RESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy in need of new therapeutic options. Using unbiased analyses of super-enhancers (SEs) as sentinels of core genes involved in cell-specific function, here we uncover a druggable SE-mediated RNA-binding protein (RBP) cascade that supports PDAC growth through enhanced mRNA translation. This cascade is driven by a SE associated with the RBP heterogeneous nuclear ribonucleoprotein F, which stabilizes protein arginine methyltransferase 1 (PRMT1) to, in turn, control the translational mediator ubiquitin-associated protein 2-like. All three of these genes and the regulatory SE are essential for PDAC growth and coordinately regulated by the Myc oncogene. In line with this, modulation of the RBP network by PRMT1 inhibition reveals a unique vulnerability in Myc-high PDAC patient organoids and markedly reduces tumor growth in male mice. Our study highlights a functional link between epigenetic regulation and mRNA translation and identifies components that comprise unexpected therapeutic targets for PDAC.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Masculino , Animales , Ratones , ARN , Epigénesis Genética , Secuencias Reguladoras de Ácidos Nucleicos , Neoplasias Pancreáticas/genética , Carcinoma Ductal Pancreático/genética , Metiltransferasas , Proteínas de Unión al ARN/genéticaRESUMEN
Breast cancers exhibit intratumoral heterogeneity associated with disease progression and therapeutic resistance. To define the sources and the extent of heterogeneity, we performed an in-depth analysis of the genomic architecture of three chemoradiation-naïve breast cancers with well-defined clinical features including variable ER, PR, ERBB2 receptor expression and two distinct pathogenic BRCA2mut genotypes. The latter included a germ line carrier and a patient with a somatic variant. In each case we combined DNA content-based flow cytometry with whole exome sequencing and genome wide copy number variant (CNV) analysis of distinct populations sorted from multiple (4-18) mapped biopsies within the tumors and involved lymph nodes. Interrogating flow-sorted tumor populations from each biopsy provided an objective method to distinguish fixed and variable genomic lesions in each tumor. Notably we show that tumors exploit CNVs to fix mutations and deletions in distinct populations throughout each tumor. The identification of fixed genomic lesions that are shared or unique within each tumor, has broad implications for the study of tumor heterogeneity including the presence of tumor markers and therapeutic targets, and of candidate neoepitopes in breast and other solid tumors that can advance more effective treatment and clinical management of patients with disease.
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Neoplasias de la Mama/genética , Carcinoma Ductal de Mama/genética , Evolución Molecular , Aneuploidia , Biomarcadores de Tumor , HumanosRESUMEN
Adenosquamous cancer of the pancreas (ASCP) is a subtype of pancreatic cancer that has a worse prognosis and greater metastatic potential than the more common pancreatic ductal adenocarcinoma (PDAC) subtype. To distinguish the genomic landscape of ASCP and identify actionable targets for this lethal cancer, we applied DNA content flow cytometry to a series of 15 tumor samples including five patient-derived xenografts (PDX). We interrogated purified sorted tumor fractions from these samples with whole-genome copy-number variant (CNV), whole-exome sequencing, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analyses. These identified a variety of somatic genomic lesions targeting chromatin regulators in ASCP genomes that were superimposed on well-characterized genomic lesions including mutations in TP53 (87%) and KRAS (73%), amplification of MYC (47%), and homozygous deletion of CDKN2A (40%) that are common in PDACs. Furthermore, a comparison of ATAC-seq profiles of three ASCP and three PDAC genomes using flow-sorted PDX models identified genes with accessible chromatin unique to the ASCP genomes, including the lysine methyltransferase SMYD2 and the pancreatic cancer stem cell regulator RORC in all three ASCPs, and a FGFR1-ERLIN2 fusion associated with focal CNVs in both genes in a single ASCP. Finally, we demonstrate significant activity of a pan FGFR inhibitor against organoids derived from the FGFR1-ERLIN2 fusion-positive ASCP PDX model. Our results suggest that the genomic and epigenomic landscape of ASCP provide new strategies for targeting this aggressive subtype of pancreatic cancer. SIGNIFICANCE: These data provide a unique description of the ASCP genomic and epigenomic landscape and identify candidate therapeutic targets for this dismal cancer.
