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This chapter provides an outline of a pattern approach to the interpretation of breast fine needle aspiration biopsy direct smears. A number of different low and medium power patterns are described that suggest certain lesions. High power is then used to assess nuclear and other features, and low and high power key features are integrated into a diagnosis or a differential diagnosis. Details of the key diagnostic cytological features are provided in the various chapters of the atlas.

BACKGROUND Recently a new system for reporting salivary gland fine-needle aspiration (FNA) cytology was proposed, the Milan System for Reporting Salivary Gland Cytopathology (MSRSGC). Herein, we evaluated diagnostic accuracy of salivary gland FNA, comparing the system previously used in our hospital with the Milan system. METHODS Salivary gland specimens obtained between 2011 and 2017 were reclassified according to MSRSGC. Risk of malignancy for each diagnostic category was determined. Diagnostic yield of both classifications was evaluated. RESULTS The cases (n = 388) were classified according to the old system: nondiagnostic (n = 28), benign (n = 246), atypical (n = 36), neoplastic (n = 57), suspicious for malignancy (n = 7) and malignant (n = 14). The lesions were distributed according to the MSRSGC: nondiagnostic (n = 28), non-neoplastic (n = 89), atypia of undetermined significance (n = 39), benign neoplasm (n = 156), neoplasm of uncertain malignant potential (n = 55), suspicious for malignancy (n = 7) and malignant (n = 14). When considering only benign and malignant cases, both classifications showed the same sensitivity (62.5%), specificity (100%) and similar accuracy (95.8%). Comparison between the two systems showed no significant difference. CONCLUSIONS Salivary gland FNA has high diagnostic accuracy and assists clinical management independently of the reporting system used, however, in some cases, the use of Milan system could be beneficial, since it allows an enhanced category stratification.

The frequency of cancer presents an increasing trend in humans and companion animals, and despite recent advances in diagnosis and treatment, it remains a major cause of morbidity and mortality in human and veterinary medicine. The epidemiological and clinicopathological similarities between spontaneous tumours of companion animals and their human counterparts make them suitable natural models for human cancer research. Moreover, the faster progression of cancer in dogs and cats in comparison with humans, associated with the shorter life span of companion animals, enables faster data retrieval than in human malignancies. Furthermore, the health effects associated with exposure to environmental hazardous materials, including cancer, occur similarly in companion animals and humans; consequently, in an epidemiological context, dogs and cats can also be useful as sentinels of human malignancies. For these reasons, comparative oncology, which can be defined as the study of spontaneous cancers in animals as models for human disease, has gained increasing importance over the last decades. Breast cancer represents the most prevalent cancer among women worldwide and the leading cause of cancer-related mortality in women. Mammary gland tumours are also among the most frequent tumours in female dogs and cats. Canine and feline mammary tumours present similar incidence, relative age of onset, risk factors, biological behaviour, metastatic pattern, histological, molecular, and genetic features, and response to therapy to human breast cancer; thus, they are recognized as suitable natural models for human breast cancer studies. The comparative “One Health” approach allows advances in knowledge of the diseases in order to obtain an improvement in clinical outcomes for affected humans and animals.

Background: The presence of highly cellular stromal fragments in breast fine needle aspirates (FNA) suggests some classical differential diagnoses such as cellular fibroadenoma, phyllodes tumour (PT), metaplastic carcinomas, and some mesenchymal/myoepithelial proliferations. The other components of the smears can help in the differential diagnosis, but the presence of a low-grade epithelial proliferation does not always represent a fibro-epithelial lesion as we demonstrate in these two cases. Cases: We discuss two cases of breast FNA, previously presented in a slide seminar at the 29th European Congress of Pathology in Amsterdam, where the common cytological finding was the presence of stromal cellular fragments together with an epithelial component. One case is a typical PT and the other is a case of a mammary carcinoma with osteoclast-like giant cells. Conclusion: Mammary carcinoma with osteoclast-like giant cells is an unusual type of breast carcinoma that should be included in the differential diagnosis of breast lesions containing cellular stroma. Since the associated carcinoma is usually low grade, careful evaluation for malignant cells on cytological smears is necessary for an accurate differential diagnosis with PT where the epithelial component is benign.

BACKGROUND Molecular testing of cytological lung cancer specimens includes, beyond epidermal growth factor receptor (EGFR), emerging predictive/prognostic genomic biomarkers such as Kirsten rat sarcoma viral oncogene homolog (KRAS), neuroblastoma RAS viral [v-ras] oncogene homolog (NRAS), B-Raf proto-oncogene, serine/threonine kinase (BRAF), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA). Next-generation sequencing (NGS) and other multigene mutational assays are suitable for cytological specimens, including smears. However, the current literature reflects single-institution studies rather than multicenter experiences. METHODS Quantitative cytological molecular reference slides were produced with cell lines designed to harbor concurrent mutations in the EGFR, KRAS, NRAS, BRAF, and PIK3CA genes at various allelic ratios, including low allele frequencies (AFs; 1%). This interlaboratory ring trial study included 14 institutions across the world that performed multigene mutational assays, from tissue extraction to data analysis, on these reference slides, with each laboratory using its own mutation analysis platform and methodology. RESULTS All laboratories using NGS (n = 11) successfully detected the study's set of mutations with minimal variations in the means and standard errors of variant fractions at dilution points of 10% (P = .171) and 5% (P = .063) despite the use of different sequencing platforms (Illumina, Ion Torrent/Proton, and Roche). However, when mutations at a low AF of 1% were analyzed, the concordance of the NGS results was low, and this reflected the use of different thresholds for variant calling among the institutions. In contrast, laboratories using matrix-assisted laser desorption/ionization–time of flight (n = 2) showed lower concordance in terms of mutation detection and mutant AF quantification. CONCLUSIONS Quantitative molecular reference slides are a useful tool for monitoring the performance of different multigene mutational assays, and this could lead to better standardization of molecular cytopathology procedures. Cancer Cytopathol 2017;125:615-26. © 2017 American Cancer Society.

