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BecauseofmarkedvariationinthediagnosticterminologyofthyroidFNA,therecentBethesdaSystemforReporting Thyroid Cytopathology (TBSRTC) was introduced and has been widely accepted and imple-mented. Although the majority of the diagnostic categories in TBSRTC are fairly straightforward, by farthe most problematic is the category of ‘‘atypia of undetermined significance’’ (AUS) or ‘‘follicular lesionof undetermined significance’’ (FLUS). According to TBSRTC, the AUS/FLUS category is reserved forthosecasesthatfallinbetweenthebenignandsuspiciouscategories

Background Targeted next-generation sequencing has the potential to stratify a tumor by molecular subtype and aid the development of a biomarker profile for prognostic risk stratification and theranostic potential. Objective To assess the frequency and distribution of pathogenic alterations in malignant lymph node cytology specimens. Design Multigene molecular profiling of archived malignant EUS-FNA lymph node cytology specimens using the Ion Ampliseq Cancer Hotspot Panel v2, which targets at least 2855 possible mutations within 50 cancer-associated genes. Setting Single tertiary referral center. Patients Sporadic, treatment naive, locally advanced primary rectal cancer by EUS-FNA (n = 76) who subsequently completed neoadjuvant therapy with on-site oncologic surgery. Main Outcome Measurements The frequency and distribution of pathogenic alterations in malignant lymph node cytology specimens by the mitogen-activated protein kinase (MAPK) or phosphoinositide 3-kinase (PI3K) signaling pathways, by KRAS or NRAS wild-type lymph node status, by extramesenteric lymph node status, and by a complete pathologic response status. Results Eleven patients (14.5%) were 50-gene panel wild-type. Sixty-five patients had 139 pathogenic alterations (2 [1-3] per patient) in 13 of 50 evaluated genes. The following represent a spectrum of identified alterations: TP53 (n = 52; 68.4%), APC (n = 36; 47.4%), KRAS (n = 22; 28.9%), FBXW7 (n = 8; 10.5%), NRAS (n = 6; 7.9%), PIK3CA (n = 4; 5.3%), SMAD4 (n = 3; 3.9%), and BRAF (n = 3; 3.9%). Pathogenic alterations were identified in the MAPK and PI3K signaling pathways in 41% and 5% of patients, respectively. Limitations Findings were limited to a 50 cancer-associated gene analysis. Conclusions Molecular EUS lymph node assessments using cancer "hotspot" panels can identify pathogenic alteration frequency and distribution and have theranostic potential for individualized patient care.

Gastric gastrointestinal stromal tumors (GISTs) usually contain the mast/stem cell growth factor receptor Kit gene ( KIT ) or platelet-derived growth factor receptor A ( PDGFRA ) mutations that can be targeted by, or mediate resistance to, imatinib. Diagnostic material often is obtained by endoscopic ultrasound–guided fine-needle aspiration, which often is unsuitable for molecular analysis. We investigated whether targeted next-generation sequencing (NGS) can be used in multiplex genotype analysis of cytology samples collected by endoscopic ultrasound–guided fine-needle aspiration. We used the Ion AmpliSeq V2 Cancer Hotspot NGS Panel (Life Technologies, Carlsbad, CA) to identify mutations in more than 2800 exons from 50 cancer-associated genes in GIST samples from 20 patients. We identified KIT mutations in 58% of samples (91% in exon 11 and 9% in exon 17) and PDGFRA mutations in 26% (60% in exon 18 and 40% in exon 12); 16% of samples had no mutations in KIT or PDGFRA. No pathogenic alterations were found in PIK3CA , BRAF , KRAS , NRAS , or FGFR3 . We predicted that 32% of patients would have primary resistance to imatinib, based on mutations in exon 17 of KIT , exon 18 of PDGFRA (D842V), or no mutation in either gene. Targeted NGS of cytology samples from GISTs is feasible and provides clinically relevant data about kinase genotypes that can help guide individualized therapy.

New natural products for drug discovery may be accessed by heterologous expression of bacterial biosynthetic pathways in metagenomic DNA libraries. However, a “universal” host is needed for this experiment. Herein, we show that Myxococcus xanthus is a potential “universal” host for heterologous expression of polyketide biosynthetic gene clusters.

Many DNA-probe assays utilize oligonucleotide-coated microparticles for capture of complementary nucleic acids from solution. During development of these assays, as well as in other particle-based nucleic acid applications, it is useful to know both the amount of duplex formation expected under various experimental conditions and the coating density of the capture oligonucleotide on the particle surface. We examined the simplest form of a DNA-probe microparticle assay: hybridization of a particle-bound capture oligonucleotide to its solution-phase complement. Fluorescein-labeled solution-phase oligonucleotide was hybridized to varying amounts of particles, and the amount of labeled oligonucleotide remaining in solution at equilibrium was measured. We present a simple two-state, all-or-none model for bimolecular hybridization of non-self-complementary sequences that can be used to calculate the equilibrium dissociation constant ( Kd ) from hybridization data. With experimental conditions where both the Kd value and the concentration of capture probe in the reaction are small relative to the concentration of labeled complementary oligonucleotide in the reaction, density of the capture probe on the particle's surface can also be determined. Kd values for particle-based hybridization were different from those obtained from solution-phase thermodynamic parameters. At higher temperatures, hybridization on particles was more efficient than hybridization in solution.

This article is an up-to-date review of thyroid fine needle aspiration. Special emphasis is placed on pitfalls and differential diagnoses of common and uncommon thyroid lesions.

Renal medullary carcinoma is an aggressive neoplasm of the renal pelvis arising in patients with a history of sickle-cell trait. The authors report a case of renal medullary carcinoma with positive urinary cytology. Due to the location of the tumor in the renal pelvis and the loosely cohesive nature of poorly differentiated neoplasms, the presence of renal medullary carcinoma in a urinary cytology specimen is not surprising. The cytologic characteristics as well as the ultrastructural features are described.

