Development of a uniform algorithmic approach to indeterminate thyroid FNAs is important in providing the most appropriate risk stratification and care to patients with thyroid nodules. Although further clinical studies will help refine guidelines for molecular testing of thyroid samples, the currently recommended panel, highlighted in this article, has shown good sensitivity and excellent specificity when used in the appropriate clinical context. Adjunctive molecular testing should have a growing role in the individualised and multidisciplinary care of patients with thyroid nodules, to better enable accurate diagnosis and appropriate therapy.
by Dr Ericka Olgaard and Dr Lewis A. Hassell
Thyroid carcinoma has been increasing in incidence over the past 30 years, in part due to increased detection by fine needle aspiration (FNA) biopsy of small thyroid nodules . FNA biopsy with cytological examination is currently the most accurate tool for diagnosis or triage of thyroid nodules. Still, a significant portion of FNA biopsies are indeterminate and require further work-up . The introduction of the Bethesda System for reporting thyroid cytopathology in 2010 provided a structure for the diagnosis ,reporting and to a degree the management of thyroid nodules .The categories of ‘atypia of undetermined significance’ (AUS) and ‘follicular lesion of undetermined significance’ (FLUS) are reserved for FNA smears that show insufficient architectural or cytological, atypia thus precluding assignment to a more definitive Bethesda diagnostic category such as benign, suspicious for malignancy (SFM), or malignant . Currently, following the thyroid nodule of interest with imaging and a repeat FNA is the standard of practice in cases of AUS, FLUS and SFM, of which up to 40% will be malignant . Most patients with indeterminate FNAs undergo surgery for histologic diagnosis of the thyroid lesion. However, the addition of molecular analysis of these lesions may reduce needless surgeries in these
Molecular pathology of thyroid cancer
Most thyroid cancers are of follicular cell origin, consisting of papillary thyroid carcinoma (PTC), accounting for 80% of thyroid malignancies, follicular thyroid carcinoma (FTC), which accounts for 15%, poorly-differentiated carcinoma, and anaplastic carcinoma . Advances in molecular pathology have identified several mutations associated with thyroid carcinomas. The majority of these are due to somatic mutations, acquired alterations in cells that are passed on by cell division. These are the focus of this review.
The most common somatic mutations are involved with the mitogen-activated protein kinase (MAPK) pathway, which controls cell proliferation, differentiation and survival [Figure 1], . Included in the pathway are BRAF, RET, and RAS genes. BRAF mutations have been identified in 45% of PTCs, consisting of two main mutations, BRAFV600found in classic and tall cell variants of PTC [Figure 2] and BRAFK601E found in the follicular variant of PTC (as well as some benign follicular adenomas). BRAF mutations are associated with more aggressive tumors (advanced stage, metastases, and extrathyroidal extension), higher recurrence rates, and often poor response to radioactive iodine therapy .
Mutations of the RET gene (RET/PTC) account for 20% – 30% of PTCs, are associated with classic or solid variants, and have been correlated with a history of ionizing radiation, as well as a younger age and smaller lesions (microcarcinomas) . Point mutations in the RAS genes account for approximately 10% of PTCs, usually of follicular variant.
Most FTCs are also associated with somatic mutations involved in the MAPK pathway, most commonly in the RAS genes (40% – 50% of cases) and the gene fusion of PAX8/PPARγ1 (30% – 40% of cases) . FTCs with RAS mutations often show a more indolent course while FTCs with PAX8/PPARγ1 fusion have demonstrated a higher propensity for vascular invasion. Poorly differentiated (insular) and anaplastic thyroid carcinomas may have molecular alterations as described above. Medullary carcinomas, both sporadic and familial, are derived from parafollicular C cells and have been associated with RET gene mutations.
Clinical utility of molecular testing
There are currently no widely used algorithms for molecular testing in cytological analysis of thyroid lesions. Because somatic mutations overlap in different types of thyroid lesions, molecular testing cannot replace cytological or histologic examination in the diagnosis of thyroid lesions. The question becomes ‘when should molecular testing supplement FNA triage and when should it be performed on resected tissue?’
There is 0% – 3% risk of malignancy in FNAs diagnosed as ‘benign’ . Comparably, the risk of malignancy in FNAs categorised as ‘malignant’ is 97% – 99%. In these two groups, it seems additional molecular testing to confirm the diagnosis with the presence or absence of one of the somatic mutations described above would be of little added value.
In contrast, up to 40% of indeterminate FNAs are malignant. The additional cost of molecular testing should be weighed against the potential savings of avoided surgery and associated complications. Samples showing mutations of BRAF or RET/PTC are reasonably specific for PTC. Although RAS mutations may be found rarely in benign thyroid neoplasms, a positive test plus cytological atypia may be more suggestive of malignancy and aid in therapeutic decisions. A panel of BRAF (both V600E and K601E), RET/PTC, RAS (KRAS, NRAS and HRAS), and PAX8/PPARγ1 was recently recommended by the American Thyroid Association for potential use in indeterminate FNA cases and will detect most mutations in thyroid carcinoma . However, upwards of 30% of thyroid malignancies may not demonstrate a detectable mutation, either due to absence of a known mutation, a rare mutation not included in the molecular testing panel, or insufficient sensitivity of current assays . Additionally, clinical studies have demonstrated that 7% of AUS/FLUS lesions with a negative molecular analysis will actually prove malignant . This number in clinical practice may be significantly higher, posing a difficult dilemma for surgeons and patients in choosing between surgery or clinical follow-up.
