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,

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...

2 Dozens of recurrent chromosomal rearrangements have been described in acute leukaemia, where they often result in the fusion of two genes and in the expression of hybrid proteins.These genetic markers often delineate distinct clinical entities, and have been incorporated into the World Health Organisation classification. Today, their evaluation at diagnosis thus provides crucial information for risk stratification, treatment decisions and residual disease evaluation 1,2 .Conventional cytogenetics is usually regarded as the gold standard method for the detection of these rearrangements. It is often considered mandatory at diagnosis but it requires a high level of expertise, is time consuming, and is not suitable for the detection of many cryptic rearrangements 3,4 . Complementary approaches such as fluorescent in situ hybridisation (FISH) and RT-PCR are thus often needed 5 , but the cost of a comprehensive analysis can rapidly become prohibitive. Many markers that may provide important clinical information are therefore never tested, lowering the chances for the patients to benefit from the optimal treatments. To address this issue, we have developed a rapid and parsimonious ligationdependant RT-PCR amplification assay (LD-RTPCR) 6 that allows for the detection of dozens of gene rearrangements (Figure 1).We created a mix of 153 LD-RTPCR probes to target more than 50 recurrent fusion genes and three NPM1 mutations ( Figure 1B and C). The procedure is detailed in supplemental methods, and representative results are provided in supplemental figure 1. As shown in supplemental figure 2A, the results inform on the identity of the two partner genes, but also on the localisation of the breakpoint, guiding the choice of an appropriate assay for residual disease evaluation. The analysis of serial dilutions showed that approximately 3.10 3 copies of transcripts are sufficient for the identification of the two partners (Supplemental figure 2B).To validate this method, we applied it to a retrospective cohort of 540 patients treated in our institutions ( Figure 2 and supplemental table 2). Sixty eight rearrangements were detected in the 180 childhood ALL cases we tested. Fifty two (76.5%) had been identified using RT-PCR at diagnosis and 20 (29.4%) using cytogenetics. Sixty seven of these rearrangements (98.5%) were detected using LD-RTPCR. As expected in this cohort, the most common were the ETV6-RUNX1 fusion (33 cases), rearrangements of the MLL gene (8 cases, four with AFF1, two with MLLT1, one with AFF4 and one with MLLT3) 7 , and the BCR-ABL1 (six cases), TCF3-PBX1 (five cases) and STIL-TAL1 fusions (four cases) 2 . The LD-RTPCR assay only failed to detect one t(4;11)(q21;q23), which was identified at diagnosis using cytogenetics but which could not be confirmed using RT-PCR. The breakpoint of this rearrangement may thus be unusual, or involve genes other than MLL and AFF1. It also validated four junctions which © 2015 Macmillan Publishers Limited. All rights reserved.3 were detected using cytogenetics, but which had not...