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Over the last decades, rapid technological and scientific advances have led to a merge of molecular sciences and clinical medicine, resulting in a better understanding of disease mechanisms and the development of novel therapies that exploit specific molecular lesions or profiles driving disease. Precision oncology is here used as an example, illustrating the potential of precision/personalized medicine that also holds great promise in other medical fields. Real-world implementation can only be achieved by dedicated healthcare connected centers which amass and build up interdisciplinary expertise reflecting the complexity of precision medicine. Networks of such centers are ideally suited for a nation-wide outreach offering access to precision medicine to patients independent of their place of residence. Two of these multicentric initiatives, Genomic Medicine Sweden (GMS) and the Centers for Personalized Medicine (ZPM) initiative in Germany have teamed up to present and share their views on core concepts, potentials, challenges, and future developments in precision medicine. Together with other initiatives worldwide, GMS and ZPM aim at providing a robust and sustainable framework, covering all components from technology development to clinical trials, ethical and legal aspects as well as involvement of all relevant stakeholders, including patients and policymakers in the field.

The somatic hypermutation (SHM) status of the clonotypic immunoglobulin heavy variable (IGHV) gene is a critical biomarker for assessing the prognosis of patients with chronic lymphocytic leukemia (CLL). Importantly, independent studies have documented that IGHV SHM status is also a predictor of responses to therapy, including both chemoimmunotherapy (CIT) and novel, targeted agents. Moreover, immunogenetic analysis in CLL has revealed that different patients may express (quasi)identical, stereotyped B cell receptor immunoglobulin (BcR IG) and are classified into subsets based on this common feature. Patients in certain stereotyped subsets display consistent biology, clinical presentation, and outcome that are distinct from other patients, even with concordant IGHV gene SHM status. All of the above highlights the relevance of immunogenetic analysis in CLL, which is considered a cornerstone for accurate risk stratification and clinical decision making. Recommendations for robust immunogenetic analysis exist thanks to dedicated efforts by ERIC, the European Research Initiative on CLL, covering all test phases, from the pre-analytical and analytical to the post-analytical, pertaining to the analysis, interpretation, and reporting of the findings. That said, these recommendations apply to Sanger sequencing, which is increasingly being superseded by next generation sequencing (NGS), further underscoring the need for an update. Here, we present an overview of the clinical utility of immunogenetics in CLL and update our analytical recommendations with the aim to assist in the refined management of patients with CLL.

Medulloblastoma is one of the most common types of brain tumors in children. During and after treatment with surgery, chemotherapy, and/or radiotherapy, children with this disease are monitored with imaging and cerebrospinal fluid analysis for the detection of tumor cells. These methods are not always sensitive or specific enough to confirm or rule out residual disease. Here, we develop a laboratory test based on the genetic makeup of medulloblastomas in 12 children. We analyze liquid biopsies (cerebrospinal fluid and blood plasma) for specific genetic fragments leaking from the individual tumors and find molecular traces of disease in 75% (9/12) of children overall. None of the children had malignant cells in the cerebrospinal fluid. We propose that this test could open up new technical possibilities to track measurable residual disease in children with medulloblastoma in order to further risk-adapt treatment, but first, larger studies of the approach at standardized time points are warranted.Medulloblastoma is a malignant embryonal tumor of the central nervous system (CNS) that mainly affects infants and children. Prognosis is highly variable, and molecular biomarkers for measurable residual disease (MRD) detection are lacking. Analysis of cell-free DNA (cfDNA) in cerebrospinal fluid (CSF) using broad genomic approaches, such as low-coverage whole-genome sequencing, has shown promising prognostic value. However, more sensitive methods are needed for MRD analysis. Here, we show the technical feasibility of capturing medulloblastoma-associated structural variants and point mutations simultaneously in cfDNA using multiplexed droplet digital PCR (ddPCR). Assay sensitivity was assessed with a dilution series of tumor in normal genomic DNA, and the limit of detection was below 100 pg of input DNA for all assays. False positive rates were zero for structural variant assays. Liquid biopsies (CSF and plasma, n = 47) were analyzed from 12 children with medulloblastoma, all with negative CSF cytology. MRD was detected in 75% (9/12) of patients overall. In CSF samples taken before or within 21 days of surgery, MRD was detected in 88% (7/8) of patients with localized disease and in one patient with the metastasized disease. Our results suggest that this approach could expand the utility of ddPCR and complement broader analyses of cfDNA for MRD detection.

Genetic diagnostics of hematological malignancies has evolved dramatically over the years, from chromosomal banding analysis to next-generation sequencing, with a corresponding increased capacity to detect clinically relevant prognostic and predictive biomarkers. In diagnostics of patients with chronic lymphocytic leukemia (CLL), we currently apply fluorescence in situ hybridization (FISH)-based analysis to detect recurrent chromosomal aberrations (del(11q), del(13q), del(17p) and trisomy 12) as well as targeted sequencing (IGHV and TP53 mutational status) for risk-stratifying purposes. These analyses are performed before start of any line of treatment and assist in clinical decision-making including selection of targeted therapy (BTK and BCL2 inhibitors). Here, we present the current view on the genomic landscape of CLL, including an update on recent advances with potential for clinical translation. We discuss different state-of-the-art technologies that are applied to enable precision diagnostics in CLL and highlight important genomic markers with current prognostic and/or predictive impact as well as those of prospective clinical relevance. In the coming years, it will be important to develop more comprehensive genomic analyses that can capture all types of relevant genetic aberrations, but also to develop highly sensitive assays to detect minor mutations that affect therapy response or confer resistance to targeted therapies. Finally, we will bring up the potential of new technologies and multi-omics analysis to further subclassify the disease and facilitate implementation of precision medicine approaches in this still incurable disease.

PURPOSE The combination of whole-genome and transcriptome sequencing (WGTS) is expected to transform diagnosis and treatment for patients with cancer. WGTS is a comprehensive precision diagnostic test that is starting to replace the standard of care for oncology molecular testing in health care systems around the world; however, the implementation and widescale adoption of this best-in-class testing is lacking. METHODS Here, we address the barriers in integrating WGTS for cancer diagnostics and treatment selection and answer questions regarding utility in different cancer types, cost-effectiveness and affordability, and other practical considerations for WGTS implementation. RESULTS We review the current studies implementing WGTS in health care systems and provide a synopsis of the clinical evidence and insights into practical considerations for WGTS implementation. We reflect on regulatory, costs, reimbursement, and incidental findings aspects of this test. CONCLUSION WGTS is an appropriate comprehensive clinical test for many tumor types and can replace multiple, cascade testing approaches currently performed. Decreasing sequencing cost, increasing number of clinically relevant aberrations and discovery of more complex biomarkers of treatment response, should pave the way for health care systems and laboratories in implementing WGTS into clinical practice, to transform diagnosis and treatment for patients with cancer.

