23 results on '"Nikoletta Sidiropoulos"'
Search Results
2. Most Frequently Cited Accreditation Inspection Deficiencies for Clinical Molecular Oncology Testing Laboratories and Opportunities for Improvement
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Nikoletta Sidiropoulos, Sarah K. Daley, Marian Briggs, Helen Fernandes, Christina M. Lockwood, Amer Z. Mahmoud, Jason D. Merker, Patricia Vasalos, Lynnette M. Wielgos, Joel T. Moncur, and Daniel H. Farkas
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Medical Laboratory Technology ,education ,Humans ,General Medicine ,Clinical Laboratory Services ,Laboratories ,Medical Oncology ,Societies, Medical ,Pathology and Forensic Medicine ,Accreditation - Abstract
Context.— The College of American Pathologists (CAP), a laboratory accreditation organization with deemed status under the Clinical Laboratories Improvement Amendments of 1988 administers accreditation checklists. Checklists are used by laboratories to ensure regulatory compliance. Peer-level laboratory professionals audit laboratory records during inspections to assess compliance. Objective.— To identify the most frequently cited deficiencies for molecular oncology laboratories undergoing CAP accreditation inspections and describe laboratory improvement opportunities. Design.— The CAP Molecular Oncology Committee (MOC), which is involved in maintaining the Molecular Pathology checklist, reviewed data and inspector comments associated with the most frequently observed citations related to molecular oncology testing from laboratories inspected by the CAP during a 2-year period (2018–2020). Results.— Of 422 molecular oncology laboratories that underwent accreditation inspections, 159 (37.7%) were not cited for any molecular oncology–related deficiencies. For the All Common (COM) and Molecular Pathology checklists, there were 364 and 305 deficiencies, corresponding to compliance rates of 98.8% and 99.6%, respectively. The most frequently cited deficiencies are described. The COM checklist deficiencies were associated most often with the analytic testing phase; the MOL checklist deficiencies were more evenly distributed across the preanalytic, analytic, and postanalytic phases of testing. Conclusions.— Molecular oncology laboratories demonstrated excellent compliance with practices that support high-quality results for patients and the health care providers who use those test results in patient management. This review includes a critical assessment of opportunities for laboratories to improve compliance and molecular oncology testing quality.
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- 2021
3. MET Exon 14 Skipping Mutations: Essential Considerations for Current Management of Non-Small-Cell Lung Cancer
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Kurtis D, Davies, Lauren L, Ritterhouse, Anthony N, Snow, and Nikoletta, Sidiropoulos
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Lung Neoplasms ,Carcinoma, Non-Small-Cell Lung ,Mutation ,Humans ,Exons - Published
- 2021
4. Functional assessment of somatic STK11 variants identified in primary human non-small cell lung cancers
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Jenna G Eaton, Tyler C Hogan, Nikoletta Sidiropoulos, Cai L McCann, Hailey M. Sarausky, Kenneth J. Hampel, Gopika Nandagopal, David J. Seward, Paula B. Deming, Meagan A Lebeau, Margaret P Cameron, Jordan Armstrong, Sean M. Lenahan, and Liam Donnelly
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Cancer Research ,Lung Neoplasms ,In silico ,AcademicSubjects/MED00710 ,DNA Mutational Analysis ,STK11 ,Mutation, Missense ,Computational biology ,Biology ,AMP-Activated Protein Kinase Kinases ,Carcinoma, Non-Small-Cell Lung ,medicine ,Biomarkers, Tumor ,Missense mutation ,Coding region ,Humans ,Genetic Predisposition to Disease ,Phosphorylation ,Loss function ,Monoclonal antibody therapy ,Cancer Biomarkers and Molecular Epidemiology ,Gene Editing ,General Medicine ,medicine.disease ,Prognosis ,Gene Expression Regulation, Neoplastic ,Alternative Splicing ,Editor's Choice ,Mutation ,Mutagenesis, Site-Directed ,Adenocarcinoma ,Biomarker (medicine) ,Disease Susceptibility ,RNA Splice Sites ,CRISPR-Cas Systems - Abstract
Serine/Threonine Kinase 11 (STK11) encodes an important tumor suppressor that is frequently mutated in lung adenocarcinoma. Clinical studies have shown that mutations in STK11 resulting in loss of function correlate with resistance to anti-PD-1 monoclonal antibody therapy in KRAS-driven non-small cell lung cancer (NSCLC), but the molecular mechanisms responsible remain unclear. Despite this uncertainty, STK11 functional status is emerging as a reliable biomarker for predicting non-response to anti-PD-1 therapy in NSCLC patients. The clinical utility of this biomarker ultimately depends upon accurate classification of STK11 variants. For nonsense variants occurring early in the STK11 coding region, this assessment is straightforward. However, rigorously demonstrating the functional impact of missense variants remains an unmet challenge. Here we present data characterizing four STK11 splice-site variants by analyzing tumor mRNA, and 28 STK11 missense variants using an in vitro kinase assay combined with a cell-based p53-dependent luciferase reporter assay. The variants we report were identified in primary human NSCLC biopsies in collaboration with the University of Vermont Genomic Medicine group. Additionally, we compare our experimental results with data from 22 in silico predictive algorithms. Our work highlights the power, utility and necessity of functional variant assessment and will aid STK11 variant curation, provide a platform to assess novel STK11 variants and help guide anti-PD-1 therapy utilization in KRAS-driven NSCLCs., Functional assessment of under- and undescribed clinical STK11 variants highlights the limitations of current in silico predictive algorithms. Successful implementation of personalized genomic medicine will rely on rapid and accurate variant classification.
