p53 expression in cytology samples may represent a marker of early-stage cancer.

BACKGROUND: TP53 gene plays a major role in the negative control of cell proliferation and in the regulation of signaling cascades. TP53 mutation may have a relevant role in the malignant transformation of thyroid cells as well as thyroid tumor progression. TP53 mutation has been detected only in few well differentiated thyroid carcinomas and is absent in benign conditions. METHODS: A total of 162 prospective thyroid cytology and corresponding histological samples diagnosed from atypia of indeterminate significance (AUS) to malignant, were studied via immunocytochemistry for p53. Hence, 50 benign lesions (B) were used as negative control. Molecular analysis for p53 only was performed. RESULTS: The cytology resulted in 50 B, 48 AUS, 40 follicular neoplasms (FNs), 23 suspicious for malignancy (SFM), and 1 malignant (M) case. The authors reported 102 negative and 60 positive p53 cases. The 60 positive cases included 27 cases with weak and/or focal cytoplasmic positivity (+1) and 33 with cases moderate (2+) to strong (3+) cytoplasmic and/or nuclear expression. Overall, 71 cases had histology (2 B, 11 AUS, 37 FN, 20 SFM, and 1 M) including 61.7% benign and 38.2% malignant diagnoses. Only 16 of 71 (5 FN, 10 SFM, and 1 M) were p53-positive. Furthermore, 100% AUS and 86.5% FN cases were p53-negative, none of which had malignant histology. All p53-positive cases were associated with a larger nodule size, tall-cell variant subtype, multifocality, extra thyroidal infiltration, and nodal metastases. Noninvasive follicular thyroid neoplasm with papillary like nuclear features were negative for p53. Few discrepancies in p53 intensity were observed on histology; there were no differences with the molecular testing. CONCLUSIONS: p53 might be useful in discriminating thyroid follicular lesions. p53 is likely to be a useful diagnostic marker in recognizing indeterminate lesions that are well-differentiated thyroid cancers.

Expression of vascular endothelial growth factor in follicular cell-derived lesions of the thyroid: Is NIFTP benign or precancerous?

Objective: Vascular endothelial growth factor (VEGF) is an angiogenic factor that plays an important role in physiological and pathological angiogenesis of the thyroid. The aim of the current study was to determine the expression characteristics of VEGF in follicular cell-derived lesions of the thyroid and to assess whether a new entity noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) is precancerous. Material and Methods: Patients diagnosed with 33 follicular adenomas (FA), 41 invasive follicular variant papillary thyroid cancer (IN-FVPTC), and 40 NIFTP in surgical resection materials were evaluated retrospectively. Immunostaining was performed on 5-µm paraffin tissue sections. The percentages of immunostaing for VEGF were evaluated on pathological materials. We used a percentage of labeled thyrocytes score (0, no labeling; 1, <30%; 2, 31-60%; 3, >60%) and an intensity score (0, no staining; 1, weak; 2, intermediate; 3, strong). The sum of two scores were accepted as the total score. Results: Mean ages of the FA, IN-FVPTC, and NIFTP groups were 44.7 ± 11.7 years, 46.9 ± 13.6 years, 43.2 ± 15.4 years, respectively and the mean VEGF immunostaining scores were 44.7 ± 29.3, 50.2 ± 32.54, 4 ± 26.3 respectively. Although there was no statistically significant difference (p= 0.347), the total score of the NIFTPs was higher than the scores of the FA (mean= 3.9 ± 1.8) and IN-FVPTC(mean= 4.3 ± 1.9) groups with a mean value of 4.6 ± 1.7. This result was remarkable. There was no statistically significant difference between tumor diameters and staining percentages (p= 0.750). Conclusion: Even if there were no statistical differences for VEGF immunostaining, it was high in NIFTPs. Since we know the role of VEGF in tumorigenesis, we can hypothesize that NIPTP can be precancerous. Our argue should be corroborated by a large prospective study.

Can Repeat Biopsies Change the Prognoses of AUS/FLUS Nodule?

OBJECTIVE: Experience with atypia of undetermined significance/follicular lesions of undetermined significance (AUS/FLUS) showed that this category exhibited a marked variability in incidence and malignant outcome in resection specimens. We aimed to determine the utility of repeated fine-needle aspiration biopsies (FNABs) and ultrasonography to determine the malignancy rate in AUS/FLUS nodules. METHODS: 23,587 nodules were biopsied, and 1,288 had at least one AUS/FLUS cytology. Ultrasonographic features including solid hypoechoic status, irregular margins, microcalcifications, nodule taller than wider, or an extrathyroidal extension were also recorded. Nodules for which only 1 FNAB revealed AUS/FLUS cytology were termed Group 1; nodules that underwent 2, 3, and 4 FNABs were termed Groups 2, 3 and 4, respectively. We compared these groups according to malignancy rates. RESULTS: 576 of nodules underwent only 1 FNAB (Group 1); 505, 174, and 33 underwent 2 (Group 2), 3 (Group 3), and 4 FNABs (Group 4), respectively. Fifty-six (30.6%), 45 (27.3%), 18 (30%), and 5 (33.3%) of Groups 1-4 were malignant, respectively. The risk of malignancy was similar in each group (p > 0.05). Suspicious ultrasonographic features were encountered in malignant nodules more than benign nodules (p < 0.05, for each). CONCLUSION: Repeat biopsy of AUS/FLUS nodules did not enhance the identification of malignancy. Ultrasonographic features may be a better guide for the decision of either surveillance or diagnostic surgery.

Semiquantitative assessment of cytomorphologic features can predict mutation status of thyroid nodules with indeterminate cytologic diagnosis.

Molecular diagnostics increasingly direct the management of thyroid nodules with an indeterminate cytologic diagnosis. This study was undertaken to correlate cytomorphologic features with the molecular profiles in an effort to identify features predictive of molecular aberrations. One hundred eighty-nine thyroid nodules with an indeterminate thyroid cytology diagnosis (atypia of undetermined significance, suspicious for follicular lesion, and suspicious for malignancy) with an adequate sample submitted for targeted mutation detection by polymerase chain reaction or next-generation sequencing were assessed semiquantitatively for the following cytomorphologic parameters: cellularity, Hurthle cell changes, microfollicles, nuclear elongation, nuclear grooves, nuclear enlargement, nuclear atypia, extent of atypia, and colloid. Based on this evaluation, a cumulative cytomorphologic score (CCS) and a more simplified overall atypia score (OAS) were assigned to each case. Associations among mutational status and each of the aforementioned parameters, CCS, and OAS were determined. Of the 189 nodules with indeterminate cytology, 63 (33.3%) harbored at least 1 mutation. RAS and BRAF were the most common mutations, found in 34 (18.0%) and 13 (6.9%) cases, respectively. Both CCS and OAS were highly associated with the presence of all mutations (P < .0001) and with the presence of BRAF and RAS mutations in particular (all P < .01). Semiquantitative assessment of various cytomorphologic features in indeterminate thyroid cytology cases showed a strong association of higher OAS and CCS and incidence of BRAF and RAS mutations. Using a more objective approach to thyroid cytology can potentially decrease the overall number of indeterminate diagnoses, leading to fewer repeat procedures and unnecessary surgical procedures.

Processing the entire capsule in thyroid follicular lesions improves diagnostic accuracy.

BACKGROUND: There is contradictory evidence in literature with respect to diagnosis and management of follicular lesions of the thyroid gland. From surgical pathology stand point, pathologists require submission and processing of entire capsule for microscopic evaluation. This can be extremely challenging especially in larger lesions. METHOD: We studied the impact of submitting entire capsule on final pathologic diagnosis in cases on which only representative sections were submitted initially and entire capsule was submitted subsequently. RESULTS: A total of 80 specimens were identified. Mean size of the nodule in these cases was 4.4 ±â€¯1.9 cm. Mean initial tissue sections submitted were 11.6 ±â€¯3.6. Entire capsule was submitted subsequently in an additional 12.6 ±â€¯13.3 sections. Submission of entire capsule contributed to final diagnosis in 3 (3.8%) cases whereby foci of capsular microinvasion were identified. There was no significant difference in the requirement of subsequent sections in specimens grossed by residents compared to those grossed by pathologist assistants (10.4 ±â€¯10.8 vs. 14.4 ±â€¯14.9, p = 0.18). The processing cost of additional sections of capsule was $ 4143 in these cases. CONCLUSION: Processing of entire capsule in thyroid follicular lesions has a definitive yield that comes at a high cost. Thin slicing and looking for areas of gross abnormality such as mushrooming may be more practical especially in larger lesions.

The predictive value of sonographic images of follicular lesions - a comparison with nodules unequivocal in FNA - single centre prospective study.

BACKGROUND: To determine the diagnostic efficacy of ultrasonographic malignancy risk features (UMRFs) in follicular lesions (FL) in a population with low risk of malignancy in FL and to compare it with a similar analysis in a group of patients with unequivocal cytology (UC): benign lesion (BL) or malignant neoplasm (MN). METHODS: Presence of UMRFs (hypoechogenicity, solid echostructure, taller-than-wide shape, pathological vascularization, irregular margins, microcalcifications and macrocalcifications) and their sets were assessed in 322 FL: 202 follicular lesions of undetermined significance (FLUS) and 120 suspicious for follicular neoplasm (SFN) and 300 nodules with UC: 200 BL and 100 MN, subsequently evaluated histopathologically. RESULTS: Cancers were confirmed in 100% nodules in MN group (89.0% of them were papillary carcinomas - PTC), in 6.4% FLUS nodules (69.2% PTC), and in 10.8% SFN nodules (30.8% PTC). In the UC group all UMRFs occurred more frequently in cancers than in benign lesions. In the FL group only calcifications were found in cancers more frequently - macro and microcalcifications together: 34.6 vs. 11.5% (p = 0.001) and isolated macrocalcifications: 26.0 vs. 6.8% (p = 0.001); the presence of those features increased the basic risk of malignancy in FL more than 2 times. The presence of at least 2 of the following URMFs: hypoechogenicity, solid echostructure, any type of calcifications and suspected shape, additionally improved sensitivity. CONCLUSIONS: Evaluation of UMRFs in FLs is less effective than in nodules with UC, and its effectiveness decreases parallel to the decrease in percentage of PTCs among malignant neoplasms and to the increase of the percentage of adenomas among benign nodules. The presence of macrocalcifications in such FLs significantly increases the basic risk of malignancy in these nodules.

Frozen section analysis in the post-Bethesda era.

BACKGROUND: The utility of frozen section (FS) for indeterminate thyroid nodules is controversial. In 2009, the Bethesda System for Reporting Thyroid Cytopathology was established to further subcategorize indeterminate fine-needle aspiration results (follicular lesions, FL) into Bethesda category 3 (BC3) and Bethesda category 4 (BC4). We hypothesize that FS will have less utility in the evaluation of BC3 lesions when compared to BC4. MATERIALS AND METHODS: A total of 479 patients who underwent thyroid lobectomy from January 2008 to July 2014 were retrospectively reviewed. Patients without appropriate Bethesda categorization were excluded. A total of 135 patients (65 FL, 45 BC3, 25 BC4) comprised the study groups. The sensitivity and specificity of FS within these three categories were determined. RESULTS: In the FL group, 6 of 65 patients were found to have thyroid cancer. Three were identified on frozen section (FS) resulting in a sensitivity and specificity of 50% and 100%, respectively. Thus, FS changed the operation in 3 of 65 cases (4.6%). In the BC3 group, 5 of 45 patients were found to have cancer. One was identified on FS resulting in a sensitivity and specificity of 20% and 100%, respectively. Thus, FS changed the operation in 1 of 45 patients (2.2%). In the BC4 group, 4 of 25 patients were found to have cancer. Two were identified on FS resulting in a sensitivity and specificity of 50% and 100% respectively. Thus, FS changed the operation in 2 of 25 patients (8%). CONCLUSIONS: There is improved utility of FS in BC 4 patients as 8% avoided reoperation. However, this benefit hinges on surgeon practice regarding the management of differentiated thyroid cancer >1 cm and <4 cm.

