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UNIVERSITY OF PISA
DEPARTMENT OF
SURGERY, MEDICAL,
MOLECULAR AND
CRITICAL AREA
PATHOLOGY
PHD IN CLINICAL PATHOPHYSIOLOGY DIRECTOR PROF F.BASOLOGraduation year 2017
“CITOLOGICALLY INDETERMINATE
SINGLE THYROID NODULE: IMPACT
OF MUTATIONAL TEST ON SURGICAL
APPROACH ”
PhD Sudent: Tutor:
~ 2 ~ INDEX 1. INTRODUCTION……….…..PAG.3 2. THYROID NODULES………PAG.5 3. ULTRASOUND SONOGRAPHY………..PAG.10 4. NODULE’S CLASSIFICATION………..PAG.19 4.1 NODULES REPORTING SYSTEM………....PAG.20 4.2 CYTO-HISTOLOGICAL COMPARISON IN INDETERMINATE DIAGNOSES………..……PAG.25 5. MUTATIONAL TESTS……….……...PAG.29 5.1 BRAF MUTATION………..PAG.33 5.2 NRAS MUTATION………..PAG.35 6. SURGICAL COMPLICATIONS………..……PAG.39 6.1 GENERAL SURGICAL COMPLICATIONS…….…….PAG.40 6.2 SPECIFIC SURGICAL COMPLICATIONS….………..PAG.42 7. RETROSPECTIVE STUDY……….PAG 47 7.1 MATERIAL AND METHODS………PAG 47 7.2 RESULTS………..PAG 49 7.3 DISCUSSION………...………PAG 51 8. PROSPECTIVE STUDY………...………PAG 61 8.1 MATERIAL AND METHODS……….……..PAG 62 8.2 RESULTS……….PAG 64 8.3 DISCUSSION………..…………PAG 67 9. CONCLUSION……….……….…PAG 69 10. BIBLIOGRAPHY………PAG 71
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1.INTRODUCTION
Palpable thyroid nodules are common in the adult population, with an estimated prevalence in the United States of 4–7%, resulting in 10 –18 million affected individuals (1–3). The incidence of thyroid nodules detectable by ultrasonography is even higher and may exceed 50% in patients over 65 years old (2, 3). The vast majority of thyroid nodules are benign and can be managed conservatively, whereas approximately 5–15% of nodules examined by ultrasound and fine-needle aspiration (FNA) cytology are malignant. A challenge facing the physician is to distinguish between benign nodules and malignant tumors to ensure that each patient receives timely and appropriate treatment, while minimizing the risk of unnecessary intervention. Fine needle aspiration cytology (FNAC) is the gold-standard modality for determining the nature of a thyroid nodule. However, it fails to yield a conclusive result in a subset of patients, labeling them as having an ‘‘indeterminate diagnosis.’’ In doing so, it compromises the proper triaging of patients to appropriate surgery. This places patients at risk of non-optimal initial surgery: an overly radical total thyroidectomy, or an unnecessary two-stage operation. The aim of this study was to assess the impact of combining gene mutation testing and
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ultrasonographic (US) features preoperatively on predicting the risk of malignancy in patients with indeterminate nodules, with the goal of offering patients the most appropriate tailored initial surgical intervention.
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2. THYROID NODULES
The basis of thyroid nodule management is the use of high-resolution ultrasonography (US), sensitive thyrotropin (TSH, formerly thyroid-stimulating hormone) assay, and fine-needle aspiration (FNA) biopsy, together with clinical findings. Thyroid scintigraphy is not necessary for diagnosis in most cases; however, it may be warranted in patients with a low serum TSH value or a multinodular gland to detect functional autonomy, most common in iodine-deficient areas. Measurement of serum TSH is the best initial laboratory test of thyroid function and should be followed by measurement of free thyroxine (FT4) and free triiodothyronine (FT3) when the TSH value is decreased, and measurement of thyroid peroxidase antibodies (TPOAbs) and FT4 when the TSH value is above the reference range.
A single, nonstimulated serum calcitonin measurement should be performed only when medullary thyroid carcinoma (MTC) is suspected due to FNA results or history. Thyroid nodules are a common finding because they are detected in up to 50 to 60% of healthy people. In most cases, they appear in euthyroid persons and cause neither compressive symptoms nor cosmetic concerns. Accordingly, the main clinical challenge in the treatment of these patients is to rule out malignancy. Most
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patients with thyroid nodules are asymptomatic, but the absence of symptoms does not rule out malignancy. Thus, clinical and US risk factors for malignant disease should always be reviewed (Table 1).
All patients with a palpable thyroid nodule or clinical risk factors should undergo US examination. Thyroid FNA should always be performed under US guidance because it makes the procedure safer, more reliable, and more accurate. In light of the low clinical risk, nodules <5 mm should always be monitored with US rather than biopsied. FNA should be considered for nodules with a major diameter ≤5-10 mm only when suspicious US signs are present (high US risk thyroid lesions) in association with pathologic lymph nodes or extrathyroidal spread. FNA is also appropriate in cases where the patient has a personal or family history of thyroid cancer or of coexistent suspicious clinical or imaging findings. FNA should be performed on nodules >10 mm that are devoid of suspicious US and/or clinical findings yet do not show a definite benign appearance (intermediate US risk thyroid lesions). Finally, FNA should be performed on spongiform, isohyperechoic, or predominantly (>50%) cystic nodules in the absence of suspicious US findings (low US risk thyroid lesions) only when nodules are ≥20 mm or progressively increasing in size. Nodules that are functioning on scintigraphy and devoid of suspicious US features can be excluded from FNA.
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A classification scheme of 5 cytologic diagnostic categories and 2 subcategories is recommended for the cytologic report: non-diagnostic, benign, indeterminate, suspicious for malignancy, or malignant. Indeterminate lesions are further subdivided into 2 subclasses with significantly different estimated risks of cancer, to better stratify the risk of malignancy associated with the “indeterminate” nodules.
Non-diagnostic aspirates composed of pure colloid and obtained from a nodule that is completely cystic on US should be labeled as compatible with a colloid cyst and require clinical and US follow-up. Solid, persistently non-diagnostic nodules may be considered for US-guided core-needle biopsy (CNB) for microhistologic assessment. Alternatively, those with clearly favorable clinical and US findings can be monitored with close surveillance, whereas suspicious lesions should be surgically resected.
Nodules with benign cytologic characteristics should undergo clinical and US follow-up. A repeat FNA should be performed in the case of suspicious clinical and/or US findings or with substantial and progressive nodule enlargement, defined as a volume increase >50% (greater than the interobserver coefficient of variation). Most patients with benign thyroid nodules do not require any treatment; levothyroxine (LT4) suppressive therapy is not recommended in euthyroid patients. In iodine-deficient geographic regions, iodine supplementation is recommended, and
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a trial of non- TSH-suppressive treatment with LT4 may be considered in young patients with a small nodular goiter. Symptomatic goiters, whether euthyroid or hyperthyroid, may be treated surgically or with radioiodine. Percutaneous ethanol injection (PEI) is the first-line treatment of relapsing benign cystic thyroid lesions. In patients with solid or complex, symptomatic or progressively enlarging benign thyroid nodules, US-guided thermal ablation treatments may effectively control nodule growth and local symptoms.
Malignant or suspicious nodules should be treated surgically. Preoperative evaluation with US, FNA, and, if needed, further imaging techniques, is recommended for appropriate surgical planning.
In nodules with indeterminate cytologic results, no single cytochemical or genetic marker is specific or sensitive enough to rule out malignancy with certainty. However, the use of molecular markers may be considered together with the cytologic subcategories and data from US to obtain additional information for management of these patients.
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Table 1. Features Suggesting Increased Risk of Malignant Potential
• History of head and neck irradiation
• Family history of medullary thyroid carcinoma, multiple endocrine neoplasia type 2, or papillary thyroid carcinoma
• Age < 14 ore > 70 years
• Male sex
• Growth of the nodule
• Firm or hard nodule consistency
• Cervical adenopathy • Fixed nodule
• Persistent dysphonia, dysphagia, or dyspnea
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Ultrasonography (US) is the most common and most useful way to image the thyroid gland and its pathology, as recognized in guidelines for managing thyroid disorders published by the American thyroid Association (4) and other authoritative bodies. In addition to facilitating the diagnosis of clinically apparent nodules, the widespread use of US has resulted in uncovering a multitude of clinically imperceptible thyroid nodules, the overwhelming majority of which are benign.
The most frequent use of US is to refine the diagnosis of a thyroid nodule. US can identify thyroid nodules, even when they are too small to palpate. Sonography can demonstrate nodules that have an enhanced risk of malignancy with the best sensitivity of any non-invasive technique, but with only fair specificity. In addition, US can be used clinically to enhance the clinical value of FNA of nodules.
The ultrasonic appearance of a thyroid nodule does not reliably differentiate a benign thyroid lesion and cancer (4, 5) but it does offer strong clues that help the clinician in the process of triage. Never the less, sonography cannot identify a specific kind of tumor such as a Hurthle cell lesion. The most reliable sonographic indicator that a nodule is malignant is observing vascular invasion by tumor, which is rarely seen. However, there are distinctions in echodensity, calcifications, distortions of the
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rim, and vascularity that favor a benign or malignant diagnosis (6-7). These characteristics are summarized in Table 2. But, understand that the features described reflect statistical probabilities and not dependable criteria.
The important thing is that single or even a few US features are inadequate to select nodules for FNA or to reliably assess the risk of thyroid cancer. In contrast, selections based on multiple characteristics that are associated with elevated cancer risk are more dependable indicators of probable malignancy. Microcalcifications, a taller than wide shape, irregular margins, and/or absence of elasticity-will probably identify nodules with a clinically meaningful increased, perhaps even high risk for malignancy (8).
1. ECHOGENICITY: Thyroid malignancies tend to be hypoechoic when compared with the rest of the thyroid (9). Since most benign thyroid nodules, which are far more common than malignancies, are also hypoechoic, this finding is not particularly useful except that it is reasonably safe to conclude that hyper-dense nodules are probably not cancerous. One group of investigators has concluded that hyperechogenic lesions occurring in thyroiditis-affected thyroid glands bear no-clinical relevance. Therefore, they advocate that aspiration biopsy of these nodules is not advisable and many clinicians follow that practice.
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2. CALCIFICATIONS : The presence of calcification is also not a straightforward diagnostic aid. Micro calcifications are relatively more common in malignant lesions than benign and may represent psammoma bodies. Micro calcifications have been reported as demonstrating a 95.2% specificity for thyroid cancer, but a low sensitivity of 59.3 % and a diagnostic accuracy of 83.8% (10). B-flow ultrasonic imaging may be particularly sensitive in detecting micro calcifications by demonstrating “twinkling” in some nodules. However, large coarse calcifications and calcifications along the rim of nodule are common in all types of nodules and reflect previous hemorrhage and degenerative changes. Thus, since some cancers may have been chronic and have undergone degenerative change, they may demonstrate peripheral or coarse internal calcification. Therefore, diagnostic FNA biopsy may be appropriate even when there are large, coarse, or eggshell calcifications to avoid missing a cancer (11). Indeed, in one investigation, among 64 thyroid nodules with peripheral calcifications 19 (30%) were benign, and 45 (70%) were malignant. Interruption and thickening of peripheral calcifications and decreased internal echogenicity of a thyroid nodule with peripheral calcifications were associated with malignancy in this study (12). In one study, the highest incidence of calcification was found in thyroid cancer (54%), followed by multinodular goiter (40%),
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solitary nodular goiter (14%), and follicular adenomas (12%). The authors reported that calcifications in a "solitary" nodule in a person younger than 40 years person should raise a strong suspicion of malignancy: relative cancer risk of 3.8 versus 2.5 in patients older than 40 years. In contrast to the prior statements, it is important to note that large calcifications are seen with increased frequency in medullary thyroid carcinoma (13).
3. HALO: A halo around the nodule may be seen with benign or malignant conditions. It suggests that there is an acoustic interface around the nodule that does not reflect the ultrasound. It implies that there are two different types of histology in the region: the nodule and the surrounding thyroid. Some observers have suggested that cancer should be suspected when the periphery of a halo has a blurred appearance.
• NODULE BOARDER: There have been investigations of a possible correlation between the degree of definition of the boarder of a nodule and the likelihood of malignancy and even of the predictability of aggressive characteristics of a papillary cancer. In one series of 155 cases, poor definition of a nodule’s edge was observed in 21.5% of patients, all of whom showed worse disease-free survival (p = 0.0477) than those with a well-defined edge. Furthermore, this finding was directly linked to US-diagnosed lateral node
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metastasis (p = 0.0001) (14).
• HEMODYNAMIC CHARACTERISTICS: Increased blood flow in the central part of a nodule is more likely associated with cancer than when the vascularity is along the periphery. An analysis of the hemodynamic characteristics of a nodule by high resolution pulsed and power Doppler ultrasonography also may offer valuable preoperative diagnostic insights. For example, one study of 25 follicular adenomas and 10 follicular carcinomas compared the vascular pattern and the velocimetric parameters (such as peak systolic velocity), end-diastolic velocity, pulsatility index or resistance index.. Eight of 10 patients with follicular carcinomas showed moderate increase of intra-nodular vascularity using “Power Doppler”. In contrast, the 21 out of 25 follicular adenomas showed only a peripheral rim of color flow. Furthermore, the velocimetric analyses were significantly higher in the patients with cancer than those with adenomas (15).
• SHAPE: There have been observations that some cancers tend to have a non-globular, “tall” shape, as if growing in one
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plane. Nodules that are tall rather than wide should be viewed with considerable suspicion.
• CYSTIC SPACES: Especially large benign or malignant thyroid nodules tend to undergo hemorrhagic or cystic degenerative changes. It has been reported that features associated with cancer in a cystic thyroid nodule include more than 50% solid tissue, eccentricity of the cystic space, and micro-calcifications.
