Novel immunotherapies (IO) show mixed responses, particular in context of brain metastases (BM). We therefore aimed to investigate the density of tumor-associated macrophages (TAM) in primary tumors (PT) and matched BM tissue samples. We retrospectively identified patients who received resection of PT and BM between 01/1990 and 10/2022. Immunohistochemical staining and density quantification (positive cells/mm2) of TAMs (CD68+ & CD163+) was performed. Generated heatmaps were analyzed for heterogeneity as quantified by Shannon Entropy. We included 62 patients (61.3% male, 38.7% female) with matched samples: 46/62 non-small cell lung cancer, 8/62 breast cancer (BC), 8/62 renal cell carcinoma. Median CD163+ TAM density was 733 cells/mm2 (range 105-5110 cells/mm2) in BM and 407 cells/mm2 (range 52-1786 cells/mm2) in PT. Median CD68+ TAM density was 924 cells/mm2 (range 119-5521 cells/mm2) in BM and 744 cells/mm2 (range 99-2164 cells/mm2) in PT. CD163+ TAM density was significantly higher in BM compared to PT over all entities (p<.05). For CD68+ TAM, a significant difference was only observed in BC with higher density in BM than in PT (p=.016). Median difference in CD163+ TAM density between PT and BM was 163 cells/mm2 (range -1258-3602 cells/mm2) and 348 cells/mm2 (range -1390-4390 cells/mm2) in CD68+ TAM. No correlation between TAM density difference and clinical outcome (time to BM and OS from BM) was observed (p>.05). Median entropy for CD163+ TAM in BM was 15.8 (range 1.5-76.2) and 14.3 (range 0.5-45.2) in PT. Concerning CD68+ TAM, median entropy was 19.9 (range 1.8-88.7) in BM and 23.9 (range 1.9-54.9) in PT. A significant difference in entropy was only observed in BC as primary breast cancer CD68+ TAM showed lower entropy indices (p=.02). Median difference in entropy between PT and BM was 3.4 (range -34.8-41.3) in CD163+ TAM and 1.41 (range -34.4-39.9) in CD68+ TAM. No correlation of TAM entropy and clinical outcome (time to BM and OS from BM) was observed (p>.05). TAM density is significantly higher in BM compared to PT. In BC, higher CD68+ TAM entropy was observed in BM, indicating higher herterogeneity. Further analyses with enlarged cohort and additional immune cell markers are ongoing.

Brain metastases (BM) are a clinical challenge in oncology. Further insight on pathobiology is the basis for new treatment approaches. Recently, S100A9 has been postulated as a novel potential biomarker for therapy resistance to radiotherapy in preclinical BM models. Therefore, we aimed to explore S100A9 levels in a real-life BM patient cohort. S100A9 protein expression was measured in the serum of patients treated at the Medical University of Vienna between 2019-2023 using an enzyme-linked immunosorbent assay (ELISA) to correlate its levels with clinicopathological parameters. Sample concentrations below the lower limit of quantification (LOQ) were treated as 50% of LOQ´s concentration and considered as non-quantifiable. Samples with a concentration above the upper limit of quantification were remeasured in dilutions. 80 patients (39% male; 61% female) with BM (n=69) and without BM (n=11) were included in this pre-liminary analysis. Median serum level of S100A9 was 23 pg/mL (IQR 16-97 pg/mL). No difference in median S100A9 levels were observed between patients with BM (23 pg/mL, IQR 16-114) and without BM (23 pg/mL, IQR 15-41) at time of blood sampling (p=0.7). Furthermore, no difference was found in median S100A9 levels in BM patients according to primary tumor’s origin (lung cancer 23 pg/mL vs. breast cancer 16 pg/mL, p=0.6) or radiated versus non-radiated BM (37 pg/mL vs. 23 pg/mL, p=0.3). Median overall survival of patients with quantifiable S100A9 levels were significantly longer than patients with non-quantifiable S100A9 serum levels (HR 2.05; p=0.05). Based on the results of these pre-liminary data, S100A9 levels did neither correlate with BM diagnosis, primary tumor’s origin nor previously applied radiotherapy. However, detected S100A9 levels correlated with survival analysis in this real-life BM patients. Further studies are currently ongoing to further evaluate S100A9 as a diagnostic and predictive biomarker in larger real-world patient cohorts.

