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Aims: In the time of COVID-19, predictive molecular pathology laboratories must still timely select oncological patients for targeted treatments. However, the need to respect social distancing measures may delay results generated by laboratory-developed tests based on sequential steps a long hands-on time. Laboratory workflows should now be simplified., Methods: The organisation of the University of Naples Federico II predictive pathology laboratory was assessed before (March-April 2019) and during (March-April 2020) the Italian lockdown., Results: The number of patients undergoing single or multiple biomarker testing was similar in 2019 (n=43) and in 2020 (n=45). Considering adequate samples for molecular testing, before the outbreak, next-generation sequencing was mostly used (35/42, 83.3%). Testing six genes had a reagent cost of €98/patient. Conversely, in 2020, almost all cases (38/41, 92.7%) were analysed by automated testing. This latter had for any single assay/gene a significant reagent cost (€95-€136) and a faster mean turnaround time (5.3 vs 7.9 working days)., Conclusion: In the times of coronavirus, laboratory fully automated platforms simplify predictive molecular testing. Laboratory staff may be more safely and cost-effectively managed., Competing Interests: Competing interests: UM reports personal fees (as speaker bureau or advisor) from Boehringer Ingelheim, AstraZeneca, Roche, MSD, Amgen, Merck and BMS. GT reports personal fees (as speaker bureau or advisor) from Roche, MSD and Pfizer., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Over the past decade, molecular cytopathology has emerged as a relevant area of modern pathology. Notably, in patients with advanced-stage cancer, cytological samples could be the only material available for diagnosis and molecular biomarker testing to identify patients suitable for targeted therapies. As a result, the contemporary cytopathologist's role extends beyond morphological assessments to include critical skills such as evaluating the adequacy of the cytological samples and managing these specimens for molecular testing. This case collection can be a valuable source of insight, especially for young pathologists, who should learn to combine the opportunities offered by molecular biology with the basis of morphological evaluation. [ABSTRACT FROM AUTHOR]

Aims: To date, precision medicine has played a pivotal role in the clinical administration of solid-tumor patients. In this scenario, a rapidly increasing number of predictive biomarkers have been approved in diagnostic practice or are currently being investigated in clinical trials. A pitfall in molecular testing is the diagnostic routine sample available to analyze predictive biomarkers; a scant tissue sample often represents the only diagnostical source of nucleic acids with which to conduct molecular analysis. At the sight of these critical issues, next-generation sequencing (NGS) platforms emerged as referral testing strategies for the molecular analysis of predictive biomarkers in routine practice, but the need for highly skilled personnel and extensive working time drastically impacts the widespread diffusion of this technology in diagnostic settings. Here, we technically validate a fully integrated NGS platform on diagnostic routine tissue samples previously tested with an NGS-based diagnostic workflow by a referral institution. Methods: A retrospective series of n = 64 samples (n = 32 DNA, n = 32 RNA samples), previously tested using a customized NGS assay (SiRe™ and SiRe fusion), was retrieved from the internal archive of the University of Naples Federico II. Each sample was tested by adopting an Oncomine Precision Assay (OPA), which is able to detect 2769 molecular actionable alterations [hotspot mutations, copy number variations (CNV) and gene fusions] on fully integrated NGS platforms (Genexus, Thermo Fisher Scientific (Waltham, MA, USA). The concordance rate between these technical approaches was determined. Results: The Genexus system successfully carried out molecular analysis in all instances. A concordance rate of 96.9% (31 out of 32) was observed between the OPA and SiRe™ panels both for DNA- and RNA-based analysis. A negative predictive value of 100% and a positive predictive value of 96.9% (62 out of 64) were assessed. Conclusions: A fully automatized Genexus system combined with OPA (Thermo Fisher Scientific) may be considered a technically valuable, time-saving sequencing platform to test predictive biomarkers in diagnostic routine practice. [ABSTRACT FROM AUTHOR]

