BL-MOL-AR Project, Preliminary Results about Liquid Biopsy: Molecular Approach Experience and Research Activity in Oncological Settings

Abstract

Background Liquid biopsy is mainly used to identify tumor cells in pulmonary neoplasms. It is more often used in research than in clinical practice. The BL-MOL-AR study aims to investigate the efficacy of next-generation sequencing (NGS) and clinical interpretation of the circulating free DNA (cfDNA) levels. This study reports the preliminary results from the first samples analyzed from patients affected by various neoplasms: lung, intestinal, mammary, gastric, biliary, and cutaneous. Methods The Biopsia Liquida-Molecolare-Arezzo study aims to enroll cancer patients affected by various malignancies, including pulmonary, intestinal, advanced urothelial, biliary, breast, cutaneous, and gastric malignancies. Thirty-nine patients were included in this preliminary report. At time zero, a liquid biopsy is executed, and two types of NGS panels are performed, comprising 17 genes in panel 1, which is already used in the routine tissue setting, and 52 genes in panel 2. From the 7th month after enrollment, 10 sequential liquid biopsies are performed up to the 17th month. The variant allele frequency (%) and cfDNA levels (ng/mL) are measured in every plasmatic sample. Results The NGS results obtained by different panels are similar even though the number of mutations is more concordant for lung pathologies. There are no significant differences in the actionability levels of the identified variants. Most of the molecular profiles of liquid biopsies reflect tissue data. Conclusions Preliminary data from this study confirm the need to clarify the limitations and potential of liquid biopsy beyond the lung setting. Overall, parameters related to cfDNA levels and variant allele frequency could provide important indications for prognosis and disease monitoring.

Keywords: NGS; cfDNA; liquid biopsy.

Fig. 1

Workflow concerning the analysis of cfDNA on enrolled patients. The NGS analysis, performed with Panels 1 and 2, is executed on the circulating DNA of the first sampling at time zero (T0). The sequential collecting starts from the seventh month to monitor the variants of clinical significance found in the NGS analysis. The quantification of cfDNA is performed on all liquid biopsies. cfDNA, circulating free DNA; ddPCR, droplet digital polymerase chain reaction; NGS, next-generation sequencing.

Fig. 2

Distribution of tumor origin in the study population.

Fig. 3

The mean number of mutations among the three cancer population cohorts (intestine, lung, and another origin). These values are calculated for every next-generation sequencing (NGS) panel type.

Fig. 4

ESMO Scale for Clinical Actionability of molecular Targets (ESCAT) tiers of the identified mutations in the plasma of patient cohort ( n  = 39). ( a ) The results are classified according to the NGS panel results. All the mutations are included among I to IV and X ESCAT tiers. ( b,c ) The pie charts show the percentual incidence of these variant types by each NGS panel result.

Fig. 5

The graphics of the two sections show the trend of the molecular response in terms of percentage change in variant allele frequency (VAF) compared to the previous measurement (VAR% VAF) starting from T1 for ( a ) case 6 INT and ( b ) case 4 INT. The graphics at the bottom of the two sections show the trend of the molecular response in terms of percentage change in the levels of cfDNA compared to the previous measurement (VAR% cfDNA). At the various monitoring points, the global clinical status of the patient is indicated. LOST, patient's death; PR: partial response; PD, disease progression; SD, stable disease.

Fig. 6

The graphics above of the two sections show the VAR % VAF starting from T1 for case 2INT ( a ) and case 3INT ( b ). The graphics at the bottom of the two sections show VAR % cfDNA. At the various monitoring points, the global clinical status of the patient is indicated. cfDNA, circulating free DNA; LOST, patient's death; PD, disease progression; SD, stable disease; VAR. variant).