Abstract

Background: Circulating tumor DNA (ctDNA) is a promising non-invasive biomarker in cancer management. We aimed to assess the dynamic evolution of ctDNA in patients with hepatocellular carcinoma (HCC).

Methods: A total of 832 plasmas collected in 173 patients with HCC and 56 patients with chronic liver diseases without HCC were studied. We evaluated the quantity of cell free DNA (cfDNA) and search for mutations in TERT promoter (TERTp.), CTNNB1, TP53, PIK3CA and NFE2L2 by ultra-deep next generation sequencing and for TERTp. by digital droplet PCR. 250 tumor samples from the same patients were sequenced for 39 driver genes.

Results: Among the 173 HCC patients, 82% were male (median age 63), 73% had cirrhosis. The etiologies of the underlying liver diseases were hepatitis B (28%), hepatitis C (42%), NAFLD (26%) and chronic alcohol intake (40%). Tumors were classified BCLC 0 (23%), BCLC A (44%), BCLC B (21%) and BCLC C (12%). Tumor sequencing identified mutations in TERTp. (71%), TP53 (28%), CTNNB1 (27%), ATM (15%), ARID1A(13%), ARID2 (8%), NFE2L2 (2%) and PIK3CA (2%).

Among the 776 plasmas of patients with HCC, 502 were collected in patients with an active HCC (aHCC), 158 24 hours after a locoregional treatment and 116 in patients with a past history of HCC but without active HCC at sampling (iHCC). Median cfDNA quantity was higher in aHCC than iHCC (0.27 vs 0.16 ng/µL, p<0.001). Within the 502 plasmas of patients with an aHCC we identified the following mutations in 46% of them (24% patients had normal serum AFP): TP53 (29%), TERTp. (27%), CTNNB1 (13%), PIK3CA (0.4%) and NFE2L2(0.2%). CfDNA mutation rate increased across tumor stages (16% BCLC 0, 25% BCLC A, 42% BCLC B and 58% BCLC C; p<0.001). The presence of mutations, particularly in the TERTp. and TP53, was associated with overall survival and recurrence/progression-free survival, both when considering all plasma samples and in the analysis of various treatment subgroups.

Among 116 iHCC plasmas, 26 (22%) had mutations; in 21 cases mutations were concordant with those observed in a previous plasma sample or in a tumor from the same patient.

Regarding plasmas obtained before and 24 hours after loco-regional treatments an increase in cfDNA level (0.19 before vs 0.63 after, p<0.001) and in mutation rate (31% vs 44%, p<0.001) was observed at H24.

Finally, a total of 179 plasmas in 50 patients with unresectable HCC treated by atezolizumab/bevacizumab were analyzed. Baseline cfDNA mutations were observed in 49% of cases; serum AFP was normal in 24% of these patients. Mutations in cfDNA observed at baseline disappeared in all patients with a radiological response at 12 weeks. In contrast, persistence of mutations under treatment was significantly associated with radiological progression (p=0.005).

Conclusion: Circulating tumor DNA offers dynamic information about tumor biology representing a non-invasive tool potentially useful to guide HCC clinical management.