LAUSR

Venetoclax is an oral, highly selective BCL2 inhibitor with significant clinical efficacy in a range of B-cell lymphoproliferative disorders, including chronic lymphocytic leukaemia (CLL) (Roberts et al, 2016; Seymour et al, 2017), mantle cell lymphoma (MCL) (Davids et al, 2017), multiple myeloma (MM) (Moreau et al, 2017) and follicular lymphoma (FL) (Davids et al, 2017). Whilst various mechanisms of potential resistance to this targeted agent have been reported in model systems (Fresquet et al, 2014; Punnoose et al, 2016; Jayappa et al, 2017), to date only upregulation of BCL-xL (also termed BCL2L1) (Agarwal et al, 2019) or acquisition of a specific BCL2 mutation (Gly101Val) (Blombery et al, 2019) have been observed in samples from patients with progressive MCL or CLL respectively during therapy and confirmed as causing clinical resistance. Venetoclax monotherapy induces objective responses in 38% of patients with FL, including 14% complete responses (Davids et al, 2017). To date, mechanisms of secondary resistance in FL patients treated with venetoclax remain undefined. We describe a patient with relapsed/refractory FL treated with venetoclax who, after an initial response, developed progressive disease on venetoclax therapy through the emergence of a subclone harbouring multiple acquired BCL2 mutations, including a novel candidate resistance mutation which reduced venetoclax binding – BCL2 Phe104Ile. A 55-year-old man was enrolled on a Phase 1 clinical trial of single agent venetoclax (NCT01328626; Roberts et al, 2016) for treatment of symptomatic relapsed FL. At study entry he had bilateral fluorodeoxyglucose (FDG)-avid inguinal lymphadenopathy and right tonsillar involvement on FDG-positron emission tomography (PET) scan. After approximately four months on venetoclax (1200 mg/day) he achieved a complete metabolic response on PET scan. After 29 months on therapy he had progressive FDG-avid right inguinal lymphadenopathy which was biopsied, confirming recurrent FL without evidence of large cell transformation. He subsequently ceased venetoclax. Given the presence of an activating EZH2 mutation he was treated with an EZH2 inhibitor (tazemetostat) and is in ongoing complete response at 34 months on therapy. Targeted next generation sequencing (Supplementary Methods) was performed on FL biopsy samples immediately prior to venetoclax treatment and at disease progression on venetoclax (Table SI). In addition to typical pathogenic mutations observed in FL (CREBBP, KMT2D and EZH2) there was evidence of aberrant somatic hypermutation (aSHM) of the BCL2 gene with presence of multiple mutations in the 5’untranslated region and the first coding exon. Copy number variation (CNV) and structural variation (SV) analysis identified a focal copy number loss of TP53 and an IGH-BCL2 translocation present in both the preand post-venetoclax samples. No new CNVs or SVs were detected in the post-venetoclax sample. In the post-venetoclax sample, seven BCL2 mutations were detected that were not detectable in the pre-venetoclax biopsy (sensitivity 3–5% variant allele frequency [VAF]). Five of the newly acquired BCL2 mutations were predicted to result in amino acid changes in the BCL2 protein and one mutation (c.585+13G>A) was predicted to result in a Gly200Ser in a noncanonical BCL2 transcript (NM_000657 2). Strikingly, one of the mutations acquired during venetoclax treatment was predicted to result in a phenylalanine substitution to an isoleucine at amino acid 104 of the BCL2 protein, which is located at the venetoclax binding site of BCL2. The VAF of the Phe104Ile mutation (12 3%) was the highest of the newly acquired BCL2 mutations in the postvenetoclax sample (estimated cancer cell fraction approximately 50% based on immunohistology) consistent with its presence in a significant proportion of the tumour compartment. The mutation could not be detected by droplet digital polymerase chain reaction (sensitivity 0 1%) in the pre-venetoclax sample. Whilst the Phe104Ile has not been described before in cancer (COSMIC) or population databases (gnomAD), two other substitutions at this position (Phe104Leu and Phe104Cys) have been previously reported in a lymphoma cell line rendered resistant through continuous exposure to venetoclax. Functional evidence to date for these BCL2 mutations indicates resistance to the drug due to reduced venetoclax binding (Fresquet et al, 2014). Another acquired BCL2 mutation in this patient (Gly33Arg) has been described in FL but it is unrelated to venetoclax therapy and experimental data to date suggests it has increased affinity for the pro-apoptotic proteins BIM (BCL2L11) and PUMA (BBC3) in protein binding studies and in cellular models (Correia et al, 2015). Phase analysis of paired sequencing read data from the preand post-venetoclax samples were consistent with the emergence of a new clonal lineage, characterized by the presence of Gly33Arg and Phe104Ile mutations within the same allele, arising from a Leu23Val negative stem clone (Fig 1). Based on the previous observation that BCL2 mutations are highly enriched in the IGH translocated allele (Correia et al, Correspondence