S1.(a) Distribution of tumor vs. normal lung tissue in K, KE, KP and KPE mice at 4 weeks post tumor induction (top). Distribution of murine lung adenocarcinoma grades (G1, G2, G3) in corresponding K, KE, KP and KPE mice at 4 weeks post tumor induction (bottom). (b) Anti-Ercc1 immunostaining of lung sections of KE mice (n = 2) at 4 weeks post tumor induction (mouse KE.1 shown at two magnifications, mouse KE.2 shown at two magnifications) (top). Anti-Ercc1 immunostaining of lung sections of KE mice (n = 2) at 12 weeks post tumor induction (mouse KE.3 shown at two magnifications, mouse KE.4 shown at two magnifications) (bottom). (c) Anti-Ercc1 immunostaining of lung sections of KPE mice (n = 5) at 4 weeks post tumor induction (shown at different magnifications) and of KPE mice (n = 5) at 12 weeks post tumor induction (shown at different magnifications).

S4. (a) Representative immunostaining of Pt-(GpG) adducts in KP and KPE cell lines after cisplatin exposure (left). Quantification of Pt-(GpG) adduct staining in KP (n = 5) and KPE (n = 6) cells after cisplatin exposure (right). P values were calculated using two-tailed t-test. (b) Clonogenic survival assay of mock-treated KP and cisplatin-treated KP cells, mock-treated KPE and cisplatin-treated KPE cells, mock-treated KPE post cis and cisplatin-treated KPE post cis cells, mock-treated KPE post cis clone 6 (*6) and cisplatin-treated KPE post cis clone 6 (*6). (c) Detailed representation of Fig. 4c.

S3. Anti-Ercc1 immunostaining of lung sections of KPE mice (n = 8) after cisplatin treatment shown at different magnifications.

You have accessJournal of UrologyCME1 Apr 2023MP50-01 VALIDATION OF AGR2 AND KRT19 AS SPECIFIC PROTEINS BEING SIGNIFICANTLY AND DIFFERENTIALLY EXPRESSED IN TERATOMA COMPARED TO NECROSIS IN RETROPERITONEAL LYMPH NODE RESECTIONS AFTER CHEMOTHERAPY (PCRPLND) Tim Nestler, Lara Kremer, Melanie Von Brandenstein, Maike Wittersheim, Svenja Wagener-Ryczek, Pia Paffenholz, Stefan Mueller, Alexander Quaas, Martin Hellmich, Margarete Odenthal, David Pfister, and Axel Heidenreich Tim NestlerTim Nestler More articles by this author , Lara KremerLara Kremer More articles by this author , Melanie Von BrandensteinMelanie Von Brandenstein More articles by this author , Maike WittersheimMaike Wittersheim More articles by this author , Svenja Wagener-RyczekSvenja Wagener-Ryczek More articles by this author , Pia PaffenholzPia Paffenholz More articles by this author , Stefan MuellerStefan Mueller More articles by this author , Alexander QuaasAlexander Quaas More articles by this author , Martin HellmichMartin Hellmich More articles by this author , Margarete OdenthalMargarete Odenthal More articles by this author , David PfisterDavid Pfister More articles by this author , and Axel HeidenreichAxel Heidenreich More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003298.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: 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. In 2022 we reported AGR2 and KRT19 to be significantly and differentially expressed in teratoma compared to necrosis in pcRPLND tissue on protein and mRNA level. The aim of this study was to validate these proteins on an independent cohort. METHODS: All consecutive pcRPLND patients from 2021 who were treated in the University Hospital of Cologne, Germany, were selected (n=66). All patients had residual masses >1 cm and normalized or plateaued tumor markers. For immunohistochemistry, the monoclonal antibodies KRT19 and AGR2 were applied to representative Formalin-Fixed Paraffin-Embedded tissue. To quantify the results, the H-score was used. The investigators were blinded to the final pathohistological results. RESULTS: The cohort was composed of 66 patients, 23 patients with T, 24 patients with V and 19 patients with N. Significantly higher H-scores were shown for AGR2 and KRT19 when comparing T vs. N and T vs. V (both p<0.0001). The discriminatory ability of the two proteins AGR2 and KRT19 was calculated by AUCs for T vs. N and was 1.0 in each case with a corresponding sensitivity and specificity for T of 100%. CONCLUSIONS: With AGR2 and KRT19, we could validate the two proteins that are significantly and differentially expressed in the pcRPLND specimen in the clinically relevant groups T vs. N. 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. Source of Funding: This work was supported by the German Ministry of Defense © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e685 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Tim Nestler More articles by this author Lara Kremer More articles by this author Melanie Von Brandenstein More articles by this author Maike Wittersheim More articles by this author Svenja Wagener-Ryczek More articles by this author Pia Paffenholz More articles by this author Stefan Mueller More articles by this author Alexander Quaas More articles by this author Martin Hellmich More articles by this author Margarete Odenthal More articles by this author David Pfister More articles by this author Axel Heidenreich More articles by this author Expand All Advertisement PDF downloadLoading ...

