J. Mol. Pathol., Volume 2, Issue 2 (June 2021) – 12 articles

Immunotherapy in the form of ICIs has revolutionized advanced NSCLC treatment algorithms, with ICI-containing combination treatments being the latest addition to approved regimens. However, PD-L1 still represents the only routinely assessed and validated biomarker apart from genetic drivers testing, impairing our capacity to personalize and guide treatment. Therefore, this paper aims to analyze the most promising emerging predictive biomarkers that could help us in the near future to select patients more effectively. Full article

The molecular pathways which promote lung cancer cell features have been broadly explored, leading to significant improvement in prognostic and diagnostic strategies. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have dramatically altered the treatment approach for patients with metastatic non-small cell lung cancer (NSCLC). Latest investigations by using next-generation sequencing (NGS) have shown that other oncogenic driver mutations, believed mutually exclusive for decades, could coexist in EGFR-mutated NSCLC patients. However, the exact clinical and pathological role of concomitant genomic aberrations needs to be investigated. In this systematic review, we aimed to summarize the recent data on the oncogenic role of concurrent genomic alterations, by specifically evaluating the characteristics, the pathological significance, and their potential impact on the treatment approach. Full article

The discovery and clinical validation of biomarkers predictive of the response of non-squamous non-small-cell lung carcinomas (NS-NSCLC) to therapeutic strategies continue to provide new data. The evaluation of novel treatments is based on molecular analyses aimed at determining their efficacy. These tests are increasing in number, but the tissue specimens are smaller and smaller and/or can have few tumor cells. Indeed, in addition to tissue samples, complementary cytological and/or blood samples can also give access to these biomarkers. To date, it is recommended and necessary to look for the status of five genomic molecular biomarkers (EGFR, ALK, ROS1, BRAFV600, NTRK) and of a protein biomarker (PD-L1). However, the short- and more or less long-term emergence of new targeted treatments of genomic alterations on RET and MET, but also on others’ genomic alteration, notably on KRAS, HER2, NRG1, SMARCA4, and NUT, have made cellular and blood samples essential for molecular testing. The aim of this review is to present the interest in using cytological and/or liquid biopsies as complementary biological material, or as an alternative to tissue specimens, for detection at diagnosis of new predictive biomarkers of NS-NSCLC. Full article

The past decade has witnessed significant advances in the application of molecular diagnostics for the pre-operative risk-stratification of cytologically indeterminate thyroid nodules. The tests that are currently marketed in the United States for this purpose combine aspects of tumor genotyping with gene and/or microRNA expression profiling. This review compares the general methodology and clinical validation studies for the three tests currently offered in the United States: ThyroSeq v3, Afirma GSC and Xpression Atlas, and ThyGeNEXT/ThyraMIR. Full article

Somatic copy number variations (CNV; i.e., amplifications and deletions) have been implicated in the origin and development of multiple cancers and some of these aberrations are designated targets for therapies. Although FISH is still considered the gold standard for CNV detection, the increasing number of potentially druggable amplifications to be assessed makes a gene-by-gene approach time- and tissue-consuming. Here we investigated the potential of next generation sequencing (NGS) custom panels to simultaneously determine CNVs across FFPE solid tumor samples. DNA was purified from cell lines and FFPE samples and analyzed by NGS sequencing using a 20-gene custom panel in the GeneReader Platform^®. CNVs were identified using an in-house algorithm based on the UMI read coverage. Retrospective validation of in-house algorithm to identify CNVs showed 97.1% concordance rate with the NGS custom panel. The prospective analysis was performed in a cohort of 243 FFPE samples from patients arriving at our hospital, which included 74 NSCLC tumors, 148 CRC tumors, and 21 other tumors. Of them, 33% presented CNVs by NGS and in 14 cases (5.9%) the CNV was the only alteration detected. We have identified CNV alterations in about one-third of our cohort, including FGFR1, CDK6, CDK4, EGFR, MET, ERBB2, BRAF, or KRAS. Our work highlights the need to include CNV testing as a part of routine NGS analysis in order to uncover clinically relevant gene amplifications that can guide the selection of therapies. Full article

Salivary gland masses are often encountered in the everyday practice of cytopathology. It is commonly known that the cytologic interpretation of these lesions can pose diagnostic problems due to overlapping cytomorphologic features. Fine needle aspiration (FNA) of salivary lesions shows good to excellent sensitivity and specificity in differentiating a neoplastic from a non-neoplastic process and in diagnosing common tumors such as pleomorphic adenoma. However, its value is limited in diagnosing specific neoplastic entities especially those with well-differentiated morphology. In light of this gap, an international group of pathologists has proposed a management-oriented, tiered classification for reporting salivary gland FNA specimens, “The Milan System for Reporting Salivary Gland Cytopathology (MSRSGC)”. Similar to other classification systems, the MSRSGC scheme comprises six diagnostic categories, which were linked with a specific risk of malignancy (ROM) and management. In this review article, the author evaluated the published literature on FNA in diagnosing salivary gland lesions with the adoption of the Milan system since its introduction in the daily practice of salivary cytopathology. Full article

Since the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak was declared a pandemic, the magnitude of coronavirus disease 2019 (COVID-19) has continued to grow, putting an unprecedented strain on all medical fields. Its effects on cytopathology workloads have been dramatic. Indeed, despite the implementation of several laboratory biosafety recommendations, cytological screening activities and cytological sampling of patients at low risk of malignancy have been postponed to limit the risk of contagion and to lessen the strain on overwhelmed hospital facilities. In this scenario, a drastic reduction in the total number of cytological specimens has been observed worldwide. This review summarizes the current evidence of the impact of the COVID-19 pandemic on cytopathology practice by focusing on its impact on cytological sample workload. Full article