Formalin-fixed, paraffin-embedded (FFPE) specimens are an underutilized resource in medical research, particularly in the setting of transcriptome sequencing, as RNA from these samples is often degraded. We took advantage of an exome capture-based RNA-sequencing protocol to explore global gene expression in paired fresh-frozen (FF) and FFPE samples from 16 diffuse large B-cell lymphoma (DLBCL) patients. While FFPE samples generated fewer mapped reads compared to their FF counterparts, these reads captured the same library complexity and had a similar number of genes expressed on average. Furthermore, gene expression demonstrated a high correlation when comparing housekeeping genes only or across the entire transcriptome (r = 0.99 for both comparisons). Differences in gene expression were primarily seen in lowly expressed genes and genes with small or large coding sequences. Using cell-of-origin classifiers and clinically relevant gene expression signatures for DLBCL, FF, and FFPE samples from the same biopsy paired nearly perfectly in clustering analysis. This was further confirmed in a validation cohort of 50 FFPE DLBCL samples. In summary, we found the biological differences between tumors to be far greater than artifacts created as a result of degraded RNA. We conclude that exome capture transcriptome sequencing data from archival samples can confidently be used for cell-of-origin classification of DLBCL samples.

Recent studies of chronic lymphocytic leukemia (CLL) have reported recurrent mutations in the RPS15 gene, which encodes the ribosomal protein S15 (RPS15), a component of the 40S ribosomal subunit. Despite some evidence about the role of mutant RPS15 (mostly obtained from the analysis of cell lines), the precise impact of RPS15 mutations on the translational program in primary CLL cells remains largely unexplored. Here, using RNA sequencing and ribosome profiling, a technique that involves measuring translational efficiency, we sought to obtain global insight into changes in translation induced by RPS15 mutations in CLL cells. To this end, we evaluated primary CLL cells from patients with wild-type or mutant RPS15 as well as MEC1 CLL cells transfected with mutant or wild-type RPS15. Our data indicate that RPS15 mutations rewire the translation program of primary CLL cells by reducing their translational efficiency, an effect not seen in MEC1 cells. In detail, RPS15 mutant primary CLL cells displayed altered translation efficiency of other ribosomal proteins and regulatory elements that affect key cell processes, such as the translational machinery and immune signaling, as well as genes known to be implicated in CLL, hence highlighting a relevant role for RPS15 in the natural history of CLL.

Autologous stem-cell transplantation (ASCT) consists of infusion of previously harvested CD34+ hematopoietic stem cells after high-dose chemotherapy. This treatment can be curative for patients wit...

Cancer treatment has been transformed by checkpoint blockade therapies, with the highest anti-tumor activity of anti-programmed death 1 (PD-1) antibody therapy seen in Hodgkin lymphoma. Disappointingly, response rates have been low in the non-Hodgkin lymphomas, with no activity seen in relapsed/refractory chronic lymphocytic leukemia (CLL) with PD-1 blockade. Thus, identifying more powerful combination therapy is required for these patients. Here, we preclinically demonstrate enhanced anti-CLL activity following combinational therapy with anti-PD-1 or anti-PD-1 ligand (PD-L1) and avadomide, a cereblon E3 ligase modulator (CELMoD). Avadomide induced type I and II interferon (IFN) signaling in patient T cells, triggering a feedforward cascade of reinvigorated T-cell responses. Immune modeling assays demonstrated that avadomide stimulated T-cell activation, chemokine expression, motility and lytic synapses with CLL cells, as well as IFN-inducible feedback inhibition through upregulation of PD-L1. Patient-derived xenograft tumors treated with avadomide were converted to CD8+ T cell-inflamed tumor microenvironments that responded to anti-PD-L1/PD-1-based combination therapy. Notably, clinical analyses showed increased PD-L1 expression on T cells, as well as intratumoral expression of chemokine signaling genes in B-cell malignancy patients receiving avadomide-based therapy. These data illustrate the importance of overcoming a low inflammatory T-cell state to successfully sensitize CLL to checkpoint blockade-based combination therapy.

Purpose: Investigation of signaling pathways altered by recurrent gene mutations and their clinical impact in a consecutive cohort of patients with newly diagnosed chronic lymphocytic leukemia (CLL). The heterogeneous clinical course and genetic complexity of CLL warrant improved molecular prognostication. However, the prognostic value of recurrent mutations at the time of diagnosis remains unclear. Experimental Design: We sequenced samples from 314 consecutive, newly diagnosed patients with CLL to investigate the clinical impact of 56 recurrently mutated genes assessed by next-generation sequencing. Results: Mutations were identified in 70% of patients with enrichment among IGHV unmutated cases. With 6.5 years of follow-up, 15 mutated genes investigated at the time of diagnosis demonstrated significant impact on time to first treatment (TTFT). Carrying driver mutations was associated with shorter TTFT and poor overall survival. For outcome from CLL diagnosis, the number of signaling pathways altered by driver mutations stratified patients better than the number of driver mutations. Moreover, we demonstrated gradual impact on TTFT with increasing number of altered pathways independent of CLL-IPI risk. Thus, a 25-gene, pathway-based biomarker assessing recurrent mutations refines prognostication in CLL, in particular for CLL-IPI low- and intermediate-risk patients. External validation emphasized that a broad gene panel including low burden mutations was key for the biomarker based on altered pathways. Conclusions: We propose to include the number of pathways altered by driver mutations as a biomarker together with CLL-IPI in prospective studies of CLL from time of diagnosis for incorporation into clinical care and personalized follow-up and treatment.

