Overview on WHO-HAEM5 and the diagnostic relevance of genetic alterations for the classification.

The landscape of haematological malignancies is constantly evolving, driven by advances in our understanding of their genetic basis. This has cumulated within the 5th Edition of the World Health Organization (WHO) Classification of Haematolymphoid Tumours published in short form in 2022 [1, 2] and being available in full length both as "Blue Book" (in print expected early 2024) as well as web-based classification (see: https://tumourclassification.iarc.who.int/welcome/). Similarly, the importance of genetic alterations for the classification is highlighted in other classification systems related to haematologic neoplasms [3-5]. In this special issue of the Medizinische Genetik, we present a comprehensive overview of the genetic alterations contributing to the classification of haematolymphoid neoplasms in the 5th Edition of the WHO classification (WHO-HAEM5) and its diagnostic relevance in the context of various haematological malignancies.

Appraisal of current technologies for the study of genetic alterations in hematologic malignancies with a focus on chromosome analysis and structural variants.

During the last five decades, chromosome analysis identified recurring translocations and inversions in leukemias and lymphomas, which led to cloning of genes at the breakpoints that contribute to oncogenesis. Such molecular cytogenetic methods as fluorescence in situ hybridization (FISH), copy number (CN) arrays or optical genome mapping (OGM) have augmented standard chromosome analysis. The use of both cytogenetic and molecular methods, such as reverse transcription-polymerase chain reaction (RT-PCR) and next generation sequencing (NGS), including whole-genome sequencing (WGS), discloses alterations that not only delineate separate WHO disease entities but also constitute independent prognostic factors, whose use in the clinic improves management of patients with hematologic neoplasms.

Genetic alterations in mature B- and T-cell lymphomas - a practical guide to WHO-HAEM5.

The identification of recurrent genomic alterations in tumour cells has a significant role in the classification of mature B- and T-cell lymphomas. Following the development of new technologies, such as next generation sequencing and the improvement of classical technologies such as conventional and molecular cytogenetics, a huge catalogue of genomic alterations in lymphoid neoplasms has been established. These alterations are relevant to refine the taxonomy of the classification of lymphomas, to scrutinize the differential diagnosis within different lymphoma entities and to help assessing the prognosis and clinical management of the patients. Consequently, here we describe the key genetic alterations relevant in mature B- and T-cell lymphomas.

Genetic alterations in chronic lymphocytic leukemia and plasma cell neoplasms - a practical guide to WHO HAEM5.

The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours (WHO-HAEM5) provides a revised classification of lymphoid malignancies including chronic lymphocytic leukemia (CLL) and plasma cell myeloma/multiple myeloma (PCM/MM). For both diseases the descriptions of precursor states such as monoclonal B-cell lymphocytosis and monoclonal gammopathy of uncertain significance (MGUS) have been updated including a better risk stratification model. New insights on mutational landscapes and branching evolutionary pattern were embedded as diagnostic and prognostic factors, accompanied by a revised structure for the chapter of plasma cell neoplasms. Thus, the WHO-HAEM5 leads to practical improvements of biological and clinical relevance for pathologists, clinicians, geneticists and scientists in the field of lymphoid malignancies. The present review gives an overview on the landscape of genetic alterations in CLL and plasma cell neoplasms with a focus on their impact on classification and treatment.

Tumor microenvironment of Burkitt lymphoma: different immune signatures with different clinical behavior.

