Differential Diagnosis of Chronic Pancreatitis and Pancreatic ......ticular tetrazolium salt, NT, is...

[CANCER RESEARCH 59, 5732–5736, November 15, 1999]

Differential Diagnosis of Chronic Pancreatitis and Pancreatic Cancer in BrushCytology Specimens

Bernard E. M. Van Driel, Thomas M. Van Gulik, Patrick D. J. Sturm, G. Johan A. Offerhaus, Dirk J. Gouma, andCornelis J. F. Van Noorden1

Academic Medical Center, University of Amsterdam, Departments of Cell Biology and Histology [B. E. M. V. D., C. J. F. V. N.], Surgery [T. M. V. G., D. J. G.], and Pathology[P. D. J. S., G. J. A. O.], 1100 DE Amsterdam, the Netherlands

ABSTRACT

Discrimination between chronic pancreatitis and pancreatic carcinomacan be complicated, particularly in brush cytology specimens. Previousstudies have shown that the oxygen insensitivity of the histochemicalreaction to detect glucose-6-phosphate dehydrogenase activity based onneotetrazolium reduction can be used for discriminating malignant cellsfrom nonmalignant cells. In the present study, we investigated the value ofthe assay for differential diagnosis between the two pancreatic diseases.Oxygen insensitivity in ductal epithelial cells in normal human pancreas,chronic pancreatitis, and pancreatic carcinoma was determined by quan-titative image analysis in sections of biopsies and in brush cytology prep-arations. In sections, the reaction in the absence of oxygen was a properreflection of glucose-6-phosphate dehydrogenase activity, whereas in thepresence of oxygen only malignant cells showed a significant reaction. Of39 brush cytology specimens, diagnosis of all 11 cases of pancreatitis and28 cases of cancer with the oxygen insensitivity test were in agreementwith independent measures of chronic pancreatitis and cancer. The oxy-gen insensitivity test is a simple and valuable tool in addition to conven-tional pathology for differential diagnosis between pancreatitis and pan-creatic cancer, both in biopsies and in brush cytology specimens.

INTRODUCTION

The clinical presentation of chronic pancreatitis often resemblesthat of pancreatic cancer (1, 2). Differential diagnosis cannot alwaysbe obtained with certainty (3–5). Moreover, it can be difficult todiscriminate cells in biopsies or brush cytology specimens of chronicpancreatitis from those of pancreatic cancer on the basis of the numberof nucleoli, nuclear contour irregularity, nuclear hyperchromasia,coarse chromatin, and nucleus:cytoplasm ratio (6). Therefore, morereliable preoperative discriminators between chronic pancreatitis andpancreatic malignancy are needed.

Activity of G6PDH2 increases in early stages of carcinogenesis andoften precedes morphological changes (7–9). G6PDH is a housekeep-ing enzyme that regulates the pentose phosphate pathway, and itsmain role in metabolism is to provide AADPH for biosynthesis anddetoxification (10). G6PDH activity can be demonstrated histochem-ically in cryostat sections or cytology specimens by using a tetrazo-lium salt as final electron acceptor to produce intensely coloredformazan (10). Because G6PDH activity can be high in any prolifer-ating cell (11), its activityper secannot be taken as a parameter todiscriminate malignant and nonmalignant cells (9, 12). When a par-ticular tetrazolium salt, NT, is used in an atmosphere of 100% oxygen,formazan production is negligible in normal epithelial cells, whereasformazan is produced in carcinomas of the colon (9, 11–13), stomach(11), breast (14), and bronchus (15). The chemical backgrounds of the

oxygen insensitivity of carcinomas are not completely elucidated yet,but there is evidence that formation of oxygen radicals is involved.Oxygen radicals ultimately cause rapid inactivation of G6PDH duringthe histochemical reaction in nonmalignant cells but not in malignantcells (16).

