RESUMO
INTRODUCTION: Tyrosinemia type 1 is a rare disease with autosomal recessive inheritance, featuring various clinical manifestations. These may encompass acute neonatal liver failure, neonatal cholestatic syndrome, chronic hepatitis, cirrhosis, hepatocellular carcinoma, and, alternatively, kidney disorders like renal tubular acidosis, Fanconi syndrome, hypophosphatemic rickets, among other alterations. Diagnosis relies on detecting toxic metabolites in the blood and urine, ideally confirmed through molecular testing. METHOD: A consensus was reached with experts in the field of inborn errors of metabolism (EIM), including eight pediatric gastroenterologists, two EIM specialists, two geneticists, three pediatric nutritionists specialized in EIM, and a pediatric surgeon specializing in transplants. Six working groups were tasked with formulating statements and justifications, and 32 statements were anonymously voted on using the Likert scale and the Delphi method. The first virtual vote achieved an 80% consensus, with the remaining 20% determined in person. RESULTS: The statements were categorized into epidemiology, clinical presentation, diagnosis, nutritional and medical treatment, and genetic counseling. CONCLUSIONS: This consensus serves as a valuable tool for primary care physicians, pediatricians, and pediatric gastroenterologists, aiding in the prompt diagnosis and treatment of this disease. Its impact on the morbidity and mortality of patients with tyrosinemia type 1 is substantial.
INTRODUCCIÓN: La tirosinemia tipo 1 es una enfermedad rara, con herencia autosómica recesiva, con múltiples manifestaciones clínicas, que pueden comprender desde falla hepática aguda neonatal, síndrome colestásico neonatal, hepatitis crónica, cirrosis o hepatocarcinoma, hasta alteraciones renales como acidosis tubular renal, síndrome de Fanconi o raquitismo hipofosfatémico, entre otras. El diagnóstico se basa en la presencia de metabolitos tóxicos en la sangre y la orina, idealmente con la confirmación molecular de la enfermedad. MÉTODO: Se realizó un consenso con expertos en el área de los errores innatos del metabolismo (EIM): ocho gastroenterólogos pediatras, dos médicos especialistas en EIM, dos genetistas, tres nutriólogas pediatras especializadas en EIM y un cirujano pediatra especialista en trasplantes. Se formaron seis mesas de trabajo encargadas de desarrollar los enunciados con sus justificaciones y fueron votados anónimamente 32 enunciados en una escala Likert con un método Delphi. La primera votación fue virtual, obteniendo consenso del 80% de los enunciados, y la segunda fue presencial, obteniendo el 20% restante. RESULTADOS: Los enunciados fueron divididos en epidemiología, cuadro clínico, diagnóstico, tratamiento nutricional y médico, y consejo genético. CONCLUSIONES: Este consenso constituye una valiosa herramienta para los médicos de atención primaria, pediatras y gastroenterólogos pediátricos, ya que ayuda a diagnosticar y tratar rápidamente esta enfermedad. Su impacto en la morbilidad y mortalidad de los pacientes con tirosinemia tipo 1 es sustancial.
Assuntos
Consenso , Tirosinemias , Humanos , Tirosinemias/diagnóstico , Tirosinemias/terapia , México , Recém-Nascido , Técnica Delphi , Aconselhamento GenéticoRESUMO
Background: Primary central nervous system (CNS) lymphoma is a very rare extranodal non-Hodgkin lymphoma. The bilateral pattern, as we call it "mirror type", has been identified in other CNS lesions such as gliomas, metastases, and demyelinating lesions, so the differential diagnosis includes imaging studies such as magnetic resonance imaging contrasted with spectroscopy, ruling out immunodeficiency or metastatic disease. Case Description: A 65-year-old female presented progressing headache, loss of memory and language alterations, as well as sensory alterations. Neuroimaging showed the presence of two equidistant periventricular lesions at the level of both ventricular atria, a spectroscopy study suggestive of malignancy. Serological studies showed no evidence of immunodeficiency or the presence of positive tumor markers; however, a biopsy was performed, which revealed a histopathological result of primary lymphoma of the CNS. Conclusion: In neuro-oncology, primary CNS tumors with multiple lesions are rare, even more, the "mirror type" lesions. Lymphomas are lesions that can present in different ways on imaging and clinical presentation. These tumors that present a vector effect due to their size, perilesional edema, or that lead to loss of neurological function are highly discussed in diagnostic and surgical treatment. Due to their prognosis, action on diagnosis and treatment must be taken as quickly as hospital resources allow.
