RESUMEN
Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder due to pathogenic variants in Fibrillin-1 (FBN1) affecting nearly one in every 10,000 individuals. We report a 16-month-old female with early-onset MFS heterozygous for an 11.2 kb de novo duplication within the FBN1 gene. Tandem location of the duplication was further confirmed by optical genome mapping in addition to genetic sequencing and chromosomal microarray. This is the third reported case of a large multi-exon duplication in FBN1, and the only one confirmed to be in tandem. As the vast majority of pathogenic variants associated with MFS are point mutations, this expands the landscape of known FBN1 pathogenic variants and supports consistent use of genetic testing strategies that can detect large, indel-type variants.
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Síndrome de Marfan , Humanos , Femenino , Lactante , Fibrilina-1/genética , Mutación , Síndrome de Marfan/diagnóstico , Síndrome de Marfan/genética , Síndrome de Marfan/patología , Pruebas Genéticas , Mutación Puntual , Fibrilinas/genética , Adipoquinas/genéticaRESUMEN
Recurrent GLI1 gene fusions have been recently described in a subset of soft tissue tumors showing a distinct monotonous epithelioid morphology with a rich capillary network and frequent S100 protein expression. Three different fusion partners-ACTB, MALAT1, and PTCH1-have been reported with the PTCH1-GLI1 fusion from 2 patients only, both with head and neck tumors. Herein, we report for the first time a PTCH1-GLI1 fusion in a primary ovarian tumor from a female patient aged 54 years who presented with a 21-cm right ovarian mass and mesenteric metastasis. The tumor was diagnosed as "favor malignant melanoma" based on histologic examination and extensive immunohistochemistry studies. The patient received 4 cycles of pembrolizumab and 2 cycles of trabectedin but developed multiple metastases. A next-generation sequencing-based assay detected a PTCH1-GLI1 fusion, which led to a revised pathologic diagnosis and a change of the patient's management. The patient was switched to the tyrosine kinase inhibitor (TKI) pazopanib to target the sonic hedgehog pathway. Her disease was stable 49 months post TKI therapy. Our case report is the first to show that a tumor with GLI1 oncogenic activation was sensitive to a TKI. The morphologic and immunohistochemistry similarities of our patient's tumor to other recently described tumors harboring GLI1 fusions suggest that these tumors may all belong to the same entity of GLI1 fusion-positive neoplasms and may be treated similarly.
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Proteínas Hedgehog , Neoplasias Ováricas , Femenino , Fusión Génica , Humanos , Indazoles , Persona de Mediana Edad , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Inhibidores de Proteínas Quinasas , Pirimidinas , Sulfonamidas , Proteína con Dedos de Zinc GLI1/genética , Proteína con Dedos de Zinc GLI1/metabolismoRESUMEN
Primary aneurysmal bone cyst (ABC) is a neoplastic process due to recurrent translocations involving the USP6 gene. By fluorescence in situ hybridization, up to 69% of primary ABCs harbored USP6 translocations; no USP6 translocation was found in secondary ABC or giant cell tumor of bone (GCT). GCT can recur locally, metastasize to the lungs in some cases, and rarely undergo malignant transformation. Differentiating primary ABC from its mimics is important for treatment and prognosis. We evaluated USP6 fusion and expression in 13 cases of primary and 1 case of secondary ABC, and 9 cases of GCT using nucleic acid extracted from formalin-fixed, paraffin-embedded tissue and a next generation sequencing (NGS)-based assay. USP6 fusions including 7 novel fusions and USP6 transcripts were identified in all 13 primary ABCs. Nine cases with strong evidence of fusions showed high levels of USP6 transcripts by reverse transcription-PCR (RT-PCR). The remaining four had no detectable USP6 expression by a first-round of RT-PCR but the presence of USP6 transcripts was identified by a second-round, nested PCR. The major fusions were confirmed by RT-PCR followed by Sanger sequencing. No USP6 fusion or transcript was detected in any of the GCTs or the case of secondary ABC by NGS or by two rounds of PCR. All USP6 translocations resulted in fusion of the entire USP6 coding sequence with promoters of the fusion gene leading to upregulation of USP6 transcription, which is likely the underlying mechanism for ABC oncogenesis. © 2016 Wiley Periodicals, Inc.