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- 2021
5. Single-cell proteogenomics reveals that SLAM/SAP signaling regulates the development of innate-like γδ T cell subsets with distinct TCR repertoires
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Jonathan E Boyson, Emma Andretta, Kenneth J. Hampel, Nikoletta Sidiropoulos, Oliver Dienz, Devdoot Majumdar, and Somen Mistri
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Immunology ,Immunology and Allergy - Abstract
γδ T cells are non-conventional T cells that are highly enriched in the mucosal tissues where they play critical roles in immunity. We recently demonstrated that the SLAM/SAP signaling pathway regulates the thymic development of innate-like γδT17 and γδTIFN subsets, in addition to γδNKT cells. Here, we utilized a single-cell proteogenomics approach coupled with γδ V(D)J profiling to define the transcriptional landscape and developmental checkpoints of SAP-dependent γδ T cells. This analysis not only confirmed our previous finding that SLAMF1 and SLAMF6 expression marks γδT17 and γδTIFN subsets, respectively, it also identified SLAMF7 as a novel marker of the SAP-dependent innate-like CD44+ CD45RB+ γδTIFN cells in both the thymus and periphery. Next, our data indicated that disruption of SAP-dependent signaling impaired γδT17 development at a very early (CD24high CD73−) stage, and was associated with the decreased expression of critical regulators of γδT17 development such as Blk and c-Maf. In contrast, while SAP also impaired γδTIFN development at an early (CD24high CD73+) stage, SAP-deficient γδTIFN cells exhibited increased expression of genes associated with TCR signaling and thymic export such as Prkch, Dgka, Klf2, and S1p1r. Finally, our analysis revealed significant alterations in the γδT17 TCR repertoire in both embryonic/neonatal thymus and the lung, as well as the presence of a SAP-dependent IFN-γ-producing lung Vγ4 population that preferentially utilized TRDV7. Altogether, these data suggest that SLAM/SAP signaling acts during the very early stages of γδ T cell development where it regulates critical pathways in both γδT17 and γδTIFN development, and influences the development of the innate-like γδ TCR repertoire. Supported by NIH (R03AI153902, P30GM118228), AAI Careers in Immunology Fellowship
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- 2022
6. Single-cell proteogenomics reveals that SLAM/SAP signaling regulates the development of innate-like γδT cell subsets with distinct TCR repertoires
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Somen K Mistri, Kenneth J Hampel, Nikoletta Sidiropoulos, Emma Andretta, Oliver Dienz, and Jonathan Boyson
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Immunology ,Immunology and Allergy - Abstract
Innate-like T cells are unusual T cells that are enriched in mucosal tissues, and which constitute a prominent source of pro-inflammatory cytokines like IL-17 and IFN-γ. While it is known that SLAM/SAP signaling is required for the development of innate-like iNKT and MAIT αβ T cells, far less is known about the role of this pathway in the development and function of γδ T cells. Here, we utilized a single-cell proteogenomics approach coupled with γδ V(D)J profiling to define the transcriptional landscape and developmental checkpoints of SAP-dependent γδ T cells. We found that SAP-dependent γδNKT TCRs utilized TRGV1 paired with TRAV15N-1 or TRAV15-1/DV6-1, and we identified two distinct developmental γδNKT stages in the neonatal thymus that were distinguished by SLAMF6 and SLAMF7 expression. Moreover, we found that a significant fraction of SLAMf1+innate-like γδT17 cells utilized TRGV4 and TRGV6, γ-chains paired with TRDV2 or TRDV5 δ-chains of limited diversity. Examination of SAP-deficient neonatal thymus revealed decreased numbers of mature SLAMF1+ γδT17 cells, which was associated with lower numbers of an invariant germline-encoded TRGV4/TRDV5 clonotype. Accordingly, we observed significant alterations in the SLAMF1+ γδT17 TCR repertoire in adult lung. We found that lung γδTIFN were CD44+CD45RB+CD27+ cells that co-expressed SLAMF6and SLAMF7, and that these cells predominantly utilized a diverse TRDV7 paired with TRGV4. Interestingly, we observed a specific decrease of these TRDV7+ γδTIFN cells in SAP-deficient mice, which we confirmed using qPCR. Altogether, these data indicate a crucial link between SLAM/SAP signaling and the development of functionally distinct innate-like γδ TCR clonotypes.
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- 2021
7. Preanalytic Variables and Tissue Stewardship for Reliable Next-Generation Sequencing (NGS) Clinical Analysis
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Carlo Bifulco, Solange Peters, Giuseppe Palmieri, Paolo A. Ascierto, and Nikoletta Sidiropoulos
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0301 basic medicine ,Standardization ,Genomics ,Computational biology ,DNA sequencing ,Pathology and Forensic Medicine ,Specimen Handling ,03 medical and health sciences ,0302 clinical medicine ,Neoplasms ,Humans ,Precision Medicine ,Gene Library ,business.industry ,NGS analysis ,High-Throughput Nucleotide Sequencing ,Sequence Analysis, DNA ,Precision medicine ,Biomarker (cell) ,030104 developmental biology ,030220 oncology & carcinogenesis ,Genomic Profile ,Molecular Medicine ,Identification (biology) ,Personalized medicine ,business - Abstract
An enduring goal of personalized medicine in cancer is the ability to identify patients who are likely to respond to specific therapies. Our growing understanding of the biology and molecular signatures of individual tumor types has facilitated the identification of predictive biomarkers and has led to an increasing number of diagnostic tests to be performed, often as serial and distinct assays on limited tumor specimens. The biomarker diagnostics field has been revolutionized by next-generation sequencing (NGS), which provides a comprehensive overview of the genomic profile of a tumor. Many preanalytic variables can influence the accuracy and reliability of NGS results. Standardization of preanalytic variables is, however, complicated by the plethora of specimen acquisition and processing methods. Variables across the tissue journey, including specimen acquisition, specimen fixation, and sectioning, as well as postfixation processing, such as nucleic acid extraction, library preparation, and choice of sequencing methods, are critical for the reliability of NGS analysis; thus, standardization would be beneficial. In this article, each step in the tissue journey is outlined, with specific focus on preanalytic variables that can influence NGS results. Practical considerations for standardization of these variables are provided to facilitate accurate, reliable, and reproducible NGS-based molecular characterization of tumors, ultimately informing diagnosis and guiding treatment.