RESUMO
RESUMEN Las estrategias para comunicar la información han experimentado muchos cambios, los cuales se han evidenciado en la pandemia. La idea es lograr que se pueda conectar con el estudiante y que la información la reciba de manera clara, oportuna y pertinente, de modo que tendrá los saberes previos para lograr integrar la información de la teoría y la práctica, con el fin de enfrentar con éxito la actividad simulada, así como cuando interactúe con el paciente. En ese contexto, el aula invertida es una estrategia didáctica que brinda videos, podcast, entre otros, para que sean revisados con anticipación, de tal forma que cuando el estudiante realice la actividad presencial o virtual se profundice y se hagan precisiones sobre los conceptos más importantes, usando metodologías activas de participación para lograr el siguiente propósito: El aprendizaje invertido busca un cambio en la dinámica del aprendizaje que apunte como eje del mismo al estudiante. El objetivo de la presente contribución docente es compartir las bondades de esta estrategia usada en estudiantes de la carrera de Odontología dentro del marco del curso de Clínica Estomatológica del Adulto I, dictado en 2023 y que pertenece al quinto ciclo de la carrera de Estomatología en la Universidad Peruana Cayetano Heredia.
ABSTRACT Strategies for communicating information have undergone many changes, which have been evidenced in the pandemic. The idea is to be able to connect with the student and that the information is received in a clear, timely and relevant manner, so that he/she will have the prior knowledge to integrate information from theory and practice, in order to successfully face the simulated activity, as well as when interacting with the patient. In this context, the inverted classroom is a didactic strategy that provides videos, podcasts, among others, to be reviewed in advance, so that when the student performs the face-to-face or virtual activity, the most important concepts are deepened and clarified, using active participation methodologies to achieve the following objective: flipped learning seeks a change in the dynamics of learning that points to the student as its axis. The objective of this teaching contribution is to share the benefits of this strategy used with students of the Dentistry degree program within the framework of the Adult Stomatology Clinic I course, taught in 2023 and which belongs to the fifth semester of the Stomatology degree program at the Universidad Peruana Cayetano Heredia.
RESUMO As estratégias de comunicação de informação sofreram muitas alterações, que são evidentes na pandemia. A ideia é conseguir conectar-se com o aluno e que a informação seja recebida de forma clara, oportuna e relevante, para que ele tenha o conhecimento prévio para poder integrar informações da teoria e da prática, a fim de enfrentar com sucesso a atividade simulada, bem como ao interagir com o paciente. Neste contexto, a sala de aula invertida é uma estratégia didática que disponibiliza vídeos, podcasts, entre outros, para serem revistos previamente, de modo a que, quando o aluno realiza a atividade presencial ou virtual, os conceitos mais importantes sejam aprofundados e clarificados, utilizando metodologias de participação ativa para atingir o seguinte objetivo: A aprendizagem investida procura uma mudança na dinâmica da aprendizagem que aponta para o aluno como eixo da mesma. O objetivo desta contribuição didática é partilhar os benefícios desta estratégia utilizada com os alunos da disciplina de Medicina Dentária no âmbito do curso de Clínica de Estomatologia de Adultos I, ministrada em 2023 e que pertence ao quinto semestre da disciplina de Estomatologia da Universidad Peruana Cayetano Heredia.
RESUMO
Woodchips in stand-off pads for wintering cows have been applied in countries like Ireland and New Zealand. Their primary role is to protect soils by effectively filtering nutrients during wet conditions, while ensuring a healthy and comfortable environment for the cows. The stand-off pad concept has the potential to be adopted in Canada to provide year-long outdoor access to tie-stall dairy cows. The objective of this study was to evaluate the effect of alternative filtering materials and bed aeration under controlled laboratory conditions. Twelve biofilter columns (0.3 m in diameter and 1-m high) were installed in 12 environmentally-controlled chambers (1.2-m wide by 2.4-m long), and divided into four treatments: a bed of conventional woodchips or an alternative mix of organic materials (sphagnum peat moss, woodchips and biochar) with and without aeration (flux rate set at 0.6 m3/min/m2). Approximately 0.6 L of semi-synthetic dairy manure and 1 L of tap water were poured on the biofilters during two experimental periods of 4 weeks, simulating the effect of either winter or summer conditions (room temperature below or over 10 °C) on the retention of nutrients and fecal bacteria. Results showed that the alternative biofilters under both summer and winter conditions were more efficient in removing COD, SS, TN, and NO3-N than conventional biofilters (maximum efficiencies of 97.6%, 99.7%, 96.4%, and 98.4%, respectively). Similarly for E. coli, they achieved a minimum concentration of 1.8 Log10 CFU/100 ml. Conventional biofilters were more efficient for PO4-P removal with a maximum efficiency of 88.2%. Aeration did not have any significant effect under the tested temperature conditions. Additional factors such as media adaptation time as well as aeration flow during this period should be considered.
