BCL3 rearrangements in B-cell lymphoid neoplasms occur in two breakpoint clusters associated with different diseases.

The t(14;19)(q32;q13) often juxtaposes BCL3 with immunoglobulin heavy chain (IGH) resulting in overexpression of the gene. In contrast to other oncogenic translocations, BCL3 rearrangement (BCL3-R) has been associated with a broad spectrum of lymphoid neoplasms. Here we report an integrative whole-genome sequence, transcriptomic, and DNA methylation analysis of 13 lymphoid neoplasms with BCL3-R. The resolution of the breakpoints at single base-pair revealed that they occur in two clusters at 5' (n=9) and 3' (n=4) regions of BCL3 associated with two different biological and clinical entities. Both breakpoints were mediated by aberrant class switch recombination of the IGH locus. However, the 5' breakpoints (upstream) juxtaposed BCL3 next to an IGH enhancer leading to overexpression of the gene whereas the 3' breakpoints (downstream) positioned BCL3 outside the influence of the IGH and were not associated with its expression. Upstream BCL3-R tumors had unmutated IGHV, trisomy 12, and mutated genes frequently seen in chronic lymphocytic leukemia (CLL) but had an atypical CLL morphology, immunophenotype, DNA methylome, and expression profile that differ from conventional CLL. In contrast, downstream BCL3-R neoplasms were atypical splenic or nodal marginal zone lymphomas (MZL) with mutated IGHV, complex karyotypes and mutated genes typical of MZL. Two of the latter four tumors transformed to a large B-cell lymphoma. We designed a novel fluorescence in situ hybridization assay that recognizes the two different breakpoints and validated these findings in 17 independent tumors. Overall, upstream or downstream breakpoints of BCL3-R are mainly associated with two subtypes of lymphoid neoplasms with different (epi)genomic, expression, and clinicopathological features resembling atypical CLL and MZL, respectively.

A framework for the clinical implementation of optical genome mapping in hematologic malignancies.

Optical Genome Mapping (OGM) is rapidly emerging as an exciting cytogenomic technology both for research and clinical purposes. In the last 2 years alone, multiple studies have demonstrated that OGM not only matches the diagnostic scope of conventional standard of care cytogenomic clinical testing but it also adds significant new information in certain cases. Since OGM consolidates the diagnostic benefits of multiple costly and laborious tests (e.g., karyotyping, fluorescence in situ hybridization, and chromosomal microarrays) in a single cost-effective assay, many clinical laboratories have started to consider utilizing OGM. In 2021, an international working group of early adopters of OGM who are experienced with routine clinical cytogenomic testing in patients with hematological neoplasms formed a consortium (International Consortium for OGM in Hematologic Malignancies, henceforth "the Consortium") to create a consensus framework for implementation of OGM in a clinical setting. The focus of the Consortium is to provide guidance for laboratories implementing OGM in three specific areas: validation, quality control and analysis and interpretation of variants. Since OGM is a complex technology with many variables, we felt that by consolidating our collective experience, we could provide a practical and useful tool for uniform implementation of OGM in hematologic malignancies with the ultimate goal of achieving globally accepted standards.

NOTCH1 mutation in chronic lymphocytic leukaemia is associated with an enhanced cell cycle G1/S transition and specific cyclin overexpression: Preclinical ground for targeted inhibition.

Studies prior to next-generation sequencing (NGS) showed that the frequent indolent course of chronic lymphocytic leukaemia (CLL) is related to most cells remaining quiescent in the G0 -G1 cell cycle phase, due to the expression of dysregulated cyclin genes. Of note, the activating nature of the NOTCH1 mutation in T lymphoblastic leukaemia also drives the dysregulation of cell cycle genes. Our goal was to comprehensively revisit the cell cycle in NOTCH1-mutated CLL (NOTCH1MUT ) to test for potential therapeutic targets. Among 378 NGS-annotated CLL cases, NOTCH1MUT cells displayed a unique transcriptome profile of G0 -G1 cell cycle components, with an overexpression of early-phase effectors, reaching a 38-, 27- and ninefold change increase for the complex elements CCND3, CDK4 and CDK6, respectively. This NOTCH1MUT cells' profile was related to more cells traversing through the cell cycle. In-vitro targeted inhibition of NOTCH1 gamma-secretase and CDK4/6 reversed the distribution of cells through the cycle phases and enhanced the killing of NOTCH1MUT CLL cells, suggesting new therapeutic approaches.

