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Radiotherapy can act as an in situ vaccine thereby activating tumor-specific immune responses that prevent tumor outgrowth in treated patients. While carbon ion radiotherapy has shown superior biophysical properties over conventional photon irradiation, the immunological effects induced have remained largely uncovered. The combination of radiotherapy with immune checkpoint inhibition (radioimmunotherapy) aims at further enhancement of anti-tumor immunity; however, studies on the immune cell composition in irradiated and distant tumors following radioimmunotherapy with carbon ions are scarce. We have established a bilateral tumor model by time shifted transplantation of murine, Her2+ EO771 tumor cells onto the flanks of immune competent mice followed by selective irradiation of the primal tumor, while sparing the consecutive tumor. We demonstrate that αCTLA4-but not αPD-L1-based radioimmunotherapy induces complete tumor rejection in our model. Intriguingly, local tumor control caused in situ immunization resulting even in eradication of non-irradiated, distant tumors. Moreover, cured mice were protected against EO771 rechallenge indicative of long lasting, tumor-protective immunological memory. Deconvolution of the treatment induced immunological effects by single cell RNA-sequencing (scRNA-seq) and concomitant flow cytometric analyses revealed in irradiated tumors predominating myeloid cells that developed into distinct tumor-associated macrophage clusters with upregulated expression of TNF and IL1 responsive genes, as well as activation of NK cells. Non-irradiated tumors showed higher frequencies of naïve T cells in irradiated mice, which were activated when combined with CTLA4 blockade. In conclusion, radioimmunotherapy with carbon ions plus CTLA4 inhibition reshapes the tumor-infiltrating immune cell composition and can induce complete rejection even of non-irradiated tumors. Our data present a rationale to combine radiotherapy approach with CTLA4 blockade to achieve durable anti-tumor immunity. Evaluation of future radioimmunotherapy approaches should thus not only focus on the immunological impacts at the site of irradiation but should also consider systemic immunological effects that might affect outgrowth of non-irradiated tumors.

Objectives Direct toxic effects of transthyretin amyloid in patient plasma upon cardiomyocytes are discussed. However, no data regarding the relevance of this putative effect for clinical outcome are available. In this monocentric prospective study, we analyzed cellular hypertrophy after phenylephrine stimulation in vitro in the presence of patient plasma and correlated the cellular growth response with phenotype and prognosis. Methods and results Progress in automated microscopy and image analysis allows high-throughput analysis of cell morphology. Using the InCell microscopy system, changes in cardiomyocyte’s size after treatment with patient plasma from 89 patients suffering from transthyretin amyloidosis and 16 controls were quantified. For this purpose, we propose a novel metric that we named Hypertrophic Index, defined as difference in cell size after phenylephrine stimulation normalized to the unstimulated cell size. Its prognostic value was assessed for multiple endpoints (HTX: death/heart transplantation; DMP: cardiac decompensation; MACE: combined) using Cox proportional hazard models. Cells treated with plasma from healthy controls and hereditary transthyretin amyloidosis with polyneuropathy showed an increase in Hypertrophic Index after phenylephrine stimulation, whereas stimulation after treatment with hereditary cardiac amyloidosis or wild-type transthyretin patient plasma showed a significantly attenuated response. Hypertrophic Index was associated in univariate analyses with HTX (hazard ratio (HR) high vs low: 0.12 [0.02–0.58], p = 0.004), DMP: (HR 0.26 [0.11–0.62], p = 0.003) and MACE (HR 0.24 [0.11–0.55], p Conclusions Attenuated cardiomyocyte growth response after stimulation with patient plasma in vitro is an independent risk factor for adverse cardiac events in ATTR patients

