ALBERT

Description: Key Points Whole-genome integrative analyses in FL reveal that genes strongly influenced by copy number are highly enriched for NF-kB pathway regulators. Subsignatures of the NF-kB targets predict transformation in FL.

Description: Background In patients with follicular lymphoma (FL) the clinical international prognostic index FLIPI is well established, but recently the impact of a number of gene mutations was evaluated. The m7-FLIPI prognostic model, derived from the FLIPI score combined with ECOG and the mutational status of seven specific genes, was shown to stratify FL patients into "low-risk" and "high-risk" with respect to 5-year failure-free survival after first-line immunochemotherapy (R-CHOP or R-CVP) (Pastore, Lancet Oncol 2015). The Nordic Lymphoma Group (NLG) performed two trials (accrual 1998-1999 and 2002-2008, respectively) including a total of 439 patients with symptomatic/progressing indolent B-cell lymphoma (79% FL) randomized 1:1 to treatment with single rituximab or rituximab plus interferon-α2a (Kimby, Leuk Lymphoma 2008, 2015). A follow-up study after 10 years found 36% still chemotherapy-free (Lockmer, JCO 2018). Aim: As part of a long-term follow-up of we aimed to investigate whether the m7-FLIPI prognostic model could be validated in a FL patient cohort treated with first-line-immunotherapy only. Methods Fresh-frozen diagnostic lymph node biopsies with sufficient material for gene sequencing were available from 88 patients with verified FL grades 1-3a and 7 patients with indolent B-cell lymphoma not otherwise specified. A targeted Lymphochip panel including the seven genes of the m7-FLIPI, EZH2, ARID1A, MEF2B, EP300, FOXO1, CREBBP and CARD11, was used. Clinical data were retrieved from the databases of the two trials and from follow-up medical records. Variables assessed included all post-trial therapies, the last dates of follow-up and causes of death. The m7-FLIPI score was calculated according to the published sum of predictor algorithm with respect to high-risk FLIPI, ECOG 〉1 and occurrence of non-silent mutations. Time to treatment failure (TTF), the primary endpoint, was defined as the interval between start of trial therapy and either initiation of new lymphoma therapy due to relapse or intolerance, or death from any cause. TTF was chosen rather than the date of stable or progressive disease described in the Pastore report, as the date of new therapy is less affected by assessment frequency and quality. The Pastore cut-off score of ≥ 0.84 for discrimination between the m7-FLIPI high- vs low-risk group was applied. TTF and overall survival (OS) were estimated with the Kaplan-Meier method and the log-rank test applied for comparison between risk groups. Results Mutational status was obtained from 95 patients, four of them with two biopsies each. In two of the doublets mutations differed between samples in which case the earliest dated samples was chosen for risk analyses. Three patients (two FL) could not be risk-stratified according to the m7-FLIPI score due to missing clinical information. Patient characteristics and gene mutations are shown in Table 1. The mutation frequency distribution was broadly similar to the Pastore report cohorts, mutations in the epigenetic modifier CREBBP by far the most common. With a median follow-up after randomization of 10.6 (range 0.6-18.3) years, 72 (76%) patients were alive. Of all, 28% had never required any new therapy, and 39% had not required any chemotherapy. For 13 out of 23 patients the cause of death was lymphoma-related i.e. due to progression of disease or complications of therapy. No difference in TTF nor OS was found between the m7-FLIPI high- vs low-risk group (fig 1a-b), but in the high-risk FLIPI group OS was shorter (fig 1d). Mutation in the EZH2 gene, coding for the catalytic subunit of a histone methyltransferase, was associated with longer TTF (p=0.04) also within FLIPI risk groups but not with OS (p=0.63). Conclusion The m7-FLIPI prognostic clinicogenetic model was not valid in our cohort with FL patients treated with only immunotherapy. Mutation in the gene EZH2 was associated with longer TTF and high-risk FLIPI with shorter OS. Disclosures Wahlin: Gilead: Consultancy, Honoraria, Research Funding; Roche: Research Funding. Kimby:AbbVie: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria; Roche: Honoraria.

Description: Introduction: Follicular lymphoma (FL) is an indolent malignancy, characterized by multiple relapses during the disease course. Annually around 2-3% of patients experience transformation to aggressive disease (tFL), most commonly to diffuse large B-cell lymphoma (DLBCL). Both transformation and progression of disease within 2 years (POD24) are associated with poor prognosis, yet the molecular events underlying these processes are not well understood. The existence of common progenitor cells (CPCs) has been inferred from genetic analyses of longitudinal biopsies. Improved characterization of genetic alterations associated with CPCs in cases with transformation and POD24 may improve our understanding of disease progression, and reveal molecular markers for high-risk disease. Methods: We performed whole-exome sequencing of 97 serial tumor biopsies and matched normal samples purified from peripheral blood from 44 FL patients. An average sequencing coverage of 700X was achieved for both tumor and normal samples, which ensured high data quality and ability to detect SNVs and InDels using our bench-marked bioinformatics pipeline. SNP6.0 data was available for 93 sequenced tumors and used to infer allele-specific copy number alterations. Several computational tools were applied to identify potential cancer driver genes (MutSig2CV, 2020plus), mutational signatures (MutationalPatterns), and to study clonal evolution (PyClone, ClonEvol). Results: Twenty-two of the 44 FL patients experienced relapses without transformation (referred to as the nFL group), and 22 patients experienced transformation (the tFL group). Nineteen patients (including both groups) experienced POD24. Both transformation and POD24 were associated with inferior overall survival. The median non-synonymous mutational burden was 96 per biopsy (range 10-326). Pre-treatment FL biopsies from the tFL group had significantly higher mutational burden compared to the nFL group (p

