Human pancreatic cancer fusion 2 (HPC2) 1-B3: A novel monoclonal antibody to screen for pancreatic ductal dysplasia

Authors

  • Terry K. Morgan MD, PhD,

    Corresponding author
    1. Department of Pathology, Oregon Health and Science University, Portland, Oregon
    • Department of Pathology, Oregon Health and Science University, Mail Code L471, 3181 SW Sam Jackson Park Rd, Portland, OR 97239

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    • Fax: (503) 494-6787

  • Karin Hardiman MD, PhD,

    1. Department of Surgery, Oregon Health and Science University, Portland, Oregon
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  • Christopher L. Corless MD, PhD,

    1. Department of Pathology, Oregon Health and Science University, Portland, Oregon
    2. Knight Cancer Institute, Oregon Health and Science University, Portland, Oregon
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  • Sandra L. White MD,

    1. Department of Pathology, Oregon Health and Science University, Portland, Oregon
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  • Robert Bonnah PhD,

    1. Family Pediatric Research Institute, Department of Pediatrics, Oregon Health and Science University, Portland, Oregon
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  • Henry Van de Vrugt PhD,

    1. Family Pediatric Research Institute, Department of Pediatrics, Oregon Health and Science University, Portland, Oregon
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  • Brett C. Sheppard MD,

    1. Department of Surgery, Oregon Health and Science University, Portland, Oregon
    2. Knight Cancer Institute, Oregon Health and Science University, Portland, Oregon
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  • Markus Grompe MD,

    1. Knight Cancer Institute, Oregon Health and Science University, Portland, Oregon
    2. Family Pediatric Research Institute, Department of Pediatrics, Oregon Health and Science University, Portland, Oregon
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  • Ediz F. Cosar MD,

    1. Department of Pathology, University of Massachusetts, Worcester, Massachusetts
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  • Philip R. Streeter PhD

    1. Knight Cancer Institute, Oregon Health and Science University, Portland, Oregon
    2. Family Pediatric Research Institute, Department of Pediatrics, Oregon Health and Science University, Portland, Oregon
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Abstract

BACKGROUND.

Pancreatic ductal adenocarcinoma is rarely detected early enough for patients to be cured. The objective of the authors was to develop a monoclonal antibody to distinguish adenocarcinoma and precancerous intraductal papillary mucinous neoplasia (IPMN) from benign epithelium.

METHODS.

Mice were immunized with human pancreatic adenocarcinoma cells and monoclonal antibodies were screened against a panel of archived pancreatic tissue sections, including pancreatitis (23 cases), grade 1 IPMN (16 cases), grade 2 IPMN (9 cases), grade 3 IPMN (13 cases), and various grades of adenocarcinoma (17 cases). One monoclonal antibody, human pancreatic cancer fusion 2 (HPC2) 1-B3, which specifically immunostained adenocarcinoma and all grades of IPMN, was isolated. Subsequently, HPC2 1-B3 was evaluated in a retrospective series of 31 fine-needle aspiration (FNA) biopsies from clinically suspicious pancreatic lesions that had long-term clinical follow-up.

RESULTS.

HPC2 1-B3 was negative in all 31 cases of chronic pancreatitis that were tested. In contrast, HPC2 1-B3 immunostained the cytoplasm and luminal surface of all 16 well- to moderately differentiated pancreatic ductal adenocarcinomas. It demonstrated only weak focal staining of poorly differentiated carcinomas. All high-grade IPMNs were found to be positive for HPC2 1-B3. The majority of low-grade to intermediate-grade IPMNs were positive (66% of cases). Immunostaining a separate series of pancreatic FNA cell blocks for HPC2 1-B3 demonstrated that the relative risk for detecting at least low-grade dysplasia (2.0 [95% confidence interval, 1.23-3.26]) was statistically significant (P = .002 by the Fisher exact test).

CONCLUSIONS.

To reduce the mortality of pancreatic cancer, more effective early screening methods are necessary. The data from the current study indicate that a novel monoclonal antibody, HPC2 1-B3, may facilitate the diagnosis of early pancreatic dysplasia. Cancer (Cancer Cytopathol) 2013;121:37–46 © 2012 American Cancer Society.

INTRODUCTION

Pancreatic ductal adenocarcinoma is usually lethal, because it is often not diagnosed until after it has already metastasized.1, 2 Nearly 40,000 people in the United States will be diagnosed with pancreatic cancer in 2012 and the majority of these patients will die of the disease.2 Improved patient survival may depend on detecting pancreatic cancer in its early stages.

