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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.

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.

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.

AimsIn 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.MethodsThe 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.ResultsNexthyro 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.ConclusionOur 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.

The indeterminate thyroid nodules diagnosed by fine-needle aspiration cytology (FNAC)represents a problem for both cytopathologists and clinicians. The former sometimes use this diagnostic category as a sort of basket, putting in cases that they do not know exactly how to classify. The latter are faced with a highly variable risk of malignancy and consequently the management remains a challenge. On the histopathological side, the new WHO classification of tumors of the thyroid introduced the concept of tumors with uncertain and low malignant potential, and the concept of non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), whose prognosis and management are still to be completely elucidated. While the risk of malignancy of the indeterminate diagnostic category has decreased due to the re-classification of certain types of papillary thyroid carcinomas of the follicular variant into a low malignant potential form (the NIFTP), cases diagnosed cytologically as indeterminate will probably increase in the future to avoid false positive diagnosis. Thus, the indeterminate thyroid diagnostic category still remains a challenge, both at the diagnostic level and for its management. The new version of the Bethesda system for reporting thyroid cytopathology suggests managing these patients with a repeat FNA, diagnostic lobectomy and/or molecular testing.

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., The reduction in the cytological sample workload during the postlockdown period 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.

AimsLung cancer predictive biomarker testing is essential to select advanced-stage patients for targeted treatments and should be carried out without delays even during health emergencies, such as the coronavirus (COVID-19) outbreak.MethodsFifteen molecular laboratories from seven different European countries compared 4 weeks of national lockdown to a corresponding period in 2019, in terms of tissue and/or plasma-based molecular test workload, analytical platforms adopted, number of cases undergoing programmed death-ligand1 (PD-L1) expression assessment and DNA-based molecular tests turnaround time.ResultsIn most laboratories (80.0%), tissue-based molecular test workload was reduced. In 40.0% of laboratories (6/15), the decrease was >25%, and in one, reduction was as high as 80.0%. In this instance, a concomitant increase in liquid biopsy was reported (60.0%). Remarkably, in 33.3% of the laboratories, real-time PCR (RT-PCR)-based methodologies increased, whereas highly multiplexing assays approaches decreased. Most laboratories (88.9%) did not report significant variations in PD-L1 volume testing.ConclusionsThe workload of molecular testing for patients with advanced-stage lung cancer during the lockdown showed little variations. Local strategies to overcome health emergency-related issues included the preference for RT-PCR tissue-based testing methodologies and, occasionally, for liquid biopsy.

Immune-checkpoint inhibitors (ICIs) play a key role in the treatment of advanced stage colorectal cancer (CRC) patients featuring a deficient DNA mismatch repair (dMMR) system or a high microsatellite instability (MSI-H) profile. However, beyond the established role in CRC patients, ICIs have highly proven efficacy in other solid tumors featuring MSI-H/dMMR status represented by endometrial, gastric, ovarian, prostatic, and pancreatic carcinomas (EC, GC, OC, PrC, and PaC). Our aim was to compare the concordance rates among the Idylla™ MSI test, TapeStation 4200, and immunohistochemical (IHC) analysis in assessing MSI-H/dMMR status in EC, GC, OC, PrC, and PaC patients. The Sanger sequencing-based Titano MSI test was used in discordant cases. One hundred and eighty-five cases (n = 40 PrC, n = 39 GC, n = 38 OC, n = 35 PaC, and n = 33 EC) were retrospectively selected. MMR protein expression was evaluated by IHC. After DNA quality and quantity evaluations, the IdyllaTM and TapeStation 4200 platforms were adopted for the evaluation of MSI status. Remarkably, compared to IHC, the Idylla™ platform achieved a global concordance rate of 94.5% (154/163) for the microsatellite stable (MSS)/proficient MMR (pMMR) cases and 77.3% (17/22) for the MSI-H/dMMR cases. Similarly, a global concordance rate of 91.4% (149/163) and 68.2% (15/22) for MSS/pMMR and MSI-H/dMMR cases was also identified between IHC and the TapeStation 4200 microfluidic system. In addition, a global concordance of 93.1% (148/159) and 69.2% (18/26) for MSS/pMMR and MSI-H/dMMR cases was observed between the Idylla™ and TapeStation 4200 platforms. Discordant cases were analyzed using the Titano MSI kit. Overall, our data pinpointed a central role for molecular techniques in the diagnostic evaluation of dMMR/MSI-H status not only in CRC patients but also in other types of solid tumors.

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.

