PBMCs collected before vaccination show low/no response to candidate vaccine peptides. (A) The nomenclature of the N-peptides and D-neopeptides. I or II indicate if the peptides were chosen/designed for HLA class I or -II binding; MUT/RNA/GBM indicates a naturally mutated peptide (MUT), derivation from the over-/highly expressed genes (RNA) or from known glioblastoma (GBM) targets; X indicates the number of the peptide; 0 or 1 indicate if the peptides had been artificially mutated (1) or not (0); (’) indicates that peptides contained the naturally mutated amino acid. (B) PBMCs collected before vaccination were stimulated with 4 μg/mL Tetanus toxoid or 2 μM CEF II peptide pool for 7 days, and proliferation was measured by ³H-thymidine incorporation assay. The proliferation strength is depicted as counts per minute (cpm). In each group, the N-peptides and their corresponding D-neopeptides are included, and the designed mutated positions are highlighted in the red box. The blue dotted line indicates the mean value of the no peptide control. The red dotted line indicates the mean value plus three standard deviations of the no peptide control. Values above the red dotted line were considered positive. (C) PBMCs collected before vaccination were stimulated with 5 μM predicted class I-peptides, including N-peptides and D-neopeptides, for 7 days, and proliferation was measured by ³H-thymidine incorporation assay. 10-15 replicate wells were tested for proliferation, and responses were depicted as cpm. (D) PBMCs collected before vaccination were stimulated with 5 μM predicted class II-peptides, including N-peptides and D-neopeptides, for 7 days, and proliferation was measured by ³H-thymidine incorporation assay. 10-15 replicate wells were tested for proliferation, and responses were depicted as cpm.

Designed mutations increase the stimulatory strength of D-neopeptides above N-peptides. (A) TILs were stimulated with 5 μM class I-peptides, including N-peptides and D-neopeptides, for 5 days. 5 replicate wells were tested for proliferation, and proliferation was measured by 3H-thymidine incorporation assay. In each group, the N-peptides and their corresponding D-neopeptides are included, and the designed mutated positions are highlighted in the red box. The blue dotted line indicates the mean value of the no peptide control. The red dotted line indicates the mean value plus three standard deviations of the no peptide control. Values above the red dotted line were considered positive. (#) indicates the peptide that was used in the 1st to 4th vaccinations. (B) TILs were stimulated with 5 μM class II-peptides, including N-peptides and D-neopeptides, for 5 days. 5 replicate wells were tested for proliferation, and proliferation was measured by 3H-thymidine incorporation assay.

Over-/highly expressed genes in the primary glioblastoma when compared with glioblastoma cohort in TCGA Database.

Schematic outline of the personalized peptide vaccination. (A) Timeline of clinical events for a patient with glioblastoma in whom we applied a highly personalized peptide vaccination. I. Vaccine peptide design. Surgically resected primary glioblastoma and PBMCs were collected. Somatic mutants and over-/highly expressed genes in glioblastoma were identified as TSAs and TAAs by WES and RNA-seq, and the HLA genotype were identified with PBMCs by DNA sequencing. Potential vaccine peptides were predicted and selected from TSAs and TAAs based on HLA binding prediction. Peptide cocktails containing ~25 peptides for HLA class I or II molecules were prepared for vaccination. II. Peptide cocktail vaccination and testing. The patient was s.c. vaccinated with the peptide cocktails, and Imiquimod or XS15 + MontanideTM were used as the adjuvant. Meanwhile, the patient received treatment with pembrolizumab and bevacizumab. No dexamethasone was given throughout the periods of vaccination. PBMCs were collected after vaccination at the indicated time points and tested with vaccine peptides to examine the effect of immunotherapy. Surgically resected recurrent glioblastoma was collected after four vaccinations, and tumor-infiltrating lymphocytes (TILs) were isolated and tested with vaccine peptides. (B) HLA genotypes of the patient with glioblastoma, including HLA-A, HLA-B, HLA-DR, HLA-DP, and HLA-DQ alleles. n.a. indicates not available.

