Abstract Ewing sarcoma is a rare, fusion-driven malignancy with poor prognosis in the metastatic setting, for which no established immunotherapeutic treatment is currently available. Fusion breakpoints are rational precision immunotherapy targets, yet clinical evidence of immunogenicity is scarce. We administered an off-the-shelf multi-peptide vaccine spanning the type 1 EWSR1–FLI1 breakpoint to a patient with high-burden metastatic Ewing sarcoma following multimodal therapy. Vaccinations were combined with GM-CSF and topical imiquimod. Longitudinal immune monitoring by in vitro peptide stimulation and intracellular cytokine staining revealed de novo polyfunctional CD4⁺ T-cell responses against all four fusion-derived peptides, first detectable by month 7 and persisting beyond two years. Treatment was well tolerated with only grade 1 local reactions. Durable disease stability was maintained for more than 26 months. These first-in-human data support the feasibility, safety, and immunogenicity of a fusion-derived peptide vaccine and warrant further evaluation of precision immunotherapy in sarcomas driven by recurrent gene fusions.

Isocitrate dehydrogenase (IDH) mutant glioma is a malignant primary brain tumor diagnosed in adults. In recent years, there has been significant progress in understanding the molecular pathogenesis and biology of these tumors. The first targeted IDH-inhibitor was approved by the US Food and Drug Administration in August 2024 for grade 2 gliomas, in light of results of a phase III trial which showed significant advantages in progression-free survival. However, biologic therapy is not curative, and subsequent treatment options offer only limited clinical benefit and often result in long-term toxicities. In addition, targeted treatment options for grade 3 and grade 4 IDH-mutant gliomas are still missing. In this study, we present n=52 patients with glioma (grade 2, 3 and 4) with confirmed IDH1 mutation (mutIDH1) in the newly diagnosed and recurrent setting who, in addition to standard-of-care, received a personalized neoantigen-targeting peptide vaccine. Each tumor was initially analyzed for somatic mutations by whole exome sequencing, and a peptide vaccine containing potential neoepitopes was designed, manufactured and vaccinated. Each vaccine consisted of peptides derived from numerous somatic mutations, including at least one peptide targeting the mutIDH1.Vaccine immunogenicity was determined by intracellular cytokine staining and simultaneous measurement of four T-cell activation markers (Interferon-γ, Tumor Necrosis Factor, Interleukin-2, CD154) after 12-day in vitro expansion of pre and post vaccination peripheral blood mononuclear cells. Extracellular CD154 staining was used to sort mutIDH1-specific CD4+T cells.Immunomonitoring revealed that the vaccines were immunogenic and induced mainly CD4 but also CD8 T cell responses. Vaccine-induced immune responses were robust and polyfunctional. Immunogenicity against mutIDH1 was high (89%). We implemented an assay which allowed us to isolate functional antigen-specific CD4+T cells in an HLA-independent manner. Subsequent T cell receptor (TCR) repertoire sequencing revealed that CD4+T cells reacting on mutIDH1 stimulation were polyclonal. Strikingly, we detected two mutIDH1-specific TCRβ candidate sequences in three different patients. These three patients had the same human leukocyte antigen (HLA) DQA-DQB alleles. The obtained TCRβ sequences could be tracked in autologous ex-vivo single-cell transcriptomic data. Our results provide a rationale for pursuing vaccination and T cell transfer strategies targeting IDH1. Furthermore, our findings indicate that personalized neoantigen-targeting vaccines might be considered for the treatment of IDH1-mutant gliomas.

