Advances in Anatomic Pathology:
Thyroid Fine Needle Aspirate: A Post-Bethesda Update
Bose, Shikha MD; Walts, Ann E. MD
Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, CA
The authors have no funding or conflicts of interest to disclose.
Reprints: Shikha Bose, MD, Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, 8700 Beverly Blvd. Suite 8709, Los Angeles, CA 90048 (e-mail: email@example.com).
Figure 2 can be viewed online in color at http://www.anatomicpathology.com.
The Bethesda system for reporting thyroid cytopathology formulated in 2007 has standardized reporting of thyroid cytology specimens and streamlined management algorithms. Although 3 of the categories (benign, malignant, and nondiagnostic) are standardized and improved, the remaining 3 (follicular lesion of undetermined significance, follicular neoplasm, and suspicious for malignancy) remain fraught with interobserver variability and uncertainty regarding management algorithms. Recent and ongoing morphologic and molecular studies that aim to resolve these issues are summarized.
Thyroid carcinoma is the most common endocrine malignancy although it comprises only 1% of all cancer diagnoses. It occurs in all age groups, has a female to male ratio of 3 to 1, and is rapidly gaining importance in public health and research into novel therapeutics. In 2012, the National Cancer Institute estimates 56,460 new cases of thyroid cancer with 1780 disease-related deaths.1 The worldwide incidence of thyroid cancer has been increasing and has almost tripled in the last 30 years.2 This has been attributed to increased detection after the advent of high-resolution imaging, increased diagnosis consequent to widespread use of fine needle aspirate (FNA), and decreased stringency in histologic criteria for the diagnosis of papillary thyroid cancer (PTC).1 Given that relative survival from thyroid cancer substantially declines after age 65, and that the population older than 65 years of age is expected to double in the next 20 years, an increase in deaths from thyroid cancer can be expected unless significant improvements in the treatment of metastatic disease can be achieved.3,4 It is estimated that as many as 7% of adults have at least 1 palpable thyroid nodule and up to 50% of adults have thyroid nodule(s) detectable by ultrasonography.5 Currently FNA provides the most cost-effective method to diagnose thyroid carcinoma, select/stratify thyroid nodules for surgical management, and/or confirm the presence of metastatic thyroid cancer. Low morbidity, high levels of patient acceptance, inexpensive equipment, and rapid reporting of results have helped make FNA a key component in the evaluation of virtually all thyroid nodules.
The Bethesda System for Reporting Thyroid Cytopathology (TBST) was developed to provide uniform terminology and diagnostic criteria for reporting thyroid FNAs and to relate these cytologic diagnoses to clinical management. It was formulated by a multidisciplinary group of thyroid experts who met in Bethesda, Maryland in October 2007.6 TBST describes 6 categories for the diagnosis and reporting of thyroid FNAs, each with an assigned “risk of malignancy” and associated recommendations for clinical management (Table 1).6,7 TBST terminology was subsequently incorporated into the 2009 revised guidelines of the American Thyroid Association (ATA) for management of (patients with) thyroid nodules and differentiated thyroid cancer.8 Several studies have now shown that implementation of TBST improves the quality of reporting by decreasing the number of ambiguous reports, increasing the positive predictive value (PPV) of malignancy in thyroid glands that are operated, and decreasing the rates of surgery for benign thyroid nodules.9–11 TBST does not appear to have affected the diagnostic accuracy or the false-positive rates of thyroid FNA or the frequency of intraoperative consultations.10 Three of the TBST categories (benign, nondiagnostic, and malignant) have been widely accepted, and their diagnostic criteria are straightforward and the recommendations for clinical management are clear. In contrast, issues/controversies regarding the 3 remaining categories [follicular lesion of undetermined significance (FLUS), follicular neoplasm (FN), and suspicious for malignancy] persist, underscoring the need for further refinement of TBST.
