Despite the well-established adverse impact of del(11q) in chronic lymphocytic leukemia (CLL), the prognostic significance of somatic ATM mutations remains uncertain. We evaluated the effects of ATM aberrations (del(11q) and/or ATM mutations) on time-to-first-treatment (TTFT) in 3631 untreated patients with CLL, in the context of IGHV gene mutational status and mutations in nine CLL-related genes. ATM mutations were present in 246 cases (6.8%), frequently co-occurring with del(11q) (112/246 cases, 45.5%). ATM-mutated patients displayed a different spectrum of genetic abnormalities when comparing IGHV-mutated (M-CLL) and unmutated (U-CLL) cases: M-CLL was enriched for SF3B1 and NFKBIE mutations, whereas U-CLL showed mutual exclusivity with trisomy 12 and TP53 mutations. Isolated ATM mutations were rare, affecting 1.2% of Binet A patients and <1% of M-CLL cases. While univariable analysis revealed shorter TTFT for Binet A patients with any ATM aberration compared to ATM-wildtype, multivariable analysis identified only del(11q), trisomy 12, SF3B1, and EGR2 mutations as independent prognosticators of shorter TTFT among Binet A patients and within M-CLL and U-CLL subgroups. These findings highlight del(11q), and not ATM mutations, as a key biomarker of increased risk of early progression and need for therapy, particularly in otherwise indolent M-CLL, providing insights into risk-stratification and therapeutic decision-making.

High genomic complexity is linked to poor prognosis in chronic lymphocytic leukaemia (CLL), but its independent prognostic value remains uncertain amid emerging biomarkers. We analysed copy number alterations (CNA) in 495 treatment-naïve patients from three randomized trials (CLL4, ADMIRE, ARCTIC), incorporating IGHV status, telomere length (TL), targeted sequencing, and DNA-methylation subtypes. Patients harboured low (LGC, 0-2 CNAs; n=334), intermediate (IGC, 3-4 CNAs; n=97), or high (HGC, >5 CNAs; n=64) genomic complexity. U-CLL (81%, p<0.001) and short TL (61%, p<0.05) were enriched in HGC, and TL inversely correlated with CNA burden (τ=-0.147, p<0.001). 62% of HGC patients were n-CLL. TP53 dysfunction was associated with HGC (36%, p<0.001). Trisomy 12 and NOTCH1 mutations, were enriched in LGC (p<0.001). HGC predicted shorter progression-free and overall survival in all univariate models but only remained independently prognostic for OS only in CLL4 (HR=1.61, p=0.02). Of 64 HGC patients, 23 had TP53 dysfunction; 92% of TP53 wild-type cases had other high-risk features (TL-S, U-CLL, or n-CLL). HGC is associated with adverse outcomes but may reflect underlying biological risk rather than serve as an independent biomarker. Its interplay with telomere attrition, immunogenetics, and epigenetic subtype warrants further validation in targeted therapy-treated cohorts. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by funded by Bloodwise (11052, 12036), the Kay Kendall Leukaemia Fund (873), Cancer Research UK (ECRIN-M3 accelerator award C42023/A29370, Southampton Experimental Cancer Medicine Centre grant C24563/A15581, Cancer Research UK Southampton Centre grant C34999/A18087, and programme C2750/A23669) and the Bournemouth Leukaemia Fund. The LRF CLL4 trial was funded by a core grant from Leukaemia and Lymphoma Research. Patient material was obtained from the UK CLL Trials Biobank, University of Liverpool, which is funded by Blood Cancer UK. ME acknowledges the support by The Arbib Charitable Fund. LC received a PhD studentship funded by Cancer Research UK and the Medical Research Council. The Baird lab is funded by Cancer Research UK programme C17199/A29202. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Somerset Regional Ethics Committee for the University of Southampton gave ethical approval for this work I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

In chronic lymphocytic leukemia (CLL), the role of complex karyotype (CK) for prognostic stratification remains a topic of debate, and the impact of specific cytogenetic abnormalities is still unclear. This study aims to investigate the clinical and biological features of CLL with t(14;19)(q32;q13) (tCLL) involving the BCL3 gene. Patients with tCLL were younger and more commonly presented unmutated IGHV gene, subset #8 stereotypy, trisomy of chromosome 12, and complex karyotype than other patients without t(14;19) (oCLL). The presence of t(14;19) was associated with a shorter time to treatment and overall survival compared to oCLL. Gene expression analysis revealed a unique transcriptome profile in tCLL, characterized by the upregulation of BCL3 and the activation of B-cell receptor, PI3K-Akt. Conversely, apoptosis-related pathways were suppressed in tCLL. While the BTK gene was upregulated, the BCL2L11 gene, coding for the pro-apoptotic protein BIM, was downregulated. Notably, patients with tCLL were characterized by a trend (p = 0.058) for a longer time to the next treatment with BTK inhibitors (BTKi) compared to those treated with a venetoclax-based (Ven-based) regimen. We underscore the adverse outcomes of tCLL, its distinct molecular features and gene expression patterns. Therefore, our data suggest that identifying tCLL could help tailor therapeutic approaches.

