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.

Schlüsselwörter

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

Key words

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

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