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Biomarcadores de Tumor/genética , Quistes Óseos Aneurismáticos/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Proteínas de Fusión Oncogénica/genética , Proteínas Proto-Oncogénicas/genética , Ubiquitina Tiolesterasa/genética , Adolescente , Adulto , Anciano , Quistes Óseos Aneurismáticos/patología , Niño , Femenino , Estudios de Seguimiento , Humanos , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Regiones Promotoras Genéticas , Estudios Retrospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adulto JovenRESUMEN
Struma ovarii accounts for 5% of ovarian teratomas. Malignant transformation occurs in <0.3%, however, the underlying molecular mechanism is unknown. We report a patient with follicular variant and tall cell variant of papillary thyroid carcinoma (PTC) arising from struma ovarii and coexisting incidental PTC in the thyroid. Mutation analysis by next-generation sequencing identified a novel germline mutation, KIT p.V530I mutation in the tumors and normal ovarian and thyroid tissue. Immunohistochemical staining showed loss of KIT expression in the PTCs. Activating mutations in KIT play an important role in diagnosis and prognosis of multiple malignancies including mastocytosis, gastrointestinal stromal tumors, and a subset of melanoma and acute myeloid leukemia. The p.V530I mutation has only been reported in 3 previous cases: acute myeloid leukemia, aggressive fibromatosis, and adenocarcinoma of the colon. In the case of aggressive fibromatosis, the patient responded well to imatinib treatment. KIT mutations have never been reported in thyroid carcinomas. This is the first case of PTC-harboring KIT mutation. Although more work needs to be done to elucidate the significance of this germline mutation, the response of the fibromatosis patient to imatinib may shed light on targeted therapy in PTC harboring this mutation.
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Antineoplásicos/uso terapéutico , Carcinoma/genética , Mesilato de Imatinib/uso terapéutico , Neoplasias Ováricas/genética , Proteínas Proto-Oncogénicas c-kit/genética , Teratoma/genética , Neoplasias de la Tiroides/genética , Carcinoma/diagnóstico , Carcinoma/tratamiento farmacológico , Carcinoma/patología , Carcinoma Papilar , Análisis Mutacional de ADN , Femenino , Mutación de Línea Germinal , Humanos , Persona de Mediana Edad , Neoplasias Ováricas/diagnóstico , Neoplasias Ováricas/patología , Pronóstico , Estruma Ovárico/patología , Teratoma/diagnóstico , Teratoma/patología , Cáncer Papilar Tiroideo , Neoplasias de la Tiroides/diagnóstico , Neoplasias de la Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/patologíaRESUMEN
Background: Exon 20 (ex20) in-frame insertions or duplications (ins/dup) in epidermal growth factor receptor (EGFR) and its analog erb-b2 receptor tyrosine kinase 2 (ERBB2) are each detected in 1.5% of non-small cell lung cancer (NSCLC). Unlike EGFR p.L858R or ex19 deletions, ex20 ins/dup is associated with de novo resistance to classic EGFR inhibitors, lack of response to immune checkpoint inhibitors, and poor prognosis. US Food and Drug Administration has approved mobocertinib and amivantamab for targeting tumors with this aberration, but the number of comprehensive studies on ex20 ins/dup NSCLC is limited. We identified 18 cases of NSCLCs with EGFR/ERBB2 ex20 ins/dup and correlated the findings with clinical and morphologic information including programed death-ligand 1 (PD-L1) expression. Methods: A total of 536 NSCLC cases tested at our institution between 2014 and 2023 were reviewed. A custom-designed 214-gene next-generation sequencing panel was used for detecting DNA variants, and the FusionPlex CTL panel (ArcherDx) was used for the detection of fusion transcripts from formalin-fixed, paraffin-embedded tissue. Immunohistochemistry (IHC)for PD-L1 was performed using 22C3 or E1L3N clones. Results: Nine EGFR and nine ERBB2 ex20 ins/dup variants were identified from an equal number of men and women, 14 were non- or light smokers, and 15 had stage IV disease. All 18 cases were adenocarcinomas. Seven of the 11 cases with available primary tumors had acinar predominant pattern, two had lepidic predominant pattern, and the remainder had papillary (one case) and mucinous (one case) patterns. Ex20 ins/dup variants were heterogenous in-frame one to four amino acids spanning A767-V774 in EGFR and Y772-P780 in ERBB2 and were clustered in the loop following the C-helix and α C-helix. Twelve cases (67%) had co-existing TP53 variants. Copy number variation in CDK4 amplification was identified in one case. No fusion or microsatellite instability was identified in any case. PD-L1 was positive in two cases, low positive in four cases, and negative in 11 cases. Conclusions: NSCLCs harboring EGFR/ERBB2 ex20 ins/dup are rare and tend to be acinar predominant, negative for PD-L1, more frequent in non- or light smokers, and mutually exclusive with other driver mutations in NSCLC. The correlation of different EGFR/ERBB2 ex20 ins/dup variants and co-existing mutations with response to targeted therapy and the possibility of developing resistant mutations after mobocertinib treatment warrants further investigation.
