Accelerated DNA replication fork speed due to loss of R-loops in myelodysplastic syndromes with SF3B1 mutation.

Myelodysplastic syndromes (MDS) with mutated SF3B1 gene present features including a favourable outcome distinct from MDS with mutations in other splicing factor genes SRSF2 or U2AF1. Molecular bases of these divergences are poorly understood. Here we find that SF3B1-mutated MDS show reduced R-loop formation predominating in gene bodies associated with intron retention reduction, not found in U2AF1- or SRSF2-mutated MDS. Compared to erythroblasts from SRSF2- or U2AF1-mutated patients, SF3B1-mutated erythroblasts exhibit augmented DNA synthesis, accelerated replication forks, and single-stranded DNA exposure upon differentiation. Importantly, histone deacetylase inhibition using vorinostat restores R-loop formation, slows down DNA replication forks and improves SF3B1-mutated erythroblast differentiation. In conclusion, loss of R-loops with associated DNA replication stress represents a hallmark of SF3B1-mutated MDS ineffective erythropoiesis, which could be used as a therapeutic target.

Divergent local and systemic antitumor response in primary uveal melanomas.

Uveal melanoma (UM) is the most common cancer of the eye. The loss of chromosome 3 (M3) is associated with a high risk of metastases. M3 tumors are more infiltrated by T-lymphocytes than low-risk disomic-3 (D3) tumors, contrasting with other tumor types in which T cell infiltration correlates with better prognosis. Whether these T cells represent an antitumor response and how these T cells would be primed in the eye are both unknown. Herein, we characterized the T cells infiltrating primary UMs. CD8+ and Treg cells were more abundant in M3 than in D3 tumors. CD39+PD-1+CD8+ T cells were enriched in M3 tumors, suggesting specific responses to tumor antigen (Ag) as confirmed using HLA-A2:Melan-A tetramers. scRNAseq-VDJ analysis of T cells evidenced high numbers of proliferating CD39+PD1+CD8+ clonal expansions, suggesting in situ antitumor Ag responses. TCRseq and tumor-Ag tetramer staining characterized the recirculation pattern of the antitumor responses in M3 and D3 tumors. Thus, tumor-Ag responses occur in localized UMs, raising the question of the priming mechanisms in the absence of known lymphatic drainage.

Familial uveal melanoma and other tumors in 25 families with monoallelic germline MBD4 variants.

BACKGROUND: Monoallelic germline MBD4 pathogenic variants were recently reported to cause a predisposition to uveal melanoma, associated with a specific tumor mutational signature and good response to immunotherapy. Monoallelic tumor pathogenic variants have also been described in brain tumors, breast cancers, and myxofibrosarcomas, whereas biallelic germline MBD4 pathogenic variants have been involved in a recessive hereditary adenomatous polyposis and a specific type of acute myeloid leukemia. METHODS: We analyzed MBD4 for all patients with a diagnosis of uveal melanoma at Institut Curie since July 2021 and in the 3240 consecutive female probands explored at the Institut Curie for suspicion of predisposition to breast cancer between July 2021 and February 2023. RESULTS: We describe 25 families whose probands carry a monoallelic germline pathogenic variant in MBD4. Eighteen of these families presented with uveal melanoma (including a case patient with multiple uveal melanoma), and 7 families presented with breast cancer. Family histories showed the first familial case of uveal melanoma in monoallelic MBD4 pathogenic variant carriers and other various types of cancers in relatives, especially breast, renal, and colorectal tumors. CONCLUSIONS: Monoallelic MBD4 pathogenic variant may explain some cases of familial and multiple uveal melanoma as well as various cancer types, expanding the tumor spectrum of this predisposition. Further genetic testing in relatives combined with molecular tumor analyses will help define the tumor spectrum and estimate each tumor's risk.

SF3B1 hotspot mutations confer sensitivity to PARP inhibition by eliciting a defective replication stress response.