Similar to cervical cancer, there are precancerous pancreatic lesions. For example, there is accumulating evidence that intraductal papillary mucinous neoplasia (IPMN) and microscopic pancreatic intraepithelial neoplasia (PanIN) are precursor lesions leading to invasive ductal carcinoma.3-7 IPMNs involve the main ducts, whereas PanINs involve the smaller ducts. Both have various grades of dysplasia such as PanIN of grades 1 to 3 and low-grade, intermediate-grade, and high-grade IPMN. The actual prevalence of pancreatic dysplasia and the long-term risk of progression to invasive adenocarcinoma are essentially unknown, but a few studies have suggested that approximately one-third of these lesions may progress to adenocarcinoma within 10 years.8-11 Unfortunately, unlike cervical cancer, to the best of our knowledge there is currently no reliable screening method with which to detect these precancerous lesions. Serological markers for invasive pancreatic cancer are in the early stages of development and to date there are no reliable markers for the detection of precancerous lesions.12 Radiographic features of precancerous and early invasive pancreatic cancer are not specific.3 Endoscopic ultrasound (EUS)-guided fine-needle aspiration (FNA) biopsies are becoming the standard of care for the evaluatation of patients with pancreatic symptoms.13-19 The advantage of this evaluation strategy is that it is minimally invasive and can sample small pancreatic masses, as well as provide tissue for cytology. A challenge of this approach is that it is highly dependent on the skill and experience of both the endoscopist and cytopathologist.15 In experienced hands, EUS-guided pancreatic FNAs have good accuracy in diagnosing adenocarcinoma (80%),15 but only moderate accuracy in the diagnosis of IPMNs (50%).16 Therefore, most clinicians supplement pancreatic FNA cytology by measuring carcinoembryonic antigen (CEA) levels in cystic fluid (positive test is > 200 ng/mL) to improve the negative predictive value (NPV).6, 16, 20

New markers with excellent positive predictive value (PPV) are needed to supplement pancreatic fluid cytology. Markers such as the K homology domain containing protein Overexpressed in Cancer (KOC)21-23 have shown promise in diagnosing pancreatic adenocarcinoma, but not IPMNs.21 Gene expression profiling of IPMNs has also yielded a list of potential candidate genes,24 and new commercially available genetic assays have demonstrated clinical promise.20 However, these molecular tests are more complex and expensive than routine immunoassays. In the current study, we describe a novel monoclonal antibody, human pancreatic cancer fusion 2 (HPC2) 1-B3, which appears to specifically detect both pancreatic ductal adenocarcinoma and precancerous IPMNs.

MATERIALS AND METHODS

Human Pancreatic Cancer Cells for Hybridoma Generation

The human tissues used for immunizations and the testing of hybridoma supernatant fluids were obtained from the Oregon Pancreatic Tumor Registry and the Department of Pathology at the Oregon Health and Science University (OHSU) using an Institutional Review Board-approved protocol with informed patient consent. Mouse hybridomas were generated using standard techniques,25 and all work with mice was conducted under a protocol approved by the OHSU Institutional Animal Care and Use Committee. BALB/C mice were immunized every 3 weeks for 3 months (3-4 total injections) with enzyme-dissociated fresh human pancreatic cancer cells. Approximately 1 × 106 cells were administered intraperitoneally with each immunization using Imject Alum as an aduvant/carrier (Thermo Scientific, Rockford, Ill). Four days after the final immunization, animals were euthanized and their spleens were removed for hybridoma generation. Splenocytes were fused with SP2/0-Ag14 myeloma cells25 and successfully fused cells were cloned using ClonaCell-HY media (STEMCELL Technologies Inc, Vancouver, BC, Canada). No more than 600 isolated clones per fusion were transferred into liquid media and distributed into 96-well plates. After clonal expansion, hybridoma supernatant fluids were screened by flow cytometry using PANC-1 cell cultures and immunohistochemistry using a preselected panel of pancreatic tissue sections.

