Advertisement

Detection of Circulating Pancreas Epithelial Cells in Patients With Pancreatic Cystic Lesions

  • Andrew D. Rhim
    Correspondence
    Reprint requests Address requests for reprints to: Andrew D. Rhim, MD, Division of Gastroenterology, Department of Internal Medicine, University of Michigan Medical School, 109 Zina Pitcher Place, BSRB 2061, Ann Arbor, Michigan 48108. fax: (734) 763-2162.
    Affiliations
    Division of Gastroenterology, Department of Internal Medicine, Pancreatic Cancer Center, University of Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan

    Gastroenterology Division, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

    Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
    Search for articles by this author
  • Fredrik I. Thege
    Affiliations
    Department of Biomedical Engineering, Cornell University, Ithaca, New York
    Search for articles by this author
  • Steven M. Santana
    Affiliations
    Sibley School of Mechanical and Aerospace Engineering, Cornell University, Ithaca, New York
    Search for articles by this author
  • Timothy B. Lannin
    Affiliations
    Sibley School of Mechanical and Aerospace Engineering, Cornell University, Ithaca, New York
    Search for articles by this author
  • Trisha N. Saha
    Affiliations
    Division of Gastroenterology, Department of Internal Medicine, Pancreatic Cancer Center, University of Michigan Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan

    Gastroenterology Division, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
    Search for articles by this author
  • Shannon Tsai
    Affiliations
    Gastroenterology Division, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

    Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
    Search for articles by this author
  • Lara R. Maggs
    Affiliations
    Gastroenterology Division, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

    Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
    Search for articles by this author
  • Michael L. Kochman
    Affiliations
    Gastroenterology Division, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

    Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

    Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
    Search for articles by this author
  • Gregory G. Ginsberg
    Affiliations
    Gastroenterology Division, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

    Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

    Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
    Search for articles by this author
  • John G. Lieb
    Affiliations
    Gastroenterology Division, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

    Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
    Search for articles by this author
  • Vinay Chandrasekhara
    Affiliations
    Gastroenterology Division, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

    Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
    Search for articles by this author
  • Jeffrey A. Drebin
    Affiliations
    Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

    Department of Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
    Search for articles by this author
  • Nuzhat Ahmad
    Affiliations
    Gastroenterology Division, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
    Search for articles by this author
  • Yu–Xiao Yang
    Affiliations
    Gastroenterology Division, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
    Search for articles by this author
  • Brian J. Kirby
    Correspondence
    Brian J. Kirby, PhD, Sibley School of Mechanical and Aerospace Engineering, Cornell University, 238 Upson Hall, Ithaca, New York 14853. fax: (607) 255-1222.
    Affiliations
    Sibley School of Mechanical and Aerospace Engineering, Cornell University, Ithaca, New York

    Department of Medicine, Division of Hematology/Medical Oncology, Weill Cornell Medical College, New York, New York
    Search for articles by this author
  • Ben Z. Stanger
    Correspondence
    Ben Z. Stanger, MD, PhD, Division of Gastroenterology, Department of Medicine, University of Pennsylvania 421 Curie Boulevard, 512 BRB II/III, Philadelphia, Pennsylvania 19104. fax: (215) 573-2486.
    Affiliations
    Gastroenterology Division, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

    Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

    Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
    Search for articles by this author
Published:December 16, 2013DOI:https://doi.org/10.1053/j.gastro.2013.12.007
      Hematogenous dissemination is thought to be a late event in cancer progression. We recently showed in a genetic model of pancreatic ductal adenocarcinoma that pancreas cells can be detected in the bloodstream before tumor formation. To confirm these findings in humans, we used microfluidic geometrically enhanced differential immunocapture to detect circulating pancreas epithelial cells in patient blood samples. We captured more than 3 circulating pancreas epithelial cells/mL in 7 of 21 (33%) patients with cystic lesions and no clinical diagnosis of cancer (Sendai criteria negative), 8 of 11 (73%) with pancreatic ductal adenocarcinoma, and in 0 of 19 patients without cysts or cancer (controls). These findings indicate that cancer cells are present in the circulation of patients before tumors are detected, which might be used in risk assessment.

