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Genomic studies have recently identified RPS15 as a new driver gene in aggressive and chemorefractory cases of chronic lymphocytic leukemia (CLL). RPS15 encodes a ribosomal protein whose conserved C-terminal domain extends into the decoding center of the ribosome. We demonstrate that mutations in highly conserved residues of this domain affect protein stability, by increasing its ubiquitin-mediated degradation, and cell-proliferation rates. On the other hand, we show that mutated RPS15 can be loaded into the ribosomes, directly impacting on global protein synthesis and/or translational fidelity in a mutation-specific manner. Quantitative mass spectrometry analyses suggest that RPS15 variants may induce additional alterations in the translational machinery, as well as a metabolic shift at the proteome level in HEK293T and MEC-1 cells. These results indicate that CLL-related RPS15 mutations might act following patterns known for other ribosomal diseases, likely switching from a hypo- to a hyperproliferative phenotype driven by mutated ribosomes. In this scenario, loss of translational fidelity causing altered cell proteostasis can be proposed as a new molecular mechanism involved in CLL pathobiology.

Purpose: Germ-line mutations in the exonuclease domains of POLE and POLD1 have been recently associated with polyposis and colorectal cancer (CRC) predisposition. Here, we aimed to gain a better understanding of the phenotypic characteristics of this syndrome to establish specific criteria for POLE and POLD1 mutation screening and to help define the clinical management of mutation carriers. Genet Med 18 4, 325–332. Methods: The exonuclease domains of POLE and POLD1 were studied in 529 kindred, 441 with familial nonpolyposis CRC and 88 with polyposis, by using pooled DNA amplification and massively parallel sequencing. Genet Med 18 4, 325–332. Results: Seven novel or rare genetic variants were identified. In addition to the POLE p.L424V recurrent mutation in a patient with polyposis, CRC and oligodendroglioma, six novel or rare POLD1 variants (four of them, p.D316H, p.D316G, p.R409W, and p.L474P, with strong evidence for pathogenicity) were identified in nonpolyposis CRC families. Phenotypic data from these and previously reported POLE/POLD1 carriers point to an associated phenotype characterized by attenuated or oligo-adenomatous colorectal polyposis, CRC, and probably brain tumors. In addition, POLD1 mutations predispose to endometrial and breast tumors. Genet Med 18 4, 325–332. Conclusion: Our results widen the phenotypic spectrum of the POLE/POLD1-associated syndrome and identify novel pathogenic variants. We propose guidelines for genetic testing and surveillance recommendations. Genet Med 18 4, 325–332.

Background: Genetic characterization is becoming relevant to predict risk of progression in smoldering MM and is fundamental to estimate survival in active MM. Thus, patients undergo multiple bone marrow (BM) aspirates for genetic screening that beyond painful, may not be fully representative due to patchy BM involvement, spatial genomic heterogeneity, or extramedullary disease. Accordingly, cell-free DNA has been investigated and showed high concordance with BM aspirates, but information is typically restricted to a few recurrent mutations since comprehensive genetic characterization (eg. whole-exome sequencing, WES) is applicable to Aim: To compare the genetic landscape of CTCs vs matched BM clonal plasma cells (PCs) and extramedullary (EM) plasmacytomas, and validate standardized assays for CTCs' detection, isolation and genetic characterization. Methods: We used EuroFlow next-generation flow (NGF) cytometry to detect and isolate peripheral blood (PB) CTCs and matched BM clonal PCs from 38 MM patients (25 at diagnosis and 13 at relapse). In 8 cases, clonal PCs from EM plasmacytomas were also FACSorted. PB T cells were always used as matched germline control. In the training set, we performed custom WES (preceded by triplicates of whole-genome amplification) in matched CTCs, BM and EM clonal PCs from the 8 patients with all three spatially distributed clones. Only those mutations present in 2/3 libraries analyzed per sample were considered positive. In the validation set, we compared mutations, copy number alterations (CNA) and translocations present in CTCs and BM clonal PCs using the Chromium Exome Solution for low DNA-input (n=8), and solely CNA using the Affymetrix CytoScan HD platform (n=22). Read mapping, variant and structural calling were performed with the Multisample Exome (Dreamgenics) and LongRanger (10XGenomics) pipelines. The Chromosome Analysis Suite software (Affymetrix) was used to analyze CNA. Only those mutations with ≥10% variant allele frequency (VAF) and CNA larger than 1Mb were considered. Results: In the training set, 193/226 (85%) and 231/269 (86%) of total mutations present in BM and EM clonal PCs, respectively, were detectable on CTCs. All MM recurrent mutations (eg. BRAF) found in BM or EM clonal PCs were present in CTCs. Of note, there were 39 mutations in EM plasmacytomas that were detectable in CTCs but absent in BM clonal PCs. Furthermore, up to 50 mutations were present in CTCs while undetectable in BM clonal PCs (n=44) or EM plasmacytomas (n=6). After showing that CTCs harbor most mutations present in both medullary and extramedullary disease and even unveil mutations undetectable in single BM aspirates or individual EM plasmacytomas, we sought to evaluate the performance of standardized assays suitable to screen mutations and/or CNA from low cell numbers (ie. CTCs). Using 10XGenomics, 250/266 (94%) of total mutations and 17/17 (100%) of MM recurrent mutations present in BM clonal PCs were detectable on CTCs (eg. KRAS, BRAF, TP53 or FAM46C). The VAF of private mutations ranged between 0.1 and 0.3, suggesting these were subclonal in their respective spatial regions. Using 10XGenomics, 101/119 (85%) CNA and 2/2 (100%) IgH Tx present in BM clonal PCs were detectable in CTCs. Using the Cytoscan HD, there was 100% concordance between CNA in CTCs and BM clonal PCs, both at the chromosomal arm and interstitial levels. All mutations in TP53 were detectable in CTCs. Furthermore, +1q, del(1p), del(17p) or t(4;14) were always detected in CTCs whenever present in BM clonal PCs, and confirmed by FISH. Conversely, such comprehensive genetic characterization unveiled innumerous CNA and translocations not tested by routine FISH panels [eg. MYC amplification or t(6;14)]. Conclusions: Using two different standardized methods, we showed in the largest series in which CTCs were genetically characterized, that these are a reliable surrogate of MM patients' genetic landscape inside and outside the BM. Because NGF is broadly used, quantification, isolation and genetic characterization of CTCs may emerge as an optimal and standardized approach for non-invasive risk-stratification of MM patients. Disclosures Rios: Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. San-Miguel:Amgen, Bristol-Myers Squibb, Celgene, Janssen, MSD, Novartis, Roche, Sanofi, and Takeda: Consultancy, Honoraria. Paiva:Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche and Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene, Janssen, Sanofi and Takeda: Consultancy.