Context.—Characteristic cytologic features have been identified that distinguish cases that are always identified from those that are sometimes missed in the College of American Pathologists Gynecologic Cytology Program for a variety of different lesions and preparations.Objective.—To compare the cytologic features of cases of squamous cell carcinoma in conventional smears that perform poorly and well.Design.—The cytologic features of 8 conventional smear cases of squamous cell carcinoma that performed poorly in the College of American Pathologists Interlaboratory Comparison Program were compared with 17 cases that performed extremely well.Results.—A total of 2387 individual interpretations were recorded. Of the 86 incorrect responses, 6.2% were for repair, and 0.8% were for Trichomonas. Cases that performed well were significantly more likely to have greater than 1000 dysplastic cells (16/17 vs 4/8, P = .02) and be keratinized (13/17 vs 1/8, P = .007). Obscuring inflammation and cell size were not significant.Conclusion.—Conventional smears with a diagnosis of squamous cell carcinoma that were always identified were significantly more likely to have greater than 1000 cells and be keratinized than cases that performed poorly.

The cytologic diagnosis of urothelial carcinoma (UC) of the upper urothelial tract (UT) is challenging. Using the UroVysion probe set adds diagnostic value for the detection of bladder cancer in voided urine. In instrumented UT specimens, the authors encountered positive UT cytology and fluorescence in situ hybridization (FISH) cases that did not demonstrate subsequent UT carcinoma.The performance of cytology and FISH in the presence or absence of concomitant bladder cancer within 2 years was compared in 61 patients (112 samples) from 2003 through 2009. The mean follow-up was 3.2 years. The authors also compared the performance of near-tetrasomy versus hypertetrasomy. Biopsy confirmation of UTUC in 21 patients was considered the gold standard.Cytology alone was found to be poorly sensitive (38%) but highly specific (89%) for the detection of UTUC. FISH was found to increase the sensitivity of cytology and decrease specificity. Tetrasomy FISH resulted in many false-positive cases. Other false-positive FISH results were likely due to the presence of bladder cancer cells contaminating the UT specimen.Caution should be used when evaluating instrumented urine specimens of the UT from patients with a previous history of bladder carcinoma, and tetrasomy FISH results should not be interpreted as abnormal because it significantly lowers the specificity of the test. The combination of cytology and FISH appears to have good specificity while maintaining good sensitivity in evaluating UTUC when using modified scoring criteria for the appropriate patient population.

Transthoracic fine needle aspiration (TFNA)/core needle biopsy (CNB) under computed tomography (CT) guidance has proved useful in the assessment of pulmonary nodules. We sought to determine the TFNA false-negative (FN) rate at our institution and identify potential causes of FN diagnoses. Medical records were reviewed from 1,043 consecutive patients who underwent CT-guided TFNA with or without CNB of lung nodules over a 5-year time period (2003-2007). Thirty-seven FN cases of "negative" TFNA/CNB with malignant outcome were identified with 36 cases available for review, of which 35 had a corresponding CNB. Cases were reviewed independently (blinded to original diagnosis) by three pathologists with 15 age- and sex-matched positive and negative controls. Diagnosis (i.e., nondiagnostic, negative or positive for malignancy, atypical or suspicious) and qualitative assessments were recorded. Consensus diagnosis was suspicious or positive in 10 (28%) of 36 TFNA cases and suspicious in 1 (3%) of 35 CNB cases, indicating potential interpretive errors. Of the 11 interpretive errors (including both suspicious and positive cases), 8 were adenocarcinomas, 1 squamous cell carcinoma, 1 metastatic renal cell carcinoma, and 1 lymphoma. The remaining 25 FN cases (69.4%) were considered sampling errors and consisted of 7 adenocarcinomas, 3 nonsmall cell carcinomas, 3 lymphomas, 2 squamous cell carcinomas, and 2 renal cell carcinomas. Interpretive and sampling error cases were more likely to abut the pleura, while histopathologically, they tended to be necrotic and air-dried. The overall FN rate in this patient cohort is 3.5% (1.1% interpretive and 2.4% sampling errors).

Metastases to the thyroid gland are not as unusual as previously believed. This study reports the largest number of patients with metastatic disease of the thyroid to date, confirms the accuracy of fine-needle aspiration (FNA) in diagnosing metastasis, and reviews the incidence and management through our institutional experience.This study entailed review of all thyroid FNAs performed at Mayo Clinic, Rochester during the period 1980 to 2010 and identified 97 patients with a metastatic solid neoplasm of the thyroid gland.Frequent primary tumor sites included kidney (22%), lung (22%), and head and neck (12%). The median age at discovery of thyroid metastasis was 63 years. The time from diagnosis of primary tumor to metastasis to the thyroid gland was most considerable for renal cell carcinoma (mean 113 mo). Forty-one patients underwent thyroid resection with an average tumor size of 3 cm. Median survival in all patients with metastases was 20 months (range, 1 to 228 mo). Patients who underwent thyroid resection had a median survival of 30 months (range, 3 to 171 mo), whereas survival in patients without thyroid surgery was 12 months (range, 1 to 228 mo, log-rank test P=0.09).Our experience over the last 30 years confirms that FNA remains a sensitive and specific method to detect metastases to the thyroid. In any patient with a history of a malignancy, a new thyroid mass should be promptly evaluated for recurrent malignancy as early diagnosis and surgical resection resulted in a nonstatistically significant increased median survival.

Studies have shown that fluorescence in situ hybridization (FISH) testing increases lung cancer detection on cytology specimens in peripheral nodules. The goal of this study was to determine whether a predictive model using clinical features and routine cytology with FISH results could predict lung malignancy after a nondiagnostic bronchoscopic evaluation. Patients with an indeterminate peripheral lung nodule that had a nondiagnostic bronchoscopic evaluation were included in this study (N = 220). FISH was performed on residual bronchial brushing cytology specimens diagnosed as negative (n = 195), atypical (n = 16), or suspicious (n = 9). FISH results included hypertetrasomy (n = 30) and negative (n = 190). Primary study end points included lung cancer status along with time to diagnosis of lung cancer or date of last clinical follow-up. Hazard ratios (HRs) were calculated using Cox proportional hazards regression model analyses, and P values.05 were considered statistically significant. The mean age of the 220 patients was 66.7 years (range, 35-91), and most (58%) were men. Most patients (79%) were current or former smokers with a mean pack year history of 43.2 years (median, 40; range, 1-200). After multivariate analysis, hypertetrasomy FISH (HR = 2.96, P.001), pack years (HR = 1.03 per pack year up to 50, P = .001), age (HR = 1.04 per year, P = .02), atypical or suspicious cytology (HR = 2.02, P = .04), and nodule spiculation (HR = 2.36, P = .003) were independent predictors of malignancy over time and were used to create a prediction model (C-statistic = 0.78). These results suggest that this multivariate model including test results and clinical features may be useful following a nondiagnostic bronchoscopic examination.

