RESUMEN
Although treatment of non-small cell lung cancer (NSCLC) with immune checkpoint inhibitors (ICIs) can produce remarkably durable responses, most patients develop early disease progression. Furthermore, initial response assessment by conventional imaging is often unable to identify which patients will achieve durable clinical benefit (DCB). Here, we demonstrate that pre-treatment circulating tumor DNA (ctDNA) and peripheral CD8 T cell levels are independently associated with DCB. We further show that ctDNA dynamics after a single infusion can aid in identification of patients who will achieve DCB. Integrating these determinants, we developed and validated an entirely noninvasive multiparameter assay (DIREct-On, Durable Immunotherapy Response Estimation by immune profiling and ctDNA-On-treatment) that robustly predicts which patients will achieve DCB with higher accuracy than any individual feature. Taken together, these results demonstrate that integrated ctDNA and circulating immune cell profiling can provide accurate, noninvasive, and early forecasting of ultimate outcomes for NSCLC patients receiving ICIs.
Asunto(s)
Biomarcadores Farmacológicos/sangre , ADN Tumoral Circulante/análisis , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Adulto , Antineoplásicos Inmunológicos/farmacología , Antígeno B7-H1/inmunología , Antígeno B7-H1/metabolismo , Biomarcadores de Tumor/genética , Linfocitos T CD8-positivos/patología , Carcinoma de Pulmón de Células no Pequeñas/patología , ADN Tumoral Circulante/genética , Femenino , Humanos , Inhibidores de Puntos de Control Inmunológico/inmunología , Inhibidores de Puntos de Control Inmunológico/metabolismo , Inmunoterapia/métodos , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Receptor de Muerte Celular Programada 1/metabolismoRESUMEN
Accurate prediction of long-term outcomes remains a challenge in the care of cancer patients. Due to the difficulty of serial tumor sampling, previous prediction tools have focused on pretreatment factors. However, emerging non-invasive diagnostics have increased opportunities for serial tumor assessments. We describe the Continuous Individualized Risk Index (CIRI), a method to dynamically determine outcome probabilities for individual patients utilizing risk predictors acquired over time. Similar to "win probability" models in other fields, CIRI provides a real-time probability by integrating risk assessments throughout a patient's course. Applying CIRI to patients with diffuse large B cell lymphoma, we demonstrate improved outcome prediction compared to conventional risk models. We demonstrate CIRI's broader utility in analogous models of chronic lymphocytic leukemia and breast adenocarcinoma and perform a proof-of-concept analysis demonstrating how CIRI could be used to develop predictive biomarkers for therapy selection. We envision that dynamic risk assessment will facilitate personalized medicine and enable innovative therapeutic paradigms.
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Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/patología , Linfoma de Células B Grandes Difuso/patología , Medicina de Precisión , Algoritmos , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/sangre , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/mortalidad , ADN Tumoral Circulante/sangre , Femenino , Humanos , Estimación de Kaplan-Meier , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/mortalidad , Terapia Neoadyuvante , Pronóstico , Supervivencia sin Progresión , Modelos de Riesgos Proporcionales , Medición de Riesgo , Resultado del TratamientoRESUMEN
The scarcity of malignant Hodgkin and Reed-Sternberg cells hampers tissue-based comprehensive genomic profiling of classic Hodgkin lymphoma (cHL). By contrast, liquid biopsies show promise for molecular profiling of cHL due to relatively high circulating tumour DNA (ctDNA) levels1-4. Here we show that the plasma representation of mutations exceeds the bulk tumour representation in most cases, making cHL particularly amenable to noninvasive profiling. Leveraging single-cell transcriptional profiles of cHL tumours, we demonstrate Hodgkin and Reed-Sternberg ctDNA shedding to be shaped by DNASE1L3, whose increased tumour microenvironment-derived expression drives high ctDNA concentrations. Using this insight, we comprehensively profile 366 patients, revealing two distinct cHL genomic subtypes with characteristic clinical and prognostic correlates, as well as distinct transcriptional and immunological profiles. Furthermore, we identify a novel class of truncating IL4R mutations that are dependent on IL-13 signalling and therapeutically targetable with IL-4Rα-blocking antibodies. Finally, using PhasED-seq5, we demonstrate the clinical value of pretreatment and on-treatment ctDNA levels for longitudinally refining cHL risk prediction and for detection of radiographically occult minimal residual disease. Collectively, these results support the utility of noninvasive strategies for genotyping and dynamic monitoring of cHL, as well as capturing molecularly distinct subtypes with diagnostic, prognostic and therapeutic potential.
