Molecular testing in cytology

In the era of personalized medicine, molecular testing plays a critical role in patient care. The rapid advance of molecular techniques, especially next-generation sequencing, makes molecular diagnosis feasible in daily practice. Molecular testing can be used as a valuable ancillary test to increase diagnostic sensitivity and specificity, especially in small biopsy or cytology samples. In addition, molecular testing plays an important role in selecting patients for appropriate treatment by detecting therapeutic and predictive biomarkers in tissue or cytology samples. Molecular studies can be applied in all cytology samples, sometimes with better results than histology. As molecular testing has become essential for patient care and is often requested to be performed in cytology samples, it is critical for cytopathologists to understand the basics of molecular diagnostic methods, indications for molecular testing, and how to best utilize different cytologic samples for this purpose. In this special issue, experts in various areas of cytopathology and molecular pathology review the literature and discuss the basics of molecular techniques and the application of molecular testing in various types of cytology samples. It is our hope that after reading the articles in this special issue, the readers can know better about the possibilities of molecular cytology, a very exciting field of pathology.     

Electron Microscopy Renders the Diagnostic Capabilities of Cytopathology More Precise: An Approach to Everyday Practice

Cytology is a powerful diagnostic tool but to make definitive diagnoses, the use of ancillary techniques is imperative. By combining immunohistochemistry (IHC) and electron microscopy (EM), cytologic diagnoses can be as precise as those of surgical pathology. In the authors' daily practice of cytopathology they use all ancillary techniques available to them: histochemistry, IHC, EM, flow cytometry, and molecular pathology. IHC is frequently used as an ancillary technique in their daily practice but EM is many times their technique of choice. By the use of EM the authors can make specific final diagnoses, make the diagnosis more definitive, narrow the differential diagnosis, or determine the origin of a neoplasm with unknown primary site. Specimens obtained by fine-needle aspiration as well as all body fluids are suitable for EM. The limiting factor is to obtain the appropriate material with the diagnostic cells for ultrastructural examination. The common diagnostic dilemmas in the everyday practice of cytology are the following: mesothelioma vs. adenocarcinoma, neuroendocrine differentiation or not, the distinction of melanoma from adenocarcinoma and sarcoma, hepatocellular carcinoma vs. adenocarcinoma, and the origin of adenocarcinomas of unknown primary. The authors discuss how they approach these diagnostic problems in their everyday practice and how they incorporate EM in solving them.     

Cytopathology of lung cancer: moving from morphology to molecular

The recent discovery and availability of new targeted therapies for lung cancer has presented new challenges to pathologists. Since many lung cancers are diagnosed by aspiration or exfoliative cytology specimens in the primary or metastatic setting, these new therapies have had an impact on the practice of respiratory cytopathology. In particular, accurate subclassification of non-small cell carcinomas and acquisition of sufficient material for molecular studies is crucial. Balancing the need of adequate material for accurate diagnosis with the demands of clinicians to do more with less tissue has been a challenge and continues to impact the way lung cancers are approached in cytopathology. This review focuses on the changes and impact of the molecular era on the diagnosis of lung cancer in cytopathology.     

“SMART” cytology: The next generation cytology for precision diagnosis

Cytology plays an important role in diagnosing and managing human diseases, especially cancer, as it is often a simple, low cost yet effective, and non-invasive or minimally invasive diagnostic tool. However, traditional morphology-based cytology practice has limitations, especially in the era of precision diagnosis. Recently there have been tremendous efforts devoted to apply computational tools and to perform molecular analysis on cytological samples for a variety of clinical purposes. Now is probably the appropriate juncture to integrate morphology, machine learning, and molecular analysis together and transform cytology from a morphology-driven practice to the next level – “SMART” Cytology. In this article we will provide a rather brief review of the relevant works for computational analysis on cytology samples, focusing on single-cell-based multiplex quantitative analysis of biomarkers, and introduce the conceptual framework of “SMART (Single cell, Multiplex, AI-driven, and Real Time)” Cytology.     