Almost one-third of all splenic marginal zone lymphoma (SMZL) cases express B-cell receptor immunoglobulin (BcR IG) encoded by the IGHV1-2*04 gene, implicating antigen selection in disease ontogeny. Evidence supporting this notion mostly derives from low-throughput sequencing approaches, which have limitations in capturing the full complexity of the BcR IG gene repertoire. This hinders the comprehensive assessment of the subclonal architecture of SMZL as shaped by antigen selection. To address this, we conducted a high-throughput immunogenetic investigation of SMZL aimed at the comprehensive characterization of the somatic hypermutation (SHM) and intraclonal diversification within the IG genes. We identified significant differences in the SHM and ID profiles between cases expressing the IGHV1-2*04 gene and those expressing other IGHV genes. Specifically, IGHV1-2*04 cases displayed (i) targeted SHM resulting in recurrent replacement SHMs, and (ii) significantly more pronounced intraclonal diversification, reflecting ongoing antigen selection. Overall, our findings suggest that SMZL cases expressing the IGHV1-2*04 gene have a distinct immunogenetic signature shaped by microenvironmental pressure on the clonotypic BcR IG, corroborating the idea that this group may represent a distinct molecular variant of SMZL.

In Chronic Lymphocytic Leukemia (CLL), t(14;19)(q32;q13), leading to the overexpression of BCL3, is found in ∼1

In chronic lymphocytic leukemia, the reliability of next-generation sequencing (NGS) to detect TP53 variants ≤10% allelic frequency (low-VAF) is debated. We tested the ability to detect 23 such variants in 41 different laboratories using their NGS method of choice. The sensitivity was 85.6%, 94.5%, and 94.8% at 1%, 2%, and 3% VAF cut-off, respectively. While only one false positive (FP) result was reported at >2% VAF, it was more challenging to distinguish true variants <2% VAF from background noise (37 FPs reported by 9 laboratories). The impact of low-VAF variants on time-to-second-treatment (TTST) and overall survival (OS) was investigated in a series of 1092 patients. Among patients not treated with targeted agents, patients with low-VAF TP53 variants had shorter TTST and OS versus wt-TP53 patients, and the relative risk of second-line treatment or death increased continuously with increasing VAF. Targeted therapy in ≥2 line diminished the difference in OS between patients with low-VAF TP53 variants and wt-TP53 patients, while patients with high-VAF TP53 variants had inferior OS compared to wild type-TP53 cases. Altogether, NGS-based approaches are technically capable of detecting low-VAF variants. No strict threshold can be suggested from a technical standpoint, laboratories reporting TP53 mutations should participate in a standardized validation set-up. Finally, whereas low-VAF variants affected outcomes in patients receiving chemoimmunotherapy, their impact on those treated with novel therapies remains undetermined. Our results pave the way for the harmonized and accurate TP53 assessment, which is indispensable for elucidating the role of TP53 mutations in targeted treatment.

