Differentiation of ductal adenocarcinoma (DAC) of the

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1 Original Article Reevaluation and Identification of the Best Immunohistochemical Panel (pvhl, Maspin, S100P, IMP-3) for Ductal Adenocarcinoma of the Pancreas 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 N Context. Differentiation of ductal adenocarcinoma of the pancreas from nonneoplastic pancreatic tissues can be challenging, especially in small biopsy and fine-needle aspiration specimens. Objective. To investigate the utility of 26 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. Design. Immunohistochemical staining for these markers was performed in 60 cases of pancreatic ductal adenocarcinoma on routine and tissue microarray sections. In addition, immunohistochemical staining for maspin, S100P, IMP-3, and pvhl was performed on cell blocks from 67 pancreatic fine-needle aspiration cases, including 44 cases of pancreatic ductal adenocarcinoma. Results. The results demonstrated that (1) more than 90% of cases of ductal adenocarcinoma were positive for maspin, S100P, and IMP-3; (2) nearly all adenocarcinoma cases were negative for pvhl, whereas nonneoplastic ducts and acini were positive for pvhl in all cases; (3) normal/reactive pancreatic ducts were frequently positive for CK7, CK19, MUC1, MUC6, CA19-9, MOC31, PSCA, mesothelin, annexin 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 adenocarcinomas were negative for DPC4/SMAD4; and (6) strong background staining was frequently seen with fascin, PSCA, and annexin A8. Conclusions. pvhl, maspin, S100P, and IMP-3 constitute the best diagnostic panel of immunomarkers for confirming the diagnosis of pancreatic ductal adenocarcinoma in both surgical and fine-needle aspiration specimens. (Arch Pathol Lab Med. 2012;136: ; doi: / arpa oa) 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, Accepted for publication September 8, 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 Reprints: Fan Lin, MD, PhD, Department of Laboratory Medicine, MC 01-31, Geisinger Medical Center, 100 N Academy Ave, Danville, PA ( Flin1@geisinger.edu). 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 , including 10 Arch Pathol Lab Med Vol 136, June 2012 Best Immunohistochemical Panel for Pancreatic Adenocarcinoma Liu et al 601

