Combining fine needle aspiration with brushing cytology has improved yields in diagnosing pancreatic ductal adenocarcinoma.

The aim of this retrospective study is to evaluate the diagnostic yields of combining fine needle aspiration (FNA) with brushing cytology (BC) in clinical work-up of pancreatic ductal adenocarcinoma. The study included a total of 97 patients who underwent both FNA and BC along with histologic/clinical follow-up (F/U). Cytologic diagnoses were categorized as negative for neoplasm (NEG), atypical/favor neoplasm (AN), and suspicious or positive for neoplasm (POS). Based on the cytologic diagnoses, the cohort was divided as follows: 23 had concordant FNA and BC diagnoses of POS/AN, all were neoplasms on F/U; 34 had disconcordant (POS/AN vs. NEG) FNA and BC diagnoses, all but 2 were neoplasms on F/U; The remaining 40 were NEG on both FNA and BC, F/U revealed that 10 were neoplasms and 30 were chronic pancreatitis. Overall, FNA rendered more true positive diagnoses than BC. However, BC but not FNA detected neoplasms in 10 patients. Most of the neoplasms identified on F/U were ductal adenocarcinoma (59 of 65). Diagnostic sensitivity, specificity, positive predictive value, negative predictive value and accuracy were 69.2, 93.8, 95.7, 60, and 77.3% for FNA alone, 50.8, 100, 100, 50.0, and 67.0% for BC alone, and 84.6, 100, 100, 76.2, and 89.7% for combining FNA with BC. In conclusion, both EUS-guided FNA and BC are valuable modalities in the preoperative diagnosis of pancreatic ductal adenocarcinoma. When used in combination, the two modalities complement each other and achieve better diagnostic yield in pancreatic ductal adenocarcinoma than either FNA or BC alone.

Computer-assisted image analysis of amyloid deposits in abdominal fat pad aspiration biopsies.

Amyloidosis is a heterogeneous group of diseases with a common outcome: deposition of insoluble protein in the visceral organs and tissues. Primary amyloidosis is a consequence of different plasma cell disorders, and it is the most common form of amyloidosis in the United States with an estimated 2,000 new cases annually. Other forms of amyloidosis include chronic inflammatory processes, familial type of amyloidosis, and localized forms like Alzheimer's disease.The diagnosis of amyloidosis is based on the clinical picture and demonstration of amyloid deposit in tissues with Congo-red stain. In our article, we describe a simple methodology for image analysis of fat pad biopsies for amyloidosis using a commercially available software Adobe Photoshop CS3(c) Extended Edition. The principle is based on calculation of the mean gray value of each blue and green channel and comparison of their ratios. As a negative control, we have used samples from heart, scar tissue, and skin with their representative control. Fibrous tissue often gives a white:blue to blue:green birefringence, which often is confused with the apple: green birefringence of the amyloid stain; however, we were successful in discriminating these colors using the methodology described in this article. We also analyzed 22 patients with at least 2 years follow-up in our institution. The specificity and the sensitivity of the computer-assisted image analysis were calculated to be 75% and 100%, respectively. These results are in agreement with the published papers (references here); however, caution should be exercised before drawing firm conclusions because of the small sample size presented here.

Utility of cytology microarray constructed from effusion cell blocks for immunomarker validation.