High-throughput RNA sequencing of the T cell receptor alpha and beta chains for simultaneous clonality and biological analyses in Sezary syndrome.

BACKGROUND: Previous investigations pointed out a role for antigen stimulation in Sezary syndrome (SS). High-throughput sequencing of the T cell receptor (TR) offers several applications beyond diagnostic purposes, including the study of T cell pathogenesis. METHODS: We performed high-throughput RNA sequencing of the TR alpha (TRA) and beta (TRB) genes focusing on the complementarity-determining region 3 (CDR3) in 11 SS and one erythrodermic mycosis fungoides (MF) patients. Five psoriasis patients were employed as controls. Peripheral blood CD4+ cells were isolated and RNA sequenced (HiSeq2500). High-resolution HLA typing was performed in neoplastic patients. RESULTS: Highly expanded predominant TRA and TRB CDR3 were only found in SS patients (median frequency: 94.4% and 93.7%). No remarkable CDR3 expansions were observed in psoriasis patients (median frequency of predominant TRA and TRB CDR3: 0.87% and 0.69%, p < 0.001 compared to SS). CDR3 almost identical to the predominant were identified within each SS patient and were exponentially correlated with frequencies of the predominant CDR3 (R2 = 0.918, p < 0.001). Forty-six different CDR3 were shared between SS patients displaying HLA similarities, including predominant TRA and TRB CDR3 in one patient that were found in other three patients. Additionally, 351 antigen matches were detected (Cytomegalovirus, Epstein-Barr, Influenza virus, and self-antigens), and the predominant CDR3 of two different SS patients matched CDR3 with specificity for Influenza and Epstein-Barr viruses. CONCLUSIONS: Besides detecting clonality, these findings shed light on the nature of SS-related antigens, pointing to RNA sequencing as a useful tool for simultaneous clonality and biological analysis in SS.

Do GWAS-Identified Risk Variants for Chronic Lymphocytic Leukemia Influence Overall Patient Survival and Disease Progression?

Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults worldwide. Although genome-wide association studies (GWAS) have uncovered the germline genetic component underlying CLL susceptibility, the potential use of GWAS-identified risk variants to predict disease progression and patient survival remains unexplored. Here, we evaluated whether 41 GWAS-identified risk variants for CLL could influence overall survival (OS) and disease progression, defined as time to first treatment (TTFT) in a cohort of 1039 CLL cases ascertained through the CRuCIAL consortium. Although this is the largest study assessing the effect of GWAS-identified susceptibility variants for CLL on OS, we only found a weak association of ten single nucleotide polymorphisms (SNPs) with OS (p < 0.05) that did not remain significant after correction for multiple testing. In line with these results, polygenic risk scores (PRSs) built with these SNPs in the CRuCIAL cohort showed a modest association with OS and a low capacity to predict patient survival, with an area under the receiver operating characteristic curve (AUROC) of 0.57. Similarly, seven SNPs were associated with TTFT (p < 0.05); however, these did not reach the multiple testing significance threshold, and the meta-analysis with previous published data did not confirm any of the associations. As expected, PRSs built with these SNPs showed reduced accuracy in prediction of disease progression (AUROC = 0.62). These results suggest that susceptibility variants for CLL do not impact overall survival and disease progression in CLL patients.

Balanced and unbalanced translocations in a multicentric series of 2843 patients with chronic lymphocytic leukemia.

