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The Editor-in-Chief of Acta Cytologica, Prof. Kari J. Syrjänen, M.D., Ph.D., FIAC will celebrate his 75th Birthday on March 20, 2023, and we would like to offer a tribute to the 3rd Editor of Acta Cytologica and one of the pioneers in HPV research.

DICER1 protein is a member of the ribonuclease (RNAse) III family with a key role in the biogenesis of microRNAs (miRNA) and in microRNA processing, potentially affecting gene regulation at the post-transcriptional level. The role of DICER1 and its relevance to thyroid cellular processes and tumorigenesis have only recently been explored, following the acknowledgement that DICER1 germline and somatic changes can contribute not only to non-toxic multinodule goiter (MNG) lesions detected in individuals of affected families but also to a series of childhood tumours, including thyroid neoplasms, which can be identified from early infancy up until the decade of 40s. In a context of DICER1 germline gene mutation, thyroid lesions have recently been given importance, and they may represent either an index event within a syndromic context or the isolated event that may trigger a deeper and broader genomic analysis screening of individuals and their relatives, thereby preventing the consequences of a late diagnosis of malignancy. Within the syndromic context MNG is typically the most observed lesion. On the other hand, in a DICER1 somatic mutation context, malignant tumours are more common. In this review we describe the role of DICER protein, the genomic events that affect the DICER1 gene and their link to tumorigenesis as well as the frequency and pattern of benign and malignant thyroid lesions and the regulation of DICER1 within the thyroidal environment.

“Small-cell malignancies of thyroid” is an unsolved dilemma. This term represents an umbrella terminology in thyroid, encompassing for a small group of tumors in which some of them are well-recognized tumors like medullary thyroid carcinoma, poorly differentiated thyroid carcinoma, and primary thyroid lymphomas and teratoma, whereas the remaining are less known as primary neuroendocrine carcinoma of thyroid, primary extraskeletal Ewing family tumors, and adamantinoma-like Ewing sarcoma. When the issue comes to evaluate a cytological sample predominantly composed of small-cell morphology, metastatic small-cell carcinomas to thyroid also should be excluded. In this review, our group focused on the main cytomorphological and clinical clues of each entity that help to set up a correct differential diagnosis. The literature discussions were also included for the entities that are not yet recognized by the mother publication WHO. A key point of the issue’s simple algorithm based on FNAC with small-cell morphology of thyroid was suggested by the authors.

Anaplastic thyroid carcinoma (ATC) is a rare cancer accounting for 40% of thyroid cancer-specific deaths. In the last 5 years, improved insights into molecular pathways led the Food and Drug Administration to license BRAF/MEK inhibitors (B/Mi) in BRAF

Contains fulltext : 287669.pdf (Publisher’s version ) (Open Access) The incidence of differentiated thyroid cancer (DTC) has been increasing worldwide, mostly, as an increase in the incidental detection of micro papillary thyroid carcinomas (microPTCs), many of which are potentially overtreated, as suggested by the unchanged mortality. Several international guidelines have suggested a less aggressive approach. More recently, it has been shown that active surveillance or minimally invasive treatments (MIT) are good alternatives for the management of these patients. In this context, patient participation in the decision-making process is paramount. The Endocrine Task Force of the European Organisation for Research and Treatment of Cancer (EORTC) has undertaken the task to establish consensus and define its position based on the scientific evidence concerning, 1) the current state of diagnostic and management options in microPTCs, including the current opinion of physicians about shared decision making (SDM), 2) the available evidence concerning patients' needs and the available decision instruments, and 3) to provide practical suggestions for implementation of SDM in this context. To improve SDM and patients' participation, knowledge gaps and research directions were highlighted.

Background High-risk human papilloma virus (HR HPV) testing and liquid-based cytology are used for primary cervical screening. Digital cytology, based on whole-slide scanned samples, is a promising technique for teaching and diagnostic purposes. The aim of our study was to evaluate the interobserver and intraobserver variation in low-grade squamous lesions, HR HPV status bias, and the use of whole-slide scanned digital cervical cytology slides. Methods Fifteen expert cytopathologists evaluated 71 digitalized ThinPrep slides (31 atypical squamous cells of undetermined significance [ASC-US], 21 negative for intraepithelial lesion or malignancy, and 19 low-grade squamous intraepithelial lesion cases). HR HPV data were accessible only in the second round. Results In interobserver analysis, Kendalls coefficient of concordance was 0.52 in the first round and 0.58 in the second round. Fleiss kappa values were 0.29 in the first round and 0.31 in the second round. In the ASC-US category, Fleiss kappa increased from 0.19 to 0.22 in the second round and the increase was even higher expressed by Kendalls coefficient: from 0.42 to 0.52. In intraobserver analysis, personal scores were higher in the second round. Conclusions The interobserver and intraobserver variability in low-grade squamous lesions was within fair agreement values in the present study, in line with previous works. The comparison of two rounds showed that expert cytopathologists are generally unbiased by the knowledge of HR HPV data, but that being informed of the HR HPV status leads to a better agreement. Stain quality and back discomfort were highlighted as factors affecting digital cytopathology use. Funding Agencies|European Federation of Cytology Societies