Twenty years after landmark publications, there is a consensus that the somatic hypermutation (SHM) status of the clonotypic immunoglobulin heavy variable (IGHV) gene is an important cornerstone for accurate risk stratification and therapeutic decision-making in patients with chronic lymphocytic leukemia (CLL). The IGHV SHM status has traditionally been determined by conventional Sanger sequencing. However, NGS has heralded a new era in medical diagnostics and immunogenetic analysis is following this trend. There is indeed a growing demand for shifting practice and using NGS for IGHV gene SHM assessment, although it is debatable whether it is always justifiable, at least taking into account financial considerations for laboratories with limited resources. Nevertheless, as this analysis impacts on treatment decisions, standardization of both technical aspects, and data interpretation becomes essential. Also, the need for establishing new recommendations and providing dedicated education and training on NGS-based immunogenetics is greater than ever before. Here we address potential and challenges of NGS-based immunogenetics in CLL. We are convinced that this perspective helps the hematological community to better understand the pros and cons of this new technological development for CLL patient management.

Patients with CLL with mutated IGHV genes (M-CLL) have better outcomes than patients with unmutated IGHVs (U-CLL). Since U-CLL usually express immunoglobulins (IGs) that are more autoreactive and more effectively transduce signals to leukemic B cells, B-cell receptor (BCR) signaling is likely at the heart of the worse outcomes of CLL cases without/few IGHV mutations. A corollary of this conclusion is that M-CLL follow less aggressive clinical courses because somatic IGHV mutations have altered BCR structures and no longer bind stimulatory (auto)antigens and so cannot deliver trophic signals to leukemic B cells. However, the latter assumption has not been confirmed in a large patient cohort. We tried to address the latter by measuring the relative numbers of replacement (R) mutations that lead to non-conservative amino acid changes (Rnc) to the combined numbers of conservative (Rc) and silent (S) amino acid R mutations that likely do not or cannot change amino acids, “(S+Rc) to Rnc IGHV mutation ratio”. When comparing time-to-first-treatment (TTFT) of patients with (S+Rc)/Rnc ≤ 1 and >1, TTFTs were similar, even after matching groups for equal numbers of samples and identical numbers of mutations per sample. Thus, BCR structural change might not be the main reason for better outcomes for M-CLL. Since the total number of IGHV mutations associated better with longer TTFT, better clinical courses appear due to the biologic state of a B cell having undergone many stimulatory events leading to IGHV mutations. Analyses of larger patient cohorts will be needed to definitively answer this question.

Key processes in the onset and evolution of chronic lymphocytic leukemia (CLL) are thought to include chronic (antigenic) activation of mature B cells through the B cell receptor (BcR), signals from the microenvironment, and acquisition of genetic alterations. Here we describe three families in which two or more siblings were affected by CLL. We investigated whether there are immunogenetic similarities in the leukemia-specific immunoglobulin heavy (IGH) and light (IGL/IGK) chain gene rearrangements of the siblings in each family. Furthermore, we performed array analysis to study if similarities in CLL-associated chromosomal aberrations are present within each family and screened for somatic mutations using paired tumor/normal whole-genome sequencing (WGS). In two families a consistent IGHV gene mutational status (one IGHV-unmutated, one IGHV-mutated) was observed. Intriguingly, the third family with four affected siblings was characterized by usage of the lambda IGLV3-21 gene, with the hallmark R110 mutation of the recently described clinically aggressive IGLV3-21R110 subset. In this family, the CLL-specific rearrangements in two siblings could be assigned to either stereotyped subset #2 or the immunogenetically related subset #169, both of which belong to the broader IGLV3-21R110 subgroup. Consistent patterns of cytogenetic aberrations were encountered in all three families. Furthermore, the CLL clones carried somatic mutations previously associated with IGHV mutational status, cytogenetic aberrations and stereotyped subsets, respectively. From these findings, we conclude that similarities in immunogenetic characteristics in familial CLL, in combination with genetic aberrations acquired, point towards shared underlying mechanisms behind CLL development within each family.

Genetics is an integral part of the clinical diagnostics of lymphomas that improves disease subclassification and patient risk-stratification. With the introduction of high-throughput sequencing technologies, a rapid, in-depth portrayal of the genomic landscape in major lymphoma entities was achieved. Whilst a few lymphoma entities were characterized by a predominant gene mutation (e.g. Waldenstrom’s macroglobulinemia and hairy cell leukemia), the vast majority demonstrated a very diverse genetic landscape with a high number of recurrent gene mutations (e.g. chronic lymphocytic leukemia and diffuse large B cell lymphoma), indeed reflecting the great clinical heterogeneity among lymphomas. These studies have allowed better understanding of the ontogeny and evolution of different lymphomas, while also identifying new genetic markers that can complement lymphoma diagnostics and improve prognostication. However, despite these efforts, there is still a limited number of gene mutations with predictive impact that can guide treatment selection. In this review, we will highlight clinically relevant diagnostic, prognostic and predictive markers in lymphomas that are used today in routine diagnostics. We will also discuss how comprehensive genomic characterization using broad sequencing panels, allowing for the simultaneous detection of different types of genetic aberrations, may aid future development of precision diagnostics in lymphomas. This may in turn pave the way for the implementation of tailored precision therapy strategies at the individual patient level.

Mantle cell lymphoma (MCL), a malignancy of B-lymphocytes, has a poor prognosis. It is thus necessary to improve the understanding of the pathobiology of MCL and identify factors contributing to its aggressiveness. Our studies, based on Affymetrix data from 17 MCL biopsies, real-time quantitative polymerase chain reaction data from 18 sorted primary MCL cells and 108 MCL biopsies compared to non-malignant tissue, reveals that GNAZ expression predicts poor clinical outcome of MCL patients (Cox regression, P = 0·014) and lymphocytosis (Mann-Whitney, P = 0·011). We show that GNAZ translates to Gαz protein - a signalling molecule within the G-protein coupled receptor network. Our findings suggest that GNAZ/Gαz contribute to the MCL pathobiology.

Chronic lymphocytic leukemia (CLL) displays remarkable clinical heterogeneity, likely attributed to the underlying biological diversity. This claim is supported by the fact that certain immunogenetic and/or genomic features identify subgroups of CLL patients with distinct prognosis and outcome.

Interrogating the tumor genome in its entirety by whole-genome sequencing (WGS) offers an unprecedented insight into the biology and pathogenesis of cancer, with potential impact on diagnostics, prognostication and therapy selection. WGS is able to detect sequence as well as structural variants and thereby combines central domains of cytogenetics and molecular genetics. Given the potential of WGS in directing targeted therapeutics and clinical decision-making, we envision a gradual transition of the method from research to clinical routine. This review is one out of three within this issue aimed at facilitating this effort, by discussing in-depth analytical validation, clinical interpretation and clinical utility of WGS. The review highlights the requirements for implementing, validating and maintaining a clinical WGS pipeline to obtain high-quality patient-specific data in accordance with the local regulatory landscape. Every step of the WGS pipeline, which includes DNA extraction, library preparation, sequencing, bioinformatics analysis, and data storage, is considered with respect to its logistics, necessities, potential pitfalls, and the required quality management. WGS is likely to drive clinical diagnostics and patient care forward, if requirements and challenges of the technique are recognized and met.

