RESUMEN
Immune checkpoint blockade has provided a paradigm shift in cancer therapy, but the success of this approach is very variable; therefore, biomarkers predictive of clinical efficacy are urgently required. Here, we show that the frequency of PD-1+CD8+ T cells relative to that of PD-1+ regulatory T (Treg) cells in the tumor microenvironment can predict the clinical efficacy of programmed cell death protein 1 (PD-1) blockade therapies and is superior to other predictors, including PD ligand 1 (PD-L1) expression or tumor mutational burden. PD-1 expression by CD8+ T cells and Treg cells negatively impacts effector and immunosuppressive functions, respectively. PD-1 blockade induces both recovery of dysfunctional PD-1+CD8+ T cells and enhanced PD-1+ Treg cell-mediated immunosuppression. A profound reactivation of effector PD-1+CD8+ T cells rather than PD-1+ Treg cells by PD-1 blockade is necessary for tumor regression. These findings provide a promising predictive biomarker for PD-1 blockade therapies.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Inhibidores de Puntos de Control Inmunológico/farmacología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/genética , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Antígenos/química , Antígenos/inmunología , Biomarcadores de Tumor , Antígenos CD28/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Humanos , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunomodulación , Activación de Linfocitos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Terapia Molecular Dirigida , Metástasis de la Neoplasia , Estadificación de Neoplasias , Neoplasias/tratamiento farmacológico , Neoplasias/etiología , Neoplasias/metabolismo , Neoplasias/mortalidad , Péptidos/química , Péptidos/inmunología , Pronóstico , Receptor de Muerte Celular Programada 1/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T Reguladores/efectos de los fármacos , Resultado del Tratamiento , Microambiente Tumoral/inmunologíaRESUMEN
Only a small percentage of patients afflicted with gastric cancer (GC) respond to immune checkpoint blockade (ICB). To study the mechanisms underlying this resistance, we examined the immune landscape of GC. A subset of these tumors was characterized by high frequencies of regulatory T (Treg) cells and low numbers of effector T cells. Genomic analyses revealed that these tumors bore mutations in RHOA that are known to drive tumor progression. RHOA mutations in cancer cells activated the PI3K-AKT-mTOR signaling pathway, increasing production of free fatty acids that are more effectively consumed by Treg cells than effector T cells. RHOA mutant tumors were resistant to PD-1 blockade but responded to combination of PD-1 blockade with inhibitors of the PI3K pathway or therapies targeting Treg cells. We propose that the metabolic advantage conferred by RHOA mutations enables Treg cell accumulation within GC tumors, generating an immunosuppressive TME that underlies resistance to ICB.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Inhibidores de Puntos de Control Inmunológico/farmacología , Neoplasias Gástricas/genética , Linfocitos T Reguladores/metabolismo , Proteína de Unión al GTP rhoA/genética , Animales , Recuento de Linfocito CD4 , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Quimiocina CXCL10/biosíntesis , Quimiocina CXCL11/biosíntesis , Ácidos Grasos no Esterificados/biosíntesis , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Receptor de Muerte Celular Programada 1/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/inmunología , Neoplasias Gástricas/inmunología , Neoplasias Gástricas/patología , Linfocitos T Reguladores/inmunología , Serina-Treonina Quinasas TOR/metabolismo , Microambiente Tumoral/inmunologíaRESUMEN
An aneurysmal bone cyst (ABC) is a benign bone neoplasm that typically occurs during the first and second decades of life. ABC usually presents as a rapidly growing intramedullary expansile mass with multiple blood-filled cysts in the metaphysis of the long tubular bones. Here, we report a case of a periosteal solid ABC that was initially diagnosed as a high-grade surface osteosarcoma. A 10-year-old male was referred to our hospital for swelling and tenderness of the left upper arm. Radiography revealed periosteal mass without fluid-fluid levels. On performing open biopsy, the tumor showed hypercellular proliferation of uniform spindle to epithelioid cells with brisk mitotic activity (up to 12/2 mm2) and lace-like osteoid formation, which was diagnosed as a high-grade surface osteosarcoma. After one course of chemotherapy using adriamycin and cisplatin, peripheral sclerosis was conspicuous, which led to pathological review and revision of diagnosis as "possibly osteoblastoma." The patient was disease-free for 4 years after marginal resection and curettage. Retrospective nanopore DNA sequencing unexpectedly detected a PAFAH1B1::USP6 rearrangement. The fusion gene was further validated using reverse transcription-polymerase chain reaction and the diagnosis was revised to ABC. Chromothripsis involving chromosome 17 has also been identified. Methylation analysis classified the present tumor as an ABC or non-ossifying fibroma using t-distributed stochastic neighbor embedding and unsupervised hierarchical clustering. This case report highlights the utility of nanopore DNA sequencing for soft tissue and bone tumor diagnosis.