Hybridoma Screening by Flow Cytometry

Flow cytometry was used to screen a commercially available human pancreatic cancer cell line (PANC-1 cells [CRL-1469]; American Type Culture Collection, Manassas, Va) for marker expression. Cultured cells were trypsinized for removal from flasks, washed, and resuspended in 100 μL of Dulbecco modified Eagle medium (DMEM) with 5% fetal bovine serum (FBS) (DMEM/FBS). Resuspended cells were combined with an equal volume of hybridoma supernatant fluid and incubated at 4°C for 25 minutes. After washing with DMEM/FBS (4°C), the cells were resuspended in 100 μL of DMEM/FBS containing 5% normal rat serum, a 1:500 dilution of propidium iodide, and a 1:200 dilution of R-phycoerythrin–conjugated goat-antimouse immunoglobulin G (Jackson ImmunoResearch Laboratories, West Grove, Pa). An isotype-matched mouse monoclonal antibody was used as a negative control.

Hybridoma Screening of Pancreatic Tissue Specimens

A total of 78 surgical resections of the pancreas were identified in the pathology archives at OHSU for the period between 2002 through 2008. This was a retrospective analysis of archived tissue specimens and therefore informed patient consent was not required. Indications for surgery included obstructive pancreatitis (23 cases), invasive ductal adenocarcinoma (17 cases), and various grades of isolated IPMN (38 cases) without associated adenocarcinoma. None of the IPMNs were cystic mucinous neoplasms with ovarian stroma. Some of the IPMNs had associated PanINs, but these were rare and were not graded separately for the current study. The IPMNs were graded by 2 independent surgical pathologists as low grade (16 cases), intermediate grade (9 cases), and high grade (13 cases) according to accepted criteria.1, 5, 26 Low-grade lesions were characterized by flat or papillary epithelial lesions comprised of tall columnar cells with small, round, basally located nuclei and abundant mucinous cytoplasm. Intermediate-grade lesions had similar architecture with a low mitotic index but moderate nuclear atypia. High-grade lesions had complex or solid architecture with nuclear atypia, including nucleoli, frequent mitoses, and necrosis, but no evidence of invasion into surrounding pancreatic tissue. Formalin-fixed and paraffin-embedded pancreatic tissue sections underwent antigen retrieval in citrate buffer solution (pH 6.0) (Target Retrieval Solution; Dako North America Inc, Carpinteria, Calif). They were then incubated with hybridoma supernatant fluid at a 1:10 dilution (10 μg/mL) in phosphate-buffered saline (PBS) for 1 hour at room temperature, followed by ImmPRESS (Vector Laboratories, Burlingame, Calif.): http://www.vectorlabs.com/catalog.aspx?catID=436 for 30 minutes. They were washed and incubated with diaminobenzidine chromagen, yielding a brown positive signal. Sections were counterstained with hematoxylin. Lesional cells were considered positive if >10% of the neoplasm was immunostained for HPC2 1-B3. Positive cells may have cytoplasmic staining (cancer) or demonstrate only luminal brush border staining (low-grade to intermediate-grade IPMNs).

HPC2 1-B3 Test Evaluation Using a Series of EUS-Guided FNA Biopsy Specimens

To test the diagnostic predictive value of HPC2 1-B3, we immunostained a retrospective series of 31 EUS-guided FNA biopsy specimens of the pancreas. These cases represented the only pancreatic FNAs in the cytopathology archives of OHSU (2001-2007) with sufficient cell block material for immunohistochemical analysis. No other exclusion criteria were used. All 31 cases had either surgical follow-up or at least 4 years of negative clinical follow-up. Indications for FNA were pancreatitis or symptoms of ductal obstruction, with an identifiable pancreatic mass. The cytologic diagnosis was either negative for malignancy (8 cases), positive for mucinous neoplasm (17 cases), or positive for ductal adenocarcinoma (6 cases). Notably, low-grade mucinous biopsy samples from the pancreatic body or tail had been previously stained for alcian yellow and alcian blue to exclude gastric contamination. Long-term clinical follow-up confirmed pancreatitis in all 8 negative cases and adenocarcinoma in all 6 malignant cases. The surgically resected mucinous neoplasms were classified as IPMNs with high-grade dysplasia (4 cases) or low-grade to intermediate-grade dysplasia (13 cases) for statistical analysis. Paraffin-embedded sections of the archived FNA biopsy cell block were then immunostained for HPC2 1-B3 using a 1:10 dilution (10 μg/mL). Staining was considered positive if the ductal epithelium demonstrated either cytoplasmic or cell surface signals. Notably, the background debris was often strongly positive when ductal cells were positive. Histologic sections of the cell blocks were also immunostained for KOC21-23 using a commercially available antibody (monoclonal antihuman IMP3, clone 69.1; Dako North America) according to the manufacturer's instructions. KOC positivity highlights the cytoplasm. The follow-up surgical resections of these FNA specimens were also immunostained and were found to be in agreement with the FNA results, but were not included in the panel data presented in Table 1.

