Liquid-based Cytology Specimens Research Articles

Handling of Thyroid FNA Samples During the COVID-19 Pandemic

Handling of Thyroid FNA Samples During the COVID-19 Pandemic

Comparison of the SuperARMS and ARMS for detecting EGFR mutations in liquid-based cytology specimens from NSCLC patients

BackgroundNon-surgical cytological specimens are adequate not only for accurate histological subtyping but also for molecular profiling. A modified amplification refractory mutation system polymerase chain reaction (ARMS PCR), known as SuperARMS PCR, was improved by optimizing the primers designation, which provides a higher sensitivity and specificity approach for free plasma DNA detection. It is unclear whether SuperARMS PCR detects epidermal growth factor receptor (EGFR) mutations in cytology samples. The aim of this study was to compare the EGFR mutations detected by ARMS PCR and SuperARMS PCR in cytology samples derived from advanced non-small cell lung cancer (NSCLC) patients.MethodsFrom March 2016 to March 2018, a total of 234 cytological samples were obtained from primary or metastatic lesions of NSCLC, including 144 fine-needle aspirations (FNAs), 36 endobroncheal ultrasonography (EBUS) FNAs, 36 transbronchial needle aspirations (TBNAs) and 18 pleural effusion (PLEs). EGFR mutations were simultaneously detected using an ADx-ARMS EGFR kit (Amoy Diagnostics CO., ltd., Xiamen, China) and an ADx-SuperARMS EGFR kit (Amoy Diagnostics CO., ltd., Xiamen, China). Digital droplet PCR (ddPCR) and next-generation sequencing (NGS) were further used to verify the EGFR mutant inconsistent samples.ResultsAll of the 234 patients with advanced or recurrent NSCLC were diagnosed and assessed by two cytopathologists, and their EGFR mutation statuses were successfully detected by ARMS and SuperARMS. Importantly, the SuperARMS and ARMS methods showed a highly concordant result of 94.0% (220/234) (95%CI: 85.0, 95.0%). The positive rate of the SuperARMS was higher than the ARMS in the cytology samples for EGFR detection (46.2% vs. 40.2%). The specific EGFR mutation sites in 16 samples (6.8%) were not completely consistent between the SuperARMS and ARMS. A total of 14 patients showed EGFR mutations when detected by SuperARMS, but by ARMS there were EGFR wild-type. Two patients were detected as having one more EGFR mutation site by SuperARMS than by ARMS. ddPCR and NGS were used to further confirm the EGFR mutations in these inconsistent samples. Eight samples had the same mutation results as the SuperARMS, and 6 samples were not verified because the remaining DNA was insufficient. A total of 78 EGFR mutation patients received Tyrosine Kinase Inhibitor (TKI) treatment. The overall objective response rate (ORR) was 88.5% (69/78) for EGFR TKI treatment.ConclusionSuperARMS showed a high sensitivity and specificity for EGFR detection and thus, is expected to become a routine test in the clinic to be used as a widely available, easy-to-operate and sensitive method for EGFR mutation detection in liquid-based cytology samples.

EGFR mutation genotyping and ALK status determination in liquid-based cytology samples of non-small cell lung cancer.

Personalised medicine for primary lung cancers (PLCs) requires molecular analysis of cancer tissue or cells. The primary objective of the present prospective study was to assess the concordance between epidermal growth factor receptor (EGFR) gene mutation detection and echinoderm microtubule-associated protein-like (EML) 4-anaplastic lymphoma kinase protein (ALK) expression using liquid-based cytology (LBC) samples and matched histology samples of PLC patients. A total of 117 patients who underwent surgical resection of non-small cell PLC were enrolled. Cytological specimens scratched from the resected PLC lesion were fixed in CytoRich Red. DNA extracted from LBC samples was examined for EGFR gene mutations. Anaplastic lymphoma kinase arrangement was analysed by immunostaining and fluorescence in situ hybridisation. Our patient cohort comprised 93 cases of adenocarcinoma, 16 squamous cell carcinoma, three adenosquamous carcinoma, two large cell neuroendocrine carcinoma, one pleomorphic carcinoma and two other cases. Sixty-six (58.4%) LBC samples harboured EGFR gene mutations. The overall concordance rate in EGFR gene mutation status, including minor mutations, between histologic and paired LBC specimens (N = 105) was 100%. The overall concordance rate of EGFR gene mutation status, including minor mutations and ALK status according to immunostains between histologic and paired LBC specimens, was 100% (105/105) and 100% (48/48), respectively. Genotyping and protein expression studies can be reliably performed using LBC samples prepared with CytoRich Red. Analysis of such samples may guide individual therapy in PLC patients.

