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A letter to the editor in response to the article "The proportion of CD34+CD38low or neg myeloblasts, but not side population frequency, predicts initial response to induction therapy in patients with newly diagnosed acute myeloid leukemia" published in a previous 2013 issue is presented.

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Comprehensive genomic sequencing is becoming a critical component in the assessment of hematologic malignancies, with broad implications for patients' management. In this context, unequivocally discriminating somatic from germline events is challenging but greatly facilitated by matched analysis of tumor:normal pairs of samples. In contrast to solid tumors, in hematologic malignancies conventional sources of normal control material (peripheral blood, buccal swabs, saliva) could be highly involved by the neoplastic process, rendering them unsuitable. In this work we describe our real-world experience using cell-free DNA (cfDNA) isolated from nail clippings as an alternate source of normal control material, through the dedicated review of 2,610 tumor:nail pairs comprehensively sequenced by MSK-IMPACT-heme. Overall, we found that nail cfDNA is a robust germline control for paired genomic studies. In a subset of patients, nail DNA may be contaminated by tumor DNA, reflecting unique attributes of the hematologic disease and transplant history. Contamination is generally low level, but significantly more common among patients with myeloid neoplasms (20.5%; 304/1,482) than among those with lymphoid diseases (5.4%; 61/1,128) and particularly enriched in myeloproliferative neoplasms with marked myelofibrosis. When identified in patients with lymphoid and plasma-cell neoplasms, mutations commonly reflected a myeloid profile and correlated with a concurrent/evolving clonal myeloid neoplasm. Donor DNA was identified in 22% (11/50) of nails collected after allogeneic stem-cell transplantation. In this cohort, an association with a recent history of graft-versus-host disease was identified. These findings should be considered as a potential limitation to the use of nails as a source of normal control DNA but could also provide important diagnostic information regarding the disease process.

Purpose: Classic Hodgkin lymphoma (cHL) is a B-cell lymphoma that occurs primarily in young adults and, less frequently, in elderly individuals. A hallmark of cHL is the exceptional scarcity (1%-5%) of the malignant Hodgkin Reed-Sternberg (HRS) cells within a network of nonmalignant immune cells. Molecular determinants governing the relationship between HRS cells and their proximal microenvironment remain largely unknown., Experimental Design: We performed spatially resolved multiplexed protein imaging and transcriptomic sequencing to characterize HRS cell states, cellular neighborhoods, and gene expression signatures of 23.6 million cells from 36 newly diagnosed Epstein-Barr virus (EBV)-positive and EBV-negative cHL tumors., Results: We show that MHC-I expression on HRS cells is associated with immune-inflamed neighborhoods containing CD8+ T cells, MHC-II+ macrophages, and immune checkpoint expression (i.e., PD1 and VISTA). We identified spatial clustering of HRS cells, consistent with the syncytial variant of cHL, and its association with T-cell-excluded neighborhoods in a subset of EBV-negative tumors. Finally, a subset of both EBV-positive and EBV-negative tumors contained regulatory T-cell-high neighborhoods harboring HRS cells with augmented proliferative capacity., Conclusions: Our study links HRS cell properties with distinct immunophenotypes and potential immune escape mechanisms in cHL., (©2024 The Authors; Published by the American Association for Cancer Research.)

Aims: B lymphoblastic leukaemia/lymphoma (B-ALL) is thought to originate from Pro/Pre-B cells and the genetic aberrations largely reside in lymphoid-committed cells. A recent study demonstrated that a proportion of paediatric B-ALL patients have BCR::ABL1 fusion in myeloid cells, suggesting a chronic myeloid leukaemia (CML)-like biology in this peculiar subset of B-ALL, although it is not entirely clear if the CD19-negative precursor compartment is a source of the myeloid cells. Moreover, the observation has not yet been extended to other fusion-driven B-ALLs., Methods and Results: In this study we investigated a cohort of KMT2A-rearranged B-ALL patients with a comparison to BCR::ABL1-rearranged B-ALL by performing cell sorting via flow cytometry followed by FISH (fluorescence in situ hybridization) analysis on each of the sorted populations. In addition, RNA sequencing was performed on one of the sorted populations. These analyses showed that (1) multilineage involvement was present in 53% of BCR::ABL1 and 36% of KMT2A-rearranged B-ALL regardless of age, (2) multilineage involvement created pitfalls for residual disease monitoring, and (3) HSPC transcriptome signatures were upregulated in KMT2A-rearranged B-ALL with multilineage involvement., Conclusions: In summary, multilineage involvement is common in both BCR::ABL1-rearranged and KMT2A-rearranged B-ALL, which should be taken into consideration when interpreting the disease burden during the clinical course., (© 2024 John Wiley & Sons Ltd.)

