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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., (© 2022 American Cancer Society.)

Aim: ALK , ROS1 , NTRK  and RET gene fusions and MET exon 14 skipping alterations represent novel predictive biomarkers for advanced non-small-cell lung cancer (NSCLC). Therefore, testing patients for these genetic variants is crucial for choosing the best selective treatment. Over the last couple of decades, next-generation sequencing (NGS) platforms have emerged as an extremely useful tool for detecting these variants. Materials & methods: In the present study, we report our NGS molecular records produced during a year of diagnostic activity. Results: Overall, our in-house developed NGS workflow successfully analyzed n = 116/131 (88.5%) NSCLC samples. Of these, eight (6.8%) and five (4.3%) out of 116 patients harbored ALK and RET gene rearrangements, respectively: one case harbored ROS1 gene fusion (0.7%). Conclusion: Our results highlight that an RNA-based NGS analysis can reliably detect gene fusion alterations, thereby playing a pivotal role in the management of NSCLC patients.

Thyroid cancer is the most common type of endocrine cancer, and its prevalence continue to rise. Non-metastatic thyroid cancer patients are successfully treated. However, looking for new therapeutic strategies is of great importance for metastatic thyroid cancers that still lead to death. With respect to this, the tumor microenvironment (TME), which plays a key role in tumor progression, should be considered as a new promising therapeutic target to hamper thyroid cancer progression. Indeed, thyroid tumors consist of cancer cells and a heterogeneous and ever-changing niche, represented by the TME, which contributes to establishing most of the features of cancer cells. The TME consists of extracellular matrix (ECM) molecules, soluble factors, metabolites, blood and lymphatic tumor vessels and several stromal cell types that, by interacting with each other and with tumor cells, affect TME remodeling, cancer growth and progression. Among the thyroid TME components, cancer-associated fibroblasts (CAFs) have gained more attention in the last years. Indeed, recent important evidence showed that thyroid CAFs strongly sustain thyroid cancer growth and progression by producing soluble factors and ECM proteins, which, in turn, deeply affect thyroid cancer cell behavior and aggressiveness. Hence, in this article, we describe the thyroid TME, focusing on the desmoplastic stromal reaction, which is a powerful indicator of thyroid cancer progression and an invasive growth pattern. In addition, we discuss the origins and features of the thyroid CAFs, their influence on thyroid cancer growth and progression, their role in remodeling the ECM and their immune-modulating functions. We finally debate therapeutic perspectives targeting CAFs.

Aims: In thyroid cytopathology, the undetermined diagnostic categories still pose diagnostic challenges. Although next-generation sequencing (NGS) is a promising technique for the molecular testing of thyroid fine-needle aspiration (FNA) specimens, access to such technology can be difficult because of its prohibitive cost and lack of reimbursement in countries with universal health coverage. To overcome these issues, we developed and validated a novel custom NGS panel, Nexthyro, specifically designed to target 264 clinically relevant mutations involved in thyroid tumourigenesis. Moreover, in this study, we compared its analytical performance with that of our previous molecular testing strategy., Methods: The panel, which includes 15 genes ( BRAF, EIF1AX, GNAS, HRAS, IDH1, KRAS, NF2, NRAS, PIK3CA, PPM1D, PTEN, RET, DICER1, CHEK2, TERT promoter), was validated with a cell-line derived reference standard and 72 FNA archival samples previously tested with the 7-gene test., Results: Nexthyro yielded 100% specificity and detected mutant alleles at levels as low as 2%. Moreover, in 5/72 (7%) FNAs, it detected more clinically relevant mutations in BRAF and RAS genes compared with the 7-gene test. Nexthyro also revealed better postsequencing metrics than the previously adopted commercial 'generic' NGS panel., Conclusion: Our comparative analysis indicates that Nexthyro is a reliable NGS panel. The study also implies that a custom-based solution for routine thyroid FNA is sustainable at the local level, allowing patients with undetermined thyroid nodules affordable access to NGS., Competing Interests: Competing interests: EV has received personal fees (as consultant and/or speaker bureau) from Diaceutics, unrelated to the current work. UM has received personal fees (as consultant and/or speaker bureau) from Boehringer Ingelheim, Roche, MSD, Amgen, Thermo Fisher Scientifics, Diaceutics, GSK, Merck and AstraZeneca, unrelated to the current work. GT reports personal fees (as speaker bureau or advisor) from Roche, MSD, Pfizer and Bayer, unrelated to the current work., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: In a previous worldwide survey, the authors showed a drastic reduction in the number of cytological specimens processed during the coronavirus disease 2019 "lockdown" period along with an increase in malignancy rates. To assess the continued impact of the pandemic on cytological practices around the world, they undertook a second follow-up worldwide survey collecting data from the post-lockdown period (2020)., Methods: Participants were asked to provide data regarding their cytopathology activity during the first 12 weeks of their respective national post-lockdown period (2020), which ranged from April 4 to October 31. Differences between the post-lockdown period and the corresponding 2019 period were evaluated, and the authors specifically focused on rates of malignant diagnoses., Results: A total of 29 respondents from 17 countries worldwide joined the survey. Overall, a lower number of cytological specimens (n = 236,352) were processed in comparison with the same period in 2019 (n = 321,466) for a relative reduction of 26.5%. The overall malignancy rate showed a statistically significant increase (12,442 [5.26%] vs 12,882 [4.01%]; P < .001) during the same time period. Similar results were obtained if both malignancy and suspicious for malignancy rates were considered together (15,759 [6.58%] vs 16,011 [4.98%]; P < .001)., Conclusions: The data showed a persistent reduction in the cytological specimen volume during the post-lockdown period (2020). However, the relative increase in the cytological workload in the late part of the post-lockdown is a promising finding of a slow return to normality., (© 2022 American Cancer Society.)