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- 2019
8. Recommendations for Ancillary Testing
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Nikoletta Sidiropoulos and Sinchita Roy-Chowdhuri
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Oncology ,medicine.medical_specialty ,business.industry ,Molecular pathology ,Papanicolaou stain ,Guideline ,medicine.disease ,Cytopathology ,Cytology ,Internal medicine ,medicine ,Carcinoma ,Biomarker (medicine) ,Lung cancer ,business - Abstract
Prior to the emergence of targeted therapeutics, conventional ancillary testing in respiratory cytology has included microbiologic culture for infectious agents, special stains and/or immunocytochemistry (ICC) for establishing a cytomorphologic diagnosis, and flow cytometry and/or fluorescence in situ hybridization (FISH) for hematolymphoid neoplasms. With the increasing use of molecular diagnostic assays for predictive, prognostic, and therapeutic purposes in lung carcinoma patients, the need for ancillary testing of lung cytology specimens has also expanded. Biomarker testing for tyrosine kinase inhibitors (TKIs) and immunotherapies using molecular and immunohistochemical methods now play a pivotal role in the management of non-small cell lung carcinoma (NSCLC) patients (Lindeman, Cagle, Aisner, Arcila, Beasley, Bernicker, et al. Updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors: guideline from the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology. Arch Pathol Lab Med, 2018). Since a large fraction of these patients present at an advanced stage of disease with unresectable tumors, cytology samples are often the only source of tissue for diagnosis and biomarker testing. Therefore, ancillary testing in lung cytology has expanded beyond its conventional role into a variety of molecular (nucleic acid and protein based) assays that have been integrated into routine cytopathology practice and play a key role in patient care. The Papanicolaou Society of Cytopathology System for Reporting Respiratory Cytology has therefore developed some recommendations for the use of ancillary testing in lung cytology (Layfield, Roy-Chowdhuri, Baloch, Ehya, Geisinger, Hsiao, et al. Utilization of ancillary studies in the cytologic diagnosis of respiratory lesions: the Papanicolaou Society of Cytopathology consensus recommendations for respiratory cytology. Diagn Cytopathol 44:1000–9, 2016).
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- 2018
9. Utilization of ancillary studies in the cytologic diagnosis of respiratory lesions: The papanicolaou society of cytopathology consensus recommendations for respiratory cytology
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Lester J. Layfield, Fernando Schmitt, Nikoletta Sidiropoulos, Susan J. Hsiao, Sinchita Roy-Chowdhuri, Neal I. Lindeman, Kim Geisinger, Hormoz Ehya, Oscar Lin, Zubair W. Baloch, Michael Roh, and Paul A. VanderLaan
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0301 basic medicine ,Gynecology ,medicine.medical_specialty ,Histology ,medicine.diagnostic_test ,business.industry ,Papanicolaou stain ,Sputum examination ,General Medicine ,Guideline ,Pathology and Forensic Medicine ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Fine-needle aspiration ,Bronchial washing ,Cytopathology ,030220 oncology & carcinogenesis ,Cytology ,Diagnosis management ,medicine ,Medical physics ,business - Abstract
The Papanicolaou Society of Cytopathology has developed a set of guidelines for respiratory cytology including indications for sputum examination, bronchial washings and brushings, CT-guided FNA and endobronchial ultrasound guided fine needle aspiration (EBUS-FNA), as well as recommendations for classification and criteria, ancillary testing and post-cytologic diagnosis management and follow-up. All recommendation documents are based on the expertise of committee members, an extensive literature review, and feedback from presentations at national and international conferences. The guideline documents selectively present the results of these discussions. The present document summarizes recommendations for ancillary testing of cytologic samples. Ancillary testing including microbiologic, immunocytochemical, flow cytometric, and molecular testing, including next-generation sequencing are discussed. Diagn. Cytopathol. 2016;44:1000-1009. © 2016 Wiley Periodicals, Inc.
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- 2016
10. Active Surveillance for Medically Inoperable Stage IA Lung Cancer in the Elderly
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Hyunsoo J. No, Steven H. Lin, Garth W. Garrison, David J. Seward, Christopher J. Anker, Carl J. Nelson, C. Matthew Kinsey, Nataniel H. Lester-Coll, Havaleh M. Gagne, and Nikoletta Sidiropoulos
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medicine.medical_specialty ,Pulmonology ,medicine.medical_treatment ,Disease ,elderly ,03 medical and health sciences ,0302 clinical medicine ,Internal medicine ,Medicine ,030212 general & internal medicine ,Stage (cooking) ,Lung cancer ,non-small cell lung cancer ,Medically inoperable ,business.industry ,active surveillance ,General Engineering ,Nodule (medicine) ,Histology ,medicine.disease ,Miscellaneous ,Radiation therapy ,Oncology ,030220 oncology & carcinogenesis ,Adenocarcinoma ,medicine.symptom ,business - Abstract
Objectives Treatment for stage IA lung cancer may be too aggressive an approach in elderly patients with competing co-morbidities. We report outcomes for those electing active surveillance (AS) and investigate factors that may predict indolent disease. Materials and methods Retrospective review was performed for 12 consecutive patients, ≥70 years old, with medically inoperable stage IA, T1N0M0 lung cancer and significant co-morbidities, who chose AS with radiation therapy (RT) reserved for clear disease progression. Collected data included Charlson-Deyo Comorbidity Index (CDCI) grades, histology, and tumor size changes. Volume doubling time (VDT) calculations used a modified Schwartz equation. Results Fifteen nodules underwent AS in 12 patients; three patients had more than one nodule. Median age of all patients was 78 (range, 71–85). All patients’ CDCI grades were ≥1, 7 were ≥2. Eleven of 12 patients were deemed to be at high-risk for falls. Twelve nodules in 12 patients were biopsied; adenocarcinoma the prevailing common (47%) histology. The median, one, two and three year patient freedom-from-RT values were 21.4 months (95% CI: 11.6-not reached), 81%, 43%, and 29%, respectively. Median VDT of treated vs. untreated nodules was 189 days (range, 62-infinite) vs. 1153 days (range, 504-infinite), respectively. No patient progressed regionally or distantly, and there have been no cancer-related deaths. Due to cardiovascular events, two patients died and one remains on hospice. Median duration of AS for those still continuing computed tomography (CT) surveillance is 35.1 months. Conclusion Selected elderly patients with stage IA lung cancer and significant co-morbidities may undergo AS without detriment in outcome. Prospective AS studies are warranted.