To date, several emerging biomarkers have gained considerable interest in the field of predictive molecular oncology. The advent of precision medicine has led to the development of innovative drugs targeting rare molecular pathways independently from histology, defined as tissue-agnostic drugs. Although there is a lot of promise for this new tissue-agnostic model in the oncological scenario, crucial issues from both the diagnostic and therapeutic standpoint are emerging. This review aims to critically examine the role of tissue-agnostic biomarkers in different solid tumors, focusing on the prevalence and methods of detection of agnostic biomarkers together with drug approvals to guide clinicians in this evolving landscape. To strengthen the framework for tissue-agnostic approvals, the dialogue between regulatory, industrial, and academic parties should be intensified. Critical questions include the development of an efficient network system that can overcome the heterogeneity of patients' inclusion criteria along with the increasingly difficult interpretation of next-generation sequencing (NGS) profiling technologies. Cost-effectiveness and risk–benefit studies are needed in the national context considering the modalities of access to diagnostic tests and reimbursement of treatments. [ABSTRACT FROM AUTHOR]

Somatic mutations in PIK3CA are present in ~40% breast cancers (BC); their detection in hormone receptor (HR)+/HER2− tumors allows for selecting patients with advanced disease eligible for PIK3CA targeting with alpelisib. The choice of what type of PIK3CA testing approach to adopt and which tissue sample to analyze is a new task in breast pathology. In this methodological study, we sought to assess the performance of next-generation sequencing (NGS) and RT-PCR for PIK3CA testing on archival formalin-fixed paraffin-embedded (FFPE) primary tumors and corresponding metastases. Sixteen HR+/HER2− BC with known PIK3CA-mutated status (ex. 7, 9, and 20) on metastatic samples by means of amplicon-based targeted NGS were selected, and n = 13 of these samples were re-tested with a commercially available CE-IVD RT-PCR assay. All available primary tumors (n = 8) were tested with both methods. NGS detected mutations in all samples, while RT-PCR in n = 2 sample-pairs and overall, in n = 5/8 (62.5%) primary tumors and 7/13 (53.8%) metastases (κ = 0.09; 95% CI, −0.69–0.87). Slight agreement (κ = 0; 95% CI, −0.59–0.59) was observed between NGS and RT-PCR, with the former being generally more sensitive in cases with low DNA quality and quantity. Post hoc visual inspection of the RT-PCR data increased the concordance to 76.9%. Targeted NGS offers reliable and robust PIK3CA testing on both tumor and metastasis FFPE samples; the accuracy of RT-PCR depends on the DNA quantity and quality. In HR+/HER2− BC, both the selection of the PIK3CA testing strategy of FFPE tissues and which sample to analyze should consider several technical parameters and should be tailored for each case. [ABSTRACT FROM AUTHOR]

BACKGROUND: The molecular characterization of thyroid nodules in cytological samples has so far been focused on discriminating between benign and malignant forms in a purely diagnostic setting. The evidence on the impact of molecular biomarkers to determine the risk of aggressiveness in cytologically "neoplastic" lesions is limited to genomic alterations (such as BRAF and TERT mutations). The aim of our study was to assess the preoperative role of microRNAs (miRNAs) in predicting the nodal status of patients with papillary thyroid cancer. METHODS: A pilot series of histological samples of papillary thyroid carcinoma with (6 cases) or without (6 cases) lymph node metastases, matched for other major clinical and pathological features, was analyzed for global miRNA expression in a screening phase. A set of miRNAs was then validated in a series of 63 consecutive cytological samples of papillary carcinomas: 48 pN‐negative and 15 pN‐positive at histology. RESULTS: Unsupervised cluster analysis segregated surgical pN‐negative and pN‐positive samples, except for 1 case. The 45 differentially expressed miRNAs in pN‐positive versus pN‐negative cases were predicted to regulate a wide range of cellular pathways, enriched for Wnt, gonadotropin‐releasing hormone receptor, and cerulein/cholecystokinin receptor signaling. In agreement with their profiles in surgical samples, 4 miRNAs of the 10 selected for validation (miR‐154‐3p, miR‐299‐5p, miR‐376a‐3p, and miR‐302E) had a significant differential expression in cytological samples of papillary carcinoma with lymph node metastases and predicted the positive nodal status with a relatively good performance. CONCLUSIONS: MiRNA profiling is a potential promising strategy to define papillary carcinoma aggressiveness in the preoperative setting. ; Preoperative biomarkers predictive of aggressive behavior in thyroid cancer are needed. Our study indicate that a microRNA‐based approach is a feasible, methodologically robust, and accurate strategy to identify biomarkers to predict the presence of lymph node metastases in papillary thyroid carcinoma in the preoperative setting. [ABSTRACT FROM AUTHOR]