Asunto(s)
Quistes Óseos Aneurismáticos , Cromotripsis , Secuenciación de Nanoporos , Osteosarcoma , Ubiquitina Tiolesterasa , Humanos , Masculino , Quistes Óseos Aneurismáticos/genética , Quistes Óseos Aneurismáticos/patología , Quistes Óseos Aneurismáticos/diagnóstico , Osteosarcoma/genética , Osteosarcoma/patología , Osteosarcoma/diagnóstico , Ubiquitina Tiolesterasa/genética , Niño , Secuenciación de Nanoporos/métodos , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Neoplasias Óseas/diagnóstico , Reordenamiento GénicoRESUMEN
Dermatofibroma (DF) is a benign tumor that forms pedunculated lesions ranging in size from a few millimeters to 2 cm, usually affecting the extremities and trunks of young adults. Histopathologically, DF is characterized by the storiform proliferation of monomorphic fibroblast-like spindle cells. In addition to neoplastic cells, secondary elements such as foamy histiocytes, Touton-type giant cells, lymphoplasmacytes, and epidermal hyperplasia are characteristic histological features. Several histological variants, including atypical, cellular, aneurysmal, and lipidized variants, have been reported; cases with variant histologies are sometimes misdiagnosed as sarcomas. We present a case of metastasizing aneurysmal DF that was initially diagnosed as an angiosarcoma on biopsy. A 26-year-old woman was referred to our hospital with a gradually enlarging subcutaneous mass in her lower left leg. Positron emission tomography-computed tomography revealed high fluorodeoxyglucose uptake not only in the tumor but also in the left inguinal region. On biopsy, ERG and CD31-positive atypical spindle cells proliferated in slit-like spaces with extravasation, leading to the diagnosis of angiosarcoma. Histology of the wide-resection specimen was consistent with DF, and lymph node metastasis was also observed. Nanopore DNA sequencing detected CD63::PRKCD fusion and copy number gain, although CD63 was not included in the target region of adaptive sampling. This report highlights the importance of recognizing the unusual clinical, radiological, and pathological features of DF to avoid misdiagnosis, and the potential diagnostic utility of nanopore sequencer.
Asunto(s)
Hemangiosarcoma , Histiocitoma Fibroso Benigno , Secuenciación de Nanoporos , Proteínas de Fusión Oncogénica , Adulto , Femenino , Humanos , Hemangiosarcoma/genética , Hemangiosarcoma/diagnóstico , Hemangiosarcoma/patología , Histiocitoma Fibroso Benigno/genética , Histiocitoma Fibroso Benigno/diagnóstico , Histiocitoma Fibroso Benigno/patología , Secuenciación de Nanoporos/métodos , Proteínas de Fusión Oncogénica/análisis , Proteínas de Fusión Oncogénica/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/diagnóstico , Tetraspanina 30/genética , Tetraspanina 30/metabolismoRESUMEN
By taking advantage of forward genetic analysis in mice, we have demonstrated that Pak1 plays a crucial role during DMBA/TPA skin carcinogenesis. Although Pak1 has been considered to promote cancer development, its overall function remains poorly understood. To clarify the functional significance of Pak1 in detail, we sought to evaluate the possible effect of an allosteric inhibitor against PAK1 (NVS-PAK1-1) on a syngeneic mouse model. To this end, we established two cell lines, 9AS1 and 19AS1, derived from DMBA/TPA-induced squamous cell carcinoma (SCC) that engrafted in FVB mice. Based on our present results, NVS-PAK1-1 treatment significantly inhibited the growth of tumors derived from 9AS1 and 19AS1 cells in vitro and in vivo. RNA-sequencing analysis on the engrafted tumors indicates that NVS-PAK1-1 markedly potentiates the epidermal cell differentiation and enhances the immune response in the engrafted tumors. Consistent with these observations, we found an expansion of Pan-keratin-positive regions and potentially elevated infiltration of CD8-positive immune cells in NVS-PAK1-1-treated tumors as examined by immunohistochemical analyses. Together, our present findings strongly suggest that PAK1 is tightly linked to the development of SCC, and that its inhibition is a promising therapeutic strategy against SCC.
