Methylation Analysis of Urinary Sample in Non-Muscle-Invasive Bladder Carcinoma: Frequency and Management of Invalid Result.

BACKGROUND: Numerous studies showed that methylation analysis represents a newly developed urinary marker based on DNA methylation changes in a panel of genomic biomarkers and it could represent a valid tool in terms of the diagnosis and prediction of high-grade urothelial carcinoma recurrences. One of the limits of the use of this new molecular method during a follow-up is represented by the number of invalid tests in routine practice. METHOD: A total of 782 patients with a diagnosis of non-muscle-invasive high-grade carcinoma (NMIBC) was studied. The Bladder EpiCheck test (BE) was performed together with cytology in all cases within 1 year after the end of treatment. In 402 patients, the urinary samples were voided urine (UV), while, in 380 cases, the samples were collected after bladder washing (IU). For all the patients with invalid BE results, a second BE test was performed following the instructions for use that indicated the test should be repeated with a new urinary sample in the case of an invalid result. RESULTS: Analyzing the two different groups (UV and IU), we found the invalid BE results seemed to be not related to urinary samples (p = 0.13 Fisher's exact test), suggesting that the collection method was not relevant in order to reduce the number of invalid tests. CONCLUSIONS: In the follow-up for NMIBC, for patients for whom a BE test is planned, a combined approach of cytology and a methylation test is recommended in order to repeat the BE test with an invalid result only in those cases with a cytological diagnosis of atypical urothelial cells (AUC) suspicious for high-grade urothelial carcinoma (SHGUC) and high-grade urothelial carcinoma (HGUC).

Impact on clinical follow-up of the Milan System for salivary gland cytology: A comparison with a traditional diagnostic classification.

INTRODUCTION: No universally accepted classification exists for salivary gland FNA. The proposed Milan System for Reporting Salivary Gland Cytopathology (MSRSGC) offers a uniform classification with management recommendations. We compared FNA diagnoses from a prior study with specific diagnoses with corresponding MSRSGC diagnoses. METHODS: One-hundred and sixty-four cases from a prior cytological study with histological follow-up were re-reviewed by one of the authors and assigned to one of the MSRSGC categories. The original and MSRSCG diagnoses were compared, as were follow-up recommendations. RESULTS: The MSRSGC system classified 29 specimens as non-diagnostic (seven histologically shown to be benign salivary gland, two non-mucinous cysts, 14 sialadenitis, one pleomorphic adenoma, one haemangioma, one lymphoma, one adenoid cystic carcinoma, one squamous carcinoma and one benign lymphoid proliferation). The original study diagnosed these lesions as: seven benign cysts, 15 benign salivary gland tissue, one benign neoplasm and two insufficient for diagnosis. In seven cases, MSRSGC disagreed with original diagnoses and surgical resection showed lesions where optimal follow-up was more consistent with original cytological diagnosis. In 10 cases with disagreement, the MSRSGC was associated with a more appropriate follow-up based on the surgical diagnosis. Malignancy risks for the Milan categories were: non-diagnostic (12%), non-neoplastic (5%), atypia of undetermined significance (19%), neoplasm, benign (5%), neoplasm (40%), suspicious for malignancy (60%) and malignant (93%). CONCLUSION: MSRSGC was comparable with the original reported diagnoses in the majority of cases. Both systems had high accuracy for distinguishing benign from malignant lesions and both were associated with appropriate follow-up in most cases.

Morphology combined with ancillary techniques: An algorithm approach for thyroid nodules.