BACKGROUND: Tissue microarray allows rapid and efficient evaluation of gene expression at the protein level and of immunochemical markers. To our knowledge, there has been no report of constructing cytology microarray using effusion cell blocks and testing its utility in immunochemical marker validation. METHODS: A total of 23 malignant effusions (primary tumor of breast [5], GI tract [5], lung [5] and ovary [8]) were used to construct a cytology microarray so that 3 cores of 0.6 mm in diameter were taken from the original cell blocks. Antibodies including AE1/AE3, EMA, and Ki-67 were applied to all cases, and CK7, CK20, TTF-1, WT-1, ER, and PR antibodies were used for selected cases. The cellularity, composition of cells, the staining pattern, and the intensity of each antibody were compared between corresponding cell block sections and CMA cores. RESULTS: The composition of tumor cells in the original block and the cores (including Sections 1 and 45) on cytology microarray were similar, ranging from 5% to 90%. Immunostains of AE1/AE3 and EMA were all positive and 100% concordant between the originals and cytology microarray. Similarly, CK7, CK20, ER, PR, TTF-1, and WT-1 stained both original blocks and cytology microarray with a high level of agreement with respect to percentage of positive cells, staining pattern (cytoplasm or nuclear), and intensity. Ki-67 stain showed slightly lower concordance (84%) with a few cases not in agreement because of low tumor burden in the original block coupled with low percentage of staining by antibody. CONCLUSIONS: Three 0.6 mm cores of cytology microarray are representative of the original cell block with cellularity and antibody staining pattern, intensity, and percentage. Therefore, CMA has a great potential in clinical research and practice as it allows rapid validation of immunocytochemical markers.

Fine-needle aspiration biopsy of metastatic malignant melanoma resembling a malignant peripheral nerve sheath tumor.

We report a case of metastatic malignant melanoma resembling a malignant peripheral sheath tumor, which posed a significant diagnostic challenge. The patient is a 76-year-old male, who presented in the emergency room with bilateral chest pain exacerbated by inspiration. The pain was present for 3 week and was not exacerbated by physical exercise. The diagnostic workup revealed bilateral parenchymal pulmonary infiltrates. The CT-scan guided fine-needle aspiration and the core biopsies of the largest pulmonary lesion revealed high-grade spindle cell neoplasm with individual cell apoptosis and necrosis. The immunohistochemical profile on the cell block showed that the cells are positive for Vimentin. The S-100 stain showed only focal positivity. The immunohistochemical stains for HMB45, Melan A, pancytokeratin, and smooth muscle actin were negative. Five years ago the patient was diagnosed with melanoma on the back with Clark level of IV. The melanoma was excised with clear margins and sentinel lymph nodes were negative. Careful examination of patient's previous slides revealed an area of spindle cell melanoma adjacent to a nodular type melanoma. Based on the patient's previous history, current clinico-pathologic presentation and immunohistochemical profile, the diagnosis of metastatic malignant melanoma resembling peripheral nerve sheath tumor was favored over the diagnosis of metastatic malignant spindle cell neoplasm of unknown primary site, which by itself is very rare clinical scenario.

Restoring satisfactory status in ThinPrep Pap test specimens with too few squamous cells and containing microscopic red blood cells.

Treatment of specimens that contain excessive blood can effectively reduce the unsatisfactory rate; however, a considerable number of unsatisfactory specimens remain. We evaluated the effectiveness of reprocessing unsatisfactory specimens that had too few squamous cells and contained microscopic red blood cells (TFSQRBC).Out of the 688 unsatisfactory specimens at microscopic screening, 197 (28.63%) were TFSQRBC that were reprocessed by treatment of glacial acetic acid (GAA). Red blood cells were observed clogging the pores in the filter of the ThinPrep device. After reprocessing, 129 (68.48%) yielded a satisfactory diagnosis, which accounted for a reduction of the unsatisfactory rate by 18.25%. In the restored satisfactory specimens, abnormal diagnoses of 1 high-grade squamous intraepithelial lesion (HSIL) (0.78%), 3 atypical glandular cells (AGC) (2.33%), and 13 atypical squamous cells of undetermined significance (ASCUS) (10.08%) were made. The abnormal diagnoses in this group of patients were significantly higher than that in the general population screened.Reprocessing unsatisfactory ThinPrep (TP) specimens of TFSQRBC can reduce the unsatisfactory rate of the TP Pap test significantly and is a cost-effective measure. The initially unsatisfactory specimens are more likely to represent cases with an abnormal diagnosis, which also justifies the effort of reprocessing this group of specimens. Adjustment of the pore size on the ThinPrep filter device may reduce the interference of red blood cells.