Background/Objectives: Fully personalized peptide vaccines targeting tumor-specific mutations are a promising treatment option for patients in an adjuvant but also advanced/metastatic disease situation in addition to non-personalized standard therapies. Here, we report a patient’s case with advanced metastatic colorectal cancer (mCRC) who was treated with a neoantigen-derived multi-peptide vaccine in addition to standard of care. Methods: Tumor-specific mutations were identified by whole exome and transcriptome sequencing. An individualized peptide vaccine was designed using an in-house developed epitope prediction and vaccine design platform. In this case, the vaccine consisted of 20 peptides targeting 18 distinct mutations. The vaccine was administered according to a prime-boost scheme for a total of 12 vaccinations. Vaccine immunogenicity was determined by stimulation of patient T cells with vaccinated peptides and subsequent intracellular cytokine staining (ICS). Tumor-infiltrating lymphocytes (TIL) were analyzed by ICS and T cell receptor beta chain (TCRβ) sequencing. Results: The patient survived for 41 months since initial diagnosis despite continuous disease progression under all therapeutic interventions. The vaccination induced multiple neoantigen-specific T cell responses in the patient without notable side effects. Two liver metastases were resected five months after the start of vaccination, and TIL were extracted and cultured. Analysis of TIL cultures revealed tumor infiltration by vaccine-induced neoantigen-specific T cells in only one of the metastases. TCRβ sequencing of neoantigen-specific T cells and tumor tissues supported this finding. Vaccine-targeted variants were reduced or absent in the metastasis with vaccine-specific T cell infiltration. Conclusions: This case demonstrates immunogenicity of a neoantigen-derived peptide vaccine and highlights tumor-infiltrating capabilities and potential cytotoxicity of vaccine-induced T cells in mCRC.

Abstract Current treatment outcome of patients with glioblastoma (GBM) remains poor. Following standard therapy, recurrence is universal with limited survival. GBM tumors from 173 patients were analyzed for somatic mutations to generate a personalized peptide vaccine targeting tumor-specific neoepitopes. In agreement with their treating physician, patients added a personalized peptide vaccine to their treatment as an individual healing attempt. Patients were monitored from October 2015 until August 2023. We retrospectively evaluated their clinical courses and the results of their immune monitoring data. Among all patients, including 70 treated prior to progression (primary) and 103 treated after progression (recurrent), the median overall survival from first diagnosis was 31.9 months (95% CI: 25.0-36.5). Adverse events were infrequent and were predominantly grade 1 or 2. An immune response to at least one of the vaccinated peptides was detected in blood samples of 87 of 97 (90%) monitored patients. T-cell responses to vaccinated neoepitope peptides were durable in most patients. Significantly prolonged survival was observed for patients with multiple vaccine-induced immune responses (53 months) compared to those with no/low induced responses (27 months; P=0.03). Altogether, our results highlight that the application of personalized neoantigen-targeting peptide vaccine is feasible and represents a promising potential treatment option for GBM patients. This is the largest real-world observation involving GBM patients safely treated with a personalized peptide vaccine to date. This real-world observation will be translated into a clinical trial to evaluate the specific contribution of the neoantigen vaccine.

Ovarian cancer is one of the most common cancers among women and the most lethal malignancy of all gynecological cancers. Surgery is promising in the early stages; however, most patients are first diagnosed in the advanced stages, where treatment options are limited. Here, we present a 49-year-old patient who was first diagnosed with stage III ovarian cancer. After the tumor progressed several times under guideline therapies with no more treatment options available at that time, the patient received a fully individualized neoantigen-derived peptide vaccine in the setting of an individual healing attempt. The tumor was analyzed for somatic mutations via whole exome sequencing and potential neoepitopes were vaccinated over a period of 50 months. During vaccination, the patient additionally received anti-PD-1 therapy to prevent further disease progression. Vaccine-induced T-cell responses were detected using intracellular cytokine staining. After eleven days of in vitro expansion, four T-cell activation markers (namely IFN-ɣ, TNF-α, IL-2, and CD154) were measured. The proliferation capacity of neoantigen-specific T-cells was determined using a CFSE proliferation assay. Immune monitoring revealed a very strong CD4+ T-cell response against one of the vaccinated peptides. The vaccine-induced T-cells simultaneously expressed CD154, TNF, IL-2, and IFN-ɣ and showed a strong proliferation capacity upon neoantigen stimulation. Next-generation sequencing, as well as immunohistochemical analysis, revealed a loss of Beta-2 microglobulin (B2M), which is essential for MHC class I presentation. The results presented here implicate that the application of neoantigen-derived peptide vaccines might be considered for those cancer stages, where promising therapeutic options are lacking. Furthermore, we provide more data that endorse the intensive investigation of B2M loss as a tumor escape mechanism in clinical trials using anti-cancer vaccines together with immune-checkpoint inhibitors.