Background: Pediatric salivary gland fine-needle aspiration (FNA) is uncommon with a higher frequency of inflammatory lesions and a small proportion of malignancies. This international, multi-institutional cohort evaluated the application of the Milan System for Reporting Salivary Gland Cytopathology (MSRSGC) and the risk of malignancy (ROM) for each diagnostic category., Methods: Pediatric (0- to 21-year-old) salivary gland FNA specimens from 22 international institutions of 7 countries, including the United States, England, Italy, Greece, Finland, Brazil, and France, were retrospectively assigned to an MSRSGC diagnostic category as follows: nondiagnostic, nonneoplastic, atypia of undetermined significance (AUS), benign neoplasm, salivary gland neoplasm of uncertain malignant potential (SUMP), suspicious for malignancy (SM), or malignant. Cytology-histology correlation was performed where available, and the ROM was calculated for each MSRSGC diagnostic category., Results: The cohort of 477 aspirates was reclassified according to the MSRSGC as follows: nondiagnostic, 10.3%; nonneoplastic, 34.6%; AUS, 5.2%; benign neoplasm, 27.5%; SUMP, 7.5%; SM, 2.5%; and malignant, 12.4%. Histopathologic follow-up was available for 237 cases (49.7%). The ROMs were as follows: nondiagnostic, 5.9%; nonneoplastic, 9.1%; AUS, 35.7%; benign neoplasm, 3.3%; SUMP, 31.8%; SM, 100%; and malignant, 100%. Mucoepidermoid carcinoma was the most common malignancy (18 of 237; 7.6%), and it was followed by acinic cell carcinoma (16 of 237; 6.8%). Pleomorphic adenoma was the most common benign neoplasm (95 of 237; 40.1%)., Conclusions: The MSRSGC can be reliably applied to pediatric salivary gland FNA. The ROM of each MSRSGC category in pediatric salivary gland FNA is relatively similar to the ROM of each category in adult salivary gland FNA, although the reported rates for the different MSRSGC categories are variable across institutions., (© 2022 American Cancer Society.)

Background: Rearranged during transfection ( RET ) gene fusions are detected in 10-20% of thyroid cancer patients. Recently, RET fusion-positive metastatic thyroid cancers have attracted much attention owing to the FDA approval of two highly selective anti-RET tyrosine kinase inhibitors, namely, selpercatinib, and pralsetinib., Areas Covered: This review summarizes the available evidence on the biological and predictive role of RET gene fusions in thyroid carcinoma patients and the latest screening assays currently used to detect these genomic alterations in histological and cytological specimens., Expert Opinion: Management of advanced thyroid carcinoma has significantly evolved over the last decade thanks to the approval of three multikinase inhibitors, i.e. sorafenib, lenvatinib, cabozantinib, and of two selective RET-tyrosine inhibitors, i.e. selpercatinib and pralsetinib. In this setting, the detection of RET-fusions in advanced thyroid cancer specimens through the use of next-generation sequencing has become a commonly used strategy in clinical practice to select the best treatment options.

