RESUMO
Post-transplant lymphoproliferative disorders (PTLD) are iatrogenic immune deficiency-associated lymphoid/plasmacytic proliferations developing due to immunosuppression in solid organ or hematopoietic stem cell allograft patients. PTLD are characterized by abnormal proliferation of lymphoid cells and have a heterogeneous clinical behavior. We profiled expression of >700 tumor microenvironment (TME)-related genes in 75 post-transplant aggressive B-cell lymphomas (PTABCL). Epstein-Barr virus (EBV)-positive PT-ABCL clustered together and were enriched for type I interferon pathway and antiviral-response genes. Additionally, a cytotoxicity gene signature associated with EBV-positivity and favorable overall survival (OS) (hazard ratio =0.61; P=0.019). In silico immunophenotyping revealed two subgroups with distinct immune cell compositions. The inflamed subgroup with higher proportions of immune cells had better outcome compared to noninflamed subgroup (median OS >200.0 vs. 15.2 months; P=0.006). In multivariable analysis with EBV status, International Prognostic Index, and rituximab-containing treatment, inflamed TME remained as an independent predictor for favorable outcome. We also compared TME between post-transplant and immunocompetent host diffuse large B-cell lymphomas (n=75) and discovered that the proportions of T cells were lower in PT-diffuse large B-cell lymphomas. In conclusion, we provide a comprehensive phenotypic characterization of PT-ABCL, highlighting the importance of immune cell composition of TME in determining the clinical behavior and prognosis of PT-ABCL.
Assuntos
Infecções por Vírus Epstein-Barr , Linfoma Difuso de Grandes Células B , Transtornos Linfoproliferativos , Humanos , Infecções por Vírus Epstein-Barr/complicações , Herpesvirus Humano 4 , Microambiente Tumoral , Rituximab/uso terapêutico , Transtornos Linfoproliferativos/patologia , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/complicaçõesRESUMO
While the abundance and phenotype of tumor-infiltrating lymphocytes are linked with clinical survival, their spatial coordination and its clinical significance remain unclear. Here, we investigated the immune profile of intratumoral and peritumoral tissue of clear cell renal cell carcinoma patients (n = 64). We trained a cell classifier to detect lymphocytes from hematoxylin and eosin stained tissue slides. Using unsupervised classification, patients were further classified into immune cold, hot and excluded topographies reflecting lymphocyte abundance and localization. The immune topography distribution was further validated with The Cancer Genome Atlas digital image dataset. We showed association between PBRM1 mutation and immune cold topography, STAG1 mutation and immune hot topography and BAP1 mutation and immune excluded topography. With quantitative multiplex immunohistochemistry we analyzed the expression of 23 lymphocyte markers in intratumoral and peritumoral tissue regions. To study spatial interactions, we developed an algorithm quantifying the proportion of adjacent immune cell pairs and their immunophenotypes. Immune excluded tumors were associated with superior overall survival (HR 0.19, p = 0.02) and less extensive metastasis. Intratumoral T cells were characterized with pronounced expression of immunological activation and exhaustion markers such as granzyme B, PD1, and LAG3. Immune cell interaction occurred most frequently in the intratumoral region and correlated with CD45RO expression. Moreover, high proportion of peritumoral CD45RO+ T cells predicted poor overall survival. In summary, intratumoral and peritumoral tissue regions represent distinct immunospatial profiles and are associated with clinicopathologic characteristics.