Aim To evaluate the intraobserver and interobserver reproducibility of the HER2 in-situ hybridization (ISH) test in breast cancer by measuring the impact of counting different numbers of invasive cancer cells. Methods and results A cohort of 101 primary invasive breast cancer cases were evaluated for HER2 gene amplification by silver ISH, and the concordance among four observers with different levels of experience, counting different numbers of invasive cancer cells, was determined. The evaluation of the samples included scoring 20 nuclei, in three different areas. The cases were scored twice, with a washout interval of at least 2 weeks. We observed an increase in the intraobserver concordance rate between the first and second evaluations with an increase in cell count. A count of 60 invasive cells was needed to obtain a concordance rate near 95% and an agreement rate greater than 0.80 by all observers. The interobserver concordance rate of the HER2 test also increased with the increase in cell count, reaching at least a 90% concordance rate with a count of 60 invasive cells. The median variability of both the HER2/CEP17 ratio and the average HER2 copy number between different evaluations decreased with the increase in cell count, being statistically higher in HER2-positive cases. Conclusions The minimal cell number recommended in current guidelines should be raised to at least 40, and preferably 60, invasive cells. Moreover, cases with amplification levels close to the threshold should be subjected to a dual count from an experienced observer.

Aim The present work aims to evaluate the presence of stromal tumour-infiltrating lymphocytes (TILs) and programmed cell death-ligand 1 (PDL1) expression in breast carcinomas and their correlation with available clinicopathological features. Methods Two independent series of invasive breast cancer (IBC), one including ductal carcinoma in situ (DCIS) pair-matched cases, were selected, and quantification of TILs was accomplished in each case. Immunohistochemistry was also performed to evaluate the expression of PDL1. Results In both cohorts evaluated, increased stromal TILs and PDL1 expression were present in about 10% of IBCs, being significantly associated with each other and both with grade 3 and triple-negative subtype. We observed a similar distribution of stromal TILs and PDL1 expression between DCIS and IBC. Finally, we observed that increased stromal TILs and PDL1 expression were significantly associated with cancer stem cell (CSC) markers, basal cell markers and vimentin expression. Interestingly, in IBC cases with vimentin expression, increased stromal TILs, as well as decreased PDL1 expression, disclosed a better clinical outcome, independently of the main classical BC prognostic factors. Conclusions We have confirmed the association of stromal TILs and PDL1 expression with aggressive forms of BC and that both are already found in in situ stages. We also showed that stromal TILs and PDL1 expression are associated with clinical outcome in cases enriched for a mesenchymal immunophenotype. We describe for the first time a close relationship between CSC markers and PDL1 expression.

Background In 2014, liquid-based cytology with HPV triage replaced conventional cytology. The aim of our study was to compare conventional and liquid-based cytology (LBC), estimate the prevalence of abnormal cervical cytology and high risk HPV (hrHPV) infection and their correlation, among screened women in Luxembourg. Methods Between the first January 2013 and 31st December 2015, 315,868 cervical samples from 150,815 women (mean age 42.2 years) were investigated by the national cytology laboratory. Slides were prepared and screened according to European Guidelines. All cytological results were classified according to the Bethesda 2001 system terminology. Results The prevalence of abnormal cervical lesions was as follows: atypical squamous cells of undetermined significance (ASC-US), 1.3%; low-grade squamous intraepithelial lesion (LSIL), 1.9%; high-grade squamous intraepithelial lesion (HSIL), 0.4%. The detection rate of cytological lesions was significantly higher with LBC than with conventional cytology. Based on 11,838 samples with concomitant cytology and HPV testing, hrHPV was detected in 9.5, 45.3, 70.0, and 92.6% of women with negative cytology, ASC-US, LSIL, and HSIL, respectively. Conclusion More cervical lesions were identified using LBC compared to conventional cytology. HrHPV infection was correlated with the severity of intraepithelial lesions. The current findings provide important information to evaluate the prevention of cervical cancer in Luxembourg and for monitoring the future impact of HPV vaccination. Diagn. Cytopathol. 2017;45:384–390. © 2017 Wiley Periodicals, Inc.