Sputum cytology: a limited role.

AIMS: To determine the cost and sensitivity of sputum cytology in routine use and to determine when sputum cytology is most appropriate. METHODS: A retrospective study, based on all sputum cytology requests received in five histopathology/cytopathology laboratories in Yorkshire from 1 January to 31 December 1993. Cytology findings were correlated with histological diagnosis or clinical outcome, and related to the speciality of the referring clinician. RESULTS: Laboratory practice and performance was similar in all five centres. The average laboratory cost of sputum cytology was 26.93. The mean absolute sensitivity was 36% and the specificity was 99.6%. The majority of specimens was submitted by general physicians or geriatricians. The largest proportion of positive specimens were submitted by chest physicians. CONCLUSIONS: Often sputum cytology is used inappropriately as a screening investigation on, or soon after, admission. In addition, it is used inappropriately before bronchoscopy. Sputum cytology should be limited to individuals in whom a histological diagnosis is desired, but in whom bronchoscopy is inappropriate or unsuccessful.     

Evolution of guidelines for respiratory cytology by the Papanicolaou Society of Cytopathology

Cytology serves a fundamental role in the evaluation of the lower respiratory tract. Cytological specimens are often the first diagnostic attempt for the evaluation of radiographic alterations. The categorization of the pathological process as infectious, inflammatory or neoplastic is critical in guiding further clinical management of the affected patient. Therefore it is imperative that cytopathologists use a standardized approach to sample handling and reporting in order to establish clear communication with the treating physician. The Papanicolaou Society of cytopathology has been an active leader in this field and has proposed several guidelines for sampling, handling, and reporting of lower respiratory tract cytology. These guidelines have been updated to incorporate new emerging concepts and technologies in the field. Respiratory medicine is a fast growing area with constant new challenges, thus requiring an ever demanding adaptation from cytopathologists and cytology specific guidelines.     

Nichtkleinzellige Lungenkarzinome

The diagnosis and treatment of non-small cell lung cancer (NSCLC) have been revolutionized over the last few years. Requirements for cytopathologists in lung cancer diagnosis have therefore changed. The general diagnostic category of NSLC is no longer sufficient. In addition cytological specimens need to be evaluated for adequacy regarding predictive marker analyses. Accurate NSCLC subtyping with a distinction of adenocarcinoma from squamous cell carcinoma is crucial for treatment decisions as the subtype will decide on the chemotherapy regimen and the choice of predictive marker analyses for targeted treatment. In the majority of cases, the subtype can be diagnosed by morphology alone. Cytology is equally well suited as biopsy specimens for the assessment of molecular predictive markers. The best results are achieved when both cytology and biopsy specimens are compared to choose the most appropriate specimen for morphological subtyping and molecular testing. In this paper, we discuss special issues of NSCLC subtyping and currently recommended predictive molecular marker analyses.     

The use of electron microscopy to refine diagnoses in the daily practice of cytopathology

Conceived as a screening tool, cytology is a field that since the 1980s has become more diagnostic in its scope. The advent of the fine-needle aspiration biopsy (FNAB) is responsible for cytology's new place in pathology. In the everyday practice of cytopathology, about 85-90% of the nongynecologic cases can be diagnosed with the use of routine stains (i.e., Papanicolaou and Diff Quik). The other 10-15% of the cases require the use of ancillary diagnostic techniques for a precise diagnosis. Immunohistochemistry helps solve approximately 50% of these cases, and the other half of these challenging cases are best approached and diagnosed by using electron microscopy (EM). In their practice, the authors obtain cytologic samples for EM routinely in difficult cases. Unfortunately, a percentage of these cases collected for ultrastructural evaluation do not have enough cells after processing, and others only have a few diagnostic cells available. In the cases in which at least a handful of cells are available, EM is almost invariably helpful in one way or another, either making a definitive diagnosis or refining the diagnosis. A sampling of FNAB cases from the authors' everyday practice is prevented to illustrate the use of EM in the practice of cytopathology. The cases have been selected from among the most common diagnostic challenges to highlight the important role that ultrastructural evaluation plays in a busy cytology practice. In our practice ultrastructural evaluation is a piece of the puzzle, which, along with the clinical history, clinical impression, light microscopic/cytologic features, and other ancillary techniques (IHC, flow cytometry, and molecular pathology), help compile an accurate diagnosis. Many times EM is the most important component of the diagnostic algorithm.     

