Expression of the Tumor Suppressor Gene Maspin in Human Pancreatic Cancers1

812 Vol. 7, 812–817, April 2001 Clinical Cancer Research

Nicolai Maass,2 Takashi Hojo, Michael Ueding, identified in normal mammary epithelium by subtractive hybrid- Jutta Lu¨ttges, Gu¨nter Klo¨ppel, Walter Jonat, and ization on the basis of its expression at the mRNA level (1). It Koichi Nagasaki was shown to have tumor suppressive activity attributable to inhibition of breast cancer cell motility, invasion, and metastasis Departments of Gynecologic Oncology [N. M., M. U., W. J., K. N.], and Pathology [J. L., G. K.], University of Kiel, 24105 Kiel, (2–4). Maspin is a Mr 42,000 protein with sequence homology Germany, and Growth Factor Division, National Cancer Center to other inhibitory serpins (2, 5). Maspin, which is located at the Research Institute, Tokyo 104-0045, Japan [T. H.] cell membrane and the extracellular matrix, does not act as a classical inhibitory serpin with antiprotease activity against trypsin-like serine proteases (6–8). ABSTRACT Maspin is expressed in normal human mammary and pros- The tumor suppressor gene maspin, a unique member tate epithelial cells but down-regulated during cancer progres- of the serpin superfamily, inhibits cell motility, invasion, and sion. The loss of maspin gene expression with increasing ma- metastasis in breast and prostate cancers. Maspin is ex- lignancy is regulated at the transcriptional level (9). Recent pressed in normal human mammary and prostate epithelial publications have discussed the participation of cytosine meth- cells but down-regulated during cancer progression. In this ylation and chromatin condensation in the down-regulation of study, we analyzed the expression of maspin in various maspin expression during neoplastic progression (10). human cancer cells by means of Northern blot and immu- Although at present the molecular and biological mecha- nohistochemistry. Maspin gene expression proved to be up- nisms of the function(s) of maspin remain unknown there is regulated in pancreatic cancer. Maspin expression was not evidence that maspin interacts with the p53 tumor suppressor detected in any of 6 gastric cancers, 4 melanomas, or 6 of 7 pathway and may function as an inhibitor of angiogenesis in breast cancer cell lines examined. In contrast, 5 of 9 pan- vitro and in vivo (11, 12). Using Northern blot analysis, reverse creatic cancer cell lines showed maspin expression, although transcription PCR and immunohistochemistry, we found further maspin expression was not detected in normal pancreatic evidence of decreasing maspin expression with increasing ma- tissue. Furthermore, maspin was expressed in 23 of 24 tu- lignancy in human breast cancer tissues (13). Pemberton et al. mor specimens obtained from pancreatic cancer patients as (14) demonstrated the presence of maspin in the epithelium of well as all high-grade precancerous lesions (PanIN3 and several normal human organs (such as prostate, thymus, testis, intraductal carcinoma extension). In contrast, no expression small intestine, and colon) and particularly in the myoepithelium was observed in normal and low-grade precancerous lesions. of the breast, where it is localized and probably functions both Our results show that maspin is a new factor associated with intra- and extracellularly. Because the maspin gene is expressed pancreatic cancer. In addition, the detection of maspin in in the epithelium of other glands, it is conceivable that it may pancreatic tumor tissues and its lack of expression in all play a similar role in the pancreas as well. For this reason, we normal pancreatic tissues suggests that maspin may be a were interested in determining whether the tumor suppressor useful marker of primary human pancreatic cancer. function described for maspin in mammary carcinomas can also be detected in pancreatic cancers. Interestingly, our data re- INTRODUCTION vealed a different pattern of maspin gene expression from that in Maspin (mammary serpin) is a serine protease inhibitor breast cancer cells. Maspin was not expressed in normal human related to the serpin family (1). The maspin gene was originally pancreatic cells but showed strong expression in pancreatic cancer cells as well as a weaker but detectable expression in precancerous pancreatic lesions. These results suggest that maspin is a new factor associated with pancreatic cancer. Received 10/2/00; revised 1/12/01; accepted 1/19/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked MATERIALS AND METHODS advertisement in accordance with 18 U.S.C. Section 1734 solely to Cell Culture and Clinical Specimens. The following indicate this fact. human cancer cell lines were studied: breast cancer (MCF-7, 1 Supported in part by Dr. Mildred Scheel Stiftung, Deutsche Kreb- ZR-75–1, SK-BR-3, BT-20, T47D, MDA-MB-231, MDA-MB- shilfe, and by the grant-in-aid from the Ministry of Health and Welfare, Japan, for the 2nd Term Comprehensive 10-Year Strategy for Cancer 468), pancreatic cancer (BxPC-3, AsPC-1, MIAPaCa-2, CA- Control and for Cancer Research (9-32 and 10-28). PAN-1, CAPAN-2, PANC-1, PSN-1, KP2, FA-6), gastric can- 2 To whom requests for reprints should be addressed, at Department of cer (MKN-1, MKN-7, MKN-28, MKN-45, MKN-74, KATO Gynecologic Oncology, University of Kiel, Michaelisstrasse 16, 24105 III), and melanoma (SEKI, G361, A375, MeWo). All of the Kiel, Germany. Phone: 49-431-597-2100; Fax: 49-431-86200; E-mail: [email protected]. cancer cell lines were cultured in RPMI 1640 supplemented 3 The abbreviations used are: HMEC, human mammary epithelial cell; with 10% FCS. HMECs were purchased from Clonetics (San PanIN, pancreatic intraductal neoplasia. Diego, CA), maintained according to supplier’s instructions, and

