Targeted genotyping of circulating tumor DNA for classical Hodgkin lymphoma monitoring: a prospective study

Abstract

The relevance of circulating tumor DNA (ctDNA) analysis as a liquid biopsy and minimal residual disease tool in the management of classical Hodgkin Lymphoma (cHL) patients was demonstrated in retrospective settings and remains to be confirmed in a prospective setting. We developed a targeted Next-Generation sequencing (NGS) panel for fast analysis (AmpliSeq technology) of nine commonly mutated genes in biopies and ctDNA of cHL patients. We then conducted a prospective trial to assess ctDNA follow up at diagnosis and after 2 cycles of chemotherapy (C2). Sixty cHL patients treated by first line conventional chemotherapy (BEACOPPescalated [21.3%], ABVD/ABVD-like [73.5%] and other regimens [5.2%, for elderly patients] were assessed in this non-interventional study. Median age of the patients was 33.5 years (range 20-86). Variants were identified in 42 (70%) patients. Mutations of NFKBIE, TNFAIP3, STAT6, PTPN1, B2M, XPO1, ITPKB, GNA13 and SOCS1 were found in 13.3%, 31.7%, 23.3%, 5%, 33.3%, 10%, 23.3%, 13.3% and 50% of patients, respectively. ctDNA concentration and genotype are correlated with clinical characteristics and presentation. Regarding early therapeutic response, 45 patients (83%, NA=6) had a negative positron emission tomography (PET) after C2 (Deauville Score 1-3). Mean of DeltaSUVmax after C2 was -78.8%. We analyzed ctDNA after C2 for 54 patients (90%). ctDNA became rapidly undetectable in all cases after C2. Variant detection in ctDNA is suitable to depict the genetic features of cHL at diagnosis and may help to assess early treatment response, in association with PET. Clinical Trial reference: NCT02815137.

Figures

Figure 1.

Number and types of somatic variants identified by gene and patient both in biopsy and circulating tumor DNA. (A) Heatmap representing somatic variants detected in circulating tumor DNA (ctDNA) in the mutated patients (the "Burden" track represents the mutational load [“burden”] of a patient based on the maximum/minimum number of mutations found in the complete cohort). (B) Unsupervised hierarchical clustering performed among the nine genes to represent the association of alterations (numbers within the clustering represent the number of observed associations between two genes).

Figure 2.

Prevalence of somatic mutations detected in DNA extracted from the 31 available biopsies and 60 plasma samples of the patients at time of diagnosis. The "genomic DNA (gDNA) and/or circulating tumor DNA (ctDNA) " column corresponds to the patients considered mutated in the biopsy and/or the plasma samples.

Figure 3.

Distribution of circulating tumor DNA median variant allelic frequency for the 41 mutated patients with positive plasma at diagnosis. ctDNA: circulating tumor DNA; VAF: variant allelic frequency

Figure 4.

Longitudinal assessment of mutation abundance in plasma circulating tumor DNA upon treatment. Evolution of median circulating tumor DNA (ctDNA) variant allelic frequency (VAF) for each patient (with detectable ctDNA mutation at diagnosis) throughout treatment (at diagnosis [“diag”] n=41) and after two cycles (C2) of chemotherapy (“post C2”, n=31). UPN: universal patient identification number.

Figure 5.

Example of possible application of liquid biopsy analysis. Patient (UPN38) presenting a positive positron emission tomography (PET) scan with residual fixation of a left supraclavicular lymph node (Deauville Scale 5) after two cycles (C2) of adriamycin (doxorubicin), bleomycin, vinblastine, dacarbazine (ABVD): the biopsy showed a reactive lymph node, indicating a false positive PET scan. Plasma circulating tumor DNA (ctDNA) sample was negative at time of the PET post C2 of ABVD with a clearance of all baseline mutations that were present at diagnosis. UPN: universal patient identification number.