AimsFollowing the development for liquid biopsies of the SiRe next-generation sequencing (NGS) panel that covers 568 clinical relevant mutations in EGFR, KRAS, NRAS, BRAF, cKIT and PDGFRa genes, in this current study, we apply this small NGS panel on tissue samples of lung cancer.MethodsA total of 322 specimens were prospectively tested. Technical parameters were analysed on both cytological and histological samples. In a subset of 75 samples, the EGFR SiRe results were compared with those generated by the European Community (CE)–IVD EGFR assay on Idylla platform. Clinical outcomes of 11 patients treated, on the basis of SiRe results, were also evaluated.ResultsOnly 28 (8.7%) specimens failed to produce a library; out of the 294 remaining samples, a total of 168 somatic mutations were found. In nearly all instances (74/75–99%), the EGFR SiRe results were confirmed by Idylla. In general, SiRe analytical parameters were excellent. However, histological and cytological specimens differed in relation to average reads for sample, mean number of mapped reads, median read length and average reads for amplicon. Treatment outcome evaluation in 11 patients showed a partial response in 82 % (9/11) patients with a median progression-free survival of 340 days.ConclusionsThe small gene panel SiRe is a clinically relevant tool useful to widespread the adoption of NGS in predictive molecular pathology laboratories.

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.

In the era of personalized medicine, cytopathology reports must be standardized to provide clinicians with clear information regarding relevant diagnostic, prognostic, and predictive tumor features. Since 2010, the international Molecular Cytopathology Meeting has been held annually in Naples, Italy, to disseminate recent breakthroughs and ongoing investigations in the field of modern cytopathology. In particular, the seventh annual meeting, which took place on November 26 through 27, 2018, focused on novel cytological and molecular classifications and novel DNA sequencing techniques, as well as on the possibility of using cytological rather than tissue specimens for lung cancer biomarker testing. The present review aims to update cytopathologists on the more recent achievements in modern cytopathology and to identify key research questions that still remain unanswered.

The College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC), and the Association for Molecular Pathology (AMP) International joint committee has recently updated the previous 2013 molecular guidelines version. Here we summarize these changes which demonstrate that molecular cytopathology is here to stay.

Objective: In our Pathology Department, fine needle aspiration (FNA) of palpable thyroid nodules isperformed by cytopathologists who ensure correct sample management and rapid on-site evaluation (ROSE).Conversely, ultrasound (US)-guided FNAs have traditionally been carried out by endocrinologists andradiologists in outside clinics, where the presence of a cytopathologist is not always feasible. To overcome thislimitation, cytopathologists have started to perform US-guided FNAs themselves. This study retrospectivelyevaluates 1 year of this novel practice.Methods: A total of 2225 US-guided FNAs were performed in our clinic by cytopathologists, whereas 1490aspirates were taken by a group of non-cytopathologists. Among these, 756 FNAs were taken by a singleexperienced endocrinologist. The distribution of the Bethesda classification categories was evaluated in each ofthese groups.Results: FNAs performed by cytopathologists were more often diagnostic and better prepared than thosetaken by non-cytopathologists, including those taken by the experienced endocrinologist (P < 0.01). The latteroperator yielded a higher rate of suspicious and malignant FNAs, reflecting a more appropriate clinical triageof worrisome nodules.Conclusion: Although the endocrinologist’s evaluation is crucial to select clinically relevant thyroid nodules,cytopathologists can reliably perform US guidance in addition to their traditional expertise in sampling,specimen preparation and ROSE.Keywords: ultrasound-guided fine needle aspiration, thyroid, rapid on-site evaluation, interventionalpathologistIntroductionThyroid fine needle aspiration (FNA) is a reliableand cost-effective diagnostic tool, as long as high-quality samples, representative of the lesion, areobtained for accurate microscopic interpretation. Theideal physician to perform thyroid FNA should beone who is skilled in obtaining representative mate-rial and in the preparation of slides. As these skillsare strengthened by the microscopic observation ofsamples, it is not surprising that the best results onpalpable nodules are obtained when the FNA is per-formed or triaged by the same cytopathologist whowill eventually make the diagnosis.

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.