Assuntos
Algoritmos , Biomarcadores Tumorais/análise , Carcinoma de Células Renais/imunologia , Técnicas de Apoio para a Decisão , Imuno-Histoquímica , Imunofenotipagem , Neoplasias Renais/imunologia , Antígenos Comuns de Leucócito/análise , Linfócitos do Interstício Tumoral/imunologia , Linfócitos T/imunologia , Microambiente Tumoral/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/terapia , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Neoplasias Renais/genética , Neoplasias Renais/mortalidade , Neoplasias Renais/terapia , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas Nucleares/genética , Fenótipo , Valor Preditivo dos Testes , Prognóstico , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genéticaRESUMO
Common variable immunodeficiency and other late-onset immunodeficiencies often co-manifest with autoimmunity and lymphoproliferation. The pathogenesis of most cases is elusive, as only a minor subset harbors known monogenic germline causes. The involvement of both B and T cells is however implicated. To study whether somatic mutations in CD4+ and CD8+ T cells associate with immunodeficiency, we recruited 17 patients and 21 healthy controls. Eight patients had late-onset common variable immunodeficiency and nine patients other immunodeficiency and/or severe autoimmunity. In total, autoimmunity occurred in 94% and lymphoproliferation in 65%. We performed deep sequencing of 2533 immune-associated genes from CD4+ and CD8+ cells. Deep T-cell receptor beta sequencing was used to characterize CD4+ and CD8+ T-cell receptor repertoires. The prevalence of somatic mutations was 65% in all immunodeficiency patients, 75% in common variable immunodeficiency and 48% in controls. Clonal hematopoiesis-associated variants in both CD4+ and CD8+ cells occurred in 24% of immunodeficiency patients. Results demonstrated mutations in known tumor suppressors, oncogenes, and genes that are critical for immune- and proliferative functions, such as STAT5B (two patients), C5AR1 (two patients), KRAS (one patient), and NOD2 (one patient). Additionally, as a marker of T-cell receptor repertoire perturbation, common variable immunodeficiency patients harbored increased frequencies of clones with identical complementarity determining region 3 sequences despite unique nucleotide sequences when compared to controls. In conclusion, somatic mutations in genes implicated for autoimmunity and lymphoproliferation are common in CD4+ and CD8+ cells of patients with immunodeficiency. They may contribute to immune dysregulation in a subset of immunodeficiency patients.
Assuntos
Síndromes de Imunodeficiência , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Regiões Determinantes de Complementaridade/genética , Humanos , Mutação , Receptores de Antígenos de Linfócitos T alfa-beta/genéticaRESUMO
BACKGROUND: CCAAT enhancer-binding protein epsilon (C/EBPε) is a transcription factor involved in late myeloid lineage differentiation and cellular function. The only previously known disorder linked to C/EBPε is autosomal recessive neutrophil-specific granule deficiency leading to severely impaired neutrophil function and early mortality. OBJECTIVE: The aim of this study was to molecularly characterize the effects of C/EBPε transcription factor Arg219His mutation identified in a Finnish family with previously genetically uncharacterized autoinflammatory and immunodeficiency syndrome. METHODS: Genetic analysis, proteomics, genome-wide transcriptional profiling by means of RNA-sequencing, chromatin immunoprecipitation (ChIP) sequencing, and assessment of the inflammasome function of primary macrophages were performed. RESULTS: Studies revealed a novel mechanism of genome-wide gain-of-function that dysregulated transcription of 464 genes. Mechanisms involved dysregulated noncanonical inflammasome activation caused by decreased association with transcriptional repressors, leading to increased chromatin occupancy and considerable changes in transcriptional activity, including increased expression of NLR family, pyrin domain-containing 3 protein (NLRP3) and constitutively expressed caspase-5 in macrophages. CONCLUSION: We describe a novel autoinflammatory disease with defective neutrophil function caused by a homozygous Arg219His mutation in the transcription factor C/EBPε. Mutated C/EBPε acts as a regulator of both the inflammasome and interferome, and the Arg219His mutation causes the first human monogenic neomorphic and noncanonical inflammasomopathy/immunodeficiency. The mechanism, including widely dysregulated transcription, is likely not unique for C/EBPε. Similar multiomics approaches should also be used in studying other transcription factor-associated diseases.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Mutação com Ganho de Função/genética , Síndromes de Imunodeficiência/genética , Inflamassomos/genética , Inflamação/genética , Macrófagos/metabolismo , Neutrófilos/fisiologia , Idoso , Caspases/genética , Caspases/metabolismo , Células Cultivadas , Feminino , Perfilação da Expressão Gênica , Humanos , Inflamassomos/metabolismo , Macrófagos/patologia , Masculino , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Linhagem , Análise de Sequência de RNA , Regulação para CimaRESUMO
PURPOSE: Recent advances in machine learning have enabled better understanding of large and complex visual data. Here, we aim to investigate patient outcome prediction with a machine learning method using only an image of tumour sample as an input. METHODS: Utilising tissue microarray (TMA) samples obtained from the primary tumour of patients (N = 1299) within a nationwide breast cancer series with long-term-follow-up, we train and validate a machine learning method for patient outcome prediction. The prediction is performed by classifying samples into low or high digital risk score (DRS) groups. The outcome classifier is trained using sample images of 868 patients and evaluated and compared with human expert classification in a test set of 431 patients. RESULTS: In univariate survival analysis, the DRS classification resulted in a hazard ratio of 2.10 (95% CI 1.33-3.32, p = 0.001) for breast cancer-specific survival. The DRS classification remained as an independent predictor of breast cancer-specific survival in a multivariate Cox model with a hazard ratio of 2.04 (95% CI 1.20-3.44, p = 0.007). The accuracy (C-index) of the DRS grouping was 0.60 (95% CI 0.55-0.65), as compared to 0.58 (95% CI 0.53-0.63) for human expert predictions based on the same TMA samples. CONCLUSIONS: Our findings demonstrate the feasibility of learning prognostic signals in tumour tissue images without domain knowledge. Although further validation is needed, our study suggests that machine learning algorithms can extract prognostically relevant information from tumour histology complementing the currently used prognostic factors in breast cancer.
Assuntos
Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Imuno-Histoquímica , Aprendizado de Máquina , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Feminino , Seguimentos , Humanos , Processamento de Imagem Assistida por Computador , Estimativa de Kaplan-Meier , Microscopia , Pessoa de Meia-Idade , Gradação de Tumores , Metástase Neoplásica , Estadiamento de Neoplasias , Prognóstico , Análise de Sobrevida , Carga Tumoral , Fluxo de TrabalhoRESUMO
Felty syndrome is a rare disease defined by neutropenia, splenomegaly, and rheumatoid arthritis. Sometimes the differential diagnosis between Felty syndrome and large granular lymphocyte leukemia is problematic. Recently, somatic STAT3 and STAT5B mutations were discovered in 30-40% of patients with large granular lymphocyte leukemia. Herein, we aimed to study whether these mutations can also be detected in Felty syndrome, which would imply the existence of a common pathogenic mechanism between these two disease entities. We collected samples and clinical information from 14 Felty syndrome patients who were monitored at the rheumatology outpatient clinic for Felty syndrome. Somatic STAT3 mutations were discovered in 43% (6/14) of Felty syndrome patients with deep amplicon sequencing targeting all STAT3 exons. Mutations were located in the SH2 domain of STAT3, which is a known mutational hotspot. No STAT5B mutations were found. In blood smears, overrepresentation of large granular lymphocytes was observed, and in the majority of cases the CD8+ T-cell receptor repertoire was skewed when analyzed by flow cytometry. In bone marrow biopsies, an increased amount of phospho-STAT3 positive cells was discovered. Plasma cytokine profiling showed that ten of the 92 assayed cytokines were elevated both in Felty syndrome and large granular lymphocyte leukemia, and three of these cytokines were also increased in patients with uncomplicated rheumatoid arthritis. In conclusion, somatic STAT3 mutations and STAT3 activation are as frequent in Felty syndrome as they are in large granular lymphocyte leukemia. Considering that the symptoms and treatment modalities are also similar, a unified reclassification of these two syndromes is warranted.
Assuntos
Síndrome de Felty/genética , Leucemia Linfocítica Granular Grande/genética , Fator de Transcrição STAT3/genética , Adulto , Idoso , Citocinas/análise , Análise Mutacional de DNA , Diagnóstico Diferencial , Síndrome de Felty/classificação , Síndrome de Felty/diagnóstico , Síndrome de Felty/patologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Leucemia Linfocítica Granular Grande/classificação , Leucemia Linfocítica Granular Grande/diagnóstico , Leucemia Linfocítica Granular Grande/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Fenótipo , Fosforilação , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5 , Domínios de Homologia de src/genéticaRESUMO
The signal transducer and activator of transcription (STAT) family of transcription factors orchestrate hematopoietic cell differentiation. Recently, mutations in STAT1, STAT5B, and STAT3 have been linked to development of immunodysregulation polyendocrinopathy enteropathy X-linked-like syndrome. Here, we immunologically characterized 3 patients with de novo activating mutations in the DNA binding or dimerization domains of STAT3 (p.K392R, p.M394T, and p.K658N, respectively). The patients displayed multiorgan autoimmunity, lymphoproliferation, and delayed-onset mycobacterial disease. Immunologically, we noted hypogammaglobulinemia with terminal B-cell maturation arrest, dendritic cell deficiency, peripheral eosinopenia, increased double-negative (CD4(-)CD8(-)) T cells, and decreased natural killer, T helper 17, and regulatory T-cell numbers. Notably, the patient harboring the K392R mutation developed T-cell large granular lymphocytic leukemia at age 14 years. Our results broaden the spectrum of phenotypes caused by activating STAT3 mutations, highlight the role of STAT3 in the development and differentiation of multiple immune cell lineages, and strengthen the link between the STAT family of transcription factors and autoimmunity.
Assuntos
Agamaglobulinemia , Doenças Autoimunes , Doenças Genéticas Inatas , Leucemia Linfocítica Granular Grande , Mutação de Sentido Incorreto , Infecções por Mycobacterium , Fator de Transcrição STAT3 , Adolescente , Adulto , Agamaglobulinemia/genética , Agamaglobulinemia/imunologia , Agamaglobulinemia/patologia , Substituição de Aminoácidos , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Linfócitos B/imunologia , Linfócitos B/patologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Células Dendríticas/patologia , Feminino , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/imunologia , Doenças Genéticas Inatas/patologia , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Leucemia Linfocítica Granular Grande/genética , Leucemia Linfocítica Granular Grande/imunologia , Leucemia Linfocítica Granular Grande/patologia , Infecções por Mycobacterium/genética , Infecções por Mycobacterium/imunologia , Infecções por Mycobacterium/patologia , Estrutura Terciária de Proteína , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Células Th17/imunologia , Células Th17/patologiaRESUMO
Although loss of epithelial integrity is a hallmark of advanced cancer, it remains poorly understood whether genetic alterations corrupting this integrity causally facilitate tumorigenesis. We show that conditional deletion of tumor suppressor gene Lkb1 (Par-4) in the mammary gland compromises epithelial integrity manifested by mislocalization of cell polarity markers, lateralization of tight junctions, deterioration of desmosomes and basement membrane (BM), and hyperbranching of the mammary ductal tree. We identify the desmosomal BM remodelling serine protease Hepsin as a key factor mediating Lkb1 loss-induced structural alterations in mammary epithelium and BM fragmentation. Although loss of Lkb1 alone does not promote mammary tumorigenesis, combination of Lkb1 deficiency with oncogenic c-Myc leads to dramatic acceleration in tumor formation. The results coupling Lkb1 loss-mediated epithelial integrity defects to mislocalization of serine protease Hepsin and to oncogenic synergy with c-Myc imply that Lkb1 loss facilitates oncogenic proliferation by releasing epithelial cells from structural BM boundaries.
Assuntos
Genes Supressores de Tumor , Glândulas Mamárias Animais/enzimologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Quinases Ativadas por AMP , Animais , Células Epiteliais/citologia , Feminino , Deleção de Genes , Genes myc , Glândulas Mamárias Animais/citologia , Camundongos , Proteínas Serina-Treonina Quinases/genéticaRESUMO
BACKGROUND: Combining cytotoxic chemotherapy or novel anticancer drugs with T-cell modulators holds great promise in treating advanced cancers. However, the response varies depending on the tumor immune microenvironment (TIME). Therefore, there is a clear need for pharmacologically tractable models of the TIME to dissect its influence on mono- and combination treatment response at the individual level. METHODS: Here we establish a patient-derived explant culture (PDEC) model of breast cancer, which retains the immune contexture of the primary tumor, recapitulating cytokine profiles and CD8+T cell cytotoxic activity. RESULTS: We explored the immunomodulatory action of a synthetic lethal BCL2 inhibitor venetoclax+metformin drug combination ex vivo, discovering metformin cannot overcome the lymphocyte-depleting action of venetoclax. Instead, metformin promotes dendritic cell maturation through inhibition of mitochondrial complex I, increasing their capacity to co-stimulate CD4+T cells and thus facilitating antitumor immunity. CONCLUSIONS: Our results establish PDECs as a feasible model to identify immunomodulatory functions of anticancer drugs in the context of patient-specific TIME.
