Seven major histocompatibility complex (MHC) class I peptides and three longer MHC class II peptides were chosen that were then used for the production of the personalized vaccine (table 1). of next-generation sequencing and digital droplet PCR (ddPCR) might be a promising method to fill this gap. Here, we present the case of a 55-year-old man who was diagnosed with high-risk metastatic UTUC in 2015. After initial surgery and palliative chemotherapy, he developed recurrence of the tumor. Genetic analysis revealed a high TMB of 41.2 mutations per megabase suggesting a potential success of immunotherapy. Therefore, in 2016, off-label treatment with the checkpoint-inhibitor ITGB6 pembrolizumab was started leading to strong regression of the disease. This therapy was then discontinued Apocynin (Acetovanillone) due to side effects and treatment with a previously Apocynin (Acetovanillone) produced PNMV was started that induced strong T cell responses. During both treatments, plasma Liquid Biopsies (pLBs) were performed to measure the number of mutated molecules per mL plasma (MM/mL) of a known tumor-specific variant in the gene by ddPCR Apocynin (Acetovanillone) for longitudinal monitoring. Under treatment, MM/mL was constantly zero. A few months after all therapies had been discontinued, an increase of MM/mL was detected that persisted in the following pLBs. When MRI scans proved tumor recurrence, treatment with pembrolizumab was started again leading to a rapid decrease of MM/mL in the pLB to again zero. Treatment response was then also confirmed by MRI. This case shows that use of immunotherapy and PNMV might be a promising treatment option for patients with high-risk metastatic UTUC. Furthermore, measurement of individually known tumor mutations in plasma ctDNA by the use of pLB could be a very sensitive biomarker to longitudinally monitor disease. gene (MLH1 c.883A T; p.Ser295Cys). The gene encodes for the MutL homolog protein 1, which plays an important role in the mismatch repair (MMR). Defects in are associated with MSI, which was also predicted in our patient. For more details of the technical methods, please see the online supplemental material. Supplementary datajitc-2020-001406supp001.pdf Even though PD-L1 testing was not established at this time, immunotherapy was recommended based on DNA MMR deficiency and high TMB in the tumor genome, and in April 2016 off-label therapy with the ICI pembrolizumab (2 mg/kg body weight every 3 weeks) was started. The patient generally tolerated the immunotherapy well, but his fifth cycle of pembrolizumab in July 2016 was delayed for 10 days due to subclinical elevations in liver transaminases. Furthermore, because of the high TMB, the tumor was considered likely to have many mutated antigens present on its surface providing a rationale for a treatment attempt with PNMVs. In order to produce such vaccines, the tumor specimen was analyzed by WES, followed by prediction of neoepitopes and selection of peptides (see online supplemental material). Seven major histocompatibility complex (MHC) class I peptides and three longer MHC class II peptides were chosen that were then used for the production of the personalized vaccine (table 1). The vaccine was produced Apocynin (Acetovanillone) as an individual healing attempt in Tuebingen, Germany, in May 2016, but following the patients wish was not yet applied at this time. Table 1 Overview of the immune monitoring results gene was measured by ddPCR. The variant was chosen to be measured in pLB as it is considered one of the tumors driver mutations and was not part of the 10 peptides used in the vaccine. Under the first course of treatment with pembrolizumab, the amount of MM/mL observed in the pLB was Apocynin (Acetovanillone) always zero (figure 2). Open in a separate window Figure 2 Timeline of the patients treatment course. Starting in February 2017, MM/mL were measured via pLB and remained at undetectable levels until July 2019. Circled numbers indicated successive MRI scans: (1) and (2) no detected changes; (3) unspecific lymph node enlargement; (4) assured tumor progression and (5) tumor regression. ctDNA, circulating tumor DNA; MM/mL, mutated molecules per ml plasma; pLB, plasma Liquid Biopsy; TMB, tumor mutational burden. Further CT scans from April 2017 until April 2018 showed stable disease. In October 2017, after the 26th cycle of pembrolizumab in total, the immune therapy was discontinued due to the development of unclear urticaria. In February 2018, under stable disease conditions and after long discussions with the patient and his physicians, the patient decided to receive his first PNMV. Some concerns existed regarding the fact that the PNMV had been produced based on 2 year old WES data. Nonetheless, having in mind the possibility of a shifted tumor mutational profile, the PNMV was applied after long consideration as it still presented a valuable treatment option at that time. The vaccine was constantly injected intracutaneously in the remaining thigh followed by a subcutaneous injection of an adjuvant.