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.

Depression is a serious comorbidity in cancer patients and is associated with increased morbidity and mortality. We investigated blood DNA methylation profiling as a potential biomarker for monitoring the likelihood of developing depressive symptoms during systemic anticancer treatment in order to early detect vulnerable patient populations. Lung cancer patients receiving systemic anticancer therapy at the Medical University of Vienna were included in this analysis. Leukocyte DNA methylation profiling from whole blood samples at timepoint of inclusion was performed with Infinium Methylation EPIC microarrays. Patients completed the EORTC QLQ-C30 questionnaire at inclusion and at every re-staging. The development of clinically relevant depressive symptoms was defined by an emotional functioning (EF) subscore below 80. The lowest EF score during the observation period per patient was used for the analyses. Seventy-three lung cancer patients (29/73 female, 39.7%; 44/73 male, 60.3%; median age 65 years, range 36-83 years) were included in this analysis. Six of 73 patients received chemotherapy, 34/73 immunotherapy and 33/73 chemoimmunotherapy. At least one EF score was available from each patient (median of 3 available scores, range 1-12 available scores). Fifty-seven of 73 (78.1%) patients developed relevant depressive symptoms during anticancer treatment while 16/73 (21.9%) patients maintained EF scores ≥80. DNA methylation differences defined by statistical tests as well as effect size were calculated between patients with EF scores <80 and patients with EF scores ≥80. Both patient groups formed well-separated clusters based on the methylation patterns of the top 500 differentially methylated CpG sites, independent of gender, the type of received systemic therapy or histologic subtype. DNA methylation profiling from peripheral blood leukocytes may identify lung cancer patients at risk for the development of depressive symptoms during systemic cancer therapy. Larger studies and biological interrogations are needed to substantiate our finding.

Cancer-associated cachexia (CAC) is an ongoing loss of skeletal muscle which cannot be fully abolished by nutritional interventions. While ESMO suggests precise CAC definitions, pre-cachexia definition is vague. Patients with solid cancers and lack of CAC at baseline, enrolled in the TRU Biobanking Program of the Medical University of Vienna between March 2019 and February 2022, were analyzed. At inclusion and each restaging, the prevalence of modified Glasgow Prognostic Score ≥1 (mGPS), weight loss ≥2% (WL), physical functioning <80 (PF2) and fatigue >30 (FA) was assessed clinically and with the EORTC QLQ-C30 questionnaire. In addition, we evaluated the burden of nutritional risk symptoms (diarrhea, nausea, vomiting, lack of appetite, constipation) with a score (NR) of 1-34. Pre-cachexia criteria were correlated with clinical parameters (age, gender, therapy status). Occurrence of CAC was evaluated using ESMO definitions. For analysis, 391 patients (47% female, 53% male, median age 59 years) were available. Median observation time was 4 months (range 0-46) with a median of 2 observations (range 1-13). Within the observed period 12% of patients developed CAC. Pre-cachexia as defined by mGPS was found in 52% of patients, by WL in 53%, by PF2 in 69%, by FA in 84%, and by NR ≥7 in 51% of patients. Pre-cachexia by WL was found less often in female (43%) than in male patients (62%; OR 2.14, 95% CI 1.43-3.23; p<0.001) and more often in treatment-naive patients (60%) than in those with prior systemic therapy (48%; OR 1.62, 95% CI 1.08-2.43, p=0.019). Patients with pre-cachexia by NR were younger (median 58, IQR 50-65) compared to the other patients (median 60, IQR 53-70; p=0.018). All other pre-cachexia definitions did not correlate with clinical characteristics (p>0.05). All five pre-cachexia definitions were fulfilled by 15% of patients, four by 27%, three by 29%, two by 14%, and one by 14%. No pre-cachexia definition was fulfilled by 2.3%. None of the investigated pre-cachexia definitions reliably identified patients with subsequent CAC development (p>0.05). Further research on pre-cachexia-defining parameters is needed for the development of standardized screening methods as current definitions fail to identify patients with high CAC risk.

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.