Although gene fusions occur rarely in non-small-cell lung cancer (NSCLC) patients, they represent a relevant target in treatment decision algorithms. To date, immunohistochemistry and fluorescence in situ hybridization are the two principal methods used in clinical trials. However, using these methods in routine clinical practice is often impractical and time consuming because they can only analyze single genes and the quantity of tissue material is often insufficient. Thus, novel technologies, able to test multiple genes in a single run with minimal sample input, are being under investigation. Here, we discuss the utility of next-generation sequencing and nCounter technologies in detecting simultaneous gene fusions in NSCLC patients.

Purpose: The efficacy of lenvatinib for advanced and progressive radioactive iodine refractory differentiated thyroid cancer is well established. Herein, we retrospectively evaluated the long-term safety and efficacy of lenvatinib in 23 patients treated at a single Institution., Methods: Clinical data of all patients treated for a differentiated thyroid cancer with lenvatinib from April 2015 to September 2020 were retrospectively analyzed., Results: A total of 23 patients were included. In all, 21 patients received lenvatinib as first-line systemic therapy. Median age at initiation of lenvatinib treatment was 68 (44-90) years. Median duration of the study from initiation of lenvatinib to study end was 23 (2-65) months. The indication for lenvatinib treatment was documented progression of distant metastases in 20 patients and of locally advanced disease in the other 3 and median duration of lenvatinib therapy was 15 (2-64) months. Best treatment responses were: partial response in 6 patients, stable disease in 14, progressive disease in 1, and not evaluable in 2. Median progression-free survival was 25 months (95% CI: 12-40) and median overall survival was 46 months (95% CI: 28-65). Three patients had to discontinue lenvatinib treatment due to serious adverse events and no drug-related death was observed. Ten patients continued lenvatinib for more than 24 months and the only newly registered adverse event after this period of time was one case of G2 proteinuria. Six patients continued lenvatinib treatment beyond documented tumor progression due to oligoprogression or slowly progressive disease (median time 18.5 months, 8-42 months). A total of 14 patients were alive at the end of the study: 11 showed partial response/stable disease on lenvatinib, including 3 who had a stable disease after local ablative therapy for oligoprogressive metastases; 3 had to change treatment, including 2 for lenvatinib-related serious adverse events and 1 for progressive disease., Conclusions: Long-term lenvatinib treatment is safe and some patients may experience persistent long-term control of the disease. Late treatment-related AEs rarely occurred. Oligoprogressive and slowly progressive disease can be managed without treatment withdrawal as long as there are some clinical benefits.

Background: During the current coronavirus disease 2019 (COVID-19) pandemic, the cytopathology workload has decreased remarkably worldwide as all screening and elective procedures have been postponed to prioritize the clinical management of patients at high oncological risk. In the current study, the authors provide data on the lasting impact of COVID-19 on cytopathology practice during the initial phases of the Italian postlockdown period., Methods: The percentages of the cytological sample types processed at the University of Naples Federico II during the first 12 weeks of the Italian postlockdown period were compared with those of the same period in 2019. The study period was divided into four 3-week periods. Differences in the rates of malignant diagnoses were also assessed., Results: During the 12-week study period, the overall cytological sample workload decreased by 41.6% in comparison with 2019. In particular, the workload significantly declined for each sample type: Pap smears, -33.3%; urine, -42.8%; serous fluids, -14.4%; thyroid, -54.5%; breast, -43%; lymph node, -27.3%; and salivary gland, -61%. By contrast, the overall malignancy rate was significantly increased (P = .0011)., Conclusions: The reduction in the cytological sample workload during the postlockdown period still represents an ongoing effect of the COVID-19 pandemic. On the other hand, the rise in the overall malignancy rate reflects the importance of prioritizing diagnostic procedures for patients at high oncological risk., (© 2021 American Cancer Society.)

