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. Such biomarkers must be clinically validated before they can be effectively used to manage patients in clinical studies or clinical practice. This can be most efficiently accomplished by analyzing archived clinical samples with well-characterized clinical outcomes. Doing studies of this type requires reoptimization of traditional molecular expression profiling techniques to analyze RNA from fixed paraffin-embedded tissues. We have modified molecular methods for RNA extraction, RNA quantification, reverse transcription, and quantitative PCR to work optimally in archived clinical samples in order to develop a clinically validated assay for breast cancer prognosis and prediction of patient response to hormonal and chemotherapy.

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. Cases (n = 220) were patients who died from breast cancer. Controls (n = 570) were breast cancer patients who were individually matched to cases with respect to age, race, adjuvant tamoxifen, medical facility and diagnosis year, and were alive at the date of death of their matched case. Using an RT-PCR assay, archived tumor tissues were analyzed for expression levels of 16 cancer-related and five reference genes, and a summary risk score (the Recurrence Score) was calculated for each patient. Conditional logistic regression methods were used to estimate the association between risk of breast cancer death and Recurrence Score. RESULTS: After adjusting for tumor size and grade, the Recurrence Score was associated with risk of breast cancer death in ER-positive, tamoxifen-treated and -untreated patients (P = 0.003 and P = 0.03, respectively). At 10 years, the risks for breast cancer death in ER-positive, tamoxifen-treated patients were 2.8% (95% confidence interval [CI] 1.7-3.9%), 10.7% (95% CI 6.3-14.9%), and 15.5% (95% CI 7.6-22.8%) for those in the low, intermediate and high risk Recurrence Score groups, respectively. They were 6.2% (95% CI 4.5-7.9%), 17.8% (95% CI 11.8-23.3%), and 19.9% (95% CI 14.2-25.2%) for ER-positive patients not treated with tamoxifen. In both the tamoxifen-treated and -untreated groups, approximately 50% of patients had low risk Recurrence Score values. CONCLUSION: In this large, population-based study of lymph node-negative patients not treated with chemotherapy, the Recurrence Score was strongly associated with risk of breast cancer death among ER-positive, tamoxifen-treated and -untreated patients.

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. 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.

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. Human placenta was selected as a model specimen for these studies since it is relatively easily obtained and has proliferative and invasive qualities similar to solid tumors. In addition, each specimen is relatively homogeneous and large enough to provide sufficient tissue to systematically compare a range of fixation conditions and reagents, thereby avoiding the variability inherent in studying collections of tumor tissue specimens. Since anatomical pathology laboratories generally offer hundreds of different tissue-fixation protocols, we focused on fixation reagents and conditions used to process the most common solid tumors for primary cancer diagnosis. Fresh placentas donated under an IRB-approved protocol were collected at delivery and immediately submerged in cold saline for transport to a central pathology laboratory for processing. RNA was extracted from each specimen, quantified, and analyzed for size distribution and analytical performance using a panel of 24 RT-PCR gene expression assays. We found that different tissue-fixation reagents and tissue-processing conditions resulted in widely varying RNA extraction yields and extents of RNA fragmentation. However, the RNA extraction method and RT-PCR assays could be optimized to achieve successful gene expression analysis for nearly all fixation conditions represented in these studies.

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. We therefore investigated the analytical performance of the assay. METHODS: Assays used a pooled RNA sample from fixed paraffin-embedded tissues to evaluate the analytical performance of a 21-gene panel with respect to amplification efficiency, precision, linearity, and dynamic range, as well as limits of detection and quantification. Performance variables were estimated from assays carried out with sample dilutions. In addition, individual patient samples were used to test the optimized assay for reproducibility and sources of imprecision. RESULTS: Assay results defined acceptable operational performance ranges, including an estimated maximum deviation from linearity of <1 cycle threshold (C(T)) units over a > or =2000-fold range of RNA concentrations, with a mean quantification bias of 0.3% and CVs of 3.2%-5.7%. An analysis of study design showed that assay imprecision contributed by instrument, operator, reagent, and day-to-day baseline variation was low, with SDs of <0.5 C(T). CONCLUSION: The analytical and operational performance specifications defined for the Oncotype DX assay allow the reporting of quantitative RS values for individual patients with an SD within 2 RS units on a 100-unit scale.

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. 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.