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Clinical Cancer Research Vol. 7, 1976-1981, July 2001
© 2001 American Association for Cancer Research

Regular Articles

Diagnosis of Pancreatic Adenocarcinoma by Detection of Human Telomerase Reverse Transcriptase Messenger RNA in Pancreatic Juice with Sample Qualification1

Keiichi Seki, Takeshi Suda2, Yutaka Aoyagi, Satoshi Sugawara, Masaaki Natsui, Hirotaka Motoyama, Yoshio Shirai, Teruo Sekine, Hirokazu Kawai, Yusaku Mita, Nobuo Waguri, Takashi Kuroiwa, Masato Igarashi and Hitoshi Asakura

Department of Molecular Genetics [K. S., T. S., Y. A., S. S., M. N., H. M., H. K., Y. M., N. W., T. K., M. I., H. A.], Department of Regenerative and Transplant Medicine [Y. S.], Graduate School of Medical and Dental Sciences, Niigata University, Niigata 951-8122, and The Division of Internal Medicine, Shibata Prefecture Hospital, Shibata 957-0052 [T. S.], Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: We evaluated the diagnostic efficacy of detection of human telomerase reverse transcriptase (hTERT) message, a catalytic domain of human telomerase, in endoscopic retrograde pancreatography (ERP)-derived pancreatic juice.

Experimental Design: Both hTERT and CD25 expression were detected by reverse transcription-PCR (RT-PCR) in 17 patients with pancreatic adenocarcinoma (PC), 12 patients with chronic pancreatitis (CP), and 7 patients with no ERP abnormality (N). In the same patients, ß-actin message was semiquantified by competitive RT-PCR. K-ras codon 12 mutations were concomitantly analyzed by enriched PCR-SSCP in 11 and 7 PC and CP cases, respectively.

Results: Expression of hTERT was detected in 88% of PC cases and 17% of CP cases but not in the normal control (N). Alterations in K-ras were detected in 73% of PC cases and 57% of CP cases, respectively. ß-Actin mRNA was expressed in >3.0 x 101 copies/µl in all but two PC cases in which hTERT mRNA was not detected. CD25-positive and -negative peripheral lymphocytes were isolated from a normal volunteer using a fluorescent activating cell sorter. The hTERT message was detected in CD25-positive peripheral lymphocytes and in 18, 25, and 0% of the pancreatic juice samples from PC, CP, and N cases, respectively. All CP cases expressing hTERT message were also CD25 positive.

Conclusions: These results suggest that detection of hTERT mRNA in pancreatic juice is a powerful tool to discriminate PC from CP, particularly when the samples are qualified against ß-actin mRNA levels and contaminating CD25-positive lymphocytes.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PC3 is one of the most aggressive known cancers. The 5-year survival rate of PC is one of the lowest of all types of cancers (1) . The poor prognosis results from both the difficulty of diagnosis in the early clinical stages and the highly metastatic and/or invasive nature of this tumor. It is believed that early diagnosis may drastically improve the survival rate. A common challenge for physicians is the differential diagnosis between PC and CP, which is assumed to be a risk factor for PC development (2) , despite the availability of advanced endoscopic, radiological, and ultrasonographic techniques. This is particularly problematic when a solid tumor is forming. Although a cytological examination of the pancreatic juice collected under ERP facilitates the diagnosis, the sensitivity of this method varies from 30 to 80% (3 , 4) . Detection of molecular abnormalities commonly associated with PC is also applied in the clinical diagnosis, including K-ras and p53 gene mutations (5 , 6) . These mutations are also detected in noncancerous tissues, such as in adenoma and pancreatitis (7, 8, 9, 10) , and are therefore of limited value in the differential diagnosis of PC and CP.