In the current era of personalized medicine, liquid biopsy has acquired a relevant importance in patient management of advanced stage non-small cell lung cancer (NSCLC). As a matter of fact, liquid biopsy may supplant the problem of inadequate tissue for molecular testing. The term 'liquid biopsy' refers to a number of different biological fluids, but is most clearly associated with plasma-related platforms. It must be taken into account that pre-analytical processing and the selection of the appropriate technology according to the clinical context may condition the results obtained. In addition, novel clinical applications beyond the evaluation of the molecular status of predictive biomarkers are currently under investigation. This review summarizes the available evidence on pre-analytical issues and different clinical applications of liquid biopsies in NSCLC patients. Liquid biopsy should be considered not only as a valid alternative but as complementary to tissue-based molecular approaches. Careful attention should be paid to the optimization and standardization of all phases of liquid biopsy samples management in order to determine a significant improvement in either sensitivity or specificity, while significant reducing the number of 'false negative' or 'false positive' molecular results. [ABSTRACT FROM AUTHOR]

The application of next generation sequencing (NGS) technology to cytological samples has significantly modified molecular cytopathology practice. Cytological samples represent a valid source of high‐quality DNA for NGS analysis, especially for predicting patients' response to targeted treatments and for refining the risk of malignancy in indeterminate cytological diagnoses. However, several pre‐analytical factors may influence the reliability of NGS clinical analysis. Here, we briefly review the challenges of NGS in cytology practice, focusing on those pre‐analytical factors that may negatively affect NGS success rates and routine diagnostic applications. Finally, we address the future directions of the field. Molecular cytopathology is a rapidly evolving field. Modern molecular cytopathologists play a key role in bridging the gap between conventional microscopy and novel molecular technologies. Cytological samples represent a valid source of high‐quality DNA for next generation sequencing (NGS) analysis, especially for predicting patients' response to targeted treatments and for refining the risk of malignancy in indeterminate cytological diagnosis. [ABSTRACT FROM AUTHOR]

Background: Next-generation sequencing (NGS) needs to be validated and standardized to ensure that cancer patients are reliably selected for target treatments. In Italy, NGS is performed in several institutions and harmonization of wet and dry procedures is needed. To this end, a consortium of five different laboratories, covering the most part of the Italian peninsula, was constituted. A narrow gene panel (SiRe®) covering 568 clinically relevant mutations in six different genes (EGFR, KRAS, NRAS, BRAF, cKIT , and PDGFR α) with a predictive role for therapy selection in non-small cell lung cancer (NSCLC), gastrointestinal stromal tumor, colorectal carcinoma (CRC), and melanoma was evaluated in each participating laboratory. Methods: To assess the NGS inter-laboratory concordance, the SiRe® panel, with a related kit and protocol for library preparation, was used in each center to analyze a common set of 20 NSCLC and CRC routine samples. Concordance rate, in terms of mutation detected and relative allelic frequencies, was assessed. Then, each institution prospectively analyzed an additional set of 40 routine samples (for a total of 160 specimens) to assess the reproducibility of the NGS run parameters in each institution. Results: An inter-laboratory agreement of 100% was reached in analyzing the data obtained from the 20 common sample sets; the concordance rate of allelic frequencies distribution was 0.989. The prospective analysis of the run metric parameters obtained by each center locally showed that the analytical performance of the SiRe® panel in the different institutions was highly reproducible. Conclusions: The SiRe® panel represents a robust diagnostic tool to harmonize the NGS procedure in different Italian laboratories. [ABSTRACT FROM AUTHOR]

