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/BACKGROUND: Samples 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 METHODS: This 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. RESULTS: The 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. CONCLUSION: Both 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.

High-risk human papillomavirus testing in cytology aspiration samples from the head and neck part 2: a survey of the American Society of Cytopathology community.

INTRODUCTION: High-risk human papillomavirus (HR-HPV) status is critical in the diagnosis of oropharyngeal squamous cell carcinoma, informing prognosis and choice of therapy. HR-HPV status additionally plays a key role in the evaluation of squamous cell carcinoma of unknown origin metastatic to cervical lymph nodes. Thus, HR-HPV testing of fine needle aspirate (FNA) specimens from the head and neck is invaluable for accurate diagnosis, prognostication, and treatment planning. MATERIALS AND METHODS: American Society of Cytopathology members were surveyed to understand the current state of HR-HPV testing on FNA samples from the head and neck. The survey focused on 3 main topic areas: practice setting of respondents, methods of collection and processing of aspirate specimens for HR-HPV testing, and validation of HR-HPV testing methodologies on aspirate samples. RESULTS: The survey reveals that laboratories employ various methods to detect HR-HPV in FNA samples, most commonly p16 immunohistochemical staining of cell block sections. Although some laboratories have independently validated their HR-HPV detection method, such validation is not universal. Finally, not all respondents currently have HR-HPV testing available, but approximately half of those without a testing method desire to make HR-HPV testing of FNA samples available. CONCLUSIONS: Survey responses highlight that various testing modalities are utilized for HR-HPV detection in aspirate samples. However, internal laboratory validation of HR-HPV testing for FNA specimens is not ubiquitous despite professional society recommendations.

Cross-contamination in cytology processing: a review of current practice.

INTRODUCTION: New cytopreparatory technologies decrease the need for direct smears in favor of an increased use of liquid-based cytology methods. Despite these practice changes, Clinical Laboratory Improvement Amendments continue to require that cytopathology laboratories have procedures to prevent cross-contamination (CC). While the incidence of CC is not well documented, specific cytologic preparations and specimens with a high potential for CC have not been generally defined by professional guidelines or consensus. The American Society of Cytopathology Clinical Practice Committee surveyed cytology practitioners to better understand current practice related to CC in cytology. MATERIALS AND METHODS: The survey focused on four topics: (1) practice settings and demographic data; (2) current practice for meeting CC requirements; (3) practice for rapid on-site evaluation; and (4) preparation types considered high risk for CC. The survey was sent to all American Society of Cytopathology and American Society for Cytotechnology members from July 1 to August 14, 2020. RESULTS: Ninety-eight percent of laboratories had a written CC policy, with 66.18% of the policies addressing rapid on-site evaluation CC procedures. Documented cases of CC were rare. Alcohol-fixed, direct smears of Pap-stained fluids were deemed the most likely to be impacted by CC. Cell block contamination during the histologic processing were reported by 56.20% of respondents. CONCLUSIONS: Changes in practice has resulted in decreased preparation types associated with a high potential for CC. Laboratories should follow a risk-based approach to define these cases. Knowledge of practice patterns among laboratories can guide the development and refinement of policy and procedures.

The utility of adaptive eLearning in cervical cytopathology education.

BACKGROUND: Adaptive eLearning allows students to experience a self-paced, individualized curriculum based on prior knowledge and learning ability. METHODS: The authors investigated the effectiveness of adaptive online modules in teaching cervical cytopathology. eLearning modules were created that covered basic concepts in cervical cytopathology, including artifacts and infections, squamous lesions (SL), and glandular lesions (GL). The modules used student responses to individualize the educational curriculum and provide real-time feedback. Pathology trainees and faculty from the authors' institution were randomized into 2 groups (SL or GL), and identical pre-tests and post-tests were used to compare the efficacy of eLearning modules versus traditional study methods (textbooks and slide sets). User experience was assessed with a Likert scale and free-text responses. RESULTS: Sixteen of 17 participants completed the SL module, and 19 of 19 completed the GL module. Participants in both groups had improved post-test scores for content in the adaptive eLearning module. Users indicated that the module was effective in presenting content and concepts (Likert scale [from 1 to 5], 4.3 of 5.0), was an efficient and convenient way to review the material (Likert scale, 4.4 of 5.0), and was more engaging than lectures and texts (Likert scale, 4.6 of 5.0). Users favored the immediate feedback and interactivity of the module. Limitations included the inability to review prior content and slow upload time for images. Learners demonstrated improvement in their knowledge after the use of adaptive eLearning modules compared with traditional methods. CONCLUSIONS: Overall, the modules were viewed positively by participants. Adaptive eLearning modules can provide an engaging and effective adjunct to traditional teaching methods in cervical cytopathology. Cancer Cytopathol 2018;126:129-35. © 2017 American Cancer Society.

