RESUMEN
Nuclear hormone receptors (NRs) are ligand-binding transcription factors that are widely targeted therapeutically. Agonist binding triggers NR activation and subsequent degradation by unknown ligand-dependent ubiquitin ligase machinery. NR degradation is critical for therapeutic efficacy in malignancies that are driven by retinoic acid and estrogen receptors. Here, we demonstrate the ubiquitin ligase UBR5 drives degradation of multiple agonist-bound NRs, including the retinoic acid receptor alpha (RARA), retinoid x receptor alpha (RXRA), glucocorticoid, estrogen, liver-X, progesterone, and vitamin D receptors. We present the high-resolution cryo-EMstructure of full-length human UBR5 and a negative stain model representing its interaction with RARA/RXRA. Agonist ligands induce sequential, mutually exclusive recruitment of nuclear coactivators (NCOAs) and UBR5 to chromatin to regulate transcriptional networks. Other pharmacological ligands such as selective estrogen receptor degraders (SERDs) degrade their receptors through differential recruitment of UBR5 or RNF111. We establish the UBR5 transcriptional regulatory hub as a common mediator and regulator of NR-induced transcription.
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Cromatina , Factores de Transcripción , Humanos , Ligandos , Cromatina/genética , Factores de Transcripción/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Ubiquitinas , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Chromatin-binding proteins are critical regulators of cell state in haematopoiesis1,2. Acute leukaemias driven by rearrangement of the mixed lineage leukaemia 1 gene (KMT2Ar) or mutation of the nucleophosmin gene (NPM1) require the chromatin adapter protein menin, encoded by the MEN1 gene, to sustain aberrant leukaemogenic gene expression programs3-5. In a phase 1 first-in-human clinical trial, the menin inhibitor revumenib, which is designed to disrupt the menin-MLL1 interaction, induced clinical responses in patients with leukaemia with KMT2Ar or mutated NPM1 (ref. 6). Here we identified somatic mutations in MEN1 at the revumenib-menin interface in patients with acquired resistance to menin inhibition. Consistent with the genetic data in patients, inhibitor-menin interface mutations represent a conserved mechanism of therapeutic resistance in xenograft models and in an unbiased base-editor screen. These mutants attenuate drug-target binding by generating structural perturbations that impact small-molecule binding but not the interaction with the natural ligand MLL1, and prevent inhibitor-induced eviction of menin and MLL1 from chromatin. To our knowledge, this study is the first to demonstrate that a chromatin-targeting therapeutic drug exerts sufficient selection pressure in patients to drive the evolution of escape mutants that lead to sustained chromatin occupancy, suggesting a common mechanism of therapeutic resistance.
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Resistencia a Antineoplásicos , Leucemia , Mutación , Proteínas Proto-Oncogénicas , Animales , Humanos , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Sitios de Unión/efectos de los fármacos , Sitios de Unión/genética , Cromatina/genética , Cromatina/metabolismo , Resistencia a Antineoplásicos/genética , Leucemia/tratamiento farmacológico , Leucemia/genética , Leucemia/metabolismo , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismoRESUMEN
ABSTRACT: Small molecules that target the menin-KMT2A protein-protein interaction (menin inhibitors) have recently entered clinical trials in lysine methyltransferase 2A (KMT2A or MLL1)-rearranged (KMT2A-r) and nucleophosmin-mutant (NPM1c) acute myeloid leukemia (AML) and are demonstrating encouraging results. However, rationally chosen combination therapy is needed to improve responses and prevent resistance. We have previously identified IKZF1/IKAROS as a target in KMT2A-r AML and shown in preclinical models that IKAROS protein degradation with lenalidomide or iberdomide has modest single-agent activity yet can synergize with menin inhibitors. Recently, the novel IKAROS degrader mezigdomide was developed with greatly enhanced IKAROS protein degradation. In this study, we show that mezigdomide has increased preclinical activity in vitro as a single-agent in KMT2A-r and NPM1c AML cell lines, including sensitivity in cell lines resistant to lenalidomide and iberdomide. Further, we demonstrate that mezigdomide has the greatest capacity to synergize with and induce apoptosis in combination with menin inhibitors, including in MEN1 mutant models. We show that the superior activity of mezigdomide compared with lenalidomide or iberdomide is due to its increased depth, rate, and duration of IKAROS protein degradation. Single-agent mezigdomide was efficacious in 5 patient-derived xenograft models of KMT2A-r and 1 NPM1c AML. The combination of mezigdomide with the menin inhibitor VTP-50469 increased survival and prevented and overcame MEN1 mutations that mediate resistance in patients receiving menin inhibitor monotherapy. These results support prioritization of mezigdomide for early phase clinical trials in KMT2A-r and NPM1c AML, either as a single agent or in combination with menin inhibitors.
