RESUMO
Fibrolamellar carcinoma is a distinct subtype of hepatocellular carcinoma that predominantly affects young patients without underlying cirrhosis. A recurrent DNAJB1-PRKACA fusion has recently been reported in fibrolamellar carcinomas. To determine the specificity of this fusion and to develop routinely available clinical methods of detection, we developed an RT-PCR assay for paraffin-embedded tissues and a FISH probe for detection of the rearrangements of the PRKACA locus. We also developed an RNA in situ hybridization assay to assess expression levels of the total chimeric transcript and wild-type transcripts. A total of 106 primary liver tumors were studied by RT-PCR, including 26 fibrolamellar carcinomas (4 of which were metastases to the abdominal wall or lymph nodes), 25 conventional hepatocellular carcinomas, 25 cholangiocarcinomas, 25 hepatic adenomas, and 5 hepatoblastomas. RT-PCR was successful in 92% of tested fibrolamellar carcinoma cases (24/26) and the DNAJB1-PRKACA fusion transcript was found in all fibrolamellar carcinomas but not in other tumor types. FISH was tested in 19 fibrolamellar carcinomas and in 6 scirrhous hepatocellular carcinomas, which can closely mimic fibrolamellar carcinoma. Rearrangements of the PRKACA locus was seen in all 19 fibrolamellar carcinoma specimens, but in none of the scirrhous hepatocellular carcinomas. Finally, a RNA in situ hybridization strategy was positive in 7/7 successfully hybridized cases, and showed mRNA over-expression in all of the fibrolamellar carcinomas. In addition, the stromal cells embedded in the characteristic intratumoral fibrosis of fibrolamellar carcinomas and the background liver tissues were negative for the DNAJB1-PRKACA fusion by all tested methods. In conclusion, detection of DNAJB1-PRKACA is a very sensitive and specific finding in support of the diagnosis of fibrolamellar carcinoma.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Proteínas de Choque Térmico HSP40/genética , Proteínas de Fusão Oncogênica/análise , Adolescente , Adulto , Biomarcadores Tumorais/análise , Carcinoma Hepatocelular/patologia , Feminino , Humanos , Hibridização In Situ/métodos , Masculino , Pessoa de Meia-Idade , Proteínas de Fusão Oncogênica/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade , Adulto JovemAssuntos
Gastroenteropatias/diagnóstico , Gastroenteropatias/genética , Fusão Gênica , Janus Quinase 2/genética , Transtornos Linfoproliferativos/diagnóstico , Transtornos Linfoproliferativos/genética , Fator de Transcrição STAT3/genética , Linfócitos T/metabolismo , Gastroenteropatias/metabolismo , Rearranjo Gênico , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Janus Quinase 2/metabolismo , Transtornos Linfoproliferativos/metabolismo , Fator de Transcrição STAT3/metabolismo , Linfócitos T/patologiaAssuntos
Proteínas de Fusão bcr-abl/genética , Hibridização in Situ Fluorescente , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Estudos RetrospectivosRESUMO
Adoptive T cell therapy has been proven effective against melanoma in mice and humans. However, because most responses are incomplete or transient, cures remain rare. To maximize the efficacy of this therapy, it will be essential to gain a better understanding of the processes which result in tumor relapse. We studied these processes using B16ova murine melanoma and adoptive transfer of OT-I T cells. Transfer of T cells as a single therapy provided a significant survival benefit for mice with established subcutaneous tumors. However, tumors which initially regressed often recurred. By analyzing tumors which emerged in the presence of a potent OT-I response, we identified a novel tumor escape mechanism in which tumor cells evaded T cell pressure by undergoing major genomic changes involving loss of the gene encoding the target tumor antigen. Furthermore, we show that these in vivo processes can be recapitulated in vitro using T cell/tumor cell co-cultures. A single round of in vitro co-culture led to significant loss of the ova gene and a tumor cell population with rapidly induced and diverse karyotypic changes. Although these current studies focus on the model OVA antigen, the finding that T cells can directly promote genomic instability has important implications for the development of adoptive T cell therapies.
Assuntos
Transferência Adotiva , Genômica , Melanoma Experimental/imunologia , Linfócitos T/citologia , Evasão Tumoral/genética , Animais , Sequência de Bases , Técnicas de Cocultura , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Instabilidade Genômica , Hibridização in Situ Fluorescente , Ativação Linfocitária , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase em Tempo Real , Linfócitos T/imunologiaRESUMO
Heterochromatin Protein 1 α (HP1α) associates with members of the chromosome passenger complex (CPC) during mitosis, at centromeres where it is required for full Aurora Kinase B (AURKB) activity. Conversely, recent reports have identified AURKB as the major kinase responsible for phosphorylation of HP1α at Serine 92 (S92) during mitosis. Thus, the current study was designed to better understand the functional role of this posttranslationally modified form of HP1α. We find that S92-phosphorylated HP1α is generated in cells at early prophase, localizes to centromeres, and associates with regulators of chromosome stability, such as Inner Centromere Protein, INCENP. In mouse embryonic fibroblasts, HP1α knockout alone or reconstituted with a non-phosphorylatable (S92A) HP1α mutant results in mitotic chromosomal instability characterized by the formation of anaphase/telophase chromatin bridges and micronuclei. These effects are rescued by exogenous expression of wild type HP1α or a phosphomimetic (S92D) variant. Thus, the results from the current study extend our knowledge of the role of HP1α in chromosomal stability during mitosis.