Chromosomal translocations in chronic lymphocytic leukemia (CLL) are very rare, and therefore systematic analysis of large series of cases is needed to allow the identification of recurrent rearrangements, breakpoints involved, and target genes. The aims of the present study were to identify new translocations and their clinical impact and to establish their frequency in a large cohort of 2843 CLL patients. By conventional cytogenetics 250 translocations were identified in 215 (7.5%) patients, 186 (74%) were apparently balanced and 64 (26%) were unbalanced. All chromosomes were involved in translocations, except Y chromosome. The chromosomes more frequently translocated were in decreasing frequency chromosomes 14, 18, 13, 17, 1, 6, 2, 3, 8, and 11. Translocations were found in the karyotypes either as the unique chromosomal abnormality (27%), associated with another alteration (24%), or as a part of a complex karyotype (CK, 48%). A large proportion of rearranged breakpoints involved genes related to CLL such as IGH (14q32), RB1, MIR15A, MIR16-1 (13q14), BCL2 (18q21), IGL (22q11.2), TP53 (17p13), IRF4 (6p25-p23), ATM (11q22), and CDK6 (7q21). Overall, 76 novel CLL translocations were identified, including a recurrent t(8;11)(p21;q21-23). Whole-genome sequencing and/or copy-number microarray data of 24 cases with translocations confirmed all rearrangements, enabled refinement of 3 karyotypes and all breakpoints at gene level. The projected survival and time to first treatment significantly decreased linearly with the number of translocations. In summary, this study allowed to establish the frequency of translocations (7.5%) and to identify new translocations in a cohort of 2843 CLL patients.

Chromosome banding analysis and genomic microarrays are both useful but not equivalent methods for genomic complexity risk stratification in chronic lymphocytic leukemia patients.

Genome complexity has been associated with poor outcome in patients with chronic lymphocytic leukemia (CLL). Previous cooperative studies established five abnormalities as the cut-off that best predicts an adverse evolution by chromosome banding analysis (CBA) and genomic microarrays (GM). However, data comparing risk stratification by both methods are scarce. Herein, we assessed a cohort of 340 untreated CLL patients highly enriched in cases with complex karyotype (CK) (46.5%) with parallel CBA and GM studies. Abnormalities found by both techniques were compared. Prognostic stratification in three risk groups based on genomic complexity (0-2, 3- 4 and ≥5 abnormalities) was also analyzed. No significant differences in the percentage of patients in each group were detected, but only a moderate agreement was observed between methods when focusing on individual cases (κ=0.507; P<0.001). Discordant classification was obtained in 100 patients (29.4%), including 3% classified in opposite risk groups. Most discrepancies were technique-dependent and no greater correlation in the number of abnormalities was achieved when different filtering strategies were applied for GM. Nonetheless, both methods showed a similar concordance index for prediction of time to first treatment (TTFT) (CBA: 0.67 vs. GM: 0.65) and overall survival (CBA: 0.55 vs. GM: 0.57). High complexity maintained its significance in the multivariate analysis for TTFT including TP53 and IGHV status when defined by CBA (hazard ratio [HR] 3.23; P<0.001) and GM (HR 2.74; P<0.001). Our findings suggest that both methods are useful but not equivalent for risk stratification of CLL patients. Validation studies are needed to establish the prognostic value of genome complexity based on GM data in future prospective studies.

Cytogenetic complexity in chronic lymphocytic leukemia: definitions, associations, and clinical impact.

