Diagnostic efficacy and molecular testing by combined fine-needle aspiration and core needle biopsy in patients with a lung nodule.

BACKGROUND: Combined image-guided fine-needle aspiration biopsy (FNA) and core needle biopsy (CNB) has become the standard of care for diagnosis and/or molecular testing for patients with a solitary lung nodule at our institution. Our purpose was to evaluate the efficacy of this practice. METHODS: We identified patients who underwent combined lung FNA/CNB during 2012 at our institution. A total of 667 patients who underwent 682 combined lung FNA/CNB procedures were included in the study, including 355 men and 312 women. Combined lung FNA/CNB procedures were performed by a radiologist. The adequacy of FNA specimens was assessed immediately by a cytopathologist. The FNA and CNB specimens were interpreted separately by a cytopathologist and a surgical pathologist, respectively. The diagnostic accuracy of the combined technique was determined. RESULTS: The rate of diagnostic consistency between FNA and CNB was 83.4%, and the rate of diagnostic accuracy for malignancy was 98.5% for combined FNA/CNB. Combined FNA/CNB showed a high diagnostic efficacy for malignancy (sensitivity, 97.6%; specificity, 100%). Combined FNA/CNB had a lower false-negative rate for malignancy (2.2%) than either FNA (7.2%) or CNB (6.2%) alone. FNA contributed to 10.3% of molecular analyses as a complementary tissue source. CONCLUSIONS: Combined lung FNA/CNB has high diagnostic efficacy for malignancy and a lower false-negative rate than either procedure alone. FNA was a valuable complement to CNB for molecular testing, potentially reducing patient inconvenience and morbidity associated with repeated lung needle biopsy.

Cervical cytology reproducibility and associated clinical and demographic factors.

BACKGROUND: Although the Pap test has been the standard screening method for cervical precancer/cancer detection, it has been criticized for having a relatively low sensitivity and a low reproducibility between pathologists. There is limited knowledge about inter-rater agreement and what clinical and demographic factors are associated with disagreements between pathologists reading the same Pap smear. METHODS: This study aimed to assess inter- and intra- rater agreement of the Pap smear in 1619 cytologic slides with biopsy confirmation, using kappa statistics. Clinical and demographic factors associated with higher odds of inter-rater agreement were also examined and stratified by histologic diagnosis grade. RESULTS: Using a five grade classification system, the overall kappa statistics for total, inter-rater, and intra-rater samples were 0.62, 0.57, and 0.88 (unweighted) and 0.83, 0.81, and 0.95 (weighted), respectively. In stratified analyses by histologic grade, total kappas ranged from 0.40 (atypia) to 0.64 (human papilloma virus/CIN 1). Factors such as referral for abnormal Pap test (diagnostic vs screening population), recruiting site, and parity were found to be associated with higher agreement between the two cytologic readings. CONCLUSIONS: We observed relatively higher levels of agreement compared with other studies. However, variability was considerable and agreement was generally moderate, suggesting that cervical screening test accuracy and reproducibility needs to be improved.

Incorporation of Cervista Human Papillomavirus 16/18 Assay Into Algorithms for Classifying Human Papillomavirus Status in Formalin-Fixed, Paraffin-Embedded Head and Neck Squamous Carcinoma Specimens.

CONTEXT.­: Human papillomavirus (HPV) DNA in situ hybridization (ISH) assay and p16 immunohistochemistry (IHC) are used to determine high-risk HPV status in formalin-fixed, paraffin-embedded (FFPE) tissues in oropharyngeal squamous cell carcinoma (SCC). Although high sensitivity and specificity for HPV can be obtained by combined p16 IHC and HPV DNA ISH, the occasional discrepancy between these assays has prompted evaluation of Cervista HPV assays in FFPE tissue from patients with oropharyngeal SCC. OBJECTIVE.­: To compare the efficacy of Cervista HPV 16/18 and Cervista HPV HR assay to that of HPV DNA ISH assay and p16 IHC in FFPE tissue in head and neck squamous cell carcinoma of oropharyngeal origin. DESIGN.­: Archived FFPE tissue from 84 patients with SCC of oropharyngeal origin and available HPV DNA ISH and p16 IHC test results were tested with the Cervista HPV 16/18 assay and further verified by polymerase chain reaction (PCR)-based HPV16/18 genotyping tests in cases with discrepancy. RESULTS.­: Of the 84 specimens, 75% (63 of 84) were positive and 16% (13 of 84) had discrepant or equivocal findings by p16 IHC and HPV DNA ISH testing. Use of Cervista HPV assays, either to clarify discrepant/equivocal findings or as confirmation after initial p16 IHC/HPV DNA ISH tests, identified 81% (68 of 84) of HPV-positive cases without equivocal HPV results. Five of 13 cases with discrepancy or equivocal HPV DNA ISH results tested positive for HPV16 or HPV18 by Cervista HPV 16/18 assay, which was further confirmed by PCR-based HPV 16/18 genotyping. CONCLUSIONS.­: The Cervista HPV assays are a reasonable alternative to HPV DNA ISH in determining HPV status in FFPE tissue specimens from patients with oropharyngeal SCC.