Asunto(s)
ADN Tumoral Circulante , Genoma Humano , Genómica , Enfermedad de Hodgkin , Humanos , Enfermedad de Hodgkin/sangre , Enfermedad de Hodgkin/clasificación , Enfermedad de Hodgkin/diagnóstico , Enfermedad de Hodgkin/genética , Mutación , Células de Reed-Sternberg/metabolismo , Microambiente Tumoral , ADN Tumoral Circulante/sangre , ADN Tumoral Circulante/genética , Análisis de Expresión Génica de una Sola Célula , Genoma Humano/genéticaRESUMEN
BACKGROUND: The risk of second tumors after chimeric antigen receptor (CAR) T-cell therapy, especially the risk of T-cell neoplasms related to viral vector integration, is an emerging concern. METHODS: We reviewed our clinical experience with adoptive cellular CAR T-cell therapy at our institution since 2016 and ascertained the occurrence of second tumors. In one case of secondary T-cell lymphoma, a broad array of molecular, genetic, and cellular techniques were used to interrogate the tumor, the CAR T cells, and the normal hematopoietic cells in the patient. RESULTS: A total of 724 patients who had received T-cell therapies at our center were included in the study. A lethal T-cell lymphoma was identified in a patient who had received axicabtagene ciloleucel therapy for diffuse large B-cell lymphoma, and both lymphomas were deeply profiled. Each lymphoma had molecularly distinct immunophenotypes and genomic profiles, but both were positive for Epstein-Barr virus and were associated with DNMT3A and TET2 mutant clonal hematopoiesis. No evidence of oncogenic retroviral integration was found with the use of multiple techniques. CONCLUSIONS: Our results highlight the rarity of second tumors and provide a framework for defining clonal relationships and viral vector monitoring. (Funded by the National Cancer Institute and others.).
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Antineoplásicos Inmunológicos , Inmunoterapia Adoptiva , Linfoma de Células B Grandes Difuso , Linfoma de Células T , Neoplasias Primarias Secundarias , Receptores Quiméricos de Antígenos , Femenino , Humanos , Persona de Mediana Edad , Productos Biológicos/efectos adversos , Productos Biológicos/uso terapéutico , Hematopoyesis Clonal , Herpesvirus Humano 4/inmunología , Herpesvirus Humano 4/genética , Inmunoterapia Adoptiva/efectos adversos , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/inmunología , Linfoma de Células B Grandes Difuso/terapia , Linfoma de Células T/etiología , Linfoma de Células T/genética , Linfoma de Células T/inmunología , Linfoma de Células T/terapia , Neoplasias Primarias Secundarias/genética , Neoplasias Primarias Secundarias/etiología , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/uso terapéutico , Antineoplásicos Inmunológicos/efectos adversos , Antineoplásicos Inmunológicos/uso terapéutico , Integración ViralRESUMEN
Radiologic screening of high-risk adults reduces lung-cancer-related mortality1,2; however, a small minority of eligible individuals undergo such screening in the United States3,4. The availability of blood-based tests could increase screening uptake. Here we introduce improvements to cancer personalized profiling by deep sequencing (CAPP-Seq)5, a method for the analysis of circulating tumour DNA (ctDNA), to better facilitate screening applications. We show that, although levels are very low in early-stage lung cancers, ctDNA is present prior to treatment in most patients and its presence is strongly prognostic. We also find that the majority of somatic mutations in the cell-free DNA (cfDNA) of patients with lung cancer and of risk-matched controls reflect clonal haematopoiesis and are non-recurrent. Compared with tumour-derived mutations, clonal haematopoiesis mutations occur on longer cfDNA fragments and lack mutational signatures that are associated with tobacco smoking. Integrating these findings with other molecular features, we develop and prospectively validate a machine-learning method termed 'lung cancer likelihood in plasma' (Lung-CLiP), which can robustly discriminate early-stage lung cancer patients from risk-matched controls. This approach achieves performance similar to that of tumour-informed ctDNA detection and enables tuning of assay specificity in order to facilitate distinct clinical applications. Our findings establish the potential of cfDNA for lung cancer screening and highlight the importance of risk-matching cases and controls in cfDNA-based screening studies.