Recent evidence suggests that complex karyotype (CK) defined by the presence of ≥3 chromosomal aberrations (structural and/or numerical) identified by using chromosome-banding analysis (CBA) may be relevant for treatment decision-making in chronic lymphocytic leukemia (CLL). However, many challenges toward the routine clinical application of CBA remain. In a retrospective study of 5290 patients with available CBA data, we explored both clinicobiological associations and the clinical impact of CK in CLL. We found that patients with ≥5 abnormalities, defined as high-CK, exhibit uniformly dismal clinical outcomes, independently of clinical stage, TP53 aberrations (deletion of chromosome 17p and/or TP53 mutations [TP53abs]), and the expression of somatically hypermutated (M-CLL) or unmutated immunoglobulin heavy variable genes. Thus, they contrasted with CK cases with 3 or 4 aberrations (low-CK and intermediate-CK, respectively) who followed aggressive disease courses only in the presence of TP53abs. At the other end of the spectrum, patients with CK and +12,+19 displayed an exceptionally indolent profile. Building upon CK, TP53abs, and immunoglobulin heavy variable gene somatic hypermutation status, we propose a novel hierarchical model in which patients with high-CK exhibit the worst prognosis, whereas those with mutated CLL lacking CK or TP53abs, as well as CK with +12,+19, show the longest overall survival. Thus, CK should not be axiomatically considered unfavorable in CLL, representing a heterogeneous group with variable clinical behavior. High-CK with ≥5 chromosomal aberrations emerges as prognostically adverse, independent of other biomarkers. Prospective clinical validation is warranted before ultimately incorporating high-CK in risk stratification of CLL.

Genomic arrays identify high-risk chronic lymphocytic leukemia with genomic complexity: a multi-center study.

Complex karyotype (CK) identified by chromosome-banding analysis (CBA) has shown prognostic value in chronic lymphocytic leukemia (CLL). Genomic arrays offer high-resolution genome-wide detection of copy-number alterations (CNAs) and could therefore be well equipped to detect the presence of a CK. Current knowledge on genomic arrays in CLL is based on outcomes of single center studies, in which different cutoffs for CNA calling were used. To further determine the clinical utility of genomic arrays for CNA assessment in CLL diagnostics, we retrospectively analyzed 2293 arrays from 13 diagnostic laboratories according to established standards. CNAs were found outside regions captured by CLL FISH probes in 34% of patients, and several of them including gains of 8q, deletions of 9p and 18p (p<0.01) were linked to poor outcome after correction for multiple testing. Patients (n=972) could be divided in three distinct prognostic subgroups based on the number of CNAs. Only high genomic complexity (high-GC), defined as ≥5 CNAs emerged as an independent adverse prognosticator on multivariable analysis for time to first treatment (Hazard ratio: 2.15, 95% CI: 1.36-3.41; p=0.001) and overall survival (Hazard ratio: 2.54, 95% CI: 1.54-4.17; p<0.001; n=528). Lowering the size cutoff to 1 Mb in 647 patients did not significantly improve risk assessment. Genomic arrays detected more chromosomal abnormalities and performed at least as well in terms of risk stratification compared to simultaneous chromosome banding analysis as determined in 122 patients. Our findings highlight genomic array as an accurate tool for CLL risk stratification.

A high proportion of cells carrying trisomy 12 is associated with a worse outcome in patients with chronic lymphocytic leukemia.

The prognosis of chronic lymphocytic leukemia (CLL) patients displaying trisomy 12 (+12) remains unclear. In this study, we analyzed the influence of the proportion of cells with +12, and other clinical and biologic factors, in time to first therapy (TTFT) and overall survival (OS), in 289 patients diagnosed with CLL carrying +12. Median OS was 129 months. One hundred seventy-four patients (60.2%) presented +12 in <60% of cells. TTFT and OS for this subgroup were longer than for the subgroup with +12 in ≥60% of cells, with a median TTFT of 49 months (CI95%, 39-58) vs 30 months (CI95%, 22-38) (P = 0.001); and a median OS of 159 months (CI95%, 119-182), vs 96 months (CI95%, 58-134) (P = 0.015). Other factors associated with a shorter TTFT were: Binet stage, B symptoms, lymphadenopathy, splenomegaly, high lymphocyte count, 11q-, high ß2 microglobulin, and high LDH. In the multivariate analysis, clinical stage, +12 in ≥60% of cells, high lymphocyte count, B symptoms, and 11q- in addition, resulted of significance in predicting shorter TTFT. Significant variables for OS were: Binet stage, lymphadenopathy, splenomegaly, high LDH, high ß2 microglobulin, 11q-, and CD38. In the multivariate analysis, only Binet stage, 11q-, and high ß2microglobulin significantly predicted shorter OS. CLL with +12 entails a heterogeneous group with intermediate prognosis. However, a high proportion of cells carrying +12 separates a subgroup of patients with poor outcome. Copyright © 2015 John Wiley & Sons, Ltd.

