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Molecular Detection of Micrometastases in Pancreatic Cancer
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3 Molecular Detection of Micrometastases in Pancreatic Cancer Marco Niedergethmann, Stefanie Knob, and Stefan Post
Introduction Despite advances in surgical oncology, local recurrence and distant metastases are the major problems in treating pancreatic cancer. Only 25% of all pancreatic cancers are resectable, and only patients who have been curatively resected (R0) enjoy a favorable outcome (Richter et al., 2003). However, the survival after surgery still remains poor. Most surgeons and oncologists treating pancreatic cancer believe that local recurrence represents the growth of residual tumor (Pantel et al., 2000). Despite histologically confirmed curative resection (no residual tumor: R0) and tumor-free lymph nodes, most patients will suffer from postoperative local recurrence or distant metastases (Trede et al., 2001). Occult dissemination of tumor cells beyond resection margins is of paramount importance for prognosis. This is impressively shown by a 50% recurrence rate within 2 years after surgery for pancreatic cancer (Richter et al., 2003; Trede et al., 2001). In recent years, numerous new techniques to detect minimal residual disease (MRD), including immunohistochemical and molecular assays, have been introduced Handbook of Immunohistochemistry and in situ Hybridization of Human Carcinomas, Volume 3: Molecular Genetics, Liver Carcinoma, and Pancreatic Carcinoma
to reveal the accurate resection status (R0 versus R1) and enrich the routine histopathology (Niedergethmann et al., 2002). These techniques detect micrometastases and disseminated tumor cells not only in lymph nodes that had appeared tumor free in routine histology but also in body compartments such as bone marrow, peritoneal cavity, and blood (Vogel et al., 2001). From the current data, it has been concluded that routine histopathology often underestimates the true tumor stage. It is well known that micrometastases of a diameter of 3 cells are diagnosed only in 1% based on serial 5-µm sections (Keene and Demeure, 2001). Diagnosing micrometastases with routine histopathology can be accomplished only on extensive serial sections and extensive lymph node dissection, which are not useful in pancreatic cancer. However, occult metastases can be more easily detected by immunohistochemical or molecular methods designed to recognize certain tumorassociated antigens, lineage-specific markers, or distinct tumor-related gene mutations. Immunohistochemical methods use antibodies against a variety of epithelial cell markers such as cytokeratins, CA 19.9, carcinoembryonic antigen (CEA), or Ber-Ep 4. These markers are
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Copyright © 2005 by Elsevier (USA) All rights reserved.
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314 often not specific because nonmalignant cells are also able to express them and thus might deliver falsepositive results. It has been demonstrated that CA 19.9 antibodies can be detected in 60% of regional lymph nodes dissected as a result of chronic pancreatitis (Ridwelski et al., 2001). As in histopathology, the limiting factor is the analysis of tissue in stepwise serial sections and not as a whole specimen. As shown in other gastrointestinal malignancies, specific genetic disorders, e.g., mutations, can be used in pancreatic cancer to detect micrometastases in different body compartments (Pantel and von KnebelDoeberitz, 2000). According to various analyses, mutated K-ras gene has been found in a range from 70% to 95% of pancreatic adenocarcinomas, and the site of mutation is restricted to codon 12 of the K-ras gene (Almoguera et al.,1988; Löhr et al., 2000). Therefore, mutant K-ras is one of the most promising genetic alterations in ductal adenocarcinoma to detect malignant cells by molecular techniques. In several studies, micrometastases have been detected at the molecular level by sensitive polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) assays for the detection of mutant codon 12 K-ras allele (Banerjee et al., 1997; Niedergethmann et al., 2002). Nevertheless, it has been discussed whether micrometastases are of prognostic significance or if these are only “dormant” tumor cells arrested in G0-phase (Pantel and von Knebel-Doeberitz, 2000; Keene et al., 2001). This chapter attempts to answer these questions and provides detailed information of immunohistochemical and molecular methods to detect lymph node micrometastases in pancreatic cancer. The following investigations were carried out with 69 specimens of resected ductal pancreatic adenocarcinomas (Department of Surgery, University-Hospital Mannheim). In all cases, corresponding paraaortic lymph nodes were obtained by en bloc dissection from the suprarenal paraaortic region. All cases had tumor-free margins. We used nine surgical specimens of patients diagnosed for adenomas of Vater’s papilla (n = 4), chronic pancreatitis (n = 4), and one cystadenoma with corresponding paraaortic lymph nodes as controls. Normal pancreatic tissue served as a negative control for each individual subject. Tumor, normal tissue, and paraaortic lymph nodes were used for histopathology and immunohistochemistry, and extended deoxyribonucleic acid (DNA) investigations were prepared. Each paraaortic lymph node was divided into two portions, one of which was formalinfixed and paraffin-embedded for immunohistochemistry, and the other one was stored frozen at −80°C until PCR analysis.
III Pancreatic Carcinoma
MATERIALS Immunohistochemistry for Pan-Cytokeratin 1. Buffered formalin (4%): 100 ml phosphate buffer saline (PBS) buffer (PBS-buffer: 200 g Natriumhydrogenphosphat, 325 g Dinatiumhydrogenphosphat, bring volume to 5 L with distilled water), 100 ml formalin (37%), bring volume to 800 ml with distilled water. 2. Paraffin wax. 3. Microtome. 4. Xylene. 5. Alcohol 100%, 96%, and 80%. 6. Brood-cupboard. 7. 0.1% Trypsine solution: 100 mg trypsine, 100 mg calciumchloride, bring volume to 100 ml with distilled water. 8. Deionized water. 9. Blocking-solution (Zymed, Bad Homburg, Germany). 10. Primary anti-pan-cytokeratin antibody (Zymed; NCC-pan-ck, dilution 1:50). 11. Tris-buffered saline (TBS) (pH 7.6): 6.055 g Tris buffer, 8.52 g NaCl, 37 ml 1 N HCl, bring volume to 1 L with distilled water, 0.5 ml Tween 20. 12. Secondary biotinylated antibody (Zymed). 13. Streptavidin-labeled immunoalkaline phosphatase (Zymed, Bad Homburg, Germany). 14. Substrate-chromogen-mixture (SCM): 0.121 g Tris-buffer and 0.5 ml 1 N HCl, bring volume to 10 ml distilled water, 2 mg Naphtol AS-MX Phosphat, 2.4 mg Levamisole hydrochloride, and 10 mg Fast Red TR Salt. 15. Hematoxylin. 16. Kaiser’s glycerine gelatin (Merck, Darmstadt, Germany).