Table 1. Screening Panel of Surgically Resected Pancreatic Tissues Immunostained for the HPC2 Antigen
Staining ResultChronic Pancreatitis (n = 23)Low-Grade IPMN (n = 16)Intermediate-Grade IPMN (n=9)High-Grade IPMN (n = 13)Invasive Adenocarcinoma (n = 17)
  • Abbreviations: HPC2, human pancreatic cancer fusion 2; IPMN, intraductal papillary mucinous neoplasm.

  • a

    Indicates all 6 invasive adenocarcinomas were poorly differentiated and showed at most weak focal staining (<10% of cells).

Positive staining010 (63%)8 (89%)13 (100%)11 (65%)
Negative staining23 (100%)6 (37%)1 (11%)06a (35%)

Western Blot Analyses

Western blot analyses were used to define the molecular mass of the HPC2 1-B3 antigen and to determine whether PANC-1 and HeLa cells (CCL-2; American Type Culture Collection) shed or secrete the antigen. PANC-1 cells and HeLa cervical cancer cells were grown in DMEM with 10% fetal calf serum (FCS) until they were approximately 80% confluent. The media was removed and replaced with DMEM with 3% FCS. After a 24-hour incubation, the supernatant fluids from these cultures was harvested to test for the presence of the HPC2 1-B3 antigen. Supernatant fluids were centrifuged at low speed to remove nonadherent cells and cellular debris and then concentrated approximately 25-fold using an Amicon Ultra Centrifugation filter with a molecular weight cutoff of 10 kilodaltons (kD) (Ultracel-10K MWCO; EMD Millipore Corporation, Billerica, Mass). The concentrate was then microfuged at high speed for 2 minutes to remove any remaining debris and the supernatant fluid was collected. The samples were then separated by electrophoresis using a Criterion XT Precast 12% Bis-Tris Gel (Bio-Rad, Hercules, Calif) and electrophoretically transferred to Immobilon polyvinylidene difluoride membranes (Millipore Corporation). The blot was blocked with PBS containing 10% skim milk and 1% bovine serum albumin. It was then incubated overnight at 4°C in a 1:500 dilution of HPC2 1-B3 hybridoma supernatant fluid. After washing in PBS plus 0.1% Tween 20, horseradish peroxidase-conjugated antimouse IgG was added at a dilution of 1:2000 for 1 hour at room temperature. The membrane was washed and developed using Western Lightning Plus ECL enhanced luminol reagent (Perkin Elmer, Waltham, Mass) and visualized using Kodak X-OMAT Blue (XB) film (Eastman Kodak Company, Rochester, NY).

Statistical Analysis

Immunohistochemical data were compared by chi-square analysis using the Fisher exact test and SAS statistical software (version 9.1.3; SAS Institute Inc, Cary, NC). Test performance of HPC2 1-B3 and KOC immunostaining, including sensitivity, specificity, PPV, NPV, and relative risk (RR) for at least low-grade dysplasia, were calculated using 2 × 2 contingency tables with binomial 95% confidence intervals (95% CIs).

RESULTS

HPC2 1-B3 in Pancreatic Cancer

Hybridoma supernatant fluids from approximately 900 colonies were screened for reactivity by flow cytometry and our preselected panel of pancreatic tissue sections. Screening yielded 1 monoclonal antibody, HPC2 1-B3, which stained PANC-1 cells by flow cytometry (Fig. 1), was negative in chronic pancreatitis, and was positive in pancreatic ductal adenocarcinoma and precancerous IPMNs on immunohistochemistry (Fig. 2).

Figure 1.

Human pancreatic cancer fusion 2 (HPC2) 1-B3 antigen is a cell surface marker. (A) Flow cytometric analysis of viable PANC-1 human pancreas cancer cells was the first step in screening for hybridoma. It demonstrated a strong signal for the HPC2 1-B3 antigen on intact cells, which supports a cell surface localization. (B) An isotype-matched negative control did not react with PANC-1 cells.

Figure 2.