Context.—Gynecologic cytopathology is a heavily regulated field, with Clinical Laboratory Improvement Amendments of 1988 mandating the collection of many quality metrics. There is a lack of consensus regarding methods to collect, monitor, and benchmark these data and how these data should be used in a quality assurance program. Furthermore, the introduction of human papilloma virus testing and proficiency testing has provided more data to monitor.Objective.—To determine good laboratory practices in quality assurance of gynecologic cytopathology.Data Sources.—Data were collected through a written survey consisting of 98 questions submitted to 1245 Clinical Laboratory Improvement Amendments–licensed or Department of Defense laboratories. There were 541 usable responses. Additional input was sought through a Web posting of results and questions on the College of American Pathologists Web site. Four senior authors who authored the survey and 28 cytopathologists and cytotechnologists were assigned to 5 working groups to analyze data and present statements on good laboratory practices in gynecologic cytopathology at the College of American Pathologists Gynecologic Cytopathology Quality Consensus Conference. Ninety-eight attendees at the College of American Pathologists Gynecologic Cytopathology Quality Consensus Conference discussed and voted on good laboratory practice statements to obtain consensus.Conclusions.—This paper describes the rationale, background, process, and strengths and limitations of a series of papers that summarize good laboratory practice statements in quality assurance in gynecologic cytopathology.

Controversy exists about whether thyroid fine-needle aspirates (FNAs) should be processed with conventional smears or liquid-based preparations (LBPs).To compare the performance of conventional smears to LBPs for thyroid FNA slides circulated in the College of American Pathologists Interlaboratory Comparison Program in Non-Gynecologic Cytology.Participant responses for thyroid FNA slides were compared with the reference diagnosis at the level of 3 general diagnostic categories: negative, suspicious (which included only follicular and Hürthle cell neoplasm), and malignant. For specific reference diagnoses of benign/goiter and papillary thyroid carcinoma, the participants' specific diagnoses were analyzed and poorly performing slides were rereviewed.The 47, 076 thyroid FNA slide responses, between 2001 and 2009, included 44, 478 responses (94%) for conventional smears and 2598 responses (6%) for LBPs. For the general reference category negative, participant responses were discrepant in 14.9% of conventional smears compared with 5.9% for LBPs (P.001). The specific reference diagnosis of benign/goiter was misdiagnosed as a follicular neoplasm in 7.8% of conventional smears, compared with 1.3% of LBP. For the general reference category of malignant, participant responses were discrepant in 7.3% of conventional smears compared with 14.7% of LBPs (P.001). The specific reference diagnosis of papillary thyroid carcinoma was misdiagnosed as benign/goiter in 7.2% of LBPs, compared with 4.8% of conventional smears (p.001).LBPs performed worse than conventional smears for cases with a reference diagnosis of papillary thyroid carcinoma. However, LBPs performed better than conventional smears for cases with a benign reference diagnosis. Specific features in thyroid FNAs that may improve the diagnostic accuracy of LBPs and conventional smears are described.

The terminology for human papillomavirus (HPV)–associated squamous lesions of the lower anogenital tract has a long history marked by disparate diagnostic terms derived from multiple specialties. It often does not reflect current knowledge of HPV biology and pathogenesis. A consensus process was convened to recommend terminology unified across lower anogenital sites. The goal was to create a histopathologic nomenclature system that reflects current knowledge of HPV biology, optimally uses available biomarkers, and facilitates clear communication across different medical specialties. The Lower Anogenital Squamous Terminology (LAST) Project was cosponsored by the College of American Pathologists and the American Society for Colposcopy and Cervical Pathology and included 5 working groups; 3 work groups performed comprehensive literature reviews and developed draft recommendations. Another work group provided the historical background and the fifth will continue to foster implementation of the LAST recommendations. After an open comment period, the draft recommendations were presented at a consensus conference attended by LAST work group members, advisors, and representatives from 35 stakeholder organizations including professional societies and government agencies. Recommendations were finalized and voted on at the consensus meeting. The final, approved recommendations standardize biologically relevant histopathologic terminology for HPV-associated squamous intraepithelial lesions and superficially invasive squamous carcinomas across all lower anogenital tract sites and detail the appropriate use of specific biomarkers to clarify histologic interpretations and enhance diagnostic accuracy. A plan for disseminating and monitoring recommendation implementation in the practicing community was also developed. The implemented recommendations will facilitate communication between pathologists and their clinical colleagues and improve accuracy of histologic diagnosis with the ultimate goal of providing optimal patient care.

a b s t r a c t Introduction: Sulindac represents a promising candidate agent for lung cancer chemoprevention, but clinical trial data have not been previously reported. We conducted a randomized, phase II chemo- prevention trial involving current or former cigarette smokers (≥30 pack-years) utilizing the multi-center, inter-disciplinary infrastructure of the Cancer Prevention Network (CPN). Methods: At least 1 bronchial dysplastic lesion identified by fluorescence bronchoscopy was required for randomization. Intervention assignments were sulindac 150 mg bid or an identical placebo bid for 6 months. Trial endpoints included changes in histologic grade of dysplasia (per-participant as primary endpoint and per lesion as secondary endpoint), number of dysplastic lesions (per-participant), and Ki67 labeling index. Results: Slower than anticipated recruitment led to trial closure after randomizing participants (n = 31 and n = 30 in the sulindac and placebo arms, respectively). Pre- and post-intervention fluorescence bron- choscopy data were available for 53/61 (87%) randomized, eligible participants. The median (range) of dysplastic lesions at baseline was 2 (1-12) in the sulindac arm and 2 (1-7) in the placebo arm. Change in dysplasia was categorized as regression:stable:progression for 15:3:8 (58%:12%:31%) subjects in the sulindac arm and 15:2:10 (56%:7%:37%) subjects in the placebo arm; these distributions were not statis- tically different (p = 0.85). Median Ki67 expression (% cells stained positive) was significantly reduced in both the placebo (30 versus 5; p = 0.0005) and sulindac (30 versus 10; p = 0.0003) arms, but the difference between arms was not statistically significant (p = 0.92). Conclusions: Data from this multi-center, phase II squamous cell lung cancer chemoprevention trial do not demonstrate sufficient benefits from sulindac 150 mg bid for 6 months to warrant additional phase III testing. Investigation of pathway-focused agents is necessary for lung cancer chemoprevention. © 2012 Elsevier Ireland Ltd. All rights reserved.