Molecular Detection by Mutated K-Ras 1. Human pancreatic adenocarcinoma cell line Pa-Tu-8902 (DSMZ: German Department of Human and Animal Cell Cultures, Mainz, Germany). 2. QIAamp DNA mini Kit (Qiagen, Hilden, Germany). 3. Photometer. 4. Thermocycler. 5. 0.5-ml tubes (for PCR) and 1.5-ml/2-ml tubes (for DNA extraction) (Eppendorf, Hamburg, Germany). 6. Thermocycler (Perkin-Elmer Inc., Norfolk, VA). 7. 25 mM MgCl2. 8. 5 mM of each dNTP (deoxyribonucleotidetriphosphate). 9. 0.3 µM 3′-primer (5′-GTC CTG CAC CAG AAA TAT TGC-3′) and 5′-primer (5′-ACT GAA TAT AAA CTT GTG GTA GTT GGA CCT-3′) (MWG Biotech AG, Ebersberg, Germany).
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3 Molecular Detection of Micrometastases in Pancreatic Cancer 10. Two units of Taq polymerase in PCR-buffer containing 500 mM KCl, 15 mM MgCl2, and 100 mM Tris-HCl (Amersham Pharmacia Biotech, Freiburg, Germany). 11. Ampermeter. 12. Agarose (ultra PURE, Paisley, UK). 13. Tris-acetat-ethylenediamine tetraacetic acid (TAE)-buffer: dilution 1:10 (Sigma Aldrich Chemie, Steinheim, Germany). 14. Ethidium bromide (Invitrogen Corporation, Paisley, Scotland). 15. Electrophoresis-box. 16. Loading buffer (Invitrogen Corporation, Paisley, Scotland). 17. DNA-ladder (Invitrogen Corporation, Paisley, Scotland). 18. Gel documentation system (Bio-Rad Laboratories GmbH, München, Germany). 19. Endonuclease Mva I system containing Mva I and incubation-buffer (Roche, Mannheim, Germany).
METHODS Immunohistochemistry for Pan-Cytokeratin 1. Fix tissue blocks in 4% buffered formalin for 24 hr and embed in paraffin. 2. Cut serial sections of 5 µm thickness of paraaortal lymph nodes with microtome. 3. Deparaffinizing and rehydration: Rinse the samples for 5 min in xylene, then for 2 mm in 100% alcohol; repeat the procedure with 96% alcohol and 80% alcohol. 4. Dry the samples for 12 hr in brood-cupboard at 37°C. 5. Incubate the samples for 40 min in 0.1% trypsine solution at 37°C for antigen retrieval. 6. Rinse sections in deionized water for 5 min. 7. For immunohistological detection the biotinstreptavidin-amplified indirect immunoalkaline phosphatase method (Zymed, Bad Homburg, Germany) was used (as described in manufacturer’s instructions). 8. Add 100 µl of blocking solution to each sample and incubate for 15 min at room temperature; rinsing is not necessary. 9. Thereafter, the primary anti-pan-cytokeratin antibody is applied (100 µl) for 1 hr at room temperature. 10. Control sections are incubated with TBS instead of the primary antibody. 11. Rinse all samples for 5 min in TBS. 12. The sections are incubated for 10 min with the secondary biotinylated antibody (100 µl). 13. Rinse the samples for 5 min in TBS. 14. Streptavidin-labeled immunoalkaline phosphatase is added for 10 min (2 drops or 100 µl).
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15. Rinse the samples for 5 min in TBS. 16. The SCM-staining is applied for 30 min at room temperature. 17. Sections are washed in tap water. 18. Counterstain the samples with hematoxylin for 1–3 min at room temperature, and mount them in Kaiser’s Glycerine Gelatin.
Molecular Detection by Mutated K-Ras 1. The human pancreatic adenocarcinoma cell line Pa-Tu-8902 from DSMZ (German Department of Human and Animal Cell Cultures) serves as a positive control. 2. Distilled water without tissue is used as an internal negative control. 3. For DNA extraction, the scheme of the QIAamp DNA mini Kit is used (as described in manufacturer’s instructions). 4. After DNA extraction, the optical density (OD) at 260 and 280 nm, respectively, is measured. 5. Aliquots corresponding to 200 ng of genomic DNA are submitted to PCR. 6. The PCR is performed using 0.5 ml tubes and a thermocycler. 7. Reactions are carried out in a total volume of 50 µl containing final concentrations of 20 µl aliquot of genomic DNA; 5 µl PCR buffer (see earlier steps); 1 µl 25 mM MgC12; 2 µl with 5 mM of each dNTP; 3 µl 0.3 µM of each 3′- and 5′-primer, respectively; 0.4 µl with 2 units of Taq polymerase; and 15.6 µl distilled water. 8. Experiments using the technique of mismatch PCR are carried out starting with an initial step for denaturation at 95°C for 5 min; then, 35 cycles are run including denaturing at 95°C for 45 sec, annealing at 59°C for 45 sec, and DNA synthesis at 72°C for 90 sec, followed by a final step at 72°C for 10 min. The primers used are leading to a PCR product of 147 base pairs (bp) in length. 9. Successful PCR is shown by ethidium bromide agarose gel (2%) electrophoresis. 10. Bring 1 g agarose in 50 ml TAE buffer, and heat the solution until agarose is completely dissolved. 11. Add 2.5 µl ethidium bromide after 2 min cooling of the solution. 12. Pour the solution into the electrophoresis box and bring into the gel-comb. 13. After cooling for 30 min at room temperature, fill TAE buffer until complete coverage of the gel is obtained. 14. After, bring 10 µl DNA aliquot and 2 µl loading buffer into the gel-pouch. 15. Fill reference line with 5 µl DNA ladder and 2 µl loading buffer.