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Carcinoma Adenoescamoso/genética , Cromatina/genética , Epigenoma , Mutación , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas p21(ras) , Carcinoma Adenoescamoso/tratamiento farmacológico , Carcinoma Adenoescamoso/patología , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Cromatina/metabolismo , Humanos , Organoides , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Análisis de la Célula Individual , Proteína Smad4/genética , Secuenciación del ExomaRESUMEN
Lynch syndrome (LS) arises in patients with pathogenic germline variants in DNA mismatch repair genes. LS is the most common inherited cancer predisposition condition and confers an elevated lifetime risk of multiple cancers notably colorectal and endometrial carcinomas. A distinguishing feature of LS associated tumors is accumulation of variants targeting microsatellite repeats and the potential for high tumor specific neoepitope levels. Recurrent somatic variants targeting a small subset of genes have been identified in tumors with microsatellite instability. Notably these include frameshifts that can activate immune responses and provide vaccine targets to affect the lifetime cancer risk associated with LS. However the presence and persistence of targeted neoepitopes across multiple tumors in single LS patients has not been rigorously studied. Here we profiled the genomic landscapes of five distinct treatment naïve tumors, a papillary transitional cell renal cell carcinoma, a duodenal carcinoma, two metachronous colorectal carcinomas, and multi-regional sampling in a triple-negative breast tumor, arising in a LS patient over 10 years. Our analyses suggest each tumor evolves a unique complement of variants and that vaccines based on potential neoepitopes from one tissue may not be effective across all tumors that can arise during the lifetime of LS patients.
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Biomarcadores de Tumor/genética , Neoplasias Colorrectales Hereditarias sin Poliposis/patología , Epítopos/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Proteínas de Unión al ADN/genética , Neoplasias Duodenales/patología , Epítopos/inmunología , Femenino , Mutación de Línea Germinal , Humanos , Inestabilidad de Microsatélites , Persona de Mediana Edad , Proteína 2 Homóloga a MutS/genética , Ploidias , Polimorfismo de Nucleótido Simple , Neoplasias de la Mama Triple Negativas/patología , Neoplasias Uterinas/patologíaRESUMEN
BACKGROUND: Elevated PD-L1 expression on tumor cells, a context associated with an adaptive immune response, has been linked to the total burden of copy number variants (CNVs) in aneuploid tumors, to microsatellite instability (MSI), and to specific genomic driver lesions, including loss of PTEN, MYC amplification, and activating mutations in driver oncogenes such as KRAS and PIK3CA. Triple-negative breast cancers (TNBCs) typically have high levels of CNVs and diverse driver lesions in their genomes. Thus, there is significant interest in exploiting genomic data to develop predictive immunotherapy biomarkers for patients with TNBC. METHODS: Whole tissue samples from 55 resected TNBCs were screened by immunohistochemistry (IHC) for PD-1 and PD-L1 by using validated antibodies and established scoring methods for staining of tumor and non-tumor cells. In parallel, we interrogated biopsies from each resection with DNA content flow cytometry and sorted the nuclei of diploid, tetraploid, and aneuploid cell populations. CNVs were mapped with CNV oligonucleotide arrays by using purified (>95%) tumor populations. We generated whole exome data for 12 sorted tumor samples to increase the resolution within loci of interest and to incorporate somatic mutations into our genomic signatures. RESULTS AND CONCLUSIONS: PD-L1 staining was detected on tumor cells in 29 out of 54 (54%) evaluable cases and was associated with increased overall survival (P = 0.0024). High levels of PD-1 and PD-L1 (IHC ≥4) were present in 11 out of 54 (20%) and 20 out of 54 (37%) cases with staining of PD-L1 primarily on tumor cells for 17 out of 20 (85%) cases. The latter included tumors with both high (>50) and low (<20) numbers of CNVs. Notably, homozygous deletion of PTEN (n = 6) or activating mutation in PIK3CA (n = 1) was not associated with increased expression of either immune checkpoint activator in TNBC. In contrast, two treatment-naïve cases with EGFR driver amplicons had high PD-L1 tumor staining. High mutational load and predicted neoepitopes were observed in MSI+ and high CNV burden TNBCs but were not associated with high PD-L1 expression on tumor cells. Our results challenge current models of genomic-based immunotherapy signatures yet suggest that discrete genomic lesions may complement existing biomarkers to advance immune checkpoint therapies for patients with TNBC.