INTRODUCTION: Several authors have underlined the limits of morphological analysis mostly in the diagnosis of follicular neoplasms (FN). The application of ancillary techniques, including immunocytochemistry (ICC) and molecular testing, contributes to a better definition of the risk of malignancy (ROM) and management of FN. According to literature, the application of models, including the evaluation of ICC, somatic mutations (ie, BRAFV600E ), micro RNA analysis is proposed for FNs. This study discusses the validation of a diagnostic algorithm in FN with a special focus on the role of morphology then followed by ancillary techniques. METHODS: From June 2014 to January 2016, we enrolled 37 FNs with histological follow-up. In the same reference period, 20 benign nodules and 20 positive for malignancy were selected as control. ICC, BRAFV600E mutation and miR-375 were carried out on LBC. RESULTS: The 37 FNs included 14 atypia of undetermined significance/follicular lesion of undetermined significance and 23 FN. Specifically, atypia of undetermined significance/follicular lesion of undetermined significance resulted in three goitres, 10 follicular adenomas and one NIFTP whereas FN/suspicious for FN by seven follicular adenomas and 16 malignancies (nine non-invasive follicular thyroid neoplasms with papillary-like nuclear features, two invasive follicular variant of papillary thyroid carcinoma [PTC] and five PTC). The 20 positive for malignancy samples included two invasive follicular variant of PTC, 16 PTCs and two medullary carcinomas. The morphological features of BRAFV600E mutation (nuclear features of PTC and moderate/abundant eosinophilic cytoplasms) were associated with 100% ROM. In the wild type cases, ROM was 83.3% in presence of a concordant positive ICC panel whilst significantly lower (10.5%) in a negative concordant ICC. High expression values of MirR-375 provided 100% ROM. CONCLUSIONS: The adoption of an algorithm might represent the best choice for the correct diagnosis of FNs. The morphological detection of BRAFV600E represents the first step for the identification of malignant FNs. A significant reduction of unnecessary thyroidectomies is the goal of this application.

Incidence, malignancy rates of diagnoses and cyto-histological correlations in the new Italian Reporting System for Thyroid Cytology: An institutional experience.

OBJECTIVE: FNA biopsy is considered as the most accurate method for the selection of patients with thyroid nodules that need for surgery or for the wait and see management. The aim of the present study is to clarify the risk of malignancy for the cytological data classified according to the 2014 Italian reporting system. METHODS: We report a retrospective analysis of 4043 patients in our institution's experience during the period April 2014 through December 2016 with the Italian reporting system for thyroid cytology. RESULTS: The diagnostic incidences of the 4043 cases were as follows: 9.8% TIR1; 1.3% TIR1C; 70% TIR2; 6.6% TIR3A; 4.5% TIR3B; 2.4% TIR4; 5.2% TIR5. A repeated aspiration was carried out in 68 out of 269 cases (25%) classified as TIR3A. A total of 407 cases with cytology underwent surgical resection. A malignant neoplasm was detected in 261 out of 407 (64%) cases. Regarding TIR3B, surgical excision was undertaken in 109 cases, which included 42 high-risk lesions and 67 Hürthle cell neoplasms. The risk of malignancy was significantly higher in the former compared to the latter (50% vs 9%; P<.05). CONCLUSIONS: This investigation emphasises the reliability of the 2014 Italian Reporting System concerning the mutual frequency of the diagnostic categories. The risk of malignancy is perfectly within the range of the estimated values.

Morphological features that can predict BRAFV600E -mutated carcinoma in paediatric thyroid cytology.

OBJECTIVE: BRAFV600E represents the most common diagnostic marker in papillary thyroid carcinoma (PTC). A few papers have demonstrated the correlation between BRAFV600E and specific morphological findings on PTCs in the adult population. This is the first reported series investigating cytological morphological parameters in paediatric thyroid carcinoma. METHODS: One hundred and nineteen paediatric samples (56 male and 63 female patients), diagnosed in the period between April 2013 and July 2015, were enrolled in the study. Fifteen patients with inadequate results were excluded. Cytological cases were processed with liquid-based cytology (LBC). BRAFV600E and immunocytochemistry for the VE1 antibody were performed on LBC. RESULTS: The diagnostic series included 10 mutated and 94 wild-type (WT) cases. Twenty two percent surgical samples showed 96% cytohistological concordance. The morphological analysis revealed plump cells (abundant eosinophilic cytoplasm and PTC nuclei) in all 10 mutated cases with only four cases showing a focal (less than 20% of the cells) plump component. None of the WT showed plump cells. A sickle nuclear shape was seen only in the mutated cases. VE1 yielded 100% positivity on mutated cases with three cytohistological discrepancies. CONCLUSIONS: The BRAFV600E mutation is also seen in paediatric cytology and the morphological features showed a high accuracy as both predictive mutational parameters and a helpful aid in management mainly of the aggressive BRAFV600E mutated carcinomas.

The cytological diagnosis of a 'benign thyroid lesion': is it a real safe diagnosis for the patient?