RESUMEN
While molecular testing of hematologic malignancies is now standard of care, there is variability in practice and testing capabilities between different academic laboratories, with common questions arising on how to best meet clinical expectations. A survey was sent to hematopathology subgroup members of the Genomics Organization for Academic Laboratories consortium to assess current and future practice and potentially establish a reference for peer institutions. Responses were received from 18 academic tertiary-care laboratories regarding next-generation sequencing (NGS) panel design, sequencing protocols and metrics, assay characteristics, laboratory operations, case reimbursement, and development plans. Differences in NGS panel size, use, and gene content were reported. Gene content for myeloid processes was reported to be generally excellent, while genes for lymphoid processes were less well covered. The turnaround time (TAT) for acute cases, including acute myeloid leukemia, was reported to range from 2 to 7 calendar days to 15 to 21 calendar days, with different approaches to achieving rapid TAT described. To help guide NGS panel design and standardize gene content, consensus gene lists based on current and future NGS panels in development were generated. Most survey respondents expected molecular testing at academic laboratories to continue to be viable in the future, with rapid TAT for acute cases likely to remain an important factor. Molecular testing reimbursement was reported to be a major concern. The results of this survey and subsequent discussions improve the shared understanding of differences in testing practices for hematologic malignancies between institutions and will help provide a more consistent level of patient care.
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Objetivos , Neoplasias Hematológicas , Humanos , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/genética , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodosRESUMEN
OBJECTIVES: To identify therapeutic targets and correlate with clinical outcomes from mutation profiling of metastatic uveal melanoma (UM) using next-generation sequencing (NGS). METHODS: Melanoma cases that were tested using DNA-based NGS panels of 25 and/or 214 genes were evaluated retrospectively (263 cases) and identified 27 UM cases. BAP1 expression was examined by immunohistochemistry. RESULTS: Mutations in GNA11 (14) and GNAQ (12) were found in 96% (nâ =â 27) of cases of UM, and most had coexisting BAP1 (17) or SF3B1 (4) mutations. Coexisting GNAQ/11-SF3B1 mutations correlated with a longer average time to first metastasis compared with GNAQ/11-BAP1 mutations (99.7 vs 38.5 months, Pâ =â .047). Three patients with BAP1 mutations received trametinib; two are still alive (15 months; 23 months), and one died (32 months). In non-UMs, only 4.2% (nâ =â 236) had BAP1 and 3.8% had SF3B1 mutations; none had coexisting GNAQ/11 mutations. CONCLUSIONS: Coexisting BAP1/SF3B1 and GNAQ/11 mutations were unique to UM. SF3B1 mutations were reported to be UM-specific in melanoma and associated with rare/no metastasis. The finding of mutated SF3B1 in 14.8% (nâ =â 27) of UMs suggests its role should be further evaluated. The correlation of BAP1/SF3B1 mutation with survival also warrants investigation.
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Melanoma , Neoplasias Primarias Secundarias , Neoplasias de la Úvea , Análisis Mutacional de ADN , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Genómica , Humanos , Melanoma/patología , Mutación , Fosfoproteínas/genética , Pronóstico , Factores de Empalme de ARN/genética , Estudios Retrospectivos , Proteínas Supresoras de Tumor/genética , Ubiquitina Tiolesterasa/genética , Neoplasias de la Úvea/genéticaRESUMEN
Trillions of microbes such as bacteria, fungi, and viruses exist in the healthy human gut microbiome. Although gut bacterial dysbiosis has been extensively studied in multiple sclerosis (MS), the significance of the fungal microbiome (mycobiome) is an understudied and neglected part of the intestinal microbiome in MS. The aim of this study was to characterize the gut mycobiome of patients with relapsing-remitting multiple sclerosis (RRMS), compare it to healthy controls, and examine its association with changes in the bacterial microbiome. We characterized and compared the mycobiome of 20 RRMS patients and 33 healthy controls (HC) using Internal Transcribed Spacer 2 (ITS2) and compared mycobiome interactions with the bacterial microbiome using 16S rRNA sequencing. Our results demonstrate an altered mycobiome in RRMS patients compared with HC. RRMS patients showed an increased abundance of Basidiomycota and decreased Ascomycota at the phylum level with an increased abundance of Candida and Epicoccum genera along with a decreased abundance of Saccharomyces compared to HC. We also observed an increased ITS2/16S ratio, altered fungal and bacterial associations, and altered fungal functional profiles in MS patients compared to HC. This study demonstrates that RRMS patients had a distinct mycobiome with associated changes in the bacterial microbiome compared to HC. There is an increased fungal to bacterial ratio as well as more diverse fungal-bacterial interactions in RRMS patients compared to HC. Our study is the first step towards future studies in delineating the mechanisms through which the fungal microbiome can influence MS disease.
