Spatial transcriptomics reveals profound subclonal heterogeneity and T-cell dysfunction in extramedullary myeloma.

Extramedullary disease (EMD) is a high-risk feature of multiple myeloma (MM) and remains a poor prognostic factor even in the era of novel immunotherapies. Here we applied spatial transcriptomics (tomo-seq [n=2] and 10X Visium [n=12]), and single-cell RNA sequencing (scRNAseq [n=3]) to a set of 14 EMD biopsies to dissect the three-dimensional architecture of tumor cells and their microenvironment. Overall, the infiltrating immune and stromal cells showed both intra- and inter-patient variation with no uniform distribution over the lesion. We observed substantial heterogeneity at the copy number level within plasma cells, including the emergence of new subclones in circumscribed areas of the tumor, consistent with genomic instability. We further identified spatial expression differences of GPRC5D and TNFRSF17, two important antigens for bispecific antibody therapy. EMD masses were infiltrated by various immune cells, including T-cells. Notably, exhausted TIM3+/PD-1+ T-cells diffusely co-localized with MM cells, whereas functional and activated CD8+ T-cells showed a focal infiltration pattern along with M1 macrophages in otherwise tumor-free regions. This segregation of fit and exhausted T-cells was resolved in the case of response to T-cell engaging bispecific antibodies. MM cells and microenvironment cells were embedded in a complex network that influenced immune activation and angiogenesis, and oxidative phosphorylation represented the major metabolic program within EMD lesions. In summary, spatial transcriptomics has revealed a multicellular ecosystem in EMD with checkpoint inhibition and dual targeting as potential new therapeutic avenues.

Influence of TP53 Gene Mutations and its Allelic Status in Myelodysplastic Syndromes with Isolated 5q Deletion.

Mutations in the TP53 gene, particularly multihit alterations, have been associated with unfavorable clinical features and prognosis in patients diagnosed with myelodysplastic syndrome (MDS). Despite this, the role of TP53 gene aberrations in MDS with isolated deletion of chromosome 5 [MDS-del(5q)] remains unclear. This study aimed to assess the impact of TP53 gene mutations and their allelic state in patients with MDS-del(5q). To that end, a comprehensive analysis of TP53 abnormalities, examining both TP53 mutations and allelic imbalances, in 682 patients diagnosed with MDS-del(5q) was conducted. Twenty-four percent of TP53-mutated patients exhibited multihit alterations, while the remaining patients displayed monoallelic mutations. TP53 multihit alterations were predictive of an increased risk of leukemic transformation. The impact of monoallelic alterations was dependent on the variant allele frequency (VAF); patients with TP53 monoallelic mutations and VAF <20% exhibited behavior similar to TP53 wild type, and those with TP53 monoallelic mutations and VAF ≥20% presented outcomes equivalent to TP53 multihit patients. This study underscores the importance of considering TP53 allelic state and VAF in the risk stratification and treatment decision-making process for patients with MDS-del(5q).

Genetic studies in clonal haematopoiesis, myelodysplastic neoplasms and acute myeloid leukaemia - a practical guide to WHO-HAEM5.

In recent years, technology developments and increase in knowledge have led to profound changes in the diagnostics of haematologic neoplasms, particularly myeloid neoplasms. Therefore an updated, fifth edition of the World Health Organization (WHO) classification of haematolymphoid neoplasms (WHO-HAEM5) will be issued in 2024. In this context, we present a practical guide for analysing the genetic aspects of clonal haematopoiesis of indeterminate potential (CHIP), clonal cytopenia of undetermined significance (CCUS), myelodysplastic neoplasms (MDS), and acute myeloid leukaemia (AML) based on WHO-HAEM5. This guide navigates through the genetic abnormalities underlying myeloid neoplasms which are required to be detected for classification according to WHO-HAEM5 and provides diagnostic algorithms.

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.

Prognostic impact of CEBPA mutational subgroups in adult AML.