Burkitt lymphoma (BL) is characterized by tumor microenvironment (TME) in which macrophages represent the main component, determining a distinct histological appearance known as "starry sky" pattern. However, in some instances, BL may exhibit a granulomatous reaction that has been previously linked to a favorable prognosis and spontaneous regression. The aim of our study was to deeply characterize the immune landscape of 7 cases of EBV + BL with granulomatous reaction compared to 8 cases of EBV + BL and 8 EBV- BL, both with typical "starry sky" pattern, by Gene expression profiling performed on the NanoString nCounter platform. Subsequently, the data were validated by multiplex and combined immunostaining. Based on unsupervised clustering of differentially expressed genes, BL samples formed 3 distinct clusters differentially enriched in BL with a diffuse granulomatous reaction (cluster 1), EBV+ BL with typical starry sky pattern (Cluster 2), EBV - BL with typical "starry sky" (cluster 3). We observed variations in the immune response signature among BL with granulomatous reaction and BL with typical "starry sky", both EBV + and EBV -. The TME signature in BL with diffuse granulomatous reaction showed a proinflammatory response, while BLs with "starry sky" were characterized by up-regulation of M2- polarization and pro-tumor response. Moreover, the analysis of additional signatures revealed an up-regulation of Dark zone-signature and epigenetic-signature in BL with typical "starry sky". Tumor associated macrophages (TAM) and epigenetic regulators may be promising targets for additional therapies in BL lymphoma opening novel immunotherapeutic strategies.

SOX11 expression is restricted to EBV-negative Burkitt lymphoma and associates with molecular genetic features.

SRY-related HMG-box gene 11 (SOX11) is a transcription factor overexpressed in mantle cell lymphoma (MCL), a subset of Burkitt lymphomas (BL) and precursor lymphoid cell neoplasms but is absent in normal B-cells and other B-cell lymphomas. SOX11 has an oncogenic role in MCL but its contribution to BL pathogenesis remains uncertain. Here, we observed that the presence of Epstein-Barr virus (EBV) and SOX11 expression were mutually exclusive in BL. SOX11 expression in EBV- BL was associated with an IG∷MYC translocation generated by aberrant class switch recombination, while in EBV-/SOX11- tumors the IG∷MYC translocation was mediated by mistaken somatic hypermutations. Interestingly, EBV- SOX11 expressing BL showed higher frequency of SMARCA4 and ID3 mutations compared to EBV-/SOX11- cases. By RNA-sequencing, we identified a SOX11-associated gene expression profile, with functional annotations showing partial overlap with the SOX11 transcriptional program of MCL. Contrary to MCL, no differences on cell migration or BCR signaling were found between SOX11- and SOX11+ BL cells. However, SOX11+ BL showed higher adhesion to VCAM-1 than SOX11- BL cell lines. Here we demonstrate that EBV- BL comprises two subsets of cases based on SOX11 expression. The mutual exclusion of SOX11 and EBV, and the association of SOX11 with a specific genetic landscape suggest a role of SOX11 in the early pathogenesis of BL.

Influence of analytical procedures on miRNA expression analyses in saliva samples.

Aim of this study was to demonstrate the influence of different analytical procedures and techniques on the resulting miRNA expression profile in healthy control subjects and tumor patients using the oral squamous cell carcinoma (OSCC) model and to demonstrate the technical and biological reproducibility. Body fluids such as saliva are suitable for non-invasive miRNA analysis because ubiquitously circulating miRNA can be found in them. It was technically possible to distinguish between healthy and diseased samples based on the miRNA expression profile found. Regardless of the methodology used, good technical reproducibility of the results seems to be achievable. On the other hand, biological reproducibility was inadequate, which is why prompt sampling and sequencing is recommended. The data indicate that malignant lesions can be detected using miRNA signatures extracted from saliva. This could stimulate further research to establish standardized protocols and kits for sample collection, miRNA extraction, sequencing and interpretation of results.