Assuntos
Escherichia coli , Esterco , Feminino , Animais , Bovinos , Temperatura , Fezes , Nutrientes , Filtração/métodosRESUMO
T-cell acute lymphoblastic leukemia (T-ALL) is a T-cell malignancy characterized by cell subsets and enriched with leukemia-initiating cells (LICs). ß-Catenin modulates LIC activity in T-ALL. However, its role in maintaining established leukemia stem cells remains largely unknown. To identify functionally relevant protein interactions of ß-catenin in T-ALL, we performed coimmunoprecipitation followed by liquid chromatography-mass spectrometry. Here, we report that a noncanonical functional interaction of ß-catenin with the Forkhead box O3 (FOXO3) transcription factor positively regulates LIC-related genes, including the cyclin-dependent kinase 4, which is a crucial modulator of cell cycle and tumor maintenance. We also confirm the relevance of these findings using stably integrated fluorescent reporters of ß-catenin and FOXO3 activity in patient-derived xenografts, which identify minor subpopulations with enriched LIC activity. In addition, gene expression data at the single-cell level of leukemic cells of primary patients at the time of diagnosis and minimal residual disease (MRD) up to 30 days after the standard treatments reveal that the expression of ß-catenin- and FOXO3-dependent genes is present in the CD82+CD117+ cell fraction, which is substantially enriched with LICs in MRD as well as in early T-cell precursor ALL. These findings highlight key functional roles for ß-catenin and FOXO3 and suggest novel therapeutic strategies to eradicate aggressive cell subsets in T-ALL.
Assuntos
Leucemia Mieloide Aguda , Leucemia-Linfoma Linfoblástico de Células T Precursoras , beta Catenina , Humanos , beta Catenina/metabolismo , Leucemia Mieloide Aguda/patologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologiaRESUMO
Follicular lymphoma (FL) is an indolent cancer of mature B-cells but with ongoing risk of transformation to more aggressive histology over time. Recurrent mutations associated with transformation have been identified; however, prognostic features that can be discerned at diagnosis could be clinically useful. We present here comprehensive profiling of both tumor and immune compartments in 155 diagnostic FL biopsies at single-cell resolution by mass cytometry. This revealed a diversity of phenotypes but included two recurrent patterns, one which closely resembles germinal center B-cells (GCB) and another which appears more related to memory B-cells (MB). GCB-type tumors are enriched for EZH2, TNFRSF14, and MEF2B mutations, while MB-type tumors contain increased follicular helper T-cells. MB-type and intratumoral phenotypic diversity are independently associated with increased risk of transformation, supporting biological relevance of these features. Notably, a reduced 26-marker panel retains sufficient information to allow phenotypic profiling of future cohorts by conventional flow cytometry.
Assuntos
Linfoma Folicular , Humanos , Linfoma Folicular/genética , Células B de Memória , Centro Germinativo , Linfócitos B , MutaçãoRESUMO
Immune checkpoint inhibitors (ICI) are used in the treatment of urothelial and renal cell cancers. While some patients may have exceptional responses, better predictive biomarkers are needed. We profiled the circulating immune compartment of patients receiving ICI to identify possible immune markers associated with immunotherapy response or resistance. Peripheral blood samples were collected prior to, and 3 weeks after initiation of ICI. Using mass cytometry, 26 distinct immune populations were identified. Responders to immune checkpoint inhibitors had higher frequencies of naïve CD4+ T-cells, and lower frequencies of CD161+ Th17 cells and CCR4+ Th2 cells. Non-responders had a higher frequency of circulating PD1+ T-cells at baseline; there was a subsequent decrease in frequency with exposure to ICI with a concomitant increase in Ki67 expression. Flow cytometry for cytokines and chemokine receptors showed that CD4+ T cells of non-responder patients expressed less CXCR4 and CCR7. In addition, their PD1- CD4+ T cells had higher TNFα and higher CCR4 expression, while their PD1+ CD4+ T cells had higher interferon γ and lower CCR4 expression. The role of γ/δ T-cells was also explored. In responders, these cells had higher levels of interferon γ, TNFα and CCR5. One patient with a complete response had markedly higher frequency of γ/δ T-cells at baseline, and an expansion of these cells after treatment. This case was analyzed using single-cell gene expression profiling. The bulk of the γ/δ T cells consisted of a single clone of Vγ9/Vδ2 cells both before and after expansion, although the expansion was polyclonal. Gene expression analysis showed that exposure to an ICI led to a more activated phenotype of the γ/δ T cells. In this study, the circulating immune compartment was shown to have potential for biomarker discovery. Its dynamic changes during treatment may be used to assess response before radiographic changes are apparent, and these changes may help us delineate mechanisms that underpin both response and resistance to ICI. It also hypothesizes a potential role for γ/δ T cells as effector cells in some cases.