412 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. In 2022 we reported AGR2 and KRT19 to be significantly and differentially expressed in teratoma compared to necrosis in pcRPLND tissue on protein and mRNA level. The aim of this study was to validate these proteins on an independent cohort. Methods: All consecutive pcRPLND patients from 2021 who were treated in the University Hospital of Cologne, Germany, were selected (n=66). All patients had residual masses > 1 cm and normalized or plateaued tumor markers. For immunohistochemistry, the monoclonal antibodies KRT19 and AGR2 were applied to representative Formalin-Fixed Paraffin-Embedded tissue. To quantify the results, the H-score was used. The investigators were blinded to the final pathohistological results. Results: The cohort was composed of 66 patients, 23 patients with T, 24 patients with V and 19 patients with N. Significantly higher H-scores were shown for AGR2 and KRT19 when comparing T vs. N and T vs. V (both p < 0.0001). The discriminatory ability of the two proteins AGR2 and KRT19 was calculated by AUCs for T vs. N and was 1.0 in each case with a corresponding sensitivity and specificity for T of 100%. Conclusions: With AGR2 and KRT19, we could validate the two proteins that are significantly and differentially expressed in the pcRPLND specimen in the clinically relevant groups T vs. N. 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.

S2.(a) µCT-based monitoring of tumor response in K and KE mice prior to and after cisplatin treatment (left). Quantification of tumor volume in K and KE mice prior to and after cisplatin treatment (right). P values were calculated using two-tailed t-test. (b) Representative immunostaining of Pt-(GpG) adducts in tumor lung sections of KP and KPE mice after single cisplatin treatment (left). Quantification of Pt-(GpG) adduct staining in of KP (n = 3) and KPE mice (n = 4) after single cisplatin treatment (right). P values were calculated using two-tailed t-test. (c) Survival of chemo-naive vs. cisplatin-treated KrasLSL.G12D/wt (K) mice (top left), chemo-naive vs. cisplatin-treated KrasLSL.G12D/wt;Ercc1fl/fl (KE) mice (top right) and comparison of cisplatin-treated KrasLSL.G12D/wt (K) and cisplatin-treated KrasLSL.G12D/wt;Ercc1fl/fl (KE) mice (bottom). P values were calculated using log-rank test.

S5. (a-e) µCT-based monitoring of KPE and KPE post cis allograft tumor response in KPE syngeneic mice (n = 4) prior to and after etoposide treatment (Mice 2-5 shown). Allograft tumors are marked with "T".

Background Testicular germ cell tumours (TGCTs) have a high metastasis rate. However, the mechanisms related to their invasion, progression and metastasis are unclear. Therefore, we investigated gene expression changes that might be linked to metastasis in seminomatous testicular germ cell tumour (STGCT) patients. Methods Defined areas [invasive tumour front (TF) and tumour centre (TC)] of non-metastatic (with surveillance and recurrence-free follow-up >2 years) and metastatic STGCTs were collected separately using laser capture microdissection. The expression of 760 genes related to tumour progression and metastasis was analysed using nCounter technology and validated with quantitative real-time PCR and enzyme-linked immunosorbent assay. Results Distinct gene expression patterns were observed in metastatic and non-metastatic seminomas with respect to both the TF and TC. Comprehensive pathway analysis showed enrichment of genes related to tumour functions such as inflammation, angiogenesis and metabolism at the TF compared to the TC. Remarkably, prominent inflammatory and cancer-related pathways, such as interleukin-6 (IL-6) signalling, integrin signalling and nuclear factor-κB signalling, were significantly upregulated in the TF of metastatic vs non-metastatic tumours. Conclusions IL-6 signalling was the most significantly upregulated pathway in metastatic vs non-metastatic tumours and therefore could constitute a therapeutic target for future personalised therapy. In addition, this is the first study showing intra- and inter-tumour heterogeneity in STGCT.