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ADN Tumoral Circulante/análisis , ADN Tumoral Circulante/genética , Detección Precoz del Cáncer/métodos , Genoma Humano/genética , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Mutación , Estudios de Cohortes , Femenino , Hematopoyesis/genética , Humanos , Pulmón/metabolismo , Pulmón/patología , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Reproducibilidad de los ResultadosRESUMEN
Signaling via the methylation of lysine residues in proteins has been linked to diverse biological and disease processes, yet the catalytic activity and substrate specificity of many human protein lysine methyltransferases (PKMTs) are unknown. We screened over 40 candidate PKMTs and identified SETD6 as a methyltransferase that monomethylated chromatin-associated transcription factor NF-κB subunit RelA at Lys310 (RelAK310me1). SETD6-mediated methylation rendered RelA inert and attenuated RelA-driven transcriptional programs, including inflammatory responses in primary immune cells. RelAK310me1 was recognized by the ankryin repeat of the histone methyltransferase GLP, which under basal conditions promoted a repressed chromatin state at RelA target genes through GLP-mediated methylation of histone H3 Lys9 (H3K9). NF-κB-activation-linked phosphorylation of RelA at Ser311 by protein kinase C-ζ (PKC-ζ) blocked the binding of GLP to RelAK310me1 and relieved repression of the target gene. Our findings establish a previously uncharacterized mechanism by which chromatin signaling regulates inflammation programs.
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Artritis Reumatoide/inmunología , FN-kappa B/metabolismo , Proteína Metiltransferasas/metabolismo , Factor de Transcripción ReIA/metabolismo , Artritis Reumatoide/genética , Artritis Reumatoide/metabolismo , Ensamble y Desensamble de Cromatina/genética , Metilación de ADN , Células HEK293 , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Humanos , Inflamación , Lisina/metabolismo , FN-kappa B/genética , FN-kappa B/inmunología , Unión Proteica/genética , Proteína Metiltransferasas/genética , Proteína Metiltransferasas/inmunología , ARN Interferente Pequeño/genética , Transducción de Señal/genética , Transducción de Señal/inmunología , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/inmunologíaRESUMEN
BACKGROUND: Viral infection is an oncogenic factor in many hematolymphoid malignancies. We sought to determine the diagnostic yield of aligning off-target reads incidentally obtained during targeted hematolymphoid next-generation sequencing to a large database of viral genomes to screen for viral sequences within tumor specimens. METHODS: Alignment of off-target reads to viral genomes was performed using magicBLAST. Localization of Merkel cell polyomavirus (MCPyV) RNA was confirmed by RNAScope in situ hybridization. Integration analysis was performed using Virus-Clip. RESULTS: Four cases of post-cardiac-transplant folliculotropic mycosis fungoides (fMF) and one case of peripheral T-cell lymphoma (PTCL) were positive in off-target reads for MCPyV DNA. Two of the four cases of posttransplant fMF and the case of PTCL showed localization of MCPyV RNA to malignant lymphocytes, whereas the remaining two cases of posttransplant fMF showed MCPyV RNA in keratinocytes. CONCLUSIONS: Our findings raise the question of whether MCPyV may play a role in rare cases of T-lymphoproliferative disorders, particularly in the skin and in the heavily immunosuppressed posttransplant setting.