Background: Familial nontoxic multinodular goiter (MNG) is a rare disease. One of the associated genes is Kelch-like ECH-associated protein 1 (KEAP1), which encodes the main inhibitor of nuclear factor erythroid 2-related transcription factor 2 (Nrf2), a central mediator of antioxidant responses. The association of KEAP1 with familial MNG is based on only two loss-of-function mutations identified in two families, only one of which included proper phenotyping and adequate demonstration of co-segregation of the phenotype and the mutation. There is no experimental evidence from model organisms to support that decreased Keap1 levels can, indeed, cause goiter. This study used mice hypomorphic for Keap1 to test whether decreased Keap1 expression can cause goiter, and to characterize the activation status of Nrf2 in their thyroid. Methods: C57BL/6J Keap1 flox/flox (Keap1 knock-down [Keap1 KD ]) mice were studied at 3 and 12 months of age. Plasma and thyroid glands were harvested for evaluation of thyroid function tests and for gene and protein expression by real-time polymerase chain reaction and immunoblotting, respectively. Results: Keap1 KD mice showed diffuse goiter that began to develop in early adult life and became highly prominent and penetrant with age. The goiter was characterized by a markedly increased size of thyroid follicles, most notably of the colloid compartment, and by absence of thyroid nodules or hyperplasia. Keap1 KD mice also showed decreased T4 levels in early adult life that were eventually well compensated over time by increased thyrotropin (TSH) levels. Nrf2 was activated in the thyroid of Keap1 KD mice. Despite a known stimulatory effect of Nrf2 on thyroglobulin (Tg) gene transcription and Tg protein abundance, the expression levels were decreased in the thyroid of Keap1 KD mice. No clear patterns were observed in the expression profiles of other thyroid hormone synthesis-specific factors, with the exception of Tg-processing and Tg-degrading cathepsins, including an increase in mature forms of cathepsins D, L, and S. Conclusions: Keap1 KD mice develop age-dependent diffuse goiter with elevated TSH levels. The precise mechanism accounting for the thyroidal phenotype remains to be elucidated, but it may involve enhanced Tg solubilization and excessive lysosomal Tg degradation.

BACKGROUND: To the authors' knowledge, the impact of the coronavirus disease 2019 (COVID-19) pandemic on cytopathology practices worldwide has not been investigated formally. In the current study, data from 41 respondents from 23 countries were reported. METHODS: Data regarding the activity of each cytopathology laboratory during 4 weeks of COVID-19 lockdown were collected and compared with those obtained during the corresponding period in 2019. The overall number and percentage of exfoliative and fine-needle aspiration cytology samples from each anatomic site were recorded. Differences in the malignancy and suspicious rates between the 2 periods were analyzed using a meta-analytical approach. RESULTS: Overall, the sample volume was lower compared with 2019 (104,319 samples vs 190,225 samples), with an average volume reduction of 45.3% (range, 0.1%-98.0%). The percentage of samples from the cervicovaginal tract, thyroid, and anorectal region was significantly reduced (P < .05). Conversely, the percentage of samples from the urinary tract, serous cavities, breast, lymph nodes, respiratory tract, salivary glands, central nervous system, gastrointestinal tract, pancreas, liver, and biliary tract increased (P < .05). An overall increase of 5.56% (95% CI, 3.77%-7.35%) in the malignancy rate in nongynecological samples during the COVID-19 pandemic was observed. When the suspicious category was included, the overall increase was 6.95% (95% CI, 4.63%-9.27%). CONCLUSIONS: The COVID-19 pandemic resulted in a drastic reduction in the total number of cytology specimens regardless of anatomic site or specimen type. The rate of malignancy increased, reflecting the prioritization of patients with cancer who were considered to be at high risk. Prospective monitoring of the effect of delays in access to health services during the lockdown period is warranted. ispartof: CANCER CYTOPATHOLOGY vol:128 issue:12 pages:885-894 ispartof: location:United States status: published

This review article summarises systems for categorisation of diagnostic errors in pathology and cytology with regard to diagnostic accuracy and the published information on human factors in pathology to date. A 12 point event-based checklist for error in diagnostic accuracy in histopathology and cytopathology is proposed derived from Dupont’s ‘Dirty Dozen’ human factor checklist, as utilised in the aerospace industry for aircraft maintenance. This human factor checklist comprises 12 human factors implicated in defects in aircraft maintenance; (i) failure of communication, (ii) complacency, (iii) lack of knowledge, (iv) distractions, (v) lack of teamwork, (vi) fatigue, (vii) lack of resources, (viii) pressure, (ix) lack of assertiveness, (x) stress, (xi) norms, and (xii) lack of awareness. The accompanying article explains practical examples of how each of these 12 human factors may cause errors in diagnostic accuracy in pathology. This checklist could be used as a template for analysis of accuracy and risks of diagnostic error in pathology either retrospectively ‘after the event’ or prospectively at the time of diagnosis. There is a need for further evaluation and validation of this proposed 12 point human factor checklist and similar systems for categorisation of diagnostic errors and diagnostic accuracy in pathology based on human factor principles.

Background Variability in preanalytical and analytical steps for immunocytochemistry (ICC) on cytology samples is poorly defined. The objective of this study was to evaluate current practices for ICC on cytology samples in European laboratories. Methods A link to an online survey with 19 questions about ICC practices was distributed to cytology laboratories through national representatives in the European Federation of Cytology Societies. Results In total, 245 laboratories responded to the survey by January 30, 2019. Cell blocks, cytospins, liquid-based cytology (LBC) preparations, and smears alone or in combination with other preparations were used for ICC in 38%, 22%, 21%, and 19% of laboratories, respectively. In general, various combinations of preparations were used for ICC in greater than one-half of laboratories (147 of 245; 60%), whereas only 1 specific type of cytology preparation was used in the remaining 98 of 245 laboratories (40%) laboratories. The majority of laboratories (217 of 226; 96%) performed ICC on automated platforms using protocols that were the same as those used for formalin-fixed, paraffin-embedded samples (238 of 527 laboratories; 45%), either optimized (138 of 527 laboratories; 26%) or optimized and validated (151 of 527 laboratories; 29%) for cytology preparations. Positive control slides, negative control slides, and external quality control were used in 174 of 223 (78%), 112 of 223 (50%), and 111 of 120 (50%) laboratories, respectively. Greater than 1000 ICC tests were performed yearly in 34% of laboratories (65 of 191; average, 1477 tests; median, 500 tests). Conclusions ICC is extensively performed in European laboratories using variously prepared cytology preparations on automated platforms, mostly without quality-assurance measures.

