Approach to FNA of Pancreatic Cysts.

Pancreatic cysts are increasingly detected on imaging studies. Accurate determination of the type of cyst is important to provide appropriate care for patients. It is also very clear that not one single modality can provide adequate diagnostic information for pancreatic cysts. A multimodal approach to the diagnosis of pancreatic cyst is the key. This review will highlight how to approach to fine-needle aspiration of pancreatic cysts. The review will also highlight salient features of common neoplastic pancreatic cysts along with the use of ancillary testing which includes biochemical testing, commonly utilized molecular tests, and/or immunohistochemical tests to provide an accurate diagnosis.

Comparing Needles and Methods of Endoscopic Ultrasound-Guided Fine-Needle Biopsy to Optimize Specimen Quality and Diagnostic Accuracy for Patients With Pancreatic Masses in a Randomized Trial.

BACKGROUND & AIMS: Given the lack of procedure standardization, findings vary from analyses of pancreatic tissues collected by endoscopic ultrasound-guided fine-needle biopsy. It is not clear which needle and technique yield the best specimen for analysis. We compared the specimen quality and accuracy of diagnoses made from samples collected by fine-needle biopsy needles using different collection techniques. METHODS: Patients found to have pancreatic masses during imaging (n = 129) were assigned randomly to groups from whom pancreatic tissue samples were collected by reverse-bevel, Menghini-tip, franseen, or fork-tip needles. A second randomization determined the technical sequence of biopsies in each patient (suction, no suction, and stylet retraction). Two independent pathologists, blinded to the type of needle and sampling technique, analyzed all the samples. Final diagnoses of malignancy were made based on surgical resection, death from cancer progression, or findings from radiology or clinical follow-up evaluations (reference standard). The primary objective was to compare the cellularity of the samples collected, defined as the proportion of core tissue in the biopsy sample. Secondary objectives were to compare the accuracy of diagnoses made from biopsy samples and identify factors associated with high cellularity. RESULTS: One-hundred and nine patients had a final diagnosis of malignancy (84.5%) and 20 patients had benign disease (15.5%). Samples collected by fork-tip or franseen needles had significantly higher cellularity than samples collected by reverse-bevels or Menghini-tip needles (P < .001). Neoplasias were identified with greater than 90% accuracy using samples collected by fork-tip or franseen needles (P < .001 compared with other needles). On multivariable regression analysis, use of franseen needles (odds ratio [OR], 4.42; 95% CI, 2.58-7.58; P < .001) or fork-tip needles (OR, 3.86; 95% CI, 2.24-6.64; P < .001), stylet retraction (OR, 2.13; 95% CI, 1.21-3.72; P = .008), no suction (OR, 2.74; 95% CI, 1.57-4.80; P < .001), and pancreatic mass larger than 3 cm (OR, 1.92; 95% CI, 1.21-3.05; P = .005) were associated with high cellularity of the sample. CONCLUSIONS: In patients with suspected pancreatic cancer, samples with the highest degree of cellularity in a single biopsy, resulting in a diagnostic accuracy of 90% of higher, were collected by fine-needle biopsy using the franseen or fork-tip needle. Clinicaltrials.gov no: NCT04085055.

Cytoplasmic PARP-1 promotes pancreatic cancer tumorigenesis and resistance.

The poly(ADP-ribose) polymerases (PARP) play important roles in repairing damaged DNA during intrinsic cell death. We recently linked PARP-1 to death receptor (DR)-activated extrinsic apoptosis, the present studies sought to elucidate the function of cytoplasmic PARP-1 in pancreatic cancer tumorigenesis and therapy. Using human normal and pancreatic cancer tissues, we analyzed the prevalence of cytoplasmic PARP-1 expression. In normal human pancreatic tissues, PARP-1 expression was present in the nucleus; however, cytoplasmic PARP-1 expression was identified in pancreatic cancers. Therefore, cytoplasmic PARP-1 mutants were generated by site-direct mutagenesis, to determine a causative effect of cytoplasmic PARP-1 on pancreatic cancer tumorigenesis and sensitivity to therapy with TRA-8, a humanized DR5 antibody. PARP-1 cytoplasmic mutants rendered TRA-8 sensitive pancreatic cancer cells, BxPc-3 and MiaPaCa-2, more resistant to TRA-8-induced apoptosis; whereas wild-type PARP-1, localizing mainly in the nucleus, had no effects. Additionally, cytoplasmic PARP-1, but not wild-type PARP-1, increased resistance of BxPc-3 cells to TRA-8 therapy in a mouse xenograft model in vivo. Inhibition of PARP enzymatic activity attenuated cytoplasmic PARP-1-mediated TRA-8 resistance. Furthermore, increased cytoplasmic PARP-1, but not wild-type PARP-1, was recruited into the TRA-8-activated death-inducing signaling complex and associated with increased and sustained activation of Src-mediated survival signals. In contrast, PARP-1 knockdown inhibited Src activation. Taken together, we have identified a novel function and mechanism underlying cytoplasmic PARP-1, distinct from nuclear PARP-1, in regulating DR5-activated apoptosis. Our studies support an innovative application of available PARP inhibitors or new cytoplasmic PARP-1 antagonists to enhance TRAIL therapy for TRAIL-resistant pancreatic cancers.