Chlamydia Trachomatis Infection in High-Risk Human Papillomavirus Based on Cervical Cytology Specimen.

Objective:High-risk human papillomavirus (HR HPV) was associated with the development of cervical cancer. Asymptomatic Chlamydia trachomatis (C. trachomatis) infection is the most common bacterial, sexually-transmitted infection. This study aimed to investigate the association of C. trachomatis in positive HR HPV and the cytological results from liquid-based cytology (LBC). Methods:150 residual LBC specimens were collected; all of which had undergone cytology and HPV testing by Cobas. The samples were established as C. trachomatis using real-time PCR (RT-PCR) with Cryptic F/Cryptic R primers. Results:Of 150 positive HPV findings, the most common (72.7%, 109/150) were the 12 other HR HPVs (viz., 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68). The cervical cytology of those positive HR HPVs were mostly negative (70.0%, 105/150). The C. trachomatis infections in positive HR HPV were 16% (24/150) HPV. The analysis of the abnormal cytology revealed that 41.6% had C. trachomatis co-infection (C. trachomatis and HPV infection) viz., LSIL (20.8%), HSIL (12.5%), and ASC-US (8.3%). A comparison with positive HPV without C. trachomatis co-infection revealed that the highest prevalence was for LSIL, ASC-US, and HSIL (11.1%, 10.3%, and 6.4%, respectively). There was no difference between the abnormalities and negative cervical cytology with negative and positive C. trachomatis co-infection in HR HPV positive (p = 0.174). Conclusion: C. trachomatis infection was not significantly associated HR-HPV and abnormal cytology. This study confirms the increasing rate of C. trachomatis infection in asymptomatic women so routine screening for these infections has been suggested to (a) prevent complications such as the chronic pelvic pain associated with prolong infection and (b) reduce sexual transmission of the infection.

Analytic performance of PANArray HPV and HPV 9G DNA chip tests for genotyping of high-risk human papillomavirus in cervical ThinPrep PreservCyt samples.

The detection of high-risk human papillomavirus (HR-HPV) is important for early diagnosis of precancerous cervical lesion. The distribution of HR-HPV genotypes in East Asia is different from that in Western countries. HR-HPVs non-16/18 including HPV-58 are highly prevalent in East Asia. Thus, a variety of HPV tests that could identify individual genotypes have been widely used. HPV 9G DNA is a deoxyribonucleic acid-based chip test, while PANArray HPV chip is a peptide nucleic acid-based array. We compared the analytic performance of these two chips for detecting and genotyping HR-HPV using 356 liquid-based cytology specimens and evaluated their diagnostic accuracies based on direct sequencing. For identifying HR-HPV, PANArray HPV and HPV 9G DNA chips agreed with each other for 85.1% of samples. Overall strength of agreement between the two tests was substantial (k = 0.68). Specifically, these two tests almost perfectly agreed for detecting several types of HR-HPV, including HPV-16, -18, -35, -52, -58, and -59 (k>0.81 for all). According to direct sequencing, PANArray HPV produced consistently higher sensitivities for detecting HR-HPV than HPV 9G DNA for either overall or individual genotypes of HR-HPV. Sensitivities and specificities for detecting HPV-58 were perfect (100%) with PANArray HPV. In conclusion, PANArray HPV is more effective than HPV 9G DNA in detecting HR-HPV. It is more useful for regions with high prevalent HPV-58 infection.

Risk of malignancy and clinical outcomes of cyst fluid only nodules in the thyroid based on ultrasound and aspiration cytology.