Fine-needle aspiration cytology of endometriosis.

Endometriosis commonly involves the pelvis, but may also present as a palpable mass in extrapelvic sites, such as the abdominal wall or inguinal region, where it can be evaluated by fine needle aspiration (FNA). In this report, we illustrate the findings seen in seven cases of endometriosis diagnosed by FNA in patients with a chief complaint of pain associated with an abdominal wall or pelvic mass, occurring in a setting of prior pelvic surgery. The most common previous surgery was Cesarean section (n = 6), followed by hysterectomy (n = 2), and hernia repair (n = 1). In all cases, cytologic examination revealed a glandular component composed largely of orderly fragments of cohesive epithelial cells, a spindle cell stromal component presenting either as loosely organized tissue fragments or single cells, and rare hemosiderin-laden macrophages. Four cases showed focal cytologic atypia in the glandular component with extreme nuclear atypia identified in two of these cases. Atypical features included nuclear crowding and disorganization, nuclear enlargement, hyperchromasia with irregular chromatin distribution and anisonucleosis, raising the possibility of a coexistent malignancy and recommendation for excision. Although malignancy was not identified in follow-up surgical excision specimens, the wide range of cytomorphologic changes that can be seen in FNA specimens of endometriosis should be recognized. Diagn. Cytopathol. 2017;45:359-363. © 2016 Wiley Periodicals, Inc.

Subtype distribution of human papillomavirus in HIV-infected women with cervical intraepithelial neoplasia stages 2 and 3 in Botswana.

Human papillomavirus (HPV) vaccines containing types 16 and 18 are likely to be effective in preventing cervical cancer associated with these HPV types. No information currently exists in Botswana concerning the HPV types causing precancerous or cancerous lesions. Our goal was to determine the prevalence of HPV types associated with precancerous cervical intraepithelial neoplasia (CIN) stages 2 and 3 in HIV-infected women in Gaborone, Botswana. HIV-infected women referred to our clinic with high-grade intraepithelial lesion on the Pap smear were enrolled in the study. HPV typing was only performed if the histopathology results showed CIN stage 2 or 3 disease using linear array genotyping (CE-IVD, Roche Diagnostics). One hundred HIV-infected women were identified with CIN stages 2 or 3 between August 11, 2009 and September 29, 2010. Eighty-two of 100 women enrolled had coinfection by multiple HPV subtypes (range, 2 to 12). Of the remaining 18 women, 14 were infected with a single high-risk subtype and 4 had no HPV detected. Overall, 92 (92%) women were infected with at least 1 high-risk HPV subtype, and 56 were coinfected with more than 1 high-risk HPV type (range, 2 to 5). Fifty-one (51%) women had HPV subtypes 16, 18, or both. HPV 16 and 18 are the most common types in HIV-infected women with CIN 2 or 3 in Gaborone, Botswana, suggesting that the implementation of HPV vaccination programs could have a significant impact on the reduction of cervical cancer incidence. However, given the relative lack of knowledge on the natural history of cervical cancer in HIV-infected women and the significant prevalence of infection and coinfection with other high-risk HPV types in our sample, the true impact and cost-effectiveness of such vaccination programs need to be evaluated.

Vaginal intraepithelial neoplasia (VAIN) after radiation therapy for gynecologic malignancies: a clinically recalcitrant entity.

OBJECTIVES: Vaginal dysplasia is associated with prior radiation therapy (RT) for gynecologic malignancies. We reviewed our institution's experience with VAIN in patients who were treated with radiation therapy for a gynecologic malignancy. METHODS: A retrospective review of patients treated for VAIN was performed. All cases of patients followed and treated for VAIN after radiation therapy were identified (n=10), along with a cohort of patients with VAIN who did not have radiation therapy (n=23). RESULTS: Mean follow-up after initial diagnosis of VAIN was 37.6 months (range: 12 to 72). Cytologic screening events after diagnosis of VAIN (n=105) showed that patients with prior RT were more than twice as likely to have recurrent dysplasia (OR 3.625, 95% CI=from 1.454 to 9.0376) after treatment. Of patients who recurred, the mean time to first recurrence was 12.3 months in cases and 15.3 months in controls, which was not statistically significant (p=0.31). Screening practices at our institution ranged from 3 month to 12 month intervals. 3 patients in the RT group and 1 patient in the control group developed invasive squamous cell cancer of the vagina. CONCLUSIONS: Vaginal dysplasia after radiation therapy is more refractory to treatment than dysplasia not associated with radiation therapy, more likely to recur after surgical and ablative therapy, and may also be more likely to progress to invasive cancer. These data support the need for further study to determine the optimal follow-up screening interval and whether aggressive surgical or ablative treatment stems disease progression in this clinical scenario.