Response rates of immune checkpoint inhibitors (ICI) differ between primary tumors and brain metastases (BM). Here, we aimed to analyze infiltration patterns of inflammatory cells within the tumor microenvironment (TME) of primary tumors and matched BM tissue samples. We retrospectively identified patients who received resection of primary tumor and BM between 01/1990 and 10/2022. Immunohistochemical FFPE tissue staining, density quantification of tumor-associated macrophages (TAMs; CD68+, CD163+) and tumor-infiltrating lymphocytes (TILs; CD3+, CD8+, CD45Ro+, FoxP3+) was performed. Images were processed with QuPath software, heterogeneity of generated heatmaps was measured by Shannon entropy. 74 patients with matched samples were analyzed: 58/74 (78%) non-small cell lung cancer (NSCLC) , 8/74 (11%) breast cancer (BC), 8/74 (11%) renal cell carcinomas (RCC) (61.3% males, 38.7% females; median age 57 years). Median OS after primary diagnosis was 176.4 weeks (range 9.4-829.1). Median time to BM was 58.2 weeks (range -24.9 -520.6). CD163+ density was significantly higher in BM, whereas CD8+ was significantly lower in BM over all entities (p<.05) compared to primary tumors. CD3+ density was lower in BM of NSCLC and BC (p<.001, p=.008, respectively). CD68+ showed higher density in BC BM (p=.016) and FoxP3+ was higher in NSCLC primary tumors (p<.001; all Wilcoxon rank sum test). Cell density and entropy of FoxP3+ correlated in matched samples (p=.002, ρ=.37 and p<.001, ρ=.65), while CD3+ (p=.002, ρ=.37) and CD8+ (p<.001, ρ=.47) did only in BM. In primary NSCLC, FoxP3+ and CD163+ showed lower entropy indices, therefore more homogeneous distribution compared to matched BM (p=.002, p=.004, respectively). Multivariable Cox regression model demonstrated significant associations of CD3+ density (p=.006) and CD45Ro+ entropy (p=.001) in primary tumor with time to BM diagnosis. Cell densities of CD163+-TAMs within the TME of BM are significantly higher compared to primary tumors, whereas CD8+ and CD3+ TILs are less present in BM. Differences in the inflammatory cell infiltration pattern could explain different ICI responses between primary tumor and BM.

BACKGROUND:There is lack of consensus whether neoadjuvant chemoradiotherapy (CHT/RT) is superior to neoadjuvant chemotherapy (CHT) alone in patients with potentially resectable stage III/N2 non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS:We retrospectively evaluated clinical parameters and outcomes in patients with clinical stage III/N2 NSCLC treated with neoadjuvant CHT/RT versus CHT followed by surgery. Nearest-neighbor propensity score (PS) matching was used to correct for pretreatment differences. RESULTS:A total of 84 patients were enrolled. Thirty-four (40%) and 50 (60%) patients received CHT/RT or CHT followed by curative-intent surgery, respectively. Overall 90-day mortality and morbidity were 0% versus 0.04% and 21% versus 18%, respectively, with no significant difference between the CHT/RT and the CHT-alone cohorts (P = 0.51 and P = 0.70). In the PS-matched cohort, complete pathological response was recorded in 25% after CHT/RT versus 0% after CHT at the time of surgery. Patients receiving neoadjuvant CHT/RT exhibited significantly better 5-year disease-free survival (DFS) [45% versus 16% CHT group; hazard ratio (HR) 0.43, P = 0.04]; 5-year overall survival (OS) was 75% after CHT/RT and 21% after CHT (HR 0.37, P = 0.001). CHT/RT more often induced pathological mediastinal downstaging (P = 0.007), but CHT/RT remained the only independent factor for DFS and OS and did not depend on mediastinal downstaging. CONCLUSIONS:In this retrospective PS-matched long-term analysis, neoadjuvant CHT/RT conferred improved DFS and OS compared with CHT alone in stage III/N2 NSCLC. These highly challenging results require confirmation in well-designed randomized controlled trials conducted at highly specialized thoracic oncology centers.