2 Antibody Name and Synonyms Table 1. Summary of Antibody Information and Staining Conditions Vendor Catalog No. Approved Use Clonality Host Animal Dilution Incubation Time (min) AR Method/Time/ Temp, 6C/pH Localization Annexin A8 SantaC a Sc RUO H60 Rabbit 1:50 40 ND C CA19-9 CellMq b 399M-15 IVD 121SLE Mouse 1:50 30 EDTA/15/100/8 C CEA Biogx c MU009-UC IVD B M-P Mouse 1: ProtK/9/100/7.5 C CDX-2 CellMq b 235R-15 IVD EPR2764Y Rabbit 1: EDTA/15/100/8 N Claudin 18 Invitro d ASR Polyclonal Rabbit 1: AR/15/100/6.0 C Claudin 4 Invitro d ASR 3E2C1 Mouse 1: EDTA/15/100/8.0 C CK17 DAKO e M7046 IVD E3 (1) Mouse 1:80 30 EDTA/15/100/8.0 C CK19 Ventana f IVD (A53-B/ Mouse 1: EDTA/15/100/8.0 C A2.26) CK20 CellMq b 320M-15 IVD Ks20.8 Mouse 1: EDTA/15/100/8.0 C CK7 CellMq b 307M-95 IVD OV-TL12/30 Mouse 1: ProtK/9/ambient/7.5 C CAM 5.2 BD g IVD CAM5.2 Mouse 1:4 30 None C DPC4/ SantaC a Sc-7699 RUO B-8 Mouse 1: EDTA N SMAD4 Fascin DAKO e M3567 RUO 55K-2 Mouse 1: EDTA/15/100/8.0 C IMP-3 DAKO e M3626 IVD 69.1 Mouse 1:50 40 EDTA/15/100/8.0 C Maspin BD g RUO G Mouse 1: EDTA/15/100/8.0 N, C Mesothelin Vector h VP-M-649 RUO 5B2 Mouse 1:20 60 EDTA M, C MOC31 DAKO e M3525 IVD MOC-31 Mouse 1: TRS/20/99/6.1 M, C MUC1 Vector h VP-M654 RUO Ma 552 Mouse 1: TRS/20/99/6.1 M, C MUC2 Vector h VP-M656 RUO Ccp 58 Mouse 1: EDTA/15/100/8.0 C MUC4 Sigma i HPA RUO Polyclonal Rabbit 1: AR/15/100/6.0 M, C MUC5AC Vector h VEC.VP- RUO CLH2 Mouse 1:50 60 Hi ph/20/99/9.9 C M657 MUC6 Vector h VP-M658 RUO CLH5 Mouse 1:50 60 Hi ph/20/99/9.9 C p53 Biogx c MU195-UC IVD BP53-12 Mouse 1: AR/15/100/6.0 N PSCA NeoMK j RB9098-P1 IVD IG8 Rabbit 1:25 30 AR/15/100/6.0 C S100P BD g RUO Clone 16 Mouse 1: ProtK/12/ambient/7.5 N, C pvhl SantaC a Sc-5575 RUO Polyclonal Rabbit 1:50 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. 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) was recorded as negative (,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 72uC with a proprietary detergent solution, followed by blocking for endogenous peroxidase. Automated antigen retrieval was performed for 36 minutes at 95uC with a proprietary solution at ph 8.0. Anti-maspin antibody (1:200) was applied to the tissue sections, and slides were incubated at 37uC for 40 minutes. Slides were subsequently rinsed in a proprietary buffer, and visualization with the ultraview DAB 602 Arch Pathol Lab Med Vol 136, June 2012 Best Immunohistochemical Panel for Pancreatic Adenocarcinoma Liu et al

3 Table 2. Summary of Immunostaining Results for Ductal Adenocarcinoma and Normal Pancreatic Tissue Antibody Ductal Adenocarcinoma, %(N= 60) Normal Pancreatic Ducts, % (N = 40) Normal Pancreatic Acini, % (N = 40) Normal Pancreatic Islets, % (N = 40) Maspin S100P IMP-3 a pvhl CAM CK7 b CK CK CK mcea CA MOC Annexin A8 a ND Claudin 4 a Claudin 18 a,c PSCA Mesothelin a MUC MUC MUC MUC5AC MUC DPC4/SMAD p53 a,d CDX2 a,d Fascin e 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. Detection Kit was achieved. Antibody denaturation was performed by heating the slides to 90uC 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 37uC. 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 , 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 Arch Pathol Lab Med Vol 136, June 2012 Best Immunohistochemical Panel for Pancreatic Adenocarcinoma Liu et al 603

4 Figure 1. Photomicrographs showing ductal adenocarcinoma positive for maspin (A), S100P (B), insulin-like growth factor 2 messenger RNA binding protein 3 (IMP-3) (C), cytokeratin 17 (CK17) (D), and MUC5AC (E) and negative for von Hippel-Lindau tumor suppressor gene protein (pvhl) (F). Only nuclear positivity or both nuclear and cytoplasmic positivity for maspin and S100P is regarded as positive expression. Normal pancreatic ducts and acini are generally negative for maspin, S100P, IMP-3, CK17, and MUC5AC (maspin, original magnification 400 [A]; S100P, original magnification 3400 [B]; IMP-3, original magnification 3400 [C]; CK17, original magnification 3400 [D]; MUC5AC, original magnification 3400 [E]; pvhl, original magnification 3200 [F]). 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, 604 Arch Pathol Lab Med Vol 136, June 2012 MUC1, MUC6, CA19-9, MOC31, PSCA, mesothelin, annexin 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 adenocarcinoma cases showed loss of expression of DPC4/SMAD4; Best Immunohistochemical Panel for Pancreatic Adenocarcinoma Liu et al