POSITIVE FOR MALIGNANCY
Since adopting TBST most centers have witnessed a substantial increase in the PPV of thyroid surgery for malignancy, malignancy rates now routinely exceed 90%.11 These high PPVs are to a large extent attributable to the fact that about 80% of thyroid malignancies are PTC and that most aspirates from PTC exhibit features that have been well described in the literature and provide a high sensitivity and a high specificity for the diagnosis of conventional PTC (Fig. 1A). Difficulties in recognizing the follicular variant of papillary carcinoma (FVPTC) in FNAs are reflected in the lower sensitivity and specificity of cytodiagnosis for this variant.12 Although the overall prognosis for PTC is good, approximately 10% to 15% of PTC exhibit aggressive behavior with frequent local recurrences and distant metastases. These tumors are often indistinguishable from well-differentiated PTC in aspirate smears. Few of the aggressive PTC have 1 or more morphologic attributes that while characteristic can nevertheless be difficult to diagnose in an FNA (eg, tall cells in the tall cell variant of PTC can be confused with Hurthle cell lesions or with the so-called columnar cell variant of PTC). The role of molecular markers in identifying the more aggressive PTC subsets is currently under intense investigation.
Follicular thyroid carcinomas (FTC) comprise 5% to 15% of thyroid cancers and, along with the other follicular-patterned lesions of the thyroid, require histologic examination to assess capsular integrity and lymph-vascular invasion for definitive diagnosis. These tumors present challenges for FNA and TBST and are discussed with the FLUS, FN, and suspicious for malignancy categories below.
Medullary carcinomas comprise about 4% of thyroid cancers, arise from the parafollicular C-cells, and are often associated with multiple endocrine neoplasia syndromes. Although the tumor cells can exhibit a variety of epithelioid, plasmacytoid, and/or spindle cell features and occasional nuclear pseudoinclusions, the presence of highly cellular smears exhibiting features that are not typical of PTC, appropriate clinical history, and confirmatory immunostain for calcitonin can provide an accurate diagnosis in most cases (Fig. 1B). In a recent study, FNA had a sensitivity of 83% and a PPV of 100% for the diagnosis of medullary carcinoma.13 The sensitivities and PPVs of FNA for the diagnosis of other uncommon thyroid malignancies including anaplastic carcinoma, lymphoma, and metastatic carcinoma ranged from 80% to 100% in that study.
TBST has had a major impact on the management of benign lesions of the thyroid as evidenced in the marked decrease in the percentage of benign glands excised.9,10 Accurate cytologic diagnosis of most lesions in the “benign” category is usually not problematic. However, the presence of focal Hurthle/Hurthle-like change in the absence of Hashimoto thyroiditis and the unequal and heterogeneous distribution of the Hurthle cell and/or lymphoid component(s) in Hashimoto thyroiditis can make it difficult to distinguish chronic thyroiditis and focal Hurthle-like change in nodular goiter (each categorized as benign by TBST) from Hurthle cell neoplasm (categorized as FN by TBST). Although it is understood that the risk of malignancy for a benign diagnosis by thyroid FNA is impacted by the percentage of benign nodules that are excised, this level of risk is estimated at <5%.6,7,14
The inclusion within TBST of precise requirements for FNA adequacy (at least 6 well-preserved and well-stained follicular epithelial cell groups, each containing at least 10 cells) and the absence of a “negative for malignancy” category (that could encompass aspirates diagnosed as benign and those that are nondiagnostic) emphasize that “benign” and “nondiagnostic” are 2 distinct diagnoses in the Bethesda system, each with its own recommendations for follow-up. This clarification has decreased the frustration previously experienced by clinicians attempting to translate the variable and often convoluted phrasing that appeared in earlier reports into patient management. Excluding FNAs performed by inexperienced aspirators, the majority of nondiagnostic thyroid aspirates come from nodules that are either cystic, <5 mm, difficult to palpate, and/or located in the posterior aspect of the gland. Accordingly, ATA guidelines8 encourage the use of ultrasound guidance for aspirations of difficult to palpate nodules and nodules with >25% cystic component. The guidelines also emphasize the importance of sampling the solid component of these mixed (solid and cystic) lesions as a means to reduce the incidence of nondiagnostic and false-negative FNAs.