Background: E-cadherin has been awarded a key role in the aetiology of both sporadic and hereditary forms of gastric cancer. In this study, we aimed to identify molecular interactors that influence the expression and function of E-cadherin associated to cancer. Methods: A data mining approach was used to predict stomach-specific candidate genes, uncovering S100P as a key candidate. The role of S100P was evaluated through in vitro functional assays and its expression was studied in a gastric cancer tissue microarray (TMA). Results: S100P was found to contribute to a cancer pathway dependent on the context of E-cadherin function. In particular, we demonstrated that S100P acts as an E-cadherin positive regulator in a wild-type E-cadherin context, and its inhibition results in decreased E-cadherin expression and function. In contrast, S100P is likely to be a pro-survival factor in gastric cancer cells with loss of functional E-cadherin, contributing to an oncogenic molecular program. Moreover, expression analysis in a gastric cancer TMA revealed that S100P expression impacts negatively among patients bearing Ecad- tumours, despite not being significantly associated with overall survival on its own. Conclusions: We propose that S100P has a dual role in gastric cancer, acting as an oncogenic factor in the context of E-cadherin loss and as a tumour suppressor in a functional E-cadherin setting. The discovery of antagonist effects of S100P in different E-cadherin contexts will aid in the stratification of gastric cancer patients who may benefit from S100P-targeted therapies. This work was financed by FEDER funds through the Operational Programme for Competitiveness Factors (COMPETE 2020), Programa Operacional de Competitividade e Internacionalização (POCI), Programa Operacional Regional do Norte (Norte 2020) and by National Funds through the Portuguese Foundation for Science and Technology (FCT), under the projects PTDC/BIMONC/0171/2012, PTDC/BIM-ONC/0281/2014, NORTE-01-0145-FEDER-000029, PTDC/MED-GEN/30356/2017, PTDC/BTM-SAL/30383/2017; doctoral grant SFRH/BD/114687/2016-AMM; post-doctoral grant SFRH/BPD/87705/2012-JF. We acknowledge the IFCT Program for funding JP and SV.

The present report reviews the current problems associated with the routine use of breast fine needle aspiration biopsy (FNAB) and discusses the potential impact that the new International Academy of Cytology (IAC) Yokohama Reporting System and the use of rapid on-site evaluation (ROSE) should have on reducing these problems to optimize breast care for patients. The recently reported IAC System aims to establish the best practice guidelines for breast FNAB, emphasizing the importance of the FNAB technique and the skillful preparation of direct smears. The IAC System proposes a standardized report and established clear terminology for defined reporting categories, each of which has a risk of malignancy and is linked to management options. The FNAB techniques that will optimize the biopsy specimen and reduce poor quality smears are reviewed and the benefits of ROSE are discussed. FNAB can diagnose accurately the vast majority of breast lesions, and ROSE has been recommended whenever possible to reduce the rate of insufficient/inadequate cases and increase the number of specific benign and malignant diagnoses. ROSE performed by a cytopathologist provides a provisional diagnosis, reducing patient anxiety and facilitating management through cost-effective immediate triage and patient selection for ancillary testing. Thus, patients can be selected for immediate core needle biopsy, as required.

Introduction: The Acibadem Health Group (AHG) has been using telepathology/digital pathology stations since 2006. In 2013, the system was changed from videoconferencing to digital pathology (whole-slide imaging) utilizing 3DHISTECH scanners and software. In 2017, digital cytology started to be used for routine cytopathologic diagnosis for thyroid fine-needle aspiration (FNA) cases. Material and Methods: Two hundred and twenty-seven thyroid cases were received for analysis using telecytology (TC) during the period from November 2017 to May 2018. Rapid on-site evaluation was performed at the Atakent Hospital of the AHG by a cytotechnologist and scanned on the same day. For every case, there were Diff-Quik- and Papanicolaou-stained FNA smears. Each glass slide was digitized with a 3DHISTECH whole-slide scanner in 1 focal Z-plane at ×40 magnification. Results: Two hundred and twenty-seven thyroid FNA specimens were retrieved, of which 25 had histologic follow-up. Samples were classified as nondiagnostic in 3%, benign in 74%, atypia of undetermined significance/follicular lesion of undetermined significance in 13%, suspicious for follicular neoplasia/follicular neoplasia in 3%, suspicious for malignancy in 4%, and malignant in 3%. When only the “suspicious for malignancy” and “malignancy” categories were considered positive tests, cytology sensitivity and specificity using TC for diagnosis was 100%. Conclusions: Our data demonstrate that TC is suitable to provide a primary diagnosis in daily routine cytology practice. Despite the promising results, there were some challenges stemming from the novelty of using TC for the primary diagnosis. The study also addresses both advantages and disadvantages of TC in daily practice to increase the efficiency of the technique in primary diagnosis.

Increased glycolytic activity with accumulation of extracellular lactate is regarded as a hallmark of cancer. In lymphomas, FDG-PET has undeniable diagnostic and prognostic value, corroborating that these tumours are avid for glucose. However, the role of glycolytic metabolism-related molecules in lymphoma is not well known. Here, we aimed to evaluate the clinical and prognostic significance of a panel of glycolytic metabolism-related molecules in primary non-Hodgkin lymphomas (NHL) and to test in vitro the putative therapeutic impact of lactate transport inhibition., NORTE-01-0145-FEDER- 000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020) under the Portugal Partnership Agreement, through the European Regional Development Fund (FEDER), and through the Competitiveness Factors Operational Programme (COMPETE) and by National funds, through the Foundation for Science and Technology (FCT), under the scope of project POCI-01-0145-FEDER-007038. JA received a fellowship from FCT, ref. SFRH/BPD/116784/2016