• MISCELLANEOUS CHARACTERISTICS: Ultrasonographers
have observed that colloid nodules, which are benign with high probability, have a more or less characteristic appearance of a “stack of pancakes”, “puff pastry like a Napoleon”, or sponge. There may be a small, echogenic, bright spot with “comet-tail shadowing” associated with colloid that must be differentiated from a pin-point bright spot. There seems to be triage- merit to these characteristics, which will require critical scrutiny. Furthermore, it is important to be aware that a cancer may co-occur in an otherwise nodular or colloid goiter.
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US Features of Benign or Malignant Thyroid Nodules US features indicative of benign nodule
Isoechoic spongiform appearance (microcystic spaces comprising >50% of the nodule)
Simple cyst with thin regular margins
Mostly cystic (>50%) nodules containing colloid (hyperechoic spots with comet-tail sign)
Regular “eggshell” calcifications around the periphery of a nodule
Us features indicative of a malignant nodule
Papillary carcinoma
Solid hypoechoic (relative to prethyroid muscles) nodule, wich may contain hyperechoic foci without posterior shadowing (i.e., microcalcifications)
Solid hypoechoic nodule, with intranodular vascularity and absence of periphereal halo
“Taller-tha-wide” nodule (AP>TR diameter when imaged in the trasverse plan
Hypoechoic nodule with speculated or lobulated margins
Hypoechoic mass with a broken calcified rim and tissue extension beyond the calcified margin
Follicular Neoplasm (either follicular adenoma or carcinoma) Isoechoic or mildly hypoechoic homogeneous nodule with intranodular vascularization and well-defined halo
Indeterminate US features
Isoechoic or hyperechoic nodule with hypoechoic halo
Mild hypoechoic (relative to surrounding parenchyma) nodule with smooth margin
Peripheral vascularization Intranodular macrocalcification
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Figure 1. Macrocalcifications
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Figure 3. Taller than wide shape
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4. NODULE’S CLASSIFICATION
Nodular lesions of the thyroid represent a very common problem for the clinician as well as a diagnostic challenge for the pathologist. Up to 5% of the general population has a palpable thyroid nodule although only approximately 5% of these clinically apparent thyroid nodules actually harbor malignancy (16, 17). The real challenge, faced by the general practitioners, endocrinologists, surgeons, and pathologists, is to reach an accurate preoperative diagnosis of malignancy and to ensure that patient receives a timely and appropriate treatment. Fine-needle aspiration (FNA) is the only test that can provide a definitive preoperative diagnosis of malignancy. The sensitivity and specificity of FNA are reported to be 68–98% and 56–100%, respectively (18).
The main purpose of the management of patients with thyroid nodules is the distinction of those who are candidate to surgery in respect to those who can simply be clinically followed. This decision primarily relies on the morphologic classification of cytologic samples obtained by fine-needle aspiration, complemented by clinical and imaging findings and, in selected cases, by molecular analysis. Various 4- to 6-tiered reporting
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schemes for thyroid cytology have been proposed and their validity has been demonstrated in several studies. The most controversial points concern the definition of the “indeterminate” cytology and the consequent non negligible rate of unnecessary diagnostic surgery.
Fine-needle aspiration biopsy (FNAB) is also regarded as the most accurate method for selection of the patients with thyroid nodules for surgery or for the ‘wait and see’ management, and it is a very cost-effective diagnostic test.
4.1 NODULES REPORTING SYSTEM
The previous SIAPEC-IAP reporting system (Table 3) was a five-tiered classification which included the following categories: TIR 1 - non diagnostic; TIR 2 - negative for neoplasia; TIR 3 - indeterminate/follicular proliferation; TIR 4 - suspicious for malignant neoplasm; and TIR 5 - positive for malignancy. The latest Italian Reporting System (Table 3) introduces the additional subgroup of TIR 1C (cystic) in the non-diagnostic group and the subdivision of the indeterminate category (TIR 3) into TIR 3A (low-risk indeterminate lesion) and TIR 3B (high-risk indeterminate lesion) (Table 4).
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Table 3. First Italian reporting system for thyroid cytology
Code Diagnostic Category Histological Correspondence
Suggested Actions
TIR1 Non diagnostic Non defined Repeat US-guided FNA TIR2 Non malignant/benign Goiter, thyroiditis Follow-up
TIR3 Indeterminate (Follicular Proliferation) Follicular adenoma Follicular carcinoma Follicular variant of papillary carcinoma Surgery
TIR4 Suspicious of Malignancy
Mostly follicular variant of papillary carcinoma Surgery TIR5 Malignant Papillary, medullary and anaplastic carcinoma Surgery
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Table 4. New Italian reporting system for thyroid cytology
Code Diagnostic Category Risk of malignancy (%)
Suggested Actions
TIR1 Non diagnostic Non defined Repeat US-guided FNA
TIR1C Non diagnostic-cystic
Low (variable on the basis of clinical
findings)
Repeat US-guided FNA
TIR2 Non malignant/benign < 3% Follow-up
TIR3A Low-risk indeterminate
lesion (LRIL) < 10%
Repeat FNA Follow Up
TIR3B High-risk indeterminate
lesion (HRIL) 15-30% Surgery
TIR4 Suspicious of Malignancy 60-80% Surgery
TIR5 Malignant > 95% Surgery
The almost contemporary American Bethesda Reporting System for Thyroid Cytology (TBRSTC) (19) identified six categories, of them the non-diagnostic, benign and malignant diagnostic groups are similar to, whereas the indeterminate lesions are different from the Italian and British classifications; the indeterminate lesions in TBRSTC are classified into three categories: 1) atypia of undetermined significance and follicular lesions of
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undetermined significance (AUS/FLUS); 2) follicular neoplasm or suspicious for follicular neoplasm (FN/SFN); and 3) suspicious for malignancy (SM) (20).
The British Thyroid Association together with the Royal College of Pathologists immediately followed the TBSRTC in subclassifying the follicular neoplasms (FN) into the two subgroups of Thy3a (atypia), corresponding to the Bethesda AUS/FLUS, and Thy3f (follicular neoplasm), corresponding to the FN/SFN of the NCI Conference, with a good diagnostic agreement among cytopathologists (21,22). It should be noted that in the British system, all cases categorized either as indeterminate or suspicious should be referred to the multidisciplinary team in order to establish a correct management.
Though the most recent updates, the Italian and British reporting systems have joined the Bethesda philosophy in devising a 6-tiered scheme, and another important institution, the Japan Thyroid Association, has recently published in English its national guidelines (Table 5). The latter includes a suggestion for reporting the cytology of thyroid nodules from a minimum of six to a maximum of eight categories, depending on whether or not the cytopathologist choses to subdivide the follicular neoplasm/indeterminate group (23).
