Immune monitoring and TCR sequencing of CD4 T cells in a long term responsive patient with metastasized pancreatic ductal carcinoma treated with individualized, neoepitope-derived multipeptide vaccines : a case report

Abstract Background Cancer vaccines can effectively establish clinically relevant tumor immunity. Novel sequencing approaches rapidly identify the mutational fingerprint of tumors, thus allowing to generate personalized tumor vaccines within a few weeks from diagnosis. Here, we report the case of a 62-year-old patient receiving a four-peptide-vaccine targeting the two sole mutations of his pancreatic tumor, identified via exome sequencing. Methods Vaccination started during chemotherapy in second complete remission and continued monthly thereafter. We tracked IFN-γ+ T cell responses against vaccine peptides in peripheral blood after 12, 17 and 34 vaccinations by analyzing T-cell receptor (TCR) repertoire diversity and epitope-binding regions of peptide-reactive T-cell lines and clones. By restricting analysis to sorted IFN-γ-producing T cells we could assure epitope-specificity, functionality, and TH1 polarization. Results A peptide-specific T-cell response against three of the four vaccine peptides could be detected sequentially. Molecular TCR analysis revealed a broad vaccine-reactive TCR repertoire with clones of discernible specificity. Four identical or convergent TCR sequences could be identified at more than one time-point, indicating timely persistence of vaccine-reactive T cells. One dominant TCR expressing a dual TCRVα chain could be found in three T-cell clones. The observed T-cell responses possibly contributed to clinical outcome: The patient is alive 6 years after initial diagnosis and in complete remission for 4 years now. Conclusions Therapeutic vaccination with a neoantigen-derived four-peptide vaccine resulted in a diverse and long-lasting immune response against these targets which was associated with prolonged clinical remission. These data warrant confirmation in a larger proof-of concept clinical trial

Fatty acid-induced apoptosis: The role of toll-like receptors 2 and 4 in insulin-secreting cells

Toll-like Rezeptoren sind als Rezeptoren des angeborenen Immunsystems bekannt. Sie gehören zur Gruppe der Mustererkennungsrezeptoren. Neben der Erkennung von körperfremden Pathogenen scheinen diese Rezeptoren auch durch andere molekulare Muster - wie zum Beispiel freie Fettsäuren - aktiviert zu werden. Dass sie an der Pathogenese des Typ 2 Diabetes in Zusammenhang mit Fettsäure-induzierter peripherer Insulinresistenz beteiligt sind, legen Ergebnisse mehrerer Gruppen nahe. Der Typ 2 Diabetes mellitus führt aber nicht nur durch die Entwicklung einer peripheren Insulinresistenz zum Auftreten klinischer Erscheinungen. Auch der Untergang von beta-Zellmasse, also eine gesteigerte Apoptoserate dieser Zellen, ist Teil der Pathogenese. Mit dieser Studie wurde untersucht, ob die Stimulation von Toll-like Rezeptor 2 und Toll-like Rezeptor 4 Apoptose in insulinsezernierenden Zellen auslösen kann und ob die Fettsäure Palmitinsäure spezifisch über diese Rezeptoren wirkt.Toll-like receptors are known as receptors of the innate immune system. They are part of the family of pattern recognition receptors. Apart from recognizing foreign pathogens, these receptors seem to be activated by other molecular clusters – such as free fatty acids. Data of several groups show that these receptors are involved in the pathogenesis of free fatty acid-induced peripheral insulin resistance. However, type 2 Diabetes mellitus does not only enter into clinical appearance by peripheral insulin resistance. The decrease of beta-cell mass, that means an elevated apoptosis rate of these cells, is part of the pathogenesis as well. This study examines whether the stimulation of toll-like receptor 2 and toll-like receptor 4 leads to apoptosis in insulin-secreting cells and if the fatty acid palmitic acid acts specifically via these receptors

Molecular tumor analysis and liquid biopsy: a feasibility investigation analyzing circulating tumor DNA in patients with central nervous system lymphomas

Abstract Background Central nervous system lymphomas (CNSL) is a devastating disease. Currently, a confirmatory biopsy is required prior to treatment. Objective Our investigation aims to prove the feasibility of a minimally-invasive diagnostic approach for the molecular characterization of CNSL. Methods Tissue biopsies from 6 patients with suspected CNSL were analyzed using a 649gene next-generation sequencing (NGS) tumor panel (tumor vs. reference tissue (EDTA-blood)). The individual somatic mutation pattern was used as a basis for the digital PCR analyzing circulating tumor DNA (ctDNA) from plasma and cerebrospinal fluid (CSF) samples, identifying one selected tumor mutation during this first step of the feasibility investigation. Results NGS-analysis of biopsy tissue revealed a specific somatic mutation pattern in all confirmed lymphoma samples (n = 5, NGS-sensitivity 100%) and none in the sample identified as normal brain tissue (NGS-specificity 100%). cfDNA-extraction was dependent on the extraction-kit used and feasible in 3 samples, in all of which somatic mutations were detectable (100%). Analysis of CSF-derived cfDNA was superior to plasma-derived cfDNA and routine microscopic analysis (lymphoma cells: n = 2, 40%). One patient showed a divergent molecular pattern, typical of Burkitt-Lymphoma (HIV+, serologic evidence of EBV-infection). Lumbar puncture was tolerated without complications, whereas biopsy caused 3 hemorrhages. Conclusions Our investigation provides evidence that analysis of cfDNA in central nervous system tumors is feasible using the described protocol. Molecular characterization of CNSL could be achieved by analysis of CSF-derived cfDNA. Knowledge of a tumor’s specific mutation pattern may allow initiation of targeted therapies, treatment surveillance and could lead to minimally-invasive diagnostics in the future

Case Report: Long-Term Survival of a Patient with Cerebral Metastasized Ovarian Carcinoma Treated with a Personalized Peptide Vaccine and Anti-PD-1 Therapy

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