Differentially expressed mRNA/proteins can distinguish viable germ cell tumors and teratomas from necrosis in retroperitoneal lymph node resections after chemotherapy (pcRPLND).
Authors: Nestler Tim , Kremer Lara , Wagener-Ryczek Svenja , Wittersheim Maike , von Brandenstein Melanie , Paffenholz Pia , Mueller Stefan , Quaas Alexander , Hellmich Martin , Odenthal Margarete , Pfister David , Heidenreich Axel 
Published: February 2022

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Background: Metastatic non-seminomatous testicular tumor patients with residual retroperitoneal tumor masses > 1cm after chemotherapy are treated with pcRPLND. The goal of pcRPLND is to remove viable tumors (V) and teratoma (T), which are present in approximately 10% and 40% of cases, respectively. However, histopathologically, only scar/necrosis (N) is identified in the remaining 50% of patients. In those patients, surgical therapy is not necessary, resulting in a relevant overtreatment. So far, no adequate distinction between the histologies exists preoperatively. Recently, the first biomarker was described with miR371a-3p in serum, which is highly specific for V, but not for T. Therefore, our goal was to identify mRNAs and proteins that are differentially expressed between V/T vs N, in particular between T and N, in pcRPLND resected cells. Methods: Forty-eight patients were identified, n = 16 each with T/V/N. Representative regions of T/V/N were microdissected and subsequently mRNA was extracted. Initially, 770 genes were analyzed using the nCounter PanCancer Progression Panel (Nanostring). For each group comparison, genes with a fold change of < -2/ > 2 and a p-value of < 0.05 were identified. Hereafter, quantitative protein analysis (proteomics) was performed on the same samples. Finally, the proteins of the 5 mRNAs with the most different and significant expression levels between T vs. N were validated by immunohistochemistry and H-score calculation. Results: By Nanostring, we identified 84 significantly differentially expressed mRNAs for the group comparisons of T vs. N, 63 for V vs. N, and 189 for T vs. V. Quantitative protein analysis revealed 25 significantly differentially expressed proteins in T vs. N, 254 in V vs. N, and 134 between T vs. V. By immunohistochemistry, all 5 antibodies showed significantly increased H scores when comparing T vs. N and T vs. V. In accordance with the objective, we found two proteins, AGR2 and KRT19, with their corresponding genes that showed significantly differential expressions for the comparison of T vs. N in both, quantitative protein analysis and Nanostring mRNA analysis, and were successfully validated by immunohistochemistry. Conclusions: With AGR2 and KRT19, we have identified two proteins with their corresponding genes that are significantly and differentially expressed in the pcRPLND specimen in the clinically relevant groups T vs. N. Both were successfully validated by immunohistochemistry. In addition, further group differences (T vs. V/ V vs. N) were revealed depending on the analytical method. In perspective, these proteins could be targeted by radiolabeled ligands as a tracer in order to reliably distinguish patients with teratoma from those with necrosis by means of functional imaging. Thus, overtreatment with pcRPLND of patients with N could be safely reduced.