RESUMO
Multiplexed immune cell profiling of the tumor microenvironment (TME) in cancer has improved our understanding of cancer immunology, but complex spatial analyses of tumor-immune interactions in lymphoma are lacking. Here, we used imaging mass cytometry (IMC) on 33 cases of diffuse large B-cell lymphoma (DLBCL) to characterize tumor and immune cell architecture and correlate it to clinicopathological features such as cell of origin, gene mutations, and responsiveness to chemotherapy. To understand the poor response of DLBCL to immune checkpoint inhibitors (ICI), we compared our results to IMC data from Hodgkin lymphoma, a cancer highly responsive to ICI, and observed differences in the expression of PD-L1, PD-1, and TIM-3. We created a spatial classification of tumor cells and identified tumor-centric subregions of immune activation, immune suppression, and immune exclusion within the topology of DLBCL. Finally, the spatial analysis allowed us to identify markers such as CXCR3, which are associated with penetration of immune cells into immune desert regions, with important implications for engineered cellular therapies. This is the first study to integrate tumor mutational profiling, cell of origin classification, and multiplexed immuno-phenotyping of the TME into a spatial analysis of DLBCL at the single-cell level. We demonstrate that, far from being histopathologically monotonous, DLBCL has a complex tumor architecture, and that changes in tumor topology can be correlated with clinically relevant features. This analysis identifies candidate biomarkers and therapeutic targets such as TIM-3, CCR4, and CXCR3 that are relevant for combination treatment strategies in immuno-oncology and cellular therapies.
Assuntos
Doença de Hodgkin , Linfoma Difuso de Grandes Células B , Receptor Celular 2 do Vírus da Hepatite A , Humanos , Linfoma Difuso de Grandes Células B/patologia , Análise Espacial , Microambiente Tumoral/genéticaRESUMO
The human immunological mechanisms defining the clinical outcome of SARS-CoV-2 infection remain elusive. This knowledge gap is mostly driven by the lack of appropriate experimental platforms recapitulating human immune responses in a controlled human lung environment. Here, we report a mouse model (i.e., HNFL mice) co-engrafted with human fetal lung xenografts (fLX) and a myeloid-enhanced human immune system to identify cellular and molecular correlates of lung protection during SARS-CoV-2 infection. Unlike mice solely engrafted with human fLX, HNFL mice are protected against infection, severe inflammation, and histopathological phenotypes. Lung tissue protection from infection and severe histopathology associates with macrophage infiltration and differentiation and the upregulation of a macrophage-enriched signature composed of 11 specific genes mainly associated with the type I interferon signaling pathway. Our work highlights the HNFL model as a transformative platform to investigate, in controlled experimental settings, human myeloid immune mechanisms governing lung tissue protection during SARS-CoV-2 infection.
Assuntos
COVID-19 , Animais , COVID-19/genética , Modelos Animais de Doenças , Humanos , Imunidade Inata , Pulmão/patologia , Macrófagos , Camundongos , SARS-CoV-2RESUMO
Lymphocyte-rich classic Hodgkin lymphoma (LR-CHL) is a rare subtype of Hodgkin lymphoma. Recent technical advances have allowed for the characterization of specific cross-talk mechanisms between malignant Hodgkin Reed-Sternberg (HRS) cells and different normal immune cells in the tumor microenvironment (TME) of CHL. However, the TME of LR-CHL has not yet been characterized at single-cell resolution. Here, using single-cell RNA sequencing (scRNA-seq), we examined the immune cell profile of 8 cell suspension samples of LR-CHL in comparison to 20 samples of the mixed cellularity (MC, 9 cases) and nodular sclerosis (NS, 11 cases) subtypes of CHL, as well as 5 reactive lymph node controls. We also performed multicolor immunofluorescence (MC-IF) on tissue microarrays from the same patients and an independent validation cohort of 31 pretreatment LR-CHL samples. ScRNA-seq analysis identified a unique CD4+ helper T cell subset in LR-CHL characterized by high expression of Chemokine C-X-C motif ligand 13 (CXCL13) and PD-1. PD-1+CXCL13+ T cells were significantly enriched in LR-CHL compared to other CHL subtypes, and spatial analyses revealed that in 46% of the LR-CHL cases these cells formed rosettes surrounding HRS cells. MC-IF analysis revealed CXCR5+ normal B cells in close proximity to CXCL13+ T cells at significantly higher levels in LR-CHL. Moreover, the abundance of PD-1+CXCL13+ T cells in the TME was significantly associated with shorter progression-free survival in LR-CHL (P = 0.032). Taken together, our findings strongly suggest the pathogenic importance of the CXCL13/CXCR5 axis and PD-1+CXCL13+ T cells as a treatment target in LR-CHL.