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Leveraging endogenous tumor-resident T-cells for immunotherapy using bispecific antibodies (BsAb) targeting CD20 and CD3 has emerged as a promising therapeutic strategy for patients with B-cell non-Hodgkin lymphomas. However, features associated with treatment response or resistance are unknown. To this end, we analyzed data from patients treated with epcoritamab-containing regimens in the EPCORE NHL-2 trial (NCT04663347). We observed downregulation of CD20 expression on B-cells following treatment initiation both in progressing patients and in patients achieving durable complete responses (CR), suggesting that CD20 downregulation does not universally predict resistance to BsAb-based therapy. Single-cell immune profiling of tumor biopsies obtained following one cycle of therapy revealed substantial clonal expansion of cytotoxic CD4+ and CD8+ T-cells in patients achieving CR, and an expansion of follicular helper and regulatory CD4+ T-cells in patients whose disease progressed. These results identify distinct tumor-resident T-cell profiles associated with response or resistance to BsAb therapy., Competing Interests: L.F. Has received research funding from Genmab A/S, AbbVie, Inc., F. Hoffmann-La Roche AG, Genentech Inc., Innate Pharma S.A.; has provided consulting services for Genmab A/S, AbbVie, Inc., F. Hoffmann-La Roche AG, Genentech, Inc., Evolveimmune Therapeutics, Inc., Sanofi, S.A.; has served as an advisor for AbbVie, Inc., Seagen, Inc., Ipsen Biopharmaceuticals, Inc., ADC therapeutics S.A.; and has received travel reimbursement from Genmab A/S, AbbVie, Inc. S.A.V. previously served as an advisor for Immunai, has provided consulting services for ADT Therapeutics and Koch Disruptive Technologies, and has received funding from BMS. G.S. has received financial compensation for consulting services in the last 12 months from: Abbvie, Atb Therapeutics, Beigene, BMS, Genentech/Roche, Genmab, Janssen, Innate Pharma, Incyte, Ipsen, Kite/Gilead, Merck, Modex, Molecular Partners, Novartis, Nurix, Orna Therapeutics, and Treeline. He is a shareholder of Owkin. He has received research support from Abbvie, Genentech, Genmab, Janssen, Ipsen, and Nurix, which his institution managed.