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Ascomicetos , Esclerosis Múltiple , Micobioma , Ascomicetos/genética , Bacterias/genética , Disbiosis/microbiología , Hongos/genética , Humanos , Micobioma/genética , ARN Ribosómico 16S/genéticaRESUMEN
NK cell-mediated resistance to murine cytomegalovirus (MCMV) is controlled by allelic Ly49 receptors, including activating Ly49H (C57BL/6 strain) and inhibitory Ly49I (129 strain), which specifically recognize MCMV m157, a glycosylphosphatidylinositol-linked protein with homology to MHC class I. Although the Ly49 receptors retain significant homology to classic carbohydrate-binding lectins, the role of glycosylation in ligand binding is unclear. Herein, we show that m157 is expressed in multiple, differentially N-glycosylated isoforms in m157-transduced or MCMV-infected cells. We used site-directed mutagenesis to express single and combinatorial asparagine (N)-to-glutamine (Q) mutations at N178, N187, N213, and N267 in myeloid and fibroblast cell lines. Progressive loss of N-linked glycans led to a significant reduction of total cellular m157 abundance, although all variably glycosylated m157 isoforms were expressed at the cell surface and retained the capacity to activate Ly49H(B6) and Ly49I(129) reporter cells and Ly49H(+) NK cells. However, the complete lack of N-linked glycans on m157 destabilized the m157-Ly49H interaction and prevented physical transfer of m157 to Ly49H-expressing cells. Thus, glycosylation on m157 enhances expression and binding to Ly49H, factors that may impact the interaction between NK cells and MCMV in vivo where receptor-ligand interactions are more limiting.
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Fibroblastos/metabolismo , Infecciones por Herpesviridae/metabolismo , Muromegalovirus/inmunología , Células Mieloides/metabolismo , Proteínas Virales/metabolismo , Animales , Línea Celular , Fibroblastos/inmunología , Fibroblastos/patología , Glicosilación , Infecciones por Herpesviridae/genética , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/virología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/patología , Activación de Linfocitos/genética , Ratones , Muromegalovirus/patogenicidad , Mutagénesis Sitio-Dirigida , Mutación/genética , Células Mieloides/inmunología , Células Mieloides/patología , Subfamilia A de Receptores Similares a Lectina de Células NK/inmunología , Subfamilia A de Receptores Similares a Lectina de Células NK/metabolismo , Unión Proteica/genética , Isoformas de Proteínas/genética , Transgenes/genética , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
Multiple sclerosis (MS) is an autoimmune disease of the CNS in which the interaction between genetic and environmental factors plays an important role in disease pathogenesis. Although environmental factors account for 70% of disease risk, the exact environmental factors associated with MS are unknown. Recently, gut microbiota has emerged as a potential missing environmental factor linked with the pathobiology of MS. Yet, how genetic factors, such as HLA class II gene(s), interact with gut microbiota and influence MS is unclear. In the current study, we investigated whether HLA class II genes that regulate experimental autoimmune encephalomyelitis (EAE) and MS susceptibility also influence gut microbiota. Previously, we have shown that HLA-DR3 transgenic mice lacking endogenous mouse class II genes (AE-KO) were susceptible to myelin proteolipid protein (91-110)-induced EAE, an animal model of MS, whereas AE-KO.HLA-DQ8 transgenic mice were resistant. Surprisingly, HLA-DR3.DQ8 double transgenic mice showed higher disease prevalence and severity compared with HLA-DR3 mice. Gut microbiota analysis showed that HLA-DR3, HLA-DQ8, and HLA-DR3.DQ8 double transgenic mice microbiota are compositionally different from AE-KO mice. Within HLA class II transgenic mice, the microbiota of HLA-DQ8 mice were more similar to HLA-DR3.DQ8 than HLA-DR3. As the presence of DQ8 on an HLA-DR3 background increases disease severity, our data suggests that HLA-DQ8-specific microbiota may contribute to disease severity in HLA-DR3.DQ8 mice. Altogether, our study provides evidence that the HLA-DR and -DQ genes linked to specific gut microbiota contribute to EAE susceptibility or resistance in a transgenic animal model of MS.
