary of Antibody Information and Staining Conditions

Antibody Name Vendor Catalog Approved
 and Synonyms No. Use

Annexin A8 SantaC (a) Sc-28825 RUO
CA19-9 CellMq (b) 399M-15 IVD
CEA Biogx (c) MU009-UC IVD

CDX-2 CellMq (b) 235R-15 IVD
Claudin 18 Invitro (d) 38-8100 ASR
Claudin 4 Invitro (d) 32-9400 ASR
CK17 DAKO (e) M7046 IVD
CK19 Ventana (f) 760-4281 IVD

CK20 CellMq (b) 320M-15 IVD
CK7 CellMq (b) 307M-95 IVD
CAM 5.2 BD (g) 349205 IVD
DPC4/ SantaC (a) Sc-7699 RUO
 SMAD4
Fascin DAKO (e) M3567 RUO
IMP-3 DAKO (e) M3626 IVD
Maspin BD (8) 554292 RUO
Mesothelin Vector (h) VP-M-649 RUO
MOC31 DAKO (e) M3525 IVD
MUC1 Vector (h) VP-M654 RUO
MUC2 Vector (h) VP-M656 RUO
MUC4 Sigma (i) HPA005895 RUO
MUC5AC Vector (h) VEC.VP- RUO
 M657
MUC6 Vector (h) VP-M658 RUO
p53 Biogx (c) MU195-UC IVD
PSCA NeoMK (j) RB9098-P1 IVD
S100P BD (g) 610307 RUO
pVHL SantaC (a) Sc-5575 RUO

Antibody Name Clonality Host Dilution
 and Synonyms Animal

Annexin A8 H60 Rabbit 1:50
CA19-9 121SLE Mouse 1:50
CEA B01-94- Mouse 1:350
 11M-P
CDX-2 EPR2764Y Rabbit 1:100
Claudin 18 Polyclonal Rabbit 1:100
Claudin 4 3E2C1 Mouse 1:100
CK17 E3 (1) Mouse 1:80
CK19 (A53-B/ Mouse 1:100
 A2.26)
CK20 Ks20.8 Mouse 1:500
CK7 OV-TL12/30 Mouse 1:200
CAM 5.2 CAM5.2 Mouse 1:4
DPC4/ B-8 Mouse 1:100
 SMAD4
Fascin 55K-2 Mouse 1:200
IMP-3 69.1 Mouse 1:50
Maspin G167-70 Mouse 1:200
Mesothelin 5B2 Mouse 1:20
MOC31 MOC-31 Mouse 1:100
MUC1 Ma 552 Mouse 1:100
MUC2 Ccp 58 Mouse 1:100
MUC4 Polyclonal Rabbit 1:150
MUC5AC CLH2 Mouse 1:50

MUC6 CLH5 Mouse 1:50
p53 BP53-12 Mouse 1:100
PSCA IG8 Rabbit 1:25
S100P Clone 16 Mouse 1:100
pVHL Polyclonal Rabbit 1:50

Antibody Name Incubation AR Method/Time/ Localization
 and Synonyms Time (min) Temp, [degrees]C/pH

Annexin A8 40 ND C
CA19-9 30 EDTA/15/100/8 C
CEA 30 ProtK/9/100/7.5 C

CDX-2 40 EDTA/15/100/8 N
Claudin 18 50 AR/15/100/6.0 C
Claudin 4 60 EDTA/15/100/8.0 C
CK17 30 EDTA/15/100/8.0 C
CK19 30 EDTA/15/100/8.0 C

CK20 30 EDTA/15/100/8.0 C
CK7 30 ProtK/9/ambient/7.5 C
CAM 5.2 30 None C
DPC4/ 40 EDTA N
 SMAD4
Fascin 30 EDTA/15/100/8.0 C
IMP-3 40 EDTA/15/100/8.0 C
Maspin 40 EDTA/15/100/8.0 N, C
Mesothelin 60 EDTA M, C
MOC31 30 TRS/20/99/6.1 M, C
MUC1 60 TRS/20/99/6.1 M, C
MUC2 60 EDTA/15/100/8.0 C
MUC4 30 AR/15/100/6.0 M, C
MUC5AC 60 Hi pH/20/99/9.9 C

MUC6 60 Hi pH/20/99/9.9 C
p53 30 AR/15/100/6.0 N
PSCA 30 AR/15/100/6.0 C
S100P 30 ProtK/12/ambient/7.5 N, C
pVHL 30 ProtK/9/ambient/7.5 M, C

Abbreviations: AR, antigen retrieval; ASR, analyte-specific
reagent; C, cytoplasmic; CA19-9, cancer antigen 19-9; CEA,
carcinoembryonic antigen; CK, cytokeratin; E, extracellular; Hi
pH, high pH; IMP-3, insulin-like growth factor 2 messenger RNA
binding protein 3; IVD, in vitro diagnostic use; M, membranous; N,
nuclear; ND, data incomplete due to the strong background
staining; ProtK, proteinase K; PSCA, prostate stem cell antigen;
pVHL, von Hippel-Lindau tumor suppressor gene protein; RUO, for
research use only; Temp, temperature; TRS, tris buffer.

