Thyroid FNAC: Causes of false-positive results.

In this paper, we aim to focus on false positive results in the evaluation of thyroid aspirations, covering cystic, inflammatory, follicular and oncocytic lesions, papillary carcinoma, and medullary carcinoma of thyroid. The recently described entity noninvasive follicular thyroid neoplasm with papillary-like nuclear features is also discussed detailing the impact of its introduction on the sensitivity and specificity of thyroid FNA, as well as the use of molecular tests for diagnostics. Medicolegal issues in relation to current practice in English law are also described.

Molecular pathology and thyroid FNA.

This review summarises molecular pathological techniques applicable to thyroid FNA. The molecular pathology of thyroid tumours is now fairly well understood. Molecular methods may be used as a rule-in test for diagnosis of malignancy in thyroid nodules, eg BRAF V600E point mutation, use of a seven-gene mutational panel (BRAF V600E, RAS genes, RET/PTC or PAX8/PPARG rearrangement), or as a comprehensive multigene next-generation sequencing panel, eg ThyroSeq v2. Molecular methods can also be applied as rule-out tests for malignancy in thyroid nodules, eg Afirma or ThyroSeq v2 or as markers of prognosis, eg TERT promoter mutation or other gene mutations including BRAF V600E, TP53 and AKT1, and as tests for newly defined tumour entities such as non-invasive follicular thyroid neoplasm with papillary like nuclei, or as a molecular marker(s) for targeted therapies. This review describes practical examples of molecular techniques as applied to thyroid FNA in routine clinical practice and the value of molecular diagnostics in thyroid FNA. It describes the range of molecular abnormalities identified in thyroid nodules and thyroid cancers with some practical applications of molecular methods to diagnosis and prognosis of thyroid nodules and thyroid cancer.

Recent Developments in the Pathology of Thyroid Cancer.

The histopathological features of thyroid cancers can be used to risk stratify patients, allowing prognostication and treatment decisions. A detailed accurate histological assessment by experienced pathologists working within a multidisciplinary context is crucial. Experience is also essential for interpretation of preoperative thyroid cytology specimens, which can be challenging. There is now more international harmonisation of numerical reporting systems for thyroid cytology. Understanding of the molecular basis of thyroid cancer has increased dramatically in recent years. Preoperative molecular pathology testing, when available, can refine cytological diagnosis to rule in or out for surgery, as well as assisting prognostication and enabling targeted treatment for thyroid tumours.

BRAF V600 co-testing is technically feasible in conventional thyroid fine needle aspiration (FNA) cytology smears and can reduce the need for completion thyroidectomy.

INTRODUCTION: While fine needle aspiration cytology (FNAC) is the mainstay of diagnosis in thyroid nodules, molecular markers of thyroid cancer have recently been shown to be of value in improving the diagnosis and reducing the rates of unnecessary surgery. METHOD: A technical method is presented for the assessment of the BRAF V600 gene mutation in thyroid cancer using a simple adaptation of a commercially available kit. After standard preparation and reporting of conventionally stained alcohol-fixed Papanicolaou or air-dried Giemsa-stained slides the coverslip is removed from one slide, the DNA is extracted and submitted for PCR analysis. RESULTS: Assessment of the BRAF V600 mutational status is feasible in very small quantities of DNA, requiring just greater than 5 ng per case from a single pre-stained FNA slide using this method. From the 14 cases examined thus far, one Thy4/Bethesda Class V case (suspicious of malignancy) has been identified with a BRAF V600 mutation and this patient, after multidisciplinary discussion, received a total thyroidectomy. CONCLUSION: Based on this methodology and other published results for the BRAF mutation, we believe that it is now feasible and cost effective for the UK NHS to BRAF co-test all Thy4/Bethesda Class V thyroid FNAs, as the additional cost of BRAF testing will still be much less than the cost of submitting all Thy4 (Bethesda Class V) patients to a partial and then a later completion thyroidectomy.

Rapid on-site assessment of specimens by biomedical scientists improves the quality of head and neck fine needle aspiration cytology.