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Encefalomielitis Autoinmune Experimental/genética , Microbioma Gastrointestinal/genética , Antígenos HLA-D/genética , Antígenos de Histocompatibilidad Clase II/genética , Esclerosis Múltiple/genética , Polimorfismo Genético , Animales , Bacterias/clasificación , Bacterias/genética , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/metabolismo , Predisposición Genética a la Enfermedad/genética , Antígenos HLA-D/metabolismo , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/metabolismo , Antígeno HLA-DR3/genética , Antígeno HLA-DR3/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Esclerosis Múltiple/metabolismo , FenotipoRESUMEN
The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome's role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.
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Microbioma Gastrointestinal/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Reacción en Cadena de la Polimerasa/métodos , ARN Ribosómico 16S/genética , Análisis de Secuencia de ARN/métodos , Animales , Heces/química , Heces/microbiología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microbiota/genética , ARN Ribosómico 16S/análisisRESUMEN
Although BRAF mutations are commonly identified in many solid tumors and the response of BRAF p.V600E-positive tumors to targeted therapy is well documented, BRAF rearrangements are less frequent and are predominantly found in low-grade glioma, melanoma, lung, colorectal, and thyroid carcinoma. Preclinical and clinical studies have demonstrated effectiveness of multiple therapies (RAF-targeted, ERK-targeted, or MEK-targeted) targeting BRAF-fusion harboring tumors. We report a rare NRF1-BRAF fusion with novel breakpoints, identified by next-generation sequencing-based assay, from a 69-year-old man with metastatic urothelial carcinoma (UC) of the renal pelvis and his initial clinical response to a second-generation MEK inhibitor, trametinib, before stopping the medication because of adverse side effects. The NRF1-BRAF fusion has only been reported in a single case of anaplastic pleomorphic xanthoastrocytoma, and BRAF rearrangement has never been reported in UC.
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Carcinoma/genética , Neoplasias Renales/genética , Factor Nuclear 1 de Respiración/genética , Proteínas Proto-Oncogénicas B-raf/genética , Anciano , Carcinoma/diagnóstico por imagen , Carcinoma/patología , Humanos , Neoplasias Renales/diagnóstico por imagen , Neoplasias Renales/patología , Pelvis Renal/diagnóstico por imagen , Pelvis Renal/patología , Hígado/patología , Masculino , Metástasis de la NeoplasiaRESUMEN
BACKGROUND: Molecular testing for epidermal growth factor receptor (EGFR) mutation and anaplastic lymphoma kinase (ALK) and ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) fusion is routinely performed in patients with stage IV lung adenocarcinoma to assess their eligibility for targeted therapy. Fine-needle aspiration (FNA)-derived material frequently is the only pathologic material available. The identification of genomic aberrations in thyroid nodules from FNA smears may help stratify cancer risk and spare patients from a second surgery. In the current study, the authors tested nucleic acid extracted from the cytology smears of lung and thyroid carcinomas for simultaneous detection of single-nucleotide variant, insertion/deletion, and gene fusion using an RNA-based next-generation sequencing assay. METHODS: A total of 27 cases (17 lung and 10 thyroid carcinomas, the majority of which had known variants) were tested. Areas of interest were scrapped from stained smears using a scalpel. Total nucleic acid was extracted. Gene fusion and mutational analysis was performed using the Comprehensive Thyroid and Lung FusionPlex Assay. Data were analyzed using the analysis pipeline provided by the vendor. Eleven cases with available formalin-fixed, paraffin-embedded (FFPE) tissue were tested in parallel. RESULTS: Gene fusions were detected in 6 cases; common single-nucleotide variants in EGFR, RAS, and BRAF in 14 cases; and in-frame deletions within EGFR in 3 cases. A concordance rate of 100% was observed between FNA and FFPE tissue. CONCLUSIONS: Cytology preparations can be a reliable source for the detection of both DNA and RNA aberrations. The ability to simultaneously detect multiple types of genomic variants is crucial for patients with advanced cancer and maximizes the usefulness of cytology specimens. Cancer Cytopathol 2018;126:158-69. © 2018 American Cancer Society.