Next-generation sequencing (NGS)-based measurable residual disease (MRD) monitoring in post-treatment settings can be crucial for relapse risk stratification in patients with B-cell and plasma cell neoplasms. Prior studies have focused on validation of various technical aspects of the MRD assays, but more studies are warranted to establish the performance characteristics and enable standardization and broad utilization in routine clinical practice. Here, the authors describe an NGS-based IGH MRD quantification assay, incorporating a spike-in calibrator for monitoring B-cell and plasma cell neoplasms based on their unique IGH rearrangement status. Comparison of MRD status (positive or undetectable) by NGS and flow cytometry (FC) assays showed high concordance (91%, 471/519 cases) and overall good linear correlation in MRD quantitation, particularly for chronic lymphocytic leukemia and B-lymphoblastic leukemia/lymphoma (R = 0.85). Quantitative correlation was lower for plasma cell neoplasms, where underestimation by FC is a known limitation. No significant effects on sequencing efficiency by the spike-in calibrator were observed, with excellent inter- and intra-assay reproducibility within the authors' laboratory, and in comparison to an external laboratory, using the same assay and protocols. Assays performed both at internal and external laboratories showed highly concordant MRD detection (100%) and quantitation (R = 0.97). Overall, this NGS-based MRD assay showed highly reproducible results with quantitation that correlated well with FC MRD assessment, particularly for B-cell neoplasms., Competing Interests: Disclosure Statement M.E.A. has served as a consultant and received honoraria from Biocartis US, Inc., Invivoscribe, Inc. Janssen Global Services, Bristol Myers Squibb, AstraZeneca, Roche, and Merck. C.H. has received honoraria from Blueprint Medicines, Hematopathology Advisory Board, and is an employee of Loxo Oncology, Inc. Y.H. and J.M. were employees of Invivoscribe, Inc., which developed and sells the commercial assay used in this paper. K.P.-D. received an honorarium from Invivoscribe not related to this study. M.R. has served as a consultant for BD Biosciences, Agios, and Celgene, as well as received contract research funding for Agios, Roche, BMS, and Bayer. A.D. has received consulting fees from Physicians' Education Resource, Seattle Genetics, Takeda, Roche, EUSA Pharma, Peerview Institute, Corvus Pharmaceuticals, and AbbVie, as well as research support from Roche and Takeda. C.V. has received consulting fees from DocDoc Pte. Ltd. and Paige.AI, Inc., (Copyright © 2024 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is an uncommon mature T-cell neoplasm occurring in patients with textured breast implants, typically after 7-10 years of exposure. Although cytopathologic or histopathologic assessment is considered the gold standard diagnostic method for BIA-ALCL, flow cytometry (FC)-based immunophenotyping is recommended as an adjunct test. However, the diagnostic efficacy of FC is not well reported. We reviewed 290 FC tests from breast implant pericapsular fluid and capsule tissue from 182 patients, including 16 patients with BIA-ALCL over a 6-year period, calculating diagnostic rates and test efficacy. FC showed an overall sensitivity of 75.9%, specificity of 100%, and negative and positive predictive values of 95.4% and 100%, respectively. Blinded expert review of false-negative cases identified diagnostic pitfalls, improving sensitivity to 96.6%. Fluid samples had better rates of adequate samples for FC testing compared with tissue samples. Paired with FC testing of operating room (OR)-acquired fluid samples, capsulectomy FC specimens added no diagnostic value in patients with concurrent fluid samples; no cases had positive capsule FC with negative fluid FC. Fluid samples are adequate for FC testing more often than tissue. Capsule tissue FC specimens do not improve FC efficacy when paired with OR-acquired fluid FC samples and are often inadequate samples. FC is 100% specific for BIA-ALCL and can serve as a confirmatory test but should not be the sole diagnostic method. Awareness of sample-specific diagnostic pitfalls greatly improves the sensitivity of BIA-ALCL testing by FC., (© 2024 International Clinical Cytometry Society.)

Despite improving outcomes, 40% of patients with newly diagnosed multiple myeloma treated with regimens containing daratumumab, a CD38-targeted monoclonal antibody, progress prematurely. By integrating tumor whole-genome and microenvironment single-cell RNA sequencing from upfront phase 2 trials using carfilzomib, lenalidomide and dexamethasone with daratumumab ( NCT03290950 ), we show how distinct genomic drivers including high APOBEC mutational activity, IKZF3 and RPL5 deletions and 8q gain affect clinical outcomes. Furthermore, evaluation of paired bone marrow profiles, taken before and after eight cycles of carfilzomib, lenalidomide and dexamethasone with daratumumab, shows that numbers of natural killer cells before treatment, high T cell receptor diversity before treatment, the disappearance of sustained immune activation (that is, B cells and T cells) and monocyte expansion over time are all predictive of sustained minimal residual disease negativity. Overall, this study provides strong evidence of a complex interplay between tumor cells and the immune microenvironment that is predictive of clinical outcome and depth of treatment response in patients with newly diagnosed multiple myeloma treated with highly effective combinations containing anti-CD38 antibodies., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Genomic profiling of hematologic malignancies has augmented our understanding of variants that contribute to disease pathogenesis and supported development of prognostic models that inform disease management in the clinic. Tumor only sequencing assays are limited in their ability to identify definitive somatic variants, which can lead to ambiguity in clinical reporting and patient management. Here, we describe the MSK-IMPACT Heme cohort, a comprehensive data set of somatic alterations from paired tumor and normal DNA using a hybridization capture-based next generation sequencing platform. We highlight patterns of mutations, copy number alterations, and mutation signatures in a broad set of myeloid and lymphoid neoplasms. We also demonstrate the power of appropriate matching to make definitive somatic calls, including in patients who have undergone allogeneic stem cell transplant. We expect that this resource will further spur research into the pathobiology and clinical utility of clinical sequencing for patients with hematologic neoplasms., (© 2023. The Author(s).)

