Molecular Markers Emerge in the Diagnosis of Thyroid Cancer

Virginia LiVolsi, MD

Although prognostic biomarkers have become increasingly important in the treatment of many types of cancer, no such markers have yet been identified and validated for thyroid cancer. In order to guide treatment decisions, molecular testing will undoubtedly become an important part of thyroid cancer treatment as targeted therapies, such as sorafenib, become available and are shown to confer a benefit in patient survival.¹

There is evidence that molecular testing may be of benefit far earlier in the course of treatment, at the diagnostic stage. “The word ‘evolution’ really comes to mind,” said Virginia LiVolsi, MD, professor of pathology and laboratory medicine at the University of Pennsylvania, explaining that a series of published articles reported howBRAFmutation testing was going to be predictive of more aggressive behavior in differentiated papillary thyroid cancer (PTC).^2,3

Lori J. Wirth, MD, on Unmet Needs in the Treatment of Thyroid Cancer

Wirth is a professor at Harvard Medical School.

In her recent presentation at the 2014 Annual Meeting of the American Head and Neck Society (AHNS), LiVolsi described how molecular testing for genetic alterations such asBRAFandRASmutation and RET/PTC translocation has begun to influence diagnosis with fine needle aspiration biopsy (FNAB) specimens, which are typically categorized pathologically according to the 6-category Bethesda classification.

She noted that there are essentially 2 main questions that one would want to ask regarding molecular testing for a patient with indeterminate cytology by FNAB: “Is it neoplastic or not?” and “If neoplastic, is it benign or malignant?”

In the case of a specimen that is atypical, suspicious, and categorized pathologically as a category 3 or 4, for example, results from molecular testing could have an impact on the overall diagnosis and management plan, according to LiVolsi. “If you do testing for molecular markers commonly found in malignant thyroid cancer, and found such a marker—the most notable being BRAF—you could be 99% sure, almost as close to certain as you could be, that this was indeed a papillary carcinoma.”

With this type of result, initial therapy would be that used for an unequivocally known papillary carcinoma, which in most cases would be a total thyroidectomy, with or without the central node compartment, she explained. Using this information, “You could then assure the patient that he or she does indeed require a total thyroidectomy, without need of doing a separate surgical procedure,” LiVolsi said.

For patients with higher categories of suspicion for malignancy (category 5 or 6), LiVolsi explained that they would already be candidates for a total thyroidectomy, and there could be some debate about whether or not molecular testing would be helpful. She noted that for clinicians who believe that positive testing forBRAFindicates a more aggressive lesion, this might lead to a more aggressive initial surgical approach, such as removal of the central compartment and evaluation of the lateral nodes. “When I say more aggressive lesion, I don't mean that the lesion is going to metastasize to the lungs tomorrow. I mean that there might be a greater risk of local recurrence,” LiVolsi added.

LiVolsi also described how pathologists now have at least 2 multigene molecular tests that can begin to answer these questions, including an RNA-based test that divides lesions into benign or not benign categories using sample-derived RNA and a panel of 167 genes. This test has a 95% negative predictive value, which could reassure patients that a lesion was in fact a benign lesion that could be watched, without surgical intervention.

A second molecular test is used to determine whether or not a lesion has a molecular signature known to be malignant; this is a DNA-based test that detects the presence of multiple mutations and translocations and has a high positive predictive value that could be used to inform clinicians of the need for aggressive intervention if any such mutations and/or translocations are found. Such information might also be used in the future to guide the use of targeted therapies that are aimed at a specific genetic lesion.

LiVolsi noted recent developments, such as results from The Cancer Genome Atlas for PTC, which will also factor into the role of molecular testing in the near future. That study will soon be published by Giordano and colleagues and describes results of whole-genome sequencing for some 500 PTC cases. The findings have identified 2 main families at the molecular level, and a third family classified as “Other.”

The first family, the BRAF family, is predominantly papillary carcinoma, with either classic type histology or the tall cell variant. These tumors have multifocality in the gland and tend to metastasize to the lymph node; with the exception of the tall cell variant, they tend to be quite indolent.