Detection of metabolites discriminating subtypes of thyroid cancer: Molecular profiling of FFPE samples using the GC/MS approach.

One of the critical issues in thyroid cancer diagnostic is differentiation between follicular adenoma, follicular carcinoma and the follicular variant of papillary carcinoma, which in some cases is not possible based on histopathological features only. In this paper we performed molecular profiling of thyroid tissue aiming to identify metabolites characteristic for different types of thyroid cancer. FFPE tissue specimens were analysed from 5 different types of thyroid malignancies (follicular, papillary/classical variant, papillary/follicular variant, medullary and anaplastic cancers), benign follicular adenoma and normal thyroid. Extracted metabolites were identified and semi-quantified using the GC/MS approach. There were 28 metabolites identified, whose abundances were significantly different among different types of thyroid tumours, including lipids, carboxylic acids, and saccharides. We concluded, that multi-component metabolome signature could be used for classification of different subtypes of follicular thyroid lesions. Moreover, potential applicability of the GC/MS-based analysis of FFPE tissue samples in diagnostics of thyroid cancer has been proved.

Diagnostic accuracy of fine needle aspiration cytology of thyroid and evaluation of discordant cases.

INTRODUCTION: The main role of fine needle aspiration cytology (FNAC) lies in differentiating between a malignant and benign thyroid nodule. It greatly influences the treatment decision. The current study was undertaken to evaluate the cytology-histopathology correlation and to analyze the cause of diagnostic errors with an eventual aim to improve diagnostic accuracy. MATERIALS AND METHODS: This is a retrospective study comparing cytology and corresponding histopathology report in 724 thyroid cases. The statistical analysis included false positive rate, false negative rate, sensitivity, specificity, positive predictive value, negative predictive value and accuracy. RESULTS: On cytological examination, 635/724 were reported as benign, 68 malignant and 21 suspicious. On histopathological examination, 626/635 cases were confirmed as benign but there were 9 discordant cases. Among the other cases histopathology diagnosis of malignancy matched in 66/68 and 11/21 cases. Diagnosis correlated in 703/724 cases (97%) [p<0.001]. False positive and false negative rates were 1.9% and 10.5%, respectively. The sensitivity and specificity were 89.5% and 98%, respectively. The positive predictive value was 84.6% and negative predictive value was 98.6%. Accuracy of FNA was 97%. CONCLUSION: In spite of high accuracy of FNAC in differentiating between a benign and malignant lesion, certain pitfalls should be kept in mind. The common false negative diagnoses were follicular pattern cases which constitute a 'gray zone', cystic papillary thyroid carcinoma (PTC) and papillary microcarcinoma. The reason for false positive diagnoses was the occurrence of nuclear features characteristic of PTC in other thyroid lesions. Awareness of pathologist regarding these pitfalls can minimize false negative/positive diagnoses.

Laser capture microdissection is a valuable tool in the preoperative molecular screening of follicular lesions of the thyroid: an institutional experience.

OBJECTIVES: The application of molecular tests to thyroid fine needle aspiration (FNA) has been shown to be a valuable tool to better refine the pre-operative malignant risk of patients with indeterminate cytology results. In this study, we investigated the feasibility of using the laser capture microdissection (LCM) technique to obtain DNA and RNA for molecular tests in routine thyroid FNA smears. METHODS: Nine coupled FNA and histological retrospective cases and 31 prospective FNA cases with a follicular neoplasm/suspicious for a follicular neoplasm (FN/SFN) diagnosis were included in this study. Both cytological and histological specimens were investigated by direct sequencing and reverse transcription-polymerase chain reaction (RT-PCR) for BRAF and RAS mutations and for PAX8/PPARG and RET/PTC rearrangements, respectively. RESULTS: LCM yielded good DNA and RNA quality in all cases (100%) in both series, irrespective of the staining used (Giemsa, Papanicolaou, immunostain for thyroglobulin) and the cytology technique (conventional or liquid-based preparations). Total mutations found in the FNA and in the corresponding histological specimen in both series were: one PAX8/PPARG rearrangement in a follicular carcinoma (FC), four NRAS mutations [in two FCs, one papillary carcinoma and one follicular adenoma (FA)] and one HRAS mutation in one FA. The sensitivity was 67% and the specificity was 91%. CONCLUSIONS: LCM is a valuable tool to obtain good quality DNA and RNA for molecular tests in cytological material from thyroid FNA, and can be a useful option in the management of patients with an FN/SFN FNA diagnosis.

