RESUMO
Cells with distinct phenotypes including stem-cell-like properties have been proposed to exist in normal human mammary epithelium and breast carcinomas, but their detailed molecular characteristics and clinical significance are unclear. We determined gene expression and genetic profiles of cells purified from cancerous and normal breast tissue using markers previously associated with stem-cell-like properties. CD24+ and CD44+ cells from individual tumors were clonally related but not always identical. CD44+ cell-specific genes included many known stem-cell markers and correlated with decreased patient survival. The TGF-beta pathway was specifically active in CD44+ cancer cells, where its inhibition induced a more epithelial phenotype. Our data suggest prognostic relevance of CD44+ cells and therapeutic targeting of distinct tumor cell populations.
Assuntos
Neoplasias da Mama/metabolismo , Células-Tronco/metabolismo , Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Antígeno CD24/metabolismo , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Linhagem da Célula , Células Cultivadas , Receptor de Proteína C Endotelial , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Receptores de Hialuronatos/metabolismo , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Gravidez , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Células-Tronco/patologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
To identify genetic changes involved in the progression of breast carcinoma, we did cDNA array comparative genomic hybridization (CGH) on a panel of breast tumors, including 10 ductal carcinoma in situ (DCIS), 18 invasive breast carcinomas, and two lymph node metastases. We identified 49 minimal commonly amplified regions (MCRs) that included known (1q, 8q24, 11q13, 17q21-q23, and 20q13) and several uncharacterized (12p13 and 16p13) regional copy number gains. With the exception of the 17q21 (ERBB2) amplicon, the overall frequency of copy number alterations was higher in invasive tumors than that in DCIS, with several of them present only in invasive cancer. Amplification of candidate loci was confirmed by quantitative PCR in breast carcinomas and cell lines. To identify putative targets of amplicons, we developed a method combining array CGH and serial analysis of gene expression (SAGE) data to correlate copy number and expression levels for each gene within MCRs. Using this approach, we were able to distinguish a few candidate targets from a set of coamplified genes. Analysis of the 12p13-p12 amplicon identified four putative targets: TEL/ETV6, H2AFJ, EPS8, and KRAS2. The amplification of all four candidates was confirmed by quantitative PCR and fluorescence in situ hybridization, but only H2AFJ and EPS8 were overexpressed in breast tumors with 12p13 amplification compared with a panel of normal mammary epithelial cells. These results show the power of combined array CGH and SAGE analysis for the identification of candidate amplicon targets and identify H2AFJ and EPS8 as novel putative oncogenes in breast cancer.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Cromossomos Humanos Par 12/genética , Progressão da Doença , Feminino , Amplificação de Genes , Perfilação da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Análise de Sequência com Séries de Oligonucleotídeos/métodosRESUMO
BACKGROUND: Familial cardiac myxomas occur in the hereditary syndrome Carney complex. Although PRKAR1A mutations can cause the Carney complex, the disorder is genetically heterogeneous. To identify the cause of a Carney complex variant associated with distal arthrogryposis (the trismus-pseudocamptodactyly syndrome), we performed clinical and genetic studies. METHODS: A large family with familial cardiac myxomas and the trismus-pseudocamptodactyly syndrome (Family 1) was identified and clinically evaluated along with two families with trismus and pseudocamptodactyly. Genetic linkage analyses were performed with the use of microsatellite polymorphisms to determine a locus for this Carney complex variant. Positional cloning and mutational analyses of candidate genes were performed to identify the genetic cause of disease in the family with the Carney complex as well as in the families with the trismus-pseudocamptodactyly syndrome. RESULTS: Clinical evaluations demonstrated that the Carney complex cosegregated with the trismus-pseudocamptodactyly syndrome in Family 1, and genetic analyses demonstrated linkage of the disease to chromosome 17p12-p13.1 (maximum multipoint lod score, 4.39). Sequence analysis revealed a missense mutation (Arg674Gln) in the perinatal myosin heavy-chain gene (MYH8). The same mutation was also found in the two families with the trismus-pseudocamptodactyly syndrome. Arg674 is highly conserved evolutionarily, localizes to the actin-binding domain of the perinatal myosin head, and is close to the ATP-binding site. We identified nonsynonymous MYH8 polymorphisms in patients with cardiac myxoma syndromes but without arthrogryposis. CONCLUSIONS: We describe a novel heart-hand syndrome involving familial cardiac myxomas and distal arthrogryposis and demonstrate that these disorders are caused by a founder mutation in the MYH8 gene. Our findings demonstrate novel roles for perinatal myosin in both the development of skeletal muscle and cardiac tumorigenesis.