Since the introduction of the Papanicolaou Society of Cytopathology System for Reporting Pancreaticobiliary Cytology, much experience has been gained and published concerning the utility of the diagnostic categories, malignancy risk of the categories and reproducibility of the system. This new information has resulted in modifications to the system which will become part of the World Health Organization (WHO) System for Reporting Pancreatic Cytology. Herein we report our experience with the system and information from the published literature. Full article

The mismatch repair (MMR) system has a key role in supporting the DNA polymerase proofreading function and in maintaining genome stability. Alterations in the MMR genes are driving events of tumorigenesis, tumor progression, and resistance to therapy. These genetic scars may occur in either hereditary or sporadic settings, with different frequencies across tumor types. Appropriate characterization of the MMR status is a crucial task in oncologic pathology because it allows for both the tailored clinical management of cancer patients and surveillance of individuals at risk. The currently available MMR testing methods have specific strengths and weaknesses, and their application across different tumor types would require a tailored approach. This article highlights the indications and challenges in MMR status assessment for molecular pathologists, focusing on the possible strategies to overcome analytical and pre-analytical issues. Full article

Thyroid nodules are a common finding in the adult population including the fact that more than 50% of individuals, over the age of 60, have thyroid nodules. The majority have been mostly detected with ultrasonography and 10% by palpation. The majority of these nodules are benign, whereas 5–15% of them are malignant. The pre-operative diagnosis of cancer is a critical challenge in order to ensure that each patient can be treated with the best tailored management with a reduction of unnecessary surgery for benign lesions. Fine needle aspiration cytology (FNAC) represents the first and most important diagnostic tool for the evaluation of thyroid lesions. According to the literature, FNAC is able to render a conclusive diagnosis in up to 70–80% of all cases. For the remaining 20–30% of nodules, cytological diagnoses fall into the category of indeterminate lesions mostly due to the lack of specific morphological features. According to the Bethesda system for reporting thyroid cytopathology (TBSRTC), indeterminate lesions can be sub-stratified into three different subcategories including “atypia of undetermined significance/follicular lesion of undetermined significance-AUS/FLUS”; “follicular or Hürthle cell neoplasm/suspicious for follicular or Hürthle cell neoplasm-FN/SFN”; and “suspicious for malignancy-SFM”. Many of these indeterminate lesions undergo repetition or diagnostic lobectomy. Nonetheless, the majority of these cases will have a benign diagnosis due to the fact that the rate of cancer ranges between 6 and 30%. It stands to reason that the application of ancillary technique, mostly molecular testing, emerged as a critical additional tool for those thyroid indeterminate lesions. Since the early 1990s, material collected from cytological samples yields sufficient and adequate cells for the detection of point mutation or gene fusions. Nonetheless, the further availability of new sequencing technologies such as next-generation sequencing (NGS) has led to more comprehensive molecular applications adopted now in clinical use. The current review investigates the multiple advances in the field of molecular testing applied in thyroid cytology. Full article

Serous effusion cytology is widely employed in the initial evaluation of the etiology of effusions with a high diagnostic sensitivity. To standardize practices, The International System for Reporting Serous Fluid Cytology (TIS) was developed following best international practices, the most up-to-date literature, and expert consensus. In the context of this system, ancillary techniques play an important role. Besides defining basic principles in laboratory specimen handling, adequacy criteria, and a standardized reporting terminology with five diagnostic categories, TIS provides an actionable framework for using immunohistochemical and molecular testing in effusion samples, namely, in atypical, suspicious of malignant samples. For diagnostic purposes, these tests may be employed to distinguish between a primary and secondary neoplasm, to confirm a diagnosis of malignant mesothelioma vs. reactive mesothelial hyperplasia, and to correctly classify and determine the primary location of a metastasis. Theranostic molecular tests may also be used for these samples to evaluate potential therapeutic targets. Pathologists play a central role in guiding this process by determining adequacy and selecting appropriate ancillary tests. The activity in this area of research should increase in the near future as new therapeutic targets are discovered and new drugs enter the clinical practice. Full article

In the era of personalized medicine, there is an increasing demand for comprehensive and complex diagnosis using minimally invasive techniques. Nowadays, it is mandatory to integrate biomarkers in the diagnostic process, as well as in the treatment and clinical management of many cancer patients. Patients with non-small cell lung cancer (NSCLC), for instance, are frequently diagnosed in advanced stages, at a point when only cytological material or small biopsies can be obtained. This pathology constitutes an interesting challenge for the testing of biomarkers in cytology. Furthermore, there is a growing development of imaging techniques that guide non-invasive approaches to obtain small biopsies or cytological samples. This has allowed fine needle aspiration cytology and fine needle aspiration biopsy (FNAC, FNAB) to become front-line procedures in the management of patients with NSCLC. It is well known that the list of biomarkers to be tested in these patients continues to increase. Nevertheless, there are several of essential biomarkers that should always be analyzed in all patients with NSCLC, not only in non-squamous but also in some squamous carcinomas (SqCC). Some of them, such as PDL1, are tested by immunocytochemistry (ICC), while others, mainly ALK and ROS1, can be tested by ICC and confirmed using other techniques such a Fluorescence In Situ Hybridization (FISH). Other biomarkers, namely EGFR and BRAF mutations, are currently evaluated by polymerase chain reaction (PCR)-based techniques including Next-Generation Sequencing (NGS). In this review, we will address the particularities and challenges that ICC and FISH pose in different types of cytological samples from an eminently practical point of view. Full article