Liquid Biopsy - Blut-basierte Biomarker zur molekularen Charakterisierung von Tumoren

Tissue analysis is the current gold standard for cancer diagnosis and characterization, although it may neither fully represent spatial tumor heterogeneity nor clonal evolution under treatment pressure. The analysis of circulating tumor cells (CTCs) and cell-free DNA (cfDNA) holds great potential to partially overcome this limitation. One major uncertainty is, however, whether both constituents (CTC vs. cfDNA) provide clinical informative value in a competitive or complementary way. Therefore, reflection of the mutational profile of tumor tissue in CTCs and cfDNA was investigated. Applicability of cfDNA-based mutation analysis in colorectal cancer (CRC) patients in relation to disease stage was systematically investigated using Droplet Digital™ PCR. From 65 patients, the KRAS and BRAF gene status was assessed in plasma and compared to tumor tissue. In 17 of 25 (68%) patients with stage IV tumors, the tissue status was displayed in plasma. In patients with stage I-III tumors, concordance was only 43% (12 of 28 patients). Interestingly, in one stage II patient, cfDNA analysis revealed a different mutation compared to the respective colon cancer. Instead, the KRAS mutation of the synchronous stage IV tumor of the pancreas was detected in plasma. This case indicated the ability of liquid biopsy (LB) to identify the predominant cancer in patients with simultaneous malignancies. In contrast to cfDNA levels, CTC detection rate in the CRC cohort was independent of tumor stage, indicating complementarity of cfDNA and CTCs. To investigate this hypothesis in neoplasms with different metastatic organotropisms, possibly affecting the clinical value of LB, panel sequencing of cfDNA and CTCs from patients with advanced CRC, head and neck squamous cell carcinoma (HNSCC) and melanoma (MEL) was performed. Only one of seven CTC samples isolated from four of 18 patients reflected the status of the solid tumor. In contrast, 78% of tissue mutations were displayed in high input cfDNA samples (30-100 ng, N=8). Highest concordance was observed in MEL and CRC with 100% and 92%, respectively, compared to only 50% in HNSCC. These results emphasized that, when analyzing cancer patients in the advanced setting, cfDNA is superior to CTCs with respect to sample handling and mutation concordance. CTCs implicated clinical use in earlier cancer stages and for the analysis of tumor heterogeneity. Overall, clinical value of LB analysis was demonstrated in special patient cases by detecting tumor heterogeneity and clonal dynamics under selective pressure, which represent the main drivers of acquired resistance and subsequent treatment failure.Gewebeanalysen stellen den gegenwärtigen Goldstandard für die Krebsdiagnose und -charakterisierung dar, obwohl dadurch weder die räumliche Tumorheterogenität noch die klonale Evolution unter selektivem Druck einer Therapie vollständig widergespiegelt werden. Die Analyse zirkulierender Tumorzellen (CTCs) und zellfreier DNA (cfDNA) besitzt ein hohes Potential diese Limitation partiell zu überwinden. Allerdings besteht Ungewissheit darüber, ob beide Bestandteile der Flüssigbiopsie (LB) Informationen von klinischer Relevanz übermitteln und ob diese von kompetitiver oder komplementärer Natur sind. Folglich wurde untersucht, inwiefern CTCs und cfDNA das Mutationsprofil des soliden Tumorgewebes repräsentieren. Die Eignung der cfDNA-basierten Mutationsanalyse in Relation zum Krankheitsstatus von Patienten mit Kolorektalkarzinom (CRC) wurde mittels Droplet Digital™ PCR systematisch untersucht. Der KRAS und BRAF Genstatus wurde in Plasmaproben von 65 Patienten ermittelt und mit dem im Gewebe verglichen. In 17 von 25 (68%) Patienten mit Tumoren im Stadium IV, hat das Plasma den bekannten Gewebestatus wiedergegeben. In Patienten mit Krebsstadium I-III lag die Konkordanz bei nur 43% (12 von 28 Patienten). Interessanterweise, wurde in einem Patienten ein zum Tumorgewebe (Stadium II) widersprüchliches Ergebnis in der cfDNA offenbart. Stattdessen wurde im Plasma der KRAS-Status des synchronen Pankreastumors (Stadium IV) widergespiegelt. Dieser Fall implizierte den Anwendungsbereich, anhand der LB den vorherrschenden Tumor in Patienten mit multiplen Krebserkrankungen zu identifizieren. Im Gegensatz zum cfDNA-Level war die CTC-Detektionsrate in der CRC-Kohorte un-abhängig vom Tumorstadium, was auf eine Komplementarität der Komponenten hin-deutete. Zur Untersuchung dieser Hypothese in Neoplasien mit unterschiedlichem Organotropismus wurde eine Panel-Sequenzierung von cfDNA und CTCs von Patienten mit fortgeschrittenem CRC, Kopf-Hals-Karzinom und Melanom durchgeführt. Nur eine von sieben CTC-Proben, die von vier der 18 Patienten isoliert wurde, spiegelte das Mutationsprofil des soliden Tumors wider. Im Gegensatz dazu, wurden 78% der Gewebemutationen in hoch konzentrierten cfDNA-Proben detektiert (30-100 ng, N=8). Die höchste Übereinstimmung lag bei 100% und 92% in Patienten mit Melanom und CRC, verglichen zu 50% bei den Kopf-Hals-Karzinomen. Diese Ergebnisse zeigten, dass cfDNA-Analysen in Patienten mit fortgeschrittenem Tumorstadium bezüglich der Probenhandhabung und Repräsentation des Tumormu-tationsprofils den CTC-Analysen überlegen sind. CTCs schienen hingegen zur Unter-suchung der Tumorheterogenität als auch in früheren Krebsstadien einen klinischen Nutzen zu versprechen. Insgesamt wurde in einigen speziellen Patientenfällen der klinische Stellenwert der LB-Analyse demonstriert, indem Tumorheterogenität und klonale Dynamiken unter selektiven Therapiedruck detektiert wurden, welche die Haupttreiber erworbener Resistenz und folgendem Therapieversagen repräsentieren