BackgroundThe number of extensive studies focusing on cyst fluid only (CFO) thyroid nodules is limited, and the risk of malignancy (ROM) in CFO nodules has not been well‐established. Thus, the purpose of this study was to investigate CFO nodules using cytology and ultrasound. In addition, we sought to define the ROM and determine the recommended clinical management of CFO nodules.MethodsWe retrospectively reviewed cytological preparations of 678 nodules that were originally identified as CFO nodules, including conventional specimens in 209 nodules, liquid based cytology (LBC) specimens in 221 nodules, and both conventional and LBC specimens in 248 nodules. Ultrasound reports with representative photographs were also reviewed.ResultsOf the 678 CFO nodules, 214 (31.6%) were reclassified into other categories, including non‐diagnostic/unsatisfactory (ND/UNS) except for CFO (n = 15), benign (n = 198), and malignant (n = 1). Conventional preparations (33.5%) were more frequently reclassified than LBC preparations (13.6%; P < .0001). Re‐aspiration for diagnosis was performed for only one calcified nodule. The rates of surgical resection and malignancy were 3.0% and 0.2%, respectively. Based on American Thyroid Association guidelines and the Kuma Hospital ultrasound classification, worrisome sonographic features were identified in 5.8% and 0% of CFO nodules, respectively.ConclusionWe propose that CFO nodules should be classified as separate from ND/UNS nodules; they should be categorized as a subtype of benign nodules. However, it is essential that fine‐needle aspiration cytology be performed under ultrasound‐guided real‐time visualization of needle placement in the target nodule in all cases.

PLEOMORPHIC RHABDOMYOSARCOMA PRESENTING AS AN ANTERIOR MEDIASTINAL MASS IN AN ADULT

SESSION TITLE: Tuesday Fellows Case Report Posters SESSION TYPE: Fellow Case Report Posters PRESENTED ON: 10/22/2019 01:00 PM - 02:00 PM INTRODUCTION: Rhabdomyosarcoma (RMS) is a rare malignancy in adults. Data regarding treatment regimens is limited, making management of this malignancy difficult. CASE PRESENTATION: A 61 y/o female presented for evaluation of an anterior mediastinal mass. She was experiencing a dry cough, chest discomfort and a 25-pound weight loss for 2 years. Exam was unremarkable with an O2 saturation of 96% on room air. Chest CT revealed a 10.8cm anterior mediastinal mass with central necrosis (Figure 1). She underwent CT guided biopsy of the mediastinal mass and pathology reported a pleomorphic spindle cell lesion with focal myogenic differentiation. While awaiting outside pathology report, the patient developed worsening SOB and was hospitalized. She was intubated after admission for respiratory distress. CXR showed enlargement in her mediastinal mass (Figure 2). After intubation she required vasopressor support and temporary prone ventilation. CT surgery took the patient for emergent mediastinal exploration with excision of her mass. The mass was abutting the posterior sternum and growing into the pericardium. Most of the mass was removed with decompression of the airway and surrounding mediastinal structures. Final surgical pathology reported pleomorphic rhabdomyosarcoma (RMS). Further imaging did not reveal any metastatic disease. Given the rarity of this tumor and its unique location, the patient was referred to higher level of care for treatment planning. She eventually received one round of chemotherapy but continued to decline and died a few days after treatment. DISCUSSION: RMS are primarily pediatric neoplasms, although they can rarely occur in adults.1 Histologically, the WHO divides RMS into 4 sub-types: embryonal, alveolar, pleomorphic and spindle cell/sclerosing.1 Pleomorphic RMS (PRMS) are quite rare, accounting for 3% of soft tissue sarcomas and less than 1% of adult malignancies.2 Mediastinal PRMS are extraordinarily rare with well under 100 reported cases.3 PRMS has a tendency to metastasize to the lungs, lymph nodes and bone marrow with metastatic disease often present at initial diagnosis.2 Standard treatment is surgical resection with wide margins. When surgery is incomplete or unable to be performed, chemotherapy and/or radiation can be pursued.2 Given the rarity of this tumor, there is limited data to guide chemotherapy, with many studies reporting it is refractory to standard chemotherapy medications. PRMS is one of the most aggressive types of RMS with a reported 5-year mortality rate of 27-44%.2 CONCLUSIONS: PRMS in adults is a rare, aggressive malignancy with few guidelines for treatment. As our case illustrates, it can present in atypical locations, making treatment even more complex. As with our patient, appropriate preparation regarding proper positioning, medications, ventilation strategy and volume status must also be considered when managing patients with mediastinal masses. Reference #1: Rudzinski, Erin R., et al. “The World Health Organization Classification of Skeletal Muscle Tumors in Pediatric Rhabdomyosarcoma: A Report From the Children's Oncology Group.” Archives of Pathology & Laboratory Medicine, vol. 139, no. 10, 2015, pp. 1281–1287., https://doi.org/10.5858/arpa.2014-0475-oa. Reference #2: Xi, Shiwen, and Weihua Tong. “Pleomorphic Rhabdomyosarcoma Metastasis to Small Intestine Causing Intussusception.” Medicine, vol. 97, no. 51, 2018, https://doi.org/10.1097/md.0000000000013648. Reference #3: Nishijima, Yoshimi, et al. “Pleomorphic Rhabdomyosarcoma Arising in the Anterior Mediastinum: A Case Report with Cytological Features of Imprint and Liquid-Based Cytology Specimens.” Diagnostic Cytopathology, vol. 45, no. 4, 2016, pp. 333–338., https://doi.org/10.1002/dc.23654. DISCLOSURES: My spouse/partner as a Speaker/Speaker's relationship with Salix Please note: $1001 - $5000 Added 03/13/2019 by Ebtesam Islam, source=Web Response, value=Speaker fee No relevant relationships by Audra Schwalk, source=Web Response