Adeno-associated virus-2 (AAV-2) causes trophoblast dysfunction, and placental AAV-2 infection is associated with preeclampsia.

Shallow invasion by extravillous trophoblast cells into the uterine wall reduces placental perfusion and causes placental dysfunction, but the one or more causes of shallow placental invasion are unknown. We hypothesized that infection with adeno-associated virus-2 (AAV-2) inhibits trophoblast invasion and is associated with preeclampsia, which is a common obstetric complication resulting from placental dysfunction. We determined that transformed extravillous trophoblast (HTR-8/SVneo) cells were susceptible to AAV-2 infection in the presence or absence of adenovirus, which provides helper function for AAV-2 replication, and that AAV-2 infection reduced invasion of HTR-8/SVneo cells through an extracellular matrix before cytopathic effects were detected. In a case-control study, AAV-2 DNA was found more frequently in trophoblast cells from cases of severe preeclampsia (22/40) than from normal term deliveries (5/27, P = 0.002). These results indicate that AAV-2 infection is a previously unidentified cause of placental dysfunction. Additional studies to determine the susceptibility of extravillous trophoblast to other viruses, and the mechanisms by which viral infection impairs placental function, are warranted.

Rapid detection of the t(11;14) translocation in mantle cell lymphoma by interphase fluorescence in situ hybridization on archival cytopathologic material.

BACKGROUND: The cytomorphologic diagnosis of mantle cell lymphoma (MCL) can be difficult and requires ancillary studies for accurate subclassification. More than 95% of MCLs are known to carry the t(11;14) chromosomal translocation. However, traditional cytogenetic studies on cytologic material can be both difficult technically and time consuming. Interphase fluorescence in situ hybridization (FISH) can be a powerful tool for detecting chromosomal changes in individual tumor cells. The authors evaluated the utility of interphase FISH for the rapid detection of t(11;14) in archival cytologic material. METHODS: The cytopathology data bases at two institutions were searched for patients with well characterized MCL (biopsy, immunophenotyping). Ten patients with MCL (8 fine-needle aspiration samples and 2 body cavity fluid samples) were identified. The area of interest on the cytology slides was marked and hybridized with two-color, locus-specific identifier DNA probes. A dual-fusion probe signal was used to detect the juxtaposition of the immunoglobulin heavy-chain (IgH) (14q32) locus with cyclin D1 (CCND1) gene sequences (11q13). Samples with tumor cell nuclei that showed at least one yellow fusion signal in addition one green signal (IgH) and one orange signal (CCND1) were interpreted as positive. Positive and negative controls were used. RESULTS: The t(11;14) translocation was detected by FISH in 10 of 10 patients (100%) with MCL. CONCLUSIONS: The cytomorphology of small-to-intermediate cell lymphomas, including MCL, follicular lymphoma, and marginal zone/mucosa-associated lymphoid tissue lymphoma, can show overlapping cytomorphologic features with one another as well as with reactive lymphoid proliferations. In selected samples in which specific classification is not possible or when confirmation is required on a small sample size, molecular analysis and cytogenetics may be helpful in arriving at an unambiguous cytodiagnosis and subclassification. Distinction of MCL from other lymphomas is important, because the clinical course is aggressive, and response to conventional chemotherapy is poor. This study showed that the detection of t(11;14) by FISH can be performed rapidly and easily on archival cytologic material for the molecular diagnosis of MCL.

Hypoxia-inducible erythropoietin signaling in squamous dysplasia and squamous cell carcinoma of the uterine cervix and its potential role in cervical carcinogenesis and tumor progression.

Tissue hypoxia is a characteristic property of cervical cancers that makes tumors resistant to chemo- and radiation therapy. Erythropoietin (Epo) is a hypoxia-inducible stimulator of erythropoiesis. Acting via its receptor (EpoR), Epo up-regulates bcl-2 and inhibits apoptosis of erythroid cells and rescues neurons from hypoxic damage. In addition to human papillomavirus infection, increased bcl-2 expression and decreased apoptosis are thought to play a role in the progression of cervical neoplasia. Using reverse transcriptase-polymerase chain reaction and Western blotting we showed that HeLa and SiHa cervical carcinoma cells and human cervical carcinomas express EpoR, and that hypoxia enhances EpoR expression. Exogenous Epo stimulated tyrosine phosphorylation and inhibited the cytotoxic effect of cisplatin in HeLa cervical carcinoma cells. Using immunohistochemistry, we examined the expression of Epo, EpoR, p16, hypoxia-inducible factor (HIF)-1alpha, and bcl-2 in benign and dysplastic cervical squamous epithelia and invasive squamous cell carcinomas (ISCCs). EpoR expression in benign epithelia was confined to the basal cell layers, whereas in dysplasias it increasingly appeared in more superficial cell layers and showed a significant correlation with severity of dysplasia. Diffuse EpoR expression was found in all ISCCs. Expression of Epo and HIF-1alpha was increased in dysplasias compared to benign epithelia. Focal Epo and HIF-1alpha expression was seen near necrotic areas in ISCCs, and showed correlation in their spatial distribution. Significant correlation was found between expression of EpoR, and p16 and bcl-2 in benign and dysplastic squamous epithelia. Our results suggest that increased expression of Epo and EpoR may play a significant role in cervical carcinogenesis and tumor progression. Hypoxia-inducible Epo signaling may play a significant role in the aggressive behavior and treatment resistance of hypoxic cervical cancers.