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Leucemia Mieloide Aguda , Morfolinas , Proteína de la Leucemia Mieloide-Linfoide , Ftalimidas , Piperidonas , Humanos , Lenalidomida/uso terapéutico , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Factores de Transcripción/genética , MutaciónRESUMEN
The interaction between menin and histone-lysine N-methyltransferase 2A (KMT2A) is a critical dependency for KMT2A- or nucleophosmin 1 (NPM1)-altered leukemias and an emerging opportunity for therapeutic development. JNJ-75276617 is a novel, orally bioavailable, potent, and selective protein-protein interaction inhibitor of the binding between menin and KMT2A. In KMT2A-rearranged (KMT2A-r) and NPM1-mutant (NPM1c) AML cells, JNJ-75276617 inhibited the association of the menin-KMT2A complex with chromatin at target gene promoters, resulting in reduced expression of several menin-KMT2A target genes, including MEIS1 and FLT3. JNJ-75276617 displayed potent anti-proliferative activity across several AML and ALL cell lines and patient samples harboring KMT2A- or NPM1-alterations in vitro. In xenograft models of AML and ALL, JNJ-75276617 reduced leukemic burden and provided a significant dose-dependent survival benefit accompanied by expression changes of menin-KMT2A target genes. JNJ-75276617 demonstrated synergistic effects with gilteritinib in vitro in AML cells harboring KMT2A-r. JNJ-75276617 further exhibited synergistic effects with venetoclax and azacitidine in AML cells bearing KMT2A-r in vitro, and significantly increased survival in mice. Interestingly, JNJ-75276617 showed potent anti-proliferative activity in cell lines engineered with recently discovered mutations (MEN1M327I or MEN1T349M) that developed in patients refractory to the menin-KMT2A inhibitor revumenib. A co-crystal structure of menin in complex with JNJ-75276617 indicates a unique binding mode distinct from other menin-KMT2A inhibitors, including revumenib. JNJ-75276617 is being clinically investigated for acute leukemias harboring KMT2A or NPM1 alterations, as a monotherapy for relapsed/refractory (R/R) acute leukemia (NCT04811560), or in combination with AML-directed therapies (NCT05453903).
RESUMEN
Valencia-Sánchez et al. have demonstrated that two histone post-translational modifications (PTMs) - H4K16 acetylation (H4K16ac) and H2BK120 ubiquitination (H2Bub) - enhance the methylation of H3K79 (H3K79me) by Dot1. This breakthrough indicates crosstalk between H4Kac/H2Bub/H3K79me and may improve our understanding of the role that Dot1/Dot1L plays in developmental processes and disease, including MLL1/KMT2A(MLL-r) leukemia.
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Histonas , Procesamiento Proteico-Postraduccional , Acetilación , Histonas/metabolismo , Metilación , UbiquitinaciónRESUMEN
The GATA and PAX-SIX-EYA-DACH transcriptional networks (PSEDNs) are essential for proper development across taxa. Here, we demonstrate novel PSEDN roles in vivo in Drosophila hematopoiesis and in human erythropoiesis in vitro Using Drosophila genetics, we show that PSEDN members function with GATA to block lamellocyte differentiation and maintain the prohemocyte pool. Overexpression of human SIX1 stimulated erythroid differentiation of human erythroleukemia TF1 cells and primary hematopoietic stem-progenitor cells. Conversely, SIX1 knockout impaired erythropoiesis in both cell types. SIX1 stimulation of erythropoiesis required GATA1, as SIX1 overexpression failed to drive erythroid phenotypes and gene expression patterns in GATA1 knockout cells. SIX1 can associate with GATA1 and stimulate GATA1-mediated gene transcription, suggesting that SIX1-GATA1 physical interactions contribute to the observed functional interactions. In addition, both fly and human SIX proteins regulated GATA protein levels. Collectively, our findings demonstrate that SIX proteins enhance GATA function at multiple levels, and reveal evolutionarily conserved cooperation between the GATA and PSEDN networks that may regulate developmental processes beyond hematopoiesis.