Celiac Disease: Updates on Pathology and Differential Diagnosis.

Celiac disease is a gluten-triggered immune-mediated disorder, characterized by inflammation of the enteric mucosa following lymphocytic infiltration and eventually resulting in villous blunting. There have been many developments in refining diagnostic laboratory tests for celiac disease in the last decade. Biopsy-sparing diagnostic guidelines have been proposed and validated in a few recent prospective studies. However, despite these developments, histologic evaluation of duodenal mucosa remains one of the most essential diagnostic tools as it helps in the diagnosis of celiac disease in individuals who do not fulfill the biopsy-sparing diagnostic criteria and in those not responding to a gluten-free diet. Histologic evaluation also allows for the assessment of mucosal recovery after treatment and in the identification of concurrent intestinal diseases. Therefore, pathologists should be familiar with the histologic spectrum of celiac disease and need to be aware of other disorders with similar symptoms and histopathology that may mimic celiac disease. This review aims to provide pathologists with updates on celiac laboratory testing, biopsy-sparing diagnostic criteria, histopathology, complications, and differential diagnoses of celiac disease.

Modeling Esophagitis Using Human Three-Dimensional Organotypic Culture System.

Esophagitis, whether caused by acid reflux, allergic responses, graft-versus-host disease, drugs, or infections, is a common condition of the gastrointestinal tract affecting nearly 20% of the US population. The instigating agent typically triggers an inflammatory response. The resulting inflammation is a risk factor for the development of esophageal strictures, Barrett esophagus, and esophageal adenocarcinoma. Research into the pathophysiology of these conditions has been limited by the availability of animal and human model systems. Three-dimensional organotypic tissue culture (OTC) is an innovative three-dimensional multicellular in vitro platform that recapitulates normal esophageal epithelial stratification and differentiation. We hypothesized that this platform can be used to model esophagitis to better understand the interactions between immune cells and the esophageal epithelium. We found that human immune cells remain viable and respond to cytokines when cultured under OTC conditions. The acute inflammatory environment induced in the OTC significantly affected the overlying epithelium, inducing a regenerative response marked by increased cell proliferation and epithelial hyperplasia. Moreover, oxidative stress from the acute inflammation induced DNA damage and strand breaks in epithelial cells, which could be reversed by antioxidant treatment. These findings support the importance of immune cell-mediated esophageal injury in esophagitis and confirms the utility of the OTC platform to characterize the underlying molecular events in esophagitis.

Surveying the serologic proteome in a tissue-specific kras(G12D) knockin mouse model of pancreatic cancer.

We have applied a serologic proteomic workflow involving three complementary MS approaches to a tissue-specific Kras(G12D) -knockin mouse model of pancreatic cancer that consistently forms precancerous lesions by 4 months of age. The three proteomics applications were highly complementary and allowed us to survey the entire range of low to high molecular weight serologic proteins. Combined, we identified 121 (49↓, 72↑) unique and statistically relevant serologic biomarkers with 88% previously reported to be associated with cancer and 38% specifically correlated with pancreatic cancer. Four markers, lysozyme C2, cytokeratin 19, Serpina1A and Pcf11, were further verified by Western blotting. When applying systems analysis, the top-associated gene ontology functions were tied to wound healing, RXR signaling, growth, differentiation and innate immune activation through the JAK/STAT pathway. Upon further investigation of the apparent immune response using a multiplex cytokine screen, we found that IFN-γ, VEGF and GM-CSF were significantly increased in serum from the Kras(G12D) animals compared to littermate controls. By combining three complementary MS applications, we were able to survey the native intact peptidome and the global proteome in parallel, unveiling pathways that may be biologically relevant to promotion of pancreatic cancer progression and serologic markers of noninvasive early-stage neoplasia.