Genome-wide DNA methylation-analysis of blastic plasmacytoid dendritic cell neoplasm identifies distinct molecular features.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) constitutes a rare and aggressive malignancy originating from plasmacytoid dendritic cells (pDCs) with a primarily cutaneous tropism followed by dissemination to the bone marrow and other organs. We conducted a genome-wide analysis of the tumor methylome in an extended cohort of 45 BPDCN patients supplemented by WES and RNA-seq as well as ATAC-seq on selected cases. We determined the BPDCN DNA methylation profile and observed a dramatic loss of DNA methylation during malignant transformation from early and mature DCs towards BPDCN. DNA methylation profiles further differentiate between BPDCN, AML, CMML, and T-ALL exhibiting the most striking global demethylation, mitotic stress, and merely localized DNA hypermethylation in BPDCN resulting in pronounced inactivation of tumor suppressor genes by comparison. DNA methylation-based analysis of the tumor microenvironment by MethylCIBERSORT yielded two, prognostically relevant clusters (IC1 and IC2) with specific cellular composition and mutational spectra. Further, the transcriptional subgroups of BPDCN (C1 and C2) differ by DNA methylation signatures in interleukin/inflammatory signaling genes but also by higher transcription factor activity of JAK-STAT and NFkB signaling in C2 in contrast to an EZH2 dependence in C1-BPDCN. Our integrative characterization of BPDCN offers novel molecular insights and potential diagnostic applications.

What is new in the 5th edition of the World Health Organization classification of mature B and T/NK cell tumors and stromal neoplasms?

The classification of tumors is essential in the diagnosis and clinical management of patients with malignant neoplasms. The World Health Organization (WHO) provides a globally applicable classification scheme of neoplasms and it was updated several times. In this review, we briefly outline the cornerstones of the upcoming 5th edition of the World Health Organization Classification of Haematolymphoid Tumours on lymphoid neoplasms. As is adopted throughout the 5th edition of the WHO classification of tumors of all organ systems, entities are listed by a hierarchical system. For the first time, tumor-like lesions have been included in the classification, and modifications of nomenclature for some entities, revisions of diagnostic criteria or subtypes, deletion of certain entities, and introduction of new entities are presented along with mesenchymal lesions specific to the stroma of lymph nodes and the spleen. In addition to specific outlines on constitutional and somatic genetic changes associated with given entities, a separate chapter on germline predisposition syndromes related to hematologic neoplasms has been added.

Effects of tofersen treatment in patients with SOD1-ALS in a "real-world" setting - a 12-month multicenter cohort study from the German early access program.

Background: In April 2023, the antisense oligonucleotide tofersen was approved by the U.S. Food and Drug Administration (FDA) for treatment of SOD1-amyotrophic lateral sclerosis (ALS), after a decrease of neurofilament light chain (NfL) levels had been demonstrated. Methods: Between 03/2022 and 04/2023, 24 patients with SOD1-ALS from ten German ALS reference centers were followed-up until the cut-off date for ALS functional rating scale revised (ALSFRS-R), progression rate (loss of ALSFRS-R/month), NfL, phosphorylated neurofilament heavy chain (pNfH) in cerebrospinal fluid (CSF), and adverse events. Findings: During the observation period, median ALSFRS-R decreased from 38.0 (IQR 32.0-42.0) to 35.0 (IQR 29.0-42.0), corresponding to a median progression rate of 0.11 (IQR -0.09 to 0.32) points of ALSFRS-R lost per month. Median serum NfL declined from 78.0 pg/ml (IQR 37.0-147.0 pg/ml; n = 23) to 36.0 pg/ml (IQR 22.0-65.0 pg/ml; n = 23; p = 0.02), median pNfH in CSF from 2226 pg/ml (IQR 1061-6138 pg/ml; n = 18) to 1151 pg/ml (IQR 521-2360 pg/ml; n = 18; p = 0.02). In the CSF, we detected a pleocytosis in 73% of patients (11 of 15) and an intrathecal immunoglobulin synthesis (IgG, IgM, or IgA) in 9 out of 10 patients. Two drug-related serious adverse events were reported. Interpretation: Consistent with the VALOR study and its Open Label Extension (OLE), our results confirm a reduction of NfL serum levels, and moreover show a reduction of pNfH in CSF. The therapy was safe, as no persistent symptoms were observed. Pleocytosis and Ig synthesis in CSF with clinical symptoms related to myeloradiculitis in two patients, indicate the potential of an autoimmune reaction. Funding: No funding was received towards this study.