Before postchemotherapy retroperitoneal lymph node dissection (pcRPLND), in patients with metastasized germ cell tumors (GCTs), those harboring necrosis (NEC) cannot be distinguished from those who have teratoma (TER), resulting in relevant overtreatment, whereas microRNA‐371a‐3p may be predictive for viable GCT. The purpose of this study was to explore messenger RNA (mRNA) and proteins to distinguish TER from NEC in pcRPLND tissue.

408 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.

based on HPV status, HPV-positive tumors were associated with lower cT stages, less LVI, but more cN þ disease. Etiologic classi fi cation based on HPV did not appear to impact survival.

Objective: We attempted to analyze whether early presentation with brain metastases (BM) represents a poor prognostic factor in patients with non-small cell lung cancer (NSCLC), which should guide the treatment team towards less intensified therapy. Patients and methods: In a retrospective bi-centric analysis, we identified patients receiving surgical treatment for NSCLC BM. We collected demographic-, tumor-, and treatment-related parameters and analyzed their influence on further survival. Results: We included 377 patients. Development of BM was precocious in 99 (26.3%), synchronous in 152 (40.3%), and metachronous in 126 (33.4%) patients. The groups were comparable in terms of age (p = 0.76) and number of metastases (p = 0.11), and histology (p = 0.1); however, mutational status significantly differed (p = 0.002). The precocious group showed the worst clinical status as assessed by Karnofsky performance score (KPS) upon presentation (p < 0.0001). Resection followed by postoperative radiotherapy was the predominant treatment modality for precocious BM, while in syn- and metachronous BM surgical and radio-surgical treatment was balanced. Overall survival (OS) did not differ between the groups (p = 0.76). A good postoperative clinical status (KPS ≥ 70) and the application of any kind of adjuvant systemic therapy were independent predictive factors for OS. Conclusion: Early BM presentation was not associated with worse OS in NSCLC BM patients.

Objective To investigate the diagnostic performance of gallium‐68 prostate‐specific membrane antigen positron emission tomography/computed tomography ( 68 Ga‐PSMA PET/CT) in patients with recurrent prostate cancer with regard to the presence of lymph node metastases (LNM) and local recurrences after primary radiotherapy. Patients and methods We retrospectively reviewed 142 patients following salvage radical prostatectomy (sRP), 50 of which had a 68 Ga‐PSMA PET/CT performed as a preoperative staging module. Predictive clinical parameters were analysed in a multivariate Cox regression analysis. Sensitivity, specificity, positive (PPV) and negative predictive values (NPV) and the accuracy of 68 Ga‐PSMA PET/CT were analysed with regard to LNM and local recurrence. Results In all, 613 lymph nodes were resected in 40 patients and 23 lymph nodes had metastatic deposits in 14 patients. In all patients local recurrence could have been found with 68 Ga‐PSMA PET/CT. Sensitivity, specificity, PPV and NPV and accuracy on a per lymph node basis were 34.78% (16.38–57.2%), 100% (99.38–100%), 100%, 97.52% (96.69–98.15%) and 97.55% (96.00–98.62%). For detecting local recurrence, the sensitivity and PPV were both 100% with an accuracy of 100% (92.89–100%). Conclusion 68 Ga‐PSMA PET/CT should be the standard imaging in biochemical recurrent prostate cancer. With this imaging module one detects first local recurrence and can detect locoregional and distant metastases more precisely than standard CT and bone scan.

Key Clinical MessageFacial nerve paralysis due to the infiltration by a lymphoma is rare and the prognosis remains poor. If perineural spread and meningeosis are suspected, quick interdisciplinary diagnostic work‐up is recommended. It should include magnetic resonance imaging, biopsy of the lesion, bone marrow biopsy, and lumbar puncture. Therapy should be initiated immediately.

Abstract Background Brain metastases (BM) frequently cause focal neurological deficits leading to a reduced Karnofsky performance score (KPS). Since KPS is routinely used to guide the choice of adjuvant therapy, we hypothesized that improving KPS by surgical resection may improve the chance for adjuvant treatment and ultimately result in better survival. We therefore analyzed the course of a large cohort undergoing resection of symptomatic brain metastases in the context of further treatment and clinical outcome. Patients and methods In a bi‐centric retrospective analysis we retrieved baseline, clinical, and treatment‐related parameters of patients operated on BM between 2010 and 2019. Survival was calculated using Kaplan‐Meier estimates; prognostic factors for survival were analyzed by Log‐rank test and Cox proportional hazards. Results We included 750 patients with a median age of 61 (19‐87) years. The functional status was significantly improved by surgical resection, with a median preoperative (KPS) of 80 (10‐100) increasing to 90 (0‐100) after surgery (P < .0001). Moreover, surgery improved the RTOG recursive partitioning analysis (RPA) class from III to I/II in 82 patients. Postoperative local radiotherapy and systemic treatment were associated with significantly longer survival (P < .0001 for each). Systemic treatment was provided significantly more frequently in patients with a fair postoperative clinical status (KPS ≥ 70; P < .0001). The postoperative clinical status, postoperative radiotherapy, systemic treatment, controlled systemic disease and < 4 BM were independent predictors for survival. Conclusion The resection of symptomatic BM may restore clinical status, so enhancing the likelihood of receiving adjuvant treatment, and therefore leading to improved overall survival.