Background and purpose: Glioblastoma multiforme (GBM) is a stroma rich immune cold tumor with still dismal prognosis of despite multimodal therapy consisting of postoperative surgery and radiochemotherapy. Consequently, all randomized Phase III clinical GBM trials attempting to integrate immune checkpoint blockade (ICB) have failed to demonstrate efficacy. We have recently shown that tumor immune microenvironment (TIME) reprograming via Bintrafusp alpha (Anti-PD-L1/TGF-beta bispecific molecule) combined with conventional photon radiotherapy elicits beneficial effects and prolong survival in experimental models (Lan, Knoll, Moustafa et al. Cancer Cell 2021). Moreover, eradication of radioresistant i.e., stem cell like, invasive and hypoxic tumor subpopulations by high linear energy transfer (LET) carbon ion radiotherapy (CIRT) was shown to generate an immune permissive niche in experimental GBM models (Chiblak et al. JCI Insight 2019, Tang et al. IJRBP 2022). Therefore, we aimed to systematically study the effect of CIRT vs. conventional photon radiotherapy (RT) in two (GL261 and SB28) orhotopic syngeneic mouse GBM models. Methods: Tumor growth monitoring was conducted longitudinally by means of bioluminescence and small animal MR-imaging. Mice were irradiated in five fractions (Fx) with a total dose of 15Gy RT vs. 5, 10 and 15Gy CIRT. Results: Intriguingly, all GL261 harbouring mice were cured after 5 × 3Gy Fx CIRT with no sign of tumor recurrence up to 80 days post irradiation. In contrast, no tumor control was found after 5 × 3Gy Fx RT led despite a significant prolongation of OS. The tumor control probability 50% (TCP50) for CIRT in GL261 was estimated to be 10Gy in 5 Fx. To asses if the curative effect was related to tumor eradication or reconstitution of immune surveillance. GL261 tumor cells were implanted in the contralateral hemisphere of cured mice. Single cell and Bulk RNAseq as well as immunostaining were conducted to deconvolute TIME effects post CIRT. Conclusions: In line with mechanistic studies, lack of tumor growth indicating persistent immunity against GL261 was found in CIRT cured mice. Similar experiments are conducted in SB28 model and will be presented. Together, these encouraging data indicate potential benefits for high-LET CIRT in immune modulation of GBM in experimental models that warrants for study and optimization towards clinical translation. Citation Format: Federica Ciamarone, Sarah Meister, Jennifer Furkel, Maximilian Knoll, Christian Schwager, Jovana Bojcevski, Yannik Streibel, Christel Herold-Mende, Michael Breckwoldt, Juergen Debus, Ivana Dokic, Amir Abdollahi, Katharina Schregel. Carbon ion radiotherapy eradicates glioblastoma via tumor immune environment reprograming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1105.

BackgroundSelective uptake of (18)F-fluoro-ethyl-tyrosine (18F-FET) is used in high-grade glioma (HGG) to assess tumor metabolic activity via positron emission tomography (PET). We aim to investigate its value for target volume definition, as a prognosticator, and associations with whole-blood transcriptome liquid biopsy (WBT lbx) for which we recently reported feasibility to mirror tumor characteristics and response to particle irradiation in recurrent HGG (rHGG).Methods18F-FET-PET data from n = 43 patients with primary glioblastoma (pGBM) and n = 33 patients with rHGG were assessed. pGBM patients were irradiated with photons and sequential proton/carbon boost, and rHGG patients were treated with carbon re-irradiation (CIR). WBT (Illumina HumanHT-12 Expression BeadChips) lbx was available for n = 9 patients from the rHGG cohort. PET isocontours (40%–70% SUVmax, 10% steps) and MRI-based treatment volumes (MRIvol) were compared using the conformity index (CI) (pGBM, n = 16; rHGG, n = 27). Associations with WBT lbx data were tested on gene expression level and inferred pathways activity scores (PROGENy) and from transcriptome estimated cell fractions (CIBERSORT, xCell).ResultsIn pGBM, median SUVmax was higher in PET acquired pre-radiotherapy (4.1, range (R) 1.5–7.8; n = 20) vs. during radiotherapy (3.3, R 1.5–5.7, n = 23; p = 0.03) and in non-resected (4.7, R 2.9–7.9; n = 11) vs. resected tumors (3.3, R 1.5–7.8, n = 32; p = 0.01). In rHGG, a trend toward higher SUVmax values in grade IV tumors was observed (p = 0.13). Median MRIvol was 32.34 (R 8.75–108.77) cm3 in pGBM (n = 16) and 20.77 (R 0.63–128.44) cm3 in rHGG patients (n = 27). The highest median CI was observed for 40% (pGBM, 0.31) and 50% (rHGG, 0.43, all tumors) isodose, with 70% (40%) isodose in grade III (IV) rHGG tumors (median CI, 0.38 and 0.49). High SUVmax was linked to shorter survival in pGBM (>3.3, p = 0.001, OR 6.0 [2.1–17.4]) and rHGG (>2.8, p = 0.02, OR 4.1 [1.2–13.9]). SUVmax showed associations with inferred monocyte fractions, hypoxia, and TGFbeta pathway activity and links to immune checkpoint gene expression from WBT lbx.ConclusionThe benefits of 18F-FET-PET imaging on gross tumor volume (GTV) definition for particle radiotherapy warrant further evaluation. SUVmax might assist in prognostic stratification of HGG patients for particle radiotherapy, highlights heterogeneity in rHGG, and is positively associated with unfavorable signatures in peripheral whole-blood transcriptomes.