Asunto(s)
Carcinoma de Células Escamosas , Modelos Animales de Enfermedad , Neoplasias Cutáneas , Quinasas p21 Activadas , Animales , Quinasas p21 Activadas/metabolismo , Quinasas p21 Activadas/genética , Quinasas p21 Activadas/antagonistas & inhibidores , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/genética , Ratones , Línea Celular Tumoral , 9,10-Dimetil-1,2-benzantraceno/toxicidad , Femenino , Diferenciación Celular/efectos de los fármacos , Acetato de Tetradecanoilforbol , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Proliferación Celular/efectos de los fármacosRESUMEN
The genetic basis of leukemogenesis in adults with B-cell acute lymphoblastic leukemia (B-ALL) is largely unclear, and its clinical outcome remains unsatisfactory. This study aimed to advance the understanding of biological characteristics, improve disease stratification, and identify molecular targets of adult B-ALL. Adolescents and young adults (AYA) (15 to 39 years old, n = 193) and adults (40 to 64 years old, n = 161) with Philadelphia chromosome-negative (Ph-) B-ALL were included in this study. Integrated transcriptomic and genetic analyses were used to classify the cohort into defined subtypes. Of the 323 cases included in the RNA sequencing analysis, 278 (86.1%) were classified into 18 subtypes. The ZNF384 subtype (22.6%) was the most prevalent, with 2 novel subtypes (CDX2-high and IDH1/2-mut) identified among cases not assigned to the established subtypes. The CDX2-high subtype (3.4%) was characterized by high expression of CDX2 and recurrent gain of chromosome 1q. The IDH1/2-mut subtype (1.9%) was defined by IDH1 R132C or IDH2 R140Q mutations with specific transcriptional and high-methylation profiles. Both subtypes showed poor prognosis and were considered inferior prognostic factors independent of clinical parameters. Comparison with a previously reported pediatric B-ALL cohort (n = 1003) showed that the frequencies of these subtypes were significantly higher in AYA/adults than in children. We delineated the genetic and transcriptomic landscape of adult B-ALL and identified 2 novel subtypes that predict poor disease outcomes. Our findings highlight the age-dependent distribution of subtypes, which partially accounts for the prognostic differences between adult and pediatric B-ALL.
Asunto(s)
Isocitrato Deshidrogenasa/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras , Enfermedad Aguda , Adolescente , Adulto , Factor de Transcripción CDX2/genética , Factor de Transcripción CDX2/metabolismo , Niño , Humanos , Isocitrato Deshidrogenasa/metabolismo , Persona de Mediana Edad , Mutación , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Pronóstico , Transcriptoma , Adulto JovenRESUMEN
Myxoid liposarcoma (MLPS) is a rare sarcoma, typically arising in deep soft tissues during the fourth to fifth decades of life. Histologically, MLPS is composed of uniform oval cells within a background of myxoid stroma and chicken-wire capillaries. Genetically, MLPS is characterized by the FUS/EWSR1::DDIT3 fusion gene, which generally results from balanced interchromosomal translocation and is detectable via DDIT3 break-apart fluorescence in situ hybridization (FISH). Here, we report an unusual intra-articular MLPS case, negative for DDIT3 break-apart FISH but positive for EWSR1::DDIT3. An 18-year-old female was referred to our hospital complaining of an intra-articular mass in the right knee joint. Histologically, the tumor was mainly composed of mature adipocytes, brown fat-like cells, and lipoblasts. Nanopore sequencing detected DNA rearrangements between EWSR1 and DDIT3 and clustered complex rearrangements involving multiple chromosomes, suggesting chromoplexy. Methylation classification using random forest, t-distributed stochastic neighbor embedding, and unsupervised hierarchical clustering correctly classified the tumor as MLPS. The copy number was almost flat. The TERT promoter C-124T was also detected. This report highlights, for the first time, the potential value of a fast and low-cost nanopore sequencer for diagnosing sarcomas.
RESUMEN
Hyalinizing clear cell carcinoma (HCCC) is a rare indolent malignant tumor of minor salivary gland origin with EWSR1::ATF1 rearrangement. Pathologically, the tumor cells possess a clear cytoplasm in a background of hyalinized stroma. Generally, the tumor cells are positive for p63 and p40 and negative for s100 and α-smooth muscle actin, suggesting that they differentiate into squamous epithelium and not into myoepithelium. In this study, we performed a detailed histopathological and genomic analysis of 6 cases of HCCC, including 2 atypical subtypes-a case of "high-grade transformation" and 1 "possessing a novel partner gene for EWSR1." We performed a sequential analysis of the primary and recurrent tumor by whole-exome sequencing, RNA sequencing, Sanger sequencing, and fluorescence in situ hybridization to investigate the effect of genomic changes on histopathology and clinical prognosis. A fusion gene involving the EWSR1 gene was detected in all cases. Five cases, including the "high-grade transformation," harbored a known EWSR1::ATF1 fusion gene; however, 1 case harbored a novel EWSR1::LARP4 fusion gene. This novel EWSR1::LARP4-fused HCCC has a SOX10-positive staining, which is different from the EWSR1::ATF1-fused HCCC. According to whole-exome sequencing and fluorescence in situ hybridization analysis, the "whole-genome doubling" and focal deletion involving CDKN2A, CDKN2B, and PTEN were detected in HCCC with "high-grade transformation." Conclusively, we identified a novel partner gene for EWSR1, LARP4, in indolent HCCC. Importantly, "high-grade transformation" and poor prognosis were caused by whole-genome doubling and subsequent genomic aberrations.