INTRODUCTION The UK Royal College of Pathologists (RCPath) Thy terminology is an internationally recognised system for reporting thyroid fine needle aspiration. The terminology has been used throughout the UK and Ireland, in some parts of Italy and Switzerland, and elsewhere in the world. There is no systematic review of the literature specifically addressing the use of the non-diagnostic for cytological diagnosis-Thy1/Thy 1c category in the UK RCPath terminology. METHODS A comprehensive literature search of online databases was conducted in October 2019 specifically examining overall reported rates of Thy1 and Thy1c in aspirates classified according to the UK Thy terminology. RESULTS Twenty-five articles were identified showing a Thy1 rate of 13.4% (2540/18 920). The studies were then stratified according to whether or not the patients underwent rapid on-site evaluation (ROSE): 6.0% (353/5841; range 3.0%-10.9%) of ROSE aspirates were Thy1 whereas 18.5% (2072/11 204; range 7.9%-43.3%) of non-ROSE patients were Thy1; (P

Background: In a previous worldwide survey, the authors showed a drastic reduction in the number of cytological specimens processed during the coronavirus disease 2019 “lockdown” period along with an increase in malignancy rates. To assess the continued impact of the pandemic on cytological practices around the world, they undertook a second follow-up worldwide survey collecting data from the post-lockdown period (2020). Methods: Participants were asked to provide data regarding their cytopathology activity during the first 12 weeks of their respective national post-lockdown period (2020), which ranged from April 4 to October 31. Differences between the post-lockdown period and the corresponding 2019 period were evaluated, and the authors specifically focused on rates of malignant diagnoses. Results: A total of 29 respondents from 17 countries worldwide joined the survey. Overall, a lower number of cytological specimens (n = 236,352) were processed in comparison with the same period in 2019 (n = 321,466) for a relative reduction of 26.5%. The overall malignancy rate showed a statistically significant increase (12,442 [5.26%] vs 12,882 [4.01%]; P

Context.— Solid pseudopapillary neoplasm of the pancreas is a low-grade malignant tumor generally associated with a good prognosis. Solid pseudopapillary neoplasms show peculiar morphologic features, but sometimes the differential diagnosis with other pancreatic neoplasms (ie, pancreatic neuroendocrine tumors) can be a challenging task, especially in cytologic or biopsy specimens. In these cases immunohistochemistry is a useful tool, but the diagnostic utility of several proposed immunohistochemical markers is questionable. In recent years, despite several attempts to characterize the pathogenetic, molecular, and prognostic features of solid pseudopapillary neoplasms, they still remain unclear. Objective.— To give the reader a comprehensive update on this entity. Data Sources.— The PubMed database (US National Library of Medicine) was searched using the following string: pseudopapillary tumor [AND/OR] neoplasm [AND/OR] pancreas. All articles written in English were included. In addition, because a heterogeneous terminology has been used in the past to define solid pseudopapillary neoplasms, the reference lists of each paper selected in the PubMed database were also reviewed. Conclusions.— This review gives a comprehensive update on the pathologic, clinical, and molecular features of solid pseudopapillary neoplasms, particularly addressing issues and challenges related to diagnosis. In addition, we have tried to correlate the molecular alterations with the morphologic and clinical features.

BACKGROUND Thyroid fine-needle aspiration (FNA) is a reliable and cost-effective diagnostic tool for establishing the nature of thyroid nodules, although up to 30% of FNAs are still classified as "indeterminate." Molecular testing of FNAs could improve preoperative diagnosis, thereby reducing unnecessary surgery. In this multicenter prospective study the authors investigated, using a 7-gene assay, the distribution and diagnostic impact of BRAF, RAS, RET/PTC, and PAX8/PPARg, the most frequent genomic alterations occurring during thyroid oncogenesis. METHODS In total, of 1172 routine FNAs from 7 centers in southern Italy were classified according to the Bethesda System for Reporting Thyroid Cytopathology. Each specimen was tested, and molecular data were compared with available histology or cytologic follow-up. RESULTS In particular, for atypia of undetermined significance/follicular lesion of undetermined significance cases, the 7-gene test confirmed the high positive predictive value of BRAFV600E and BRAF-like mutations (80%) and the moderate positive predictive value of RAS-like alterations (32.4%), suggesting different surgical management, depending on the type of mutation. The rate of mutation-positive FNAs was strictly related to the risk of malignancy of each diagnostic class, supporting the identification of prognostically relevant diagnostic categories. CONCLUSIONS The 7-gene panel test improves the preoperative risk stratification of indeterminate thyroid FNAs, especially when considering the biologic significance of the different types of mutations. Moreover, the rate of mutation-positive FNAs is related to the risk of malignancy of each diagnostic class.

