Use of plasma ctDNA as a potential biomarker for longitudinal monitoring of a patient with metastatic high-risk upper tract urothelial carcinoma receiving pembrolizumab and personalized neoepitope-derived multipeptide vaccinations: a case report.

Upper tract urothelial carcinoma (UTUC) is often diagnosed late and exhibits poor prognosis. Only limited data are available concerning therapeutic regimes and potential biomarkers for disease monitoring. Standard therapies often provide only insufficient treatment options. Hence, immunotherapies and complementary approaches, such as personalized neoepitope-derived multipeptide vaccine (PNMV), come into focus. In this context, genetic analysis of tumor tissue by whole exome sequencing represents an essential diagnostic step in order to calculate tumor mutational burden (TMB) and to reveal tumor-specific neoantigens. Furthermore, disease progression is essential to be monitored. Longitudinal screening of individually known mutations in plasma circulating tumor DNA (ctDNA) by the use 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 pembrolizumab was started leading to strong regression of the disease. This therapy was then discontinued due to side effects and treatment with a previously 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 MLH1 gene by ddPCR 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.

Tumor mutation burden and circulating tumor DNA in combined CTLA-4 and PD-1 antibody therapy in metastatic melanoma - results of a prospective biomarker study.

BACKGROUND: Metastasized or unresectable melanoma has been the first malignant tumor to be successfully treated with checkpoint inhibitors. Nevertheless, about 40-50% of the patients do not respond to these treatments and severe side effects are observed in up to 60%. Therefore, there is a high need to identify reliable biomarkers predicting response. Tumor Mutation Burden (TMB) is a debated predictor for response to checkpoint inhibitors and early measurement of ctDNA can help to detect treatment failure to immunotherapy in selected melanoma patients. However, it has not yet been clarified how TMB and ctDNA can be used to estimate response to combined CTLA-4 and PD-1 antibody therapy in metastatic melanoma. PATIENTS AND METHODS: In this prospective biomarker study, we included 35 melanoma patients with ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) therapy. In all patients, a tumor panel of 710 tumor-associated genes was applied (tumor vs. reference tissue comparison), followed by repetitive liquid biopsies. Cell-free DNA was extracted and at least one driver mutation was monitored. Treatment response was evaluated after about three months of therapy. RESULTS: TMB was significantly higher in responders than in nonresponders and TMB > 23.1 Mut/Mb (TMB-high) was associated with a survival benefit compared to TMB ≤ 23.1 Mut/Mb (TMB-low or TMB-intermediate). Furthermore, a > 50% decrease of cell-free DNA concentration or undetectable circulating tumor DNA (ctDNA), measured by tumor-specific variant copies/ml of plasma at first follow-up three weeks after treatment initiation were significantly associated with response to combined immunotherapy and improved overall survival, respectively. It is noticeable that no patient with TMB ≤ 23.1 Mut/Mb and detectable or increasing ctDNA at first follow-up responded to immunotherapy. CONCLUSION: High TMB, > 50% decrease of cell-free DNA concentration, and undetectable ctDNA at first follow-up seem to be associated with response and overall survival under combined immunotherapy. The evaluation of ctDNA and cell-free DNA three weeks after treatment initiation may be suitable for early assessment of efficacy of immunotherapy.

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

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.