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Proteínas de Drosophila/metabolismo , Eritropoyesis/genética , Redes Reguladoras de Genes , Hematopoyesis/genética , Animales , Línea Celular Tumoral , Drosophila , Factores de Transcripción GATA/metabolismo , Técnicas de Inactivación de Genes , Proteínas de Homeodominio/metabolismo , Humanos , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción Paired Box/metabolismoRESUMEN
Nonreceptor tyrosine kinases (NRTKs) represent an important class of signaling molecules driving diverse cellular pathways. Aberrant expression and hyperphosphorylation of TNK2, an NRTK, have been implicated in multiple cancers. However, the exact proteins and cellular events that mediate phenotypic changes downstream of TNK2 are unclear. Biological systems that employ proximity-dependent biotinylation methods, such as BioID, are being increasingly used to map protein-protein interactions, as they provide increased sensitivity in discovering interaction partners. In this study, we employed stable isotope labeling with amino acids in cell culture and BioID coupled to the biotinylation site identification technology (BioSITe) method that we recently developed to quantitatively explore the interactome of TNK2. By performing a controlled comparative analysis between full-length TNK2 and its truncated counterpart, we were able to not only identify site-level biotinylation of previously well-established TNK2 binders and substrates including NCK1, NCK2, CTTN, and STAT3, but also discover several novel TNK2 interacting partners. We also performed co-immunoprecipitation and immunofluorescence analysis to validate the interaction between TNK2 and CLINT1, a novel TNK2 interacting protein. Overall, this work reveals the power of the BioSITe method coupled to BioID and highlights several molecules that warrant further exploration to assess their functional significance in TNK2-mediated signaling.
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Proteínas Tirosina Quinasas , Transducción de Señal , Biotinilación , Unión Proteica , Proteínas Tirosina Quinasas/genéticaRESUMEN
KRAS is one of the most frequently mutated genes across all cancer subtypes. Two of the most frequent oncogenic KRAS mutations observed in patients result in glycine to aspartic acid substitution at either codon 12 (G12D) or 13 (G13D). Although the biochemical differences between these two predominant mutations are not fully understood, distinct clinical features of the resulting tumors suggest involvement of disparate signaling mechanisms. When we compared the global phosphotyrosine proteomic profiles of isogenic colorectal cancer cell lines bearing either G12D or G13D KRAS mutation, we observed both shared as well as unique signaling events induced by the two KRAS mutations. Remarkably, while the G12D mutation led to an increase in membrane proximal and adherens junction signaling, the G13D mutation led to activation of signaling molecules such as nonreceptor tyrosine kinases, MAPK kinases, and regulators of metabolic processes. The importance of one of the cell surface molecules, MPZL1, which was found to be hyperphosphorylated in G12D cells, was confirmed by cellular assays as its knockdown led to a decrease in proliferation of G12D but not G13D expressing cells. Overall, our study reveals important signaling differences across two common KRAS mutations and highlights the utility of our approach to systematically dissect subtle differences between related oncogenic mutants and potentially lead to individualized treatments.
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Neoplasias Colorrectales , Proteínas Proto-Oncogénicas p21(ras) , Alelos , Neoplasias Colorrectales/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Mutación , Fosfoproteínas , Fosfotirosina , Proteómica , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
Biotin-based labeling strategies are widely employed to study protein-protein interactions, subcellular proteomes and post-translational modifications, as well as, used in drug discovery. While the high affinity of streptavidin for biotin greatly facilitates the capture of biotinylated proteins, it still presents a challenge, as currently employed, for the recovery of biotinylated peptides. Here we describe a strategy designated Biotinylation Site Identification Technology (BioSITe) for the capture of biotinylated peptides for LC-MS/MS analyses. We demonstrate the utility of BioSITe when applied to proximity-dependent labeling methods, APEX and BioID, as well as biotin-based click chemistry strategies for identifying O-GlcNAc-modified sites. We demonstrate the use of isotopically labeled biotin for quantitative BioSITe experiments that simplify differential interactome analysis and obviate the need for metabolic labeling strategies such as SILAC. Our data also highlight the potential value of site-specific biotinylation in providing spatial and topological information about proteins and protein complexes. Overall, we anticipate that BioSITe will replace the conventional methods in studies where detection of biotinylation sites is important.