Assisting cytopathology training in medically under-resourced countries: defining the problems and establishing solutions

Cytology is able to deliver rapid accurate diagnoses with minimal equipment and laboratory infrastructure at minimal cost, and this is especially so for fine needle biopsy (FNB), which is a powerful diagnostic tool in medically resource-poor environments, where histopathology laboratories are small in number and poorly supported financially. The crucial element in the development of cytology services is to train a sufficient number of well trained cytopathologists and cytotechnologists to create a 'critical mass' of personnel who not only provide routine diagnostic services, but also can train an ever expanding number of pathologists, cytotechnologists, and health workers. A review of practical programs to train cytopathologists and cytotechnologists in their own countries will be presented, including a recent series of FNB and cytology tutorials run in sub Saharan Africa. The need for local cytopathology programs and the potential for both local and visiting cytopathologists to provide a faculty will be discussed, as well as a range of possible programs which can bring African pathologists and trainee pathologists to Western institutions for periods of their training. Ideally, the regional Societies of Cytology, including the recently formed West African Society of Cytology, will establish their own diagnostic protocols, training programs, syllabuses, examinations and accreditation and career pathways for both cytopathologists and cytotechnologists, and organize tutorials where they will invite overseas faculty to contribute. Crucially, these new societies will empower cytopathologists and cytotechnologists to approach health services and governments to state the need for cytology services as a cost-effective accurate diagnostic service that enhances patient care.     

The minefield of indeterminate thyroid nodules: could artificial intelligence be a suitable diagnostic tool?

Artificial intelligence (AI) is assuming a central role in anatomic pathology for ancillary diagnosis in histology and cytology. AI techniques can analyse large amounts of data and identify patterns that may not be visible to the human eye.Several studies have explored the potential of such techniques to improve the accuracy and efficiency of thyroid nodule diagnosis and to increase the sensitivity and specificity of thyroid cytopathology. Specifically, the indeterminate categories of ‘the Bethesda system for reporting thyroid cytopathology’ (TBSRTC) represent a major diagnostic challenge, and articles reported in this review highlight the potential of new AI technologies in improving the accuracy and standardisation of the cytological diagnosis of indeterminate thyroid nodules.Although a large amount of data supports AI's utility in thyroid cytopathology, further research is needed to integrate and standardise AI-based diagnostic systems in clinical workflows.     

Advances in cytology of lung cancer

Cytopathology has emerged as a promising platform in precision oncology especially after the revolutionary change in our understanding of the concept of lung cancer etiopathogenesis. With increasing use of minimally invasive techniques for sample acquisition, it becomes almost mandatory to utilize precious cytology samples maximally and judiciously by appropriate triaging of the specimen and timely action of the cytopathology team. Existing patient management protocols require accurate morphologic and molecular diagnosis of the lung cancer specimens which needs knowledge about evolving techniques related to specimen procurement, updates of genomic variants of lung cancer and recently developed molecular testing platforms and algorithms which are capable enough to use even miniscule amount of diagnostic material. This review provides a brief knowledge about advances in cytology of lung cancer which are helpful for developing correct clinical management strategies.     