Assuntos
Aurora Quinase B/metabolismo , Instabilidade Cromossômica , Proteínas Cromossômicas não Histona/metabolismo , Animais , Aurora Quinase B/antagonistas & inibidores , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Aberrações Cromossômicas , Células HeLa , Heterocromatina/metabolismo , Humanos , Cinetocoros/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitose , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologiaRESUMO
OBJECTIVE: To test the hypothesis that chromosomal rearrangements (CRs) can distinguish low risk of progression (LRP) from intermediate and high risk of progression (IHRP) to prostate cancer (PCa) and if these CRs have the potential to identify men with LRP on needle biopsy that harbor IHRP PCa in the prostate gland. PATIENTS AND METHODS: Mate pair sequencing of amplified DNA from pure populations of Gleason patterns in 154 frozen specimens from 126 patients obtained between August 14, 2001, and July 15, 2011, was used to detect CRs including abnormal junctions and copy number variations. Potential CR biomarkers with higher incidence in IHRP than in LRP to cancer and having significance in PCa biology were identified. Independent validation was performed by fluorescence in situ hybridization in 152 specimens from 124 patients obtained between February 12, 2002, and July 12, 2008. RESULTS: The number of abnormal junctions did not distinguish LRP from IHRP. Loci corresponding to genes implicated in PCa were more frequently altered in IHRP. Integrated analysis of copy number variations and microarray data yielded 6 potential markers that were more frequently detected in Gleason pattern 3 of a Gleason score 7 of PCa than in Gleason pattern 3 of a Gleason score 6 PCa. Five of those were cross-validated in an independent sample set with statistically significant areas under the receiver operating characteristic curves (AUCs) (P≤.01). Probes detecting deletions in PTEN and CHD1 had AUCs of 0.87 (95% CI, 0.77-0.97) and 0.73 (95% CI, 0.60-0.86), respectively, and probes detecting gains in ASAP1, MYC, and HDAC9 had AUCs of 0.71 (95% CI, 0.59-0.84), 0.82 (95% CI, 0.71-0.93), and 0.77 (95% CI, 0.66-0.89), respectively (for expansion of gene symbols, use search tool at www.genenames.org). CONCLUSION: Copy number variations in regions encompassing important PCa genes were predictive of cancer significance and have the potential to identify men with LRP PCa by needle biopsy who have IHRP PCa in their prostate gland.
Assuntos
Biomarcadores Tumorais/genética , DNA de Neoplasias/genética , Estadiamento de Neoplasias , Próstata/patologia , Neoplasias da Próstata/genética , Idoso , Biomarcadores Tumorais/metabolismo , Biópsia por Agulha , Variações do Número de Cópias de DNA , Progressão da Doença , Seguimentos , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Próstata/metabolismo , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/metabolismo , Curva ROC , Estudos Retrospectivos , Fatores de RiscoRESUMO
OBJECTIVES: MYB rearrangement is observed in approximately 28% to 86% of adenoid cystic carcinomas (ACCs). Also, ACC features a p63+/p40+ immunophenotype in greater than 90% of cases, compared with p63+/p40- polymorphous low-grade adenocarcinoma (PLGA). Our aim was to investigate the incidence of (1) MYB rearrangement and (2) p63/p40 immunoreactivity in ACC and PLGA of minor salivary glands (MSGs). STUDY DESIGN: Seven cases of ACC as well as five of PLGA were evaluated by using a MYB (6 q23.3) break-apart fluorescence in situ hybridization (FISH) probe. In addition, all cases were immunohistochemically stained with p63 and p40 antibodies. RESULTS: All five successfully hybridized ACCs featured MYB rearrangement, whereas PLGAs did not show MYB rearrangement. Interestingly, one case of PLGA demonstrated a single intact copy of MYB in greater than 88% of the neoplastic cells. All ACCs exhibited consistent p63+/p40+ staining, whereas PLGAs demonstrated a p63+/p40- immunophenotype. CONCLUSIONS: (1) MYB rearrangement is encountered in ACCs but not PLGAs of MSGs; (2) MYB aberrations, for example, monosomy or deletion, can be seen in PLGAs; (3) combined p63/p40 immunostaining can be used to differentiate ACC from PLGA in incisionally biopsied specimens; and (4) performance of either FISH or p63/p40 immunohistochemistry is expected to be able to confirm the diagnosis of ACC or PLGA in small intraoral biopsies, since both techniques appeared to be diagnostically accurate in this pilot study.