Introduction: Collection of tumor samples is not always feasible in non-small cell lung cancer (NSCLC) patients, and circulating free DNA (cfDNA) extracted from blood represents a viable alternative. Different sensitive platforms have been developed for genetic cfDNA testing, some of which are already in clinical use. However, several difficulties remain, particularly the lack of standardization of these methodologies. Areas covered: Here, the authors present a review of the literature to update the applicability of cfDNA for diagnosis and monitoring of NSCLC patients. Expert commentary: Detection of somatic alterations in cfDNA is already in use in clinical practice and provides valuable information for patient management. Monitoring baseline alterations and emergence of resistance mutations is one of the most important clinical applications and can be used to non-invasively track disease evolution. Today, different technologies are available for cfDNA analysis, including whole-genome or exome sequencing and targeted methods that focus on a selection of genes of interest in a specific disease. In the case of Next Generation Sequencing (NGS) approaches, in depth coverage of candidate mutation loci can be achieved by selecting a limited number of targeted genes. [ABSTRACT FROM PUBLISHER]

Simple Summary: The role of serum Cyfra 21.1 as a biomarker in thyroid cancer has yet to be validated. This study investigated the diagnostic or prognostic role of serum Cyfra 21.1 in thyroid cancer. In the present analysis, we found that serum Cyfra 21.1 was increased in thyroid cancer with distant metastasis compared with thyroid cancer without metastasis. Furthermore, in progressive disease when thyroglobulin was undetectable or thyroglobulin monitoring was useless because of thyroglobulin antibody, serial follow-up based on serum Cyfra 21.1 levels might be used as an alternative biomarker for disease monitoring. Background: Serum Cyfra 21.1, the soluble fragment of CK19, has been used as a prognostic tumor marker in various cancers, indicating poor tumor differentiation and increased metastasis. Methods: We analyzed the serum Cyfra 21.1 level in 51 consecutive patients with thyroid cancer manifesting distant metastasis treated with prior total thyroidectomy. Serum Cyfra 21.1 levels of 26 thyroid cancer patients without metastasis and 50 healthy individuals were used for comparison. Results: Higher serum Cyfra 21.1 levels were detected in thyroid cancer patients with distant metastasis compared with healthy subjects and thyroid cancer patients without metastasis (p = 0.012). Serum Cyfra 21.1 levels were significantly increased in patients with positive BRAF V600E mutation (p = 0.019), undergoing Tyrosine Kinase Inhibitor (TKI) therapy (p = 0.008), with radioiodine-refractory status (p = 0.047), and in disease progression compared with those manifesting stable disease (p = 0.007). In progressive disease with undetectable or unmonitored thyroglobulin because of thyroglobulin antibody, serum Cyfra 21.1 was useful as a biomarker for follow-up of disease course. Conclusion: Serum Cyfra 21.1 in thyroid cancer patients might represent an alternative biomarker predicting tumor progression, especially in cases not associated with serum Tg levels. [ABSTRACT FROM AUTHOR]