Abstract Background Immunotherapy (IO) has changed the treatment landscape of metastatic cancer patients, however, treatment resistance is frequent. We aimed to characterize the inflammatory tumor microenvironment in brain metastases (BM) after IO to gain a deeper understanding of immunologic escape mechanisms. Material and Methods Solid cancer patients who had BM resection after IO progression (IO cohort) were retrospectively identified. We analyzed tumor-infiltrating immune cell subsets (CD3, CD8, CD45RO, FOXP3) and expression of immune checkpoint molecules (PD-L1, PD-1, LAG-3) by immunohistochemistry. A control cohort of BM tissue samples without prior IO served for comparison (no immunotherapy cohort, NIO). Results Twenty-eight IO patients (12/28, 42.9% females; 16/28, 57.1% males; median 61 years; 14/28, 50% lung cancer; 5/28, 17.9% melanoma; 4/28, 14.3% renal cell carcinoma; 1/28, 3.6% breast cancer; 4/28, 14.3% other cancer entities) and 57 NIO patients (28/57, 49.1% females; 29/57, 50.9% males; median 58 years; 35/57, 61.4% lung cancer; 9/57, 15.8% breast cancer; 4/57, 7.0% melanoma; 3/57, 5.3% renal cell carcinoma; 6/57, 10.5% other cancer entities) were included. IO patients had a median of one (range 0-4) systemic therapy line prior to IO. Median time from last IO application until BM resection was 5.6 months (range 0.2-49.8 months). Patients received a median number of 7 (range 1-56) IO applications (14/28, 50% PD-1-targeting IO; 8/28, 28.6% PD-L1; 2/28, 7.1% CTLA4; 4/28, 14.3% CTLA4+PD-1; 3/28, 10.7% IO+chemotherapy). No statistically significant differences in the densities of investigated TILs or PD-L1 expression between the IO and the NIO cohort were observed. Patients of the IO cohort showed higher PD-L1 expression compared to the NIO cohort (57.1 vs. 42.1%, Chi-square, p>0.05). Overall survival (OS) was similar in both cohorts, with a median OS of 11.0 months (range 5.0-17.0) in the IO cohort and 11.0 months (range 5.5-16.5) in the NIO cohort. Conclusion Our findings show an upregulation of PD-L1 in BM occurring after prior IO therapy in the absence of other overt changes in the inflammatory microenvironment. Ongoing analyses in this cohort are investigating possible molecular driver of resistance by analyzing DNA methylation profiles of pre-and post-IO tissue samples of the IO cohort to potentially gain insights on inflammatory IO resistance mechanisms in BM patients.

Considering the rising detection of asymptomatic brain metastases (BM) during screening and staging procedures over the last decades, we aimed to evaluate the incidence, clinical characteristics and the prognostic value of neurological symptoms in BM from breast cancer. Patients with newly diagnosed BM from breast cancer (BC) were identified from the Vienna Brain Metastasis Registry. Patients were grouped into the three main subtypes: hormone receptor-positive/HER2 (human epidermal growth factor receptor 2)-negative breast cancer (HR BC), HER2 overexpressing BC (HER2 BC) and triple-negative BC (TN BC). A retrospective chart review and statistical outcome analyses were performed. 968 patients with BM from breast cancer (34.4% HR BC; 28.9% HER2 BC; 21.8% TN BC; 14.9% unknown receptor status) were included in the analysis. 81.6% (790/968) of the patients presented with neurological symptoms at BM diagnosis including focal deficits (695/790;88.0%), signs of increased intracranial pressure (388/790;49.1%), epileptic seizures (126/790;15.9%) and neuropsychological symptoms (133/790;16.8%). Patients with HER2 BC (66/280;23.6%) were significantly more often diagnosed with BM in absence of neurological symptoms during screening procedures compared to HR BC (62/333;18.6%) and TN BC (31/211;14.7%; chi-square test; p=0.043). Among all BC subtypes, asymptomatic patients presented with a significant longer median overall survival (OS) from diagnosis of BM compared to symptomatic patients (p<0.05; log-rank test; HR BC 20 vs. 9 months; HER2 BC 20 vs. 11 months; TN 11 vs. 5 months). In multivariate analysis, the breast cancer-specific Graded Prognostic Assessment (Breast-GPA: HR:1.4; 95% CI, 1.27-1.49; p<0.001) and the presence of neurological symptoms (HR:1.6; 95% CI, 1.35-1.95; p<0.001) presented as independently associated with survival prognosis from time of BM diagnosis respectively. Neurological symptoms are independently associated with overall survival in patients with BM from breast cancer. Our results highlight the need to incorporate neurological symptoms into the prognostic assessment of patients with BM from breast cancer.

