Sequencing-Based Analysis of Clonal Evolution of 25 Mantle Cell Lymphoma Patients at Diagnosis and after Failure of Standard Immunochemotherapy

Mantle cell lymphoma (MCL) is a rare chronically relapsing subtype of aggressive B-cell non-Hodgkin lymphoma characterized by the canonical chromosomal translocation t (11;14) and other recurrent molecular cytogenetic aberrations. Front-line therapy of MCL is still based on immunochemotherapy. While the mutational landscape of newly diagnosed MCL has been repeatedly published, our knowledge of clonal development and genetic aberrations (i.e., mutations and copy number alterations - CNA) associated with MCL relapse (REL) remain limited.

We implemented whole-exome sequencing (WES) of 25 MCL patients at diagnosis (DG) and first REL after the failure of standard R-CHOP-based immunochemotherapy. GISTIC algorithm was used for the evaluation of significantly changed regions of CNAs (i.e., amplifications and deletions). The detected mutational and CNA changes were filtered out against a pre-assembled gene list encompassing genes frequently and recurrently altered in hematological malignancies. By the design of our study and patient selection, our cohort comprised prognostically adverse patients according to MCL international prognostic index (MIPI), morphology, or proliferation rate by Ki-67.

The most frequently mutated gene at DG was TP53 (48% of patients). Mutations of five other genes, including ATM, KMT2D, CCND1, LRP1B, and SP140 were found in at least four patients (≥ 16%). According to CNA prediction, TP53 deletion was detected in 5 patients (isolated TP53 deletion in 2 patients) further increasing the percentage of patients with TP53 inactivation to 56%. The most significant CNAs at DG were the loss of the 9p21.3 region encompassing the CDKN2A gene (12% of patients) and gains of the 13q31.3 region containing the MIR17HG gene (12% of patients).

A mean count of mutations per patient was significantly higher at REL (34) compared to DG (27). The most frequently mutated gene at REL was LRP1B (3 new patients). Other newly detected variants comprised mutations of KMT2D, HOXD9, CDC27, RYR2, and FLNA genes (each newly mutated in 2 patients). Concerning TP53, all mutations detected at DG were also detected at REL. Even though no new mutations of TP53 were detected at REL, the median variant allele frequency of the pre-existing TP53 mutations increased from 0.35 at DG to 0.76 at REL. Furthermore, six new patients with predicted TP53 loss were found at REL. In total, the percentage of patients with TP53 inactivation increased from 56% at DG to 72% at REL.

The most significant CNA at REL was the deletion of the 9p21.3 region encompassing CDKN2A/2B genes (48% of patients; q value = 9.61e^-18). Other significant CNAs newly detected at REL included deleted 8p21.3, and 6q21 regions with predicted losses of BIN3, EGR3, SORBS3, ARID1B, FOXO3, PRDM1, TAB2, and TNFAIP3 genes (16 - 24 % of patients). The most significant amplified regions at REL comprised 13q31.3,3q26.32, and 12q13.3 regions with predicted gains of MIR17HG (in 24% of patients), BCL6, ETV5, SOX2 (44% of patients), and PIK3CA (48% of patients) genes. With no exception, all regions altered at DG were also affected at REL. Compared to DG, however, the CNAs detected at REL were more frequent and affected larger areas (i.e., they had more significant weight, Figure 1A, and 1B).

In summary, our results demonstrate a drastic clonal evolution in MCL after the failure of standard front-line immunochemotherapy. The WES data suggest that the resistant clones detected at REL were already present at DG. The data also suggest that these clones frequently harbored the inactivation of genes involved in DNA damage pathways (e.g., TP53, CDKN2A/2B). Frequent gains of PIK3CA and MIR17HG, and frequent losses of FOXO3 suggest enhanced dependence on the PI3K/AKT pathway at REL compared to DG. A significantly higher mean count of mutations per patient and significantly more frequent and larger CNAs at REL compared to DG both demonstrate a marked increase in MCL heterogeneity after the failure of frontline immunochemotherapy. The increased lymphoma heterogeneity at REL would further increase the probability of failure of salvage therapy based on chemotherapy, which underlines the necessity of innovative treatments based on targeted drugs and immunotherapy in all relapsed MCL patients.

Trněný:Zentiva: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Gilead Sciencis: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Morphosys: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria.