Fine-needle Aspiration Samples Research Articles

An Overview of 10 Years of Activity of a Molecular Laboratory for Buruli Ulcer Diagnosis at a Field Hospital in Benin.

Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans. Early diagnosis is crucial to prevent morbidity. In November 2012, a field laboratory fully equipped for the rapid on-site quantitative PCR (qPCR) diagnosis of M. ulcerans was established at the Buruli ulcer treatment center (CDTLUB) center in Pobè Benin, a region where BU is endemic. We describe its first 10 years of activity and its gradual evolution into an expert laboratory for BU diagnosis. From 2012 to 2022, the laboratory analyzed 3,018 samples from patients attending consultations for suspected BU at the CDTLUB in Pobè. Ziehl-Neelsen staining and qPCR targeting the IS2404 sequence were performed. Since 2019, the laboratory has also received and analyzed 570 samples from other centers. The laboratory confirmed the diagnosis of BU by qPCR for 39.7% samples: M. ulcerans DNA was detected in 34.7% of swabs, 47.2% of all fine needle aspiration samples (FNA) and 44.6% of all skin biopsy specimens. Positive Ziehl-Neelsen staining results were obtained for 19.0% samples. Bacterial load, estimated by qPCR, was significantly greater for the Ziehl-Neelsen-positive samples than for Ziehl-Neelsen-negative samples, and detection rates were highest for FNA samples. Overall, 26.3% of the samples received from other centers were positive for BU. Most of these samples were sent by the CDTLUBs of Lalo, Allada, and Zagnanado, Benin. The establishment of the laboratory in the CDTLUB of Pobè has been a huge success. Optimal patient care depends on the close proximity of a molecular biology structure to BU treatment centers. Finally, FNA should be promoted among caregivers. IMPORTANCE Here, we describe the first 10 years of activity at a field laboratory established at the Buruli ulcer treatment center (CDTLUB) in Pobè, Benin, a country in which Mycobacterium ulcerans is endemic. Between 2012 and 2022, the laboratory analyzed 3,018 samples from patients consulting the CDTLUB of Pobè with a suspected clinical BU. Ziehl-Neelsen staining and qPCR targeting the IS2404 sequence were performed. In total, 39.7% of samples tested positive by qPCR and 19.0% tested positive by Ziehl-Neelsen staining. Detection rates were highest for FNA samples, and the bacterial loads estimated by qPCR were significantly higher for Ziehl-Neelsen-positive samples than for Ziehl-Neelsen-negative samples. Since 2019, the laboratory has also analyzed 570 samples received from outside the CDTLUB of Pobè, 26.3% of which were positive for BU. Most of these samples were sent by the CDTLUBs of Lalo, Allada, and Zagnanado in Benin. The establishment of the laboratory in the CDTLUB of Pobè has been a huge success, with major benefits for both the medical staff and patients. Our findings illustrate that the usefulness and feasibility of having a diagnostic center in rural Africa, where the disease is endemic, is a key part of optimal patient care, and that FNA should be promoted to increase detection rates.

Abstract #1384628: An Incidental Thyroid Lesion on FDG-PET Scan in a Patient with Long History of Hypothyroidism and Radiation Exposure

An 81-year-old woman with history of lung cancer and hypothyroidism with an incidental thyroid lesion on (FDG)-positron emission tomography (PET) scan is subsequently diagnosed with primary thyroid lymphoma (PTL) using flow cytometry. An 81-year-old woman with a history of lung cancer and hypothyroidism presented to the endocrinology office for evaluation of a thyroid lesion. Primary hypothyroidism was diagnosed 30 years prior to presentation and was controlled with levothyroxine 88 mcg daily. Lung cancer was diagnosed 8 years prior, and treated with radiation therapy. Patient had an FDG-PET scan obtained for the evaluation of lung nodules, and a hypermetabolic focus was noted in the left thyroid region with a max SUV of 18. On thyroid ultrasound, the left thyroid lobe appeared heterogenous with solid nodular areas. The physical exam revealed a smooth but firm thyroid gland, without cervical lymphadenopathy. Laboratory workup showed thyroid stimulating hormone and free thyroxine in the normal range. Microsomal antibody was 4.2 IU/mL (< 5.6 IU/mL), and thyroglobulin antibody was 356.4 IU/mL (< 14.4 IU/mL). Fine needle aspiration (FNA) biopsy showed a polymorphous lymphoid sample. Given the high SUV on PET-scan and longstanding history of Hashimoto’s thyroiditis, repeat FNA biopsy with flow cytometry was subsequently performed and showed a lymphoid population consistent with a non-Hodgkin lymphoma (NHL). The patient was referred to oncology and diagnosed with indolent NHL. Further staging revealed interval growth in lung nodules, with biopsy showing recurrence of adenocarcinoma, but no other metastatic disease. The patient underwent radiation therapy for both her lung adenocarcinoma and primary thyroid lymphoma (PTL). Primary thyroid lymphoma accounts for less than 5% of thyroid cancers. It presents with a female predominance in the 7th decade of life, with Hashimoto’s thyroiditis as the main risk factor. Patients may present with an enlarging neck mass, with or without compressive symptoms, and cervical lymphadenopathy. Diagnosis of PTL can be made through FNA biopsy, core biopsy, or open surgical biopsy. It is important to note that because these lesions have few pathognomonic features, diagnosis with FNA biopsy alone can be difficult, and sensitivity is increased to 97% with the use of flow cytometry, as was demonstrated in our case. Treatment is with radiation therapy or combined chemoradiotherapy. Surgery has a role limited to the management of airway compromise, as surgical resection has not proven to improve overall survival. This case highlights the utility of including flow cytometry with FNA sample when diagnosing PTL.

Molecular Profiling of 50 734 Bethesda III-VI Thyroid Nodules by ThyroSeq v3: Implications for Personalized Management.

Comprehensive genomic analysis of thyroid nodules for multiple classes of molecular alterations detected in a large series of fine needle aspiration (FNA) samples has not been reported. To determine the prevalence of clinically relevant molecular alterations in Bethesda categories III-VI (BCIII-VI) thyroid nodules. This retrospective analysis of FNA samples, tested by ThyroSeq v3 using Genomic Classifier and Cancer Risk Classifier at UPMC Molecular and Genomic Pathology laboratory, analyzed the prevalence of diagnostic, prognostic, and targetable genetic alterations in a total of 50 734 BCIII-VI nodules from 48 225 patients. Among 50 734 informative FNA samples, 65.3% were test-negative, 33.9% positive, 0.2% positive for medullary carcinoma, and 0.6% positive for parathyroid. The benign call rate in BCIII-IV nodules was 68%. Among test-positive samples, 73.3% had mutations, 11.3% gene fusions, and 10.8% isolated copy number alterations. Comparing BCIII-IV nodules with BCV-VI nodules revealed a shift from predominantly RAS-like alterations to BRAF V600E-like alterations and fusions involving receptor tyrosine kinases (RTK). Using ThyroSeq Cancer Risk Classifier, a high-risk profile, which typically included TERT or TP53 mutations, was found in 6% of samples, more frequently BCV-VI. RNA-Seq confirmed ThyroSeq detection of novel RTK fusions in 98.9% of cases. In this series, 68% of BCIII-IV nodules were classified as negative by ThyroSeq, potentially preventing diagnostic surgery in this subset of patients. Specific genetic alterations were detected in most BCV-VI nodules, with a higher prevalence of BRAF and TERT mutations and targetable gene fusions compared to BCIII-IV nodules, offering prognostic and therapeutic information for patient management.

Unraveling the significance of TSHR mutations in indeterminate thyroid cytology specimens.

We investigated the clinical significance of thyroid-stimulating hormone receptor (TSHR) mutations detected in thyroid fine needle aspiration (FNA) specimens. The pathology archives at our institution were reviewed between 2018 and 2021 for indeterminate (Bethesda category III and IV) specimens with Thyroseq® analysis showing TSHR mutations. A total of 2184 cases diagnosed as atypia/follicular lesion of undetermined significance (AUS/FLUS), and 2625 diagnosed as follicular neoplasm/suspicious for follicular neoplasm (FN/SFN) were identified. A total of 1735 AUS/FLUS and 2339 SFN/FN underwent Thyroseq® analysis; 69 showed TSHR mutations (1.6%, 59 female, 10 male, average age: 55 years). Ten cases showed oncocytic features. Twelve patients underwent radionuclide scans within 1 year of FNA:11 were hyperfunctioning. Nodule size and TSH levels were weakly correlated. Twenty-two different TSHR mutations were identified (most common: M453T). A second mutation was found in five cases (EZH1 n = 2, and EIF1AX n = 3). The expression of sodium-iodide transporter (NIS) mRNA was in the range of 0.01%-62.43% out of all sequencing reads, and was elevated in 49 (71%) cases. Surgical pathology follow-up was available in five cases (all benign except one). On follow-up available for 38 cases (mean: 24 months; range: 7-47 months), 34 (89.5%) nodules remained stable and 3 (8%) increased in size. TSHR mutations in thyroid FNA samples classified as indeterminate are rare, generally benign, and commonly associated with autonomy on scan if performed.

Multiplex Digital PCR Assay to Detect Multiple KRAS and GNAS Mutations Associated with Pancreatic Carcinogenesis from Minimal Specimen Amounts

Digital PCR (dPCR) allows for highly sensitive quantification of low-frequency mutations and facilitates early detection of cancer. However, low-throughput targeting of single hotspots in dPCR hinders variant specification when multiple probes are used. We developed a dPCR method to simultaneously identify major variants related to pancreatic carcinogenesis. Using a two-dimensional plot of droplet fluorescence under the optimized concentration of two fluorescent probe pools, the absolute quantification of different KRAS and GNAS variants was determined. Successful detection of the multiple driver mutations was verified in 24 surgically resected tumor samples from 19 patients and 22 fine-needle aspiration samples from patients with pancreatic ductal adenocarcinoma. Precise quantification of the variant allele frequency was optimized by using template DNA at a concentration as low as 1 to 10 ng. Furthermore, amplicons targeting multiple hotspots were successfully enriched with fewer false-positive findings using high-fidelity polymerase, allowing for the detection of various KRAS and GNAS mutations with high probability in small amount of cell/tissue specimens. Using this target enrichment, mutations at a rate of 90% in small residual tissues, such as the fine-needle aspiration needle flush and microscopic lesions in resected specimens, were successfully identified. The proposed method allows for low-cost, accurate detection of driver mutations to diagnose cancers, even with minimal tissue collection.

Multiplex protein analysis and ensemble machine learning methods of fine needle aspirates from prostate cancer patients reveal potential diagnostic signatures associated with tumour grade.

Improved molecular diagnosis is needed in prostate cancer (PC). Fine needle aspiration (FNA) is a minimally invasive biopsy technique, less traumatic compared to core needle biopsy, and could be useful for diagnosis of PC. Molecular biomarkers (BMs) in FNA-samples can be assessed for prediction, eg of immunotherapy efficacy before treatment as well as at treatment decision time points during disease progression. In the present pilot study, the expression levels of 151 BM proteins were analysed by proximity extension assay in FNA-samples from 16 patients, including benign prostate lesions (n= 3) and cancers (n= 13). An ensemble data analysis strategy was applied using several machine learning models. Twelve potentially predictive BM proteins correlating with International Society of Urological Pathology grade groups were identified, among them vimentin, tissue factor pathway inhibitor 2, and integrin beta-5. The validity of the results was supported by network analysis that showed functional associations between most of the identified putative BMs. We also showed that multiple immune checkpoint targets can be assessed (eg PD-L1, CD137, and Galectin-9), which may support the selection of immunotherapy in advanced PC. Results are promising but need further validation in a larger cohort. Our pilot study represents a "proof of concept" and shows that multiplex profiling of potential diagnostic and predictive BM proteins is feasible on tumour material obtained by FNA sampling of prostate cancer. Moreover, our results demonstrate that an ensemble data analysis strategy may facilitate the identification of BM signatures in pilot studies when the patient cohort is limited.

Integrated gene profiling of fine-needle aspiration sample improves lymph node metastasis risk stratification for thyroid cancer.

Lymph node metastasis risk stratification is crucial for the surgical decision-making of thyroid cancer. This study investigated whether the integrated gene profiling (combining expression, SNV, fusion) of Fine-Needle Aspiration (FNA) samples can improve the prediction of lymph node metastasis in patients with papillary thyroid cancer. In this retrospective cohort study, patients with papillary thyroid cancer who went through thyroidectomy and central lymph node dissection were included. Multi-omics data of FNA samples were assessed by an integrated array. To predict lymph node metastasis, we built models using gene expressions or mutations (SNV and fusion) only and an Integrated Risk Stratification (IRS) model combining genetic and clinical information. Blinded histopathology served as the reference standard. ROC curve and decision curve analysis was applied to evaluate the predictive models. One hundred and thirty two patients with pathologically confirmed papillary thyroid cancer were included between 2016-2017. The IRS model demonstrated greater performance [AUC=0.87 (0.80-0.94)] than either expression classifier [AUC=0.67 (0.61-0.74)], mutation classifier [AUC=0.61 (0.55-0.67)] or TIRADS score [AUC=0.68 (0.62-0.74)] with statistical significance (p < 0.001), and the IRS model had similar predictive performance in large nodule [>1cm, AUC=0.88 (0.79-0.97)] and small nodule [≤1cm, AUC=0.84 (0.74-0.93)] subgroups. The genetic risk factor showed independent predictive value (OR=10.3, 95% CI:1.1-105.3) of lymph node metastasis in addition to the preoperative clinical information, including TIRADS grade, age, and nodule size. The integrated gene profiling of FNA samples and the IRS model developed by the machine-learning method significantly improve the risk stratification of thyroid cancer, thus helping make wise decisions and reducing unnecessary extensive surgeries.

Comparison between Target Sample Check Illuminator and White Light Observation in Discriminating the Presence of Target Specimen for Endoscopic Ultrasound-Guided Fine Needle Aspiration Sample

Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) is an essential endoscopic tissue sampling method for diagnosing pancreatobiliary diseases; however, determining the presence of target specimens mixed in the blood by conventional observation is challenging due to the small size of the obtained sample. This study investigated the usefulness of a target sample check illuminator (TSCI) that emits a specific wavelength of light to determine the presence of target specimens. Twenty-seven patients who underwent EUS-FNA at our hospital were included. Conventional white light observation was performed for the collected samples, followed by TSCI; six people evaluated the presence of the target specimen on a 5-point scale. The target specimen discrimination score using TSCI (median: 5) was significantly higher than that using conventional white light observation (median: 1) (p < 0.001). No significant difference was observed in the discrimination score between the evaluator (novice vs. expert, p = 0.162) and puncture needle (22G vs. 25G, p = 0.196). The discriminability of TSCI in the samples obtained using EUS-FNA was significantly higher than that of conventional observation. TSCI does not depend on the evaluator or puncture needle for the identification of the target specimen; hence, it can provide a good pathological specimen and may contribute to the improvement of the diagnostic ability.

Persons with HIV Develop Spike-Specific Lymph Node Germinal Center Responses following SARS-CoV-2 Vaccination.

COVID-19 disproportionately affects persons with HIV (PWH) in worldwide locations with limited access to SARS-CoV-2 vaccines. PWH exhibit impaired immune responses to some, but not all, vaccines. Lymph node (LN) biopsies from PWH demonstrate abnormal LN structure, including dysregulated germinal center (GC) architecture. It is not clear whether LN dysregulation prevents PWH from mounting Ag-specific GC responses in the draining LN following vaccination. To address this issue, we longitudinally collected blood and draining LN fine needle aspiration samples before and after SARS-CoV-2 vaccination from a prospective, observational cohort of 11 PWH on antiretroviral therapy: 2 who received a two-dose mRNA vaccine series and 9 who received a single dose of the Ad26.COV2.S vaccine. Following vaccination, we observed spike-specific Abs, spike-specific B and T cells in the blood, and spike-specific GC B cell and T follicular helper cell responses in the LN of both mRNA vaccine recipients. We detected spike-specific Abs in the blood of all Ad26.COV2.S recipients, and one of six sampled Ad26.COV2.S recipients developed a detectable spike-specific GC B and T follicular helper cell response in the draining LN. Our data show that PWH can mount Ag-specific GC immune responses in the draining LN following SARS-CoV-2 vaccination. Due to the small and diverse nature of this cohort and the limited number of available controls, we are unable to elucidate all potential factors contributing to the infrequent vaccine-induced GC response observed in the Ad26.COV2.S recipients. Our preliminary findings suggest this is a necessary area of future research.

Study of Extramedullary Lymphoblastic Lymphoma (LBL) Diagnosed by Flowcytometric Immunophenotyping (FCI) on Fine Needle Aspirate (FNA) Sample-A Case Series of 18 Cases.

Lymphoblastic lymphoma (LBL) accounts for about 2% of all lymphomas. Recognition of T/B-LBL albeit of their rareness is very important as they present as localized diseases with low tumor burden. They can present both at nodal and extranodal sites. Limitation of diagnosis and classification of lymphoma on fine needle aspirate (FNA) can be minimized by clubbing it with flowcytometric immunophenotyping (FCI) to ensure diagnostic accuracy rapidly. Study of a series of 18 cases of LBL to assess the utility of FCI on FNA and effusion samples in extramedullary LBL. FCI was done on FNA and effusion samples from 130 morphologically diagnosed/suspicious cases of lymphoreticular malignancy, followed by peripheral blood and bone marrow (BM) examination. The patients diagnosed to have B/T LBL, based on WHO 2017 classification, were selected for further analyses. FCI of 130 cases showed 91 mature and 18 precursor lymphoid neoplasms. These 18 cases were from lymph nodes (11), pleural fluid (03), and soft tissue masses (04). Peripheral blood and BM of 15/18 cases were normal of which FCI revealed T-LBL (11) and B-LBL (04). Two cases (both T LBL) showed BM involvement (<25%), while one case of B-LBL which was misdiagnosed as mature lymphoma by immunohistochemistry (IHC) evolved as B ALL. Diagnosis of extramedullary B/T-LBL needs comprehensive evaluation of clinical presentation, cytomorphology, and immunophenotyping. Rapid and accurate diagnosis by FCI on FNA and effusion samples allows early therapeutic decisions, thereby avoiding leukemic dissemination.

Diagnostic Value of Preparing Additional Liquid-Based Cytology Slides and Cell Blocks from Residue Material in Thyroid fine Needle Aspiration.

After liquid-based cytology (LBC) technique developed for cervical cytology, it has been used for nongynecological samples and has been very successful. It offers having extra slides of the samples for further examination and ancillary tests. Moreover, cell blocks can be formed from the residue material. The study aimed to evaluate the importance of preparing a second LBC slide or a cell block from the residue material of thyroid fine needle aspiration (FNA) samples to reach a definitive diagnosis in cases diagnosed as nondiagnostic (ND) after the first slide. Seventy five cases diagnosed as ND after the first slide were included in study. For 50 cases, the second LBC slides were prepared (LBC group); for 25 cases, cell block was performed from residue material (CB group). Two groups were compared in terms of reaching a definitive diagnosis. At the end of secondary procedures, a definitive diagnosis was reached in a total of 24 cases (32%). Twenty of 50 cases (40%) in LBC group reached a definitive diagnosis while four cases (16%) in the CB group reached a definitive diagnosis. Achieving a definitive diagnosis was found statistically higher in the LBC group in which the second slide was formed compared to the CB group (P =0.036). Preparing a second slide with LBC method is more purposive than preparing a cell block from the residue material of thyroid FNA samples. Reducing the percentage of ND cases will protect patients from complications and morbidity that may arise from repeated FNA.

The Similarities and Differences between Liquid-based and Conventional Methods in Evaluation of Thyroid Fine Needle Aspiration Cytologies.

Thyroid nodules are one of the most frequent medical issues in endocrinology in our country and around the world. The appropriate evaluation of the nodule is critical in the management of patients. Fine needle aspiration cytology (FNAC) is one of the most accurate tools for evaluating these nodules. Conventional and liquid-based (LB) methods are available for thyroid FNAC. In this paper, we aim to determine the best cytological method for the evaluation of thyroid fine needle aspiration materials. Fine needle aspiration (FNA) materials obtained and prepared by conventional and LB methods from 310 patients were evaluated. These slides were compared in terms of 12 cytological parameters in a three-tiered system, and the kappa coefficient was calculated. Two slides were prepared from FNA samples, and the rest of the material was left in protective solution for LB cytology. Surepath was used as a LB technique. Conventional slides (CS) were left to dry in the air and stained with May Grunwald Giemsa. Slides prepared by two different techniques were compared in 12 cytomorphological parameters, and Kappa coefficients and correlations were calculated. Kappa statistics. In comparison of LB and CS, the highest kappa coefficient was in chromatin texture (ĸ:0.738) and inflammatory cells (ĸ: 0.482). On other parameters, fair or poor agreement was observed. Although there are some superiorities to LB cytology, co-application of these two methods is more favorable in thyroid fine needle aspiration.