Assuntos
Antineoplásicos , Neoplasias da Mama , Compostos Bicíclicos Heterocíclicos com Pontes , Metformina , Sulfonamidas , Humanos , Feminino , Complexo I de Transporte de Elétrons/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Células Dendríticas , Metformina/farmacologia , Metformina/uso terapêutico , Microambiente TumoralRESUMO
Interleukin (IL)-21 is the most recently recognized of the cytokines that share the common cytokine receptor gamma chain (gamma(c)), which is mutated in humans with X-linked severe combined immunodeficiency. We now report that IL-21 synergistically acts with IL-15 to potently promote the proliferation of both memory (CD44high) and naive (CD44low) phenotype CD8+ T cells and augment interferon-gamma production in vitro. IL-21 also cooperated, albeit more weakly, with IL-7, but not with IL-2. Correspondingly, the expansion and cytotoxicity of CD8+ T cells were impaired in IL-21R-/- mice. Moreover, in vivo administration of IL-21 in combination with IL-15 boosted antigen-specific CD8+ T cell numbers and resulted in a cooperative effect on tumor regression, with apparent cures of large, established B16 melanomas. Thus, our studies reveal that IL-21 potently regulates CD8+ T cell expansion and effector function, primarily in a synergistic context with IL-15.
Assuntos
Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Interleucina-15/farmacologia , Interleucinas/farmacologia , Melanoma Experimental/terapia , Animais , Proliferação de Células/efeitos dos fármacos , Testes Imunológicos de Citotoxicidade , Sinergismo Farmacológico , Citometria de Fluxo , Fluoresceínas , Proteína gp160 do Envelope de HIV , Memória Imunológica/imunologia , Imunoterapia Adotiva , Interferon gama/metabolismo , Interleucina-15/metabolismo , Interleucina-15/uso terapêutico , Interleucina-7/metabolismo , Interleucinas/metabolismo , Interleucinas/uso terapêutico , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , SuccinimidasRESUMO
In myelodysplastic syndrome (MDS) and myeloproliferative neoplasm (MPN), bone marrow (BM) histopathology is assessed to identify dysplastic cellular morphology, cellularity, and blast excess. Yet, other morphologic findings may elude the human eye. We used convolutional neural networks to extract morphologic features from 236 MDS, 87 MDS/MPN, and 11 control BM biopsies. These features predicted genetic and cytogenetic aberrations, prognosis, age, and gender in multivariate regression models. Highest prediction accuracy was found for TET2 [area under the receiver operating curve (AUROC) = 0.94] and spliceosome mutations (0.89) and chromosome 7 monosomy (0.89). Mutation prediction probability correlated with variant allele frequency and number of affected genes per pathway, demonstrating the algorithms' ability to identify relevant morphologic patterns. By converting regression models to texture and cellular composition, we reproduced the classical del(5q) MDS morphology consisting of hypolobulated megakaryocytes. In summary, this study highlights the potential of linking deep BM histopathology with genetics and clinical variables. SIGNIFICANCE: Histopathology is elementary in the diagnostics of patients with MDS, but its high-dimensional data are underused. By elucidating the association of morphologic features with clinical variables and molecular genetics, this study highlights the vast potential of convolutional neural networks in understanding MDS pathology and how genetics is reflected in BM morphology. See related commentary by Elemento, p. 195.
Assuntos
Síndromes Mielodisplásicas , Doenças Mieloproliferativas-Mielodisplásicas , Medula Óssea/patologia , Humanos , Aprendizado de Máquina , Mutação/genética , Síndromes Mielodisplásicas/diagnóstico , Doenças Mieloproliferativas-Mielodisplásicas/genéticaRESUMO
Ras proteins play a causal role in human cancer by activating multiple pathways that promote cancer growth and invasion. However, little is known about how Ras induces the first diagnostic features of invasion in solid tumors, including loss of epithelial integrity and breaching of the basement membrane (BM). In this study, we found that oncogenic Ras strongly promotes the activation of hepsin, a member of the hepsin/TMPRSS type II transmembrane serine protease family. Mechanistically, the Ras-dependent hepsin activation was mediated via Raf-MEK-ERK signaling, which controlled hepsin protein stability through the heat shock transcription factor-1 stress pathway. In Ras-transformed three-dimensional mammary epithelial culture, ablation of hepsin restored desmosomal cell-cell junctions, hemidesmosomes, and BM integrity and epithelial cohesion. In tumor xenografts harboring mutant KRas, silencing of hepsin increased local invasion concomitantly with accumulation of collagen IV. These findings suggest that hepsin is a critical protease for Ras-dependent tumorigenesis, executing cell-cell and cell-matrix pathologies important for early tumor dissemination. SIGNIFICANCE: These findings identify the cell-surface serine protease hepsin as a potential therapeutic target for its role in oncogenic Ras-mediated deregulation of epithelial cell-cell and cell-matrix interactions and cohesion of epithelial structure.