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Acetilglucosamina/metabolismo , Biotina/química , Química Clic/métodos , Péptidos/aislamiento & purificación , Procesamiento Proteico-Postraduccional , Estreptavidina/química , Acetilglucosamina/química , Secuencia de Aminoácidos , Animales , Anticuerpos Inmovilizados/química , Linfocitos B/química , Biotinilación , Línea Celular , Cromatografía Liquida , Células HEK293 , Humanos , Ratones , Péptidos/química , Proteolisis , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: Myelodysplastic syndromes (MDS) are a heterogenous collection of clonal bone marrow diseases characterized by cytopenias, abnormal karyotypes, molecular abnormalities, and dysplasia by flow cytometry and/or morphology. The progression of MDS to severe cytopenias and/or overt leukemia is associated with the accumulation of additional cytogenetic abnormalities, suggesting clonal evolution. The impact of these accumulated abnormalities on myeloid maturation and the severity of the disease is poorly understood. METHODS: Bone marrow specimens from 16 patients with cytogenetic abnormalities were flow cytometrically sorted into three myeloid populations: progenitors, immature myeloid cells, and mature myeloid cells. Fluorescence in situ hybridization analysis was performed on each to determine the distribution of chromosomal abnormalities during myeloid maturation. RESULTS: Our findings revealed three distinct distributions of cytogenetic abnormalities across myeloid maturation, each of which corresponded to specific cytogenetic abnormalities. Group 1 had continuous distribution across all maturational stages and contained patients with a single cytogenetic aberration associated with good-to-intermediate prognosis; Group 2 had accumulation of abnormalities in immature cells and contained patients with high-risk monosomy 7; and Group 3 had abnormalities defining the founding clone equally distributed across maturational stages while subclonal abnormalities were enriched in progenitor cells and contained patients with multiple, non-monosomy 7, abnormalities with evidence of clonal evolution. CONCLUSIONS: Our findings demonstrate that low-risk abnormalities (e.g., del(20q) and trisomy 8) occurring in the founding clone display a markedly different disease etiology, with respect to myeloid maturation, than monosomy 7 or abnormalities acquired in subclones, which result in a disruption of myeloid cell maturation in MDS.
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Síndromes Mielodisplásicos , Humanos , Hibridación Fluorescente in Situ , Citometría de Flujo , Síndromes Mielodisplásicos/genética , Aberraciones Cromosómicas , Fenotipo , Genotipo , Células MieloidesRESUMEN
Gastrointestinal stromal tumor (GIST) is commonly characterized by activating mutations in the receptor tyrosine kinase KIT. Tyrosine kinase inhibitors are the only approved therapy for GIST, and complementary treatment strategies are urgently needed. As GIST lacks oncogene amplification and relies upon an established network of transcription factors, we hypothesized that unique chromatin-modifying enzymes are essential in orchestrating the GIST epigenome. We identified through genome-scale CRISPR screening that MOZ and Menin-MLL chromatin regulatory complexes are cooperative and unique dependencies in GIST. These complexes were enriched at GIST-relevant genes and regulated their transcription. Inhibition of MOZ and Menin-MLL complexes decreased GIST cell proliferation by disrupting interactions with transcriptional/chromatin regulators, such as DOT1L. MOZ and Menin inhibition caused significant reductions in tumor burden in vivo, with superior effects observed with combined Menin and KIT inhibition. These results define unique chromatin regulatory dependencies in GIST and identify potential therapeutic strategies for clinical application. SIGNIFICANCE: Although many malignancies rely on oncogene amplification, GIST instead depends upon epigenetic regulation of KIT and other essential genes. Utilizing genome-scale CRISPR dependency screens, we identified complementary chromatin-modifying complexes essential to GIST and characterize the consequences of their disruption, elucidating a novel therapeutic approach to this disease. This article is highlighted in the In This Issue feature, p. 1599.