Interstitial 13q14 deletions detected in the karyotype and translocations with concomitant deletion at 13q14 in chronic lymphocytic leukemia: different genetic mechanisms but equivalent poorer clinical outcome.

Deletion of 13q14 as the sole abnormality is a good prognostic marker in chronic lymphocytic leukemia (CLL). Nonetheless, the prognostic value of reciprocal 13q14 translocations [t(13q)] with related 13q losses has not been fully elucidated. We described clinical and biological characteristics of 25 CLL patients with t(13q), and compared with 62 patients carrying interstitial del(13q) by conventional G-banding cytogenetics (CGC) [i-del(13q)] and 295 patients with del(13q) only detected by fluorescence in situ hybridization (FISH) [F-del(13q)]. Besides from the CLL FISH panel (D13S319, CEP12, ATM, TP53), we studied RB1 deletions in all t(13q) cases and a representative group of i-del(13q) and F-del(13q). We analyzed NOTCH1, SF3B1, and MYD88 mutations in t(13q) cases by Sanger sequencing. In all, 25 distinct t(13q) were described. All these cases showed D13S319 deletion while 32% also lost RB1. The median percentage of 13q-deleted nuclei did not differ from i-del(13q) patients (73% vs. 64%), but both were significantly higher than F-del(13q) (52%, P < 0.001). Moreover, t(13q) patients showed an increased incidence of biallelic del(13q) (52% vs. 11.3% and 14.9%, P < 0.001) and higher rates of concomitant 17p deletion (37.5% vs. 8.6% and 7.2%, P < 0.001). RB1 involvement was significantly higher in the i-del(13q) group (79%, P < 0.001). Two t(13q) patients (11.8%) carried NOTCH1 mutations. Time to first treatment in t(13q) and i-del(13q) was shorter than F-del(13q) (67, 44, and 137 months, P = 0.029), and preserved significance in the multivariate analysis. In conclusion, t(13q) and del(13q) patients detected by CGC constitute a subgroup within the 13q-deleted CLL patients associated with a worse clinical outcome.

Spontaneous Regression Accompanied by Concomitant Immune Alterations in a Patient with Chronic Lymphocytic Leukemia.

Spontaneous regression (SR) of chronic lymphocytic leukemia (CLL) is a rare event (0.2% - 1%). Some advances have been made in understanding the tumor genetic characteristics of such patients, although the immunological mechanisms leading to SR remain unclear. We describe a series of immunological events related to regression dynamics, allowing the identification of a SR phase (associated with >99% reduction of CLL cells in peripheral blood and adenopathy resolution in less than one year, concurrently with a nine-fold increase in monocyte counts, high B2M and the appearance of an oligoclonal serum IgG band), followed by a persistent regression (PR) phase that was maintained for ≥17 months. Our observations highlight a role of monocytes and B2M in SR, potentially related to immune activation. The oligoclonal IgG band detected during SR was maintained in PR, suggesting either a change in the ability of malignant cells (IgM+IgD+IgG‒) to differentiate into IgG-secreting cells, or an anti-tumor humoral response from normal B cells. These findings imply immune and molecular mechanisms required to eliminate malignant cells and might suggest new immunotherapies for CLL.

Complex Karyotype Detection in Chronic Lymphocytic Leukemia: A Comparison of Parallel Cytogenetic Cultures Using TPA and IL2+DSP30 from a Single Center.