The use of cytological material in melanoma for programmed death ligand 1 immunostaining.

OBJECTIVE: Interest in immune therapies has exploded since the 2014 approval of first-generation programmed cell death 1 blocking antibodies for use in advanced melanoma. Clinical trials have focused primarily on histological material as the gold standard for evaluating programmed death ligand 1 (PD-L1) by immunoperoxidase (IPOX) studies. Studies validating the use of cytological specimens in the assessment of PD-L1 by IPOX staining are needed to optimise tissue utilisation in complementary diagnostic testing. METHODS: Twenty-three melanoma surgical biopsies (SBx) with an IPOX stain for PD-L1 clone 28-8, and a corresponding cytological specimen from the same patient, adequate for PD-L1 evaluation, were selected. Cell-transfer cell blocks (CBs) and conventional CBs were used to perform PD-L1 testing. Tumour proportion scores (TPS) were generated and the results were correlated with the corresponding SBx. RESULTS: Overall agreement (OA) using a ≥1% TPS cut-off for SBx compared to CB was 88.9%, positive percent agreement (PPA) was 87.5%, and negative percent agreement (NPA) was 100%, OA using a ≥5% TPS cut-off was 55.6%, PPA was 42.9%, and NPA was 100%. SBx compared to cell-transfer CB using a ≥1% TPS cut-off had an OA of 65.2%, a PPA of 55.6%, and a NPA of 100%, while a ≥5% TPS cut-off generated an OA of 52.2%, a PPA of 35.7%, and a NPA of 77.8%. CONCLUSION: Our results demonstrate that cytological material, particularly conventional CB, is a viable alternative for evaluating PD-L1 in melanoma cases and suggest that a lower threshold (≥1%) may be beneficial when evaluating cytological material.

Accuracy of diagnosing mantle cell lymphoma and identifying its variants on fine-needle aspiration biopsy.

BACKGROUND: Mantle cell lymphoma (MCL) is an incurable B-cell lymphoma portending an aggressive clinical course; the blastoid and pleomorphic morphological variants have an even worse prognosis. In addition, patients with classic morphology and a high proliferation index (HPI), also have reduced survival. Although variants have been defined, to the authors' knowledge the ability to detect these subtypes by fine-needle aspiration biopsy (FNAB) has not been described. METHODS: MCL cases diagnosed by lymph node FNAB with concurrent core needle biopsy were reviewed from 146 patients, accounting for 172 specimen pairs. FNAB and core needle biopsy diagnoses were compared to determine concordance rates. Flow cytometric immunophenotype and Ki-67 rates were evaluated. RESULTS: The classic subtype was diagnosed in 58% of cases (99 of 172 pairs) and variant morphology was diagnosed in 42% of cases (73 of 172 pairs) by histology. Twenty-nine patients presented with variant morphology whereas 28 underwent transformation. A nontraditional immunophenotype including loss of CD5 or FMC-7 and expression of CD23 and CD10 was found in 44% of variants (29 of 66 variants) and 19% of classic subtypes (18 of 94 classic subtypes) (P = .0008). Ki-67 rates averaged from 56% to 76% for blastoid and pleomorphic cases, 53% to 55% for MCL-HPI cases, and 17% to 19% for classic cases. The sensitivity and specificity to detect MCL variants by FNAB were 74% and 93%, respectively. CONCLUSIONS: The accuracy of diagnosing MCL is high when adequate samples for cytomorphology and flow cytometry are obtained. Subtyping variants by cytomorphology alone has challenges, but overall demonstrates high sensitivity and specificity. The performance of Ki-67 on cytology specimens is useful for detecting MCL with HPI.