Measurable residual disease (MRD), defined as the population of cancer cells that persist following therapy, serves as the critical reservoir for disease relapse in acute myeloid leukemia and other malignancies. Understanding the biology enabling MRD clones to resist therapy is necessary to guide the development of more effective curative treatments. Discriminating between residual leukemic clones, preleukemic clones, and normal precursors remains a challenge with current MRD tools. Here, we developed a single-cell MRD (scMRD) assay by combining flow cytometric enrichment of the targeted precursor/blast population with integrated single-cell DNA sequencing and immunophenotyping. Our scMRD assay shows high sensitivity of approximately 0.01%, deconvolutes clonal architecture, and provides clone-specific immunophenotypic data. In summary, our scMRD assay enhances MRD detection and simultaneously illuminates the clonal architecture of clonal hematopoiesis/preleukemic and leukemic cells surviving acute myeloid leukemia therapy.

Gene fusions involving tumor protein p63 gene (TP63) occur in multiple T and B cell lymphomas and portend a dismal prognosis for patients. The function and mechanisms of TP63 fusions remain unclear, and there is no target therapy for patients with lymphoma harboring TP63 fusions. Here, we show that TP63 fusions act as bona fide oncogenes and are essential for fusion-positive lymphomas. Transgenic mice expressing TBL1XR1::TP63, the most common TP63 fusion, develop diverse lymphomas that recapitulate multiple human T and B cell lymphomas. Here, we identify that TP63 fusions coordinate the recruitment of two epigenetic modifying complexes, the nuclear receptor corepressor (NCoR)-histone deacetylase 3 (HDAC3) by the N-terminal TP63 fusion partner and the lysine methyltransferase 2D (KMT2D) by the C-terminal TP63 component, which are both required for fusion-dependent survival. TBL1XR1::TP63 localization at enhancers drives a unique cell state that involves up-regulation of MYC and the polycomb repressor complex 2 (PRC2) components EED and EZH2. Inhibiting EZH2 with the therapeutic agent valemetostat is highly effective at treating transgenic lymphoma murine models, xenografts, and patient-derived xenografts harboring TP63 fusions. One patient with TP63 -rearranged lymphoma showed a rapid response to valemetostat treatment. In summary, TP63 fusions link partner components that, together, coordinate multiple epigenetic complexes, resulting in therapeutic vulnerability to EZH2 inhibition.