SF3B1 hotspot mutations are associated with a poor prognosis in several tumor types and lead to global disruption of canonical splicing. Through synthetic lethal drug screens, we identify that SF3B1 mutant (SF3B1MUT) cells are selectively sensitive to poly (ADP-ribose) polymerase inhibitors (PARPi), independent of hotspot mutation and tumor site. SF3B1MUT cells display a defective response to PARPi-induced replication stress that occurs via downregulation of the cyclin-dependent kinase 2 interacting protein (CINP), leading to increased replication fork origin firing and loss of phosphorylated CHK1 (pCHK1; S317) induction. This results in subsequent failure to resolve DNA replication intermediates and G2/M cell cycle arrest. These defects are rescued through CINP overexpression, or further targeted by a combination of ataxia-telangiectasia mutated and PARP inhibition. In vivo, PARPi produce profound antitumor effects in multiple SF3B1MUT cancer models and eliminate distant metastases. These data provide the rationale for testing the clinical efficacy of PARPi in a biomarker-driven, homologous recombination proficient, patient population.

Circulating Tumor DNA as a Prognostic Factor in Patients With Resectable Hepatic Metastases of Uveal Melanoma.

OBJECTIVE: We report here the results of a prospective study of circulating tumor DNA (ctDNA) detection in patients undergoing uveal melanoma (UM) liver metastases resection (NCT02849145). BACKGROUND: In UM patients, the liver is the most common and often only site of metastases. Local treatments of liver metastases, such as surgical resection, have a likely benefit in selected patients. METHODS: Upon enrollment, metastatic UM patients eligible for curative liver surgery had plasma samples collected before and after surgery. GNAQ / GNA11 mutations were identified in archived tumor tissue and used to quantify ctDNA by droplet digital polymerase chain reaction which was then associated with the patient's surgical outcomes. RESULTS: Forty-seven patients were included. Liver surgery was associated with a major increase of cell-free circulating DNA levels, with a peak 2 days after surgery (∼20-fold). Among 40 evaluable patients, 14 (35%) had detectable ctDNA before surgery, with a median allelic frequency of 1.1%. These patients experienced statistically shorter relapse-free survival (RFS) versus patients with no detectable ctDNA before surgery (median RFS: 5.5 vs 12.2 months; hazard ratio=2.23, 95% CI: 1.06-4.69, P =0.04), and had a numerically shorter overall survival (OS) (median OS: 27.0 vs 42.3 months). ctDNA positivity at postsurgery time points was also associated with RFS and OS. CONCLUSIONS: This study is the first to report ctDNA detection rate and prognostic impact in UM patients eligible for surgical resection of their liver metastases. If confirmed by further studies in this setting, this noninvasive biomarker could inform treatment decisions in UM patients with liver metastases.

Noncanonical splicing junctions between exons and transposable elements represent a source of immunogenic recurrent neo-antigens in patients with lung cancer.

Although most characterized tumor antigens are encoded by canonical transcripts (such as differentiation or tumor-testis antigens) or mutations (both driver and passenger mutations), recent results have shown that noncanonical transcripts including long noncoding RNAs and transposable elements (TEs) can also encode tumor-specific neo-antigens. Here, we investigate the presentation and immunogenicity of tumor antigens derived from noncanonical mRNA splicing events between coding exons and TEs. Comparing human non-small cell lung cancer (NSCLC) and diverse healthy tissues, we identified a subset of splicing junctions that is both tumor specific and shared across patients. We used HLA-I peptidomics to identify peptides encoded by tumor-specific junctions in primary NSCLC samples and lung tumor cell lines. Recurrent junction-encoded peptides were immunogenic in vitro, and CD8+ T cells specific for junction-encoded epitopes were present in tumors and tumor-draining lymph nodes from patients with NSCLC. We conclude that noncanonical splicing junctions between exons and TEs represent a source of recurrent, immunogenic tumor-specific antigens in patients with NSCLC.

Epigenetically controlled tumor antigens derived from splice junctions between exons and transposable elements.