Current CLL guidelines recommend a two parallel cultures assessment using TPA and IL2+DSP30 mitogens for complex karyotype (CK) detection. Studies comparing both mitogens for CK identification in the same cohort are lacking. We analyzed the global performance, CK detection, and concordance in the complexity assessment of two cytogenetic cultures from 255 CLL patients. IL2+DSP30 identified more altered karyotypes than TPA (50 vs. 39%, p = 0.031). Moreover, in 71% of those abnormal by both, IL2+DSP30 identified more abnormalities and/or abnormal metaphases. CK detection was similar for TPA and IL2+DSP30 (10% vs. 11%). However, 11/33 CKs (33%) were discordant, mainly due to the detection of a normal karyotype or no metaphases in the other culture. Patients requiring treatment within 12 months after sampling (active CLL) displayed significantly more CKs than those showing a stable disease (55% vs. 12%, p < 0.001). Disease status did not impact cultures' concordance (κ index: 0.735 and 0.754 for stable and active). Although CK was associated with shorter time to first treatment (TTFT) using both methods, IL2+DSP30 displayed better accuracy than TPA for predicting TTFT (C-index: 0.605 vs. 0.580, respectively). In summary, the analysis of two parallel cultures is the best option to detect CKs in CLL. Nonetheless, IL2+DSP30 could be prioritized above TPA to optimize cytogenetic assessment in clinical practice.

Biallelic losses of 13q do not confer a poorer outcome in chronic lymphocytic leukaemia: analysis of 627 patients with isolated 13q deletion.

Losses in 13q as a sole abnormality confer a good prognosis in chronic lymphocytic leukaemia (CLL). Nevertheless, its heterogeneity has been demonstrated and the clinical significance of biallelic 13q deletions remains controversial. We compared the clinico-biological characteristics of a series of 627 patients harbouring isolated 13q deletions by fluorescence in situ hybridization (FISH), either monoallelic (13q × 1), biallelic (13q × 2), or the coexistence of both clones (13qM). The most frequent 13q deletion was 13q × 1 (82·1%), while 13q × 2 and 13qM represented 8·6% and 9·3% of patients respectively. The median percentage of altered nuclei significantly differed across groups: 55%, 72·5% and 80% in 13q × 1, 13q × 2 and 13qM (P < 0·001). However, no significant differences in the clinical outcome among 13q groups were found. From 84 patients with sequential FISH studies, eight patients lost the remaining allele of 13q whereas none of them changed from 13q × 2 to the 13q × 1 group. The percentage of abnormal cells detected by FISH had a significant impact on the five-year cumulative incidence of treatment and the overall survival, 90% being the highest predictive power cut-off. In conclusion, loss of the remaining 13q allele is not enough to entail a worse prognosis in CLL. The presence of isolated 13q deletion can be risk-stratified according to the percentage of altered cells.

Leukemic Involvement Is a Common Feature in Waldenström Macroglobulinemia at Diagnosis.

Waldenström Macroglobulinemia (WM) is a lymphoplasmacytic lymphoma with bone marrow (BM) involvement and IgM monoclonal gammopathy. To date, no studies have focused specifically on peripheral blood (PB) involvement. In this study, 100 patients diagnosed with WM according to the World Health Organization (WHO) criteria were included based on the demonstration of MYD88mut in BM and the availability of PB multiparametric flow cytometry (MFC) analysis. Leukemic involvement by MFC was detected in 50/100 patients. A low percentage of mature small lymphocytes in PB smears was observed in only 15 cases. MYD88mut by AS-qPCR was detected in PB in 65/100 cases. In cases with leukemic expression by MFC, MYD88mut was detected in all cases, and IGH was rearranged in 44/49 cases. In 21/50 patients without PB involvement by MFC, molecular data were consistent with circulating disease (MYD88mut by AS-qPCR 3/50, IGH rearranged 6/50, both 12/50). Therefore, PB involvement by standard techniques was detected in 71/100 patients. MYD88mut was detected in PB by dPCR in 9/29 triple negative cases. Overall, 80% of the patients presented PB involvement by any technique. Our findings support the role of PB MFC in the evaluation of patients with IgM monoclonal gammopathy and provide reliable information on correlation with molecular features. The development of a feasible MFC assay may stand as an objective tool in the classification of mature B cell neoplasms presenting with IgM monoclonal gammopathy.