(a) Santa Cruz Biotechnology, Inc, Santa Cruz, California.

(b) Cell Marque Corporation, Rocklin, California.

(c) BioGenex Laboratories Inc, San Ramon, California.

(d) Invitrogen Corporation, Carlsbad, California.

(e) Dako North America, Inc, Carpinteria, California.

(f) Ventana Medical Systems, North American Corporate
Headquarters, Tucson, Arizona.

(g) Becton Dickinson Immunocytometry Systems
(BD Biosciences), San Jose, California.

(h) Vector Laboratories, Inc, Burlingame, California.

(i) Sigma-Aldrich Corporation, Saint Louis, Missouri.

(j) Neomarkers, Inc, Fremont, California.

Table 2. Summary of Immunostaining Results for
Ductal Adenocarcinoma and Normal Pancreatic Tissue

Antibody Ductal Normal Normal Normal
 Adenocar- Pancreatic Pancreatic Pancreatic
 cinoma, Ducts, % Acini, % Islets, %
 %(N = 60) (N = 40) (N = 40) (N = 40)

Maspin 100 0 0 0
S100P 95 0 0 0
IMP-3 (a) 90 8 0 95
pVHL 3 100 100 100
CAM 5.2 70 100 100 100
CK7 (b) 96 100 70 0
CK20 15 0 0 0
CK19 75 95 3 0
CK17 60 0 0 0
mCEA 85 0 0 0
CA19-9 75 100 40 0
MOC31 97 100 100 100
Annexin A8 (a) ND 70 40 0
Claudin 4 (a) 94 100 100 83
Claudin 18 (a,c) 80 50 40 100
PSCA 56 90 100 100
Mesothelin (a) 57 70 0 0
MUC1 95 50 0 0
MUC2 4 0 0 0
MUC4 50 0 0 0
MUC5AC 67 0 0 0
MUC6 17 90 20 0
DPC4/SMAD4 41 100 100 100
p53 (a,d) 60 10 0 0
CDX2 (a,d) 5 10 10 0
Fascin (e) 85 100 100 0

Abbreviations: CA19-9, cancer antigen 19-9; CK, cytokeratin; IMP-3,
insulin-like growth factor 2 messenger RNA binding protein 3; mCEA,
monoclonal carcinoembryonic antigen; ND, no data collected due to
background staining; PSCA, prostate stem cell antigen; pVHL, von
Hippel-Lindau tumor suppressor gene protein.

(a) Weak staining for IMP-3, annexin A8, claudin 4, claudin 18,
mesothelin, p53, and CDX2 was observed in normal pancreatic ducts
and acini.

(b) Focal (1 +) and weak staining for CK7 was noted in pancreatic
acini.

(c) Claudin 18 was diffusely (4+) and strongly positive in normal
pancreatic islets.

(d) Only focal (1 +) staining for p53 and CDX2 was noted.

(e) Fascin also stained endothelial cells and stromal cells.

Table 3. Summary of Immunostaining Results for pVHL,
Maspin, S100P, and IMP-3 in Ductal Adenocarcinomas

Antibody Negative 1+ 2+ 3+ 4+ Total Positive
 Cases, No.
 (%) (N = 60)

pVHL 58 2 0 0 0 2/60 (3)
Maspin 0 1 2 7 50 60/60 (100)
S100P 3 5 6 10 36 57/60 (95)
IMP-3 6 8 5 13 28 54/60 (90)

Abbreviations: IMP-3, insulin-like growth factor 2 messenger
RNA binding protein 3;pVHL, von Hippel-Lindau tumor suppressor
gene protein.

Table 4. Summary of Immunostaining Results for Fine-Needle
Aspiration Biopsy Specimens

Group Maspin, S100P,
 No. (%) No. (%)

Pancreatic DAC 42/42 (a) (100) 44/44 (100)
Suspicious 13/13 (100) 13/13 (100)
Benign 1/10 (10) 2/10 (20)

Group IMP-3, pVHL,
 No. (%) No. (%)

Pancreatic DAC 37/40 (b) (93) 0/42 (a) (0)
Suspicious 10/13 (77) 0/13 (0)
Benign 1/10 (10) 7/10 (70)

Abbreviations: DAC, ductal adenocarcinoma; IMP-3,
insulin-like growth factor 2 messenger RNA binding
protein 3; pVHL, von Hippel-Lindau tumor suppressor
gene protein.

(a) Two cases did not contain tumor cells in the
deeper sections.

(b) Four cases did not contain tumor cells in the
deeper sections.
COPYRIGHT 2012 College of American Pathologists
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2012 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:Original Article
Author:Liu, Haiyan; Shi, Jianhui; Anandan, Vasuki; Wang, Hanlin L.; Diehl, David; Blansfield, Joseph; Gerha
Publication:Archives of Pathology & Laboratory Medicine
Date:Jun 1, 2012
Words:6053
Previous Article:Evaluation of an automated digital imaging system, Nextslide Digital Review network, for examination of peripheral blood smears.
Next Article:CD68 immunostaining in the evaluation of chronic histiocytic intervillositis.
Topics:

Terms of use | Privacy policy | Copyright © 2018 Farlex, Inc. | Feedback | For webmasters