Immunostaining of histologic sections of chronic pancreatitis, intraductal papillary mucinous neoplasms (IPMNs), and ductal adenocarcinoma for human pancreatic cancer fusion 2 (HPC2) 1-B3 and K homology domain containing protein overexpressed in cancer (KOC) is shown. (A-C) Representative sections of chronic pancreatitis demonstrating negative immunostaining for HPC2 1-B3 and KOC are shown. Normal ducts also were negative (data not shown). (D-F) Intraductal papillary mucinous neoplasms demonstrated apical membranous immunostaining (indicated by the arrow) for (E) HPC2 1-B3 but (F) KOC was reproducibly negative in low-grade and intermediate-grade IPMNs. (G-I) Invasive ductal adenocarcinoma demonstrated both (H inset) luminal and (H) patchy cellular staining for HPC2 1-B3, whereas (I) KOC was observed to be diffusely positive in tumor cell cytoplasm. Panels A, D, and G were stained with hematoxylin and eosin. The remaining panels were immunostained with brown chromagen representing a positive stain. Sections were counterstained with hematoxylin. Panels D to F were photographed using a × 10 objective; panels A to C and G to I were photographed using a × 20 objective (Leica; Wetzlar, Germany).

Invasive adenocarcinomas stained strongly and diffusely for HPC2 1-B3. The antigen predominantly localized to the cytoplasm of moderately differentiated cancers (Fig. 2) and the apical border of well-differentiated ductal adenocarcinomas (Fig. 2H, Inset). HPC2 1-B3 staining of poorly differentiated adenocarcinomas demonstrated only a weak focal cytoplasmic signal, although all 6 of these cases stained strongly for the KOC marker (Fig. 2) (Table 1).

Both HPC2 1-B3 and KOC immunostained EUS-guided FNAs of pancreatic cancer (5 of 6 cases and 4 of 6 cases, respectively), but not chronic pancreatitis (Table 2). HPC2 1-B3 was observed in carcinoma cells and in the surrounding mucinous debris (Fig. 3). In contrast, KOC stained carcinoma cells but not the surrounding debris (Fig. 3). Pancreatitis was reproducibly negative for HPC2 1-B3 (8 cases), suggesting the antigen is not expressed by reactive pancreatic ductal epithelium (Fig. 3).

Figure 3.

Immunostaining of histologic sections of pancreatic fine-needle aspiration (FNA) cell blocks for human pancreatic cancer fusion 2 (HPC2) 1-B3 and K homology domain containing protein overexpressed in cancer (KOC) is shown. (A-C) Representative sections of reactive ductal epithelium from a case subsequently diagnosed as chronic pancreatitis are shown. Both HPC2 1-B3 and KOC immunostaining of the FNA cell block were negative. (D-F) Intraductal papillary mucinous neoplasms often demonstrated apical membranous staining, whereas KOC staining was negative unless the cytology was high grade. (G-I) Invasive ductal adenocarcinoma was usually positive with both antibodies, although HPC2 1-B3 usually demonstrated apical staining and (H) an abundant signal in surrounding debris. (I) In contrast, KOC staining was restricted to tumor cell cytoplasm. Panels A, D, and G were stained with hematoxylin and eosin. The remaining panels were immunostained with brown chromagen representing a positive stain. Sections were counterstained with hematoxylin. Panels A to I were photographed using a × 20 objective (Leica; Wetzlar, Germany).

Table 2. Surgical Outcome Compared With Immunostaining FNAs for HPC2 and KOCa
ImmunostainingBenign Pancreatitis (n = 8)Low-Grade IPMN (n = 11)Intermediate- Grade IPMN (n = 2)High-Grade IPMN (n = 4)Invasive Adenocarcinoma (n = 6)
  • Abbreviations: FNAs, fine-needle aspirations; HPC2, human pancreatic cancer fusion 2; IPMN, intraductal papillary mucinous neoplasm; KOC, K homology domain containing protein overexpressed in cancer.

  • a

    Relative risk (RR) and Fisher exact testing to detect at least low-grade dysplasia using HPC2 were significant (RR, 2.0; 95% confidence interval [95% CI], 1.23-3.26 [P = .002]). KOC detected high-grade dysplasia, but not low-grade or intermediate-grade dysplasia (RR, 1.5; 95% CI, 1.13-1.99 [P = .15]).