Rapid On-Site Evaluation (ROSE): A Microfluidic Approach.

Rapid on-site evaluation (ROSE) increases the diagnostic accuracy of fine-needle aspiration (FNA) samples from cysts, a sack-like fluid-containing tissue that sometimes can be precancerous, but is highly dependent on the skills and availability of cytopathologists. We present a semiautomated sample preparation device for ROSE. The device consists of a smearing tool and a capillary-driven chamber that allow smearing and staining of an FNA sample in a single platform. Here, we show the capability of the device to prepare samples for ROSE, using a human pancreatic cancer cell line (PANC-1) and liver, lymph node, and thyroid FNA model samples. Using microfluidics, the device reduces the equipment needed in an operating room for FNA sample preparation, which may lead to a wider implementation of ROSE in healthcare centers.

Reducing non diagnostic thyroid cytology using on-site adequacy assessment performed by junior medical staff

Objective:To assess efficacy of bedside cytology assessment of thyroid fine needle aspirate samples performed by junior medical staff compared to cytologists. Methods: Retrospective analysis was performed of 1490 thyroid fine needle aspirates. Samples were divided into three groups: cellular adequacy assessment performed by cytologists, interns, and no onsite adequacy assessment (blind). A 45-minute training session was provided to medical interns with the aim of identifying whether adequate cellular material was demonstrated, as defined by the Bethesda criteria. The primary outcome was the rate of nondiagnostic samples, and the secondary outcome was the number of fine needle aspirate passes required to reach sample adequacy. Results: The incidence of non-diagnostic samples for the cytologist, blind, and intern groups were 6.90%, 17.1% and 14.0% respectively (p&lt;0.001). There was no statistically significant difference in non-diagnostic rate between the blind and intern groups. Significantly more aspirates required more than two passes in the cytologist group (74.8%) and blind (94.7%) compared to the intern group (58.7%, p&lt;0.001). Conclusions: On-site adequacy assessment performed by cytopathologists is the gold standard for minimising non-diagnostic thyroid FNAs, however, medical staff with minimal anatomical pathology experience can be trained to perform on-site adequacy assessment and improve outcomes with the implementation of a simple targeted training program.

Intrathyroidal parathyroid adenomas: Scoping review on clinical presentation, preoperative localization, and surgical treatment.

Intrathyroidal parathyroid adenomas (IPAs) are a rare cause of primary hyperparathyroidism. They are often difficult to localize preoperatively and intraoperatively, making diagnosis and treatment challenging. Current data on IPAs are sparse and fragmented in the literature. This makes it difficult to compare the effectiveness of different imaging and surgical techniques. To address this issue, this scoping review maps the literature on IPAs, focusing on four domains: clinical presentation, current localization methods, different surgical techniques, and histopathological features. A search of MEDLINE, Embase, and the Cochrane Library was conducted, with 19 studies meeting the inclusion criteria. The characteristics of IPAs on ultrasound, fine-needle aspiration, CT, MRI, sestamibi-based techniques, and selective venous sampling are summarized. Emerging imaging modalities, including autofluorescence, are introduced. Surgical methods and intraoperative factors that correlate with high success rates for removal are highlighted. This review also identifies gaps in knowledge to guide further research into this area.

P53 immunolabeling in EUS-FNA biopsy can predict low resection rate and early recurrence in resectable or borderline resectable pancreatic cancer treated with neoadjuvant therapy.

KRAS, P16, TP53, and SMAD4/DPC4 mutations are common in pancreatic ductal adenocarcinoma (PDAC). The study aimed to evaluate the association between gene mutations in pre-treatment endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) samples and clinical outcomes of patients with PDAC. There were 43 patients with resectable (R) PDAC and 41 patients with borderline resectable (BR) PDAC. CDKN2A/p16, TP53, and SMAD4/DPC4 were evaluated through immunohistochemistry (IHC) of pretreatment EUS-FNA (n=84) and resected specimens (n=71). All patients received neoadjuvant therapy. IHC of EUS-FNA specimens revealed p16 loss in 61 (73%), abnormal p53 in 61 (73%), and Smad4 loss in 38 (45%) patients. Abnormal p53 was associated with a lower resection rate (p=.017). Abnormal p53 and Smad4 loss were associated with recurrence within 6 months post-pancreatectomy (p=.03, p=.03, respectively). Univariate Cox regression analysis was conducted to reveal that abnormal p53 (p=.07), p16 loss and abnormal p53 (p=.04), and Smad4 and p16 loss (p=.03) were associated with poor prognosis. Pre-treatment abnormal labeling of p53 in EUS-FNA specimen was associated with a lower resection rate and an early recurrence in R or BR PDAC cases.