OBJECTIVE: In fine needle aspiration cytology (FNAC), the category of benign thyroid lesions (BTL), which constitutes 65-70% of all thyroid FNAC, and can be correctly diagnosed by morphology alone, is an important entity. A diagnosis of BTL denotes a lesion managed with follow-up unless found in conjunction with compressive symptoms. Although this diagnosis can be quite simple, there are cases in which the scant cellular or colloid component may pose diagnostic issues. Herein, we describe the experiences of evaluating BTL at two large academic institutions. We evaluated the clinical importance of a correct diagnosis of BTL to define the exact inherent risk of a false-negative result (FNR). METHODS: From January 2008 through to June 2013, 506 (3.6%) out of 15 850 patients with BTL underwent surgery. All nodules were sampled under sonographic guidance (US) and processed either with liquid-based cytology (LBC), Diff-Quik® smears or alcohol-Papanicolaou staining methods. RESULTS: The histological follow-up of 506 BTL series included 493 benign and 13 malignant lesions. The latter group included four follicular carcinomas (FC), two classic variants of papillary thyroid carcinoma (PTC), one macrofollicular PTC and six follicular variants of PTC (FVPC). The malignancy rate for the BTL category was 2.5%. CONCLUSIONS: When diagnosed by expert cytopathologists, BTL represents a robust diagnosis and might reduce the number of FNR. Additional diagnostic experience and a large case series could enable cytopathologists to recognise all the morphological entities of BTL. An important additional aid is the extensive sampling of the lesions to reduce issues related to a low cellularity.

DIAGNOSIS OF ENDOCRINE DISEASE: High-yield thyroid fine-needle aspiration cytology: an update focused on ancillary techniques improving its accuracy.

Thyroid fine-needle aspiration (FNA) cytology is a fast growing field. One of the most developing areas is represented by molecular tests applied to cytological material. Patients that could benefit the most from these tests are those that have been diagnosed as 'indeterminate' on FNA. They could be better stratified in terms of malignancy risk and thus oriented with more confidence to the appropriate management. Taking in to consideration the need to improve and keep high the yield of thyroid FNA, professionals from various fields (i.e. molecular biologists, endocrinologists, nuclear medicine physicians and radiologists) are refining and fine-tuning their diagnostic instruments. In particular, all these developments aim at increasing the negative predictive value of FNA to improve the selection of patients for diagnostic surgery. These advances involve terminology, the application of next-generation sequencing to thyroid FNA, the use of immunocyto- and histo-chemistry, the development of new sampling techniques and the increasing use of nuclear medicine as well as molecular imaging in the management of patients with a thyroid nodule. Herein, we review the recent advances in thyroid FNA cytology that could be of interest to the 'thyroid-care' community, with particular focus on the indeterminate diagnostic category.

The potential of liquid-based cytology in lymph node cytological evaluation: the role of morphology and the aid of ancillary techniques.

OBJECTIVES: Our aim was to evaluate the feasibility and diagnostic accuracy of liquid-based cytology (LBC) on lymph node fine needle aspiration (FNA). FNA may fulfil a challenging role in the evaluation of the majority of primary (benign and malignant) diagnoses as well as metastatic lymph node lesions. Although the morphological features may be quite easily recognized, cytological samples with a scant cellular component may raise some issues. METHODS: We appraised 263 cytological lymph nodes from different body regions analysed between January and December 2013, including 137 male and 126 female patients, and processed with LBC. RESULTS: The cytological diagnoses included 160 benign and 103 malignant lesions. We reported 35 benign and 73 malignant lesions from 108 with surgical follow-up. The latter malignant series included 68 metastatic lesions, four suspicious for malignancy and one inadequate sample. The cytological diagnoses were supported by 62 conclusive immunocytochemical and 28 molecular analyses. Of the 108 cases, we documented 35 true negatives, 72 true positives, one false negative and no false positives, resulting in 98.6% sensitivity, 100% specificity, 99% diagnostic accuracy, 97.2% negative predictive value and 100% positive predictive value. CONCLUSIONS: FNA represents the first diagnostic tool in lymph node management and a reliable approach in order to avoid an excision biopsy. Furthermore, LBC is a feasible method for ancillary tests for which methanol-fixed samples are suitable, such as immunocytochemistry and molecular analysis.

Large non-functioning parathyroid cysts: our institutional experience of a rare entity and a possible pitfall in thyroid cytology.