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- 2018
11. Small-cell transformation of ALK-rearranged non-small-cell adenocarcinoma of the lung
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Kenneth J. Hampel, Nikoletta Sidiropoulos, Farrah Khan, Agnes Balla, and Dara L. Aisner
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0301 basic medicine ,Adult ,Lung Neoplasms ,Oncogene Proteins, Fusion ,medicine.medical_treatment ,Biopsy ,Targeted therapy ,03 medical and health sciences ,0302 clinical medicine ,hemic and lymphatic diseases ,Carcinoma, Non-Small-Cell Lung ,medicine ,Adenocarcinoma of the lung ,Anaplastic lymphoma kinase ,Humans ,Anaplastic Lymphoma Kinase ,Epidermal growth factor receptor ,Molecular Targeted Therapy ,Neoplasm Metastasis ,neoplasm of the lung ,Protein Kinase Inhibitors ,Chromosomal inversion ,Neoplasm Staging ,Gene Rearrangement ,biology ,Oncogene ,business.industry ,General Medicine ,Exons ,medicine.disease ,respiratory tract diseases ,030104 developmental biology ,Cell Transformation, Neoplastic ,030220 oncology & carcinogenesis ,Cancer research ,biology.protein ,Adenocarcinoma ,Female ,business ,Tomography, X-Ray Computed ,Tyrosine kinase ,Rapid Cancer Communication - Abstract
Anaplastic lymphoma kinase (ALK) gene rearrangements are present in ∼5% of non-small-cell lung cancers (NSCLCs). These rearrangements occur because of a chromosomal inversion within the short arm of Chromosome 2, which results in the formation of the echinoderm microtubule-associated protein-like 4 (EML4)–ALK fusion oncogene. Whereas NSCLC transformation to SCLC is a rare phenomenon described in epidermal growth factor receptor (EGFR) mutant cancers primarily after treatment with targeted therapy, it is exceedingly rare in ALK-rearranged adenocarcinomas. It is currently unclear what the therapeutic significance of the rearrangement is in this transformed tumor as there is a paucity of medical literature describing follow-up care and outcomes of patients in this rare scenario. We describe a unique case in which a patient with ALK-rearranged adenocarcinoma underwent small-cell transformation at a metastatic site with retained ALK rearrangement and was provided clinical follow-up after treatment with second-generation tyrosine kinase inhibiter (TKI) therapy.
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- 2018
12. Comprehensive Validation of Cytology Specimens for Next-Generation Sequencing and Clinical Practice Experience
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Ken J. Hampel, Nikoletta Sidiropoulos, Agnes Balla, Mukesh K. Sharma, and Catherine E. Cottrell
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0301 basic medicine ,medicine.medical_specialty ,DNA sequencing ,Pathology and Forensic Medicine ,Specimen Handling ,03 medical and health sciences ,0302 clinical medicine ,Targeted ngs ,Cytology ,Neoplasms ,Biopsy ,Medicine ,Humans ,medicine.diagnostic_test ,business.industry ,High-Throughput Nucleotide Sequencing ,Reproducibility of Results ,Cell Biology ,DNA, Neoplasm ,Molecular analysis ,Clinical Practice ,030104 developmental biology ,030220 oncology & carcinogenesis ,Surgical biopsy ,Molecular Medicine ,Radiology ,business - Abstract
Biopsy specimens are subjected to an expanding portfolio of assays that regularly include mutation profiling via next-generation sequencing (NGS). Specimens derived via fine-needle aspiration, a common biopsy technique, are subjected to a variety of cytopreparatory methods compared with surgical biopsies that are almost uniformly processed as formalin-fixed, paraffin-embedded tissue. Therefore, the fine-needle aspiration–derived specimens most commonly accepted for molecular analysis are cell blocks (CBs), because they are processed most similarly to surgical biopsy tissue. However, CB preparations are fraught with challenges that risk unsuccessful sequencing and repeat biopsies, with the potential to further increase health care costs and delay clinical care. The diversity of cytopreparations and the resource-intensive clinical validation of NGS pose significant challenges to more consistent use of non-CB (NCB) cytology specimens. As part of clinical validation of a targeted NGS assay, DNA subjected to nine cytopreparatory methods was evaluated for sequencing performance and was shown to be uniformly acceptable for clinical NGS. Of the 379 clinical cases analyzed after validation, the majority (56%) were derived from NCB cytology specimens. This specimen class had the lowest DNA insufficiency rate (1.5%) and showed equivalent sequencing performance to surgical and CB formalin-fixed, paraffin-embedded tissue. NCB cytology specimens are valuable sources of tumor nucleic acid and are the preferred specimen type for clinical NGS at our institution.
