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Long-range interactions between regulatory elements and promoters are key in gene transcriptional control; however, their study requires large amounts of starting material, which is not compatible with clinical scenarios nor the study of rare cell populations. Here we introduce low input capture Hi-C (liCHi-C) as a cost-effective, flexible method to map and robustly compare promoter interactomes at high resolution. As proof of its broad applicability, we implement liCHi-C to study normal and malignant human hematopoietic hierarchy in clinical samples. We demonstrate that the dynamic promoter architecture identifies developmental trajectories and orchestrates transcriptional transitions during cell-state commitment. Moreover, liCHi-C enables the identification of disease-relevant cell types, genes and pathways potentially deregulated by non-coding alterations at distal regulatory elements. Finally, we show that liCHi-C can be harnessed to uncover genome-wide structural variants, resolve their breakpoints and infer their pathogenic effects. Collectively, our optimized liCHi-C method expands the study of 3D chromatin organization to unique, low-abundance cell populations, and offers an opportunity to uncover factors and regulatory networks involved in disease pathogenesis. Here the authors present the low input capture Hi-C (liCHi-C) method, a cost-effective, flexible method to map and robustly compare promoter interactomes at high resolution. liCHi-C identifies new disease-associated genes and structural variants to ultimately illuminate their pathogenic effects.

Mantle cell lymphoma (MCL) is genetically characterized by the IG :: CCND1 translocation mediated by an aberrant V(D)J rearrangement. CCND1 translocations and overexpression have been identified in occasional aggressive B-cell lymphomas with unusual features for MCL. The mechanism generating CCND1 rearrangements in these tumors and their genomic profile are not known. We have reconstructed the IG :: CCND1 translocations and the genomic profile of 13 SOX11-negative aggressive B-cell lymphomas using whole genome/exome and target sequencing. The mechanism behind the translocation was an aberrant V(D)J rearrangement in three tumors and by an anomalous IGH class-switch recombination (CSR) or somatic hypermutation (SHM) mechanism in ten. The tumors with a V(D)J-mediated translocation were two blastoid MCL and one high-grade B-cell lymphoma. None of them had a mutational profile suggestive of DLBCL. The ten tumors with CSR/SHM-mediated IGH :: CCND1 were mainly large B-cell lymphomas, with mutated genes commonly seen in DLBCL and BCL6 rearrangements in 6. Two cases, which transformed from marginal zone lymphomas, carried mutations in KLF2 , TNFAIP3 and KMT2D . These findings expand the spectrum of tumors carrying CCND1 rearrangement that may occur as a secondary event in DLBCL mediated by aberrant CSR/SHM and associated with a mutational profile different from that of MCL.

The presence of a monoclonal protein detected by serum immunofixation electrophoresis (sIFE) has been reported as an adverse prognostic factor in chronic lymphocytic leukemia (CLL). However, the genetic underpinning of this finding has not been studied. We retrospectively studied 97 CLL patients with simultaneous information on sIFE and genetic alterations detected by next-generation sequencing. sIFE was positive in 49 patients. The most common isotypes were IgG κ (27%) and bi/triclonal (25%). A +sIFE was associated with a higher number of mutated genes [median 2 (range 0–3) vs. 0 (range 0–2), p = 0.006], and a higher frequency of unmutated IGHV status (60 vs. 29%, p = 0.004). An IgM monoclonal protein was associated with TP53 mutations (36% in IgM +sIFE vs. 12% in non-IgM +sIFE or –sIFE, p = 0.04), and bi/triclonal proteins with NOTCH1 mutations (33% in bi/triclonal vs. 9% in monoclonal +sIFE or –sIFE, p = 0.04). These data suggest an association between a +sIFE and a higher mutational burden, and some monoclonal isotypes with specific mutations.

Chronic lymphocytic leukaemia (CLL) is a frequent disease in which the genetic alterations determining the clinicobiological behaviour are not fully understood. Here we describe a comprehensive evaluation of the genomic landscape of 452 CLL cases and 54 patients with monoclonal B-lymphocytosis, a precursor disorder. We extend the number of CLL driver alterations, including changes in ZNF292 , ZMYM3 , ARID1A and PTPN11 . We also identify novel recurrent mutations in non-coding regions, including the 3′ region of NOTCH1 , which cause aberrant splicing events, increase NOTCH1 activity and result in a more aggressive disease. In addition, mutations in an enhancer located on chromosome 9p13 result in reduced expression of the B-cell-specific transcription factor PAX5. The accumulative number of driver alterations (0 to ≥4) discriminated between patients with differences in clinical behaviour. This study provides an integrated portrait of the CLL genomic landscape, identifies new recurrent driver mutations of the disease, and suggests clinical interventions that may improve the management of this neoplasia. Genomic approaches in more than 500 patients are used to extend the number of chronic lymphocytic leukaemia (CLL) driver alterations, and also identify novel recurrent mutations in non-coding regions, including an enhancer of PAX5 and the 3′ untranslated region of NOTCH1 , which lead to aberrant splicing events, increased NOTCH1 protein stability and activity, and an adverse clinical outcome. Driver genes in chronic lymphocytic leukaemia This paper from the International Cancer Genomic Consortium reports the results of a number of genomic approaches used to characterize chronic lymphocytic leukaemia and its precursor in more than 500 patients. As well as extending the number of chronic lymphocytic leukaemia driver alterations found in the coding portion of the genome, the study identies novel recurrent mutations in non-coding regions, including the 3′ UTR of NOTCH1 , which cause aberrant splicing events, an increase in NOTCH1 activity and more aggressive disease, as well as mutations in an enhancer which result in reduced expression of the B-cell-specific transcription factor PAX5.