Fine needle aspiration (FNA), although very reliable for cytologically benign and malignant thyroid nodules, has much lower predictive value in the case of suspicious or indeterminate nodules. We aimed to identify clinical predictors of malignancy in the subset of patients with suspicious FNA cytology.We reviewed the electronic medical records of 462 patients who had FNA of thyroid nodules at our institution with a suspicious cytological diagnosis, and underwent surgery at Mayo Clinic between January 2004 and September 2008. Demographic data including age, gender, history of exposure to radiation and use of thyroid hormone was collected. The presence of single versus multiple nodules by ultrasonography, nodule size, and serum thyroid-stimulating harmone (TSH) level before thyroid surgery were recorded. Analysis of the latter was limited to patients not taking thyroid hormone or antithyroid drugs at the time of FNA.Of the 462 patients, 327 had lesions suspicious for follicular neoplasm (S-FN) or Hürthle cell neoplasm (S-HCN), 125 had cytology suspicious for papillary carcinoma (S-PC) and 10 had a variety of other suspicious lesions (medullary cancer, lymphoma and atypical). Malignancy rate for suspicious neoplastic lesions (FN+HCN) was ∼15%, whereas malignancy rate for lesions S-PC was 77%. Neither age, serum TSH level, or history of radiation exposure were associated with increased malignancy risk. The presence of multiple nodules (41.1% vs. 26.4%, p=0.0014) or smaller nodule size (2.6±1.8 cm vs. 2.9±1.6 cm, p=0.008) was associated with higher malignancy risk. In patients with cytology suspicious for neoplasm (FN, HCN) malignancy risk was higher in those receiving thyroid hormone therapy than in nonthyroid hormone users (37.7% vs. 16.5%, p=0.0004; odds ratio: 3.1), although serum TSH values did not differ significantly between thyroid hormone users and nonusers.In patients with cytologically suspicious thyroid nodules, the presence of multiple nodules or smaller nodule size was associated with increased risk of malignancy. In addition, our study demonstrates for the first time, an increased risk of malignancy in patients with nodules suspicious for neoplasm who are taking thyroid hormone therapy. The reason for this association is unknown.

Context.—In 2006, the first gynecologic cytology proficiency tests were offered by the College of American Pathologists. Four years of data are now available using field-validated slides, including conventional and liquid-based Papanicolaou tests.Objective.—To characterize the pattern of error types that resulted in initial proficiency-test failure for cytotechnologists, primary screening pathologists, and secondary pathologists (those whose slides are prescreened by cytotechnologists).Design.—The results of 37 029 initial College of American Pathologists Papanicolaou proficiency tests were reviewed from 4 slide-set modules: conventional, ThinPrep, SurePath, or a module containing all 3 slide types.Results.—During this 4-year period, cytotechnologists were least likely to fail the initial test (3.4%; 614 of 18 264), followed by secondary pathologists (ie, those reviewing slides already screened by a cytotechnologist) with a failure rate of 4.2% (728 of 17 346), and primary pathologists (ie, those screening their own slides) having the highest level of failure (13.7%; 194 of 1419). Failure rates have fallen for all 3 groups over time. Pathologists are graded more stringently on proficiency tests, and more primary pathologists would have passed if they had been graded as cytotechnologists. There were no significant differences among performances using different types of slide sets. False-positive errors were common for both primary (63.9%; 124 of 194 errors) and secondary (55.6%; 405 of 728 errors) pathologists, whereas automatic failures were most common for cytotechnologists (75.7%; 465 of 614 errors).Conclusions.—The failure rate is decreasing for all participants. The failures for primary pathologist screeners are due to false-positive responses. Primary screening cytotechnologists and secondary pathologists have automatic failures more often than do primary screening pathologists.

To assess Hybrid Capture 2 (HC2) and fluorescence in situ hybridization (FISH) for the detection of cervical intraepithelial neoplasia 2 or worse (CIN 2+) in patients with a cytologic diagnosis of low grade squamous intraepithelial lesion (LSIL).Residual samples from 115 LSIL-diagnosed cervical cytology specimens were evaluated by high-risk human papillomavirus (HR-HPV) HC2 testing and FISH using biotin-labeled probes to HR-HPV and chromosomal probes to 3q26 (TERC) and 8q24 (CMYC). A cervical biopsy diagnosis of CIN 2+ was considered as evidence of high grade disease.The positive and negative predictive values of HC2 and FISH for detecting patients with CIN 2+ were 32% vs. 37% and 100% vs. 93%, respectively. The sensitivities of HC2 and FISH for CIN 2+ were not significantly different (100% vs. 90%, p = 0.25), while the specificity of HC2 was significantly lower than that of FISH (28% vs. 48%, p=0.003). FISH diagnosed fewer specimens as positive as compared to HC2 (62% vs. 79%).These preliminary data suggest that FISH testing may be useful for determining which patients with LSIL are most likely to have CIN 2+ on clinical follow-up.

BACKGROUND: Up to 80% of thyroid nodules with an indeterminate diagnosis on fine-needle aspiration (FNA) (eg, “suspicious for follicular neoplasm”) prove to be benign at the time of surgical resection. Ancillary tests in current use are limited in their ability to improve the preoperative detection of malignant follicular thyroid nodules. Studies using paraffin-embedded tissue have indicated that high mobility group AT-hook 2 (HMGA2) overexpression is present in a high percentage of malignant thyroid neoplasms but not in benign thyroid neoplasms. In the current study, the ability of HMGA2 overexpression analysis to preoperatively distinguish benign from malignant thyroid nodules by reverse transcriptase–polymerase chain reaction (RT-PCR) on suspicious cytologic smears was evaluated. METHODS: Patients who underwent thyroid FNA and subsequent thyroid resection from 2001 through 2007 were identified. A subset of these patients who had a cytologic diagnosis of “suspicious” underwent HMGA2 expression analysis. HMGA2 expression was detected on cells scraped from cytologic smears with 1-step, real-time quantitative RT–PCR. RESULTS: Of the 125 cases identified, RNA extraction and HMGA2 analysis were successful in 115 cases. With an HMGA2 overexpression change of 5.9-fold or greater compared with a thyroid tumor cell line as a positive cutoff, the test was found to have the following overall performance for detecting malignant nodules: sensitivity of 71%, specificity of 97%, positive predictive value of 94%, and negative predictive value of 84%. HMGA2 overexpression was found to have low sensitivity for detecting Hurthle cell carcinoma (33%). CONCLUSIONS: HMGA2 mRNA expression analysis can be performed on cytologic smears and demonstrates a high specificity and positive predictive value and relatively high sensitivity and negative predictive value for detecting malignancy in “suspicious” thyroid aspirate specimens. Cancer (Cancer Cytopathol) 2010. © 2010 American Cancer Society.