Programmed death ligand 1 testing in non-small cell lung carcinoma cytology cell block and aspirate smear preparations.

BACKGROUND: Immune checkpoint inhibitors targeting the programmed cell death 1 (PD-1) receptor and its ligand, programmed death ligand 1 (PD-L1), have emerged as a therapeutic approach for patients with non-small cell lung carcinoma (NSCLC). PD-L1 expression, assessed by immunohistochemistry (IHC), is used to select patients for PD-1/PD-L1 inhibitor therapy. Most studies have been performed with histology specimens, with limited data available on the performance in cytology specimens. This study evaluated PD-L1 in cytology specimens and compared the results with those from paired core-needle biopsy for concordance. METHODS: Forty-one NSCLC fine-needle aspiration cases that had paired core-needle biopsy specimens with PD-L1 IHC were selected. A Papanicolaou-stained direct smear and a cell block section from each case were stained with a Dako PD-L1 pharmDx antibody (clone 22C3). Only slides with 100 or more tumor cells (37 smears and 38 cell blocks) were evaluated. Tumor proportion scores (TPS) were assessed on the basis of the partial/complete membranous staining of tumor cells and were correlated with those of paired core-needle biopsy. RESULTS: All 9 smears that were negative for PD-L1 staining showed 100% concordance with the paired core-needle biopsy, whereas 28 smears with PD-L1 expression showed a similar TPS, except for 1 smear that was discordant. In contrast, 10 negative paired core-needle biopsy cases corresponded to 9 concordant negative cell blocks, whereas 1 cell block had a TPS of 1% to 5%. The remaining 28 cell blocks demonstrated PD-L1 expression, with 22 cases showing a TPS similar to that of the paired core-needle biopsy, whereas 6 cell blocks were discordant, likely because of intratumoral heterogeneity. CONCLUSIONS: The results show that NSCLC cytology samples evaluated for PD-L1 have high concordance with paired core-needle biopsy samples and can be used for assessing PD-L1 expression. Cancer Cytopathol 2018;126:342-52. © 2018 American Cancer Society.

Salvaging the supernatant: next generation cytopathology for solid tumor mutation profiling.

With the expanding role of targeted therapy in patients with solid tumors, pathologists face the daunting task of having to maximize limited volume tissue obtained by fine needle aspiration for a variety of molecular tests. While most molecular studies on fine needle aspiration samples have been reported using cellular material, recent studies have shown that a substantial amount of DNA can be retrieved from the supernatant fluid of aspirate needle rinses after cell pelleting for cytospin or cell block preparations. In routine clinical workflow, the supernatant is discarded; however this fluid may provide a complementary source of DNA for tumor mutational profiling. In this study, we evaluated the post-centrifuged supernatant from 25 malignant and 10 benign fine needle aspiration needle rinses. The mean and median DNA yields from the supernatants were 445 ng and 176.4 ng (range, 15.1-2958 ng), respectively. Next generation sequencing using the Ion AmpliSeq Cancer Hotspot Panel v2 detected somatic mutations in all 25 malignant samples. No mutations were detected in any of the benign samples tested. When available, mutations detected in the supernatant fluid were compared to the next generation sequencing analysis performed on a prior or concurrent surgical specimen from the same patient and showed 100% concordance. In a subset of cases (n = 19) mutations in EGFR, KRAS, BRAF, PIK3CA, and NRAS were successfully confirmed by droplet digital PCR, providing an orthogonal platform for mutation analysis. In summary, in this study we show that post centrifuged supernatants from fine needle aspiration needle rinses can provide a robust substrate for expanded mutation profiling by next generation sequencing, as well as hotspot mutation testing by droplet digital PCR. The ability to detect somatic mutations from otherwise discarded supernatant fluids offers the ability to triage and effectively utilize limited volume fine needle aspiration samples when multiple molecular tests are requested, without the need to re-biopsy for additional tissue samples.

Granulomatous/sarcoid-like lesions associated with checkpoint inhibitors: a marker of therapy response in a subset of melanoma patients.