The hyalinizing trabecular adenoma/tumor is a rare and poorly characterized follicular-derived thyroid neoplasm recently shown to harbor recurrent PAX8-GLIS1 or PAX8-GLIS3 gene fusions. Here we sought to define the repertoire of genetic alterations of hyalinizing trabecular tumors, and whether PAX8-GLIS3 fusions are pathognomonic for hyalinizing trabecular tumors. A discovery series of eight hyalinizing trabecular tumors was subjected to RNA-sequencing (n = 8), whole-exome sequencing (n = 3) or targeted massively parallel sequencing (n = 5). No recurrent somatic mutations or copy number alterations were identified in hyalinizing trabecular tumor, whereas RNA-sequencing revealed the presence of a recurrent genetic rearrangement involving PAX8 (2q14.1) and GLIS3 (9p24.2) genes in all cases. In this in-frame fusion gene, which comprised exons 1-2 of PAX8 and exons 3-11 of GLIS3, GLIS3 is likely placed under the regulation of PAX8. Reverse transcription RT-PCR and/or fluorescence in situ hybridization analyses of a validation series of 26 hyalinizing trabecular tumors revealed that the PAX8-GLIS3 gene fusion was present in all hyalinizing trabecular tumors (100%). No GLIS1 rearrangements were identified. Conversely, no PAX8-GLIS3 gene fusions were detected in a cohort of 237 control thyroid neoplasms, including 15 trabecular thyroid lesions highly resembling hyalinizing trabecular tumor from a morphological standpoint, as well as trabecular/solid follicular adenomas, solid/trabecular variants of papillary carcinoma, and Hurthle cell adenomas or carcinomas. Our data provide evidence to suggest that the PAX8-GLIS3 fusion is pathognomonic for hyalinizing trabecular tumors, and that the presence of the PAX8-GLIS3 fusion in thyroid neoplasms may be used as an ancillary marker for the diagnosis of hyalinizing trabecular tumor, thereby avoiding overtreatment in case of misdiagnoses with apparently similar malignant tumors.

Background: Thyroid cancer accounts for 1% of cancer cases in developed countries, in which papillary thyroid carcinoma (PTC) is the most common type. There are multiple variants of PTC described to date, some of them with aggressive behavior and poor clinical outcome. These variants are well described and accepted in recent guidelines of many international societies, and the prognostic and management implications are well laid out. Due to their established clinical importance and to guide appropriate surgical management, it is now imperative in clinical practice, including cytopathology, to differentiate aggressive variants from nonaggressive ones. This review aims to describe the variants of PTC and to provide a practical algorithmic approach to facilitate the cytological diagnosis of these variants. Summary: Subtyping PTC variants on fine needle aspiration cytology (FNAC) is challenging even for the most experienced cytopathologist. To facilitate a correct subtyping on FNAC, we propose a stepwise approach that is mainly designed for conventional smear methodology. This approach requires first to stratify the lesions into oncocytic and nononcocytic features before analyzing further details in cell morphology and pattern. Key Messages: (1) Subtyping in PTC is possible on cytopathology. (2) The main aim of the cytopathologist is to differentiate aggressive from nonaggressive variants. (3) The subtyping of PTC can help in the surgical management of the patients.

Objective The second version of The Bethesda System for Reporting Thyroid Cytopathology endorsed the introduction of non-invasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTP) as a distinct entity with low malignant potential into clinical practice. Consequently, the risk of malignancy (ROM) of cytological diagnoses has changed, but the magnitude of the change remains uncertain. The present systematic review was undertaken to obtain more robust information about the true impact of NIFTP on the ROM among patients undergoing surgery following a fine-needle aspiration cytology (FNAC) diagnosis of suspicious for malignancy (Bethesda V) or malignant (Bethesda VI). As they are managed surgically, these two diagnostic categories are the primary entities that are clinically impacted by the advent of NIFTP. Design Systematic review and meta-analysis. Methods A comprehensive literature search of online databases was performed in November 2018. The search was conducted looking for data of histologically proven NIFTP with preoperative FNAC. Results One-hundred fifty-seven articles were identified and nine were included in the study. Overall, there were 13,752 thyroidectomies with a cancer prevalence of 45.7%. When NIFTP was considered non-malignant, the pooled risk difference for ROM was 5.5%. Applying meta-analysis, the pooled prevalence of NIFTP among nodules with FNAC of Bethesda V or Bethesda VI was 14 and 3%, respectively. Conclusion This meta-analysis shows that the inclusion of NIFTP leads to a reduction in the ROM for the Bethesda V and Bethesda VI FNAC diagnostic categories by 14 and 3%, respectively. Clinicians should be aware of these data to avoid overtreatment.