Following the National Cancer Institute Thyroid Fine Needle Aspiration State of the Science Conference, the thyroid fine-needle aspiration biopsy (FNAB) practice at Mayo Clinic, Rochester, Minnesota, conducted retrospective analyses correlating cytologic and histologic evaluations of thyroid nodules. Cytologic and histologic reports were retrieved for patients with thyroid nodules who underwent thyroid FNAB between January 2001 and December 2007, with subsequent surgical thyroid resection. Cases were classified by major cytologic and histologic diagnosis and specific diagnostic subcategories. Of 1,945 FNAB cytologic results, 180 (9.3%) were nondiagnostic; 512 (26.3%) were negative for malignancy; 27 (1.4%) were atypical; 729 (37.5%) were suspicious for malignancy; and 497 (25.6%) were positive for malignancy. Histology was benign in 1,179 (60.6%) and malignant in 766 (39.4%). For thyroid malignancy as the disease outcome, at cytologic thresholds of atypical, suspicious, and positive, overall sensitivity of thyroid FNAB was 94.5%, 94.1%, and 65.0%, respectively, and specificity was 46.0%, 48.3%, and 98.5%, respectively. Positive predictive value for all malignancies was 97.0%, and negative predictive value was 92.0%. When separated by specific malignant outcomes, diagnoses of papillary carcinoma, medullary carcinoma, and lymphoma had specificity of suspicious FNAB diagnoses ranging from 90.5% to 99.6%; positive predictive value ranged from 87.5% to 91.4%. For follicular or Hurthle carcinoma, suspicious FNAB diagnoses had a specificity of 52.5% and a positive predictive value of 5.9%. Sensitivity of indeterminate FNAB diagnoses ranged from 72.7% to 95.3%. For follicular or Hurthle pattern malignancies, indeterminate cytologic diagnoses should be interpreted with caution by the clinician considering surgical management. Diagn. Cytopathol. 2012;40:E27-E32. © 2010 Wiley Periodicals, Inc.

Objective It is broadly accepted that the false positive (FP) rate for endoscopic ultrasound fine needle aspiration (EUS FNA) is 0–1%. It was hypothesised that the FP and false suspicious (FS) rates for EUS FNA are greater than reported. A study was undertaken to establish the rate and root cause of discordant interpretation. Design Using a prospectively maintained endoscopic database, cytohistological discordant EUS FNA examinations from 30 July 1996 to 31 December 2008 were identified retrospectively. Setting Tertiary referral centre. Main outcome measures Discordant FNA was defined by positive or suspicious FNA cytology in the absence of malignancy or neoplasm in the subsequent surgical pathology specimen, specifically in the absence of neoadjuvant therapy. Three cytopathologists conducted a blinded review of randomised discordant and matched specimens. Results FNA was performed in 5667/18 066 (31.4%) patients undergoing EUS, of whom 2547 had cytology results interpreted as ‘positive’ or ‘suspicious’ or ‘atypical’ for malignancy or neoplasm. Subsequent surgical resection without prior neoadjuvant therapy was performed in 377 patients with positive or suspicious cytology. The FP rate was 20/377 (5.3%) and increased to 27/377 (7.2%) when FS cases were included. The incidence of discordance was consistent over time (1996–2002: 10/118 (8.6%) vs 2003–2008: 17/259 (6.6%); p=0.5) and was higher in non-pancreatic FNA (15%) than pancreatic FNA (2.2%; p=0.0001). Two-thirds of the non-pancreatic FP cases involved sampling of perioesophageal or perirectal nodes in patients with luminal neoplasms or Barrett9s oesophagus. Following pathological re-review, discordance was attributed to translocated cell contamination/sampling error (50%) or cytopathologist interpretive error (50%). Conclusions These findings refute the accepted paradigm that FP cytology rarely occurs with EUS FNA. Further investigation revealed that FP FNA developed secondary to endosonographer technique or initial cytological misinterpretation, and is particularly likely when perioesophageal or perirectal nodes are aspirated in the setting of a luminal neoplasm or Barrett9s oesophagus. Further study is needed to determine the significance of these findings and potential impact on the performance of FNA and patient outcomes.

Bronchoscopically collected cytology specimens are commonly used to obtain a diagnosis of cancer in patients with pulmonary lesions. However, the sensitivity of cytology is suboptimal, especially for peripheral lesions less than 2 cm in diameter.We assessed the performance of a testing algorithm using cytology and fluorescence in situ hybridization (FISH) as part of clinical practice.Bronchial brushing specimens (n = 343) were obtained from patients undergoing bronchoscopy for indeterminate pulmonary lesions. Routine cytology was performed and specimens without a positive diagnosis (n = 294) were analyzed by FISH, using residual brushing material. Pathology-confirmed lung cancer or clinical/radiographic evidence of disease was considered diagnostic of malignancy.Routine cytology had a sensitivity and specificity of 41% (23 of 56) and 100% (45 of 45) for central lesions and 20% (26 of 133) and 100% (109 of 109) for peripheral nodules, respectively. FISH detected an additional 32% of lung cancers (18 central and 43 peripheral) not detectable by cytology alone, while producing false positive diagnoses in 22% (10 of 45) and 6% (6 of 109) benign central and peripheral lesions, respectively. In peripheral nodules, FISH detected (relative to routine cytology) an additional 44% (15 of 34) and 28% (25 of 91) of lung cancers less than 2 cm and 2 cm or more in size, respectively. A positive FISH result had a likelihood ratio of 1.45 and 5.87 for central and peripheral lesions and 3.44 and 15.38 for peripheral nodules less than 2 cm and 2 cm or more in size, respectively.FISH testing significantly increases the detection of lung cancer over routine cytology alone. It is especially useful for peripheral nodules.

