Background:When tumour tissue is unavailable, cell-free DNA (cfDNA)can serve as a surrogate for genetic analyses. Because mutated alleles in cfDNA are usually below 1%, next-generation sequencing (NGS)must be narrowed to target only clinically relevant genes. In this proof-of-concept study, we developed a panel to use in ultra-deep sequencing to identify such mutations in cfDNA.Methods:Our panel (‘SiRe') covers 568 mutations in six genes (EGFR, KRAS, NRAS, BRAF, cKIT and PDGFRα)involved in non-small-cell lung cancer (NSCLC), gastrointestinal stromal tumour, colorectal carcinoma and melanoma. We evaluated the panel performance in three steps. First, we analysed its analytical sensitivity on cell line DNA and by using an artificial reference standard with multiple mutations in different genes. Second, we analysed cfDNA from cancer patients at presentation (n=42), treatment response (n=12) and tumour progression (n=11); all patients had paired tumour tissue and cfDNA previously genotyped with a Taqman-derived assay (TDA). Third, we tested blood samples prospectively collected from NSCLC patients (n=79) to assess the performance of SiRe in clinical practice.Results:SiRe had a high analytical performance and a 0.01% lower limit of detection. In the retrospective series, SiRe detected 40 EGFR, 11 KRAS, 1 NRAS and 5 BRAF mutations (96.8% concordance with TDA). In the baseline samples, SiRe had 100% specificity and 79% sensitivity relative to tumour tissue. Finally, in the prospective series, SiRe detected 8.7% (4/46) of EGFR mutations at baseline and 42.9% (9/21) of EGFR p.T790M in patients at tumour progression.Conclusions:SiRe is a feasible NGS panel for cfDNA analysis in clinical practice.

EGFR mutations detected on cytology specimens by a centralized laboratory can predict TKI treatment response equally well as mutations identified on histology samples.

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Quantitative molecular reference slides are a useful tool for monitoring the performance of different multigene mutational assays, and this could lead to better standardization of molecular cytopathology procedures. Cancer Cytopathol 2017;125:615-26. © 2017 American Cancer Society.

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.

Cytologic sampling is the mainstay of diagnosing advanced lung cancer. Moreover, to select patients for personalized first-line or second-line treatment, epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) and c-ros oncogene 1 (ROS1) rearrangements are tested on cytologic preparations. Commercially available fluorescence in situ hybridization (FISH) and immunocytochemistry (ICC) assays have primarily been used for the identification of cells harboring ALK or ROS1 gene fusions on histologic rather than cytologic preparations. However, it is now recognized that FISH and ICC also can be applied on cytologic samples provided the cytopathologist is aware that FISH and ICC results are not always concordant and that the performance of ICC largely depends on antibody clones, signal detection systems, and scoring systems. Notably, the routine clinical use of FISH and ICC may be replaced by emerging next-generation sequencing and digital, color-coded barcode technologies, which have the advantage of simultaneously evaluating ALK, ROS1, and EGFR alterations in a single analysis. Although their use in clinical cytologic practice remains to be fully established, it is conceivable that this technology will replace both FISH and ICC analyses in future diagnostic algorithms. Here, the authors review studies devoted to testing ALK and ROS1 on cytology specimens in an attempt to provide an update for the cytopathologist regarding current and evolving practice. Cancer Cytopathol 2017;125:817-30. © 2017 American Cancer Society.

AimsIn 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.MethodsThe 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.ResultsThe 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).ConclusionIn the times of coronavirus, laboratory fully automated platforms simplify predictive molecular testing. Laboratory staff may be more safely and cost-effectively managed.

IntroductionLung cancer is the most frequent cause of cancer death, worldwide (1). In the last years, several clinical trials have defined the pivotal role of the molecular assessment of different biomarkers, such as epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), ROS proto-oncogene 1 receptor tyrosine kinase (ROS1), v-Raf murine sarcoma viral oncogene homolog B (BRAF), and programmed death-ligand 1 (PD-L1), in order to administrate either tyrosine kinase inhibitors (TKIs) or immune-checkpoint inhibitors to improve survival and quality of life of advanced stage non-small cell lung cancer (NSCLC) patients (2-14).