Tissue Microarray from Cell Block Material (cbTMA)—An Additional Shot for Cytology in the Predictive Pathology Era: The PD-L1 Experience

Generally, predictive biomarker tests are clinically validated on histological formalin-fixed, paraffin-embedded (FFPE) samples. In addition to FFPE samples, cytological samples have also emerged as a useful approach to detect predictive biomarkers. However, as of today, despite the promising results reported in the recent literature, their full implementation in routine clinical practice is still lagging owing to a lack of standardized preparatory protocols, challenging assessments of cyto-histological correlation, and variable inter-observer agreement. The aim of this report was to explore the possibility of implementing a large-scale validation of predictive biomarker testing on cytological material. To this aim, we evaluated the technical feasibility of PD-L1 assessment on a cell block (CB)-derived tissue microarray (cbTMA). Consecutive and unselected CBs prepared from metastatic lymph node fine-needle cytology (FNC) samples were retrospectively collected and used for TMA construction. PD-L1 immunohistochemistry (IHC) was carried out on cbTMA sections with the companion diagnostic kit SP263 assay. TMA contained 33 CB-derived cores. A total of 20 sections were hematoxylin and eosin (H&E) stained. Overall, 29 (88%) samples were visible at least in one H&E-stained slide. Four cases out of five sections stained with the SP263 assay (4/29, 13.8%) showed PD-L1 positivity in neoplastic and/or immune cells; remarkably, no unspecific background was observed. Although our study was based on a limited and non-selected series, our findings do provide proof of concept for the use of cbTMA in predictive biomarker testing on cytological material in large-scale post-clinical trial validation studies, multicenter studies, and quality control programs.

The Multifaceted Profile of Thyroid Disease in the Background of DICER1 Germline and Somatic Mutations: Then, Now and Future Perspectives

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

Fine-Needle Aspiration Is Suitable for Breast Cancer BRCA Molecular Assessment: A Case Report

Breast cancer is the most common cause of cancer-related deaths in the female population worldwide. To the best of our knowledge, breast cancer (BRCA)1/2 gene mutations have not been described yet on breast cancer cytological specimens. Here we describe the case of a 38-year old woman with a family and personal history for breast cancer, who underwent a fine needle aspiration (FNA) procedure for a novel 30 mm lesion located in the external quadrants of the contralateral (left) breast. Cytological findings and ancillary immunostaining confirmed the diagnosis of a triple negative NST carcinoma. BRCA1/2 molecular assessment was carried out on DNA extracted from cytological (November 2020), biopsy (December 2014) and surgical resection (July 2015) specimens, as well as on the resection of a benign fibroadenoma, by using a next generation sequencing approach. Molecular analysis showed a pathogenic BRCA1 insertion (c.5266dupC; p.Q1756PfsTer74) in the cytological specimen (allelic fraction 92.0%), biopsy (allelic fraction 84.2%), surgical resection (allelic fraction 87.8%) and fibroadenoma (58.9%), demonstrating a germinal BRCA mutated status.

From Information Overload to Actionable Insights: Digital Solutions for Interpreting Cancer Variants from Genomic Testing

Given the increase in genomic testing in routine clinical use, there is a growing need for digital technology solutions to assist pathologists, oncologists, and researchers in translating variant calls into actionable knowledge to personalize patient management plans. In this article, we discuss the challenges facing molecular geneticists and medical oncologists in working with test results from next-generation sequencing for somatic oncology, and propose key considerations for implementing a decision support software to aid the interpretation of clinically important variants. In addition, we review results from an example decision support software, NAVIFY Mutation Profiler. NAVIFY Mutation Profiler is a cloud-based software that provides curation, annotation, interpretation, and reporting of somatic variants identified by next-generation sequencing. The software reports a tiered classification based on consensus recommendations from AMP, ASCO, CAP, and ACMG. Studies with NAVIFY Mutation Profiler demonstrated that the software provided timely updates and accurate curation, as well as interpretation of variant combinations, demonstrating that decision support tools can help advance implementation of precision oncology.