Introduction: Coexisting trisomies of chromosomes 12 and 19 define a cytogenetic subgroup of CLL (+12+19 CLL) with shared clinicobiological characteristics. +12+19 CLL cases often (~70%) also carry trisomy 18; most cases (~85%) are CD38+ with high percentages of CD38+ cells; all cases with available FACS data express surface IgG with mutated IGHV genes; and, the clinical course is very indolent. Here, we sought to better understand +12+19 CLL through a thorough genomic and transcriptomic analysis. Methods and Results: Whole-genome sequencing in +12+19 CLL (n=10, 7 also harbored +18) revealed 3 cases with BIRC3 pathogenic variants: 2 with an identical c.1639delC frameshift deletion and 1 with a stop-gain SNV. We sought to validate this finding in a series of 47 independent +12+19 CLL cases (29/47 also harbored +18) using a target enrichment panel including 205 genes known as putative drivers in hematologic malignancies, including CLL, as well as a backbone of SNPs across the genome allowing the detection of copy-number alterations. We found 24/46 (52%) cases harboring BIRC3 pathogenic variants, for a total of 8 different variants that were located within the intervening region between the CARD and RING domains. The most frequent was c.1639delC, p.Q547Nfs*21 (VAF range: 3.9-42%), present in 19/24 (79%) BIRC3mut cases; in 7 cases it coexisted with other BIRC3 variants. To investigate the functional consequences of BIRC3 c.1639delC, we lentivirally expressed it in MEC1 cells using a doxycycline-inducible expression vector (pCW57-GFP-2A-MCS), resulting in >96% GFP+ cells. Western analysis confirmed a lower molecular weight band below the endogenous full-length protein, consistent with the predicted truncated form (p.Q547Nfs*21). The truncated protein was overexpressed compared to the full-length WT protein, suggesting dominant mutant expression. RNA-seq of MEC1 cells (biological triplicates) expressing BIRC3 c.1639delC versus empty-vector identified 2525 significantly deregulated genes (padj<0.05), 1547 up- (log₂FC>2) and 978 down-regulated (log₂FC<–2). Pronounced disruption of cell cycle regulatory networks was noted, with downregulation of key G1/S transition drivers (e.g. CCND1, CCNE1, CCNE2, CDC6, E2F1, E2F2) and core mitotic regulators (e.g. CDK1, CDK6, MAD2L1, PLK1), alongside upregulation of the cyclin-dependent kinase inhibitor CDKN1A. BIRC3mut MEC1 cells also showed upregulation of anti-apoptotic regulatory genes (BCL2L10, BCL2L1) and downregulation of pro-apoptotic mediators (FAS, CASP7, CASP8AP2). To better understand the hierarchy of genomic aberrations in relation to IG class switching, we profiled high-purity (>98%) IgM+ and IgG+ cell fractions obtained by FACS-sorting from 11 CLL cases with +12+19 CLL (5/11 also carried +18) using the gene panel mentioned above. In all cases, IgM+ cells represented a very minor population, ranging from 0.1-1.6% of total CD19+ cells. Eight cases (73%) harbored identical trisomies in both fractions, while 3/11 were negative for trisomies in the IgM+ fraction. BIRC3 c.1639delC was detected in both fractions of 4 cases, with higher VAF in IgG+ cells; in only IgG+ cells of 3 cases; and, in only IgM+ cells in 1 case. Clonally related mu and gamma IGHV-IGHD-IGHJ transcripts were detected in the IgM+ and IgG+ fractions, respectively, of 3 cases with available RNA. We also investigated the transcriptome of +12+19 CLL (n=16, 13 also harbored +18) by RNA-seq using as a comparator CLL stereotyped subset #4 (n=7) on the grounds that these two subgroups share IgG expression, mutated IGHV genes and indolent clinical courses. Compared to subset #4, +12+19 CLL displayed upregulation of genes associated with signaling, inflammation, cytokines, transcription, translation and metabolism. Prompted by this result, we studied the signaling capacity of +12+19 CLL (n=10) through BcR crosslinking with anti-IgG. However, this stimulation did not induce changes in either the phosphorylation status of ERK and PLCG2 or the calcium influx in B cells, as assessed by flow cytometry, indicating attenuated BcR signaling. Conclusion: We report a remarkable enrichment of BIRC3 mutations in +12+19 CLL. In this cytogenetic CLL subgroup, IgG+ cells coexist with infrequent IgM+ cells bearing shared genomic aberrations. On these grounds, class switching to IgG was apparently selected for in the clonal progenitors.

Recurrent mutations in the third base of U1 spliceosomal RNA responsible for marked splicing and expression abnormalities have been described in chronic lymphocytic leukemia (CLL) and some solid tumors. However, the clinical significance of these mutations in large and independent CLL cohorts as well as their presence in other B-cell neoplasms is unknown. Here we characterized U1 mutations in 1670 CLL and 363 mature B-cell lymphomas. We confirmed that the g.3A>C U1 mutation is found in 3.5% of CLL, which conferred rapid disease progression independently of the main biological and clinical prognostic markers of the disease. Additionally, a recurrent g.9C>T mutation was found in 1.5% of CLL causing downstream splicing alterations and associated with adverse prognosis. We also identified a g.4C>T mutation in 10% of diffuse large B-cell lymphomas of the germinal center subtype and a g.7A>G mutation in 30% of EBV-negative Burkitt lymphomas, both of which altered the splicing pattern of multiple genes. This study reveals novel, recurrent, and tumor-specific U1 mutations in mature B-cell neoplasms with biological and prognostic implications, thus establishing U1 as a novel pan-B-cell malignancy driver gene.