Introduction Indeterminate diagnoses are rendered on 15%-30% of thyroid fine-needle aspirates (FNA). Thus, a second diagnostic opinion given by an outside expert pathologist is a common practice that facilitates a more appropriate clinical management. Conversely, the role of an intra-institutional second opinion diagnosis (iSOD), which is usually informally performed in-house, has not been well established. Methods To assess the contribution of iSOD, a retrospective series of 34 thyroid FNA diagnosed as follicular neoplasm/suspicious follicular neoplasm (FN/SFN) with matched histological follow-up and a malignancy rate of 17.6% was selected and independently reviewed by two cytopathologists (CYT1 and 2). Cases with discrepant diagnoses were referred to a third in-house senior cytopathologist for the iSOD. The malignancy rates (MR) obtained after single independent reviews and iSOD were compared. Results MR obtained after CYT1 and CYT2 re-screening was similar (14.28% and 19.04%, respectively) and did not improve the original MR (17.64%). Conversely, after the iSOD of discrepant diagnoses, the overall malignancy rate increased up to the 27.27%, potentially sparing unnecessary surgical procedures. Conclusions Intra-institutional second opinion practice for “indeterminate” thyroid FNA avoids unnecessary surgeries and maximises the detection of malignant cases diagnosed as FN/SFN.

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 ofEGFRp.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 anEGFRmutated 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 aPIK3CAp.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.

BackgroundAnalysis of K- and N-RAS mutations is mandatory before planning treatment of metastatic colorectal cancer, because only RAS wild-type (WT) patients can benefit from treatment with anti-EGFR monoclonal antibodies (cetuximab and panitumumab).Case reportHere we report the case of a 69-year-old male patient affected by metastatic sigmoid cancer. He underwent left hemicolectomy, and histology diagnosed a well-differentiated, pT4, node-positive adenocarcinoma; KRAS analysis performed with direct sequencing identified a mutation in exon 2 of the KRAS gene (GGT->GTT). After first-line chemotherapy with FOLFOX6 plus bevacizumab, the patient underwent surgical resection of residual liver metastases. Histology showed metastatic deposits from colic adenocarcinoma with extensive coagulative necrosis. Mutational analysis of the KRAS gene was also performed on liver metastases by pyrosequencing assay, and no mutation was identified. Due to the discordant results (GGT->GTT exon 2 KRAS mutation in the primary tumor, and KRAS-WT in the liver metastases), mutational analysis on liver metastasis was repeated using next-generation sequencing and enriching the sample in tumor cells by manual microdissection; the same type of mutation of the primary tumor (GGT->GTT exon 2 KRAS gene) was confirmed.ConclusionsAccurate tissue sampling and adequately sensitive assays are essential to correctly identify colorectal cancer patients who can be treated with an anti-EGFR monoclonal antibody.

Metastatic colorectal cancer harbouring a mutation in codon 12 or 13 of the KRAS gene does not benefit from therapy with antibodies targeting the epidermal growth factor receptor (EGFR). The implementation of community KRAS testing is generating a rapid flow of new data that have implications for the pathologist and testing guidelines besides the physician. Therefore, it seems timely to draw together the threads of this large body of information in order that pathologists can be knowledgeable partners in the multidisciplinary process of targeted cancer therapy and to help refine current testing guidelines. This review addresses (1) the most relevant methodological and technical aspects of KRAS testing in terms of sample site (primary/metastatic), test specimens (resection/biopsy/cytology) and the diverse molecular methods available; (2) the issues related to daily practice, namely, the timing of the test, its turnaround time and the quality control procedures; and (3) the evidence related to the relationship between KRAS genetic intratumoural heterogeneity, clinical sensitivity of mutational detection tools and anti-EGFR treatment outcome. Hopefully, in the near future, elucidation of the potential of biomarker panels and of the mechanisms underlying primary and acquired resistance to anti-EGFR therapy will refine even further personalised treatment regimens for patients with metastatic colorectal cancer.

The advent of genomic based personalized medicine has led to multiple advances in the molecular characterization of many tumor types, such as non-small cell lung cancer (NSCLC). NSCLC is diagnosed in most cases on small tissue samples that may be not always sufficient for EGFR mutational assessment to select patients for first and second generations’ tyrosine kinase inhibitors (TKIs) therapy. In patients without tissue availability at presentation, the analysis of cell free DNA (cfDNA) derived from liquid biopsy samples, in particular from plasma, represent an established alternative to provide EGFR mutational testing for treatment decision making. In addition, a new paradigm for TKIs resistance management was recently approved by Food and Drug Administration, supporting the liquid biopsy based genotyping prior to tissue based genotyping for the detection of T790M mutation to select patients for third generation TKIs. In these settings, real time PCR (RT-PCR) and digital PCR ’targeted’ methods, which detect known mutations by specific probes, have extensively been adopted. Taking into account the restricted reference range and the limited multiplexing power of these targeted methods, the performance of liquid biopsy analyses may be further improved by next generation sequencing (NGS). While most tissue based NGS genotyping is well established, liquid biopsy NGS application is challenging, requiring a careful validation of the whole process, from blood collection to variant calling. Here we review this evolving field, highlighting those methodological points that are crucial to accurately select NSCLC patients for TKIs treatment administration by NGS on cfDNA.

