Gene mutation and rearrangement testing are based upon decades of characterization of the molecular alterations responsible for driving thyroid tumorigenesis . These studies have culminated in recent large-scale sequencing projects, such as The Cancer Genome Atlas (TCGA) sequencing study , and have resulted in a comprehensive profile of the landscape of alterations in thyroid tumors.

The TCGA study of papillary thyroid cancer examined single-nucleotide variants, small indels, copy number alterations, rearrangements, mRNA expression, miRNA expression, and DNA methylation of 496 papillary thyroid carcinomas [13]. Driver alterations were identified by this analysis for 96.5% of cases [13]. Thus, driver mutations that account for nearly all papillary thyroid cancers have now been described. The majority of alterations seen in papillary thyroid carcinoma involve the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K) pathways. The knowledge gained through prior studies and large-scale sequencing projects have guided the design of gene mutation/rearrangement panels.

Sensitivity measures the proportion of actual positives which are true positives, and specificity measures the proportion of negatives which are true negatives. Sensitivity and specificity reflect the performance characteristics of a test. PPV and NPV, however, will vary depending on the prevalence of disease. For example, a patient population with higher incidence of thyroid cancer than the population where the test was validated will have a lower NPV . Another factor that may affect observed NPV and PPV may be institutional differences in the malignancy rates for each indeterminate cytology category. Published test performance characteristics are summarized in Table 15.2.

Strengths of gene mutation/rearrangement panels are their high specificity and PPV for malignancy. In general, these tests are being utilized to “rule in” malignancy. Strengths of gene expression classifiers are their high sensitivity and NPV. This test is being used to “rule out” malignancy. The ideal diagnostic test would have high sensitivity, specificity, NPV, and PPV and would be able to both rule in and rule out malignancy. Combination testing is one approach being pursued: for example, addition of the ThyraMIR test to the ThyGenX test gives a sensitivity of 89%, specificity of 85%, NPV of 94%, and PPV of 74%. Also, addition of the Afirma MTC or Afirma BRAF malignancy classifiers may add some specificity to the Afirma GEC , although no data regarding this has been published yet.

Of the currently commercially available tests, the ThyroSeq v2 test shows much promise as a stand-alone test that could be used to both rule in and rule out malignancy, with a NPV of 96% and PPV of 83% in FN/SFN nodules and a NPV of 97.2% and PPV of 76.9% in AUS/FLUS nodules . Further understanding of thyroid pathogenesis and the potential role of cooperating genes or mutations may help in further refining the NPV and PPV of the panel. In particular, RAS alterations, which may be seen in carcinomas as well as benign/indolent neoplasms such as follicular adenomas or NIFTP , confer a lower risk of malignancy of 74 to 87% than other alterations such as the BRAF V600E mutation which has a >95% risk of malignancy [12, 36, 37]. Knowledge of the differences in risk based on the specific gene mutation present can guide patient management. Furthermore, use of large, NGS-based mutation panels would allow for the discovery of co-occurring mutations (e.g., in TP53 or EIF1AX) that may prove to be associated with increased risk.

Test performance characteristics have formed the basis of clinical algorithms to guide the use of molecular testing in guiding perioperative decision making [81]. For a positive result on seven-gene mutation/rearrangement panels, which have high specificity and high PPV, indeterminate cytology thyroid nodules may be managed with oncologic thyroidectomy. A negative result on a seven-gene mutation/rearrangement panel may be managed by observation or diagnostic thyroid lobectomy for AUS/FLUS nodules and by diagnostic thyroid lobectomy for FN/SFN or SMC nodules. Gene expression classifiers have high sensitivity and high NPV. For a benign result, observation or diagnostic thyroid lobectomy would be appropriate, and for a suspicious result on AUS/FLUS or FN/SFN nodules, at least a diagnostic lobectomy should be considered.

For the ThyroSeq v2 panel , which has good overall sensitivity, specificity, NPV, and PPV, the following clinical algorithm can be considered (Fig. 15.1). When test is negative for all alterations, in nodules with Bethesda III and IV cytology (and pretest cancer probability is that expected for these Bethesda categories), the residual probability of cancer is expected to be 3–4%. According to the NCCN guidelines, these patients can be followed by observation, similar to the recommendations for benign cytology nodules. For nodules with a positive ThyroSeq result, the type of mutation and other test results allow one to predict the probability of cancer in the nodule and estimate how aggressive the cancer is, helping to define the most appropriate surgical approach. An isolated RAS or RAS-like mutation predicts a high probability (~80%) of either low-risk cancer or a precancerous tumor, NIFTP . Many of these patients can be treated with therapeutic lobectomy, which is currently recommended for low-risk thyroid cancers. Isolated BRAF V600E or other BRAF V600E-like mutations confer a very high (>95%) probability of cancer of intermediate risk for disease recurrence. The risk may be further modified by clinical parameters; for example, small (<1 cm) tumors may still be of low risk. These patients can be treated with total thyroidectomy or lobectomy. Test positivity for multiple driver mutations is virtually diagnostic of cancer and predicts a significant risk of disease recurrence and tumor-related mortality. These patients would benefit from total thyroidectomy, with consideration for central compartment lymph node dissection.