Detektion von Mikrometastasen nach kurativer Resektion duktaler Adenokarzinome des Pankreas

Zusammenfassung

Einleitung: Nach vermeintlich kurativen Resektionen von Adenokarzinomen des Pankreas zeigen sich in vielen Fällen frühzeitige lokoregionäre Rezidive oder Metastasen. Okkulte Mikrometastasen entgehen oft dem histologischen Nachweis. Eine Ergänzung der Diagnostik durch sensitivere Methoden zum Nachweis einer minimal residualen Krebserkrankung oder „minimal residual disease” (MRD) ist wünschenswert. Mutationen im Kodon 12 des K-ras-Gens bieten sich aufgrund der hohen Mutationsfrequenzen als molekularer Marker an. Ziel dieser Arbeit war der Nachweis einer MRD in paraaortalen Lymphknoten nach kurativer Resektion beim duktalen Adenokarzinom des Pankreas mittels Nachweis von Mutationen im K-ras-Gen.
Methodik: Zwischen Januar 1999 und September 2000 wurden prospektiv von Patienten mit Pankreaskopftumor (n = 51) neben Tumor- und Normalgewebe auch paraaortale Lymphknoten asserviert. Bei Patienten mit duktalen Adenokarzinomen (Studiengruppe, n = 40) oder anderen Tumoren (Kontrollgruppe, n = 11) waren die paraaortalen Lymphknoten histopathologisch tumorfrei. Aus diesen Gewebeproben wurde DNA extrahiert und Mutationen im K-ras-Gen mittels einer Mismatch-PCR (Polymerase Chain Reaction) detektiert. Als Positivkontrolle diente in allen Fällen die Pankreaskarzinomzell-Linie PaTu-8902.
Ergebnisse: In 73 % (29/40) der Fälle fand sich im Primärtumor eine Mutation im K-ras-Gen. In 17 % dieser Patienten (5/29) zeigte sich diese K-ras-Mutation auch in Proben paraaortaler Lymphknoten, die folglich als Mikrometastase respektive „MRD” bewertet wurden. Die Identität der Mutation konnte mittels mutationsspezifischer Primer bewiesen werden. In den Proben der Kontrollgruppe konnte keine K-ras-Mutation detektiert werden.
Zusammenfassung: Mittels beschriebener PCR-Analytik kann sensitiv Tumorzell-DNA in Tumorgewebe und Lymphknoten nachgewiesen werden. Eine Detektion okkulter Metastasen im Sinne einer „MRD” stellt eine Ergänzung der Routinepathologie dar und ermöglicht die Definition prognostisch relevanter Subgruppen nach kurativer Resektion.

Summary

Objectives: Despite apparently curative resection adenocarcinomas of the pancreas early recur. Thus, the pathological examination should be enriched by sensitive methods to detect minimal residual disease (MRD). Mutant K-ras is the most promising genetic alteration in ductal adenocarcinoma and may serve to detect malignant cells by polymerase chain reaction (PCR) based techniques. Therefore, we set out to detect K-ras mutations by PCR for evaluation of MRD in patients after curative resection of pancreatic adenocarcinoma.
Patients and Methods: Tumor tissue and corresponding paraaortic lymph nodes were obtained from 51 patients, who underwent surgery for pancreatic head tumors. The paraaortic lymph nodes were staged as tumor-free by routine histopathology in all cases diagnosed for ductal adenocarcinoma (study group, n = 40) or other tumors (control group, n = 11). Therefore, DNA of both primary tumors and lymph nodes was extracted and analysed by a PCR-based assay with respect to mutated K-ras. As a positive control the human pancreatic cancer cell line PaTu-8902 was used.
Results: K-ras mutations were detected in 73 % (29/40) of primary tumors of ductal adenocarcinomas and in 17 % (5/29) in the corresponding paraaortic lymph nodes, which were diagnosed as tumor-free by routine pathology. The identical type of point mutation was found in primary tumors and corresponding lymph nodes by use of sequence specific primers. In the control group no K-ras mutation was detected.
Conclusion: Tumor cell DNA can be detected sensitively in tumor- and lymph node specimen with the described method. Routinely assessed, this method is able to detect MRD and could enrich the pathological examination, in order to determine prognostic relevant subgroups of patients.

