Whole transcriptome RNA-Seq analysis of breast cancer recurrence risk using formalin-fixed paraffin-embedded tumor tissue.

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.

Rt-PCR gene expression profiling of RNA from paraffin-embedded tissues prepared using a range of different fixatives and conditions.

Although RNA is isolated from archival fixed tissues routinely for reverse transcription polymerase chain reaction (RT-PCR) and microarray analyses to identify biomarkers of cancer prognosis and therapeutic response prediction, the sensitivity of these molecular profiling methods to variability in pathology tissue processing has not been described in depth. As increasing numbers of expression analysis studies using fixed archival tumor specimens are reported, it is important to examine how dependent these results are on tissue-processing methods.We carried out a series of studies to systematically evaluate the effects of various tissue-fixation reagents and protocols on RNA quality and RT-PCR gene expression profiles.

Tumor marker discovery by expression profiling RNA from formalin fixed paraffin embedded tissues.

Clear identification among early-stage cancer patients of those at highest risk of having metastatic disease would be of great benefit in treatment planning and management. Considerable additional benefit would accrue to high-risk patients if their responses to specific therapeutic alternatives could be predicted. Molecular biomarkers in the form of gene expression profiles are proving to be more effective tools for both prognostic and predictive patient stratification than more traditional methods such as patient demographics and histopathology indicators.

Analytical validation of the Oncotype DX genomic diagnostic test for recurrence prognosis and therapeutic response prediction in node-negative, estrogen receptor-positive breast cancer.

Background: Oncotype DX is a clinically validated, high-complexity, multianalyte reverse transcription-PCR genomic test that predicts the likelihood of breast cancer recurrence in early-stage, node-negative, estrogen receptor-positive breast cancer. The Recurrence Score (RS) provides a more accurate, reproducible measure of breast cancer aggressiveness and therapeutic responsiveness than standard measures. Individualized patient management requires strict performance criteria for clinical laboratory tests.

A population-based study of tumor gene expression and risk of breast cancer death among lymph node-negative patients.

Introduction: The Oncotype DX assay was recently reported to predict risk for distant recurrence among a clinical trial population of tamoxifen-treated patients with lymph node-negative, estrogen receptor (ER)-positive breast cancer. To confirm and extend these findings, we evaluated the performance of this 21-gene assay among node-negative patients from a community hospital setting.

Methods: A case-control study was conducted among 4,964 Kaiser Permanente patients diagnosed with node-negative invasive breast cancer from 1985 to 1994 and not treated with adjuvant chemotherapy.

Measurement of gene expression in archival paraffin-embedded tissues: development and performance of a 92-gene reverse transcriptase-polymerase chain reaction assay.

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.