Background: Several single-nucleotide polymorphisms (SNPs) are known to increase the risk of Hashimoto's thyroiditis (HT); such SNPs reside in thyroid-specific genes or in genes related to autoimmunity, inflammation, and/or cellular defense to stress. The transcription factor Nrf2, encoded by NFE2L2, is a master regulator of the cellular antioxidant response. This study aimed to evaluate the impact of genetic variation in NFE2L2 on the risk of developing HT. Methods: In a case-control candidate gene association study, functional SNPs in the NFE2L2 promoter (rs35652124, rs6706649, and rs6721961) were examined either as independent risk factors or in combination with a previously characterized HT risk allele (rs28665122) in the gene SELENOS, encoding selenoprotein S (SelS). A total of 997 individuals from the north of Portugal (Porto) were enrolled, comprising 481 HT patients and 516 unrelated healthy controls. SELENOS and NFE2L2 SNPs were genotyped using TaqMan ® assays and Sanger sequencing, respectively. Odds ratios (ORs) were calculated using logistic regression, with adjustment for sex and age. Expression of SelS was analyzed by immunohistochemistry in thyroid tissue from HT patients and control subjects. Molecular interactions between the Nrf2 and SelS pathways were investigated in thyroid tissues from mice and in rat PCCL3 thyroid follicular cells. Results: When all three NFE2L2 SNPs were considered together, the presence of one or more minor alleles was associated with a near-significant increased risk (OR = 1.43, p = 0.072). Among subjects harboring only major NFE2L2 alleles, there was no increased HT risk associated with heterozygosity or homozygosity for the SELENOS minor allele. Conversely, in subjects heterozygous or homozygous for the SELENOS risk allele, the presence of an NFE2L2 minor allele significantly increased HT risk by 2.8-fold (p = 0.003). Immunohistochemistry showed reduced expression of SelS in thyroid follicular cells of HT patients. In Nrf2 knockout mice, there was reduced expression of SelS in thyroid follicular cells; conversely, in PCCL3 cells, reducing SelS expression caused reduced activity of Nrf2 signaling. Conclusions: The NFE2L2 promoter genotype interacts with the SELENOS promoter genotype to modulate the risk of HT in a Portuguese population. This interaction may be due to a bidirectional positive feedback between the Nrf2 and SelS pathways.

The indeterminate thyroid nodules diagnosed by fine-needle aspiration cytology (FNAC)represents a problem for both cytopathologists and clinicians. The former sometimes use this diagnostic category as a sort of basket, putting in cases that they do not know exactly how to classify. The latter are faced with a highly variable risk of malignancy and consequently the management remains a challenge. On the histopathological side, the new WHO classification of tumors of the thyroid introduced the concept of tumors with uncertain and low malignant potential, and the concept of non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), whose prognosis and management are still to be completely elucidated. While the risk of malignancy of the indeterminate diagnostic category has decreased due to the re-classification of certain types of papillary thyroid carcinomas of the follicular variant into a low malignant potential form (the NIFTP), cases diagnosed cytologically as indeterminate will probably increase in the future to avoid false positive diagnosis. Thus, the indeterminate thyroid diagnostic category still remains a challenge, both at the diagnostic level and for its management. The new version of the Bethesda system for reporting thyroid cytopathology suggests managing these patients with a repeat FNA, diagnostic lobectomy and/or molecular testing.

Acinar cell carcinomas (ACCs) of the pancreas are a heterogeneous group of neoplasms showing a wide spectrum of morphological features including acinar, solid, glandular, and trabecular architecture. In addition, uncommon cytological aspects have recently been described and include oncocytic, spindle, clear, and pleomorphic cell types. This wide histological spectrum represents a challenge in the diagnostic task for pathologists. Molecular mechanisms involved in the onset and progression of ACCs are not completely known, but, in general, they differ from those observed in ductal adenocarcinomas or neuroendocrine neoplasms of the pancreas and frequently include alterations in the APC/β-catenin pathway. In the present paper, we describe a new variant of ACC showing thyroid-like follicular features and CTNNB1 mutation. This phenotype needs to be included in the spectrum of morphological presentation of ACC.

Cytology is a modern, fast, secure, and cost-effective method to render a diagnosis of neoplastic processes throughout the body, including neuroendocrine neoplasms. Fine-needle aspiration and exfoliative cytology specimens show a good sensitivity and specificity in detecting neuroendocrine neoplasms. Increased imaging has led to an increased and earlier detection of neuroendocrine neoplasms. In addition, the easy accessibility of both endoscopic procedures and a fast-diagnostic workup by cytology allow for earlier diagnoses on less invasively obtained material, increasingly in an outpatient setting. Thus, cytology has contributed to the decrease in the mortality of neuroendocrine neoplasms. This chapter focuses on general and site-specific cytology of neuroendocrine neoplasms to make the reader familiar with the spectrum of the cytology of neuroendocrine neoplasms. In addition, differential diagnoses and the use of ancillary techniques such as immunocytochemisty and immunohistochemistry are discussed. Finally, the widely accepted organ system-based, specific classification systems to facilitate the conversation among members of multidisciplinary endocrine oncology team are discussed.

Background: Familial nontoxic multinodular goiter (MNG) is a rare disease. One of the associated genes is Kelch-like ECH-associated protein 1 (KEAP1), which encodes the main inhibitor of nuclear factor erythroid 2-related transcription factor 2 (Nrf2), a central mediator of antioxidant responses. The association of KEAP1 with familial MNG is based on only two loss-of-function mutations identified in two families, only one of which included proper phenotyping and adequate demonstration of co-segregation of the phenotype and the mutation. There is no experimental evidence from model organisms to support that decreased Keap1 levels can, indeed, cause goiter. This study used mice hypomorphic for Keap1 to test whether decreased Keap1 expression can cause goiter, and to characterize the activation status of Nrf2 in their thyroid. Methods: C57BL/6J Keap1flox/flox (Keap1 knock-down [Keap1KD]) mice were studied at 3 and 12 months of age. Plasma and thyroid glands were harvested for evaluation of thyroid function tests and for gene and protein expression by real-time polymerase chain reaction and immunoblotting, respectively. Results: Keap1KD mice showed diffuse goiter that began to develop in early adult life and became highly prominent and penetrant with age. The goiter was characterized by a markedly increased size of thyroid follicles, most notably of the colloid compartment, and by absence of thyroid nodules or hyperplasia. Keap1KD mice also showed decreased T4 levels in early adult life that were eventually well compensated over time by increased thyrotropin (TSH) levels. Nrf2 was activated in the thyroid of Keap1KD mice. Despite a known stimulatory effect of Nrf2 on thyroglobulin (Tg) gene transcription and Tg protein abundance, the expression levels were decreased in the thyroid of Keap1KD mice. No clear patterns were observed in the expression profiles of other thyroid hormone synthesis-specific factors, with the exception of Tg-processing and Tg-degrading cathepsins, including an increase in mature forms of cathepsins D, L, and S. Conclusions: Keap1KD mice develop age-dependent diffuse goiter with elevated TSH levels. The precise mechanism accounting for the thyroidal phenotype remains to be elucidated, but it may involve enhanced Tg solubilization and excessive lysosomal Tg degradation.