Variant ALK-fusion positive anaplastic large cell lymphoma (ALCL): A population-based paediatric study of the NHL-BFM study group.

Frequency, distribution and prognostic meaning of ALK-partner genes other than NPM1 in ALK-positive anaplastic large-cell lymphoma (ALCL) are unknown. Forty-nine of 316 ALCL diagnosed in the NHL-BFM study group showed no nuclear ALK expression suggestive of a variant ALK-partner; 41 were analysed by genomic capture high-throughput sequencing or specific RT-PCRs. NPM1::ALK was detected in 13 cases. Among the 28 patients with a non-NPM1::ALK-fusion partner, ATIC (n = 8; 29%) and TPM3 (n = 9; 32%) were the most common. Five of eight patients with ATIC::ALK-positive ALCL relapsed, none of nine with TPM3::ALK. Variant ALK-partners are rare and potentially associated with different prognoses.

The fifth edition of the WHO classification of mature B-cell neoplasms: open questions for research.

The fifth edition of the World Health Organization Classification of Haematolymphoid Tumours (WHO-HAEM5) is the product of an evidence-based evolution of the revised fourth edition with wide multidisciplinary consultation. Nonetheless, while every classification incorporates scientific advances and aims to improve upon the prior version, medical knowledge remains incomplete and individual neoplasms may not be easily subclassified in a given scheme. Thus, optimal classification requires ongoing study, and there are certain aspects of some entities and subtypes that require further refinements. In this review, we highlight a selection of these challenging areas to prompt more research investigations. These include (1) a 'placeholder term' of splenic B-cell lymphoma/leukaemia with prominent nucleoli (SBLPN) to accommodate many of the splenic lymphomas previously classified as hairy cell leukaemia variant and B-prolymphocytic leukaemia, a clear new start to define their pathobiology; (2) how best to classify BCL2 rearrangement negative follicular lymphoma including those with BCL6 rearrangement, integrating the emerging new knowledge on various germinal centre B-cell subsets; (3) what is the spectrum of non-IG gene partners of MYC translocation in diffuse large B-cell lymphoma/high-grade B-cell lymphoma and how they impact MYC expression and clinical outcome; how best to investigate this in a routine clinical setting; and (4) how best to define high-grade B-cell lymphoma not otherwise specified and high-grade B-cell lymphoma with 11q aberrations to distinguish them from their mimics and characterise their molecular pathogenetic mechanism. Addressing these questions would provide more robust evidence to better define these entities/subtypes, improve their diagnosis and/or prognostic stratification, leading to better patient care. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The 5th edition of the World Health Organization Classification of mature lymphoid and stromal tumors - an overview and update.

The purpose of this review is to give an overview on the conceptual framework and major developments of the upcoming 5th edition of the World Health Organization (WHO) Classification of Haematolymphoid tumours (WHO-HAEM5) and to highlight the most significant changes made in WHO-HAEM5 compared with the revised 4th edition (WHO-HAEM4R) of lymphoid and stromal neoplasms. The changes from the revised 4th edition include the reorganization of entities by means of a hierarchical system that is realized throughout the 5th edition of the WHO classification of tumors of all organ systems, a modification of nomenclature for some entities, the refinement of diagnostic criteria or subtypes, deletion of certain entities, and introduction of new entities. For the first time, tumor-like lesions, mesenchymal lesions specific to lymph node and spleen, and germline predisposition syndromes associated with the lymphoid neoplasms are included in the classification.

Current Molecular and Clinical Landscape of ATRT - The Link to Future Therapies.

