Clinical specimens
MSI, tumor, Tumor, cancer, Cancer
TUMOR, TUMOR, CANCER, MICROSATELLITE INSTABILITY, CPS, ONCOLOGY, PATHOLOGY, CANCER
The diagnostic FFPE tumor tissue samples were used to perform a comprehensive baseline genomic profiling of metastatic lesion at the Center of Molecular Pathology (MPZ), Institute of Pathology, University of Heidelberg, Germany. Using the large-scale TruSight Oncology 500 (TSO500, Illumina) panel that covers 521 cancer relevant genes and comprises a total of 1.94 megabases (Mb) of the genome, the genomic profiles included not only potentially clinically relevant tumor mutations but also a statement about tumor mutational burden (TMB) and microsatellite instability (MSI). DNA extraction and libraries for the capture-based TSO500 were prepared as described previouslyPD-L1 expression status had been determined as part of the diagnostic workup prior to the inclusion to the CheCUP trial in ten cases. Additional five patients had FFPE tumor tissue available for PD-L 1 immunohistochemistry, which was subsequently performed at the Institute of Pathology Heidelberg according to local standards. In brief, 3 μm thick paraffin sections were prepared. Deparaffinization and tissue staining were performed using a Ventana Benchmark Ultra device (Roche Diagnostics, Mannheim, Germany). Slides were deparaffinized and incubated with cell conditioning solution (Cell Conditioning 1 [CC1], Roche Diagnostics) at 95 C for 64 min. IHC staining was performed according to standard protocols (Ventana PD-L1 assay, clone SP263; Roche, Mannheim, Germany; Material number: 07208162001; Part Number: 740-4907; incubation time: 16 minutes). Hematoxylin was used for counterstaining of cell nuclei. IHC stainings were evaluated by a specialist in pathology and scoring of PD-L1 was performed according to standardized scoring criteria. PD-L1 positivity was defined as either Combined positivity score (CPS) or Tumor proportion score (TPS) Plasma samples were longitudinally collected from 34 CUP patients (median: 1; range: 1-10 samples per patient) starting at screening period prior ICI treatment and afterwards on day 1 of every second treatment cycle (approximately every three months after treatment initiation). In addition, 19 plasma samples from healthy donors were collected at the German Cancer Research Center of Heidelberg, Germany. All healthy donors provided written informed consent for the use of biological material for all study-related purposes by consenting to the preapproved Molecularly Aided Stratification for Tumor Eradication Research (NCT-MASTER) program (ClinicalTrials.gov ID NCT05852522). Plasma was isolated from whole blood two or 24 hours after venipuncture using the recommended double spin protocol for maximum plasma recovery. Briefly, 20 ml whole blood collected and stored in STRECK BCT tubes (Streck) was first centrifuged at 1600 x g for 10 min at room temperature. The upper plasma layer was again centrifuged at 4600 x g for 10 min at room temperature to remove cell debris, and directly utilized for ccfDNA isolation as detailed below.