Assuntos
Artrogripose/genética , Neoplasias Cardíacas/genética , Mutação de Sentido Incorreto , Cadeias Pesadas de Miosina/genética , Mixoma/genética , Transtornos da Pigmentação/genética , Trismo/genética , Análise Mutacional de DNA , Feminino , Dedos/anormalidades , Genótipo , Mutação em Linhagem Germinativa , Humanos , Escore Lod , Masculino , Cadeias Pesadas de Miosina/química , Neoplasias Primárias Múltiplas/genética , Linhagem , SíndromeRESUMO
Aneurysmal bone cyst (ABC) is a locally aggressive osseous lesion that typically occurs during the first two decades of life. ABC was regarded historically as a nonneoplastic process, but recent cytogenetic data have shown clonal rearrangements of chromosomal bands 16q22 and 17p13, indicating a neoplastic basis in at least some ABCs. Herein we show that a recurring ABC chromosomal translocation t(16;17)(q22;p13) creates a fusion gene in which the osteoblast cadherin 11 gene (CDH11) promoter region on 16q22 is juxtaposed to the entire ubiquitin-specific protease USP6 (Tre2) coding sequence on 17p13. CDH11-USP6 fusion transcripts were demonstrated only in ABC with t(16;17) but other ABCs had CDH11 or USP6 rearrangements resulting from alternate cytogenetic mechanisms. CDH11 is expressed strongly in bone, and our findings implicate a novel oncogenic mechanism in which deregulated USP6 transcription results from juxtaposition to the highly active CDH11 promoter.
Assuntos
Cistos Ósseos Aneurismáticos/genética , Caderinas/genética , Endopeptidases/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Oncogênicas/genética , Translocação Genética , Adolescente , Cistos Ósseos Aneurismáticos/diagnóstico , Criança , Cromossomos Humanos Par 16 , Cromossomos Humanos Par 17 , Feminino , Rearranjo Gênico , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Masculino , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas , Ubiquitina TiolesteraseRESUMO
We determined, by serial analysis of gene expression (SAGE) analysis of normal and DCIS (ductal carcinoma in situ) mammary epithelial cells, that psoriasin and several other genes implicated in psoriasis are aberrantly expressed in high-grade, comedo DCIS. Real-time PCR, mRNA in situ hybridization, and immunohistochemical analysis of breast carcinomas confirmed that psoriasin is frequently overexpressed in estrogen receptor-negative tumors. To gain insight into regulatory pathways that control psoriasin expression, we developed polyclonal and monoclonal antibodies and investigated mechanisms that may account for elevated levels of psoriasin in DCIS. Here, we report that loss of attachment to extracellular matrix, growth factor deprivation, and confluent conditions dramatically up-regulate psoriasin expression in MCF10A mammary epithelial cells. All of these conditions are characteristic of high-grade DCIS and psoriatic skin lesions; therefore, the same mechanisms may be responsible for increased expression of psoriasin in vitro and in vivo.