Different prognostic impact of recurrent gene mutations in chronic lymphocytic leukemia depending on IGHV gene somatic hypermutation status: a study by ERIC in HARMONY.

Recent evidence suggests that the prognostic impact of gene mutations in patients with chronic lymphocytic leukemia (CLL) may differ depending on the immunoglobulin heavy variable (IGHV) gene somatic hypermutation (SHM) status. In this study, we assessed the impact of nine recurrently mutated genes (BIRC3, EGR2, MYD88, NFKBIE, NOTCH1, POT1, SF3B1, TP53, and XPO1) in pre-treatment samples from 4580 patients with CLL, using time-to-first-treatment (TTFT) as the primary end-point in relation to IGHV gene SHM status. Mutations were detected in 1588 (34.7%) patients at frequencies ranging from 2.3-9.8% with mutations in NOTCH1 being the most frequent. In both univariate and multivariate analyses, mutations in all genes except MYD88 were associated with a significantly shorter TTFT. In multivariate analysis of Binet stage A patients, performed separately for IGHV-mutated (M-CLL) and unmutated CLL (U-CLL), a different spectrum of gene alterations independently predicted short TTFT within the two subgroups. While SF3B1 and XPO1 mutations were independent prognostic variables in both U-CLL and M-CLL, TP53, BIRC3 and EGR2 aberrations were significant predictors only in U-CLL, and NOTCH1 and NFKBIE only in M-CLL. Our findings underscore the need for a compartmentalized approach to identify high-risk patients, particularly among M-CLL patients, with potential implications for stratified management.

Chronic lymphocytic leukaemia with 17p deletion: a retrospective analysis of prognostic factors and therapy results.

Patients with chronic lymphocytic leukaemia (CLL) whose tumour cells harbour a 17p deletion (17p-) are universally considered to have a poor prognosis. The deletion can be detected at diagnosis or during the evolution of the disease, particularly in patients who have received chemotherapy. We sought to evaluate the natural history of patients with 17p- CLL, identify predictive factors within this prognostic subgroup, and evaluate the results of different therapeutic approaches. Data from 294 patients with 17p- CLL followed up at 20 different institutions was retrospectively collected and analysed. Median age was 68 (range 27-98) years at the time of fluorescence in situ hybridization analysis. After 17p- documentation, 52% received treatment, achieving an overall response rate of 50%. Median overall survival was 41 months, and was significantly shorter in patients with elevated beta(2)-microglobulin concentration (P < 0·001), B symptoms (P = 0·016), higher percentage of cells with deletion (P < 0·001), and acquired deletions (P = 0·012). These findings suggest that patients with 17p- CLL have a variable prognosis that can be refined using simple clinical and laboratory features, including 17p- clone size, beta2-microglobulin concentration, presence of B symptoms and type of deletion (de novo versus acquired).

Outcomes and molecular profile of oligomonocytic CMML support its consideration as the first stage in the CMML continuum.

Patients with oligomonocytic chronic myelomonocytic leukemia (OM-CMML) are currently classified according to the 2017 World Health Organization myelodysplastic syndromes classification. However, recent data support considering OM-CMML as a specific subtype of chronic myelomonocytic leukemia (CMML), given their similar clinical, genomic, and immunophenotypic profiles. The main purpose of our study was to provide survival outcome data of a well-annotated series of 42 patients with OM-CMML and to compare them to 162 patients with CMML, 120 with dysplastic type (D-CMML), and 42 with proliferative type (P-CMML). OM-CMML had significantly longer overall survival (OS) and acute myeloid leukemia-free survival than did patients with CMML, considered as a whole group, and when compared with D-CMML and P-CMML. Moreover, gene mutations associated with increased proliferation (ie, ASXL1 and RAS-pathway mutations) were identified as independent adverse prognostic factors for OS in our series. We found that at a median follow-up of 53.47 months, 29.3% of our patients with OM-CMML progressed to D-CMML, and at a median follow-up of 46.03 months, 28.6% of our D-CMML group progressed to P-CMML. These data support the existence of an evolutionary continuum of OM-CMML, D-CMML, and P-CMML. In this context, we observed that harboring more than 3 mutated genes, carrying ASXL1 mutations, and a peripheral blood monocyte percentage >20% significantly predicted a shorter time of progression of OM-CMML into overt CMML. These variables were also detected as independent adverse prognostic factors for OS in OM-CMML. These data support the consideration of OM-CMML as the first evolutionary stage within the proliferative continuum of CMML.