Assuntos
Linfócitos B/metabolismo , Quimiocina CXCL13/metabolismo , Doença de Hodgkin/patologia , Receptores CXCR5/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Antígeno B7-H1/metabolismo , Imunofluorescência , Humanos , Linfonodos/citologia , Receptor de Morte Celular Programada 1/metabolismo , RNA-Seq , Células de Reed-Sternberg/patologia , Análise de Célula Única , Microambiente Tumoral/imunologiaRESUMO
PURPOSE: The efficacy of EZH2 inhibition has been modest in the initial clinical exploration of diffuse large B-cell lymphoma (DLBCL), yet EZH2 inhibitors are well tolerated. Herein, we aimed to uncover genetic and pharmacologic opportunities to enhance the clinical efficacy of EZH2 inhibitors in DLBCL. EXPERIMENTAL DESIGN: We conducted a genome-wide sensitizing CRISPR/Cas9 screen with tazemetostat, a catalytic inhibitor of EZH2. The sensitizing effect of IKZF1 loss of function was then validated and leveraged for combination treatment with lenalidomide. RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing analyses were performed to elucidate transcriptomic and epigenetic changes underlying synergy. RESULTS: We identified IKZF1 knockout as the top candidate for sensitizing DLBCL cells to tazemetostat. Treating cells with tazemetostat and lenalidomide, an immunomodulatory drug that selectively degrades IKAROS and AIOLOS, phenocopied the effects of the CRISPR/Cas9 screen. The combined drug treatment triggered either cell-cycle arrest or apoptosis in a broad range of DLBCL cell lines, regardless of EZH2 mutational status. Cell-line-based xenografts also showed slower tumor growth and prolonged survival in the combination treatment group. RNA-seq analysis revealed strong upregulation of interferon signaling and antiviral immune response signatures. Gene expression of key immune response factors such as IRF7 and DDX58 were induced in cells treated with lenalidomide and tazemetostat, with a concomitant increase of H3K27 acetylation at their promoters. Furthermore, transcriptome analysis demonstrated derepression of endogenous retroviruses after combination treatment. CONCLUSIONS: Our data underscore the synergistic interplay between IKAROS degradation and EZH2 inhibition on modulating epigenetic changes and ultimately enhancing antitumor effects in DLBCL.
Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste , Linfoma Difuso de Grandes Células B , Apoptose/genética , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Humanos , Lenalidomida , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologiaRESUMO
PURPOSE: Tumor-infiltrating lymphocytes (TIL) are strongly associated with survival in most cancers; however, the tumor-reactive subset that drives this prognostic effect remains poorly defined. CD39, CD103, and PD-1 have been independently proposed as markers of tumor-reactive CD8+ TIL in various cancers. We evaluated the phenotype, clonality, and prognostic significance of TIL expressing various combinations of these markers in high-grade serous ovarian cancer (HGSC), a malignancy in need of more effective immunotherapeutic approaches. EXPERIMENTAL DESIGN: Expression of CD39, CD103, PD-1, and other immune markers was assessed by high-dimensional flow cytometry, single-cell sequencing, and multiplex immunofluorescence of primary and matched pre/post-chemotherapy HGSC specimens. RESULTS: Coexpression of CD39, CD103, and PD-1 ("triple-positive" phenotype) demarcated subsets of CD8+ TIL and CD4+ regulatory T cells (Treg) with a highly activated/exhausted phenotype. Triple-positive CD8+ TIL exhibited reduced T-cell receptor (TCR) diversity and expressed genes involved in both cytolytic and humoral immunity. Triple-positive Tregs exhibited higher TCR diversity and a tumor-resident phenotype. Triple-positive TIL showed superior prognostic impact relative to TIL expressing other combinations of these markers. TIGIT was uniquely upregulated on triple-positive CD8+ effector cells relative to their CD4+ Treg counterparts. CONCLUSIONS: Coexpression of CD39, CD103, and PD-1 demarcates highly activated CD8+ and CD4+ TIL with inferred roles in cytolytic, humoral, and regulatory immune functions. Triple-positive TIL demonstrate exceptional prognostic significance and express compelling targets for combination immunotherapy, including PD-1, CD39, and TIGIT.