BACKGROUND: Immune checkpoint therapy has dramatically changed the landscape of cancer therapy, providing an efficacious and durable therapeutic option for patients with advanced-stage disease. However, dermatologic toxicities are a well-recognized side effect in patients receiving this therapy. A spectrum of immune related adverse events (irAEs) involving the skin can occur and include immunobullous disorders, lichenoid dermatitis, and vitiligo. Granulomatous/sarcoid-like lesions are now being recognized with the current class of checkpoint inhibitors (CPIs) that involve the dermis, the subcutaneous tissue (panniculitis), and lymph nodes. CASE PRESENTATION: We report 3 patients who developed granulomatous/sarcoid-like lesions while being treated with immune checkpoint therapy for advanced-stage melanoma, and we provide a comprehensive review of the literature in which similar cases are described. To date, 26 patients (including the 3 from this report) have been described with a median age of 57 years who developed granulomatous/sarcoid-like lesions associated with CPIs (median onset 6 months), of which 77% of patients had melanoma as primary tumor. To manage this adverse side effect, therapy was withheld in 38% of patients and 44% of the patients were treated with systemic steroids and 8% patients with localized therapy (one patient with intralesional triamcinolone). 96% of patients demonstrated either resolution or improvement of granulomatous/sarcoid-like lesions associated with CPIs irrespective of medical intervention. Therapeutic response, stable disease, or remission of primary malignancy was observed in 71% of reported patients who developed granulomatous/sarcoid-like lesions associated with CPIs over a median follow-up of 11.5 months since initiation of treatment. CONCLUSIONS: The development of granulomatous/sarcoid-like lesions associated with CPIs is a recognized manifestation with the current class of immune checkpoint therapy that may clinically and radiographically mimic disease recurrence. Awareness of this type of toxicity is important for appropriate management and possible measurement of therapeutic response in a subset of patients who manifest this type of immune-mediated reaction.

Efficacy of telecytopathology for preliminary assessment of fine-needle aspirations performed at a remote facility.

INTRODUCTION: The need for real time anatomic pathology services has grown as healthcare systems, traditionally found at large medical centers, expand into smaller communities. The placement of a pathologist is not cost-, time-, or resource-efficient. Telecytopathology can provide rapid offsite evaluation of cytology tissues. This study evaluated the accuracy rate of rendered preliminary assessments for telecytopathology of ultrasound (US)-guided fine-needle aspirations (FNAs) for an offsite facility by comparing preliminary assessment results with the final diagnosis. MATERIALS AND METHODS: The pathology database was searched for telecytopathology US-guided FNAs with rapid offsite evaluation performed at a regional care center from August 2014 to June 2016. A total of 674 consecutive US-guided FNAs from 444 patients were obtained. FNA sites included lymph node (345 cases), breast (178 cases), thyroid gland (71 cases), and others (80 cases). RESULTS: Preliminary assessments of the 674 FNAs were adequate/benign in 275 (41%) cases, adequate/malignant in 182 (27%) cases, adequate/further review needed in 162 (24%) cases, indeterminate/borderline cellularity in 37 (5%) cases, and nondiagnostic in 18 (3%) cases. Final FNA diagnoses rendered included 391 (58%) negative for malignancy, 205 (30%) malignant, 34 (5%) atypical/suspicious for malignancy, 26 (4%) indeterminate cellularity-favor benign, and 18 (3%) nondiagnostic specimens. Concurrent core biopsy was performed in 42 cases and 83 cases were triaged for ancillary studies. The majority (99%) of US-guided FNAs demonstrated concordant preliminary assessments with the final diagnoses. A major discrepancy occurred in 1 case; 5 cases had minor discrepancies. CONCLUSIONS: Remote facility telecytopathology can be utilized as an accurate modality in guiding appropriate tissue acquisition and final diagnosis.

The usefulness of various cytologic specimen preparations for PD-L1 immunostaining in non-small cell lung carcinoma.