Minimal residuale Krebserkrankung - K-ras Mutation - Adenokarzinom Pankreas - Kurative Resektion

Minimal residual disease - K-ras mutation - Pancreatic adenocarcinoma - Curative resection

Literatur

• 1 Almoguera C, Shibata D. et al . Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes.  Cell. 1988;  53 549-555
• 2 Ando N, Nakao A, Nomoto S. et al . Detection of mutant K-ras in dissected paraaortic lymph nodes of patients with pancreatic adenocarcinoma.  Pancreas. 1997;  15 374-378
• 3 Aoki Y, Hosaka S. et al . Reassesment of K-ras mutationsat codon 12 by direct PCR and sequencing from tissue microdissection in human pancreatic adenocarcinomas.  Pancreas. 2000;  21 152-157
• 4 Banerjee S K, Makdisi W F. et al . A two-step enriched nested PCR technique enhances sensitivity for detection of codon 12 K-ras mutations in pancreatic adenocarcinoma.  Pancreas. 1997;  15 16-24
• 5 Berndt C, Haubold K, Wenger F. et al . K-ras mutations in the stools and tissue samples from patients with malignant and nonmalignant pancreatic diseases.  Clin Chem. 1998;  44 2103-2107
• 6 Bos J L. The ras gene family and human carcinogenesis.  Mutat Res. 1988;  195 255-271
• 7 Brown D C, Purushotham A D. et al . Detection of intraoperative tumor cell dissemination in patients with breast cancer by use of reverse transcription and polymerase chain reaction.  Surgery. 1995;  117 96-101
• 8 Caldas C, Hahn S A, Hruban R H. et al . Detection of K-ras mutations in the stool of patients with pancreatic adenocarcinoma and pancreatic ductal hyperplasia.  Cancer Res. 1994;  54 3568-3573
• 9 Daus H, Bürger B. et al . Ki-ras mutation as molecular marker for carcinoma of the pancreas.  Dtsch Med Wschr. 1995;  120 821-825
• 10 Demeure M J, Doffek K M, Komorowski R A, Wilson S D. Adenocarcinoma of the pancreas.  Cancer. 1998;  83 1328-1334
• 11 Elsässer H P, Lehr U. et al . Structural analysis of a new highly metastatic cell line PaTu 8902 from a primary human pancreatic adenocarcinoma.  Virchows Archiv B Cell Pathol. 1993;  64 201-207
• 12 Eskelinen M J, Haglund U H. Prognosis of human pancreatic adenocarcinoma.  Eur J Surg. 1999;  165 292-306
• 13 Gebhardt C, Meyer W, Reichel M, Wünsch P H. Prognostic factors in the operative treatment of ductal pancreatic carcinoma.  Langenbeck's Arch Surg. 2000;  385 14-20
• 14 Grünewald K, Lyons J. et al . High frequency of Ki-ras codon 12 mutations in pancreatic adenocarcinoma.  Int J Cancer. 1989;  43 1037-1041
• 15 Hardingham J E, Kotasek D, Farmer, B, Butler R N, Mi J X. Immunobead-PCR.  Cancer Res. 1993;  53 3455-3458
• 16 Hermanek P. Pathology and biology of pancreatic ductal adenocarcinoma.  Langenbeck's Arch Surg. 1998;  383 274-281
• 17 Hohenberger W, Kastl S. Neoadjuvant and adjuvant therapy of ductal pancreatic carcinoma.  Zentralbl Chir. 2000;  125 348-355
• 18 Janes R H, Niederhuber J E. et al . National patterns of care for pancreatic cancer. Results of a survey by the Commission on Cancer.  Ann Surg. 1996;  223 261-272
