ANALYTICAL VERIFICATION OF A NOVEL GENE EXPRESSION TEST WHICH
IDENTIFIES BENIGN THYROID NODULES IN PATIENTS WITH INDETERMINATE
FINE NEEDLE-ASPIRATION (FNA) CYTOLOGY
Authors: Sean Walsh, Jonathan Wilde, Ed Tom, Jessica Reynolds, Daphne Chen, Jim
Veitch, Darya Chudova, Eric Wang, Lyssa Friedman, Rob Monroe, David Steward,
Mark Lupo, Richard Lanman and Giulia C. Kennedy
Keywords: thyroid, FNA, cytopathology, molecular analysis, gene expression, nodules
A novel multigene expression (mRNA) test was developed to identify benign thyroid
nodules in a subgroup of patients with indeterminate cytologic FNA. A high negative
predictive value would enable physicians to safely avoid surgery for patients classified
with benign nodules by the gene expression signature.
Approximately one thousand thyroid FNAs were collected either in the clinic or
preoperatively (after anesthesia, before incision) in the operating room. FNA material (1
or 2 passes) was placed directly into an FNA preservative solution. The samples were
shipped to the reference laboratory, where total RNA was extracted and a wholetranscript amplification procedure was used to prepare the sample for hybridization onto
a custom gene expression microarray. The performance of this gene expression test
was evaluated in a series of reagent and analytical verification studies, as summarized
below.
FNA preservative solution was tested for its ability to preserve the integrity of FNA
material under stressed time and temperature conditions, with results yielding high
quality and quantity RNA after multiple days of storage at room temperature, and high
tolerance for sample shipping conditions.
Extraction of total RNA from FNA samples yielded high quality total RNA typically in the
range of 25-500 ng. A custom manufactured tissue lysate control reagent, used as a
process control for the RNA extraction step, was tested over multiple weeks of
independent runs across multiple lots, demonstrating consistent test performance.
Custom manufactured total RNA control samples were developed and tested for use as
in-process controls for the gene expression test (one benign and one malignant total
RNA). These controls were tested alongside real FNA samples over multiple lots,
operators, runs, and in the background of variation in other reagent lots. In all cases,
controls and test FNA samples classified correctly, demonstrating high reproducibility in
the test classification/result for repeated tests on the same sample.
Human blood and genomic DNA were tested separately as potential interfering
substances on the gene expression test. Neither substance was found to interfere with
extraction or amplification steps of the assay. Malignant FNAs were highly resistant to
classification changes, including up to 90% blood content.
In summary, extensive reagent and analytical performance studies were conducted to
evaluate the reliability and reproducibility of a novel gene expression test for thyroid
FNA samples. The results indicate that the test produces a clinically robust and
reproducible result under a variety of experimental and clinical conditions.