Although allogeneic hematopoietic cell transplant (allo-HCT) is curative for high-risk pediatric acute myeloid leukemia (AML), disease relapse remains the primary cause of posttransplant mortality. To identify pressures imposed by allo-HCT on AML cells that escape the graft-versus-leukemia effect, we evaluated immune signatures at diagnosis and posttransplant relapse in bone marrow samples from 4 pediatric patients using a multimodal single-cell proteogenomic approach. Downregulation of major histocompatibility complex class II expression was most profound in progenitor-like blasts and accompanied by correlative changes in transcriptional regulation. Dysfunction of activated natural killer cells and CD8+ T-cell subsets at relapse was evidenced by the loss of response to interferon gamma, tumor necrosis factor α signaling via NF-κB, and interleukin-2/STAT5 signaling. Clonotype analysis of posttransplant relapse samples revealed an expansion of dysfunctional T cells and enrichment of T-regulatory and T-helper cells. Using novel computational methods, our results illustrate a diverse immune-related transcriptional signature in posttransplant relapses not previously reported in pediatric AML., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Accurate classification and risk stratification are critical for clinical decision making in patients with acute myeloid leukemia (AML). In the newly proposed World Health Organization and International Consensus classifications of hematolymphoid neoplasms, the presence of myelodysplasia-related (MR) gene mutations is included as 1 of the diagnostic criteria for AML, AML-MR, based largely on the assumption that these mutations are specific for AML with an antecedent myelodysplastic syndrome. ICC also prioritizes MR gene mutations over ontogeny (as defined in the clinical history). Furthermore, European LeukemiaNet (ELN) 2022 stratifies these MR gene mutations into the adverse-risk group. By thoroughly annotating a cohort of 344 newly diagnosed patients with AML treated at the Memorial Sloan Kettering Cancer Center, we show that ontogeny assignments based on the database registry lack accuracy. MR gene mutations are frequently observed in de novo AML. Among the MR gene mutations, only EZH2 and SF3B1 were associated with an inferior outcome in the univariate analysis. In a multivariate analysis, AML ontogeny had independent prognostic values even after adjusting for age, treatment, allo-transplant and genomic classes or ELN risks. Ontogeny also helped stratify the outcome of AML with MR gene mutations. Finally, de novo AML with MR gene mutations did not show an adverse outcome. In summary, our study emphasizes the importance of accurate ontogeny designation in clinical studies, demonstrates the independent prognostic value of AML ontogeny, and questions the current classification and risk stratification of AML with MR gene mutations., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Flow cytometry plays a critical role in the diagnosis, prognostication, therapy response evaluation, and clinical management of plasma cell neoplasms. The review summarizes how flow cytometry is used in the initial evaluation to distinguish primary and secondary clonal plasma cell populations from each other and from reactive plasma cells. We further illustrate the kinds of prognostic information the assessment can provide at diagnosis and disease follow-up of primary plasma cell neoplasms. Technical requirements for MRD assays and their use in therapy efficacy assessment and clinical decision-making in multi-myeloma are discussed., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Non-Hodgkin lymphoma (NHL) is a heterogeneous disease, encompassing a wide variety of individually distinct neoplastic entities of mature B-, T-, and NK-cells. While they constitute a broad category, they are the most common hematologic malignancies in the world. The distinction between different neoplastic entities requires a multi-modal approach, such as flow cytometric immunophenotyping, which can exclude a neoplastic proliferation and help narrow the differential diagnosis. This article describes a flow cytometric test developed at Memorial Sloan Kettering Cancer Center to assess B-, T-, and NK-cells in a single tube, 21-antibody, 19-color assay. The assay can identify most B- and T-cell NHLs with high specificity and sensitivity and significantly narrow the differential when a specific diagnosis cannot be made. The basic protocol provides a detailed operational procedure for sample processing, staining, and cytometric acquisition. The support protocol provides typical steps and caveats for data analysis in lymphoproliferative disorders and in discriminating a variety of specific disease entities from each other and normal lymphoid populations. © 2023 Wiley Periodicals LLC. Basic Protocol: Processing, staining, and cytometric analysis of samples for B- and T-cell assessment Support Protocol: Analysis and interpretation of the B- and T-cell lymphocyte assay., (© 2023 Wiley Periodicals LLC.)