      Keywords

      Abbreviations used in this paper:

      CEC (circulating epithelial cell), DAPI (4′,6-diamidino-2-phenylindole), GEDI (geometrically enhanced differential immunocapture), IPMN (intraductal papillary mucinous neoplasm), PDAC (pancreatic ductal adenocarcinoma), Pdx-1 (pancreatic and duodenal homeobox protein-1.), CK (cytokeratin 19)
      See Covering the Cover synopsis on page 595.
      A widely accepted paradigm in cancer biology is that epithelial cancers progress in a linear manner whereby cancer-defining properties are acquired sequentially.
      • Weinberg R.A.
      In this model, cancer cells acquire metastatic potential after large primary tumors are established. However, in pancreatic ductal adenocarcinoma (PDAC), the linear progression model cannot be reconciled with clinical observations. A number of patients undergoing pancreatectomy for chronic pancreatitis will develop disseminated PDAC, although only precancerous pancreatic intraepithelial neoplasias, but no tumors, are found on histologic analysis.
      • Sakorafas G.H.
      • et al.
      In addition, in patients with small primary tumors (<2 cm) who have no clinical evidence of metastatic disease, 5-year survival after pancreatectomy is less than 18% owing to recurrent metastatic disease.
      • Agarwal B.
      • et al.
      These data suggest that metastatic seeding may occur before the formation of large primary tumors. Moreover, we recently showed that hematogenous dissemination occurs before tumor formation, in a lineage-labeled genetic model of PDAC,
      • Rhim A.D.
      • et al.
      at which time the pancreas contained only pancreatic intraepithelial neoplasias. Based on the clinical characteristics of PDAC and our findings within a recapitulative mouse model, we hypothesized that bloodstream seeding of pancreas-derived epithelial cells can occur in patients with clinical evidence of only precancerous lesions of the pancreas and no detectable invasive carcinoma.
      To test our hypothesis, we performed a blinded prospective pilot study of 3 cohorts, as follows: (1) patients with no history of cancer presenting for average-risk, age-appropriate colonoscopy screening and no adenomas detected; (2) patients with precancerous cystic lesions (intraductal papillary mucinous neoplasm [IPMN] or mucinous cystic neoplasms) of the pancreas with no evidence of tumor or metastasis on computerized tomography or magnetic resonance imaging, who did not qualify for surgery using the Sendai criteria
      • Tanaka M.
      • et al.
      (including no evidence of dysplasia or cancer on fine-needle aspiration, if performed); and (3) patients with cytology-confirmed PDAC. Peripheral blood was obtained from patients who consented before the procedure. We analyzed blood samples using geometrically enhanced differential immunocapture (GEDI), a microfluidic platform that has been shown to detect circulating tumor cells from patients with prostate cancers with high sensitivity.
      • Gleghorn J.P.
      • et al.
      • Kirby B.J.
      • et al.
      Here, we functionalized the GEDI device using antibodies to epithelial cell adhesion molecule to capture circulating epithelial cells (CECs). Captured cells then were stained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize nuclei and fluorescently conjugated antibodies to CD45, a universal marker of leukocytes, and cytokeratin 19 (CK), a marker of epithelial-derived cells or pancreatic and duodenal homeobox protein-1 (Pdx-1), a pancreas-specific transcription factor. A blinded observer (B.J.K.) enumerated CECs using the following 2 definitions: (1) CD45-, DAPI+, and (2) CK+,CD45-,DAPI+ using a fluorescence microscope. Definition 1 was confirmed retrospectively with automated cell enumeration and 4-color immunofluorescence for epithelial and pancreas-specific markers (Supplementary Figures 1 and 2, Supplementary Materials and Methods).
      We prospectively enrolled 48 patients (Table 1). Cyst- and cancer-free patients tended to be younger compared with the cystic lesion and PDAC cohorts (P = .003). However, there were no differences in other demographics. Most (85%) cystic lesions were classified as side-branch IPMNs. The size of cystic lesions varied from 5 to 28 mm. Patients with PDAC had a wide range of primary tumor diameters (15–91 mm) and tumor stages (I–IV).
      Table 1Patient Characteristics
      Age, yRaceSexFHxBMISmokingEtOH, avg/wkCA19-9 serumCEA serumCECSize of cyst/tumor, mmCyst type/cancer stage
      Cancer-free controls (n = 19)53CaucM35.7Never00
      40CaucM26.3Never10
      64CaucF28.3Never00
      61CaucM31.0Never10
      48CaucF21.0Never00
      62AAMF35.4Never00
      74CaucF19.8Never00
      56AAMF24.7Never0.50
      70CaucM26.4Previous00 (CK)
      51AAMF56.6Never00 (CK)
      60CaucM29.5Never00 (CK)
      66CaucM21.9Never1.50 (CK)
      84AAMF29.3Never00 (CK)
      53AAMF46.1Never00 (CK)
      59AAMF26.6Never01 (CK)
      50AAMF36.6Never1.52 (CK)
      58AAMF30.2Never03 (CK)
      73AAMF26.6Never00 (CK)
      50CaucF24.0Never00 (CK)
      Mean59.630.30.3 ± 0.8
      Cystic lesion (n = 21)67CaucF31.6Never009Side-branch IPMN, multiple
      62CaucFY20.2Never3008Side-branch IPMN
      64CaucM34.9Never586.7016MCN
      75CaucM24.3Never0015Side-branch IPMN
      65CaucFY22.4Never069.5Side-branch IPMN
      60CaucM28.3Never01.9221614Side-branch IPMN
      72CaucM21.3Current3<12210MCN
      81CaucM27.7Never6015Side-branch IPMN
      58CaucM20.7Current2005Side-branch IPMN
      64CaucFY18.0Never5463.8020Side-branch IPMN
      73CaucF24.6Never0414Side-branch IPMN
      69CaucF27.8Previous40 (CK)11Side-branch IPMN
      68CaucM26.3Previous70 (CK)3Side-branch IPMN
      74CaucF26.6None019 (CK)6.5Side-branch IPMN