Immunogenetic evidence implicates the B cell receptor immunoglobulin (BcR IG) in the natural history of splenic marginal zone lymphoma (SMZL). Indeed, SMZL carries distinct features of somatic hypermutation (SHM) amongst cases utilizing particular IGHV genes in their BcR IG. Moreover, the BcR IG gene repertoire in SMZL is restricted, whereby ~30% of cases utilize the IGHV1-2*04 gene and allele, notable for carrying a tryptophan (W) residue at position VH FR3-75 instead of the arginine (R) residue that is encoded by all remaining IGHV1-2 gene alleles and almost all other human IGHV genes and alleles. IGHV1-2*04 cases display heterogeneous CDR3 features and light chains, as well as pronounced intraclonal diversification within the heavy chain gene rearrangements. Overall, this constellation of features argues for heavy chain dominance in this major immunogenetic subgroup of SMZL. Against that, scant information exists regarding the functional and structural properties of the clonotypic BcR IG in SMZL, while the relevance of the IGHV1-2*04 overuse remains an enigma. In order to address this knowledge gap, we performed immunological, biochemical and crystallographic studies of the clonotypic BcR IG from SMZL cases that we expressed as recombinant monoclonal antibodies (rmAbs). ELISA against molecules that are common antigenic targets of naturally-occurring autoantibodies and disease-occurring/related pathological autoantibodies, namely DNA, actin, myosin, β-amyloid, TNP, thyroglobulin, IgG F(ab)'2 fragments, and trinitrophenyl (TNP), revealed that SMZL rmAbs (n=42, of which 14 utilized the IGHV1-2*04) were poly/autoreactive, regardless of the expressed IGHV gene and their SHM status. Of note, comparison with CLL rmAbs (n=35), known for their poly/autoreactivity, revealed significantly (p<0.05) stronger binding of the SMZL rmAbs to all tested autoantigens on autoantigen protein microarrays. Autoreactivity was corroborated by flow cytometry showing that 38/42 SMZL rmAbs bound viable MEC1, HEK293 and HS-5 cells; and, by immunohistochemistry using the SMZL rmAbs as primary Abs, which documented binding to human tissues (appendix, tonsil, lymph nodes and kidney), albeit with distinct profiles depending on the expressed IGHV gene. Next, in order to assess the functional relevance of the W residue in IGHV1-2*04 BcR IG, we used PCR-based site-directed mutagenesis for modifying W75 to R (IGHV1-2*04 W75R, n=10), documenting significant (p<0.05) differences in antigen reactivity profile between the authentic IGHV1-2*04 rmAbs vs the respective IGHV1-2*04 W75R rmAbs. Further, in order to test the hypothesis of heavy chain dominance, we coupled the IGHV1-2*04 heavy chains with random light chains and compared their antigen reactivity profile versus the authentic rmAbs, observing no differences. Finally, we crystallized two different IGHV1-2*04 SMZL BcR Fabs and determined their structures to high resolution. In all crystals obtained, regardless of pH and precipitating agent, pairs of Fab molecules were found to interact homotypically through a symmetrical stacking cation-π interaction mediated by the allele-specific W75 residue and side chain of R95 in the IGHV1-2*04 heavy chain, burying an extensive surface of ~650 Å2. In silico analysis with FOLDX highlighted the high energetic contribution of these residues in promoting the formation of the homodimers in the crystals. In conclusion, we document pronounced poly/autoreactivity in SMZL suggesting that SMZL likely derives from a progenitor B-cell population with highly restricted BcR IG structures and autoreactive potential. We also provide experimental support to the hypothesis of heavy chain dominance in the IGHV1-2*04 subgroup which raises the intriguing possibility that the IGHV1-2*04 heavy chain may represent a target for immunotherapeutic interventions in a sizeable fraction of SMZL.

