IntroductionIn the recent years, the Austrian general population has faced a confluence of multiple crises. This study investigates the support wishes and mental health parameters of the Austrian general population aiming to comprehending the unmet needs and providing guidance for future psychosocial interventions and research endeavors.Methods1,031 participants attended the online survey and one third (n = 332) wished for further support to improve mental well-being in April 2022. A total of 280 participants accompanied their support wish with written accounts. Participants’ mental health status was evaluated using the PHQ-9 (depression), GAD-7 (anxiety), ISI (insomnia), PSS-10 (perceived stress), CAGE (alcohol abuse), WHO-5 (well-being), and the SCOFF (eating disorder) questionnaires. Data analysis employed a mixed-methods approach.ResultsThe preeminent support wish identified was the need for professional mental support (29.3%), followed by communication (21.6%), other professional support except mental and medical support (13.9%). In line with these findings, participants expressing a support wish experienced increased mental health distress across all assessed parameters.ConclusionThe findings indicate the presence of a vulnerable population within the Austrian general population, which may benefit from targeted support interventions. Consequently, this study contributes to the identification of unmet support needs among the Austrian populace during times of crisis, facilitating the development and enhancement of precisely tailored intervention strategies.

The mental health of school students has been severely impacted by the aftermath of the COVID-19 pandemic. The present study used a mixed methods approach to assess students' mental health and examine their wishes for support to improve their psychological well-being. We further investigated gender and age group differences in the amount of clinically relevant mental health problems and the roles that mental health and gender had on desired support. Between April and May 2022, a total of 616 Austrian students aged between 14 and 20 participated in a cross-sectional online survey (77.4% female; 19.8% male; 2.8% non-binary) assessing wishes for support regarding mental well-being and mental health indicators (depression: PHQ-9; anxiety: GAD-7; insomnia: ISI; stress: PSS-10; eating disorders: SCOFF; alcohol abuse: CAGE). A wish for support was expressed by 46.6% of the students. Qualitative content analysis revealed that the two most important categories of desired support types were "professional help" and "someone to talk to". The group of students with a wish for support in general significantly more often showed clinically relevant depression, anxiety, insomnia, eating disorders, or high stress symptoms. Students that wished for professional help significantly more often exceeded the cut-off for clinically relevant depression, anxiety, and high stress. Those who wished for someone to talk to significantly more often exceeded the cut-off for clinically relevant eating disorders. The results indicate a great need for support for young people's mental health problems and that this need is even more urgent for students.

The small-molecule E26 transformation-specific (ETS) factor inhibitor YK-4-279 was developed for therapy of ETS/EWS fusion-driven Ewing's sarcoma. Here we aimed to identify molecular factors underlying YK-4-279 responsiveness in ETS fusion-negative cancers. Cell viability screenings that deletion of P53 induced hypersensitization against YK-4-279 especially in the BRAFV600E-mutated colon cancer model RKO. This effect was comparably minor in the BRAF wild-type HCT116 colon cancer model. Out of all ETS transcription factor family members, especially ETS1 overexpression at mRNA and protein level was induced by deletion of P53 specifically under BRAF-mutated conditions. Exposure to YK-4-279 reverted ETS1 upregulation induced by P53 knock-out in RKO cells. Despite upregulation of p53 by YK-4-279 itself in RKOp53 wild-type cells, YK-4-279-mediated hyperphosphorylation of histone histone H2A.x was distinctly more pronounced in the P53 knock-out background. YK-4-279-induced cell death in RKOp53-knock-out cells involved hyperPARylation of PARP1, translocation of the apoptosis-inducible factor AIF into nuclei, and induction of mitochondrial membrane depolarization, all hallmarks of parthanatos. Accordingly, pharmacological PARP as well as BRAFV600E inhibition showed antagonistic activity with YK-4-279 especially in the P53 knock-out background. Taken together, we identified ETS factor inhibition as a promising strategy for the treatment of notoriously therapy-resistant p53-null solid tumours with activating MAPK mutations.