The expression of programmed death-ligand 1 (PD-L1) is an established prerequisite for the administration of checkpoint inhibitor therapy and is of prognostic value in several cancer types. Data concerning the potential effect of PD-L1 on the prognosis of thyroid carcinoma are limited. Therefore, this study aimed to provide a systematic review of the published data on this topic. The literature was reviewed to gather and quantify evidence on the prognostic role of PD-L1 in follicular epithelial derived thyroid carcinomas and determine its association with clinicopathological parameters. A meta-analysis was performed using the DerSimonian-Laird random-effects model. The quality of studies was evaluated with the Newcastle-Ottawa Scale and a modified GRADE approach used to rate the quality of evidence. Out of 445 papers, 18 were included and 15 provided adequate data for meta-analysis. The quality of evidence ranged from low to high. PD-L1 expression was significantly associated with a reduced disease-free survival (DFS) (RR 1.63, CI 1.04–2.56, p = 0.03, I2 68%, τ2 0.19 and HR 1.90, CI 1.33–2.70, p

Background: Familial non-toxic multinodular goiter (MNG) is a rare disease. KEAP1 gene (Kelch-like ECH-associated protein 1) that encodes the main inhibitor of nuclear factor erythroid 2-related transcription factor 2 (Nrf2), a central mediator of antioxidant responses, has been found to be one of the mutated genes that lead to familial MNG. The proposed association of KEAP1 with familial MNG is based on only two loss-of-function mutations in respective Japanese families, only one of which included proper phenotyping and demonstration of co-segregation of phenotype and mutation. To date, there is no experimental evidence from model organisms to support that decreased Keap1 levels can cause goiter. Hypothesis: We hypothesized that enhanced Nrf2 signaling induced by loss of Keap1 function in mice can lead to goiter. Methods: To this end, male Keap1 hypomorphic C57BL/6J mice that express ~80% less Keap1 in their tissues (Keap1 knockdown mice:“Keap1KD”) were studied at 3 and 12 months of age and compared to wild-type mice (WT). Plasma, thyroids and pituitary glands were collected for assessment of thyroid function by radioimmunoassays and for histology as well as gene and protein expression by quantitative PCR and immunoblotting respectively. Results: Keap1KD showed diffuse goiter that began to develop in early adult life and became highly prominent at the age of 12 months when the thyroids of Keap1KD were 6-fold heavier than WT. Histomorphometry assessment of thyroids showed that Keap1KD had ~3-fold larger follicle area and colloid compartment but no thyroid nodules or hyperplasia was detected. Keap1KD also showed primary hypothyroidism already in early adult life that was eventually well-compensated over time by increased TSH levels (at age of 12 months: WT TSH=47.7±9.1 mU/L, Keap1KD TSH=460±74 mU/L). This was also reflected in the pituitary gland of Keap1KD where Tshb mRNA was ~3-fold higher than WT. Despite a known stimulatory effect of Nrf2 on Tg gene transcription and Tg protein abundance, these measures were decreased in the thyroid of Keap1KD mice. No clear patterns were observed in the expression profiles of other thyroid hormone synthesis-specific factors, such as Duox1, Duoxa1, Duox2, Duoxa2, Tpo, Nis, Dio1, Dio2, Dehal1 mRNA levels, with the exception of Tg-processing and Tg-degrading cathepsins, including an increase in mature forms of cathepsins D, L and S. Conclusions: Keap1KD mice showed age-dependent diffuse goiter and compensated hypothyroidism. The precise mechanism accounting for the thyroidal phenotype remains to be elucidated, but it may involve enhanced Tg solubilization and excessive lysosomal Tg degradation. This study unravels novel roles of the druggable Keap1/Nrf2 pathway in thyroid function and economy. Subclinical hypothyroidism in Keap1KD mice may have broader implications regarding their use in metabolic research.

Italian consensus for the classification and reporting of thyroid cytology has proposed to discriminate the cancer prevalence of high (Tir 3B) vs. low (Tir 3A) risk indeterminate nodules. To obtain more robust evidence on this topic, we performed a meta-analysis of the Odds Ratio (OR) of malignancy of Tir 3B vs. Tir 3A nodules. A comprehensive literature exploration of online databases was conducted until May 2018. Original articles reporting histology of nodules cytologically classified as Tir 3A and Tir 3B were eligible. Pooled cancer prevalence in Tir 3A and Tir 3B, and OR of Tir 3B vs. Tir 3A were calculated. The search revealed 95 articles, and 10 were included for the meta-analysis. Overall, 1168 indeterminate lesions were reported (441 Tir 3A and 727 Tir 3B), of which 391 were cancers. The pooled cancer prevalence was 17% in Tir 3A and 47% in Tir 3B. The OR of Tir 3B vs. Tir 3A was 4.24 (95% CI 2.75 to 6.53) with mild heterogeneity and without publication bias. When we considered non-invasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTP) as non-malignant, cancer rate was lower, and OR of Tir 3B against Tir 3A was 2.93 (95% CI 1.60 to 5.37), with no heterogeneity but with publication bias. The Italian system for thyroid cytology is reliable to assess indeterminate lesions at low and high risk, being Tir 3B associated with a cancer risk significantly higher than Tir 3A, also when considering NIFTP as non-malignant entity.