Mantle cell lymphoma (MCL) is an aggressive tumor, but a subset of patients may follow an indolent clinical course. To understand the mechanisms underlying this biological heterogeneity, we performed whole-genome and/or whole-exome sequencing on 29 MCL cases and their respective matched normal DNA, as well as 6 MCL cell lines. Recurrently mutated genes were investigated by targeted sequencing in an independent cohort of 172 MCL patients. We identified 25 significantly mutated genes, including known drivers such as ataxia-telangectasia mutated (ATM), cyclin D1 (CCND1), and the tumor suppressor TP53; mutated genes encoding the anti-apoptotic protein BIRC3 and Toll-like receptor 2 (TLR2); and the chromatin modifiers WHSC1, MLL2, and MEF2B. We also found NOTCH2 mutations as an alternative phenomenon to NOTCH1 mutations in aggressive tumors with a dismal prognosis. Analysis of two simultaneous or subsequent MCL samples by whole-genome/whole-exome (n = 8) or targeted (n = 19) sequencing revealed subclonal heterogeneity at diagnosis in samples from different topographic sites and modulation of the initial mutational profile at the progression of the disease. Some mutations were predominantly clonal or subclonal, indicating an early or late event in tumor evolution, respectively. Our study identifies molecular mechanisms contributing to MCL pathogenesis and offers potential targets for therapeutic intervention.

