Comparing malignant monocytosis across the updated WHO and ICC classifications of 2022.

ABSTRACT: The World Health Organization (WHO) classification of hematolymphoid tumors and the International Consensus Classification (ICC) of 2022 introduced major changes to the definition of chronic myelomonocytic leukemia (CMML). To assess its qualitative and quantitative implications for patient care, we started with 3311 established CMML cases (according to WHO 2017 criteria) and included 2130 oligomonocytosis cases fulfilling the new CMML diagnostic criteria. Applying both 2022 classification systems, 356 and 241 of oligomonocytosis cases were newly classified as myelodysplastic (MD)-CMML (WHO and ICC 2022, respectively), most of which were diagnosed as myelodysplastic syndrome (MDS) according to the WHO 2017 classification. Importantly, 1.5 times more oligomonocytosis cases were classified as CMML according to WHO 2022 than based on ICC, because of different diagnostic criteria. Genetic analyses of the newly classified CMML cases showed a distinct mutational profile with strong enrichment of MDS-typical alterations, resulting in a transcriptional subgroup separated from established MD and myeloproliferative CMML. Despite a different cytogenetic, molecular, immunophenotypic, and transcriptional landscape, no differences in overall survival were found between newly classified and established MD-CMML cases. To the best of our knowledge, this study represents the most comprehensive analysis of routine CMML cases to date, both in terms of clinical characterization and transcriptomic analysis, placing newly classified CMML cases on a disease continuum between MDS and previously established CMML.

Tumor necrosis factor α promotes clonal dominance of KIT D816V+ cells in mastocytosis: role of survivin and impact on prognosis.

ABSTRACT: Systemic mastocytosis (SM) is defined by the expansion and accumulation of neoplastic mast cells (MCs) in the bone marrow (BM) and extracutaneous organs. Most patients harbor a somatic KIT D816V mutation, which leads to growth factor-independent KIT activation and accumulation of MC. Tumor necrosis factor α (TNF) is a proapoptotic and inflammatory cytokine that has been implicated in the clonal selection of neoplastic cells. We found that KIT D816V increases the expression and secretion of TNF. TNF expression in neoplastic MCs is reduced by KIT-targeting drugs. Similarly, knockdown of KIT or targeting the downstream signaling cascade of MAPK and NF-κB signaling reduced TNF expression levels. TNF reduces colony formation in human BM cells, whereas KIT D816V+ cells are less susceptible to the cytokine, potentially contributing to clonal selection. In line, knockout of TNF in neoplastic MC prolonged survival and reduced myelosuppression in a murine xenotransplantation model. Mechanistic studies revealed that the relative resistance of KIT D816V+ cells to TNF is mediated by the apoptosis-regulator BIRC5 (survivin). Expression of BIRC5 in neoplastic MC was confirmed by immunohistochemistry of samples from patients with SM. TNF serum levels are significantly elevated in patients with SM and high TNF levels were identified as a biomarker associated with inferior survival. We here characterized TNF as a KIT D816V-dependent cytokine that promotes clonal dominance. We propose TNF and apoptosis-associated proteins as potential therapeutic targets in SM.

2021 Update on MRD in acute myeloid leukemia: a consensus document from the European LeukemiaNet MRD Working Party.

Measurable residual disease (MRD) is an important biomarker in acute myeloid leukemia (AML) that is used for prognostic, predictive, monitoring, and efficacy-response assessments. The European LeukemiaNet (ELN) MRD Working Party evaluated standardization and harmonization of MRD in an ongoing manner and has updated the 2018 ELN MRD recommendations based on significant developments in the field. New and revised recommendations were established during in-person and online meetings, and a 2-stage Delphi poll was conducted to optimize consensus. All recommendations are graded by levels of evidence and agreement. Major changes include technical specifications for next-generation sequencing-based MRD testing and integrative assessments of MRD irrespective of technology. Other topics include use of MRD as a prognostic and surrogate end point for drug testing; selection of the technique, material, and appropriate time points for MRD assessment; and clinical implications of MRD assessment. In addition to technical recommendations for flow- and molecular-MRD analysis, we provide MRD thresholds and define MRD response, and detail how MRD results should be reported and combined if several techniques are used. MRD assessment in AML is complex and clinically relevant, and standardized approaches to application, interpretation, technical conduct, and reporting are of critical importance.

Higher-order connections between stereotyped subsets: implications for improved patient classification in CLL.

Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins. Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR immunoglobulin stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR immunoglobulin stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. To address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29 856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed "satellites," were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL.

Dark-matter matters: Discriminating subtle blood cancers using the darkest DNA.

