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Keywords:

  • intraductal papillary-mucinous tumor of the pancreas;
  • telomerase activity;
  • endoscopic retrograde pancreatic juice aspiration;
  • malignant

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

BACKGROUND

It has been reported that, in patients with intraductal papillary-mucinous tumor (IPMT) of the pancreas, it is difficult to distinguish adenoma from carcinoma preoperatively. Recently, it has also been reported that telomerase activity was detected in many patients with carcinoma. In this report, the authors used the method of telomerase repeat amplification protocol (TRAP) assay on pancreatic juice retrieved by endoscopic retrograde pancreatic juice aspiration (ERP aspiration).

METHODS

Pancreatic juice was collected from 28 patients (13 with intraductal carcinoma and 15 with adenoma) using ERP aspiration at either Hiroshima University Hospital or its affiliated hospitals. Two samples of pancreatic juice were collected from each patient. Each sample was examined by cytology for Papanicolaou staining and TRAP assay.

RESULTS

Four of 13 IPMT patients (31%) with intraductal carcinoma were diagnosed accurately by cytology. Seven of nine patients who were classified with benign tumors by cytologic assessment had tumors that expressed telomerase activity. Overall, 11 of 13 IPMT patients (85%) with intraductal carcinoma were diagnosed correctly by cytology associated with telomerase activity. All of the IPMT patients with adenoma were classified with benign tumors by cytologic assessment, and telomerase activity was not expressed.

CONCLUSIONS

In this study, the authors found that telomerase activity was expressed with a comparatively high probability in intraductal carcinoma. These results suggest that telomerase activity in pancreatic juices may be used as an adjunct to cytologic diagnosis and may aid further in distinguishing between benign IPMT and malignant IPMT of the pancreas preoperatively. Cancer 2001;91:35–41. © 2001 American Cancer Society.

Since intraductal papillary-mucinous tumor (IPMT) was first reported as a mucin-producing tumor by Ohashi et al. in 1982, many investigators have verified these findings.1–4 In 1997, the Armed Forces Institute of Pathology (AFIP) classified IPMT into four types of tumors: adenoma, tumor with borderline malignant potential, intraductal carcinoma, and papillary-mucinous carcinoma.5 However, it has been reported that, in IPMT, it is difficult to distinguish benign species from malignant species.2, 6–11 Furthermore, strong pancreatic duct dilatation and large cysts with mural nodules are indicative of the malignant potential of IPMT.9, 10 We examined the pancreatic juice using cytologic methods to distinguish between adenoma and intraductal carcinoma. We found that the cytologic accuracy rate was 31% in patients with intraductal carcinoma.

Conversely, telomerase is responsible for cell immortality and is known to be activated in most human malignancies,12–14 such as hepatocellular carcinoma, lung carcinoma, and gastric carcinoma.15–17 In this report, we examined whether the measurement of telomerase activity in pancreatic juice was an accurate indicator of malignancy in patients with IPMT.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Patients

Endoscopic retrograde pancreatic juice aspiration (ERP aspiration) was performed on a total of 150 patients at Hiroshima University Hospital or its affiliated hospitals between 1997 and 1999. Of these 150 patients, we examined IPMT specimens from 28 patients. There were 23 men and 5 women ranging in age from 52 years to 77 years (mean age, 66.9 years). Overall, there were 13 patients with intraductal carcinoma and 15 patients with adenoma with mucin hypersecretion. All patients underwent surgical resection and were diagnosed histologically according to the classification of the AFIP. All specimens were reviewed by experienced pathologists.

ERP Aspiration

Conventional ERP was first performed by injection of an iodinated contrast agent (meglumine iotalamate, 60% weight/volume; Daiichi Pharmaceuticals Co., Ltd., Tokyo, Japan). After selective ERP,18, 19 immediately after an intravenous injection of 1 unit/kg body weight of secretin (Eisai, Tokyo, Japan), pancreatic juice was collected for approximately 10 minutes with a catheter. A sample was divided into two sections: one for use in the cytologic assessment and the other for the telomerase repeat amplification protocol (TRAP) assay.

Cytology and Cytologic Criteria

The cytology sample was immediately smeared on at least three slides, fixed in 95% ethanol, and stained with the Papanicolaou technique. All specimens were reviewed by three experienced pathologists and cytologists. The cytologic criteria used in the current study were based on the report by Robins et al.20 Cytologic criteria were described previously.19, 20, 22 To diagnose carcinoma, either two or more major criteria and one minor criterion or one major criterion and three minor criteria were required.