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316 16. To run gel, use ampermeter (70–100 V) for 1 hr. Digital analysis is recommended. 17. After electrophoresis digest, aliquots of genomic DNA with restriction endonuclease Mva I according to the manufacturer’s instructions (Roche, Mannheim, Germany). 18. Reactions are carried out in a total volume of 20 µl containing final concentrations of 10 µl DNA aliquot after PCR, 1 µl Mva I, 2 µl incubations buffer, and 7 µl distilled water; incubate the aliquots for 5 hr at 37°C. 19. Analyze PCR again in ethidium bromide agarose gel electrophoresis (2%) as described in Steps 9–12, then fill in 20 µl aliquot with 3 µl loading buffer into the gel-pouch and run gel using ampermeter (70–100 V) for 90 min. 20. Each individual sample is analyzed in triplicate manner.
RESULTS
Figure 46. Occult tumor cells in a paraaortic lymph node of a patient with pancreatic adenocarcinoma after curative pancreatico-duodenectomy: Positive immunoreactions of carcinoma cells within the lymphoreticular tissue of a paraaortic node using anti-pan-cytokeratin antibodies; counterstained with hematoxylin (magnification: 200X).
Immunohistochemical Examination Using the pan-cytokeratin, antibody-positive immunoreaction of single or grouped carcinoma cells within the lymphoreticular tissue of the nodes was found. Positive immunoreactivity was observed in 5 out of 69 specimens (7.2%). As a consequence, these five cases were diagnosed for micrometastases in paraaortic lymph nodes. In Figure 46 a strong cytoplasmic immunoreaction of carcinoma cells within the lymphoreticular tissue of a paraaortic node is demonstrated. No positive staining was found except in cancer cells. In comparison, using routine histology with hematoxylin and eosin staining, one could find occult tumor cells in paraaortic lymph nodes only in 3 of 69 specimens (4%).
in no other case of the control group. In addition, no mutation was detected in all individual negative controls (normal pancreatic tissue). In pancreatic adenocarcinoma specimen, 12 cases (12/69, 17.4%) showed mutant K-ras gene in corresponding paraaortic lymph nodes. Therefore, in subjects with K-ras–positive primary tumors, micrometastases could be found in 29% (12/42). Immunohistological staining was able to detect tumor cells in lymph nodes in only five of these subjects. Mutated K-ras, an indicator for tumor-cell DNA, that is found in paraaortic lymph nodes, can be set equivalent with micrometastases. There was no mutated K-ras found in paraaortic lymph node specimen of the control group.
Molecular Detection of Micrometastases The use of the described mismatch primers results in amplification of a 147-bp product. A restriction site for Mva I is created in wild-type (wt), but not in mutated K-ras using the 5′-primer, whereas 3′-primer inserts a restriction site in both, serving as an internal control for a successful Mva I digestion. Thus, a wildtype allele, is detected by the appearance of a 107-bp band, whereas in cells harboring K-ras mutations, an additional band of 136 bp can be visualized. Forty-two of 69 (61%) ductal adenocarcinomas harboring the K-ras mutation in primary tumor were identified, visualized by appearence of the additional 136-bp band after Mva I digestion and subsequent gel electrophorsis. In the control specimen K-ras mutation was found in 1 of 4 (25%) adenomas of Vater’s papilla but
DISCUSSION Ductal adenocarcinoma of the pancreas is associated with the worst 5-year survival rate of any form of gastrointestinal cancer, even after curative resection (Hartel et al., 2002; Richter et al., 2003). However, until now, the question of why some patients die within a few months after curative resection because of metastases or local recurrence, whereas others enjoy a more favorable outcome has not been sufficiently answered. We argue that the present histologic sectioning may lack sufficient sensitivity for assessing lymph nodes and surgical resection margins for tumor involvement and minimal residual cancer, respectively. Therefore, highly sensitive methods have been investigated to assess patients with other types of
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3 Molecular Detection of Micrometastases in Pancreatic Cancer gastrointestinal cancers for minimal residual disease (Vogel et al., 2001) In 2001, PCR-based assays were used for detecting MRD in histologically negative lymph nodes from patients with gastric and colorectal cancer (Keene and Demeure, 2001). For pancreatic cancer, it has been suggested that immunohistochemical antibodies be used against epithelial- or tumor-associated antigens, such as cytokeratin (CK) or CEA in various body compartments for the detection of occult micrometastases at the time of surgery (Niedergethmann et al., 2002). One group suggested combining routine histopathology with CK immunohistology for detection of MRD after surgery because the latter method increases the sensitivity for micrometastases (Ridweiski et al., 2001). Because mutant K-ras is the most evident genetic alteration in pancreatic adenocarcinoma, several investigators have performed analyses for this gene as a marker for occult tumor cells, e.g., in stool, blood, or tissue specimens (Tada et al., 1991). It has been demonstrated that detection of K-ras mutation in
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regional and paraaortic lymph nodes is superior to routine pathologic examination, and it has been suggested that using this for prognostic reevaluation (Demeure et al., 1998). Tamagawara et al. (1997) could reveal that evidence of MRD in regional lymph nodes by detection of mutant K-ras was a predictive marker for recurrence in patients with pancreatic adenocarcinoma. From our investigations the following conclusions can be drawn: 1) patients with positive K-ras mutations in paraaortic lymph nodes will have a significantly worse prognosis than those without, 2) K-ras positive paraaortic lymph nodes are an independent prognostic marker after curative resection, and 3) patients with K-ras mutations in paraaortic lymph nodes will suffer significantly earlier from recurrent cancer (Niedergethmann et al., 2002). A summary of recent methods to detect minimal residual disease in pancreatic cancer is given in Table 20. With the described method one could find K-ras mutations in a similar range, as described by other authors
Table 20. Minimal Residual Disease in Pancreatic Adenocarcinoma: Summary of Detection Methods and Prognostic Implications
Author/year
Patients [n]
Compartment
Method
Ando 1997 Bilchik 2000
15 33
Paraaortic LN Blood
PCR PCR
Demeure 1998 Funaki 1998 Hosch 1997 Niedergethmann 2002
29 3 18 69
Reg. LN Blood Reg. LN Paraaortic LN
PCR PCR IHC Histology IHC
Ridwelski 2000
15
Reg. LN
PCR IHC
Roder 1999
48
Bone marrow
IHC
Tamagawara 1997 Vogel 1999
12 80
Reg. LN Bone marrow
PCR IHC
Warshaw 1991 Yamada 2000 Yamaguchi 2000
40 25 31
Peritoneum Peritoneum Reg. LN Paraaortic LN
Cytology IHC PCR
Z’graggen 2001
105
Blood
IHC
Bone marrow
Marker K-ras (DNA) MET, Gal-Nac-T, βhCG K-ras (DNA) CEA (mRNA) Ber-EP4 Hematoxylin and eosin Cytokeratin (pan CK) K-ras (DNA) Cytokeratin (AE1, AE3) Cytokeratin (CKKL- 1, CK2, A45-B/B3) K-ras (DNA) CA19.9, C1P83, Ra96, Kl−1 – K-ras (DNA) K-ras, P53 (DNA) Cytokeratin (AE1, AE3)
Detection of Micrometastases (%)
Prognostic Correlation
42 94
n.a. n.a.