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Neoplasias Gastrointestinales , Tumores del Estroma Gastrointestinal , Histona Acetiltransferasas/metabolismo , Cromatina/genética , Epigénesis Genética , Neoplasias Gastrointestinales/tratamiento farmacológico , Neoplasias Gastrointestinales/genética , Tumores del Estroma Gastrointestinal/tratamiento farmacológico , Tumores del Estroma Gastrointestinal/genética , Humanos , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Tirosina Quinasas Receptoras/genética , Factores de Transcripción/genéticaRESUMEN
Acute myeloid leukemia (AML) remains difficult to treat and requires new therapeutic approaches. Potent inhibitors of the chromatin-associated protein MENIN have recently entered human clinical trials, opening new therapeutic opportunities for some genetic subtypes of this disease. Using genome-scale functional genetic screens, we identified IKAROS (encoded by IKZF1) as an essential transcription factor in KMT2A (MLL1)-rearranged (MLL-r) AML that maintains leukemogenic gene expression while also repressing pathways for tumor suppression, immune regulation and cellular differentiation. Furthermore, IKAROS displays an unexpected functional cooperativity and extensive chromatin co-occupancy with mixed lineage leukemia (MLL)1-MENIN and the regulator MEIS1 and an extensive hematopoietic transcriptional complex involving homeobox (HOX)A10, MEIS1 and IKAROS. This dependency could be therapeutically exploited by inducing IKAROS protein degradation with immunomodulatory imide drugs (IMiDs). Finally, we demonstrate that combined IKAROS degradation and MENIN inhibition effectively disrupts leukemogenic transcriptional networks, resulting in synergistic killing of leukemia cells and providing a paradigm for improved drug targeting of transcription and an opportunity for rapid clinical translation.
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Leucemia Mieloide Aguda , Cromatina , Expresión Génica , Humanos , Factor de Transcripción Ikaros/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/genética , Factores de Transcripción/genéticaRESUMEN
The nuclear lamina is a proteinaceous network of filaments that provide both structural and gene regulatory functions by tethering proteins and large domains of DNA, the so-called lamina-associated domains (LADs), to the periphery of the nucleus. LADs are a large fraction of the mammalian genome that are repressed, in part, by their association to the nuclear periphery. The genesis and maintenance of LADs is poorly understood as are the proteins that participate in these functions. In an effort to identify proteins that reside at the nuclear periphery and potentially interact with LADs, we have taken a two-pronged approach. First, we have undertaken an interactome analysis of the inner nuclear membrane bound LAP2ß to further characterize the nuclear lamina proteome. To accomplish this, we have leveraged the BioID system, which previously has been successfully used to characterize the nuclear lamina proteome. Second, we have established a system to identify proteins that bind to LADs by developing a chromatin-directed BioID system. We combined the BioID system with the m6A-tracer system which binds to LADs in live cells to identify both LAD proximal and nuclear lamina proteins. In combining these datasets, we have further characterized the protein network at the nuclear lamina, identified putative LAD proximal proteins and found several proteins that appear to interface with both micro-proteomes. Importantly, several proteins essential for LAD function, including heterochromatin regulating proteins related to H3K9 methylation, were identified in this study.
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Lámina Nuclear/metabolismo , Proteoma/metabolismo , Animales , Línea Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/metabolismo , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Genoma , Heterocromatina/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Ratones , Células 3T3 NIH , Lámina Nuclear/genética , Lámina Nuclear/patología , Proteínas Nucleares/genética , Unión Proteica/fisiología , Dominios Proteicos/fisiología , Proteoma/genética , Proteómica/métodosRESUMEN
An international working group within the European LeukemiaNet gathered, aiming to determine the role of flow cytometry (FC) in myelodysplastic syndromes (MDS). It was agreed that FC has a substantial application in disease characterization, diagnosis and prognosis. FC may also be useful in predicting treatment responses and monitoring novel and standard therapeutic regimens. In this article the rationale is discussed that flow cytometry should be integrated as a part of diagnostic and prognostic scoring systems in MDS.