Telomerase is an enzyme implicated in the de novo synthesis of GGTTAG telomeric DNA onto chromosomal ends to stabilize telomeres, concomitant with immortality in cancer cells (11, 12, 13) . Telomerase consists of three components, human telomerase RNA, an RNA template complementary to GGTTAG (14) , hTERT, a catalytic domain (15) , and the other protein component, TP1 (16) . Although hTR is expressed ubiquitously in both cancerous and noncancerous tissues (17) , hTERT expression is rate limiting for telomerase activity (18) . Detection of telomerase activity using a TRAP assay of pancreatic juice is more likely to diagnose PC than detection of K-ras codon 12 mutations (19, 20, 21) . Several problems remain to be overcome before this technique can be introduced in clinics. These include the use of radioisotopes and the possibility of false positives attributable to contaminating lymphocytes, which can show telomerase activity without malignant transformation (22 , 23) . The present study aimed to determine whether the detection of hTERT mRNA in pancreatic juice would be a useful diagnostic tool for pancreatic cancer and attempted to decrease the risk of false-positive and -negative diagnosis using this strategy.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Pancreatic Juice.
Thirty-six patients were referred to our clinic for ERP. Informed consent was obtained from each patient, and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki, as reflected in a priori approval by the institution’s human research committee. The pancreatic juice was collected through a catheter placed selectively into the pancreatic duct after an i.v. injection of secretin. After discarding the first secretion, which included the contrast medium, the pancreatic juice was collected in 2.5-ml serum tubes, snap frozen in liquid nitrogen, and stored at -80°C until use. Seventeen patients, cases 1 to 17, had PC proven by histological examination of surgically resected specimens or other clinical diagnostic modalities: ultrasonography, ERP including cytological examination, dynamic computed tomography, and magnetic resonance imaging. Twelve patients had a clinical diagnosis of chronic pancreatitis void of cancer (cases 18–29). No abnormal findings were detected in 7 patients (cases 30–36).

RT-PCR.
Approximately 2 ml of pancreatic juice were diluted in 15 ml of phosphate buffer saline prepared with diethyl pyrocarbonate-treated water. Cells were collected by centrifugation. Total RNA was isolated using IsoGen (Wako, Osaka, Japan) according to the manufacturer’s manual and resuspended in 20 µl of TE [10 mM Tris-HCl (pH 7.5), 0.1 mM EDTA]. cDNA synthesis was performed using 5.6-µl aliquots in a 20-µl reaction mixture, which included random hexa-oligonucleotides and RAV-2 reverse transcriptase (Takara, Kyoto, Japan). hTERT cDNA was amplified by nested PCR. The first PCR amplified a 499-bp (10–508 nucleotides) fragment, which included exons 1 and 2, a region without alternative splicing (24) . One µl of a 250-fold dilution of the first PCR reaction was subjected to a second PCR. Nested primers were used to amplify a 429-bp fragment. A 491-bp {alpha} chain of interleukin 2 receptor cDNA was amplified in a 10-µl reaction mixture using primers 5'-AATGCACAAGCTCTGCCACTC (sense) and 5'-GGCCACTGCTACCTGGTACTC (antisense). To quantify the amount of mRNA recovered, ß-actin cDNA was coamplified with a constant amount of competitor DNA, which could be amplified with the same primers but produced 100-bp shorter products. PCR was terminated at 30 cycles, at which the products were linearly generated. After separation through 6% polyacrylamide gel, the products were visualized by ethidium bromide staining. The image was digitally captured with a DC40 digital camera (Eastman Kodak, Rochester, NY), and intensity ratios of native and competitor DNA products were calculated using ScionImage (Scion, Frederick, MD). The number of cDNAs was calculated by transforming the ratio along the standard curve, which was deduced from the dilution experiments using known amounts of native and competitor DNAs.

Enriched PCR-SSCP.
Mutations in K-ras exon 1 codon 12 were analyzed by a two-step PCR method with restriction enzyme digestion, followed by nonradioisotopic SSCP analysis (25) . Briefly, a 157-bp fragment containing K-ras codon 12 was amplified from genomic DNA extracted from frozen pancreatic juice. The PCR product was digested with BstNI overnight at 60°C. The 250-fold dilution of the digested PCR product was amplified with nested primers to generate a 135-bp fragment. Aliquots of the second PCR reaction products were diluted with a loading buffer consisting of 90% deionized formamide, 20 mM EDTA, and 0.05% bromphenol blue and xylene cyanol. After denaturation at 80°C for 5 min, the samples were electrophoresed through 15% polyacrylamide gels. The fragments were visualized under UV irradiation after staining with GelStar (Takara).