Precision diagnostics is one of the two pillars of precision medicine. Sequencing efforts in the past decade have firmly established cancer as a primarily genetically driven disease. This concept is supported by therapeutic successes aimed at particular pathways that are perturbed by specific driver mutations in protein-coding domains and reflected in three recent FDA tissue agnostic cancer drug approvals. In addition, there is increasing evidence from studies that interrogate the entire genome by whole-genome sequencing that acquired global and complex genomic aberrations including those in non-coding regions of the genome might also reflect clinical outcome. After addressing technical, logistical, financial and ethical challenges, national initiatives now aim to introduce clinical whole-genome sequencing into real-world diagnostics as a rational and potentially cost-effective tool for response prediction in cancer and to identify patients who would benefit most from 'expensive' targeted therapies and recruitment into clinical trials. However, so far, this has not been accompanied by a systematic and prospective evaluation of the clinical utility of whole-genome sequencing within clinical trials of uniformly treated patients of defined clinical outcome. This approach would also greatly facilitate novel predictive biomarker discovery and validation, ultimately reducing size and duration of clinical trials and cost of drug development. This manuscript is the third in a series of three to review and critically appraise the potential and challenges of clinical whole-genome sequencing in solid tumors and hematological malignancies.

The tumor cells in diffuse large B‐cell lymphomas (DLBCL) are considered to originate from germinal center derived B‐cells (GCB) or activated B‐cells (ABC). Gene expression profiling (GEP) is preferably used to determine the cell of origin (COO). However, GEP is not widely applied in clinical practice and consequently, several algorithms based on immunohistochemistry (IHC) have been developed. Our aim was to evaluate the concordance of COO assignment between the Lymph2Cx GEP assay and the IHC‐based Hans algorithm, to decide which model is the best survival predictor. Both GEP and IHC were performed in 359 homogenously treated Swedish and Danish DLBCL patients, in a retrospective multicenter cohort. The overall concordance between GEP and IHC algorithm was 72%; GEP classified 85% of cases assigned as GCB by IHC, as GCB, while 58% classified as non‐GCB by IHC, were categorized as ABC by GEP. There were significant survival differences (overall survival and progression‐free survival) if cases were classified by GEP, whereas if cases were categorized by IHC only progression‐free survival differed significantly. Importantly, patients assigned as non‐GCB/ABC both by IHC and GEP had the worst prognosis, which was also significant in multivariate analyses. Double expression of MYC and BCL2 was more common in ABC cases and was associated with a dismal outcome. In conclusion, to determine COO both by IHC and GEP is the strongest outcome predictor to identify DLBCL patients with the worst outcome.

Introduction: The SAM domain and HD domain 1 (SAMHD1) protein is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase, which depletes the intracellular dNTP substrates and thus protects the host (human) cells from replication of viruses such as HIV. Mutations of SAMHD1 gene have been linked to Aicardi-Goutières syndrome (AGS). In lymphoid malignancies, SAMHD1 gene mutations have been detected in a subset of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) resulting in decreased SAMHD1 mRNA levels and also mantle cell lymphoma (MCL) among B-cell neoplasms as well as in a subset (20%) of T-prolymphocytic leukemia (T-PLL). Therefore, SAMHD1 may play a role in oncogenesis as a tumor suppressor. In addition, SAMHD1 may confer resistance to cytarabine by hydrolysing their active triphosphate metabolites and its high protein levels correlate with poorer clinical outcome in acute myeloid leukemia. The mutation status of SAMHD1 gene and its expression patterns in peripheral T-cell lymphoma types is not known yet. The purpose of this study was to investigate SAMHD1 gene alterations using next generation sequencing and SAMHD1 protein expression in common types of PTCL. Methods: The study group included 81 adult patients with peripheral T-cell lymphomas (PTCL) including 26 patients with ALK+ anaplastic large cell lymphoma (ALCL), 20 ALK- ALCL, 13 angioimmunoblastic T-cell lymphomas (AILT) and 22 PTCL, not otherwise specified (NOS) with pre-treatment, formalin-fixed, paraffin-embedded (FFPE) tumor tissues available for immunohistochemical analysis. Double immunostaining (SAMHD1/CD68) was used to distinguish CD68+ histiocytes from the neoplastic T-cells. The Ventana autostainer and a previously validated monoclonal antibody for SAMHD1 (#A303-691A; Bethyl Laboratories, San Antonio, TX, USA) was utilized. The percentage of SAMHD1-positive cells was calculated by counting at least 500 tumor cells in each case. In a subset of 28 PTCLs, next generation sequencing (NGS) was performed using FFPE tissues and an enriched custom TruSight gene panel of 52 genes relevant to lymphoma biology. In addition, 3 control tissue samples were included in the analysis. The analysis pipeline was based on GATK best practices guidelines and all variants were annotated using Ensembl VEP v94.5. Freedom from progression (FFP) and overall survival (OS), were the clinical endpoints. Survival analyses were performed using the Kaplan-Meier method (log-rank test). Results: The expression level of SAMHD1 (percentage of positive neoplastic T-cells) varied significantly with AILT showing the highest level (median percentage 80%) as compared to ALK+ ALCL that showed the lowest level (median percentage 40%) of SAMHD1 expression (p=0.019, Kruskall-Wallis test). SAMHD1 mutations were detected for the first time in a subset of PTCL including 4/11 (36%) ALK+ ALCL, 1/5 (20%) ALK- ALCL, 3/6 (50%) AILT and 2/5 (40%) PTCL, NOS. The SAMHD1 gene alterations included missense mutations, nonsense (stopcodon) and splice region mutations. Importantly, reduced level (low percentage of positive tumor cells) of SAMHD1 protein expression was significantly associated with the presence of SAMHD1 mutations. More specifically, the median percentage of SAMHD1+ neoplastic T-cells was 80% in the PTCL group with wild-type SAMHD1 gene compared to 30% in the PTCL group with mutated SAMHD1 gene (p=0.01, Mann-Whitney U test), thus suggesting that alterations of SAMHD1 gene may represent a mechanism of SAMHD1 protein downregulation in a subset of PTCL. SAMHD1 expression or gene alterations did not correlate with FFP or OS in any PTCL histologic type, although the number of patients included in each group was not adequate to draw definite conclusions for prognostic significance. Conclusions: SAMHD1 gene mutations are frequently detected in a subset of PTCL and are associated with reduced expression of SAMHD1 protein. These findings reveal a novel mechanism (SAMHD1 mutations) of SAMHD1 downregulation in PTCL, and further support the tumor suppressor function of SAMHD1 gene in lymphomas. Disclosures Rosenquist Brandell: AbbVie: Honoraria; AstraZeneca: Honoraria; Illumina: Honoraria; Janssen: Honoraria; Roche: Honoraria.