Comprehensive validation of liquid-based cytology specimens for next-generation sequencing in cancer genome analysis.

In addition to conventional cytology, liquid-based cytology (LBC) is also used for immunocytochemistry and gene analysis. However, an appropriate method to obtain high quality DNA for next-generation sequencing (NGS) using LBC specimens remains controversial. We determined the optimal conditions for fixation with an alcohol-based fixative for LBC and DNA extraction using cultured cancer cell lines and clinical specimens. The extracted DNA was processed for NGS after the DNA quality was confirmed based on the DNA concentration and degree of degradation. The optimal conditions for cultured cells to obtain high quality DNA were to fix the cells at a density of 6 × 103 or 2 × 104 cells/mL and to use the magnetic bead-based DNA extraction method. Even after storing the fixed cells for 90 days, DNA extracted using the above and other extraction kits, including membrane-based methods, did not undergo degradation. Furthermore, 5-year-old residual LBC samples demonstrated high DNA quality that was suitable for NGS. Furthermore, a cancer genome panel analysis was successfully performed with DNA extracted from cultured cells fixed at 6 × 103 cells/mL for 90 days, and with DNA from residual LBC samples even after 1 year of storage. Residual LBC samples may be a useful source of DNA for clinical NGS to promote genome-based cancer medicine.

The Evolving Field of Cytopathology and Its Expanding Role in Pathologic Practice.

Cytopathology is one of the most rapidly developing fields in pathology, with significant impact on the diagnostic practice of all organ systems. Its impressive progress not only relies on its minimally invasive nature and cost-effectiveness, but also stems from the clear relationship of its diagnostic categories with risk of malignancy and clinical management, as well as the successful integration of molecular/genetic tests into daily practice. With the increasing clinical demand for less tissue and more specific diagnoses, cytopathology will lead future pathologic development in years to come. In this special section, we capture selected topics in recent cytopathologic progress, including both original studies and timely reviews.One of the most important breakthroughs in cytopathology in 2018 was the establishment and publication of the Milan System for Reporting Salivary Gland Cytopathology.1 The Milan system is based on the studies of many institutions around the globe. In this series, we publish the original article from Temple University (Philadelphia, Pennsylvania) by Luo et al describing their logistics and efforts to build an institutional 6-tier classification system to report salivary gland cytopathology. Slides of 107 salivary gland fine-needle aspirations (FNAs) with subsequent histology slides were reviewed and reclassified using the 6-tier system. The FNA diagnoses were generally consistent with histologic diagnoses for the disease spectrum. The sensitivity, specificity, positive predictive value, and negative predictive value for diagnosing malignancies with this system were 86% (12 of 14), 93% (40 of 43), 80% (12 of 15), and 95% (40 of 42), respectively. The risk of malignancy increased from 0% (0 of 13) for negative for neoplasm to 7% (2 of 29) for benign neoplasm, 67% (2 of 3) for suspicious for malignancy, and 83% (10 of 12) for positive for malignancy. This study points out the rationale and helps to pave the way for the adoption of the Milan System for Reporting Salivary Gland Cytopathology.Cytopathologic materials are a great source for molecular tests critical to guide therapeutic decision-making and accurate diagnoses.2 Doxtader et al describe their experience at the Cleveland Clinic with epidermal growth factor receptor (EGFR) mutation testing by next-generation sequencing and anaplastic lymphoma kinase (ALK) gene rearrangement testing by fluorescence in situ hybridization (FISH) in non–small cell lung carcinomas. At their institution, liquid-based cytology specimens are the primary resource used for molecular testing. In most instances, adequate DNA could be extracted from the residual cell pellets for next-generation sequencing, and ThinPrep slides could be used reliably for FISH testing for ALK gene rearrangements. In occasional cases where the cell pellet material was not adequate for molecular testing, cell blocks and/or surgical pathology specimens were secondary options. In a different original study using an immunocytologic approach, Ren and Klump showed that 87.6% of carcinomas of Müllerian origin in ascites and pleural fluids were positive for PAX8; 19.2%, 18.1%, 23.4%, and 8.5% of high-grade serous carcinomas were positive for GATA3, SOX2, uroplakin II, and SALL4, respectively. Increasing acceptance of molecular tests into cytopathologic practice is also highlighted by a recent recommendation from the International Mesothelioma Interest Group to conduct p16 (by FISH) and BAP1 (by immunohistochemistry) staining in cytologic specimens suspicious for mesothelioma.3The diagnostic power of cytopathology for infectious disease in the lower respiratory tract is highlighted by Baldassarri et al in this series. Cytomorphologic analysis helps to triage and plays a critical supporting role to the microbiology laboratory in prompt and accurate identification of causative organisms, particularly in immunosuppressed patients. Again, cytopathologic materials are perfect for molecular tests including polymerase chain reaction, immunochromatographic assay, and direct antigen detection in identification of bacteria, viruses, and fungal elements.Tremendous progress has been made in the past few years in cytopathology, from the standardized reporting system to FNA sample techniques to molecular tests of tumor-specific markers and specific infectious agents. The continued improvement of cytopathologic practice will undoubtably expand its role in the clinical management of patients.