EM disease in MM increases from newly-diagnosed into relapsed patients, and typically predicts for inferior survival. In fact, no treatment seems to be effective in cases with plasma cell (PC) leukemia, which represents the most aggressive form of EM MM. Unfortunately, the mechanisms of extramedullary spread in MM are not well understood, and there is almost no data about the genetic landscape of both forms of EMD (plasmacytomas and peripheral blood CTCs). Here, we performed whole exome sequencing (WES) to analyze the genomic profiles of highly purified FACS sorted BM and EM clonal PCs from 6 patients with relapsed MM. In 5/6 cases we had all three tissue specific clones, whereas in the remaining case no CTCs were detectable. Depending on the amount of genomic DNA from each clone, whole-genome amplification was performed and in such cases, triplicates of 10 ng of DNA were amplified up to 10 µg using the Illustra GenomiPhi HY DNA amplification kit (GE healthcare). Enrichment of exonic sequences was performed for each library using the SureSelectXT Human All Exon V5+UTRs capture kit (Agilent). To identify somatic mutations we used the mutation caller named Varscan. Variants potentially affecting protein function, including non-synonymous variants, frameshifts in the coding sequence, stop codon-introducing (nonsense) or variants potentially affecting splicing, were analyzed. Only those mutations present in 2/3 libraries analyzed per sample were considered positive. Overall, a median of 89 (67 - 474) somatic mutations were detected. EM plasmacytomas showed the highest mutation load, followed by CTCs and BM clonal PCs (85 vs 77 vs 75, respectively; P=.07), supporting a higher genomic instability/evolution of EM clones. Strikingly, all 6 cases showed lack of concordance in the mutation profiles of the three tissue related clones; even 2x2 comparisons between BM clonal PCs vs CTCs or plasmacytomas, or between the two forms of EM MM (ie. plasmacytomas vs CTCs) showed lack of 100% concordance in every single patient. Despite high inter-tissue heterogeneity, it should be noted that whenever present, recurrent and potentially actionable mutations in genes such as KRAS (n=3), KDM4A (n=2), KMT2A (n=2), ARID5B (n=2), TRIO (n=2), BRAF (n=1) or CCND1 (n=1) were detected in all three clones, with the exception of one patient in which CTCs lacked a KRAS mutation. Only one and less frequent actionable mutation in the EPHB2 gene (eg. Herceptin) was exclusively noted in the EM plasmacytoma from one patient. In order to understand the cellular origin of the three tissue related subclones, we then investigated the degree of similarity between each pair of clones at the individual patient level. The pair of clones showing the highest similarity in their mutation profiles were EM plasmacytomas with CTCs (60%, 30% - 94%), followed by BM clonal PCs with CTCs (58%, 35% - 92%), and by BM clonal PCs with EM plasmacytomas (57%, 37% - 91%). Thus, these data suggests that while CTC clones may represent a cellular bridge in between BM and EM plasmacytomas, there is continuous genomic evolution once the different clones have seeded in their respective tissue niches. Accordingly, EM plasmacytomas showed the highest number of specific mutations, followed by BM clonal PCs and CTCs (medians of 9, 4 and 3, respective). We then looked for recurrent mutations specifically present in EM clones, and found that CTCs showed exclusive and recurrent mutations in the CEP152, FSIP2, SYNE1 and TENM1 and ZNF585A genes, whereas EM plasmacytomas showed exclusive and recurrent mutations in the PITRM1 (Metalloprotease 1) gene. Furthermore, ATP7B, MTOR, TBC1D21 and ZNF717 mutations were present in both CTCs and plasmacytomas. In summary, we compared for the first time the genomic profiles of BM vs EM CTCs and plasmacytoma clones, and showed at the individual patient level, the existence of high mutation heterogeneity in between all three tissue related clones. However, actionable mutations in genes such as KRAS or BRAF were typically shared by BM and EM clones. According to the level of similarity between each clone, CTCs would be the most plausible precursor of EM plasmacytomas. Nevertheless, based on the number of specific mutations present in EM clones, it is likely that EM spreading followed by continuous clonal evolution starts much earlier prior to its clinical detection, which emphasizes the need for sensitive techniques to capture the early stages of EM spreading. Disclosures Paiva: Celgene: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria, Research Funding; Sanofi: Consultancy, Research Funding; EngMab: Research Funding; Amgen: Honoraria; Binding Site: Research Funding.

Here we perform whole-exome sequencing of samples from 105 individuals with chronic lymphocytic leukemia (CLL), the most frequent leukemia in adults in Western countries. We found 1,246 somatic mutations potentially affecting gene function and identified 78 genes with predicted functional alterations in more than one tumor sample. Among these genes, SF3B1, encoding a subunit of the spliceosomal U2 small nuclear ribonucleoprotein (snRNP), is somatically mutated in 9.7% of affected individuals. Further analysis in 279 individuals with CLL showed that SF3B1 mutations were associated with faster disease progression and poor overall survival. This work provides the first comprehensive catalog of somatic mutations in CLL with relevant clinical correlates and defines a large set of new genes that may drive the development of this common form of leukemia. The results reinforce the idea that targeting several well-known genetic pathways, including mRNA splicing, could be useful in the treatment of CLL and other malignancies.

This paper is distributed under the terms of the Creative Commons Attribution-Non-Commercial-Share Alike licence.-- et al., Chronic lymphocytic leukaemia (CLL), the most frequent leukaemia in adults in Western countries, is a heterogeneous disease with variable clinical presentation and evolution(1,2). Two major molecular subtypes can be distinguished, characterized respectively by a high or low number of somatic hypermutations in the variable region of immunoglobulin genes(3,4). The molecular changes leading to the pathogenesis of the disease are still poorly understood. Here we performed whole-genome sequencing of four cases of CLL and identified 46 somatic mutations that potentially affect gene function. Further analysis of these mutations in 363 patients with CLL identified four genes that are recurrently mutated: notch 1 (NOTCH1), exportin 1 (XPO1), myeloid differentiation primary response gene 88 (MYD88) and kelch-like 6 (KLHL6). Mutations in MYD88 and KLHL6 are predominant in cases of CLL with mutated immunoglobulin genes, whereas NOTCH1 and XPO1 mutations are mainly detected in patients with unmutated immunoglobulins. The patterns of somatic mutation, supported by functional and clinical analyses, strongly indicate that the recurrent NOTCH1, MYD88 and XPO1 mutations are oncogenic changes that contribute to the clinical evolution of the disease. To our knowledge, this is the first comprehensive analysis of CLL combining whole-genome sequencing with clinical characteristics and clinical outcomes. It highlights the usefulness of this approach for the identification of clinically relevant mutations in cancer., This work was funded by the Spanish Ministry of Science and Innovation (MICINN) through the Instituto de Salud Carlos III (ISCIII) and Red Temática de Investigación del Cáncer (RTICC) del ISCIII. C.L.-O. is an Investigator of the Botin Foundation and D.T., of the ICREA program. We thank E. Santos for his support of this project, A. Carracedo and J. Benítez for genotyping studies, C. Fortuny for the supply of samples and N. Villahoz and M. C. Muro for their work in the coordination of the CLL-ICGC Consortium.