The epiCMIT (epigenetically determined Cumulative MIToses) mitotic clock traces B-cell mitotic history via DNA methylation changes in heterochromatin and H3K27me3-containing chromatin. While high scores correlated with poor outcomes in CLL and MCL, its prognostic significance in SMZL remains unknown. Derived from 142 SMZL cases using DNA methylation microarrays, epiCMIT values were correlated with genomic, transcriptomic, and clinical data. EpiCMIT as a continuous variable was significantly higher in females (p=0.02), patients with IGHV1-2*04 allele usage (p<0001), intermediate IGHV somatic hypermutation load (97-99.9% identity, p=0.04), elevated mutational burden (25 vs. 17 mut/Mb, p=0.001), driver gene mutations [KLF2 (p<0.001), NOTCH2 (p<0.01), TP53 (p=0.01), KMT2D (p<0.001)], and del(7q) (p=0.01). Negative correlation between epiCMIT and telomere length (r=-0.29 p<0.001) supported the association between cumulated proliferation and telomere attrition. While univariate analysis highlighted epiCMIT as robust predictor of shorter treatment-free survival (TFS), multivariate analysis confirmed epiCMIT as an independent marker for shorter TFS. In summary, our matched multi-omic datasets facilitate the clinico-biological characterization of SMZL and introduces epiCMIT as a strong prognostic marker, identifying high-risk patients and predicting reduced treatment-free survival, hence providing a new tool for risk-adapted patient management.### Competing Interest StatementR.R. had received honoraria from AbbVie, AstraZeneca, Illumina, Janssen and Roche. L.S. had received consultancy from AbbVie, AstraZeneca, BeiGene, Janssen and Lilly. P.G had received honoraria , consultancy and research funding from AbbVie, AstraZeneca, BeiGene, BMS, Lilly, Janssen, MSD and Roche. D.R. had received honoraria and research funding from AbbVie, AstraZeneca, BeiGene, BMS, Gilead, Janssen, Lilly and Kyte. M.A had received research funding from Pfizer. F.F had received honoraria from AbbVie, AstraZeneca, BeiGene and Janssen. R.W had received honoraria from AbbVie, AstraZeneca, BeiGene, Janssen and Secura bio. C.T. had received honoraria and/or consultancy from BMS, Janssen, Novartis, Roche, Abbvie, Gilead, Hospira, Amgen, Cellectis, Kyte, Takeda, Incyte, Bayer. All other authors have declared no competing interests.### Funding StatementThe study was funded by research grants from the Kay Kendall Leukemia Fund (873, 1104), and Cancer Research UK (ECRIN-M3 accelerator award C42023/A29370). MDF is supported by a postdoctoral grant from the Spanish Association Against Cancer. R.R is supported by grants from the Swedish Cancer Society, the Swedish Research Council, Region Stockholm, and Radiumhemmets Forskningsfonder, Stockholm. ### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:NHS South Central- Hampshire B Research ethics committee granted ethical approval for this workI confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors

Introduction: The introduction of targeted agents has radically changed the management of patients with chronic lymphocytic leukemia (CLL). Randomized controlled trials have shown the superiority of targeted agents over chemoimmunotherapy (CIT) in patients with relapsed/refractory (R/R) CLL. Following these trials, the treatment paradigm of CLL in daily practice shifted from CIT to targeted agents in any line of treatment. However, it is largely unknown if this has improved the overall survival (OS) of patients with R/R CLL in the real world. Methods: This is a retrospective, observational study that aimed to explore differences in the OS of patients with CLL in a real-world setting. Patients who received second-line treatment for CLL between 2010 and 2019 in five European countries (Czech Republic, Greece, Italy, Spain, and the United Kingdom) were eligible for the study. The patients were allocated to two different groups (CIT-era and BTKi-era groups) according to the actual use of BTKis in R/R settings in each country. The year when the use of BTKis surpassed 30% in any line was defined as the cutoff year for each country. Cases treated before the cutoff year were allocated to the CIT-era group, and cases treated after the cutoff year were allocated to the BTKi-era group. We compared the OS from the initial date of second line treatment between the two groups. The analysis was also done after adjusting for age at second line treatment through propensity score matching. Results: A total of 1877 patients (1384 in the CIT-era group and 493 in the BTKi-era group) from 38 centers were eligible for the study. The cutoff year was 2019, 2018, 2017, 2016, and 2014 for the Czech Republic, Greece, Italy, Spain, and the United Kingdom, respectively. In both groups, most patients were males [919 (66.4%) in the CIT-era and 337 (68.4%) in the BTKi-era group, p=0.461]. Patients in the CIT-era group were younger at both diagnosis [64 years (interquartile range (IQR)=57-71) vs 66 (IQR=59-74), p=0.001 for CIT-era and BTKi-era groups, respectively] and the start of second-line treatment [69 years (IQR=63-76) vs 73 (IQR=65, 79) p=0.001 for the CIT-era and BTKi-era groups, respectively]. The CIRS score was higher in the BTKi-era group [3 (IQR=1.25-6) vs 4 (IQR=2-6), p=0.053 for the CIT-era and BTKi-era groups, respectively]. Unfavorable disease biomarkers were similarly distributed among the two groups [del(11q): 215 (27.7%) vs. 80 (24.2%), p=0.26 | del(17p): 117 (14.7%) vs. 54 (15.7%), p=0.7 | TP53 mutations: 94 (19%) vs. 41 (17.3%), p=0.66 | unmutated immunoglobulin heavy variable (IGHV) gene status: 792 (78.8%) vs 276 (76.2%), p=0.35 for the CIT-era and BTKi-era groups, respectively]. The follow-up time from diagnosis (153 months [95% confidence intervals (CI):149-160) vs 106.6 (95% CI:98-115) for CIT-era and BTKi-era groups, respectively] and second-line treatment [81 months (95% CI:77-86) vs 30 (95% CI:27- 33) for CIT-era and BTKi-era groups, respectively] were longer for the CIT-era group. Patients in the BTKi-era group had statistically significantly better OS compared to the CIT-era group (Hazard ratio (HR): 0.74 95% CI: 0.61-0.91, p=0.003). This difference was even more pronounced when comparing OS using propensity score matching (HR: 0.71, 95% CI: 0.58-0.88, p=0.001). The median OS for the CIT-era group was 65 months (95% CI=57-71), while the median OS for the BTKi-era group was not reached (95% CI=not estimable, not estimable). Finally, we performed a univariable and multivariable analysis (MVA) to assess the risk factors for death in the entire population. TP53 aberrations and unmutated IGHV gene status remained the only statistically significant risk factors for death in the MVA (HR: 1.7, 95% CI: 1.03-3.13, p=0.039, HR: 2.48, 95% CI: 1.08-5.66, p=0.032, respectively). The only protective factor against death was treatment with BTKis in the R/R setting (HR: 0.5, 95% CI: 0.28-0.88, p=0.017). Conclusion: Our findings demonstrated that the availability of BTKis has improved the OS of patients with R/R CLL in the real-world setting.