Assuntos
Biomarcadores Tumorais/biossíntese , Neoplasias da Mama/metabolismo , Mama/metabolismo , Proteínas de Ligação ao Cálcio/biossíntese , Carcinoma in Situ/metabolismo , Carcinoma Ductal de Mama/metabolismo , Sequência de Aminoácidos , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Mama/citologia , Mama/fisiologia , Neoplasias da Mama/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Carcinoma in Situ/genética , Carcinoma Ductal de Mama/genética , Citoplasma/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Dados de Sequência Molecular , Psoríase/genética , Psoríase/metabolismo , Psoríase/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína A7 Ligante de Cálcio S100 , Proteínas S100 , Frações Subcelulares/metabolismo , Células Tumorais CultivadasRESUMO
Uterine leiomyomata are one of several benign tumors characterized by frequent chromosomal rearrangement involving 12q15. The 12q15 rearrangement in leiomyomata typically is manifested as t(12;14)(q15;q23-24), which has been hypothesized to create pathobiologically significant fusion transcripts derived from HMGA2 and RAD51L1. To explore further this hypothesis, we mapped chromosomal breakpoints in 38 uterine leiomyomata with rearrangements involving 12q15 using fluorescence in situ hybridization. Most tumors (n = 26) harbored der(14)t(12;14)(q15;q23-24), whereas chromosomes 1, 5, 8, and 10 were involved in rearrangements with 12q15 in six myomas. An additional six cases had more complex rearrangements, including breakpoints other than 12q15 or 14q23-24, inversions of chromosome 12, insertions of 12q15 into chromosome 14, or additional translocation partners. Breakpoints were mapped either 5' (centromeric) or 3' (telomeric) in the HMGA2 locus in 24 and nine cases, respectively; one tumor was a mosaic of cells with either 5' or 3' breakpoints. Breakpoints flanking the gene in both 5' and 3' regions were found in six cases. Analysis of one tumor by 3' rapid amplification of cDNA ends showed altered transcripts in which either exons 1-3 of HMGA2 were aberrantly spliced to cryptic sites in chromosome 12 or transcripts encompassing the full coding sequence of HMGA2 through a portion of the 3' untranslated region were fused to sequence from chromosome 14. A panel of 10 uterine leiomyomata with t(12;14) was specifically tested for fusion transcripts. RAD51L1-HMGA2 transcripts were not detected. HMGA2-RAD51L1 transcripts, however, were detected in four tumors; two of these tumors had uncommon rearrangements in the 3' region of HMGA2 and two had 5' rearrangements. Although the mechanism of fusion transcripts derived from tumors with 5' breakpoints is unclear, these findings indicate that formation of a fusion transcript is not the principle pathobiological mechanism in uterine leiomyomata. The pattern of rearrangements suggests dysregulated expression of HMGA2, most often by translocation of chromosome 14 sequence 5' to this gene.
Assuntos
Cromossomos Humanos Par 12/genética , Proteína HMGA2/genética , Leiomioma/genética , Proteínas de Fusão Oncogênica/genética , Neoplasias Uterinas/genética , Processamento Alternativo , Quebra Cromossômica/genética , Proteínas de Ligação a DNA/genética , Feminino , Rearranjo Gênico , Humanos , Hibridização in Situ Fluorescente , RNA Mensageiro/genética , Rad51 Recombinase , Telômero/genéticaRESUMO
This appendix, developed by the staff at the Center for Advanced Molecular Diagnostics in the Department of Pathology at the Brigham and Women's Hospital, includes a comprehensive list of current "macros" or standardized statements used to facilitate reporting of cytogenetic results. These are provided as a useful reference for other laboratories. The statements are organized under the general categories of constitutional or acquired abnormalities and subdivided into analysis type (GTG-banding, FISH, or chromosomal microarray). Multi-specimen usage macros are included that can be applied to two or more specimen types.
Assuntos
Aberrações Cromossômicas , Análise Citogenética/métodos , Escrita Médica/normas , Diagnóstico Pré-Natal/normas , Aborto Espontâneo/genética , Bandeamento Cromossômico/métodos , Bandeamento Cromossômico/normas , Análise Citogenética/normas , Citogenética/métodos , Feminino , Aconselhamento Genético , Humanos , Hibridização in Situ Fluorescente/métodos , Hibridização in Situ Fluorescente/normas , Masculino , Diagnóstico Pré-Natal/métodosRESUMO
Maturity-onset diabetes of the young (MODY) is a subtype of diabetes defined by an autosomal dominant inheritance and a young onset. Six MODY genes have been discovered to date. To identify additional MODY loci, we conducted a genome scan in 21 extended U.S. families (15 white and 6 from minorities, for a total of 237 individuals) in which MODY was not caused by known MODY genes. Seven chromosomal regions (1q42, 2q24, 2q37, 4p13, 8p23, 11p15, and 19q12) had a parametric heterogeneity logarithm of odds (HLOD) > or =1.00 or a nonparametric logarithm of odds (LOD) > or =0.59 (P < or = 0.05) in the initial screen. After typing additional markers at these loci to reduce the spacing to 2-3 cM, significant linkage was detected on 8p23 (HLOD = 3.37 at D8S1130 and nonparametric LOD = 3.66; P = 2 x 10(-5) at D8S265), where a 4.7-Mb inversion polymorphism is located. Thirty percent of the families (6 of 21) were linked with this region. Another linkage peak on chromosome 2q37 with an HLOD of 1.96 at D2S345/D2S2968 accounted for diabetes in an additional 25% of families (5 of 21). All 6 minority families were among the 11 families linked to these loci. None of the other loci followed up had an HLOD exceeding 1.50. In summary, we have identified a MODY locus on 8p23 that accounts for diabetes in a substantial proportion of MODY cases unlinked to known MODY genes. Another novel MODY locus may be present on 2q37. Cloning these new MODY genes may offer insights to disease pathways that are relevant to the cause of common type 2 diabetes.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 8/genética , Diabetes Mellitus Tipo 2/genética , Adolescente , Adulto , Criança , Cromossomos Humanos Par 2/genética , Diabetes Mellitus Tipo 2/etnologia , Ligação Genética , Marcadores Genéticos , Genoma Humano , Humanos , Escore Lod , Pessoa de Meia-Idade , Grupos Minoritários , Fenótipo , População Branca/genéticaRESUMO
Numerical and structural chromosome abnormalities can be accurately detected in cells from archived tissues using fluorescence in situ hybridization (FISH). This unit describes two common approaches to performing FISH in formalin-fixed, paraffin-embedded tissue. The first approach utilizes 4 to 6 µm tissue sections in cases for which preserving tissue morphology is necessary, and the second involves extraction of intact nuclei from 50-µm tissue sections. To interpret FISH results using 4 to 6 µm sections, an adequate number of nuclei must be evaluated to perform statistical analysis. Evaluation of 30 to 50 nuclei from the single-cell suspension generally gives an interpretable result.