After a nondiagnostic FNA, repeat aspiration utilizing ultrasound guidance has been reported to yield a diagnostic FNA in as many as 75% of solid nodules and 50% of cystic nodules.15 In another study, a second benign FNA reduced the overall false-negative rate of thyroid FNAs from 10.2% to 4.5%.16 Noting that 90% of their cases with a false-negative FNA showed increased vascularity or suspicious features on ultrasound, these authors echo the ATA in recommending correlation with sonography as a means of prioritizing nodules that yield nondiagnostic FNAs for reaspiration. It is also well known that on-site assessment of aspirate adequacy substantially decreases the incidence of nondiagnostic FNAs.16–18 To date, the time-consuming nature of on-site adequacy assessment has prohibited its implementation in some cytology laboratories. However, it is expected that continued advances in telepathology will help make this service available to many more aspirators in the near future. About 7% of thyroid nodules (including some that are malignant on excision) remain nondiagnostic on repeat aspirations.19,20
FOLLICULAR LESION OF UNDETERMINED SIGNIFICANCE/FOLLICULAR NEOPLASM/SUSPICIOUS FOR MALIGNANCY
These 3 TBST categories, collectively referred to as “indeterminate diagnoses,” comprise from 5% to 42% of thyroid FNAs, and consist mainly of “follicular-patterned lesions.” Diagnosis is on the basis of the relative proportion of follicular cells with or without atypia, colloid, and a microfollicular pattern. Using the current Bethesda system, FNAs from a variety of follicular-patterned lesions (histologically confirmed as hyperplastic nodules, follicular adenomas, FTC, and/or FVPTC) can be diagnosed as FLUS, FN, or suspicious for malignancy. Inconsistent application of subjective and overlapping criteria for FLUS, FN, and suspicious for malignancy is reflected in low levels of intraobserver and interobserver diagnostic agreement within and across these categories and in differences in the risk of malignancy reported for each category by different laboratories.11,21–26 FLUS, with a TBST assigned 5% to 15% risk of malignancy, is the most frequent abnormal diagnosis rendered in thyroid FNAs. Although it is recommended that FLUS does not exceed 7% of a pathologist’s or a laboratory’s thyroid FNA diagnoses,6,27 this reported percentage varies from 3.0% to 29% across laboratories and from 2.5% to 28.6% across cytopathologists.11,21,24,28 FN, which has an assigned 15% to 30% risk of malignancy, is the second most frequent abnormal diagnosis rendered in thyroid cytology.
Histologic evaluation is traditionally considered as the gold standard against which FNA cytology is compared. The difficult task of refining TBST to reduce the number of indeterminate thyroid FNAs and to improve the cytohistologic correlation of follicular-patterned lesions is further complicated by the substantial variability in intraobserver and interobserver histologic evaluation of follicular-patterned lesions and controversy regarding the criteria that best define vascular invasion.29 In a study where 6 “experts” in surgical pathology of the thyroid reviewed sections from 15 follicular-patterned lesions, the intraobserver diagnostic agreement ranged from 17% to 100% and the unanimous interobserver agreement on benign and malignant diagnoses was as low as 27%.30
Review of the recent cytology literature indicates that several approaches are being explored to increase the clinical utility of TBST with respect to these indeterminate cytodiagnoses. These approaches involve 1 or more of the following: (a) reducing the number of diagnostic categories in TBST by combining or deleting categories in the current system; (b) determining whether cytologic/nuclear or architectural features are more predictive of malignancy and quantitating the amount or extent of a “diagnostic” feature that should be required for inclusion in the suspicious for malignancy category; (c) assessing the value of repeat aspirations in indeterminate nodules; (d) requiring consensus review before diagnosing a thyroid FNA in one of the indeterminate categories; (e) assessing the potential role of molecular studies as adjuncts to cytodiagnosis.