Splenic marginal zone lymphoma (SMZL) is a rare, predominantly indolent B-cell lymphoma constituting fewer than 2% of lymphoid neoplasms. However, around 30% of patients have a shorter survival despite currently available treatments and the prognosis is especially poor for the 5–15% of cases that transform to a large cell lymphoma. Mounting evidence suggests that the molecular pathogenesis of SMZL is critically shaped by microenvironmental triggering and cell-intrinsic aberrations. Immunogenetic investigations have revealed biases in the immunoglobulin gene repertoire, indicating a role of antigen selection. Furthermore, cytogenetic studies have identified recurrent chromosomal abnormalities such as deletion of the long arm of chromosome 7, though specific disease-associated genes remain elusive. Our knowledge of SMZL’s mutational landscape, based on a limited number of cases, has identified recurring mutations in KLF2, NOTCH2, and TP53, as well as genes clustering within vital B-cell differentiation pathways. These mutations can be clustered within patient subgroups with different patterns of chromosomal lesions, immunogenetic features, transcriptional signatures, immune microenvironments, and clinical outcomes. Regarding SMZL epigenetics, initial DNA methylation profiling has unveiled epigenetically distinct patient subgroups, including one characterized by elevated expression of Polycomb repressor complex 2 (PRC2) components. Furthermore, it has also demonstrated that patients with evidence of high historical cell division, inferred from methylation data, exhibit inferior treatment-free survival. This review provides an overview of our current understanding of SMZL’s molecular basis and its implications for patient outcomes. Additionally, it addresses existing knowledge gaps, proposes future research directions, and discusses how a comprehensive molecular understanding of the disease will lead to improved management and treatment choices for patients.