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Neoplasias Pulmonares/genética , Mutación , ARN Neoplásico/genética , Análisis de Secuencia de ARN/métodos , Neoplasias de la Tiroides/genética , Fusión Génica , Humanos , Mutación INDEL , Hibridación Fluorescente in Situ , Neoplasias Pulmonares/patología , Polimorfismo de Nucleótido Simple , Proto-Oncogenes Mas , Neoplasias de la Tiroides/patologíaRESUMEN
Histone deacetylase inhibitors (HDACI) are potential therapeutic agents that inhibit tumor cell growth and survival. Although there are several publications regarding the effects of HDACIs on prostate cancer cell growth, their mechanism(s) of action remains undefined. We treated several human prostate cancer cell lines with the HDACI trichostatin A and found that trichostatin A induced cell death in androgen receptor (AR)-positive cell lines to higher extent compared with AR-negative cell lines. We then discovered that trichostatin A and other HDACIs suppressed AR gene expression in prostate cancer cell lines as well as in AR-positive breast carcinoma cells and in mouse prostate. Trichostatin A also induced caspase activation, but trichostatin A-induced AR suppression and cell death were caspase independent. In addition, we found that doxorubicin inhibited AR expression, and p21 protein completely disappeared after simultaneous treatment with trichostatin A and doxorubicin. This effect may be attributed to the induction of protease activity under simultaneous treatment with these two agents. Further, simultaneous treatment with trichostatin A and doxorubicin increased cell death in AR-positive cells even after culturing in steroid-free conditions. The protease/proteasome inhibitor MG132 protected AR and p21 from the effects of trichostatin A and doxorubicin and inhibited trichostatin A-induced cell death in AR-positive prostate cells. Taken together, our data suggest that the main mechanism of trichostatin A-induced cell death in AR-positive prostate cancer is inhibition of AR gene expression. The synergistic effect of simultaneous treatment with trichostatin A and doxorubicin is mediated via inhibition of AR expression, induction of protease activity, increased expression of p53, and proteolysis of p21.
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Apoptosis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Ácidos Hidroxámicos/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Receptores Androgénicos/metabolismo , Animales , Antibióticos Antineoplásicos/farmacología , Western Blotting , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Caspasas/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Doxorrubicina/farmacología , Sinergismo Farmacológico , Quimioterapia Combinada , Activación Enzimática/efectos de los fármacos , Humanos , Leupeptinas/farmacología , Luciferasas , Masculino , Ratones , Regiones Promotoras Genéticas , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Androgénicos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
It has been suggested in many studies that combined treatment with chemotherapeutic agents and apoptosis-inducing ligands belonging to TNFR family is a more effective strategy for cancer treatment. However, the role of androgen regulation of TNFR family-induced apoptosis in prostate cancer is poorly understood. In this study, we investigated the dose-dependent effects of androgen on TNF-alpha and TRAIL-mediated apoptosis in LNCaP. To investigate the interaction between the androgen receptor (AR) and the caspase-2 gene, chromatin immunoprecipitation analysis was used, and we are the first to identify that AR interacts in vivo with an androgen-responsive elements in intron 8 of caspase-2 gene. We have found that DHT inhibited apoptosis in dose-dependent manner. There is a direct, androgen-dependent correlation between the levels of activated Akt and caspase activation after treatment with TNF-alpha and TRAIL. We have also found that there are at least two different regulatory mechanisms of p53 expression by androgen: at the gene and protein levels. At the same time, the level of AR was found to be higher in LNCaP-si-p53 compared to LNCaP-mock cells. These data indicate that there is a mutual regulation of expression between p53 and AR. Our study suggests that androgen-dependent outcome of apoptotic treatment can occur, at least in part, via the caspase-2, Akt and p53-mediated pathways.