Purpose: Nowadays, the clinical management of thyroid nodules needs to be multi-disciplinary. In particular, the crosstalk between endocrinologists and cytopathologists is key. When FNAs are properly requested by endocrinologists for nodules characterised by relevant clinical and ultrasound features, cytopathologists play a pivotal role in the diagnostic work-up. Conversely, improper FNA requests can lead to questionable diagnostic efficiency. Recently, recommendations to delay all non-urgent diagnostic procedures, such as thyroid FNAs, to contain the spread of COVID-19 infection, have made the interplay between endocrinologists and cytopathologists even more essential. The objective of this study was to assess the impact of COVID-19 pandemic on our practice by evaluating the total number of FNAs performed and the distribution of the Bethesda Categories before, during, and after the lockdown., Methods: We analysed the FNA trends before (1st January 2019 to March 13th 2020), during (March 14th to May 15th), and after (May 16th to July 7th) the lockdown., Results: Although the total number of weekly FNAs dropped from 62.1 to 23.1, our referring endocrinologists managed to prioritise patients with high-risk nodules. In fact, in the post-lockdown, the weekly proportion of benign diagnoses dropped on average by 12% and that of high-risk diagnoses increased by 6%., Conclusions: The lesson we have learned so far from this pandemic is that by applying safety protocols to avoid contagion and by increasing the threshold for FNA requests for thyroid nodules, we can continue to guarantee our services to high-risk patients even in times of a health crisis.

Objectives: to evaluate the implementation of an integrated care model for thyroid disease on thyroid surgery at the University Hospital "Federico II" of Naples (Campania Region, Southern Italy)., Design: quasi-experimental design employing an interrupted time series analysis., Setting and Participants: all subjects who were admitted to the University Hospital "Federico II" for thyroid surgery between January 2008 and December 2018. The integrated care model for thyroid disease was implemented starting from January 2016., Main Outcome Measures: rate of partial thyroidectomies over all thyroidectomies; rate of diagnosed thyroid cancers over all diagnosed thyroid tumours; length of stay (LOS). Differences pre- and post-interventions were assessed employing Poisson (for count outcomes) and linear (for continuous outcomes) regression models. Models were adjusted for age, gender, tumour diagnosis (none, benign, malignant), Charlson index, and discharge month., Results: data on 4,233 thyroidectomies were included. There was no difference between pre- and post-intervention trends for the rate of partial thyroidectomies over all thyroidectomies (pre-intervention: IRR 1.00; 95%CI 0.99;1.00 - post-intervention: IRR 1.00; 95%CI 0.98;1.02) and for the rate of diagnosed thyroid cancers over all thyroid tumours (pre-intervention IRR 0.99; 95%CI 0.99;1.00 - post-intervention IRR 1.00; 95%CI 0.99;1.01). On the contrary, the LOS reduced from 4.5 (±4.3) days in 2008 to 3.2 (±3.2) days in 2018. The multivariate analysis confirmed this reduction, estimated to be 1.1 days on average in the pre-intervention eight-year period (pre-intervention coefficient -0.01; 95%CI -0.02;-0.01), followed by an even greater reduction in the post-intervention three-year period which was estimated to be 1.1 day (post-intervention: coefficient -0.03; 95%CI -0.05;-0.01)., Conclusions: the implementation of an integrated care model for thyroid disease contributed to reduce the LOS for thyroidectomies, improving the efficiency in the management of thyroid disease. However, this intervention had no impact in reducing the rate of total thyroidectomies.

Background: Artificial genomic reference standards in a cytocentrifuge/cytospin format with well-annotated genomic data are useful for validating next-generation sequencing (NGS) on routine cytopreparations. Here, reference standards were optimized to be stained by different laboratories before DNA extraction and to contain a lower number of cells (2 × 10 5 ). This was done to better reflect the clinical challenge of working with insufficient cytological material., Methods: A total of 17 worldwide laboratories analyzed customized reference standard slides (slides A-D). Each laboratory applied its standard workflow. The sample slides were engineered to harbor epidermal growth factor receptor (EGFR) c.2235&#95;2249del15 p.E746&#95;A750delELREA, EGFR c.2369C>T p.T790M, Kirsten rat sarcoma viral oncogene homolog (KRAS) c.38G>A p.G13D, and B-Raf proto-oncogene, serine/threonine kinase (BRAF) c.1798&#95;1799GT>AA p.V600K mutations at various allele frequencies (AFs)., Results: EGFR and KRAS mutation detection showed excellent interlaboratory reproducibility, especially on slides A and B (10% and 5% AFs). On slide C (1% AF), either the EGFR mutation or the KRAS mutation was undetected by 10 of the 17 laboratories (58.82%). A reassessment of the raw data in a second-look analysis highlighted the mutations (n = 10) that had been missed in the first-look analysis. BRAF c.1798&#95;1799GT>AA p.V600K showed a lower concordance rate for mutation detection and AF quantification., Conclusions: The data show that the detection of low-abundance mutations is still clinically challenging and may require a visual inspection of sequencing reads to detect. Genomic reference standards in a cytocentrifuge/cytospin format are a valid tool for regular quality assessment of laboratories performing molecular studies on cytology with low-AF mutations., (© 2019 American Cancer Society.)