(A) Reduce the Number of Diagnostic Categories in The Bethesda System for Reporting Thyroid Cytopathology
In a retrospective study using TBST, we identified considerable overlap in the diagnosis and in the assigned malignancy risk estimates for the FLUS and FN categories and for the suspicious for malignancy and malignant categories and proposed a simplified version of the Bethesda System for reporting thyroid FNAs that provided 4 nonoverlapping, statistically significant, and more clinically relevant diagnostic categories: unsatisfactory/nondiagnostic, benign, FLUS/FN, and suspicious for malignancy/malignant.31 Using this 4-category simplified version of TBST intraobserver and interobserver diagnostic agreement improved.32 However, in a study of 40 cases originally reported as FLUS by 2 “experienced” pathologists and reclassified as benign, FN, suspicious for malignancy, or malignant, Shi et al,33 found that elimination of the FLUS category decreased the sensitivity of thyroid FNA for detecting PTC from 100% to 27%, increased the false-positive and false-negative rates for detecting cancer, and decreased intraobserver and interobserver diagnostic agreement. Tissue follow-up revealed 37% of the reclassified as benign cases to be PTC and 38% of the reclassified as FN and suspicious cases to be benign. The authors concluded that the FLUS category should not be eliminated although they advocated minimizing its use. In a multi-institutional study involving the elimination of the FLUS category and retrospective reclassification of FNAs originally reported as FLUS, no significant differences were observed in the negative predictive value for the benign category or in the PPV for the FN and malignant categories when the 5- and 6-category systems were compared.34 In that study the most significant differences between the 5-tiered and the 6-tiered systems were the percentage of cases classified as benign and as FNs. Singh and Wang35 suggest that the FLUS category could be eliminated without decreasing the PPV of the remaining TBST categories because aspirates with scant cellularity and/or preparation artifact are often misdiagnosed as FLUS rather than as nondiagnostic.
(B) Assess the Importance of Cytologic Versus Architectural Features as Predictors of Malignancy and Quantitate the Amount or Extent of a Diagnostic Feature That Should be Required for Inclusion in the Suspicious for Malignancy Category
Attempts to determine whether architectural atypia (microfollicular pattern) (Fig. 1C) or atypical nuclear/cytologic features (Fig. 1D) are more predictive of malignancy have not been conclusive. In a study that evaluated the predictive value of 24 cytomorphologic features for “neoplasia” (defined as adenoma or malignancy in excised lesions) in FNAs that were originally diagnosed as FLUS, several nuclear/cytologic features (irregular nuclear membranes, nuclear overlapping, coarse chromatin) and several architectural features (syncytial tissue fragments, isolated microfollicles, follicles with scalloped borders) were each positively correlated with “neoplasia,” whereas honeycombing, colloid, and histiocytes were inversely correlated with “neoplasia.”36 In another study of FLUS, cases subclassified into 5 groups using combinations of nuclear/cytologic and architectural atypia, Renshaw37 concluded that nuclear/cytologic and architectural features are both important risk factors for malignancy and that microfollicles, even in the absence of cytologic atypia, should be reported as FLUS. He suggests that aspirates with cytologic atypia alone, scant aspirates with so-called “atrophic” follicles, and cellular aspirates with a mix of macrofollicles and microfollicles without significant cytologic atypia have similar risks of malignancy. In contrast, Abele and Levine38 suggested that microfollicles and cytologic atypia (ie, nuclear enlargement) should both be required for a diagnosis of FLUS and that in the absence of significant cytologic atypia (ie, nuclear enlargement), FNAs with microfollicles should be diagnosed as benign. VanderLaan et al39 found that FNAs with cytologic atypia, both cytologic and architectural atypia (microfollicles), or “unspecified” atypia carried twice the risk of malignancy as compared with FNAs exhibiting architectural atypia alone. They noted that 90% of the cancers that followed a diagnosis of FLUS were PTC of which 85% were FVPTC. Architectural atypia alone was more likely to predict follicular adenoma than PTC. In a retrospective review of FNAs diagnosed as “suspicious for follicular neoplasm (FN),” Lubitz et al40 calculated the predicted probability of malignancy as 88.4% when the nodule was ≥4 cm and nuclear grooves and a transgressing vessel were both identified in the aspirate. If the authors excluded cases diagnosed as PTC on histology from the study, anisokaryosis and presence of nucleolus replaced the presence of nuclear grooves as significant predictors of malignancy, and then nodule size (≥4 cm), a transgressing vessel and anisokaryosis lacking a nucleolus had a predicted probability of malignancy of 96.5%.
