Fine-needle aspiration cytology (FNAC) is a well-accepted procedure for the diagnosis and biological characterization of breast carcinoma. Estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) status have a strong prognostic and predictive value in invasive breast carcinoma (IBC). ThinPrep (TP) cytology, which uses an alcohol-based fixative, is increasingly being used for immunocytochemistry. In this study, the authors compared the immunocytochemical evaluation of hormone receptors (HR) and HER2 on TP-processed FNAC with the immunohistochemical analysis performed on the corresponding formalin-fixed paraffin-embedded (FFPE) breast tumor specimens, which are considered the gold standard.

FNACs were performed on 116 primary IBCs at the time of diagnosis and subjected to immunocytochemical evaluation of HR and HER2 using the TP method. The same markers were immunohistochemical evaluated on the corresponding FFPE tissue specimens. HER2 fluorescent in situ hybridization analysis was performed only on the equivocal immunohistochemical results.

The HR results of the TP cytology specimens showed a very good agreement with those of the corresponding FFPE tissue samples (Cohen kappa test = 0.92; concordance rate = 98%) for estrogen receptor, and a good agreement (kappa = 0.76; concordance rate = 90.9%) for progesterone receptor. A perfect agreement (kappa = 1) was observed between TP and FFPE tissue samples in evaluating HER2 status.

Alcohol-based fixation seems not to affect the immunocytochemical evaluation of HR and HER2. Considering the high levels of agreement between the evaluation of HR and HER2, on both cytology specimens and on the corresponding FFPE tissue samples, the authors concluded that the TP technique can be routinely used for the biological characterization of IBC. Cancer (Cancer Cytopathol) 2012. © 2012 American Cancer Society.

Arginase-I is a key urea cycle metalloenzyme that has been used as an immunohistochemistry (IHC) marker for hepatocellular carcinoma (HCC). Previous studies have demonstrated the efficacy of HepPar-1 and glypican-3 (GPC-3) IHC in liver fine needle aspiration (FNA) cytology.

Arginase-1 IHC was performed on FNA cell blocks, and its performance characteristics were compared with HepPar-1 and GPC-3. Ninety-two formalin-fixed, paraffin-embedded cell blocks were selected (HCC [n = 44], cirrhosis [n = 2], focal nodular hyperplasia [n = 3], hepatic adenomas [n = 2], dysplastic nodules [n = 6], and metastatic carcinomas [n = 35]). IHC staining with antibodies directed against arginase-1, HepPar-1, and GPC-3 was performed with appropriate positive and negative controls.

Arginase-1 positivity was demonstrated in 37 of 44 (84.1%) cases of HCC, compared with 32 of 44 cases (72.7%) and 25 of 44 cases (56.8%) for HepPar-1 and GPC-3, respectively. Arginase-1 and GPC-3 expression were not observed in any cases of metastatic carcinoma (0%), whereas HepPar-1 expression was present in 1 case of metastatic carcinoma. In addition, both arginase-1 and HepPar-1 expression were present in all 13 cases (100%) of nonmalignant hepatocellular lesions, whereas GPC-3 expression was absent in all 13 cases (0%).

This study demonstrates that both arginase-1 and HepPar-1 are effective IHC markers of hepatocellular differentiation. Furthermore, arginase-1 demonstrates superior sensitivity compared with GPC-3 and HepPar-1 in the diagnosis of HCC, whereas GPC-3 demonstrates superior specificity, as staining is not observed in benign hepatocellular lesions. Hence, use of arginase-1 with HepPar-1 and GPC-3 can aid in the diagnosis of HCC and separating from metastatic carcinoma. Cancer (Cancer Cytopathol) 2012. © 2012 American Cancer Society.

Intrapancreatic accessory spleen (IPAS) is a rare benign lesion of the pancreas that frequently clinically and radiographically mimics a solid neoplasm. Very rarely, epidermoid cysts may form in IPAS and be mistaken for a cystic neoplasm of the pancreas on radiographic imaging. IPAS and epidermoid cyst involving intrapancreatic cyst (ECIPAS) are benign, and, if recognized, do not require surgical intervention. There are few reports of the cytopathologic features of IPAS diagnosed by fine needle aspiration (FNA).