Separation of CD25-positive and -negative Peripheral Lymphocytes.
Peripheral blood was obtained from a healthy human volunteer with approval to investigate hTERT expression. PBLs were collected by Ficoll-Isopaque (1.077) gradient centrifugation. After lysing the erythrocytes with ammonium chloride solution (155 mM NH4Cl + 10 mM KHCO3, 1 mM EDTA, 0.2 M Tris-HCl, pH 7.6), the isolated PBLs were washed twice and suspended in RPMI 1640 supplemented with 10% FCS. The surface phenotype of the cells was identified using phycoerythrin-conjugated anti-CD25 monoclonal antibody (B1.49.9; Immunotech, Westbrook, CT). CD25 and interleukin 2 receptor {alpha} are nomenclatures for the same molecule. The fractions of CD25+ PBLs and CD25- PBLs were sorted by FACStar II plus (Becton Dickinson, Lincoln Park, NJ). Purity was >98%.

TRAP Assay.
Telomerase activity in the sorted lymphocytes was analyzed by the TRAP, which included an internal control (TRAPeze; Intergen Co., Purchase, NY). Amplified products were electrophoresed through 12% polyacrylamide gels in TBE (0.09 M Tris-borate, 0.02 M EDTA) and visualized using GelStar (Takara).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
hTERT Expression and Detection of K-ras Codon 12 Mutations in Pancreatic Juice.
The expression of hTERT in pancreatic juice was measured using reverse transcription coupled with nested PCR (see "Materials and Methods"). Message was detected in 15 of 17 patients with PC (Table 1)Citation but in only 2 of 12 patients with CP devoid of cancer. No message was detected in any of the 7 cases with no pancreatic abnormalities as detected by ERP (N; Table 2Citation ). The sensitivity, specificity, and overall accuracy of the diagnosis of PC based on the presence of hTERT message in pancreatic juices were 88, 83, and 86%, respectively. Because there was not enough pancreatic juice collected in all of the cases for analysis of both hTERT message and K-ras codon 12 mutations, K-ras analysis was carried out in only 11 and 7 cases of PC and CP, respectively. Alterations of K-ras codon 12 were detected in 8 of 11 PC cases using the enriched PCR-SSCP method (Table 1)Citation . Mutations were also detected in 4 of 7 CP cases (Table 2)Citation . The sensitivity, specificity, and overall accuracy of the diagnosis of PC based on the alterations in K-ras codon 12 detected in pancreatic juices were 73, 43, and 61%, respectively. All cancer cases with K-ras mutations were also hTERT positive.


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  		Table 1 Detection of several messages and K-ras mutations in pancreatic juice from pancreatic cancer cases

 

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  		Table 2 Detection of several messages and K-ras mutations in pancreatic juice from cases without pancreatic cancer

 
Quantification of ß-Actin mRNA in Pancreatic Juice.
To define the minimum amount of mRNA suitable for hTERT evaluation, the amount of ß-actin message in pancreatic juice was quantified by competitive RT-PCR (Fig. 1)Citation . This level varied between samples from <3.0 x 101 to >3.0 x 103 copies in every 1 µl of the reverse-transcribed products. More than 3.0 x 101 copies/µl were recovered in all but 2 PC cases, cases 16 and 17, in which hTERT message could not be detected, even in the presence of clinical evidence for advanced PC. These findings suggest that a sample in which <3.0 x 101 copies/µl of ß-actin mRNA are detected should be discarded without evaluation. We therefore evaluated only those CP cases in which >3.0 x 101 copies/µl of ß-actin message was detected. In 2 cases, cases 18 and 19, hTERT message was detected without any evidence of PC, and 6.7 x 102 and >3.0 x 103 copies/µl of ß-actin message, respectively, were detected.



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  		Fig. 1. Semiquantitative RT-PCR with human ß-actin cDNA. A, serial dilutions of cDNA from pancreatic juice were amplified with 100 copies of competitor DNA, which has exactly the same sequence as ß-actin cDNA except for removal of a 100-bp fragment between the primers. A, ß-actin cDNAs were coamplified from 30 to 3000 copies with 100 copies of the competitor for 30 cycles and separated through 6% polyacrylamide gels. {circ} and •, positions of the ß-actin cDNA and competitor products, respectively. B, the ratio between the ß-actin cDNA and competitor DNA products was logarithmically plotted against a known amount of ß-actin cDNA subjected to the PCR. A linear standard curve was obtained from 30 to 950 copies of ß-actin cDNA. The function of this line was expressed as F(x) = 66.1X1.33. C, representative electrophoresis patterns for the semiquantification of ß-actin cDNA. In cases 16 and 17, Lanes 1 and 3, respectively, the band intensity of ß-actin cDNA ({circ}) was ~2.5-fold weaker than that of the 100 copies of the competitor DNA (•). Because coamplification of 30 copies of ß-actin cDNA and 100 copies of the competitor DNA produced the bands with a 0.59:1 intensity ratio, the amount of ß-actin cDNA amplified in cases 16 and 17 was <30 copies. In contrast, in case 4 (Lane 2), the intensity of the ß-actin cDNA band was 3.2-fold higher than that of the competitor DNA. Lane M, 100-bp ladder including spike at 1000 bp. Lanes 4 and 5 revealed independent amplification of the competitor DNA or ß-actin cDNA, respectively.