ATRT is a highly aggressive and rare pediatric CNS tumor of very young children. Its genetic hallmark is bi-allelic inactivation of SMARCB1 encoding INI1. Rarely SMARCA4 encoding BRG1 is affected. Up to 30% are associated with constitutional heterozygous pathogenic variants in one of the two genes, giving rise to the Rhabdoid-Tumor-Predisposition-Syndromes (RTPS) 1 and 2. Characteristic DNA methylation profiles distinguish ATRT from other SMARCB1-deficient entities. Three distinct subtypes ATRT-MYC, -TYR, and -SHH are on record. ATRT-SHH may be further divided into the subgroups ATRT-SHH1A, -SHH1B, and -SHH2. The cure of ATRT remains challenging, notwithstanding an increasing understanding of molecular pathomechanisms and genetic background. The implementation of multimodal institutional treatment protocols has improved prognosis. Regardless of treatment approaches, clinical risk factors such as age, metastases, and DNA methylation subtype affect survival probability. We provide a critical appraisal of current conventional multimodal regimens and emerging targeted treatment approaches investigated in clinical trials and entity-specific registries. Intense treatment approaches featuring radiotherapy (RT) and high-dose chemotherapy (HDCT) face the difficulty of balancing tumor control and treatment-related toxicity. Current approaches focus on minimizing radiation fields by proton beam therapy or to withhold RT in HDCT-only approaches. Still, a 40-75% relapse rate upon first-line treatment reveals the need for novel treatment strategies in primary and even more in recurrent/refractory (r/r) disease. Among targeted treatments, immune checkpoint inhibitors and epigenetically active agents appear most promising. Success remains limited in single agent approaches. We hypothesize that mechanism-informed combination therapy will enhance response, as the low mutational burden of ATRT may contribute to acquiring resistance to single targeted agents. As DNA methylation group-specific gene expression profiles appear to influence response to distinct agents, the future treatment of ATRT should respect clinical and biological heterogeneity in risk group adjusted treatment protocols.

Mosaic chromosomal alterations in peripheral blood leukocytes of children in sub-Saharan Africa.

In high-income countries, mosaic chromosomal alterations in peripheral blood leukocytes are associated with an elevated risk of adverse health outcomes, including hematologic malignancies. We investigate mosaic chromosomal alterations in sub-Saharan Africa among 931 children with Burkitt lymphoma, an aggressive lymphoma commonly characterized by immunoglobulin-MYC chromosomal rearrangements, 3822 Burkitt lymphoma-free children, and 674 cancer-free men from Ghana. We find autosomal and X chromosome mosaic chromosomal alterations in 3.4% and 1.7% of Burkitt lymphoma-free children, and 8.4% and 3.7% of children with Burkitt lymphoma (P-values = 5.7×10-11 and 3.74×10-2, respectively). Autosomal mosaic chromosomal alterations are detected in 14.0% of Ghanaian men and increase with age. Mosaic chromosomal alterations in Burkitt lymphoma cases include gains on chromosomes 1q and 8, the latter spanning MYC, while mosaic chromosomal alterations in Burkitt lymphoma-free children include copy-neutral loss of heterozygosity on chromosomes 10, 14, and 16. Our results highlight mosaic chromosomal alterations in sub-Saharan African populations as a promising area of research.

Transcriptional reprogramming by mutated IRF4 in lymphoma.

Disease-causing mutations in genes encoding transcription factors (TFs) can affect TF interactions with their cognate DNA-binding motifs. Whether and how TF mutations impact upon the binding to TF composite elements (CE) and the interaction with other TFs is unclear. Here, we report a distinct mechanism of TF alteration in human lymphomas with perturbed B cell identity, in particular classic Hodgkin lymphoma. It is caused by a recurrent somatic missense mutation c.295 T > C (p.Cys99Arg; p.C99R) targeting the center of the DNA-binding domain of Interferon Regulatory Factor 4 (IRF4), a key TF in immune cells. IRF4-C99R fundamentally alters IRF4 DNA-binding, with loss-of-binding to canonical IRF motifs and neomorphic gain-of-binding to canonical and non-canonical IRF CEs. IRF4-C99R thoroughly modifies IRF4 function by blocking IRF4-dependent plasma cell induction, and up-regulates disease-specific genes in a non-canonical Activator Protein-1 (AP-1)-IRF-CE (AICE)-dependent manner. Our data explain how a single mutation causes a complex switch of TF specificity and gene regulation and open the perspective to specifically block the neomorphic DNA-binding activities of a mutant TF.