Assuntos
Neoplasias da Mama/patologia , Células Epiteliais/patologia , Fatores de Transcrição de Choque Térmico/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Serina Endopeptidases/metabolismo , Animais , Membrana Basal/citologia , Membrana Basal/patologia , Mama/patologia , Neoplasias da Mama/genética , Carcinogênese/patologia , Comunicação Celular , Linhagem Celular Tumoral , Colágeno Tipo IV/metabolismo , Desmossomos/patologia , Células Epiteliais/citologia , Feminino , Técnicas de Silenciamento de Genes , Fatores de Transcrição de Choque Térmico/genética , Humanos , Sistema de Sinalização das MAP Quinases/genética , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Transgênicos , Mutação , Invasividade Neoplásica/patologia , Cultura Primária de Células , Estabilidade Proteica , Proteínas Proto-Oncogênicas p21(ras)/genética , Serina Endopeptidases/genética , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Breast cancer is now globally the most frequent cancer and leading cause of women's death. Two thirds of breast cancers express the luminal estrogen receptor-positive (ERα + ) phenotype that is initially responsive to antihormonal therapies, but drug resistance emerges. A major barrier to the understanding of the ERα-pathway biology and therapeutic discoveries is the restricted repertoire of luminal ERα + breast cancer models. The ERα + phenotype is not stable in cultured cells for reasons not fully understood. We examine 400 patient-derived breast epithelial and breast cancer explant cultures (PDECs) grown in various three-dimensional matrix scaffolds, finding that ERα is primarily regulated by the matrix stiffness. Matrix stiffness upregulates the ERα signaling via stress-mediated p38 activation and H3K27me3-mediated epigenetic regulation. The finding that the matrix stiffness is a central cue to the ERα phenotype reveals a mechanobiological component in breast tissue hormonal signaling and enables the development of novel therapeutic interventions. Subject terms: ER-positive (ER + ), breast cancer, ex vivo model, preclinical model, PDEC, stiffness, p38 SAPK.
Assuntos
Neoplasias da Mama/genética , Receptor alfa de Estrogênio/genética , Mecanotransdução Celular/genética , Transcriptoma , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Estudos de Casos e Controles , Linhagem Celular Tumoral , Cinamatos/farmacologia , Colágeno/química , Colágeno/farmacologia , Combinação de Medicamentos , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Feminino , Fulvestranto/farmacologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Histonas/genética , Histonas/metabolismo , Humanos , Indazóis/farmacologia , Laminina/química , Laminina/farmacologia , Glândulas Mamárias Humanas/efeitos dos fármacos , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , Fenótipo , Proteoglicanas/química , Proteoglicanas/farmacologia , Tamoxifeno/farmacologia , Técnicas de Cultura de Tecidos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Signal transducer and activator of transcription (STAT) proteins are latent transcription factors that mediate a wide range of actions induced by cytokines, interferons, and growth factors. We now report the development of thymic T cell lymphoblastic lymphomas in transgenic mice in which Stat5a or Stat5b is overexpressed within the lymphoid compartment. The rate of lymphoma induction was markedly enhanced by immunization or by the introduction of TCR transgenes. Remarkably, the Stat5 transgene potently induced development of CD8+ T cells, even in mice expressing a class II-restricted TCR transgene, with resulting CD8+ T cell lymphomas. These data demonstrate the oncogenic potential of dysregulated expression of a STAT protein that is not constitutively activated, and that TCR stimulation can contribute to this process.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas do Leite , Leucemia-Linfoma Linfoblástico de Células Precursoras/etiologia , Receptores de Antígenos de Linfócitos T/fisiologia , Transativadores/fisiologia , Animais , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Fator de Transcrição STAT3 , Fator de Transcrição STAT5RESUMO