Immunoglobulin gene sequence analysis in chronic lymphocytic leukemia : updated ERIC recommendations

Purpose: We sought to investigate whether B cell receptor immunoglobulin (BcR IG) stereotypy is associated with particular clinicobiological features among chronic lymphocytic leukemia (CLL) patients expressing mutated BcR IG (M-CLL) encoded by the IGHV4-34 gene, and also ascertain whether these associations could refine prognostication. Experimental Design: In a series of 19,907 CLL cases with available immunogenetic information, we identified 339 IGHV4-34–expressing cases assigned to one of the four largest stereotyped M-CLL subsets, namely subsets #4, #16, #29 and #201, and investigated in detail their clinicobiological characteristics and disease outcomes. Results: We identified shared and subset-specific patterns of somatic hypermutation (SHM) among patients assigned to these subsets. The greatest similarity was observed between subsets #4 and #16, both including IgG-switched cases (IgG-CLL). In contrast, the least similarity was detected between subsets #16 and #201, the latter concerning IgM/D-expressing CLL. Significant differences between subsets also involved disease stage at diagnosis and the presence of specific genomic aberrations. IgG subsets #4 and #16 emerged as particularly indolent with a significantly (P < 0.05) longer time-to-first-treatment (TTFT; median TTFT: not yet reached) compared with the IgM/D subsets #29 and #201 (median TTFT: 11 and 12 years, respectively). Conclusions: Our findings support the notion that BcR IG stereotypy further refines prognostication in CLL, superseding the immunogenetic distinction based solely on SHM load. In addition, the observed distinct genetic aberration landscapes and clinical heterogeneity suggest that not all M-CLL cases are equal, prompting further research into the underlying biological background with the ultimate aim of tailored patient management. Clin Cancer Res; 23(17); 5292–301. ©2017 AACR.

We recently reported a truncating deletion in the NFKBIE gene, which encodes IκBε, a negative feedback regulator of NF-κB, in clinically aggressive chronic lymphocytic leukemia (CLL). Because preliminary data indicate enrichment of NFKBIE aberrations in other lymphoid malignancies, we screened a large patient cohort (n = 1460) diagnosed with different lymphoid neoplasms. While NFKBIE deletions were infrequent in follicular lymphoma, splenic marginal zone lymphoma, and T-cell acute lymphoblastic leukemia (

In this issue of Blood, Bretones et al expand knowledge of the functional consequences of recurrent mutations in RPS15, a gene that encodes a ribosomal protein of the 40S subunit and is enriched in patients with clinically aggressive chronic lymphocytic leukemia (CLL).1 By transfecting RPS15 mutants and applying different technologies to assess ribosome activity and efficiency in combination with high-throughput proteome profiling, they were able to demonstrate reduced half-life of RPS15, impaired translational fidelity, and changes in the expressed proteome in mutant vs wild-type RPS15.

The activated B cell (ABC-like) subtype of diffuse large B cell lymphoma (DLBCL) is characterized by chronic activation of signaling initiated by immunoglobulin μ (IgM). By analyzing the DNA copy number profiles of 1000 DLBCL tumors, we identified gains of 18q21.2 as the most frequent genetic alteration in ABC-like DLBCL. Using integrative analysis of matched gene expression profiling data, we found that the TCF4 (E2-2) transcription factor gene was the target of these alterations. Overexpression of TCF4 in ABC-like DLBCL cell lines led to its occupancy on immunoglobulin (IGHM) and MYC gene enhancers and increased expression of these genes at the transcript and protein levels. Inhibition of TCF4 activity with dominant-negative constructs was synthetically lethal to ABC-like DLBCL cell lines harboring TCF4 DNA copy gains, highlighting these gains as an attractive potential therapeutic target. Furthermore, the TCF4 gene was one of the top BRD4-regulated genes in DLBCL cell lines. BET proteolysis-targeting chimera (PROTAC) ARV771 extinguished TCF4, MYC, and IgM expression and killed ABC-like DLBCL cells in vitro. In DLBCL xenograft models, ARV771 treatment reduced tumor growth and prolonged survival. This work highlights a genetic mechanism for promoting immunoglobulin signaling in ABC-like DLBCL and provides a functional rationale for the use of BET inhibitors in this disease.

Chronic lymphocytic leukemia (CLL) stereotyped subsets #6 and #8 include cases expressing unmutated B cell receptor immunoglobulin (BcR IG) (U-CLL). Subset #6 (IGHV1-69/IGKV3-20) is less aggressive compared to subset #8 (IGHV4-39/IGKV1(D)-39) which has the highest risk for Richter's transformation among all CLL. The underlying reasons for this divergent clinical behavior are not fully elucidated. To gain insight into this issue, here we focused on epigenomic signatures and their links with gene expression, particularly investigating genome-wide DNA methylation profiles in subsets #6 and #8 as well as other U-CLL cases not expressing stereotyped BcR IG. We found that subset #8 showed a distinctive DNA methylation profile compared to all other U-CLL cases, including subset #6. Integrated analysis of DNA methylation and gene expression revealed significant correlation for several genes, particularly highlighting a relevant role for the TP63 gene which was hypomethylated and overexpressed in subset #8. This observation was validated by quantitative PCR, which also revealed TP63 mRNA overexpression in additional nonsubset U-CLL cases. BcR stimulation had distinct effects on p63 protein expression, particularly leading to induction in subset #8, accompanied by increased CLL cell survival. This pro-survival effect was also supported by siRNA-mediated downregulation of p63 expression resulting in increased apoptosis. In conclusion, we report that DNA methylation profiles may vary even among CLL patients with similar somatic hypermutation status, supporting a compartmentalized approach to dissecting CLL biology. Furthermore, we highlight p63 as a novel prosurvival factor in CLL, thus identifying another piece of the complex puzzle of clinical aggressiveness. What's new? In chronic lymphocytic leukemia (CLL), cases with unmutated immunoglobulin receptors (U-CLL) are generally associated with inferior outcome, albeit still displaying considerable heterogeneity. Might such differences in CLL progression be explained by epigenetics? In this study, the authors found that an unusually aggressive subset of CLLs called subset #8 has a distinctive DNA-methylation profile. They also found that p63 is a novel pro-survival factor for CLL cells. These molecular studies may lead to new prognostic biomarkers, and possibly new therapeutic targets, for CLL.