Assuntos
Separação Celular/métodos , Aberrações Cromossômicas , Hibridização in Situ Fluorescente/métodos , Inclusão em Parafina/métodos , Fixação de Tecidos/métodos , Fracionamento Celular/instrumentação , Fracionamento Celular/métodos , Núcleo Celular/química , Núcleo Celular/ultraestrutura , Separação Celular/instrumentação , Sondas de DNA/síntese química , Fixadores , Formaldeído , Humanos , Hibridização in Situ Fluorescente/instrumentação , Microtomia/instrumentação , Microtomia/métodos , Inclusão em Parafina/instrumentação , Análise de Célula Única/instrumentação , Análise de Célula Única/métodosRESUMO
Genetically activated kinases have been attractive therapeutic targets in cancer due to the relative ease of developing tumor-specific treatment strategies for them. To discover novel putative oncogenic kinases, we identified 26 genes commonly amplified and overexpressed in breast cancer and subjected them to a lentiviral shRNA cell viability screen in a panel of breast cancer cell lines. Here, we report that CLK2, a kinase that phosphorylates SR proteins involved in splicing, acts as an oncogene in breast cancer. Deregulated alternative splicing patterns are commonly observed in human cancers but the underlying mechanisms and functional relevance are still largely unknown. CLK2 is amplified and overexpressed in a significant fraction of breast tumors. Downregulation of CLK2 inhibits breast cancer growth in cell culture and in xenograft models and it enhances cell migration and invasion. Loss of CLK2 in luminal breast cancer cells leads to the upregulation of epithelial-to-mesenchymal transition (EMT)-related genes and a switch to mesenchymal splice variants of several genes, including ENAH (MENA). These results imply that therapeutic targeting of CLK2 may be used to modulate EMT splicing patterns and to inhibit breast tumor growth.
Assuntos
Processamento Alternativo , Neoplasias da Mama/enzimologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Tirosina Quinases/fisiologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular , Forma Celular , Transição Epitelial-Mesenquimal , Feminino , Expressão Gênica , Humanos , Camundongos , Transplante de NeoplasiasRESUMO
Mesenchymomas are neoplasms in which there are at least two types of differentiated cells of mesenchymal derivation other than fibrous tissue. Chondrolipoangioma is a rare type of mesenchymoma composed predominantly of cartilage and adipose tissue with vascular elements and myxoid tissue present in lesser proportions. Cytogenetic analysis was performed on a case of chondrolipoangioma and revealed a t(12;15) (q13;q26) as the sole chromosome abnormality in 40 metaphases analyzed. However, using fluorescence in situ hybridization (FISH) analysis, a complex rearrangement was found involving chromosomes 2, 12, and 15, with a cryptic rearrangement of the gene ( HMGI-C; HMGA2) coding for high-mobility group I protein. This finding suggests a role for the HMGI-C gene also in the pathogenesis of this uncommon benign tumor type, in addition to its well-established role in the pathogenesis of common benign tumors such as lipomas, uterine leiomyomas, pulmonary chondroid hamartomas, and endometrial polyps.