Oncogenesis often implicates epigenetic alterations, including derepression of transposable elements (TEs) and defects in alternative splicing. Here, we explore the possibility that noncanonical splice junctions between exons and TEs represent a source of tumor-specific antigens. We show that mouse normal tissues and tumor cell lines express wide but distinct ranges of mRNA junctions between exons and TEs, some of which are tumor specific. Immunopeptidome analyses in tumor cell lines identified peptides derived from exon-TE splicing junctions associated to MHC-I molecules. Exon-TE junction-derived peptides were immunogenic in tumor-bearing mice. Both prophylactic and therapeutic vaccinations with junction-derived peptides delayed tumor growth in vivo. Inactivation of the TE-silencing histone 3-lysine 9 methyltransferase Setdb1 caused overexpression of new immunogenic junctions in tumor cells. Our results identify exon-TE splicing junctions as epigenetically controlled, immunogenic, and protective tumor antigens in mice, opening possibilities for tumor targeting and vaccination in patients with cancer.

Functional characterization of 5p15.33 risk locus in uveal melanoma reveals rs452384 as a functional variant and NKX2.4 as an allele-specific interactor.

The TERT/CLPTM1L risk locus on chromosome 5p15.33 is a pleiotropic cancer risk locus in which multiple independent risk alleles have been identified, across well over ten cancer types. We previously conducted a genome-wide association study in uveal melanoma (UM), which uncovered a role for the TERT/CLPTM1L risk locus in this intraocular tumor and identified multiple highly correlated risk alleles. Aiming to unravel the biological mechanisms in UM of this locus, which contains a domain enriched in active chromatin marks and enhancer elements, we demonstrated the allele-specific enhancer activity of this risk region using reporter assays. In UM, we identified the functional variant rs452384, of which the C risk allele is associated with higher gene expression, increased CLPTM1L expression in UM tumors, and a longer telomere length in peripheral blood mononuclear cells. Electrophoretic mobility shift assays and quantitative mass spectrometry identified NKX2.4 as an rs452384-T-specific binding protein, whereas GATA4 preferentially interacted with rs452384-C. Knockdown of NKX2.4 but not GATA4 resulted in increased TERT and CLPTM1L expression. In summary, the UM risk conferred by the 5p locus is at least partly due to rs452384, for which NKX2.4 presents strong differential binding activity and regulates CLPTM1L and TERT expression. Altogether, our work unraveled some of the complex regulatory mechanisms at the 5p15.33 susceptibility region in UM, and this might also shed light on shared mechanisms with other tumor types affected by this susceptibility region.

MBD4 deficiency is predictive of response to immune checkpoint inhibitors in metastatic uveal melanoma patients.

BACKGROUND: MBD4 mutations have been reported in uveal melanomas, acute myeloid leukemias, colorectal adenocarcinomas, gliomas, and spiradenocarcinomas and cause a hypermutated phenotype. Although metastatic uveal melanomas (mUM) are usually resistant to immune checkpoint inhibitors (ICI), the first reported MBD4-mutated (MBD4m) patient responded to ICI, suggesting that MBD4 mutation may predict response to ICI. METHODS: Retrospective cohort of mUM patients treated with ICI. MBD4 was sequenced in a subset of these patients. RESULTS: Three hundred mUM patients were included. Median follow-up was 17.3 months. Ten patients with an objective response and 20 cases with stable disease for >12 months were observed, corresponding to an objective response rate of 3.3% and a clinical benefit (i.e., responder patients and stable disease) rate of 10%. Of the 131 tumors sequenced for MBD4, five (3.8%) were mutated. MBD4 mutation was associated with a better objective response rate as three out of five MBD4m versus 4% of MBD4 wild-type patients responded (p < 0.001). Of these five responders, three presented progressive disease at 2.8, 13.9, and 22.3 months. Median PFS was 4.0 months in MBD4 wild-type and 22.3 months in MBD4m patients (HR = 0.22; p = 0.01). Median OS in MBD4def patients was unreached as compared to 16.6 months in MBD4pro (HR = 0.11; 95% CI: 0.02-0.86; log-rank p-test = 0.04; Fig. 2e). CONCLUSIONS: In mUM patients, MBD4 mutation is highly predictive for the response, PFS, and overall survival benefit to ICI. MBD4 could be a tissue-agnostic biomarker and should be sequenced in mUM, and other tumor types where MBD4 mutations are reported.