HPC2 positive05 (45%)1 (50%)4 (100%)5 (83%)
HPC2 negative8 (100%)6 (55%)1 (50%)01 (17%)
KOC positive0003 (75%)4 (67%)
KOC negative8 (100%)11 (100%)2 (100%)1 (25%)2 (33%)

HPC2 1-B3 Staining of IPMNs

Immunostaining of surgically resected IPMNs revealed diffuse, strong, apical border staining for HPC2 1-B3 in the majority of low-grade precancerous lesions (10 of 16 cases; 63%), nearly all of the intermediate-grade lesions (8 of 9 cases; 89%), and all of the high-grade IPMNs with severe dysplasia (13 of 13 cases; 100%). In addition, the HPC2 1-B3 antigen was reproducibly detected in the ductal lumens of IPMNs, suggesting it is also secreted by these precancerous lesions (Fig. 2E). A few tissue sections also contained foci of grade 3 PanIN, which stained for HPC2 1-B3 (data not shown). Only rare foci of grade 1 PanIN were identified in the sections examined in the current study; therefore, although they were negative for HPC2 1-B3, further testing will be required in a larger cohort. HPC2 1-B3 immunostained all 4 high-grade IPMNs (100%) and approximately one-half of the FNA biopsies from low-grade to intermediate-grade IPMNs (6 of 13 cases; 46%) (Table 2). Positive staining was predominantly observed along the apical border of dysplastic cells and in the surrounding mucinous debris (Fig. 3). KOC stained the majority of high-grade cases (3 of 4 cases; 75%), but none of the low-grade to intermediate-grade IPMNs (0 of 13 cases) (Table 2).

Predictive Value of Immunostaining EUS-Guided FNA Cell Blocks for HPC2 1-B3

In the current series, HPC2 1-B3 was found to detect more cases of severe dysplasia and invasive adenocarcinoma than the KOC marker (9 of 10 cases vs 7 of 10 cases). All 8 cases of pancreatitis were negative (Table 2). HPC2 1-B3 was more sensitive than KOC in identifying precancerous pancreatic ductal lesions (65% vs 30%). The PPV of both markers was 100%, although the 95% CI was wide given the relatively limited sample size. The RR and Fisher exact test of detecting at least low-grade dysplasia using HPC2 1-B3 was significant (RR, 2.0; 95% CI, 1.23-3.26 [P = .002]. In contrast, KOC did not detect low-grade dysplasia and had an overall RR of 1.5 (95% CI, 1.13-1.99; P = .15 using the Fisher exact test).

Western Blot Analysis and HPC2 1-B3 Secretion

Western blot analysis of cultured PANC-1 and HeLa tumor cells revealed HPC2 1-B3 protein in both the cell lysates and culture supernatant fluid (Fig. 4). Human HeLa cervical cancer cells were chosen because we identified high levels of HPC2 1-B3 protein in this cell line. The data from the current study suggest that HPC2 1-B3 is approximately 55 kD to 65 kD in size and is secreted or shed by both tumor cell lines into culture supernatant fluid. PANC-1 cells secrete less HPC2 1-B3 than HeLa cells, but together with our immunohistochemical analyses of IPMNs and pancreatic adenocarcinomas (Figs. 2 and 3), these Western blot data support the hypothesis that the HPC2 1-B3 antigen is likely secreted by dysplastic and neoplastic pancreatic ductal cells.

Figure 4.

Human pancreatic cancer fusion 2 (HPC2) 1-B3 antigen is a secreted protein. Western blot analysis demonstrated immunostaining for the HPC2 1-B3 antigen at 55 kilodaltons (kDa) to 65 kDa in lysates of both HeLa cells (lane 1) and PANC-1 cells (lane 2). HeLa cells expressed significantly more HPC2 1-B3 antigen than PANC-1 cells. Analysis of culture supernatant fluids from HeLa cells (lane 3) revealed the presence of HPC2 1-B3 antigen, suggesting the marker may be secreted by tumor cells. PANC-1 secretion (lane 4) was barely detectable (arrow).

DISCUSSION

To reduce the mortality of pancreatic cancer, early screening methods similar to those used for cervical cancer will be necessary. The data from the current study indicate that a novel monoclonal antibody, HPC2 1-B3, may facilitate the diagnosis of early pancreatic ductal dysplasia, which we suspect could be more likely to progress to invasive adenocarcinoma. The detection of these early lesions is vital to reducing patient mortality.