68 100 72 4
+ n.a. + +
7 17 100
+ + (IPF) n.a.
52
+
83 38 (BM)
+ +
39 (P) 30 52 33
+ + +
26
−
24
−
BM; bone marrow; IHC; immunohistochemistry; IPF; independent prognostic factor (multivariate analysis); P; peritoneal cavity; Paraaortic LN; paraaortic lymph nodes; PCR, Polymerase chain reaction; n.a.; not applicable; Reg. LN; regional lymph nodes.
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318 (Demeure et al., 1998; Tamagawa et al., 1997). Minimal residual disease in paraaortic lymph nodes after curative resection can be detected in 7% by cytokeratin immunohistochemistry and in 17% by the PCR-based assay to detect mutant K-ras, underlining the sensitivity of the presented molecular detection system. In comparison, in routine histology with hematoxylin and eosin staining, occult tumor cells in those lymph nodes can only be diagnosed in 4%. Therefore, we believe, in accordance with other authors (Demeure et al.,1998; Tamagawa et al., 1997; Yamada et al., 2000), that the identification of K-ras mutations for the detection of MRD in lymph nodes is superior to the morphologic approach of the pathologic examination and that cancer cells may have already spread to lymph nodes that were histologically diagnosed as negative. In conclusion, the routine, stepwise sectioning pathologic examination lacks the detection of micrometastases in many patients, whereas PCR-based assays to screen for mutated K-ras analyzes tumor cell DNA of the whole sample reveals a higher sensitivity. This can enrich the routine histopathologic examination in resectable pancreatic cancer and might precisely identify the “real” tumor stage.
References Almoguera, C., Shibata, D., Forrester, K., Martin, J., Arnheim, N., and Perucho, M. 1988. Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell 53:549–555. Ando, N., Nakao, A., Nomoto, S., Takeda, S., Kaneko, T., Kurokawa, T., Nonami, T., and Takagi, H. 1997. Detection of mutant K-ras in dissected paraaortic lymph nodes of patients with pancreatic adenocarcinoma. Pancreas 15:374–378. Banerjee, S.K., Makdisi, W.F., Weston, A.P., and Campbell, D.R. 1997. A two-step enriched nested PCR technique enhances sensitivity for detection of codon 12 K-ras mutations in pancreatic adenocarcinoma. Pancreas 15:16–24. Bilchik, A., Miyashiro, M., Kelley, M., Kuo, C., Fujiwara, Y., Nakamori, S., Monden, M., and Hoon, D.S. 2000. Molecular detection of metastatic pancreatic carcinoma cells using a multimarker reverse transcriptase-polymerase chain reaction assay. Cancer 88:1037–1044. Demeure, M.J., Doffek, K.M., Komorowski, R.A., and Wilson, S.D. 1998. Adenocarcinoma of the pancreas: Detection of occult metastases in regional lymph nodes by a polymerase chain reaction based assay. Cancer 83:1328–1334. Elsässer, H.P., Lehr, U., Agricola, B., and Kern, H.F. 1993. Structural analysis of a new highly metastatic cell line PaTu 8902 from a primary human pancreatic adenocarcinoma. Virch. Arch. B Cell Pathol. 64:201–207. Funaki, N.O., Tanaka, J., Hosotani, R., Kogire, M., Suwa, H., Imamura, M. 1998. Quantitative analysis of carcinoembryonic antigen messenger RNA in peripheral venous blood and portal blood of patients with pancreatic ductal adenocarcinoma. Clin. Cancer Res. 4:855–860. Hartel, M., Niedergethmann, M., Farag-Soliman, M., Sturm, J.W., Richter, A., Trede, M., and Post, S. 2002. Benefit of venous resection for ductal adenocarcinoma of the pancreatic head. Eur. J. Surg. 168:707–712.