INTRODUCTION: Evaluation of programmed cell death ligand-1 (PD-L1) on formalin-fixed paraffin-embedded (FFPE) histologic specimens is required for immunotherapy with pembrolizumab in non-small cell lung carcinoma (NSCLC). A significant percent of patients may be diagnosed on cytology samples alone; however, FFPE tissue may not always be available. Here, we evaluated the feasibility of PD-L1 staining on a variety of cytologic preparations including conventional cell blocks (CB), cell-transfer cell blocks (CTCB), and direct smears. MATERIALS AND METHODS: We identified 30 NSCLC cytology cases that had PD-L1 status evaluated on a concurrent core needle biopsy. Papanicolaou-stained smears (PS) were reviewed and a moderately cellular smear was selected per case to prepare a CTCB. CTCB, CB, and PS (when available) were stained with PD-L1 22C3 and tumor proportion scores (TPS) were compared across all preparations with the concurrent core biopsies. RESULTS: Concordance of PD-L1 positivity in CB and PS versus core biopsy was high, 80% (12 of 15) and 94.4% (17 of 18), respectively. CTCB versus core biopsy concordance was lower in comparison, 62% (13 of 21) for PD-L1 positivity. Overall, TPS was lower in CTCB compared with CB and PS. Among the 3 preparations, CTCB and CB sections were easier to interpret as PS displayed various artifactual staining patterns. CONCLUSIONS: In summary, our study demonstrates that CB and PS preparations are suitable surrogates for histologic FFPE tissue for determining PD-L1 status in NSCLC patients. CTCBs, on the other hand, show high discordance and are not optimal specimens. Pre-analytic factors in unconventional cytology preparations may need optimization and negative results should be interpreted with caution.

Utility of endoscopic ultrasound-guided fine-needle aspiration of regional lymph nodes that are proximal to and far from the primary distal esophageal carcinoma.

Implications of assessing the proximal and far para-tracheal or sub-carinal nodes (para-tracheal [PTN] or sub-carinal [SCN]) associated with lower primary esophageal carcinomas (ECs) are unclear. To evaluate the value of endoscopic ultrasound guided fine-needle aspiration (EUS-FNA) for PTN and SCN, we analyzed results by positron emission tomography (PET) avidity, 4 EUS node malignancy features, and EUS-FNA results in all patients with Siewert's I or II EC. Of 133 patients (PTN, n=102; SCN, n=31) with EUS-FNA, 47 (35%) patients had malignant node, leading to treatment modifications. EUS-FNA diagnosed significantly more patients with malignant nodes (p=0.02) even when PET and EUS features were combined. Among 94 PET-negative and EUS-negative patients, 9 (10%) had malignant EUS-FNA. At a minimum follow-up of 1 year, only 3 (5%) of 62 patients with benign EUS-FNA had evidence of malignancy in the nodal area of prior EUS-FNA. Patients with malignant EUS-FNA independently had a much shorter overall survival (OS) than those with benign EUS-FNA (p<0.001). Our data suggest that a benign EUS-FNA is highly accurate and need not be pursued further. However, malignant EUS-FNA of PTN/SCN was independently prognostic, conferred a shorter OS, and altered the management of 35% of patients.

Utility of BRCA1-associated protein 1 immunoperoxidase stain to differentiate benign versus malignant mesothelial proliferations in cytologic specimens.

BACKGROUND: Loss of BAP1 has recently been described as a highly specific marker for distinguishing malignant mesotheliomas (MM) from benign mesothelial proliferations (BMP). The aim of this study was to evaluate the utility of BAP1 in cytospin and cell block (CB) preparations. METHODS: We searched the database at our institution for cases of MM (n = 21) and BMP (n = 11). A Papanicolaou stained cytospin preparation and CB section from each case were selected for BAP1 staining. The results were scored as nuclear BAP1 staining, absent or present. Positive staining in non-neoplastic cells was noted as internal control. RESULTS: In the study group, MM cells showed a loss of BAP1 staining in 18/21(86%) cytospin and 19/21(90%) CB preparations. All cytospins showed appropriate positive nuclear staining in the "internal control" inflammatory cells. However, only 17/21(81%) of CB sections showed good internal control with positive nuclear staining in inflammatory cells. The intensity of BAP1 nuclear staining varied across cases. Overall, the intensity of staining was higher in cytospins, providing sharper contrast between the malignant cells with loss of stain and the benign inflammatory cells with retained stain in cases of MM. Sensitivity/specificity for cytospins when compared with CB was 86/100 and 88/100%, respectively. CONCLUSIONS: Cytospin preparations have comparable sensitivity and specificity to CB with greater intensity and better contrast between negative and positive nuclei making the interpretation easier. We advise to interpret BAP1 IPOX stain results with caution with careful attention to staining of background nonmalignant "internal control" cells. Diagn. Cytopathol. 2017;45:312-319. © 2016 Wiley Periodicals, Inc.