Pancreatic Ductal Dysplasia

Dysplastic pancreatic ductal lesions include IPMNs in the larger ducts and PanINs in the smaller ducts.4, 5 Similar to cervical cancer, many cases of pancreatic ductal neoplasia begin as low-grade dysplasia and progress over time into high-grade dysplasia and invasive adenocarcinoma.8-11 Unfortunately, unlike cervical cancer, to our knowledge there are currently no effective screening methods that are both reliable and relatively inexpensive. For example, cytology alone identifies pancreatic ductal adenocarcinoma and high-grade dysplasia, but it often misses low-grade to intermediate-grade IPMNs. In our experience, a potential pitfall with the cytologic diagnosis of low-grade IPMNs is gastric contamination. EUS-guided FNAs of the body or tail of the pancreas are performed through the gastric wall, rather than the duodenum. This is significant because duodenal contamination is easily distinguished by the presence of goblet cells in cytologic preparations, but gastric mucosa is morphologically very similar to low-grade IPMNs. Some authors have suggested using the immunohistochemical evaluation of mucin gene expression profiles to address this issue.27-29 Gastric mucosa and duodenal mucosa are negative for HPC2 1-B3 (data not shown).

Molecular Markers to Screen for Pancreatic Duct Dysplasia

Serological markers for invasive pancreatic cancer currently are in the early stages of development, but markers12 and radiographic features of precancerous lesions are not reliably accurate.9 Aside from aspirate CEA levels, few immunohistochemical markers to date have been validated to screen EUS-guided FNA specimens. For example, in a recent study of 36 cases (7 cases of pancreatitis, 15 IPMNs, and 14 ductal adenocarcinomas), Toll et al reported that KOC was specific for pancreatic cancer and did not stain pancreatitis or IPMNs.21 Similarly, Zhao et al have shown that KOC is positive in 88% of pancreatic ductal cancers diagnosed by EUS-guided FNA (35 of 40 cases).23 Yantiss et al reported positive KOC staining in surgically resected ductal adenocarcinomas and in a subset of IPMNs with high-grade dysplasia (4 of 10 cases), but not in IPMNs with low-grade to intermediate-grade dysplasia (2 cases and 3 cases, respectively).22 Given the NPV provided by CEA testing,6, 16 the additional PPV for high-grade dysplasia and adenocarcinoma provided by KOC is impressive.21-23 However, cytopathologists are already reliable at identifying high-grade pancreatic ductal lesions15; therefore, the diagnostic problem is detecting low-grade to intermediate-grade IPMNs that may progress to invasive ductal adenocarcinoma. This is a weakness of the KOC marker.

Clinical Utility of HPC2 1-B3

The HPC2 1-B3 marker is only in the earliest stages of validation, but the reported sample size in the current pilot study is comparable to that published by others investigating similar antibody-based markers for pancreatic cancer.21-23 The added value of using HPC2 1-B3 is not only its ability to immunostain pancreatic ductal adenocarcinoma and high-grade dysplasia but also its value in detecting low-grade to intermediate-grade IPMNs. The marker appears to be specific, because it was negative in all 23 cases of chronic pancreatitis in our screening panel and was negative in all 8 cases of pancreatitis identified in the current series of EUS-guided FNA specimens. HPC2 1-B3 detected more cases of high-grade dysplasia and invasive adenocarcinoma than the KOC marker and HPC2 1-B3 identified approximately one-half of the low-grade to intermediate-grade IPMNs that underwent biopsy using EUS-guided FNA. In surgically resected cases, HPC2 1-B3 identified the majority of low-grade to intermediate-grade IPMNs and all of the high-grade cases. It did not diffusely stain 6 of 17 adenocarcinomas, but all 6 of these cases represented the poorly differentiated carcinomas that were inserted into this primary screening panel and they did demonstrate weak focal staining.