Hosch, S.B., Knoefel, W.T., Metz, S., Stoecklein, N, Niendorf, A., Broelsch, C.E., and Izicki, J.R. 1997. Early lymphatic tumor cell dissemination in pancreatic cancer: Frequency and prognostic significance. Pancreas 15:152–159. Keene, S.A., and Demeure, M.J. 2001. The clinical significance of micrometastases and molecular metastases. Surgery 129:1–5. Löhr, M., Maisonneuve, P., and Lowenfels, A.B. 2000. K-Ras mutations and benign pancreatic disease. Int. J. Pancreatol. 27:93–103. Niedergethmann, M., Rexin, M., Hildenbrand, R., Knob, S., Sturm, J.W., Richter, A., and Post, S. 2002. Prognostic implications of routine, immunohistological, and molecular staging in resectable pancreatic adenocarcinoma. Am. J. Surg. Pathol. 26:1578–1587. Pantel, K., and von Knebel-Doeberitz, M. 2000. Detection and clinical relevance of micrometastatic cancer cells. Curr. Opin. Oncol. 12:95–101. Richter, A., Niedergethmann, M., Hartel, M., Sturm, J.W., Lorenz, D., Trede, M., and Post, S. 2003. Longterm results of partial pancreaticoduodenectomy for ductal adenocarcinoma of the pancreatic head: A 25-year experience. World J. Surg. 27:324–329. Ridwelski, K., Meyer, F., Fahlke, J., Kasper, U., Roessner, A., and Lippert, H. 2001. Value of cytokeratin and Ca 19-9 antigen in immunohistological detection of disseminated tumor cells in lymph nodes in pancreas carcinoma. Chirurg 72:920–926. Roder, J.D., Thorban, S., Pantel, K., and Siewert, J.R. 1999. Micrometastases in bone marrow: Prognostic indicators for pancreatic cancer. World J. Surg. 23:888–891. Tada, M., Omata, M., and Otho, M. 1991. Clinical application of ras gene mutation for diagnosis of pancreatic adenocarcinoma. Gastroenterology 100:233–238. Tamagawa, E., Ueda, M., Sugano, K., Mukai, K., Ogata, S., and Kitajima, M. 1997. Pancreatic lymph nodal and plexus micrometastasis detected by enriched polymerase chain reaction and nonradioisotopic single-trans conformation polymorphism analysis: A new predictive factor for recurrent pancreatic carcinoma. Clin. Cancer Res. 3:2143–2149. Trede, M., Richter, A., and Wendl, K. 2001. Personal observations, opinions, and approaches to cancer of the pancreas and the periampullary area. Surg. Clin. North Am. 81:595–610. Vogel, I., Kruger, U., Marxsen, J., Soeth, E., Kalthoff, H., Henne-Bruns, D., Kremer, B., and Juhl, H. 1999. Disseminated tumor cells in pancreatic cancer patients detected by immunocytology: A new prognostic factor. Clin. Cancer Res. 5:593–599. Vogel, I., and Kalthoff, H. 2001. Disseminated tumour cells: Their detection and significance for prognosis of gastrointestinal and pancreatic carcinomas. Virch. Arch. 439:109–117. Warshaw, A.L. 1991. Implications of peritoneal cytology for staging of early pancreatic cancer. Am. J. Surg. 161:26–29. Yamada, T., Nakamori, S., Ohzato, H., Higaki, N., Aoki, T., Oshimda, S., Shiozaki, K., Okami, J., Hayashi, N., Nagano, H., Dono, K., Umeshita, K., Sakon, M., and Monden, M. 2000. Outcome of pancreatic cancer patients based on genetic lymph node staging. Int. J. Oncol. 16:1165–1171. Yamaguchi, K., Chijiiwa, K., Torato, N., Kinoshita, M., and Tanaka, M. 2000. Ki-ras codon 12 point and P53 mutations: A molecular examination of the main tumor, liver, portal vein, peripheral arterial blood and para-aortic lymph node in pancreatic cancer. Am. J. Gastroenterol. 95:1939–1945. Z’graggen, K., Centeno, B.A., Fernandez-del Castillo, C., Jimenez, R.E., Werner, J., and Warshaw, A.L. 2001. Biological implications of tumor cells in blood and bone marrow of pancreatic cancer patients. Surgery 129:537–546.
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3 Molecular Detection of Micrometastases in Pancreatic Cancer Marco Niedergethmann, Stefanie Knob, and Stefan Post
Introduction Despite advances in surgical oncology, local recurrence and distant metastases are the major problems in treating pancreatic cancer. Only 25% of all pancreatic cancers are resectable, and only patients who have been curatively resected (R0) enjoy a favorable outcome (Richter et al., 2003). However, the survival after surgery still remains poor. Most surgeons and oncologists treating pancreatic cancer believe that local recurrence represents the growth of residual tumor (Pantel et al., 2000). Despite histologically confirmed curative resection (no residual tumor: R0) and tumor-free lymph nodes, most patients will suffer from postoperative local recurrence or distant metastases (Trede et al., 2001). Occult dissemination of tumor cells beyond resection margins is of paramount importance for prognosis. This is impressively shown by a 50% recurrence rate within 2 years after surgery for pancreatic cancer (Richter et al., 2003; Trede et al., 2001). In recent years, numerous new techniques to detect minimal residual disease (MRD), including immunohistochemical and molecular assays, have been introduced Handbook of Immunohistochemistry and in situ Hybridization of Human Carcinomas, Volume 3: Molecular Genetics, Liver Carcinoma, and Pancreatic Carcinoma
to reveal the accurate resection status (R0 versus R1) and enrich the routine histopathology (Niedergethmann et al., 2002). These techniques detect micrometastases and disseminated tumor cells not only in lymph nodes that had appeared tumor free in routine histology but also in body compartments such as bone marrow, peritoneal cavity, and blood (Vogel et al., 2001). From the current data, it has been concluded that routine histopathology often underestimates the true tumor stage. It is well known that micrometastases of a diameter of 3 cells are diagnosed only in 1% based on serial 5-µm sections (Keene and Demeure, 2001). Diagnosing micrometastases with routine histopathology can be accomplished only on extensive serial sections and extensive lymph node dissection, which are not useful in pancreatic cancer. However, occult metastases can be more easily detected by immunohistochemical or molecular methods designed to recognize certain tumorassociated antigens, lineage-specific markers, or distinct tumor-related gene mutations. Immunohistochemical methods use antibodies against a variety of epithelial cell markers such as cytokeratins, CA 19.9, carcinoembryonic antigen (CEA), or Ber-Ep 4. These markers are
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Copyright © 2005 by Elsevier (USA) All rights reserved.