Peripheral T-cell lymphomas (PTCL) are a heterogeneous, and often aggressive group of non-Hodgkin lymphomas. Recent advances in the molecular and genetic characterization of PTCLs have helped to delineate differences and similarities between the various subtypes, and the JAK/STAT pathway has been found to play an important oncogenic role. Here, we aimed to characterize the JAK/STAT pathway in PTCL subtypes and investigate whether the activation of the pathway correlates with the frequency of STAT gene mutations. Patient samples from AITL (n = 30), ALCL (n = 21) and PTCL-NOS (n = 12) cases were sequenced for STAT3, STAT5B, JAK1, JAK3, and RHOA mutations using amplicon sequencing and stained immunohistochemically for pSTAT3, pMAPK, and pAKT. We discovered STAT3 mutations in 13% of AITL, 13% of ALK+ ALCL, 38% of ALK- ALCL and 17% of PTCL-NOS cases. However, no STAT5B mutations were found and JAK mutations were only present in ALK- ALCL (15%). Concurrent mutations were found in all subgroups except ALK+ ALCL where STAT3 mutations were always seen alone. High pY-STAT3 expression was observed especially in AITL and ALCL samples. When studying JAK-STAT pathway mutations, pY-STAT3 expression was highest in PTCLs harboring either JAK1 or STAT3 mutations and CD30+ phenotype representing primarily ALK- ALCLs. Further investigation is needed to elucidate the molecular mechanisms of JAK-STAT pathway activation in PTCL.
RESUMO
The immunologic microenvironment in various solid tumors is aberrant and correlates with clinical survival. Here, we present a comprehensive analysis of the immune environment of acute myeloid leukemia (AML) bone marrow (BM) at diagnosis. We compared the immunologic landscape of formalin-fixed paraffin-embedded BM trephine samples from AML (n = 69), chronic myeloid leukemia (CML; n = 56), and B-cell acute lymphoblastic leukemia (B-ALL) patients (n = 52) at diagnosis to controls (n = 12) with 30 immunophenotype markers using multiplex immunohistochemistry and computerized image analysis. We identified distinct immunologic profiles specific for leukemia subtypes and controls enabling accurate classification of AML (area under the curve [AUC] = 1.0), CML (AUC = 0.99), B-ALL (AUC = 0.96), and control subjects (AUC = 1.0). Interestingly, 2 major immunologic AML clusters differing in age, T-cell receptor clonality, and survival were discovered. A low proportion of regulatory T cells and pSTAT1+cMAF- monocytes were identified as novel biomarkers of superior event-free survival in intensively treated AML patients. Moreover, we demonstrated that AML BM and peripheral blood samples are dissimilar in terms of immune cell phenotypes. To conclude, our study shows that the immunologic landscape considerably varies by leukemia subtype suggesting disease-specific immunoregulation. Furthermore, the association of the AML immune microenvironment with clinical parameters suggests a rationale for including immunologic parameters to improve disease classification or even patient risk stratification.
Assuntos
Medula Óssea/metabolismo , Leucemia Mieloide Aguda/imunologia , Receptores de Antígenos de Linfócitos T/genética , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sobrevida , Adulto JovemRESUMO
RMRP was the first non-coding nuclear RNA gene implicated in a disease. Its mutations cause cartilage-hair hypoplasia (CHH), an autosomal recessive skeletal dysplasia with growth failure, immunodeficiency, and a high risk for malignancies. This study aimed to gain further insight into the role of RNA Component of Mitochondrial RNA Processing Endoribonuclease (RMRP) in cellular physiology and disease pathogenesis. We combined transcriptome analysis with single-cell analysis using fibroblasts from CHH patients and healthy controls. To directly assess cell cycle progression, we followed CHH fibroblasts by pulse-labeling and time-lapse microscopy. Transcriptome analysis identified 35 significantly upregulated and 130 downregulated genes in CHH fibroblasts. The downregulated genes were significantly connected to the cell cycle. Multiple other pathways, involving regulation of apoptosis, bone and cartilage formation, and lymphocyte function, were also affected, as well as PI3K-Akt signaling. Cell-cycle studies indicated that the CHH cells were delayed specifically in the passage from G2 phase to mitosis. Our findings expand the mechanistic understanding of CHH, indicate possible pathways for therapeutic intervention and add to the limited understanding of the functions of RMRP.