HPC2 1-B3 immunostaining of low-grade to intermediate-grade IPMNs raises challenging management issues. Some clinicians may choose to follow patients with low-grade IPMNs and only excise higher grade lesions that are more likely to progress to invasive adenocarcinoma5, 14 In our practice, we excise any large duct IPMN, regardless of its grade. In contrast, side-branch PanINs without high-grade atypia are excised only if they create mass lesions measuring > 3 cm. Therefore, it is possible that HPC2 1-B3 may detect side-branch PanINs, which are not necessary to excise. However, we reject this scenario as a weakness because imaging studies should be able to distinguish main duct and side-branch lesions. Therefore, we believe positive HPC2 1-B3 immunostaining would be a benefit solely based on the increased sensitivity and specificity of the cytologic diagnosis. Moreover, we hypothesize that positive HPC2 1-B3 immunostaining may potentially discriminate between low-grade IPMNs that may progress to carcinoma and those that do not progress. In future studies, our group intends to correlate HPC2 1-B3 staining with KRAS mutations14, 30, 31 and long-term risk of disease progression. In contrast, the KOC marker cannot be used for this purpose because it does not stain low-grade IPMNs. Another potential strength of the HPC2 1-B3 antibody may be its ability to detect secreted antigen in FNA biopsy specimens or potentially in pancreatic duct fluid by enzyme-linked immunoadsorbent assay (ELISA). Duct fluid screening would be preferred because it could be performed periodically as a screening test for pancreatic ductal dysplasia. Indeed, the HPC2 1-B3 antigen was observed in the supernatant fluid of the cell cultures in the current study and was also noted in the luminal debris of IPMNs and invasive adenocarcinomas that were immunostained for HPC2 1-B3. The combination of ELISA and cytology would be most effective, because the cytopathologist could then distinguish low-grade from high-grade nuclear features. The decision to perform surgical resection on an IPMN or PanIN depends on its location, size, and grade. Therefore, maximizing diagnostic information for the surgeon is best.

This may be especially important when distinguishing PanINs. Many surgeons resect large duct IPMNs, but may opt to follow patients with side-branch PanINs if they are of low grade. Others may follow large patients with duct IPMNs if they are of low grade, but perform surgical resection for high-grade lesions. High-grade PanINs that were identified in the tissue sections in the current study immunostained for HPC2 1-B3. There were few low-grade PanINs in the current study, but the rare cases of grade 1 PanIN were negative. This is important, because low-grade PanINs have a low risk of progression into invasive cancer.

For now, the diagnostic value of HPC2 1-B3 should be viewed with caution until larger prospective studies are completed. HPC2 1-B3 appears to be specific for pancreatic ductal dysplasia and it was recently shown that it can distinguish between metastatic pancreatic adenocarcinoma and primary cholangiocarcinoma in liver biopsies.32 Our long-term objective is to determine whether HPC2 1-B3 and CEA levels in pancreatic duct fluid will complement each other to provide improved predictive value as a relatively inexpensive screening “Pap [Papanicolaou] smear of the pancreas.” Unfortunately, we did not have CEA levels available in our pilot study.

We have not yet identified the antigen targeted by the HPC2 1-B3 monoclonal antibody. However, once it is isolated, it will be interesting to compare this antigen with other potential IPMN markers of a similar size (55 kD-65 kD) suggested by cDNA microarray analysis.24 A better understanding of the antigen and its role in pancreatic dysplasia may provide significant insights into the progressive pathophysiology of this malignant disease.

Acknowledgements

We thank Maria Grompe, Yong-Ping Zhong, and Pamela Canaday for their assistance with flow cytometric data acquisition and analyses; Stephanie Abraham and Kelsea Lanxon-Cookson for support in the generation of human pancreatic cancer fusion 2 (HPC2) 1-B3; Carolyn Gendron, Cara Poage, and Dornald Myles for outstanding histology support; and Miriam Douthit for management of the Oregon Pancreatic Tumor Registry. We would also like to acknowledge the Flow Cytometry and Monoclonal Antibody Shared Resources within the Knight Cancer Institute and Oregon Stem Cell Center.

FUNDING SUPPORT

Dr. Morgan's contribution was funded by the Office of Research on Women's Health and the National Institute of Child Health and Human Development, Oregon BIRCWH HD043488-08. Dr. Streeter was supported by the Oregon Stem Cell Center, the Oregon Health and Science University Knight Cancer Institute, and a generous gift from Randy and Mary Huebner. Supported in part by the Oregon Clinical and Translational Research Institute, grant number UL1RR024140 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH) and NIH Roadmap for Medical Research and the Oregon Health and Science University Knight Cancer Institute, and grant number P30 CA 069533 from the National Cancer Institute.

CONFLICT OF INTEREST DISCLOSURES

Several of the authors (T.K.M., M.G., C.C., and P.S.) are listed on a patent application for the antibody HPC2 1-B3.

Ancillary