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- 2017
13. Identification of tumor-reactive B cells and systemic IgG in breast cancer based on clonal frequency in the sentinel lymph node
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Stephanie C. Pero, William N. Voss, Nikoletta Sidiropoulos, Jimmy Gollihar, David N. Krag, Yuri Tanno, Sebastian Schaetzle, Jared W. Ellefson, Chang-Han Lee, Girja S. Shukla, George Georgiou, Yu-Jing Sun, Christopher C. Krag, Seth P. Harlow, Jonathan R. McDaniel, Andrew P. Horton, and Gregory C. Ippolito
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0301 basic medicine ,Cancer Research ,medicine.medical_treatment ,Immunology ,Sentinel lymph node ,Sequence Homology ,Breast Neoplasms ,Immunoglobulin G ,Article ,03 medical and health sciences ,Breast cancer ,0302 clinical medicine ,Immune system ,Antigen ,Cancer immunotherapy ,Antigens, Neoplasm ,Immunology and Allergy ,Medicine ,Humans ,Amino Acid Sequence ,Lymph node ,Cells, Cultured ,B-Lymphocytes ,biology ,business.industry ,Cancer ,Antibodies, Monoclonal ,medicine.disease ,Clone Cells ,030104 developmental biology ,medicine.anatomical_structure ,Oncology ,030220 oncology & carcinogenesis ,Cancer research ,biology.protein ,Female ,Antibody ,Sentinel Lymph Node ,business - Abstract
A better understanding of antitumor immune responses is key to advancing the field of cancer immunotherapy. Endogenous immunity in cancer patients, such as circulating anticancer antibodies or tumor-reactive B cells, has been historically yet incompletely described. Here, we demonstrate that tumor-draining (sentinel) lymph node (SN) is a rich source for tumor-reactive B cells that give rise to systemic IgG anticancer antibodies circulating in the bloodstream of breast cancer patients. Using a synergistic combination of high-throughput B-cell sequencing and quantitative immunoproteomics, we describe the prospective identification of tumor-reactive SN B cells (based on clonal frequency) and also demonstrate an unequivocal link between affinity-matured expanded B-cell clones in the SN and antitumor IgG in the blood. This technology could facilitate the discovery of antitumor antibody therapeutics and conceivably identify novel tumor antigens. Lastly, these findings highlight the unique and specialized niche the SN can fill in the advancement of cancer immunotherapy.SIGNIFICANCEUsing high-throughput molecular cloning and antibody proteomics to study coordinated antitumor immunity in breast cancer patients, we simultaneously demonstrate that the sentinel lymph node is a localized source of expanded antitumor B cells undergoing affinity maturation and that their secreted antibodies are abundant as systemic IgG circulating in blood.
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- 2017
14. Variant call concordance between two laboratory-developed, solid tumor targeted genomic profiling assays using distinct workflows and sequencing instruments
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Ken J. Hampel, Nikoletta Sidiropoulos, Francine B. de Abreu, Jason D. Peterson, and Gregory J. Tsongalis
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0301 basic medicine ,Concordance ,Clinical Biochemistry ,Genomics ,Biology ,Polymorphism, Single Nucleotide ,Sensitivity and Specificity ,DNA sequencing ,Pathology and Forensic Medicine ,03 medical and health sciences ,Nucleic acid thermodynamics ,0302 clinical medicine ,Neoplasms ,Humans ,Indel ,Molecular Biology ,Illumina dye sequencing ,Alleles ,Genetics ,Gene Expression Profiling ,High-Throughput Nucleotide Sequencing ,Nucleic Acid Hybridization ,Ion semiconductor sequencing ,DNA, Neoplasm ,Exons ,Sequence Analysis, DNA ,Introns ,Gene expression profiling ,030104 developmental biology ,030220 oncology & carcinogenesis - Abstract
Targeted genomic profiling (TGP) using massively parallel DNA sequencing is becoming the standard methodology in clinical laboratories for detecting somatic variants in solid tumors. The variety of methodologies and sequencing platforms in the marketplace for TGP has resulted in a variety of clinical TGP laboratory developed tests (LDT). The variability of LDTs is a challenge for test-to-test and laboratory-to-laboratory reliability. At the University of Vermont Medical Center (UVMMC), we validated a TGP assay for solid tumors which utilizes DNA hybridization capture and complete exon and selected intron sequencing of 29 clinically actionable genes. The validation samples were run on the Illumina MiSeq platform. Clinical specificity and sensitivity were evaluated by testing samples harboring genomic variants previously identified in CLIA-approved, CAP accredited laboratories with clinically validated molecular assays. The Molecular Laboratory at Dartmouth Hitchcock Medical Center (DHMC) provided 11 FFPE specimens that had been analyzed on AmpliSeq Cancer Hotspot Panel version 2 (CHPv2) and run on the Ion Torrent PGM. A Venn diagram of the gene lists from the two institutions is shown. This provided an excellent opportunity to compare the inter-laboratory reliability using two different target sequencing methods and sequencing platforms. Our data demonstrated an exceptionally high level of concordance with respect to the sensitivity and specificity of the analyses. All clinically-actionable SNV and InDel variant calls in genes covered by both panels (n=17) were identified by both laboratories. This data supports the proposal that distinct gene panel designs and sequencing workflows are capable of making consistent variant calls in solid tumor FFPE-derived samples.