OBJECTIVE: Immediate rapid on-site assessment (ROSA) of fine needle aspiration cytology (FNAC) specimens by biomedical scientists (BMS), the UK equivalent of cytotechnologists, or by pathologists may improve specimen quality and cellular adequacy rates for lymph node, head and neck and thyroid FNAC. The aim of this study was to evaluate the effect of introducing ROSA by BMS in an outpatient clinic setting. METHODS: The adequacy rate and sensitivity of histological diagnosis for lymph node, thyroid and salivary gland FNAC samples were determined before and after the introduction of BMS ROSA. The additional financial costs and time required to perform this service were also estimated. RESULTS: Thirty-one patients underwent ultrasound (US)-guided FNAC with ROSA and 151 without. ROSA reduced the number of FNAC insufficient in quality for diagnosis from 43% to 19% (P = 0.0194). The estimated additional cost for pathology per patient for ROSA was between £52.05 and £70.74, equivalent to €65.40/US $83.90 and €88.89/US $114.0, respectively, an increase of between 28% and 49% from the original cost. ROSA necessitated an additional 6 minutes clinic time per patient, reducing the number of patients that could be seen in an average clinic from 13 to 10 as well as requiring increased laboratory time for FNAC slide assessment. CONCLUSION: ROSA by suitably trained biomedical scientists and with appropriate consultant pathologist support can improve the quality of FNAC sampling for head and neck lesions. Although ROSA resulted in both additional financial and time costs, these are more than likely to be offset by a reduction in patients returning to clinic for repeat FNAC or undergoing unnecessary surgery.

Diagnostic terminology for reporting thyroid fine needle aspiration cytology: European Federation of Cytology Societies thyroid working party symposium, Lisbon 2009.

A European Federation of Cytology Societies (EFCS) working party of 28 members from 14 European countries met at the European Congress of Cytology in Lisbon in September 2009, with two observers from the USA, to discuss the need for standardising thyroid FNA nomenclature in the light of the National Institute of Cancer (NCI) recommendations resulting from the State of the Science conference in Bethesda in 2007. The data were obtained through two questionnaires sent by email and a transcript of the live discussion at the congress, which is presented in full. The surveys and discussion showed that there were currently no national terminologies for reporting thyroid FNA in the different European countries except in Italy and the UK. Personal, 'local', surgical pathology and descriptive terminologies were in use. All but one of the working party members agreed that thyroid FNA reporting should be standardised. Whilst almost a third would adopt the NCI Bethesda terminology, which offers the advantages of a 'risk of cancer' correlation and is linked to clinical recommendations, more than half favoured a translation of local terminology as the first step towards a unified nomenclature, as has been done recently in the UK. There was some disagreement about the use of: a) the six-tiered as opposed to four or five-tiered systems, b) the use of an indeterminate category and c) the 'follicular neoplasm' category, which was felt by some participants not to be different from the 'suspicious of malignancy' category. The conclusions will be passed to the different national societies of cytology for discussion, who will be asked to map their local terminologies to the Bethesda classification, observe its acceptance by clinicians and audit its correlation with outcome.

Individual crypt genetic heterogeneity and the origin of metaplastic glandular epithelium in human Barrett's oesophagus.

OBJECTIVES: Current models of clonal expansion in human Barrett's oesophagus are based upon heterogenous, flow-purified biopsy analysis taken at multiple segment levels. Detection of identical mutation fingerprints from these biopsy samples led to the proposal that a mutated clone with a selective advantage can clonally expand to fill an entire Barrett's segment at the expense of competing clones (selective sweep to fixation model). We aimed to assess clonality at a much higher resolution by microdissecting and genetically analysing individual crypts. The histogenesis of Barrett's metaplasia and neo-squamous islands has never been demonstrated. We investigated the oesophageal gland squamous ducts as the source of both epithelial sub-types. METHODS: Individual crypts across Barrett's biopsy and oesophagectomy blocks were dissected. Determination of tumour suppressor gene loss of heterozygosity patterns, p16 and p53 point mutations were carried out on a crypt-by-crypt basis. Cases of contiguous neo-squamous islands and columnar metaplasia with oesophageal squamous ducts were identified. Tissues were isolated by laser capture microdissection and genetically analysed. RESULTS: Individual crypt dissection revealed mutation patterns that were masked in whole biopsy analysis. Dissection across oesophagectomy specimens demonstrated marked clonal heterogeneity, with multiple independent clones present. We identified a p16 point mutation arising in the squamous epithelium of the oesophageal gland duct, which was also present in a contiguous metaplastic crypt, whereas neo-squamous islands arising from squamous ducts were wild-type with respect to surrounding Barrett's dysplasia. CONCLUSIONS: By studying clonality at the crypt level we demonstrate that Barrett's heterogeneity arises from multiple independent clones, in contrast to the selective sweep to fixation model of clonal expansion previously described. We suggest that the squamous gland ducts situated throughout the oesophagus are the source of a progenitor cell that may be susceptible to gene mutation resulting in conversion to Barrett's metaplastic epithelium. Additionally, these data suggest that wild-type ducts may be the source of neo-squamous islands.