Oxygen insensitivity of the G6PDH assay develops during thetransition from premalignancy to malignancy in both human andmurine colon epithelium (9). The development of oxygen insensitivityfits well in the concept of the dysplasia-carcinoma sequence in colo-rectal carcinogenesis (17). In fact, prognosis based on a combinationof oxygen insensitivity of the G6PDH assay and conventional clinicalpathological parameters was far better than prognosis on the basis ofclinical pathological parameters alone for colorectal cancer patients(16, 18). Therefore, the objectives of the present study were thefollowing: (a) to investigate whether oxygen insensitivity of theG6PDH assay in sections of biopsies in combination with morphologyresults in a better differential diagnosis between chronic pancreatitisand pancreatic malignancy than diagnosis based on morphologyalone; and (b) whether malignant epithelial cells can be discriminatedfrom normal and hyperplastic epithelial cells in brush cytology spec-imens.

MATERIALS AND METHODS

Tissue Specimens.Biopsies of chronic pancreatitis, pancreatic carcinoma,and normal pancreas were obtained from the Department of Surgery in theAcademic Medical Center (Amsterdam, the Netherlands). Seven specimens ofnormal pancreas (Fig. 1A) were obtained from patients who underwent partialpancreatectomy during resection of other types of lesions. Surgical specimensfrom 23 patients with chronic pancreatitis and from 17 patients with pancreaticcancer who underwent pancreatic resections between 1995 and 1997 werereviewed microscopically by a pathologist (G. J. A. O.) in a blinded fashion forthe presence of (atypical) hyperplasia or malignancy (Fig. 1B). Ductal epithe-lial hyperplasia was defined as an abnormal increase in the number of cellslining pancreatic ducts, which are more than twice as tall as normal cells (19).Hyperplasia varied from simple to atypical. Simple ductal hyperplasia wascharacterized by basally located uniform nuclei, cellular crowding, with focalpseudostratification of the nuclei. Papillary mucinous hyperplasia was recog-nized by papillary structures with tufting of epithelial fronts into the ductallumen and a core of fine fibrovascular tissue derived from the lamina propria.Atypical hyperplasia was established when all columnar epithelium formed apapillary structure without a fibrous core and was considered as a precancerouscondition or carcinomain situ (19). Simple ductal hyperplasia was observed in12 specimens of chronic pancreatitis and 4 specimens of pancreatic carcinoma.Papillary hyperplastic ducts were observed in three specimens of chronicpancreatitis and three specimens of pancreatic carcinoma. In these threespecimens of chronic pancreatitis, atypical hyperplasia was observed as well.Features that characterize chronic pancreatitis (Fig. 1C), i.e., atrophy of exo-crine acinar tissue and abundant fibrosis and connective tissue, were observedin the vicinity of almost every carcinoma (Fig. 1B).

Brush cytology preparations were obtained postoperatively from 39 patientswith proven or suspected carcinoma of the pancreatic head region who under-went a Whipple resection (subtotal pancreatico-duodenectomy) between 1996and 1998. The distal common bile duct and the main pancreatic duct werebrushed with an endocervical brush (Cervibrush; Cellpath, Hemel Hempstead,United Kingdom) immediately after arrival of the resection specimens at the

Received 4/26/99; accepted 9/23/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Academic Medical Center,Departments of Cell Biology and Histology, Meibergdreef 15, 1105 AZ Amsterdam, theNetherlands. Phone: 31-20-566-4970; Fax: 31-20-697-4156; E-mail: [email protected].

2 The abbreviations used are: G6PDH, glucose-6-phosphate dehydrogenase; NT,neotetrazolium.

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pathology laboratory. The cytology specimens were collected in a blindedfashion, and results of the oxygen insensitivity test were compared afterwardwith the diagnosis of the pathologist of the cytology specimens on the one handand histology on the other. Because carcinomas of the ampulla of Vater havea better prognosis than the other types of cancer in the pancreatic head region(pancreas or distal bile duct; Ref. 20), the residual activity of brush cytologyspecimens was analyzed both as one group and when subdivided in groupsdependent on tumor origin.