Abstract The 2022 WHO revision and the ICC classification have recently modified the diagnostic criteria for chronic myelomonocytic leukemia (CMML) and acute myeloid leukemia. However, there is no consensus on whether CMML with NPM1 mutation (NPM1mut) should be diagnosed as AML. Nowadays, it is a subject of discussion because of its diagnostic and therapeutic implications. Therefore, we describe a case of a patient diagnosed with CMML NPM1mut and briefly review the literature to highlight the uncertainty about how to classify a CMML with NPM1 mutation. We emphasize the importance of a comprehensive molecular study, which is crucial to optimize the individualized treatment of patients, enabling them to access targeted therapies. This brief communication describes the case of a patient diagnosed with chronic myelomonocytic leukemia with NPM1 mutation and briefly review the literature to highlight the uncertainty about how to classify this condition.

The prognostic significance of lymphocyte doubling time (LDT) in chronic lymphocytic leukemia (CLL) was identified when the biology of the disease was poorly understood and therapy was not effective. We assessed the clinical and biological significance of LDT in 848 CLL patients in a real-life setting and the context of new biomarkers and effective therapy. A short LDT (≤12 months) was enriched for adverse biomarkers. Patients with a rapid LDT did need therapy shortly after diagnosis (median 23 months vs. not reached; p  < 0.001) and had a poorer overall survival (median 95 months vs. not reached p  < 0.001). LDT, IGHV mutational status, Beta-2 microglobulin, and Rai clinical stage were independent predictors for time to first treatment in the whole series and in Binet stage A patients. No correlation was observed between LDT and response to chemoimmunotherapy. However, a short LDT along with age ≥65 years, high-risk FISH (del(17p), del(11q)), unmutated IGHV, increased Beta-2 microglobulin, and TP53 mutations predicted short survival. Moreover, the prognostic significance of LDT was independent of the CLL-IPI and the Barcelona/Brno prognostic model. LDT remains an important outcome marker in the modern CLL era and should be incorporated into the clinical assessment and stratification of CLL patients.

Two new diagnostic classifications of acute myeloid leukemia (AML) were published in 2022 to update current knowledge on disease biology. In previous 2017-edition categories of AML with myelodysplasia-related changes, AML was not otherwise specified, but AML with mutated RUNX1 experienced profound changes. We performed whole exome sequencing on a cohort of 69 patients with cytogenetic intermediate-risk AML that belonged to these diagnostic categories to correlate their mutational pattern and copy-number alterations with their new diagnostic distribution. Our results show that 45% of patients changed their diagnostic category, being AML myelodysplasia-related the most enlarged, mainly due to a high frequency of myelodysplasia-related mutations (58% of patients). These showed a good correlation with multilineage dysplasia and/or myelodysplastic syndrome history, but at the same time, 21% of de novo patients without dysplasia also presented them. RUNX1 was the most frequently mutated gene, with a high co-occurrence rate with other myelodysplasia-related mutations. We found a high prevalence of copy-neutral loss of heterozygosity, frequently inducing a homozygous state in particular mutated genes. Mild differences in current classifications explain the diagnostic disparity in 10% of patients, claiming a forthcoming unified classification.

Despite emerging molecular information on chronic myelomonocytic leukemia (CMML), patient outcome remains unsatisfactory and little is known about the transformation to acute myeloid leukemia (AML). In a single-center cohort of 219 CMML patients, we explored the potential correlation between clinical features, gene mutations, and treatment regimens with overall survival (OS) and clonal evolution into AML. The most commonly detected mutations were TET2, SRSF2, ASXL1, and RUNX1. Median OS was 34 months and varied according to age, cytogenetic risk, FAB, CPSS and CPSS-Mol categories, and number of gene mutations. Hypomethylating agents were administered to 37 patients, 18 of whom responded. Allogeneic stem cell transplantation (alloSCT) was performed in 22 patients. Two-year OS after alloSCT was 60.6%. Six patients received targeted therapy with IDH or FLT3 inhibitors, three of whom attained a long-lasting response. AML transformation occurred in 53 patients and the analysis of paired samples showed changes in gene mutation status. Our real-world data emphasize that the outcome of CMML patients is still unsatisfactory and alloSCT remains the only potentially curative treatment. However, targeted therapies show promise in patients with specific gene mutations. Complete molecular characterization can help to improve risk stratification, understand transformation, and personalize therapy.

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.