The presence of TP53 aberrations ( TP53ab) and/or unmutated IGHV genes (U-CLL) helps select initial treatment for CLL patients (Hallek 2018 & Walewska 2022). However, many other biomarkers identified in recent years have failed to impact clinical decisions due to their coexistence with poor-risk indicators and uncertainty in their predictive abilities, often without suitable validation in long-term Phase II/III trials with comprehensive molecular analysis. We evaluated the clinical significance of two biomarkers, DNA methylation-based epitype (DME) and telomere length (TL), in (immuno-)chemotherapy clinical trials using samples from UKCLL4 (n=304) and ARCTIC/ADMIRE (ARC/ADM) (n=215). To our knowledge, DME and TL have not been assessed in a single study. Therefore, we utilized previously published DME (Wojdacz, 2019) and TL data (Strefford, 2015 & Norris, 2019), supplemented with new MMQ-PCR data (n=60). TL cut-offs of short (TL-S, <2.92kb), intermediate (TL-I, 2.92-3.57kb), and long (TL-L, >3.57kb) and DME classifications of n-CLL (naive B-cell-like), i-CLL (intermediate B-cell-like) and m-CLL (memory B-cell-like) were employed. 73% of m-CLL and 50% of n-CLL patients harboured TL-L and TL-S, respectively (p<0.001, Figure 1). Additionally, n-CLL and TL-S were associated with poor-risk indicators, such as TP53ab and del(11q) (p<0.05, Figure 1). We then assessed the impact of 10 clinico-biological features, including DME, TL, age and treatment arm, on progression-free survival (PFS) and overall survival (OS) in CLL4 and ARC/ADM cohorts using univariate analysis (UA). The n-CLL group exhibited the shortest PFS and OS in the ARC/ADM cohort (PFS hazard ratio (HR):4.47, 95% confidence interval (CI):2.56-7.82 & OS HR:3.95, CI:1.66-9.35, p<0.01), greater than the presence of TP53ab (PFS HR:4.39, CI:2.69-7.17 & OS HR:3.77, CI:1.94-7.33, p<0.001). In CLL4, whilst TP53ab was the strongest predictor of PFS and OS (PFS HR:3.61, CI:2.39-5.44 & OS HR: 3.66, CI:2.4-5.57, p<0.001), both n-CLL (PFS HR:1.96, CI:1.32-2.9 & OS HR:2.8, CI:1.81-4.34, p<0.001) and TL-S (PFS HR:2.36, CI:1.7-3.29 & OS HR:2.66, CI:1.87-3.76, p<0.001) were in the top five predictors of PFS and OS along with U-CLL and biallelic ATM inactivation ( biATM). Next, we performed Kaplan-Meier subgroup analysis, investigating DME in TL subgroups and vice versa. Examination of both biomarkers in the opposing subgroups in the ARC/ADM cohort, showed that TL could further stratify the i-CLL subgroup, with TL-L predicting longer PFS (median:6.12 years) compared to TL-S (HR:5.78, CI:2.34-14.33, median:3.8 years, p<0.001) or TL-I (HR:3.29, CI:1.4-7.76, median:4.35 years, p<0.01). As this pairwise analysis suggested that DME and TL may differentially contribute to outcome, we performed a multivariate cox regression, whilst controlling for confounding variables such as TP53ab and U-CLL. Covariates that were significant in UA were included in a stepwise backwards elimination process until a final model was reached. The CLL4 models were based on 246 subjects with 221 PFS and 205 OS events, ARC/ADM models were based on 138 and 176 subjects with 86 and 45 events for PFS and OS, respectively. For PFS models, TL-S emerged as significant (CLL4 HR:2.14, CI:1.39-3.3, p<0.001 & ARC/ADM HR:2.18, CI:1.17-4.05, p<0.01) with a HR lower than TP53ab (CLL4 HR:3.38, CI:2.13-5.37 & ARC/ADM HR:4.94, CI:2.58-9.48, p<0.001). DME emerged as significant for PFS in the CLL4 cohort (n-CLL HR:2.35, CI:1.37-4.05, p<0.01), along with SF3B1 mutation and treatment arm (Figure 2). In ARC/ADM, U-CLL, TP53ab, TL-S, del11q and biATM emerged as significant predictors of PFS (Figure 2). For OS, in both cohorts, TP53ab (CLL4 HR:2.77, CI:1.74-4.4, p<0.001 & ARC/ADM HR:3.28, CI:1.64-6.55, p<0.001) and DME (CLL4 HR:2.07, CI:1.15-3.73, p<0.05 & ARC/ADM, HR:3.4, CI:1.14-10.12, p<0.05) were found to predict shorter survival (Figure 2). Currently, we are integrating additional IGHV/IGLV data into our analysis, including the presence of IGLV3-21R110. In conclusion, by assessing the individual contribution of DME and TL to disease survival, we found that both variables offer valuable independent prognostic information when included in statistical models with poor-risk genomic lesions. TL and DME could help identify IGHV-mutated patients destined to respond poorly to (immuno-)chemotherapy, that might be more favourably treated with targeted agents.