Concurrent fine needle aspirations and core needle biopsies: a comparative study of substrates for next-generation sequencing in solid organ malignancies.

Minimally invasive procedures, such as fine needle aspiration and core needle biopsy, are commonly used for the diagnosis in solid organ malignancies. In the era of targeted therapy, it is crucial for molecular testing to be performed on these limited volume specimens. Although several recent studies have demonstrated the utility of small biopsy specimens for molecular testing, there remains debate as to whether core needle biopsy specimens are more reliable than fine needle aspiration for molecular studies. In this study, we reviewed concurrently acquired fine needle aspiration and core needle biopsy samples (n=24), and compared overall cellularity, tumor fraction, and the results of next-generation sequencing. All somatic mutations detected in core needle biopsy samples were also detected in fine needle aspiration samples. The estimated tumor fraction was significantly higher in fine needle aspiration smears than core needle biopsy samples (P=0.003), whereas the overall DNA yield from smears was significantly lower than that obtained from the core needle biopsy specimens (P=0.01). The normalized average amplicon coverage for the genes analyzed was significantly higher in cytology smears than paired core needle biopsy samples, with lower numbers of failed amplicons and higher overall mutation allelic frequencies seen in the former. We further evaluated 100 malignant fine needle aspiration and core needle biopsy samples, acquired concurrently, for overall cellularity and tumor fraction. Overall cellularity and tumor fraction of fine needle aspiration samples was significantly higher than concurrently acquired core needle biopsy samples (P<0.001). In conclusion, we show that fine needle aspiration samples frequently provide better cellularity, higher tumor fraction, and superior sequencing metrics than concurrently acquired core needle biopsy samples. Cytologic specimens, therefore, should be better integrated into routine molecular diagnostics workflow to maximize limited tissues for clinically relevant genomic testing.

Prognosis of gastric adenocarcinoma patients with various burdens of peritoneal metastases.

BACKGROUND: Peritoneal metastases (PM) in patients with gastric adenocarcinoma (GAC) may be identified by diagnostic laparoscopy (DL) or imaging (I). Although prognosis is poor, some patients have excellent outcome. We compared the overall survival (OS) of patients in 3 groups: those with positive cytology (CY+) by DL (DL-CY+), those with gross PM (GPM) by DL (DL-GPM+) and with GPM obvious on I (I-GPM+). METHODS: 146 GAC patients were identified. The Kaplan-Meier analysis, univariate, and multivariate Cox proportional hazards regression models were employed. RESULTS: Patients were primarily men (67%), with good ECOG scores (0-1; 89%), had DL (84%), had poorly differentiated GAC (92%), and had received chemotherapy (89%). The median OS for all patients was 15 months (5% CI, 12.9-18.2 months). The DL-CY+ group had median OS of 22.5 months (95% CI, 15-29.3 months). Patients with I-GPM+ had four times the risk of death than those with DL-CY+ (P < 0.001) and patients with DL-GPM+ had two times the risk of death than those with DL-CY+ (P = 0.001). At 36 months, all DL-GPM+ and I-GPM+ had died but 8 patients with DL-CY+ remained alive. CONCLUSIONS: Some GAC patients with DL-CY+ have long OS; therefore, novel strategies to further prolong their OS are needed.

Optimizing the DNA yield for molecular analysis from cytologic preparations.

BACKGROUND: Cytology smears and cytospin preparations are increasingly being used for molecular testing. With these limited samples, optimizing tissue extraction to maximize the DNA yield is, therefore, critical. This study examined 2 common methods of tissue extraction and compared DNA yields from different types of glass slides. METHODS: The H226 lung cancer cell line and 5 clinical samples of cellular effusions were used to prepare Diff-Quik-stained cytospins on 4 types of glass slides: fully frosted (FF), nonfrosted (NF), positively charged (PC), and silane-coated (SC). Tissue extraction was performed by either scalpel-blade scraping or cell lifting with the Pinpoint Slide DNA Isolation System (Zymo Research). DNA was extracted with the QIAamp DNA Mini Kit (Qiagen) and was quantified with the Quant-iT PicoGreen Kit (Life Technologies). RESULTS: The DNA yield in cell-line cytospins was significantly lower from FF slides versus NF, PC, and SC slides with both scraping and cell-lifting methods. In addition, scraping yielded significantly more DNA than cell lifting (P = .005). DNA yields from 5 clinical effusion cases with FF and NF slides showed results similar to the results for cell-line samples, with scraping consistently yielding more DNA than cell lifting and with NF slides outperforming FF slides. CONCLUSIONS: Optimizing the DNA yield extracted from cytology specimens maximizes the chances of successful molecular testing and is critical in cases of low or marginal cellularity. This study demonstrates the following: 1) scraping yields more DNA than cell lifting, and 2) NF slides yield more DNA than FF slides.