TRAP Assay

The TRAP assay was described previously.21, 22 In brief, the pancreatic juice sample was centrifuged at ×2000 g for 5 minutes. The cell pellet was washed three times in diethyl pyrocarbonate-phosphate buffered saline at ×2000 g for 5 minutes. Cells were then counted and stored at −80 °C until they were assayed for telomerase. The cell pellet was homogenized in CHAPS lysis buffer to a final density of 103 cells/μL. After 25 minutes of incubation on ice, the lysates were centrifuged at ×16,000 g for 20 minutes at 4 °C, and the supernatant was snap frozen in liquid nitrogen and stored at −80 °C. A cell extract of 1 μL was used for each TRAP assay.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Diagnosis of IPMT by ERP

In the patients with adenoma, the greatest dimension of the cystic lesion was <20 mm, and that of the pancreatic duct was <8 mm (Table 2). However, those findings also were seen in six patients with intraductal carcinoma (Table 1, Patients 3, 4, 8–10, and 13). Figure 1 shows samples from Patient 4 in Table 1. This patient had a cystic lesion measuring 8 mm and dilatation of main pancreatic duct that measured 5 mm with ERP (Fig. 1a). Cytology revealed that the tumor was benign (Fig. 1c) and that telomerase activity was expressed (Fig. 3, lane 2), and the pathologic diagnosis was intraductal carcinoma (Fig. 1d). Figure 2 shows samples from Patient 3 in Table 2. This patient had a cystic lesion that measured 5 mm and dilatation of the main pancreatic duct that measured 7 mm with ERP (Fig. 2a). Cytology revealed that the tumor was benign (Fig. 2c) and that there was no expression of telomerase activity (Fig. 3, lane 10), and the pathologic diagnosis was adenoma (Fig. 2d).

Table 1. Telomerase Activity and Cytology in Patients with Intraductal Carcinoma
PatientAge (yrs)GenderDiagnosisaCyst sizebPancreatic duct diameter (mm)CytologycTelomerase activitydFollow up (months)e
  • M: male; F: female.

  • a

    Carcinoma means intraductal carcinoma. All patients were diagnosed histologically according to the classification system of the Armed Forces Institute of Pathology.

  • b

    The greatest dimension of the cystic lesion (mm).

  • c

    Cytologic criteria used in the current study were based on the report by Robins et al.20

  • d

    Telomerase activity was investigated with the telomeric repeat amplification protocol assay (+, expression; −, no expression).

  • e

    These patients had no recurrence and no metastatic disease.

176MCarcinoma13BenignAlive (33)
272MCarcinoma20BenignAlive (30)
368MCarcinoma182Benign+Alive (40)
468MCarcinoma85Benign+Alive (38)
552MCarcinoma383Benign+Alive (29)
674MCarcinoma257Benign+Alive (27)
774MCarcinoma25Benign+Alive (41)
862MCarcinoma188Benign+Alive (37)
975FCarcinoma187Benign+Alive (37)
1074MCarcinoma172Malignant+Alive (11)
1171MCarcinoma12Malignant+Alive (11)
1256FCarcinoma226Malignant+Alive (42)
1374FCarcinoma142Malignant+Alive (24)
Table 2. Telomerase Activity and Cytology in Patients with Adenoma
PatientAge (yrs)GenderDiagnosisaCyst sizebPancreatic duct diameter (mm)CytologyaTelomerase activitydFollow up (months)e
  • M: male; F: female.

  • a

    All patients were diagnosed histologically according to the classification of system of the Armed Forces Institute of Pathology.5

  • b

    The greatest dimension of the cystic lesion (mm).

  • c Cytologic criteria used in the current study were based on the report by Robins et al.20

  • d

    Telomerase activity was investigated for with the telomerase repeat amplification protocol assay (−, no expression).

  • e

    These patients had no disease recurrence and no metastatic disease. However, patient 15 died with cerebral infarction.

159MAdenoma52BenignAlive (26)
272MAdenoma5BenignAlive (34)
363MAdenoma57BenignAlive (35)
467MAdenoma166BenignAlive (37)
557MAdenoma185BenignAlive (22)
654FAdenoma174BenignAlive (33)
755FAdenoma158BenignAlive (35)
861MAdenoma124BenignAlive (29)
963MAdenoma8BenignAlive (26)
1071MAdenoma185BenignAlive (17)
1173MAdenoma206BenignAlive (25)
1265MAdenoma188BenignAlive (26)
1364MAdenoma167BenignAlive (36)
1469MAdenoma157BenignAlive (30)
1577MAdenoma187BenignDead (28)
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Figure 1. The diagnostic procedure used for Patient 4 in Table 1. (a) Endoscopic retrograde pancreatic juice aspiration demonstrated mild dilatation of the main pancreatic duct and a small cystic lesion with some filling defects within the duct and the cyst. (b) Papilla of Vater showed no open enlargement. (c) Cytologic findings showed epithelial cells with mild atypia. (d) Histopathologic findings showed intraductal carcinoma (hematoxylin and eosin staining; original magnification, ×200).

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thumbnail image

Figure 2. The diagnostic procedure used for Patient 3 in Table 2. (a) Endoscopic retrograde pancreatic juice aspiration demonstrated mild dilatation of the main pancreatic duct and a small cystic lesion with some filling defects within the duct and the cyst. (b) Papilla of Vater showed a mild open enlargement. (c) Cytologic findings showed epithelial cells with mild atypia. (d) Histopathologic findings showed adenoma (hematoxylin and eosin staining; original magnification, ×200).