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314 often not specific because nonmalignant cells are also able to express them and thus might deliver falsepositive results. It has been demonstrated that CA 19.9 antibodies can be detected in 60% of regional lymph nodes dissected as a result of chronic pancreatitis (Ridwelski et al., 2001). As in histopathology, the limiting factor is the analysis of tissue in stepwise serial sections and not as a whole specimen. As shown in other gastrointestinal malignancies, specific genetic disorders, e.g., mutations, can be used in pancreatic cancer to detect micrometastases in different body compartments (Pantel and von KnebelDoeberitz, 2000). According to various analyses, mutated K-ras gene has been found in a range from 70% to 95% of pancreatic adenocarcinomas, and the site of mutation is restricted to codon 12 of the K-ras gene (Almoguera et al.,1988; Löhr et al., 2000). Therefore, mutant K-ras is one of the most promising genetic alterations in ductal adenocarcinoma to detect malignant cells by molecular techniques. In several studies, micrometastases have been detected at the molecular level by sensitive polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) assays for the detection of mutant codon 12 K-ras allele (Banerjee et al., 1997; Niedergethmann et al., 2002). Nevertheless, it has been discussed whether micrometastases are of prognostic significance or if these are only “dormant” tumor cells arrested in G0-phase (Pantel and von Knebel-Doeberitz, 2000; Keene et al., 2001). This chapter attempts to answer these questions and provides detailed information of immunohistochemical and molecular methods to detect lymph node micrometastases in pancreatic cancer. The following investigations were carried out with 69 specimens of resected ductal pancreatic adenocarcinomas (Department of Surgery, University-Hospital Mannheim). In all cases, corresponding paraaortic lymph nodes were obtained by en bloc dissection from the suprarenal paraaortic region. All cases had tumor-free margins. We used nine surgical specimens of patients diagnosed for adenomas of Vater’s papilla (n = 4), chronic pancreatitis (n = 4), and one cystadenoma with corresponding paraaortic lymph nodes as controls. Normal pancreatic tissue served as a negative control for each individual subject. Tumor, normal tissue, and paraaortic lymph nodes were used for histopathology and immunohistochemistry, and extended deoxyribonucleic acid (DNA) investigations were prepared. Each paraaortic lymph node was divided into two portions, one of which was formalinfixed and paraffin-embedded for immunohistochemistry, and the other one was stored frozen at −80°C until PCR analysis.
III Pancreatic Carcinoma
MATERIALS Immunohistochemistry for Pan-Cytokeratin 1. Buffered formalin (4%): 100 ml phosphate buffer saline (PBS) buffer (PBS-buffer: 200 g Natriumhydrogenphosphat, 325 g Dinatiumhydrogenphosphat, bring volume to 5 L with distilled water), 100 ml formalin (37%), bring volume to 800 ml with distilled water. 2. Paraffin wax. 3. Microtome. 4. Xylene. 5. Alcohol 100%, 96%, and 80%. 6. Brood-cupboard. 7. 0.1% Trypsine solution: 100 mg trypsine, 100 mg calciumchloride, bring volume to 100 ml with distilled water. 8. Deionized water. 9. Blocking-solution (Zymed, Bad Homburg, Germany). 10. Primary anti-pan-cytokeratin antibody (Zymed; NCC-pan-ck, dilution 1:50). 11. Tris-buffered saline (TBS) (pH 7.6): 6.055 g Tris buffer, 8.52 g NaCl, 37 ml 1 N HCl, bring volume to 1 L with distilled water, 0.5 ml Tween 20. 12. Secondary biotinylated antibody (Zymed). 13. Streptavidin-labeled immunoalkaline phosphatase (Zymed, Bad Homburg, Germany). 14. Substrate-chromogen-mixture (SCM): 0.121 g Tris-buffer and 0.5 ml 1 N HCl, bring volume to 10 ml distilled water, 2 mg Naphtol AS-MX Phosphat, 2.4 mg Levamisole hydrochloride, and 10 mg Fast Red TR Salt. 15. Hematoxylin. 16. Kaiser’s glycerine gelatin (Merck, Darmstadt, Germany).
Molecular Detection by Mutated K-Ras 1. Human pancreatic adenocarcinoma cell line Pa-Tu-8902 (DSMZ: German Department of Human and Animal Cell Cultures, Mainz, Germany). 2. QIAamp DNA mini Kit (Qiagen, Hilden, Germany). 3. Photometer. 4. Thermocycler. 5. 0.5-ml tubes (for PCR) and 1.5-ml/2-ml tubes (for DNA extraction) (Eppendorf, Hamburg, Germany). 6. Thermocycler (Perkin-Elmer Inc., Norfolk, VA). 7. 25 mM MgCl2. 8. 5 mM of each dNTP (deoxyribonucleotidetriphosphate). 9. 0.3 µM 3′-primer (5′-GTC CTG CAC CAG AAA TAT TGC-3′) and 5′-primer (5′-ACT GAA TAT AAA CTT GTG GTA GTT GGA CCT-3′) (MWG Biotech AG, Ebersberg, Germany).
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3 Molecular Detection of Micrometastases in Pancreatic Cancer 10. Two units of Taq polymerase in PCR-buffer containing 500 mM KCl, 15 mM MgCl2, and 100 mM Tris-HCl (Amersham Pharmacia Biotech, Freiburg, Germany). 11. Ampermeter. 12. Agarose (ultra PURE, Paisley, UK). 13. Tris-acetat-ethylenediamine tetraacetic acid (TAE)-buffer: dilution 1:10 (Sigma Aldrich Chemie, Steinheim, Germany). 14. Ethidium bromide (Invitrogen Corporation, Paisley, Scotland). 15. Electrophoresis-box. 16. Loading buffer (Invitrogen Corporation, Paisley, Scotland). 17. DNA-ladder (Invitrogen Corporation, Paisley, Scotland). 18. Gel documentation system (Bio-Rad Laboratories GmbH, München, Germany). 19. Endonuclease Mva I system containing Mva I and incubation-buffer (Roche, Mannheim, Germany).