 
hTERT Message in CD25-positive Peripheral Lymphocytes.
The pancreatic juice samples in which hTERT message was detected without any evidence of PC were collected immediately after acute aggravation of chronic pancreatitis. We therefore assumed that the activated lymphocytes in the pancreatic juice may account for the hTERT message in these cases. To evaluate this possibility, we first analyzed whether hTERT message was preferentially detected in activated lymphocytes circulating in the peripheral blood stream. The peripheral lymphocytes from a normal volunteer were separated into CD25-positive and -negative groups by a fluorescence activated cell sorter, using an antibody against the {alpha} chain of the interleukin 2 receptor (Fig. 2)Citation . Telomerase activity was not detected in either group of lymphocytes using a nonisotopic TRAP assay on half of the recovered cells (5 x 105 cells). However, analysis of the remaining cells detected hTERT message in CD25-positive lymphocytes but not in the negative cells.



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  		Fig. 2. hTERT expression in CD25-positive lymphocytes. A, separation pattern of CD25-positive and -negative lymphocytes isolated using a fluorescence-activated cell sorter. The area indicated by the horizontal bar was collected as CD25-positive lymphocytes, which comprised 22% of the total lymphocytes. B, TRAP assay on the 5 x 105 sorted lymphocytes. The products were visualized by UV irradiation after GelStar staining. In both CD25-positive and -negative lymphocytes (Lanes 3 and 4, respectively), telomerase activity was not detected. An internal standard (arrowhead) was included in Lane 1, and an external standard, TSR8, was coamplified with an internal standard in Lane 2. C, amplification of ß-actin, CD25, and hTERT messages by RT-PCR. ß-Actin message was detected in both CD25-positive and -negative lymphocytes, whereas CD25 and hTERT message was detected only in CD25-positive lymphocytes.

 
CD25 mRNA in Pancreatic Juice.
CD25 message was detected in 3 of 17 PC cases, 3 of 12 CP cases, and in none of the 7 normal controls. Intriguingly, this message was detected in both of the 2 cases expressing hTERT message in the absence of PC (Fig. 3)Citation . Taken together with the quantification of ß-actin message, the detection of hTERT mRNA in pancreatic juice to diagnose PC should be limited in its application to samples from which sufficient mRNA is recovered and which have a negligible level of activated lymphocyte contamination. Finally, the overall accuracy of this strategy reached 100% in the present study, when there were 28 sample cases in total: 12, 9, and 7 cases with PC, CP and N, respectively.



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  		Fig. 3. Representative electrophoretic pattern of RT-PCR products for hTERT and CD25 using pancreatic juice samples. RT-PCR products for hTERT and CD25 were separated through 6% polyacrylamide gels and visualized by ethidium bromide staining. Lanes 1–3 were results for CP cases, cases 20, 19, and 24, respectively, and Lanes 4–6 were PC cases, cases 4, 11, and 16, respectively. ß-Actin was sufficiently detected in all cases except case 16, in which <3 x 101 copies were detected in 1 µl of the reverse-transcribed products. CD25 message was detected only in case 19, in which hTERT message was detected without any evidence for PC. Lane M, 100-bp ladders including a spike at 1000 or 500 bp for hTERT or CD25, respectively.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Detection of telomerase activity in pancreatic juice has been proposed as a useful tool in the diagnosis of PC (19, 20, 21) . Unfortunately, several problems exist when applying this technique clinically. The method necessitates the use of radioisotopes. We tried unsuccessfully to detect TRAP products from the pancreatic juice of 10 patients with definite PC on polyacrylamide gel with GelStar staining, which provides a 10-fold higher sensitivity than ethidium bromide (data not shown). It was clear that the sensitivity of GelStar is insufficient because telomerase activity could not be detected, even in 5 x 105 CD25-positive lymphocytes in which hTERT message was detected (Fig. 2)Citation . Radioisotopic analysis of normal adult peripheral mononuclear cells detected a low level of telomerase activity in as low as 104 cells (26) . Although TRAP products were detected in pancreatic juice by staining with SYBR Green, the bands were too weak to use as a diagnostic indicator in clinics (21) .