TILs do not respond to N-class I-peptides. TILs were stimulated with 5- or 10 μM N-class I-peptides for 5 days. 5 replicate wells were tested for proliferation, and proliferation was measured by 3H-thymidine incorporation assay. The proliferation strength is depicted as SI. (#) indicates the peptide that was used in the 1st to 4th vaccinations. Data are expressed as mean ± SEM.

Background The mitochondrial metabolism in prostate cancer (PCa) is of great importance due the unique metabolic shift from glycolysis to oxidative phosphorylation. In this study, we aimed to analyze the expression level of mitochondrial markers TOM20, DRP1 and OPA1 in benign and malignant tissue, to assess if these markers are associated with different grade and stage of PCa. Materials and Methods This study assessed TOM20, DRP1, and OPA1 expression in formalin-fixed, paraffin-embedded prostate tissue samples, including benign and malignant tissue specimen. Immunohistochemistry on tissue microarrays was conducted, with staining intensities scored semi-quantitatively. Statistical analyses evaluated associations with PCa grade and stage. A survival analysis for biochemical recurrence (RFS), overall survival (OS) and disease specific survival (DSS) was performed using multivariate Cox regression analysis to assess prognostic properties of the markers. Results In total, 527 patients were included in our analysis, which composed of 45 (8.5%) benign prostate hyperplasia (BPH) and 482 (91.5%) PCa samples (436 localized (90.5%) and 46 (9.5%) metastatic). Immunoreactivity for TOM20, DRP1 and OPA1 was strong in 2 of 43 (4.7%), 1 of 43 (2.3%) and 0 of 43 (0%) of BPH control tissue. Strong marker expression was significantly increased in radical prostatectomy specimen (TOM20: 111/371 (29.9%), DRP1: 89/373 (23.9%), OPA1: 60/371 (16.2%), p<0.001) and in metastatic tissue (TOM20: 22/42 (52.4%), DRP1: 14/42 (33.3%), OPA1: 21/41 (51.2%), p<0.001). None of the markers demonstrated prognostic properties for RFS, OS, and DSS. Conclusion A strong association between the expression of the mitochondrial markers TOM20, DRP1 and OPA1 and PCa aggressiveness was demonstrated. However, these markers were not found to be prognostic regarding RFS, OS and DSS. Future studies are needed focusing on the underlying mechanisms of the upregulation of mitochondrial metabolism in aggressive PCa and evaluate potential therapeutic implications.

Activated T lymphocytes in recurrent tumor interact with macrophages. (A) Expression of T cell activating markers HLA-DR and Tim-3 in the perivascular areas and peritumoral areas of the primary and recurrent tumor was detected by IMC. (B) CD3-CD7+ NK cells infiltrated in the perivascular areas and peritumoral areas of the primary and recurrent tumor was detected by IMC. (C) Pro-inflammatory CD11c+ CD68+ macrophages, which had infiltrated the perivascular areas and peritumoral areas of the primary and recurrent tumor, were detected by IMC. (D) Colocalization analysis of CD68+ macrophages and CD3+ T cells in the perivascular areas and peritumoral areas of the primary and recurrent tumor were detected by IMC. Scale bars are indicated in the figures.

Increased T lymphocyte infiltration in the recurrent compared to the primary tumor. (A) CD3 immunohistochemical staining in the primary and recurrent tumor. (B) Three fields of view were selected and counted CD3+ T cells in both perivascular and peritumoral areas of the primary and recurrent tumors using ImageJ. Scale bars are indicated in the figures.