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Carcinoma de Células de Merkel , Poliomavirus de Células de Merkel , Micosis Fungoide , Infecciones por Polyomavirus , Poliomavirus , Neoplasias Cutáneas , Infecciones Tumorales por Virus , Humanos , Poliomavirus de Células de Merkel/genética , Carcinoma de Células de Merkel/patología , Neoplasias Cutáneas/patología , Infecciones por Polyomavirus/complicaciones , Infecciones por Polyomavirus/patología , ADN Viral/análisis , Hibridación in Situ , Infecciones Tumorales por Virus/patología , Poliomavirus/genéticaRESUMEN
Cancer somatic mutations can generate neoantigens that distinguish malignant from normal cells. However, the personalized identification and validation of neoantigens remains a major challenge. Here we discover neoantigens in human mantle-cell lymphomas by using an integrated genomic and proteomic strategy that interrogates tumour antigen peptides presented by major histocompatibility complex (MHC) class I and class II molecules. We applied this approach to systematically characterize MHC ligands from 17 patients. Remarkably, all discovered neoantigenic peptides were exclusively derived from the lymphoma immunoglobulin heavy- or light-chain variable regions. Although we identified MHC presentation of private polymorphic germline alleles, no mutated peptides were recovered from non-immunoglobulin somatically mutated genes. Somatic mutations within the immunoglobulin variable region were almost exclusively presented by MHC class II. We isolated circulating CD4+ T cells specific for immunoglobulin-derived neoantigens and found these cells could mediate killing of autologous lymphoma cells. These results demonstrate that an integrative approach combining MHC isolation, peptide identification, and exome sequencing is an effective platform to uncover tumour neoantigens. Application of this strategy to human lymphoma implicates immunoglobulin neoantigens as targets for lymphoma immunotherapy.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos de Neoplasias/inmunología , Región Variable de Inmunoglobulina/inmunología , Linfoma de Células del Manto/inmunología , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Linfocitos T CD4-Positivos/inmunología , Citotoxicidad Inmunológica , Análisis Mutacional de ADN , Epítopos de Linfocito T/inmunología , Exoma/genética , Genómica , Antígenos HLA-D/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/genética , Inmunoterapia/tendencias , Linfoma de Células del Manto/genética , Linfoma de Células del Manto/patología , Linfoma de Células del Manto/terapia , Mutación , ProteómicaRESUMEN
BACKGROUND & AIMS: Biomarkers are needed to risk stratify after chemoradiotherapy for localized esophageal cancer. These could improve identification of patients at risk for cancer progression and selection of additional therapy. METHODS: We performed deep sequencing (CAncer Personalized Profiling by deep Sequencing, [CAPP-Seq]) analyses of plasma cell-free DNA collected from 45 patients before and after chemoradiotherapy for esophageal cancer, as well as DNA from leukocytes and fixed esophageal tumor biopsy samples collected during esophagogastroduodenoscopy. Patients were treated from May 2010 through October 2015; 23 patients subsequently underwent esophagectomy, and 22 did not undergo surgery. We also sequenced DNA from blood samples from 40 healthy control individuals. We analyzed 802 regions of 607 genes for single-nucleotide variants previously associated with esophageal adenocarcinoma or squamous cell carcinoma. Patients underwent imaging analyses 6-8 weeks after chemoradiotherapy and were followed for 5 years. Our primary aim was to determine whether detection of circulating tumor DNA (ctDNA) after chemoradiotherapy is associated with risk of tumor progression (growth of local, regional, or distant tumors, detected by imaging or biopsy). RESULTS: The median proportion of tumor-derived DNA in total cell-free DNA before treatment was 0.07%, indicating that ultrasensitive assays are needed for quantification and analysis of ctDNA from localized esophageal tumors. Detection of ctDNA after chemoradiotherapy was associated with tumor progression (hazard ratio, 18.7; P < .0001), formation of distant metastases (hazard ratio, 32.1; P < .0001), and shorter disease-specific survival times (hazard ratio, 23.1; P < .0001). A higher proportion of patients with tumor progression had new mutations detected in plasma samples collected after chemoradiotherapy than patients without progression (P = .03). Detection of ctDNA after chemoradiotherapy preceded radiographic evidence of tumor progression by an average of 2.8 months. Among patients who received chemoradiotherapy without surgery, combined ctDNA and metabolic imaging analysis predicted progression in 100% of patients with tumor progression, compared with 71% for only ctDNA detection and 57% for only metabolic imaging analysis (P < .001 for comparison of either technique to combined analysis). CONCLUSIONS: In an analysis of cell-free DNA in blood samples from patients who underwent chemoradiotherapy for esophageal cancer, detection of ctDNA was associated with tumor progression, metastasis, and disease-specific survival. Analysis of ctDNA might be used to identify patients at highest risk for tumor progression.