Background Targeted immunotherapies are of growing interest in the treatment of various cancers. B7 homolog 3 protein (B7-H3), a member of the co-stimulatory/-inhibitory B7-family, exerts immunosuppressive and pro-tumorigenic functions in various cancer types and is under evaluation in ongoing clinical trials. Unfortunately, interaction partner(s) remain unknown which restricts the druggability. Methods Aiming to identify potential binding partner(s) of B7-H3, a yeast two-hybrid and a mass spectrometry screen were performed. Potential candidates were evaluated by bimolecular fluorescence complementation (BiFC) assay, co-immunoprecipitation (co-IP), and functionally in a 3H-thymidine proliferation assay of Jurkat cells, a T-cell lineage cell line. Prognostic value of angio-associated migratory cell protein (AAMP) and B7-H3 expression was evaluated in isocitrate dehydrogenase 1 wildtype (IDH1wt) glioblastoma (GBM) patients from The Cancer Genome Atlas (TCGA)-GBM cohort. Results Of the screening candidates, CD164, AAMP, PTPRA, and SLAMF7 could be substantiated via BiFC. AAMP binding could be further confirmed via co-IP and on a functional level. AAMP was ubiquitously expressed in glioma cells, immune cells, and glioma tissue, but did not correlate with glioma grade. Finally, an interaction between AAMP and B7-H3 could be observed on expression level, hinting toward a combined synergistic effect. Conclusions AAMP was identified as a novel interaction partner of B7-H3, opening new possibilities to create a targeted therapy against the pro-tumorigenic costimulatory protein B7-H3.

Summary Cellular morphology has the capacity to serve as a surrogate for cellular state and functionality. However, primary cardiomyocytes, the standard model in cardiovascular research, are highly heterogeneous cells and therefore impose methodological challenges to analysis. Hence, we aimed to devise a robust methodology to deconvolute cardiomyocyte morphology on a single-cell level: C-MORE (cellular morphology recognition) is a workflow from bench to data analysis tailored for heterogeneous primary cells using our R package cmoRe. We demonstrate its utility in proof-of-principle applications such as modulation of canonical hypertrophy pathways and linkage of genotype-phenotype in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). In our pilot study, exposure of cardiomyocytes to blood plasma prior to versus after aortic valve replacement allows identification of a disease fingerprint and reflects partial reversibility following therapeutic intervention. C-MORE is a valuable tool for cardiovascular research with possible fields of application in basic research and personalized medicine., Graphical abstract, Highlights C-MORE deconvolutes cardiomyocyte morphology on single-cell level C-MORE could be utilized for high-throughput pharmacological screens C-MORE detects pathological phenotypes caused by genetic alterations C-MORE liquid biopsy setup reflects disease state in individuals with aortic stenosis, Furkel et al. present an integrative single-cell morphology-based strategy (C-MORE) to detect cardiomyocyte activation status and response to pharmacological treatment. Deconvolution of disease state in cardiomyocyte cultures exposed to liquid biopsies and in genetically engineered models by C-MORE may pave the way for development of next-generation personalized cardiovascular medicine.