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Antígeno B7-H1/genética , Biomarcadores de Tumor/genética , Variaciones en el Número de Copia de ADN/genética , Receptor de Muerte Celular Programada 1/genética , Neoplasias de la Mama Triple Negativas/genética , Anciano , Aneuploidia , Fosfatidilinositol 3-Quinasa Clase I/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Genoma Humano/genética , Humanos , Estimación de Kaplan-Meier , Linfocitos Infiltrantes de Tumor/patología , Inestabilidad de Microsatélites , Persona de Mediana Edad , Mutación , Fosfohidrolasa PTEN/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Neoplasias de la Mama Triple Negativas/patología , Secuenciación del ExomaRESUMEN
BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) is a lethal cancer with complex genomes and dense fibrotic stroma. This study was designed to identify clinically relevant somatic aberrations in pancreatic cancer genomes of patients with primary and metastatic disease enrolled and treated in two clinical trials. METHODS: Tumour nuclei were flow sorted prior to whole genome copy number variant (CNV) analysis. Targeted or whole exome sequencing was performed on most samples. We profiled biopsies from 68 patients enrolled in two Stand Up to Cancer (SU2C)-sponsored clinical trials. These included 38 resected chemoradiation naïve tumours (SU2C 20206-003) and metastases from 30 patients who progressed on prior therapies (SU2C 20206-001). Patient outcomes including progression-free survival (PFS) and overall survival (OS) were observed. RESULTS: We defined: (a) CDKN2A homozygous deletions that included the adjacent MTAP gene, only its' 3' region, or excluded MTAP; (b) SMAD4 homozygous deletions that included ME2; (c) a pancreas-specific MYC super-enhancer region; (d) DNA repair-deficient genomes; and (e) copy number aberrations present in PDA patients with long-term (⩾ 40 months) and short-term (⩽ 12 months) survival after surgical resection. CONCLUSIONS: We provide a clinically relevant framework for genomic drivers of PDA and for advancing novel treatments.
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Secuencia de Bases , Carcinoma Ductal Pancreático/genética , Neoplasias Pancreáticas/genética , Eliminación de Secuencia , Adulto , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biopsia , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/secundario , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/genética , Variaciones en el Número de Copia de ADN , Análisis Mutacional de ADN , Reparación del ADN/genética , Supervivencia sin Enfermedad , Elementos de Facilitación Genéticos , Exoma , Femenino , Genes myc , Homocigoto , Humanos , Malato Deshidrogenasa/genética , Masculino , Proteínas Asociadas a Microtúbulos/genética , Persona de Mediana Edad , Páncreas/patología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Purina-Nucleósido Fosforilasa/genética , Proteína Smad4/genética , Tasa de Supervivencia , Proteína p53 Supresora de Tumor/genéticaRESUMEN
PURPOSE: Vorinostat is a histone deacetylase inhibitor (HDACi). Based on a confirmed partial response (PR) in an adenoid cystic carcinoma (ACC) patient treated with vorinostat in a prior phase 1 trial, we initiated this phase 2 trial. METHODS: Vorinostat was administered orally 400 mg daily, 28 day cycles. The primary objective was to evaluate response rate (RR). Exploratory studies included whole exome sequencing (WES) of selected patients. RESULTS: Thirty patients were enrolled. Median age of patients was 53 years (range 21-73). Median number of cycles was 5 (range 1-66). Lymphopenia (n = 5), hypertension (n = 3), oral pain (n = 2), thromboembolic events (n = 2) and fatigue (n = 2) were the only grade 3 adverse events (AEs) that occurred in more than 1 patient. Eleven patients were dose reduced secondary to drug-related AEs. Two patients had a partial response (PR), with response durations of 53 and 7.2 months. One patient had a minor response with a decrease in ascites (for 19 cycles). Stable disease was the best response in 27 patients. Targeted and WES of 8 patients in this trial identified mutations in chromatin remodeling genes highlighting the role of the epigenome in ACC. CONCLUSION: Vorinostat demonstrated efficacy in patients with ACC supporting the inclusion of HDACi in future studies to treat ACC.