The second problem is the difficulty in evaluating sample suitability for telomerase analysis. Confidence in the sample reliability is a requirement for clinical use in diagnosis in addition to the higher sensitivity and specificity. The reliability depends not only on the methodology itself but also on sample quality. For the diagnosis of PC using pancreatic juice, it appears that insufficient recovery and/or degradation of the ductal cells (27) , because the ductal cells are collected under remote operation of ERP and in a suspension of pancreatic juice containing active digestive enzymes. To determine the quality and quantity of each sample with respect to recovery of mRNA, we quantified the ß-actin message as an internal control. This analysis revealed that samples expressing <3.0 x 101 copies/µl of ß-actin message in the reverse-transcribed products were not suitable for evaluation of hTERT. In such samples, hTERT message could not be detected, even in patients with advanced PC. This study, therefore, indicated that sample quality assessment is easier for detection of hTERT message than it is for TRAP analysis.

PC is quite resistant to all anticancer drugs currently available (28) . Surgical resection is deemed as the only effective method to treat PC. This approach, however, is sometimes quite radical and drastically reduces the quality of the patient’s life (29) . In this context, the risk of a false-positive diagnosis should be minimized at all costs. It has been reported that weak telomerase activity may be detected in CP devoid of cancer (19) . It was suspected that an increased number of infiltrating lymphocytes might be the cause of this activity. An up-regulation of telomerase activity without transformation was reported with presentation of antigen to T cells. In addition, the samples showing hTERT mRNA expression in the absence of cancer cells were obtained from patients with acute aggravation of CP. Furthermore, hTERT message was detected in CD25-positive but not in CD25-negative peripheral lymphocytes from a normal volunteer. Thus, we propose that removal of samples exhibiting CD25 expression from the evaluation would decrease the number of false positives. This is supported by the findings in this study that all hTERT-positive pancreatic juice samples without cancer expressed CD25 mRNA. Together, these observations strongly suggest that detection of hTERT message in pancreatic juice expressing CD25 is not suitable for cancer diagnosis by detection of hTERT message. It may be difficult to assess this type of contamination using protein in pancreatic juice.

It was reported by several others that the detection of K-ras mutations in exon 1 codon 12 is useful in the diagnosis of PC (30 , 31) . This mutation is, however, represented with substantial frequency in hyperplastic foci and pancreatic juice from patients with chronic pancreatitis in the absence of cancer (7, 8) . In fact, a K-ras mutation was detected in 4 of 7 CP cases in this study. Consequently, the specificity of detection of K-ras mutations fell to 43%. This value is consistent with that published in the literature. The detection of K-ras mutations in pancreatic juice is therefore lower in both sensitivity and specificity than the detection of hTERT message. If the samples were controlled for CD25 expression, hTERT mRNA was not detected in any of the patients with CP without cancer. To maximize the accurate and efficient diagnosis of PC, we recommend the detection of hTERT mRNA, particularly after evaluation of the sample with respect to mRNA recovery and contamination of activated lymphocytes.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by Grant-in-Aid 09670521 for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, a grant-in-aid from the Hamanako Fund, and a grant-in-aid from the Yujin Fund of Niigata University School of Medicine. Back

2 To whom requests for reprints should be addressed, at Department of Molecular Genetics, Graduate School of Medical and Dental Sciences, Niigata University, 1-757 Asahimachi-dori, Niigata 951-8122, Japan. Fax: 81-25-227-0776; E-mail: mailto:suda@med.niigata-u.ac.jp. Back

3 The abbreviations used are: PC, pancreatic adenocarcinoma; CP, chronic pancreatitis; N, patient with no ERP abnormality; ERP, endoscopic retrograde pancreatography; PBL, peripheral blood lymphocyte; hTERT, human telomerase reverse transcriptase; RT-PCR, reverse transcription-PCR: SSCP, single-strand conformation polymorphism; TRAP, telomeric repeat amplification protocol. Back