Background: ETS factors are an evolutionary conserved family of transcription factors with a wide range of cell molecular functions including embryonic development and immune cell function. YK-4-279 is a novel ETS-factor inhibitor originally designed to inhibit the transcriptional activity of the EWS-Fli1 fusion protein in Ewing sarcoma. Analysis of several other human cancer entities revealed promising activity also in human melanoma cells, often overexpressing ETS factors. Drug resistance development, the main obstacle for sustained clinical response, involves changes both in tumor cells as well as the tumor environment including tumor-associated macrophages.

The BRAF gene and the TERT promoter are among the most frequently altered genomic loci in low-grade (LGG) and high-grade-glioma (HGG), respectively. The coexistence of BRAF and TERT promoter aberrations characterizes a subset of aggressive glioma. Therefore, we investigated interactions between those alterations in malignant glioma. We analyzed co-occurrence of BRAF(V600E) and TERT promoter mutations in our clinical data (n = 8) in addition to published datasets (n = 103) and established a BRAF(V600E)-positive glioma cell panel (n = 9) for in vitro analyses. We investigated altered gene expression, signaling events and TERT promoter activity upon BRAF- and E-twenty-six (ETS)-factor inhibition by qRT-PCR, chromatin immunoprecipitation (ChIP), Western blots and luciferase reporter assays. TERT promoter mutations were significantly enriched in BRAF(V600E)-mutated HGG as compared to BRAF(V600E)-mutated LGG. In vitro, BRAF(V600E)/TERT promoter double-mutant glioma cells showed exceptional sensitivity towards BRAF-targeting agents. Remarkably, BRAF-inhibition attenuated TERT expression and TERT promoter activity exclusively in double-mutant models, while TERT expression was undetectable in BRAF(V600E)-only cells. Various ETS-factors were broadly expressed, however, only ETS1 expression and phosphorylation were consistently downregulated following BRAF-inhibition. Knock-down experiments and ChIP corroborated the notion of a functional role for ETS1 and, accordingly, all double-mutant tumor cells were highly sensitive towards the ETS-factor inhibitor YK-4-279. In conclusion, our data suggest that concomitant BRAF(V600E) and TERT promoter mutations synergistically support cancer cell proliferation and immortalization. ETS1 links these two driver alterations functionally and may represent a promising therapeutic target in this aggressive glioma subgroup.

Abstract Metal‐driven self‐assembly afforded a multitude of fascinating supramolecular coordination complexes (SCCs) with applications as catalysts, host–guest, and stimuli‐responsive systems. However, the interest in the biological applications of SCCs is only starting to emerge and thorough characterization of their behavior in biological milieus is still lacking. Herein, we report on the synthesis and detailed in‐cell tracking of a Pt2L2 metallacycle. We show that our hexagonal supramolecule accumulates in cancer cell nuclei, exerting a distinctive blue fluorescence staining of chromatin resistant to UV photobleaching selectively in nucleolar G4‐rich regions. SCC co‐localizes with epitopes of the quadruplex‐specific antibody BG4 and replaces other well‐known G4 stabilizers. Moreover, the photophysical changes accompanying the metallacycle binding to G4s in solution (fluorescence quenching, absorption enhancement) also take place intracellularly, allowing its subcellular interaction tracking.

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers and a major cause of cancer mortality worldwide. At late stage of the disease CRC often shows (multiple) metastatic lesions in the peritoneal cavity which cannot be efficiently targeted by systemic chemotherapy. This is one major factor contributing to poor prognosis. Oxaliplatin is one of the most commonly used systemic treatment options for advanced CRC. However, drug resistance - often due to insufficient drug delivery - is still hampering successful treatment. The anticancer activity of oxaliplatin includes besides DNA damage also a strong immunogenic component. Consequently, the aim of this study was to investigate the effect of bacterial ghosts (BGs) as adjuvant immunostimulant on oxaliplatin efficacy. BGs are empty envelopes of gram-negative bacteria with a distinct immune-stimulatory potential. Indeed, we were able to show that the combination of BGs with oxaliplatin treatment had strong synergistic anticancer activity against the CT26 allograft, resulting in prolonged survival and even a complete remission in this murine model of CRC carcinomatosis. This synergistic effect was based on an enhanced induction of immunogenic cell death and activation of an efficient T-cell response leading to long-term anti-tumor memory effects. Taken together, co-application of BGs strengthens the immunogenic component of the oxaliplatin anticancer response and thus represents a promising natural immune-adjuvant to chemotherapy in advanced CRC.