• 19 Kawesha A, Ghanneh P. et al . K-ras Oncogene subtype mutations are associated with survival but not expression of P53, p16^INK4A, P21^WAF-1, Cyclin D1, ERBB-2 and ERBB-3 in resected pancreatic ductal adenocarcinoma.  Int J Cancer. 2000;  89 469-474
• 20 Kondo H, Sugano K. et al . Detection of point mutations in the K-ras oncogene at codon 12 in pure pancreatic juice for diagnosis of pancreatic carcinoma.  Cancer. 1994;  73 1589-1594
• 21 Mori M, Mimori K, Ueo H. et al . Molecular detection of circulating solid carcinoma cells in the peripheral blood: the concept of early systemic disease.  Int J Cancer. 1996;  68 739-743
• 22 Niedergethmann M, Hildenbrand R. et al . Angiogenesis and Cathepsin expression are prognostic factors in pancreatic cancer after curative resection.  Int J Pancreatol. 2000;  28 33-41
• 23 Niedergethmann M, Richter A. et al . Rare indications for a Kausch-Whipple procedure.  Eur J Surg. 2001;  167 115-119
• 24 Nitecki S S, Sarr M G, Colby T V, van Heerden J A. Long term survival after resection for ductal adenocarcinoma of the pancreas. Is it really improving?.  Ann Surg. 1995;  221 59-66
• 25 Noguchi S, Aihara R. et al . Detection of breast cancer micrometases in axillary lymph nodes by means of reverse transcriptase-polymerase chain reaction.  Am J Pathol. 1996;  148 649-656
• 26 Pantel K, von Knebel-Doeberitz M. Detection and clinical relevance of micrometastatic cancer cells.  Current Opinon Oncol. 2000;  12 95-101
• 27 Pantel K, von Knebel-Doeberitz M, Izbicki J R, Riethmüller G. Disseminierte Tumorzellen.  Chirurg. 1997;  68 1241-1250
• 28 Schoenfeld A, Luqmani Y. et al . Detection of breast cancer micormetastases in axillary lymph nodes by using polymerase chain reaction.  Cancer Res. 1994;  54 2986-2990
• 29 Smit V THB, Boo A JM. et al . K-ras codon 12 mutations occur very frequently in pancreatic adenocarcinoma.  Nucl Acids Res. 1988;  16 7773-7782
• 30 Tada M, Omata M, Otho M. Clinical application of ras gene mutation for diagnosis of pancreatic adenocarcinoma.  Gastroenterology. 1991;  100 233-238
• 31 Tada M, Teratani T, Komatsu Y, Kawabe T, Shiratori Y, Omata M. Quantitative analysis of ras gene mutation in pancreatic juice for diagnosis of pancreatic adenocarcinoma.  Dig Dis Sci. 1998;  43 15-20
• 32 Tamagawa E, Ueda M, Sugano K, Mukai K, Ogata S, Kitajima M. Pancreatic lymph nodal and plexus micrometastasis detected by enriched polymerase chain reaction and nonradioisotopic single-strans conformation polymorphism analysis.  Clin Canc Research. 1997;  3 2143-2149
• 33 Trede M. Surgical options for pancreatic cancer. In: Trede M, Carter DC (Hrsg). Surgery of the pancreas. 2. Ausgabe. Churchill Livingston, New York 1997; 471-481
• 34 Trede M, Schwall G, Saeger H D. Survival after pancreaticoduodenectomy.  Ann Surg. 1990;  211 447-458
• 35 Van der Velde-Zimmermann D, Roijers J F. et al . Molecular test for the detection of tumor cells in blood and sentinel nodes of melanoma patients.  Am J Pathol. 1996;  149 759-764
• 36 Washaw A L, del Castillo C. Pancreatic carcinoma.  New Engl J Med. 1992;  326 455-465
• 37 Yeo C J, Cameron J L. Prognostic factors in ductal pancreatic cancer.  Langenbecks Arch Surg. 1998;  383 129-133