Glycolysis Dependency as a Hallmark of SF3B1-Mutated Cells.

SF3B1 mutations are recurrent in cancer and result in aberrant splicing of a previously defined set of genes. Here, we investigated the fate of aberrant transcripts induced by mutant SF3B1 and the related functional consequences. We first demonstrate that mutant SF3B1 does not alter global nascent protein synthesis, suggesting target-dependent consequences. Polysome profiling revealed that 35% of aberrantly spliced transcripts are more translated than their corresponding canonically spliced transcripts. This mostly occurs in genes with enriched metabolic functions. Furthermore, LC-MS/MS analysis showed that mutant SF3B1 impacts the abundance of proteins involved in metabolism. Functional metabolic characterization revealed that mutant SF3B1 decreases mitochondrial respiration and promotes glycolysis to compensate for defective mitochondrial metabolism. Hence, mutant SF3B1 induces glycolysis dependency, which sensitizes cells to glycolysis inhibition. Overall, we provide evidence of the oncogenic involvement of mutant SF3B1 in uveal melanoma through a metabolic switch to glycolysis, revealing vulnerability to glycolysis inhibitors as a promising therapeutic strategy.

Different Pigmentation Risk Loci for High-Risk Monosomy 3 and Low-Risk Disomy 3 Uveal Melanomas.

BACKGROUND: Uveal melanoma (UM), a rare malignant tumor of the eye, is predominantly observed in populations of European ancestry. UMs carrying a monosomy 3 (M3) frequently relapse mainly in the liver, whereas UMs with disomy 3 (D3) are associated with more favorable outcome. Here, we explored the UM genetic predisposition factors in a large genome-wide association study (GWAS) of 1142 European UM patients and 882 healthy controls . METHODS: We combined 2 independent datasets (Global Screening Array) with the dataset described in a previously published GWAS in UM (Omni5 array), which were imputed separately and subsequently merged. Patients were stratified according to their chromosome 3 status, and identified UM risk loci were tested for differential association with M3 or D3 subgroups. All statistical tests were 2-sided. RESULTS: We recapitulated the previously identified risk locus on chromosome 5 on CLPTM1L (rs421284: odds ratio [OR] =1.58, 95% confidence interval [CI] = 1.35 to 1.86; P = 1.98 × 10-8) and identified 2 additional risk loci involved in eye pigmentation: IRF4 locus on chromosome 6 (rs12203592: OR = 1.76, 95% CI = 1.44 to 2.16; P = 3.55 × 10-8) and HERC2 locus on chromosome 15 (rs12913832: OR= 0.57, 95% CI = 0.48 to 0.67; P = 1.88 × 10-11). The IRF4 rs12203592 single-nucleotide polymorphism was found to be exclusively associated with risk for the D3 UM subtype (ORD3 = 2.73, 95% CI = 1.87 to 3.97; P = 1.78 × 10-7), and the HERC2 rs12913832 single-nucleotide polymorphism was exclusively associated with risk for the M3 UM subtype (ORM3 = 2.43, 95% CI = 1.79 to 3.29; P = 1.13 × 10-8). However, the CLPTM1L risk locus was equally statistically significant in both subgroups. CONCLUSIONS: This work identified 2 additional UM risk loci known for their role in pigmentation. Importantly, we demonstrate that UM tumor biology and metastatic potential are influenced by patients' genetic backgrounds.

Definition of Biologically Distinct Groups of Conjunctival Melanomas According to Etiological Factors and Implications for Precision Medicine.