Argyrophilic nucleolar organizing region associated protein synthesis for cytologic discrimination of follicular thyroid lesions.

Fine needle aspiration biopsy (FNAB) of the thyroid gland is an important tool for preoperative diagnosis; however, its benefit is limited for follicular lesions. Nucleolar organizer regions (NORs) are ribosomal gene regions that stain with silver (Ag) when they are active. These regions can be used to differentiate neoplastic and non-neoplastic lesions. We used a new AgNOR technique to investigate FNAB of cases diagnosed as follicular adenoma and carcinoma. Fourteen cases of follicular thyroid carcinoma (FTC) and 28 cases of thyroid follicular adenomas (FA) were stained using the silver NOR-associated protein (AgNOR) technique. One hundred nuclei per sample were examined, AgNORs were counted, and the total AgNOR area/nuclear area (TNORa/Na) ratio of each cell was calculated. We found that cases with FTC had significantly higher TNORa/Na than cases of FA. Also, cases with FTC had significantly higher AgNOR counts than cases with FA. AgNOR counting may help discriminate FTC and FA by routine cytopathology before surgery.

Proteomics for the diagnosis of thyroid lesions: preliminary report.

OBJECTIVE: Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a unique proteomic technology that explores the spatial distribution of biomolecules directly in situ, thus integrating molecular and morphological information. The possibility of correlating distribution maps of multiple analyses with cytological features makes it an ideal research tool for discovering new diagnostic markers. A previous study showed that MALDI-IMS could help discrimination between different types of thyroid lesions, especially papillary thyroid carcinoma (PTC); the present feasibility study on ex vivo fine needle aspiration (FNA) smears describes its potential in detecting new proteomic targets of other thyroid lesions (follicular lesions, medullary carcinoma). METHODS: MALDI-IMS was conducted on ex vivo FNAs obtained from surgical specimens and corresponding in vivo samples. Differences between proteomic profiles of different thyroid lesions were compared. RESULTS: Comparing the protein profiles of hyperplastic nodules obtained from three different patients with each other, and with a new PTC, showed a high degree of concordance, indicating good reproducibility of the IMS technology on cytological samples, suggesting its potential as a tool for biomarker discovery. Furthermore, comparison of the average proteomic profiles of hyperplastic nodules with a Hürthle cell adenoma revealed significant differences, underlying the capability of MALDI-IMS to distinguish between different thyroid lesions. Finally, the proteomic profile of medullary thyroid carcinoma was also characterized. CONCLUSIONS: Our results confirmed the possible role of MALDI-IMS in the search for diagnostic targets of PTC and follicular lesions, which could be applied in larger trials aimed at the identification of proteins, convertible to cost-effective diagnostic tools such as immunohistochemistry. These tests could be used to analyse in vivo cytological smears, improving the preoperative diagnosis of indeterminate thyroid nodules.

Accurate diagnosis of thyroid follicular lesions from nuclear morphology using supervised learning.

Follicular lesions of the thyroid remain significant diagnostic challenges in surgical pathology and cytology. The diagnosis often requires considerable resources and ancillary tests including immunohistochemistry, molecular studies, and expert consultation. Visual analyses of nuclear morphological features, generally speaking, have not been helpful in distinguishing this group of lesions. Here we describe a method for distinguishing between follicular lesions of the thyroid based on nuclear morphology. The method utilizes an optimal transport-based linear embedding for segmented nuclei, together with an adaptation of existing classification methods. We show the method outputs assignments (classification results) which are near perfectly correlated with the clinical diagnosis of several lesion types' lesions utilizing a database of 94 patients in total. Experimental comparisons also show the new method can significantly outperform standard numerical feature-type methods in terms of agreement with the clinical diagnosis gold standard. In addition, the new method could potentially be used to derive insights into biologically meaningful nuclear morphology differences in these lesions. Our methods could be incorporated into a tool for pathologists to aid in distinguishing between follicular lesions of the thyroid. In addition, these results could potentially provide nuclear morphological correlates of biological behavior and reduce health care costs by decreasing histotechnician and pathologist time and obviating the need for ancillary testing.