Fig. 1 Northern blot analysis of maspin in cancer cell lines and human tissues. A, HMECs and seven human breast cancer cell lines. B, nine pancreatic cancer cell lines. C, six gastric cancer cell lines. D, four melanoma cell lines. Each lane con- tains 10 ␮g of total RNA. E, expression of maspin in normal tissues. The blot containing 2 ␮g poly(A) RNA per lane was purchased from Clontech. The blots were hybridized with 2.5 kb maspin cDNA probe. 36B4 was used as loading and transfer control.

assessed at early passages. Total RNA was extracted from cells 23). The human multiple tissue Northern (MTN) blots (Clon- when cultures reached 80% confluence, as described previously tech, Palo Alto, CA) were used to determine the tissue distri- (15, 16). bution. Whipple resection specimens were obtained from 24 pa- Immunohistochemistry. A mouse antihuman maspin tients [14 female and 10 male; mean age, 69.6 years (range monoclonal antibody was purchased from PharMingen Interna- 46–76)] with ductal adenocarcinoma of the pancreas head from tional (San Diego, CA). For the staining of HMECs and cancer cell a series of 70 pancreatic resections performed in the years lines, cells were cultured in chamber slides for 24 h to 60–70% 1996–1999 in the Department of Surgery, University of Kiel confluency, fixed with 4% paraformaldehyde in PBS and perme- (Kiel, Germany). Histological classification and grading were ablized with methanol/3% H2O2 before blocking with 10% fetal performed according to the criteria of WHO 1996 and Lu¨ttges et bovine serum for 30 min. Cells were incubated with antihuman al. (17, 18). Of the carcinomas, one was classified as grade 1, 15 maspin antibody (diluted 1:75) according to the manufacturer’s as grade 2, and 8 as grade 3. One tumor was staged as stage I instructions. Peroxidase-conjugated sheep antimouse IgG was used disease, 6 as stage II, 14 as stage III, and 3 as stage IV. Ductal as secondary antibody at a dilution of 1:75 and was color-devel- lesions were classified according to the recently proposed PanIN oped using diaminobenzidine. Cells were then counterstained with classification (19–21). These samples where obtained from dif- hematoxylin, dehydrated, and mounted. In addition, 5-␮m sections ferent areas of the same surgical specimens as the carcinomas. of formalin-fixed, paraffin-embedded tissue samples from pancre- In addition, three surgical specimens of normal pancreas (from atic cancers, normal pancreatic tissues, and precancerous pancreatic 1 male and 2 female patients) were also investigated and served lesions were analyzed. After microwave-based antigen retrieval as control tissues. The staining of the carcinomas, normal pan- with 0.05 M Tris buffer (pH 9.0) for 15 min, the sections were creatic tissues, intraductal tumor extensions, and hyperplastic incubated with the antihuman maspin monoclonal antibody (dilut- duct epithelia was evaluated. The cytoplasmic staining intensity ed 1:75) for 12 h. Bound antibodies were detected using the was scored as follows: 0, no staining; 1, faint; 2, moderate; and avidin-biotin complex technique. New Fuchsin/Naphtol AS-Bi 3, strong. The cytospin specimens were scored in the same phosphate was used as a substrate and hematoxylin was used for manner. counterstaining. RNA Isolation and Northern Blot. Maspin cDNA was kindly provided by Dr. Ming Zhang (Baylor College of Medi- cine, Houston, TX). Total cell RNA was isolated from the RESULTS cancer cell lines using the RNeasy Mini kit (QIAGEN, Hilden, Expression of Maspin in Human Pancreatic Cancer Germany). A 2.5-kb EcoRI/XhoI fragment from the maspin Cell Lines. To evaluate the expression of maspin in cancer cDNA plasmid (pMZ-32) was labeled with 32P using a Re- cells several human cancer cell lines and normal tissues were diprime DNA labeling system (Amersham Life Science, Arling- investigated by means of Northern blot. Maspin gene expression ton Heights, IL) and used in Northern analyses of total RNA as was not detected in any of the six gastric cancer, four melanoma described previously (15, 16). For standardization, membranes and seven breast cancer cell lines with the exception of the were stripped and reprobed with the probe 36B4 under similar MDA-MB-468 breast cancer cell line (Fig. 1, A, C, and D). In conditions to assess RNA loading and transfer efficiency (22, contrast, maspin mRNA expression was observed in five of nine