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., (© 2017 American Cancer Society.)

This review is focused on the challenges in standardising and optimising molecular testing workflow in cytopathology. Although cytological samples yield optimal quality DNA, whose minimal amounts in most cases suffice even for multigene mutational profiling, the success of molecular testing is strongly dependent on standardised preanalytical protocols for maximising DNA yield and quality. Sample cytopreparation influences, even more, the quality of RNA and consequently the potential success of reverse transcription-PCR. Here, the educational and technical involvement of the cytopathologist as a relevant component of a multidisciplinary team, in the issues related to test request, specimen collection, fixation, processing, staining, tumour fraction enrichment, DNA quality/quantity assessment and storage conditions is discussed. In addition, the specific sample requirements related to more recent technological developments are examined, underlining the modern role of the cytopathologist, whose continuous education is crucial to meet the opportunities of molecular medicine., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Background: Non small cell lung cancer (NSCLC) is diagnosed in most cases on small tissue samples, such as cytological preparations and histological biopsies; these limited tissue specimens may be not always sufficient for testing epidermal growth factor receptor ( EGFR ) mutations and other relevant predictive biomarkers. Cell-free DNA (cfDNA) can be used as a surrogate for EGFR mutational testing, whenever tissue is unavailable. However, the detection of gene mutations on cfDNA is challenging; in fact, the extremely low concentration of circulating tumor DNA requires the implementation of highly sensitive and validated next generation techniques., Methods: Thus, we have recently validated a novel next generation sequencing (NGS) assay, employing the SiRe ® gene panel to detect on cfDNA mutations of EGFR and KRAS, NRAS, BRAF, cKIT and PDGFR genes. In this current study, we report on a series of NSCLC patients, without available tissue for EGFR testing, who prospectively underwent SiRe ® NGS analysis., Results: The results confirm the high clinical performance, in terms of success rate and mutation detection, of NGS based analysis of cfDNA., Conclusions: SiRe ® NGS panel represent an effective diagnostic tool in cfDNA analysis setting., Competing Interests: Conflicts of Interest: The authors have no conflicts of interest to declare.

Non-small cell lung carcinoma harbouring epidermal growth factor receptor ( EGFR ) mutation, usually progress after an initial response to tyrosine-kinase inhibitors (TKI). Liquid biopsy enables with a simple blood draw the accurate detection of EGFR p.T790M mutation, the most common resistance mechanism, avoiding the more invasive tissue re-biopsy. However, in a subset of cases, resistance mechanisms are more complex featuring both genetic and morphological changes. Here we report the case of a 67 years-old woman, affected by an EGFR mutated lung adenocarcinoma and treated by TKI. At disease progression, the patient developed a morphological transition to squamous cell carcinoma in association to the arising of a PIK3CA p.E542K mutant subclone. This case illustrates that, even in the "liquid biopsy" era, cytology can have still a role by providing an overall assessment of both morphology and genetic TKI resistance mechanisms., Competing Interests: Competing interests: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