Vitamin deficiencies and supplement intake is discussed controversially in oncology. Therefore, we aimed to investigate vitamin D, A and E levels and their correlation with clinical characteristics and symptomatic burden in a large pan-cancer cohort. 25-hydroxy-vitamin D, vitamin A, and vitamin E levels were measured at treatment start in patients receiving palliative cancer treatment at the Medical University of Vienna (MUV) included in the “Biobanking Program for Personalized Immunotherapy” between May 2019 and March 2022. Measurements were performed using a chemiluminescence immunoassay (vitamin D) and high performance liquid chromatography (vitamin A, vitamin E) established at the Department of Laboratory Medicine (MUV). Cut off values defining the normal range were predefined as follows: vitamin D (75-250nmol/L), vitamin A (1.05-2.45μmol/L), vitamin E (12.0-42.0μmol/L). Symptom burden was assessed using the EORTC quality of life questionnaire QLQC-30 and its subscore assessing fatigue. Vitamin D, A, and E levels were measured in a total of 231 patients (92 females, 139 males, median age: 64 years [range: 23-89]). Median vitamin levels were as follows: vitamin D 66.9nmol/l (range: 10.4-186.0nmol/l), vitamin A 1.64μmol/l (range: 0.29-4.22μmol/l), and vitamin E 35.8μmol/l (range: 20.2-76.8μmol/l). Patients presented with vitamin D deficiencies in 138/231 (59.7%) and vitamin A deficiencies in 34/231 cases (14.7%). No vitamin E deficiencies were detected, however, 25/231 patients (24.2%) presented with vitamin E levels above normal. Vitamin A levels were increased in 26/231 patients (11.3%), while vitamin D levels above the upper limit of normal in 1/231 patient (0.4%). No correlations between vitamin levels and clinical characteristics at sample collection such as sex, age, tumor origin, symptom burden or fatigue were observed (p>0.05). Additionally, no correlation between vitamin levels and overall survival could be detected (p>0.05). Even though vitamin deficiencies are common in patients with cancer, no influences of vitamin levels on symptom burden, fatigue or survival could be detected.

Immunotherapy (IO) has become an important component of systemic treatment in early-stage triple-negative breast cancer (eTNBC). Considering toxicity and financial burden, identifying reliable biomarkers of response is urgently warranted. Therefore, we investigated blood cell DNA methylation profiles as biomarker for achieving a pathologic complete response (pCR) at surgery after neoadjuvant chemoimmunotherapy. eTNBC patients receiving systemic chemoimmunotherapy before surgery at the Medical University of Vienna were included in this analysis. DNA from peripheral blood mononuclear cells (PBMCs) was isolated from whole blood samples collected at start of neoadjuvant therapy and methylation profiling was performed with Infinium Methylation EPIC microarrays. Routine histologic work-up from surgery determined a pCR or non-pCR according to pathologic standard procedures. Seven eTNBC patients (all women), median age of 63 years (range 40-83 years), were available for this analysis. All patients received neoadjuvant chemoimmunotherapy with pembrolizumab plus weekly paclitaxel/carboplatin followed by pembrolizumab plus epirubicin/cyclophosphamide. Three out of 7 (42.9%) patients achieved a pCR, while 4/7 (57.1%) had residual disease at surgery. Notably, when focusing on their methylation patterns, patients with pCR and non-pCR could be clearly segregated into two distinct groups. This distinction was based on the methylation profiles of the top 500 differentially methylated CpG sites. Our results revealed differentially methylated genes between patients w/o pCR that might play a role in response to chemoimmunotherapy. Blood cell methylation profiles might serve as predictive biomarker for pCR in eTNBC and larger patient cohorts are needed to validate our findings.

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.

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.

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.

Immunotherapy (IO) is an important pillar in the treatment of various advanced solid cancers, but resistance is frequent. We aimed to characterize the inflammatory tumor microenvironment of patients with brain metastasis (BM) progression after IO to gain insight on potential inflammatory resistance mechanisms.

Patients with chronic active Crohn's disease are dependent on systemic glucocorticosteroids. The aim of the study was to investigate the efficacy of budesonide, a topically selective glucocorticosteroid, as therapy in these patients. We investigated 20 patients with chronic active Crohn's disease. During the last 6 months prior to the study the patients had a median Crohn's disease activity index (CDAI) of 193 (interquartile range: 122-230) (monthly controls) with a median prednisolone dosage of 14 mg per day (9-20). Budesonide was given 3 to 6 mg daily and prednisolone was weaned within one month. The patients were seen monthly for 6 months. Treatment was considered not successful, if under budesonide therapy CDAI was above 200 and increased more than 60 points despite weaning of prednisolone. Only 5 patients remained in the study for 6 months without deterioration. All other patients (75%) dropped out. The reasons for drop out of the study were worsening in 11 cases, the occurrence of extraintestinal manifestations without signs of severe intestinal inflammation in one case and noncompliance in 3 cases. Worsening could be confirmed by an increase not only of CDAI but also of biochemical parameters of inflammation in all cases. Our data show clearly, that in the dosage investigated budesonide was not effective in chronic active Crohn's disease. Further investigations are needed to evaluate higher dosages of budesonide versus conventional glucocorticosteroids.