Pericardial cytopathology

Cytopathology diagnosis of three hundred and thirty three pericardial effusions performed between September 1999 and August 2006 were systematically analyzed and compared with the biopsy in 71 cases. We described the clinical circumstances, the etiologies, the cytomorphologic features of the effusions, the role of liquid based cytology and ancillary studies such as immunocytochemistry and the sensitivity of cytopathology compared with histopathology. Among the 57 pericardial tumors, 51 (89%) were metastatic carcinomas--included 42 (73%) adenocarcinomas--, 5 (9%) were malignant lymphomas and one (2%) was a malignant mesothelioma. Cytology of pericardial effusion with ancillary studies enables rapid cytodiagnosis as specific as histodiagnosis provided by biopsy.     

Mesenchymal neoplasms: Is it time for cytology? New perspectives for the pre-operative diagnosis of soft tissue tumors in the molecular era

Soft tissue tumors comprise a great variety of common and rare entities with overlapping features. Their diagnosis is based on the evaluation of several histological parameters which are difficult to assess on small incisional biopsies. Useful diagnostic markers in the field of soft tissue tumors include: 1) molecular biomarkers detecting pathogenetically relevant, distinctive alterations; 2) immunohistochemical surrogate biomarkers of pathogenetically relevant, distinctive molecular alterations; 3) highly specific immunohistochemical biomarkers indicating tumor differentiation. Their introduction in clinical practice has revolutionized the pre-operative diagnosis of soft tissue tumors. Cytology has long been considered inadequate as a first-line approach in this setting. However, since the implementation of new immunohistochemical and molecular tests with high diagnostic specificity, fine needle aspiration cytology (FNAC) is starting to gain acceptance for the pre-operative assessment of soft tissue tumors. FNAC represents a versatile, poorly expensive and well-tolerated diagnostic strategy with relevant advantages over histological biopsies. Moreover, evidences suggest that, in expert hands, FNAC can also aim at a definite diagnosis, especially if a cell block is prepared, allowing the application of multiple ancillary techniques.     

Electron microscopy renders the diagnostic capabilities of cytopathology more precise: an approach to everyday practice

Cytology is a powerful diagnostic tool but to make definitive diagnoses, the use of ancillary techniques is imperative. By combining immunohistochemistry (IHC) and electron microscopy (EM), cytologic diagnoses can be as precise as those of surgical pathology. In the authors' daily practice of cytopathology they use all ancillary techniques available to them: histochemistry, IHC, EM, flow cytometry, and molecular pathology. IHC is frequently used as an ancillary technique in their daily practice but EM is many times their technique of choice. By the use of EM the authors can make specific final diagnoses, make the diagnosis more definitive, narrow the differential diagnosis, or determine the origin of a neoplasm with unknown primary site. Specimens obtained by fine-needle aspiration as well as all body fluids are suitable for EM. The limiting factor is to obtain the appropriate material with the diagnostic cells for ultrastructural examination. The common diagnostic dilemmas in the everyday practice of cytology are the following: mesothelioma vs. adenocarcinoma, neuroendocrine differentiation or not, the distinction of melanoma from adenocarcinoma and sarcoma, hepatocellular carcinoma vs. adenocarcinoma, and the origin of adenocarcinomas of unknown primary. The authors discuss how they approach these diagnostic problems in their everyday practice and how they incorporate EM in solving them.     

Shared Perspectives from Cytology and Microbiology

Cytology, also known as cytopathology, and microbiology laboratories reside in hospitals, clinics, or free-standing laboratories, in which scientists are employed to diagnose many medical conditions, including infection, inflammation, or cancer. There is overlap in the types of clinical samples shared by cytology and microbiology laboratories. Specimens include swabs, sputum, and fine-needle biopsy specimens or aspirates. When laboratories share clinical samples, there can be similarities and differences in the proper transport media and preservatives used. When specimens or functions are shared, there is an opportunity for each laboratory to learn from each other. Increased awareness of the roles and processes in cytology and microbiology can decrease the number of lost or improperly preserved specimens. Improved communication and collaboration between laboratory sections can increase diagnostic accuracy and reduce patient harm. This review summarizes laboratory career choices, sample collection devices, shared specimens, and preservatives associated with shared specimens. Cytology and microbiology laboratories can work together to improve pre-analytic processes and diagnostic accuracy.     