Introduction: Although relapses after radiotherapy are common in prostate cancer (PCA) patients, those with a high risk for radioresistance cannot be identified prior to treatment yet. Therefore, this proof-of-concept study was performed to compare protein expression profiles of patients with radio-recurrent PCA to patients treated with primary radical prostatectomy separated by Gleason risk groups. We hypothesized that radio-recurrent PCA have a similar protein expression as high-risk Gleason PCA. Methods: Patient cohorts consisted of (i) 31 patients treated with salvage prostatectomy for locally recurrent PCA after primary radiotherapy and (ii) 94 patients treated with primary prostatectomy split into a Gleason high-risk (≥4 + 3; n = 42 [44.7%]) versus a low-risk group (≤3 + 4; n = 52 [55.3%]). Immunohistochemistry was performed using 15 antibodies with known association to radioresistance in PCA in vitro. ELISA was used for validation of selected markers in serum. Results: Androgen receptor (AR) was overexpressed in most radio-recurrent PCA (89.7%) and in most primary high-risk Gleason PCA (87.8%; p = 0.851), while only 67.3% of the low-risk group showed an expression (p = 0.017). Considering the highest Gleason pattern in primary PCA, aldo-keto reductase family 1 member C3 (AKR1C3) was most similarly expressed by patients with radio-recurrent PCA and patients with Gleason patterns 4 and 5 (p = 0.827 and p = 0.893) compared to Gleason pattern 3 (p = 0.20). These findings were supported by ELISA. Conclusion: This is the first study to evaluate protein markers in order to predict radioresistance in PCA. Our results point to AR and AKR1C3 as the most promising markers that might help stratify patients for radiotherapy.

The proximal DNA damage response kinase ATM is frequently inactivated in human malignancies. Germline mutations in the ATM gene cause Ataxia-telangiectasia (A-T), characterized by cerebellar ataxia and cancer predisposition. Whether ATM deficiency impacts on tumor initiation or also on the maintenance of the malignant state is unclear. Here, we show that Atm reactivation in initially Atm-deficient B- and T cell lymphomas induces tumor regression. We further find a reduced T cell abundance in B cell lymphomas from Atm-defective mice and A-T patients. Using T cell-specific Atm-knockout models, as well as allogeneic transplantation experiments, we pinpoint impaired immune surveillance as a contributor to cancer predisposition and development. Moreover, we demonstrate that Atm-deficient T cells display impaired proliferation capacity upon stimulation, due to replication stress. Altogether, our data indicate that T cell-specific restoration of ATM activity or allogeneic hematopoietic stem cell transplantation may prevent lymphomagenesis in A-T patients.

57 Background: Although relapses after radiotherapy are common in prostate cancer (PCa) patients, there are no clinical models or markers to identify patients at high risk for radioresistance. So far, only in vitro studies and xenograft models have been performed to identify gene expression patterns associated with radioresistance. However, studies which address the protein pattern to predict radioresistance in humans are completely missing. In order to determine potential biomarkers for radioresistance, we compared protein expression profiles of radioresistant PCa patients with PCa of primary prostatectomized patients. Methods: Two study groups consisting of: I) 30 patients who were treated by salvage prostatectomy and II) 94 patients treated by primary prostatectomy were formed. Tissue microarrays were constructed and immunostained for 15 proteins which are suggested to be associated with radioresistance by in vitro findings. Kruskal-Wallis test was used for multiple group comparison and followed by Dunn-Bonferroni-Test to detect intergroup differences. Cohen’s d was used to calculate the intergroup effect size. Results: Most proteins studied did not show any relevant differences between radioresistant PCa and primary PCa, except for two (AR and AKR1C3). On comparing immunostaining patterns between radioresistant PCa and primary PCa separated by Gleason risk groups, we observed only AR (androgen receptor) to be most expressed in radioresistant PCa (89.7%) and, in 87.8% of primary PCa of the high-risk group ( > 7a) (p = 0.851, Cohen’s d = 0.05), while only 67.3% PCa of the low-risk group (≤7a) (p = 0.017, Cohen’s d = 0.55) were positive. Considering the highest Gleason pattern per patient, only AKR1C3 (Aldo-Keto Reductase Family 1 Member C3) was seen to be similarly expressed in radiation-resistant PCa and patients with Gleason patterns 4 and 5 (p = 0.827, Cohen’s d = 0.05 and p = 0.893, Cohen’s d = 0.10) as compared to Gleason pattern 3 (p = 0.20, Cohen’s d = 0.69) in primary PCa. Conclusions: This is the first study evaluating protein expression profiles to predict radioresistance in PCa, where AR and AKR1C3 were identified to be the most promising protein markers.