In the advanced stages of non-small cell lung cancer (NSCLC), molecular testing is often performed on archival cytological smears. The nCounter system (NanoString Technologies) is a new promising multiplex digital colour-coded barcode technology. However, its feasibility to evaluate the RNA expression of clinical relevant biomarkers on routine cytological smears is still uncertain. To this end, RNA was extracted from 12 NSCLC routine stained cytological smears, and nCounter analysis performed by using a 48-gene panel. Overall, 11/12 (92%) of the smears were adequate for the secondary analysis, fulfilling the quality check parameter analysis of nSolver software. This pilot study shows that RNA nCounter analysis is feasible on routine cytological smears preparing the field for the implementation of this technology in the routine setting., Competing Interests: Competing interests: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Background: Molecular testing of cytological lung cancer specimens includes, beyond epidermal growth factor receptor (EGFR), emerging predictive/prognostic genomic biomarkers such as Kirsten rat sarcoma viral oncogene homolog (KRAS), neuroblastoma RAS viral [v-ras] oncogene homolog (NRAS), B-Raf proto-oncogene, serine/threonine kinase (BRAF), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA). Next-generation sequencing (NGS) and other multigene mutational assays are suitable for cytological specimens, including smears. However, the current literature reflects single-institution studies rather than multicenter experiences., Methods: Quantitative cytological molecular reference slides were produced with cell lines designed to harbor concurrent mutations in the EGFR, KRAS, NRAS, BRAF, and PIK3CA genes at various allelic ratios, including low allele frequencies (AFs; 1%). This interlaboratory ring trial study included 14 institutions across the world that performed multigene mutational assays, from tissue extraction to data analysis, on these reference slides, with each laboratory using its own mutation analysis platform and methodology., Results: All laboratories using NGS (n = 11) successfully detected the study's set of mutations with minimal variations in the means and standard errors of variant fractions at dilution points of 10% (P = .171) and 5% (P = .063) despite the use of different sequencing platforms (Illumina, Ion Torrent/Proton, and Roche). However, when mutations at a low AF of 1% were analyzed, the concordance of the NGS results was low, and this reflected the use of different thresholds for variant calling among the institutions. In contrast, laboratories using matrix-assisted laser desorption/ionization-time of flight (n = 2) showed lower concordance in terms of mutation detection and mutant AF quantification., Conclusions: 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., (© 2017 American Cancer Society.)

Highly sensitive genotyping techniques are useful to detect epidermal growth factor receptor (EGFR) mutations on lung cancer cytological samples, when these specimens feature only few neoplastic cells. This study aimed to validate digital PCR (dPCR) methodology on cytological material. In plasmid model system, dPCR allowed for the detection of a minimal percentage (1%) of EGFR mutant alleles. Cytological samples (n = 30), with neoplastic cell percentage ranging from 10% to 80% and yielding a quantity of extracted DNA ranging from 1.75 to 60 ng/µL were selected. Results previously generated by fragment length and TaqMan assays (n = 8 exon 19 deletions, n = 2 L858R mutations and n = 20 wild-type DNA) were compared with those obtained by dPCR. Data were highly concordant (96.6%). However, dPCR detected an additional L858R mutation that had been missed by TaqMan assay on a paucicellular smear. This mutation was confirmed by cloning PCR products and sequencing. Thus, dPCR can reliably be used to increase EGFR mutation detection rate on scarcely cellular lung cancer smears., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Colorectal cancer (CRC) is one of the most frequent and deadly malignancies worldwide. Despite the progresses made in diagnosis and treatment, the identification of tumor markers is still a strong clinical need, because current treatments are efficacious only in a subgroup of patients. UbcH10 represents a potential candidate biomarker, whose expression levels could be employed to predict response or resistance to chemotherapy or targeted agents. UbcH10 mRNA and protein expression levels have been evaluated in a large group of CRC patients and correlated with clinico-pathological characteristics, including KRAS mutations. Moreover, the endogenous levels of UbcH10 and its role on cell growth have been evaluated in CRC cells. Finally, to investigate the impact of UbcH10 protein expression on the response to irinotecan, its active metabolite SN-38 and cetuximab treatment, UbcH10 silencing experiments were carried-out on two colon carcinoma cell lines, Caco-2, and DLD1. Overexpression of UbcH10 mRNA and protein was observed in the vast majority of patients analyzed. UbcH10 suppression decreased CRC cell growth rate (at least in part through deregulation of Cyclin B and ERK1) and sensitized them to pharmacological treatments with irinotecan, SN-38 and cetuximab (at least in part through a down-regulation of AKT). Taken together, these findings indicate that UbcH10 expression regulates CRC growth and could play an important role in the personalization of the therapy of CRC patients., (© 2015 Wiley Periodicals, Inc.)

Spindle epithelial tumor with thymus-like differentiation (SETTLE) is a very rare tumor of the thyroid gland. An algorithm for the diagnosis and treatment of SETTLE has yet to be established. The aim of this study was to identify all case reports of SETTLE and to compare the clinical-pathological features and therapy of the cases identified. We performed a PubMed search for case reports of SETTLE in English published up to November 2014 in which "SETTLE" and "Spindle epithelial tumor with thymus-like differentiation" were keywords. We identified 35 articles for a total of 42 cases. We found that SETTLE usually occurs in children and adolescents as an asymptomatic neck mass. Thyroid function tests and tumor markers are invariably within normal range in all patients, and fine needle aspiration biopsy is rarely diagnostic for SETTLE. All 42 patients had undergone thyroidectomy. After surgical resection, chemotherapy (adjuvant or first/second-line treatment) and/or radiotherapy were administered to control tumor growth in cases with metastatic involvement. Although SETTLE presents a low-grade malignancy, it can metastasize to lymph nodes, the mediastinum, lung, vertebrae, and kidney even many years after the initial diagnosis. SETTLE may have a good prognosis if appropriately treated at initial presentation and if patients undergo long-term monitoring with regular clinical and morphological evaluations.