Optical Genome Mapping: A Promising New Tool to Assess Genomic Complexity in Chronic Lymphocytic Leukemia (CLL).

Novel treatments in chronic lymphocytic leukemia (CLL) have generated interest regarding the clinical impact of genomic complexity, currently assessed by chromosome banding analysis (CBA) and chromosomal microarray analysis (CMA). Optical genome mapping (OGM), a novel technique based on imaging of long DNA molecules labeled at specific sites, allows the identification of multiple cytogenetic abnormalities in a single test. We aimed to determine whether OGM is a suitable alternative to cytogenomic assessment in CLL, especially focused on genomic complexity. Cytogenomic OGM aberrations from 42 patients were compared with CBA, FISH, and CMA information. Clinical−biological characteristics and time to first treatment (TTFT) were analyzed according to the complexity detected by OGM. Globally, OGM identified 90.3% of the known alterations (279/309). Discordances were mainly found in (peri-)centromeric or telomeric regions or subclonal aberrations (<15−20%). OGM underscored additional abnormalities, providing novel structural information on known aberrations in 55% of patients. Regarding genomic complexity, the number of OGM abnormalities had better accuracy in predicting TTFT than current methods (C-index: 0.696, 0.602, 0.661 by OGM, CBA, and CMA, respectively). A cut-off of ≥10 alterations defined a complex OGM group (C-OGM, n = 12), which included 11/14 patients with ≥5 abnormalities by CBA/CMA and one patient with chromothripsis (Kappa index = 0.778; p < 0.001). Moreover, C-OGM displayed enrichment of TP53 abnormalities (58.3% vs. 3.3%, p < 0.001) and a significantly shorter TTFT (median: 2 vs. 43 months, p = 0.014). OGM is a robust technology for implementation in the routine management of CLL patients, although further studies are required to define standard genomic complexity criteria.

TP53 Abnormalities Are Underlying the Poor Outcome Associated with Chromothripsis in Chronic Lymphocytic Leukemia Patients with Complex Karyotype.

Chromothripsis (cth) has been associated with a dismal outcome and poor prognosis factors in patients with chronic lymphocytic leukemia (CLL). Despite being correlated with high genome instability, previous studies have not assessed the role of cth in the context of genomic complexity. Herein, we analyzed a cohort of 33 CLL patients with cth and compared them against a cohort of 129 non-cth cases with complex karyotypes. Nine cth cases were analyzed using optical genome mapping (OGM). Patterns detected by genomic microarrays were compared and the prognostic value of cth was analyzed. Cth was distributed throughout the genome, with chromosomes 3, 6 and 13 being those most frequently affected. OGM detected 88.1% of the previously known copy number alterations and several additional cth-related rearrangements (median: 9, range: 3-26). Two patterns were identified: one with rearrangements clustered in the region with cth (3/9) and the other involving both chromothriptic and non-chromothriptic chromosomes (6/9). Cases with cth showed a shorter time to first treatment (TTFT) than non-cth patients (median TTFT: 2 m vs. 15 m; p = 0.013). However, when stratifying patients based on TP53 status, cth did not affect TTFT. Only TP53 maintained its significance in the multivariate analysis for TTFT, including cth and genome complexity defined by genomic microarrays (HR: 1.60; p = 0.029). Our findings suggest that TP53 abnormalities, rather than cth itself, underlie the poor prognosis observed in this subset.