Molecular pathology of non-small cell lung cancer: a practical guide

The traditional distinction between small cell lung cancer and non-small cell lung cancer (NSCLC) is no longer sufficient for treatment planning. It is advised to handle small diagnostic specimens prudently because they are often the only specimen available for molecular analysis. Pathologists are experiencing pressure to subclassify lung carcinoma based on extremely small tumor samples, because NSCLC tumor subtyping is now essential to determine molecular testing strategies. Evaluation for EGFR mutations and ALK rearrangements are now considered to be the standard of care in advanced-stage pulmonary adenocarcinomas. Immunohistochemical stains can aid in subclassifying NSCLC, but performing these ancillary studies can significantly reduce the quantity of tissue available for molecular tests, requiring careful balancing of these 2 needs. The pathologist plays a pivotal role in facilitating clear and timely communication between the clinical oncology care team and the molecular laboratory to ensure that the appropriate tests are ordered and optimal material is submitted for testing.     

Cytopathology in the diagnosis,treatment and management of malignant respiratory disease

Lung carcinomas are almost always diagnosed using small samples of cellular material. Diagnostic cytopathology has a major role and, with efficient and thoughtful processing and examination of fine needle aspiration or fluid specimens, it is possible to accurately diagnose and perform a full range of predictive testing. This short piece begins by placing cytopathology within an overall umbrella of cellular pathology and briefly describes recent developments in lung cancer diagnosis and treatment. Cytopathological features of the main relevant tumour types and their differential diagnoses are discussed together with their appearance in commonly encountered specimen types such as those from endobronchial ultrasound. There are suggestions for efficient workflow and illustrations of diagnostic pitfalls. Although some trainees are wary of cytopathology, there is no magic (good or bad) involved and any time spent acquiring a facility with interpretation of these specimens as well as their formalin-fixed counterparts will enhance diagnostic skill and, ultimately, patient care.     

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.     

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.     

Update on cytology samples for the use of molecular pathology and other ancillary tests in the move towards next-generation sequencing

The use of molecular pathology is becoming more evident every year, with 92% of pathologists stating that they use molecular pathology on a regular basis when reporting diagnostic work. This growth has been led not just through service development and improvement, but also with the introduction of new treatments offering patients significant improvement and overall better survival from cancer. These new treatments tend to require a targeted genomic test to identify specific cohorts of patients that will benefit. This has led to a reform of genomic and molecular pathology testing across the four nations of the United Kingdom, with the introduction of new regional testing hubs. These centres mainly use next generation sequencing platforms, to test for both the inherited and somatic mutations which drive disease. Next generation sequencing offers high-throughput cost effective sequencing of DNA, either via sequencing; individual genes, groups of genes, coding DNA known as Exome sequencing and whole genome sequencing to analysis every base pair in the patients DNA. This technology comes with new ways of working within the laboratory and relays heavily on the data analysis via bioinformatic analysis to; analyse, manipulate, quality control and interpret the vast about of data from these platforms. With the regionalization of testing to the genomic hubs, and with the increased movement of diagnostic cytopathology towards near patient sampling, this opens an opportunity for cytopathology departments to work in partnership with the new genetic testing hubs, to make sure that both parties can avail off the superior benefits offered by cytology samples; safe procurement, formalin free, high diagnostic value, rapid, minimally invasive and a high DNA yield. Cytopathology can also offer a rapid report based on sample suitability for molecular testing based on cellular content, but this to will change with the possible presence of cell-free DNA (cfDNA) which has been reported to be present in abundance in serous fluid supernatants. This supernatant that is normally discarded in the preparation of samples may hold vast diagnostic potential in the move to NGS testing.     

Pulmonary small cell carcinoma: Review,common and uncommon differentials,genomics and management

Lung cancer is the leading cause of cancer‐related death worldwide. It is divided into sub‐categories based upon morphology, immunostaining pattern, biology, molecular profile, and/or treatment options. Up until the early 2000s when driver mutations with targeted therapies were identified in a subset of adenocarcinomas, the most critical distinction of lung carcinomas was driven by differences in treatment between small cell carcinoma (SCC) and nonsmall cell lung carcinoma (NSCLC). The distinction between SCC and NSCLC remains critical in the 21st century for management, especially for advanced stage cancer. In the vast majority of cases, morphological features are sufficient to separate SCC from other types of lung cancers. In some instances, however, cytomorphological features and immunohistochemical overlap with other tumors, limited sample availability, and/or crush artifact pose diagnostic challenges. The aim of this review is to highlight salient features of SCC and ancillary studies to distinguish it from common and uncommon potential mimickers, as well as provide updates in genomics and management.     