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- 2017
15. Clinical Genomic Testing
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Debra G.B. Leonard, Ken J. Hampel, and Nikoletta Sidiropoulos
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Translational bioinformatics ,Scope (project management) ,business.industry ,Genomics ,Biology ,Bioinformatics ,Data science ,Genome ,Variety (cybernetics) ,ComputingMethodologies_PATTERNRECOGNITION ,Informatics ,Human genome ,Personalized medicine ,business - Abstract
Increasingly, the experience and knowledge gleaned via research-based interrogation of the human genome is being leveraged and translated for use in the clinical realm to inform medical decision making. The clinical implementation of genome-based diagnostics is fraught with a variety of challenges that include but are not limited to the scope of: knowledge and the medical significance of genome-based data, scalable technology and infrastructure, informatics, and regulatory requirements. This chapter will provide an overview of state-of-the-art genomic technologies with a focus on DNA-based Next-Generation Sequencing. Herein is a review of the clinical applications of genomic testing and the common considerations for developing this testing in the clinical laboratory setting. The common challenges to incorporating genomics into clinical care for the laboratory, clinicians, and patients are also addressed.
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- 2017
16. List of Contributors
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Joanne Armstrong, Katrina Armstrong, Samuel J. Aronson, Adam C. Berger, Zhaohui Chen, Andrew Dervan, Katherine Donigan, Edward S. Dove, Eric Faulkner, Benjamin French, Elyse Galloway, Geoffrey S. Ginsburg, Elizabeth A. Grice, Ken J. Hampel, N. Katsanis, S.H. Katsanis, Stephen E. Kimmel, Bartha M. Knoppers, Graeme T. Laurie, Liis Leitsalu, Debra G.B. Leonard, Elizabeth Mansfield, Daniel R. Masys, Robert McDonough, Jacquelyn S. Meisel, Andres Metspalu, Michael F. Murray, Lori A. Orlando, Philip R.O. Payne, Alanna Kulchak Rahm, Arti K. Rai, Timothy E. Reddy, Shelby D. Reed, Laura Lyman Rodriguez, R. Ryanne Wu, Jay Shendure, Nikoletta Sidiropoulos, Steven Tjoe, Jennifer E. Van Eyk, David L. Veenstra, Deepak Voora, Robyn Ward, Huntington F. Willard, and Marc S. Williams
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- 2017
17. Clinical trials for pharmacogenomics testing for warfarin dosing: Relevance to general community practices
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Alan H B Wu and Nikoletta Sidiropoulos
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medicine.medical_specialty ,business.industry ,Treatment outcome ,Warfarin ,MEDLINE ,Clinical trial ,Pharmacogenomics ,medicine ,heterocyclic compounds ,Relevance (information retrieval) ,cardiovascular diseases ,Dosing ,business ,Intensive care medicine ,Genetics (clinical) ,Pharmacogenetics ,medicine.drug - Abstract
Clinical trials for pharmacogenomics testing for warfarin dosing: Relevance to general community practices
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- 2011
18. 29. Integrating ClinGen somatic cancer variant description standards into crowdsourced curation technology via CIViC database for ClinVar submission
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Gordana Raca, Shruti Rao, Kilannin Krysiak, Arpad Danos, Subha Madhavan, Erica K. Barnell, Xuan Shirley Li, Matthew McCoy, Deborah I. Ritter, Susanna Kiwala, Dara L. Aisner, Nikoletta Sidiropoulos, Adam C. Coffman, Angshumoy Roy, Christine M. Micheel, Joshua F. McMichael, Lynzey Kujan, Annette Leon, Simina M. Boca, Obi L. Griffith, Shashi Kulkarni, Dmitriy Sonkin, Malachi Griffith, and Alex H. Wagner
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World Wide Web ,Cancer Research ,Somatic cell ,Genetics ,medicine ,Cancer ,Biology ,medicine.disease ,Molecular Biology - Published
- 2018
19. Quality Improvement by Standardization of Procurement and Processing of Thyroid Fine-Needle Aspirates in the Absence of On-site Cytological Evaluation
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Larry J. Dumont, Francis L. Quinlisk, Vijayalakshmi Padmanabhan, Nikoletta Sidiropoulos, Jorge L. Gonzalez, and Allan C. Golding
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Adult ,Male ,Quality Control ,Thyroid nodules ,medicine.medical_specialty ,Quality management ,Adolescent ,Standardization ,Endocrinology, Diabetes and Metabolism ,Sample (material) ,Biopsy, Fine-Needle ,Cohort Studies ,Young Adult ,Endocrinology ,Odds Ratio ,medicine ,Humans ,Thyroid Nodule ,Aged ,Retrospective Studies ,Aged, 80 and over ,business.industry ,Thyroid ,Nodule (medicine) ,Middle Aged ,medicine.disease ,Surgery ,Aspiration cytology ,Treatment Outcome ,medicine.anatomical_structure ,Cytopathology ,Female ,Radiology ,medicine.symptom ,business - Abstract
Thyroid nodules are relatively common and are routinely evaluated by fine-needle aspiration cytology, usually performed by clinicians. We noticed qualitative and/or quantitative variability in samples submitted to the cytopathology laboratory from clinicians, for example, the number of glass slides submitted (2-25) and air-dried smears versus alcohol-fixed slides, with variability in specimen adequacy and interpretability. The objective of this study was to standardize the preanalytic variables to determine if there is an improvement in the specimen quality.We standardized the method of collection (ultrasound-guided, 25-gauge needle, four passes) and preparation of samples (four total smears: two air-dried, two fixed, with liquid-based preparation and/or cell block) and personnel involved.Standardization of thyroid nodule fine-needle aspiration and sample preparation by clinical staff resulted in an overall improvement in the quality of sample (odds ratio = 3.82, 95% confidence interval 2.02-7.24, p0.0001) with an increased proportion of satisfactory samples from 67% prestandardization to 89% poststandardization.Standardization resulted in a significant improvement in specimen interpretability.
