HIF1a — Expression im FFPE-Gewebe aus Weichteil-Sarkomen: Signifikante Bedeutung für Prognose, histopathologische Differentialdiagnose und Co-Expression verschiedener Angiogenesemarker

Abstract

Introduction: Though there have been some achievements in understanding the tumor biology and especially the genetic profile of Malignant Soft Tissue Sarcomas there are still some issues that need to be addressed. First and foremost there is the necessity to find markers that would help to get upfront information on the effectiveness of radiotherapy and afterwards on the expected prognosis. Furthermore an effective and specific chemotherapeutic treatment for soft tissue sarcomas with a relatively low toxicity is yet to find. Particularly the results of Imatinib in the treatment of GIST-Tumors directed to targeted therapy as a possible solution for that problem. Among other genes out of the angiogenic signal-transduction pathway HIF1a seems to be a very promising marker. This pilot study was intended for getting expression profiles of angiogenic markers in malignant fibrous histiocytomas (MFH) and neurogenic sarcomas (NS) and for correlating the results to overall survival. Methodology: FFPE samples were gathered from 44 patients with either malignant fibrous histiocytoma (18/44, 41 %) or neurogenic sarcoma (26/44, 59 %) with a median age of 57 (MFH: 70, 39–85; NS: 53, 16–72) years at time of operation who were scheduled for primary surgical resection. After a review of representative H&E stained slides of the FFPE blocks by a pathologist, determining quality of the sample and estimating the tumor load per sample, section slides of 10 µm thickness were obtained for laser captured microdissection (P.A.L.M. Microlaser Technologies AG, Munich, Germany). The Isolation of RNA from the gained tumor tissue was performed in accordance with a patented procedure at Response Genetics Inc (Los Angeles, CA; US patent No. 6248,535). The cDNA-Preparation steps were accomplished as previously described. To quantify mRNA expression levels we used an internal reference gene (beta-actin) and our gene set on a method based on real-time fluorescence detection of amplified cDNA (ABI PRISM 7900 Sequence Detection System [TaqMan] Perkin-Elmer Applied Biosystem, Foster City, CA). Results: All patient samples showed measurable amount of mRNA. The correlation of HIF1a with the other evaluated genes was tested with Spearman’s test for bivariate correlations. The correlation reached the level of significance at VEGF-A (p = 0,02, < 0,05), VEGF-C (p = 0,00004), VEGFR-1 (p = 0,009), PDGFR-A (p = 0,0006) and HB-EGF (p = 0,002). Univariate analysis showed a significant difference in the HIF1a gene expression levels between MFH and NS [median: 3,5030 (range 1,529 – 8,689) vs. 1,2974, (range 0,408 – 4,170); p < 0,001]. To evaluate independent prognostic factors associated with survival multivariate Cox proportional hazards regression analysis with stepwise selection was used with the gene set, age, pT and the UICC Stage as covariates. In the group of patients with NS the low expression (< 0.9, 25^th percentile) of HIF1a showed a significant survival benefit (p = 0.008) besides a low UICC Stage (p = 0.04; overall model fit p = 0.01). The patients with MFH had a survival benefit with high HIF1a expression (> 5.1, 70^th percentile) besides a low UICC stage (overall model fit p = 0.004). Conclusions: This pilot study shows that HIF1a expression in MFH and NS may have a significant impact on prognosis. We generated the hypothesis that either a very low or a very high HIF1a expression may be beneficial at certain tumors. Further studies with more patients seem to be warranted to assess this hypothesis.