Evasion to new treatments of advanced melanoma is still associated with a poor prognosis. Choosing the best combination of agents that can bypass resistance mechanisms remains a challenge. Sphaeropsidin A (Sph A) is a fungal bioactive secondary metabolite previously shown to force melanoma cells to undergo apoptosis via cell volume dysregulation. This work studied its in vitro combination with cytotoxic chemotherapeutics in a rational manner.

Supramolecular Pt(ii) quadrangular boxes bind native and G-quadruplex DNA motifs in a size-dependent fashion. Three Pt molecular squares of distinct size show biological activity against cancer cells and heavily influence the expression of genes known to form G-quadruplexes in their promoter regions. The smallest Pt-box displays less activity but more selectivity for a quadruplex formed in the c-Kit gene.

Aim We aimed to characterize angiogenesis and proliferation and their correlation with clinical characteristics in a large brain metastasis ( BM ) series. Methods K i67 proliferation index, microvascular density ( MVD ) and hypoxia‐inducible factor 1 alpha ( HIF ‐1 alpha) index were determined by immunohistochemistry in BM and primary tumour specimens. Results Six hundred thirty‐nine BM specimens of 639 patients with lung cancer (344/639; 53.8%), breast cancer (105/639; 16.4%), melanoma (67/639; 10.5%), renal cell carcinoma ( RCC ; 52/639; 8.1%) or colorectal cancer ( CRC ; 71/639; 11.1%) were available. Specimens of the corresponding primary tumour were available in 113/639 (17.7%) cases. Median K i67 index was highest in CRC BM and lowest in RCC BM ( P < 0.001). MVD and HIF ‐1 alpha index were both highest in RCC BM and lowest in melanoma BM ( P < 0.001). Significantly higher K i67 indices, MVD and HIF ‐1 alpha indices in the BM than in matched primary tumours were observed for breast cancer, non‐small cell lung cancer ( NSCLC ) and CRC . Correlation of tissue‐based parameters with overall survival in individual tumour types showed a favourable and independent prognostic impact of low K i67 index [hazard ratio ( HR ) 1.015; P < 0.001] in NSCLC BM and of low K i67 index ( HR 1.027; P = 0.008) and high angiogenic activity ( HR 1.877; P = 0.002) in RCC . Conclusion Our data argue for differential pathobiological and clinical relevance of K i67 index, HIF 1‐alpha index and MVD between primary tumour types in BM patients. An independent prognostic impact of tissue‐based characteristics was observed in patients with BM from NSCLC and RCC , supporting the incorporation of these tissue‐based parameters into diagnosis‐specific prognostic scores.

In primary melanoma, ETV1 transcription factor was suggested to be activated mainly by gene amplification and to promote tumor growth in cooperation with BRAF V600E . Aim of this study was to investigate ETV1 expression in human melanoma with a focus on brain metastases. We investigated ETV1 in 68 human melanoma brain metastases using FISH for ETV1 gene (located at chromosome 7p21) and centromere chromosome 7 and immunohistochemistry for ETV1, BRAF V600E , and ETV1/BRAF associated proteins pMSK1, pRSK1, pp38, pMEK1/2, MAPKAP kinase 2, CIC, HIF-1alpha and Ki-67. We further studied ETV1 copy number variations in 32 melanoma cell lines from primary and metastatic lesions using array CGH. The influence of the MAP kinase pathway activity on ETV1 mRNA and protein expression under BRAF wild-type and BRAF V600E conditions were determined in melanoma cell lines using qRT-PCR and Western Blot. No ETV1 high grade amplifications were observed in tissue samples, but low grade ETV1 gene amplifications were found in 7 (10.3 %) melanoma brain metastases. ETV1 protein expression in tissue samples (15 %) correlated with BRAF V600E status (p = 0.007) and HIF-1alpha expression (p = 0.049), but not with ETV1 gene dose. Application of the BRAFV600E-specific inhibitor vemurafenib and the BRAFV6ooE/V600K-inhibitor dabrafenib revealed predominant regulation of ETV-1 mRNA and protein via MAPK-pathway. ETV1 expression is a rare event in human melanoma and seems to be rather based on hyperactivation of MAPK signals, by BRAF V600E mutation, than on ETV1 gene amplification. Consequently, therapeutic inhibition of BRAF and the downstream MAPK pathway also down-regulates oncogenic ETV1 expression.