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Carcinoma Adenoide Quístico/tratamiento farmacológico , Inhibidores de Histona Desacetilasas/administración & dosificación , Ácidos Hidroxámicos/administración & dosificación , Recurrencia Local de Neoplasia/tratamiento farmacológico , Neoplasias de las Glándulas Salivales/tratamiento farmacológico , Adulto , Anciano , Carcinoma Adenoide Quístico/genética , Ensamble y Desensamble de Cromatina , Femenino , Redes Reguladoras de Genes , Inhibidores de Histona Desacetilasas/efectos adversos , Humanos , Ácidos Hidroxámicos/efectos adversos , Masculino , Persona de Mediana Edad , Mutación , Metástasis de la Neoplasia , Recurrencia Local de Neoplasia/genética , Variantes Farmacogenómicas , Neoplasias de las Glándulas Salivales/genética , Vorinostat , Secuenciación del Exoma , Adulto JovenRESUMEN
We used DNA content flow cytometry followed by oligonucleotide array based comparative genomic hybridization to survey the genomes of 326 tumors, including 41 untreated surgically resected triple negative breast cancers (TNBC). A high level (log2ratio ≥ 1) 9p24 amplicon was found in TNBC (12/41), glioblastomas (2/44), and colon carcinomas (2/68). The shortest region of overlap for the amplicon targets 9p24.1 and includes the loci for PD-L1, PD-L2, and JAK2 (PDJ amplicon). In contrast this amplicon was absent in ER+ (0/8) and HER2+ (0/15) breast tumors, and in pancreatic ductal adenocarcinomas (0/150). The PDJ amplicon in TNBCs was correlated with clinical outcomes in group comparisons by two-sample t-tests for continuous variables and chi-squared tests for categorical variables. TNBC patients with the PDJ amplicon had a worse outcome with worse disease-free and overall survival. Quantitative RT-PCR confirmed that the PDJ amplicon in TNBC is associated with elevated expression of JAK2 and of the PD-1 ligands. These initial findings demonstrate that the PDJ amplicon is enriched in TNBC, targets signaling pathways that activate the PD-1 mediated immune checkpoint, and identifies patients with a poor prognosis.
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Antígeno B7-H1/genética , Biomarcadores de Tumor/genética , Cromosomas Humanos Par 9 , Sitios Genéticos , Janus Quinasa 2/genética , Proteína 2 Ligando de Muerte Celular Programada 1/genética , Neoplasias de la Mama Triple Negativas/genética , Adulto , Anciano , Carcinoma Ductal Pancreático/genética , Neoplasias Colorrectales/genética , Hibridación Genómica Comparativa , Supervivencia sin Enfermedad , Femenino , Citometría de Flujo , Dosificación de Gen , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Glioblastoma/genética , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias Pancreáticas/genética , Fenotipo , Factores de Tiempo , Resultado del Tratamiento , Neoplasias de la Mama Triple Negativas/enzimología , Neoplasias de la Mama Triple Negativas/mortalidad , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/terapia , Regulación hacia ArribaRESUMEN
BACKGROUND: Chromosomal breakage followed by faulty DNA repair leads to gene amplifications and deletions in cancers. However, the mere assessment of the extent of genomic changes, amplifications and deletions may reduce the complexity of genomic data observed by array comparative genomic hybridization (array CGH). We present here a novel approach to array CGH data analysis, which focuses on putative breakpoints responsible for rearrangements within the genome. RESULTS: We performed array comparative genomic hybridization in 29 primary tumors from high risk patients with breast cancer. The specimens were flow sorted according to ploidy to increase tumor cell purity prior to array CGH. We describe the number of chromosomal breaks as well as the patterns of breaks on individual chromosomes in each tumor. There were differences in chromosomal breakage patterns between the 3 clinical subtypes of breast cancers, although the highest density of breaks occurred at chromosome 17 in all subtypes, suggesting a particular proclivity of this chromosome for breaks. We also observed chromothripsis affecting various chromosomes in 41% of high risk breast cancers. CONCLUSIONS: Our results provide a new insight into the genomic complexity of breast cancer. Genomic instability dependent on chromosomal breakage events is not stochastic, targeting some chromosomes clearly more than others. We report a much higher percentage of chromothripsis than described previously in other cancers and this suggests that massive genomic rearrangements occurring in a single catastrophic event may shape many breast cancer genomes.