Lenalidomide is an effective component of induction and maintenance therapy for multiple myeloma, though with a risk of secondary malignancies, including acute lymphoblastic leukemia (ALL). In contrast to therapy-related myeloid neoplasia, lenalidomide-associated lymphoblastic neoplasia remains poorly characterized. We conducted a dual institution retrospective study of 32 ALL cases that arose after lenalidomide maintenance (all B-lineage, 31/32 BCR::ABL-negative). B-cell ALL (B-ALL) was diagnosed at median 54 months (range, 5-119) after first exposure to lenalidomide and after median 42 months of cumulative lenalidomide exposure (range, 2-114). High incidence of TP53 mutations (9/19 evaluable cases) and low hypodiploidy (8/26 patients) were identified. Despite median age of 65 years and poor-risk B-ALL features observed in the cohort, rates of complete response (CR) or CR with incomplete hematologic recovery were high (25/28 patients receiving treatment). Median event-free survival was 35.4 months among treated patients (not reached among those undergoing allogeneic hematopoietic cell transplantation [HCT]). Sixteen patients remain alive without evidence of B-ALL after HCT or extended maintenance therapy. We also describe regression of B-ALL or immature B-cell populations with B-ALL immunophenotype after lenalidomide discontinuation in 5 patients, suggesting lenalidomide may drive leukemic progression even after initiation of lymphoblastic neoplasia and that lenalidomide withdrawal alone may be an appropriate first-line intervention in selected patients. Monitoring for early B-ALL-like proliferations may offer opportunities for lenalidomide withdrawal to prevent progression. Established combination chemotherapy regimens, newer surface antigen-targeted approaches, and allogeneic HCT are effective in many patients with lenalidomide-associated B-ALL and should be offered to medically fit patients., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

The optimal induction strategy for mixed phenotype acute leukemia (MPAL) is unknown, though retrospective data has shown improved remission rates and overall survival with acute lymphoblastic leukemia (ALL)-based regimens. At Memorial Sloan Kettering Cancer Center (MSKCC), the most utilized induction regimen for MPAL is high dose cytarabine plus mitoxantrone ("ALL-2"), though outcomes with this regimen are not well described. In this study, outcomes to first-line induction chemotherapy in 24 patients at MSKCC with MPAL classified by 2016 World Health Organization criteria are reported. The overall response rate was 94 % (16 of 17) in patients receiving ALL-2, including 86 % (6 of 7) in patients with extramedullary disease. Thirteen patients who received ALL-2 induction proceeded to allogeneic hematopoietic cell transplant (allo-HCT). The most common toxicity associated with ALL-2 was febrile neutropenia, documented in 12 patients. With a median follow-up of 37 months, median overall survival was not reached in the ALL-2 cohort, and 3-year overall survival was 62 %. In multivariate analysis, age ≥ 60 years and MPAL with isolated extramedullary disease were associated with significantly worse overall survival (P = .009 and P = .01, respectively). These results support further prospective investigation of ALL-2 as a front-line induction regimen for adults with MPAL., Competing Interests: Declaration of Competing Interest T.M.W is employed by Blueprint Medicines Corporation. E.A, A.D, P.K.G, W.X, J.G, S.D.W, M.R, R.S, and S.E.S declare no competing financial interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Measurement of minimal/measurable residual disease (MRD) in B-lymphoblastic leukemia/lymphoma (B-ALL) has become a routine clinical evaluation tool and remains the strongest predictor of treatment outcome. In recent years, new targeted anti-CD19 and anti-CD22 antibody-based and cellular therapies have revolutionized the treatment of the high-risk B-ALL. The new treatments raise challenges for diagnostic flow cytometry, which relies on the presence of specific surface antigens to identify the population of interest. So far, reported flow cytometry-based assays are developed to either achieve a deeper MRD level or to accommodate the loss of surface antigens post-target therapies, but not both., Methods: We developed a single tube flow cytometry assay (14-color-16-parameters). The method was validated using 94 clinical samples as well as spike-in and replicate experiments., Results: The assay was well suited for monitoring response to targeted therapies and reached a sensitivity below 10 -5 with acceptable precision (coefficient of variation < 20%), accuracy, and interobserver variability (κ = 1)., Conclusions: The assay allows for sensitive disease detection of B-ALL MRD independent of CD19 and CD22 expression and allows uniform analysis of samples regardless of anti-CD19 and CD22 therapy., (© 2023 International Clinical Cytometry Society.)

