Feasibility of Next-generation Sequencing of Liquid Biopsy (Circulating Tumor DNA) Samples and Tumor Tissue from Patients with Metastatic Prostate Cancer in a Real-world Clinical Setting in Germany.

BACKGROUND AND OBJECTIVE: With European Medicines Agency approval of PARP inhibitors in metastatic castration-resistant prostate cancer and ongoing trials in metastatic hormone-sensitive prostate cancer, detection of genetic alterations in BRCA1/2 and other homologous recombination repair genes has gained an important role. Our aim was to investigate the feasibility and comparability of comprehensive next-generation sequencing (NGS) of liquid biopsy (LB; circulating tumor DNA) and tumor tissue (TT) samples in a real-world clinical setting. METHODS: The study cohort consisted of 50 patients with metastatic prostate cancer (mPC) who had TT NGS performed for BRCA1/2 alterations and consent for additional LB NGS. The Oncomine Comprehensive Assay v3 (Thermo Fisher Scientific, Waltham, MA, USA) was used for TT NGS. The Guardant360 83-gene assay (Guardant Health, Palo Alto, CA, USA) was used for LB NGS, including all types of somatic alterations, microsatellite instability, and blood tumor mutational burden. We calculated BRCA1/2 alteration rates and the negative percentage agreement (NPA) and positive percentage agreement (PPA) between TT and LB results. KEY FINDINGS AND LIMITATIONS: TT NGS was successful in 44/50 patients (88%), with pathogenic BRCA1/2 alterations detected in four (9%). LB NGS was successful in all 50 patients (100%), with BRCA1/2 alterations detected in ten (20%). In a subgroup analysis for the 44 patients with successful TT NGS, NPA was 85% and PPA was 50%. The median time between TT sample collection and blood sampling for NGS was 132 wk (IQR 94-186). The limited sample size and differences in the time of NGS assessment are limitations. CONCLUSIONS AND CLINICAL IMPLICATIONS: LB NGS resulted in a higher detection rate for BRCA1/2 alterations in comparison to conventional TT NGS (20% vs 9%). Ideally, BRCA1/2 testing should be based on both approaches to identify all patients with mPC eligible for PARP inhibitor therapy. PATIENT SUMMARY: Our study shows that genetic tests for both tumor tissue and blood samples results in higher rates of detection of BRCA1/2 gene alterations in patients with metastatic prostate cancer.

Heterogeneity of small duct- and large duct-type intrahepatic cholangiocarcinoma.

AIMS: The histological subtype of intrahepatic cholangiocarcinoma (iCCA) is associated with different mutational characteristics that impact clinical management. So far, data are lacking on the presence of small duct iCCA (SD-iCCA) and large duct iCCA (LD-iCCA) in a single patient. The aim of the current study was to determine the presence and degree of intratumoural heterogeneity of SD- and LD-iCCA features in different tumour regions. METHODS AND RESULTS: All patients treated with surgically resected iCCA at Frankfurt University Hospital between December 2005 and March 2023 were retrospectively analysed. Histomorphological features of SD- and LD-iCCA were evaluated by an expert hepatobiliary pathologist. Tissue samples suspicious for subtype heterogeneity were further investigated. Immunohistochemistry for N-cadherin, S100P, MUC5AC, MUC6, TFF1 and AGR2 and mutational profiling with the Illumina TruSight Oncology 500 (TSO500) assay were performed separately for the SD- and LD-iCCA regions. Of 129 patients with surgically resected iCCA, features of either SD- or LD-iCCA were present in 67.4% (n = 87) and 24.8% of the patients (n = 32), respectively; 7.8% (n = 10) had histomorphological features of both SD- and LD-iCCA, seven patients (5.4%) of which had sufficient formalin-fixed, paraffin-embedded tissue for further analysis. Heterogeneity of both subtypes could be confirmed with immunohistochemistry. In five of seven (71.4%) patients, molecular profiling revealed intratumoural differences in genetic alterations between the SD- and LD-iCCA region. In one patient, a BRAF mutation (p.V600E) was found in the SD-iCCA but not in the LD-iCCA region of the tumour. CONCLUSIONS: A marked portion of patients with iCCA exhibits both SD- and LD-iCCA in different tumour regions. In case of the presence of histopathological heterogeneity, mutational profiling should be considered to avoid missing therapeutically relevant genetic alterations.

A Comparison of Two Different FFPE Tissue Dissection Methods for Routine Diagnostics in Molecular Pathology: Manual Macrodissection versus Automated Microdissection Using the Roche "AVENIO Millisect" System.

Currently, in routine diagnostics, most molecular testing is performed on formalin-fixed, paraffin-embedded tissue after a histomorphological assessment. In order to find the best possible and targeted individual therapy, knowing the mutational status of the tumour is crucial. The "AVENIO Millisect" system Roche introduced an automation solution for the dissection of tissue on slides. This technology allows the precise and fully automated dissection of the tumour area without wasting limited and valuable patient material. In this study, the digitally guided microdissection was directly compared to the manual macrodissection regarding the precision and duration of the procedure, their DNA concentrations as well as DNA qualities, and the overall costs in 24 FFPE samples. In 21 of 24 cases (87.5%), the DNA yields of the manually dissected samples were higher in comparison to the automatically dissected samples. Shorter execution times and lower costs were also benefits of the manual scraping process. Nevertheless, the DNA quality achieved with both methods was comparable, which is essential for further molecular testing. Therefore, it could be used as an additional tool for precise tumour enrichment.

Sequencing of BRCA1/2-alterations using NGS-based technology: annotation as a challenge.

In this study, the molecular profile of different BRCA-associated tumor types was assessed with regard to the classification and annotation of detected BRCA1/2 variants. The aim was to establish guidelines in order to facilitate the interpretation of BRCA1/2 alterations in routine diagnostics. Annotation of detected variants was evaluated compared to background mutations found in normal tissue samples and manually reviewed according to distinct online databases. This retrospective study included 48 samples (45 tumors, three non-tumors), which were sequenced with the GeneReader (QIAGEN). Thereof ten samples were additionally analyzed with the Ion S5™ (Thermo Fisher) and 20 samples with the MiSeq™ (Illumina®) to compare the different NGS devices, as well as the sequencing results and their quality. The analysis showed that the individual NGS platforms detected different numbers of BRCA1/2 alterations in the respective tumor sample. In addition, the GeneReader revealed variability in the detection and classification of pathogenic alterations within the platform itself as well as in comparison with the other platforms or online databases. The study concluded that the Ion S5™ in combination with the Oncomine™ Comprehensive Assay v3 is most recommendable for current and prospective requirements of molecular analysis in routine diagnostics. In addition to the two BRCA1/2 genes, a broad number of other genes (BRCAness genes and genes involved in the repair pathway) is covered by the panel, which may open up new treatment options for patients depending on the respective eligibility criteria.