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Neoplasias de la Mama/genética , Rotura Cromosómica , Inestabilidad Genómica/genética , Neoplasias de la Mama/patología , Cromosomas Humanos/genética , Hibridación Genómica Comparativa , Predisposición Genética a la Enfermedad/genética , Genómica , Humanos , Persona de Mediana Edad , Clasificación del TumorRESUMEN
The aggressiveness of pancreatic cancer is associated with the acquisition of mesenchymal characteristics by a subset of pancreatic cancer cells. The factors driving the development of this subset are not well understood. In this study, we tested the hypothesis that acquisition of a mesenchymal phenotype occurs selectively in tumor cells that harbor specific enabling genetic alterations. We obtained whole-genome comparative genomic hybridization (CGH) measurements on pancreatic cancer cell lines that have either an epithelial-like (17 cell lines) or a mesenchymal-like (9 cell lines) phenotype in vitro. The total amounts of amplifications and deletions were equivalent between the epithelial and mesenchymal groups, but 20 genes showed a major difference between the groups in prevalence of alterations. All 20 alterations (18 deletions and 2 amplifications) were more prevalent in the mesenchymal group, confirming the advanced nature of this cellular subtype. CDKN2A was altered in more than 50% of both groups, but co-deletions in neighboring genes, and concomitant loss of gene expression, were more prevalent in the mesenchymal group, suggesting that the size of the loss around CDKN2A affects cell phenotype. Whole-genome CGH on 11 primary cancer tissues revealed that the 20 genes were altered at a higher prevalence (up to 55% of the cases for certain genes) than randomly selected sets of 20 genes, with the same direction of alteration as in the cell lines. These findings support the concept that specific genetic alterations enable phenotype plasticity and provide promising candidate genes for further research.
Asunto(s)
Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Páncreas/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Línea Celular Tumoral , Hibridación Genómica Comparativa , Dosificación de Gen , Genómica , Humanos , Páncreas/citologíaRESUMEN
BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) is a highly lethal cancer characterized by complex aberrant genomes. A fundamental goal of current studies is to identify those somatic events arising in the variable landscape of PDA genomes that can be exploited for improved clinical outcomes. METHODS: We used DNA content flow sorting to identify and purify tumor nuclei of PDA samples from 50 patients. The genome of each sorted sample was profiled by oligonucleotide comparative genomic hybridization and targeted resequencing of STAG2. Transposon insertions within STAG2 in a KRAS (G12D)-driven genetically engineered mouse model of PDA were screened by RT-PCR. We then used a tissue microarray to survey STAG2 protein expression levels in 344 human PDA tumor samples and adjacent tissues. Univariate Kaplan Meier analysis and multivariate Cox Regression analysis were used to assess the association of STAG2 expression relative to overall survival and response to adjuvant therapy. Finally, RNAi-based assays with PDA cell lines were used to assess the potential therapeutic consequence of STAG2 expression in response to 18 therapeutic agents. RESULTS: STAG2 is targeted by somatic aberrations in a subset (4%) of human PDAs. Transposon-mediated disruption of STAG2 in a KRAS (G12D) genetically engineered mouse model promotes the development of PDA and its progression to metastatic disease. There was a statistically significant loss of STAG2 protein expression in human tumor tissue (Wilcoxon-Rank test) with complete absence of STAG2 staining observed in 15 (4.3%) patients. In univariate Kaplan Meier analysis nearly complete STAG2 positive staining (>95% of nuclei positive) was associated with a median survival benefit of 6.41 months (P = 0.031). The survival benefit of adjuvant chemotherapy was only seen in patients with a STAG2 staining of less than 95% (median survival benefit 7.65 months; P = 0.028). Multivariate Cox Regression analysis showed that STAG2 is an independent prognostic factor for survival in pancreatic cancer patients. Finally, we show that RNAi-mediated knockdown of STAG2 selectively sensitizes human PDA cell lines to platinum-based therapy. CONCLUSIONS: Based on these iterative findings we propose that STAG2 is a clinically significant tumor suppressor in PDA.