Somatic hypermutation status of the IGHV gene is essential for treating patients with chronic lymphocytic leukemia/small lymphocytic lymphoma. Unlike the conventional low-throughput method, assessment of somatic hypermutation by next-generation sequencing (NGS) has potential for uniformity and scalability. However, it lacks standardization or guidelines for routine clinical use. We critically assessed the performance of an amplicon-based NGS assay across 458 samples. Using a validation cohort (35 samples), the comparison of two platforms (Ion Torrent versus Illumina) and two primer sets [leader versus framework region 1 (FR1)] in their ability to identify clonotypic IGHV rearrangement(s) revealed 97% concordance. The mutation rates were identical by both platforms when using the same primer set (FR1), whereas a slight overestimation bias (+0.326%) was found when comparing FR1 with leader primers. However, for nearly all patients this did not affect the stratification into mutated or unmutated categories, suggesting that use of FR1 may provide comparable results if leader sequencing is not available and allowing for a simpler NGS laboratory workflow. In routine clinical practice (423 samples), the productive rearrangement was successfully detected by either primer set (leader, 97.7%; FR1, 94.7%), and a combination of both in problematic cases reduced the failure rate to 1.2%. Higher sensitivity of the NGS-based analysis also detected a higher frequency of double IGHV rearrangements (19.1%) compared with traditional approaches., (Copyright © 2023 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Patients treated with cytotoxic therapies, including autologous stem cell transplantation, are at risk for developing therapy-related myeloid neoplasms (tMN). Preleukemic clones (ie, clonal hematopoiesis [CH]) are detectable years before the development of these aggressive malignancies, although the genomic events leading to transformation and expansion are not well defined. Here, by leveraging distinctive chemotherapy-associated mutational signatures from whole-genome sequencing data and targeted sequencing of prechemotherapy samples, we reconstructed the evolutionary life-history of 39 therapy-related myeloid malignancies. A dichotomy was revealed, in which neoplasms with evidence of chemotherapy-induced mutagenesis from platinum and melphalan were hypermutated and enriched for complex structural variants (ie, chromothripsis), whereas neoplasms with nonmutagenic chemotherapy exposures were genomically similar to de novo acute myeloid leukemia. Using chemotherapy-associated mutational signatures as temporal barcodes linked to discrete clinical exposure in each patient's life, we estimated that several complex events and genomic drivers were acquired after chemotherapy was administered. For patients with prior multiple myeloma who were treated with high-dose melphalan and autologous stem cell transplantation, we demonstrate that tMN can develop from either a reinfused CH clone that escapes melphalan exposure and is selected after reinfusion, or from TP53-mutant CH that survives direct myeloablative conditioning and acquires melphalan-induced DNA damage. Overall, we revealed a novel mode of tMN progression that is not reliant on direct mutagenesis or even exposure to chemotherapy. Conversely, for tMN that evolve under the influence of chemotherapy-induced mutagenesis, distinct chemotherapies not only select preexisting CH but also promote the acquisition of recurrent genomic drivers., (© 2023 by The American Society of Hematology.)

Minimal/measurable residual disease (MRD) is the most important independent prognostic factor for patients with B-lymphoblastic leukemia (B-LL). MRD post therapy has been incorporated into risk stratification and clinical management, resulting in substantially improved outcomes in pediatric and adult patients. Currently, MRD in B-ALL is most commonly assessed by multiparametric flow cytometry and molecular (polymerase chain reaction or high-throughput sequencing based) methods. The detection of MRD by flow cytometry in B-ALL often begins with B cell antigen-based gating strategies. Over the past several years, targeted immunotherapy directed against B cell markers has been introduced in patients with relapsed or refractory B-ALL and has demonstrated encouraging results. However, targeted therapies have significant impact on the immunophenotype of leukemic blasts, in particular, downregulation or loss of targeted antigens on blasts and normal B cell precursors, posing challenges for MRD detection using standard gating strategies. Novel flow cytometric approaches, using alternative strategies for population identification, sometimes including alternative gating reagents, have been developed and implemented to monitor MRD in the setting of post targeted therapy., (© 2023 International Clinical Cytometry Society.)