Sclerosing epithelioid fibrosarcoma of the pancreas.

Sclerosing epithelioid fibrosarcoma (SEF) is a distinctive variant of fibrosarcoma characterized by epithelioid tumor cells arranged in nests, cords, or sheets embedded within a sclerotic collagenous matrix. It is a relatively newly described malignant fibroblastic tumor, with only fewer than 100 cases reported in English literature. Most cases are located in the lower extremities and limb girdles. Here, we present a case of SEF of the pancreas in a 67-year-old white man and provide a review of literature to date, with emphasis on the differential diagnosis. To our knowledge, this is the first reported case of SEF involving the pancreas.

The Critical Value of Telepathology in the COVID-19 Era.

Background: Telepathology, which includes the use of telecommunication links, helps enable transmission of digital pathology images for primary diagnosis, quality assurance, education, research, or second opinion diagnoses. Observations: This review covers all aspects of telepathology implementation, including the selection of platforms, budgets and regulations, validation, implementation, education, quality monitoring, and the potential to improve practice. Considering the long-term trends, the lessons of the COVID-19 pandemic, and the potential for future pandemics or other disasters, the validation and implementation of telepathology remains a reasonable choice for laboratories looking to improve their practice. Conclusions: Though barriers to implementation exist, there are potential benefits, such as the wide spectrum of uses like frozen section, telecytology, primary diagnosis, and second opinions. Telepathology represents an innovation that may transform the future of pathology practice.

Caspase-11 promotes high-fat diet-induced NAFLD by increasing glycolysis, OXPHOS, and pyroptosis in macrophages.

Introduction: Non-alcoholic fatty liver disease (NAFLD) has a global prevalence of 25% of the population and is a leading cause of cirrhosis and hepatocellular carcinoma. NAFLD ranges from simple steatosis (non-alcoholic fatty liver) to non-alcoholic steatohepatitis (NASH). Hepatic macrophages, specifically Kupffer cells (KCs) and monocyte-derived macrophages, act as key players in the progression of NAFLD. Caspases are a family of endoproteases that provide critical connections to cell regulatory networks that sense disease risk factors, control inflammation, and mediate inflammatory cell death (pyroptosis). Caspase-11 can cleave gasdermin D (GSDMD) to induce pyroptosis and specifically defends against bacterial pathogens that invade the cytosol. However, it's still unknown whether high fat diet (HFD)-facilitated gut microbiota-generated cytoplasmic lipopolysaccharides (LPS) activate caspase-11 and promote NAFLD. Methods: To examine this hypothesis, we performed liver pathological analysis, RNA-seq, FACS, Western blots, Seahorse mitochondrial stress analyses of macrophages and bone marrow transplantation on HFD-induced NAFLD in WT and Casp11-/- mice. Results and Discussion: Our results showed that 1) HFD increases body wight, liver wight, plasma cholesterol levels, liver fat deposition, and NAFLD activity score (NAS score) in wild-type (WT) mice; 2) HFD increases the expression of caspase-11, GSDMD, interleukin-1ß, and guanylate-binding proteins in WT mice; 3) Caspase-11 deficiency decreases fat liver deposition and NAS score; 4) Caspase-11 deficiency decreases bone marrow monocyte-derived macrophage (MDM) pyroptosis (inflammatory cell death) and inflammatory monocyte (IM) surface GSDMD expression; 5) Caspase-11 deficiency re-programs liver transcriptomes and reduces HFD-induced NAFLD; 6) Caspase-11 deficiency decreases extracellular acidification rates (glycolysis) and oxidative phosphorylation (OXPHOS) in inflammatory fatty acid palmitic acid-stimulated macrophages, indicating that caspase-11 significantly contributes to maintain dual fuel bioenergetics-glycolysis and OXPHOS for promoting pyroptosis in macrophages. These results provide novel insights on the roles of the caspase-11-GSDMD pathway in promoting hepatic macrophage inflammation and pyroptosis and novel targets for future therapeutic interventions involving the transition of NAFLD to NASH, hyperlipidemia, type II diabetes, metabolic syndrome, metabolically healthy obesity, atherosclerotic cardiovascular diseases, autoimmune diseases, liver transplantation, and hepatic cancers.