Here we report a series of 6 cases of endoscopic ultrasound (EUS)-guided FNA of IPAS, 3 of which had histological confirmation, including 1 case of histologically confirmed ECIPAS.

Cytomorphologic features of IPAS include a polymorphous population of hematopoietic cells, including lymphocytes, eosinophils, histiocytes, plasma cells, and red blood cells, admixed with numerous small blood vessels representing splenic sinusoids. CD8 immunostaining of cell block or core biopsy material highlights splenic endothelial cells and confirms the diagnosis. FNA of ECIPAS reveals predominantly macrophages and proteinaceous debris.

Diagnostic pitfalls include pancreatic neuroendocrine tumor. If IPAS is recognized as a diagnostic consideration on EUS-FNA, unnecessary surgical resection may be avoided. Cancer (Cancer Cytopathol) 2012. © 2012 American Cancer Society.

Novel high-throughput molecular technologies have made the collection and storage of cells and small tissue specimens a critical issue. The FTA card provides an alternative to cryopreservation for biobanking fresh unfixed cells. The current study compared the quality and integrity of the DNA obtained from 2 types of FTA cards (Classic and Elute) using 2 different extraction protocols (“Classic” and “Elute”) and assessed the feasibility of performing multiplex mutational screening using fine-needle aspiration (FNA) biopsy samples.

Residual material from 42 FNA biopsies was collected in the cards (21 Classic and 21 Elute cards). DNA was extracted using the Classic protocol for Classic cards and both protocols for Elute cards. Polymerase chain reaction for p53 (1.5 kilobase) and CARD11 (500 base pair) was performed to assess DNA integrity.

Successful p53 amplification was achieved in 95.2% of the samples from the Classic cards and in 80.9% of the samples from the Elute cards using the Classic protocol and 28.5% using the Elute protocol (P = .001). All samples (both cards) could be amplified for CARD11. There was no significant difference in the DNA concentration or 260/280 purity ratio when the 2 types of cards were compared. Five samples were also successfully analyzed by multiplex MassARRAY spectrometry, with a mutation in KRAS found in 1 case.

High molecular weight DNA was extracted from the cards in sufficient amounts and quality to perform high-throughput multiplex mutation assays. The results of the current study also suggest that FTA Classic cards preserve better DNA integrity for molecular applications compared with the FTA Elute cards. Cancer (Cancer Cytopathol) 2012. © 2012 American Cancer Society.

Cell block (CB) preparation during the endobronchial ultrasound-guided transbronchial fine-needle aspiration (EBUS-TBNA) procedure plays an important role in the diagnosis of lung cancer and recovery of cellular material for molecular characterization of the tumor. However, the efficiency of the conventional method of CB preparation is suboptimal.

In the current study, the “tissue coagulum clot” cell block (TCC-CB) method was used to prepare the CBs and its efficiency was compared with that of the conventional saline rinse cell block (NR-CB) method. A total of 84 consecutive TCC-CBs (106 lymph nodes [LNs] and 14 lung lesions) and 28 consecutive cases of NR-CB (39 LNs and 3 lung lesions) obtained within the same time period were included in the current study.

In the TCC-CB specimens, 94 of 106 LN cases (88.7%) yielded sufficient diagnostic material, as did 11 of 14 lung lesions (78.6%). In the NR-CB group, which was used as the control, 22 of 39 LN specimens (56.4%) and none of 3 lung specimens (0%) were found to provide sufficient diagnostic material. Although the average size of the LNs in the study group were not significantly different from those in the control group (1.76 cm vs 1.82 cm; P > .05), the overall nondiagnostic rates in the TCC-CB and NR-CB groups were 11.2% and 43.6%, respectively (P < .001). The nondiagnostic rates of the lung specimens were 15.4% in the TCC-CB group and 100% in the NR-CB group (P < .05). In addition, immunohistochemistry studies and epidermal growth factor receptor (EGFR)/KRAS mutational analyses were performed in 26 and 14 TCC-CB cases, respectively. With the exception of 1 case, all of them had satisfactory results.

The data from the current study demonstrate that the TCC-CB method significantly increases the cellular yield of CB preparations without compromising cytomorphological characterization of tumor cells. Cancer (Cancer Cytopathol) 2011;. © 2011 American Cancer Society.