Tissue and Cell Processing.Fresh tissue biopsies were immediately fro-zen in liquid nitrogen. The frozen material was stored at280°C until furtheruse. Serial sections (8mm thick) were cut at225°C on a motor-driven cryostat(Bright, Huntingdon, United Kingdom) fitted with a retraction microtome at alow but constant speed to minimize variation in section thickness (21). Sec-tions were stored at280°C until use. Brush cytology preparations of epithelialcells on glass slides were made by smearing pancreatic duct brushings of freshpancreatic tissue and were stored at280°C until use.

Oxygen Insensitivity Test Based on the Histochemical Assay of G6PDHActivity. Incubation media to demonstrate G6PDH activity were prepared asdescribed previously in detail (16, 21). Incubation media were poured intoglass vials and equilibrated for 10 min in an atmosphere of either 100% oxygenor 100% nitrogen using a tonometer to avoid formation of gas bubbles in theviscous media. Meanwhile, sections and cytological smears were air dried (5min at 37°C). Plastic rings were placed around sections or cells and filled with

media. Coverslips were placed upon the rings to avoid loss of gas from themedia during incubation. Serial sections and cell smears were incubated intriplicate in the presence of oxygen or nitrogen. Serial sections were obtainedfrom opposite sides of all biopsies. In this way, we were able to establish thatvariation in oxygen insensitivity within biopsies was negligible. In case ofbrush cytology specimens, part of each cell smear was incubated in thepresence of oxygen, and the remainder was incubated in the absence ofoxygen. After 10 min of incubation, sections or smears were rinsed thoroughlyand mounted in glycerol jelly. Reproducibility of the histochemical reactionwas tested by inclusion of at least two samples in a run that were shown to beoxygen insensitive in a previous run. Control reactions were performed byomitting substrate and coenzyme from the incubation media (21). No ornegligible amounts of formazan were produced in these controls, which im-plied that the quantitative histochemical method for the detection of G6PDHactivity was specific.

Image Analysis and Processing.End point absorbance measurements offormazan production by G6PDH activity in epithelial cells were performedwith image analysis as described previously in detail (22, 23) using a Vanox-Tphotomicroscope (Olympus, Tokyo, Japan) with a32 objective (N.A. 0.08),monochromatic light of 585 nm, a Cohu 4913 CCD camera (Cohu, San Diego,CA), an 8-bit monochrome frame grabber (LG-3; Scion, Frederick, MD), anda Power Macintosh 8100/110 computer (Apple, Cupertino, CA) using thepublic domain NIH Imaging software program (written by Wayne Rasband;

Fig. 1. Photomicrographs of serial sections of normal pan-creas (A, D,andG), pancreatic carcinoma (B, E,andH), andchronic pancreatitis (C, F, andI) stained with H&E (A, B,andC) or stained for activity of G6PDH in the absence (D, E, andF) or presence (G, H,and I) of oxygen. Staining inD, E, andF represents total activity of the enzyme, whereas lack ofstaining inG and I reflects oxygen sensitivity of ductal epi-thelial cells in normal pancreas and chronic pancreatitis, re-spectively. Staining inH reflects oxygen insensitivity of cancercells.360.

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DIFFERENTIAL DIAGNOSIS OF PANCREATIC LESIONS



NIH).3 Gray values were converted into absorbance values by using a set ofneutral density filters (Kodak, Rochester, NY; Ref. 23). Absorbance values ofcontrol reactions were subtracted from test values to obtain specific activity(21). The measurement procedure was as follows. G6PDH activity in thepresence of oxygen was determined in each of three serial sections or cellsmears. In each section, five ducts and ductules with the highest content offormazan were selected. Subsequently, corresponding areas after incubation inthe absence of oxygen were measured. In each cell smear, three to five singlecells with highest activity were analyzed for both conditions. Pilot experimentshave shown that determination of residual activity of a smear was most reliableon the basis of formazan content of the darkest cells in that smear. It appearedthat three to five cells/smear was a sufficient sampling size, because residualactivity of the three smears of each pancreas was similar. Residual G6PDHactivity was calculated as a percentage of formazan produced in the presenceof oxygen as compared with that produced in the absence of oxygen after 10min of incubation in the same areas in serial sections. We considered cells tobe oxygen sensitive when the residual activity was,20% and oxygen insen-sitive when the residual activity was.20% (9, 13).