Attempts to quantitate cytologic and architectural features that are qualitative and subjective are fraught with potential errors and have poor reproducibility. In a recent study designed to determine whether cytologic or architectural atypia could further stratify FLUS to predict carcinoma, we eliminated the confounding effects of subjective and semiquantitative terms such as “rare,” “few,” “some,” “focal,” “probable,” “possible” which were used differently across cytopathologists and scored each feature on a binary scale (present or absent) with possible and probable considered as present. In our review of 83 consecutive FNAs diagnosed as FLUS and subsequently excised, comparison of the FNAs from the benign and malignant cases showed significant differences (P<0.05) in the frequencies of nuclear grooves/irregular nuclear membranes, nuclear overlap/crowding, nuclear pseudoinclusions, and Hurthle change. The presence of nuclear overlap/crowding was strongly correlated with the presence of nuclear grooves/irregular membranes, and nuclear pseudoinclusions and malignancy was present in 61% of the cases whose FNAs exhibited one or more of these features indicating that these FNAs are more appropriately diagnosed as suspicious for malignancy (the category to which TBST assigns a risk of malignancy of 50% to 75%). No significant differences were observed in the frequencies of cell sheets, microfollicles, nuclear enlargement, colloid, lymphocytes, bland/compact chromatin, or cell aggregates/clusters reported in aspirates from the benign and malignant cases.41
(C) Assess the Role of Repeat Fine Needle Aspirate After an Indeterminate Diagnosis on Fine Needle Aspirate
Although there is general agreement that repeat FNA is helpful in the clinical management of FLUS cases, repeat FNA is generally not recommended for nodules diagnosed as FN or suspicious for malignancy as these nodules are best excised.22,37
(D) Consensus Review as a Prerequisite for a Diagnosis of Follicular Lesion of Undetermined Significance
By conducting a group consensus review of 50 thyroid aspirates originally reported as FLUS, Jing et al42 achieved a 78% reduction in the number of FNAs reported as FLUS, provided optimal interobserver diagnostic agreement, and substantially improved cytohistologic concordance. In the study, approximately 50% of the cases originally reported as FLUS were reclassified as benign. Although the ability of group consensus review to improve diagnostic accuracy is recognized, this activity is time consuming and not feasible for a large number of multislide cases on a routine basis.
(E) Molecular Studies as Adjuncts
Because differences in case selection, study design, and other confounding factors make it difficult to compare/reconcile results from morphology-based studies and/or to reach definitive conclusions on how best to improve the diagnosis and management of cases that yield indeterminate cytologic diagnoses, molecular technologies (genomics, proteomics, and metabolomics) are actively being explored as potential diagnostic, therapeutic, and prognostic adjuncts to thyroid FNA.