A large series of hyalinizing trabecular tumors: Cytomorphology and ancillary techniques on fine needle aspiration.

Hyalinizing trabecular tumors (HTTs) are rare, essentially benign, follicular cell-derived thyroid neoplasms characterized by a trabecular growth pattern and nuclear pseudoinclusions. Their cytological findings are misleading, because these tumors are often misinterpreted on fine needle aspirate cytology as malignant lesions, such as papillary thyroid cancer and/or medullary thyroid cancer, leading to unnecessary total thyroidectomy. The aim of this study was to analyze the cytomorphological features and application of ancillary techniques in a series of HTTs. Of 26 histological cases of HTT collected from September 2001 to December 2018, 18 cases had concomitant cytopathology. Cytological cases were processed with liquid-based cytology (LBC). Immunocytochemistry for HBME-1 and galectine-3 as well as molecular testing for BRAFV600E mutation were performed on both LBC and histological specimens. The 18 lesions with fine needle aspirate cytology ranged in size from 5 to 45mm. Cytological diagnoses included: 1 benign lesion favoring goiter (5.5%), 4 atypia of undetermined significance (22.2%), 6 follicular neoplasms (33.3%), 5 suspicious for malignancy favoring papillary thyroid cancer (28%), and 2 malignant (11%). Hence, 89% HTT had a negative concordant immunopanel, and they were 100% wild-type BRAFV600E . The majority of our HTTs (83.3%) were diagnosed in the indeterminate Bethesda categories, suggesting that their cytomorphological features pose issues for reaching a conclusive cytological diagnosis. The ancillary test results in our series support the fact that HTT is a benign neoplasm.

Genome-wide profiling of human papillomavirus DNA integration in liquid-based cytology specimens from a Gabonese female population using HPV capture technology

Human papillomavirus (HPV) is recognised as the cause of precancerous and cancerous cervical lesions. Furthermore, in high-grade lesions, HPV is frequently integrated in the host cell genome and associated with the partial or complete loss of the E1 and E2 genes, which regulate the activity of viral oncoproteins E6 and E7. In this study, using a double-capture system followed by high-throughput sequencing, we determined the HPV integration status present in liquid-based cervical smears in an urban Gabonese population. The main inclusion criteria were based on cytological grade and the detection of the HPV16 genotype using molecular assays. The rate of HPV integration in the host genome varied with cytological grade: 85.7% (6/7), 71.4% (5/7), 66.7% (2/3) 60% (3/5) and 30.8% (4/13) for carcinomas, HSIL, ASCH, LSIL and ASCUS, respectively. For high cytological grades (carcinomas and HSIL), genotypes HPV16 and 18 represented 92.9% of the samples (13/14). The integrated form of HPV16 genotype was mainly found in high-grade lesions in 71.4% of samples regardless of cytological grade. Minority genotypes (HPV33, 51, 58 and 59) were found in LSIL samples, except HPV59, which was identified in one HSIL sample. Among all the HPV genotypes identified after double capture, 10 genotypes (HPV30, 35, 39, 44, 45, 53, 56, 59, 74 and 82) were detected only in episomal form. Our study revealed that the degree of HPV integration varies with cervical cytological grade. The integration event might be a potential clinical prognostic biomarker for the prediction of the progression of neoplastic lesions.