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- 2009
20. Circulating tumor cells in hepatocellular carcinoma: a pilot study of detection, enumeration, and next-generation sequencing in cases and controls
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Eric A. Collisson, Jimmy Hwang, Kimberley J. Evason, Elizabeth M. Wayne, Tim Butler, Francis Y. Yao, John W. Park, Alan P. Venook, Nikoletta Sidiropoulos, Bilal Hameed, Ryan M. McWhirter, Mark Jesus M. Magbanua, and Robin Kate Kelley
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Male ,Cancer Research ,Hepatocellular carcinoma ,Kaplan-Meier Estimate ,Neoplastic Cells ,Circulating tumor cells (CTC) ,chemistry.chemical_compound ,0302 clinical medicine ,Circulating tumor cell ,Surgical oncology ,80 and over ,Circulating ,Sequencing ,Neoplasm Metastasis ,Cancer ,Aged, 80 and over ,0303 health sciences ,screening and diagnosis ,Liver Diseases ,Liver Disease ,Liver Neoplasms ,High-Throughput Nucleotide Sequencing ,Epithelial cell adhesion molecule ,Single Nucleotide ,Middle Aged ,Neoplastic Cells, Circulating ,Prognosis ,Epithelial Cell Adhesion Molecule ,3. Good health ,Detection ,Oncology ,030220 oncology & carcinogenesis ,Public Health and Health Services ,Female ,Stem cell ,Research Article ,Liver Cancer ,Carcinoma, Hepatocellular ,Epithelial-Mesenchymal Transition ,Oncology and Carcinogenesis ,Polymorphism, Single Nucleotide ,DNA sequencing ,03 medical and health sciences ,Rare Diseases ,Antigens, Neoplasm ,Clinical Research ,medicine ,Genetics ,Humans ,Oncology & Carcinogenesis ,Hepatocellular carcinoma (HCC) ,Antigens ,Polymorphism ,neoplasms ,030304 developmental biology ,Aged ,business.industry ,Carcinoma ,Human Genome ,Circulating tumor cells ,Hepatocellular ,medicine.disease ,digestive system diseases ,4.1 Discovery and preclinical testing of markers and technologies ,Circulating biomarkers ,chemistry ,EpCAM ,Cancer research ,Neoplasm ,business ,Digestive Diseases ,Cell Adhesion Molecules - Abstract
Background Circulating biomarkers are urgently needed in hepatocellular carcinoma (HCC). The aims of this study were to determine the feasibility of detecting and isolating circulating tumor cells (CTCs) in HCC patients using enrichment for epithelial cell adhesion molecule (EpCAM) expression, to examine their prognostic value, and to explore CTC-based DNA sequencing in metastatic HCC patients compared to a control cohort with non-malignant liver diseases (NMLD). Methods Whole blood was obtained from patients with metastatic HCC or NMLD. CTCs were enumerated by CellSearch then purified by immunomagnetic EpCAM enrichment and fluorescence-activated cell sorting. Targeted ion semiconductor sequencing was performed on whole genome-amplified DNA from CTCs, tumor specimens, and peripheral blood mononuclear cells (PBMC) when available. Results Twenty HCC and 10 NMLD patients enrolled. CTCs ≥ 2/7.5 mL were detected in 7/20 (35%, 95% confidence interval: 12%, 60%) HCC and 0/9 eligible NMLD (p = 0.04). CTCs ≥ 1/7.5 mL was associated with alpha-fetoprotein ≥ 400 ng/mL (p = 0.008) and vascular invasion (p = 0.009). Sequencing of CTC DNA identified characteristic HCC mutations. The proportion with ≥ 100x coverage depth was lower in CTCs (43%) than tumor or PBMC (87%) (p
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- 2015
21. Concurrent driver mutations in non-small cell lung cancer (NSCLC) patients (p) on targeted therapy uncovered by comprehensive molecular profiling
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Boris C. Bastian, Matthew A. Gubens, Collin M. Blakely, Luping Lin, Trever G. Bivona, Nikoletta Sidiropoulos, Saurabh Asthana, Barry S. Taylor, and Tyrrell A. Nelson
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Cancer Research ,business.industry ,medicine.medical_treatment ,Egfr tki resistance ,non-small cell lung cancer (NSCLC) ,medicine.disease ,Bioinformatics ,EGFR Tyrosine Kinase Inhibitors ,respiratory tract diseases ,Targeted therapy ,Oncology ,Egfr mutation ,Cancer research ,medicine ,business ,neoplasms - Abstract
11004 Background: NSCLC p with EGFR mutations respond initially to EGFR tyrosine kinase inhibitors (TKIs) but invariably develop acquired EGFR TKI resistance. Prior studies identified the EGFR T790M mutation and activation of MET, PI3K, AXL, HER2 and the MAPK pathway as drivers of acquired EGFR TKI resistance. To date, comprehensive molecular profiling to identify actionable modifiers of EGFR TKI response has not been conducted in NSCLC p on therapy. Methods: We performed next generation sequencing (NGS) using a 263-gene Nimblegen custom cancer panel on DNA isolated from primary patient lung adenocarcinoma FFPE specimens prior to initiating standard erlotinib treatment and upon the development of acquired erlotinib resistance after only 3 months of therapy. Results: In the pretreatment sample, we confirmed the presence of the EGFRL858R mutation in 95% of the sequencing reads and discovered a concurrent BRAF V600E mutation with a frequency of ~ 6%. NGS performed on the acquired erlotinib resistance sample revealed acquisition of the EGFR T790M mutation with a frequency of ~ 14%. Notably, the frequency of the BRAF V600E mutation increased 10-fold upon acquired erlotinib resistance from ~ 6% in the pretreatment tumor to ~ 60% in the recurrent tumor. We found that overexpression of BRAF V600E in H3255 human NSCLC, which harbor EGFR L858R (but not BRAF V600E) and are erlotinib sensitive, caused resistance to erlotinib treatment (10-fold increase in erlotinib IC50). BRAF V600E-mediated erlotinib resistance was reversed by treatment with the BRAF inhibitor vemurafenib. Additional functional studies are ongoing and the complete dataset will be presented. Conclusions: These results indicate that EGFR-mutant NSCLC can harbor additional oncogenic driver mutations in BRAF at low frequencies prior to therapy. EGFR TKI treatment can lead to expansion of BRAF V600E expressing tumor cells, resulting in acquired EGFR TKI resistance that can be reversed by BRAF inhibitor treatment. The data demonstrate the utility of routine molecular profiling of NSCLC p on targeted therapy and offer unprecedented insight into the genetic basis of therapeutic resistance.