Statistics. Kruskal-Wallis nonparametric one-way ANOVA tests were ap-plied to determine whether mean G6PDH activity in the absence of oxygen andmean residual activity in biopsies of normal pancreas, chronic pancreatitis, andpancreatic carcinoma differed significantly from each other. In case of signif-icant differences, Dunn’s multiple comparisons tests were applied to determinewhich groups differed from each other.

In addition, mean residual activity in brush cytology specimens of chronicpancreatitis and pancreatic carcinoma were compared with the use of a two-tailed Mann-Whitney nonparametric test. The medians differed significantlyfrom each other whenP was,0.05.

RESULTS

Biopsies.The histochemical assay in the absence of oxygenshowed that ductal epithelial cells contained highest G6PDH activityof all cell types present in the biopsies (Fig. 1,D–F). G6PDH activityin ductular epithelial cells was not significantly different in normalpancreas (mean units6 SD, 3.26 1.1), chronic pancreatitis (meanunits6 SD, 4.86 3.0), and pancreatic carcinomas (mean units6 SD,4.5 6 1.6). In the presence of 100% oxygen, residual activity wasalways,20% in epithelial cells of ducts in normal pancreas (meanpercentage6 SD, 4.46 3.2; Figs. 1G and 2). Residual activity inepithelial cells of ducts in chronic pancreatitis was also,20% in 20of 23 biopsies (mean percentage6 SD, 8.16 5.2; Figs. 1I and 2).Residual activity in pancreatic carcinoma (mean percentage6 SD,57.2 6 23.6; Figs. 1Hand 2) was significantly higher than that innormal pancreas and chronic pancreatitis (P, 0.001). In fact, allcarcinomas showed residual activity$35% (Fig. 2).

The 20 biopsies of chronic pancreatitis with residual activity,20%contained only histologically normal and simple hyperplastic ductsbut no papillary hyperplastic ducts. Each biopsy of the three remain-ing chronic pancreatitis patients (patients A, B, and C) showed resid-ual activity.20% in a single atypical hyperplastic duct (Fig. 2). In thebiopsy of patient A, an atypical hyperplastic duct was the onlyoxygen-insensitive duct, with a residual activity of 38%. In the biopsyof patient B, one oxygen-insensitive atypical hyperplastic duct andone oxygen-insensitive papillary hyperplastic duct were found withresidual activities of 35 and 72%, respectively (Fig. 3). The biopsy ofpatient C contained one atypical hyperplastic duct with residual ac-tivity of 30% and a single mucinous cystadenoma with a residualactivity of 46%. The remainder of the biopsies of patients A, B, andC was oxygen-sensitive, similar to other biopsies of chronic pancre-atitis.

Patients A and B appeared to have pancreatic malignancy after-ward. Malignancy was not found by the pathologist in the biopsies

that were used for the oxygen insensitivity test. Patient A died shortlyafter diagnostic confirmation of pancreatic cancer. All other 22 pa-tients with chronic pancreatitis were still alive up to 2 years offollow-up. Patient B is considered to be an exception, because variousneoplasms, such as colorectal carcinoma and adenomatous polyps,were demonstrated at different time points during the last 20 years. Insummary, oxygen insensitivity was only observed in malignant ductalepithelium or in hyperplastic epithelium in patients with provenneoplasms.

Brush Cytology Preparations. The oxygen insensitivity test wasapplied in a blinded fashion to brush cytology preparations of 39patients with proven or suspected carcinoma in the pancreatic headregion (Fig. 4). All patients that were diagnosed on the basis of brushcytology specimens and histology to have a malignancy after pancre-atico-duodenectomy (n 5 27) showed oxygen-insensitive neoplasticepithelial cells [mean residual activity (%)6 SD, 73.16 16.6]. Nodifferences in residual activity were observed between carcinomas inthe pancreas (mean percentage6 SD, 70.66 17.3), distal bile ducts(mean percentage6 SD, 74.06 16.8), and ampulla of Vater (meanpercentage6 SD, 76.66 17.7).