Lung adenocarcinoma and papillary thyroid carcinoma (PTC) share a number of microscopic and immunophenotypical features. Thus, patients presenting with thyroid and lung synchronous neoplasms may be difficult on fine-needle aspiration (FNA) samples to define the site of origin of the malignancy. In the case reported here, inherent to a 57-years-old man presenting with a right lung mass and a large (44 mm) thyroid nodule, an integrated cytological, immunocytochemical and molecular approach enabled to clarify the primary nature of the neoplasm. FNA cytology showed in both sites papillary structures and nuclear changes reminiscent of PTC. The lung origin of the neoplasm was suggested on cell-block immunocytochemistry showing thyroid transcription factor-1 positive and PAX8 and TGB negative neoplastic cells. Next generation sequencing performed on the Ion Torrent platforms by the Ion Ampliseq Colon and Lung Cancer panel showed a similar genomic profile in both neoplastic sites with a concurrent KRAS G12C mutation. An integrated approach on FNA biospecimen is safe, cost effective, and may be coupled effectively with modern ancillary molecular techniques that may be useful, besides their predictive value, as a adjunctive diagnostic tool when the synchronous occurrence of lesions featuring overlapping morphologies challenge the cytopathologist., (© 2015 Wiley Periodicals, Inc.)

Until a few years ago, only lung cancer histological specimens were considered suitable for testing epidermal growth factor receptor (EGFR) mutations. Then, several retrospective studies were designed to test EGFR mutation on a sizeable number of parallel cytological and histological samples obtained from the same patients and, even more recently, several institutions reported their prospective clinical experiences on routine specimens. Basing on these studies the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology have recently considered cytological samples suitable for EGFR testing. Therefore, it seems timely to draw together the threads of this large body of information in order that cytopathologists can be knowledgeable partners in the multidisciplinary process of targeted cancer therapy and to help refine current testing guidelines. This review addresses (1) the more common proposed techniques including the use of direct cytologic smears cell blocks and liquid based cytology; (2) the issues related to current practice, which in Europe is external centralized testing that is usually done on samples containing very few cells; and (3) the future directions based on the implementation on lung cytology of next generation sequencing approaches., (© 2014 Wiley Periodicals, Inc.)

Both experimental and clinical studies have suggested that any potential treatment of polycystic kidney disease (PKD) should start early and last for a long time to be effective, with unavoidable side reactions and considerable costs. The aim of the present study was to test how low doses of rapamycin (RAPA; 0.15 mg/kg ip for 4 days/wk), tolvaptan (TOLV; 0.005% in diet), or AEZ-131 (AEZ; a novel ERK inhibitor, 30 mg/kg for 3 days/wk by gavage), alone and in association, affect the progression of polycystic renal disease in PCK rats. Rats were treated for 8 wk starting at 4-6 wk of age. The efficacy of low doses of such drugs in inhibiting their respective targets was confirmed by immunoblot experiments. Compared with rats in the control (CON) group, RAPA treatment caused a significant reduction in cyst volume density (CVD; -19% vs. the CON group) and was numerically similar to that in TOLV-treated rats (-18%, not significiant), whereas AEZ treatment was not effective. RAPA + TOLV treatment resulted in a significantly lower CVD (-49% vs. the CON group) and was associated with a striking decrease in cAMP response element-binding protein phosphorylation, and similar data were detected in RAPA + AEZ-treated rats (-42%), whereas TOLV + AEZ treatment had virtually no effect. RAPA administration significantly lessened body weight gain, whereas TOLV administration resulted a mild increase in diuresis and a significant increase in cAMP urinary excretion. Histological data of tubular proliferation were in full agreement with CVD data. In conclusion, this study demonstrates that the association of low doses of RAPA, TOLV, and AEZ slows the progression of PKD with limited side effects, suggesting the use of combined therapies also in clinical trials., (Copyright © 2014 the American Physiological Society.)