      81CaucM27.5Previous71 (CK)25Side-branch IPMN
      58AAMF32.9None014 (CK)5Side-branch IPMN
      74CaucF21.7None00 (CK)25Side-branch IPMN
      65CaucF31.4None00 (CK)28MCN
      79ASAMM21.5None012 (CK)23Side-branch IPMN
      77CaucF28.5None0191.30 (CK)28Side-branch IPMN
      80CaucM29.9None00 (CK)25Side-branch IPMN
      Mean69.826.14.5 ± 7.3
      PDAC (n = 11)92CaucM
      65CaucM27.0Never08058.2052Stage I
      76CaucM33.5Never01261Stage IV
      69CaucM29.2Current3764465991Stage II
      65CaucM26.1Current1127393333Stage IV
      59AAMM37.3Previous01256323Stage IIB
      70CaucF24.8Previous0912615Stage III
      73AAMM19.9Previous0857917Stage IV
      62CaucM22.0Never0410040Stage IV
      69CaucM29.4Current18622416Stage IV
      83AAMF25.5Previous14 (CK)40Stage IV
      27.3Never05 (CK)27Stage IV
      Mean71.227.414.1 ± 18.1
      NOTE. CK, denotes quantification of CECs using definition 2, CK+/DAPI+/CD45-. Otherwise, CECs were quantified using definition 1 (DAPI+/CD45-).
      AAM, African American; ASAM, Asian American; Cauc, Caucasian; CEA, carcinoembryonic antigen; FHx, family history; MCN, mucinous cyst neoplasm.
      Sixteen of 19 cancer-free controls had no CECs by either definition (Figure 1B). When CECs were detected, there were no more than 3/mL. Seven of 9 (78%) patients with PDAC had detectable CECs, with an average of 16.2 ± 19.5 CEC/mL blood (P < .0001 compared with cancer-free patients by the Mann–Whitney test). Eight of 21 (40%) patients with cystic lesions of the pancreas had detectable CECs, averaging 4.5 ± 7.3 CECs/mL blood (P = .022 compared with cancer-free patients), and there was a significant difference in CECs across the 3 groups by 1-way analysis of variance (P = .015). Interestingly, there was no significant difference in the number of CECs detected among cyst lesion patients based on the immunofluorescence definition used (Figure 1B; black denotes definition 1, red denotes CEC analysis from different patients using definition 2); that is, a similar percentage of cyst lesion patients contained CECs by either definition, and, when CECs were detected in these patients, a similar concentration was found. We found no correlation with CEC count and tumor or cyst size, cancer stage or serum carbohydrate antigen 19-9 (CA-19-9) and carcinoembryonic antigen.
      Figure thumbnail gr1
      Figure 1Detection of CECs in patients using GEDI. (A) Depiction of the GEDI device. (B) Vertical scatterplots of CEC concentrations (per milliliter blood) for cancer-free patients (control), patients with cystic lesions of the pancreas without dysplasia or tumor (cystic lesion), and patients with PDAC. Bars indicate statistically significant differences using the Mann–Whitney test. Representative images of individual GEDI-captured nucleated cells from (C) PI34 cells in culture, (D) control human blood spiked with PI34 cells and (E) blood from a patient with PDAC. Cells were stained for CD45 (green), Pdx-1 (red), and DNA (DAPI, blue). Scale bar: 20 μm.
      To confirm the pancreas origin of CECs, we stained cells for Pdx-1, a pancreas-specific transcription factor, expressed in up to 60% of all CECs in mouse models of PDAC
      • Rhim A.D.
      • et al.
      (Figure 1B). Adherent and GEDI-captured primary PDAC cells also expressed nuclear Pdx-1 (21% of PI34 and 10.7% of Panc-01; Figure 1C). However, no nuclear Pdx-1 was detected within human breast (MCF-7) or prostate (LNCaP, CWR22Rv1) cancer cells or CD45+ leukocytes (data not shown). These data suggest that Pdx-1 is a specific marker of pancreas-derived cells. In our analyses, 29% of all CECs showed nuclear Pdx-1 staining (Figure 1D). These data confirm that at least a portion of all GEDI-captured epithelial cells derive from the pancreas.
      In conclusion, we report that pancreas epithelial cells can enter the bloodstream in patients with cystic lesions of the pancreas before the clinical diagnosis of cancer. By using state-of-the-art microfluidic technology
      • Kirby B.J.
      • et al.
      and immunofluorescence staining, we confirmed the pancreas origin of captured CECs. Thus, these findings suggest that the ability to seed the bloodstream may precede the formation of detectable tumors, supporting our findings in genetic mouse models of PDAC.
      • Rhim A.D.
      • et al.
      These data are supported by the recent finding that 24.6% of resected side-branch IPMNs that do not satisfy Sendai criteria contain regions of high-grade dysplasia or invasive carcinoma.
      • Fritz S.
      • et al.
      Data from our mouse model predict that these cells represent early, occult cancer cells,
      • Rhim A.D.
      • et al.
      although we do not yet have evidence to support this in human beings. Studies are underway to interrogate the genomic signature of CECs from cystic lesion patients—if these cells represent the earliest forms of cancer, we predict that they would contain a complement of somatic mutations associated with PDAC. Genomic analyses of CECs represent a technical challenge that recently was addressed elegantly using massively parallel sequencing of RNA from captured tumor cells from patients with PDAC
      • Yu M.
      • et al.
      ; however, cyst lesion patients contain many fewer CECs, complicating genomic analysis. Furthermore, it is still unknown if patients with CECs are destined to form tumors. If associated with subsequent tumor formation, CEC detection could be used as a biomarker for cancer risk stratification in patients at risk for PDAC. Studies underway in this regard will prospectively follow GEDI-analyzed cystic lesion patients to determine if CEC number or genomic analysis are predictive of an eventual diagnosis of PDAC.

