Page 147 - Haematologica - Vol. 105 n. 6 - June 2020

g”). The “MIPI-g” improved the model discrimination ability compared to the MIPI-c alone, defining three risk groups: i) low-risk patients (4-year progression free survival and overall survival of 72.0% and 94.5%); ii) inter- mediate-risk patients (4-year progression free survival and overall survival of 42.2% and 65.8%) and iii) high- risk patients (4-year progression free survival and overall survival of 11.5% and 44.9%). Our results: i) confirm that TP53 disruption identifies a high-risk population characterized by poor sensitivity to conventional or intensified chemotherapy; ii) provide the pivotal evidence that patients harboring KMT2D mutations share the same poor outcome as patients harboring TP53 disruption; and iii) allow to develop a tool for the identi- fication of high-risk MCL patients for whom novel therapeutic strategies need to be investigated. (Trial regis- tered at clinicaltrials.gov identifier: NCT02354313).
KMT2D mutations and TP53 disruptions in MCL
Introduction
The introduction of high dose cytarabine-containing chemoimmunotherapeutic regimens and autologous transplantation (ASCT) have considerably improved the outcome of young fit mantle cell lymphoma (MCL) patients. Nonetheless, approximately 20-25% of MCL patients demonstrate inadequate efficacy of intensified chemoimmunotherapy as they are either primary refrac- tory or relapse within 2 years from ASCT.1-5
Clinical and pathological scores, including the MCL international prognostic index (MIPI),6 the Ki-67 prolifera- tive index,7 and their combination in the MIPI-c score, stratify MCL patients in groups at different risk of relapse.8 However, none of these tools has sufficient positive pre- dictive value to trigger the development of tailored sched- ules specifically designed for high risk patients.9
Several recurrent mutations have been described in MCL, affecting DNA repair genes and cell cycle regulators (TP53, ATM, CCND1), epigenetic regulation genes (KMT2D, WHSC1) and cell-signaling pathways genes (NOTCH1-2, BIRC3, TRAF2).10-12 The proof of principle that MCL genetics can impact on disease outcome stems from studies that have focused on the TP53 tumor sup- pressor gene, including both mutations and 17p dele- tions.13-17
We prospectively assessed the clinical impact of a panel of genomic alterations in a cohort of young MCL patients treated with high dose chemoimmunotherapy and ASCT from the Fondazione Italiana Linfomi (FIL) MCL0208 phase 3 trial.18 The results document that KMT2D muta- tions associate with poor outcome in MCL and, along with TP53 mutations and 17p deletions, might be integrat- ed in a new prognostic score to segregate a subgroup of patients who obtain minimal or no benefit from intensive chemoimmunotherapy. The prognostic score was validat- ed in an independent series of cases.
Methods
Patients series
The FIL-MCL0208 (NCT02354313) is a phase 3, multicenter, open-label, randomized, controlled study, designed to determine the efficacy of lenalidomide as maintenance versus observation in young (18-65 years old), fit, advanced stage (Ann arbor II-IV) MCL patients after first line intensified and high-dose chemo- immunotherapy followed by ASCT. Cases of non-nodal MCL were excluded.19 The clinical trial, as well as the ancillary muta- tional study, were approved by the Ethical Committees of all the enrolling Centers. All patients provided written informed consent for the use of their biological samples for research purposes, in
accordance with Institutional Review Boards requirements and the Helsinki's declaration. Clinical results of the fist interim analy- sis of the trial were already presented.18 Further information are supplied in the Online Supplementary Materials and Methods.
Biological samples
Tumor cells were sorted from the baseline bone marrow (BM) samples by immunomagnetic beads (CD19 MicroBeads,human- Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and stocked as dry pellets.
Tumor DNA was extracted according to DNAzol protocol (Life Technologies). Germline DNA was obtained from peripheral blood (PB) mononuclear cells collected under treatment and proven to be tumor free by minimal residual disease (MRD) analy- sis. Further information are supplied in the Online Supplementary Materials and Methods.
Next generation sequencing (NGS)
A targeted resequencing panel (target region: 37’821 bp) (Online Supplementary Table S1) including the coding exons and splice sites of seven genes (ATM, TP53, CCND1, WHSC1, KMT2D, NOTCH1 exon 34, BIRC3) that are recurrently mutated in ≥5% of MCL tumors was specifically designed.10-12 We also included in the panel TRAF220 and CXCR4.21 NGS libraries preparation was performed using TruSeq Custom Amplicon sequencing assay according to manufacturer’s protocol (Illumina, Inc., San Diego, CA, USA). Multiplexed libraries (n=48 per run) were sequenced using 300-bp paired-end runs on an Illumina MiSeq sequencer, (median depth of coverage 2,356x). A robust and previously validated bioinformatics pipeline was used for variant calling (Online Supplementary Materials and Methods). Copy number variation analysis methods22,23 are sup- plied in the Online Supplementary Materials and Methods.
MRD analysis
For MRD purposes, MCL diagnostic BM and PB samples were investigated for immunoglobulin heavy chain (IGH) gene rearrangements and BCL1/IGH MTC by qualitative PCR.24-26 Both BM and PB samples were analyzed for MRD at specific time points during and after treatment. Further information are sup- plied in the Online Supplementary Materials and Methods.
Statistical analysis
The primary outcome of the clinical study was progression-free survival (PFS) and secondary outcomes included overall survival (OS).27 The adjusted effects of mutations and exposure variables (MIPI-c and blastoid variant) on PFS and OS were estimated by Cox regression. To compare clinical baseline features between patients enrolled in the molecular study and patients not included in the analysis, we used Mann-Whitney test for continuous variables and Pearson’s χ2 test for categorical variables. Statistical analyses were performed using Stata 13.0 and R 3.4.1. Further information are sup- plied in the Online Supplementary Materials and Methods.
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