Expression of gamma-H2AX in melanocytic lesions.

gamma-H2AX is a marker of activated DNA damage and is overexpressed in many malignancies and their precursor lesions. Previous studies have demonstrated the expression of gamma-H2AX in melanoma and dysplastic nevus, but its diagnostic and prognostic utility in a full range of melanocytic lesions has not been fully studied. In the current study, we investigated gamma-H2AX expression in a total of 162 melanocytic lesions. We found that gamma-H2AX was observed at higher levels (percentage and intensity of staining) in melanoma in situ (12/13), primary cutaneous melanoma (32/33; with the exception of desmoplastic melanoma), and metastatic melanoma (58/62), which was statistically different from that in benign nevus (7/9), dysplastic nevus (6/10), and Spitz nevus (5/9) considered together (P < .0001). Of note, desmoplastic melanoma (20/26) demonstrated weak or negative gamma-H2AX staining. The expression of gamma-H2AX did not show significant correlation with many melanoma prognostic factors, including Breslow depth, mitotic rate, and sentinel lymph node status. Except for desmoplastic melanoma, no difference in gamma-H2AX levels was observed among various melanoma subtypes. The overexpression of gamma-H2AX in melanoma as opposed to nevus indicates its possible role in melanomagenesis. Based on the overlap in subsets of nevi and melanomas, the potential clinical utility of this antibody remains uncertain until further studies have been carried out in a larger cohort of melanocytic lesions, including borderline cases.

Comparison of PAX-2, RCC antigen, and antiphosphorylated H2AX antibody (gamma-H2AX) in diagnosing metastatic renal cell carcinoma by fine-needle aspiration.

Diagnosing metastatic renal cell carcinoma (RCC) by fine-needle aspiration (FNA) can be challenging. Existing antibodies supporting a diagnosis of RCC, including CD10 and RCC-Ma, have problems with specificity and interpretation. In this report, we evaluate the use of two newer immunostains, PAX-2 and gamma-H2AX, which to our knowledge have not been studied in FNA material, in the diagnosis of metastatic RCC and in comparison with RCC-Ma. 29 cases of metastatic RCC were identified as well as a TMA of an additional 30 RCC cases. In the case cohort, RCC-Ma in a membranous pattern of staining identified 15/27 (56%) metastatic RCC, although interpretation was made difficult in many cases due to focality of staining and non-specific cytoplasmic staining. PAX-2 stained 23/29 (79%) of tumors in a nuclear stain, most strongly. Gamma-H2AX stained 19/26 (73%) of metastatic RCC strongly in a nuclear stain. In the TMA, strong, diffuse nuclear staining with gamma-H2AX was present in 22/30 RCC (73%). If weak staining was also included as positive, 26/30 (87%) were positive. PAX-2 stained RCC TMA with a lower percentage at 56%, including weaker staining intensity. Both PAX-2 and gamma-H2AX demonstrated patchy staining of normal renal tubules, PAX-2 to a greater extent. Both PAX-2 and gamma-H2AX are sensitive markers for the diagnosis of metastatic RCC, with improved ease of interpretation when compared with RCC-Ma. A combination of all 3 markers identified 87% of cases, and failure to stain for both PAX-2 and gamma-H2AX suggests against, but does not disprove, a diagnosis of RCC.

Utility of antiphosphorylated H2AX antibody (gamma-H2AX) in diagnosing metastatic renal cell carcinoma.