506 Background: Not much is known about the molecular mechanisms resulting in tumor progression and finally leading to metastasis in testicular germ cell tumor (TGCT). Only a few studies in some other tumor types have identified a limited set of genes, related to invasion, progression or metastases to be distinctly upregulated at the invasive tumor front in metastasized patients. However, systematic investigations are missing. Therefore, regional differences in the TGCT subtype seminomas were investigated to achieve a better understanding of the mechanisms involved in the metastatic process. Methods: Formalin-fixed paraffin embedded (FFPE) tissue samples of patients with clinical stage I disease, no adjuvant therapy and a relapse-free survival of at 2 years (n = 21), and patients showing metastasis (n = 14) were selected for the study. The tumor front (TF) and tumor center (TC) regions of each patient were determined and separately collected using laser capture microdissection. RNA was extracted and a multiplex gene expression analysis was performed on all TF and TC samples using nCounter technology of Nanostring. A panel of 770 transcripts was analyzed using the PanCancer Progression panel. Different bioinformatics tools were employed for analyzing the expression data. Results: Differential gene expression patterns were observed in the metastatic and non-metastatic patients, with respect to both the tumor front and tumor center regions. Ingenuity pathway analysis on the differentially expressed genes showed enrichment of tumor functions like migration, invasion, and angiogenesis at the TF as compared to the TC. Remarkably, prominent inflammatory and cancer related pathways such as IL-6 signaling, acute phase response signaling, NF-κB signaling and, dendritic cell maturation were significantly upregulated in the metastatic versus non-metastatic tumors (z-score > ± 2 and p-value < 0.05). Conclusions: This is the first study showing tumor heterogeneity in TGCTs. Evidently, IL-6 signalling was the most significantly upregulated pathway in the metastatic versus the non-metastatic patients, that could serve as a therapeutic target for personalized therapy.

528 Background: Up to 40% of pure seminomatous germ cell tumors (GCTs) are clinically metastasized at initial diagnosis. Additionally, up to 20% of patients have occult metastasis but are not diagnosed due to missing markers. We hypothesize that pure seminomatous GCTs differ from metastasized to non-metastasized patients, possibly owing to differences in tumor heterogeneity. Since in few other tumor types genes linked to metastasis are shown to be more often upregulated at the tumor front, we investigated different regions of seminomas in non-metastasized and metastasized patients. Methods: Seminoma patient samples without metastasis, with no adjuvant therapy, and with recurrence-free follow-up of at least two years (n = 21), and patients showing metastasis (n = 14) were selected for the study. Based on formalin-fixed paraffin embedded tissues, the tumor front (TF) and tumor center (TC) regions of each patient were determined and separately collected using laser capture microdissection. RNA was extracted and a multiplex gene expression analysis was performed on all TF and TC samples using nCounter technology of Nanostring. A panel of 770 transcripts was analyzed using the PanCancer Progression panel. Different bioinformatics tools were employed for analyzing the expression data. Results: Hierarchical cluster analysis showed no differential gene clustering between metastasized and non-metastasized patients. Comparing TF and TC in the metastasized group more genes (29 genes) were significantly differentially expressed (log2 fold change > 1.5, FDR < 0.05) compared to cSI patients (12 genes). Pairwise comparison of TF and TC for each individual patient showed differential expression of genes in these two regions. Lasso regression analysis of TC could not reveal any signature to predict metastasis. However, with respect to the TF a signature of only six genes was sufficient to predict metastasis with a specificity of 100%, and a sensitivity of 92.9%. Conclusions: This study describes tumor heterogeneity in seminomas and identifies a gene signature that could predict metastasis and has to be validated on occult metastasized seminomatous patients.