Background Programmed cell death ligand 1 (PD-L1) is a predictive biomarker of the response of immunotherapy in some types of cancer. In the last years it was described its expression in breast cancer, namely in triple-negative breast cancer (TNBC) cases. In order to better understand the prognostic value of PD-L1 in breast cancer, this study analysed its expression in a series of primary tumours and respective nodal metastases. Methods PD-L1 expression was evaluated by immunohistochemistry in a series of 52 breast cancer cases and paired lymph node metastasis and correlate with the clinicopathological features of the primary tumour. The TNBC cases were re-classified using morphological and immunohistochemistry surrogate markers and the expression of PD-L1 was correlate with the different subtypes. Results It was observed that the majority of the cases with PDL-1 positive in the nodal metastasis did not express PD-L1 in the primary tumour (90.0%, 10 out of 11 cases). In addition, from the cases with a negative PD-L1 expression in the primary tumour, 23.8% expressed PD-L1 in the metastasis (10 out of 42 cases).No relationship was found between the PD-L1 expression in nodal metastasis and the clinicopathological features of the primary tumour. Finally, basal-like immunosuppressed (BLIS) TN tumours seem to be less prone to express PD-L1. Conclusion Although without statistical significance, there is a gain in terms of the PD-L1 expression in the nodal metastasis when compared to the primary tumour. This may have therapeutic implications on immunotherapy that blocks the PD-1/PD-L1 pathway.

Background: Artificial genomic reference standards in a cytocentrifuge/cytospin format with well-annotated genomic data are useful for validating next-generation sequencing (NGS) on routine cytopreparations. Here, reference standards were optimized to be stained by different laboratories before DNA extraction and to contain a lower number of cells (2 × 10 5 ). This was done to better reflect the clinical challenge of working with insufficient cytological material. Methods: A total of 17 worldwide laboratories analyzed customized reference standard slides (slides A-D). Each laboratory applied its standard workflow. The sample slides were engineered to harbor epidermal growth factor receptor (EGFR) c.2235_2249del15 p.E746_A750delELREA, EGFR c.2369C>T p.T790M, Kirsten rat sarcoma viral oncogene homolog (KRAS) c.38G>A p.G13D, and B-Raf proto-oncogene, serine/threonine kinase (BRAF) c.1798_1799GT>AA p.V600K mutations at various allele frequencies (AFs). Results: EGFR and KRAS mutation detection showed excellent interlaboratory reproducibility, especially on slides A and B (10% and 5% AFs). On slide C (1% AF), either the EGFR mutation or the KRAS mutation was undetected by 10 of the 17 laboratories (58.82%). A reassessment of the raw data in a second-look analysis highlighted the mutations (n=10) that had been missed in the first-look analysis. BRAF c.1798_1799GT>AA p.V600K showed a lower concordance rate for mutation detection and AF quantification. Conclusions: The data show that the detection of low-abundance mutations is still clinically challenging and may require a visual inspection of sequencing reads to detect. Genomic reference standards in a cytocentrifuge/cytospin format are a valid tool for regular quality assessment of laboratories performing molecular studies on cytology with low-AF mutations.

Fine needle aspiration (FNA) of thyroid has been used for years in a multidisciplinary approach, to diagnose different entities, in preventing over- or under-treatment of thyroid nodules. This method has a widespread use around the globe. In this chapter, we aimed to focus on false-positive results in the evaluation of thyroid aspirations, covering cystic, inflammatory lesions, follicular lesions, oncocytic lesions, papillary carcinoma, and medullary carcinoma of thyroid. The recently described entity, “noninvasive follicular thyroid neoplasm with papillary-like nuclear features” (NIFTP), is also covered as well as the discussion about the impact of its introduction in the sensitivity and specificity of thyroid FNA.

Ductal Carcinoma in Situ is an entity which bears the risk of progression into Invasive Breast Carcinoma of No Special Type (IBC of NST). Evidence suggests that the malignant potential is already present in the non invasive period. Currently, the tumor microenvironment interaction has gained importance since the genetic and translational modifications of the DCIS lesion itself does not inform about the probability of malignancy sufficient for the risk stratification concerning the prognosis of the entity. Recent evidence underlines the interaction of the surrounding cells as affecting the fate of DCIS. Reproducibility of a diagnosis and grading of DCIS is another problem which is tried to be overcome with the incorporation of deep learning convolutional neural network analyses and various gene expression assays. This summarizes the findings of the recent studies to elucidate the transition of DCIS to IBC of NST regarding the histopathology, molecular biology while reflecting on the current prognostic data of DCIS with the treatment methods that are in application.

In the late stages of non-small cell lung cancer, the detection of sensitizing mutations of the epidermal growth factor receptor (EGFR) is mandatory to select patients’ treatment with first- or second-generation tyrosine kinase inhibitors (TKIs). In patients showing progressive disease, the assessment of the EGFR exon 20 resistance point mutation p.T790M is required for third-generation TKI administration. However, molecular analysis does not capture all the different mechanisms of resistance against these molecules. A variety of morphological changes associated with acquired resistance have also been described. Since an altered morphology may be the only clue to acquired resistance, cytopathology still plays a relevant role in this setting. In this comprehensive review, we have focused on the relevance of squamous cell carcinoma, small cell lung cancer and large-cell neuroendocrine carcinoma transitions from adenocarcinoma resistant to TKIs.