Targeting oncogenic TERT promoter variants by allele-specific epigenome editing.

BACKGROUND: Activation of dominant oncogenes by small or structural genomic alterations is a common driver mechanism in many cancers. Silencing of such dominantly activated oncogenic alleles, thus, is a promising strategy to treat cancer. Recently, allele-specific epigenome editing (ASEE) has been described as a means to reduce transcription of genes in an allele-specific manner. In cancer, specificity to an oncogenic allele can be reached by either targeting directly a pathogenic single-nucleotide variant or a polymorphic single-nucleotide variant linked to the oncogenic allele. To investigate the potential of ASEE in cancer, we here explored this approach by targeting variants at the TERT promoter region. The TERT promoter region has been described as one of the most frequently mutated non-coding cancer drivers. RESULTS: Sequencing of the TERT promoter in cancer cell lines showed 53% (41/77) to contain at least one heterozygous sequence variant allowing allele distinction. We chose the hepatoblastoma cell line Hep-G2 and the lung cancer cell line A-549 for this proof-of-principle study, as they contained two different kinds of variants, namely the activating mutation C228T in the TERT core promoter and the common SNP rs2853669 in the THOR region, respectively. These variants were targeted in an allele-specific manner using sgRNA-guided dCas9-DNMT3A-3L complexes. In both cell lines, we successfully introduced DNA methylation specifically to the on-target allele of the TERT promoter with limited background methylation on the off-target allele or an off-target locus (VEGFA), respectively. We observed a maximum CpG methylation gain of 39% and 76% on the target allele when targeting the activating mutation and the common SNP, respectively. The epigenome editing translated into reduced TERT RNA expression in Hep-G2. CONCLUSIONS: We applied an ASEE-mediated approach to silence TERT allele specifically. Our results show that the concept of dominant oncogene inactivation by allele-specific epigenome editing can be successfully translated into cancer models. This new strategy may have important advantages in comparison with existing therapeutic approaches, e.g., targeting telomerase, especially with regard to reducing adverse side effects.

A quantum physics layer of epigenetics: a hypothesis deduced from charge transfer and chirality-induced spin selectivity of DNA.

BACKGROUND: Epigenetic mechanisms are informational cellular processes instructing normal and diseased phenotypes. They are associated with DNA but without altering the DNA sequence. Whereas chemical processes like DNA methylation or histone modifications are well-accepted epigenetic mechanisms, we herein propose the existence of an additional quantum physics layer of epigenetics. RESULTS: We base our hypothesis on theoretical and experimental studies showing quantum phenomena to be active in double-stranded DNA, even under ambient conditions. These phenomena include coherent charge transfer along overlapping pi-orbitals of DNA bases and chirality-induced spin selectivity. Charge transfer via quantum tunneling mediated by overlapping orbitals results in charge delocalization along several neighboring bases, which can even be extended by classical (non-quantum) electron hopping. Such charge transfer is interrupted by flipping base(s) out of the double-strand e.g., by DNA modifying enzymes. Charge delocalization can directly alter DNA recognition by proteins or indirectly by DNA structural changes e.g., kinking. Regarding sequence dependency, charge localization, shown to favor guanines, could influence or even direct epigenetic changes, e.g., modification of cytosines in CpG dinucleotides. Chirality-induced spin selectivity filters electrons for their spin along DNA and, thus, is not only an indicator for quantum coherence but can potentially affect DNA binding properties. CONCLUSIONS: Quantum effects in DNA are prone to triggering and manipulation by external means. By the hypothesis put forward here, we would like to foster research on "Quantum Epigenetics" at the interface of medicine, biology, biochemistry, and physics to investigate the potential epigenetic impact of quantum physical principles on (human) life.