Mixed neuroendocrine-nonneuroendocrine neoplasm (MiNEN) consisting of adenoma and well-differentiated neuroendocrine tumor (NET) has been recently defined as "MANET." However, the clinico-pathologic and pathogenetic features of this entity are not thoroughly studied. We examined the clinico-pathologic features of 12 MANETs by expanding their p53 and β-catenin expression profiles as well as the presence of microsatellite instability and KRAS, BRAF, and PIK3CA mutations in the 2 tumor components. In all cases, the adenomatous component represented the larger part of the lesions and the NET was localized in the deep central portion of polyps. In 9 cases the latter was represented by NET G1, in 2 by NET G2, and in 1 by NET G3. In all cases, the glandular and NET components were intimately admixed, with zone of transition between the tumor components. The NET component was p53 negative in all cases and 3 of 8 cases showed variable nuclear positivity for β-catenin. All patients with follow-up data were alive and free of disease after a mean follow-up time of 9 years. No mutations in KRAS, BRAF, and PIK3CA genes and no microsatellite instability were found in both tumor components. Review of the literature also identified 59 previously reported MANETs and no tumor-related death has been found. Like mixed adenoneuroendocrine carcinomas, a high-grade malignant form of MiNENs with a poorly differentiated neuroendocrine carcinoma component, a common origin for both tumor constituents may be hypothesized. Moreover, the current series provides evidence that MANET is an indolent disease that needs to be distinguished from aggressive high-grade MiNENs.

OBJECTIVE In 2016, there were an estimated 56 870 new cases of thyroid cancer (TC) in the USA. Fine needle aspiration cytology (FNAC) is the most safe, accurate and cost-effective method for the initial investigation of thyroid nodules. FNAC is limited by the inability to diagnose malignancy in follicular-patterned lesions accurately and, as a result, 20%-30% of cases under investigation for TC are classified as cytologically indeterminate, illustrating a problem with current FNAC procedure. Raman spectroscopy has shown promising results for the detection of many cancers; however, to date there has been no report on the performance of Raman spectroscopy on thyroid cytological samples. The aim of this study was to examine whether Raman spectroscopy could be used to correctly classify cell lines representing benign thyroid cells and various subtypes of TC. METHODS A benign thyroid cell line and seven TC cell lines were prepared as ThinPrep® cytology slides and analysed with Raman spectroscopy. Principal components analysis and linear discriminant analysis were implemented to develop effective diagnostic algorithms for classification of Raman spectra of different TC subtypes. RESULTS The spectral differences separating benign and TC cell lines were assigned to differences in the composition of nucleic acids, lipids, carbohydrates and protein in the benign and cancer cells. Good sensitivities (74%-85%), specificities (65%-93%) and diagnostic accuracies (71%-88%) were achieved for the identification of TC. CONCLUSION These findings suggest that Raman spectroscopy has potential for preoperative TC diagnosis on FNAC samples.

Objective The management of occult lymph node metastasis (LNM) in head and neck squamous cell carcinoma has been a matter of controversy for decades. The vascular density within the tumor microenvironment, as an indicator of ongoing angiogenesis, could constitute an attractive predictor of LNM. The use of the panvascular endothelial antibody CD31 as a marker of occult LNM has never been reported. The aim of this study was to assess the predictive value of CD31 microvascular density for the detection of occult LNM in squamous cell carcinoma of the oral cavity and oropharynx. Study Design Case series with chart review. Setting Tertiary university hospital. Subjects and Methods Intra- and peritumoral microvascular density values were determined in 56 cases of squamous cell carcinoma of the oral cavity (n = 50) and oropharynx (n = 6) with clinically negative necks using the CD31 marker. Statistical associations of CD31 microvascular densities with clinicopathologic data were then established. Results Peritumoral CD31 microvascular density was significantly associated with occult LNM in multivariate analysis (P < .01). Recursive partitioning analysis for this parameter found a cutoff of 19.33, which identified occult LNM with a sensitivity of 91%, a specificity of 65%, a positive predictive value of 40%, a negative predictive value of 97%, and an overall diagnostic accuracy of 71%. Conclusion Peritumoral CD31 microvascular density in primary squamous cell carcinoma of the oral cavity and oropharynx allows accurate prediction of occult LNM.

The recent update of The Bethesda System for Reporting Thyroid Cytology (TBSRTC) is a very important development in the evaluation of thyroid nodules. Clinical experience and scientific literature both show that practitioners performing thyroid fine-needle aspiration are accustomed to basing the clinical management of patients on reports using TBSRTC. Specifically, clinicians are familiar with the percent risk of malignancy corresponding to each TBSRTC diagnostic category (DC), as well as with the respective recommendation for clinical management. However, most clinicians are much less familiar with the specific considerations that lie between a given DC, on the one end, and the respective risk of malignancy and associated management recommendation, on the other end.A deeper understanding of the system can enlighten the clinician's thinking about the specific nodule under examination and can guide the decision-making process in a more meaningful way. Such an understanding can only be developed via close two-way communication between cytopathologists and clinicians. Through this type of interaction in the authors' tertiary medical center, recurring issues of particular importance for clinical practice were identified, which are reported here in the form of 16 frequently asked questions posed by the clinician to the cytopathologist.For each frequently asked question, an answer is provided based on the literature, the authors' experience, the new version of TBSRTC, and the new World Health Organization classification of tumors of endocrine organs.