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Adenocarcinoma/terapia , Biomarcadores de Tumor/sangre , Carcinoma de Células Escamosas/diagnóstico , Quimioradioterapia , ADN Tumoral Circulante/sangre , Neoplasias Esofágicas/terapia , Adenocarcinoma/sangre , Adenocarcinoma/diagnóstico , Adenocarcinoma/mortalidad , Anciano , Biomarcadores de Tumor/aislamiento & purificación , Biopsia , Carcinoma de Células Escamosas/sangre , Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/terapia , ADN Tumoral Circulante/aislamiento & purificación , Progresión de la Enfermedad , Neoplasias Esofágicas/sangre , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/mortalidad , Esófago/diagnóstico por imagen , Esófago/patología , Estudios de Factibilidad , Femenino , Voluntarios Sanos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Neoplasia Residual , Supervivencia sin Progresión , Estudios Prospectivos , Estudios Retrospectivos , Medición de Riesgo/métodos , Tomografía Computarizada por Rayos XRESUMEN
We introduce CIBERSORT, a method for characterizing cell composition of complex tissues from their gene expression profiles. When applied to enumeration of hematopoietic subsets in RNA mixtures from fresh, frozen and fixed tissues, including solid tumors, CIBERSORT outperformed other methods with respect to noise, unknown mixture content and closely related cell types. CIBERSORT should enable large-scale analysis of RNA mixtures for cellular biomarkers and therapeutic targets (http://cibersort.stanford.edu/).
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Tonsila Palatina/citología , Tonsila Palatina/metabolismo , Técnicas de Cultivo de Tejidos/métodos , Conservación de Tejido/métodos , Transcriptoma , Biomarcadores , Regulación de la Expresión Génica/fisiología , Humanos , ARN/clasificación , ARN/genética , ARN/metabolismo , Reproducibilidad de los Resultados , Programas InformáticosRESUMEN
Follicular lymphoma (FL) is incurable with conventional therapies and has a clinical course typified by multiple relapses after therapy. These tumors are genetically characterized by B-cell leukemia/lymphoma 2 (BCL2) translocation and mutation of genes involved in chromatin modification. By analyzing purified tumor cells, we identified additional novel recurrently mutated genes and confirmed mutations of one or more chromatin modifier genes within 96% of FL tumors and two or more in 76% of tumors. We defined the hierarchy of somatic mutations arising during tumor evolution by analyzing the phylogenetic relationship of somatic mutations across the coding genomes of 59 sequentially acquired biopsies from 22 patients. Among all somatically mutated genes, CREBBP mutations were most significantly enriched within the earliest inferable progenitor. These mutations were associated with a signature of decreased antigen presentation characterized by reduced transcript and protein abundance of MHC class II on tumor B cells, in line with the role of CREBBP in promoting class II transactivator (CIITA)-dependent transcriptional activation of these genes. CREBBP mutant B cells stimulated less proliferation of T cells in vitro compared with wild-type B cells from the same tumor. Transcriptional signatures of tumor-infiltrating T cells were indicative of reduced proliferation, and this corresponded to decreased frequencies of tumor-infiltrating CD4 helper T cells and CD8 memory cytotoxic T cells. These observations therefore implicate CREBBP mutation as an early event in FL evolution that contributes to immune evasion via decreased antigen presentation.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Linfoma Folicular/genética , Mutación , Células Madre Neoplásicas/patología , Proteína de Unión a CREB/genética , Cromatina/metabolismo , Citometría de Flujo , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Linfoma Folicular/inmunología , Reacción en Cadena de la PolimerasaRESUMEN