At present, thyroid fine-needle aspiration (FNA) specimens are diagnosed using a tiered classification scheme, with the most popular of these being the 5-tiered and 6-tiered systems. In this study, the authors present their institutional experiences using these 2 different systems and evaluate their efficacy based on the surgical follow-up.

Thyroid FNA specimens and their corresponding surgical resection specimens were collected between 2007 and 2009. The following diagnostic categories are used in both systems: unsatisfactory/nondiagnostic, benign, follicular neoplasm/suspicious for follicular neoplasm, suspicious for malignancy, and malignant. An additional category termed atypia of undetermined significance/follicular lesion of undetermined significance was used for atypical cases in the 6-tiered system. Statistical analysis was performed by comparing the different diagnostic categories.

The case cohort included a total of 7686 thyroid FNA specimens representing 3962 nodules and 3724 nodules, respectively, in the 5-tiered and 6-tiered systems. Negative predictive values for the benign categories (96.9% vs 97.5%; P = 1) and positive predictive values for both the follicular neoplasm categories (26.5% vs 32.1%; P = .2531) and the malignant categories (99.1% vs 99.4%; P = 1) were similar. The most significant differences between the 5-tiered and 6-tiered systems were the percentage of cases classified as benign (83.9% vs 55.4%; P < .0001) and as follicular neoplasms (4.6% vs 23.8%; P < .0001). It is interesting to note that fewer patients were referred for surgery in the 5-tiered system compared with the 6-tiered one (9.1% vs 36.5%; P < .0001).

Use of either the 5-tiered or 6-tiered reporting systems for thyroid FNA specimens can potentially affect the clinical management of patients with thyroid nodules. Cancer (Cancer Cytopathol) 2011. © 2011 American Cancer Society.

The Bethesda System (TBS) for reporting thyroid cytopathology introduced the atypia of undetermined significance/follicular lesion of undetermined significance (AUS) category, but did not provide adequate guidance for the appropriate use of this diagnosis. In the current study, the authors sought to identify an appropriate measure for AUS use based on experience to date with TBS.

The authors reviewed 8 series, including their own laboratory experiences, with a total of 30,466 thyroid aspirates classified within TBS.

The median AUS rate was 9.9% with a range of 3.0% to 18.0%. Use of the individual diagnostic categories within TBS varied up to 12.7-fold. The ratio of “suspicious for follicular neoplasm” plus “suspicious for malignancy” to “malignant” (M) diagnoses varied the least (1.8-fold). The AUS:M ratio provided a suitable measure of assessing AUS use, with a median ratio of 2.0 and a range of 0.5 to 4.9.

Based on available studies, an AUS:M ratio of 1.0 to 3.0 is recommended. AUS:M ratios > 3.0 are likely because of overdiagnosis of AUS or underdiagnosis of M. AUS:M ratios < 1.0 are mostly due to low AUS rates, at the likely expense of sensitivity. Cancer (Cancer Cytopathol) 2011;. © 2011 American Cancer Society.

Practice patterns regarding on-site assessment of the adequacy of image-guided fine needle aspiration biopsies (FNABs) vary among laboratories, but in many laboratories primary responsibility rests with the cytotechnologists. On-site evaluation provides feedback on the need for additional passes and facilitates triaging of the specimen for time-sensitive ancillary studies. Prior studies have suggested that cytotechnologists can assess the initially obtained specimens correctly, but they are few in number and limited by small size. The purpose of this study was to assess the frequency with which our cytotechnologists were able to correctly assess specimens as adequate using a large-scale database that included a wide range of image-guided FNABs.

The frequency that on-site adequacy assessments of 5241 image-guided FNABs were correct was determined by correlating the cytotechnologists' assessments of adequacy with the final cytologic interpretation. An adequacy assessment was considered correct if the FNAB was ultimately reported as satisfactory and unequivocally benign or malignant. An adequate reading on a case that was ultimately reported as unsatisfactory, atypical, or suspicious was deemed “incorrect.” The effect of imaging modality was also analyzed.

Of 5241 FNABs, 2784 (53%) were interpreted as adequate on site. Of these, 2637 (95%) were correctly considered adequate. Of the common biopsy sites sampled, the adequacy assessments for liver FNABs demonstrated the highest frequency for being correctly considered adequate (97%) and those for kidney FNABs showed the lowest (90%). Imaging modality had no effect on accuracy.