      Acknowledgments

      The authors thank Anil K. Rustgi for critical reading of the manuscript, and Emily Jacobson for aiding in patient recruitment.
      Andrew Rhim and Brian Kirby were responsible for the study concept, analysis, and interpretation of data; Andrew Rhim, Michael Kochman, and Brian Kirby were responsible for the study design; Andrew Rhim, Michael Kochman, Gregory Ginsberg, John Lieb, Vinay Chandrasekhara, Jeffrey Drebin, Nuzhat Ahmad, Trisha Saha, Shannon Tsai, and Lara Maggs were responsible for patient enrollment; Andrew Rhim, Fredrik Thege, Steven Santana, Timothy Lannin, Michael Kochman, Brian Kirby, Trisha Saha, and Shannon Tsai were responsible for the acquisition of data; Andrew Rhim and Yu–Xiao Yang were responsible for the statistical analysis; Andrew Rhim was responsible for the drafting of the manuscript; Andrew Rhim, Fredrik Thege, Michael Kochman, Gregory Ginsberg, Vinay Chandrasekhara, Jeffrey Drebin, Yu–Xiao Yang, Brian Kirby, and Ben Stanger were responsible for critical revision of the manuscript.

      Supplementary Material

      References

        • Weinberg R.A.
        Carcinogenesis. 2008; 29: 1092-1095
        • Sakorafas G.H.
        • et al.
        Dig Liver Dis. 2003; 35: 482-485
        • Agarwal B.
        • et al.
        Pancreas. 2008; 36: e15-e20
        • Rhim A.D.
        • et al.
        Cell. 2012; 148: 349-361
        • Tanaka M.
        • et al.
        Pancreatology. 2006; 6: 17-32
        • Gleghorn J.P.
        • et al.
        Lab Chip. 2010; 10: 27-29
        • Kirby B.J.
        • et al.
        PLoS One. 2012; 7: e35976
        • Fritz S.
        • et al.
        Ann Surg. 2012; 256: 313-320
        • Yu M.
        • et al.
        Nature. 2012; 487: 510-513