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

Abstract

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.

Conflict of interest statement

The authors declare no competing financial interests. MS is an employee of Alacris Theranostics GmbH.

© 2021. The Author(s).

Figures

Fig. 1. Patient characteristics in comparison to the corresponding cfDNA concentrations and total CTC counts.

For each patient, the detectable CTC count and cfDNA concentration were examined, possibly affected by the therapy status, including treatment prior to study enrollment and the time span (Δt) between the last therapy (Tx) and liquid biopsy collection (LB). CT chemotherapy, IT immunotherapy, RT radiotherapy, SURG surgery, TT targeted therapy, mo months. ‡available NGS data from whole genome amplified CTCs.

Fig. 2. Comparative analysis of tissue-derived mutations and their representation in LB samples.

Patients were categorized into two sub-groups referring to A high-input and B low-input cfDNA samples used for NGS analysis (≥30 and <30 ng, respectively). ‡Multiple mutations were detected in the same gene. FIC density gradient centrifugation-enriched CTCs, LB liquid biopsy, LR local recurrence, NGS next-generation sequencing, ROS RosetteSep™-enriched CTCs, tDNA tumor-derived DNA.

Fig. 3. Comparative analysis of cfDNA mutations and their concordance with corresponding tissue samples.

Patients were assigned to a sub-group based on A high-input and B low-input cfDNA samples used for NGS analysis (≥30 and <30 ng, respectively). FIC Ficoll-enriched CTCs, LB liquid biopsy, LR local recurrence, NGS next-generation sequencing, ROS RosetteSep™-enriched CTCs, tDNA tumor-derived DNA.

Fig. 4. Concordance of CTC-derived alterations with corresponding samples from the same individual.

Of a total count of 206 CTC-derived mutations detected in four patients, 44 (21%) were also retrieved in another CTC, cfDNA, and/or tissue sample from the same patient, whereas 162 (79%) were unique for the analyzed circulating tumor cell.

Fig. 5. Mutation profiles of corresponding CTCs, cfDNA, and tissue from a single patient reflected tumor heterogeneity, potentially affecting different cancer hallmark-related pathways.

A Shared, discordant (different mutations in the same gene), and private gene alterations were identified in tumor tissue and LB from CRC002.1 and B, C assigned to cancer hallmarks, demonstrating distinct differences of involved cancer-related pathways likely to correlate with the requirements concerning tumor growth and metastasis to distant sites. ‡Multiple mutations were detected in the same gene.

Fig. 6. Tissue and plasma genotyping of a patient with refractory melanoma to immunotherapy and BRAF-MEK-inhibition at different time points of the disease.

Schematic illustration of the clinical course, including the duration of administered treatment, therapy adaption due to side effects (flash) or progressive disease (red circles), and tumor genotyping conducted on tissue (indicated by a scalpel) or plasma (indicated by a syringe). Allele frequencies (AF) of BRAF and NRAS mutations were determined by ddPCR analysis.