Clinical Pearls

• Molecular testing will become an important part of thyroid cancer treatment as targeted therapies, such as sorafenib, become available and are shown to confer a benefit in patient survival.
• Molecular testing may be of benefit far earlier in the course of treatment, at the diagnostic stage.
• Genetic alterations such asBRAFandRASmutation and RET/PTC translocation has begun to influence diagnosis with FNAB specimens.
• Pathologists now have at least 2 multigene molecular tests, including an RNA-based test that divides lesions into benign or not benign categories using sample-derived RNA and a panel of 167 genes.
• A second molecular test may determine whether or not a lesion has a molecular signature known to be malignant; this test has a high positive predictive value that could be used to inform clinicians of the need for aggressive intervention.
• Several questions remain unanswered, including what type of molecular testing should be done; which FNAB specimens should be tested; which surgical specimens should be tested; and how such information will be used by the surgeon.

The second family, the RAS family, tends to be papillary carcinoma as defined by nuclear changes and follicular pattern, encapsulated or partly encapsulated, with less multifocality in the gland and less risk for lymph node metastases. However, ≈20% of these cancers have vascular invasion and capacity for spread to distant sites, such as the lungs and bone.

According to LiVolsi, the third family, “Other,” is “all over the map” with many different mutations and epigenetic changes. Although fewer samples corresponding to this group were studied overall, LiVolsi noted that this group has the most potential for future study that is likely to be of diagnostic or prognostic import.

The use of molecular testing in thyroid cancer diagnosis is not without controversy. For example, while some studies support the role ofBRAFmutations, such as V600E, in promoting aggressive tumor behavior,^3,4others have suggested that when factors such as extrathyroidal invasion, lymph node metastases, histologic subtype, and distant metastases are taken into account, BRAF (V600E) status was no longer independently associated with mortality.⁵

Other chromosomal translocations, such as the thyroid transcription factor PAX8/peroxisome proliferator-activated receptor gamma (PPARγ) fusion, originally thought to be specific for follicular patterned thyroid lesions,⁶have also been found to be expressed in nonlesional tissues and follicular adenomas and thus may have limited utility as a diagnostic marker.⁷

LiVolsi said there are several currently unanswered questions, including what type of molecular testing should be done (eg, isolated markers vs a panel of genes); which FNAB specimens should be tested (eg, all FNAB vs only indeterminate FNAB); which surgical specimens should be tested (eg, all PTC vs extraglandular or metastatic PTC); and how such information will be used by the surgeon (eg, lobectomy vs total thyroidectomy). She also noted that follow-up for most markers is, in most cases, only on the order of approximately 3 years; for PTC, longer follow- up will be needed. According to LiVolsi, such studies will need to be conducted retrospectively; for example, by using archived formalin fixed paraffin embedded (FFPE) specimens.

Lastly, the predictive value of these molecular markers versus classical predictors of tumor size, stage, and lymph node invasion also remains to be evaluated.

References

1. Brose MS, Nutting CM, Jarzab B, et al, on behalf of the DECISION investigators. Sorafenib in radioactive iodine-refractory, locally advanced or metastatic differentiated thyroid cancer: a randomised, double-blind, phase 3 trial.Lancet. 2014;384(9940):319-328.
2. Davies H, Bignell GR, Cox C, et al. Mutations of the BRAF gene in human cancer.Nature. 2002; 417(6892):949-954.
3. Xing M. BRAF mutation in thyroid cancer.Endocr Relat Cancer. 2005;12(2):245-262.
4. Xing M. BRAF V600E mutation and papillary thyroid cancer.JAMA. 2013;310(5):535.
5. Cappola AR, Mandel SJ. Molecular testing in thyroid cancer: BRAF mutation status and mortality.JAMA. 2013;309(14):1529-1530.
6. Kroll TG, Sarraf P, Pecciarini L, et al. PAX8-PPARgamma1 fusion oncogene in human thyroid carcinoma [corrected].Science. 2000;289(5483):1357-1360. Erratum in: Science. 2000;289(5484):1474.
7. Gustafson KS, LiVolsi VA, Furth EE, Pasha TL, Putt ME, Baloch ZW. Peroxisome proliferator-activated receptor gamma expression in follicular-patterned thyroid lesions. Caveats for the use of immunohistochemical studies.Am J Clin Pathol. 2003;120(2):175-181.