Recent studies have shown limited utility of routine surveillance imaging for diffuse large B-cell lymphoma (DLBCL) patients achieving remission. Detection of molecular disease by immunoglobulin high-throughput sequencing (Ig-HTS) from peripheral blood provides an alternate strategy for surveillance. We prospectively evaluated the utility of Ig-HTS within 311 blood and 105 tumor samples from 75 patients with DLBCL, comparing Ig-HTS from the cellular (circulating leukocytes) and acellular (plasma cell-free DNA) compartments of peripheral blood to clinical outcomes and (18)fluoro-deoxyglucose positron emission tomography combined with computed tomography (PET/CT; n = 173). Clonotypic immunoglobulin rearrangements were detected in 83% of patients with adequate tumor samples to enable subsequent monitoring in peripheral blood. Molecular disease measured from plasma, compared with circulating leukocytes, was more abundant and better correlated with radiographic disease burden. Before treatment, molecular disease was detected in the plasma of 82% of patients compared with 71% in circulating cells (P = .68). However, molecular disease was detected significantly more frequently in the plasma at time of relapse (100% vs 30%; P = .001). Detection of molecular disease in the plasma often preceded PET/CT detection of relapse in patients initially achieving remission. During surveillance time points before relapse, plasma Ig-HTS demonstrated improved specificity (100% vs 56%, P < .0001) and similar sensitivity (31% vs 55%, P = .4) compared with PET/CT. Given its high specificity, Ig-HTS from plasma has potential clinical utility for surveillance after complete remission.
Asunto(s)
Inmunoglobulinas/genética , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunoglobulinas/sangre , L-Lactato Deshidrogenasa/sangre , Linfoma de Células B Grandes Difuso/sangre , Masculino , Persona de Mediana Edad , Tomografía de Emisión de Positrones , Estudios ProspectivosRESUMEN
UNLABELLED: For practical and robust de novo identification of genomic fusions and breakpoints from targeted paired-end DNA sequencing data, we developed Fusion And Chromosomal Translocation Enumeration and Recovery Algorithm (FACTERA). Our method has minimal external dependencies, works directly on a preexisting Binary Alignment/Map file and produces easily interpretable output. We demonstrate FACTERA's ability to rapidly identify breakpoint-resolution fusion events with high sensitivity and specificity in patients with non-small cell lung cancer, including novel rearrangements. We anticipate that FACTERA will be broadly applicable to the discovery and analysis of clinically relevant fusions from both targeted and genome-wide sequencing datasets. AVAILABILITY AND IMPLEMENTATION: http://factera.stanford.edu.
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Algoritmos , Aberraciones Cromosómicas , Puntos de Rotura del Cromosoma , Fusión Génica , Carcinoma de Pulmón de Células no Pequeñas/genética , Mapeo Cromosómico , Genómica/métodos , Humanos , Neoplasias Pulmonares/genética , Análisis de Secuencia de ADN , Programas Informáticos , Translocación GenéticaRESUMEN
Follicular lymphoma (FL) is currently incurable using conventional chemotherapy or immunotherapy regimes, compelling new strategies. Advances in high-throughput sequencing technologies that can reveal oncogenic pathways have stimulated interest in tailoring therapies toward actionable somatic mutations. However, for mutation-directed therapies to be most effective, the mutations must be uniformly present in evolved tumor cells as well as in the self-renewing tumor-cell precursors. Here, we show striking intratumoral clonal diversity within FL tumors in the representation of mutations in the majority of genes as revealed by whole exome sequencing of subpopulations. This diversity captures a clonal hierarchy, resolved using immunoglobulin somatic mutations and IGH-BCL2 translocations as a frame of reference and by comparing diagnosis and relapse tumor pairs, allowing us to distinguish early versus late genetic eventsduring lymphomagenesis. We provide evidence that IGH-BCL2 translocations and CREBBP mutations are early events, whereas MLL2 and TNFRSF14 mutations probably represent late events during disease evolution. These observations provide insight into which of the genetic lesions represent suitable candidates for targeted therapies.