Conventional Transbronchial Needle Aspiration (cTBNA) and EBUS-Guided Transbronchial Needle Aspiration (EBUS-TBNA): A Retrospective Study on the Comparison of the Two Methods for Diagnostic Adequacy in Molecular Analysis

Introduction: In recent years, there has been a growing development of molecularly targeted therapies for various types of solid tumors—in particular, in non-small-cell lung cancer (NSCLC). This has required the need for greater quantities of tissue that is able to support ancillary studies, alongside cyto-histological diagnoses for the assessment of molecular targets. Conventional TBNA (cTBNA) and EBUS-guided TBNA (EBUS-TBNA) have shown a high diagnostic yield for malignant mediastinal and/or hilar lymph node enlargement and peribronchial masses; however, few studies have compared these two procedures. We retrospectively compared TBNA patients (EBUS-TBNA and cTBNA) in order to determine the diagnostic yield and material adequacy for subsequent ancillary analyses. Materials and Methods: We retrospectively evaluated 318 patients with clinical suspicion of lung cancer or with disease recurrence. All of the patients underwent TBNA (either EBUS-TBNA or cTBNA) on enlarged mediastinal and/or hilar lymph nodes and peribronchial masses between January 2017 and June 2021 at the University Hospital of Pisa, Italy. After a definitive diagnosis, molecular analyses and an evaluation of PD-L1 expression were performed in the cases of adenocarcinoma, squamous cell carcinoma, and NSCLC, not otherwise specified (NOS). Results: EBUS-TBNA was performed in 199 patients and cTBNA was performed in 119 patients with 374 and 142 lymph nodes, respectively. The overall diagnostic yield for positive diagnoses was 59% (diagnostic rate of 61% in EBUS-TBNA, and 55% in cTBNA). Adenocarcinoma (ADC) was the most frequent diagnosis in both methods. EBUS-TBNA diagnostic adequacy was 72% for molecular analysis, while it was 55.5% for cTBNA, showing a statistical trend (p = 0.08) towards the significance of EBUS. The average percentage of neoplastic cells was also statistically different between the two methods (p = 0.05), reaching 51.19 ± 22.14 in EBUS-TBNA and 45.25 ± 22.84 in cTBNA. With regard to the PD-L1 protein expression, the percentage of positivity was similar in both procedures (86% in EBUS-TBNA, 85% in cTBNA). Conclusions: Conventional TBNA (cTBNA) and EBUS-guided TBNA (EBUS-TBNA) are minimally invasive diagnostic methods that are associated with a high diagnostic yield. However, EBUS-TBNA has an improved diagnostic adequacy for molecular analysis compared to cTBNA, and is associated with a higher average percentage of neoplastic cells.

Clinical and Molecular Features of Anti-CENP-B Autoantibodies

Centromeric proteins are the foundation for assembling the kinetochore, a macromolecular complex that is essential for accurate chromosome segregation during mitosis. Anti-centromere antibodies (ACAs) are polyclonal autoantibodies targeting centromeric proteins (CENP-A, CENP-B, CENP-C), predominantly CENP-B, and are highly associated with rheumatologic disease (lcSSc/CREST syndrome). CENP-B autoantibodies have also been reported in cancer patients without symptoms of rheumatologic disease. The rise of oncoimmunotherapy stimulates inquiry into how and why anti-CENP-B autoantibodies are formed. In this review, we describe the clinical correlations between anti-CENP-B autoantibodies, rheumatologic disease, and cancer; the molecular features of CENP-B; possible explanations for autoantigenicity; and, finally, a possible mechanism for induction of autoantibody formation.