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Table 5. Synopsis of the most important reporting system
UK RCPath ITALY USA BETHESDA
JAPAN THYROID ASSOCIATION Diagnostic Category Diagnostic Category Diagnostic Category Diagnostic Category Thy1/Thy 1c Non-diagnostic for cytological diagnosis TIR1: non-diagnostic TIR 1C: Cystic
I. Non-diagnostic Normal or Benign
Thy2/Thy2c Non-neoplastic
TIR 2:
Non-malignant/benign II. Benign Normal or Benign
Thy 3a Neoplasm possible – atypia present
TIR 3A: Low-risk indeterminate lesion (LRIL) III. Atypia of undeterminated significance (AUS) or follicular lesion u.s. (FLUS) Indeterminate A. Follicular Neoplasm • A1 favor benign • A2 border-line • A3 favor malignant B. Others (atypia in non-follicular patterned lesions) Thy 3f Neoplasm possible – follicular neoplasm suspected TIR 3B: High-risk indeterminate lesion (HRIL) IV. Follicular neoplasm or suspicious for a follicular neoplasm Thy 4 Suspicious of malignancy TIR 4: Suspicious of malignancy V. Suspicious of malignany Malignancy suspected Thy 5 Diagnostic of malignancy
TIR 5: Malignant VI. Malignant Malignancy
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The main purpose of a reporting system for thyroid cytology is to distinguish the nodular lesions that should be addressed to surgery from those that can be clinically followed-up. The majority of the thyroid nodules (60-80% in different studies) show a cytological picture, which can be easily classified either as benign (nodular goiter or thyroiditis) or malignant (usually papillary carcinoma (PTC)). A smaller though variable portion of these nodules yields non-diagnostic (5-15%), and a variable amount of these lesions falls into the indeterminate category (10-25%) (24, 25). The latter represents a continuous range of morphologic pictures identified by a poor amount of colloid and an increased cellularity. These findings, at the histological level, identify three types of tumors: 1) follicular adenoma (FA), a benign tumor with a predominant microfollicular architecture and usually encased by a continuous fibrous capsule; 2) follicular carcinoma (FTC), the malignant counterpart of the FA sharing with the former the follicular structure but showing features of aggression to the fibrous capsule and the adjacent vessels; and 3) PTC, especially its follicular variant (FVPTC) that shows a histological pattern similar to the previous but is composed by
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follicular cells with clearing and prominent pleomorphism of the nuclei (26).
The possibility that a lesion exhibiting an indeterminate picture corresponds to an adenoma instead of a FTC or a FVPTC (which belongs to the group of well-differentiated thyroid carcinomas, WDTC) is of critical importance because the risk of malignant yield represents the ultimate goal of the FNA technique and affects the evaluation of its efficacy. The histological diagnosis of WDTC can be difficult in some instances and some cytological criteria (such as clear nuclei) are also poorly reproducible between experts and pathologists in such a manner that the true malignant nature of the encapsulated form of FVPTC has been discussed (26, 27). The majority of WDTC pursue an indolent clinical course, especially the FVPTC and the PTC under 1 cm of size (microPTC, mPTC), so that, according to Ito et al. (28), their evolution can be followed by periodical sonographic examinations until they become as clinically relevant as to be addressed to surgery. Although some clinicians do not agree with this strategy, the low aggressiveness of WDTC does not seem to justify the high amount of surgical procedures and is sometimes carried out for diagnostic purposes. In fact, the diagnostic lobectomy suggested for indeterminate diagnoses is necessary to sample and appropriately examine the capsule of a thyroid tumor in order to make a differential diagnosis between FA and FTC. Nonetheless, there is a progression in the atypical features of the
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follicular cells which can be used as predictor of risk of likelihood of malignancy in WDTC. There are two types of atypia of the follicular cells: i) architectural atypia that is the presence of follicular cells aggregated in microfollicles; and ii) nuclear atypia, which includes clearing, elongation and irregularity of the nuclei (20). The latter seems more strongly related to the risk of malignancy of a follicular-patterned lesion than the former (29). Based on this feature, the first published reporting systems from U.K. and Italy identified only one category of true indeterminate diagnoses (Thy 3 for BTA and TIR 3 for the Italian SIAPEC system), which represented the most debated group causing the vast majority of unnecessary surgical procedures. However, the last updates of those systems have subdivided the indeterminate category into two subgroups, which are identified, similarly to the TBSRTC, by the different weight of the architectural in respect to the nuclear atypia and by the different risk of malignant occurrence.
The project of the 2014 reporting system is different from the previous one and emphasizes some points of difformity in comparison to the Anglo-Saxon systems. The first point is the different weight of the criteria of atypicality. Though the architectural atypia (see above) remains the basis for distinguishing low-risk and high-risk lesions (TIR 3A from TIR 3B), a significant degree of nuclear atypia warrants the inclusion of a lesion in one of the high-risk categories (TIR 3B or TIR 4)
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which are being addressed to surgery. For this reason, the Italian Committee expects that the low-risk category (TIR 3A) might result in 5-10% rate of malignant occurrence at histology compared to the 5-15% of expected range for the homologous categories of the British and American systems.
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5. MUTATIONAL TESTS
Thyroid carcinoma accounts for only 1,200 deaths in the United States each year, but the cost associated with thyroid carcinoma detection is far from trivial. Largely reflecting the inability of current diagnostic tests to discern thyroid carcinoma from the overwhelming background of benign thyroid nodules, the estimated 75,000 – 80,000 thyroid surgeries performed each year in the United States is disproportionate to the prevalence of clinically relevant thyroid cancers (30). Fine needle aspiration (FNA) is highly touted as the best available diagnostic tool, but as a guide for subsequent management of thyroid nodules it is often indecisiveness. In most series, between 15% and 20% of FNAs yield indeterminate results, results that are suspicious for but not diagnostic of malignancy (31). Moreover, only 17–51% of thyroids resected in the FNA era actually harbor carcinoma (32). Recognizing that most nodules that are suspicious on cytologic grounds ultimately prove to be benign, intraoperative frozen section analysis has long been used a means of guiding the extent of surgical resection. Frozen section analysis, however, rarely impacts on the intraoperative management of suspicious nodules, bringing into question this once hallowed practice (33). Clearly, novel strategies to discern benign from malignant thyroid nodules
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are much needed. Activating mutations of the BRAF gene occur in a broad range of human cancers including thyroid cancer (34). BRAF mutations induce constitutive activation of the RAS/RAF/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signal transduction pathway, providing a potent promitogenic force that drives malignant transformation (35) (Figure 5).
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As we said before, the most common and reliable diagnostic tool for evaluation of thyroid nodules is fine-needle aspiration (FNA) cytology. FNA provides a definitive diagnosis of benign or malignant thyroid disease in most cases. However, in about 25% of nodules, FNA cytology cannot reliably exclude cancer, and such cases are placed in one of the indeterminate categories (16, 36, 37). By the current Bethesda System for Reporting Thyroid Cytopathology, the indeterminate categories include three specific cytological diagnoses: atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS), follicular or oncocytic (Hürthle cell) neoplasm/suspicious for follicular or oncocytic (Hürthle cell) neoplasm (FN/SFN), and suspicious for malignant cells (SMC), with a predicted probability of cancer of 5–15, 15–30, and 60– 75%, respectively (19). Because FNA is unable to provide a definitive diagnosis for these nodules, most patients with indeterminate cytology undergo diagnostic surgery to establish a histopathological diagnosis. However, only 10–40% of such surgically resected thyroid nodules will prove to be malignant (19, 38). These unneeded operations, with their attendant expenses and risks, may be avoided if the FNA procedure could reliably establish the presurgical diagnosis of a benign nodule. Additionally, because the standard of care is to offer a second surgery to complete the thyroidectomy once diagnostic lobectomy confirms a cancer, a more optimal surgical
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management would be a single up-front total thyroidectomy that is offered when the diagnosis of cancer is established preoperatively. Furthermore, some genetic alterations (BRAF V600E) portend substantially worse prognosis and nodal metastasis (39, 40) and thus extent of surgery may be impacted by the availability of this information preoperatively.