Immunocytochemistry in thyroid cytology and its multiple roles: a systematic review

The limits of morphological analysis, mostly encountered in the diagnosis of “follicular thyroid neoplasms (FN)”, were clearly reported in many papers. For this reason, the application of ancillary techniques, including immunocytochemistry (ICC) and molecular testing, contributes to a better definition of the risk of malignancy (ROM) and management of thyroid nodules (TN). This review discusses the diagnostic and prognostic role of the most relevant immunomarkers in TN, including HBME-1, Galectin-3, CD56, and VE1-BRAF among the others. Furthermore, we also analyzed the growing interest for the so-called next-generation immunomarkers, which represent one of the new frontiers for cancer scientific research. According to the literature, the diagnostic and prognostic relevance of their performance is able to help the morphological evaluation, especially for the indeterminate categories and controversial diagnoses. In fact, even though ancillary techniques cannot substitute the relevance of morphological evaluation of thyroid samples, they might represent a valid adjunct for those cases in which controversial features are not able to lead to a univocal diagnosis. Herein the review of literature about each of the immunomarkers in thyroid nodules.     

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.     

The role of molecular analyses in the diagnosis and treatment of non-small-cell lung carcinomas

Non-small-cell lung cancer (NSCLC) subtyping has recently been a key factor in determining patient management with novel drugs. In addition, the identification of distinct oncogenic driver mutations frequently associated with NSCLC histotype and coupled to the clinical responses to targeted therapies have revolutionized the impact of histologic type and molecular biomarkers in lung cancer. Several molecular alterations involving different genes (EGFR, KRAS, ALK, BRAF, and HER2) seem to have a remarkable predilection for adenocarcinoma and specific inhibitors of EGFR and ALK are now available for patients with adenocarcinoma harboring the relevant gene alterations. The efficacy of histology-based and molecular-targeted therapies had a deep impact in (1) re-defining classification of lung cancer (particularly adenocarcinomas) and (2) routine clinical practice of pathologists involved in optimization of handling of tissue samples in order to guarantee NSCLC subtyping with the help of immunohistochemistry and adequately preserve tumor cells for molecular analysis. In agreement with the modern multidisciplinary approach to lung cancer, we reviewed here the diagnostic and predictive value of molecular biomarkers according to the clinical, pathologic, and molecular biologist viewpoints.     

Differential diagnosis of lung carcinoma with three-dimensional quantitative molecular vibrational imaging

The advent of molecularly targeted therapies requires effective identification of the various cell types of non-small cell lung carcinomas (NSCLC). Currently, cell type diagnosis is performed using small biopsies or cytology specimens that are often insufficient for molecular testing after morphologic analysis. Thus, the ability to rapidly recognize different cancer cell types, with minimal tissue consumption, would accelerate diagnosis and preserve tissue samples for subsequent molecular testing in targeted therapy. We report a label-free molecular vibrational imaging framework enabling three-dimensional (3-D) image acquisition and quantitative analysis of cellular structures for identification of NSCLC cell types. This diagnostic imaging system employs superpixel-based 3-D nuclear segmentation for extracting such disease-related features as nuclear shape, volume, and cell-cell distance. These features are used to characterize cancer cell types using machine learning. Using fresh unstained tissue samples derived from cell lines grown in a mouse model, the platform showed greater than 97% accuracy for diagnosis of NSCLC cell types within a few minutes. As an adjunct to subsequent histology tests, our novel system would allow fast delineation of cancer cell types with minimum tissue consumption, potentially facilitating on-the-spot diagnosis, while preserving specimens for additional tests. Furthermore, 3-D measurements of cellular structure permit evaluation closer to the native state of cells, creating an alternative to traditional 2-D histology specimen evaluation, potentially increasing accuracy in diagnosing cell type of lung carcinomas.     

Current concepts on the molecular pathology of non-small cell lung carcinoma

Recent advances in the understanding of the complex biology of non-small cell lung carcinoma (NSCLC), particularly activation of oncogenes by mutation, translocation and amplification, have provided new treatment targets for this disease, and allowed the identification of subsets of NSCLC tumors, mostly with adenocarcinoma histology, having unique molecular profiles that can predict response to targeted therapy. The identification of specific genetic and molecular targetable abnormalities using tumor tissue and cytology specimens followed by the administration of a specific inhibitor to the target, are the basis of personalized lung cancer treatment. In this new paradigm, the role of a precise pathology diagnosis of lung cancer and the proper handling of tissue and cytology samples for molecular testing is becoming increasingly important. These changes have posed multiple new challenges for pathologists to adequately integrate routine histopathology analysis and molecular testing into the clinical pathology practice for tumor diagnosis and subsequent selection of the most appropriate therapy.     