Asunto(s)
Adenocarcinoma de Células Claras , Carcinoma , Neoplasias de las Glándulas Salivales , Humanos , Hibridación Fluorescente in Situ , Proteína EWS de Unión a ARN/genética , Glándulas Salivales/patología , Secuencia de Bases , Genes cdc , Proteínas de Fusión Oncogénica/genética , Neoplasias de las Glándulas Salivales/genética , Neoplasias de las Glándulas Salivales/patología , Adenocarcinoma de Células Claras/genética , Adenocarcinoma de Células Claras/patología , Factores de Transcripción SOXE/genéticaRESUMEN
The cellular origins of cervical cancer and the histological differentiation of human papillomavirus (HPV)-infected cells remain unexplained. To gain new insights into the carcinogenesis and histological differentiation of HPV-associated cervical cancer, we focused on cervical cancer with mixed histological types. We conducted genomic and transcriptomic analyses of cervical cancers with mixed histological types. The commonality of the cellular origins of these cancers was inferred using phylogenetic analysis and by assessing the HPV integration sites. Carcinogenesis was estimated by analyzing human gene expression profiles in different histological types. Among 42 cervical cancers with known HPV types, mixed histological types were detected in four cases, and three of them were HPV18-positive. Phylogenetic analysis of these three cases revealed that the different histological types had a common cell of origin. Moreover, the HPV-derived transcriptome and HPV integration sites were common among different histological types, suggesting that HPV integration could occur before differentiation into each histological type. Human gene expression profiles indicated that HPV18-positive cancer retained immunologically cold components with stem cell properties. Mixed cervical cancer has a common cellular origin among different histological types, and progenitor cells with stem-like properties may be associated with the development of HPV18-positive cervical cancer.
Asunto(s)
Infecciones por Papillomavirus , Neoplasias del Cuello Uterino , Femenino , Humanos , Neoplasias del Cuello Uterino/patología , Papillomavirus Humano 18/genética , Filogenia , Papillomaviridae/genética , ADN Viral/genéticaRESUMEN
BACKGROUND: Identifying biomarkers to predict immune checkpoint inhibitor (ICI) efficacy is warranted. Considering that somatic mutation-derived neoantigens induce strong immune responses, patients with a high tumour mutational burden reportedly tend to respond to ICIs. However, there are several conflicting data. Therefore, we focused on the original function of neoantigenic mutations and their impact on the tumour microenvironment (TME). METHODS: We evaluated 88 high-frequency microsatellite instability (MSI-H) colorectal cancers and analysed the function of the identified neoantigenic mutations and their influence on programmed cell death 1 (PD-1) blockade efficacy. The results were validated using The Cancer Genome Atlas (TCGA) datasets. RESULTS: We identified frameshift mutations in RNF43 as a common neoantigenic gene mutation in MSI-H tumours. However, loss-of-function RNF43 mutations induced noninflamed TME by activating the WNT/ß-catenin signalling pathway. In addition, loss of RNF43 function induced resistance to PD-1 blockade even in neoantigen-rich tumours. TCGA dataset analyses demonstrated that passenger rather than driver gene mutations were related to the inflamed TME in diverse cancer types. CONCLUSIONS: We propose a novel concept of "paradoxical neoantigenic mutations" that can induce noninflamed TME through their original gene functions, despite deriving neoantigens, suggesting the significance of qualities as well as quantities in neoantigenic mutations.
