Roche, SIRIC Lyric, LYSARC, National Institutes of Health, the Henry J Predolin Foundation, and the Spanish Plan Nacional de Investigacion.

Follicular lymphoma (FL) is the most frequent indolent B cell lymphoma and is still considered to be incurable. In recent years, whole-exome sequencing studies of large cohorts of patients have greatly improved our knowledge of the FL mutational landscape. Moreover, the prolonged evolution of this disease has enabled some insights regarding the early pre-lymphoma lesions as well as the clonal evolution after treatment, allowing an evolutionary perspective on lymphomagenesis. Deciphering the earliest initiating lesions and identifying the molecular alterations leading to disease progression currently represent important goals; accomplishing these could help identify the most relevant targets for precision therapy.

Splenic diffuse red pulp lymphoma is an indolent small B-cell lymphoma recognized as a provisional entity in the World Health Organization 2008 classification. Its precise relationship to other related splenic B-cell lymphomas with frequent leukemic involvement or other lymphoproliferative disorders remains undetermined. We performed whole-exome sequencing to explore the genetic landscape of ten cases of splenic diffuse red pulp lymphoma using paired tumor and normal samples. A selection of 109 somatic mutations was then evaluated in a cohort including 42 samples of splenic diffuse red pulp lymphoma and compared to those identified in 46 samples of splenic marginal zone lymphoma and eight samples of hairy-cell leukemia. Recurrent mutations or losses in BCOR (the gene encoding the BCL6 corepressor) – frameshift (n=3), nonsense (n=2), splicing site (n=1), and copy number loss (n=4) – were identified in 10/42 samples of splenic diffuse red pulp lymphoma (24%), whereas only one frameshift mutation was identified in 46 cases of splenic marginal zone lymphoma (2%). Inversely, KLF2, TNFAIP3 and MYD88, common mutations in splenic marginal zone lymphoma, were rare (one KLF2 mutant in 42 samples; 2%) or absent (TNFAIP3 and MYD88) in splenic diffuse red pulp lymphoma. These findings define an original genetic profile of splenic diffuse red pulp lymphoma and suggest that the mechanisms of pathogenesis of this lymphoma are distinct from those of splenic marginal zone lymphoma and hairy-cell leukemia.

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The histone methyltransferase EZH2 has an essential role in the development of follicular lymphoma (FL). Recurrent gain-of-function mutations in EZH2 have been described in 25% of FL patients and induce aberrant methylation of histone H3 lysine 27 (H3K27). We evaluated the role of EZH2 genomic gains in FL biology. Using RNA sequencing, Sanger sequencing and SNP-arrays, the mutation status, copy-number and gene-expression profiles of EZH2 were assessed in a cohort of 159 FL patients from the PRIMA trial. Immunohistochemical (IHC) EZH2 expression (n=55) and H3K27 methylation (n=63) profiles were also evaluated. In total, 37% of patients (59/159) harbored an alteration in the EZH2 gene (mutation n=46, gain n=23). Both types of alterations were associated with highly similar transcriptional changes, with increased proliferation programs. An H3K27me3/me2 IHC score fully distinguished mutated from wild-type samples, showing its applicability as surrogate for EZH2 mutation analysis. However, this score did not predict the presence of gains at the EZH2 locus. The presence of an EZH2 genetic alteration was an independent factor associated with a longer progression-free survival (hazard ratio 0.58, 95% confidence interval 0.36–0.93, P=0.025). We propose that the copy-number status of EZH2 should also be considered when evaluating patient stratification and selecting patients for EZH2 inhibitor-targeted therapies.

TET2 and DNMT3A mutations are frequently identified in T-cell lymphomas of T follicular helper cell origin (TCL-TFH), clonal hematopoiesis (CH), and myeloid neoplasms (MNs). The relationships among these 3 entities, however, are not well understood. We performed comprehensive genomic studies on paired bone marrow and tissue samples as well as on flow cytometry–sorted bone marrow and peripheral blood subpopulations from a cohort of 22 patients with TCL-TFH to identify shared CH-type mutations in various hematopoietic cell compartments. Identical mutations were detected in the neoplastic T-cell and myeloid compartments of 15 out of 22 patients (68%), including TET2 (14/15) and DNMT3A (10/15). Four patients developed MNs, all of which shared CH-type mutations with their TCL-TFH; additional unique genetic alterations were also detected in each patient’s TCL-TFH and MN. These data demonstrate that CH is prevalent in patients with TCL-TFH and that divergent evolution of a CH clone may give rise to both TCL-TFH and MNs.

Recent advances in next-generation sequencing (NGS) have enabled the quantitation of circulating tumour DNA (ctDNA) encoding the clonal rearranged V(D)J immunoglobulin locus. We aimed to evaluate the clonal heterogeneity of follicular lymphoma (FL) in the tumour and the plasma at diagnosis and to assess the prognostic value of the ctDNA level. Plasma samples at diagnosis were available for 34 patients registered in the PRIMA trial (NCT00140582). One tumour clonotype or more could be detected for 29 (85%) and 25 (74%) patients, respectively, in the tumour or plasma samples. In 18 patients, several subclones were detected in the tumour (2 to 71 subclones/cases) and/or in the plasma (2 to 20 subclones/cases). In more than half of the cases, the distribution of subclones differed between the tumour and plasma samples, reflecting high clonal heterogeneity and diversity in lymphoma subclone dissemination. In multivariate analysis, a high level of ctDNA was the only independent factor associated with patients’ progression-free survival (HR 4, IC 95 (1.1-37), p=.039). In conclusion, an NGS-based immunosequencing method reveals the marked clonal heterogeneity of follicular lymphoma in patients with FL, and quantification of ctDNA at diagnosis represents a potential powerful prognostic biomarker that needs to be investigated in larger cohorts.

It has been recently shown that DNA methyl transferase overexpression is correlated with unfavourable prognosis in human malignancies while methylation deregulation remains a hallmark that defines acute myeloid leukemia (AML). The oncogenic transcription factor EVI1 is involved in methylation deregulation and its overexpression plays a major role for predicting an adverse outcome. Moreover, the identification of DNMT3A mutations in AML patients has recently been described as a poor prognostic indicator. In order to clarify relationship between these key actors in methylation mechanisms and their potential impact on patient outcomes, we analysed 195 de novo AML patients for the expression of DNMT3A, 3B (and its non-catalytic variant 3BNC) and their correlations with the outcome and the expression of other common prognostic genetic biomarkers (EVI1, NPM1, FLT3ITD/TKD and MLL) in adult AML. The overexpression of DNMT3B/3BNC is (i) significantly correlated with a shorter overall survival, and (ii) inversely significantly correlated with event-free survival and DNMT3A expression level. Moreover, multivariate analysis showed that a high expression level of DNMT3B/3BNC is statistically a significant independent poor prognostic indicator. This study represents the first report showing that the overexpression of DNMT3B/3BNC is an independent predictor of poor survival in AML. Its quantification should be implemented to the genetic profile used to stratify patients for therapeutical strategies and should be useful to identify patients who may benefit from therapy based on demethylating agents.