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1.
PLoS One ; 9(4): e94202, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24727804

RESUMEN

The identification of gene fusions promises to play an important role in personalized cancer treatment decisions. Many rare gene fusion events have been identified in fresh frozen solid tumors from common cancers employing next-generation sequencing technology. However the ability to detect transcripts from gene fusions in RNA isolated from formalin-fixed paraffin-embedded (FFPE) tumor tissues, which exist in very large sample repositories for which disease outcome is known, is still limited due to the low complexity of FFPE libraries and the lack of appropriate bioinformatics methods. We sought to develop a bioinformatics method, named gFuse, to detect fusion transcripts in FFPE tumor tissues. An integrated, cohort based strategy has been used in gFuse to examine single-end 50 base pair (bp) reads generated from FFPE RNA-Sequencing (RNA-Seq) datasets employing two breast cancer cohorts of 136 and 76 patients. In total, 118 fusion events were detected transcriptome-wide at base-pair resolution across the 212 samples. We selected 77 candidate fusions based on their biological relevance to cancer and supported 61% of these using TaqMan assays. Direct sequencing of 19 of the fusion sequences identified by TaqMan confirmed them. Three unique fused gene pairs were recurrent across the 212 patients with 6, 3, 2 individuals harboring these fusions respectively. We show here that a high frequency of fusion transcripts detected at the whole transcriptome level correlates with poor outcome (P<0.0005) in human breast cancer patients. This study demonstrates the ability to detect fusion transcripts as biomarkers from archival FFPE tissues, and the potential prognostic value of the fusion transcripts detected.


Asunto(s)
Biomarcadores de Tumor/análisis , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Formaldehído/química , Proteínas de Fusión Oncogénica/análisis , Adhesión en Parafina/métodos , Femenino , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Técnicas In Vitro
2.
PLoS One ; 7(7): e40092, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22808097

RESUMEN

RNA biomarkers discovered by RT-PCR-based gene expression profiling of archival formalin-fixed paraffin-embedded (FFPE) tissue form the basis for widely used clinical diagnostic tests; however, RT-PCR is practically constrained in the number of transcripts that can be interrogated. We have developed and optimized RNA-Seq library chemistry as well as bioinformatics and biostatistical methods for whole transcriptome profiling from FFPE tissue. The chemistry accommodates low RNA inputs and sample multiplexing. These methods both enable rediscovery of RNA biomarkers for disease recurrence risk that were previously identified by RT-PCR analysis of a cohort of 136 patients, and also identify a high percentage of recurrence risk markers that were previously discovered using DNA microarrays in a separate cohort of patients, evidence that this RNA-Seq technology has sufficient precision and sensitivity for biomarker discovery. More than two thousand RNAs are strongly associated with breast cancer recurrence risk in the 136 patient cohort (FDR <10%). Many of these are intronic RNAs for which corresponding exons are not also associated with disease recurrence. A number of the RNAs associated with recurrence risk belong to novel RNA networks. It will be important to test the validity of these novel associations in whole transcriptome RNA-Seq screens of other breast cancer cohorts.


Asunto(s)
Neoplasias de la Mama/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Recurrencia Local de Neoplasia/genética , Adhesión en Parafina , Análisis de Secuencia de ARN , Fijación del Tejido , Secuencia de Bases , Biomarcadores de Tumor/genética , ADN Intergénico/genética , Femenino , Formaldehído , Humanos , Intrones/genética , Modelos de Riesgos Proporcionales , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico , Factores de Riesgo
3.
Am J Pathol ; 164(1): 35-42, 2004 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-14695316

RESUMEN

Throughout the last decade many laboratories have shown that mRNA levels in formalin-fixed and paraffin-embedded (FPE) tissue specimens can be quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) techniques despite the extensive RNA fragmentation that occurs in tissues so preserved. We have developed RT-PCR methods that are sensitive, precise, and that have multianalyte capability for potential wide use in clinical research and diagnostic assays. Here it is shown that the extent of fragmentation of extracted FPE tissue RNA significantly increases with archive storage time. Probe and primer sets for RT-PCR assays based on amplicons that are both short and homogeneous in length enable effective reference gene-based data normalization for cross comparison of specimens that differ substantially in age. A 48-gene assay used to compare gene expression profiles from the same breast cancer tissue that had been either frozen or FPE showed very similar profiles after reference gene-based normalization. A 92-gene assay, using RNA extracted from three 10- micro m FPE sections of archival breast cancer specimens (dating from 1985 to 2001) yielded analyzable data for these genes in all 62 tested specimens. The results were substantially concordant when estrogen receptor, progesterone receptor, and HER2 receptor status determined by RT-PCR was compared with immunohistochemistry assays for these receptors. Furthermore, the results highlight the advantages of RT-PCR over immunohistochemistry with respect to quantitation and dynamic range. These findings support the development of RT-PCR analysis of FPE tissue RNA as a platform for multianalyte clinical diagnostic tests.


Asunto(s)
Artefactos , Neoplasias de la Mama/genética , Perfilación de la Expresión Génica , ARN Neoplásico/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias de la Mama/metabolismo , Perfilación de la Expresión Génica/métodos , Humanos , Inmunohistoquímica , Adhesión en Parafina , Reproducibilidad de los Resultados , Estudios Retrospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Sensibilidad y Especificidad , Factores de Tiempo
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