Assuntos
Proteína HMGA2/genética , Mesenquimoma/genética , Neoplasias Musculares/genética , Translocação Genética , Actinas/análise , Biópsia , Cromossomos Humanos Par 12 , Cromossomos Humanos Par 15 , Desmina/análise , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Cariotipagem , Masculino , Mesenquimoma/patologia , Pessoa de Meia-Idade , Neoplasias Musculares/patologia , Proteínas S100/análiseRESUMO
INTRODUCTION: ALK gene rearrangements occur in approximately 5% of lung adenocarcinomas (ACAs), leading to anaplastic lymphoma kinase (ALK) overexpression and predicting response to targeted therapy. Fluorescence in situ hybridization (FISH) is the standard procedure for detection of ALK rearrangements in lung ACA but requires specialized equipment and expertise. Immunohistochemistry (IHC) for ALK protein overexpression is a promising screening modality, with reports of newer antibodies showing excellent sensitivity and specificity for ALK-rearranged lung ACA. METHODS: In this study, we analyzed ALK IHC (5A4 clone) in 186 cases from our clinical service and compared it with ALK FISH and EGFR and KRAS mutation status. RESULTS: Twelve cases had concordant ALK protein overexpression and ALK rearrangement by FISH. Three ALK-rearranged cases lacked ALK protein expression. Of these discrepant cases, one had a coexisting EGFR mutation and a subtle atypical ALK rearrangement manifested as a break in the 5' centromeric portion of the FISH probe. One case had a concurrent BRAF mutation. Follow-up testing on a metastasis revealed absence of the ALK rearrangement, with persistent BRAF mutation. In one ALK-rearranged protein negative case, very limited tissue remained for ALK IHC, raising the possibility of false negativity because of protein expression heterogeneity. Importantly, ALK protein expression was detected in one case initially thought not to have an ALK rearrangement. In this case, FISH was falsely negative because of interference by benign reactive nuclei. After correcting for these cases, ALK IHC was 93% sensitive and 100% specific as compared with FISH. CONCLUSIONS: ALK IHC improves the detection of ALK rearrangements when used together with FISH, and its use in lung ACA genetic testing algorithms should be considered.
Assuntos
Adenocarcinoma/diagnóstico , Rearranjo Gênico , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/diagnóstico , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Idoso , Algoritmos , Quinase do Linfoma Anaplásico , Biomarcadores Tumorais/genética , Receptores ErbB/genética , Feminino , Seguimentos , Testes Genéticos , Humanos , Técnicas Imunoenzimáticas , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação/genética , Estadiamento de Neoplasias , Prognóstico , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Sensibilidade e Especificidade , Taxa de Sobrevida , Proteínas ras/genéticaRESUMO
This appendix, developed by the staff at the Clinical Cytogenetics Laboratory at the Brigham and Women's Hospital, includes a comprehensive list of current "macros" or standardized statements used to facilitate reporting of cytogenetic results. These are provided as a reference for other laboratories. The statements are organized under the general categories of constitutional or acquired abnormalities and subdivided into analysis type (GTG-banding or FISH). Multi-specimen usage macros are included that can be applied to two or more specimen types.