Fig. 2 Maspin staining in HMECs and cancer cell lines. A, BxPC-3; B, AsPC-1; C, PANC-1; D, HMECs (ϫ100). Cells were probed with antihuman maspin antibody and counterstained with hematoxylin. In pancreatic cancer cell lines, maspin staining was moderate to strong in the cell lines BxPC-3 and AsPC-1 and in HMECs. PANC-1 showed no staining.

human pancreatic cancer cell lines (Fig. 1B). Maspin was highly (Fig. 3B; Table 1),which was diffusely distributed throughout expressed in BxPC-3 and AsPC-1, whereas low expression was the tumors. The staining intensity was generally strong and found in CAPAN-2, KP2, and FA-6. In the normal tissues, high varied only a little, except that cells with a broad clear cyto- expression of maspin mRNA was observed in mammary epi- plasm showed a faint positivity (Fig. 3C; Table 1). The only thelial cells (Fig. 1A), whereas none of eight other normal case that did not stain positively was a rare type of clear-cell tissues (heart, brain, placenta, lung, liver, skeletal muscle, kid- ductal adenocarcinoma (24). Intraductal non-clear-cell areas of ney, and pancreas) showed expression of maspin mRNA (Fig. this case, however, showed faint nuclear and cytoplasmic stain- 1E). It should be noted that, although a variety of pancreatic ing. Some cells also exhibited nuclear staining that was always cancer cell lines exhibited maspin mRNA expression, maspin accompanied by strong cytoplasmic staining. Intraductal exten- was not detected in normal pancreatic tissues on Northern blot sions of the carcinomas (17/17) and lesions of PanIN grade 3 analysis. (7/7) stained positive also but with lower intensity. In contrast, The expression of maspin in pancreatic cancer cells was ductal hyperplasia without dysplasia and low-grade dysplasia confirmed by immunohistochemistry. Representative staining (Table 1, 8/8; PanIN 1A, 1B, and 2) such as mucinous cell results in mammary epithelial cells and pancreatic cancer cell hypertrophy or papillary hyperplasia stained negative (Fig. 3D; lines are shown in Fig. 2. Positive staining for maspin was Table 1). In addition, foci of squamous intraductal metaplasia observed in HMECs (Fig. 2D), but no staining was seen in the (6/6) showed cytoplasmic staining (Table 1). There was no MCF-7 breast cancer cell line (data not shown). Of the pancre- correlation between the staining intensity and the histological atic cancer cell lines, BxPC-3 and AsPC-1 showed strong grade or stage of the tumors. maspin staining (Fig. 2, A and B), and a weak signal was observed in KP2 (data not shown). As shown in Fig. 2C, the pancreatic cell line PANC-1 lacked maspin expression. These DISCUSSION data are consistent with Northern blot analysis. Maspin was originally described as a tumor suppressor Expression of Maspin in Surgical Specimens. Acinar gene that affects cell motility and invasion (1). Recent findings cells and ductal epithelia from the tumor-associated pancreatic suggest that maspin is part of the p53 tumor suppressor pathway tissues as well as the normal pancreas (control cases) stained (12). Maspin expression is high in normal human mammary and negatively (Fig. 3A; Table 1). A strong cytoplasmic reaction of prostate epithelial cells but is decreased in breast and prostate all tumor cells was observed in 23 of 24 ductal adenocarcinomas cancers and lost in metastatic cells. We examined the expression

