Validation and reproducibility of a microarray-based gene expression test for tumor identification in formalin-fixed, paraffin-embedded specimens.

Tumors whose primary site is challenging to diagnose represent a considerable proportion of new cancer cases. We present validation study results for a gene expression-based diagnostic test (the Pathwork Tissue of Origin Test) that aids in determining the tissue of origin using formalin-fixed, paraffin-embedded (FFPE) specimens. Microarray data files were generated for 462 metastatic, poorly differentiated, or undifferentiated FFPE tumor specimens, all of which had a reference diagnosis. The reference diagnoses were masked, and the microarray data files were analyzed using a 2000-gene classification model. The algorithm quantifies the similarity between RNA expression patterns of the study specimens and the 15 tissues on the test panel. Among the 462 specimens, overall agreement with the reference diagnosis was 89% (95% CI, 85% to 91%). In addition to the positive test results (ie, rule-ins), an average of 12 tissues for each specimen could be ruled out with >99% probability. The large size of this study increases confidence in the test results. A multisite reproducibility study showed 89.3% concordance between laboratories. The Tissue of Origin Test makes the benefits of microarray-based gene expression tests for tumor diagnosis available for use with the most common type of histology specimen (ie, FFPE).

Molecular classification of thyroid nodules using high-dimensionality genomic data.

OBJECTIVE: We set out to develop a molecular test that distinguishes benign and malignant thyroid nodules using fine-needle aspirates (FNA). DESIGN: We used mRNA expression analysis to measure more than 247,186 transcripts in 315 thyroid nodules, comprising multiple subtypes. The data set consisted of 178 retrospective surgical tissues and 137 prospectively collected FNA samples. Two classifiers were trained separately on surgical tissues and FNAs. The performance was evaluated using an independent set of 48 prospective FNA samples, which included 50% with indeterminate cytopathology. RESULTS: Performance of the tissue-trained classifier was markedly lower in FNAs than in tissue. Exploratory analysis pointed to differences in cellular heterogeneity between tissues and FNAs as the likely cause. The classifier trained on FNA samples resulted in increased performance, estimated using both 30-fold cross-validation and an independent test set. On the test set, negative predictive value and specificity were estimated to be 96 and 84%, respectively, suggesting clinical utility in the management of patients considering surgery. Using in silico and in vitro mixing experiments, we demonstrated that even in the presence of 80% dilution with benign background, the classifier can correctly recognize malignancy in the majority of FNA samples. CONCLUSIONS: The FNA-trained classifier was able to classify an independent set of FNAs in which substantial RNA degradation had occurred and in the presence of blood. High tolerance to dilution makes the classifier useful in routine clinical settings where sampling error may be a concern. An ongoing multicenter clinical trial will allow us to validate molecular test performance on a larger independent test set of prospectively collected thyroid FNAs.

Multicenter validation of a 1,550-gene expression profile for identification of tumor tissue of origin.

PURPOSE: Malignancies found in unexpected locations or with poorly differentiated morphologies can pose a significant challenge for tissue of origin determination. Current histologic and imaging techniques fail to yield definitive identification of the tissue of origin in a significant number of cases. The aim of this study was to validate a predefined 1,550-gene expression profile for this purpose. METHODS: Four institutions processed 547 frozen specimens representing 15 tissues of origin using oligonucleotide microarrays. Half of the specimens were metastatic tumors, with the remainder being poorly differentiated and undifferentiated primary cancers chosen to resemble those that present as a clinical challenge. RESULTS: In this blinded multicenter validation study the 1,550-gene expression profile was highly informative in tissue determination. The study found overall sensitivity (positive percent agreement with reference diagnosis) of 87.8% (95% CI, 84.7% to 90.4%) and overall specificity (negative percent agreement with reference diagnosis) of 99.4% (95% CI, 98.3% to 99.9%). Performance within the subgroup of metastatic tumors (n = 258) was found to be slightly lower than that of the poorly differentiated and undifferentiated primary tumor subgroup, 84.5% and 90.7%, respectively (P = .04). Differences between individual laboratories were not statistically significant. CONCLUSION: This study represents the first adequately sized, multicenter validation of a gene-expression profile for tissue of origin determination restricted to poorly differentiated and undifferentiated primary cancers and metastatic tumors. These results indicate that this profile should be a valuable addition or alternative to currently available diagnostic methods for the evaluation of uncertain primary cancers.

Interlaboratory performance of a microarray-based gene expression test to determine tissue of origin in poorly differentiated and undifferentiated cancers.

Clinical workup of metastatic malignancies of unknown origin is often arduous and expensive and is reported to be unsuccessful in 30 to 60% of cases. Accurate classification of uncertain primary cancers may improve with microarray-based gene expression testing. We evaluated the analytical performance characteristics of the Pathwork tissue of origin test, which uses expression signals from 1668 probe sets in a gene expression microarray, to quantify the similarity of tumor specimens to 15 known tissues of origin. Sixty archived tissue specimens from poorly and undifferentiated tumors (metastatic and primary) were analyzed at four laboratories representing a wide range of preanalytical conditions (eg, personnel, reagents, instrumentation, and protocols). Cross-laboratory comparisons showed highly reproducible results between laboratories, with correlation coefficients between 0.95 to 0.97 for measurements of similarity scores, and an average 93.8% overall concordance between laboratories in terms of final tissue calls. Bland-Altman plots (mean coefficients of reproducibility of 32.48+/-3.97) and kappa statistics (kappa >0.86) also indicated a high level of agreement between laboratories. We conclude that the Pathwork tissue of origin test is a robust assay that produces consistent results in diverse laboratory conditions reflecting the preanalytical variations found in the everyday clinical practice of molecular diagnostics laboratories.

Tissue handling for genome-wide expression analysis: a review of the issues, evidence, and opportunities.

CONTEXT: Molecular diagnostic applications that use microarrays to analyze large numbers of genes simultaneously require high-quality mRNA. As these genome-wide expression assays become more commonly used in medical practice, pathologists and oncologists will benefit from understanding the importance of obtaining high-quality RNA in order to generate reliable diagnostic and prognostic information, especially as these relate to cancer. OBJECTIVE: To review the effects that different tissue preservation techniques have on RNA quality and to provide practical advice on changes in tissue acquisition and handling that may soon be needed for certain clinical situations. DATA SOURCES: A review of recent literature on RNA quality, tissue fixation, cancer diagnosis, and gene expression analysis. CONCLUSIONS: Studies have consistently shown that frozen tissue yields more intact RNA than formalin-fixed, paraffin-embedded tissue. The chemical modification, cross-linking, and fragmentation caused by formalin fixation often render RNA unsuitable for microarray analysis. Thus, when expression analysis involving hundreds or more than 1000 gene markers is contemplated, pathologists should consider freezing a specimen within half an hour (preferably within minutes) of surgical resection and storing it at -80 degrees C or below. In coming years, pathologists will need to work closely with oncologists and other clinicians to determine when saving frozen tissue for microarray expression analysis is both practical and necessary. In select cases, the benefit of implementing a few extra tissue-handling steps may improve diagnostic and prognostic capability.