Intra-institutional second opinion diagnosis can reduce unnecessary surgery for indeterminate thyroid FNA: A preliminary report on 34 cases.

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.

Cytopathologists can reliably perform ultrasound-guided thyroid fine needle aspiration: a 1-year audit on 3715 consecutive cases.

OBJECTIVE: In our Pathology Department, fine needle aspiration (FNA) of palpable thyroid nodules is performed 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 and radiologists in outside clinics, where the presence of a cytopathologist is not always feasible. To overcome this limitation, cytopathologists have started to perform US-guided FNAs themselves. This study retrospectively evaluates 1 year of this novel practice. METHODS: A total of 2225 US-guided FNAs were performed in our clinic by cytopathologists, whereas 1490 aspirates were taken by a group of non-cytopathologists. Among these, 756 FNAs were taken by a single experienced endocrinologist. The distribution of the Bethesda classification categories was evaluated in each of these groups. RESULTS: FNAs performed by cytopathologists were more often diagnostic and better prepared than those taken by non-cytopathologists, including those taken by the experienced endocrinologist (P < 0.01). The latter operator yielded a higher rate of suspicious and malignant FNAs, reflecting a more appropriate clinical triage of 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.

Challenges and opportunities of next-generation sequencing: a cytopathologist's perspective.

Molecular cytopathology has gene sequencing as its core technology. Until recently, cytological samples were only tested by sequential single-gene mutational tests. Today, with the better understanding of the molecular events involved in malignancy and the mechanisms of pharmacotherapy, larger gene panels are more informative than a single biomarker. Next-generation sequencing (NGS), matched with the multiplex capture of targeted gene regions and analysed by sophisticated bioinformatics tools, enables the simultaneous detection of multiple mutations in multiple genes. With the development of miniaturised technology and benchtop sequencers, it is not unlikely that NGS will soon be adopted for routine molecular diagnostics, including cytological samples. This review addresses (1) the most relevant methodological and technical aspects of the NGS analysis workflow and the diverse platforms available; (2) the issues related to daily practice implementation, namely, the cytological sample requirement and the validation procedures; and (3) the opportunities that NGS offers in different fields of cytopathology, to increase mutation detection sensitivity in paucicellular smears and to extend the analysis to a larger number of gene regions. Cytopathologists involvement and coordination in this rapidly evolving field is crucial for the effective implementation of NGS in the present and future cytological practice.

Outsourcing cytological samples to a referral laboratory for EGFR testing in non-small cell lung cancer: does theory meet practice?

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(™) (51.2%) or direct smears (43.2%), were more 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.

CD10 is useful to identify gastrointestinal contamination in endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) cytology from pancreatic ductal adenocarcinoma.

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.

Performance of EGFR mutant-specific antibodies in different cytological preparations: a validation study.

OBJECTIVES: Molecular testing for epidermal growth factor receptor (EGFR) mutations is required to select the most appropriate treatment for advanced-stage non-small cell lung cancer (NSCLC). In routine practice, cytological samples are often the only specimens available for testing. When the number of neoplastic cells is large, DNA-based assays are the gold standard. When cytological samples contain only a few neoplastic cells, immunocytochemistry (ICC) using anti-EGFR mutant-specific antibodies may be more effective. We aim to assess the specificity and sensitivity of IHC staining in cytological specimens using mutated cell lines subjected to different cytopreparations and staining methods. METHODS: HCC827 (exon 19 p.E746-A750 del) and H3255 (exon 21 p.L858R) cell lines were subjected to different fixation (air dried, alcohol or CytoLyt(®)), staining (Diff-Quik(®) or Papanicolaou) and preparation (smears or cell blocks) methods before ICC. In a second set of experiments, mutated cells were mixed with EGFR wild-type cells to obtain low-level (10%) mutated cytological samples. The intensity and percentage of cells stained were evaluated against validated molecular techniques. Moreover, the cell lines were subjected to poor growing conditions to simulate routine specimens that are less optimal than in vitro samples. RESULTS: The cytological preparations showing the most intense staining were formalin-fixed cell blocks and samples fixed with CytoLyt or alcohol, including Papanicolaou-destained samples. Conversely, air-dried slides showed the least intense staining. Mutant antibodies allowed the detection of mutated cells, even when representing only 10% of the total population. Although, in necrotic specimens, an aspecific background signal appeared, the viable cells still retained anti-mutant EGFR positivity. CONCLUSIONS: All cytological preparations are suitable for ICC using anti-EGFR mutant-specific antibodies, in particular formalin-fixed cell blocks and alcohol- or CitoLyt-fixed samples. the method is also validated to detect even a few mutant cells in less than optimal samples.

KRAS mutation detection by high-resolution melting analysis significantly predicts clinical benefit of cetuximab in metastatic colorectal cancer.

BACKGROUND: Anti-epidermal growth factor receptor (EGFR) monoclonal antibodies are restricted to KRAS wild-type (WT) metastatic colorectal cancers (mCRCs), usually identified by direct sequencing, that may yield false negative results because of genetic heterogeneity within the tumour. We evaluated the efficiency of high-resolution melting analysis (HRMA) in identifying KRAS-mutant (MUT) tumours. METHODS: We considered 50 mCRC patients scored as KRAS-WT by direct sequencing and treated with cetuximab-containing chemotherapy, and tested the correlations between HRMA findings and response rate (RR), progression-free (PFS) and overall survival (OS). RESULTS: Aberrant melting curves were detected in four (8%) cases; gene cloning confirmed these mutations. Response rate (RR) of HRMA KRAS-WT patients was 28.3%. There was no response in HRMA KRAS-MUT patients. Disease control rate (responsive plus stable disease) was 58.7% in HRMA KRAS-WT patients and 25% in HRMA KRAS-MUT patients. There was no correlation between HRMA KRAS status and RR (P=0.287) or disease control (P=0.219). Median PFS (4.8 vs 2.3 months; hazard ratio (HR)=0.29, P=0.02) and OS (11.0 vs 2.7 months; HR=0.11, P=0.03) were significantly longer for the HRMA KRAS-WT than for HRMA KRAS-MUT patients. CONCLUSIONS: High-resolution melting analysis identified 8% more KRAS-MUT patients not responding to cetuximab-containing regimens, suggesting that HRMA may be more effective than direct sequencing in selecting patients for anti-EGFR antibodies.

Herbal hepatotoxicity: a case of difficult interpretation.

Herbal medicines are widely used in the world and are generally considered effective and safe, although many studies have demonstrated their potential toxic effects, particularly for the liver. We present a case of a woman, who developed a mixed cholestatic/hepatocellular liver injury due to herbal products. Firstly, she was admitted to Division of Surgery for right upper abdominal pain and jaundice and, for the suspect of biliary obstruction, she underwent to cholecystectomy. For persistence of liver enzymes elevation, she was admitted to our Gastroenterology Unit. We excluded every etiologies of hepatitis and, after an intensive dialogue with the patient, we obtained a history of herbal medicines use. Then, we performed a liver biopsy which was compatible with hepatotoxic injury. Therapy with ursodeoxycholic acid (UDCA) was started. Liver function tests returned to normal in two months. We describe this clinical case to encourage the communication doctor/patient in phytotherapy area and physician knowledge about efficacy and side effects of herbal medicine to avoid delayed diagnosis.