OBJECTIVE: Large non-functioning parathyroid cysts represent a rare entity with a benign clinical course. They may be misdiagnosed as thyroid cystic neoplasms on fine needle aspiration cytology (FNAC), resulting in inappropriate surgical treatment. We evaluated our institutional experience in the diagnosis of large parathyroid cystic lesions underlining all the differential diagnoses and pitfalls. METHODS: In the period between 1998 and 2012, we reported the cytology of eight large (>2.5 cm) parathyroid cystic lesions (all female patients) with histological control. The aspirations were performed with a 25-gauge needle with ultrasonographic guidance. The aspirated material was processed with liquid-based cytology (LBC). All the patients had normal serum parathyroid hormone (PTH) and calcium. RESULTS: The cytological samples showed a fluid watery component without colloid and few or absent epithelial cells. The resulting negativity for thyroglobulin and positivity for PTH, carried out on the cystic fluids, suggested parathyroid lesions rather than either thyroid cystic lesions (including follicular thyroid neoplasm) or cystic malignant lesions. All the patients underwent surgery without complications. CONCLUSIONS: To the best of our knowledge, this is one of the largest series with cytohistological evaluation of large parathyroid cysts. The incidence of large parathyroid cysts remains controversial as most patients are asymptomatic. FNAC may be performed with conclusive results in the majority of cystic cases. The detection of PTH and calcium on the cystic liquid is likely to achieve a correct cytological diagnosis, allowing adequate treatment and ruling out a more frequent thyroid lesion.

Angiogenesis in hand chondroma: an immunohistochemical study.

Hand chondroma is a particular cartilagineous tumour, being clinically benign, but morphologically malignant. This study investigates the expression of VEGF together with other growth factors and proliferation markers such as TGFß2, Ki-67, TNF, FGF1, P53 in 8 cases of hand chondroma treated with courettage, in order to define the ethiopathogenesis of this tumour and the clinical significance of the resulting immunohistochemical profile, with particular respect to angiogenesis. VEGF was expressed in all cases; 5 cases were positive for TFGß2 and 3 for PDGF. None of the other factors was expressed. On the basis of histologic results a specific model of tumour progression based on the indicators of angiogenesis could be related to hand tumours, in which VEGF expression should be the first stadium of the tumour aggressiveness, and the following PDGF, TGF 2 expression should be accompanied with a morphological outline worsening. Nevertheless the non constant expression of these indicators and the absent expression of proliferated indicators can explain the scant tendency to the relapse in presence of accurate curettage. It is important to remember that the cellular polymorphism typical of the cartilaginous tumours does not allow the application of an only oncogenesis model.

Thyroid fine needle aspiration cytology processed by ThinPrep: an additional slide decreased the number of inadequate results.

BACKGROUND: Fine needle aspiration cytology is the most accurate tool for diagnosing thyroid nodules. Its accuracy is related to the rate of inadequate samples, which can be minimized with the adoption of on-site assessment of the adequacy of the material. The introduction of liquid-based cytology (LBC) in the thyroid does not allow checking on the adequacy of the cellularity. The possibility of making a second LBC slide for decreasing the number of non-diagnostic cases is studied. METHODS: Out of 553 cases diagnosed in a single institution from January to March 2005, 166 consecutive cases with an LBC slide reviewed by the same pathologist were evaluated. The cases were classified in a five-tiered category system (Thy1 to 5 according to the British Thyroid Association guidelines) and all but two (with cystic degeneration) were processed by LBC and stained with Papanicolaou stain. The above-mentioned categories are defined as follows: Thy1 inadequate or haemorrhagic, Thy2 non-neoplastic lesion; Thy3 follicular lesion/suspected follicular neoplasm; Thy4 suspicious for malignancy; Thy5 diagnostic of malignancy. For each case the cytological diagnosis was made on the LBC slide and the adequacy of the cellularity for a conclusive diagnosis was assessed. RESULTS: Of the 166 cases, 39 were non-diagnostic (Thy1-inadequate), ten presented features of cystic degeneration (Thy1-haemorrhagic), 90 were benign (Thy2), 22 were diagnosed as a follicular lesion (Thy3), one as suspicious of malignancy (Thy4) and four as papillary carcinoma (Thy5). Thirty-nine cases had a second LBC for achieving a definitive diagnosis with eventual re-classification. Of these cases, 23 (61.5%) led to a conclusive diagnosis (18 Thy2, five Thy3) with a 18.5% decrease of the inadequacy rate. CONCLUSIONS: The making of an additional LBC slide helps in achieving a diagnosis in cases classified as non-diagnostic on the first standard slide (52.1% recovery rate). This procedure is particularly helpful for meeting the adequacy criteria in benign and indeterminate lesions and could also be used for refining the diagnosis of suspicious for a malignant thyroid neoplasm.