News in the classification of pulmonary adenocarcinomas and potential prognostic and predictive factors in non-small lung cancer

Lung cancers are still divided into two major subgroups: small-cell and non-small cell lung cancer (NSCLC) irrespective of biological heterogeneity of NSCLC. It is a key task of the pathologist to provide an accurate classification of tumorous lesions to avoid the term NSCLC and to use it only in the vast minority of cases. Moreover, the most recent reclassification of pulmonary adenocarcinomas should be reflected in the standard biopsy protocol reporting. There is also an increasingly urgent need to provide high quality material for testing of the genetic characteristics of NSCLC, especially the presence and functional status of the EGFR receptor (epidermal growth factor receptor), as well as other potential prognostic markers. The requirement for the quality and swiftness of diagnosis puts major emphasis on the close multidisciplinary collaboration with the central role of a specialized pathologist, who coordinates the differential-diagnostic procedure. This in turn implies the necessity of accounting for the increasing financial burden of diagnostic departments.     

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.     

EGFR mutation detection in NSCLC��assessment of diagnostic application and recommendations of the German Panel for Mutation Testing in NSCLC

EGFR mutation testing in non-small cell lung cancer (NSCLC) is a novel and important molecular pathological diagnostic assay that is predictive of response to anti-epidermal growth factor receptor (EGFR) therapy. A comprehensive compilation of a large number of EGFR mutation analyses of the German Panel for Mutation Analyses in NSCLC demonstrates (a) a higher than previously reported mutation frequency outside the conventionally tested exons 19 and 21 and (b) an overall superiority of sequencing based assays over mutation-specific PCR. The implications for future diagnostic EGFR mutation testing are discussed.     

Molecular pathology of non-small cell lung cancer

Our increasing understanding of the molecular pathogenesis of non-small cell lung cancer (NSCLC), particularly adenocarcinomas, has opened the door to ‘personalised medicine’ and the advent of new therapeutic strategies. Over the last few years new drugs, or classes of drugs, have been licensed and entered clinical practice for use in advanced NSCLC. The activity of these drugs is dependent on the presence of specific molecular or protein changes in cancer cells which are usually identified using ‘companion diagnostic tests’ specifically designed for this purpose. Pathologists and Pathology Departments have had to forge new links with Clinical Scientists in order to facilitate these additional investigations on the, often limited, tissue obtained for diagnosis. This collaboration plays a critical role in providing the link that allows integration of the traditional morphological diagnosis with the results of these new ‘companion diagnostic’ tests to guide patient management.     

RcaNet:一种预测肿瘤突变负荷的深度学习模型

近年来，肺癌的发病率和死亡率不断攀升，已成为对人类生命健康威胁最大的恶性肿瘤之一，而非小细胞肺癌(NSCLC)发病率占肺癌总发病率的80%以上。由于其复杂的诊断过程和昂贵的诊断成本，NSCLC的有效诊断和治疗已成为医生面临的巨大挑战。医学相关研究发现，肿瘤突变负荷(TMB)与NSCLC免疫治疗的疗效呈正相关，同时TMB值对靶向治疗和化疗的疗效具有一定的预测作用。基于上述发现，本研究提出一种深度学习模型(RcaNet),该模型以残差网络(ResNet)为骨干网络，在网络内增加多维度特征注意和多尺度信息融合，以增强网络对肺癌病理组织切片深层特征的关注与提取能力。通过将RcaNet与主流深度学习模型在TCGA公开数据集上进行实验，实验训练样本数为925 954张。结果表明，RcaNet模型的性能平均曲线下面积(AUC)值为0.883 0,比现有结果最好的CAIM模型高出6.8%,比ResNeSt模型高出4.2%,比ResNet模型高出5.3%。研究结果对非小细胞肺癌诊断治疗有较强的指导意义，具有较高的应用价值。     

Rapid on-site evaluation for head and neck lesions

Rapid on-site evaluation (ROSE) has proven to be a valuable practice in cytopathology allowing diagnostic evaluation of head and neck lesions. It involves the immediate assessment of fine-needle aspiration samples to ensure adequacy and enable cytopathologists to make decisions regarding further diagnostic procedures and ancillary techniques. ROSE also reduces turnaround times, enabling timely treatment decisions and enhancing patient care. However, the challenges associated with ROSE include the need for an experienced on-site cytopathologist, need for extra time from the cytopathologist, need for optimal staining quality and a possible extended time for procedure. This review discusses the use of ROSE in head and neck, particularly in thyroid and salivary gland lesions, listing its advantages and disadvantages and overall impact on the diagnostic yield and patient's overall guidance.     

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.