Mitochondria-targeted ubiquinone (MitoQ) decreases ethanol-dependent micro and macro hepatosteatosis.

UNLABELLED: Chronic alcohol-induced liver disease results in inflammation, steatosis, and increased oxidative and nitrosative damage to the mitochondrion. We hypothesized that targeting an antioxidant to the mitochondria would prevent oxidative damage and attenuate the steatosis associated with alcoholic liver disease. To test this we investigated the effects of mitochondria-targeted ubiquinone (MitoQ) (5 and 25 mg/kg/day for 4 weeks) in male Sprague-Dawley rats consuming ethanol using the Lieber-DeCarli diet with pair-fed controls. Hepatic steatosis, 3-nitrotyrosine (3-NT), 4-hydroxynonenal (4-HNE), hypoxia inducible factor α (HIF1α), and the activity of the mitochondrial respiratory chain complexes were assessed. As reported previously, ethanol consumption resulted in hepatocyte ballooning, increased lipid accumulation in the form of micro and macrovesicular steatosis, and induction of cytochrome P450 2E1 (CYP2E1). MitoQ had a minor effect on the ethanol-dependent decrease in mitochondrial respiratory chain proteins and their activities; however, it did decrease hepatic steatosis in ethanol-consuming animals and prevented the ethanol-induced formation of 3-NT and 4-HNE. Interestingly, MitoQ completely blocked the increase in HIF1α in all ethanol-fed groups, which has previously been demonstrated in cell culture models and shown to be essential in ethanol-dependent hepatosteatosis. CONCLUSION: These results demonstrate the antioxidant capacity of MitoQ in alleviating alcohol-associated mitochondrial reactive oxygen species (ROS) and several downstream effects of ROS/RNS (reactive nitrogen species) production such as inhibiting protein nitration and protein aldehyde formation and specifically ROS-dependent HIF1α stabilization.

Dynamic telecytology compares favorably to rapid onsite evaluation of endoscopic ultrasound fine needle aspirates.

BACKGROUND AND AIMS: Rapid onsite evaluation (ROSE) has been demonstrated to correlate with final cytologic interpretations and improves the diagnostic yield of endoscopic ultrasound (EUS)-fine needle aspiration (FNA); however, its availability is variable across centers. The aim of this prospective study was to evaluate whether remote telecytology can substitute for ROSE. METHODS: Consecutive patients who underwent EUS-FNA for diverse indications at a high volume referral center were enrolled and all samples were prospectively evaluated by three methods. ROSE was performed by a cytopathologist in the procedure room; simultaneously dynamic telecytology was done by a different cytopathologist in a remote location at our institution. The third method, final cytologic interpretation in the laboratory, was the gold standard. Telecytology was performed using an Olympus microscope system (BX) which broadcasts live images over the Internet. Accuracy of telecytology and agreement with other methods were the principle outcome measurements. RESULTS: Twenty-five consecutive samples were obtained from participants 40-87 years old (median age 63, 48 % male). There was 88 % agreement between telecytology and final cytology (p < 0.001) and 92 % agreement between ROSE and final cytology (p < 0.001). There was consistency between telecytology and ROSE (p value for McNemar's χ(2) = 1.0). Cohen's kappa for agreement for telecytology and ROSE was 0.80 (SE = 0.11), confirming favorable correlation. CONCLUSION: Dynamic telecytology compares favorably to ROSE in the assessment of EUS acquired fine needle aspirates. If confirmed by larger trials, this system might obviate the need for onsite interpretation of EUS-FNA specimens.

Endoscopic ultrasound and endobronchial ultrasound-guided fine-needle aspiration of deep-seated lymphadenopathy: Analysis of 1338 cases.