Background: Lineage infidelity is characteristic of mixed phenotype acute leukemia and is also seen in blast phase of chronic myeloid leukemia (CML), myeloid/lymphoid neoplasia with eosinophilia and gene rearrangements, and subtypes of acute myeloid leukemia. Driver genetic events often occur in multipotent progenitor cells in myeloid neoplasms, suggesting that multilineage output may be more common than appreciated. This phenomenon is not well studied in myelodysplastic syndrome (MDS) and non-CML myeloproliferative neoplasms (MPN)., Methods: We systematically evaluated phenotypic lineage infidelity by reviewing bone marrow pathology and flow cytometry (FC) studies of 1262 consecutive patients with a diagnosis of MDS and/or non-CML MPN. We assessed B- and T-cells in these patients by FC. When abnormal B-lymphoblast (ABLB) populations were detected, we additionally evaluated immature B-cells using a high sensitivity FC assay for B-lymphoblastic leukemia/lymphoma (B-ALL)., Results: We identified 9 patients (7 MDS, 7/713, 1%; 2 non-CML MPN, 2/312, 0.6%; 0 in MDS/MPN) with low-level ABLB populations (0.012%-3.6% of WBCs in marrow) with abnormal immunophenotypes. Genetic studies on flow sorted cell populations confirmed that some ABLB populations were clonally related to myeloid blasts (4/6, 67%). On follow-up, ABLB populations in 8/9 patients remained stable or disappeared. Only 1 case progressed to B-ALL., Conclusions: These findings demonstrate that phenotypically detectable abnormal immature B lineage output occurs in MDS and non-CML MPN, albeit rarely. While presence of ABLB does not necessarily reflect blast crisis, the underlying disease biology of our findings may ultimately be relevant to patient management and warrants further investigation., (© 2021 International Clinical Cytometry Society.)

The rarity of malignant Hodgkin and Reed Sternberg (HRS) cells in classic Hodgkin lymphoma (cHL) limits the ability to study the genomics of cHL. To circumvent this, our group has previously optimized fluorescence-activated cell sorting to purify HRS cells. Using this approach, we now report the whole-genome sequencing landscape of HRS cells and reconstruct the chronology and likely etiology of pathogenic events leading to cHL. We identified alterations in driver genes not previously described in cHL, APOBEC mutational activity, and the presence of complex structural variants including chromothripsis. We found that high ploidy in cHL is often acquired through multiple, independent chromosomal gains events including whole-genome duplication. Evolutionary timing analyses revealed that structural variants enriched for RAG motifs, driver mutations in B2M, BCL7A, GNA13, and PTPN1, and the onset of AID-driven mutagenesis usually preceded large chromosomal gains. This study provides a temporal reconstruction of cHL pathogenesis., Significance: Previous studies in cHL were limited to coding sequences and therefore not able to comprehensively decipher the tumor complexity. Here, leveraging cHL whole-genome characterization, we identify driver events and reconstruct the tumor evolution, finding that structural variants, driver mutations, and AID mutagenesis precede chromosomal gains. This article is highlighted in the In This Issue feature, p. 171., (©2023 American Association for Cancer Research.)

Flow cytometry has been indispensable in diagnosing B cell lymphoma and plasma cell neoplasms. The advances in novel multicolor flow cytometry have also made this technology a robust tool for monitoring minimal/measurable residual disease in chronic lymphocytic leukemia and multiple myeloma. However, challenges using conventional gating strategies to isolate neoplastic B or plasma cells are emerging due to the rapidly increasing number of antibody therapeutics targeting single or multiple classic B/plasma cell-lineage markers, such as CD19, CD20, and CD22 in B cells and CD38 in plasma cells. This review is the first of a two-part series that summarizes the most current targeted therapies used in B and plasma cell neoplasms and proposes detailed alternative approaches to overcome post-targeted therapy analysis challenges by flow cytometry. The second review in this series (Chen et al.) focuses on challenges encountered in the use of targeted therapy in precursor B cell neoplasms., (© 2022 International Clinical Cytometry Society.)