Despite recent refinements in the diagnostic and prognostic assessment of CEBPA mutations in AML, several questions remain open, i.e. implications of different types of basic region leucin zipper (bZIP) mutations, the role of co-mutations and the allelic state. Using pooled primary data analysis on 1010 CEBPA-mutant adult AML patients, a comparison was performed taking into account the type of mutation (bZIP: either typical in-frame insertion/deletion (InDel) mutations (bZIPInDel), frameshift InDel or nonsense mutations inducing translational stop (bZIPSTOP) or single base-pair missense alterations (bZIPms), and transcription activation domain (TAD) mutations) and the allelic state (single (smCEBPA) vs. double mutant (dmCEBPA)). Only bZIPInDel patients had significantly higher rates of complete remission and longer relapse free and overall survival (OS) compared with all other CEBPA-mutant subgroups. Moreover, co-mutations in bZIPInDel patients (e.g. GATA2, FLT3, WT1 as well as ELN2022 adverse risk aberrations) had no independent impact on OS, whereas in non-bZIPInDel patients, grouping according to ELN2022 recommendations added significant prognostic information. In conclusion, these results demonstrate bZIPInDel mutations to be the major independent determinant of outcome in CEBPA-mutant AML, thereby refining current classifications according to WHO (including all dmCEBPA and smCEBPA bZIP) as well as ELN2022 and ICC recommendations (including CEBPA bZIPms).

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.

Developmental trajectories and cooperating genomic events define molecular subtypes of BCR::ABL1-positive ALL.

ABSTRACT: Distinct diagnostic entities within BCR::ABL1-positive acute lymphoblastic leukemia (ALL) are currently defined by the International Consensus Classification of myeloid neoplasms and acute leukemias (ICC): "lymphoid only", with BCR::ABL1 observed exclusively in lymphatic precursors, vs "multilineage", where BCR::ABL1 is also present in other hematopoietic lineages. Here, we analyzed transcriptomes of 327 BCR::ABL1-positive patients with ALL (age, 2-84 years; median, 46 years) and identified 2 main gene expression clusters reproducible across 4 independent patient cohorts. Fluorescence in situ hybridization analysis of fluorescence-activated cell-sorted hematopoietic compartments showed distinct BCR::ABL1 involvement in myeloid cells for these clusters (n = 18/18 vs n = 3/16 patients; P < .001), indicating that a multilineage or lymphoid BCR::ABL1 subtype can be inferred from gene expression. Further subclusters grouped samples according to cooperating genomic events (multilineage: HBS1L deletion or monosomy 7; lymphoid: IKZF1-/- or CDKN2A/PAX5 deletions/hyperdiploidy). A novel HSB1L transcript was highly specific for BCR::ABL1 multilineage cases independent of HBS1L genomic aberrations. Treatment on current German Multicenter Study Group for Adult ALL (GMALL) protocols resulted in comparable disease-free survival (DFS) for multilineage vs lymphoid cluster patients (3-year DFS: 70% vs 61%; P = .530; n = 91). However, the IKZF1-/- enriched lymphoid subcluster was associated with inferior DFS, whereas hyperdiploid cases showed a superior outcome. Thus, gene expression clusters define underlying developmental trajectories and distinct patterns of cooperating events in BCR::ABL1-positive ALL with prognostic relevance.

Impaired FADD/BID signaling mediates cross-resistance to immunotherapy in Multiple Myeloma.

The treatment landscape in multiple myeloma (MM) is shifting from genotoxic drugs to immunotherapies. Monoclonal antibodies, immunoconjugates, T-cell engaging antibodies and CART cells have been incorporated into routine treatment algorithms, resulting in improved response rates. Nevertheless, patients continue to relapse and the underlying mechanisms of resistance remain poorly understood. While Impaired death receptor signaling has been reported to mediate resistance to CART in acute lymphoblastic leukemia, this mechanism yet remains to be elucidated in context of novel immunotherapies for MM. Here, we describe impaired death receptor signaling as a novel mechanism of resistance to T-cell mediated immunotherapies in MM. This resistance seems exclusive to novel immunotherapies while sensitivity to conventional anti-tumor therapies being preserved in vitro. As a proof of concept, we present a confirmatory clinical case indicating that the FADD/BID axis is required for meaningful responses to novel immunotherapies thus we report impaired death receptor signaling as a novel resistance mechanism to T-cell mediated immunotherapy in MM.

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.

Mechanisms of antigen escape from BCMA- or GPRC5D-targeted immunotherapies in multiple myeloma.