We can use a panel of somatic mutations in thyroid FNA material and provide helpful diagnostic information. The panel includes most common mutations including that collectively occur in approximately 70% of thyroid cancer, i.e. BRAF V600E, NRAS codon 61, HRAS codon 61, and KRAS codons 12/13 point mutations and RET/PTC1, RET/PTC3, and PAX8/PPARγ rearrangements. The revised American Thyroid Association's management guidelines recommend to consider the mutational panel for nodules with indeterminate FNA cytology to help guide clinical management (4).
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5.1 BRAF MUTATION
BRAF mutations in thyroid tumors are generally restricted to PTC (40%-45%) and undifferentiated or poorly differentiated carcinomas (20%-40%), which presumably are derived from a PTC precursor (41,42). The overwhelming majority (>98%) of mutations in the BRAF gene involve codon 600 and result in a valine-to-glutamate substitution (V600E) (41,43). BRAF V600E mutations have not been identified in other thyroid neoplasms, including follicular adenoma (FA), follicular thyroid carcinoma (FTC), and medullary thyroid carcinoma (42). However, other uncommon BRAF mutations can be identified in 1.6% of different follicular neoplasms, including the follicular variant of papillary thyroid carcinoma (FVPTC), and also FA and FTC (44). The K601E mutation is most frequently associated with the non-invasive follicular variant of papillary thyroid carcinoma (NI-FVPTC; 80%), with the remaining 20% mostly being FA (45). The prevalence of BRAF mutations in PTC is highly variable and depends on the PTC subtype, the population studied and its iodine intake, and the sensitivity of the mutation assay (41) According to the Cancer Genome Atlas Research Network, PTC can be roughly subclassified molecularly into 2 groups with fundamentally different genomic, epigenomic, and proteomic
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profiles as well as distinct clinical features: a BRAFV600E-like phenotype including most classical papillary thyroid carcinoma (cPTC) and tall-cell variants of PTC and a RAS-like phenotype including most FVPTC (44). BRAF V600E mutations are present in approximately 80% of tall-cell variant PTC cases, in 40% to 60% of cPTC cases, and in only approximately 10% of FVPTC cases (41). FVPTC with infiltrative growth has a BRAF V600E mutation frequency of approximately 25%, and it has a significant rate of lymph node metastases and recurrences (46,47). In contrast, NI-FVPTC, which lacks the BRAF V600E mutation but often harbors RAS mutations (48) has little, if any, metastatic potential or recurrence risk, regardless of the quantitative or qualitative extent of the nuclear features of PTC (49). Thus, the clinical behavior and molecular alterations of infiltrative FVPTC are closer to those of cPTC, whereas NI-FVPTC is clinically and molecularly similar to FA/FTC, for which the clinical behaviour is determined by capsular and lymphovascular invasion, 2 features that cannot be assessed cytologically.
A limitation of BRAF testing in thyroid FNAB, even when it is combined with other less specific molecular markers such as RAS and RET/PTC (and PAX8/PPARg for FTC), is that the mutation/alteration is present only in a subset of PTCs, and thus a negative result does not necessarily provide useful information for clinical management because it does not rule out malignancy
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(50,51).
Furthermore, most BRAF mutations are identified in cases already classified cytologically as PTC ( 70% of cases), for which the usefulness of this finding is debated; the suspicious for malignancy category probably represents the area in which BRAF testing alone would have the greatest utility because approximately 15% to 20% of cases will be positive for the BRAF V600E mutation (50,51,52). In contrast, the rate of detection of the BRAF V600E mutation in the indeterminate diagnostic categories of AUS/FLUS and FN/SFN is only 4.6% (52,53). For these low-risk Bethesda indeterminate diagnostic categories with a low prevalence of BRAF mutations (comprising essentially FA, FVPTC, and FTC), the usefulness and cost-effectiveness of routine BRAF testing alone are doubtful (54).
5.2. NRAS MUTATION
The RAS oncogene plays a fundamental role in human tumorigenesis (55). Activating mutations in its three proto-oncogenes—HRAS, KRAS and NRAS—are found in nearly all human cancers. Thyroid cancer is one of the earliest cases where activating RAS mutations were discovered (56). Today, the role of RAS oncogene in thyroid tumorigenesis has been well
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established (57). Mutations in RAS genes occur, on average, in 30–45 % follicular thyroid cancer (FTC), 30–45 % follicular variant papillary thyroid cancer (FVPTC), 20–40 % poorly differentiated thyroid cancer (PDTC), 10–20 % anaplastic thyroid cancer, and rarely classical papillary thyroid cancer (PTC). RAS mutations also occur in 20–25 % benign follicular thyroid adenoma (FTA). Considerable interest has been drawn to the potential clinical utility of RAS mutations as diagnostic and prognostic molecular markers, among other molecular markers, for thyroid cancer (58). Given these occurrence patterns in thyroid tumors, however, RAS mutations themselves have low diagnostic sensitivities and specificities.
In the last decade, much effort has been made to apply RAS mutations as diagnostic molecular markers to FNAB of thyroid nodules, particularly those with indeterminate cytology, which currently represents a major diagnostic challenge in clinical practice.
RAS functions downstream from mitogenic growth factor receptors as a key membrane bound GTPase signal transduction protein. RAS thereby acts as a molecular switch, signaling along the MAPK, phosphatidylinositol 3-kinase/AKT, and other intracellular signaling pathways, controlling diverse cellular processes such as differentiation, proliferation, and cell survival (Figure 6) (59, 60). Three highly homologous human RAS genes, NRAS, KRAS, and HRAS, have been described (61). Oncogenic
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alleles of RAS carry mutations in specific hotspots of the 3 genes in codons 12, 13, and 61 (60). These mutations cause loss of intrinsic GTPase activity, leading to constitutive activation of RAS and increased activation of MAPK and alternate pathway signaling.
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Activating mutations of RAS in thyroid epithelial cells were first reported in the 1980s, but only recently have been directly implicated as early and frequent events in transformation and proliferation of thyroid carcinoma (62). However, the diagnostic and prognostic importance of RAS mutations has been obscured because these mutations are often reported not only in thyroid cancers but also in follicular adenomas (FAs) and histologically hyperplastic nodules (HNs) as well (63). Better understanding of the role of RAS mutation in thyroid carcinogenesis is important in light of the expanding use of mutational analysis in preoperative thyroid FNA evaluation.
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6. SURGICAL COMPLICATIONS
Mortality from thyroid and parathyroid surgery is virtually disregarded nowadays. During the eighteenth century, however, the mortality rate of thyroid surgery was as high as 40% from bleeding and sepsis. Once death from thyroid operation became an exception, specific pitfalls of the procedure, namely, injuries to the laryngeal nerves and damage to the parathyroid glands, became obvious.