Background Increased incidence of brain metastases (BM) in non-small cell lung cancer (NSCLC) with ALK translocations was postulated, however, ALK gene aberrations in NSCLC-BM have not been investigated so far. Methods We investigated ALK and EML4 gene aberrations (amplifications, translocations, inversions) by fluorescent in situ hybridization (FISH) (n = 175) and ALK and EML4 protein expression by immunohistochemistry (n = 221) in NSCLC BM and corresponding primary tumors. Results ALK translocations were found in 4/151 (2.6%; 3 of them involving EML4) of BM of adenocarcinomas (AC), 1/9 (11.1%) of adenosquamous carcinomas (ASC), 0/5 of squamous cell carcinomas (SCC) and 0/10 of large cell carcinomas (LCC). Rearrangement of ALK without involvement of EML4 was seen in 1 AC-BM and rearrangement of EML4 without involvement of ALK in 3 AC-BM, 1 ASC-BM and 1 LCC. ALK amplifications without gene rearrangements were found in BM of 16/151 (10.6%) AC, 2/5 (40%) SCC, 0/9 ASC and one LCC. ALK translocation status was constant between BM and primary tumors in 16 evaluable cases including two cases with ALK–EML4 translocations Among these 16 cases ALK amplification was seen in two BM and none of the primary tumors. All cases with translocations but not with amplifications of ALK showed protein expression. We found no association of ALK gene status with patient age, gender or overall survival time. Conclusions ALK translocations and amplifications are found in approximately 3% and 11% of NSCLC-BM, respectively. While ALK translocations appear to be constant between primary tumors and BM, amplifications seem to be more prevalent in BM. ALK translocation, but not ALK amplification is associated with ALK protein overexpression. Further studies are needed to determine whether NSCLC-BM patients with ALK gene aberrations may benefit from specific inhibitor therapy.

The ErbB receptor family has been implicated in brain metastases (BM) formation in various cancer types and specific targeted therapies are available. We investigated the overexpression of EGFR, HER2 and HER3 in BM of non-small cell lung cancer (NSCLC) patients to get a better insight on pathobiology of BM and potential drugable targets. We performed immunohistochemical analysis of EGFR, HER2 and HER3 on tissue microarrays of 131 NSCLC-BM. Fifty-one of 131 (38.9%) specimens were considered as positive for EGFR overexpression, 12/131 (9.2%) for HER2 and 27/131 (20.6%) for HER3 respectively. Sixty-nine of 131 (52.7%) of the cases showed overexpression of at least one marker. Four of 131 (3.1%) were positive for all three markers. Strong correlation was observed between HER2 and HER3 overexpression (p = 0.009; Chi-square test after Bonferroni-Holmes correction). No statistically significant correlation of EGFR, HER2 or HER3 overexpression with clinico-pathological parameters including overall survival times was observed. We observed overexpression of ErbB receptor family members, which represent established therapeutic targets in various primary tumours, in approximately half of NSCLC-BM. Further studies should investigate the role of the ErbB pathway in development of and as a therapeutic target in BM of NSCLC patients.

The ErbB receptor family has been implicated in brain metastases (BM) formation in various cancer types and specific targeted therapies are available. We investigated the overexpression of EGFR, HER2 and HER3 in BM of non-small cell lung cancer (NSCLC) patients to get a better insight on pathobiology of BM and potential drugable targets. We performed immunohistochemical analysis of EGFR, HER2 and HER3 on tissue microarrays of 131 NSCLC-BM. Fifty-one of 131 (38.9%) specimens were considered as positive for EGFR overexpression, 12/131 (9.2%) for HER2 and 27/131 (20.6%) for HER3 respectively. Sixty-nine of 131 (52.7%) of the cases showed overexpression of at least one marker. Four of 131 (3.1%) were positive for all three markers. Strong correlation was observed between HER2 and HER3 overexpression (p = 0.009; Chi-square test after Bonferroni-Holmes correction). No statistically significant correlation of EGFR, HER2 or HER3 overexpression with clinico-pathological parameters including overall survival times was observed. We observed overexpression of ErbB receptor family members, which represent established therapeutic targets in various primary tumours, in approximately half of NSCLC-BM. Further studies should investigate the role of the ErbB pathway in development of and as a therapeutic target in BM of NSCLC patients.