Assuntos
Cistadenocarcinoma Seroso/imunologia , Cistadenocarcinoma Seroso/patologia , Linfócitos do Interstício Tumoral , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Antígenos CD/biossíntese , Apirase/biossíntese , Cistadenocarcinoma Seroso/metabolismo , Feminino , Humanos , Cadeias alfa de Integrinas/biossíntese , Linfócitos do Interstício Tumoral/metabolismo , Neoplasias Ovarianas/metabolismo , Prognóstico , Receptor de Morte Celular Programada 1/biossínteseRESUMO
Primary mediastinal large B-cell lymphoma (PMBL) is a type of aggressive B-cell lymphoma that typically affects young adults, characterized by presence of a bulky anterior mediastinal mass. Lymphomas with gene expression features of PMBL have been described in nonmediastinal sites, raising questions about how these tumors should be classified. Here, we investigated whether these nonmediastinal lymphomas are indeed PMBLs or instead represent a distinct group within diffuse large B-cell lymphoma (DLBCL). From a cohort of 325 de novo DLBCL cases, we identified tumors from patients without evidence of anterior mediastinal involvement that expressed a PMBL expression signature (nm-PMBLsig+; n = 16; 5%). A majority of these tumors expressed MAL and CD23, proteins typically observed in bona fide PMBL (bf-PMBL). Evaluation of clinical features of nm-PMBLsig+ cases revealed close associations with DLBCL, and a majority displayed a germinal center B cell-like cell of origin (GCB). In contrast to patients with bf-PMBL, patients with nm-PMBLsig+ presented at an older age and did not show pleural disease, and bone/bone marrow involvement was observed in 3 cases. However, although clinically distinct from bf-PMBL, nm-PMBLsig+ tumors resembled bf-PMBL at the molecular level, with upregulation of immune response, JAK-STAT, and NF-κB signatures. Mutational analysis revealed frequent somatic gene mutations in SOCS1, IL4R, ITPKB, and STAT6, as well as CD83 and BIRC3, with the latter genes significantly more frequently affected than in GCB DLBCL or bf-PMBL. Our data establish nm-PMBLsig+ lymphomas as a group within DLBCL with distinct phenotypic and genetic features. These findings may have implications for gene expression- and mutation-based subtyping of aggressive B-cell lymphomas and related targeted therapies.
Assuntos
Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Linfoma Difuso de Grandes Células B/genética , Neoplasias do Mediastino/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos B/imunologia , Variações do Número de Cópias de DNA/genética , Análise Mutacional de DNA , Feminino , Células HEK293 , Humanos , Evasão da Resposta Imune , Imunofenotipagem , Janus Quinases/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Linfoma não Hodgkin/genética , Linfoma não Hodgkin/patologia , Masculino , Neoplasias do Mediastino/patologia , Pessoa de Meia-Idade , Mutação/genética , Receptores de Interleucina-4/genética , Fatores de Transcrição STAT/metabolismo , Hipermutação Somática de Imunoglobulina/genética , Adulto JovemRESUMO
The mutational landscape of gray zone lymphoma (GZL) has not yet been established, and differences from related entities are largely unknown. Here, we studied coding sequence mutations of 50 Epstein-Barr virus (EBV)-negative GZLs and 20 polymorphic EBV+ diffuse large B-cell lymphoma (DLBCL) not otherwise specified (poly-EBV-L) in comparison with classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), and DLBCL. Exomes of 21 GZL and 7 poly-EBV-L cases, along with paired constitutional DNA, were analyzed as a discovery cohort, followed by targeted sequencing of 217 genes in an extension cohort of 29 GZL and 13 poly-EBV-L cases. GZL cases with thymic niche involvement (anterior mediastinal mass) exhibited a mutation profile closely resembling cHL and PMBCL, with SOCS1 (45%), B2M (45%), TNFAIP3 (35%), GNA13 (35%), LRRN3 (32%), and NFKBIA (29%) being the most recurrently mutated genes. In contrast, GZL cases without thymic niche involvement (n = 18) had a significantly distinct pattern that was enriched in mutations related to apoptosis defects (TP53 [39%], BCL2 [28%], BIRC6 [22%]) and depleted in GNA13, XPO1, or NF-κB signaling pathway mutations (TNFAIP3, NFKBIE, IKBKB, NFKBIA). They also exhibited more BCL2/BCL6 rearrangements compared with thymic GZL. Poly-EBV-L cases presented a distinct mutational profile, including STAT3 mutations and a significantly lower coding mutation load in comparison with EBV- GZL. Our study highlights characteristic mutational patterns in GZL associated with presentation in the thymic niche, suggesting a common cell of origin and disease evolution overlapping with related anterior mediastinal lymphomas.