BACKGROUND The Bethesda System for Reporting Thyroid Cytopathology category of atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) includes fine-needle aspiration (FNA) specimens that cannot straightforwardly be classified as benign or malignant. To determine whether morphological subcategorization based on atypia qualifiers and molecular testing could improve malignancy risk stratification of AUS/FLUS patients, this study assessed the correlation between these qualifiers and the molecular alterations commonly harbored by thyroid neoplasms. METHODS A total of 162 AUS/FLUS cases were subcategorized by atypia qualifiers (Hurthle cell changes, architectural atypia, and cytologic atypia [CyA]) and were tested for BRAF, N-H-KRAS, RET/PTC, and paired box 8 (PAX8)/peroxisome proliferator activated receptor γ (PPARg) mutations. RESULTS CyA was observed more frequently in mutation-positive AUS/FLUS (14 of 37 [37.84%]) than mutation-negative AUS/FLUS (20 of 125 [16.00%]; P < .0084), and it specifically harbored the BRAFV600E point mutation. Malignancy was confirmed in the available follow-up. Conversely, although RAS was the most frequent mutation identified in AUS/FLUS FNA specimens (26 of 37 cases [70.27%]; P < .0001), it was distributed across various AUS/FLUS subcategories and was not significantly associated with a specific atypia qualifier or malignant outcome according to the available follow-up. Rearrangements of both RET/PTC (n = 1) and PAX8/PPARg (n = 3) were rarely retrieved in the FNA samples. CONCLUSIONS BRAF and RAS mutations are associated with different AUS/FLUS qualifiers and hence have different risks of malignancy. Consequently, a hybrid molecular and morphological subcategorization system could improve the malignancy risk stratification of thyroid FNA samples diagnosed as AUS/FLUS. Cancer Cytopathol 2018;126:317-25. © 2018 American Cancer Society.

HPV-positive head and neck squamous cell carcinoma (HNSCC) is increasingly frequent. Management is particularly debated in the case of postsurgical high-risk features, that is, positive surgical margins and extracapsular spread (ECS). In this increasingly complex emerging framework of HNSCC treatment, representative preclinical models are needed to support future clinical trials and advances in personalized medicine. Here, we present an immunocompetent mouse model based on the implantation of mouse tonsil epithelial HPV16-E6/E7-expressing cancer cells into the submental region of the floor-of-the-mouth. Primary tumors were found to replicate the patterns of human HNSCC local invasion and lymphatic dissemination. To study disease progression after surgery, tumors were removed likely leaving behind residual disease. Surgical resection of tumors was followed by a high rate of local recurrences (>90%) within the first 2-3 weeks. While only 50% of mice had lymph node metastases (LNM) at time of primary tumor excision, all mice with recurrent tumors showed evidence of LNM. To study the consecutive steps of LNM progression and distant metastasis development, LNs from tumor-bearing mice were transplanted into naive recipient mice. Using this approach, transplanted LNs were found to recapitulate all stages and relevant histological features of regional metastasis progression, including ECS and metastatic spread to the lungs. Altogether, we have developed an immunocompetent HPV-positive HNSCC mouse model of postsurgical local recurrence and regional and distant metastasis progression suitable for preclinical studies.

Enterobiasis is one of the most common human parasitic infections. It is considered an intestinal parasite, but cases of extra-intestinal affections exist, notably infections of the female genital tract. Enterobius vermicularis (EV) eggs (or ova) have been found in the cervical smears of two patients in our institute during the last 16 years. No gynaecological or gastrointestinal symptoms were reported, and there was no known intestinal infection in these two cases. A review of the available literature revealed rare cases of vaginal enterobiasis, with a wide range of clinical presentations, many patients being asymptomatic. The diagnosis may sometimes be difficult, mainly because of the lack of clinical suspicion. However, cytological identification of EV in cervico-vaginal smears is important, especially when considering the risk of ascending infections of the genital tract associated with severe complications.

Monoclonal antibodies that inhibit the interaction between PD1 and PD-L1 are approved for clinical use in several cancer types, and they are also in clinical trials for additional indications, including thyroid carcinomas. A few papers have reported on PD-L1 expression in thyroid carcinomas, including a recent large study by Ahn et al. in Endocrine-Related Cancer using tissue microarrays on differentiated and anaplastic thyroid carcinoma. However, the expression of PD-L1 in medullary thyroid carcinoma (MTC) has not been reported so far, even though ongoing clinical studies aim to test the effectiveness of checkpoint inhibitors in this rare histotype as well. We thus assessed PD-L1 expression in both tumor cells and tumor-infiltrating immune cells in a series of 16 MTC cases at a tertiary center. Tumor cells were positive in only one case, which had 5% positive cells. 1% and 2% of the inflammatory cells were stained in two cases. No correlation was evident between PD-L1 expression and survival in our series. Our results are indicative of near uniform absence of PD-L1 expression in MTC and its accompanying inflammatory cells; these results should be replicated on a larger scale in other centers. Definitive answers regarding the utility of PD1/PD-L1 immunophenotyping in MTC and of the use of checkpoint inhibitors in the treatment of this aggressive and rare thyroid cancer are expected from ongoing clinical trials, which should perform correlations with PD1/PD-L1 expression.