Received 12/20/00; revised 4/16/01; accepted 4/17/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Johnson C. Prognosis in pancreatic cancer. Lancet, 349: 1027-1028, 1997.[Medline]
  2. Bansal P., Sonnenberg A. Pancreatitis is a risk factor for pancreatic cancer. Gastroenterology, 109: 247-251, 1995.[CrossRef][Medline]
  3. Nakaizumi A., Tatsuta M., Uehara H., Yamamoto R., Takenaka A., Kishigami Y., Takemura K., Kitamura T., Okuda S. Cytologic examination of pure pancreatic juice in the diagnosis of pancreatic carcinoma. Cancer (Phila.), 70: 2610-2614, 1992.[CrossRef][Medline]
  4. Mitchell M. L., Carney C. N. Cytologic criteria for the diagnosis of pancreatic carcinoma. Am. J. Clin. Pathol., 83: 171-176, 1985.[Medline]
  5. Barton C. M., Staddon S. L., Hughes C. M., Hall. P. A., O’Sullivan C., Kloppel G., Theis B., Russell R. C., Neoptolemos J., Williamson. R. C., Lane D. P., Lemoine N. R. Abnormalities of the p53 tumour suppressor gene in human pancreatic cancer. Br. J. Cancer, 64: 1076-1082, 1991.[Medline]
  6. Ruggeri B., Zhang S. Y., Caamano J., DiRado M., Flynn S. D., Klein-Szanto A. J. Human pancreatic carcinomas and cell lines reveal frequent and multiple alterations in the p53 and Rb-1 tumor-suppressor genes. Oncogene, 7: 1503-1511, 1992.[Medline]
  7. van Laethem J. L. Ki-ras oncogene mutations in chronic pancreatitis: which discriminating ability for malignant potential? Ann. NY Acad. Sci., 880: 210-218, 1999.[CrossRef]
  8. Tada M., Ohashi M., Shiratori Y., Okudaira T., Komatsu Y., Kawabe T., Yoshida H., Machinami R., Kishi K., Omata M. Analysis of K-ras gene mutation in hyperplastic duct cells of the pancreas without pancreatic disease. Gastroenterology, 110: 227-231, 1996.[CrossRef][Medline]
  9. Gansauge S., Schmid R. M., Muller J., Adler G., Mattfeldt T., Beger H. G. Genetic alterations in chronic pancreatitis: evidence for early occurrence of p53 but not K-ras mutations. Br. J. Surg., 85: 337-340, 1998.[CrossRef][Medline]
  10. Berrozpe G., Schaeffer J., Peinado M. A., Real F. X., Perucho M. Comparative analysis of mutations in the p53 and K-ras genes in pancreatic cancer. Int. J. Cancer, 58: 185-191, 1994.[Medline]
  11. Blackburn, E. H., and Greider, C. W. Telomeres. Cold spring Harbor, NY: Cold Spring Harbor Laboratory, 1995.
  12. Morin G. B. The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats. Cell, 59: 521-529, 1989.[CrossRef][Medline]
  13. Kim N. W., Piatyszek M. A., Prowse K. R., Harley C. B., West M. D., Ho P. L., Coviello G. M., Wright W. E., Weinrich S. L., Shay J. W. Specific association of human telomerase activity with immortal cells and cancer. Science (Wash. DC), 266: 2011-2015, 1994.[Abstract/Free Full Text]
  14. Feng J., Funk W. D., Wang S. S., Weinrich S. L., Avilion A. A., Chiu C. P., Adams R. R., Chang E., Allsopp R. C., Yu J., Le S., West M. D., Harley C. B., Andrews W. H., Greider C. W., Villeponteau B. The RNA component of human telomerase. Science (Wash. DC), 269: 1236-1241, 1995.[Abstract/Free Full Text]
  15. Meyerson M., Counter C. M., Eaton E. N., Ellisen L. W., Steiner P., Caddle S. D., Ziaugra L., Beijersbergen R. L., Davidoff M. J., Liu Q., Bacchetti S., Haber D. A., Weinberg R. A. hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell, 90: 785-795, 1997.[CrossRef][Medline]
  16. Nakayama J., Saito M., Nakamura H., Matsuura A., Ishikawa F. TLP1: a gene encoding a protein component of mammalian telomerase is a novel member of WD repeats family. Cell, 88: 875-884, 1997.[CrossRef][Medline]