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Evolución Clonal/genética , ADN de Neoplasias/genética , Linfoma Folicular/genética , Mutación/fisiología , Células Clonales/metabolismo , Células Clonales/patología , Progresión de la Enfermedad , Exoma/genética , Frecuencia de los Genes , Genoma Humano/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunofenotipificación , Linfoma Folicular/patología , Tasa de Mutación , Reacción en Cadena de la Polimerasa , RecurrenciaRESUMEN
Packaging of eukaryotic genomes into chromatin has wide-ranging effects on gene transcription. Curiously, it is commonly observed that deletion of a global chromatin regulator affects expression of only a limited subset of genes bound to or modified by the regulator in question. However, in many single-gene studies it has become clear that chromatin regulators often do not affect steady-state transcription, but instead are required for normal transcriptional reprogramming by environmental cues. We therefore have systematically investigated the effects of 83 histone mutants, and 119 gene deletion mutants, on induction/repression dynamics of 170 transcripts in response to diamide stress in yeast. Importantly, we find that chromatin regulators play far more pronounced roles during gene induction/repression than they do in steady-state expression. Furthermore, by jointly analyzing the substrates (histone mutants) and enzymes (chromatin modifier deletions) we identify specific interactions between histone modifications and their regulators. Combining these functional results with genome-wide mapping of several histone marks in the same time course, we systematically investigated the correspondence between histone modification occurrence and function. We followed up on one pathway, finding that Set1-dependent H3K4 methylation primarily acts as a gene repressor during multiple stresses, specifically at genes involved in ribosome biosynthesis. Set1-dependent repression of ribosomal genes occurs via distinct pathways for ribosomal protein genes and ribosomal biogenesis genes, which can be separated based on genetic requirements for repression and based on chromatin changes during gene repression. Together, our dynamic studies provide a rich resource for investigating chromatin regulation, and identify a significant role for the "activating" mark H3K4me3 in gene repression.
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Cromatina/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Estrés Fisiológico , Cromatina/genética , Ensamble y Desensamble de Cromatina , Inmunoprecipitación de Cromatina , Diamida/farmacología , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Histonas/metabolismo , Metilación , Nucleosomas/genética , Nucleosomas/metabolismo , Fosforilación , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Eliminación de Secuencia , Especificidad por Sustrato , Factores de Tiempo , Imagen de Lapso de Tiempo/métodos , Transcripción GenéticaRESUMEN
Heterogeneity in the tumor microenvironment (TME) of follicular lymphomas (FLs) can affect clinical outcomes. Current immunotherapeutic strategies, including antibody- and cell-based therapies, variably overcome pro-tumorigenic mechanisms for sustained disease control. Modeling the intact FL TME, with its native, syngeneic tumor-infiltrating leukocytes, is a major challenge. Here, we describe an organoid culture method for cultivating patient-derived lymphoma organoids (PDLOs), which include cells from the native FL TME. We define the robustness of this method by successfully culturing cryopreserved FL specimens from diverse patients and demonstrate the stability of TME cellular composition, tumor somatic mutations, gene expression profiles, and B/T cell receptor dynamics over 3 weeks. PDLOs treated with CD3:CD19 and CD3:CD20 therapeutic bispecific antibodies showed B cell killing and T cell activation. This stable system offers a robust platform for advancing precision medicine efforts in FL through patient-specific modeling, high-throughput screening, TME signature identification, and treatment response evaluation.