Abstract Background Based on the the rising detection of asymptomatic brain metastases (BM) and the remarkable intracranial responses of targeted- and immunotherapy in specific subgroups of asymptomatic BM patients, we aimed to evaluate the clinical characteristics and prognostic value of neurological symptoms in BM from melanoma. Material and Methods 670 patients with newly diagnosed melanoma BM were identified from the Vienna Brain Metastasis Registry. We performed retrospective chart review and statistical outcome analyses. Results 370/670 (55.2%) patients presented with neurological symptoms including neurological deficits (316/370; 85.4%), signs of increased intracranial pressure (126/370; 34.1%) and epileptic seizures (53/370; 14.3%). 57.6% (80/139) of patients with BRAF mutated melanoma presented with asymptomatic BM at diagnosis. Absence of neurological symptoms at BM diagnosis was associated with a longer median overall survival after time of BM diagnosis (7 vs. 5 months; p< 0.001; log-rank test). In multivariate analysis with the diagnosis-specific graded prognostic assessment (DS-GPA: HR:1.4; 95% CI, 1.25-1.48; p<0.001) and the graded prognostic assessment for melanoma using molecular markers (Melanoma-molGPA: HR:0.8; 95% CI, 0.66 -0.92; p<0.001), the presence of neurological symptoms (HR 0.8; 95% CI, 0.69 -0.96; p=0.003/ HR:0.6; 95% CI, 0.59-0.72; p<0.001) was found to be independently associated with survival prognosis from time of BM diagnosis respectively. Conclusion: Neurological symptoms at the time of BM diagnosis are an independent and strong prognostic factor in melanoma BM patients. Our results argue for the integration of neurological symptoms into the prognostic assessment of patients with BM from melanoma.

Abstract Background Brain metastases (BM) constitute the most common central nervous system tumors. The treatment options of BM consist of surgery, radiotherapy, radiosurgery, chemotherapy, and immunotherapy. Regarding surgery in BM, the extent of resection (EOR) represents a crucial factor for patient prognosis. However, first studies using postoperative MRI demonstrated that an unexpected residual tumor after surgery of BM is not uncommon despite these tumors were considered to be well-demarcated. The aim of this study was thus to investigate in a large cohort including multiple neurosurgical centers the EOR following BM resection, potential risk factors for incomplete resection and postsurgical follow-up data. Material and Methods In the current retrospective study conducted at 5 specialized neurosurgical centers in Austria, we included patients with BM resection and available postoperative MRI. The EOR following BM resection was determined by postoperative MRI (complete vs incomplete resection). Additionally, the data on the intraoperative judgement of the EOR of the performing neurosurgeon were collected. Moreover, potential factors for incomplete resection including tumor localization, tumor volume, primary tumor, pattern of contrast media enhancement on imaging and tumor eloquence were investigated. Finally, the rate of local progression of BM after initial surgery was analyzed in the follow-up period and overall survival data were collected. Results Altogether, 548 patients with 649 surgically treated BM were included. According to postoperative MRI, complete resection was achieved in 407 (66%) of 649 BM and incomplete resection in 176 (29%) of 649 BM. Misjudgment of the EOR by the neurosurgeon was found in 25% of cases and resulted in an unexpected residual tumor which was evident on postoperative MRI in 122 (22%) BM. Preoperative tumor volume was significantly larger in incompletely resected BM. Moreover, local progression was significantly more common in cases with incompletely resected BM and was also associated with shorter overall survival. Conclusion Our data of this study including multiple centers indicate that postoperative MRI is capable to detect a relatively high rate of unexpected residual tumors following resection of BM. Since local progression was more common in BM with residual tumors and this was associated with shorter survival, special attention should be paid to achieve a complete tumor resection.

The incidence of brain-only oligometastatic diseases have been described to be increasing. Therefore, we aimed to investigate the clinical course of patients with BM as the only site of metastatic disease. 6083 patients with BM from different solid tumors treated between 1989-2021 at the Medical University of Vienna were identified from the Vienna Brain Metastasis Registry. Patients with one BM as the only metastatic site were classified as brain-only oligometastatic, whereas patients with extracranial and intracranial metastatic lesions were classified as poly-metastatic. A retrospective analyses was performed. 1299/6083 (21.4%;[56.4% male; 43.6% female; median age 62 years]) patients presented with brain-only oligometastatic disease at time of BM diagnosis. Brain-only oligometastatic disease was most frequently observed in lung cancer (67.0%) followed by breast cancer (9.0%),melanoma (4.3%), cancer of unknown primary (4.5%), colorectal cancer (4.3%), renal cell carcinoma (3.2%), and others (7.7%) (p>0.001; chi-square test). The single BM as the only metastatic site was synchronously diagnosed with the primary tumor in 43.8% (569/1299) of patients. The median time from diagnosis of primary tumor to diagnosis of BM was significantly longer in poly-metastatic patients than in brain-only oligometastatic patients (25 vs. 17 months; p-value <0.001; log-rank test). Patients with brain-only oligometastatic disease showed a significantly longer median overall survival from diagnosis of BM compared to poly-metastatic patients (11 vs. 6 months; p< 0.001; log-rank test). Furthermore, 22.0% (286/1299) of patients with brain-only oligometastatic disease had long-term survival of >24 months, while only 11.0% (526/4784) of poly-metastatic patients presented with similar favorable survival (p<0.001; chi-square test). Patients with brain only oligometastatic disease present with a more favorable survival prognosis than patients with additional extracranial involvement. Multidisciplinary treatment planning should be encouraged in patients with brain-only oligometastatic disease to address the high proportion of patients surviving more than 24 months.