Today morbidity remains a subject of concern for surgeons performing thyroid and parathyroid procedures. Injury of the recurrent laryngeal nerve and hypoparathyroidism are the most frequent complications. The key issue of an effective and safe surgical approach is a profound knowledge of specific anatomy and pathophysiology in combination with meticulous handling and dissection of tissue. The overall permanent complication rate should not exceed 1% in centers providing expertise (64).
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6.1 GENERAL SURGICAL COMPLICATIONS WOUND INFECTION
Wound infections, usually caused by Staphylococcus or
Streptococcus species are considered to be rare events, occurring
in 0.3% to 0.8% (64) of cases. Antibiotic prophylaxis is recommended only in immunocompromised patients or in those with valvular cardiac disorders. While mild neck cellulitis frequently regresses under conservative treatment, abscesses require rapid incision and evacuation. Delay of invasive treatment can result in devastating mediastinitis. Clinically evident seromas respond well to percutaneous aspiration.
BLEEDIND
The incidence of symptomatic hemorrhage requiring reintervention amounts to 0.1–1.5% (65). Postoperative bleeding will characteristically be prefaced by respiratory distress, pain, or cervical pressure, dysphagia, and increased blood drainage. No specific perioperative risk factors that would allow identification of the high-risk patient population for this potentially lethal complication are known. High surgical volume does not reduce the incidence of hematoma formation. Consequently, the key issue of prevention is attention to anatomic detail and careful hemostasis during surgery.
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In the majority of patients, symptomatic hemorrhage occurs between 6 and 12 hours after the initial operation. Since in approximately 20% of cases the onset of hematoma symptoms is reported beyond 24 hours postoperatively, ambulatory surgery with a 4- to 8-hour observation period might harbor risk of delayed intervention.
Once recognized, the wound should be deliberately reopened and the hematoma evacuated. In case of significant respiratory distress emergency bedside hematoma evacuation, if necessary in combination with endotracheal intubation, is required (Figure 7). The requirement for tracheotomy either in the emergency setting or due to persisting airway obstruction after hematoma removal is generally a rare event.
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6.2 SPECIFIC SURGICAL COMPLICATIONS
UNILATERAL INJURY TO THE RECURRENT
LARYNGEAL NERVE
Recurrent laryngeal nerve (RLN) injury is one of the most serious complications in endocrine surgery. The recurrent laryngeal nerve originates from the trunk of the vagus nerve. Upon reaching the larynx, it is renamed the inferior laryngeal nerve. It innervates all the intrinsic muscles of the same side with the exception of the cricothyroid muscles, and supplies sensory innervation to the laryngeal mucosa below the true vocal folds. While ascending, the nerve on the right and on the le side delivers branches that supply the trachea and the esophagus. The morphologic appearance and course of the recurrent laryngeal nerve are subject to great anatomic variability. In addition, it may o en be over- looked that the nerve most frequently does not consist only of a single trunk but exhibits a network of smaller branches (Figure 8). On their way to the cricothyroid muscle where they enter the larynx, both nerves run close to the capsule of the lateral aspect of the thyroid and cross the inferior thyroid artery. Several variations of the relationship between the nerve and the artery, particularly on the right side, can be observed. e nerve may pass superficially to the artery, deep to it, or between the branches of
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the vessel.
Figure 8. Anatomy of inferior laryngeal nerve
During cervical exploration the recurrent laryngeal nerve can be exposed at different levels; caudally, at the crossing with the common carotid artery, in the neighborhood of the inferior thyroid artery, and cranially, at Berry’s ligament, a dense condensation of the posterior thyroid capsule near the cricoid cartilage and upper tracheal rings.
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To alleviate the visual identification of the nerve and to provide an intraoperative tool to prove its functional integrity, diverse monitoring methods, i.e., intramuscular vocal cord electrodes inserted either through the cricothyroid membrane or placed endoscopically, endotracheal tube surface electrodes, endoscopic visualization of the vocal cords in combination with nerve stimulation (66), and palpation of the cricoarytenoid muscle with simultaneous neural stimulation (67), have been developed.
Damage to the recurrent laryngeal nerve may be caused by different mechanisms: cutting, clamping, or stretching of the nerve, nerve skeletonization, local compression of the nerve due to edema or hematoma, or thermal injury by electrocoagulation. Transient cord paresis, which is often caused by edema or axon damage by excessive nerve stretching, seldom lasts more than 4– 6 weeks. When no restitution of function is notable within 6–12 months postoperatively, permanent damage should be assumed. When a paralyzed vocal cord stays in the paramedian position the patients frequently remain asymptomatic. This phenomenon is due to compensatory overadduction of the intact cord and consecutive constriction of the glottic chink. The majority of asymptomatic patients need no special treatment but close observation.
If the paretic cord moves to the lateralized position, hoarseness or aspiration can occur.
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BILATERAL RECURRENT LARYNGEAL NERVE
INJURY
This serious complication results in a near midline position of the vocal cords and variable degrees of airway obstruction. Commonly, it will be diagnosed directly after extubation or during the early postoperative phase. The patient should be reintubated without delay and treated systemically with corticosteroids. In the presence of reversible nerve injury, extubation under controlled conditions is feasible in most cases after 24–72 hours and no further treatment is necessary. In case of persisting respiratory obstruction, reintubation and a tracheostomy must be carried out immediately. If the vocal cords fail to recuperate after a waiting period of 9–12 months, tracheostomy remains as a permanent solution or transverse laser cordotomy is performed (68).
HYPOPARATHYROIDISM
The reported rate of hypocalcemia after thyroid surgery varies from 1% to over 50% (69). While the majority of instances of postoperative hypocalcemia are transient, permanent hypoparathyroidism is decidedly unusual and should amount to
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less than 1%.
Although the pathogenesis of postthyroidectomy hypocalcemia is multifactorial, damage to the parathyroid glands in the form of direct injury, unrecognized inadvertent removal, or indirectly by devascularization of the gland are the most common causes. Other causative factors are negative calcium balance due to calcium absorption by bones in repair of osteodystrophy in hyperthyroid patients, decreased serum albumin levels caused by hemodilution, increased secretion of calcitonin during thyroid mobilization, or conditions associated with increased renal excretion of calcium.