Cytotechnologists are almost always correct when assessing initial FNAB samples as adequate. Cancer (Cancer Cytopathol) 2011. © 2011 American Cancer Society.

Recently, the Food and Drug Administration approved the use of the location-guided imaging system FocalPoint GS (FPGS), on SurePath Papanicolaou (Pap) tests for primary screening. The objective of the current study was to evaluate the impact of FPGS on the following: distribution of diagnostic categories; rate of high-risk human papillomavirus (HR-HPV)–positive ASC-US cases; and quality control (QC) data before and after FPGS implementation.

A search of the laboratory information system was performed to identify all SurePath Pap tests processed in our laboratory for the first 19 months after FPGS implementation. We also retrieved all SurePath specimens from a 16-month period prior to FPGS implementation to serve as the control. During the period from Janaury 2008 to April 2009, the FocalPoint Slide Profiler was used.

Implementation of FPGS resulted in a significantly higher percentage of LSIL and ASC-US interpretations, as well as a significant increase in the detection of candidiasis and bacterial vaginosis. The ASC-to-SIL ratio was 1.4 and 1.9 before and after FPGS implementation, respectively. There was a decrease in the HR-HPV positive rate in ASC-US cases, and a decrease in the estimated false-negative fraction after FPGS implementation.

In conclusion, our study seems to demonstrate a favorable performance of FPGS in the routine clinical setting. FPGS may have the potential to be a promising screening tool for gynecologic cytology in a low-risk patient population. Cancer (Cancer Cytopathol) 2011;. © 2011 American Cancer Society.

To the authors' knowledge, the diagnostic value of cell block (CB) as an adjunct to ThinPrep liquid-based cytology (LBC) of bronchial washing specimens in the detection and subclassification of pulmonary neoplasms has not been well evaluated. The objective of the current study was to evaluate the diagnostic utility of CB in this setting.

A total of 74 bronchial washing specimens and concurrently prepared CBs with a diagnosis of malignant or suspicious/atypical obtained from bronchoscopy procedures performed during 2009 were reviewed along with 28 randomly selected negative cases. LBC and CBs were reviewed independently. Deeper levels and ancillary studies were performed on CBs for specific tumor classification if needed. LBC and CB diagnoses were correlated with final histology and/or bronchial brushings.

Use of CBs increased the number of positive diagnoses from 18 (LBC only) to 30 (combined LBC and CB) and 36 (combined LBC and CB with ancillary studies), with increased diagnostic yields of 67% and 100%, respectively. CB without ancillary techniques detected 22 malignancies whereas LBC detected 18 malignancies and CB with ancillary techniques detected 29 malignancies. A specific tumor diagnosis was possible in 22 of 29 (76%) malignancies detected by CB. Bronchial brushings and histology confirmed malignancy in 91% and 92% of cases, respectively.

CB combined with LBC was found to improve the rate of detection of malignancy over LBC alone, especially in cases with suspicious or atypical LBC diagnoses. Increased diagnostic yield is observed when CB is used with or without ancillary studies, but the yield is higher with CB using ancillary studies. CB serves as yet another available source of diagnostic material for immunohistochemical and molecular studies. Cancer (Cancer Cytopathol) 2011;. © 2011 American Cancer Society.

A growing proportion of head and neck squamous cell carcinoma (HNSCC) is caused by the human papillomavirus (HPV). In light of the unique natural history and prognosis of HPV-related HNSCCs, routine HPV testing is being incorporated into diagnostic protocols. Accordingly, there is an escalating demand for an optimal detection strategy that is sensitive and specific, transferrable to the diagnostic laboratory, standardized across laboratories, cost-effective, and amenable to broad application across specimen types including cytologic preparations.

Cytologic preparations (fine-needle aspirates [FNAs] and brushes) were obtained from surgically resected HNSCCs and evaluated for the presence of high-risk HPV using the Hybrid Capture 2 assay. HPV analysis was also performed on the corresponding tissue sections using HPV in situ hybridization and p16 immunohistochemistry. In cases in which the immunohistochemical and in situ hybridization results were discordant, HPV status was determined by real-time polymerase chain reaction detection of E7 expression. HPV status in the tissues and corresponding cytologic samples was compared.