Eleven patients with suspected carcinomas showed oxygen-sensi-tive epithelial cells (mean percentage6 SD, 14.3 6 4.3). Thesepatients were diagnosed to have chronic pancreatitis both after exam-ination of the brush cytology specimens and histology by the pathol-ogist. The residual activity of these patients was significantly lower(P , 0.0001) than that of patients who were diagnosed to havemalignancy. One patient, who was diagnosed by the pathologist on thebasis of examination of brush cytology specimens to have chronicpancreatitis, showed a residual activity of 72.0%. This patient wasdiagnosed later on the basis of histology to have a carcinoma in thepancreatic head.

DISCUSSION

The histochemical assay with the use of NT gives a proper reflec-tion of actual G6PDH activity provided that the assay is performed inthe absence of oxygen (10, 15). Under these conditions, epithelialcells of normal, hyperplastic, and cancerous ducts showed similarG6PDH activity. In the presence of oxygen, NT-formazan productionwas inhibited in epithelial cells of histologically normal and simplehyperplastic ducts, because the residual activity was always,20%.Pancreatic carcinomas were always oxygen insensitive, because theresidual activity was.35%. Similar results were found for colorectal3 Available via Internet at http://rsb.info.nih.gov.

Fig. 2. Residual G6PDH activity in ductal epithelial cells of normal pancreas that areoxygen sensitive (n5 7; v), chronic pancreatitis that is totally oxygen-sensitive (n 5 20;‚), single ducts in biopsies of 3 chronic pancreatitis patients that are oxygen-(in)sensitive(n 5 5; f; see also Fig. 3), and pancreatic carcinomas (n 5 17; ).

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carcinomas (9). The oxygen insensitivity test thus proved to be a validdiscriminator between chronic pancreatitis and pancreatic cancer.

Oxygen insensitivity was observed in three biopsies of chronicpancreatitis in single atypical ducts only. Two of these patients hadpancreatic malignancy, and one patient had a mucinous cystadenoma.Mucinous cystadenomas have a high malignant potential, and transi-tion into cystadenocarcinomas is often observed (24, 25). The findingof oxygen-insensitive hyperplastic ducts in these three patients may beexplained in two ways: (a) the oxygen insensitivity of epithelial cellsin these atypical hyperplastic ducts is attributable to the presence ofmalignancy in the vicinity. Elevated oxygen insensitivity in hyperpla-sia adjacent to carcinomas was found in the colon as well (9); and (b)because atypical hyperplasia itself is considered to be precancerous(19, 26, 27), oxygen insensitivity in these ducts may also be caused bytheir own progression toward malignancy. Invasive carcinoma inatypical hyperplastic ducts has occasionally been described (28). Thisatypical hyperplasia of the intraductal mucinous papillary type is arelatively novel and more and more often reported entity (29).

Because oxygen insensitivity was observed in all papillary andatypical hyperplastic ducts and all carcinomas, we conclude that

oxygen insensitivity starts to occur in papillary hyperplasia. Simplehyperplastic ducts, which were frequently observed in chronic pan-creatitis and nearby carcinomas, were always oxygen sensitive. Thisdevelopment of oxygen insensitivity is similar to that in human andmurine colorectal carcinogenesis, where oxygen insensitivity devel-ops during the transition from premalignancy to malignancy (9).There is little or no evidence of progression of simple hyperplasticducts in pancreas (19) and colorectal hyperplastic polyps (28) into acarcinoma, and both are always oxygen sensitive.