Automating the Paris System for urine cytopathology-A hybrid deep-learning and morphometric approach.

The Paris System for Urine Cytopathology (the Paris System) has succeeded in making the analysis of liquid-based urine preparations more reproducible. Any algorithm seeking to automate this system must accurately estimate the nuclear-to-cytoplasmic (N:C) ratio and produce a qualitative "atypia score." The authors propose a hybrid deep-learning and morphometric model that reliably automates the Paris System. Whole-slide images (WSI) of liquid-based urine cytology specimens were extracted from 51 negative, 60 atypical, 52 suspicious, and 54 positive cases. Morphometric algorithms were applied to decompose images to their component parts; and statistics, including the NC ratio, were tabulated using segmentation algorithms to create organized data structures, dubbed rich information matrices (RIMs). These RIM objects were enhanced using deep-learning algorithms to include qualitative measures. The augmented RIM objects were then used to reconstruct WSIs with filtering criteria and to generate pancellular statistical information. The described system was used to calculate the N:C ratio for all cells, generate object classifications (atypical urothelial cell, squamous cell, crystal, etc), filter the original WSI to remove unwanted objects, rearrange the WSI to an efficient, condensed-grid format, and generate pancellular statistics containing quantitative/qualitative data for every cell in a WSI. In addition to developing novel techniques for managing WSIs, a system capable of automatically tabulating the Paris System criteria also was generated. A hybrid deep-learning and morphometric algorithm was developed for the analysis of urine cytology specimens that could reliably automate the Paris System and provide many avenues for increasing the efficiency of digital screening for urine WSIs and other cytology preparations.

Sec62/Ki67 and p16/Ki67 dual-staining immunocytochemistry in vulvar cytology for the identification of vulvar intraepithelial neoplasia and vulvar cancer: a pilot study.

The aim of this study was to analyze the diagnostic performance of a newly established immunocytochemical dual-staining protocol for the simultaneous expression of SEC62 and Ki67 in vulvar liquid-based cytology specimens for the identification of vulvar intraepithelial neoplasia (VIN) and vulvar cancer. In addition, we investigated the p16/Ki67 dual stain, which has already been established in cervical cytology. For this pilot study, residual material from liquid-based cytology was collected retrospectively from 45 women. The presence of one or more double-immunoreactive cells was considered as a positive test result for Sec62/Ki67 and p16/Ki67 dual staining. The test results were correlated with the course of histology. All cases of VIN and vulvar cancer were Sec62/Ki67 and p16/Ki67 dual-stain positive, and normal and low-grade squamous intraepithelial lesions were all negative. The sensitivity of cytology for VIN + cases was 100% (22/22), whereas punch biopsy classified one case of vulvar carcinoma as inflammation. All cases with high-intensity (grades 3 and 4) Sec62 staining in Sec62/Ki67-positive cases were carcinomas. The results of this study demonstrate that Sec62/Ki67 and p16 Ki67 dual-staining cytology could be a promising adjunctive diagnostic tool for VIN and squamous cell carcinoma, in addition to standard histology.

Technique for preparation of liquid based cytology specimens from formalin-fixed tissue

Technique for preparation of liquid based cytology specimens from formalin-fixed tissue

Sec62/Ki67 dual staining in cervical cytology specimens: a new marker for high-grade dysplasia.