The reverse pH gradient is a major feature associated with cancer cell reprogrammed metabolism. This phenotype is supported by increased activity of pH regulators like ATPases, carbonic anhydrases (CAs), monocarboxylate transporters (MCTs) and sodium–proton exchangers (NHEs) that induce an acidic tumor microenvironment, responsible for the cancer acid-resistant phenotype. In this work, we analyzed the expression of these pH regulators and explored their inhibition in breast cancer cells as a strategy to enhance the sensitivity to chemotherapy. Expression of the different pH regulators was evaluated by immunofluorescence and Western blot in two breast cancer cell lines (MDA-MB-231 and MCF-7) and by immunohistochemistry in human breast cancer tissues. Cell viability, migration and invasion were evaluated upon exposure to the pH regulator inhibitors (PRIs) concanamycin-A, cariporide, acetazolamide and cyano-4-hydroxycinnamate. Additionally, PRIs were combined with doxorubicin to analyze the effect of cell pH dynamic disruption on doxorubicin sensitivity. Both cancer cell lines expressed all pH regulators, except for MCT1 and CAXII, only expressed in MCF-7 cells. There was higher plasma membrane expression of the pH regulators in human breast cancer tissues than in normal breast epithelium. Additionally, pH regulator expression was significantly associated with different molecular subtypes of breast cancer. pH regulator inhibition decreased cancer cell aggressiveness, with a higher effect in MDA-MB-231. A synergistic inhibitory effect was observed when PRIs were combined with doxorubicin in the breast cancer cell line viability. Our results support proton dynamic disruption as a breast cancer antitumor strategy and the use of PRIs to boost the activity of conventional therapy., This research was funded by National funds, through the Foundation for Science and Technology (FCT)—project UIDB/50026/2020 and UIDP/50026/2020; and by the projects NORTE 01-0145-FEDER-000013 and NORTE-01-0145-FEDER-000023, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This work was also supported by an internal CE SPU project MetabRes_CESPU_2017. DT-V received a fellowship from FCT (ref. SFRH/BD/103025/2014).

Successful metastatic spreading relies on cancer cells with stem-like properties, glycolytic metabolism and increased antioxidant protection, allowing them to escape anoikis and to survive in circulation. The expression of P-cadherin, a poor prognostic factor in breast cancer, is associated with hypoxic, glycolytic and acidosis biomarkers. In agreement, P-cadherin-enriched breast cancer cell populations presents a glycolytic and an acid-resistance phenotype. Our aim was to evaluate whether P-cadherin expression controls the glycolytic and oxidative phosphorylation fluxes of matrix-detached breast cancer cells, acting as an antioxidant and enhancing their survival in anchorage-independent conditions. By using matrix-detached breast cancer cells, we concluded that P-cadherin increases glucose-6-phosphate dehydrogenase expression, up-regulating the carbon flux through the pentose phosphate pathway, while inhibiting pyruvate oxidation to acetyl-coA via pyruvate dehydrogenase kinase-4 (PDK-4) activation. Accordingly, P-cadherin expression conferred increased sensitivity to dichloroacetate (DCA), a PDK inhibitor. P-cadherin expression also regulates oxidative stress in matrix-detached breast cancer cells, through the control of antioxidant systems, such as catalase and superoxide dismutases (SOD)1 and 2, providing these cells with an increased resistance to doxorubicin-induced anoikis. Importantly, this association was validated in primary invasive breast carcinomas, where an enrichment of SOD2 was found in P-cadherin-overexpressing breast carcinomas. In conclusion, we propose that P-cadherin up-regulates carbon flux through the pentose phosphate pathway and decreases oxidative stress in matrix-detached breast cancer cells. These metabolic remodeling and antioxidant roles of P-cadherin can promote the survival of breast cancer cells in circulation and in metastatic sites, being a possible player in breast cancer therapeutic resistance to pro-oxidant-based interventions.

Mammary Paget's disease (PD) is characterized by eczematous changes of the nipple-areolar complex and is usually associated with an underlying breast cancer (BC). PD of the breast is rare, comprising about 1–4% of all BC. Male BC is also uncommon, representing less than 1% of all BC. Thus, male mammary PD is extremely rare. We report the case of mammary PD associated with an underlying BC in situ in a 41-year-old young man who complained of bilateral gynecomastia and mild eczematous signs in the nipple.

The Papanicolaou Society of Cytopathology has developed a set of guidelines for respiratory cytology including indications for sputum examination, bronchial washings and brushings, CT-guided FNA and endobronchial ultrasound guided fine needle aspiration (EBUS-FNA), as well as recommendations for classification and criteria, ancillary testing and post-cytologic diagnosis management and follow-up. All recommendation documents are based on the expertise of committee members, an extensive literature review, and feedback from presentations at national and international conferences. The guideline documents selectively present the results of these discussions. The present document summarizes recommendations for ancillary testing of cytologic samples. Ancillary testing including microbiologic, immunocytochemical, flow cytometric, and molecular testing, including next-generation sequencing are discussed. Diagn. Cytopathol. 2016;44:1000-1009. © 2016 Wiley Periodicals, Inc.

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.

Phyllodes tumours constitute an uncommon but complex group of mammary fibroepithelial lesions. Accurate and reproducible grading of these tumours has long been challenging, owing to the need to assess multiple stratified histological parameters, which may be weighted differently by individual pathologists. Distinction of benign phyllodes tumours from cellular fibroadenomas is fraught with difficulty, due to overlapping microscopic features. Similarly, separation of the malignant phyllodes tumour from spindle cell metaplastic carcinoma and primary breast sarcoma can be problematic. Phyllodes tumours are treated by surgical excision. However, there is no consensus on the definition of an appropriate surgical margin to ensure completeness of excision and reduction of recurrence risk. Interpretive subjectivity, overlapping histological diagnostic criteria, suboptimal correlation between histological classification and clinical behaviour and the lack of robust molecular predictors of outcome make further investigation of the pathogenesis of these fascinating tumours a matter of active research. This review consolidates the current understanding of their pathobiology and clinical behaviour, and includes proposals for a rational approach to the classification and management of phyllodes tumours.