METHODS Immunohistochemistry for Pan-Cytokeratin 1. Fix tissue blocks in 4% buffered formalin for 24 hr and embed in paraffin. 2. Cut serial sections of 5 µm thickness of paraaortal lymph nodes with microtome. 3. Deparaffinizing and rehydration: Rinse the samples for 5 min in xylene, then for 2 mm in 100% alcohol; repeat the procedure with 96% alcohol and 80% alcohol. 4. Dry the samples for 12 hr in brood-cupboard at 37°C. 5. Incubate the samples for 40 min in 0.1% trypsine solution at 37°C for antigen retrieval. 6. Rinse sections in deionized water for 5 min. 7. For immunohistological detection the biotinstreptavidin-amplified indirect immunoalkaline phosphatase method (Zymed, Bad Homburg, Germany) was used (as described in manufacturer’s instructions). 8. Add 100 µl of blocking solution to each sample and incubate for 15 min at room temperature; rinsing is not necessary. 9. Thereafter, the primary anti-pan-cytokeratin antibody is applied (100 µl) for 1 hr at room temperature. 10. Control sections are incubated with TBS instead of the primary antibody. 11. Rinse all samples for 5 min in TBS. 12. The sections are incubated for 10 min with the secondary biotinylated antibody (100 µl). 13. Rinse the samples for 5 min in TBS. 14. Streptavidin-labeled immunoalkaline phosphatase is added for 10 min (2 drops or 100 µl).
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15. Rinse the samples for 5 min in TBS. 16. The SCM-staining is applied for 30 min at room temperature. 17. Sections are washed in tap water. 18. Counterstain the samples with hematoxylin for 1–3 min at room temperature, and mount them in Kaiser’s Glycerine Gelatin.
Molecular Detection by Mutated K-Ras 1. The human pancreatic adenocarcinoma cell line Pa-Tu-8902 from DSMZ (German Department of Human and Animal Cell Cultures) serves as a positive control. 2. Distilled water without tissue is used as an internal negative control. 3. For DNA extraction, the scheme of the QIAamp DNA mini Kit is used (as described in manufacturer’s instructions). 4. After DNA extraction, the optical density (OD) at 260 and 280 nm, respectively, is measured. 5. Aliquots corresponding to 200 ng of genomic DNA are submitted to PCR. 6. The PCR is performed using 0.5 ml tubes and a thermocycler. 7. Reactions are carried out in a total volume of 50 µl containing final concentrations of 20 µl aliquot of genomic DNA; 5 µl PCR buffer (see earlier steps); 1 µl 25 mM MgC12; 2 µl with 5 mM of each dNTP; 3 µl 0.3 µM of each 3′- and 5′-primer, respectively; 0.4 µl with 2 units of Taq polymerase; and 15.6 µl distilled water. 8. Experiments using the technique of mismatch PCR are carried out starting with an initial step for denaturation at 95°C for 5 min; then, 35 cycles are run including denaturing at 95°C for 45 sec, annealing at 59°C for 45 sec, and DNA synthesis at 72°C for 90 sec, followed by a final step at 72°C for 10 min. The primers used are leading to a PCR product of 147 base pairs (bp) in length. 9. Successful PCR is shown by ethidium bromide agarose gel (2%) electrophoresis. 10. Bring 1 g agarose in 50 ml TAE buffer, and heat the solution until agarose is completely dissolved. 11. Add 2.5 µl ethidium bromide after 2 min cooling of the solution. 12. Pour the solution into the electrophoresis box and bring into the gel-comb. 13. After cooling for 30 min at room temperature, fill TAE buffer until complete coverage of the gel is obtained. 14. After, bring 10 µl DNA aliquot and 2 µl loading buffer into the gel-pouch. 15. Fill reference line with 5 µl DNA ladder and 2 µl loading buffer.
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316 16. To run gel, use ampermeter (70–100 V) for 1 hr. Digital analysis is recommended. 17. After electrophoresis digest, aliquots of genomic DNA with restriction endonuclease Mva I according to the manufacturer’s instructions (Roche, Mannheim, Germany). 18. Reactions are carried out in a total volume of 20 µl containing final concentrations of 10 µl DNA aliquot after PCR, 1 µl Mva I, 2 µl incubations buffer, and 7 µl distilled water; incubate the aliquots for 5 hr at 37°C. 19. Analyze PCR again in ethidium bromide agarose gel electrophoresis (2%) as described in Steps 9–12, then fill in 20 µl aliquot with 3 µl loading buffer into the gel-pouch and run gel using ampermeter (70–100 V) for 90 min. 20. Each individual sample is analyzed in triplicate manner.
RESULTS
Figure 46. Occult tumor cells in a paraaortic lymph node of a patient with pancreatic adenocarcinoma after curative pancreatico-duodenectomy: Positive immunoreactions of carcinoma cells within the lymphoreticular tissue of a paraaortic node using anti-pan-cytokeratin antibodies; counterstained with hematoxylin (magnification: 200X).
Immunohistochemical Examination Using the pan-cytokeratin, antibody-positive immunoreaction of single or grouped carcinoma cells within the lymphoreticular tissue of the nodes was found. Positive immunoreactivity was observed in 5 out of 69 specimens (7.2%). As a consequence, these five cases were diagnosed for micrometastases in paraaortic lymph nodes. In Figure 46 a strong cytoplasmic immunoreaction of carcinoma cells within the lymphoreticular tissue of a paraaortic node is demonstrated. No positive staining was found except in cancer cells. In comparison, using routine histology with hematoxylin and eosin staining, one could find occult tumor cells in paraaortic lymph nodes only in 3 of 69 specimens (4%).
in no other case of the control group. In addition, no mutation was detected in all individual negative controls (normal pancreatic tissue). In pancreatic adenocarcinoma specimen, 12 cases (12/69, 17.4%) showed mutant K-ras gene in corresponding paraaortic lymph nodes. Therefore, in subjects with K-ras–positive primary tumors, micrometastases could be found in 29% (12/42). Immunohistological staining was able to detect tumor cells in lymph nodes in only five of these subjects. Mutated K-ras, an indicator for tumor-cell DNA, that is found in paraaortic lymph nodes, can be set equivalent with micrometastases. There was no mutated K-ras found in paraaortic lymph node specimen of the control group.