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Andrógenos/fisiología , Apoptosis/fisiología , Transducción de Señal/fisiología , Proteínas Reguladoras de la Apoptosis/metabolismo , Secuencia de Bases , Caspasa 2 , Inhibidores de Caspasas , Caspasas/genética , Caspasas/metabolismo , Línea Celular Tumoral , Cartilla de ADN , Activación Enzimática , Humanos , Inmunoprecipitación , Masculino , Glicoproteínas de Membrana/metabolismo , Receptores Androgénicos/metabolismo , Receptores del Factor de Necrosis Tumoral/fisiología , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/metabolismo , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
The histone deacetylase inhibitor Trichostatin A (TSA) has previously been found to induce caspase activity in the human prostate cancer cell lines DU145 and LNCaP. TSA treatment resulted in the release of cytochrome c and Smac/DIABLO from mitochondria in DU145, and activation of caspase-9 in both cell lines. We concluded that TSA mediated its effect via the mitochondrial pathway. The aim of the current study was to determine how TSA initiated the caspase cascade. The results revealed that caspase-2 plays an important role in TSA-induced apoptosis. Inhibition of caspase-2 by siRNA or expression of caspase-2dn substantially decreased caspase activity after TSA treatment in both cell lines, siRNA caspase-2 also inhibited TSA-induced cell death. Caspase-2 acts upstream of caspase-8 and -9 and mediates mitochondrial cytochrome c release. Coimmunoprecipitation experiments show that caspase-2 formed protein complexes with RADD/RAIDD and PIDD. Together, these data indicate that caspase-2 initiates caspase cascade after TSA treatment and involves the formation of the PIDDosome.
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Proteínas Portadoras/metabolismo , Caspasa 2/metabolismo , Ácidos Hidroxámicos/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Proteína Adaptadora de Señalización CRADD/metabolismo , Proteínas Portadoras/genética , Caspasa 2/genética , Caspasa 8/metabolismo , Caspasa 9/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Citocromos c/metabolismo , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte , Activación Enzimática/efectos de los fármacos , Inhibidores de Histona Desacetilasas , Humanos , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , ARN Interferente Pequeño/genética , TransfecciónRESUMEN
Solitary fibrous tumor (SFT) is a mesenchymal tumor of fibroblastic origin, which can affect any region of the body. 10-15% of SFTs metastasize and metastatic tumors are uniformly lethal with no effective therapies. The behavior of SFT is difficult to predict based on morphology. Recently, an intrachromosomal gene fusion between NAB2 and STAT6 was identified as the defining driving genetic event of SFT and different fusion types correlated with tumor histology and behavior. Due to the proximity of NAB2 and STAT6 on chromosome 12, this fusion may be missed by fluorescence in-situ hybridization. We evaluated 12 SFTs from 10 patients. All tumors showed strong nuclear staining for STAT6 by immunohistochemistry (IHC). The same formalin-fixed, paraffin-embedded blocks for IHC were used for gene fusion detection by a next-generation sequencing (NGS)-based assay. Targeted RNA fusion sequencing for gene fusions was performed using the Universal RNA Fusion Detection Kit, the Archer(™) FusionPlex(™) Sarcoma Panel and the Ion Torrent PGM, and data were analyzed using the Archer Analysis Pipeline 3.3. All tumors were positive for NAB2-STAT6 fusion. Six types of fusions were detected: NAB2ex4-STAT6ex2, NAB2ex2-STAT6ex5, NAB2ex6-STAT6ex16, NAB2ex6-STAT6ex17, NAB2ex3-STAT6ex18 and NAB2intron6-STAT6Ex17. The NGS findings were confirmed by RT-PCR followed by Sanger sequencing. No STAT6 fusion was detected in selected morphologic mimics of SFT. The assay also allows for detection of novel fusions and can detect NAB2-STAT6 fusions at a single-base resolution.
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Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Proteínas de Fusión Oncogénica/genética , Análisis de Secuencia de ARN/métodos , Tumores Fibrosos Solitarios/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Femenino , Pruebas Genéticas/métodos , Humanos , Masculino , Persona de Mediana Edad , Proteínas Represoras/genética , Reproducibilidad de los Resultados , Estudios Retrospectivos , Factor de Transcripción STAT6/genéticaRESUMEN
The human prostatic carcinoma cell line DU145 has previously been found to be resistant to treatment with TNF-family ligands. However, TRAIL, TNF-alpha and anti-Fas antibodies (Ab) treatment in combination with the histone deacetylase inhibitor Trichostatin A (TSA) converted the phenotype of DU145 from resistant to sensitive. TSA induced 15% cell death but simultaneous treatment with TRAIL, TNF-alpha and anti-Fas Ab resulted in 55%, 70% and 40% cell death, respectively. Simultaneous treatment did not increase the level of TSA-induced histone acetylation, but induced the release of acetylated histones from chromatin into the cytosol. This release was caspase dependent since it was abrogated by Z-VAD-fmk. In addition, treatment with TSA induced caspase-9 activation and resulted in the release of cytochrome c and Smac/DIABLO from mitochondria. To further investigate the role of caspase-9 in TSA-mediated apoptosis we used two different approaches: (1) cells were pretreated with the caspase-9 inhibitor Z-LEHD-fmk, and (2) cells were transfected with a dominant-negative form of caspase-9. Both approaches gave similar results: cells became resistant to treatment with TSA. These data indicate that TSA mediates its effect via the mitochondrial pathway. This was confirmed by examining DU145 overexpressing Bcl-2. These transfectants were resistant to TSA treatment. Taken together, our data shows that only simultaneous treatment with TNF-family ligands and TSA in DU145 resulted in caspase activity sufficient to induce apoptosis. The combination of TSA and TNF-family ligands could potentially be the basis for the treatment of prostate cancer.