Cytomorphologic features of fine-needle aspiration of metastatic and recurrent melanoma.

Melanoma is an aggressive malignancy with a growing prevalence. Although early detection and excision offer a potential cure, recurrences and metastases are not uncommon. Fine-needle aspiration (FNA) can play a vital role in their detection as a relatively noninvasive, rapid, and economical alternative for tracking disease evolution. Prior clinical history and classic cytological features of melanoma (loosely cohesive smear pattern and single cells with large nuclei, prominent nucleoli, and melanin pigment) aid in cytological diagnoses. However, not all melanomas contain melanin pigment or characteristic cytologic features. We examined a large series of melanoma cases to determine the incidence of melanin pigment, the most common cell morphology, and the presence or absence of apoptosis/necrosis associated with this highly aggressive neoplasm.

Overexpression of p16INK4A in liquid-based specimens (SurePath) as marker of cervical dysplasia and neoplasia.

Human papillomavirus (HPV) is recognized as a causal agent for cervical carcinomas. Assimilation of HPV oncogenes E6 and E7 into the host DNA promotes upregulation of cyclin dependent kinase inhibitor (CDKI) p16(INK4A), detectable by monoclonal antibody in the developing cervical cancer cells. The aim of this study was to 1) develop a protocol for p16(INK4A) immunocytochemical staining on SurePath preparations, and 2) determine its utility as an HPV marker on a spectrum of cervical reactive and neoplastic lesions. Seventy-two specimens consisting of 28 nonneoplastic/nondysplastic cases (NN), one reactive glandular cells (RGC), 27 low-grade squamous intraepithelial lesions (LSIL), 10 high-grade squamous intraepithelial lesions (HSIL), one squamous cell carcinoma (SCCA), four atypical glandular cells (AGUS), and two adenocarcinomas (ADCA) were reprepped by SurePath and antibody to p16(INK4A) applied at 1:100 dilution using the Dako Envision + System on the Dako Autostainer. Expression of p16(INK4A) within the nucleus principally and cytoplasm of at least 10-15 cells was considered positive. All initial Papanicolaou-stained discrepant cases (p16(INK4A) positivity of NN and RGC cases and lack of reactivity in LSIL, HSIL, and AGUS) were reviewed. Nine of ten (90%) HSIL, one (100%) SCCA, 21/27 (78%) LSIL, and some reactive and inflammatory specimens demonstrated the presence of p16(INK4A). Reevaluation of discrepant cases revealed that several were underinterpreted (four NN were LSIL, one RGC was AGUS) or overinterpreted (one LSIL was NN). Following reassessment, false-positive staining was present in only 1/25 (1.4%) NN. Six of 30 (20%) LSIL lacked p16(INK4A) positivity. One of 10 (10%) HSIL had no staining. Two of four AGUS did not react with p16(INK4A) antibody. Both SCCA (1) and ADCA (2) had positive expression. This study confirms the intimate relationship between p16(INK4A) and HPV cytopathic effect. The p16(INK4A) immunocytochemical stain can be applied to liquid-based cervical preparations. This technique offers a more objective approach to deciphering "gray areas" of gynecologic cytopathology.

ASCUS in Papanicolaou smears. Problems, controversies, and potential future directions.

Each year, an estimated 2 to 3 million women in the United States receive a Papanicolaou smear diagnosis of atypical squamous cells of undetermined significance (ASCUS). There is no uniform practice for the diagnosis or management of these patients, and the annual cost of aggressively treating ASCUS lesions is estimated to be billions of dollars. Since its introduction in 1988, ASCUS has been problematic and controversial. ASCUS is a problem to define, to diagnose, to reproduce, and to manage. The following article reviews the various aspects and problems of ASCUS, its controversies regarding diagnosis, reproducibility, and management, and discusses the possible future directions of this category in light of the recent Bethesda System 2001 Workshop recommendations.