Key Points Microenvironmental stimuli affect EZH2 expression and function in CLL. Combined B-cell signaling and EZH2 inhibition showed synergistic effects on primary CLL cells.

During B cell maturation, the complex process of immunoglobulin (IG) gene V(D)J recombination coupled with somatic hypermutation (SHM) gives rise to a unique DNA sequence within each individual B cell. Since B cell malignancies result from the clonal expansion of a single cell, IG genes represent a unique molecular signature common to all the malignant cells within an individual patient; thus, IG gene rearrangements can be used as clonal markers. In addition to serving as an important clonal identifier, the IG gene sequence can act as a 'molecular timeline' since it is associated with specific developmental stages and hence reflects the history of the B cell involved in the neoplastic transformation. Moreover, for certain malignancies, in particular chronic lymphocytic leukemia (CLL), the IG gene sequence holds prognostic and potentially predictive capabilities. That said, extrapolating meaningful conclusions from IG gene sequence analysis would be impossible if robust methods and tools were not available to aid in their analysis. This article, drawing on the vast experience of the European Research Initiative on CLL (ERIC), details the technical aspects and essential requirements necessary to ensure reliable and reproducible IG gene sequence analysis in CLL, a test that is now recommended for all CLL patients prior to treatment. More specifically, the various analytical stages are described ranging from the identification of the clonotypic IG gene rearrangement and the determination of the nucleotide sequence to the accurate clinical interpretation of the IG gene sequence data. De två första författarna delar förstaförfattarskapet.

Purpose: The role of antigen(s) in shaping the T-cell repertoire in chronic lymphocytic leukemia, although relevant for understanding malignant cell interactions with cognate T cells, is largely unexplored. Experimental Design: Here we profiled the T-cell receptor β chain gene repertoire in 58 chronic lymphocytic leukemia patients, focusing on cases assigned to well-characterized subsets with stereotyped clonotypic B-cell receptor immunoglobulins, therefore those cases most evidently selected by antigen (subsets #1, #2, and #4). Results: Remarkable repertoire skewing and oligoclonality were observed, and differences between subsets were noted regarding both T-cell receptor β chain gene usage and the extent of clonality, with subset #2 being the least oligoclonal. Longitudinal analysis of subset #4 cases revealed that although the repertoire may fluctuate over time, certain clonotypes persist, thus alluding to persistent antigenic stimulation. Shared (“stereotyped”) clonotypes were found between different patients, reflecting selection by common antigenic elements. Cross-comparison of our dataset with public databases showed that some T-cell clonotypes may have expanded secondary to common viral infections; however, the majority of clonotypes proved to be disease-specific. Conclusions: Overall, the T-cell receptor β chain repertoire in chronic lymphocytic leukemia is likely shaped by antigen selection and the implicated antigenic elements may concern epitopes that also select the malignant B-cell progenitors or, more intriguingly, chronic lymphocytic leukemia–derived epitopes. Clin Cancer Res; 22(1); 167–74. ©2015 AACR.

The BCL-2 inhibitor venetoclax has only limited activity in DLBCL despite frequent BCL-2 overexpression. Since constitutive activation of the B cell receptor (BCR) pathway has been reported in both ABC and GCB DLBCL, we investigated whether targeting SYK or BTK will increase sensitivity of DLBCL cells to venetoclax. We report that pharmacological inhibition of SYK or BTK synergistically enhances venetoclax sensitivity in BCL-2-positive DLBCL cell lines with an activated BCR pathway in vitro and in a xenograft model in vivo, despite the only modest direct cytotoxic effect. We further show that these sensitizing effects are associated with inhibition of the downstream PI3K/AKT pathway and changes in the expression of MCL-1, BIM, and HRK. In addition, we show that BCR-dependent GCB DLBCL cells are characterized by deficiency of the phosphatase SHP1, a key negative regulator of the BCR pathway. Re-expression of SHP1 in GCB DBLCL cells reduces SYK, BLNK, and GSK3 phosphorylation and induces corresponding changes in MCL1, BIM, and HRK expression. Together, these findings suggest that SHP1 deficiency is responsible for the constitutive activation of the BCR pathway in GCB DLBCL and identify SHP1 and BCL-2 as potential predictive markers for response to treatment with a venetoclax/BCR inhibitor combination.

Key Points S1PR1 mutations are present in 7.8% of patients with MCL and are significantly more frequent at relapse. S1PR1 mutations reduce expression of the S1PR1 receptor, which mediates migration towards the tissue-to-blood egress factor S1P in MCL.

As new generations of targeted therapies emerge and tumor genome sequencing discovers increasingly comprehensive mutation repertoires, the functional relationships of mutations to tumor phenotypes remain largely unknown. Here, we measured ex vivo sensitivity of 246 blood cancers to 63 drugs alongside genome, transcriptome, and DNA methylome analysis to understand determinants of drug response. We assembled a primary blood cancer cell encyclopedia data set that revealed disease-specific sensitivities for each cancer. Within chronic lymphocytic leukemia (CLL), responses to 62% of drugs were associated with 2 or more mutations, and linked the B cell receptor (BCR) pathway to trisomy 12, an important driver of CLL. Based on drug responses, the disease could be organized into phenotypic subgroups characterized by exploitable dependencies on BCR, mTOR, or MEK signaling and associated with mutations, gene expression, and DNA methylation. Fourteen percent of CLLs were driven by mTOR signaling in a non-BCR-dependent manner. Multivariate modeling revealed immunoglobulin heavy chain variable gene (IGHV) mutation status and trisomy 12 as the most important modulators of response to kinase inhibitors in CLL. Ex vivo drug responses were associated with outcome. This study overcomes the perception that most mutations do not influence drug response of cancer, and points to an updated approach to understanding tumor biology, with implications for biomarker discovery and cancer care.