Molecular studies may also be helpful in therapeutic decisions, including monitoring for therapeutic success and/or recurrence of tumour and in potential directed therapy. This is where testing on resected tumours is a consideration. Treatment for papillary thyroid cancer has long involved surgical excision and radioactive iodine treatment to eradicate residual thyroidal tissue. However, BRAFV600E mutations have been associated with iodine-refractory PTC and these patients could benefit from more tailored therapy, including more extensive initial surgery, higher dose radioactive iodine treatment, and closer follow-up [5,10]. Alternative investigational therapies such as a MAPK kinase inhibitor targeted at BRAF, much like the those available for patients with metastatic melanoma show promise in phase II trials . Hence, analysis of BRAF mutations in patients with PTC may become very valuable.
Qualitative methods of detection of point mutations (BRAF and RAS) are commonly available, mostly involving PCR-based methods with excellent sensitivity, achieved by using pyrosequencing, melting curve analysis, microarrays, fragment analysis, and conventional (Sanger) sequencing [2, 12]. Detection of chromosomal rearrangements of RET/PTC and PAX8/PPARγ1 requires analysis of RNA, which is less stable than DNA. Samples that are fresh or frozen can be used in reverse-transcriptase PCR (RT-PCR), but formalin fixed paraffin-embedded samples are inappropriate for use in RT-PCR and require fluorescence in-situ hybridisation (FISH), a more labour intensive and less sensitive method.
Advances in analysis of microRNA (miRNA), small segments of non-coding RNA that help regulate gene expression, have identified several unique expression profiles in thyroid cancer . Patterns of overexpressed miRNA can distinguish between papillary, follicular, poorly-differentiated and anaplastic thyroid carcinomas. miRNA assays are currently becoming available and show tremendous potential for diagnostic and prognostic use in the future.
The cytopathologist’s role in the diagnosis of thyroid nodules is still vital, but shifting. Development of a uniform algorithmic approach to indeterminate thyroid FNAs is important in providing these patients with the most appropriate risk stratification and care . Although further clinical studies will help refine guidelines for molecular testing of thyroid samples, the currently recommended panel of BRAF, RAS, RET/PTC and PAX8/PPARγ1 has shown good sensitivity and excellent specificity when used in the appropriate clinical context. Adjunctive molecular testing appears ready for a growing role in individualised and multidisciplinary care of patients with thyroid nodules, to better enable accurate diagnosis and most efficacious therapy.
1. Nikiforov YE, Nikiforova MN. Molecular genetics and diagnosis of thyroid cancer. Nat Rev Endocrinol 2011;7:569-580.
2. Hassell LA, Gillies EM, Dunn ST. Cytologic and Molecular Diagnosis of Thyroid Cancers. Is it time for routine reflex testing? Cancer Cytopathol 2011. DOI: 10.1002/cncy.20186.
3. Cibas ES, Ali SZ. The Bethesda System for reporting thyroid cytopathology. Thyroid 2010;19:1159-1165.
4. Bongiovanni M, Krane JF, Cibas ES, Faquin WC. The atypical thyroid fine-needle aspiration: past, present, and future. Cancer Cytopathol 2011. DOI: 10.1002/cncy.20178.
5. Nikiforov YE, Ohori NP, Hodak SP et al. Impact of mutational testing on the diagnosis and management of patients with cytologically indeterminate thyroid nodules: a prospective analysis of 1056 FNA samples. J Clin Endocrinol Metab 2011;96:3390-97.
6. Kang G, Cho EY, Shin JH et al. Review of fine-needle aspiration for evaluating thyroid nodule. Cancer Cytopathol 2011. DOI: 10.1002/cncy.20179.
7. Jin L, Sebo JT, Nakamura N et al. BRAF mutation analysis in fine needle aspiration (FNA) cytology of the thyroid. Diagn Mol Pathol 2006;15(3):136-43.
8. Nikiforov YE. Molecular diagnostics of thyroid tumors. Arch Pathol Lab Med 2011;135:569-77.
9. Cooper DS et al. Revised American Thyroid Association management guidelines for patients with thyroid nodules and differentiated thyroid cancer. Thyroid 2009;19(11):1167-214.
10. Hayes DN, Lucas AS, Tanvetyanon T et al. Phase II efficiacy and pharmacogenomics study of selumetinib (AZD6244; ARRY-142886) in iodine131 refractory papillary thyroid carcinoma (IRPTC) with or without follicular elements. Clin Cancer Res 2012. DOI:10.1158/1078-0432.
11. Menon MP, Khan A. Micro-RNAs in thyroid neoplasms: molecular, diagnostic and therapeutic implications. J Clin Pathol 2009;62:978-85.
Ericka Olgaard, D.O. and
Lewis A. Hassell, M.D.
Department of Pathology
940 Stanton L. Young Blvd., Rm. 451
Oklahoma City, OK 73104
Tel+ 1 405 271 4062