Assuntos
Doença de Hodgkin/genética , Linfoma Difuso de Grandes Células B/genética , Neoplasias do Mediastino/genética , Mutação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/genética , Feminino , Doença de Hodgkin/complicações , Humanos , Linfoma Difuso de Grandes Células B/complicações , Masculino , Neoplasias do Mediastino/complicações , Pessoa de Meia-Idade , Timo/metabolismo , Adulto JovemRESUMO
When the World Health Organization defined high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) as a clinical category, rearrangements were the only structural variant (SV) incorporated. An "atypical double-hit" category has been proposed, encompassing tumors with concurrent MYC and BCL2 SVs other than cooccurring translocations (ie, copy number variations [CNVs]). Although the identification of a gene expression signature (DHITsig) shared among tumors harboring MYC and BCL2 rearrangements (HGBL-DH/TH-BCL2) has confirmed a common underlying biology, the biological implication of MYC and BCL2 CNVs requires further elucidation. We performed a comprehensive analysis of MYC and BCL2 SVs, as determined by fluorescent in situ hybridization (FISH), in a cohort of 802 de novo tumors with diffuse large B-cell lymphoma morphology. Although BCL2 CNVs were associated with increased expression, MYC CNVs were not. Furthermore, MYC and BCL2 CNVs, in the context of atypical double-hit, did not confer a similar gene expression profile as HGBL-DH/TH-BCL2. Finally, although MYC immunohistochemistry (IHC) has been proposed as a screening tool for FISH testing, 2 mechanisms were observed that uncoupled MYC rearrangement from IHC positivity: (1) low MYC messenger RNA expression; and (2) false-negative IHC staining mediated by a single-nucleotide polymorphism resulting in an asparagine-to-serine substitution at the 11th amino acid residue of MYC (MYC-N11S). Taken together, these results support the current exclusion of MYC and BCL2 CNVs from HGBL-DH/TH and highlight the ability of a molecular-based classification system to identify tumors with shared biology that FISH and IHC fail to fully capture.
Assuntos
Linfoma Difuso de Grandes Células B/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-myc/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas c-bcl-2/análise , Proteínas Proto-Oncogênicas c-myc/análise , Transcriptoma , Adulto JovemRESUMO
Diffuse large B-cell lymphoma (DLBCL) is a molecularly heterogeneous group of malignancies with frequent genetic abnormalities. G-quadruplex (G4) DNA structures may facilitate this genomic instability through association with activation-induced cytidine deaminase (AID), an antibody diversification enzyme implicated in mutation of oncogenes in B-cell lymphomas. Chromatin immunoprecipitation sequencing analyses in this study revealed that AID hotspots in both activated B cells and lymphoma cells in vitro were highly enriched for G4 elements. A representative set of these targeted sequences was validated for characteristic, stable G4 structure formation including previously unknown G4s in lymphoma-associated genes, CBFA2T3, SPIB, BCL6, HLA-DRB5 and MEF2C, along with the established BCL2 and MYC structures. Frequent genome-wide G4 formation was also detected for the first time in DLBCL patient-derived tissues using BG4, a structure-specific G4 antibody. Tumors with greater staining were more likely to have concurrent BCL2 and MYC oncogene amplification and BCL2 mutations. Ninety-seven percent of the BCL2 mutations occurred within G4 sites that overlapped with AID binding. G4 localization at sites of mutation, and within aggressive DLBCL tumors harboring amplified BCL2 and MYC, supports a role for G4 structures in events that lead to a loss of genomic integrity, a critical step in B-cell lymphomagenesis.
RESUMO
Gray zone lymphoma (GZL), a B-cell lymphoma with features intermediate between large B-cell lymphoma (LBCL) and classic Hodgkin lymphoma (cHL), is a rare and poorly defined entity. Alongside GZL, a subset of Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) has been described with polymorphic/GZL-like morphology (polymorphic-EBV-L). To fill the important gap in our understanding of the pathogenic process underlying these entities, we performed a gene expression study of a large international cohort of GZL and polymorphic-EBV-L, combined with cHL and primary mediastinal large B-cell lymphoma (PMBCL) cases. In an unsupervised principal component analysis, GZL cases presented with intermediate scores in a spectrum between cHL and PMBCL, whereas polymorphic-EBV-L clustered distinctly. The main biological pathways underlying the GZL spectrum were related to cell cycle, reflecting tumor cell content, and extracellular matrix signatures related to the cellular tumor microenvironment. Differential expression analysis and phenotypic characterization of the tumor microenvironment highlighted the predominance of regulatory macrophages in GZL compared with cHL and PMBCL. Two distinct subtypes of GZL were distinguishable that were phenotypically reminiscent of PMBCL and DLBCL, and we observed an association of PMBCL-type GZL with clinical presentation in the "thymic" anatomic niche. In summary, gene expression profiling (GEP) enabled us to add precision to the GZL spectrum, describe the biological distinction compared with polymorphic-EBV-L, and distinguish cases with and without thymic involvement as 2 subgroups of GZL, namely PMBCL-like and DLBCL-like GZL.