The mechanistic target of rapamycin (mTOR) promotes pathological remodeling in the heart by activating ribosomal biogenesis and mRNA translation. Inhibition of mTOR in cardiomyocytes is protective; however, a detailed role of mTOR in translational regulation of specific mRNA networks in the diseased heart is unknown. We performed cardiomyocyte genome‐wide sequencing to define mTOR‐dependent gene expression control at the level of mRNA translation. We identify the muscle‐specific protein Cullin‐associated NEDD8‐dissociated protein 2 (Cand2) as a translationally upregulated gene, dependent on the activity of mTOR. Deletion of Cand2 protects the myocardium against pathological remodeling. Mechanistically, we show that Cand2 links mTOR signaling to pathological cell growth by increasing Grk5 protein expression. Our data suggest that cell‐type‐specific targeting of mTOR might have therapeutic value against pathological cardiac remodeling., Genome‐wide translational profiling identifies mTORC1‐dependent genes in cardiomyocytes in response to neurohumoral stimulation. Expression of the muscle‐specific gene Cand2 is controlled by mTORC1 and Cand2 regulates cardiac function and pathological hypertrophy.

Cellular morphology has the capacity to serve as surrogate for cellular state and functionality. However, primary cardiomyocytes, the standard model in cardiovascular research, are highly heterogeneous cells and thereby impose methodological challenges to analysis. Hence, we aimed to devise a robust methodology to deconvolute cardiomyocytes’ morphology on a single cell level: C-MORE (cellular morphology recognition) is a workflow from bench to data analysis tailored for heterogeneous primary cells using our new R-package cmoRe. We demonstrate its utility in proof-of-principle applications such as modulation of canonical hypertrophy pathways and linkage of genotype-phenotypes in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). In our pilot study, exposure of cardiomyocytes to patients’ blood plasma prior vs. post aortic valve replacement allowed identification of a disease fingerprint and reflected partial reversibility following therapeutic intervention. In summary, C-MORE constitutes a valuable tool for cardiovascular research with possible fields of application in basic research and personalized medicine.

BACKGROUND Elevated interleukin (IL)-6-levels have been described in familial variant transthyretin amyloidosis (ATTRv) associated polyneuropathy and heart failure. However, IL-6 in cardiac ATTR amyloidosis (ATTR-CM) and its prognostic value have not been investigated yet. AIM We aim to study the correlation between IL-6 levels with clinical presentation (Gillmore-class) and outcome [heart transplantation or death (htx/death)], or the combined endpoint of cardiac decompensation or htx/death in ATTR-CM. METHODS IL-6 levels of 106 ATTR-CM patients [54 wild-type ATTRwt, 52 ATTRv-CM], 15 asymptomatic carriers of ATTR mutations (aATTRv-CM) and 27 healthy donors were quantified using Luminex technology. Statistical analysis was performed using parametric survival regression models. RESULTS We found that IL-6 levels from wild-type ATTR patients were significantly elevated compared to healthy controls, while aATTRv-CM carriers and ATTRv-CM patients did not show a significant difference. IL-6 levels showed significantly higher values in increasing Gillmore classes. Univariate analyses revealed association of low IL-6 levels with cardiac decompensation and htx/death [odds ratio: 0.26 (0.09-0.72), P = 0.01] and htx/death [odds ratio: 0.15 (0.04-0.58), P = 0.006]. However, in the multivariate model, no significant improvement of risk prediction was seen for IL-6, while established prognostic factors were significantly associated with outcome. CONCLUSION Raised IL-6 levels correlate with clinical presentation and are associated with worse outcome in ATTR-CM but do not improve stratification in addition to established risk factors.

The mechanistic target of rapamycin (mTOR) is a key regulator of pathological remodeling in the heart by activating ribosomal biogenesis and mRNA translation. Inhibition of mTOR in cardiomyocytes is protective, however, a detailed role of mTOR in translational regulation of specific mRNA networks in the diseased heart is largely unknown. A cardiomyocyte genome-wide sequencing approach was used to define mTOR-dependent post-transcriptional gene expression control at the level of mRNA translation. This approach identified the muscle-specific protein Cullin-associated NEDD8-dissociated protein 2 (Cand2) as a translationally upregulated gene, dependent on the activity of mTOR. Deletion of Cand2 protects the myocardium against pathological remodeling. Mechanistically, we found that Cand2 links mTOR signaling to pathological cell growth by increasing Grk5 protein expression. Our data suggest that cell-type-specific targeting of mTOR might have therapeutic value for adverse pathological cardiac remodeling.