The Performance of an Extended Next Generation Sequencing Panel Using Endobronchial Ultrasound-Guided Fine Needle Aspiration Samples in Non-Squamous Non-Small Cell Lung Cancer: A Pragmatic Study

Introduction/BackgroundSamples from endobronchial ultrasound-guided fine needle aspiration (EBUS-TBNA) are frequently used for next generation sequencing (NGS) in patients with non-small cell lung cancer (NSCLC) to look for genetic driver mutations. The objective of the current study was to evaluate the performance of extended NGS panels using EBUS-TBNA samples in a real-world setting and identify factors associated with the success of NGS. Materials and MethodsThis study included all patients who underwent EBUS and were diagnosed with non-squamous NSCLC with mediastinal metastasis from 2016 to 2019 at the University of Pennsylvania. We reviewed demographic information, imaging studies, procedure reports, pathology and NGS reports. Logistic regression was used to analyze factors associated with the success of NGS panels. ResultsThe success rates of NGS using EBUS-TBNA samples were 92.5%, and 91.5% for DNA and RNA NGS panels respectively. Samples from higher N stage (N2 and N3 lymph nodes) and with higher tumor cellularity (>25%) resulted in higher success rate for DNA NGS. The effect of tumor cellularity remained borderline significant after entering multivariable logistic regression. The short-axis diameter of the sampled lymph node on CT scan, FDG-avidity on PET CT and >3 EBUS passes per lymph node during the procedure were not associated with NGS success. ConclusionBoth DNA and RNA extended-panel NGS had high performance using EBUS-TBNA samples. Sampling more advanced nodal stations and obtaining samples with higher tumor cellularity were associated with higher success rate of DNA NGS. Other imaging or procedural factors did not affect NGS performance.

Identifying metastatic renal cell carcinoma in thyroid fine-needle aspirates by molecular testing.

Renal cell carcinoma (RCC) is the most common type of cancer found to metastasize to the thyroid gland. These tumors may represent a diagnostic challenge in cytology. However, most RCC tumors carry VHL alterations, which are rare in primary thyroid tumors. The aim of this study was to evaluate the utility of molecular testing in detecting metastatic RCC in thyroid fine-needle aspiration (FNA) samples. From November 2017 until March 2022, thyroid FNA samples with ThyroSeq v3 results showing both VHL alterations and low/absent expression of thyroid cell markers were analyzed. Eighteen samples from 15 patients met the inclusion criteria. On molecular analysis, deleterious VHL mutations were found in nine (50%) nodules, VHL copy number alteration (CNA) in two (11%), and both mutations and CNA in seven (39%). None of the cases showed mutations commonly found in thyroid tumors. The mean age of these patients was 68 (range, 49-89) years with a male to female ratio of 2:1. Eight (53%) patients had multiple thyroid nodules on ultrasound. On cytology, 14 (78%) nodules were diagnosed as Bethesda III, 2 (11%) as Bethesda IV, and 2 (11%) as Bethesda V. At the time of cytology review, the history of RCC, sometimes remote, was available for ten patients. Of the 14 patients with medical history or surgical follow-up available, all had history of RCC or renal mass or revealed metastatic RCC on thyroidectomy. This study demonstrates that molecular testing can reliably identify metastatic RCC in thyroid nodules with indeterminate cytology, which could improve patient management.