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7 RETROSPECTIVE STUDY
7.1 MATERIAL AND METHODS
Between January 2009 and July 2013, 11,300 patients underwent total thyroidectomy for various indications at the authors’ center. Total thyroidectomy was performed either conventionally or via minimally invasive video-assisted thyroidectomy (MIVAT) or via robot-assisted transaxillary thyroidectomy (RATT), when selection criteria were met for MIVAT and RATT. Among these, 258 underwent conventional thyroidectomy for indeterminate uninodular thyroid disease. An indeterminate cytology includes Bethesda categories III, IV, and V. However, the 258 patients selected for this study had single nodules reported as suspicious for a follicular neoplasm (Bethesda category IV). They included 190 females (73.7%) and 68 males (26.3%). The female:male ratio was 4:1, and the mean age at diagnosis was 44 years (range 14–78 years). The mean sonographically estimated thyroid volume (SETV) was 16mL (range 2.9–115.7mL), and the mean nodule size was 23.5 mm (range 6–71 mm). The US features of thyroid nodules and the
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presence of biochemical thyroiditis were recorded. The US features assessed by gray-scale US were: hypoechogenicity, microcalcifications, irregular margins, and a ‘‘taller than wide’’ shape. Color Doppler US was not applied. Therefore, the pattern of nodular blood flow was not reported. The presence of biochemical thyroiditis was assessed by measurements of serum levels of thyroid antibodies (antithyroglobulin and antithyroid peroxidase antibodies). Serum levels of these antibodies were available, not because they are part of the routine work-up for thyroid nodular disease, but because they were required for research purposes by endocrinologists. None of the patients underwent lymph node dissection or lymphadenectomy through ‘‘berry picking,’’ as none had suspicious lymph nodes sonographically or as an intraoperative finding.
The surgical strategy regarding nodal dissection at the center is to perform a selective compartment-oriented nodal dissection based on suspicious sonographic and/or intraoperative findings. Prophylactic central compartment nodal dissection (CCLND) is not performed, even for an established diagnosis of papillary carcinoma, as it has been demonstrated in a prospective randomized controlled study that the addition of prophylactic CCLND does not favorably affect disease-free and overall survival, and is associated with greater morbidity. The histologic evaluation of all surgical specimens was conducted by two expert pathologists (F.B. and A.P.). Tumors were classified according to
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the World Health Organization (WHO) histological criteria. BRAF and NRAS sequences were analyzed in all 258 formalin-fixed, paraffin-embedded (FFPE) tissue samples. Tumoral areas were carefully isolated from two 10 lm thick sections, and genomic DNA was purified using a QIAmp DNA Mini Kit (Qiagen) following the manufacturer’s instructions. BRAF exon 15 and NRAS exon 2 were amplified according to standard procedures, and analyzed by direct Sanger sequencing on a AbiPrism 3130 Genetic Analyzer (Applied Biosystem). The pathologists were blinded to the molecular marker status and ultrasound features when making the histologic diagnosis.
7.2 RESULTS
On final histological examination, 90/258 patients (34.9%) had a thyroid carcinoma, and 168/258 patients (65.1%) had a benign nodule. Cases showing malignancy are collectively referred to as the malignant group (MG). Those with benign lesions are referred to as the benign group (BG). Within the MG, 71/90 (78.9%) had a follicular variant of papillary carcinoma, 10/90 (11.1%) had a classical papillary carcinoma, 6/90 patients (6.7%) had a solid variant of papillary carcinoma, and 3/90 patients (3.3%) had a follicular carcinoma. According to the TNM staging system (4), 39, 34, and 17 out of the 90 patients had T1, T2, and T3 tumors,
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respectively. None had nodal or distant metastases (N0, M0). The mean age in the BG was 47 years (range 14–75 years), with a female-to-male ratio of 3:1. The mean SETV was 17.3 mL (range 3.4–60.8 mL), and the mean nodule size was 27 mm (range 6–68 mm). The MG had a mean age of 45 years (range 14–78 years), with a female-to-male ratio of 3:1 as well. The mean SETV was 15.7 mL (range 2.9–115.7 mL), and the mean nodule size was 23.5mm (range 6–71mm). Both groups were demographically comparable. Regarding mutational markers, a BRAF mutation was present in 8/258 (3.1%) of cases. A NRAS mutation was present in 31/258 (12.0%) cases. These mutations were mutually exclusive. BRAF mutations were only seen in the MG, equally divided between BRAFV600E and BRAFK601E. Thirty- one NRAS exon 2 alterations were identified: 25 (80.6%) Q61R and 6 (19.4%) Q61K mutations. NRAS mutations occurred in 9/168 patients (5.4%) of the BG and in 22/90 patients (24.4%) of the MG. The occurrence of both mutations correlated significantly with malignancy (p < 0.05; Table 1).
The US features evaluated in our series (hypoechogenicity, microcalcifications, irregular margins, and a ‘‘taller than wide’’ shape) occurred in 162, 57, 24, and 33 out of 258 nodules, respectively. Hypoechogenicity was present in 53/90 (58.9%) of the MG and in 109/168 (64.8%) of the BG. The correlation of hypoechogenicity with malignancy was not statistically significant (p=0.52). Microcalcifications were present in 17/168
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(10.1%) of the BG and in 40/90 patients (44.4%) of the MG. Irregular margins were present in 2/168 (1.2%) of the BG and in 22/90 (24.4%) of the MG. A ‘‘taller than wide’’ shape was present in 9/168 (5.4%) of the BG and in 24/90 (26.6%) of the MG. The occurrence of all these features with malignancy was statistically significant (p < 0.05; Table 1). Biochemical thyroiditis was present in 45/258 patients: 30/168 (17.8%) and 15/90 (16.6%) of the BG and MG, respectively. The correlation of biochemical thyroiditis with malignancy was not statistically significant (p = 0.48; Table 1). The risk of malignancy in patients positive for a mutational marker and one or more suspicious US feature is demonstrated in Table 2. This table also demonstrates the positive predictive value (PPV) of suspicious US features (single and in combinations). Table 3 demonstrates the use of combining the absence of mutational markers and the absence of suspicious US features (single and in combinations) in determining the likelihood of malignancy. The risk of malignancy observed in those negative for both mutational markers and all suspicious US features was only (11.8%).
7.3 DISCUSSION
Thyroid nodular disease is common in clinical practice. Its prevalence largely depends on the population being evaluated and
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the detection method used. The prevalence of palpable thyroid nodules is around 5%, whereas the prevalence of non-palpable lesions (incidentally discovered on imaging studies) is much higher. A prevalence as high as 67% has been reported with the use of high-resolution ultrasonography (70). The risk of malignancy per patient is equal for both palpable and non-palpable nodules. However, it is relatively low (71). The ultimate goal of the diagnostic evaluation of a patient with a thyroid nodule is to determine its nature, consequently providing timely and appropriate treatment.
Although determining the nature of a thyroid nodule cannot be achieved solely by US, US can display features that may aid in predicting the nature of the nodule. Thyroid nodules are usually evaluated by a gray-scale and color Doppler US. Many sonographic features have been evaluated for their ability to predict the nature of a thyroid nodule. These include size, echogenicity, margin definition, shape/ orientation, the presence and extent of a halo, the presence and type of calcifications, the pattern of blood flow, and measures of tissue elasticity (US elastography) (72). Nodule size is not predictive of malignancy. The likelihood of cancer in a thyroid nodule is the same, irrespective of the size measured on US (71,72). As for the other features, the literature has demonstrated inconsistency in their predictive values, and the combination of findings improves the positive predictive value of US to some extent (71-74). In the
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present series, the vascularity of nodules was not evaluated. It has been demonstrated that there is no additional value of color Doppler US compared to gray-scale US in predicting the nature of a thyroid nodule (75-77). The features whose presence significantly correlated with malignancy included microcalcifications, irregular margins, and a ‘‘taller than wide’’ shape. On the other hand, hypoechogenicity did not significantly correlate with malignancy in the present study, as it occurred in 58.9% and 64.8% of malignant and benign nodules, respectively. The PPV of irregular margins, ‘‘taller than wide’’ shape, and microcalcifications were 91.6%, 72.7%, and 70.1%, respectively. The sensitivities and PPV of US features are demonstrated in Tables 1 and 2, respectively. Microcalcifications are defined as punctuate tiny foci that are too small to induce posterior acoustic shadowing. A ‘‘taller than wide’’ nodule is a nodule with an anteroposterior diameter greater than its transverse diameter, and was predictive of malignancy. It is believed that this non-parallel orientation is a reflection of the centrifugal growth pattern of malignancy across normal tissue planes. In the present series, the PPV of ultrasonography improved dramatically when findings were combined. Combining at least two features always achieved a likelihood of malignancy of 100% (Table 2). However, a major limitation to the utility of US alone in diagnosing malignancy is that sonographic features with a high PPV have low occurrences in malignancy, and thus are of low sensitivity.