Asunto(s)
Neoplasias Colorrectales , Neoplasias , Humanos , Receptor de Muerte Celular Programada 1 , Microambiente Tumoral , Neoplasias/genética , Mutación , Inestabilidad de Microsatélites , Neoplasias Colorrectales/patologíaRESUMEN
BACKGROUND: Intratumor heterogeneity (ITH) in microsatellite instability-high (MSI-H) colorectal cancer (CRC) has been poorly studied. We aimed to clarify how the ITH of MSI-H CRCs is generated in cancer evolution and how immune selective pressure affects ITH. METHODS: We reanalyzed public whole-exome sequencing data on 246 MSI-H CRCs. In addition, we performed a multi-region analysis from 6 MSI-H CRCs. To verify the process of subclonal immune escape accumulation, a novel computational model of cancer evolution under immune pressure was developed. RESULTS: Our analysis presented the enrichment of functional genomic alterations in antigen-presentation machinery (APM). Associative analysis of neoantigens indicated the generation of immune escape mechanisms via HLA alterations. Multiregion analysis revealed the clonal acquisition of driver mutations and subclonal accumulation of APM defects in MSI-H CRCs. Examination of variant allele frequencies demonstrated that subclonal mutations tend to be subjected to selective sweep. Computational simulations of tumour progression with the interaction of immune cells successfully verified the subclonal accumulation of immune escape mutations and suggested the efficacy of early initiation of an immune checkpoint inhibitor (ICI) -based treatment. CONCLUSIONS: Our results demonstrate the heterogeneous acquisition of immune escape mechanisms in MSI-H CRCs by Darwinian selection, providing novel insights into ICI-based treatment strategies.
Asunto(s)
Neoplasias del Colon , Neoplasias Colorrectales , Humanos , Inestabilidad de Microsatélites , Neoplasias Colorrectales/patología , Neoplasias del Colon/genética , Mutación , Presentación de Antígeno , Repeticiones de Microsatélite/genéticaRESUMEN
BACKGROUND & AIMS: A detailed understanding of antitumor immunity is essential for optimal cancer immune therapy. Although defective mutations in the B2M and HLA-ABC genes, which encode molecules essential for antigen presentation, have been reported in several studies, the effects of these defects on tumor immunity have not been quantitatively evaluated. METHODS: Mutations in HLA-ABC genes were analyzed in 114 microsatellite instability-high colorectal cancers using a long-read sequencer. The data were further analyzed in combination with whole-exome sequencing, transcriptome sequencing, DNA methylation array, and immunohistochemistry data. RESULTS: We detected 101 truncating mutations in 57 tumors (50%) and loss of 61 alleles in 21 tumors (18%). Based on the integrated analysis that enabled the immunologic subclassification of microsatellite instability-high colorectal cancers, we identified a subtype of tumors in which lymphocyte infiltration was reduced, partly due to reduced expression of HLA-ABC genes in the absence of apparent genetic alterations. Survival time of patients with such tumors was shorter than in patients with other tumor types. Paradoxically, tumor mutation burden was highest in the subtype, suggesting that the immunogenic effect of accumulating mutations was counterbalanced by mutations that weakened immunoreactivity. Various genetic and epigenetic alterations, including frameshift mutations in RFX5 and promoter methylation of PSMB8 and HLA-A, converged on reduced expression of HLA-ABC genes. CONCLUSIONS: Our detailed immunogenomic analysis provides information that will facilitate the improvement and development of cancer immunotherapy.
Asunto(s)
Neoplasias Colorrectales/genética , Neoplasias Colorrectales/inmunología , Genes MHC Clase I/genética , Escape del Tumor/genética , Escape del Tumor/inmunología , Microglobulina beta-2/genética , Alelos , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Metilación de ADN , Epigénesis Genética , Expresión Génica , Antígenos HLA-A/genética , Antígenos HLA-A/metabolismo , Humanos , Inmunogenética , Linfocitos Infiltrantes de Tumor , Inestabilidad de Microsatélites , Complejo de la Endopetidasa Proteasomal/genética , Factores de Transcripción del Factor Regulador X/genética , Tasa de Supervivencia , Microglobulina beta-2/metabolismoRESUMEN
Macroautophagy (hereafter, autophagy) is a process that directs the degradation of cytoplasmic material in lysosomes. In addition to its homeostatic roles, autophagy undergoes dynamic positive and negative regulation in response to multiple forms of cellular stress, thus enabling the survival of cells. However, the precise mechanisms of autophagy regulation are not fully understood. To identify potential negative regulators of autophagy, we performed a genome-wide CRISPR screen using the quantitative autophagic flux reporter GFP-LC3-RFP. We identified phosphoribosylformylglycinamidine synthase, a component of the de novo purine synthesis pathway, as one such negative regulator of autophagy. Autophagy was activated in cells lacking phosphoribosylformylglycinamidine synthase or phosphoribosyl pyrophosphate amidotransferase, another de novo purine synthesis enzyme, or treated with methotrexate when exogenous levels of purines were insufficient. Purine starvation-induced autophagy activation was concomitant with mammalian target of rapamycin complex 1 (mTORC1) suppression and was profoundly suppressed in cells deficient for tuberous sclerosis complex 2, which negatively regulates mTORC1 through inhibition of Ras homolog enriched in brain, suggesting that purines regulate autophagy through the tuberous sclerosis complex-Ras homolog enriched in brain-mTORC1 signaling axis. Moreover, depletion of the pyrimidine synthesis enzymes carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase and dihydroorotate dehydrogenase activated autophagy as well, although mTORC1 activity was not altered by pyrimidine shortage. These results suggest a different mechanism of autophagy induction between purine and pyrimidine starvation. These findings provide novel insights into the regulation of autophagy by nucleotides and possibly the role of autophagy in nucleotide metabolism, leading to further developing anticancer strategies involving nucleotide synthesis and autophagy.