Peripheral blood T cell responses to v-peptides increase with repetitive vaccination. (A) PBMCs were collected 3 weeks after the 2nd peptide cocktail vaccination, and CD45RA-negative PBMCs were stimulated with 4 μg/mL Tetanus toxoid, 2 μM CEF II peptide pool or 5 μM v-peptides for 7 days. 5-10 replicate wells were tested for proliferation, and proliferation measured by 3H-thymidine incorporation assay. CD45RA-negative PBMCs collected before vaccination were used as reference. The proliferation strength is depicted as stimulation index (SI). The red dotted line indicates a stimulatory response of SI = 2, and values above the red dotted line were considered positive. (#) indicates the peptide that was used in the vaccinations. (B) PBMCs were collected 5 weeks after the 3rd peptide cocktail vaccination, and CD45RA-negative PBMCs were stimulated with 4 μg/mL Tetanus toxoid, 2 μM CEF II peptide pool or 5 μM v-peptides for 7 days. 5-10 replicate wells were tested for proliferation, and proliferation was measured by 3H-thymidine incorporation assay. CD45RA-negative PBMCs collected before vaccination were used as the control. Data are expressed as mean ± SEM, and p values were determined using an unpaired t-test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Supplementary Table 1. Somatic mutations of the patient's glioblastoma; Supplementary Table 2. See Excel File; Supplementary Table 3. Literature-derived, glioblastoma-associated TAAs; Supplementary Table 4. See Excel File; Supplementary Table 5. Over-/highly expressed genes that were chosen for the design of vaccine peptides; Supplementary Table 6. Peptide cocktails for vaccination; Supplementary Table 7. Antibodies for imaging mass cytometry.

Predicted TSA/TAA-derived N-peptides and D-neopeptides

Vaccinated D-neopeptides activate tumor-infiltrating antitumor T cells. (A) Circos plots provide an overview of the frequencies of Vβ-Jβ pairing in the primary and recurrent tumors. (B) Surface TCR β chain expression of the D-neopeptide-specific CD4+ TCCs was analyzed using FACs.

Immune checkpoint inhibitors are standard-of-care for the treatment of advanced melanoma, but their use is limited by immune-related adverse events. Proteomic analyses and multiplex cytokine and chemokine assays from serum at baseline and at the adverse event onset indicated aberrant T cell activity with differential expression of type I and III immune signatures. This was in line with the finding of an increase in the proportion of CD4+ T cells with IL-17A expression at the adverse event onset in the peripheral blood using flow cytometry. Multiplex immunohistochemistry and spatial transcriptomics on immunotherapy-induced skin rash and colitis showed an increase in the proportion of CD4+ T cells with IL-17A expression. Anti-IL-17A was administered in two patients with mild myocarditis, colitis and skin rash with resolution of the adverse events. This study highlights the potential role of type III CD4+ T cells in adverse event development and provides proof-of-principle evidence for a clinical trial using anti-IL-17A for treating adverse events.

Abstract BACKGROUND Diffuse midline glioma (DMG) is a fatal childhood brain cancer with a survival rate of less than one year from diagnosis. Pharmacological approaches and immunotherapy have failed to make a clinical impact. Incomplete understanding of the tumor microenvironment (TME) and tumor associated antigens have contributed to the observed poor prognosis. Therefore, mapping TME is urgently needed. METHODS Whole brains were collected at autopsy from 80 pediatric subjects, including patients diagnosed with DMG (n=50), GBM (n=9), non-malignant controls (n=10), ependymoma (n=5), ATRT (n=2). Up to four brain anatomical sites were selected: primary tumor, metastatic and adjacent healthy control sites, and processed for staining with hematoxylin and eosin (H&E). After reviewing the tumor and healthy regions, three punch cores were obtained resulting in a total of 918 core punches and two TMA blocks. Multiplexed immunofluorescence (MxIF) technology was used to probe 43 biomarkers on a single TMA slide, focusing on immune cell types and activation thereof. Clinical data including genomics alterations were obtained from each patient for downstream analyses. RESULTS Highest expressed immune markers across all patients were CD8 and CD3 (T-cells), CD68, Iba1 and CD163 (microglia). In DMGs, T-cells were mostly detected and increased in ONC201 and immunotherapy treated patients compared to only immunotherapy treated patients. Analysis of Iba1 confirmed a higher difference in activation in primary tumor compared to metastatic and adjacent healthy tissue, whereas in contrast, CD68 was significantly increased in metastatic sites. Patient plasma and RNA transcriptome analysis confirmed biomarkers and immune related pathways. Further, immune competent murine models were used to validate immune cell infiltration. AI deep learning technology modeling is ongoing now to, in future, be able to predict cell type composition and neighborhoods mapping. CONCLUSION We posit that this comprehensive data and characterization of the DMG TME will help to identify biology-informed targeted treatments.

