Combining germline, tissue and liquid biopsy analysis by comprehensive genomic profiling to improve the yield of actionable variants in a real-world cancer cohort

Abstract

Background: Comprehensive next-generation sequencing is widely used for precision oncology and precision prevention approaches. We aimed to determine the yield of actionable gene variants, the capacity to uncover hereditary predisposition and liquid biopsy appropriateness instead of, or in addition to, tumor tissue analysis, in a real-world cohort of cancer patients, who may benefit the most from comprehensive genomic profiling.

Methods: Seventy-eight matched germline/tumor tissue/liquid biopsy DNA and RNA samples were profiled using the Hereditary Cancer Panel (germline) and the TruSight Oncology 500 panel (tumor tissue/cfDNA) from 23 patients consecutively enrolled at our center according to at least one of the following criteria: no available therapeutic options; long responding patients potentially fit for other therapies; rare tumor; suspected hereditary cancer; primary cancer with high metastatic potential; tumor of unknown primary origin. Variants were annotated for OncoKB and AMP/ASCO/CAP classification.

Results: The overall yield of actionable somatic and germline variants was 57% (13/23 patients), and 43.5%, excluding variants previously identified by somatic or germline routine testing. The accuracy of tumor/cfDNA germline-focused analysis was demonstrated by overlapping results of germline testing. Five germline variants in BRCA1, VHL, CHEK1, ATM genes would have been missed without extended genomic profiling. A previously undetected BRAF p.V600E mutation was emblematic of the clinical utility of this approach in a patient with a liver undifferentiated embryonal sarcoma responsive to BRAF/MEK inhibition.

Conclusions: Our study confirms the clinical relevance of performing extended parallel tumor DNA and cfDNA testing to broaden therapeutic options, to longitudinally monitor cfDNA during patient treatment, and to uncover possible hereditary predisposition following tumor sequencing in patient care.

Keywords: Circulating cell-free DNA; Clinically actionable variants; Droplet digital pcr (ddPCR); Germline pathogenic variants; Next-generation sequencing; Targeted therapy.

Fig. 1

Actionable gene variants in 7 out of 16 tumor tissues analyzed. Actionable somatic variants and gene amplifications (OncoKB level 1–3) in 7/16 (43.75%) tumor tissues are reported. OncoKB level 1, 3B, 3A is indicated in green, lilac, purple, respectively. Gene amplification with OncoKB level 1 is represented with a red circle

Fig. 2

Actionable gene variants in 9 out of 23 circulating cell-free DNA samples analyzed. Actionable somatic variants and gene amplifications (OncoKB level 1–3) in 9/23 (39.1%) circulating cell-free DNA samples are reported. OncoKB level 1, 3B, 3A is indicated in green, lilac, purple, respectively. Gene amplification with OncoKB level 1 is represented with a red circle

Fig. 3

Circulating cell-free DNA mutation profiles and dynamic changes during treatment of GE01 patient. Timeline of the patient’s clinical history and longitudinal tracking of circulating cell-free DNA are shown. Time points on therapy are indicated with blue brackets. The timeline baseline is indicated as T0 and the subsequent times are calculated in months starting from baseline. UESL undifferentiated embryonal sarcoma of the liver, PD progressive disease, SD stable disease, wt wild-type, VAF variant allele frequency

Fig. 4

Workflow to improve the detection yield of actionable variants and their implications for cancer patient’s outcome and clinical management. The flow chart suggests the use of an integrated cfDNA, tumor and germline analysis by next-generation sequencing comprehensive panels in order to increase the yield of actionable variants. The suggested workflow is based on our results obtained in 23 patients with diverse and rare tumor types. For this reason, consider confirming these findings on larger cohorts and prospective clinical trials to support this approach. Our recommendation is to perform the concurrent tumor tissue and cfDNA molecular profiling, when possible. Identification of germline actionable variants is possible through a germline-focused analysis of tumor/cfDNA using a robust and sensitive bioinformatic analysis, opening to genetic counselling and secondary germline testing protocols, as well as cascade testing in family members. The figure also addresses the interpretation of discordant data between tumor tissue biopsy versus liquid biopsy and their clinical implications. While actionable variants found only in tumor DNA and both in tumor DNA and cfDNA are candidate for clinical and therapeutically management, we suggest that actionable variants found only in ctDNA need further confirmation with orthogonal sensitive methods and, if confirmed, longitudinal monitoring is indicated, prior to consideration for therapy treatment and implications on patient outcomes