In their article, "Lessons Learned from Translating Genome Sequencing to Clinical Routine: Understanding the Accuracy of a Diagnostic Pipeline", Haack et al [...].

Current treatment outcome of patients with glioblastoma (GBM) remains poor. Following standard therapy, recurrence is universal with limited survival. Tumors from 173 GBM patients are analysed for somatic mutations to generate a personalized peptide vaccine targeting tumor-specific neoantigens. All patients were treated within the scope of an individual healing attempt. Among all vaccinated patients, including 70 treated prior to progression (primary) and 103 treated after progression (recurrent), the median overall survival from first diagnosis is 31.9 months (95% CI: 25.0-36.5). Adverse events are infrequent and are predominantly grade 1 or 2. A vaccine-induced immune response to at least one of the vaccinated peptides is detected in blood samples of 87 of 97 (90%) monitored patients. Vaccine-specific T-cell responses are durable in most patients. Significantly prolonged survival is observed for patients with multiple vaccine-induced T-cell responses (53 months) compared to those with no/low induced responses (27 months; P = 0.03). Altogether, our results highlight that the application of personalized neoantigen-targeting peptide vaccine is feasible and represents a promising potential treatment option for GBM patients.

Next-generation sequencing (NGS)-based comprehensive tumor profiling from liquid biopsy samples can significantly improve diagnosis and monitoring of tumors when high-quality tissue material is difficult to obtain. In addition, it offers the potential to capture the entire complexity of the tumor, which is particularly important for highly heterogeneous or metastatic tumors. Here, we report the findings of an analytical performance evaluation of the TruSight Oncology 500 circulating tumor DNA (ctDNA) assay, a 523-gene NGS panel developed for ctDNA-based comprehensive genomic profiling of tumors, using reference and patient samples. Using 30 ng cell-free DNA, the assay showed high sensitivity and low variant detection variability for single-nucleotide variants, insertions and deletions, and fusions down to a variant allele frequency (VAF) of 0.5% in the reference samples and VAFs that were highly concordant with previous digital droplet PCR results in the patient samples. At reduced input amounts (20, 15, and 5 ng) and below VAFs of 0.5%, sensitivity was considerably lower and variant detection variability increased. Covering 523 tumor-associated genes, the assay demonstrated a convincing performance comparable to NGS-based ctDNA assays with smaller gene panels, highlighting its value to screen large numbers of different genes. (J Mol Diagn 2024, 26: 61-72; https://doi.org/ 10.1016/j.jmoldx.2023.10.001)