GENETIC ALTERATIONS IN NONMEDULLARY THYROID CANCER
The initiation and progression of nonmedullary thyroid cancer involves the accumulation of various genetic and epigenetic alterations. Although hereditary forms of PTC are known, definite susceptibility genes have not been defined.43 In contrast, FTC is known to be associated with some familial syndromes (Table 2).44–48 Familial nonmedullary thyroid carcinomas are heterogeneous diseases, with early age onset, are usually bilateral and multicentric, and comprise about 10% to 15% of thyroid cancers.43 The sporadic forms of thyroid cancer are commonly initiated by mutations in effector genes (RET, BRAF, RAS) of the mitogen-activated protein kinase (MAPK) pathway49 while progression occurs by alterations in the phosphatidylinositol 3 (PI3)-kinase-AKT pathway (Fig. 2). Point mutations and chromosomal rearrangements are the most common mechanisms of genetic alteration. Point mutations, commonly seen in BRAF and RAS genes, involve the substitution of a single nucleotide within the DNA chain and result in an altered protein that is constitutively activated. These mutations have been found to be mutually exclusive.50
Chromosomal rearrangements, as observed with RET/PTC and PAX8/PPARγ, occur as a result of breakage and transference of a segment of 1 chromosome to a new site on the same chromosome or to a nonhomologous chromosome. This transfer either approximates the promoter of 1 gene to the coding sequence of the second gene (RET/PTC) leading to excessive production of the protein or approximates 2 coding sequences resulting in the formation of a fusion protein (PAX8/PPARγ). These proteins then function as oncogenes that drive the MAPK pathway.
BRAF is an intracellular effector of the MAPK pathway. In BRAF V600E, a thymine to adenine nucleotide substitution occurs at residue 1799 in >95% of mutations resulting in a valine to glutamate replacement at position 600.49,51 BRAF mutations are detected in 40% to 45% of PTCs with the highest incidence reported in the tall cell (80%) and classic (60%) variants. BRAF mutations are infrequent in FVPTC (10%) and in PTC that occur in young individuals and/or are associated with radiation (0% to 12%).52–54 Although BRAF mutation is reported to be specific for PTC, a low rate of false positivity has been reported in studies from Korea.55,56 BRAF mutations are also identified in 20% to 40% anaplastic carcinoma.57,58
Several studies have confirmed the association of BRAF mutation with aggressive tumor behavior.59–61 In a recent meta-analysis of 27 studies (total, 5655 patients), the incidence of BRAF mutation was 49% and BRAF mutation was associated with an increased likelihood of extrathyroidal extension [odds ratio (OR), 2.14; 95% confidence interval (CI), 1.68-2.73], lymph node metastasis (OR, 1.54; 95% CI, 1.21-1.97), and advanced tumor stage (OR, 2.00; 95% CI, 1.61-2.49). Eight of the studies showed 2-fold increases in the risk of recurrent/persistent disease (95% CI, 1.67-2.74).60 BRAF mutation has also been associated with an increased incidence of tumor-related mortality.62 Studies are in progress to determine whether optimization of surgical treatment of BRAF-associated PTC will be beneficial.63
The RET/PTC translocation involves the translocation of the kinase domain of the RET gene located on chromosome 10q11.2 to the promoter of at least 15 different genes located on chromosome 10 or on various other chromosomes, resulting in aberrant ligand-independent activation of the MAPK pathway.50 The most common rearrangements are the RET/PTC1 (inv 10)(q11.2;q21.2) and RET/PTC3 (inv 10)(q11.2;q11), both of which are paracentric intrachromosomal inversions.64,65 The distribution of the RET/PTC rearrangement is heterogeneous. Clonal rearrangements occurring in >1% of tumor cells are exclusively identified in 10% to 20% of PTC.66 These mutations are commonly found in PTC of younger individuals or children and are associated with ionizing radiation.67 RET/PTC1 is seen in PTC exhibiting classic morphology whereas RET/PTC3 is commonly observed in the solid variant. Sensitive methods have also detected nonclonal rearrangements in <1% of neoplastic cells in 10% to 45% of benign lesions and adenomas.68–70
Activating point mutations are located in NRAS, HRAS, and KRAS at codons 12, 13, and 61, with NRAS and HRAS codon 61 mutations being the most common.71,72 RAS mutations are found in follicular-patterned thyroid lesions including 40% to 50% of FTC, 20% to 40% of anaplastic carcinomas, and 10% to 20% of FVPTC.73,74 RAS mutations are also found in 20% to 40% of follicular adenomas and in up to 20% of adenomatous goiters raising the possibility that these lesions might represent precursor lesions.74,75
PAX8/PPARγ translocation is identified in follicular-based lesions and involves an in-frame fusion of PAX8 (thyroid transcription factor) with PPARG1 (peroxisome proliferator-activated receptor-γ 1) gene, t(2;3)(q13;p25) resulting in overexpression of a fusion protein.76 The mechanism of oncogenesis is not yet clear. This rearrangement is identified in 20% to 70% of FTC, 0% to 20% of follicular adenomas, and 0% to 5% of PTC.77–81 Tumors with this rearrangement are associated with a microfollicular, solid, or trabecular growth pattern, a thick fibrous capsule and overexpression of galectin-3 and/or HBME-1, markers commonly used in distinguishing benign from malignant thyroid tumors. This fusion mutation has not been reported in goiters or poorly differentiated/anaplastic carcinomas.