Lentiviral modification of hematopoietic stem cells (HSCs) paved the way for in vivo experimentation and therapeutic approaches in patients with genetic disease. A disadvantage of this method is the use of a ubiquitous promoter leads not only to genetic modification of the leukocyte subset of interest e.g. T-cells, but also all other subsequent leukocyte progeny of the parent HSCs. To overcome this limitation we tested a bicistronic lentivirus, enabling subset specific modifications. Designed novel lentiviral constructs harbor a global promoter (mPGK) regulating mCherry for HSCs selection and a T-cell specific promoter upstream of eGFP. Two T-cell specific promoters were assessed: the distal Lck—(dLck) and the CD3δ-promoter. Transduced HSCs were FACS sorted by mCherry expression and transferred into sublethally irradiated C57/BL6 mice. Successful transplantation and T-cell specific expression of eGFP was monitored by peripheral blood assessment. Furthermore, recruitment response of lentiviral engineered leukocytes to the site of inflammation was tested in a peritonitis model without functional impairment. Our constructed lentivirus enables fast generation of subset specific leukocyte transgenesis as shown in T-cells in vivo and opens new opportunities to modify other HSCs derived subsets in the future.

Background Projection of future cancer incidence is of high importance for epidemiological and biomedical research. Age-Period-Cohort (APC) models are established for such projections, usually based on long-term cancer registry data (>20yrs). In many countries (including Germany), however, nationwide long-term data are not yet available. It is unclear which statistical approach should be recommended for projections using rather short-term data. Methods An R package (incAnalysis) for the comparative analysis of projection methods was developed, supporting in particular Bayesian models fitted by Integrated Nested Laplace Approximations (INLA). It was used to assess operating characteristics (bias, coverage, precision) of various statistical approaches to cancer incidence projection based on artificially shortened long-term data from three cancer registries (SEER-9, NORDCAN, Saarland). Additionally, a novel age-period INLA z-model with multivariate tensor product smoothers is described. Results Coverage was high (mostly >90%) for Bayesian APC models (BAPC), whereas less complex models showed differences in coverage dependent on projection-period. Intercept-only models yielded values below 20% for coverage. Bias increased and precision decreased for longer projection periods for all except intercept-only models. Precision was lowest for BAPC, multivariate smoother and Age-Period GLMs with interaction effect. Conclusion The incAnalysis R package allows for straightforward comparison of (cancer incidence) rate projection approaches. Of the evaluated model classes, more complex models showed an increase in coverage at the cost of precision; bias increased for longer projection intervals, mostly irrespective of the level of complexity.

Purpose: To assess the value of whole blood transcriptome data from liquid biopsy (lbx) in recurrent high-grade glioma (rHGG) patients for longitudinal molecular monitoring of tumor evolution under carbon ion irradiation (CIR). Methods: Whole blood transcriptome (WBT) analysis (Illumina HumanHT-12 Expression BeadChips) was performed in 14 patients with rHGG pre re-irradiation (reRT) with CIR and 3, 6 and 9 weeks post-CIR (reRT grade III:5, 36%, IV:9, 64%). Patients were irradiated with 30, 33, 36 GyRBE (n = 5, 6, 3) in 3GyRBE per fraction. Results: WTB analysis showed stable correlation with treatment characteristics and patients tumor grade, indicating a preserved tumor origin specific as well as dynamic transcriptional fingerprints of peripheral blood cells. Initial histopathologic tumor grade was indirectly associated with TMEM173 (STING), DNA-repair (ATM, POLD4) and hypoxia related genes. DNA-repair, chromatin remodeling (LIG1, SMARCD1) and immune response (FLT3LG) pathways were affected post-CIR. Longitudinal WTB fingerprints identified two distinct trajectories of rHGG evolution, characterized by differential and prognostic CRISPLD2 expression pre-CIR. Conclusions: Lbx based WTB analysis holds the potential for molecular stratification of rHGG patients and therapy monitoring. We demonstrate the feasibility of the peripheral blood transcriptome as a sentinel organ for identification of patient, tumor characteristics and CIR specific fingerprints in rHGG.