Assuntos
Aberrações Cromossômicas , Transtornos Cromossômicos/diagnóstico , Análise Citogenética , Testes Genéticos/normas , Diagnóstico Pré-Natal , Bandeamento Cromossômico , Feminino , Testes Genéticos/métodos , Humanos , Hibridização in Situ Fluorescente , Masculino , GravidezRESUMO
Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKI), including crizotinib, are effective treatments in preclinical models and in cancer patients with ALK-translocated cancers. However, their efficacy will ultimately be limited by the development of acquired drug resistance. Here we report two mechanisms of ALK TKI resistance identified from a crizotinib-treated non-small cell lung cancer (NSCLC) patient and in a cell line generated from the resistant tumor (DFCI076) as well as from studying a resistant version of the ALK TKI (TAE684)-sensitive H3122 cell line. The crizotinib-resistant DFCI076 cell line harbored a unique L1152R ALK secondary mutation and was also resistant to the structurally unrelated ALK TKI TAE684. Although the DFCI076 cell line was still partially dependent on ALK for survival, it also contained concurrent coactivation of epidermal growth factor receptor (EGFR) signaling. In contrast, the TAE684-resistant (TR3) H3122 cell line did not contain an ALK secondary mutation but instead harbored coactivation of EGFR signaling. Dual inhibition of both ALK and EGFR was the most effective therapeutic strategy for the DFCI076 and H3122 TR3 cell lines. We further identified a subset (3/50; 6%) of treatment naive NSCLC patients with ALK rearrangements that also had concurrent EGFR activating mutations. Our studies identify resistance mechanisms to ALK TKIs mediated by both ALK and by a bypass signaling pathway mediated by EGFR. These mechanisms can occur independently, or in the same cancer, suggesting that the combination of both ALK and EGFR inhibitors may represent an effective therapy for these subsets of NSCLC patients.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Receptores ErbB/metabolismo , Neoplasias Pulmonares/metabolismo , Mutação , Inibidores de Proteínas Quinases/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Quinase do Linfoma Anaplásico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Crizotinibe , Resistencia a Medicamentos Antineoplásicos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Modelos Moleculares , Proteínas de Fusão Oncogênica/biossíntese , Proteínas de Fusão Oncogênica/genética , Pirazóis/farmacologia , Piridinas/farmacologia , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de SinaisRESUMO
Preimplantation genetic testing, which includes preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS), is a form of a very early prenatal testing. The goal of this method is to avoid transfer of embryos affected with a specific genetic disease or condition. This unit describes the steps involved in amplifying DNA from a single blastomere and specific assays for detecting a variety of DNA mutations. For some assays, whole-genome amplification by primer-extension preamplification (PEP) is performed prior to analysis. Support protocols describe the biopsy of one or two blastomeres from the developing preimplantation embryo, isolation for further investigation of all blastomeres from embryos shown to have the mutant allele, and isolation of single lymphocytes or lymphoblastoid cells as models for single-cell DNA analysis. A procedure for FISH analysis on single interphase blastomeres is provided along with support protocols for probe preparation and probe validation, which is recommended as a preliminary step before performing any PGD or PGS FISH analysis.
Assuntos
DNA/genética , Testes Genéticos/métodos , Hibridização in Situ Fluorescente/métodos , Diagnóstico Pré-Implantação/métodos , Animais , Separação Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , HumanosRESUMO
This appendix, developed by the staff at the Clinical Cytogenetics Laboratory at the Brigham and Women's Hospital, includes a comprehensive list of current "macros" or standardized statements used to facilitate reporting of cytogenetic results. These are provided as a reference for other laboratories. The statements are organized under the general categories of constitutional or acquired abnormalities and subdivided into analysis type (GTG-banding or FISH). Multi-specimen usage macros are included that can be applied to two or more specimen types.
Assuntos
Técnicas de Laboratório Clínico , Citogenética/métodos , Citogenética/normas , Hibridização in Situ Fluorescente , Bandeamento Cromossômico , Feminino , Humanos , Cariotipagem , MasculinoAssuntos
Proteína HMGA2/genética , Lipoma/genética , Mutação , Adulto , Feminino , Humanos , Lipoma/etiologiaRESUMO
The transition of ductal carcinoma in situ (DCIS) to invasive carcinoma is a poorly understood key event in breast tumor progression. Here, we analyzed the role of myoepithelial cells and fibroblasts in the progression of in situ carcinomas using a model of human DCIS and primary breast tumors. Progression to invasion was promoted by fibroblasts and inhibited by normal myoepithelial cells. Molecular profiles of isolated luminal epithelial and myoepithelial cells identified an intricate interaction network involving TGFbeta, Hedgehog, cell adhesion, and p63 required for myoepithelial cell differentiation, the elimination of which resulted in loss of myoepithelial cells and progression to invasion.
Assuntos
Neoplasias da Mama/patologia , Carcinoma Intraductal não Infiltrante/patologia , Neoplasias da Mama/genética , Carcinoma Intraductal não Infiltrante/genética , Adesão Celular , Metilação de DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Homeostase , Humanos , Invasividade Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único , Fator de Crescimento Transformador beta/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Numerical and structural chromosome abnormalities can be accurately detected in cells from archived tissues using fluorescence in situ hybridization (FISH). This unit describes two common approaches to performing FISH in formalin-fixed, paraffin-embedded tissue. The first approach utilizes 4 to 6 microm tissue sections in cases for which preserving tissue morphology is necessary, and the second involves extraction of intact nuclei from 50 microm tissue sections. To interpret FISH results using 4 to 6 microm sections, an adequate number of nuclei must be evaluated to perform statistical analysis. Evaluation of 30 to 50 nuclei from the single cell suspension generally gives an interpretable result.