Molecular Detection of Micrometastases The use of the described mismatch primers results in amplification of a 147-bp product. A restriction site for Mva I is created in wild-type (wt), but not in mutated K-ras using the 5′-primer, whereas 3′-primer inserts a restriction site in both, serving as an internal control for a successful Mva I digestion. Thus, a wildtype allele, is detected by the appearance of a 107-bp band, whereas in cells harboring K-ras mutations, an additional band of 136 bp can be visualized. Forty-two of 69 (61%) ductal adenocarcinomas harboring the K-ras mutation in primary tumor were identified, visualized by appearence of the additional 136-bp band after Mva I digestion and subsequent gel electrophorsis. In the control specimen K-ras mutation was found in 1 of 4 (25%) adenomas of Vater’s papilla but
DISCUSSION Ductal adenocarcinoma of the pancreas is associated with the worst 5-year survival rate of any form of gastrointestinal cancer, even after curative resection (Hartel et al., 2002; Richter et al., 2003). However, until now, the question of why some patients die within a few months after curative resection because of metastases or local recurrence, whereas others enjoy a more favorable outcome has not been sufficiently answered. We argue that the present histologic sectioning may lack sufficient sensitivity for assessing lymph nodes and surgical resection margins for tumor involvement and minimal residual cancer, respectively. Therefore, highly sensitive methods have been investigated to assess patients with other types of
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3 Molecular Detection of Micrometastases in Pancreatic Cancer gastrointestinal cancers for minimal residual disease (Vogel et al., 2001) In 2001, PCR-based assays were used for detecting MRD in histologically negative lymph nodes from patients with gastric and colorectal cancer (Keene and Demeure, 2001). For pancreatic cancer, it has been suggested that immunohistochemical antibodies be used against epithelial- or tumor-associated antigens, such as cytokeratin (CK) or CEA in various body compartments for the detection of occult micrometastases at the time of surgery (Niedergethmann et al., 2002). One group suggested combining routine histopathology with CK immunohistology for detection of MRD after surgery because the latter method increases the sensitivity for micrometastases (Ridweiski et al., 2001). Because mutant K-ras is the most evident genetic alteration in pancreatic adenocarcinoma, several investigators have performed analyses for this gene as a marker for occult tumor cells, e.g., in stool, blood, or tissue specimens (Tada et al., 1991). It has been demonstrated that detection of K-ras mutation in
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regional and paraaortic lymph nodes is superior to routine pathologic examination, and it has been suggested that using this for prognostic reevaluation (Demeure et al., 1998). Tamagawara et al. (1997) could reveal that evidence of MRD in regional lymph nodes by detection of mutant K-ras was a predictive marker for recurrence in patients with pancreatic adenocarcinoma. From our investigations the following conclusions can be drawn: 1) patients with positive K-ras mutations in paraaortic lymph nodes will have a significantly worse prognosis than those without, 2) K-ras positive paraaortic lymph nodes are an independent prognostic marker after curative resection, and 3) patients with K-ras mutations in paraaortic lymph nodes will suffer significantly earlier from recurrent cancer (Niedergethmann et al., 2002). A summary of recent methods to detect minimal residual disease in pancreatic cancer is given in Table 20. With the described method one could find K-ras mutations in a similar range, as described by other authors
Table 20. Minimal Residual Disease in Pancreatic Adenocarcinoma: Summary of Detection Methods and Prognostic Implications
Author/year
Patients [n]
Compartment
Method
Ando 1997 Bilchik 2000
15 33
Paraaortic LN Blood
PCR PCR
Demeure 1998 Funaki 1998 Hosch 1997 Niedergethmann 2002
29 3 18 69
Reg. LN Blood Reg. LN Paraaortic LN
PCR PCR IHC Histology IHC
Ridwelski 2000
15
Reg. LN
PCR IHC
Roder 1999
48
Bone marrow
IHC
Tamagawara 1997 Vogel 1999
12 80
Reg. LN Bone marrow
PCR IHC
Warshaw 1991 Yamada 2000 Yamaguchi 2000
40 25 31
Peritoneum Peritoneum Reg. LN Paraaortic LN
Cytology IHC PCR
Z’graggen 2001
105
Blood
IHC
Bone marrow
Marker K-ras (DNA) MET, Gal-Nac-T, βhCG K-ras (DNA) CEA (mRNA) Ber-EP4 Hematoxylin and eosin Cytokeratin (pan CK) K-ras (DNA) Cytokeratin (AE1, AE3) Cytokeratin (CKKL- 1, CK2, A45-B/B3) K-ras (DNA) CA19.9, C1P83, Ra96, Kl−1 – K-ras (DNA) K-ras, P53 (DNA) Cytokeratin (AE1, AE3)
Detection of Micrometastases (%)
Prognostic Correlation
42 94
n.a. n.a.
68 100 72 4
+ n.a. + +
7 17 100
+ + (IPF) n.a.
52
+
83 38 (BM)
+ +
39 (P) 30 52 33
+ + +
26
−
24
−
BM; bone marrow; IHC; immunohistochemistry; IPF; independent prognostic factor (multivariate analysis); P; peritoneal cavity; Paraaortic LN; paraaortic lymph nodes; PCR, Polymerase chain reaction; n.a.; not applicable; Reg. LN; regional lymph nodes.
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318 (Demeure et al., 1998; Tamagawa et al., 1997). Minimal residual disease in paraaortic lymph nodes after curative resection can be detected in 7% by cytokeratin immunohistochemistry and in 17% by the PCR-based assay to detect mutant K-ras, underlining the sensitivity of the presented molecular detection system. In comparison, in routine histology with hematoxylin and eosin staining, occult tumor cells in those lymph nodes can only be diagnosed in 4%. Therefore, we believe, in accordance with other authors (Demeure et al.,1998; Tamagawa et al., 1997; Yamada et al., 2000), that the identification of K-ras mutations for the detection of MRD in lymph nodes is superior to the morphologic approach of the pathologic examination and that cancer cells may have already spread to lymph nodes that were histologically diagnosed as negative. In conclusion, the routine, stepwise sectioning pathologic examination lacks the detection of micrometastases in many patients, whereas PCR-based assays to screen for mutated K-ras analyzes tumor cell DNA of the whole sample reveals a higher sensitivity. This can enrich the routine histopathologic examination in resectable pancreatic cancer and might precisely identify the “real” tumor stage.
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