Pathologists have an important role in the diagnosis of infectious disease (ID). In many cases, a definitive diagnosis can be made using cytopathology alone. However, several ancillary techniques can be used on cytological material to reach a specific diagnosis by identifying the causative agent and consequently defining the management of the patient. This review aims to present the effectiveness of the application of molecular studies on cytological material to diagnose IDs and discuss the advantages and disadvantages of the various molecular techniques according to the type of cytological specimen and the infectious agents.

Clinicians are increasingly facing the decision of performing anal cancer screening in high-risk groups. Anal cytology is commonly the first approach. We systematically reviewed recommendations favoring anal cytology for anal cancer screening. Three databases were searched: PubMed, Scopus, and Embase, from January 2007 to 12 September 2019. The references cited by the retrieved articles and the websites of relevant organizations were also searched without language restrictions. Studies reporting guidelines from regional or national societies, institutes, or groups were included. Eight papers met the inclusion criteria and were selected, five were from the United States of America (USA) and three from Europe. There were no national recommendations published. There was one guideline specifically for solid-organ transplant recipients. The other seven targeted HIV-positive patients, with HIV-positive men who have sex with men (MSM) included as a screening group in all of these. Two recommendations favored screening in all HIV-positive patients. Five recommendations targeting HIV-positive patients made considerations about the cytology follow-up, recommending at least annual cytology in case of a normal result, and in case of squamous cytological abnormalities, a referral for anoscopy/high-resolution anoscopy. There were no recommendations for upper and lower age limits for screening. In conclusion, several societies recommend anal cancer screening using anal cytology in HIV-positive MSM patients. There is a lack of screening recommendations for other high-risk groups, with only one society recommending screening in transplant recipients.

It is now widely accepted that the tumor microenvironment is a pathologically active niche that shapes tumor nature, evolution and response to treatment. Close interactions between cancer cells and stroma are known to regulate several cancer pathways and thus the determination of different tumor-stromal interactions could be an important step in invasiveness. The breast cancer microenvironment is a complex combination of several different cell types and molecules and a key contributor to tumor development and progression. The microenvironment includes fibroblasts, macrophages, immune cells, tumor-infiltrating lymphocytes, endothelial cells and angiogenic vascular cells, whereas stromal cells surround and interact with tumor cells. Recent data demonstrate significant gene expression alterations in microenvironment cells during disease progression and several stromal cell types are implicated in promoting the "hallmarks of cancer", which can be explored as targets for cancer therapy. Besides identifying new therapeutic targets, the microenvironment has also been implicated in chemotherapy resistance, suggesting that the crosstalk between cancer and its microenvironment is a promising strategy to target breast cancer.

One of the promising tools to evaluate collagen in the extracellular matrix is the second-harmonic generation microscopy (SHG). This approach may shed light on the biological behavior of cancers and their taxonomy, but has not yet been applied to characterize collagen fibers in cases diagnosed as invasive breast carcinoma (BC) of histological special types (IBC-ST). Tissue sections from 99 patients with IBC-ST and 21 of invasive breast carcinoma of no special type (IBC-NST) were submitted to evaluation of collagen parameters by SHG. Tissue microarray was performed to evaluate immunohistochemical-based molecular subtype. In intratumoral areas, fSHG and bSHG (forward-SHG and backward-SHG) collagen parameters achieved their lowest values in mucinous, papillary and medullary carcinomas, whereas the highest values were found in classic invasive lobular and tubular carcinomas. Unsupervised hierarchical cluster analysis and minimal spanning tree using intratumoral collagen parameters allowed the identification of three main groups of breast cancer: group A (classic invasive lobular and tubular carcinomas); group B (IBC-NST, metaplastic, invasive apocrine and micropapillary carcinomas); and group C (medullary, mucinous and papillary carcinomas). Our findings provide further characterization of the tumor microenvironment of IBC-ST. This understanding may add information to build more consistent tumor categorization and to refine prognostication.

Background In 2016, the Papanicolaou Society of Cytopathology (PSC) issued a new classification scheme for respiratory cytology. We aim to evaluate our samples according to this classification and to assess risk of malignancy and diagnostic yield of different cytological modalities. Methods Respiratory specimens (sputum, bronchial wash/brush, BAL and FNA) obtained between 2007 and 2016 were reclassified according to PSC guidelines. Risk of malignancy for each diagnostic category was determined. Diagnostic yield was evaluated based on three-categorical approach. Results One thousand, two hundred and ninety respiratory specimens were retrieved, of which 280 had histologic follow-up. Samples were reclassified as nondiagnostic 16%, negative for malignancy 53%, atypical 5.4%, neoplastic (benign neoplasm/low-grade carcinoma) 0.4%, suspicious for malignancy 2.1% and malignant 23.1%. Risk of malignancy for each category was 64.01% for ND, 48.27% for NM, 59.09% for A, 100% for N-B-LG; 90% for SM and 89.74% for M. When only malignant cases were considered positive tests, cytology sensitivity was 55% and specificity 88%. Conclusion Our results were in line with PSC guidelines, but the use of multiple cytological techniques may cause some discrepancies in overall diagnostic yield and in estimated risks of malignancy, which is important due to the widespread utilization of different cytological procedures.