Conjunctival melanoma (ConjMel) is a potentially deadly ocular melanoma, originating from partially sunlight-exposed mucosa. We explored the mutational landscape of ConjMel and studied the correlation with etiological factors. We collected 47 primary ConjMel samples and performed next-generation sequencing of 400 genes. Hotspot mutations in BRAF, NRAS, HRAS, and KIT were observed in 16 (34%), 5 (11%), 2, and 2 cases, respectively. Patients with BRAF and CDKN2A-mutated ConjMel tended to be younger while the NF1-mutated one tended to be older. The eight tumors arising from nevi were enriched in CTNNB1 mutations (63% vs. 8%; Fisher's exact p-test = 0.001) compared to non-nevi ConjMel and five were devoid of BRAF, RAS, NF1, or KIT mutations, suggesting a specific oncogenic process in these tumors. The two KIT-mutated cases carried SF3B1 mutations and were located on sun-protected mucosa, a genotype shared with genital and anorectal mucosal melanomas. Targetable mutations were observed in ERBB2, IDH1, MET, and MAP2K1 (one occurrence each). Mutational landscape of ConjMel characterizes distinct molecular subtypes with oncogenic drivers common with mucosal and skin melanomas. CTNNB1 mutations were associated with nevus-derived ConjMel. Concomitant KIT/SF3B1 mutations in sun-protected cases suggest a common tumorigenic process with genital and anorectal mucosal melanomas.

Splicing Patterns in SF3B1-Mutated Uveal Melanoma Generate Shared Immunogenic Tumor-Specific Neoepitopes.

Disruption of splicing patterns due to mutations of genes coding splicing factors in tumors represents a potential source of tumor neoantigens, which would be both public (shared between patients) and tumor-specific (not expressed in normal tissues). In this study, we show that mutations of the splicing factor SF3B1 in uveal melanoma generate such immunogenic neoantigens. Memory CD8+ T cells specific for these neoantigens are preferentially found in 20% of patients with uveal melanoma bearing SF3B1-mutated tumors. Single-cell analyses of neoepitope-specific T cells from the blood identified large clonal T-cell expansions, with distinct effector transcription patterns. Some of these expanded T-cell receptors are also present in the corresponding tumors. CD8+ T-cell clones specific for the neoepitopes specifically recognize and kill SF3B1-mutated tumor cells, supporting the use of this new family of neoantigens as therapeutic targets. SIGNIFICANCE: Mutations of the splicing factor SF3B1 in uveal melanoma generate shared neoantigens that are uniquely expressed by tumor cells, leading to recognition and killing by specific CD8 T cells. Mutations in splicing factors can be sources of new therapeutic strategies applicable to diverse tumors.This article is highlighted in the In This Issue feature, p. 1861.

Functional and conformational impact of cancer-associated SF3B1 mutations depends on the position and the charge of amino acid substitution.

The hotspot mutations of SF3B1, the most frequently mutated splicing gene in cancers, contribute to oncogenesis by corrupting the mRNA splicing. Further SF3B1 mutations have been reported in cancers but their consequences remain unclear. Here, we screened for SF3B1 mutations in the vicinity of the hotspot region in tumors. We then performed in-silico prediction of the functional outcome followed by in-cellulo modelling of different SF3B1 mutants. We show that cancer-associated SF3B1 mutations present varying functional consequences that are loosely predicted by the in-silico algorithms. Analysis of the tertiary structure of SF3B1 mutants revealed that the resulting splicing errors may be due to a conformational change in SF3B1 N-terminal region, which mediates binding with other splicing factors. Our study demonstrates a varying functional impact of SF3B1 mutations according to the mutated codon and the amino acid substitution, implying unequal pathogenic and prognostic potentials of SF3B1 mutations in cancers.

Germline MBD4 Mutations and Predisposition to Uveal Melanoma.

