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Letter to Blood.-- Curiel-Olmo, Soraya et al., Splenic diffuse red pulp lymphoma (SDRPL) is a rare small B-cell neoplasm provisionally included in the category of unclassifiable splenic B-cell lymphomas/leukemias in the 2008 World Health Organization classification. SDRPL is characterized by a diffuse pattern of involvement of the splenic red pulp by small monomorphous B lymphocytes., This work was supported by grants from the Ministerio de Economía, Industria y Competitividad (RTICC RD06/0020/0107, RD12/0036/0060, PI 12/1682, PT13/ 0010/0007, PI16/01294, SAF2013-47416-R, CIBERONC-ISCIII, PIE15/0081) and the Asociación Española Contra el Cáncer, Spain. S.D. was supported by the Torres Quevedo subprogramme (Ministerio de Economía y Competitividad [MICINN]) under grant agreement PTQ-12-05391.

INTRODUCTION Cell free tumor DNA (cftDNA) based targeted next generation sequencing (NGS) is a novel tool that enables accurate and minimally invasive tumor genotyping. MATERIAL AND METHODS Here we have performed cftDNA targeted NGS for the molecular diagnosis of a series of 92 peripheral blood samples obtained at diagnosis in patients included in a phase II randomized clinical trial comparing standard RCHOP versus BRCAP as first line treatment in patients with poor IPI (aaIPI2-3 or aaIPI1 with high b2m, age 18-71, NCT01848132). Centralized histopathological review of diagnostic tissue samples confirmed the diagnosis: 83 DLBCL (68%, 55/81 GCB, 32%, 26/81 non-GCB, according to Hans immunohistochemical algorithm), 4 TCHRBCL, 3 PMBCL, 1 concurrent DLBCL and FL3A, 1 FL 3B. 7 out of 83 cases had DLBCL morphology and DH by FISH (6 MYC/BCL2 and 1 MYC/BCL6, all GCB-type by IHC). A targeted NGS approach using amplicon-based chemistry (Truseq, Illumina) that interrogates exonic regions of 35 relevant genes in DLBCL (1458 amplicons analyzed) was used for library generation from plasma derived DNA. Germline DNA from peripheral blood granulocytes from 89 out of 92 patients was sequenced in parallel and used as control DNA for variant calling. Only somatic variants with a read depth above 100 and Variant Allele Frequency > 5% were considered. RESULTS cftDNA concentration (hGE/mL) was associated with LDH levels (U/mL) at diagnosis. Mean cftDNA concentration was lower in patients with low LDH (U Mann whitney p= 0.005). Somatic mutations were identified in 74% (68 out of 92) patient plasma samples. 242 somatic mutations were considered after filtering. Mean of 3.6 mutations per patient. 10 genes were mutated in >10% of the patients, including: FAT2 (19.6%), ARID1A (18%), NOTCH1 (17.4%), NOTCH2 (15.2%), KMT2D (14.13%), SMARCA4 (14%), ATM (13%), EP300 (13%), EZH2 (13%), PLCG2 (13%). Recurrent mutations were found in EZH2 (2 patients). Mutations in BCR/TLR pathway genes were found in less than 10% of the cases (CARD11, 7.6%; MYD88, 4.4%; CD79A, 5.4%; CD79B, 1%; BTK, 4.4%). NOTCH1 mutations were found preferentially in GCB type DLBCL (12/55 GCB vs 1/26 nonGCB, Chi square p value DISCUSSION cftDNA targeted NGS captures the mutational profile in a significant fraction of DLBCL patients and enables tumor genotyping in the clinical trial setting. In contrast with previously published data mutations in NOTCH pathway, including NOTCH1, NOTCH2 and SGK1 were relatively frequent in our series of poor IPI patients, outnumbering the frequency of mutations in BCR/TLR pathway. NOTCH pathway mutations were also preferentially found in GCB-type DLBCL-NOS, PMBCL and 1 case of TCHRBCL. In summary cftDNA genotyping based on targeted NGS can provide potentially useful information for therapy selection in DLBCL patients. Disclosures Martín: Roche: Consultancy, Honoraria, Other: Travel expenses; Celgene: Consultancy, Honoraria, Other: Travel expenses; Servier: Honoraria, Other: Travel expenses; Janssen: Honoraria, Other: Travel expenses. Gonzalez-Barca:Roche: Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Gilead: Consultancy; Celtrion: Consultancy. Montes-Moreno:TAKEDA: Consultancy; Roche: Consultancy.