PI3Kδ (idelalisib) and BTK (ibrutinib) inhibitors have demonstrated significant clinical activity in chronic lymphocytic leukemia (CLL) interfering with the cross-talk between CLL cells and the lymph node microenviroment, yet their mechanism of action remains to be fully elucidated. Here, we developed an ex vivo model with the aim of reproducing the effects of the microenvironment that would help shed light on the in vivo mechanism of action of idelalisib and ibrutinib and predict their clinical efficacy in individual patients. First we explored the effects of various cell-extrinsic elements on CLL apoptosis and proliferation and found that the combination of CpG+IL2+HS5 stromal cell line + human serum +CLL plasma and erythrocyte fractions represented the best co-culture conditions to test the effects of the novel inhibitors. Then, using this assay, we investigated the impact of idelalisib and ibrutinib on both survival and proliferation in 30 CLL patients. While both drugs had a limited direct pro-apoptotic activity, a potent inhibition of proliferation was achieved at clinically achievable concentrations. Notably, up to 10% of CLL cells still proliferated even at the highest concentrations, likely mirroring the known difficulty to achieve complete responses in vivo. Altogether, this novel assay represents an appropriate ex vivo drug testing system to potentially predict the clinical response to novel inhibitors in particular by quantifying the antiproliferative effect., This work was supported in part by H2020 “MEDGENET, Medical Genomics and Epigenomics Network” (Grant 692298), funded by the European Union; the Swedish Cancer Society, the Swedish Research Council, and Lion’s Cancer Research Foundation; Associazione Italiana per la Ricerca sul Cancro AIRC (Investigator Grant #20246 to PG and Special Program Molecular Clinical Oncology - 5 per mille #9965), Milano, Italy, Ricerca; ERA-NET TRANSCAN-2 JTC 2016, GCH-CLL. Vivia has received unrestricted research support from Gilead.

Altres ajuts: Supported by the IMI 2 HARMONY JU under GA No 116026, this JU receives support from the EU's H2020 R&I program and EFPIA. Further supported by the EU Horizon 2020 projects MEDGENET 692298, AEGLE 644906, projects CEITEC 2020 (LQ1601), NCMG research infrastructure (LM2015091 funded by MEYS CR), project FNBr 65269705, FM MU ROZV/24/LF/2016, DFG (SFB1074, projects B1 and B2, and EU (FIRE CLL)), and the Swedish Cancer Society and the Swedish Research Council. Publication reflects only the authors' views and the Commission is not responsible for any use that may be made of the information it contains. In chronic lymphocytic leukemia (CLL), TP53 gene defects, due to deletion of the 17p13 locus and/or mutation(s) within the TP53 gene, are associated with resistance to chemoimmunotherapy and a particularly dismal clinical outcome. On these grounds, analysis of TP53 aberrations has been incorporated into routine clinical diagnostics to improve patient stratification and optimize therapeutic decisions. The predictive implications of TP53 aberrations have increasing significance in the era of novel targeted therapies, i.e., inhibitors of B-cell receptor (BcR) signaling and anti-apoptotic BCL2 family members, owing to their efficacy in patients with TP53 defects. In this report, the TP53 Network of the European Research Initiative on Chronic Lymphocytic Leukemia (ERIC) presents updated recommendations on the methodological approaches for TP53 mutation analysis. Moreover, it provides guidance to ensure that the analysis is performed in a timely manner for all patients requiring treatment and that the data is interpreted and reported in a consistent, standardized, and accurate way. Since next-generation sequencing technologies are gaining prominence within diagnostic laboratories, this report also offers advice and recommendations for the interpretation of TP53 mutation data generated by this methodology.

Our understanding of the DNA methylome and its impact on cancer evolution and disease progression is rapidly evolving. In this issue of Cancer Cell, Queiros et al. provide a detailed characterization of the DNA methylome in mantle cell lymphoma and reveal novel molecular subtypes, potentially with different cellular origins.

The activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) is characterized by the chronic activation of signaling initiated by immunoglobulin-μ (IgM). By analyzing DNA copy profiles of 1,000 DLBCLs, we identified gains of 18q21.2 as the most frequent genetic alteration in ABC-like DLBCL. We show that these alterations target the TCF4 (E2-2) transcription factor, and that over-expression of TCF4 leads to its occupancy on immunoglobulin gene enhancers and increased expression of IgM at the transcript and protein level. The TCF4 gene is one of the top BRD4-regulated genes in DLBCL. Using a BET proteolysis-targeting chimera (PROTAC) we show that TCF4 and IgM expression can be extinguished, and ABC-like DLBCL cells can be killed in vitro and in vivo. This highlights a novel genetic mechanism for promoting immunoglobulin signaling in ABC-like DLBCL and provides a functional rationale for the use of BET inhibitors in this disease.

With the introduction of next-generation sequencing, the genetic landscape of the complex group of B-cell lymphoid malignancies has rapidly been unravelled in recent years. This has provided important information about recurrent genetic events and identified key pathways deregulated in each lymphoma subtype. In parallel, there has been intense search and development of novel types of targeted therapy that ‘hit’ central mechanisms in lymphoma pathobiology, such as BTK, PI3K or BCL2 inhibitors. In this review, we will outline the current view of the genetic landscape of selected entities: follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, chronic lymphocytic leukaemia and marginal zone lymphoma. We will detail recurrent alterations affecting important signalling pathways, that is the B-cell receptor/NF-κB pathway, NOTCH signalling, JAK-STAT signalling, p53/DNA damage response, apoptosis and cell cycle regulation, as well as other perhaps unexpected cellular processes, such as immune regulation, cell migration, epigenetic regulation and RNA processing. Whilst many of these pathways/processes are commonly altered in different lymphoid tumors, albeit at varying frequencies, others are preferentially targeted in selected B-cell malignancies. Some of these genetic lesions are either involved in disease ontogeny or linked to the evolution of each disease and/or specific clinicobiological features, and some of them have been demonstrated to have prognostic and even predictive impact. Future work is especially needed to understand the therapy-resistant disease, particularly in patients treated with targeted therapy, and to identify novel targets and therapeutic strategies in order to realize true precision medicine in this clinically heterogeneous patient group.