Asunto(s)
Autofagia , Sistemas CRISPR-Cas , Amidofosforribosiltransferasa/genética , Ligasas de Carbono-Nitrógeno con Glutamina como Donante de Amida-N/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica , Células HEK293 , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/genéticaRESUMEN
Liquid biopsy analyzes the current status of primary tumors and their metastatic regions. We aimed to develop an optimized protocol for single-cell sequencing of floating tumor cells (FTCs) in pleural effusion as a laboratory test. FTCs were enriched using a negative selection of white blood cells by a magnetic-activated cell sorting system, and CD45-negative and cytokeratin-positive selection using a microfluidic cell separation system with a dielectrophoretic array. The enriched tumor cells were subjected to whole-genome amplification (WGA) followed by genome sequencing. The FTC analysis detected an EGFR exon 19 deletion in Case 1 (12/19 cells, 63.2%), and EML4-ALK fusion (17/20 cells, 85%) with an alectinib-resistant mutation of ALK (p.G1202R) in Case 2. To eliminate WGA-associated errors and increase the uniformity of the WGA product, the protocol was revised to sequence multiple single FTCs individually. An analytical pipeline, accurate single-cell mutation detector (ASMD), was developed to identify somatic mutations of FTCs. The large numbers of WGA-associated errors were cleaned up, and the somatic mutations detected in FTCs by ASMD were concordant with those found in tissue specimens. This protocol is applicable to circulating tumor cells analysis of peripheral blood and expands the possibility of utilizing molecular profiling of cancers.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Biopsia Líquida/métodos , Derrame Pleural Maligno/genética , Derrame Pleural Maligno/patología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/patología , Adulto , Anciano , Anciano de 80 o más Años , Quinasa de Linfoma Anaplásico/genética , Antineoplásicos/uso terapéutico , Carbazoles/farmacología , Separación Celular/métodos , Crizotinib/uso terapéutico , ADN/aislamiento & purificación , Resistencia a Antineoplásicos/genética , Exones/genética , Femenino , Amplificación de Genes , Eliminación de Gen , Genes erbB-1 , Humanos , Separación Inmunomagnética/métodos , Queratinas , Antígenos Comunes de Leucocito , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Mutación , Células Neoplásicas Circulantes , Proteínas de Fusión Oncogénica/genética , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
ALK, ROS1, and RET kinase fusions are important predictive biomarkers of tyrosine kinase inhibitors (TKIs) in non-small-cell lung cancer (NSCLC). Analysis of cell-free DNA (cfDNA) provides a noninvasive method to identify gene changes in tumor cells. The present study sought to use cfRNA and cfDNA for identifying fusion genes. A reliable protocol was established to detect fusion genes using cfRNA and assessed the analytical validity and clinical usefulness in 30 samples from 20 cases of fusion-positive NSCLC. The results of cfRNA-based assays were compared with tissue biopsy and cfDNA-based liquid biopsy (Guardant360 plasma next-generation sequencing [NGS] assay). The overall sensitivity of the cfRNA-based assay was 26.7% (8/30) and that of cfDNA-based assay was 16.7% (3/18). When analysis was limited to the samples collected at chemo-naïve or progressive disease status and available for both assays, the sensitivity of the cfRNA-based assay was 77.8% (7/9) and that of cfDNA-based assay was 33.3% (3/9). Fusion gene identification in cfRNA was correlated with treatment response. These results suggest that the proposed cfRNA assay is a useful diagnostic test for patients with insufficient tissues to facilitate effective administration of first-line treatment and is a useful tool to monitor the progression of NSCLC for consideration of second-line treatments.