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Proteínas Reguladoras de la Apoptosis/farmacología , Apoptosis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Ácidos Hidroxámicos/farmacología , Glicoproteínas de Membrana/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Factor de Necrosis Tumoral alfa/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Ligandos , Masculino , Modelos Biológicos , Neoplasias de la Próstata/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNFRESUMEN
It has been suggested that some nuclear transcription factors may participate in the regulation of mitochondrial functions through transcriptional control of mitochondrial DNA. Very little is known about the response of transcription factors within mitochondria to the activation of death receptors. Recent publications indicate that nuclear factor-kappaB (NF-kappaB) is localized in mitochondria of mammalian cells. Because of the critical role of mitochondria in the execution of many apoptotic pathways, we suggest that NF-kappaB-dependent mechanisms operating at the level of mitochondria contribute to its role in regulating death receptor signaling. We have found NF-kappaB p65 and p50 subunits with DNA binding activity in the mitochondria of prostatic carcinoma cell lines. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) affects DNA binding activity of mitochondria-associated NF-kappaB but does not change the amount of p65 in mitochondria, which suggests activation of mitochondrial NF-kappaB without additional translocation of NF-kappaB subunits to mitochondria. We have also shown that TRAIL decreases mitochondrial genome encoded mRNA levels and inhibition of NF-kappaB prevents this decrease. TRAIL effects on mitochondrial NF-kappaB-DNA binding and mitochondrial genome encoded mRNA levels also depend on Bcl-2 overexpression. In addition, transcription factor activator protein-1 with DNA binding activity is also found in mitochondria of prostatic carcinoma cells and TRAIL treatment affects this binding. In summary, NF-kappaB is found in mitochondria of prostatic carcinoma cells, where it is thought to regulate mitochondria genome encoded mRNA levels in response to TRAIL treatment.
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ADN Mitocondrial/metabolismo , Glicoproteínas de Membrana/metabolismo , FN-kappa B/metabolismo , Neoplasias de la Próstata , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Reguladoras de la Apoptosis , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Glicoproteínas de Membrana/genética , Mitocondrias/fisiología , Subunidad p50 de NF-kappa B , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN/metabolismo , ARN Mensajero/metabolismo , ARN Mitocondrial , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción ReIA , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Here we show that LNCaP, which is resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, becomes sensitive to TRAIL after overexpression of full-length, wild-type BAD (BAD WT). TRAIL induces caspase-dependent cleavage of BAD WT that results in generation of a M(r) 15,000 protein. LNCaP stably expressing truncated BAD (tBAD) and cells expressing mutated BAD at the caspase cleavage site were less sensitive to TRAIL treatment when compared to LNCaP expressing BAD WT. Cytochrome c and Smac/DIABLO release from mitochondria into cytosol was found after TRAIL treatment only in cells overexpressing BAD WT. Furthermore, differences in phosphorylation of serine residues for BAD WT and tBAD were identified. BAD WT was phosphorylated at positions S136 and S155, whereas tBAD was phosphorylated at positions S112, S136, and S155. LNCaP stably expressing BAD mutated at serine 112 to alanine was less sensitive to TRAIL treatment when compared to LNCaP expressing BAD WT. Lastly, recombinant BAD cleaved by caspase-3 is a more potent inducer of cytochrome c and Smac/DIABLO release than BAD WT. In summary, BAD-mediated sensitivity of LNCaP to TRAIL depends on the phosphorylation status of BAD WT and tBAD.