BACKGROUND: We retrospectively studied 1338 samples of lymph nodes obtained by endoscopic and endobronchial ultrasound-guided fine needle aspiration biopsy (EUS and EBUS-FNAB) with an objective of characterizing the utility of this diagnostic modality in the assessment of deep-seated lymphadenopathy. The secondary aims were to establish the utility in the diagnosis of lymphoma and to determine the number of passes required to obtain adequate cellularity for flow cytometric analysis. MATERIALS AND METHODS: On-site assessment was performed by a cytopathologist using Diff-Quik (American Scientific Products, McGraw Park, IL) stain. In addition, Papanicolaou and immunohistochemical stains were performed and additional samples were sent for flow cytometric analyses (n = 145). The final cytologic diagnosis was correlated with surgical pathology diagnosis and/or clinical follow-up. In select cases, fluorescence in situ hybridization analysis with specific probes was performed on Diff-Quik smears. RESULTS: Both morphology as well as ancillary studies (flow cytometry or immunohistochemical stain and/or fluorescence in situ hybridization) show that EUS and EBUS-FNA are effective techniques to detect and stage intrathoracic and intra-abdominal tumors. Operating characteristics show that these are highly sensitive (89%) and specific (100%) techniques for the diagnosis of lymphoma. At least two passes provided an average of 5.66 million cells (range, 0.12-62.32 million) for lymphoma cases. CONCLUSIONS: EUS and EBUS-FNA are powerful modalities to stage malignancies and at least two passes can provide adequate cells for flow cytometric analysis. We also demonstrate that fluorescence in situ hybridization analysis can be performed on Diff-Quik-stained and mounted smears.

TeleCyP (Telecytopathology): real-time fine-needle aspiration interpretation.

OBJECTIVE: To assess the application of TeleCyP for real-time fine-needle aspiration interpretation (RFI) necessary for case management and specimen triage. STUDY DESIGN: Twenty-two endobronchial ultrasound (EBUS)-guided mediastinal and pulmonary cases were included in the learning phase to determine the time and efficiency of TeleCyP. Slides were scanned by a cytopathology fellow in real time, and high-speed transmitted images over a secure network were interpreted by a cytopathologist while maintaining audio communication. In the validation phase, an additional 38 pancreas cases from endoscopic ultrasound (EUS) were evaluated recapitulating the RFI scenario from the learning phase. The cytopathologist was blinded to the results of the diagnosis in both phases. RESULTS: The time to provide assessment of specimen adequacy and a preliminary diagnosis was 53 s in the learning phase and 49 s in the validation phase. There was 100% correlation between RFI and TeleCyP assessment for specimen adequacy. TeleCyP particularly posed challenges in providing definitive interpretation on EUS-fine-needle aspiration of some of the pancreatic solid masses (11%, 4/36). CONCLUSION: TeleCyP can serve as a powerful alternative, time-efficient strategy to provide RFI, and for specimen triaging which is critical for personalized medicine and patient management.

Serous cavity metastasis: Evaluation of unknown primary.

Malignant effusions can occur in patients with neoplasia. Once a metastatic diagnosis is confirmed, the primary site of origin of malignancy needs to be ascertained. This task can be challenging without a prior history of malignancy. In some patients their effusion may be the initial presentation of an underlying malignancy. Metastases usually present with a dual population of mesothelial and malignant cells. Combining cytomorphologic examination with ancillary testing such as immunocytochemistry can help identify the origin of the foreign malignant cell population. Helpful architectural clues include a single cell pattern, solid cell ball pattern, single file arrangement, papillary formation, psammoma bodies and background mucin. Useful cellular features include the presence of signet ring cells, small cells, pleomorphic and multinucleated giant cells, squamous cells, spindle cells and pigmentation. Rarely, despite an extensive work-up the primary site of origin for a malignant effusion may remain unresolved. This review article will be incorporated finally as one of the chapters in CMAS (CytoJournal Monograph/Atlas Series) #2. It is modified slightly from the chapter by the initial authors in the first edition of Cytopathologic Diagnosis of Serous Fluids.

Serous fluid: Reactive conditions.

This chapter highlights the steps that would help to analyze any fluid. It highlights importance of knowing gross analysis of fluid along with biochemical information. These parameters along with clinical information are very important in arriving at a differential diagnosis. Morphologic appearances in the fluid can often become challenging and occasionally reactive conditions can reveal changes that may mimic malignancies. This chapter provides not only a framework of approach to assessment of fluid cytology but also shows how to distinguish some of the challenging reactive conditions from the diagnosis of carcinoma. The chapter also utilizes two cases to demonstrate approach to reactive conditions. This review article will be incorporated finally as one of the chapters in CMAS (CytoJournal Monograph/Atlas Series) #2. It is modified slightly from the chapter by the initial authors in the first edition of Cytopathologic Diagnosis of Serous Fluids.