Cytology has been reported to have suboptimal sensitivity for detecting pancreatobiliary tract cancer in biliary tract specimens partly as a result of low specimen cellularity and obscuring noncellular components. The goal of this study was to determine if the use of a glacial acetic acid wash prior to processing would increase the cellularity and improve the quality of ThinPrep® slides when compared to standard non-gyn ThinPrep processing. Fifty consecutive pancreatobiliary tract specimens containing 20 ml of sample/PreservCyt® were divided equally for standard non-gyn ThinPrep (STP) and glacial acetic acid ThinPrep processing (GATP). A manual drop preparation was also performed on residual STP specimen to determine the number of cells left in the vial during STP processing. Twenty-six (52%) specimens had more epithelial cell groupings with the GATP methodology while 19 (38%) had equivalent cellularity with both methods. The STP method produced more epithelial cell groupings in 5 (10%) of the specimens. Of the 26 specimens that had less cells with the STP method, 14 (54%) had ≥50 cell groupings on the manual drop slide processed from the residual STP specimen suggesting that many cells remain in the vial after STP processing. The GATP method was preferred in 25 (50%) of the specimens, the STP method in 5 (10%), while both methodologies provided similar findings in the remaining 20 (40%) of specimens. The data from this study suggests that the GATP method results in more cells being placed on the slide and was preferred over the STP method in a majority of specimens. Diagn. Cytopathol. 2010;38:627–632. © 2009 Wiley-Liss, Inc.

The 1st edition of the Bethesda System for Reporting Thyroid Cytopathology introduced six categories for reporting fine needle aspiration results, one of which was termed “follicular neoplasm (FN)” or “suspicious for a follicular neoplasm (SFN).” These are synonymous terms for the same category, and it is recommended that a laboratory chooses the term it prefers and use it exclusively for nodules that are suspicious for a follicular neoplasm. The criteria proposed in the 1st edition are reaffirmed in this 2nd edition: this category is intended for those aspirates that are at least moderately cellular and comprised of follicular cells, most of which show significant architectural abnormality in the form of microfollicles and/or trabeculae. In this 2nd edition, the criteria have been expanded, however, to include follicular-patterned aspirates with mild nuclear changes (mild enlargement, contour irregularity, and clearing); such cases can be classified as FN/SFN so long as true papillae and intranuclear pseudoinclusions are absent. In such cases, a note that some nuclear features raise the possibility of an invasive follicular variant of papillary carcinoma or the more indolent noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) can be helpful. The recommended management of a patient with a diagnosis of FN/SFN is surgical excision of the lesion, most often a hemithyroidectomy or lobectomy; molecular testing may be used to supplement risk assessment in lieu of proceeding directly to surgery.

The goal of this study was to assess the performance characteristics of the Automated Cellular Imaging System (ACIS III) for HER2 immunohistochemical analysis. The study was performed on 187 biopsy slides from patients who underwent HER2 testing between January and February 2008. Three scoring methods by the ACIS III were compared with the manual score and fluorescence in situ hybridization (FISH) results for HER2 amplification. The equal distribution score (EQD) method, in which 2 areas each of high-, moderate-, and low-intensity staining were measured, most closely matched the FISH HER2 amplification result. The numbers of immunohistochemically negative (0 or 1+)/FISH+ cases were equivalent for all methods. The EQD method had significantly fewer 2+ cases (n = 16) (P.001) vs the manual method (n = 35) and yielded a higher positive predictive value (38%) for HER2 amplification compared with the manual method (20%). The EQD method may more accurately identify FISH-amplified HER2 cases with fewer 2+ cases that would be "reflexed" to FISH compared with the manual method.

To compare a recently developed fluorescence in situ hybridization (FISH) high-risk human papillomavirus (HR-HPV) assay to Hybrid Capture 2 (HC2) (Digene Corporation, Gaithersburg, Maryland, U.S.A.) and polymerase chain reaction (PCR) for the detection of HR-HPV subtypes in cervical cytology specimens.One hundred forty-one liquid-based cytology specimens were used to produce a thin-layer slide for FISH analysis. The remaining material was sent for HC2 and PCR HR-HPV testing. Thin-layer slides were hybridized with a FISH probe set containing a biotin-labeled HR-HPV cocktail and were manually screened for HR-HPV-infected cells. Specimens withor = 1 HPV-positive cell by FISH were considered positive for HR-HPV infection.There was complete concordance between HC2, FISH and PCR in 104 (75%) specimens. FISH was concordant with HC2 and PCR in 120 (85%) and 115 (82%) specimens, respectively. HC2 and PCR were concordant in 118 (84%) specimens.The concordance of HR-HPV detection between FISH and HC2/PCR appears similar to concordances between HC2 and PCR. This suggests that FISH may be another method of detecting HR-HPV while having the potential to add additional information such as integrated/episomal staining and the ability to detect chromosomal abnormalities in individual cells.

Context.—Minimum cellular criteria for satisfactory Papanicolaou tests were established with the Bethesda System in 2001, and unsatisfactory rates are used as a quality-reporting measure.Objective.—To evaluate practices and unsatisfactory rates from laboratories responding to the 2007 College of American Pathologists supplemental questionnaire survey.Design.—In 2007, a supplemental questionnaire was mailed to 1621 laboratories enrolled in the 2006 College of American Pathologists Interlaboratory Comparison Program in Gynecologic Cytology (PAP Education), requesting data from the 2006 calendar year. Unsatisfactory rates, reasons for unsatisfactory specimens, laboratory size, and specimen preparation type were analyzed.Results.—A total of 42% of the laboratories responded to the survey. Most of those laboratories (637 of 674; 94.5%) used the Bethesda System minimum cellularity criteria. Of those laboratories responding, 79% (527 of 667) used the Bethesda System criteria for atrophic or postirradiation specimens. Unsatisfactory rates have increased since 1996. SurePath preparations were associated with the lowest unsatisfactory rate (50th percentile, 0.30; 95th percentile, 1.3), conventional Papanicolaou tests had the highest 95th percentile rates (50th percentile, 1.0; 95th percentile, 5.90), and ThinPrep specimens had the highest median percentile (50th percentile, 1.1; 95th percentile, 3.4). The most-common reason for unsatisfactory Papanicolaou tests was too few squamous cells. Air-drying artifact was the least-common reason for unsatisfactory reporting for liquid-based preparations.Conclusions.—Use of the Bethesda System criteria for unsatisfactory specimens is widespread. Unsatisfactory rates have increased since 1996; however, the median rates are 1.1% or less for all preparations. Results from the College of American Pathologists PAP Education supplemental questionnaire continue to provide valuable benchmarking data for cytologic quality-improvement programs in laboratories.