• Liquid biopsy has revolutionized the management of cancer patients. • In particular, liquid biopsy-based testing has proven to be highly beneficial for identifying actionable cancer markers, especially when solid tissue biopsies are insufficient or unattainable. • Beyond the predictive role, liquid biopsy may be a useful tool for comprehensive tumor genotyping, identification of emergent resistance mechanisms, monitoring of minimal residual disease, early detection, and cancer interception. • The application of next generation sequencing to liquid biopsy has led to the "quantum leap" of predictive molecular pathology. Liquid biopsy has revolutionized the management of cancer patients. In particular, liquid biopsy-based testing has proven to be highly beneficial for identifying actionable cancer markers, especially when solid tissue biopsies are insufficient or unattainable. Beyond the predictive role, liquid biopsy may be a useful tool for comprehensive tumor genotyping, identification of emergent resistance mechanisms, monitoring of minimal residual disease, early detection, and cancer interception. The application of next generation sequencing to liquid biopsy has led to the "quantum leap" of predictive molecular pathology. Here, we review the evolving role of liquid biopsy in lung cancer. [ABSTRACT FROM AUTHOR]

• A plethora of predictive biomarkers have been approved by international societies for the clinical management of advanced NSCLC patients. • Scant diagnostic material and testing strategies (RT-PCR, NGS) drastically impacts on the molecular profiling of NSCLC patients. • Harmonized procedures are crucial to optimize pre-analytical, analytical and data reporting. • Biomarkers ATLAS (https://biomarkersatlas.com/) knowledge-based database is a diagnostic tool supporting healthcare personnel integrating updated clinical and molecular records from patients collected in 80 referral Italian institution. To date, precision medicine has revolutionized the clinical management of Non-Small Cell Lung Cancer (NSCLC). International societies approved a rapidly improved mandatory testing biomarkers panel for the clinical stratification of NSCLC patients, but harmonized procedures are required to optimize the diagnostic workflow. In this context a knowledge-based database (Biomarkers ATLAS, https://biomarkersatlas.com/) was developed by a supervising group of expert pathologists and thoracic oncologists collecting updated clinical and molecular records from about 80 referral Italian institutions. Here, we audit molecular and clinical data from n = 1100 NSCLC patients collected from January 2019 to December 2020. Clinical and molecular records from NSCLC patients were retrospectively collected from the two coordinating institutions (University of Turin and University of Naples). Molecular biomarkers (KRAS, EGFR, BRAF, ROS1, ALK, RET, NTRK, MET) and clinical data (sex, age, histological type, smoker status, PD-L1 expression, therapy) were collected and harmonized. Clinical and molecular data from 1100 (n = 552 mutated and n = 548 wild-type) NSCLC patients were systematized and annotated in the ATLAS knowledge-database. Molecular records from biomarkers testing were matched with main patients' clinical variables. Biomarkers ATLAS (https://biomarkersatlas.com/) represents a unique, easily managing, and reliable diagnostic tool aiming to integrate clinical records with molecular alterations of NSCLC patients in the real-word Italian scenario. [ABSTRACT FROM AUTHOR]

We have previously shown that CBX7 expression is associated with a more malignant phenotype in thyroid cancer. On this basis, we decided to investigate its possible prognostic value in colorectal cancer (CRC). CBX7 expression has been analysed by immunohistochemistry in tissue microarray (TMA) specimens obtained from a large series of sporadic CRC resections (n =1420). The CBX7 expression data have been correlated with several clinico-pathological parameters. CBX7 expression is reduced or absent in a significant number of CRC samples in comparison to the normal colonic mucosa and the loss of CBX7 expression correlates with a poor outcome of CRC (p <0.001). The block of CBX7 expression seems to occur at a transcriptional level since quantitative RT-PCR analysis showed a reduced CBX7-specific mRNA levels in CRC samples versus normal counterpart tissue (up to more than 50-fold). Finally, the restoration of CBX7 expression in two CRC cell lines reduces their proliferation rate suggesting a role of the loss of CBX7 expression in the progression step of colon carcinogenesis. Therefore, the data reported here indicate that the evaluation of CBX7 expression may represent a valid tool in the prognosis of colon cancer since a reduced survival of CRC patients is associated with the loss of CBX7 expression. [Copyright &y& Elsevier]