Mutations involving TP53, CTNNB1 (β-catenin), and less frequently various genes in the PI3 kinase signaling pathway (PI3CA, PTEN, and AKT1) accumulate in poorly differentiated and anaplastic carcinomas as late events and are thought to be indicative of dedifferentiation and progression.82–86
MicroRNAs are small noncoding RNAs that function as negative regulators of protein expression. Many microRNAs have been found to be deregulated in thyroid cancer and several such as miR-146b, miR-221, and miR-222 are highly upregulated in PTC, whereas others are abnormally expressed in FTC (miR-197, miR-346, miR-155, and miR-224) and anaplastic carcinomas (miR-30d, miR-125b, miR-26a, and miR-30a-5p). Although the role of these microRNAs in tumorigenesis is currently unclear, studies are in progress to determine their utility in the diagnosis and prognosis of thyroid cancer.87–92
CLINICAL UTILITY OF MOLECULAR MARKERS
Thyroid FNA is an accurate test with good positive and negative predictive values for malignant and benign diagnoses. However, in cases where FNA results are indeterminate, the revised ATA management guidelines recommend that clinicians consider molecular testing to help guide clinical management.8
Because of its relative frequency and specificity for PTC, BRAF has been the most common gene tested in FNA samples. A meta-analysis of 22 such studies50 revealed that FNA material is reliable for molecular analysis and that the V600E mutation is highly specific for PTC. A total of 99.3% (1109/1117) of the BRAF-positive nodules were PTC on final histopathology. Five of the 8 false-positive cases (no cancer identified by histology) were reported in 1 study that used a more sensitive detection method. Thus, BRAF mutation positivity confers >99% risk of malignancy. In this study, 15% to 40% of the BRAF-mutated samples had an indeterminate FNA cytology result, indicating that the presence of BRAF mutation might be of diagnostic value in these cases.
As BRAF mutations occur in only 40% to 45% of PTC, testing for this mutation alone would miss a substantial number of PTC and other BRAF-mutation–negative thyroid cancers. Several studies have investigated the usefulness of a panel of mutations including BRAF and RAS mutations and RET/PTC and PAX8/PPARγ rearrangements for analysis of thyroid FNA samples.93–97 These studies demonstrate that in an appropriate clinical setting the presence of any mutation is a strong predictor of malignancy in thyroid nodules irrespective of the cytologic diagnosis.93–95 The presence of BRAF, RET/PTC, or PAX8/PPARγ correlates with the presence of malignancy in 100% of cases, whereas RAS mutations have a 74% to 87% PPV for cancer, consistent with the known presence of RAS mutations in some benign follicular adenomas and goiters. Despite the lower specificity for malignancy, RAS mutations have the attribute of being positive in more challenging cytologic diagnoses such as FVPTC and FTC.