Immune checkpoint inhibitors (ICIs) are standard-of-care for the treatment of advanced melanoma, but their use is limited by immune-related adverse events (irAEs). Proteomic analysis and multiplex cytokine/chemokine assay from serum at baseline and at irAEs onset in 82 patients indicated aberrant T-cell activity with differential expression of Type I and III immune signatures. This was in line with an increase in the proportions of monocytes and decrease of IL-17A producing CD4+ T-cells in the peripheral blood in single cell RNA sequencing. Multiplex immunohistochemistry and spatial transcriptomics on ICI-induced skin rash and colitis showed increase in the proportion of CD4+ T-cells with IL-17A expression. Anti-IL17A mAbs were administered in two patients with myocarditis, colitis and skin rash with resolution of the irAE. This study demonstrates the potential role of Type III CD4+ T-cells in the irAEs development and provides proof-of-principle evidence to support a clinical trial examining anti-IL17A in their management.

Abstract Diffuse midline glioma (DMG) is a fatal childhood brain cancer with a survival rate of less than one year from diagnosis. Pharmacological approaches as well as immunotherapy have failed to make a clinical impact. Incomplete understanding of the tumor microenvironment (TME) and tumor associated antigens have contributed to the observed poor prognosis. We hypothesize that characterization of the DMG TME will identify biology-informed targeted treatments. We thus obtained postmortem specimens from 70 patients diagnosed with brain cancers, including 50 DMGs, 20 other types and 10 non-CNS-cancer patients. Up to four anatomical brain locations were selected including the primary tumor, metastatic and adjacent healthy sites. Formalin-fixed-paraffin-embedded (FFPE) specimens were processed for constructing a tissue microarray (TMA). The TMA was stained for a number of markers (H&E, H3K27M, H3K27me3, KI67) and was scored by a neuropathologist. We then used a multiplexed immunofluorescence (MxIF) technology, Cell DIVE™, to iteratively probe 33 biomarkers on a single tissue slide, focusing on immune cell type profiling and activation, and histone mutation status. Analysis of biomarker density and spatial relationships are underway. The main expressed immune markers across all patients were CD163, CD68, and CD8. CD8, a cytotoxic T-cell biomarker, was highly detected in pons, cerebellum, thalamus and the frontal lobe (tumor and healthy) of DMG patients and varied according to clinical intervention, including ONC201 treatment. Furthermore, in comparison to other tumors, DMGs exhibited a higher expression of CD3, T- cell marker, and CD4, a T-helper cell biomarker. Analysis of Iba1, a microglial marker, confirmed a higher difference in microglial activation in primary tumor compared to metastatic and adjacent healthy tissue. In contrast, CD68 was significantly increased in metastatic sites. Analysis is still ongoing. We report establishment of the most comprehensive TMA for pediatric brain tumors, which provides insights into TME comparing critically important clinical variables.

PARP inhibitors (PARPi) are increasingly used in breast cancer therapy, including high-grade triple-negative breast cancer (TNBC) treatment. Varying treatment responses and PARPi resistance with relapse currently pose limitations to the efficacy of PARPi therapy. The pathobiological reasons why individual patients respond differently to PARPi are poorly understood. In this study, we analyzed expression of PARP1, the main target of PARPi, in normal breast tissue, breast cancer, and its precursor lesions using human breast cancer tissue microarrays covering a total of 824 patients, including more than 100 TNBC cases. In parallel, we analyzed nuclear adenosine diphosphate (ADP)-ribosylation as a marker of PARP1 activity and TRIP12, an antagonist of PARPi-induced PARP1 trapping. Although we found PARP1 expression to be generally increased in invasive breast cancer, PARP1 protein levels and nuclear ADP-ribosylation were lower in higher tumor grade and TNBC samples than non-TNBCs. Cancers with low levels of PARP1 and low levels of nuclear ADP-ribosylation were associated with significantly reduced overall survival. This effect was even more pronounced in cases with high levels of TRIP12. These results indicate that PARP1-dependent DNA repair capacity may be compromised in aggressive breast cancers, potentially fueling enhanced accumulation of mutations. Moreover, the results revealed a subset of breast cancers with low PARP1, low nuclear ADP-ribosylation, and high TRIP12 levels, which may compromise their response to PARPi, suggesting a combination of markers for PARP1 abundance, enzymatic activity, and trapping capabilities might aid patient stratification for PARPi therapy.