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FNAC establishes a reliable diagnosis in 70–80% of instances. However, the remaining 20–30% of cases are labeled as being indeterminate for malignancy. An indeterminate diagnosis collectively includes Bethesda categories III, IV, and V. Labeling patients as having an indeterminate diagnosis is troublesome, as it places them at risk of a non- optimal initial surgical intervention, that is, an overly radical total thyroidectomy, or an unnecessary two-stage operation. This particularly applies to Bethesda category IV, whose risk of malignancy lies in the gray zone between those of Bethesda categories III and IV. The 15–30% risk of malignancy associated with lesions suspicious for a follicular/Hurthle cell neoplasm could be considered neither low nor high enough to make a diagnostic lobectomy or a total thyroidectomy, respectively, acceptable routine options.
In an attempt to resolve the dilemma of an indeterminate diagnosis, many centers turned to molecular diagnostics. Gene mutation markers (mutational markers) are one of several molecular tools used. Gene mutations associated with thyroid cancer can be detected in both fine-needle aspirates and surgical specimens. In the present series, the mutational analysis was performed on surgical specimens from areas clearly identified as malignant. Archived cytological smears were not available to compare concordance of results. Nevertheless, absolute concordance between mutational analysis results obtained with histological and cytological samples has been demonstrated.
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BRAF mutations are the most common gene alterations in thyroid cancer, and are typically seen in papillary carcinomas but rarely in follicular carcinomas (1.4%). Many mutational variants of BRAF exist; the most common one is BRAFV600E followed by BRAFK601E. BRAF mutations are highly specific for malignancy (78).
They correlate with a malignant outcome in almost 100% of instances (81).
RAS point mutations are also common in thyroid cancer. However, they are also found in 20–40% of benign lesions. RAS mutations can occur in all three RAS genes (NRAS, HRAS, and KRAS). NRAS mutations are the most common genetic alterations detected in indeterminate nodules (80). Despite occurring in a considerable proportion of benign lesions, it has been demonstrated that RAS-mutated benign lesions are prone to malignant transformation and would benefit from surgery (78). Based on what has been mentioned, and taking cost- effectiveness into account, the mutational analysis was limited to BRAF and RAS mutations. However, as mutational panels are considered rule-in tests, including additional mutations and/or rearrangements would be useful.
In the present series, the presence of a BRAF mutation was restricted to papillary carcinomas, with a PPV of 100%. BRAFV600E and BRAFK601E were equally common. However,
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a major limitation to its diagnostic utility, as inferred from the present series, is its low occurrence (3.1% of all indeterminate nodules and 8.8% of all malignancies). An NRAS mutation was the most common gene alteration in the present series of indeterminate nodules. Its occurrence correlated significantly with a malignant outcome, and its PPV was 70.9%. These results can be inferred from Table 1.
In the present study, the detection of any combination of a mutation and a suspicious US feature was strongly predictive of malignancy. Correlation with a malignant outcome was often at 100% (Table 2). The synergistic impact of adding mutational markers to suspicious gray-scale US features in predicting malignancy becomes evident when comparing the present results with the scoring system proposed by Russ et al. (73).
Applying the diagnostic modality in the present study achieves a predictivity equivalent or higher than that of a TIRADS score of 5 with only one suspicious US feature instead of requiring more than two. On the basis of the present findings, indeterminate nodules that are positive for such a combination can be treated with total thyroidectomy as the initial surgical approach. This is particularly applicable to cases that are considered clinically significant malignancies for which total thyroidectomy would be favored over any other treatment modality according to the American Thyroid Association (ATA) guidelines (4). On the other hand, being negative for both mutations and suspicious US
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features (152 in the present series) was associated with only a 11.8% risk of malignancy (18/152; Table 3). Furthermore, of the 18 patients with malignancy, only nine patients (six and three with T2 and T3 tumors, respectively) required postoperative radioactive iodine ablation therapy. On the basis of the reduced likelihood of malignancy, particularly high-risk malignancies in this group of patients with lesions suspicious for follicular neoplasms, lobectomy could have been both diagnostic and therapeutic in 94.1% (143/152). It is also believed that with improvements of diagnostic modalities and consequent further risk reduction in this category of lesions, diagnostic lobectomy could even be avoided. Observation could become a viable option in view of the fact that thyroid cancers grow slowly and most patients have an excellent prognosis. In summary, abiding to the diagnostic modality used in this study and, consistent with ATA guidelines on the management of differentiated thyroid cancer, 55% of patients could have been spared a total thyroidectomy, which could be considered as an overly radical procedure for this condition. These 55% of cases are the sum of 52% with a benign lesion and 3% with a malignancy, in which a total lobectomy could be considered as adequate treatment.
To conclude, the utility of innovative diagnostic modalities such as mutational markers complements standard evaluation of thyroid nodules labeled as ‘‘indeterminate.’’ Furthermore, a synergistic impact is obtained when molecular markers are
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combined with combinations of suspicious sonographic findings. Improving diagnostic accuracy in this subset of nodules aids in providing patients with the optimal initial surgical intervention.
Table 1. Summary of features assessed and their correlation with
malignancy. Benign (%) Cancer (%) P Value Odds Ratio (OR) 95% CI Total 168 (65) 90 (35) BRAF mutation Positive Negative 0 (0) 168 (100) 8 (8.8) 82 (91.2) 0.0152 34.72 1.98-608.93 N-RAS mutation Positive Negative 9 (5.4) 159 (94.6) 22 (24.4) 68 (75.6) < 0.0001 5.71 2.50-13.05 Microcalcifications Present Absent 17 (10.1) 151 (89.9) 40 (44.4) 50 (55.6) < 0.0001 7.10 3.70-13.63 Irregular margin Present Absent 2 (1.2) 166 (98.8) 22 (24.4) 68 (75.6) < 0.0001 26.85 6.14-117.34 Taller than wide
Present Absent 9 (5.3) 159 (94.7) 24 (26.6) 66 (73.4) < 0.0001 6.42 2.83-14.55 Thyroiditis Present Absent 30 (17.8) 138 (82.2) 15 (16.6) 75 (83.4) 0.81 0.92 0.46-1.81