Survival is poorer in elderly colorectal cancer (CRC) pa- tients, and it is impaired by lymph node spread and distant metastases. Nevertheless, patients aged below 45 years have lower 5-year survival rates compared with those aged between 45 and 54 years, and comparable to those of patients aged 55 - 74 years. Hence, a different tumor biology may underlie CRC development in persons with different ages. It is difficult to estimate from available statistics the risk of mortality strictly related to CRC itself, rather than to confounding factors. Older patients have more comorbidities and are more frail compared with younger ones, and the role of response to treatment stress and of potential treatment harms in determining mortality ac- cording to age are difficult to be obtained. We recently observed that UbcH10 protein is overexpressed in CRC patients and has an important role in affecting tumor growth and sensitivity to treatments [ 1]. We aimed to assess the expression of UbcH10 in CRC cancer samples as compared with healthy tissue sample from the same patient according to age at surgery, and to investigate the potential implications in the clinical management of young and elderly patients.

AimsThe incidence of RAS/RAF/PI3KA and TP53 gene mutations in colorectal cancer (CRC) is well established. Less information, however, is available on other components of the CRC genomic landscape, which are potential CRC prognostic/predictive markers.MethodsFollowing a previous validation study, ion-semiconductor next-generation sequencing (NGS) was employed to process 653 routine CRC samples by a multiplex PCR targeting 91 hotspot regions in 22 CRC significant genes.ResultsA total of 796 somatic mutations in 499 (76.4%) tumours were detected. Besides RAS/RAF/PI3KA and TP53, other 12 genes showed at least one mutation including FBXW7 (6%), PTEN (2.8%), SMAD4 (2.1%), EGFR (1.2%), CTNNB1 (1.1%), AKT1 (0.9%), STK11 (0.8%), ERBB2 (0.6%), ERBB4 (0.6%), ALK (0.2%), MAP2K1 (0.2%) and NOTCH1 (0.2%).ConclusionsIn a routine diagnostic setting, NGS had the potential to generate robust and comprehensive genetic information also including less frequently mutated genes potentially relevant for prognostic assessments or for actionable treatments.

Merkel cell carcinoma (MCC) is a rare neuroendocrine carcinoma of the skin, originally described by Toker in 1972, showing highly aggressive behavior and frequent local, regional, and distant spread.1 The etiology of MCC is unknown; however, risk factors include increased age, immunosuppression, and radiation exposure.2 More recently, a viral etiology has been considered since a new polyomavirus, i.e. Merkel cell polyomavirus, was isolated from MCC tumor tissues.3 MCC metastases to the pancreas are uncommonly observed and may represent a diagnostic challenge, in particular on cytology specimens, which are frequently the only specimens available from a suspicious pancreatic mass.4, 5 Here we report a case of MCC metastatic to the body and tail of the pancreas, diagnosed by endoscopic ultrasound‐guided fine‐needle aspiration (EUS‐FNA).

In advanced non-small-cell lung carcinomas epidermal growth factor receptor (EGFR) and KRAS testing is often performed on cytology. Liquid-based cytology (LBC), which eliminates the need for slide preparation by clinicians, may be very useful. In 42 LBC DNA was extracted twice. One sample was obtained directly from CytoLyt solution, whereas the other DNA sample was derived after smear preparation and laser capture microdissection (LCM) of Papanicolaou-stained cells. EGFR and KRAS mutational analyses were performed by direct sequencing. On CytoLyt-derived DNA four EGFR (9%) and five KRAS (12%) gene mutations were found. When direct sequencing was performed after LCM, the rate of cases that displayed either EGFR or KRAS mutations increased from 21% to 40%. Although time-consuming, LCM makes direct sequencing highly sensitive even on LBC preparations containing only a few cells.