Efficacy of reflex HPV16/18 genotyping in predicting CIN3/VAIN3 in women with HPV+/Pap- results.

INTRODUCTION: Genotyping for human papillomavirus (HPV) types 16/18 has been recommended for women with HPV+/Pap- co-testing results on cervical cancer screening. In this study, we evaluated the efficacy of reflex HPV16/18 genotyping for predicting high-grade cervical and vaginal intraepithelial neoplasia (CIN3/VAIN3) in women who underwent a HPV16/18 genotyping test following HPV+/Pap- co-testing results. MATERIALS AND METHODS: We retrospectively reviewed records of 175 women who underwent reflex HPV16/18 genotyping after HPV+/Pap- co-testing results at our institution from 2011 to 2013. The HPV16/18 genotyping results using the Cervista HPV16/18 assay were correlated with follow-up data (ie, 107 biopsy and 68 Pap test) to assess the risk of CIN3/VAIN3. RESULTS: In 175 women (median age: 51 years), HPV16 and/or 18 were detected in 47 women (26.9%): 36 with HPV16, 9 with HPV18, and 2 with HPV16 and 18. The cumulative incidence of CIN3/VAIN3 was 4.6-fold higher in women with a positive HPV16/18 genotyping result (10.6%) than in women with a negative HPV16/18 result (2.3%) with a statistically significant difference (P = 0.015). The predictive value of reflex HPV16/18 genotyping for CIN3/VAIN3 was 63% (5 out of 8). CONCLUSIONS: A positive reflex HPV16/18 genotyping result predicts a significantly higher risk of CIN3/VAIN3 than a negative reflex 16/18 HPV result, supporting the need for an immediate colposcopy evaluation in these patients. For women with a negative reflex HPV16/18 genotyping result, an annual HPV/Pap cytology co-testing is a reasonable follow-up to predict non-16/18 HPV-associated CIN3/VAIN3.

Factors affecting the success of next-generation sequencing in cytology specimens.

BACKGROUND: The use of cytology specimens for next-generation sequencing (NGS) is particularly challenging because of the unconventional substrate of smears and the often limited sample volume. An analysis of factors affecting NGS testing in cytologic samples may help to increase the frequency of successful testing. METHODS: This study reviewed variables associated with all in-house cytology cases (n = 207) that were analyzed by NGS with the Ion Torrent platform during a 10-month interval. A statistical analysis was performed to measure the effects of the DNA input threshold, specimen preparation, slide type, tumor fraction, DNA yield, and cytopathologist bias. RESULTS: One hundred sixty-four of 207 cases (79%) were successfully sequenced by NGS; 43 (21%) failed because of either a low DNA yield or a template/library preparation failure. The median estimated tumor fraction and DNA concentration for the successfully sequenced cases were 70% and 2.5 ng/µL, respectively, whereas they were 60% and 0.2 ng/µL, respectively, for NGS failures. Cell block sections were tested in 91 cases, and smears were used in 116 cases. NGS success positively correlated with the DNA yield but not the tumor fraction. Cell block preparations showed a higher success rate than smears. Frosted-tip slides yielded significantly more DNA than fully frosted slides. Lowering the input DNA concentration below the manufacturer's recommended threshold of 10 ng (>0.85 ng/µL) resulted in a marked increase in the NGS success rate from 58.6% to 89.8%. CONCLUSIONS: The failure of NGS with cytology samples is usually a result of suboptimal DNA due to multiple pre-analytical factors. Knowledge of these factors will allow better selection of cytology material for mutational analysis.

Clinical performance of hybrid capture 2 human papillomavirus testing for recurrent high-grade cervical/vaginal intraepithelial neoplasm in patients with an ASC-US Papanicolaou test result during long-term posttherapy follow-up monitoring.