Asunto(s)
Quinasa de Linfoma Anaplásico/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Ácidos Nucleicos Libres de Células , Fusión Génica , Neoplasias Pulmonares/genética , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas c-ret/genética , Proteínas Proto-Oncogénicas/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Biopsia , Carbazoles/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Ácidos Nucleicos Libres de Células/aislamiento & purificación , Crizotinib/uso terapéutico , Proteínas del Citoesqueleto/genética , Progresión de la Enfermedad , Resistencia a Antineoplásicos/genética , Femenino , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Biopsia Líquida/métodos , Pulmón/patología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/aislamiento & purificación , Piperidinas/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , ARN Mensajero/aislamiento & purificación , Sensibilidad y EspecificidadRESUMEN
Triple-negative breast cancer (TNBC) cells do not express estrogen receptors, progesterone receptors, or human epidermal growth factor receptor 2. Currently, apart from poly ADP-ribose polymerase inhibitors, there are few effective therapeutic options for this type of cancer. Here, we present comprehensive characterization of the genetic alterations in TNBC performed by high coverage whole genome sequencing together with transcriptome and whole exome sequencing. Silencing of the BRCA1 gene impaired the homologous recombination pathway in a subset of TNBCs, which exhibited similar phenotypes to tumors with BRCA1 mutations; they harbored many structural variations (SVs) with relative enrichment for tandem duplication. Clonal analysis suggested that TP53 mutations and methylation of CpG dinucleotides in the BRCA1 promoter were early events of carcinogenesis. SVs were associated with driver oncogenic events such as amplification of MYC, NOTCH2, or NOTCH3 and affected tumor suppressor genes including RB1, PTEN, and KMT2C. Furthermore, we identified putative TGFA enhancer regions. Recurrent SVs that affected the TGFA enhancer region led to enhanced expression of the TGFA oncogene that encodes one of the high affinity ligands for epidermal growth factor receptor. We also identified a variety of oncogenes that could transform 3T3 mouse fibroblasts, suggesting that individual TNBC tumors may undergo a unique driver event that can be targetable. Thus, we revealed several features of TNBC with clinically important implications.
Asunto(s)
Proteína BRCA1/genética , Transcriptoma/genética , Neoplasias de la Mama Triple Negativas/genética , Proteína p53 Supresora de Tumor/genética , Células 3T3 , Animales , Metilación de ADN/genética , Exoma/genética , Femenino , Amplificación de Genes , Genoma Humano , Secuenciación de Nucleótidos de Alto Rendimiento , Recombinación Homóloga/genética , Humanos , Ratones , Mutación , Proteínas de Neoplasias/genética , Regiones Promotoras Genéticas , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
The silencing of tumor suppressor genes by promoter CpG island (CGI) methylation is an important cause of oncogenesis. Silencing of MLH1 and BRCA1, two examples of oncogenic events, results from promoter CGI methylation. Interestingly, both MLH1 and BRCA1 have a divergent promoter, from which another gene on the opposite strand is also transcribed. Although studies have shown that divergent transcription is an important factor in transcriptional regulation, little is known about its implication in aberrant promoter methylation in cancer. In this study, we analyzed the methylation status of CGI in divergent promoters using a recently enriched transcriptome database. We measured the extent of CGI methylation in 119 colorectal cancer (CRC) clinical samples (65 microsatellite instability high [MSI-H] CRC with CGI methylator phenotype, 28 MSI-H CRC without CGI methylator phenotype and 26 microsatellite stable CRC) and 21 normal colorectal tissues using Infinium MethylationEPIC BeadChip. We found that CGI within divergent promoters are less frequently methylated than CGI within unidirectional promoters in normal cells. In the genome of CRC cells, CGI within unidirectional promoters are more vulnerable to aberrant methylation than CGI within divergent promoters. In addition, we identified three DNA sequence motifs that correlate with methylated CGI. We also showed that methylated CGI are associated with genes whose expression is low in normal cells. Thus, we here provide fundamental observations regarding the methylation of divergent promoters that are essential for the understanding of carcinogenesis and development of cancer prevention strategies.