Asunto(s)
Linfoma Folicular , Humanos , Linfoma Folicular/terapia , Linfoma Folicular/diagnóstico , Linfoma Folicular/genética , Microambiente Tumoral , Linfocitos B , Receptores de Antígenos de Linfocitos T , OrganoidesRESUMEN
Cerebrospinal fluid tumor-derived DNA (CSF-tDNA) analysis is a promising approach for monitoring the neoplastic processes of the central nervous system. We applied a lung cancer-specific sequencing panel (CAPP-Seq) to 81 CSF, blood, and tissue samples from 24 lung cancer patients who underwent lumbar puncture (LP) for suspected leptomeningeal disease (LMD). A subset of the cohort (N = 12) participated in a prospective trial of osimertinib for refractory LMD in which serial LPs were performed before and during treatment. CSF-tDNA variant allele fractions (VAFs) were significantly higher than plasma circulating tumor DNA (ctDNA) VAFs (median CSF-tDNA, 32.7%; median plasma ctDNA, 1.8%; P < 0.0001). Concentrations of tumor DNA in CSF and plasma were positively correlated (Spearman's ρ, 0.45; P = 0.03). For LMD diagnosis, cytology was 81.8% sensitive and CSF-tDNA was 91.7% sensitive. CSF-tDNA was also strongly prognostic for overall survival (HR = 7.1; P = 0.02). Among patients with progression on targeted therapy, resistance mutations, such as EGFR T790M and MET amplification, were common in peripheral blood but were rare in time-matched CSF, indicating differences in resistance mechanisms based on the anatomic compartment. In the osimertinib cohort, patients with CNS progression had increased CSF-tDNA VAFs at follow-up LP. Post-osimertinib CSF-tDNA VAF was strongly prognostic for CNS progression (HR = 6.2, P = 0.009). Detection of CSF-tDNA in lung cancer patients with suspected LMD is feasible and may have clinical utility. CSF-tDNA improves the sensitivity of LMD diagnosis, enables improved prognostication, and drives therapeutic strategies that account for spatial heterogeneity in resistance mechanisms.
RESUMEN
Several gene-expression signatures predict survival in diffuse large B-cell lymphoma (DLBCL), but the lack of practical methods for genome-scale analysis has limited translation to clinical practice. We built and validated a simple model using one gene expressed by tumor cells and another expressed by host immune cells, assessing added prognostic value to the clinical International Prognostic Index (IPI). LIM domain only 2 (LMO2) was validated as an independent predictor of survival and the "germinal center B cell-like" subtype. Expression of tumor necrosis factor receptor superfamily member 9 (TNFRSF9) from the DLBCL microenvironment was the best gene in bivariate combination with LMO2. Study of TNFRSF9 tissue expression in 95 patients with DLBCL showed expression limited to infiltrating T cells. A model integrating these 2 genes was independent of "cell-of-origin" classification, "stromal signatures," IPI, and added to the predictive power of the IPI. A composite score integrating these genes with IPI performed well in 3 independent cohorts of 545 DLBCL patients, as well as in a simple assay of routine formalin-fixed specimens from a new validation cohort of 147 patients with DLBCL. We conclude that the measurement of a single gene expressed by tumor cells (LMO2) and a single gene expressed by the immune microenvironment (TNFRSF9) powerfully predicts overall survival in patients with DLBCL.
Asunto(s)
Biomarcadores de Tumor/genética , Genes Relacionados con las Neoplasias , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/mortalidad , Microambiente Tumoral/genética , Proteínas Adaptadoras Transductoras de Señales , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/fisiología , Niño , Estudios de Cohortes , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Pruebas Genéticas , Humanos , Proteínas con Dominio LIM , Linfoma de Células B Grandes Difuso/diagnóstico , Metaloproteínas/genética , Metaloproteínas/fisiología , Persona de Mediana Edad , Neoplasias/genética , Neoplasias/patología , Pronóstico , Proteínas Proto-Oncogénicas , Análisis de Supervivencia , Microambiente Tumoral/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/fisiología , Adulto JovenRESUMEN
Histone modifications affect DNA-templated processes ranging from transcription to genomic replication. In this study, we examine the cell cycle dynamics of the trimethylated form of histone H3 lysine 4 (H3K4me3), a mark of active chromatin that is viewed as "long-lived" and that is involved in memory during cell state inheritance in metazoans. We synchronized yeast using two different protocols, then followed H3K4me3 patterns as yeast passed through subsequent cell cycles. While most H3K4me3 patterns were conserved from one generation to the next, we found that methylation patterns induced by alpha factor or high temperature were erased within one cell cycle, during S phase. Early-replicating regions were erased before late-replicating regions, implicating replication in H3K4me3 loss. However, nearly complete H3K4me3 erasure occurred at the majority of loci even when replication was prevented, suggesting that most erasure results from an active process. Indeed, deletion of the demethylase Jhd2 slowed erasure at most loci. Together, these results indicate overlapping roles for passive dilution and active enzymatic demethylation in erasing ancestral histone methylation states in yeast.