RESUMEN
Liposarcoma is the most common soft tissue sarcoma, but little is known about the genomic basis of this disease. Given the low cell content of this tumor type, we utilized flow cytometry to isolate the diploid normal and aneuploid tumor populations from a well-differentiated liposarcoma prior to array comparative genomic hybridization and whole genome sequencing. This work revealed massive highly focal amplifications throughout the aneuploid tumor genome including MDM2, a gene that has previously been found to be amplified in well-differentiated liposarcoma. Structural analysis revealed massive rearrangement of chromosome 12 and 11 gene fusions, some of which may be part of double minute chromosomes commonly present in well-differentiated liposarcoma. We identified a hotspot of genomic instability localized to a region of chromosome 12 that includes a highly conserved, putative L1 retrotransposon element, LOC100507498 which resides within a gene cluster (NAV3, SYT1, PAWR) where 6 of the 11 fusion events occurred. Interestingly, a potential gene fusion was also identified in amplified DDR2, which is a potential therapeutic target of kinase inhibitors such as dastinib, that are not routinely used in the treatment of patients with liposarcoma. Furthermore, 7 somatic, damaging single nucleotide variants have also been identified, including D125N in the PTPRQ protein. In conclusion, this work is the first to report the entire genome of a well-differentiated liposarcoma with novel chromosomal rearrangements associated with amplification of therapeutically targetable genes such as MDM2 and DDR2.
Asunto(s)
Cromosomas Humanos Par 11/genética , Cromosomas Humanos Par 12/genética , Reordenamiento Génico , Inestabilidad Genómica , Liposarcoma/genética , Proteínas de Neoplasias/genética , Sinaptotagmina I/genética , ADN de Neoplasias/genética , Receptores con Dominio Discoidina , Femenino , Amplificación de Genes , Genes Relacionados con las Neoplasias , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Familia de Multigenes , Proteínas Tirosina Quinasas Receptoras , Receptores MitogénicosRESUMEN
Glioblastoma (GBM) is known to be a heterogeneous disease; however, the genetic composition of the cells within a given tumour is only poorly explored. In the advent of personalised medicine the understanding of intra-tumoural heterogeneity at the cellular and the genetic level is mandatory to improve treatment and clinical outcome. By combining ploidy-based flow sorting with array-comparative genomic hybridization we show that primary GBMs present as either mono- or polygenomic tumours (64 versus 36%, respectively). Monogenomic tumours were limited to a pseudodiploid tumour clone admixed with normal stromal cells, whereas polygenomic tumours contained multiple tumour clones, yet always including a pseudodiploid population. Interestingly, pseudodiploid and aneuploid fractions carried the same aberrations as defined by identical chromosomal breakpoints, suggesting that evolution towards aneuploidy is a late event in GBM development. Interestingly, while clonal heterogeneity could be recapitulated in spheroid-based xenografts, we find that genetically distinct clones displayed different tumourigenic potential. Moreover, we show that putative cancer stem cell markers including CD133, CD15, A2B5 and CD44 were present on genetically distinct tumour cell populations. These data reveal the clonal heterogeneity of GBMs at the level of DNA content, tumourigenic potential and stem cell marker expression, which is likely to impact glioma progression and treatment response. The combined knowledge of intra-tumour heterogeneity at the genetic, cellular and functional level is crucial to assess treatment responses and to design personalized treatment strategies for primary GBM.
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Carcinogénesis/patología , Neoplasias del Sistema Nervioso Central/genética , Neoplasias del Sistema Nervioso Central/patología , Glioblastoma/genética , Glioblastoma/patología , Células Madre Neoplásicas/patología , Fenotipo , Animales , Biopsia , Carcinogénesis/genética , Línea Celular Tumoral , Variaciones en el Número de Copia de ADN/genética , ADN de Neoplasias/genética , Citometría de Flujo , Xenoinjertos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ploidias , Estudios Retrospectivos , Análisis de la Célula IndividualRESUMEN
Sarcomas are cancers that arise in soft tissues or bone and make up a small percentage of malignancies. In an effort to identify potential genetic targets for therapy, this study explores the genomic landscape of a metastatic undifferentiated pleomorphic sarcoma (UPS) with spindle cell morphology. Thick sections (50 µm) of formalin-fixed, paraffin-embedded tissue from a primary, recurrent, and metastatic tumor were collected and processed from a single patient for DNA content-based flow-sorting and analyses. Nuclei of diploid and aneuploid populations were sorted from the malignant tissues and their genomes interrogated with array comparative genomic hybridization. The third sample was highly degraded and did not contain any intact ploidy peaks in our flow assays. A 2.5N aneuploid population was identified in the primary and recurrent sample. We detected a series of shared and unique genomic aberrations in the sorted aneuploid populations. The patterns of aberrations suggest that two similar but independent clonal populations arose during the clinical history of this rare tumor. None of these aberrations were detected in the matching sorted diploid samples. The targeted regions of interest might play a role in UPS and may lead to clinical significance with further investigation.