  17. Avilion A. A., Piatyszek M. A., Gupta J., Shay J. W., Bacchetti S., Greider C. W. Human telomerase RNA and telomerase activity in immortal cell lines and tumor tissues. Cancer Res., 56: 645-650, 1996.[Abstract/Free Full Text]
  18. Nakayama J., Tahara H., Tahara E., Saito M., Ito K., Nakamura H., Nakanishi T., Tahara E., Ide T., Ishikawa F. Telomerase activation by hTRT in human normal fibroblasts and hepatocellular carcinomas. Nat. Genet., 18: 65-68, 1998.[CrossRef][Medline]
  19. Hiyama E., Kodama T., Shinbara K., Iwao T., Itoh M., Hiyama K., Shay J. W., Matsuura Y., Yokoyama T. Telomerase activity is detected in pancreatic cancer but not in benign tumors. Cancer Res., 57: 326-331, 1997.[Abstract/Free Full Text]
  20. Suehara N., Mizumoto K., Muta T., Tominaga Y., Shimura H., Kitajima S., Hamasaki N., Tsuneyoshi M., Tanaka M. Telomerase elevation in pancreatic ductal carcinoma compared to nonmalignant pathological states. Clin. Cancer Res., 3: 993-998, 1997.[Abstract]
  21. Uehara H., Nakaizumi A., Tatsuta M., Baba M., Takenaka A., Uedo N., Sakai N., Yano H., Iishi H., Ohigashi H., Ishikawa O., Okada S., Kakizoe T. Diagnosis of pancreatic cancer by detecting telomerase activity in pancreatic juice: comparison with K-ras mutations. Am. J. Gastroenterol., 94: 2513-2518, 1999.[CrossRef][Medline]
  22. Weng N., Levine B. L., June C. H., Hodes R. J. Regulation of telomerase RNA template expression in human T lymphocyte development and activation. J. Immunol., 158: 3215-3220, 1997.[Abstract]
  23. Hu B. T., Lee S. C., Marin E., Ryan D. H., Insel R. A. Telomerase is up-regulated in human germinal center B cells in vivo and can be re-expressed in memory B cells activated in vitro. J. Immunol., 159: 1068-1071, 1997.[Abstract]
  24. Wick M., Zubov D., Hagen G. Genomic organization and promoter characterization of the gene encoding the human telomerase reverse transcriptase (hTERT). Gene (Amst.), 232: 97-106, 1999.[CrossRef][Medline]
  25. Kondo H., Sugano K., Fukayama N., Kyogoku A., Nose H., Shimada K., Ohkura H., Ohtsu A., Yoshida S., Shimosato Y. Detection of point mutations in the K-ras oncogene at codon 12 in pure pancreatic juice for diagnosis of pancreatic carcinoma. Cancer (Phila.)., 73: 1589-1594, 1994.[CrossRef][Medline]
  26. Hiyama K., Hirai Y., Kyoizumi S., Akiyama M., Hiyama E., Piatyszek M. A., Shay J. W., Ishioka S., Yamakido M. Activation of telomerase in human lymphocytes and hematopoietic progenitor cells. J. Immunol., 155: 3711-3715, 1995.[Abstract]
  27. Furui T., Ikeda M., Chao-Ming L., Okita K., Nakamura K. Protein degradation in human pure pancreatic juice analyzed by two-dimensional gel electrophoresis. Electrophoresis, 17: 797-802, 1996.[CrossRef][Medline]
  28. van Groeningen C. J. Intravenous and intra-arterial chemotherapeutic possibilities in biliopancreatic cancer. Ann. Oncol., 10 (Suppl. 4): 305-307, 1999.[Abstract/Free Full Text]
  29. Bottger T. C., Engelmann R., Junginger T. Is age a risk factor for major pancreatic surgery? An analysis of 300 resections. Hepatogastroenterology, 46: 2589-2598, 1999.[Medline]
  30. Sturm P. D., Rauws E. A., Hruban R. H., Caspers E., Ramsoekh T. B., Huibregtse K., Noorduyn L. A., Offerhaus G. J. Clinical value of K-ras codon 12 analysis and endobiliary brush cytology for the diagnosis of malignant extrahepatic bile duct stenosis. Clin. Cancer Res., 5: 629-635, 1999.[Abstract/Free Full Text]
  31. Villanueva A., Reyes G., Cuatrecasas M., Martinez A., Erill N., Lerma E., Farre A., Lluis F., Capella G. Diagnostic utility of K-ras mutations in fine-needle aspirates of pancreatic masses. Gastroenterology, 110: 1587-1594, 1996.[CrossRef][Medline]



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