Assuntos
Infecções por Vírus Epstein-Barr , Perfilação da Expressão Gênica , Doença de Hodgkin , Linfoma Difuso de Grandes Células B , Feminino , Herpesvirus Humano 4/genética , Doença de Hodgkin/genética , Humanos , Linfoma Difuso de Grandes Células B/genética , Masculino , Pessoa de Meia-Idade , Microambiente TumoralRESUMO
Hodgkin lymphoma is characterized by an extensively dominant tumor microenvironment (TME) composed of different types of noncancerous immune cells with rare malignant cells. Characterization of the cellular components and their spatial relationship is crucial to understanding cross-talk and therapeutic targeting in the TME. We performed single-cell RNA sequencing of more than 127,000 cells from 22 Hodgkin lymphoma tissue specimens and 5 reactive lymph nodes, profiling for the first time the phenotype of the Hodgkin lymphoma-specific immune microenvironment at single-cell resolution. Single-cell expression profiling identified a novel Hodgkin lymphoma-associated subset of T cells with prominent expression of the inhibitory receptor LAG3, and functional analyses established this LAG3+ T-cell population as a mediator of immunosuppression. Multiplexed spatial assessment of immune cells in the microenvironment also revealed increased LAG3+ T cells in the direct vicinity of MHC class II-deficient tumor cells. Our findings provide novel insights into TME biology and suggest new approaches to immune-checkpoint targeting in Hodgkin lymphoma. SIGNIFICANCE: We provide detailed functional and spatial characteristics of immune cells in classic Hodgkin lymphoma at single-cell resolution. Specifically, we identified a regulatory T-cell-like immunosuppressive subset of LAG3+ T cells contributing to the immune-escape phenotype. Our insights aid in the development of novel biomarkers and combination treatment strategies targeting immune checkpoints.See related commentary by Fisher and Oh, p. 342.This article is highlighted in the In This Issue feature, p. 327.
Assuntos
Doença de Hodgkin/genética , Análise de Célula Única , Transcriptoma/genética , Microambiente Tumoral/genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Doença de Hodgkin/patologia , Humanos , Masculino , Análise de Sequência de RNA , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia , Linfócitos T Reguladores/imunologia , Transcriptoma/imunologia , Microambiente Tumoral/imunologiaRESUMO
Diffuse large B-cell lymphoma (DLBCL) is the most common histologic subtype of non-Hodgkin lymphoma and is notorious for its clinical heterogeneity. Patient outcomes can be predicted by cell-of-origin (COO) classification, demonstrating that the underlying transcriptional signature of malignant B-cells informs biological behavior in the context of standard combination chemotherapy regimens. In the current study, we used mass cytometry (CyTOF) to examine tumor phenotypes at the protein level with single cell resolution in a collection of 27 diagnostic DLBCL biopsy specimens from treatment naïve patients. We found that malignant B-cells from each patient occupied unique regions in 37-dimensional phenotypic space with no apparent clustering of samples into discrete subtypes. Interestingly, variable MHC class II expression was found to be the greatest contributor to phenotypic diversity. Within individual tumors, a subset of cases showed multiple phenotypic subpopulations, and in one case, we were able to demonstrate direct correspondence between protein-level phenotypic subsets and DNA mutation-defined subclones. In summary, CyTOF analysis can resolve both intertumoral and intratumoral heterogeneity among primary samples and reveals that each case of DLBCL is unique and may be comprised of multiple, genetically distinct subclones. © 2019 International Society for Advancement of Cytometry.
Assuntos
Linfoma Difuso de Grandes Células B , Humanos , Linfoma Difuso de Grandes Células B/genética , MutaçãoRESUMO
Accurate measurement of clonal genotypes, mutational processes, and replication states from individual tumor-cell genomes will facilitate improved understanding of tumor evolution. We have developed DLP+, a scalable single-cell whole-genome sequencing platform implemented using commodity instruments, image-based object recognition, and open source computational methods. Using DLP+, we have generated a resource of 51,926 single-cell genomes and matched cell images from diverse cell types including cell lines, xenografts, and diagnostic samples with limited material. From this resource we have defined variation in mitotic mis-segregation rates across tissue types and genotypes. Analysis of matched genomic and image measurements revealed correlations between cellular morphology and genome ploidy states. Aggregation of cells sharing copy number profiles allowed for calculation of single-nucleotide resolution clonal genotypes and inference of clonal phylogenies and avoided the limitations of bulk deconvolution. Finally, joint analysis over the above features defined clone-specific chromosomal aneuploidy in polyclonal populations.