In the previous studies, we demonstrated that Sec62 is essential for tumor cell migration, epithelial-to-mesenchymal transition, and intracellular stress tolerance. An increase in Sec62 expression correlated with an increase in cervical dysplasia severity in liquid-based cytology specimens. Ki67 is an established proliferation marker. Thus, in this study, we examined a method of Sec62/Ki67 dual staining for the detection of high-grade dysplasia and cancer in cervical liquid-based cytology specimens. Sec62/Ki67 dual staining was performed on 100 cervical liquid-based cytology specimens. The staining results were correlated with cytological, immunocytological (p16/Ki67), colposcopic, and histological findings. All 56 (n = 56, 100%) cases of cervical intraepithelial neoplasia grade 3 and cervical cancer (CIN3+ lesions) were positive for Sec62/Ki67 staining, while low-grade lesions and normal cells were negative. Sec62/Ki67 staining was highly sensitive and specific for the detection of CIN2+ and CIN3+ lesions (94.37%; 100% and 100%; 84.09%, respectively). Sec62/Ki67 dual-staining immunocytochemistry is a promising cytological tool for interpreting high-grade squamous lesions in cytological specimens and for assessing the risk of progression to cancer.

Reducing DNA damage by formaldehyde in liquid-based cytology preservation solutions to enable the molecular testing of lung cancer specimens.

Liquid-based cytology (LBC) is a useful cytopathological method, and LBC lung adenocarcinoma specimens may be used for genetic analysis in the near future. In the current study, the authors determined whether LBC specimens can be used for epidermal growth factor receptor (EGFR) mutation analysis in human lung adenocarcinoma cell lines. Genomic DNA was extracted from 3 lung adenocarcinoma cell lines that were fixed in LBC preservation solution using 2 protocols (one for cultured cells and one for tissues) of a DNA extraction kit. Different fixation times were tested for each protocol: 30 minutes, 1 hour, and 1 to 9 days. As controls, cells also were fixed in 10% formalin or 95% ethanol. The authors investigated the effect of fixation time on DNA fragmentation, polymerase chain reaction (PCR) amplification, and EGFR mutation detection. The DNA yield of LBC specimens tended to decrease depending on fixation time. When using the DNA extraction protocol for tissues, PCR amplification was successful after 9 days of fixation, although extracted genomic DNA that was fixed for >1 hour demonstrated fragmentation. Mutation analyses using the Cycleave PCR method were successful after 7 days of fixation. The DNA extraction protocol for tissues was appropriate for lung adenocarcinoma cell lines that were stored for >1 day in a preservative solution. The results of the current study demonstrated that EGFR mutations can be detected on day 7 using lung adenocarcinoma cell lines fixed in CytoRich Red preservative. When LBC specimens are used for targeted molecular genetic testing, the appropriate preservative solution and extraction protocol first should be determined.

Prediction of Nondiagnostic Results in Fine-Needle Aspiration of Thyroid Nodules: utility of On-Site Gross Visual Assessment of Specimens for Liquid-Based Cytology

To explore a comprehensive approach for on-site gross visual assessments of liquid-based cytology (LBC) specimens of thyroid nodules and determine morphologic criteria that help predict nondiagnostic rates. Two-hundred nodules from 165 patients who underwent fine-needle aspiration (FNA) at our hospital were included in this prospectively designed, retrospective analysis. Specimens were visually assessed on-site for three morphologic categories (specimen color, specimen volume, and particle count) using a 5-point grading. Twenty-two nodules (11%) showed nondiagnostic results. Regarding specimen color, nondiagnostic rates tended to be higher in grades 1 (75%) and 5 (100%) than in grades 2 (18%), 3 (8%), or 4 (17%), with a significant difference between grade 1 and grade 3 ( P = .003). For specimen volume, nondiagnostic results were significantly more common in grade 1 (33%) and 5 (33%) than in grades 3 (5%) or 4 (1%) ( P<.005). There was a significant negative correlation between the grading of the particle count and the nondiagnostic rate (Spearman ρ = -1.000; P<.001). The sensitivity and specificity in the prediction of nondiagnostic results were 77% and 76%, respectively, at the optimal cutoff value of 2 (grade 2 or lower). Particle count was an important morphologic criterion that helped predict nondiagnostic rates in LBC specimens of thyroid nodules, and the specimen color and volume were also useful adjuncts. In routine practice, on-site gross visual assessment followed by resampling (if necessary) may potentially help reduce the rates of nondiagnostic results, repeat FNAs, and the number of unnecessary needle passes. FNA = fine-needle aspiration; LBC = liquid-based cytology; ROC = receiver operating characteristic; US = ultrasonography.