BACKGROUND: Uveal melanoma (UM) arises from malignant transformation of melanocytes in the uveal tract of the eye. This rare tumor has a poor outcome with frequent chemo-resistant liver metastases. BAP1 is the only known predisposing gene for UM. UMs are generally characterized by low tumor mutation burden, but some UMs display a high level of CpG>TpG mutations associated with MBD4 inactivation. Here, we explored the incidence of germline MBD4 variants in a consecutive series of 1093 primary UM case patients and a series of 192 UM tumors with monosomy 3 (M3). METHODS: We performed MBD4 targeted sequencing on pooled germline (n = 1093) and tumor (n = 192) DNA samples of UM patients. MBD4 variants (n = 28) were validated by Sanger sequencing. We performed whole-exome sequencing on available tumor samples harboring MBD4 variants (n = 9). Variants of unknown pathogenicity were further functionally assessed. RESULTS: We identified 8 deleterious MBD4 mutations in the consecutive UM series, a 9.15-fold (95% confidence interval = 4.24-fold to 19.73-fold) increased incidence compared with the general population (Fisher exact test, P = 2.00 × 10-5, 2-sided), and 4 additional deleterious MBD4 mutations in the M3 cohort, including 3 germline and 1 somatic mutations. Tumors carrying deleterious MBD4 mutations were all associated with high tumor mutation burden and a CpG>TpG hypermutator phenotype. CONCLUSIONS: We demonstrate that MBD4 is a new predisposing gene for UM associated with hypermutated M3 tumors. The tumor spectrum of this predisposing condition will likely expand with the addition of MBD4 to diagnostic panels. Tumors arising in such a context should be recognized because they may respond to immunotherapy.

Genetic alterations of SUGP1 mimic mutant-SF3B1 splice pattern in lung adenocarcinoma and other cancers.

Genes involved in 3'-splice site recognition during mRNA splicing constitute an emerging class of oncogenes. SF3B1 is the most frequently mutated splicing factor in cancer, and SF3B1 mutants corrupt branchpoint recognition leading to usage of cryptic 3'-splice sites and subsequent aberrant junctions. For a comprehensive determination of alterations leading to this splicing pattern, we performed a pan-TCGA screening for SF3B1-specific aberrant acceptor usage. While the most of aberrant 3'-splice patterns were explained by SF3B1 mutations, we also detected nine SF3B1 wild-type tumors (including five lung adenocarcinomas). Genomic profile analysis of these tumors identified somatic mutations combined with loss-of-heterozygosity in the splicing factor SUGP1 in five of these cases. Modeling of SUGP1 loss and mutations in cell lines showed that both alterations induced mutant-SF3B1-like aberrant splicing. Our study provides definitive evidence that genetic alterations of SUGP1 genocopy SF3B1 mutations in lung adenocarcinoma and other cancers.

ShallowHRD: detection of homologous recombination deficiency from shallow whole genome sequencing.

SUMMARY: We introduce shallowHRD, a software tool to evaluate tumor homologous recombination deficiency (HRD) based on whole genome sequencing (WGS) at low coverage (shallow WGS or sWGS; ∼1X coverage). The tool, based on mining copy number alterations profile, implements a fast and straightforward procedure that shows 87.5% sensitivity and 90.5% specificity for HRD detection. shallowHRD could be instrumental in predicting response to poly(ADP-ribose) polymerase inhibitors, to which HRD tumors are selectively sensitive. shallowHRD displays efficiency comparable to most state-of-art approaches, is cost-effective, generates low-storable outputs and is also suitable for fixed-formalin paraffin embedded tissues. AVAILABILITY AND IMPLEMENTATION: shallowHRD R script and documentation are available at https://github.com/aeeckhou/shallowHRD. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Prospective validation in epithelial tumors of a gene expression predictor of liver metastasis derived from uveal melanoma.