Based on benchmark HPV testing of the tissue sections, 14 HNSCCs were classified as HPV positive and 10 as HPV negative. All corresponding cytologic preparations were correctly classified using the Hybrid Capture 2 assay.

The Hybrid Capture 2 strategy, already widely used for the detection of high-risk HPV in cervical brushes, is readily transferrable to HNSCCs. Consistent accuracy in cytologic preparation suggests its potential application in FNAs from patients who present with lymph node metastases, and may eliminate the need to obtain tissue solely for the purpose of HPV testing. Cancer (Cancer Cytopathol) 2012. © 2011 American Cancer Society.

Although previous studies have shown that p16^INK4a and Ki-67 are sensitive and specific markers for high-grade lesions (≥CIN2) on cervical biopsies, limited information is available regarding the performance of a dual-staining approach as a diagnostic adjunct in cervical cytology. We evaluated a dual p16^INK4a/Ki-67 immunocytochemistry (ICC) assay to determine its sensitivity and specificity versus that of high-risk HPV (HR-HPV) in a US-based pilot cytology study.

ThinPrep specimens from 122 cervical cytology specimens encompassing 23 negative (NILM), 20 ASC-US, 22 LSIL, 17 ASCH, 22 HSIL, and 18 AGC cases were processed for multiplexed ICC staining using a CINtec Plus Kit. Dual-positive assay results were defined based on the detection of 1 or more epithelial cells that were stained for both p16^INK4a and Ki-67 without regard to cellular morphology. HR-HPV testing was performed by multiplex PCR with capillary electrophoresis genotyping.

Dual staining for p16^INK4a and Ki-67 was frequently detected in HSIL and AGC but was rarely detected in NILM cases. The HR-HPV assay showed a sensitivity of 76.2% and a specificity of 55.8% for the detection of clinically significant cervical squamous or endometrial lesions. In contrast, the colocalization of p16^INK4a plus Ki-67 maintained a high sensitivity of 81.8% and improved specificity to 81.8% for biopsy-confirmed CIN2/3, endocervical adenocarcinoma, or endometrial adenocarcinoma.

Dual staining for p16^INK4a/Ki-67 immunocytochemistry dramatically increased specificity and maintained high-level sensitivity for the diagnosis of CIN2/3 or glandular lesions compared with PCR-based testing for HR-HPV. Cancer (Cancer Cytopathol) 2012. © 2011 American Cancer Society.

Pulmonary adenocarcinoma (AD) has a variety of architectural patterns. Recently, a 3-tiered histological pattern-based grading system was developed for stage I lung AD, stratifying patients into low, intermediate, and high risk for recurrence. However, cytology may serve as the primary method for diagnosis in patients with inoperable disease. Attempts to correlate architecture between parallel cytological and histological preparations have not been successful. Therefore, we evaluated cytomorphologic features of previously histologically graded AD to identify features of potential prognostic significance.

One hundred and thirteen fine-needle aspirations with excised adenocarcinomas were reviewed. In the liquid-based preparation, we evaluated cell arrangements (flat sheets vs 3-D clusters vs single cells), nuclear features (size variability, shape, and contour), nucleoli (prominent or inconspicuous), presence of nuclear inclusions, chromatin (fine, coarse, or clumped), and quality of background. The features were tested by multivariate analysis to identify associations with histological grade and disease-free survival (DFS), and a cytological score was generated.

Nuclear size, chromatin pattern, and nuclear contours showed a significant association with histological grade and DFS. These features were included in the composite cytological score (range, 0-5). By grouping the cytological scores, we stratified the tumors into low (median DFS, 100%), intermediate (median DFS, 78%), and high (median DFS, 55%) rate of recurrence (P = .008). There was a good correlation with the histological grading system.

In liquid-based preparations, distinctive cytological features of pulmonary adenocarcinoma correlate with levels of histological differentiation and can be combined into a score with prognostic significance. Cancer (Cancer Cytopathol) 2012. © 2011 American Cancer Society.

Thyroid fine-needle aspiration (FNA) is the primary diagnostic tool used for the evaluation of thyroid nodules. Although most aspirates provide diagnostic cytology, some are classified as indeterminate. The aim of this study was to determine whether the second review of FNA cytology can improve the diagnostic values and to assess the role of proto-oncogene B-Raf (BRAF) mutation testing in the diagnosis of papillary carcinoma (PC).

