Clinical Validation of ThyroidPrint: A Gene Expression Signature for Diagnosis of Indeterminate Thyroid Nodules

Thyroid nodules are a very frequent condition reaching up to 30-40% of the adult population.
Although most thyroid nodules have little clinical significance, in many cases a fine needle
aspirate (FNA) biopsy will be performed to determine its nature. In 70% of cases, a FNA will
be reported as benign and in 10% of cases as cancer. However, the remaining 20% of cases the
thyroid nodule will be reported as indeterminate. The latter patients have a risk of
malignancy ranging from 15 to 25%, and in most cases the patient will undergo thyroid
lobectomy or total thyroidectomy to determine the final pathology, resulting in an
unacceptable number of unnecessary surgeries. This has a major health impact, including
surgical risks and permanent hormonal supplementation, as well as unwarranted health costs
estimated at 1.6 billion USD. Therefore, there is a need for diagnostic tools in order to
improve the diagnostic accuracy of the FNA and avoidance of the high rate of unnecessary
surgeries.

GeneproDx has developed a gene expression signature to improve the diagnostic accuracy of FNA
biopsy of thyroid nodules reported as indeterminate. The ThyroidPrint diagnostic measures the
expression of 10 genes in a FNA sample. It combines the results of the 10 biomarkers using a
proprietary algorithm to predict benign thyroid nodules. This assay is classified as
multi-analyte algorithm assays (MAAA).

The biomarkers consist of multiplex TaqMan® gene expression assays run on Qiagen's Rotor-Gene
Q MDx RT-PCR IVD Platform instrument, which is a FDA cleared instrument. The following 10
genes comprise the biomarker panel: CXCR3, CCR3, CXCL10, CK19, TIMP1, CLDN1, CAR, XB130, HO-1
and CCR7. Each gene run in a multiplex configuration with two reference genes. Each assay is
performed with Research Use Only (RUO) kits and reagents on a FDA cleared instrument.

ThyroidPrint has been developed using two different cohorts of samples, a training set and a
testing set. Using linear discriminant analysis, the training set identified the final
biomarker panel including; CXCR3, CCR3, CXCL10, CK19, TIMP1, CLDN1, CAR, XB130, HO-1 and
CCR7. In brief, the biomarkers have the following significance. CCR3 and CCR7 are chemokine
receptors that are highly expressed in papillary thyroid cancer tumor cells. CXCR3 is also a
chemokine receptor and along with its receptor, CXCL10, are detected in thyroid autoimmune
disease. CAR is a G-couple receptor and has been shown to be involved in cancer and has
decreased expression in parathyroid adenoma. CK19 is a keratin and has been used in thyroid
tumors to recognize papillary carcinomas. CLDN1 is a structural protein and has been shown to
be differentially expressed in tumors compared to normal tissue and has increased mRNA levels
in papillary thyroid carcinoma. XB130 is also a structural protein and its expression has
been demonstrated in papillary thyroid carcinoma. TIMP1 is a protease inhibitor with mRNA
levels increased in advanced stages of thyroid carcinoma. HO- 1 is an oxygenase and its
expression has correlates with tumor aggressiveness in thyroid cancer. In the final
classifier, the expression of each gene was ̈weighted ̈ based on its individual relative
classifying ability. The cutoff score was chosen in the ROC curve generated in the training
set based on a minimum Sensitivity of 92% to guarantee a high Negative Predictive Value
(>95%). This cutoff score offered a Specificity of 83%.An independent testing set of samples
reproduced the diagnostic performance observed in the training set and showed consistent
results in FNA samples. The assay has proven to accurately predict benign nodules in thyroid
FNA samples with a Negative Predictive Value of 96% and Specificity of 83% in both cohorts.
The definitive validation of an MAAA requires a final validation set, which analyzes samples
that will be used in the routine clinical setting; in this case indeterminate thyroid nodules
samples. In addition, in order to show clinical validity, the validation set must be
performed, as a statistically powered multi-institutional trial to assure that the data is
applicable to a broad population spectrum and has appropriate confidence intervals. A first,
statistically powered, multi-institutional trial is currently underway in Chile to prove
Clinical Validity. This trial includes 8 sites and will recruit approximately 3000 FNA to be
completed by December 2017.

Inclusion Criteria:

1. Patients undergoing FNA of a thyroid nodule.

2. Thyroid nodule greater 1cm

3. Age greater 18 years old.

Exclusion Criteria:

1. Thyroid nodule greater 1cm

2. Patients less than 18 years old,

3. Previous history of coagulation disorders and patients.

4. Ultrasound evidence of malignant cervical adenopathy.