The confluence of deep sequencing and powerful machine learning is providing an unprecedented peek at the darkest of the dark genomic matter, the non-coding genomic regions lacking any functional annotation. While deep sequencing uncovers rare tumor variants, the heterogeneity of the disease confounds the best of machine learning (ML) algorithms. Here we set out to answer if the dark-matter of the genome encompass signals that can distinguish the fine subtypes of disease that are otherwise genomically indistinguishable. We introduce a novel stochastic regularization, ReVeaL, that empowers ML to discriminate subtle cancer subtypes even from the same 'cell of origin'. Analogous to heritability, implicitly defined on whole genome, we use predictability (F1 score) definable on portions of the genome. In an effort to distinguish cancer subtypes using dark-matter DNA, we applied ReVeaL to a new WGS dataset from 727 patient samples with seven forms of hematological cancers and assessed the predictivity over several genomic regions including genic, non-dark, non-coding, non-genic, and dark. ReVeaL enabled improved discrimination of cancer subtypes for all segments of the genome. The non-genic, non-coding and dark-matter had the highest F1 scores, with dark-matter having the highest level of predictability. Based on ReVeaL's predictability of different genomic regions, dark-matter contains enough signal to significantly discriminate fine subtypes of disease. Hence, the agglomeration of rare variants, even in the hitherto unannotated and ill-understood regions of the genome, may play a substantial role in the disease etiology and deserve much more attention.

Mutational landscape of patients with acute promyelocytic leukemia at diagnosis and relapse.

Despite the high probability of cure of patients with acute promyelocytic leukemia (APL), mechanisms of relapse are still largely unclear. Mutational profiling at diagnosis and/or relapse may help to identify APL patients needing frequent molecular monitoring and early treatment intervention. Using an NGS approach including a 31 myeloid gene-panel, we tested BM samples of 44 APLs at the time of diagnosis, and of 31 at relapse. Mutations in PML and RARA genes were studied using a customized-NGS-RNA panel. Patients relapsing after ATRA-chemotherapy rarely had additional mutations (P = .009). In patients relapsing after ATRA/ATO, the PML gene was a preferential mutation target. We then evaluated the predictive value of mutations at APL diagnosis. A median of two mutations was detectable in 9/11 patients who later relapsed, vs one mutation in 21/33 patients who remained in CCR (P = .0032). This corresponded to a significantly lower risk of relapse in patients with one or less mutations (HR 0.046; 95% CI 0.011-0.197; P < .0001). NGS-analysis at the time of APL diagnosis may inform treatment decisions, including alternative treatments for cases with an unfavorable mutation profile.

Identification of prognostic parameters in CLL with no abnormalities detected by chromosome banding and FISH analyses.

Chronic Lymphocytic Leukaemia (CLL) is a heterogeneous disease with a clinical course dependent on cytogenetic features. However, in 15-20% of cases both chromosome banding and fluorescence in situ hybridisation analyses do not show any kind of abnormality. With the aim to identify dependable molecular prognostic factors in this subgroup, we performed a comprehensive analysis on 171 patients including genomic arrays (comparative genomic hybridisation and single nucleotide polymorphism), immunoglobulin heavy chain variable region genes (IGHV) status, flow cytometry and targeted sequencing. Genomic arrays detected 73 aberrations in 39 patients (23%). Most frequently, patients had 1 aberration (25/171; 15%), while 14 patients (8%) had at least 2 aberrations. IGHV status was unmutated in 53/171 (31%) patients. SF3B1 was the most frequently mutated gene (26/171 patients; 15%), followed by NOTCH1 (15/171; 9%). At univariate analysis, an adverse impact on time to treatment (TTT) was evident for SF3B1 mutations, higher white blood cell count, higher CLL cells percentage by flow cytometry, CD38 positivity, IGHV unmutated status and at least 2 genomic array abnormalities. Of these, SF3B1 mutations, CLL cells percentage, IGHV unmutated status and number of genomic array aberrations maintained their impact in multivariate analysis. In conclusion, by integrating genomic and molecular data, we identified patients at higher risk for treatment need.

Ultra-deep sequencing leads to earlier and more sensitive detection of the tyrosine kinase inhibitor resistance mutation T315I in chronic myeloid leukemia.