Predicting the risk of liver metastasis can have important prognostic and therapeutic implications, given the availability of liver-directed therapy. Uveal melanoma has a striking predisposition for liver metastasis despite the absence of anatomical proximity. Understanding its biology may uncover factors promoting liver metastasis in other malignancies. We quantified gene expression by RNAseq in 76 uveal melanomas and combined with public data in a meta-analysis of 196 patients. The meta-analysis of uveal melanoma gene expression identified 63 genes which remained prognostic after adjustment for chromosome 3 status. Two genes, PTP4A3 and JPH1, were selected by L1-penalized regression and combined in a prognostic score. The score predicted liver-specific relapse in a public pan-cancer dataset and in two public colorectal cancer datasets. The score varied between colorectal consensus molecular subtypes (CMS), as did the risk of liver relapse, which was lowest in CMS1. Additional prospective validation was done by real-time PCR in 463 breast cancer patients. The score was significantly correlated with liver relapse in hormone receptor positive tumors. In conclusion, the expression of PTP4A3 and JPH1 correlates with risk of liver metastasis in colorectal cancer and breast cancer. The underlying biological mechanism is an interesting area for further research.

A variant erythroferrone disrupts iron homeostasis in SF3B1-mutated myelodysplastic syndrome.

Myelodysplastic syndromes (MDS) with ring sideroblasts are hematopoietic stem cell disorders with erythroid dysplasia and mutations in the SF3B1 splicing factor gene. Patients with MDS with SF3B1 mutations often accumulate excessive tissue iron, even in the absence of transfusions, but the mechanisms that are responsible for their parenchymal iron overload are unknown. Body iron content, tissue distribution, and the supply of iron for erythropoiesis are controlled by the hormone hepcidin, which is regulated by erythroblasts through secretion of the erythroid hormone erythroferrone (ERFE). Here, we identified an alternative ERFE transcript in patients with MDS with the SF3B1 mutation. Induction of this ERFE transcript in primary SF3B1-mutated bone marrow erythroblasts generated a variant protein that maintained the capacity to suppress hepcidin transcription. Plasma concentrations of ERFE were higher in patients with MDS with an SF3B1 gene mutation than in patients with SF3B1 wild-type MDS. Thus, hepcidin suppression by a variant ERFE is likely responsible for the increased iron loading in patients with SF3B1-mutated MDS, suggesting that ERFE could be targeted to prevent iron-mediated toxicity. The expression of the variant ERFE transcript that was restricted to SF3B1-mutated erythroblasts decreased in lenalidomide-responsive anemic patients, identifying variant ERFE as a specific biomarker of clonal erythropoiesis.

Evolutionary Routes in Metastatic Uveal Melanomas Depend on MBD4 Alterations.

PURPOSE: Uveal melanomas (UM) are genetically simple tumors carrying few copy number alterations (CNA) and a low mutation burden, except in rare MBD4-deficient, hypermutated cases. The genomics of uveal melanoma metastatic progression has not been described. We assessed the genetic heterogeneity of primary and metastatic MBD4-proficient and -deficient uveal melanomas.Experimental Design: We prospectively collected 75 metastatic and 16 primary samples from 25 consecutive uveal melanoma patients, and performed whole-exome sequencing. RESULTS: MBD4-proficient uveal melanomas contained stable genomes at the nucleotide level, acquiring few new single nucleotide variants (SNVs; 16 vs. 13 in metastases and primary tumors, respectively), and no new driver mutation. Five CNAs were recurrently acquired in metastases (losses of 1p, 6q, gains of 1q, 8q, and isodisomy 3). In contrast, MBD4-deficient uveal melanomas carried more than 266 SNVs per sample, with high genetic heterogeneity and TP53, SMARCA4, and GNAS new driver mutations. SNVs in MBD4-deficient contexts were exploited to unveil the timeline of oncogenic events, revealing that metastatic clones arose early after tumor onset. Surprisingly, metastases were not enriched in monosomy 3, a previously defined metastatic risk genomic feature. Monosomy 3 was associated with shorter metastatic-free interval compared with disomy 3 rather than higher rate of relapse. CONCLUSIONS: MBD4-proficient uveal melanomas are stable at the nucleotide level, without new actionable alterations when metastatic. In contrast, MBD4 deficiency is associated with high genetic heterogeneity and acquisition of new driver mutations. Monosomy 3 is associated with time to relapse rather than rate of relapse, thus opening avenues for a new genetic prognostic classification of uveal melanomas.