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- 2013
22. Circulating tumor cells (CTC) in metastatic hepatocellular carcinoma (HCC): Comparison to control patients with nonmalignant liver diseases (NMLD) and exploratory characterization by next generation sequencing (NGS)
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Francis Y. Yao, Eric A. Collisson, Iche M. Siah, Nikoletta Sidiropoulos, Elizabeth M. Wayne, Alan P. Venook, Robin Katie Kelley, John W. Park, Tim Butler, Jimmy Hwang, Kimberley J. Evason, Bilal Hameed, and Mark Jesus M. Magbanua
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Whole Genome Amplification ,Cancer Research ,Pathology ,medicine.medical_specialty ,business.industry ,Cancer ,Ion semiconductor sequencing ,Cell sorting ,medicine.disease ,Peripheral blood mononuclear cell ,digestive system diseases ,Circulating tumor cell ,Oncology ,medicine ,Cancer research ,Biomarker (medicine) ,business ,neoplasms ,Whole blood - Abstract
207 Background: Noninvasive biomarkers are urgently needed to improve diagnosis, prognosis, and molecular characterization in HCC. Detection of CTC by EpCAM-enrichment methods is associated with poor outcomes in other tumors. Approximately 35% of HCC are EpCAM+. We hypothesized that CTC are detectable in a subset of metastatic HCC but not in NMLD and that CTC DNA might serve as a source of tumor DNA for biomarker detection and discovery. Methods: Whole blood was obtained from (1) patients with metastatic HCC and (2) NMLD controls. CTC were enumerated using CellSearch by blinded investigators. If > 10 CTC/7.5 mL, immunomagnetic enrichment for EpCAM followed by fluorescence-activated cell sorting (FACS) was also performed to collect highly purified CTC for whole genome amplification (WGA) by GenomePlex WGA4 kit. NGS using 46-gene AmpliSeq Cancer Panel (Ion Torrent) was performed on DNA from CTC, formalin-fixed paraffin embedded tumor (FFPE), and peripheral blood mononuclear cells (PBMC) if available. Results: 20 HCC and 10 NMLD were enrolled. FFPE was available in 7 HCC. In HCC cohort, median alpha-fetoprotein (AFP) was 492 ng/mL (range: 3.8-587,134) and vascular invasion (VI) was present in 13/20 (65%). CTC ≥ 3/7.5 mL were detected in 6/20 (30%, 95% CI: 0.08, 0.52) HCC and 0/9 (95% CI: 0, 0) eligible NMLD (p=0.07). One NMLD had CTC > 3/7.5 mL but was found to have HCC on surveillance ultrasound and excluded. CTC ≥ 3/7.5 mL were associated with AFP ≥ 400 (p=0.01) and VI (p=0.05) by Fisher’s exact test. NGS identified mutations listed in COSMIC in the majority including TP53 and CTNNB1. Variants appeared more frequent in CTC than FFPE in this small sample. Coverage was significantly lower in CTC than FFPE (coverage 20x mean 50% vs 93%, p < 0.02 by unpaired t-test). Conclusions: CTC are a promising biomarker in metastatic HCC and merit further study, including non-EpCAM methods. CTC were associated with AFP and VI. NGS of CTC WGA DNA is feasible and identified characteristic mutations but was limited by coverage bias. Updated NGS findings including 5 cases with paired CTC, FFPE, and/or PBMC will be presented.
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- 2013
23. Quality Improvement by Standardization of Procurement and Processing of Thyroid Fine-Needle Aspirates in the Absence of On-site Cytological Evaluation.
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Nikoletta Sidiropoulos, Larry J. Dumont, Allan C. Golding, Francis L. Quinlisk, Jorge L. Gonzalez, and Vijayalakshmi Padmanabhan
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NEEDLE biopsy , *CYTOLOGY , *CELLULAR pathology , *BIOLOGICAL specimens , *STANDARDIZATION , *THYROIDITIS - Abstract
Background:Thyroid nodules are relatively common and are routinely evaluated by fine-needle aspiration cytology, usually performed by clinicians. We noticed qualitative and/or quantitative variability in samples submitted to the cytopathology laboratory from clinicians, for example, the number of glass slides submitted (2–25) and air-dried smears versus alcohol-fixed slides, with variability in specimen adequacy and interpretability. The objective of this study was to standardize the preanalytic variables to determine if there is an improvement in the specimen quality.Methods:We standardized the method of collection (ultrasound-guided, 25-gauge needle, four passes) and preparation of samples (four total smears: two air-dried, two fixed, with liquid-based preparation and/or cell block) and personnel involved.Results:Standardization of thyroid nodule fine-needle aspiration and sample preparation by clinical staff resulted in an overall improvement in the quality of sample (odds ratio = 3.82, 95% confidence interval 2.02–7.24, p< 0.0001) with an increased proportion of satisfactory samples from 67% prestandardization to 89% poststandardization.Conclusions:Standardization resulted in a significant improvement in specimen interpretability. [ABSTRACT FROM AUTHOR]
- Published
- 2009
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