Assuntos
Fase G2/genética , RNA Longo não Codificante/genética , Adulto , Apoptose/genética , Regulação para Baixo/genética , Endorribonucleases/genética , Fibroblastos/fisiologia , Cabelo/anormalidades , Doença de Hirschsprung/genética , Humanos , Síndromes de Imunodeficiência/genética , Linfócitos/fisiologia , Osteocondrodisplasias/congênito , Osteocondrodisplasias/genética , Fosfatidilinositol 3-Quinases/genética , Doenças da Imunodeficiência Primária/genética , Transdução de Sinais/genética , Transcriptoma/genética , Regulação para Cima/genéticaRESUMO
As novel immunological treatments are gaining a foothold in the treatment of acute lymphoblastic leukemia (ALL), it is elemental to examine ALL immunobiology in more detail. We used multiplexed immunohistochemistry (mIHC) to study the immune contexture in adult precursor B cell ALL bone marrow (BM). In addition, we developed a multivariate risk prediction model that stratified a poor survival group based on clinical parameters and mIHC data. We analyzed BM biopsy samples of ALL patients (n = 52) and healthy controls (n = 14) using mIHC with 30 different immunophenotype markers and computerized image analysis. In ALL BM, the proportions of M1-like macrophages, granzyme B+CD57+CD8+ T cells, and CD27+ T cells were decreased, whereas the proportions of myeloid-derived suppressor cells and M2-like macrophages were increased. Also, the expression of checkpoint molecules PD1 and CTLA4 was elevated. In the multivariate model, age, platelet count, and the proportion of PD1+TIM3+ double-positive CD4+ T cells differentiated a poor survival group. These results were validated by flow cytometry in a separate cohort (n = 31). In conclusion, the immune cell contexture in ALL BM differs from healthy controls. CD4+PD1+TIM3+ T cells were independent predictors of poor outcome in our multivariate risk model, suggesting that PD1 might serve as an attractive immuno-oncological target in B-ALL.
Assuntos
Medula Óssea/imunologia , Linfócitos T CD8-Positivos/imunologia , Transplante de Células-Tronco Hematopoéticas , Macrófagos/imunologia , Células Supressoras Mieloides/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Microambiente Tumoral/imunologia , Adolescente , Adulto , Idoso , Antígeno CTLA-4 , Estudos de Casos e Controles , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida , Adulto JovemRESUMO
Mutations in several proteins functioning as endolysosomal components cause monogenic autoimmune diseases, of which pathogenesis is linked to increased endoplasmic reticulum stress, inefficient autophagy, and defective recycling of immune receptors. We report here a heterozygous TOM1 p.G307D missense mutation, detected by whole-exome sequencing, in two related patients presenting with early-onset autoimmunity, antibody deficiency, and features of combined immunodeficiency. The index patient suffered from recurrent respiratory tract infections and oligoarthritis since early teens, and later developed persistent low-copy EBV-viremia, as well as an antibody deficiency. Her infant son developed hypogammaglobulinemia, autoimmune enteropathy, interstitial lung disease, profound growth failure, and treatment-resistant psoriasis vulgaris. Consistent with previous knowledge on TOM1 protein function, we detected impaired autophagy and enhanced susceptibility to apoptosis in patient-derived cells. In addition, we noted diminished STAT and ERK1/2 signaling in patient fibroblasts, as well as poor IFN-γ and IL-17 secretion in T cells. The mutant TOM1 failed to interact with TOLLIP, a protein required for IL-1 recycling, PAMP signaling and autophagosome maturation, further strengthening the link between the candidate mutation and patient pathophysiology. In sum, we report here an identification of a novel gene, TOM1, associating with early-onset autoimmunity, antibody deficiency, and features of combined immunodeficiency. Other patient cases from unrelated families are needed to firmly establish a causal relationship between the genotype and the phenotype.