Applicability of liquid biopsies to represent the mutational profile of tumor tissue from different cancer entities

Genetic investigation of tumor heterogeneity and clonal evolution in solid cancers could be assisted by the analysis of liquid biopsies. However, tumors of various entities might release different quantities of circulating tumor cells (CTCs) and cell-free DNA (cfDNA) into the bloodstream, potentially limiting the diagnostic potential of liquid biopsy in distinct tumor histologies. Patients with advanced colorectal cancer (CRC), head and neck squamous cell carcinoma (HNSCC), and melanoma (MEL) were enrolled in the study, representing tumors with different metastatic patterns. Mutation profiles of cfDNA, CTCs, and tumor tissue were assessed by panel sequencing, targeting 327 cancer-related genes. In total, 30 tissue, 18 cfDNA, and 7 CTC samples from 18 patients were sequenced. Best concordance between the mutation profile of tissue and cfDNA was achieved in CRC and MEL, possibly due to the remarkable heterogeneity of HNSCC (63%, 55% and 11%, respectively). Concordance especially depended on the amount of cfDNA used for library preparation. While 21 of 27 (78%) tissue mutations were retrieved in high-input cfDNA samples (30-100 ng, N = 8), only 4 of 65 (6%) could be detected in low-input samples (<30 ng, N = 10). CTCs were detected in 13 of 18 patients (72%). However, downstream analysis was limited by poor DNA quality, allowing targeted sequencing of only seven CTC samples isolated from four patients. Only one CTC sample reflected the mutation profile of the respective tumor. Private mutations, which were detected in CTCs but not in tissue, suggested the presence of rare subclones. Our pilot study demonstrated superiority of cfDNA- compared to CTC-based mutation profiling. It was further shown that CTCs may serve as additional means to detect rare subclones possibly involved in treatment resistance. Both findings require validation in a larger patient cohort

Detection of mutations in circulating cell‐free DNA in relation to disease stage in colorectal cancer

Enthusiasm has emerged for the potential of liquid biopsies to provide easily accessible genetic biomarkers for early diagnosis and mutational cancer characterization. We here systematically investigated the suitability of circulating cell-free DNA (cfDNA) analysis for mutation detection in colorectal cancer (CRC) patients with respect to clinicopathological disease stage. Droplet Digital PCR (ddPCR) was performed to detect common point mutations in the KRAS and BRAF oncogenes in cfDNA from 65 patients and compared to mutations in tumor tissue. Stage of disease was classified according to UICC (Union for International Cancer Control) criteria. In tumor tissue, KRAS or BRAF mutations were present in 35 of 65 cases (44% UICC stage I, 50% stage II, 47% stage III, and 62% stage IV). Although cfDNA was detected in 100% of patients, ddPCR displayed the tumor tissue mutation in only 1 of 6 (17%) stage II patients, whereas 10 of 18 (56%) reported variants were verified in cfDNA samples of the stage IV cohort. No BRAF or KRAS mutation was detected in cfDNA from patients with wild-type tumor tissue. In one case of mutant stage II colon cancer (KRAS-G12C), the G12D variant was detected in cfDNA instead. Further workup revealed that circulating tumor-derived DNA and liver metastases originated from a synchronous KRAS-mutated cancer of the pancreas. Our results demonstrate that ddPCR-based analysis is highly specific and useful for mutation monitoring, but the sensitivity limits its usefulness for early cancer detection

Multiparametric Phenotyping of Circulating Tumor Cells for Analysis of Therapeutic Targets, Oncogenic Signaling Pathways and DNA Repair Markers

Detection of circulating tumor cells (CTCs) has been established as an independent prognostic marker in solid cancer. Multiparametric phenotyping of CTCs could expand the area of application for this liquid biomarker. We evaluated the Amnis&reg; brand ImageStream&reg;X MkII (ISX) (Luminex, Austin, TX, USA) imaging flow cytometer for its suitability for protein expression analysis and monitoring of treatment effects in CTCs. This was carried out using blood samples from patients with head and neck squamous cell carcinoma (n = 16) and breast cancer (n = 8). A protocol for negative enrichment and staining of CTCs was established, allowing quantitative analysis of the therapeutic targets PD&ndash;L1 and phosphorylated EGFR (phospho&ndash;EGFR), and the treatment response marker &gamma;H2AX as an indicator of radiation&ndash;induced DNA damage. Spiking experiments revealed a sensitivity of 73% and a specificity of 100% at a cut&ndash;off value of &ge;3 CTCs, and thus confirmed the suitability of the ISX-based protocol to detect phospho&ndash;EGFR and &gamma;H2AX foci in CTCs. Analysis of PD&ndash;L1/&ndash;L2 in both spiked and patient blood samples further showed that assessment of heterogeneity in protein expression within the CTC population was possible. Further validation of the diagnostic potential of this ISX protocol for multiparametric CTC analysis in larger clinical cohorts is warranted