Chronic myeloid leukemia cells acquire resistance to tyrosine kinase inhibitors through mutations in the ABL1 kinase domain. The T315I mutation mediates resistance to imatinib, dasatinib, nilotinib and bosutinib, whereas sensitivity to ponatinib remains. Mutation detection by conventional Sanger sequencing requires 10%-20% expansion of the mutated subclone. We studied the T315I mutation development by ultra-deep sequencing on the 454 XL+ platform (Roche) in comparison to Sanger sequencing. By ultra-deep sequencing, mutations were detected at loads of 1%-2%. We selected 40 patients who had failed first-line to third-line treatment (imatinib, dasatinib, nilotinib) and had high loads of the T315I mutation detected by Sanger sequencing. We confirmed T315I mutations by ultra-deep sequencing and investigated the mutation dynamics by backtracking earlier samples. In 20 of 40 patients, we identified the T315I three months (median) before Sanger sequencing detection limits were reached. To exclude sporadic low percentage mutation development without subsequent mutation outgrowth, we selected 42 patients without resistance mutations detected by Sanger sequencing but loss of major molecular response. Here, no mutation was detected by ultradeep sequencing. Additional non-T315I resistance mutations were found in 20 of 40 patients. Only 15% had two mutations per cell; the other cases showed multiple independently mutated clones and the T315I clone demonstrated a rapid outgrowth. In conclusion, T315I mutations could be detected earlier by ultra-deep sequencing compared to Sanger sequencing in a selected group of cases. Earlier mutation detection by ultra-deep sequencing might allow treatment to be changed before clonal increase of cells with the T315I mutation.

Oncogenic role of miR-155 in anaplastic large cell lymphoma lacking the t(2;5) translocation.

Anaplastic large cell lymphoma (ALCL) is a rare, aggressive, non-Hodgkin's lymphoma that is characterized by CD30 expression and disease onset in young patients. About half of ALCL patients bear the t(2;5)(p23;q35) translocation, which results in the formation of the nucleophosmin-anaplastic lymphoma tyrosine kinase (NPM-ALK) fusion protein (ALCL ALK(+)). However, little is known about the molecular features and tumour drivers in ALK-negative ALCL (ALCL ALK(-)), which is characterized by a worse prognosis. We found that ALCL ALK(-), in contrast to ALCL ALK(+), lymphomas display high miR-155 expression. Consistent with this, we observed an inverse correlation between miR-155 promoter methylation and miR-155 expression in ALCL. However, no direct effect of the ALK kinase on miR-155 levels was observed. Ago2 immunoprecipitation revealed miR-155 as the most abundant miRNA, and enrichment of target mRNAs C/EBPß and SOCS1. To investigate its function, we over-expressed miR-155 in ALCL ALK(+) cell lines and demonstrated reduced levels of C/EBPß and SOCS1. In murine engraftment models of ALCL ALK(-), we showed that anti-miR-155 mimics are able to reduce tumour growth. This goes hand-in-hand with increased levels of cleaved caspase-3 and high SOCS1 in these tumours, which leads to suppression of STAT3 signalling. Moreover, miR-155 induces IL-22 expression and suppresses the C/EBPß target IL-8. These data suggest that miR-155 can act as a tumour driver in ALCL ALK(-) and blocking miR-155 could be therapeutically relevant. Original miRNA array data are to be found in the supplementary material (Table S1).

Epigenetic silencing of miR-708 enhances NF-κB signaling in chronic lymphocytic leukemia.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression and their deregulation is involved in tumor development. Epigenetic gene silencing in cancer by DNA methylation contributes to the silencing of tumor-suppressor genes, including miRNAs. We have recently shown that the promoter of miR-708 is aberrantly methylated in chronic lymphocytic leukemia (CLL). To characterize the molecular signaling networks that are influenced by miR-708, we performed a luciferase-based screen evaluating the effects of ectopic miR-708 expression on leukemia-relevant signaling pathways. We found that miR-708 strongly repressed NF-κB signaling, a pathway known to be deregulated in CLL. Among the predicted miR-708 targets was IKKß (inhibitor of kappa light polypeptide gene enhancer in B cells, kinase-ß/IKBKB), a key kinase facilitating NF-κB signaling. We validated the interaction of miR-708 with the 3'-untranslated region of IKKß and found that miR-708 overexpression represses endogenous IKKß. Phosphorylation of the IKKß target IκBα and expression of known NF-κB target genes were impaired by miR-708. Furthermore, we identified an enhancer region downstream of the miR-708 promoter that displays a distinct DNA methylation status in CLL. High enhancer methylation is significantly correlated with lower miR-708 expression and is predominantly found in patients with poor prognosis and shorter time to treatment. These results demonstrate that miR-708 regulates the NF-κB pathway by targeting IKKß, and that methylation of a key enhancer region contributes to its suppression in CLL.

Identification of a specific immunophenotype associated with a consistent pattern of genetic mutations including SRFS2 and gene expression profile in MDS.