AbstractPurpose: The low mutational load of some cancers is considered one reason for the difficulty to develop effective tumor vaccines. To overcome this problem, we developed a strategy to design neopeptides through single amino acid mutations to enhance their immunogenicity. Experimental Design: Exome and RNA sequencing as well as in silico HLA-binding predictions to autologous HLA molecules were used to identify candidate neopeptides. Subsequently, in silico HLA-anchor placements were used to deduce putative T-cell receptor (TCR) contacts of peptides. Single amino acids of TCR contacting residues were then mutated by amino acid replacements. Overall, 175 peptides were synthesized and sets of 25 each containing both peptides designed to bind to HLA class I and II molecules applied in the vaccination. Upon development of a tumor recurrence, the tumor-infiltrating lymphocytes (TIL) were characterized in detail both at the bulk and clonal level. Results: The immune response of peripheral blood T cells to vaccine peptides, including natural peptides and designed neopeptides, gradually increased with repetitive vaccination, but remained low. In contrast, at the time of tumor recurrence, CD8+ TILs and CD4+ TILs responded to 45% and 100%, respectively, of the vaccine peptides. Furthermore, TIL-derived CD4+ T-cell clones showed strong responses and tumor cell lysis not only against the designed neopeptide but also against the unmutated natural peptides of the tumor. Conclusions: Turning tumor self-peptides into foreign antigens by introduction of designed mutations is a promising strategy to induce strong intratumoral CD4+ T-cell responses in a cold tumor like glioblastoma.

Metastases account for most cancer-related deaths, yet the mechanisms underlying metastatic spread remain poorly understood. Recent evidence demonstrates that senescent cells, while initially restricting tumorigenesis, can induce tumor progression. Here, we identify the metalloproteinase inhibitor TIMP1 as a molecular switch that determines the effects of senescence in prostate cancer. Senescence driven either by PTEN deficiency or chemotherapy limits the progression of prostate cancer in mice. TIMP1 deletion allows senescence to promote metastasis, and elimination of senescent cells with a senolytic BCL-2 inhibitor impairs metastasis. Mechanistically, TIMP1 loss reprograms the senescence-associated secretory phenotype (SASP) of senescent tumor cells through activation of matrix metalloproteinases (MMPs). Loss of PTEN and TIMP1 in prostate cancer is frequent and correlates with resistance to docetaxel and worst clinical outcomes in patients treated in an adjuvant setting. Altogether, these findings provide insights into the dual roles of tumor-associated senescence and can potentially impact the treatment of prostate cancer.

Abstract INTRODUCTION The relatively small size of biopsied CNS tumors has presented a historical challenge for real-time drug screens. Moreover, in vivo assessment of drug response does not often benefit patients with aggressive gliomas given the relatively long time (>8 months) of tumor engraftment in the classic mouse PDX models. Here, we aimed to develop an innovative real-time in vivo and in vitro drug screening platform capable of analyzing a minimal number (<1E6) of cells obtained at biopsy. METHODS Existing primary cells were used to test 6 different culture platforms. The top platform was selected and used to expand tumor cells obtained of DMG biopsy. Tumor cells were validated using the minION sequencing platform. Single and combination drug (n=7) screens were performed. Effective drugs were further evaluated in zebrafish PDX and non-tumor bearing models to assess efficacy and toxicity, respectively. RESULTS A total of 8 biopsies were obtained. Successful cell expansion was achieved in 6/8 (75%) and a limited drug screen in 3/6 (50%) of cases. Single and combination drug (n=7) assays identified responder and non-responders to candidate drugs. Systemic toxicity of effective drugs was tested in non-tumor bearing zebrafish. Tumor cells were engrafted in zebrafish providing the opportunity for an in vivo screen. The entire process was completed within 21 days on average. CONCLUSIONS A novel platform was developed for rapid in vitro and in vivo drug screens of tumor cells obtained at biopsy. This platform will provide the opportunity to establish personalized therapy for heterogeneous cancers including DMGs.