CONTEXT: Women who have been treated for high-grade cervical or vaginal intraepithelial neoplasia (CIN or VAIN) or invasive carcinoma are at risk for recurrent/persistent disease and require long-term monitoring. The role of human papillomavirus (HPV) testing in this setting is unclear. OBJECTIVE: To evaluate the clinical performance of the Hybrid Capture 2 (HC2) HPV test for recurrent/residual high-grade CIN or VAIN in patients with a posttherapy abnormal squamous cells of undetermined significance (ASC-US) Papanicolaou test result. DESIGN: We reviewed the follow-up data on 100 patients who had an ASC-US Papanicolaou test and HC2 HPV results after treatment for high-grade CIN/VAIN or carcinoma. Human papillomavirus genotyping was performed for women with a negative HC2 result whose follow-up biopsy revealed CIN/VAIN 2+. RESULTS: The patients' mean age was 47 years. The HC2 test result was positive in 33% of the patients. Follow-up biopsy was available for 17 of these patients (52%) and for 25 of the 67 patients (37%) with a negative HC2 result. A total of 5 of the patients (29%) with a positive HC2 result and 2 of the patients (8%) with a negative HC2 result had CIN/VAIN 3 on follow-up biopsy, a statistically insignificant difference (P = .10). Human papillomavirus 16/18 genotypes were detected in the CIN/VAIN 2+ lesions of 5 patients with a negative HC2 result. CONCLUSIONS: HC2 yielded a false-negative rate of 8% for CIN 3. HC2 testing therefore may not be sufficient for triage of patients with an ASC-US Papanicolaou test result. Patients with ASC-US during long-term posttherapy follow-up need close monitoring, with colposcopic evaluation if clinically indicated.

Analytical performance of cervista HPV 16/18 in SurePath pap specimens.

BACKGROUND: To evaluate the performance of Cervista HPV 16/18 in SurePath specimens, we compared the analytical sensitivity of Cervista HPV 16/18 with that of a previously validated PCR-based, commercially available HPV genotyping assay, EasyChip HPV Blot, in residual specimens collected after routine Pap tests at our cancer center. METHODS: We retrospectively selected 79 consecutive Cervista HPV HR (high risk)-positive SurePath residual Pap specimens. The cytology results for these specimens comprised 42 negative, 22 ASC-US/ASC-H, 10 low-grade squamous intraepithelial lesions, and 5 high-grade squamous intraepithelial lesions. HPV 16/18 genotypes of the 79 specimens were analyzed by Cervista HPV 16/18 assay and EasyChip genotyping assay and compared with the patient's follow-up results. RESULTS: Of the 79 cases, 33 (42%) were positive for HPV16/18 by Cervista HPV 16/18 and 37 (47%) were positive by EasyChip. The overall agreement between the 2 assays, at 85% (67/79), was good (kappa = 0.698, 95% CI: 0.541-0.855). In the 65 patients with follow-up results, the sensitivity for predicting cervical intraepithelial neoplasia grade 2 or higher (CIN2+) was 77% for Cervista HPV 16/18 assay and 69% for EasyChip. The predictive values for CIN2+ in cases stratified by Pap results were highly consistent between the Cervista HPV16/18 and EasyChip assays; there was one false negative HPV16 result, in a specimen identified as NILM by EasyChip. CONCLUSION: Our findings support use of the Cervista HPV 16/18 assay for HPV16/18 genotyping in SurePath Pap specimens. However, further studies of larger cohorts with clinical follow-up data are required to verify the efficacy of Cervista HPV16/18 assay in SurePath Pap specimens.

Techniques for cytologic sampling of pancreatic and bile duct lesions: The Papanicolaou Society of Cytopathology Guidelines.

The Papanicolaou Society of Cytopathology has developed a set of guidelines for pancreatobiliary cytology, including indications for endoscopic ultrasound guided fine-needle aspiration biopsy, techniques of the endoscopic retrograde cholangiopancreatography, terminology and nomenclature of pancreatobiliary disease, ancillary testing, and postbiopsy management. All documents are based on the expertise of the authors, a review of literature, discussions of the draft document at several national and international meetings over an 18 month period and synthesis of online comments of the draft document on the Papanicolaou Society of Cytopathology website [www.papsociety.org]. This document presents the results of these discussions regarding the use of sampling techniques in the cytological diagnosis of biliary and pancreatic lesions. This document summarizes the current state of the art for techniques in acquiring cytology specimens from the biliary tree as well as solid and cystic lesions of the pancreas.