BACKGROUND: Myelodysplastic syndromes (MDS) comprise a heterogeneous group of diseases classified by comprehensive diagnostics. Identification of homogeneous subgroups is desirable to understand differences in clinical course and to develop targeted treatment approaches. We identified a specific CD11b/CD16 expression pattern in granulocytes associated with reduced CD45 expression in myeloid progenitor cells (MPC) in MDS cases and assessed its genetic background by whole genome (WGS) and whole transcriptome sequencing (WTS). METHODS: The cohort consisted of 32 MDS cases with the specific aberrant immunophenotype. Since all these 32 cases were found to be SRSF2 mutated additional 51 SRSF2 mutated MDS cases without this specific immunophenotype were selected as controls. For all cases WGS and WTS were performed. RESULTS: The immunophenotype newly identified in SRSF2 mutated MDS patients is characterized (1) by a specific maturation pattern, i.e. an increase of CD11b expression without CD16 expression followed by an increase in CD16 expression without further CD11b expression and (2) by only dim CD45 expression of MPC. STAG2 mutations were exclusively found in MDS cases with the specific immunophenotype (17/32, 53% vs. 0%, p < 0.001). Hence, >50% of cases with the specific immunophenotype were characterized by co-mutations in SRSF2 and STAG2. In addition, cluster analysis revealed a specific gene expression profile of such cases. CONCLUSION: We here for the first time describe a specific immunophenotype which defines MDS cases with SRSF2 mutations and a consistent and specific mutational and gene expression profile. This comprehensive data warrants analysis of further such cases to assess the feasibility of defining a new sub-entity of MDS.

Interplay of TP53 allelic state, blast count, and complex karyotype on survival of patients with AML and MDS.

Several clinical and genetic factors impact overall survival (OS) in myelodysplastic neoplasms (MDS) and acute myeloid leukemia (AML), including complex karyotype (CK), TP53 allelic state, and blast count. We analyzed the interplay of these factors by performing Cox regression analysis and by determining the frequency of TP53 single-hit (sh) and double-hit (dh) events and OS in MDS (n = 747) with <5% blasts, with ≥5% but <10% blasts, and ≥10% but <20% blasts and AML (n = 772). MDS with <5% blasts showed the best outcome, followed by with ≥5% but <10% blasts, and ≥10% but <20% blasts, and AML (median OS: 75, 54, 27, and 18 months, respectively). The same hierarchy was observed when each subgroup was divided into TP53sh, TP53dh, and without TP53 alterations (alt), revealing a dismal outcome of TP53dh in all subgroups (17, 10, 8, and 1 month[s], respectively). MDS with <5% blasts differed from the other subgroups by showing predominantly TP53sh (76% of TP53alt cases), and by an independent adverse impact of CK on OS (hazard ratio, 5.2; P < .001). The remaining subgroups displayed many similarities, with TP53dh found at high frequencies (67%, 91%, and 71%, respectively) and only TP53alt but not CK independently influencing OS, and TP53dh showing the strongest influence. When the total cohort was split based on TP53 state, only the blast count and not CK had an independent adverse impact on OS in all subgroups. Thus, TP53dh is the strongest prognostic factor, further supporting its integration into risk stratification guidelines and classification as a separate entity. However, the blast count also influences OS independent of TP53 state, whereas CK plays a minor prognostic role.

AML classification in the year 2023: How to avoid a Babylonian confusion of languages.

In parallel to the 5th edition of the World Health Organization Classification of Haematolymphoid Tumours (WHO 2022), an alternative International Consensus Classification (ICC) has been proposed. To evaluate the impact of the new classifications on AML diagnoses and ELN-based risk classification, we analyzed 717 MDS and 734 AML non-therapy-related patients diagnosed according to the revised 4th WHO edition (WHO 2017) by whole genome and transcriptome sequencing. In both new classifications, the purely morphologically defined AML entities decreased from 13% to 5%. Myelodysplasia-related (MR) AML increased from 22% to 28% (WHO 2022) and 26% (ICC). Other genetically-defined AML remained the largest group, and the abandoned AML-RUNX1 was mainly reclassified as AML-MR (WHO 2022: 77%; ICC: 96%). Different inclusion criteria of AML-CEBPA and AML-MR (i.a. exclusion of TP53 mutated cases according to ICC) were associated with differences in overall survival. In conclusion, both classifications focus on more genetics-based definitions with similar basic concepts and a large degree of agreement. The remaining non-comparability (e.g., TP53 mutated AML) needs additional studies to definitely answer open questions on disease categorization in an unbiased way.