ProEx C and p16(INK4a) staining of cytology/histology specimens have recently been explored to help distinguish high-grade squamous intraepithelial lesions (HSIL) from benign mimics. The goal of this study was to evaluate the performance characteristics of p16 and ProEx C in tissue and patient matched ThinPrep liquid-based cytology specimens. Residual cervical ThinPrep cytology specimens and tissue blocks (N = 64) from 63 patients were stained with p16 and ProEx C. Review of immunostained material, Papanicolaou and H&E stained slides was performed by two cytopathologists. The cytology slides were evaluated for the presence or absence of squamous atypia as well as immunoreactivity. Histologic specimens were interpreted as negative, indeterminate, or positive for each immunostain. There was 86% agreement (55/64) between the p16 and ProEx C stains on tissue specimens. Eleven specimens were interpreted as positive for both stains. All had a low- or high-grade squamous lesion on the corresponding H&E section. ProEx C was able to identify four low-grade squamous intraepithelial lesion specimens that were interpreted as negative by p16. All four HSIL specimens demonstrated p16 and ProEx C staining. However, 84% of cytology negative specimens demonstrated false-positive staining. Clinical utilization of both stains, combined with morphologic features, may be beneficial for confirming HSIL on histologic specimens. ProEx C and/or p16 immunostains may lead to a false-positive result in cytology specimens due to staining of normal appearing cells.

This study was performed to identify differences in the frequency of specific cytologic diagnoses obtained between specimens prepared by the filtration and ThinPrep (Cytyc, Marlborough, MA) techniques and to assess how these cytologic diagnoses correlated with pathologic findings. Data were collected from 2,347 voided urine specimens analyzed 8 months before and after ThinPrep implementation. Urine cytologic and bladder biopsy specimens were obtained as part of clinical follow-up, and positive diagnoses were considered evidence of malignancy. After ThinPrep implementation, the proportion of specimens diagnosed as negative significantly decreased (85.5% vs 78.6%; P < .001), clusters (5.8% vs 5.6%; P = .597) and positive (2.8% vs 3.3%; P = .143) diagnoses remained similar, and atypical (3.1% vs 8.4%; P < .001) and “suspicious” diagnoses (2.7% vs 4.1%; P < .001) increased. After 1 year of follow-up, there was no significant difference in the percentage of patients diagnosed with bladder carcinoma between the 2 methods for all cytologic categories.

OBJECTIVE Esophageal squamous cell carcinoma (ESCC) is associated with very high regional mortality rates in several countries. Our initial test of esophageal cytology screening devices found them not sensitive enough for an early detection program. The current study tested a newly designed "mechanical" balloon and a traditional Chinese inflatable balloon, followed by liquid-based cytology, to detect biopsy-proven squamous dysplasia and early cancer. STUDY DESIGN Participants were randomized to a cytologic sampler, followed by endoscopy with iodine staining. For each patient, the cytologic diagnosis (test) was compared with the worst endoscopic biopsy diagnosis (truth). RESULTS Seven hundred forty subjects completed both examinations. Approximately 30% showed atypical squamous cells of undetermined significance (ASCUS), and 10% showed squamous intraepithelial lesions. Seven hundred twenty-five subjects (98%) had satisfactory biopsies, and 32% had low grade dysplasia or worse disease. Defining > ASCUS, favor neoplastic, as a positive screening test, the sensitivities/specificities of the mechanical and inflatable balloons were 39%/85% and 46%/84%, respectively, for detecting any squamous dysplasia or cancer. CONCLUSION These esophageal cell samplers performed equivalently, but the accuracy was still too low for a primary screening test. These results highlight the need to develop new cytologic criteria or molecular markers that can better detect early squamous esophageal disease [corrected]

Pancreatobiliary cancer is detected by fluorescence in situ hybridization (FISH) of pancreatobiliary brush samples with UroVysion probes, originally designed to detect bladder cancer. We designed a set of new probes to detect pancreatobiliary cancer and compared its performance with that of UroVysion and routine cytology analysis.We tested a set of FISH probes on tumor tissues (cholangiocarcinoma or pancreatic carcinoma) and non-tumor tissues from 29 patients. We identified 4 probes that had high specificity for tumor vs non-tumor tissues; we called this set of probes pancreatobiliary FISH. We performed a retrospective analysis of brush samples from 272 patients who underwent endoscopic retrograde cholangiopancreatography for evaluation of malignancy at the Mayo Clinic; results were available from routine cytology and FISH with UroVysion probes. Archived residual specimens were retrieved and used to evaluate the pancreatobiliary FISH probes. Cutoff values for FISH with the pancreatobiliary probes were determined using 89 samples and validated in the remaining 183 samples. Clinical and pathologic evidence of malignancy in the pancreatobiliary tract within 2 years of brush sample collection was used as the standard; samples from patients without malignancies were used as negative controls. The validation cohort included 85 patients with malignancies (46.4%) and 114 patients with primary sclerosing cholangitis (62.3%). Samples containing cells above the cutoff for polysomy (copy number gain of ≥2 probes) were classified as positive in FISH with the UroVysion and pancreatobiliary probes. Multivariable logistic regression was used to estimate associations between clinical and pathology findings and results from FISH.The combination of FISH probes 1q21, 7p12, 8q24, and 9p21 identified cancer cells with 93% sensitivity and 100% specificity in pancreatobiliary tissue samples and were therefore included in the pancreatobiliary probe set. In the validation cohort of brush samples, pancreatobiliary FISH identified samples from patients with malignancy with a significantly higher level of sensitivity (64.7%) than the UroVysion probes (45.9%) (P.001) or routine cytology analysis (18.8%) (P .001), but similar specificity (92.9%, 90.8%, and 100.0% respectively). Factors significantly associated with detection of carcinoma, in adjusted analyses, included detection of polysomy by pancreatobiliary FISH (P.001), a mass by cross-sectional imaging (P.001), cancer cells by routine cytology (overall P = .003), as well as absence of primary sclerosing cholangitis (P = .011).We identified a set of FISH probes that detects cancer cells in pancreatobiliary brush samples from patients with and without primary sclerosing cholangitis with higher levels of sensitivity than UroVysion probes. Cytologic brushing test results and clinical features were independently associated with detection of cancer and might be used to identify patients with pancreatobiliary cancers.