Analyzed IGH, IGK/L rearrangements. Annotation characteristics

Supplementary Table S1 from Inactivation of HOXA Genes by Hypermethylation in Myeloid and Lymphoid Malignancy is Frequent and Associated with Poor Prognosis

Improved classification of rare lymphoid neoplasms would be aided by a deeper understanding of their underlying molecular features and is important for diagnosis, prognosis and therapy. Tumor entities classified within the WHO category of splenic B cell lymphomas and leukemias often exhibit heterogenous, transecting features, and include hairy cell leukemia (HCL), splenic diffuse red pulp lymphoma (SDRPL), splenic marginal zone lymphoma (SMZL), and the newly described WHO entity, splenic B cell lymphoma/leukemia with prominent nucleoli (SBLPN); the latter including patients formerly classified as HCL-variant (HCL-V). Genome-wide epigenetic information provides a tumor cell fingerprint combining cell-of-origin and tumor-specific events. Here we used DNA methylation to perform an unbiased molecular subclassification and to explore novel biological aspects of these patients. Samples from patients with a pathological diagnosis of HCL, HCL-V, SDRPL and SMZL (made prior to the 5 th WHO revision and ICC classifications) were obtained from 19 institutions across 9 countries, totaling 367 patients. Cells were FACS-purified where necessary and DNA was analyzed by 450/850K Illumina DNA methylation arrays. Genetic mutations were assessed by whole-exome or targeted sequencing, IGHV-D-J sequences by Sanger sequencing, and copy number alterations (CNAs) by Illumina arrays. The 1000 most variable CpG methylation sites were used for k-means clustering. Recursive feature elimination/random forest algorithms were used to develop a classifier for DNA methylation-based subgroups with 98% accuracy. Unsupervised clustering of 197 patients diagnosed with HCL, HCL-V or SDRPL revealed 5 distinct DNA methylation (M) subgroups ( Figure 1). Subgroup assignment was stable throughout longitudinal sampling (including pre/post-treatment) and consistent between splenic, bone marrow and PBMC derived cells. A subgroup with universally clonal BRAF-V600E mutations and majority diagnosed as HCL was termed the M-HCL subgroup ( Table 1). Four other groups termed M-SBLPN1-4 contained all HCL-V and SDRPL diagnosed samples and were devoid of BRAF-V600E mutations. M-SBLPN1 comprised MAP2K1 mutations (91%) and was enriched for CREBBP, ARIDIA and TERT-promoter mutations. These patients displayed an HCL-like immunophenotype (64.3% CD25+) with 1/3 diagnosed as HCL. M-SBLPN2 exhibited the highest prevalence of TP53 mutations and concomitant genomic instability. Patients in M-SBLPN1,2 were enriched in unmutated IGHV4-34 rearrangements. M-SBLPN3,4 subgroups displayed an immunophenotype more dissimilar to HCL, mutated IGHV genes, and enrichment of IGLL5, SYK and BIRC3 mutations. M-SBLPN4 contained the most SDRPL samples, suggesting it may represent the SDRPL entity retained by the WHO. We next uncovered that 29/170 SMZL patients displayed DNA methylation patterns mapping to M-SBLPN2-4. These patients were phenotypically and molecularly similar to SBLPN (70% displaying villous morphology and depleted in IGHV1-2*04, NOTCH2, KLF2 mutations), likely representing SMZL patients suggested for reassignment to SBLPN in the updated WHO classification. To elucidate molecular pathways governing the biology of M-SBLPN subgroups, transcription factor motif enrichment analysis in hypomethylated genomic regions revealed selective activation of AP-1 in M-HCL along with ETS in M-SBLPN1,2. Both transcription factors are downstream of MAPK signaling, consistent with activating BRAF and MAP2K1 mutations in these subgroups. However, we observed strong ETS enrichment in the absence of MAP2K1 in M-SBLPN along with mutual exclusivity of MAP2K1 and TP53 mutations, suggesting TP53 mutations are driving ETS activation. Although lymphoid neoplasms rarely exhibit TERT promoter mutations, 83% of M-SBLPN1 patients showed the c.-124C>T mutation commonly observed in other cancers producing an ETS binding site and ectopic TERT activation. ETS activation and gain of an ETS site by mutation implies oncogenesis involves aberrant TERT activation in this subgroup. In summary, we have developed a DNA methylation-based classifier that resolves 4 SBLPN subgroups with distinct molecular features, and reclassifies a subset of SMZL and HCL patients, adding further information to the updated WHO/ICC entities. We reveal distinct biological pathways operating in M-SBLPN subgroups that may aid targeted therapy approaches.

PDF file - 442K, Functional Analysis of the ATM-p53-p21 pathway in CLL4 trial

Univariable and multivariable analysis for TTFT within M-CLL.