Es wird vermutet, dass das ösophageale Adenokarzinom (EAC) durch gastroosophagealen Reflux auf dem Boden des Barrett-Ösophagus (BE) entsteht. Bei der Tumorprogression könnten Matrix-Metalloproteasen eine wichtige Rolle spielen. Die Expression von MMP-1 und MMP-13 wurde im Ösophaguskarziom (n=41 EAC mit BE, n=19 EAC ohne BE, n=10 Plattenepithelkarzinom, ESCC) sowie im nicht-dysplastischen BE (n=18) untersucht. Die Koexpression von MMP-1 und Cdx-2 (intestinale Metaplasie) und die Koexpression von MMP-1 und Ki-67 (Proliferation) wurde mittels Immunhistochemie und auf mRNA-Ebene untersucht. Die Ergebnisse wurde mit klinisch-pathologischen Eigenschaften korreliert. Im gesunden Plattenepithel wurde weder MMP-1 noch MMP-13 exprimiert. In allen EAC ohne BE wurde MMP-1 exprimiert (100%). Im EAC mit BE, war in 95% MMP-1 im EAC nachweisbar. Die Expression von MMP-1 im BE ohne IN lag bei 56%. Das ESCC exprimierte in 60% MMP-1. Bei der quantitativen Analyse zeigten sich 48% MMP-1 positive Zellen im EAC mit BE und 35% im angrenzendem BE (p<0,05). Mit 44% MMP-1 positiver Zellen im EAC ohne BE, lag die Expression signifikant über der im BE mit EAC (p<0,05). Im ESCC (32% MMP-1 positiv) lag eine im Vergleich zu allen EACs signifikant geringere Expression vor. Im BE ohne IN waren 4% der Zellen MMP-1 positiv. Die RT-PCR bestätigte die Ergebnisse der IHC auf mRNA-Ebene. Eine Präparate waren negativ für MMP-13. Die Untersuchung der Koexpression von MMP-1 in Ki-67 positiven Zellen zeigte eine starke direkte Korrelation (r=0,943 für BE und r= 0,811 für EAC). Eine hohe MMP-1 Expression war mit einem positiven Lymphknotenstatus assoziiert aber nicht mit einem schlechterem Überleben (p=0,307). Die Ergebnisse zeigen, dass MMP-1 eine wichtige Rolle bei der Invasion und Metastasierung des Barrett assoziierten EAC spielen könnte. Die Assoziation eines positiven Lymphknotenstatus mit hoher MMP-1-Expression spricht dafür, dass MMP-1 ein wichtiger Faktor bei der malignen Progression sein könnte. / Background: Esophageal adenocarcinomas (EACs) arise due to gastroesophageal reflux, with Barrett’s esophagus (BE) regarded as precancerous lesion. Matrix metalloproteinases (MMPs) might play a role during the multistep carcinogenetic process. Methods: Expression of MMP-1 and -13 was analyzed in esophageal cancer (n = 41 EAC with BE, n = 19 EAC without BE, and n = 10 esophageal squamous-cell carcinomas, ESCC), furthermore in BE without intraepithelial neoplasia (IN) (n = 18), and the cell line OE-33. MMP-1 was co-labelled with Ki-67 (proliferation), Cdx-2 (marker for intestinal metaplasia, BE) and analyzed on mRNA level. MMP-1 staining results were correlated with clinicopatholocical parameters. Results: On protein level, MMP-1 expression was found in 39 of 41 (95%) EAC with BE, in 19 of 19 (100%) EAC without BE, in 6 of 10 (60%) ESCC, and in 10 of 18 (56%) BE without IN. No expression of MMP-13 was found in these specimens. Quantification showed 48% MMP-1 positive cells in EAC with BE, compared to 35% in adjacent BE (p < 0.05), 44% in EAC without BE, 32% in ESCC, and 4% in BE without IN. Immunofluorescence double staining experiments revealed increased MMP-1 expressing in proliferating cells (MMP-1+/Ki-67+) (r = 0.943 for BE and r = 0.811 for EAC). On mRNA-level, expression of MMP-1 was significantly higher in EAC compared to BE (p = 0.01) and confirmed immunohistochemical staining results. High MMP-1 levels were associated with lymph node metastases but not with poorer survival (p = 0.307). Conclusions: Our findings suggest that MMP-1 plays a role as preinvasive factor in BE-associated EAC. Expression of MMP-1 in proliferating BE and EAC cells suggest malignant proliferation following the clonal expansion model. MMP-13 seems to play no important role in the multistep carcinogenetic process.

The definition of clinical target volume (CTV) margins around gross tumor volume (GTV) for radiotherapy of esophageal cancer (EC) and many solid tumors is still a challenge hence the currently available in-vivo imaging techniques still fail to detect areas of microscopic tumor extension (MTE). Many parameters of the tumor microenvironment (TME), e.g., tumor cell proliferation, cancer stem cells, hypoxia, kinases, immune architecture and patient-specific parameters are hypothesized as inducers of MTE in esophageal cancer and other tumors. The correlation of these TME biomarkers with MTE before, during or after radiochemotherapy (RCHT) is crucial in the era of image-guided, adaptive high-precision photon or particle therapy. In this thesis, two study cohorts were used to assess some selected TME biomarkers and their predictive value on MTE for an improved CTV definition. The first study used immunohistochemistry analysis for the assessment of TME marker namely HIF-1α, Ki67, p53, CXCR4 and PD1 in a cohort of retrospectively collected formalin-fixed paraffin-embedded (FFPE) blocks of EC patients treated with either neoadjuvant radiochemotherapy plus resection (NRCHT+R) or resection alone (R). The subsequent study employing a multiplex-immunofluorescence technique assessed the expression of various markers, i.e., FAK, ILK, CD44, HIF-1α and Ki67, in a cohort of prospectively prepared FFPE resection specimens of EC patients with implantable fiducial gold markers at the proximal and distal tumor borders illustrating the GTV prior to NRCHT+R and correlated those markers to the MTE. The findings from our first study showed upregulation of HIF-1α, Ki67, p53, CXCR4 and PD1, within squamous cell carcinoma (SCC) and adenocarcinoma (AC) patients treated with R compared to those having undergone NRCHT+R. In the second study higher expression of FAK+, CD44+, HIF-1+, and Ki67+ cells in tumor-nests than in tumor-stroma of both SCC and AC patients was found, although ILK+ cells were higher in tumor stroma. In addition, MTE reaching up to 31 mm beyond the fiducial markers was found in three patients (all cT3N1) with a stronger expression of FAK+, CD44+ and ILK+ cells in tumor-nests in between the fiducial markers (former GTV) and beyond those (former CTV), even after NRCHT. In conclusion, there is thus far no evidence that the TME influences the CTV margin on an individual patient basis, hence differences in the TME between patients with residual tumor cells in the original CTV compared to those without were not detected.