Assuntos
Carcinoma Adenoide Cístico/genética , Rearranjo Gênico , Genes myb , Neoplasias das Glândulas Salivares/genética , Adulto , Idoso , Biomarcadores Tumorais/análise , Biópsia , Carcinoma Adenoide Cístico/patologia , Feminino , Humanos , Epitopos Imunodominantes/análise , Imuno-Histoquímica , Imunofenotipagem , Hibridização in Situ Fluorescente , Masculino , Proteínas de Membrana/análise , Pessoa de Meia-Idade , Fragmentos de Peptídeos/análise , Projetos Piloto , Neoplasias das Glândulas Salivares/patologia , Glândulas Salivares Menores/patologiaRESUMO
INTRODUCTION: A significant portion of adenoid cystic carcinoma (ACC) cases are characterized by a t(6;9)(q22-23;p23-24) translocation that originates a MYB-NFIB fusion oncogene. The MYB-NFIB fusion oncoprotein activates transcription of MYB-mediated pathways that impact cell cycle control, DNA repair, and apoptosis. This translocation seems highly specific for ACC. Moreover, therapies targeting MYB-activated pathways to treat ACC are being explored. Pulmonary ACC (PACC) has not been thoroughly studied for rearrangements of the MYB gene. METHODS: Mayo Clinic Rochester surgical pathology archives (1972-2011) were searched for PACC. All cases were reviewed and classified according to the predominant histologic pattern (cribriform, solid, and tubular) by two surgical pathologists. Fluorescence in situ hybridization (FISH) was employed using a break-apart strategy to detect MYB rearrangement (at 6q23.3). Medical records were studied. RESULTS: Forty cases of PACC were studied; tissue blocks were available for FISH analysis in 35 cases. Six cases failed to hybridize. In 12 of 29 cases (41%), the MYB gene region was disrupted, whereas 17 cases (59%) showed no evidence of rearrangement. FISH studies performed on other histologic subtypes of lung cancer (10 squamous cell carcinomas, 10 adenocarcinomas, and 10 small-cell carcinomas) failed to show MYB rearrangement. There was no significant difference in MYB rearrangement status with respect to predominant histologic pattern, clinical features, or clinical outcome. CONCLUSIONS: A MYB rearrangement was identified in 41% of PACC and was 100% specific. FISH studies for MYB may be of diagnostic utility in PACC, particularly on small biopsy specimens. MYB rearrangement in PACC does not seem to be associated with clinical features or prognosis.
Assuntos
Carcinoma Adenoide Cístico/patologia , Citogenética/métodos , Proteínas de Fusão Oncogênica/genética , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
OBJECTIVE: To use fluorescence in situ hybridization (FISH) to visualize genetic abnormalities in interphase cell nuclei (interphase FISH) of acinar cell carcinoma, ductal adenocarcinoma, and islet cell carcinoma of the pancreas. PATIENTS AND METHODS: Between April 4, 2007, and December 4, 2008, interphase FISH was used to study paraffin-embedded preparations of tissue obtained from 18 patients listed in the Mayo Clinic Biospecimen Resource for Pancreas Research with a confirmed diagnosis of acinar cell carcinoma, ductal adenocarcinoma, islet cell carcinoma, or pancreas without evidence of neoplasia. FISH probes were used for chromosome loci of APC (see glossary at end of article for expansion of all gene symbols), BRCA2, CTNNB1, EGFR, ERBB2, CDKN2A, TP53, TYMP, and TYMS. These FISH probes were used with control probes to distinguish among various kinds of chromosome abnormalities of number and structure. RESULTS: FISH abnormalities were observed in 12 (80%) of 15 patients with pancreatic cancer: 5 of 5 patients with acinar cell carcinoma, 5 of 5 patients with ductal adenocarcinoma, and 2 (40%) of 5 patients with islet cell carcinoma. All 3 specimens of pancreatic tissue without neoplasia had normal FISH results. Gains of CTNNB1 due to trisomy 3 occurred in each tumor with acinar cell carcinoma but in none of the other tumors in this study. FISH abnormalities of all other cancer genes studied were observed in all forms of pancreatic tumors in this investigation. CONCLUSION: FISH abnormalities of CTNNB1 due to trisomy 3 were observed only in acinar cell carcinoma. FISH abnormalities of genes implicated in familial cancer, tumor progression, and the 5-fluorouracil pathway were common but were not associated with specific types of pancreatic cancer.