Precision Prevention: The 2019 ASCCP Risk-Based Management Consensus Guidelines for Abnormal Cervical Cancer Screening Tests and Cancer Precursors

The approach to cervical cancer prevention has evolved significantly over the past two decades. HPV immunization has decreased the specificity of screening modalities and HPV-based testing has been replacing our previously successful morphology-only approach. Additionally, there is much more emphasis on providing precision prevention, rather than the previously used “one-fits-all” management strategies. A number of new biomarkers are entering clinical practice and being integrated into cervical cancer screening and management in order to enable a more personalized assessment of the risk for precancer/cancer for an individual patient. The 2019 ASCCP Risk-Based Management Consensus Guidelines expand on the concept of “equal management for equal risk”. They consider a patient’s history in addition to current test results to provide recommendations for increased surveillance/treatment in patients at higher risk for CIN3+ while minimizing interventions for lower-risk patients who have new versus persistent HPV infection. Clinical management decisions are based on immediate risk and 5-year risk estimates for CIN3+, which are determined by referencing an extensive risk table compiled by the National Cancer Institute (NCI). The course of action for a given patient is recommended by comparison of the risk in the risk database, to the predetermined clinical action thresholds. These guidelines address the need for simplification and offer some stability for the provider while being conducive to the incorporation of anticipated continued technologic advances in methods for cervical cancer prevention. Their enduring nature will allow for changes needed based on risk reduction as HPV vaccination uptake increases and vaccinated women reach screening age. Similarly, the design allows for the addition of new tests into the risk assessment calculations after their approval by applicable regulatory agencies and review/consensus approval by the ASCCP new technology and enduring guidelines workgroups. As cytopathologists, we must be familiar with the scientific advancements in primary and secondary prevention, evolving screening and management guidelines, and participate actively in the multidisciplinary approach for the prevention of cervical cancer.

Liquid Biopsy for Biomarker Testing in Non-Small Cell Lung Cancer: A European Perspective

The development of targeted therapies has improved survival rates for patients with advanced non-small cell lung cancer (NSCLC). However, tissue biopsy is unfeasible or inadequate in many patients, limiting biomarker testing and access to targeted therapies. The increasing numbers of established and emerging biomarkers with available targeted treatments highlights the challenges associated with sequential single-gene testing and limited tissue availability. Multiplex next-generation sequencing (NGS) offers an attractive alternative and represents a logical next step, and in cases where the tumour is inaccessible, tissue biopsy yields insufficient tumour content, or when the patient’s performance status does not allow a tissue biopsy, liquid biopsy can provide valuable material for molecular diagnosis. Here, we explore the role of liquid biopsy (i.e., circulating cell-free DNA analysis) in Europe. Liquid biopsies could be used as a complementary approach to increase rates of molecular diagnosis, with the ultimate aim of improving patient access to appropriate targeted therapies. Expert opinion is also provided on potential future applications of liquid biopsy in NSCLC, including for cancer prevention, detection of early stage and minimum residual disease, monitoring of response to therapy, selection of patients for immunotherapy, and monitoring of tumour evolution to enable optimal adaptation/combination of drug therapies.

Liquid Biopsy Analysis in Clinical Practice: Focus on Lung Cancer

Lung cancer is the leading cause of cancer death worldwide. Despite the emergence of highly effective targeted therapies, up to 30% of advanced stage non-small cell lung cancer (NSCLC) patients do not undergo tissue molecular testing because of scarce tissue availability. Liquid biopsy, on the other hand, offers these patients a valuable opportunity to receive the best treatment options in a timely manner. Indeed, besides being much faster and less invasive than conventional tissue-based analysis, it can also yield specific information about the genetic make-up and evolution of patients’ tumors. However, several issues, including lack of standardized protocols for sample collection, processing, and interpretation, still need to be addressed before liquid biopsy can be fully incorporated into routine oncology practice. Here, we reviewed the most important challenges hindering the implementation of liquid biopsy in oncology practice, as well as the great advantages of this approach for the treatment of NSCLC patients.

Molecular Testing of Thyroid Fine-Needle Aspiration: Local Issues and Solutions. An Interventional Cytopathologist Perspective

Molecular testing has acquired a relevant role for diagnostic and prognostic stratification of indeterminate thyroid nodules. Besides the available commercial solutions marketed in the United States, various local testing strategies have been developed in the last decade. In this setting, the modern interventional cytopathologist, the physician who performs the both aspirate and the morphologic interpretation plays a key role in the correct handling of fine-needle aspiration (FNA) samples not only for microscopy but also for molecular techniques. This review summarizes experiences with local approaches to the molecular testing of thyroid FNA, highlighting the role of the modern interventional cytopathologist.