Value of HPV 16/18 Genotyping and p16/Ki-67 Dual Staining to Predict Progression to HSIL/CIN2+ in Negative Cytologies From a Colposcopy Referral Population.

To assess the prognostic value of human papillomavirus (HPV) 16/18 genotyping and p16/Ki-67 dual staining cytology in high-risk HPV (hrHPV)-positive women with no lesion or minor abnormalities. We evaluated progression to high-grade squamous intraepithelial lesion/cervical intraepithelial neoplasia grades 2 to 3 or cervical cancer (HSIL/CIN2+), persistence/regression of hrHPV infection in women referred to colposcopy showing hrHPV infection, histology diagnosis different from HSIL/CIN2+, and negative cytology. HPV 16/18 genotyping and dual staining were performed in liquid-based cytologic specimens obtained on the first visit. Progression was observed in 16 (8.0%) of 200 women. Those with HPV 16/18 infection had an increased risk of progression compared with women infected by other hrHPV types, and they also showed more persistence. However, no association was observed between progression or persistence and the result of the dual staining. HPV 16/18-positive women with no lesions or minor abnormalities are at high risk of progression to HSIL/CIN2+ and hrHPV persistence.

K-ras mutation analysis of residual liquid-based cytology specimens from endoscopic ultrasound-guided fine needle aspiration improves cell block diagnosis of pancreatic ductal adenocarcinoma.

BackgroundEndoscopic ultrasound-guided fine needle aspiration (EUS-FNA) technology is widely used for the diagnosis of pancreatic masses. However, in some cases, inadequate tissue volume or difficulty of morphological diagnosis are constraining factors for adequate cytopathological evaluation. K-ras mutation is the most frequently acquired genetic abnormality, occurring in approximately 90% of all patients with pancreatic ductal adenocarcinoma (PDAC). In the present study, the clinical utility of residual liquid-based cytology (LBC) specimens obtained using EUS-FNA for K-ras mutation analysis was evaluated.MethodsIn this study, 81 patients with pancreatic lesions were examined. The cell block (CB) specimens separated from EUS-FNA samples were morphologically evaluated by hematoxylin–eosin (HE) staining. Final diagnoses were confirmed by CB specimens, surgical resection specimens, diagnostic imaging, and clinical follow-up. Genomic DNA of residual LBC specimens stored at 4°C for several months were extracted and assessed for K-ras mutations using a fluorescence resonance energy transfer-based preferential homoduplex formation assay.ResultsK-ras mutation analysis using residual LBC samples was successful in all cases. The sensitivity, specificity, and accuracy of CB examination alone were 77.4%, 100%, and 81.3%, respectively, and those of the combination of CB examination and K-ras mutation analysis were 90.3%, 92.3%, and 90.7%, respectively. Furthermore, K-ras mutations were detected in 8 (57.1%) of 14 PDAC samples for which the CB results were inconclusive.ConclusionThese findings suggest that K-ras mutation analysis using residual LBC specimens improves the diagnostic accuracy of EUS-FNA.

Molecular Testing of Non-Small Cell Lung Carcinoma Diagnosed by Endobronchial Ultrasound-Guided Transbronchial Fine-Needle Aspiration: The Cleveland Clinic Experience.

Given the increasing demand for molecular testing of non-small cell lung carcinoma specimens to guide therapeutic decision-making and the trend toward minimally invasive techniques for obtaining diagnostic tissue, cytopathology laboratories must devise strategies to maximize DNA yield for necessary molecular testing. To describe our experience at Cleveland Clinic with epidermal growth factor receptor (EGFR) mutation testing by next-generation sequencing and anaplastic lymphoma kinase (ALK) gene rearrangement testing by fluorescence in situ hybridization of non-small cell lung carcinomas diagnosed by cytology, with an emphasis on specimens obtained by endobronchial ultrasound-guided transbronchial fine-needle aspiration. Data sources include a review of the current literature, including published articles from our institution. At our institution, liquid-based cytology specimens are the primary resource used for molecular testing of non-small cell lung carcinomas; in most instances, adequate DNA can be extracted from the residual cell pellet for next-generation sequencing, and ThinPrep slides can be used reliably for fluorescence in situ hybridization testing for ALK gene rearrangements. In occasional cases where the cell pellet material is not adequate for molecular testing, cell blocks and/or surgical pathology specimens are secondary options. The cytopathologist's role in specimen handling and triage is essential to ensure that molecular testing can be carried out successfully.