The sensitivity of the test to detect cancer cells in brush cytologypreparations (Table 1) was better than that of conventional cytologicalexamination reported in the literature, which ranges from 30 to 92%(30–33). This wide range in sensitivity may be the result of variationin interpretation or numbers of cells collected (34). Furthermore,confounding may occur of atypical cells from chronic pancreatitis andbenign-appearing cells from well-differentiated pancreatic carcinomas

Fig. 4. Photomicrographs of single pancreatic carcinoma cells in a smear of which partwas incubated to demonstrate G6PDH activity in the absence of oxygen (A) and part wasincubated in the presence of oxygen (B), showing the oxygen insensitivity phenomenon onthe single-cell level in brush cytology preparations.3275.

Fig. 3. Photomicrographs of serial sections of a pap-illary hyperplastic duct (A, B,and C) and an atypicalpapillary hyperplastic duct (D, E, andF) in the pancreasof a chronic pancreatitis patient who developed pancre-atic malignancy (patient B). The sections are stainedwith H&E (A andD), and for G6PDH in the absence (BandE), or presence (C andF) of oxygen. The residualG6PDH activity in oxygen inC andF are 72 and 35%,respectively.A–C,390; D–F, 350.

Table 1 Sensitivity and specificity of diagnosis by routine cytology and the oxygeninsensitivity of brush cytology specimens of 39 patients with proven or suspected

carcinoma in the pancreatic head region

The golden standard is the diagnosis by the pathologist on the basis of histologyafterward.

Routine cytology

Histology

Chronic pancreatitis Carcinoma

Chronic pancreatitis 11 1Carcinoma 0 27

11 28 39

Sensitivity: 96%Specificity: 100%

Oxygen insensitivity testa

Histology

Chronic pancreatitis Carcinoma

,20% 11 0.20% 0 28

11 28 39Sensitivity: 100%Specificity: 100%a Oxygen insensitivity is expressed as residual activity. Residual activity,20% is

taken as nonmalignant and.20% as malignant.

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(35). Finally, pancreatic carcinomas may develop from chronic pan-creatitis (36–38), which makes differential diagnosis even more com-plicated. However, because the brush cytology preparations in ourstudy were obtained postoperatively, the sampling may have beendirected toward suspicious sites. It remains to be determined whetherthe assay has the same sensitivity in the setting of more randomlyassigned samplings (e.g.,endoscopic retrograde pancreatic juice sam-pling) prior to a decision of surgical intervention.

All brush cytology specimens of carcinomas were oxygen insensi-tive, and all chronic pancreatitis specimens showed oxygen sensitivityof the cells without exception. The sensitivity of the oxygen insensi-tivity test was demonstrated by the one patient who was diagnosed bythe pathologist on the basis of brush cytology specimens to havechronic pancreatitis but showed high activity of G6PDH in the pres-ence of oxygen (Table 1). This patient appeared to have a carcinoma,as was determined by histology afterward. Therefore, the oxygeninsensitivity test appears to be a valuable extra tool for pathologists toassess the actual pancreatic disease in brush cytology preparationsbecause single cancer cells in smears that are positive in the oxygeninsensitivity test can be detected easily without relying on morphol-ogy.

Different approaches have been described to diagnose pancreaticcancer or to predict development of pancreatic cancer from prema-lignant stages. A major role for the K-ras oncogene in ductal pancre-atic carcinogenesis has been suggested, but these mutations werefound in up to 75% of pancreatic carcinomas (39). In contrast, theoxygen insensitivity test recognized all pancreatic carcinomas withoutan exception. When other criteria are inadequate to distinguishchronic pancreatitis and pancreatic carcinomas, the oxygen insensi-tivity test should allow successful differential diagnosis.

In conclusion, oxygen insensitivity of the histochemical or cyto-chemical assay of G6PDH activity proved to be a valuable tool forsimple discrimination between pancreatic cancer and chronic pancre-atitis.

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1999;59:5732-5736. Cancer Res Bernard E. M. Van Driel, Thomas M. Van Gulik, Patrick D. J. Sturm, et al. Cancer in Brush Cytology SpecimensDifferential Diagnosis of Chronic Pancreatitis and Pancreatic

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