5 Figure 2. Photomicrograph showing normal pancreatic ducts, acini, and islets positive for von Hippel-Lindau tumor suppressor gene protein (pvhl) (A); in contrast, pvhl is not seen in most cases of adenocarcinoma, and only 2 cases show focal (1+) pvhl positivity (B) (original magnifications 3200 [A and B]). Figure 3. A and B, Double staining with maspin and von Hippel-Lindau tumor suppressor gene protein (pvhl) in a case of ductal adenocarcinoma (pvhl [purple] and maspin [brown]). Note that maspin is positively expressed in adenocarcinoma, whereas pvhl expression is negative in adenocarcinoma but positive in nonneoplastic ducts (original magnifications 3200 [A] and 3400 [B]). Figure 4. Strong p53 positivity is seen in 60% of cases of pancreatic ductal adenocarcinoma (original magnification 3400). Figure 5. Loss of DPC4/SMAD4 expression is seen in 60% of cases of pancreatic ductal adenocarcinoma (original magnification 3400). Arch Pathol Lab Med Vol 136, June 2012 Best Immunohistochemical Panel for Pancreatic Adenocarcinoma Liu et al 605

6 Table 3. Summary of Immunostaining Results for pvhl, Maspin, S100P, and IMP-3 in Ductal Adenocarcinomas Antibody Negative Total Positive Cases, No. (%) (N = 60) pvhl /60 (3) Maspin /60 (100) S100P /60 (95) IMP /60 (90) Abbreviations: IMP-3, insulin-like growth factor 2 messenger RNA binding protein 3; pvhl, von Hippel-Lindau tumor suppressor gene protein. and (6) strong background staining was frequently seen with fascin (including staining for endothelial cells and stromal cells), PSCA, and annexin A8. 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 Table 4. Summary of Immunostaining Results for Fine-Needle Aspiration Biopsy Specimens Group Maspin, No. (%) S100P, No. (%) IMP-3, No. (%) pvhl, No. (%) Pancreatic DAC 42/42 a (100) 44/44 (100) 37/40 b (93) 0/42 a (0) Suspicious 13/13 (100) 13/13 (100) 10/13 (77) 0/13 (0) Benign 1/10 (10) 2/10 (20) 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. 606 Arch Pathol Lab Med Vol 136, June 2012 Best Immunohistochemical Panel for Pancreatic Adenocarcinoma Liu et al

7 Figure 6. Photomicrographs showing adenocarcinoma on the cell block section (A) and expression of maspin (B), S100P (C), insulin-like growth factor 2 messenger RNA binding protein 3 (IMP-3) (D), and von Hippel-Lindau tumor suppressor gene protein (pvhl) (E) in pancreatic ductal adenocarcinomas in fine-needle aspiration samples on cell blocks. Note that only nuclear staining or both nuclear and cytoplasmic staining for maspin and S100P is regarded as positive. IMP-3 shows cytoplasmic staining (hematoxylin-eosin, original magnification 3400 [A]; maspin, original magnification 3600 [B]; S100P, original magnification 3600 [C]; IMP-3, original magnification 3600 [D]; pvhl, original magnification 3400 [E]). 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 Arch Pathol Lab Med Vol 136, June 2012 either negative or had only cytoplasmic positivity for S100P. 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 Best Immunohistochemical Panel for Pancreatic Adenocarcinoma Liu et al 607

8 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 5 30) of esophageal adenocarcinomas, 51 90% (N 5 38) of colorectal adenocarcinomas, and 65% (N 5 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 5 23) of gallbladder adenocarcinomas and 65% (N 5 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 5 18) of hepatocellular carcinomas and 13% (N 5 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 ultrasoundguided 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): 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. 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