Background Fine needle aspiration cytology (FNAC) is an important diagnostic tool in a range of medical settings. It is fast, quick and a highly accurate diagnostic method and can be used, in settings with minimal laboratory infrastructures. Methods In this report, we describe the experience in the use of FNAC since it is introduction in 1996 in the Anatomical Pathology Service of the Maputo Central Hospital (MCH), along with more detailed data referring to 2009-2010. Results The number of FNAC analyses increased gradually from 269 (4.1% of all pathologic tests of the Service) in 1996, when it was introduced in Mozambique, to 3234 (17% of all tests) in 2010. Lymph nodes were the organs most frequently biopsied, followed by breast and soft tissues. Inflammatory conditions, especially tuberculosis, were the most frequent diagnoses (22.2% of the cases), followed by hyperplastic conditions (20.6%), benign tumors (13.4%) and malignant tumors (12.3%). Conclusion Our results clearly demonstrate that even in an environment with poor laboratory resources, it is possible to establish a FNAC clinic that can provide a quick and precise diagnosis for clinicians to aid in early treatment interventions, especially in inflammatory diseases which were the majority of our cases.

Despite the significant biological, behavioural and management differences between ductal carcinoma in situ (DCIS) and invasive carcinoma of the breast, they share many morphological and molecular similarities. Differentiation of these two different lesions in breast pathological diagnosis is based typically on the presence of an intact barrier between the malignant epithelial cells and stroma; namely, the myoepithelial cell (MEC) layer and surrounding basement membrane (BM). Despite being robust diagnostic criteria, the identification of MECs and BM to differentiate in-situ from invasive carcinoma is not always straightforward. The MEC layer around DCIS may be interrupted and/or show an altered immunoprofile. MECs may be absent in some benign locally infiltrative lesions such as microglandular adenosis and infiltrating epitheliosis, and occasionally in non-infiltrative conditions such as apocrine lesions, and in these contexts this does not denote malignancy or invasive disease with metastatic potential. MECs may also be absent around some malignant lesions such as some forms of papillary carcinoma, yet these behave in an indolent fashion akin to some DCIS. In Paget's disease, malignant mammary epithelial cells extend anteriorly from the ducts to infiltrate the epidermis of the nipple but do not typically infiltrate through the BM into the dermis. Conversely, BM-like material can be seen around invasive carcinoma cells and around metastatic tumour cell deposits. Here, we review the role of MECs and BM in breast pathology and highlight potential clinical implications. We advise caution in interpretation of MEC features in breast pathology and mindfulness of the substantive evidence base in the literature associated with behaviour and clinical outcome of lesions classified as benign on conventional morphological examination before changing classification to an invasive lesion on the sole basis of MEC characteristics.

Metastatic disease is the main cause of death by cancer. Metastases are frequently diagnosed using cytology specimens, either using fine-needle aspiration (FNA) or effusion specimens. Over the last couple of decades, several molecular techniques have been successfully applied to cytological specimens. In situ hybridization and sequencing can nowadays be routinely performed in cytological specimens, and the application of these techniques in the metastatic cancer setting has the potential to change the landscape of patient care with advanced cancer. Additionally, as concepts like tumor clonal evolution are being translated to clinical trials and clinical care, the role and potential applications of molecular cytology are exponentially growing. Cytology, coupled with molecular techniques, has the potential to be the main source of specimens for longitudinal tracking of tumor metastasis and therefore have a central role in upcoming biomarker evaluation. In this chapter, we will review some the contemporary challenges that metastatic disease presents and how molecular cytology can help overcoming many of these, to ultimately improve patient diagnosis and care.

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. This paper review the main causes of false positives in thyroid FNA and give some tips in how to avoid these mistakes.

Lung cancer is the leading cause of cancer death in the world. Lung cytopathology is a significant part of the cytopathology practice. Less than 30% of patients with non-small cell lung cancer (NSCLC) are eligible for surgical treatment, and more than 70% of the NSCLCs have only cytological specimens available for molecular analysis. The most frequently used techniques to obtain material are bronchoscopy and fine-needle aspiration (FNA), performed under imaging guidance; EUS-guided FNA is used to stage lung cancer and to study mediastinal lesions. These materials are used to determine the origin and the nature of the lesions and to apply ancillary techniques. The development of molecular heterogeneity in NSCLC has led to the identification of molecular subgroups, which are responsive to target therapies, especially in lung adenocarcinoma that is the most common histological subtype. The clinical relevance of EGFR (epidermal growth factor receptor) and KRAS (Kirsten rat sarcoma viral oncogene) mutational status has been established, and testing these mutations by sequencing or RT-PCR (Reverse transcriptase polymerase chain reaction) methods has become a standard practice. The discovery of ALK (anaplastic lymphoma kinase) translocations and the responsiveness of these tumors to ALK inhibitors have led to study them by FISH (fluorescence in situ hybridization) and RT-PCR. Other genes such as ROS1, RET, and c-Met have been routinely assessed for targeted therapies. Recently the introduction of next-generation sequencing has focused on new gene alterations, although their clinical relevance has not been well established, yet. Recently, gene panels RNA-based to check rearrangements are under evaluation, and probably in future they will be developed also in clinical settings in order to find all molecular alterations.