Objective: Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) cytology is an effective tool to diagnose pancreatic ductal adenocarcinoma (PDA). Standard morphological criteria are usually reliable. When contaminating gastrointestinal (GI) epithelial cells are prevalent among neoplastic cells, these can be highlighted by carcinoembryonic antigen (CEA) staining. CD10 is a cell-surface metallopeptidase normally expressed by the GI epithelial apical border, whose expression is decreased or lost in PDA. We included CD10 in a panel, together with CEA, to discriminate the GI contaminant cells from PDA cells on cell blocks. Methods: Eight cases of EUS-FNA of PDA, featuring both contaminating GI cells and neoplastic cells, whose corresponding cell blocks were available for immunostaining, were selected. CD10 and CEA were stained on cell blocks by standard methods. Results: CD10 strongly labelled only the GI cells, with a well-defined apical membrane signal; conversely, GI cells did not show CEA staining; benign duodenal cells were faintly labelled in only one case. Malignant cells were positive for CEA and negative for CD10, with the exception of one case with labelled neoplastic cells with weak diffuse cytoplasmic positivity. CD10 apical membrane staining was a feature only seen in benign GI cells. Conclusions: As a loss of CD10 is a consistent feature of PDA, this marker can be useful, together with CEA, to aid the cytopathologist to identify neoplastic cells in a background rich in GI contaminant cells. © 2014 John Wiley & Sons Ltd.

Objective: Guidelines from the College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC) and the Association for Molecular Pathology (AMP) consider cytology suitable for testing epidermal growth factor receptor (EGFR) mutations in lung adenocarcinoma. The guidelines recommend that cytopathologists first discuss the possibility of testing squamous cell carcinomas (SqCC) in multidisciplinary meetings. Second, cell blocks should be analysed rather than smear preparations and, third, specimens should be sent to external molecular laboratories within three working days of receiving requests. This study monitored how these recommendations are met in practice. Methods: Our laboratory received 596 requests from cytologists from 13 different institutions. For each case, the cytological diagnosis, cytopreparation type, and time between the request and sample mailing were compared with the recommendations. Results: Of the 596 samples, 32 (5.4%) had been reported as SqCC. Three of these (9.4%) showed EGFR mutation. Cytological slides, either ThinPrep TM (51.2%) or direct smears (43.2%), weremore frequently received than cell blocks (5.7%). The mean time between the oncologist's request and specimen dispatching was 5.8 working days. Conclusions: The occurrence of mutations in samples reported as SqCC was higher than expected. This questions the reliability of the original diagnosis, which reinforced the recommendation to evaluate the opportunity for testing non-adenocarcinoma cytology on a case-by-case basis. In spite of CAP/IASLC/AMP recommendations, cell blocks were underutilized for EGFR testing, but cytological slides were suitable for DNA analyses. Significant efforts are needed to avoid delays in outsourcing cytological samples for EGFR testing.

Objectives: In this study we reviewed our practice of lung cancer epidermal growth factor receptor (EGFR) mutational testing in an academic centralized laboratory setting, where direct smears and liquid-based cytology (LBC) slides represent the most frequent cytological specimens received. The aim was to assess the differences, if any, between these sample types in terms of DNA yield, adequacy rates and overall EGFR testing performance. Study Design: A total of 362 cases were retrieved - received from January 2012 to January 2014 for EGFR testing - including 204 LBC specimens and 158 smears. Exon 19 deletions and the L858R point mutation in exon 21, detected by fragment assay and TaqMan assay, respectively, were confirmed by direct sequencing or by high-resolution melting. Results: Although the direct smears showed a higher DNA yield (60.94 vs. 23.07 ng/µl) and were more frequently cell-rich (54%) than the LBC slides (31.4%), the differences in adequacy (direct smears: 97.4%; LBCs: 94.1%) and in mutant rate (direct smears: 10.3%; LBCs: 14.0%) between the two sample types did not reach statistical significance. Conclusions: Not only direct smears but also LBC slides represent an effective preparation and storage medium for cytological material to be used for EGFR molecular testing.

Background: Warthin tumor (WT) is a common parotid lesion reliably diagnosed by fine-needle aspiration (FNA). Worrisome metaplastic changes may occur in WT. Their interpretation as mucoepidermoid carcinoma represents a diagnostic pitfall. Moreover, WT and mucoepidermoid carcinoma may coexist, making this distinction difficult. So it is worthwhile to report unusual WT features. We describe a WT with signet-ring cells (SRCs). Case: A 61-year-old male presenting with a 3.6-cm right parotid gland mass underwent FNA with rapid on-site evaluation. DiffQuik-stained smears showed groups of oncocytic cells with abundant granular cytoplasm in a background rich with debris, foamy macrophages and lymphoid cells. SRCs were observed on Papanicolaou-stained smears prepared from a second pass. A WT with SRC features was diagnosed. Histology revealed a WT with post-FNA infarctual changes. Conclusion: To avoid false-positive diagnoses, the cytopathologist should be aware that SRC features may occur in WT. The concomitant presence of oncocytes and SRCs is useful to correctly diagnose this unusual WT variant.