Summary Localized radiotherapy (RT) induces an immunogenic antitumor response that is in part counterbalanced by activation of immune evasive and tissue remodeling processes, e.g., via upregulation of programmed cell death-ligand 1 (PD-L1) and transforming growth factor β (TGF-β). We report that a bifunctional fusion protein that simultaneously inhibits TGF-β and PD-L1, bintrafusp alfa (BA), effectively synergizes with radiotherapy, leading to superior survival in multiple therapy-resistant murine tumor models with poor immune infiltration. The BA + RT (BART) combination increases tumor-infiltrating leukocytes, reprograms the tumor microenvironment, and attenuates RT-induced fibrosis, leading to reconstitution of tumor immunity and regression of spontaneous lung metastases. Consistently, the beneficial effects of BART are in part reversed by depletion of cytotoxic CD8+ T cells. Intriguingly, targeting of the TGF-β trap to PD-L1+ endothelium and the M2/lipofibroblast-like cell compartment by BA attenuated late-stage RT-induced lung fibrosis. Together, the results suggest that the BART combination has the potential to eradicate therapy-resistant tumors while sparing normal tissue, further supporting its clinical translation.

2012 Background: Identification of isocitrate dehydrogenase mutations (IDHm) and glioma CpG island methylator phenotype (G-CIMP) as well as methylation of O6-methylguanine DNA methyltransferase (MGMT) promotor has substantially improved stratification of glioma patients into prognostic subgroups. In extension of static pre-therapy diagnostic, we sought to investigate the impact of glioblastoma evolution under selection pressure of standard therapy on methylome level. Methods: For the training cohort (T), methylome (450k Illumina) data of paired samples from 50 patients with glioblastoma (GBM, 11 G-CIMP+) were analyzed, i.e., primary (P) and at the time point of recurrence (R, re-surgery) after standard therapy at NCT. For 39 pairs matching RNASeq data was analyzed. Validation cohorts consisted of Heidelberg (V1) total n = 650, GBM (n:585, 8 G-CIMP+), grade III (n:65, CIMP+ 65), Austrian GBM (V2, n = 499, 36 IDHm, pyrosequencing data) and the TCGA (V3) Lower-grade-glioma cohort (LGG, n = 477, grade III n: 247, 178 G-CIMP+, grade II n: 228, 206 G-CIMP+). Results: Limited number of consensus differentially methylated probes (DMP) were found across all P vs. R samples (nCpG = 411 CpGs, FDR < 0.05). In contrast, heterogeneity in GBM evolution was found by similarity analysis of delta-methylome data of 50 PR pairs resulting in two distinct clinical subgroups and one “intermediate” group. Intriguingly, n = 114.652 DMP (FDR < 0.05) was found by comparing the evolutionary “poor” (n = 15) vs. “good” (n = 13) GBM phenotypes. A random forest classifier was built to identify the evolutionary subgroups in P samples. The performance of “good” prognosis classifier was in T cohort HR: 0.54 [0.30-0.97], p = 0.04; V1: 0.57 [0.43-0.76], p < 0.001, V2: 0.62 [0.47-0.82], p < 0.001, LGG: 0.16 [0.08-0.32], p < 0.001. In “good” prognosis group (T), neither G-CIMP+ (n = 3) nor MGMT-STP27 (oddsratio, OR: 0.56, p = 0.47) was enriched. MGMT-STP27 OR was 0.47 (V1, p = 0.47) or 1.28 (V2, p = 0.45), respectively. The evolutionary subgroups remain prognostic independent of GCIMP status in LGG (V3). “Poor” glioma are enriched for RTKI/II methylome subtypes, and contain less frequently the mesenchymal subtype. Bevacizumab treatment showed a survival benefits only in “poor” subtype (V1+2). Conclusions: Discovery of a methylome based classifier of glioma evolution informs on “good” and “poor” prognosis subtypes and may have ramification for stratifying patients for therapy such as e.g., antiangiogenesis.