Thyroid FNAC cytology: can we do it better?

This article reviews recent developments in thyroid fine needle aspiration cytology (FNAC). While thyroid nodules are common, carcinoma is comparatively rare. Although histological assessment is used in most studies as the benchmark, the differential diagnosis on cytology or histology is not always reproducible. The literature shows wide variations in criteria for inadequate thyroid FNAC and study inclusion or exclusion criteria. In-clinic assessment of specimen adequacy and in-clinic reporting of thyroid FNAC has become popular although the costs and resource implications of in-clinic thyroid FNAC assessment and reporting are substantial. Many centres continue to use conventional techniques although liquid-based cytology and ultrasound-guided FNAC are gaining in popularity. Standardized categorical systems for FNAC reporting can make results easier to understand for clinicians and give clear indications for therapeutic action. Multidisciplinary case review is also essential, especially when there is diagnostic uncertainty. While currently of limited use, molecular pathology testing holds out some promise for the future.

Galectin-3 is not useful in thyroid FNA.

INTRODUCTION: Previous studies have suggested that galectin-3 immunohistochemistry may be useful in the fine needle aspiration (FNA) diagnosis of thyroid carcinoma as it has been reported to selectively stain carcinomas and not adenomas or goitres. METHODS: Fifty-one patients were included in a prospective study of galectin-3 in thyroid FNA; 88.2% were female and 11.8% male, mean age 53 years, range 25-87 years. Cell blocks were prepared and stained for galectin-3 if any cells were present in needle washings from the respective FNAs. RESULTS: Twelve of 51 (23.5%) of cell blocks contained epithelial cells. One benign and one inadequate FNA were negative for galectin-3 staining. One of five non-diagnostic FNA cases, a papillary carcinoma on final histology showed positive staining. Four follicular neoplasm/suspicious of carcinoma cases showed negative staining. One malignant FNA case, a papillary carcinoma showed positive staining with galectin-3 but three further carcinomas, two papillary and one follicular were galectin-3 negative. CONCLUSION: Galectin-3 immunohistochemistry does not appear to be a useful adjunct to diagnosis in thyroid FNA as it does not reliably distinguish malignant and benign lesions. Many thyroid aspirates are of low cellularity and are not suitable for cell block immunohistochemistry.

Prognostic relevance of extensive necrosis in renal cell carcinoma.

AIMS: Evidence suggests that the presence of tumour necrosis is an adverse prognostic factor in renal cell carcinoma (RCC). However, it has also been shown that tumour regression, a microscopic feature associated with necrosis, may be a favourable short term prognostic factor in RCC. METHODS: Pathology reports of 253 RCCs from 1992 to 2001 were reviewed, and identified 37 tumours with substantial macroscopic or microscopic necrosis. Microscopic pathology, TNM 1997 tumour stage, and clinical follow up were reviewed and correlated with pathological findings. Three cases were rejected because two were diagnosed at necropsy, and a third was the result of renal arterial embolisation. RESULTS: Twenty of the 34 cases showed <50% necrosis, 10 showed 50-94% tumour necrosis, and four showed >95% tumour necrosis. Follow up data were unavailable in three cases. Nine of the remaining 31 tumours progressed; six were group 3 tumours showing <50% necrosis, three were group 2 tumours showing 50-94% necrosis, and none was a group 1 tumour showing >95% necrosis. CONCLUSIONS: Extensive necrosis (>95% necrosis) is rare in RCC, accounting for only 1.6% of those diagnosed during eight years in this unselected hospital series. The microscopic pattern of necrosis was typical, requiring extensive tumour sampling for definitive tumour diagnosis. Although there were only four patients with extensive necrosis, none developed recurrent or metastatic carcinoma, or died from RCC. Although extensive (>95%) necrosis may imply better short term prognosis after adjusting for tumour pathological TNM stage, it is probably not a prognostic variable in RCC.

Are renal microgranulomas related to inflammatory tubular destruction?

Two cases of renal microgranuloma formation are reported, one in a patient with known Crohn's disease and another in a case of acute renal allograft rejection. In both cases, the microgranulomas arose as a result of inflammatory tubular destruction, in a manner analogous to that seen in patients with ulcerative colitis arising adjacent to ruptured epithelial crypts in the large intestine. Microgranulomas may occur at multiple anatomical sites in Crohn's disease, although renal microgranulomas are very rare. Non-specific inflammatory tubular destruction should be considered as a cause of renal microgranuloma formation, in addition to systemic granulomatous diseases, such as tuberculosis, sarcoid, or Crohn's disease, when granulomas are seen in the presence of inflammatory tubular destruction in renal biopsies.