Abstract BACKGROUND Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor. Despite of the current SoC, patients have a poor prognosis with a median OS of about 14-17 months.Method, RESULTS We report retrospectively analyzed data from 203 GBM patients (IDH wildtype) who added PTCV derived from patient individual tumor mutations to their treatment regimen in the setting of individual treatment attempts (ITA) in Germany. Data were analyzed re. vaccine induced T cell responses and safety acc. to CTCAE terminology version 5.0. Subsets of patients with and without hypermethylation of the MGMT promoter were further evaluated re. efficacy. Treated patients had a median age of 53 years at diagnosis, started vaccination after a median of 11 months after diagnosis, received a median of 8 immunizations with a median of 20 neoepitope peptides, and had a median follow-up time since first vaccination of 21 months. They usually received SoC (Stupp scheme), often accompanied by additional therapies. In general, the PTCV was well tolerated, and most patients experienced only Grade 1 and 2 AEs. Most frequently injection site reactions were observed (67%). Seven patients developed Grade 3 allergic or anaphylactic reactions after 7 or more vaccinations. For 119 patients T-cell responses against the vaccinated neoepitopes were investigated. Almost all patients developed vaccine induced T-cell responses (97%). 71% of patients showed both CD4+ and CD8+ T-cell responses towards vaccinated peptides. Immune responses usually increased over time peaking within 3-6 months. Median OS of the whole cohort was 26 mo (95% CI 24, 31). Primary GBM patients without recurrence events before the start of vaccination and with or without hypermethylated MGMT promoter status showed a mOS of 41 months (95% CI: 32, NA) or 23 months (95% CI: 20, NA), respectively. CONCLUSION The retrospective data analysis from a real-world cohort of GBM patients suggests that PTCV are safe, immunogenic, and may prolong the survival of patients.

Genetic diagnostics uses sequencing to find causative variants for patients' phenotypes. Several recent publications have declared large increases in diagnostic yield when using whole-genome sequencing (WGS) instead of whole-exome sequencing (WES). Reanalyzing published data, we show that the yield increase is overestimated.

Previous studies suggested that severe epilepsies, e.g., developmental and epileptic encephalopathies (DEEs), are mainly caused by ultra-rare de novo genetic variants. For milder disease, rare genetic variants could contribute to the phenotype. To determine the importance of rare variants for different epilepsy types, we analyzed a whole-exome sequencing cohort of 9,170 epilepsy-affected individuals and 8,436 control individuals. Here, we separately analyzed three different groups of epilepsies: severe DEEs, genetic generalized epilepsy (GGE), and non-acquired focal epilepsy (NAFE). We required qualifying rare variants (QRVs) to occur in control individuals with an allele count ≥ 1 and a minor allele frequency ≤ 1:1,000, to be predicted as deleterious (CADD ≥ 20), and to have an odds ratio in individuals with epilepsy ≥ 2. We identified genes enriched with QRVs primarily in NAFE (n = 72), followed by GGE (n = 32) and DEE (n = 21). This suggests that rare variants may play a more important role for causality of NAFE than for DEE. Moreover, we found that genes harboring QRVs, e.g., HSGP2, FLNA, or TNC, encode proteins that are involved in structuring the brain extracellular matrix. The present study confirms an involvement of rare variants for NAFE that occur also in the general population, while in DEE and GGE, the contribution of such variants appears more limited.

Localized prostate cancer is curable, but metastatic castration sensitive prostate cancer has a low 5-year survival rate, while broad treatment options are lacking. Here we present an mCSPC patient under remission receiving individualized neoantigen-derived peptide vaccination as recurrence prophylaxis in the setting of an individual treatment attempt. The patient was initially analyzed for somatic tumor mutations and then consecutively treated with two different peptide vaccines over a period of 33 months. The first vaccine contained predicted HLA class I binding peptides only whereas the second vaccine contained both predicted HLA class I and II binding peptides. Intracellular cytokine staining after 12 day in-vitro expansion measuring four T-cell activation markers (IFNg, TNF-α, IL-2, CD154) was used to determine vaccine-induced T-cell responses. While the first vaccine induced only one robust CD4+ T-cell response after 21 vaccinations, co-vaccination of HLA class I and II peptides induced multiple strong and durable CD4+ and CD8+ T-cell responses already after sixth vaccinations. The vaccine-induced immune responses were robust and polyfunctional. PSA remained undetectable for 51 months. The results presented here implicate that neoantigen-targeting vaccines might be considered for those cancer subtypes where therapeutic options are limited. Furthermore, our findings suggest that both HLA class I and II restricted peptides should be considered for future peptide vaccination trials.