RESUMEN
Tumors frequently arise as a result of an acquired genomic instability and the subsequent evolution of neoplastic populations with variable genomes. A barrier to the study of the somatic genetics of human solid tumors in vivo is the presence of admixtures of non-neoplastic cells with normal genomes in patient samples. These can obscure the presence of somatic aberrations including mutations, homozygous deletions, and breakpoints in biopsies of interest. Furthermore, clinical samples frequently contain multiple neoplastic populations that cannot be distinguished by morphology. Consequently, it is difficult to determine whether mutations detected in a sample of interest are concurrent in a single clonal population or if they occur in distinct cell populations in the same sample. The advent of targeted therapies increases the selection for preexisting populations. However the asymmetric distribution of therapeutic targets in clonal populations provides a mechanism for the rapid evolution of resistant disease. Thus, there is a need to not only isolate tumor from normal cells, but to also enrich distinct populations of clonal neoplastic cells in order to apply genome technologies to identify clinically relevant genomic aberrations that drive disease in patients in vivo. To address this we have applied single and multiparameter DNA content based flow assays to the study of solid tumors. Our work has identified examples of clonal resistance to effective therapies. This includes androgen withdrawal in advanced prostate cancer. In addition we demonstrate examples of co-existing clonal populations with highly aberrant genomes and ploidies in a wide variety of solid tumors. We propose that clonal analysis of tumors, based on flow cytometry and high resolution genome analyses of purified neoplastic populations, provides a unique approach to the study of therapeutic responses and the evolution of resistance.
RESUMEN
It is widely accepted that many cancers arise as a result of an acquired genomic instability and the subsequent evolution of tumor cells with variable patterns of selected and background aberrations. The presence and behaviors of distinct neoplastic cell populations within a patient's tumor may underlie multiple clinical phenotypes in cancers. A goal of many current cancer genome studies is the identification of recurring selected driver events that can be advanced for the development of personalized therapies. Unfortunately, in the majority of rare tumors, this type of analysis can be particularly challenging. Large series of specimens for analysis are simply not available, allowing recurring patterns to remain hidden. In this paper, we highlight the use of DNA content-based flow sorting to identify and isolate DNA-diploid and DNA-aneuploid populations from tumor biopsies as a strategy to comprehensively study the genomic composition and behaviors of individual cancers in a series of rare solid tumors: intrahepatic cholangiocarcinoma, anal carcinoma, adrenal leiomyosarcoma, and pancreatic neuroendocrine tumors. We propose that the identification of highly selected genomic events in distinct tumor populations within each tumor can identify candidate driver events that can facilitate the development of novel, personalized treatment strategies for patients with cancer.
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ADN/genética , Genómica/métodos , Neoplasias/genética , Neoplasias de las Glándulas Suprarrenales/genética , Glándulas Suprarrenales/metabolismo , Anciano , Canal Anal/metabolismo , Aneuploidia , Neoplasias del Ano/genética , Neoplasias de los Conductos Biliares/genética , Conductos Biliares Intrahepáticos/metabolismo , Colangiocarcinoma/genética , Diploidia , Femenino , Humanos , Leiomiosarcoma/genética , Masculino , Persona de Mediana Edad , Tumores Neuroendocrinos/genética , Páncreas/metabolismo , Neoplasias Pancreáticas/genéticaRESUMEN
Formalin fixed paraffin embedded (FFPE) tissues are a vast resource of annotated clinical samples. As such, they represent highly desirable and informative materials for the application of high definition genomics for improved patient management and to advance the development of personalized therapeutics. However, a limitation of FFPE tissues is the variable quality of DNA extracted for analyses. Furthermore, admixtures of non-tumor and polyclonal neoplastic cell populations limit the number of biopsies that can be studied and make it difficult to define cancer genomes in patient samples. To exploit these valuable tissues we applied flow cytometry-based methods to isolate pure populations of tumor cell nuclei from FFPE tissues and developed a methodology compatible with oligonucleotide array CGH and whole exome sequencing analyses. These were used to profile a variety of tumors (breast, brain, bladder, ovarian and pancreas) including the genomes and exomes of matching fresh frozen and FFPE pancreatic adenocarcinoma samples.