Cytodiagnosis of rhinoscleroma and its morphological clues: Our experience with four cases

Rhinoscleroma, a chronic progressive inflammatory disease of the upper respiratory tract, has a prolonged course and passes through three overlapping clinicopathologic stages. The histopathology findings are well illustrated in the literature; however, there is a paucity of reports explaining cytomorphological features of rhinoscleroma. It is the proliferative stage when disease presents as a nodular mass which can be targeted for cytological study. Here, the authors report fine-needle aspiration (FNA) cytology of four cases of rhinoscleroma who presented with intranasal firm, nodular lesions; its cytology revealed numerous, large, singly scattered histiocytes in a dense inflammatory background of lymphocytes, plasma cells, and few eosinophils. The microorganisms were visualized as intracytoplasmic loosely clustered, short rods within the histiocytes (Mikulicz cells) in May-Grunwald-Giemsa (MGG) smears. FNA sampling of rhinoscleroma in its nodular stage can yield classic cytomorphology with demonstration of organisms on air-dried smears which can further substantiate the cytodiagnosis. Thus, high index of suspicion and diligent search can aid in early and accurate diagnosed even on cytology with the help of conventional wet fixed Pap and air-dried MGG stain; both being complementary to each other and further can be supported with special stains such as periodic acid–Schiff stain or Gram whenever required.

Analysis of Liquid Biopsy Performance for the Detection of Lymphoma in Dogs with a Focus on Recurrent Copy Number Variants in Matched Plasma and Lymph Node Aspirates: Implications for a Comparative Oncology Model

Introduction Liquid biopsy has shown great promise for the detection and monitoring of lymphoma in humans. Recently, similar technology has been developed and made commercially available for dogs. Because the canine and human cancer genomes share such high homology (>90%), and certain lymphoma subtypes are highly similar in terms of their biologic and molecular features between the two species, the study of lymphoma in dogs affords unique opportunities for the genomic characterization of the disease, comparative oncology studies, and the development and assessment of novel therapies. The current study describes the performance of a next-generation sequencing-based liquid biopsy assay for the detection of canine lymphoma, and in a subset of patients, analyzes concordance for prognostic copy number variants between matched plasma samples and lymph node aspirate specimens. Methods As part of a larger clinical research collection program for the CANcer Detection in Dogs (CANDiD) study (Flory, 2022), blood samples were collected following diagnosis but prior to undergoing therapy from a cohort of 116 client-owned dogs with lymphoma (63 intermediate to large B-cell, 21 intermediate to large T-cell, 6 T-zone, and 26 unphenotyped). Lymphomas with inconclusive immunophenotype or other types of indolent lymphomas were excluded. A subset of dogs analyzed for the current study also had matched cytology specimens available from lymph node fine needle aspirates (FNA). Blood samples and FNA specimens were subjected to DNA extraction, library preparation, and next-generation sequencing. Sequencing data were analyzed using an internally developed bioinformatics pipeline to detect genomic alterations associated with the presence of cancer. Specific analysis focused on FNA-plasma concordance for recurrent copy number variants that have been previously identified in canine lymphoma patients and have prognostic potential (i.e., gains in chromosomes 13 and 31 and losses in chromosome 14) (Richards, 2015; Thomas, 2003). Results The overall detection rate for lymphoma by liquid biopsy was 92.2% (107/116); with detection rates of 57% for localized/regional disease (Stages I and II) and 95% for disseminated disease (Stages III, IV, and V). By immunophenotype, the detection rate for B-cell lymphoma was 97%, T-cell 91%, T-zone 50%, and unphenotyped 92%. In the subset of lymphoma-diagnosed dogs with matched plasma and FNA samples, the majority of cases had at least one of the prognostic copy number alterations previously reported in canine lymphoma patients (13G, 14L, 31G), demonstrating the potential of liquid biopsy to noninvasively detect clinically important biomarkers. A variety of genomic alterations were identified in patients with positive liquid biopsy results, including single nucleotide variants in common oncogenes and tumor suppressor genes (e.g., TP53 mutations), and copy number variants across the genome. Some of these variants are also important biomarkers in human lymphoma, in particular TP53 mutations (Chakravarty, 2017), highlighting the potential for canine liquid biopsy testing to support comparative oncology studies for the benefit of both species. Conclusion A novel next-generation sequencing-based liquid biopsy tool has demonstrated the ability to identify genomic alterations in blood samples of dogs with lymphoma, including recurrent CNVs previously described in canine lymphoma patients, and variants in genes previously described in human and canine lymphoma tissue. These findings, along with the high degree of homology between human and canine lymphomas, support the utility of liquid biopsy for the study of lymphoma in dogs as a comparative oncology model for accelerated biomarker discovery and therapeutic development in both species.