Dr. P. V. Gharpure - An eminent Indian pathologist and a pioneer in starting oral polio vaccination in India.

Dr. Purushottam Vishwanath Gharpure was an eminent Indian pathologist and an emeritus Professor of Pathology at the Grant Medical College, Bombay. He was a pioneer in carrying out the first field trial of polio vaccination which marked the beginning of the polio eradication program in India. Dr. Gharpure set an example by taking his laboratory work to the field and proving how the laboratory research can be used to better the society. The mesmerizing story of Dr. "Gharpure's life" is described in this paper.

Tamoxifen enhances therapeutic effects of gemcitabine on cholangiocarcinoma tumorigenesis.

Cholangiocarcinoma is a highly malignant tumor with limited therapeutic options. We have previously reported that tamoxifen (TMX) induces apoptosis of cholangiocarcinoma cells and reduces cholangiocarcinoma tumorigenesis in mice. In the present studies, we determined the effect of combination therapy of TMX and gemcitabine (GMT), another chemotherapeutical reagent for many cancers, on cholangiocarcinoma tumorigenesis and investigated the responsible mechanisms. GMT inhibited cell growth and induced apoptosis of cholangiocarcinoma cells in a concentration-dependent manner. TMX enhanced GMT-induced apoptosis of cholangiocarcinoma cells. Consistently, GMT (15 mg/kg) inhibited cholangiocarcinoma tumorigenesis in nude mice by 50%. TMX (15 mg/kg) enhanced the inhibitory effect of GMT on tumorigenesis by 33%. The inhibition of tumor growth correlated with enhanced apoptosis in tumor tissues. To elucidate the mechanisms underlying the additive effects of TMX on GMT-induced apoptosis, we determined the activation of caspases in cholangiocarcinoma cells exposed to GMT, TMX, or both. Activation of caspases 9 and 3, as well as cytochrome c release to the cytosol, was demonstrated in cells exposed to both reagents. In contrast, TMX activated caspase 2, whereas GMT had no effect. Inhibition of caspase 2 activation decreased TMX-, but not GMT-, induced activation of caspase 3 and apoptosis of cholangiocarcinoma cells. Similarly, activation of caspase 2 was found in tumors from TMX-treated mice, but not GMT-treated mice. Therefore, the enhanced effect of TMX on GMT-induced cholangiocarcinoma cell death is partially mediated by activation of caspase 2. TMX and GMT both induce apoptosis and inhibit cholangiocarcinoma tumorigenesis, which may be attributed to the activation of distinct apoptosis signals by TMX and GMT. Our studies provide in vivo evidence and molecular insight to support the use of TMX and GMT in combination as an effective therapy for cholangiocarcinoma.

Diagnosis of metastatic fibrolamellar hepatocellular carcinoma by endoscopic ultrasound-guided fine needle aspiration.

The fibrolamellar variant of hepatocellular carcinoma (FL-HCC) is distinguished from other hepatocellular carcinomas (HCC) by its unique clinical and pathologic features. Cytological features for this tumor on fine needle aspiration (FNA) of primary tumors have been described earlier. We present here a unique case of metastatic FL-HCC diagnosed by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) of mediastinal adenopathy. A 32-year-old woman with a history of oral contraceptive use presented with nausea and severe abdominal pain but no ascites or stigmata of cirrhosis. She had a past history of resection of a liver lesion. Serial computed tomography scans revealed mediastinal lymphadenopathy and the patient was referred for endoscopic ultrasound (EUS). A transesophageal EUS-FNA was performed and tissue was collected for cytological evaluation by an on-site pathologist with no knowledge of prior history. Based on morphology correlated with prior history received later, a final diagnosis of metastatic FL-HCC in the retrocardiac lymph node was rendered on the EUS-FNA samples. There are very few reports in the literature where a diagnosis of FL-HCC is rendered at unusual sites. This case highlights that EUS-FNA is a relatively non-invasive, rapid, accurate and effective modality in obtaining tissue from otherwise hard-to-reach areas. It also suggests that metastasis of FL-HCC can be observed in mediastinal nodes and that diagnosis based on cytological features can be rendered even when the tumor is identified at unusual locations.