Background: Epidermal growth factor receptor (EGFR) mutations are reliably detected by referral laboratories, even if most lung cancer cytology specimens sent to such laboratories contain very few cells. However, EGFR mutations may be distributed heterogeneously within tumors, thereby raising concerns that mutations detected on cytology are not representative of the entire tumor and, thus, are less reliable in predicting response to tyrosine kinase inhibitor (TKI) treatment than mutations detected on histology. To address this issue, the authors reviewed their clinical practice archives and compared the outcome of TKI treatment among patients who were selected by cytology versus patients who were selected by histology., Methods: From July 2010 to July 2012, 364 cytology samples and 318 histology samples were received. Exon 19 deletions and the L858R point mutation in exon 21, detected by fragment assay and TaqMan assay, respectively, were confirmed by direct sequencing; discrepancies were resolved by cloning polymerase chain reaction products. The response rate (RR) and progression-free survival (PFS) at 12 months (range, 3-34 months) were evaluable in 13 EGFR-mutated patients who were selected for treatment by cytology and 13 patients who were selected by histology., Results: The mutation rate was similar in histology samples (8.5%) and cytology samples (8.8%). The RR (54%) and PFS (9.2 months) were similar in histologically selected patients and cytologically selected patients (RR, 62%; PFS, 8.6 months; P = .88). The disease control rate (responsive plus stable disease) was 92% in histologically selected patients and 100% in cytologically selected patients., Conclusions: EGFR mutations detected on cytology specimens by a centralized laboratory can predict TKI treatment response equally well as mutations identified on histology samples., (© 2013 American Cancer Society.)

Background: Fine needle cytology (FNC) of thyroid nodules is not always diagnostic. Most of FNCs undeterminated for malignancy belong to the cytological class of "follicular neoplasm/suspicious for follicular neoplasm" lesions (FN). In this group only 10-30% of cases are malignant and the most appropriate surgical management is still controversial. Here, this issue was addressed and the more reliable predictive criteria of malignancy were also evaluated., Methods: We retrospectively evaluated 472 patients, surgically treated after a FN diagnosis in a tertiary care referral center. In patients affected by bilateral thyroid disease with a cytological diagnosis of FN, or when high-risk clinical features and familiarity for thyroid cancer were present, total thyroidectomy (TT) was performed. Conversely, hemithyroidectomy (HT) was preferred when the nodule was single and when the age was ≤ 45 years. Frozen section examination was not used, and if cancer was diagnosed by definitive pathology of the HT specimen, the remnant thyroid lobe was removed. Histological features, surgical complications, and long-term outcomes of the remnant lobe were reported. Clinical features predictivity was also evaluated., Results: TT was performed in 154/472 pts (32.62%), while HT was carried out in 318/472 cases (67.37%). The overall malignancy rate (MR) was 18.85% (89/472 pts), respectively 16% (51/318pts) following HT, and 24.6% (38/154pts) following TT, with a statistically significant difference. Similarly, the rates of transient and definitive hypoparathyroidism and the mean hospital stay following TT were higher than after HT (and statistically significant). Age < 45years and female gender were more frequently associated to malignancy. The rate of complications following second surgery was comparable to that of primary HT. In the HT group incidence of unexpected contralateral papillary thyroid cancer (PTC) was 9.8% and, after 88.2 ± 30.42 months mean follow-up, completion surgery for benign pathology was carried out in 6.7% of cases., Conclusions: Our data show that histology following a cytological FN diagnosis is malignant only in a low percentage of cases (89/472, 18.85%). Following TT, a MR higher than in HT was observed. Even if some clinical features are cancer associated, malignancy cannot be reliably predicted before surgery. Thus, in solitary low-risk lesions, HT is still the standard of care. Its lower complication rates makes HT the safest procedure. In case of multiglandular disease TT may be recommended. Further investigation is warranted to achieve a better preoperative diagnostic accuracy in order to reduce the amount of surgical operations with diagnostic aim.

The foamy gland pattern (FGP) may impart to pancreatic ductal adenocarcinoma (PDA) a deceptively benign appearance. Thus, its diagnosis on FNA is challenging. A case of PDA with FGP, that was suspected on Diff Quik smears, diagnosed on cell-block preparation and confirmed by ancillary stains and molecular techniques, is here reported. The diagnostic interpretation of foamy atypical cells on direct smears may benefit from cell block preparation and from ancillary techniques., (Copyright © 2012 Wiley Periodicals, Inc.)