Context.—Although the cytologic features of leukemia/ lymphoma in cerebrospinal fluid specimens are well known, the correlation of these features with the ability of cytologists to identify this tumor have not been well studied. Objective.—To identify the morphologic features of leukemia/lymphoma in cerebrospinal fluid that are associated with good performance and poor performance in an educational interlaboratory comparison program; and to identify the morphologic features associated with how well a slide performs with regard to its reference diagnosis. Design.—The performance of 147 cases of leukemia/ lymphoma in the College of American Pathologists Interlaboratory Comparison Program in Non-gynecologic Cytology was analyzed. The cytologic features of a subset of 31 cases composed of relatively equal numbers of Romanowsky-stained and Papanicolaou-stained specimens were further evaluated, and those that performed poorly (n = 12) were compared with those that performed extremely well (n = 19). Results.—For all cases of leukemia/lymphoma in the program, the rate of misclassification as benign for cases with Papanicolaou stain was significantly higher than for those with Romanowsky stain (9.5% vs 2.6%, P < .001). Compared with cases that performed well, slides that performed poorly were more likely to have less than 200 abnormal cells (42% vs 5%, P = .02). The size of the tumor cells and preservation were not significant. Conclusion.—Cases of specimens of leukemia/lymphoma in cerebrospinal fluid are more likely to be misdiagnosed as benign if they are Papanicolaou-stained or have 200 abnormal cells.

Context.—Although microfollicles are a well-known feature of follicular neoplasms in fine-needle aspirates of the thyroid, the level of agreement about classifying groups as microfollicles is not known. Objective.—To determine what features are present in groups that are consistently classified as microfollicles. Design.—We showed 45 small groups of follicular cells each composed of fewer than 50 cells from fine-needle aspirates of the thyroid to 12 members of the College of American Pathologists Cytopathology Committee, who then classified the groups into microfollicles, macrofollicles, and indeterminate groups. Results.—Twenty groups were consistently classified as microfollicles, 7 groups as macrofollicles, and 18 groups as indeterminate. Cases that were consistently classified as microfollicles were composed of fewer than 15 cells (19/ 20 cases), were arranged in a circle with a lumen (13/20 cases) that was at least two-thirds complete (12/20 cases), and were flat (18/20 cases). Cases that were classified as macrofollicular had between 8 and 35 cells, were arranged in sheets composed of at least 15 cells (5/7 cases) or rows of 8 cells (2/7 cases), and were flat (7/7 cases). In contrast, cases that were indeterminate were composed of either 3-dimensional groups (5/18 cases), flat groups of fewer than 10 cells (11/18 cases), or single cells (1/18 cases). Conclusion.—Not all small groups of follicular cells are consistently classified as microfollicles, and some are more often classified as macrofollicles. The criteria described here for reproducible microfollicles (

From the inception of TBS in 1988, it has been well received by the pathology and clinical communities. The need for a unifying system of nomenclature was so great that most of the laboratories in the United States, as well as in numerous countries worldwide, quickly adopted TBS. By 1991, only 2 years after the initial publication of the initial TBS, 57% of United States laboratories were using the system. By 1994, almost 90% of laboratories were using TBS [28]. Although explanations for such a quick acceptance of TBS are many, one of the leading reasons is, most certainly, the sound and thoughtful basis for all of the elements of TBS. These include: (1) nomenclature that provides uniform diagnostic terminology to facilitate unambiguous communication between the laboratory and the clinician; (2) diagnostic categories that incorporate the latest scientific information on the pathogenesis and prognosis of cervical lesions; and (3) incorporation of the evaluation of specimen adequacy as an integral part of the report. From the beginning, TBS was more than just a diagnostic lexicon. When Drs. Kurman and Solomon decided that something had to be done about the diagnostic mess in GYN cytology, they did not develop another system. Instead, they convened an international consensus conference to look at the problem and come up with a comprehensive, relevant solution. The input from the entire community, which lent strength to the initial TBS, has continued to the present. This is highlighted by the enormous amount of input that was received by the 2001 TBS forum groups that enabled them to fine tune the existing terminology to meet the needs of today's clinicians. The result, continues to be an evolving, comprehensive, and clinically relevant entity, that should meet the needs of pathologists and clinicians for the foreseeable future.

OBJECTIVE: To evaluate the effect of cellularity on the sensitivity of both screening and diagnosis in a liquid-based cervical sample. STUDY DESIGN: Sure-Path samples (TriPath Imagine Inc., Burlington, North Carolina, U.S.A.) with known diagnoses were selected, including 18 negative, 16 low grade squamous intracpithelial lesion (LSIL) and 12 high grade squamous intraepithelial lesion (HSIL) cases. Through a serial dilution technique, samples of varying cellularity were prepared. The 275 slides were assigned random numbers and were routinely screened by 1 of 2 senior cytotechnologists, blinded to the reference diagnosis. Specimens with a screening diagnosis of atypical squamous cells of undetermined significance (ASCUS) or higher were reviewed by two pathologists, resulting in a final consensus diagnosis. Using a grid counting system, cellularity was determined for each slide. RESULTS: There was a clear demarcation in sensitivity between specimens with a cellularity of < 5,000 or ≥ 5,000 squanious cells. This applied to both the sensitivity for screening and to the final consensus diagnosis. For cases with a reference diagnosis of LSIL +, at a cytotechnologist screening level of ASCUS or greater, sensitivity increased from 72.8% (< 5,000 cells) to 98.1% (≥ 5,000 cells) and for a reference diagnosis of HSIL from 85.7% to 100%, respectively. Similarly, for the consensus diagnosis, sensitivity rose from 78.5% (