For decades, it has been known that patients with certain autoimmune and inflammatory disorders, such as rheumatoid arthritis (RA) and primary Sjögren's syndrome (pSS), have an increased risk of developing malignant lymphoma. Although the clinico-biological reasons for this association remain largely unknown, our knowledge has improved and new insights have been obtained. First, the direct link between autoimmunity and lymphomagenesis has been strengthened by large epidemiological studies showing a consistent risk increase of lymphoma associated with certain autoimmune/inflammatory conditions in independent cohorts from different countries. Second, a number of local and systemic disease-related risk factors in these diseases have been repeatedly linked to lymphoma development, with the prime examples being disease severity and the degree of inflammatory activity. Considering the key role of B- and T-cell activation in the pathogenesis of both autoimmunity and lymphoma, it is perhaps not surprising that longstanding chronic inflammation and/or antigen stimulation have emerged as major predisposing factors of lymphoma in patients with active autoimmune disease. Finally, increasing evidence suggests that lymphomas associated with autoimmunity constitute a different spectrum of entities compared to lymphomas arising in patients without any known autoimmune or inflammatory conditions, pointing to a different pathobiology. In this review, we summarize the recent literature that supports a direct or indirect link between immune-mediated disease and lymphoma and describe the characteristics of lymphomas developing in the different diseases. We also discuss molecular, genetic and microenvironmental factors that may come into play in the pathobiology of these disorders.

The chromatin modifier EZH2 is overexpressed and associated with inferior outcome in mantle cell lymphoma (MCL). Recently, we demonstrated preferential DNA methylation of HOX genes in MCL compared with chronic lymphocytic leukemia (CLL), despite these genes not being expressed in either entity. Since EZH2 has been shown to regulate HOX gene expression, to gain further insight into its possible role in differential silencing of HOX genes in MCL vs. CLL, we performed detailed epigenetic characterization using representative cell lines and primary samples. We observed significant overexpression of EZH2 in MCL vs. CLL. Chromatin immune precipitation (ChIP) assays revealed that EZH2 catalyzed repressive H3 lysine 27 trimethylation (H3K27me3), which was sufficient to silence HOX genes in CLL, whereas in MCL H3K27me3 is accompanied by DNA methylation for a more stable repression. More importantly, hypermethylation of the HOX genes in MCL resulted from EZH2 overexpression and subsequent recruitment of the DNA methylation machinery onto HOX gene promoters. The importance of EZH2 upregulation in this process was further underscored by siRNA transfection and EZH2 inhibitor experiments. Altogether, these observations implicate EZH2 in the long-term silencing of HOX genes in MCL, and allude to its potential as a therapeutic target with clinical impact.

Chronic lymphocytic leukemia (CLL) is a clinically and biologically heterogeneous disease where the majority of patients have an indolent disease course, while others may experience a far more aggressive disease, treatment failure and poor overall survival. During the last two decades, there has been an intense search to find novel biomarkers that can predict prognosis as well as guide treatment decisions. Two of the most reliable molecular prognostic markers, both of which are offered in routine diagnostics, are the immunoglobulin heavy chain variable (IGHV) gene mutational status and fluorescence in situ hybridization (FISH) detection of prognostically relevant genomic aberrations (e.g. 11q-, 13q-, +12 and 17p-). In addition to these markers, a myriad of additional biomarkers have been postulated as potential prognosticators in CLL, on the protein (e.g. CD38, ZAP70, TCL1), the RNA (e.g. LPL, CLLU1, micro-RNAs) and the genomic (e.g. TP53, NOTCH1, SF3B1 and BIRC3 mutations) level. Efforts are now being made to test these novel markers in larger patient cohorts as well as in prospective trials, with the ultimate goal to combine the "best" markers in a "CLL prognostic index" applicable for the individual patient. Although it is clear that these studies have significantly improved our knowledge regarding both prognostication and the biology of the disease, there is still an immediate need for recognizing biomarkers that can predict therapy response, and efforts should now focus on addressing this pertinent issue. In the present article, we review the extensive literature in the field of prognostic markers in CLL, focus on the most clinically relevant markers and discuss future directions regarding biomarkers in CLL.

Over the past two decades, aberrant DNA methylation has emerged as a key player in the pathogenesis of chronic lymphocytic leukemia (CLL), and knowledge regarding its biological and clinical consequences in this disease has evolved rapidly. Since the initial studies relating DNA hypomethylation to genomic instability in CLL, a plethora of reports have followed showing the impact of DNA hypermethylation in silencing vital single gene promoters and the reversible nature of DNA methylation through inhibitor drugs. With the recognition that DNA hypermethylation events could potentially act as novel prognostic and treatment targets in CLL, the search for aberrantly methylated genes, gene families and pathways has ensued. Subsequently, the advent of microarray and next-generation sequencing technologies has supported the hunt for such targets, allowing exploration of the methylation landscape in CLL at an unprecedented scale. In light of these analyses, we now understand that different CLL prognostic subgroups are characterized by differential methylation profiles; we recognize DNA methylation of a number of signaling pathways genes to be altered in CLL, and acknowledge the role of DNA methylation outside of traditional CpG island promoters as fundamental players in the regulation of gene expression. Today, the significance and timing of altered DNA methylation within the complex epigenetic network of concomitant epigenetic messengers such as histones and miRNAs is an intensive area of research. In CLL, it appears that DNA methylation is a rather stable epigenetic mark occurring rather early in the disease pathogenesis. However, other consequences, such as how and why aberrant methylation marks occur, are less explored. In this review, we will not only provide a comprehensive summary of the current literature within the epigenetics field of CLL, but also highlight some of the novel findings relating to when, where, why and how altered DNA methylation materializes in CLL.

Recent studies have revealed recurrent mutations of the NOTCH1, SF3B1 and BIRC3 genes in chronic lymphocytic leukemia (CLL), especially among aggressive, chemorefractory cases. Nevertheless, it is currently unknown whether their presence may differ in subsets of patients carrying stereotyped B-cell receptors and also exhibiting distinct prognoses. Here, we analyzed the mutation status of NOTCH1, SF3B1 and BIRC3 in three subsets with particularly poor prognosis, that is, subset # 1, # 2 and # 8, aiming to explore links between genetic aberrations and immune signaling. A remarkably higher frequency of SF3B1 mutations was revealed in subset # 2 (44%) versus subset # 1 and # 8 (4.6% and 0%, respectively; P