Fig. 3 Maspin staining in pancreatic cancer tissues. A, normal pancreatic tissue of the control cases with negatively stained acinar and ductal cells. B, invasive ductal adenocarcinoma of the pancreas with strong cytoplasmic and nuclear staining of the tumor cells and negative hyperplastic duct epithelium. C, invasive ductal adenocarcinoma showing strong cytoplasmic staining of tumor cells with dense cytoplasm and faint staining of those with clear and vacuolated cytoplasm. D, ductal papillary hyperplasia without nuclear atypia (PanIN1b, bottom half) without expression of maspin and PanIN3 lesion (strong nuclear abnormalities, top half) showing moderate cytoplasmic and nuclear staining.

of maspin in various cancer cells using Northern blot analysis cancer cell lines examined expressed maspin mRNA. Immuno- and immunohistochemistry. histochemical staining using a monoclonal maspin antibody Only a few reports of maspin expression in cancer cells yielded identical expression patterns, indicating that up-regu- have been published (12, 25, 26). However, our Northern blot lated maspin mRNA expression is translated into protein in analysis revealed that more than one-half of the pancreatic pancreatic cancer cells. Previously presented data of maspin

Table 1 Immunohistochemically assessed expression of maspin in pancreatic ductal adenocarcinomas, associated duct lesions, and normal pancreatic tissue No. of cases Staining intensity (% of cases) Negative (0) Faint (1) Moderate (2) Strong (3) Invasive ductal pancreatic adenocarcinoma (n ϭ 24) 1a/24 (4) 3/24 (12) 4/24 (17) 16/24 (67) Intraductal carcinoma extension (n ϭ 17)b 0/17 10/17 (59) 6/17 (35) 1/17 (6) PanIN3 (n ϭ 5)b 0/7 6/7 (86) 1/7 (14) 0/7 PanIN 1A, 1B, 2 (n ϭ 8)b 8/8 (100) 0/8 0/8 0/8 Squamous metaplasia, (n ϭ 6)b 0/6 5/6 (83) 1/6 (17) 0/6 Acinar cells and duct epithelium associated with carcinoma (n ϭ 24) 24/24 (100) 0/24 0/24 0/24 Acinar cells and duct epithelium of the control cases (n ϭ 3) 3/3 (100) 0/3 0/3 0/3 a Clear cell carcinoma. b Number of cases that exhibited intraductal carcinoma extension or the various lesions.