Background: Papillary thyroid carcinoma (PTC) with prominent hobnail features is a recently recognized PTC variant. Its histological hallmark is represented by elongated cells showing a high nuclear/cytoplasmic ratio and a hobnail appearance. Few histological studies have been performed showing aggressive clinical and pathological features. Thus, a better patient management might benefit from its early diagnosis on fine needle aspiration (FNA) samples. To date, the FNA cytology of PTC with prominent hobnail features has not been described. Case Report: We retrospectively analyzed the FNA taken from a histologically proven PTC with prominent hobnail features. Although there was a certain degree of morphological overlap with the tall cell variant, some peculiar cytological features were observed. In particular, the novel ‘comet-like’ cell feature could represent the cytological counterpart of the histological hobnailing. V600E BRAF mutation was observed on a matched cytohistological specimen. Conclusion: Further investigation is required to validate our diagnostic criteria, as the recognition of PTC with prominent hobnail features on FNA may prospectively enable its preoperative diagnosis, suggesting more aggressive neck surgery.

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.

Fine-needle aspiration (FNA) diagnosis of oncocytic lesions is challenging. In fact, oncocytic changes occur in inflammatory, hyperplastic, and neoplastic settings, including both benign and malignant tumors. The rare oncocytic variant of papillary thyroid carcinoma (PTC), shows papillae composed by cells with large oncocytic granular cytoplasm featuring clear PTC nuclear features. A morphological similar, but biologically distinct lesion, is the encapsulated papillary oncocytic neoplasia. Here, we first report on FNA, its cytological features together with histological, immunohistochemical, and molecular correlates. Diagn. Cytopathol. 2011;. © 2011 Wiley-Liss, Inc.

Recent therapeutic progresses in nonsmall cell lung cancer (NSCLC) and in colorectal cancer (CRC) are based on agents that specifically target the epidermal growth factor receptor (EGFR). To identify the patients most likely to benefit from such therapies, EGFR or KRAS gene mutation tests are mandatory, respectively, in NSCLC and in CRC. In patients with locally advanced or metastatic disease, exploiting cytological samples for these tests avoids not curative surgery. Here, we review the studies that have applied gene mutation assays on cytological samples of NSCLC and CRC to select patients for anti-EGFR therapy. We argue that the standard of quality of gene mutation tests on cytological samples is closely dependent on the extent of the cytopathologist's involvement.

A 63-year-old woman underwent urgent coronary angiography after failed fibrinolysis for an inferior ST-segment elevation acute myocardial infarction. The occlusion of the obtuse marginal branch (Thrombolysis in Myocardial Infarction flow 0 to 1) was observed (Figure 1A). The aspiration with the Diver CE catheter (Invatec, Italy) resulted in a huge amount of a soft, tubular tissue (Figure 2) with eventual complete flow restoration (Thrombolysis in Myocardial Infarction flow 3) (Figure 1B). The intravascular ultrasound (iLab System, Boston Scientific, Minneapolis, Minn) revealed the presence of the intimal flap in the proximal segment of the obtuse marginal branch. The false lumen was almost completely filled by thrombus (Figure 3). The thin strut cobalt-chromium Carbofilm-coated Chrono stent (3.0×2.5 mm; CID, …

We report the history of a pitfall in thyroid histopathology of a 47-yr-old man with a euthyroid nodular goiter involving the right lobe. The ultrasound-guided fineneedle aspiration of the dominant (3 cm) nodule, showing benign-appearing follicular cells, colloid, and scattered Hurthle cells, was consistent with a benign hyperplastic nodule (1). Because the left lobe had not shown evidence of nodular disease, the patient was submitted to a right lobectomy. The pathologist described a heavy lymphoplasmacyticbackgroundwithoccasionalgerminalcenter formation, as observed in Hashimoto thyroiditis, and a microscopic ( 1cm) follicularproliferationofepithelial thyroidcellswhose nucleidisplayedmembrane irregularityandchromatinclearing with occasional grooves. Thus, a histological report of a follicular variant of papillary microcarcinoma was issued. The patient underwent a completion thyroidectomy at the Federico II UniversityofNaples.At thatoccasion, thecompletesetofslides from both surgeries was reviewed by expert pathologists in the field of thyroid disease.