The clinical and genomic landscape of patients with DDX41 variants identified during diagnostic sequencing.

Deleterious germ line variants in DDX41 are a common cause of genetic predisposition to hematologic malignancies, particularly myelodysplastic neoplasms (MDS) and acute myeloid leukemia (AML). Targeted next-generation sequencing was performed in a large cohort of sequentially recruited patients with myeloid malignancy, covering DDX41 as well as 30 other genes frequently mutated in myeloid malignancy. Whole genome transcriptome sequencing data was analyzed on a separate cohort of patients with a range of hematologic malignancies to investigate the spectrum of cancer predisposition. Altogether, 5737 patients with myeloid malignancies were studied, with 152 different DDX41 variants detected. Multiple novel variants were detected, including synonymous variants affecting splicing as demonstrated by RNA-sequencing. The presence of a somatic DDX41 variant was highly associated with DDX41 germ line variants in patients with MDS and AML, and we developed a statistical approach to incorporate the co-occurrence of a somatic DDX41 variant into germ line variant classification at a very strong level (as per the American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines). Using this approach, the MDS cohort contained 108 of 2865 (3.8%) patients with germ line likely pathogenic/pathogenic (LP/P) variants, and the AML cohort 106 of 2157 (4.9%). DDX41 LP/P variants were markedly enriched in patients with AML and MDS compared with those in patients with myeloproliferative neoplasms, B-cell neoplasm, and T- or B-cell acute lymphoblastic leukemia. In summary, we have developed a framework to enhance DDX41 variant curation as well as highlighted the importance of assessment of all types of genomic variants (including synonymous and multiexon deletions) to fully detect the landscape of possible clinically relevant DDX41 variants.

Spleen tyrosine kinase inhibition prevents chemokine- and integrin-mediated stromal protective effects in chronic lymphocytic leukemia.

The microenvironment provides essential growth and survival signals to chronic lymphocytic leukemia (CLL) cells and contributes to their resistance to cytotoxic agents. Pharmacologic inhibition of spleen tyrosine kinase (SYK), a key mediator of B-cell receptor (BCR) signaling, induces apoptosis in primary CLL cells and prevents stroma contact-mediated cell survival. This report demonstrates a role of SYK in molecularly defined pathways that mediate the CLL-microenvironmental crosstalk independent from the BCR. Chemokine and integrin stimulation induced SYK phosphorylation, SYK-dependent Akt phosphorylation, and F-actin formation in primary CLL cells. Inhibition of SYK by 2 pharmacologic inhibitors and siRNA-knockdown abrogated downstream SYK signaling and morphologic changes induced by these stimuli. CLL cell migration toward CXCL12, the major homing attractor, and CLL cell adhesion to VCAM-1, a major integrin ligand expressed on stromal cells, were markedly reduced by SYK inhibition. In combination with fludarabine, the SYK inhibitor R406 abrogated stroma-mediated drug resistance by preventing up-regulation of the antiapoptotic factor Mcl-1 in CLL cells. SYK blockade in CLL is a promising therapeutic principle not only for its inhibition of the BCR signaling pathway, but also by inhibiting protective stroma signals in a manner entirely independent of BCR signaling.

SF3B1 mutated MDS: Blast count, genetic co-abnormalities and their impact on classification and prognosis.

Recently, MDS with mutated SF3B1 and blast count <5% was proposed as distinct entity with favorable prognosis by the international working group for the prognosis of MDS (IWG-PM), the 5th edition of the WHO classification and the International Consensus Classification. To further characterize this entity with respect to the genomic landscape, AML transformation rate and clinical outcome, we analyzed 734 MDS patients by whole genome sequencing. SF3B1 mutations were identified in 31% (n = 231), most frequently accompanied by TET2 mutations (29%). 144/231 (62%) SF3B1mut samples fulfilled entity criteria proposed by IWG-PM (SF3B1ent). These cases were associated with longer survival, lower AML transformation rate, normal karyotypes and harbored less accompanying mutations compared to SF3B1mut samples not falling into the proposed SF3B1 entity (SF3B1nent). Of SF3B1mut cases 7% (15/231; SF3B1ent: 3/144 [2%]; SF3B1nent: 12/87 [14%]) progressed to AML compared to 15% SF3B1 wild-type patients (75/503). Of these 15 SF3B1mut cases, 10 (67%) showed RUNX1 mutations at MDS or AML stage. Multivariate analysis revealed that del(5q) and RUNX1 mutations were independent negative prognostic factors for overall survival, while blast count >5% was not. In conclusion, SF3B1mut MDS has a favorable prognosis independent of blast count if karyotype and RUNX1 mutations are considered.