expression in normal pancreatic tissue showed conflicting re- maspin gene to the list of possible genes involved in pancreatic sults: Pemberton et al. (14) could not detected maspin mRNA carcinogenesis. The biological role of maspin expression in expression by Northern blot but did detect maspin-like protein pancreatic cancer should be determined by further investigation. expression in glandular epithelia of the pancreas by immuno- Carcinoma of the pancreas is the fourth highest cause of staining using a polyclonal antibody. The discrepancy may be cancer-related death and shows the highest mortality rate of all attributable to different characteristics of the antimaspin anti- cancers in most Western countries (30). Several tumor-associ- bodies used, such as reaction with distinct epitopes or different ated antigens, such as CEA, CA125, and CA19–9, are used to specificity. A monoclonal antimaspin antibody was used in our monitor pancreatic cancer patients (31). However, they are not study. On the other hand, reduced or lacking mRNA expression tumor specific and are commonly expressed in normal and has been reported in breast and prostate tumor cells, whereas benign conditions (32, 33). The fact that maspin was detected in corresponding normal cells exhibited high expression (1–4, 20). pancreatic cancer but was not expressed in normal pancreas Although this difference between the pancreas and other organs tissues suggests that maspin could serve as a useful marker for needs further analysis, maspin appears be an interesting factor primary human pancreatic cancer. associated with pancreatic cancer. In conclusion, we have demonstrated that maspin may play Although cell lines often develop artificial gene changes an important role in the carcinogenesis of pancreatic cancer, in during long-term culturing, our studies showed that maspin addition to its tumor suppressor activity in breast and prostate expression occurs not only in pancreatic cancer cell lines but cancer. We have shown that maspin gene expression is up- also in clinical pancreatic cancer tissues. Interestingly, we ob- regulated in pancreatic cancer at the RNA and protein level, in served maspin staining in 23 of 24 pancreatic cancer tissues, contrast to its down-regulation in breast and prostate cancers. which suggests that maspin expression is a common event in The function of maspin as a tumor suppressor gene involved in pancreatic cancer cells. Unlike the cancer tissues, no or faint tumor invasion, metastasis, and angiogenesis may not be limited expression was observed in corresponding normal pancreatic to breast and prostate cancer. Its relationship to carcinoma of the tissues and low-grade precancerous lesions. Furthermore, pancreas opens a new angle to the discussion on its function in maspin expression seems to increase with increasing malig- cancer. nancy from normal pancreas tissue via precancerous lesions to invasive carcinomas. These findings indicate that maspin ex- ACKNOWLEDGMENTS pression is of biological relevance in vivo for the development of pancreatic cancers. Although at present, the molecular and We thank Dr. Ming Zhang for providing the maspin cDNA plasmid. biological mechanisms of maspin’s function are unknown, several authors adhere to the hypothesis that maspin functions at the level of invasion and metastasis by blocking tumor cell migra- REFERENCES tion and proliferation (2–4). Our findings, which show the 1. Zou, Z., Anisowicz, A., Hendrix, M. J. C., Thor, A., Neveu, M., up-regulation of maspin in pancreatic cancer, provide new in- Sheng, S., Rafidi, K., Seftor, E., and Sager, R. Maspin, a serpin with formation about factors that regulate tumor cell development. tumor-suppressing activity in human mammary epithelial cells. Science (Washington DC), 263: 526–529, 1994. It has been shown repeatedly that distinct genes such as the 2. Sheng, S., Pemberton, P., and Sager, R. Production, purification, and K-ras oncogene and the tumor suppressor genes p53, p16, DCC, characterization of recombinant maspin proteins. J. Biol. Chem., 269: and DPC4/SMAD4 are frequently altered in pancreatic cancer 30988–30993, 1994. and may be essential for its genesis (27, 28). Interestingly, the 3. Sheng, S., Carey, J., Seftor, E. A., Dias, L., Hendrix, M. J. C., and maspin gene is mapped on chromosome 18q21.3 in close prox- Sager, R. Maspin acts at the cell membrane to inhibit invasion and imity to the DCC and DPC4/SMAD4 genes. Losses of chromo- motility of mammary and prostatic cancer cells. Proc. Natl. Acad. Sci. USA, 93: 11669–11674, 1996. some 18q including the loci for the genes DCC and DPC4/ 4. Sheng, S., Truong, B., Fredrickson, D., Wu, R., Pardee, A. B., and SMAD4 are the most frequently identified genetic alterations in Sager, R. Tissue-type plasminogen activator is a target of the tumor pancreatic cancer (29). Although the regulatory mechanism of suppressor gene maspin. Proc. Natl. Acad. Sci. USA, 95: 499–504, maspin expression remains unknown, our study has added the 1998.