B cell maturation antigen (BCMA) target loss is considered to be a rare event that mediates multiple myeloma (MM) resistance to anti-BCMA chimeric antigen receptor T cell (CAR T) or bispecific T cell engager (TCE) therapies. Emerging data report that downregulation of G-protein-coupled receptor family C group 5 member D (GPRC5D) protein often occurs at relapse after anti-GPRC5D CAR T therapy. To examine the tumor-intrinsic factors that promote MM antigen escape, we performed combined bulk and single-cell whole-genome sequencing and copy number variation analysis of 30 patients treated with anti-BCMA and/or anti-GPRC5D CAR T/TCE therapy. In two cases, MM relapse post-TCE/CAR T therapy was driven by BCMA-negative clones harboring focal biallelic deletions at the TNFRSF17 locus at relapse or by selective expansion of pre-existing subclones with biallelic TNFRSF17 loss. In another five cases of relapse, newly detected, nontruncating, missense mutations or in-frame deletions in the extracellular domain of BCMA negated the efficacies of anti-BCMA TCE therapies, despite detectable surface BCMA protein expression. In the present study, we also report four cases of MM relapse with biallelic mutations of GPRC5D after anti-GPRC5D TCE therapy, including two cases with convergent evolution where multiple subclones lost GPRC5D through somatic events. Immunoselection of BCMA- or GPRC5D-negative or mutant clones is an important tumor-intrinsic driver of relapse post-targeted therapies. Mutational events on BCMA confer distinct sensitivities toward different anti-BCMA therapies, underscoring the importance of considering the tumor antigen landscape for optimal design and selection of targeted immunotherapies in MM.

Genomics of deletion 7 and 7q in myeloid neoplasm: from pathogenic culprits to potential synthetic lethal therapeutic targets.

Complete or partial deletions of chromosome 7 (-7/del7q) belong to the most frequent chromosomal abnormalities in myeloid neoplasm (MN) and are associated with a poor prognosis. The disease biology of -7/del7q and the genes responsible for the leukemogenic properties have not been completely elucidated. Chromosomal deletions may create clonal vulnerabilities due to haploinsufficient (HI) genes contained in the deleted regions. Therefore, HI genes are potential targets of synthetic lethal strategies. Through the most comprehensive multimodal analysis of more than 600 -7/del7q MN samples, we elucidated the disease biology and qualified a list of most consistently deleted and HI genes. Among them, 27 potentially synthetic lethal target genes were identified with the following properties: (i) unaffected genes by hemizygous/homozygous LOF mutations; (ii) prenatal lethality in knockout mice; and (iii) vulnerability of leukemia cells by CRISPR and shRNA knockout screens. In -7/del7q cells, we also identified 26 up or down-regulated genes mapping on other chromosomes as downstream pathways or compensation mechanisms. Our findings shed light on the pathogenesis of -7/del7q MNs, while 27 potential synthetic lethal target genes and 26 differential expressed genes allow for a therapeutic window of -7/del7q.

The Gene Expression Classifier ALLCatchR Identifies B-cell Precursor ALL Subtypes and Underlying Developmental Trajectories Across Age.

Current classifications (World Health Organization-HAEM5/ICC) define up to 26 molecular B-cell precursor acute lymphoblastic leukemia (BCP-ALL) disease subtypes by genomic driver aberrations and corresponding gene expression signatures. Identification of driver aberrations by transcriptome sequencing (RNA-Seq) is well established, while systematic approaches for gene expression analysis are less advanced. Therefore, we developed ALLCatchR, a machine learning-based classifier using RNA-Seq gene expression data to allocate BCP-ALL samples to all 21 gene expression-defined molecular subtypes. Trained on n = 1869 transcriptome profiles with established subtype definitions (4 cohorts; 55% pediatric / 45% adult), ALLCatchR allowed subtype allocation in 3 independent hold-out cohorts (n = 1018; 75% pediatric / 25% adult) with 95.7% accuracy (averaged sensitivity across subtypes: 91.1% / specificity: 99.8%). High-confidence predictions were achieved in 83.7% of samples with 98.9% accuracy. Only 1.2% of samples remained unclassified. ALLCatchR outperformed existing tools and identified novel driver candidates in previously unassigned samples. Additional modules provided predictions of samples blast counts, patient's sex, and immunophenotype, allowing the imputation in cases where these information are missing. We established a novel RNA-Seq reference of human B-lymphopoiesis using 7 FACS-sorted progenitor stages from healthy bone marrow donors. Implementation in ALLCatchR enabled projection of BCP-ALL samples to this trajectory. This identified shared proximity patterns of BCP-ALL subtypes to normal lymphopoiesis stages, extending immunophenotypic classifications with a novel framework for developmental comparisons of BCP-ALL. ALLCatchR enables RNA-Seq routine application for BCP-ALL diagnostics with systematic gene expression analysis for accurate subtype allocation and novel insights into underlying developmental trajectories.