Introduction SOX11 is a transcription factor (TF) aberrantly expressed in the majority of mantle cell lymphomas (MCLs), which is generally associated with aggressive clinical behaviour. No mutations, genetic aberrations or direct correlations with differential DNA methylation at the promoter related to its expression have been found in MCL. Deeper insights into its regulation can be found by considering the three-dimensional (3D) chromatin structure. It is becoming clear that the genome can be partitioned into 3D building blocks, topologically associated domains (TADs) and that enhancer regions likely regulate genes within their TADs by 3D contacts, but do not affect genes outside their own TADs. By mapping the 3D chromatin structure, we previously identified a distant putative SOX11 enhancer showing enhancer activity and 3D contacts with the SOX11 gene in the SOX11-positive MCL cell line Z-138, but not in the SOX11-negative MCL cell line JVM-2. Aims We aimed to deepen our understanding of the differential 3D contacts and enhancer activity previously observed at the putative SOX11 enhancer in SOX11-positive versus SOX11-negative MCL cell lines by addressing the following questions: (i) Do TAD boundaries around the SOX11 locus change between SOX11-positive and -negative MCLs? (ii) How do the 3D contacts and chromatin states at this region behave in primary MCL cases and normal B cells? (iii) Is the putative SOX11 enhancer involved in SOX11 expression in other tissues? Methods We have extended our experimental analyses of the putative SOX11 enhancer by performing (i) HiC-sequencing and 3D fluorescence in situ hybridization (3D FISH) in MCL cell lines Z-138 and JVM-2, (ii) 4C-sequencing, chromatin inmmunoprecipiation followed by deep sequencing (ChIP-seq) of 6 histone marks, an Assay for Transposase-Accessible Chromatin with deep sequencing (ATAC-seq) and chromatin state modeling by chromHMM (using the 6 histone marks) in primary MCL cases and normal naive and memory B-cells. Furthermore, we have explored the activity of this region in other SOX11 expressing cell lines studied within the ENCODE Consortium. Results HiC-sequencing in the cell lines Z-138 (SOX11-positive) and JVM-2 (SOX11-negative) showed that the SOX11 locus and its putative enhancer are located within the same TAD in both samples. Hence, shifts in TAD boundaries do not seem to underlie the differential 3D chromatin interactions between the SOX11 locus and its putative enhancer in these two cell lines. By ChIP-seq and chromatin state modeling we observed that the promoter of SOX11 is poised, i.e., carrying histone marks H3K4me3 and H3K27me3, in normal naive and memory B-cells and the SOX11-negative MCL primary case. Furthermore, we observed weak enhancer activity at the putative SOX11 enhancer in normal naive and memory B-cells and the SOX11-negative MCL primary case, but strong enhancer activity, marked by the presence of H3K27ac, only in SOX11-positive samples. In addition, by ATAC-seq we identified two specific chromatin accessible regions that potentially represent the transcription factor binding sites responsible for activation of this enhancer region in SOX11-positive MCLs. By 4C-sequencing we observed that the SOX11 locus and its putative enhancer show high 3D contacts in two other SOX11-positive MCL cell lines (GRANTA-519 and JEKO-1) and in a SOX11-positive primary MCL case, but not in a SOX11-negative primary MCL case. Furthermore, the differential 3D contacts at these regions in Z-138 and JVM-2 were confirmed by 3D FISH, which is currently being performed in primary MCL cases. Interestingly, no 3D contacts were observed in normal naive and memory B cells, indicating that although the SOX11 promoter is poised within these normal B-cell subpopulations, primed looping at these regions does not exist and seems not to explain the 3D contacts we observed in SOX11-positive MCL cell lines and primary cases. When investigating chromatin states in cell lines studied by ENCODE with an active SOX11 promoter (H1-hESC, HSMM and NHLF) none of them show activity in the identified region, suggesting that the putative SOX11 enhancer is de novo activated only in the context of MCL lymphomagenesis. Conclusions We provide new evidence that the activation of a distant SOX11 putative enhancer and its 3D contacts to the SOX11 gene, is a de novo event in SOX11-positive MCL cell lines and primary cases that is likely specific for this malignancy. Disclosures No relevant conflicts of interest to declare.