Abstract BACKGROUND We aimed to investigate the changing clinical characteristics of patients with brain metastases (BM) over the last three decades as the foundation for modern BM specific clinical trial planning. MATERIAL AND METHODS 6031 patients with newly diagnosed BM from different solid tumors treated between 1986–2020 were identified from the Vienna Brain Metastasis Registry. RESULTS The fraction of BM originating from the most common BM causing primary tumors (lung cancer, breast cancer and melanoma) was stable over the observation period from 1986–2020. BM from renal cell carcinoma, colorectal cancer and cancer of unknown primary (CUP) decreased over time (p<0.001). Synchronous diagnosis of BM and primary tumor was more frequently observed in lung cancer and CUP patients compared to breast cancer patients (p<0.001). An increasing fraction of patients presented with asymptomatic BM (1986–1999: 20.2% vs. 2010–2020: 30.4%; p<0.001), specifically in lung cancer (p<0.001), melanoma (p<0.001) and renal cell cancer (p=0.004). A decrease of neurosurgical procedures (1986–1999: 39.3% vs. 2010–2020: 20.4%) and an increase of radiation treatments (1986–1999: 56.5% vs. 2010–2020: 73.0%) and systemic therapies (1986–1999: 0.6% vs. 2010–2020: 2.4%; p<0.001) was observed. Furthermore, median overall survival significantly increased across entities (1986–1999: 5 months vs. 2010–2020: 7 months; p=0.001). Intracranial progression as the cause of death increased across entities (p< 0.001). The prognostic DS-GPA (Hazard ratio [HR] 1.42; p< 0.001) and the Lung-molGPA (HR 1.67; p<0.001) could be validated. CONCLUSION We observed changes of BM presentation and clinical parameters during the observation period depending on primary tumor origins. Future BM studies should follow an entity-specific approach and address the characteristics of modern BM cohorts.

Radio- and immunotherapy were postulated to have synergistic efficacy in brain metastasis (BM) treatment due to the immune modulating properties of radiation. Therefore, we aimed to investigate changes in the inflammatory microenvironment after local radiation treatments in BM specimens. Formalin fixed and paraffin embedded BM samples from treatment naïve patients (group 1) and from patients treated whole brain radiotherapy (WBRT) (group 2) or stereotactic radiosurgery (SRS) (group 3) or combined WBRT and SRS (group 4) or prophylactic cranial irradiation (group 5) before BM resection were identified from the Vienna Brain Metastasis Registry. T cell subsets (CD3+, CD8+, CD45RO+, FOXP3+ and LAG3) as well as expression of PD-L1 were investigated. Specimens from 81 patients (55 lung cancer, 15 breast cancer, 4 renal cell cancer, 1 melanoma, 1 colorectal cancer & 5 other tumor types) were available for analysis. Group 1 presented with statistically significantly higher CD3+ (median: 492.6 cells/mm2), CD8+ (median: 116.3 cells/mm2) and LAG3+ (median: 17.6 cells/mm2) TIL densities than group 2 (CD3+ median:55.5 cells/mm2; LAG3+ median: 4.6 cells/mm2), group 3 (CD3+ median: 67.7 cells/mm2; CD8+ median: 40.6 cells/mm2) and group 4 (CD3+ median: 38.28 cells/mm2; p-value <0.05; Kruskal Wallis test). No significant changes of the inflammatory microenvironment in group 5 compared to the other groups, and in PD-L1 expressions between the groups were observed (p-value >0.05; Kruskal Wallis test). Of 24/81 (29.6%) patients matched samples of initial resected BM and recurrent BM were available. All investigated T cell subsets were numerically lower in recurrent BM from patients treated with radiation therapy between resections than in recurrent BM from patients without radiation treatment between BM resections (p > 0.05; Mann-Whitney U-test). Our data indicate an immunosuppressive effect of radiotherapy on BM, as evidenced by decreased T cell infiltration in radiated versus non-radiated BM specimens. Future clinical studies should focus on the optimal timely sequencing of immune modulating therapies and radiotherapy.