Description: Background: High-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) occurs in approximately 8% of de novo diffuse large B-cell lymphoma (DLBCL) cases, with BCL2 translocations (HGBL-DH/TH-BCL2) being a germinal center B-cell like (GCB) phenomenon (Scott et al. Blood 2018). HGBL-DH/TH is associated with poor outcome when treated with standard R-CHOP-therapy, but retrospective studies suggest that more aggressive treatment may improve outcome (Petrich et al. Blood 2014; Oki et al. BJH 2014). To overcome limitations with identification of HGBL-DH/TH by fluorescence in situ hybridization (FISH), Ennishi et al. developed a gene expression signature (DHITsig) that identified tumors with a HGBL-DH/TH-BCL2 gene expression phenotype. This DHITsig was translated into a new assay (DLBCL90) for application on formalin-fixed paraffin-embedded (FFPE) biopsies (Ennishi et al. J Clin Oncol 2019), and included identification of cell-of-origin (COO) and primary mediastinal B-cell lymphoma (Scott et al. Blood 2014; Mottok et al. Blood 2018). The DHITsig roughly doubled the number of cases in the HGBL-DH/TH-group compared with FISH, and was associated with a poor prognosis in patients treated with R-CHOP. In our study we aimed to validate the DLBCL90 assay in an independent cohort of young high-risk patients treated with dose-intensive immunochemotherapy within two Nordic trials. Patients and methods: RNA was extracted from pretreatment FFPE biopsies from 88 high-risk de novo DLBCL patients treated with dose-dense immunochemotherapy with systemic CNS prophylaxis in two Nordic trials (Holte et al. Ann. Oncol. 2013; Leppä et al. 15-ICML 2019). In the first trial patients received 6 courses of R-CHOEP-14 followed by 1 course of HD-Mtx and HD-Ara-C. In the second trial 2 courses of HD-Mtx were given in combination with R-CHOP-14 at the start of treatment, followed by 4 courses of R-CHOEP-14 and 1 course of R-HD-Ara-C at the end. Liposomal Ara-C was also administered intrathecally at courses 1, 3 and 5. Digital gene expression was performed, applying the DLBCL90 assay on the NanoString platform (NanoString Technologies, Seattle, WA) and FISH break-apart probes for MYC, BCL2 and BCL6 were used for identification of HGBL-DH/TH. Results: The COO assay assigned 54%, 31% and 15% of the tumors to the GCB, activated B-cell like (ABC) and unclassified group, respectively. The ABC- and unclassified DLBCLs showed a trend towards inferior outcome when compared to the GCB-group (OS: 59%, 66%, 91%, p=0.076, PFS: 74%, 59%, 85%, p=0.122, FFS: 63%, 51%, 83%, p=0.052, respectively). The DHITsig was only seen in the GCB subtype. Of the patients with the GCB subtype, 5 (10%) and 11 patients (23%) were assigned to the DHITsig-positive and DHITsig-indeterminate group, respectively. FISH results were available for 71 samples, and 6 were identified as HGBL-DH/TH-BCL2. Four of them were assigned to the DHITsig-positive group, while 1 case was assigned to the DHITsig-indeterminate group with a 79% probability of belonging to the DHITsig-positive group (cut-off 80%). The last case was a triple-hit tumor that was assigned to the DHITsig-negative group. This patient had a favorable outcome with a complete remission after first line therapy, and was still in remission at the last follow-up 45 months after diagnosis. Overall, after a median follow-up of 64 months, 17 patients (19%) experienced relapse and 13 patients (15%) had died. Within the GCB subgroup, there were no significant differences in clinical outcome (OS, PFS, FFS) between the DHITsig positive, negative and indeterminate groups when analyzed as separate groups, or when the positive/indeterminate groups were merged (figure). Conclusion: We confirm that the DLBCL90 gene expression assay identifies HGBL-DH/TH-BCL2 in FFPE biopsies. This, together with a rapid turnaround time, makes it an attractive alternative to FISH for double-hit assignment in the clinical setting. In our cohort of young-high risk patients treated with dose-dense immunochemotherapy, the DHITsig was not associated with inferior survival. Of note, neither was the HGBL-DH/TH defined by FISH. This may be due to the intensified treatment, overcoming the poor prognostic impact of the double-hit biology. Disclosures Jørgensen: Gilead: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees. Jerkeman:Roche: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Acerta: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Scott:Roche/Genentech: Research Funding; Celgene: Consultancy; Janssen: Consultancy, Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding. Holte:Novartis: Honoraria, Other: Advisory board.