A recent prospective analysis98 of residual material from 1056 consecutive thyroid FNA samples with indeterminate cytology (ie, Bethesda categories of FLUS, FN, and suspicious for malignancy) showed that the residual material was adequate for molecular analysis in 92% of cases. A total of 479 of the patients underwent surgery. The mutation analysis was performed using a newly developed polymerase chain reaction assay for a panel of mutations: BRAF V600E, NRAS codon 61, HRAS codon 61, and KRAS codons 12/13 point mutations and RET/PTC1, RET/PTC3, and PAX8/PPARγ rearrangements and included stringent assessment for the presence of epithelial cells. Detection of any mutation conferred 88%, 87%, and 95% risk of malignancy in the FLUS, FN, and suspicious for malignancy Bethesda categories, respectively. The risk of cancer in mutation-negative nodules was 6%, 14%, and 28% in the FLUS, FN, and suspicious for malignancy categories, respectively. There were 13 mutation-negative cancers in the FLUS FNAs. Of these, only 1 lesion showed extrathyroidal extension. It is noteworthy that even with the use of this panel of mutations, substantial numbers of thyroid cancers in the FN and suspicious for malignancy groups remained mutation negative. Although the clinical management of these patients was not significantly affected, Nikiforov et al98 proposed that these patients can be offered lobectomy instead of the 1-step total thyroidectomy, which is appropriate in mutation-positive cases.
Although molecular testing is reported to decrease the false-negative rate of benign cytologic diagnoses, there is currently little impetus to perform molecular analysis on these samples as the risk of malignancy is <5%. Various studies have reported reductions in false-negative rates in FNAs diagnosed as benign from 2.1% to 0.9%95 and 10% to 6%.94 The cost-effectiveness of testing all negative cytology samples and the potential benefit from the detection of only a few cancers requires further investigation.
Similarly, it is not established whether molecular testing of FNA specimens diagnosed as malignant should be undertaken. BRAF-mutated tumors may benefit from more extensive initial surgery as it has been shown that, because of the increased incidence of cervical recurrence, these tumors undergo more frequent reoperation than tumors without the mutation.63,99 Thus, Yip et al63 suggest that knowledge of BRAF mutational status might optimize the extent of surgery and lymph node dissection. In addition, on the basis of data that suggest BRAF-mutated PTC are more resistant to radioiodine treatment because of a reduced ability to trap radioiodine,100,101 an increased dose of radioiodine, less suppression of the thyroid stimulating hormone, and closer follow-up has been suggested as initial postoperative treatment of BRAF-mutation–positive cancers.102 Knowledge of the mutation status may also provide options for targeted therapy.103
The association between BRAF V600E and aggressive disease characteristics has also been reported in papillary microcarcinomas (tumors≤1 cm).104–107 Although BRAF mutation is identified in 24% to 63% of papillary microcarcinomas, <10% to 15% of these tumors behave aggressively. Howell et al108 reported that tumor recurrence is limited to older (65 years and above) patients. Using BRAF mutation status, a set of 3 pathologic features (superficial tumor location, intraglandular tumor spread/multifocality, and tumor fibrosis), and stepwise regression analysis, Niemeier et al109 developed a combined molecular pathologic score for predicting aggressive tumor behavior. Using this score, their sensitivity and specificity for detection of extrathyroidal spread or recurrence increased from 77% to 96% and from 68% to 80%, respectively; thus indicating that BRAF mutational status could stratify papillary microcarcinomas for aggressive clinical management.
The Bethesda classification has standardized reporting of thyroid cytology specimens and streamlined management algorithms. However, it has generated considerable controversy regarding the use and management of the indeterminate category. Many studies (both morphologic and molecular) are ongoing in an effort to improve the diagnosis and management of this group of cases. Whether molecular analysis alone or in concert with morphologic evaluation will provide reliable stratification of indeterminate thyroid FNAs is not yet clear. Cost-effectiveness, management algorithms, and specific indications will also need to be determined.
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