Targeted Genotyping of Circulating Tumor Dna for Classical Hodgkin Lymphoma Monitoring: A Prospective Study

Abstract

Introduction: Previous studies have highlighted the potential of circulating tumor DNA (ctDNA) to determine the mutational profile of DLBCL, and assess the molecular changes over time and the genetic mechanisms of resistance. In addition, the quantity of ctDNA could also predict the response and outcome of the patients. The aim of this study was to analyze the mutational profile at diagnosis and its dynamics after frontline treatment and in the refractory/relapse settings. Methods:We included 65 patients (M/F 32/33, median age 64 years) diagnosed with DLBCL in a single institution between 2016 and 2018 according to the WHO criteria. All patients were treated with chemoimmunotherapy. After initial treatment, 47 patients achieved CR, 4 PR, 7 were refractory and in 7 cases the response was not evaluable. Samples were obtained both at diagnosis and at relapse/progression in refractory/relapsed patients. Tumor genomic DNA (gDNA) was isolated from formalin-fixed paraffin-embedded (FFPE) diagnostic tissue biopsies and at relapse when available. A panel of 115 genes was amplified using a hybridization capture based protocol from 10-30 ng of ctDNA and 150 ng of gDNA (SureSelectXT-Agilent Technologies) and sequenced in a MiSeq instrument (Illumina). Results: ctDNA was obtained from all patients at diagnosis (median of 34 ng; range: 10-1507 ng) and from 12 refractory/relapsed cases. At least one mutation could be detected in 88% of the cases.Median number of mutations per sample was 6 (0-21 mutations).The genes most frequently mutated at diagnosis in plasma samples were KMT2D, TP53, MYD88, TNFRSF14, PIM1, CREBBP, BCL2 and EP300. In 38 cases, paired FFPE samples were available as detailed in the figure. Sensitivity of ctDNA to detect tumor mutations at baseline FFPE samples was 75% (95%IC: 69.5-80.5). Of note, most of the cases in which mutations were not detected in the ctDNA had a primary extranodal origin. In the 12 cases with a ctDNA sample at diagnosis and at progression, 5 cases showed the same mutational profile, whereas in 6 cases a change in the number of mutated genes was observed (3 cases had fewer mutations; 3 cases had new mutations) and in one case no mutations were detected at relapse. In addition, selection of minor subclonal mutations was observed in 2 cases. Patients with high pretreatment ctDNA levels (>3 log hGE/mL) had a worse PFS and OS than those with low levels (18-month PFS 24 vs 77%; 18-month OS 58 vs 94%; p<0.004).Conclusions: ctDNA shows a good correlation with the information obtained in the tumor. Therefore it could be a valuable tool to assess the mutational profile both at diagnosis and at relapse. Pretreatment ctDNA levels have an impact on outcome.Introduction: 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. Methods:We developed a targeted Next-Generation sequencing (NGS) panel for fast analysis (Am...

Cell-Free DNA for the Management of Classical Hodgkin Lymphoma

“…The similarity (R 2 = 0.978) of mutational profiles from paired gDNA/cfDNA samples favors the capability of CAPP-seq to precisely detect low burden variants in cfDNA. In our experience, comparability between gDNA and cfDNA profiles with an NGS-limited gene panel is close to 85% [ 10 ] at the level variant. Of note, median VAF appears to be higher in cfDNA than in biopsies probable due to the common scarcity of tumor cells in cHL biopsies [ 10 ].…”

“…In our experience, comparability between gDNA and cfDNA profiles with an NGS-limited gene panel is close to 85% [ 10 ] at the level variant. Of note, median VAF appears to be higher in cfDNA than in biopsies probable due to the common scarcity of tumor cells in cHL biopsies [ 10 ]. In the study by Desch et al [ 66 ] in pediatric cHL patients, the average VAFs were 1.1% for tumor DNA (from whole tissue sections) and 11.1% for cfDNA, but all 30 variants discovered in cfDNA were then confirmed in macrodissected HRS-cell rich regions of paired tumor biopsies, confirming the reliability of cfDNA-obtained mutational profiles.…”

“…Our team also led an observational prospective study based on cfDNA testing including 60 consecutive cHL cases treated by frontline ABVD and/or escalated BEACOPP. We observed somatic variants in 42/60 (70%) patients at baseline [ 10 ]. However, this gene panel was unable to disclose variants in all of the patients, probably because the panel was too restricted and sensitivity was insufficient to reveal ultra-low abundance subclones which are close to the sequencer limit of detection (variant allele frequency (VAF) 0.1%).…”

“…In line with these tumor DNA access difficulties, several teams hypothesized that discovering tumor specific somatic alterations would be more appropriate in the bloodstream, i.e., in cell-free DNA (cfDNA) extracted from plasma. Indeed, various proof-of-concept studies have recently shown that cHL molecular analysis is feasible using cfDNA and highly sensitive methods [ 7 , 8 , 9 , 10 , 11 ]. Despite the scarcity of HRS cells and a typical lower tumor volume, many studies have indicated that success rates for cfDNA detection in cHL are close to those achieved for diffuse large B cell lymphoma (DLBCL).…”

Bringing circulating tumor DNA to the clinic in Hodgkin lymphoma

“…More recently, research efforts have focused not only on the development of such methods, but on the translation into the clinic and on defining the utility of ctDNA in various malignancies. 5-7 In this issue of Haematologica, Camus and colleagues 8 explore the utility of ctDNA detection by an amplicon-based next-generation sequencing (NGS) approach in classical Hodgkin lymphoma (cHL) in a prospective observational study.…”