'Indeterminate for malignancy' (Tir3/Thy3 in the Italian and British systems for classification) thyroid fine needle aspiration (FNA) cytology reporting: morphological criteria and clinical impact.

BACKGROUND: The British system (Thy1-5), the Bethesda system for reporting thyroid cytopathology (BSRTC) and the Italian Society of Anatomic Pathology and Cytology (SIAPEC) classification represent the most important international classifications for thyroid cytopathology. Irrespective of the system used, the 'indeterminate' categories are still debated among cytopathologists, particularly with regard to diagnostic criteria, clinical impact of subclassification and role of molecular techniques. AIM: We aimed to find answers to the following questions: Are there shared criteria in cytological preparations that allow the division of indeterminate follicular lesions into subcategories? What is the true clinical impact of this possible subclassification? METHODS: Among 1150 consecutive thyroid fine needle aspiration (FNA) specimens, 80 patients had nodules with a final cytological report of Tir3 (SIAPEC)/Thy3. These 80 cases were re-evaluated and subclassified according to morphological criteria into three groups: pure follicular proliferations, Hürthle cell follicular lesions and atypical proliferations. RESULTS: Sixteen (20%) cases were categorized as pure follicular proliferations, 40 (50%) as Hürthle cell follicular lesions and 24 (30%) as atypical proliferations. Surgery was performed in 57 cases (71%). Cyto-histological correlation showed that follicular adenoma was the most frequent final diagnosis in the cases treated by surgery (24/57, 42%). The overall malignancy rate in the Tir3 category was 28% (16/57). Atypical proliferations were more often malignant than either of the follicular groups (53% versus 19%, P = 0.019). CONCLUSIONS: A five-tiered classification, subdividing the 'indeterminate for malignancy' class into 'follicular proliferations' and 'atypical lesions' could be adopted. As a result of their higher risk of malignancy, surgical management of the atypical lesions would be justified. In future, the introduction of a genetic panel might contribute to their stratification, to the determination of a more accurate risk of malignancy of the atypical lesions and to the verification of follicular proliferations that are benign.

A systematic approach to assess the strengths and limitations of cytomorphology in the diagnosis of the follicular variant of papillary thyroid carcinoma.

OBJECTIVE: To identify the most useful cytomorphological features of follicular variant of papillary thyroid carcinoma (FVPTC). METHODS: Fine needle aspiration cytological features of seven histologically proven FVPTCs were systematically evaluated in a blinded manner for various architectural, nuclear, cytoplasmic and background features with special reference to nuclear morphology. RESULTS: Most smears were moderate to highly cellular with clustered and/or repetitive microfollicles, rare macrofollicles and minimal thick gummy colloid. Six of seven cases showed significant nuclear crowding/overlapping. Fairly uniform nucleomegaly (mostly three to five times the size of a mature lymphocyte) of intact neoplastic cells and enlarged naked nuclei were prominent features in all seven cases, whereas enlarged ovoid nuclei were seen in two cases. Chromatin was fine to coarsely granular and evenly distributed. Occasional nuclear grooves (NGs) and intranuclear cytoplasmic inclusions (INCIs) were seen in five and three cases, respectively. This refined approach led to a precise diagnosis of FVPTC in six cases, which were earlier interpreted as various follicular lesions. Follicular hyperplasia was excluded by the absence of significant amounts of colloid and atretic naked nuclei, whilst the possibility of follicular adenoma or follicular carcinoma was excluded by the presence of one or more features suggestive of papillary thyroid carcinoma. CONCLUSIONS: Our study showed a high cell yield, microfollicular pattern, nuclear overcrowding/overlapping, scanty gummy colloid and enlarged naked nuclei as the most consistent features of FVPTC. Although inconsistent, features such as enlarged ovoid nuclei and syncytial clusters were complementary to the diagnosis in the absence of NGs and INCIs.