Thyroid aspirates from 1060 patients were submitted for cytologic evaluation and BRAFV600E mutation analysis. A second cytologic review was performed by 2 cytopathologists in light of the mutation status.

Of the 313 patients who received surgery, 200 (63.9%) had been initially diagnosed as malignant by cytology. They were surgically confirmed as PCs, and the BRAFV600E mutation was detected in 82.5% of the cases. Ninety-five of 102 cases (93.1%) with indeterminate cytology turned out to be malignant, and the mutation was present in 63.3% of PCs. The sensitivity, accuracy, and negative predictive value (NPV) of the second review were better than those of initial cytologic diagnosis (P <.001). The addition of the mutation analysis significantly increased the sensitivity, accuracy, and NPV for detecting PCs compared with those of cytology alone.

Qualified cytologic diagnosis increases the effectiveness of FNA, forgoing the need for repeat biopsy or intraoperative frozen section evaluation. Preoperative BRAF mutation testing can supplement the routine cytology in the selection of thyroid nodules for surgery. Cancer (Cancer Cytopathol) 2012. © 2011 American Cancer Society.

The cytodiagnosis of melanoma in fine-needle aspiration (FNA) specimens can be challenging, often requiring the use of immunocytochemistry. As constitutively activating mutations in the BRAF oncogene are present in at least 40% of melanomas, the use of FNA material to interrogate the BRAF mutational status is likely to increase. Because cell blocks, traditionally used for these studies, can occasionally exhibit insufficient tumor cellularity, the authors investigated the utility of direct smears for immunocytochemistry and BRAF mutational analysis.

Immunocytochemistry for S-100, HMB-45, and Mart-1 was prospectively performed on direct smears in 17 FNAs of metastatic melanoma. Next, BRAF sequencing was performed using DNA isolated from archived Diff-Quik–stained direct smears for 15 cases. In parallel, sequencing was performed using DNA obtained from corresponding cell blocks.

S-100 positivity in the tumor cells was observed in all 17 cases. HMB-45 and Mart-1 positivity was noted in 81% and 88% of cases, respectively. All 3 markers were positive in 76% of cases. Next, of the 15 archived melanoma FNAs tested, BRAF mutations were observed in 8 (53%); 5 and 3 melanomas harbored the V600E and V600K mutation, respectively. Corresponding cell blocks were also tested for all 15 cases, yielding concordant BRAF results in 14 (93%); 1 cell block yielded a false-negative result.

Cytologic direct smears represent a robust and valuable source of cellular material for immunocytochemistry and molecular studies, especially in instances in which inadequate cell block cellularity is anticipated or encountered. Cancer (Cancer Cytopathol) 2012. © 2011 American Cancer Society.

Body fluid specimens may be the first and only pathologic specimen for clinical evaluation in metastatic cancer cases. The challenge of identifying the tissue of origin in metastatic cancer has led to the emergence of molecular-based assays, such as the microarray-based Pathwork Tissue of Origin gene expression test. The ability to use body fluid specimens in this test would be valuable in providing diagnoses to cancer patients without clearly identifiable primary sites. In the current study, the authors evaluated the Tissue of Origin Test for use with malignant effusion specimens.

A total of 27 metastasis-positive body fluid specimens from different body sites, including pleural, ascites, pericardial, and pelvic wash fluids, were obtained from patients with known diagnoses. Nine specimens from nonmalignant body fluids were included as controls. RNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tissue and gene expression analysis was performed with the Tissue of Origin Test.

Seventeen of 27 metastasis-positive samples were non-necrotic with ≥60% tumor and yielded sufficient RNA. Of these samples, 94.1% (16 of 17) were in agreement with the available diagnosis. Of the 9 negative control samples evaluated, 7 (77.8%) demonstrated microarray expression profiles most similar to lymphoma, which is consistent with the predominance of inflammatory cells in these specimens.

The results of the current study demonstrated that FFPE cell blocks from cytologic body fluid specimens yield adequate diagnostic material for the Pathwork test and can be used in the workup of patients with unknown primary tumors. Cancer (Cancer Cytopathol) 2012. © 2011 American Cancer Society.