Pathology of ductal carcinoma in situ of the breast: current status.

Ductal carcinoma in situ (DCIS) now accounts for around 20-25% of mammographically detected breast cancers. There is strong evidence to show that classification schemes for DCIS should be based primarily on nuclear grade and necrosis as these two features have been shown to be prognostically important as well as having high interobserver reproducibility among pathologists. Newer classifications of DCIS that employ these features, such as the Van Nuys DCIS Classification, are of prognostic importance in predicting recurrence of DCIS after breast conservation and show high levels of reproducibility. For treatment of DCIS via breast conservation a high pre-operative diagnostic rate is desirable, only achievable via needle-guided core biopsy. If local excision without radiotherapy is to be given there is strong evidence to support the requirement for a 10 mm tumour-free margin. Assurance that a margin is tumour-free requires sequential specimen processing which is both time consuming and costly, but which can be justified in cost and morbidity terms as radiotherapy may not be required for those patients with a 10 mm tumour-free margin. Other methods of specimen examination involve examination of mammographically directed tissue slices or alternative methods of excision margin assessment such as "onion skinning" of the specimen. Endocrine therapy will doubtless become more important for adjuvant therapy of DCIS as well as chemoprophylaxis in the future.

Referrals for second opinion in surgical pathology: implications for management of cancer patients in the UK.

OBJECTIVE: To compare patterns of outgoing referral practice from one large district general hospital histopathology (cellular pathology) laboratory to other pathology laboratories. DESIGN: Referral cases for the relevant years were identified via hand searching of consultant referral files and from a central laboratory referral file. A comparison was made of the number and nature of pathology case referrals made to other laboratories in year 1990 with those made in year 1998. SETTING: Large district general hospital pathology laboratory in the UK. RESULTS: A statistically significant increase in the number of cases referred for a second opinion to an outside pathologist was noted, from 60 to 128 cases, representing an increase from 0.35 to 0.56% of total laboratory specimen workload (P=0.0034). In 36 (31.0%) of 116 cases from 1998 the diagnosis was altered, or a confident diagnosis was made where previously there was no definite diagnosis. Five cases with a benign in-house diagnosis had a malignant second opinion diagnosis and five cases with a malignant in-house diagnosis had a benign second opinion diagnosis. The largest single category of referred cases was for classification/grading of malignant lymphoma, comprising 27 (23%) of cases. The mean time delay between receipt of a specimen in the laboratory and issuing of the final report was 22 days (range 7-60 days). Only 25% of the referred cases were reported within 14 days. CONCLUSIONS: Referrals are an important component of pathology practice. In the UK much of this activity is performed on a 'grace and favour' basis between laboratories despite the fact that referral cases are often complex and time consuming for the recipient pathologist and laboratory. Histopathology referrals do not seem to be adequately costed and accounted for in interinstitutional service level agreements within the UK National Health Service.

Colonic lymphoid hyperplasia in melanosis coli.

We describe the case of a patient with Rett syndrome, a syndrome characterized by progressive infant encephalopathy, developmental delay, dementia, autism, ataxia, microcephaly, spastic paraparesis, and autonomic neuropathy with constipation. At colonoscopy, multiple foci of tiny white, sessile, polypoid lesions were seen throughout the colon and rectum, mimicking the appearances of small hyperplastic or adenomatous polyps, associated with generalized melanosis coli. This is the first case to our knowledge describing melanosis coli in a patient with Rett syndrome. As melanosis pigment deposition is characteristically not seen in lymphoid tissue, the lymphoid tissue was identifiable at endoscopy as multiple white nodules mimicking generalized colonic polyposis throughout the colon. We discuss the likely mechanisms of lymphoid hyperplasia and coexistent melanosis coli in Rett syndrome.

February 2001: A 74 year old man with a history over 3 months of increasing dyspnea and malaise.

The February COM: A man of 78 years with idiopathic late-onset cerebellar ataxia developed renal failure in association with a high ESR and positive pANCA. This was complicated by a subclinical spinal subarachnoid hemorrhage which was related to necrotizing inflammation of small leptomeningeal vessels. Renal cortical infarcts were due to similar inflammation in arcuate and interlobular arteries. Spinal subarachnoid hemorrhage is rare and usually due to rupture of an arteriovenous malformation. However, an immunogenic connective tissue disorder should be considered in the differential diagnosis. In this case, the histology and results of an autoantibody screen support a diagnosis of microscopic polyangiitis.