5. Potempa, J., Korzus, E., and Travis, J. The serpin superfamily of K-ras mutations in the disease-free pancreas: analysis of type, age relation, proteinase inhibitors: structure, function, and regulation. J. Biol. Chem., and spatial distribution. Virchows Arch., 435: 461–468, 1999. 269: 15957–15960, 1994. 20. Lu¨ttges, J., and Klo¨ppel, G. Precancerous conditions of pancreatic 6. Sager, R. Function of maspin. Science (Washington DC), 165: 1893, carcinoma. J. Hepatobiliary Pancreat. Surg., 7: 568–574, 2000. 1994. 21. Hruban, R. H., Adsay, N. V., Albores-Saavedra, J., Compton, C. 7. Pemberton, P. A., Wong, D. T., Gibson, H. L., Kiefer, M. C., Garrett. E.S., Goodman, S. N., Kern, S. E., Klimstra, D., Klo¨ppel, G., Fitzpatrick, P. A., Sager, R., and Barr, P. J. The tumor suppressor Longnecker, D. S., Lu¨ttges, J., and Offerhaus, J. A. Pancreatic intraepi- maspin does not undergo the stressed to relaxed transition or inhibit thelial neoplasia (PanIN): a new nomenclature and classification system trypsin-like serine proteases. J. Biol. Chem., 270: 15832–15837, 1995. for pancreatic duct lesions. Am. J. Surg. Pathol., in press, 2001. 8. Hendrix, M. J. C., Seftor, E. A., Thomas, P. A., Sheng, S., and Seftor, 22. Masiakowski, P., Breathnach, R., Bloch, J., Gannon, F., Krust, A., R. E. B. Biological function(s) of maspin. Proc. Am. Assoc. Cancer and Chambon, P. Cloning of cDNA sequences of hormone-regulated Res., 38: 64, 1997. genes from the MCF-7 human breast cancer cell line. Nucleic Acids Res., 10: 7895–7903, 1982. 9. Zhang, M., Maass, N., Magit, D., and Sager, R. Transactivation through Ets and Ap1 transcriptional sites determines the expression of 23. Laborda, J. 36B4 cDNA used as an estradiol-independent mRNA the tumor-suppressing gene maspin. Cell Growth Differ., 8: 179–186, control is the cDNA for human acidic ribosomal phosphoprotein PO. 1997. Nucleic Acids Res., 19: 3998, 1991. 24. Lu¨ttges, J., Vogel, I., Menke, M., Henne-Bruns, D., Kremer, B., and 10. Domann, F. E., Rice, J. C., Hendrix, M. J., and Futscher, B. W. Klo¨ppel, G. Clear cell carcinoma of the pancreas: an adenocarcinoma Epigenetic silencing of maspin gene expression in human breast can- with ductal phenotype. Histopathology, 12: 444–448, 1998. cers. Int. J. Cancer, 85: 805–810, 2000. 25. Sager, R., Sheng, S., Anisowicz, A., Sotiropoulou, G., Zou, Z., 11. Zhang, M., Volpert, O., Shi, Y. H., and Bouck, N. Maspin is an Stenman, G., Swisshelm, K., Chen, Z., Hendrix, M. J., Pemberton, P., angiogenesis inhibitor. Nat. Med., 6: 196–199, 2000. Rafidi, K., and Ryan, K. RNA genetics of breast cancer: maspin as 12. Zou, Z., Gao, C., Nagaich, A. K., Connell, T., Saito, S., Moul, J. W., paradigm. Cold Spring Harb. Symp. Quant. Biol., 59: 537–546, 1994. Seth, P., Appella, E., and Srivastava, S. p53 regulates the expression of 26. Jiang, W. G., Hiscox, S., Horrobin, D. F., Bryce, R. P., and Mansel, the tumor suppressor gene maspin. J. Biol. Chem., 275: 6051–6054, R. E. ␥ linolenic acid regulates expression of maspin and the motility of 2000. cancer cells. Biochem. Biophys. Res. Commun., 237: 639–644, 1997. 13. Maass, N., Nagasaki, K., Hojo, T., Yamaguchi, K., Sager, R., and 27. Sirivatanauksorn, V., Sirivatanauksorn, Y., and Lemoine, N. R. Jonat, W. Expression of tumor suppressor gene maspin in human breast Molecular pattern of ductal pancreatic cancer. Langenbeck’s Arch. cancer tissue. Proc. Am. Soc. Clin. Oncol., 18: 634, 1999. Surg., 383: 105–115, 1998. 