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Figure 3. Telomerase repeat amplification protocol (TRAP) assay of an intraductal papillary-mucinous tumor. The TRAP assay was investigated by using 103 pancreatic duct cells with each patient. Lanes 1–7 show Patients 3–9, respectively, in Table 1. Lanes 8 and 9 show Patients 1 and 2, respectively, in Table 1. Lanes 10–13 show adenomas. Because the TRAP assay was detected as a clear band in this examination, a semifixed quantity is not shown.

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Accuracy of TRAP Assay and Cytology

Of 13 patients with intraductal carcinoma, 4 patients (31%) were diagnosed with malignant disease by cytology, and 11 patients (85%) had tumors that showed detectable expression of telomerase activity (Table 1, Fig. 3). It is noteworthy that, although seven patients with intraductal carcinoma were diagnosed with benign tumors by cytology, all patients had tumors that showed detectable telomerase activity (Table 1, Patients 3–9; Fig. 3, lanes 1–7). Of 15 patients with adenoma, all were diagnosed with benign tumors by cytology, and no expression of telomerase activity was observed (Table 2; Fig. 3, lanes 10–13).

Comparison of Telomerase Activity and Cytologic Accuracy in Pancreatic Juice as Diagnostic Tools for IPMT

A comparison of telomerase activity with cytologic accuracy in pancreatic juice is summarized in Table 3. The sensitivity, specificity, predictive value for positive results, and predictive value for negative results for IPMT with telomerase activity were 85%, 100%, 100%, and 88%, respectively. The same values for cytologic accuracy were 31%, 100%, 100%, and 63%, respectively. With regard to the examination of telomerase activity, the sensitivity was higher compared with cytologic assessment.

Table 3. Comparison of Telomerase Activity and Cytology in Pancreatic Juice for the Diagnosis of Patients with Intraductal Papillary-Mucinous Tumors
Diagnostic testSensitivitySpecificityPVPRPVNR
  1. PVPR: predictive value for positive results; PVNR: predictive value for negative results.

Telomerase activity85% (11 of 13)100% (15 of 15)100% (11 of 11)88% (15 of 17)
Cytology31% (4 of 13)100% (15 of 15)100% (4 of 4)63% (15 of 24)

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

IPMT of the pancreas has been recognized increasingly as a clinical entity that is characterized by unique clinicopathologic features distinct from those of common pancreatic ductal cell carcinoma.5 It also has been reported that IPMT indicates a favorable prognosis for patients after pancreatectomy compared with common pancreatic ductal cell carcinoma. Furthermore, a preoperative diagnosis of malignant IPMT is difficult to determine (Fig. 1).2, 6–11 Many investigators have examined whether the use of molecular biologic factors, such as RAS and p53, is helpful in distinguishing between benign IPMTs and malignant IPMTs.19, 23–27 However, there is room for argument in their results.

The usefulness of telomerase activity measurement in pancreatic juices has been investigated at many institutions in patients with pancreatic carcinoma (common pancreatic ductal cell carcinoma).22, 27, 28 In this report, we investigated the usefulness of telomerase activity in pancreatic juice as a measure of malignancy of IPMT. Our data suggest that the measurement of telomerase activity may be a useful diagnostic tool in the distinction of intraductal carcinoma and adenoma. In our investigation, telomerase activity has indicated that even the resected specimens from patients with intraductal carcinoma show telomerase expression (our unpublished results). However, there are a number of reasons for the measurement of telomerase activity, including the possibility that RNase can contaminate when washing frequency is increased due to the mucin-rich pancreatic juice. In the future, further study will be needed to accumulate sufficient numbers of patients.

In conclusion, we found that telomerase activity was correlated highly with malignancy of IPMT. Figure 4 shows the diagnostic procedure of IPMT diagnosis in our laboratory. If any abnormalities, such as pancreatic duct dilatation or cystic lesion, are observed with ultrasonography or magnetic resonance cholangiopancreatography, then cytologic assessment and pancreatic juice telomerase activity are used to differentiate adenoma from carcinoma using ERP. If the result of either method is positive, then a diagnosis of malignant IPMT is warranted. However, if both results are negative, then the patient should be monitored closely due to the possibility of carcinogenesis at a later stage.

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Figure 4. Diagnostic procedure for intraductal papillary-mucinous tumor (IPMT). US: ultrasonography; EUS: endoscopic ultrasonography; CT: computed tomography; MRCP: magnetic resonance cholangiopancreatography; ERCP: endoscopic retrograde cholangiopancreatography.

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Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The authors are grateful thank to Fumio Shimamoto, M.D., and to M. Oda, C.T., for their histocytologic interpretation and to Eiso Hiyama, M.D.; T. Sasaki, M.D.; K. Morinaka, M.D.; K. Kariya, M.D.; S. Yamamoto, M.D.; M. Murakami, M.D.; and Y. Kuwada, M.D., for helpful discussions about this work.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
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