ObjectivesFGFR1 amplifications are common in squamous cell carcinoma and rare in adenocarcinoma of the lung, but have not been investigated in brain metastases of non-small cell lung cancer (NSCLC).Materials and methodsWe performed fluorescent in situ hybridization (FISH) for FGFR1 and immunohistochemistry for pAKT, PI3K, HIF1a and Ki67 in 175 NSCLC brain metastases and 11 matched primary tumors. ALK gene rearrangement status was available from a previous study. We performed statistical correlations of clinical, histopathological and molecular data.ResultsFGFR1 amplifications were found in a total of 30/175 (17%) brain metastases: 4/21 (19%) squamous cell carcinomas, 20/130 (15.3%) adenocarcinomas, 2/12 (16.6%) adenosquamous carcinomas, 4/9 (44.4%) large cell carcinomas and 0/3 neuroendocrine large cell carcinoma. FGFR1 gene status was identical between primary tumors and brain metastases in 9/11 evaluable cases. In 2/11 cases (1 adenosquamous and 1 large cell carcinoma), FGFR1 amplifications were present only in the brain metastasis and not in the primary tumor. Furthermore, we found a significant positive correlation of ALK and FGFR1 gene amplification status in brain metastases (p<0.001, Chi square test). Patients with high-level FGFR1 amplifications had significantly higher number of visceral metastases (p<0.001, Chi square test).ConclusionOur findings argue for an enrichment of FGFR1 amplifications in brain metastases of adenocarcinomas (where they were 5-fold more frequent than reported for primary tumors) and possibly also of other non-squamous carcinomas, but not in squamous cell carcinomas of the lung. These results may be relevant for targeted therapy and prophylaxis of NSCLC brain metastases.

To analyze the prognostic value of the extent of peritumoral brain edema in patients operated for single brain metastases (BM), we retrospectively evaluated pre-operative magnetic resonance images in a discovery cohort of 129 patients and a validation cohort of 118 patients, who underwent neurosurgical resection of a single BM in two different hospitals. We recorded clinical parameters and immunohistochemically assessed the Ki67 index, the microvascularization patterns and the expression of hypoxia-induced factor 1 alpha (HIF1a) in the BM tissue specimens retrieved at neurosurgery. Statistical analysis including uni- and multivariate survival analyses were performed. Baseline characteristics were well balanced between the discovery and validation cohorts. In univariate analysis, we found a significant association of favorable overall survival time with young patient age, high Karnofsky performance score, low graded prognostic assessment (GPA) class, absence of extracranial metastases, adjuvant treatment with whole brain radiotherapy and, surprisingly, large brain edema. In multivariate analysis, only GPA and extent of brain edema remained independent prognostic parameters. The prognostic impact of the extent of brain edema was consistent in the two patient cohorts. Furthermore, we found a significant correlation of small brain edema with brain-invasive tumor growth pattern as assessed intraoperatively by the neurosurgeon, low neo-angiogenic activity and low expression of HIF1a. Extent of brain edema independently correlates with prognosis in patients operated for single BM. In conclusion, patients with small peritumoral edema have shorter survival times and their tumors are characterized by a more brain-invasive growth, lower HIF1a expression and less angiogenic activity.