14. Pemberton, P. A., Tipton, A. R., Pavloff, N., Smith, J., Erickson, 28. Tarafa, G., Villanueva, A., Farre´, L., Rodriguez, J., Musule´n, E., J. R., Mouchabeck, Z. M., and Kiefer, M. C. Maspin is an intracellular Reyes, G., Seminago, R., Olmedo, E., Paules, A. B., Peinado, M. A., serpin that partitions into secretory vesicles and is present at the cell Bachs, O., and Capell‡, G. DCC and SMAD4 alterations in human surface. J. Histochem. Cytochem., 45: 1697–1706, 1997. colorectal and pancreatic tumor dissemination. Oncogene, 19: 546–555, 15. Nagasaki, K., Maass, N., Manabe, T., Hanzawa, H., Tsukada, T., 2000. Kikuchi, K., and Yamaguchi, K. Identification of a novel gene, DAM1, 29. Schleger, C., Arens, N., Zentgraf, H., Bley, U., and Verbeke, C. amplified at chromosome 1p13.3–21 region in human breast cancer cell Identification of frequent chromosomal aberrations in ductal adenocar- lines. Cancer Lett., 140: 219–226, 1999. cinoma of the pancreas by comparative genomic hybridization (CGH). 16. Nagasaki, K., Manabe, T., Hanzawa, H., Maass, N., Tsukada, T., J. Pathol., 191: 27–32, 2000. and Yamaguchi, K. Identification of a novel gene, LDOC1, down- 30. Parker, S. L., Tong, T., Bolden, S., and Wingo, P. A. Cancer regulated in cancer cell lines. Cancer Lett., 140: 227–234, 1999. statistics. CA Cancer J. Clin., 65: 5–27, 1996. 17. Klo¨ppel, G., Solcia, E., Longnecker, D. S., Capella, C., and Sobin, 31. Sakamoto, K., Haga, Y., and Yoshimura, R. Comparative effective- L. H. Histological typing of tumors of the exocrine pancreas. In: WHO ness of the tumor diagnostics, CA 19-9, CA 125. CEA in patients with International Histological Classification of Tumors, Ed. 2. Berlin: diseases of the digestive system. Gut, 28: 323–329, 1987. Springer-Verlag, 1996. 32. Safi, F., Roscher, F. S., and Bittner, R. High sensitivity and spec- 18. Lu¨ttges, J., Schemm, S., Vogel, I., Hedderich, J., Kremer, B., and ificity of CA 19-9 for pancreatic carcinoma in comparison to chronic Klo¨ppel, G. The grade of pancreatic ductal carcinomas is an independent pancreatitis: serological and immunohistochemical findings. Pancreas, prognostic factor and is superior to the immunohistochemical assess- 2: 398–403, 1987. ment of proliferation. J. Pathol., 191: 154–161, 2000. 33. Maestranzi, S., Przemioslo, R., Mitchell, H., and Sherwood, R. A. 19. Lu¨ttges, J., Reinecke-Lu¨thge, A., Mo¨llmann, B., Menke, M. A. O. H., The effect of benign and malignant liver disease on the tumor markers Clemens, A., Klimpfinger, B., Sipos, B., and Klo¨ppel, G. Duct changes and CA 19-9 and CEA. Ann. Clin. Biochem., 35: 99–103, 1998.

Nicolai Maass, Takashi Hojo, Michael Ueding, et al.

Clin Cancer Res 2001;7:812-817.

Updated version Access the most recent version of this article at: http://clincancerres.aacrjournals.org/content/7/4/812

Cited articles This article cites 30 articles, 10 of which you can access for free at: http://clincancerres.aacrjournals.org/content/7/4/812.full#ref-list-1

Citing articles This article has been cited by 18 HighWire-hosted articles. Access the articles at: http://clincancerres.aacrjournals.org/content/7/4/812.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/7/4/812. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.