Asunto(s)
Neoplasias Colorrectales/genética , Islas de CpG/genética , Metilación de ADN/genética , Regiones Promotoras Genéticas/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Inestabilidad de Microsatélites , Fenotipo , Transcriptoma/genéticaRESUMEN
Every year, approximately 1.2 million cases of colorectal carcinoma (CRC) are newly diagnosed worldwide. Although metastases to distant organs are often fatal complications of CRC, little information is known as to how such metastatic lesions are formed. To reveal the genetic profiles for CRC metastasis, we conducted whole-exome RNA sequencing on CRC tumors with liver metastasis (LM) (group A, n = 12) and clinical stage-matched larger tumors without LM (group B, n = 16). While the somatic mutation profiles were similar among the primary tumors and LM lesions in group A and the tumors in group B, the A-to-C nucleotide change in the context of "AAG" was only enriched in the LM regions in group A, suggesting the presence of a DNA damage process specific to metastasis. Genes already known to be associated with CRC were mutated in all groups at a similar frequency, but we detected somatic nonsynonymous mutations in a total of 707 genes in the LM regions, but not in the tumors without LM. Signaling pathways linked to such "LM-associated" genes were overrepresented for extracellular matrix-receptor interaction or focal adhesion. Further, fusions of the ADAP1 (ArfGAP with dual PH domain 1) were newly identified in our cohort (3 out of 28 patients), which activated ARF6, an ADAP1-substrate. Infrequently, mutated genes may play an important role in metastasis formation of CRC. Additionally, recurrent ADAP1 fusion genes were unexpectedly discovered. As these fusions activate small GTPase, further experiments are warranted to examine their contribution to CRC carcinogenesis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Neoplasias Colorrectales/genética , Fusión Génica , Neoplasias Hepáticas/genética , Proteínas del Tejido Nervioso/genética , Adulto , Anciano , Anciano de 80 o más Años , Carcinogénesis/genética , Neoplasias Colorrectales/patología , Análisis Mutacional de ADN , Femenino , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Neoplasias Hepáticas/secundario , Masculino , Persona de Mediana Edad , Mutación Puntual , Secuenciación del ExomaRESUMEN
Obesity is a serious global health issue; however, the roles of genetics and epigenetics in the onset and progression of obesity are still not completely understood. The aim of this study was to determine the role of Kdm4b, which belongs to a subfamily of histone demethylases, in adipogenesis and fat metabolism in vivo. We established conditional Kdm4b knockout mice. Inactivation of Kdm4b in adipocytes (K4bKO) induced profound obesity in mice on a high fat diet (HFD). The HFD-fed K4bKO mice exhibited an increased volume of fat mass and higher expression levels of adipogenesis-related genes. In contrast, the genes involved in energy expenditure and mitochondrial functions were down-regulated. Supporting these findings, the energy expenditure of Kdm4b-deficient cells was markedly decreased. In addition, progression of glucose intolerance and hepatic steatosis with hepatocellular damages was observed. These data indicate that Kdm4b is a critical regulator of systemic metabolism via enhancing energy expenditure in adipocytes.
Asunto(s)
Tejido Adiposo/patología , Dieta Alta en Grasa/efectos adversos , Metabolismo Energético , Histona Demetilasas con Dominio de Jumonji/fisiología , Enfermedades Metabólicas/patología , Obesidad/patología , Adipogénesis , Tejido Adiposo/metabolismo , Animales , Células Cultivadas , Femenino , Metabolismo de los Lípidos , Masculino , Enfermedades Metabólicas/etiología , Enfermedades Metabólicas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/etiología , Obesidad/metabolismoRESUMEN
The major driver mutations of lung cancer, EGFR mutations and EML4-ALK fusion, are mainly detected in terminal respiratory unit (TRU)-type lung adenocarcinomas, which typically show lepidic and/or papillary patterns, but are rarely associated with a solid or invasive mucinous morphology. In order to elucidate the key genetic events in non-TRU-type lung cancer, we carried out whole-exome sequencing on 43 non-TRU-type lung adenocarcinomas based on morphology (17 acinar, nine solid, and two enteric adenocarcinomas, and 15 adenocarcinomas with a mucinous morphology). Our analysis identified mutations in TP53 (16/43, 37.2%), KRAS (13/43, 30.2%), and NKX2-1/TTF-1 (7/43; 16.3%) as the top three significantly mutated genes, while the EGFR mutation was rare (1/43, 2.3%) in this cohort. Eight NKX2-1/TTF-1 mutations (five frameshift, two nonsense, and one missense) were identified, with one case harboring two distinct NKX2-1/TTF-1 mutations (one missense and one frameshift). Functional assays with the NK2 homeobox 1 (NKX2-1)/thyroid transcription factor 1 (TTF-1) mutants revealed that none of them retain the activity as a transcriptional factor. Histologically, invasive mucinous adenocarcinomas accounted for most of the NKX2-1/TTF-1 mutations (five cases), as well as one enteric and one acinar adenocarcinoma. Immunohistochemistry showed that the cohort was largely divided into TTF-1-postive/hepatocyte nuclear factor 4-α (HNF4-α)-negative and TTF-1-negative/HNF4-α-positive groups. NKX2-1/TTF-1 mutations were exclusively found in the latter, in which the gastrointestinal markers, mucin 5AC and cytokeratin 20, were frequently expressed. Bisulfite sequencing revealed that the NKX2-1/TTF-1 gene body was highly methylated in NKX2-1/TTF-1-negative cases, including those without the NKX2-1/TTF-1 mutations. The genetic or epigenetic inactivation of NKX2-1/TTF-1 may play an essential role in the development and aberrant differentiation of non-TRU-type lung adenocarcinomas.