BACKGROUNDBrain metastases (BM) of gastro-oesophageal cancer are exceedingly rare and only limited data exist on their pathobiology.MATERIALS AND METHODSWe identified tissue samples of BM of gastro-oesophageal cancer and analyzed the expression of human epidermal growth factor receptor-2 (HER2), phosphorylated signal transducer and activator of transcription-3 (pSTAT3), epithelial growth factor receptor (EGFR), V600E point mutation of the v-raf murine sarcoma viral oncogene homolog-B1 (BRAF V600E), cluster of differentiation molecule-34 (CD34), hypoxia inducible factor-1α (HIF 1-α) and Ki-67 by immunohistochemical methods.RESULTSOur series comprised of twenty adenocarcinomas and one oesophageal squamous cell carcinoma. Three (14%), 7 (33%), 9 (43%), 18 (86%) and 0 BM specimens were scored positively for HER2, EGFR, pSTAT3, HIF1-α and BRAF V600E expression. The median Ki-67 index was 59%. The microvascular density was moderate-to-high and active intratumoral microvascular sprouting was evident in 20/21 (95%) of BMs. The HER2 and EGFR expression status were consistent between primary tumors and BM in all three assessable cases. HIF1-α and pSTAT3 expression were significantly higher in HER2-positive cases.CONCLUSIONTherapeutic use of agents targeting HER2, pSTAT3, EGFR and angiogenesis may be feasible for selected BM of gastro-esophageal cancer. HER2 positivity does not seem to predispose to brain colonization in gastro-esophageal cancer.

2053 Background: The sub-cohort of brain metastases (BM) patients (pts) with single BM has relatively favorable survival times, but novel prognostic factors are needed to individualize patient management. Methods: We retrospectively evaluated pre-operative magnetic resonance images in patients, who underwent neurosurgical resection of a single BM. The localization and largest diameter of the BM was recorded. The extent of brain edema (BE) was graded on contrast-enhanced T1, T2 and fluid-attenuated inversion recovery (FLAIR) images as follows: BE <1cm, BE >1 cm without affection of the contralateral hemisphere, BE >1cm with affection of the contralateral hemisphere. Further, immunohistochemically assessed expression of hypoxia-induced factor 1 alpha (HIF1a) was analyzed in each BM tissue specimen. Results: 159 pts were studied (81 male, 78 female; age range 24 to 80 years, median 58). The primary tumors were lung carcinoma in 74, breast cancer in 26, kidney cancer in 13, colorectal cancer in 11, melanoma in 9 and other tumor types in 26 cases. At univariate analysis, we found a significant positive correlation of overall survival time (OS) with young age, Karnofsky index >80, non-melanoma histology, and, surprisingly, large BE (p<0.05, log-rank test). At multivariate analysis, pts age, histological diagnosis and extent of BE remained independent prognostic parameters (Cox regression model, p<0.05). We found a significant positive correlation of extent of BE with expression of HIF1a (Mann-Whitney-U test, p=0.003). Conclusions: Large BE positively and independently correlates with favorable prognosis in pts operated for single BM. Peritumoral BE correlates with HIF1a expression, indicating a possible role of tumor hypoxia in its formation. [Table: see text]

While cancer research has been focused on tumor cells for many years, evidence is growing that the tumor stroma and in particular cancer-associated fibroblasts (CAFs) in particular play essential roles in the progression of human malignant disease. In human lung cancer, CAFs expressing the transmembrane protein podoplanin were shown to have significant influence on the patients' prognosis. In this study, we investigated the presence and prognostic role of podoplanin-expressing CAFs in a large series of patients with invasive breast cancer. Podoplanin expression was evaluated immunohistochemically in 367 breast cancers. Tumors with ≥10% distinct podoplanin staining were considered as positive (CAF+). Cytoplasmic podoplanin expression of tumor cells was considered as positive when ≥5% of tumor cells showed a distinct podoplanin expression. In normal breast tissue, no podoplanin-expressing fibrocytes were found. Thirty-three patients (9%) with breast cancer showed podoplanin expression in CAFs. In 28 patients (8%), a podoplanin expression in tumor cells was observed. A strong negative correlation of CAF+ with estrogen receptor status (p<0.001), and a significant association with higher histological grading (p<0.001) was seen. In multivariable analysis, CAFs+ was an independent prognostic factor for disease free (1.78 Hazard ratio; p=0.026) and overall survival (2.304 Hazard ratio; p=0.002) in patients with breast cancer. Podoplanin-expressing CAFs contribute to the prognosis of invasive breast cancer, indicating a highly aggressive subgroup. CAFs may present a highly selective target for anti-cancer therapies in patients with invasive breast cancer.