Asunto(s)
Formaldehído/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Adhesión en Parafina , Análisis de Secuencia de ADN/métodos , Fijación del Tejido , Adenocarcinoma/genética , Adenocarcinoma/patología , Clonación Molecular , Hibridación Genómica Comparativa , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologíaRESUMEN
BACKGROUND: Maxillary sinus carcinoma (MSC) is a rare cancer of the head and neck region. Patients are treated with surgery, radiation therapy, and chemotherapy and the treatment regimen is based on patient's age, general health condition, disease stage, and its extent of spread. There is very little information available on the genetics of this disease. DNA content based flow sorting of tumor cells followed by array comparative genomic hybridization allows for high definition global assessment of distinct clonal changes within tumor populations. METHODS: We applied this technique to primary and metastatic samples collected from a patient with radio- and chemotherapy refractory maxillary sinus carcinoma to gauge the progression of this disease. RESULTS: A clonal KIT amplicon was present in aneuploid populations sorted from the primary tumor and in divergent subclones arising in metastatic foci found in the brain, lung, and jejunum. The evolution of these subclones was associated with distinct genetic aberrations and DNA ploidies. CONCLUSION: The information presented here paves the path to understanding the development and progression of this disease.
Asunto(s)
Neoplasias del Seno Maxilar/genética , Antineoplásicos/uso terapéutico , Hibridación Genómica Comparativa , Humanos , Neoplasias del Seno Maxilar/patología , Neoplasias del Seno Maxilar/terapia , Metástasis de la Neoplasia , RadioterapiaRESUMEN
Cancers frequently arise as a result of an acquired genomic instability and the subsequent clonal evolution of neoplastic cells with variable patterns of genetic aberrations. Thus, the presence and behaviors of distinct clonal populations in each patient's tumor may underlie multiple clinical phenotypes in cancers. We applied DNA content-based flow sorting to identify and isolate the nuclei of clonal populations from tumor biopsies, which was coupled with array CGH and targeted resequencing. The results produced high-definition genomic profiles of clonal populations from 40 pancreatic adenocarcinomas and a set of prostate adenocarcinomas, including serial biopsies from a patient who progressed to androgen-independent metastatic disease. The genomes of clonal populations were found to have patient-specific aberrations of clinical relevance. Furthermore, we identified genomic aberrations specific to therapeutically responsive and resistant clones arising during the evolution of androgen-independent metastatic prostate adenocarcinoma. We also distinguished divergent clonal populations within single biopsies and mapped aberrations in multiple aneuploid populations arising in primary and metastatic pancreatic adenocarcinoma. We propose that our high-definition analyses of the genomes of distinct clonal populations of cancer cells in patients in vivo can help guide diagnoses and tailor approaches to personalized treatment.
Asunto(s)
Adenocarcinoma/genética , Evolución Molecular , Variación Genética , Metástasis de la Neoplasia/genética , Neoplasias Pancreáticas/genética , Neoplasias de la Próstata/genética , Biopsia , Células Clonales , Hibridación Genómica Comparativa , Cartilla de ADN/genética , Citometría de Flujo , Genómica/métodos , Humanos , Hibridación Fluorescente in Situ , Masculino , Análisis por Micromatrices , Reacción en Cadena de la Polimerasa , Medicina de Precisión/métodos , Análisis de Secuencia de ADNRESUMEN
Eosinophils are often dominant inflammatory cells present in the lungs of asthma patients. Nonetheless, the role of these leukocytes remains poorly understood. We have created a transgenic line of mice (PHIL) that are specifically devoid of eosinophils, but otherwise have a full complement of hematopoietically derived cells. Allergen challenge of PHIL mice demonstrated that eosinophils were required for pulmonary mucus accumulation and the airway hyperresponsiveness associated with asthma. The development of an eosinophil-less mouse now permits an unambiguous assessment of a number of human diseases that have been linked to this granulocyte, including allergic diseases, parasite infections, and tumorigenesis.