Mutational landscape of primary mediastinal b-cell lymphoma (pmbcl) by means of circulating tumor dna analysis

Background: Primary mediastinal B‐cell lymphoma (PMBCL) is a rare subtype of non‐Hodgkin lymphoma that predominantly occurs in adolescents and young adults. Biologically, PMBCL shares many similarities with classical Hodgkin lymphoma, including constitutive activation of the JAK‐STAT and NF‐kB pathways. High‐throughput sequencing of circulating tumor DNA (ctDNA) in peripheral blood (“liquid biopsy”) has recently emerged as a promising noninvasive approach in different types of neoplasms, including lymphoma. This option could be helpful in PMBCL when a biopsy specimen of the tumor is not available for molecular analysis and can also provide an insight into the overall clonal heterogeneity and noninvasively assess the molecular changes over time and the genetic mechanisms of resistance. Aims: The aim of this study was to analyze the mutational profile at diagnosis and in the refractory/relapse settings in PMBCL patients using ctDNA and to compare, when available, with genotyping from tissue biopsy DNA. Methods: We included 9 patients (M/F 4/5, median age 29 years) diagnosed with PMBCL in a single institution between 2015 and 2018 according to the WHO criteria. All patients were treated with R‐CHOP. After initial treatment, 4 patients achieved complete remission (CR), 2 partial response (PR), 3 were refractory. ctDNA extraction was performed from 1–2 mL of plasma collected in PAXgene Blood ccfDNA tubes (Qiagen). Samples were obtained both at diagnosis and at relapse/progression after chemoimmunotherapy. Tumor genomic DNA (gDNA) was isolated from formalin‐fixed paraffin‐embedded (FFPE) diagnostic tissue biopsies. Libraries were constructed with 10–30 ng of ctDNA and 150 ng of gDNA, using molecular‐barcoded library adapters and a hybridization capture based method (SureSelectXT‐Agilent Technologies). Mutation profiles were generated using a custom hybridization capture‐based panel of 115 genes and sequenced in a MiSeq instrument (Illumina, San Diego, CA). Results: ctDNA was obtained from all patients at diagnosis and from 4/5 patients in PR or who were refractory after treatment. Median amount of ctDNA at diagnosis was 46 ng (range: 10–74 ng). In all the cases, at least one mutation could be detected, with a median of 10 mutations per sample (range 1–22mutations). In 4 of the 5 cases with simultaneous tissue and peripheral blood samples, a similar number of mutations was detected with the same genes involved. The genes most frequently mutated at diagnosis were B2 M, GNA13, SOCS1, STAT6, HIST1H1C, HIST1H1E, MFHAS1, NFKBIE (figure). In 3 of the 4 cases with a ctDNA sample at diagnosis and at progression, changes in the allelic frequency of the mutations could be observed, whereas in the remaining case no mutation at progression could be detected. Of note, none of the 3 patients with NFKBIE mutation achieved a complete remission after first line treatment. Summary/Conclusion: Preliminary data on the use of ctDNA in patients with PMBCL show a good correlation with the information obtained in the tissue. Therefore, it can be a real‐time method to evaluate the mutational landscape and the appearance of clones resistant to treatment. image