Ein Pl?doyer für die Zytopathologie

Compared to other European and non-European countries the benefits of cytopathology for the diagnosis of many tumors is still underestimated in Germany for traditional reasons. Cytological methods provide excellent material from many organs for morphological, immunochemical and molecular examinations so that a definitive diagnosis is cytologically possible in many cases and the number of exploratory surgical operations could therefore be reduced. Improvements in this deplorable situation will only be possible if a standardized training period in cytology is consistently included in the training of general pathologists. This requires organizational and infrastructural changes within the institutes of pathology. In this respect, the university institutes as important training institutions should lead the way.     

Routine and specialised techniques in the diagnosis of haematological neoplasms.

The relative importance of traditional techniques used in the diagnosis of haematological neoplasms has altered during the past decade. Cytology and histology retain their central role but the importance of cytochemistry has declined, except in the diagnosis of AML. Immunophenotyping is of major importance in the diagnosis of ALL, some categories of AML and the LPDs. Cytogenetic and molecular genetic analysis are important in the diagnosis of CML and are becoming increasingly important in the diagnosis of chronic LPDs and other haematological neoplasms. Diagnostic haematology laboratories which are not specialist leukaemia centres should have ready access to all of these techniques to ensure optimal patient management. However, not all techniques need to be performed in every laboratory.     

Parathyroid lesions: Difficult diagnosis on cytology

Cytology of parathyroid lesion (PL) is often confused with that of thyroid lesions. Differentiation between thyroid and PL is very difficult on cytomorphology because of their similar features and close anatomical proximity. Three cases of PLs reported on cytology in last one year were retrieved from archives of cytology department. Their cytomorphological details were studied and were correlated with the available biochemical parameters. Histopathology was available in two cases. Radiological assistance and parathyroid hormone (PTH) assessment in our cases formed the basis of diagnosing PLs on cytology. We discuss the differential diagnosis and pitfalls in cytological diagnosis of PLs. However, histopathology remains the gold standard for diagnosis. Interpretation of PLs on cytology remains problematic due to its rarity and limited available literature. The cytomorphology combined with clinical and biochemical data supported by histopathology are necessary to improve the diagnostic sensitivity of PLs. Diagn. Cytopathol. 2016;44:704–709. © 2016 Wiley Periodicals, Inc.     

A summary of recent international standardised reporting systems in cytopathology

In recent years, there has been an increasing move towards the use of standardized reporting systems in the field of cytopathology. Their implementation aims to provide a universal language of cytology reporting, fostering better communication and understanding between pathologists and clinicians. Such standardized systems can help reduce reporting variability, provide frameworks for clinical audit and research, and guide clinical management strategies, ultimately improving patient care. This article summarises the salient and practical points set out in some of the more recently developed international reporting systems, namely The International System for Reporting Serous Fluid Cytopathology, The Milan System for Reporting Salivary Gland Cytopathology, and The Paris System for Reporting Urinary Cytology. A subsequent subsection discusses the international reporting systems available for thyroid fine-needle aspiration cytology specimens, with particular focus on the UK Royal College of Pathologists guidance.     

Interventional and EBUS cytology in Sweden

Interventional cytology was first introduced in Sweden in the late 1940ies by Sixten Franzén at the Karolinska University Hospital in Solna, Stockholm. In the early 1950ies, Nils Söderström started using the technique at the University Hospital in Lund. Cytology was successively established as common practice at the pathology departments in Sweden, and e.g. Solna and Lund today have a high rate of cytological samples. Over the years new techniques, such as endobronchial ultrasound (EBUS)-guided fine-needle aspirations, and analyses have been introduced, contributing to the maintained value of cytology as a diagnostic method. In this article, we present a brief history and the current situation of cytology in Sweden with focus on interventional and EBUS cytology.