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Citometría de Flujo/métodos , Síndromes Mielodisplásicos/diagnóstico , Síndromes Mielodisplásicos/terapia , Evaluación de Resultado en la Atención de Salud/métodos , Humanos , Agencias Internacionales , Síndromes Mielodisplásicos/clasificación , Pronóstico , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Sociedades CientíficasRESUMEN
Flow cytometric cell sorting combined with molecular gene rearrangement analysis was used to detect and to further characterize simultaneously occurring phenotypically distinct B cell monoclonal lymphoid and monoclonal plasma cell populations from 38 individual specimens. By sorting and subsequent gene rearrangement analysis, separate or identical monoclonality genotypes could be revealed and confirmed. In only 13 of 38 specimens, the B lymphoid cells and plasma cell populations showed an identical genotypic profile, while 25 had non-identical profiles (including 4 process control specimens). The majority of the genotypically identical group had a phenotype consistent with Waldenström's/lymphoplasmacytic lymphoma (WM/LPL), while WM/LPL phenotype was present in 16/25 of the non-identical cases. Proof of an identical monoclonal genotype for plasmacytic and B-lymphoid cell populations must be used to define WM/LPL as a distinct entity in the clinical setting of monoclonal lymphoid and plasma cells expressing the same light chains. Conversely, the confirmation of genotypically distinct populations can significantly improve confidence in diagnostic and prognostic decisions in specimens with B lymphoid lymphomas and a concurrent, possibly smoldering myeloma or multiple myeloma. These techniques are requisite in future clinical studies for diagnosis and prognosis in these diseases.
Asunto(s)
Cadenas Ligeras de Inmunoglobulina/metabolismo , Linfocitos/patología , Células Plasmáticas/patología , Macroglobulinemia de Waldenström/genética , Macroglobulinemia de Waldenström/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Médula Ósea/patología , Estudios de Cohortes , Análisis Citogenético , Femenino , Frecuencia de los Genes , Reordenamiento Génico de Linfocito B/fisiología , Humanos , Cadenas Ligeras de Inmunoglobulina/genética , Recuento de Linfocitos , Linfocitos/inmunología , Linfocitos/metabolismo , Masculino , Persona de Mediana Edad , Células Plasmáticas/metabolismo , Análisis de Secuencia de ADN , Macroglobulinemia de Waldenström/patologíaRESUMEN
BACKGROUND: In patients with unexplained cytopenias, abnormal karyotyping studies can be found with inconclusive light microscopic findings. Multidimensional flow cytometry (FCM) can identify myelomonocytic cells with aberrant phenotypes often not seen by standard morphology. METHODS: In 431 patients presenting with unexplained cytopenia(s) FCM results were compared to abnormal karyotyping and FISH results recognized as associated with myelodysplastic syndrome (MDS) in the 2008 WHO classification, to assess the degree of and types of phenotypic abnormalities observed using a previously reported flow cytometric scoring system (FCSS). Fluorescence activated cell sorting was also used to identify subpopulations of abnormal maturing myelomonocytic cells that carry the genotypic abnormality. RESULTS: For marrows with complex (three or more karyotypic abnormalities), two abnormalities, isolated chromosome seven anomalies, del(5q) or del(13q), 100% of cases were positive when using a FCSS cutoff of ≥ 2. Trisomy 8, del(20 q), and minus Y had flow scores ≥ 2 in 72, 60, and 18%, respectively, but in some cases the flow score was high, indicating myeloid dysplasia. Most patients (16/22) with high myeloid progenitor cells (MyPC) (> 20%) also exhibited maturing myeloid cell abnormalities by FCM. Morphology was negative in the maturing myeloid cells in many cases with phenotypically abnormal myeloid cells. CONCLUSIONS: The high correlation between genotypic and phenotypic abnormalities suggests a possible increased utility of flow cytometry in the diagnosis of patients with unexplained cytopenias and may be useful in future clinical studies and in the classification by the WHO, using the FCSS rather than simple counting of flow cytometric abnormalities.