Application of liquid-based preparation to non-gynaecologic exfoliative cytology.

Thin-layer cytology (TLC) is an automated method for processing cells harvested in a liquid solution and collected onto a single slide. The leftover material can be used for other techniques such as immunocytochemistry, molecular biology and flow cytometry. TLC has been applied with good results in exfoliative cytology of pulmonary, urinary, gastrointestinal and oral districts as well as in the evaluation of serous effusions. The main advantages of TLC over conventional techniques (CS) are: (a) simplification of the sampling technique; (b) decrease in cellular artefacts leading to a lesser amount of inadequate diagnoses; and (c) applicability of additional investigations. The limits of TLC are: (a) changes in the morphologic picture of some lesions; (b) increase of the workload for technical staff; and (c) increased cost. The application of TLC to non-gynaecologic specimens favours many innovative developments and can be regarded as an appropriate substitute for CS.

Simultaneous immunohistochemical expression of HBME-1 and galectin-3 differentiates papillary carcinomas from hyperfunctioning lesions of the thyroid.

AIMS: The histological diagnosis is critical for the postsurgical management and follow-up of thyroid malignancies. The differential diagnosis between papillary carcinoma and hyperfunctioning lesions, either with papillary hyperplasia or with a follicular architecture, can create real diagnostic difficulty. The aim of this study was to evaluate the expression of several antibodies considered to be markers of malignancy in malignant and hyperfunctioning thyroid neoplasms and to include the most effective of them in a diagnostic panel. METHODS AND RESULTS: One hundred resected thyroid nodules--58 hyperfunctioning benign lesions and 42 papillary carcinomas (14 follicular variant, 14 macrofollicular variant and 14 classic type)--were immunohistochemically studied for HBME-1, galectin-3, cytokeratin (CK) 19 and RET-proto-oncogene. HBME-1 and galectin-3 showed 92.8% and 89% sensitivity, respectively, and their coexpression was present in 36 out of 42 papillary carcinomas (85.7%) and absent in non-malignant lesions. Their association increased sensitivity to 94.7% and the diagnostic accuracy to 97.9% and involved the highest number of cases (95%) in comparison with two other panels including, respectively, three (HBME-1, galectin-3, CK19) and all four antibodies. CONCLUSION: An immunohistochemical panel consisting of HBME-1 and galectin-3 can make a correct distinction between malignant and hyperfunctioning thyroid neoplasms with high diagnostic accuracy.

Gene expression profiling of adrenal cortical tumors by cDNA macroarray analysis. Results of a preliminary study.

Adrenocortical carcinoma (ACC) are highly malignant tumors with poor prognosis. To verify if it is possible to assess their differential gene expression by a cDNA macroarray analysis using RNA extracted from paraffin sections, we analyzed two different cohorts of adrenal cortical adenoma (ACA) and ACC. Paraffin sections of seven ACC and seven ACA were analyzed. Transcriptional profiles were generated by commercially available c-DNA arrays testing 82 genes. Hybridization signals were quantified by densitometry and the intensity signal was compared for each gene between ACA and ACC cohorts. RNA was successfully extracted in only four out of 14 cases. Four genes displayed a significantly different expression (ACC/ACA ratio>1.5 or<0.6). Heat shock protein 60 (HSP-60) (ratio>2), Ciclin D1 and topoisomerase I (ratio>1.5) were overexpressed in the ACC cohort, while jun proto-oncogene was down-regulated. cDNA macroarray analysis from paraffin sections of adrenal tumors is feasible, despite with a low success rate. The different expression of HSP-60, Ciclin D1, jun proto-oncogene and topoisomerase I indicates that these genes may play a role in ACC pathogenesis and could represent potential diagnostic/prognostic/therapeutic target markers. Larger series of patients are necessary to confirm the biologic, diagnostic, prognostic and therapeutic implications of these findings.