Hypoxia is a common feature of solid tumours that promotes invasion and metastatic dissemination. Invadopodia are actin-rich membrane protrusions that direct extracellular matrix proteolysis and facilitate tumour cell invasion. Here, we show that CSRP2, an invadopodial actin bundling protein, is upregulated by hypoxia in various breast cancer cell lines, as well as in pre-clinical and clinical breast tumour specimens. We functionally characterized two hypoxia responsive elements within the proximal promoter of CSRP2 gene which are targeted by hypoxia-inducible factor-1 (HIF-1) and required for promoter transactivation in response to hypoxia. Remarkably, CSRP2 knockdown significantly inhibits hypoxia-stimulated invadopodium formation, ECM degradation and invasion in MDA-MB-231 cells, while CSRP2 forced expression was sufficient to enhance the invasive capacity of HIF-1a-depleted cells under hypoxia. In MCF-7 cells, CSRP2 upregulation was required for hypoxia-induced formation of invadopodium precursors that were unable to promote ECM degradation. Collectively, our data support that CSRP2 is a novel and direct cytoskeletal target of HIF-1 which facilitates hypoxia-induced breast cancer cell invasion by promoting invadopodia formation. The authors are grateful to Monika Dieterle, Arnaud Muller, Pter Nazarov and Muhammad Zaeem Noman (Oncology Department, LIH, Luxembourg) for technical assistance, support in statistical analyses and constructive discussions. The authors also warmly thank Sara A. Courtneidge for the gift of the Tks5-GFP construct (Oregon Health and Science University, Portland, USA). This work was mainly supported by a research grant from “Fondation Cancer” Luxembourg (FC/2016/02), and the National Research Fund (C16/ BM/11297905). Joshua Brown Clay is recipient of a Postdoctoral fellowship from “Fonds De La Recherche Scientifque” - FNRS “Télévie” (7.4512.16). Antoun Al Absi and Hannah Wurzer are recipients of PhD fellowships from the National Research Fund, Luxembourg (AFR7892325 and PRIDE15/10675146/CANBIO, respectively).

BACKGROUND: Fine-needle aspiration cytology (FNAC) of serous membrane effusions may fulfil a challenging role in the diagnostic analysis of both primary and metastatic disease. From this perspective, liquid-based cytology (LBC) represents a feasible and reliable method for empowering the performance of ancillary techniques (ie, immunocytochemistry and molecular testing) with high diagnostic accuracy. METHODS: In total, 3171 LBC pleural and pericardic effusions were appraised between January 2000 and December 2013. They were classified as negative for malignancy (NM), suspicious for malignancy (SM), or positive for malignancy (PM). RESULTS: The cytologic diagnoses included 2721 NM effusions (2505 pleural and 216 pericardic), 104 SM effusions (93 pleural and 11 pericardic), and 346 PM effusions (321 pleural and 25 pericardic). The malignant pleural series included 76 unknown malignancies (36 SM and 40 PM effusions), 174 metastatic lesions (85 SM and 89 PM effusions), 14 lymphomas (3 SM and 11 PM effusions), 16 mesotheliomas (5 SM and 11 SM effusions), and 3 myelomas (all SM effusions). The malignant pericardic category included 20 unknown malignancies (5 SM and 15 PM effusions), 15 metastatic lesions (1 SM and 14 PM effusions), and 1 lymphoma (1 PM effusion). There were 411 conclusive immunocytochemical analyses and 47 molecular analyses, and the authors documented 88% sensitivity, 100% specificity, 98% diagnostic accuracy, 98% negative predictive value, and 100% positive predictive value for FNAC. CONCLUSIONS: FNAC represents a primary diagnostic tool for effusions and a reliable approach with which to determine the correct follow-up. Furthermore, LBC is useful for ancillary techniques, such as immunocytochemistry and molecular analysis, with feasible diagnostic and predictive utility. Cancer (Cancer Cytopathol) 2015;000:000-000. V C 2015 American Cancer Society.

Objective: To identify associations between cytological criteria in fine needle aspiration (FNA) specimens and histological subtypes of lobular breast carcinoma (classical and other types). Study Design: FNA cytology and mastectomy specimens from 72 cases of invasive lobular breast carcinoma were consecutively retrieved from the files of the Amaral de Carvalho Hospital, Jaú-São Paulo, Brazil. All cases were reviewed regarding five cytological criteria: cellularity, cellular cohesion, presence of inflammation, nucleoli and nuclear atypia. The χ2 test or Fisher's exact tests with 95% confidence intervals (CI) were used. Results: The classical type showed lower initial cytological diagnosis of malignancy compared to the other variants (p = 0.017; odds ratio (OR) 0.26, 95% CI 0.89-0.80). Moderate/intense cellular cohesion (p = 0.011; OR 0.18, 95% CI 0.04-0.73) and mild atypia (p = 0.000; OR 16.15, 95% CI 3.20-81.48) were significantly associated with the classical type of lobular breast carcinoma, while the absence of inflammation (p = 0.082; OR 0.36, 95% CI 0.12-1.15) was marginally associated with the classical type. Conclusions: In cytology, the characterization of lobular carcinoma as malignant is difficult, especially the classical type. The association between cell cohesion and the classical type of lobular breast carcinoma may be one of the factors that complicate this diagnosis.