The differential diagnosis of metastatic renal cell carcinoma (RCC) includes, although is not limited to, hepatocellular carcinoma (HCC) and adrenocortical carcinoma (ACC) due to overlapping morphology. Immunohistochemical markers, including RCC marker (RCC-Ma) have been employed with varying success in the differential diagnosis of RCC. Our preliminary tissue microarray study demonstrated that gamma-H2AX, an antibody that specifically reacts with phosphorylated histone H2AX, stained many primary RCC strongly and did not stain HCC or ACC, prompting us to evaluate its utility in these tumors and to compare it with RCC-Ma. Seventy-one cases of metastatic RCC, 18 HCC, and 21 ACC were stained with gamma-H2AX and RCC-Ma and the sensitivity and specificity of each marker was compared. RCC-Ma demonstrated a membranous pattern of staining in 70% of RCC cases (50/71), and none of the ACC or HCC (100% specificity for RCC). Nuclear staining by gamma-H2AX had a similar sensitivity of 70% for RCC but a lower specificity of 77%, as it was seen in 1 of 18 HCC (5%) and 8 of 21 (38%)ACC. In metastatic RCC, 83% (39/47) of tumors with a higher nuclear grade stained with gamma-H2AX, compared with 46% (11/24) of low nuclear grade (equivalent of Fuhrman 2 and lower) tumors. RCC-Ma had a similar rate of staining in low and high-grade tumors, 75% (18/24) and 68% (32/47), respectively. More importantly, of RCCs that were negative for RCC-Ma, 14 of 21 (67%) were positive for gamma-H2AX. The results suggest gamma-H2AX is a useful adjunct in diagnosis of metastatic RCC when RCC-Ma is negative and in higher grade RCC, which are often a diagnostic challenge. A nuclear pattern of staining of gamma-H2AX has a comparable sensitivity with RCC-Ma, and the interpretation is easier and more reliable. RCC-Ma is 100% specific for RCC, but only when a membranous pattern of staining is interpreted as positive.

Fine-needle aspiration cytology of Rosai-Dorfman disease of bone.

Sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease) is a rare, benign self-limiting condition of unknown etiology. Less than a quarter of cases have only extranodal involvement and a few cases of skeletal involvement of Rosai-Dorfman disease without associated lymphadenopathy have been reported in the literature. We herein report cytohistologic findings in a case of sole skeletal Rosai-Dorfman disease in a 51-year-old woman who presented with an expansile, heterogeneous lesion at T11 with cord compression and edema. A CT-guided fine-needle aspiration of T-11 lesion was performed and the sample was processed by ThinPrep technique. The ThinPrep smear showed characteristic features of Rosai-Dorfman disease including hypercellularity with moderate number of histiocytes in a background of lymphocytes, plasma cells, and neutrophils. The histiocytes possessed abundant, pale and vacuolated cytoplasm, rounded nuclei with smooth nuclear membranes, fine chromatin, and distinct nucleoli. The histiocytes showed emperipolesis of lymphocytes and neutrophils. The diagnosis was confirmed by concurrent biopsy with immunhistochemical study. Our case highlighted the role of fine-needle aspiration with ThinPrep technique in the diagnosis of Rosai-Dorfman disease.

The clinical and diagnostic impact of using standard criteria of adequacy assessment and diagnostic terminology on thyroid nodule fine needle aspiration.

The study was aimed to investigate the impact of using standard criteria for assessing specimen adequacy and diagnostic terminology (CAST) on fine-needle aspiration (FNA) diagnosis and clinical management of thyroid nodules. The study included similar numbers of FNAs performed in 2 year before (group A) and 1.5 year after (group B) implementing the standard CAST. In comparison to group A, group B showed a significantly lower rate of nondiagnostic specimens (RND) (16.1% vs. 21.6%, P

Fine-needle aspiration cytological features of Cherubism.

Cherubism is a rare benign, non-neoplastic condition characterized by facial alteration due to symmetrically expansion of bilateral mandibles and maxillas. A fine-needle aspiration of a right cheek mass was performed in a 17-year-old teenager who was previously diagnosed Cherubism. The smears showed moderate cellularity with spindle cells mixed with multinucleated giant cells of osteoclast type. The cells appeared bland with normal nuclear/cytoplasm ratio. The nuclei were round or oval with smooth nuclear membranes and fine, evenly distributed chromatin. These cytologic features were considered consistent with but not diagnostic of Cherubism. Clinical correlations are needed in distinguishing Cherubism from other lesions containing giant cells.

Nodule heterogeneity as shown by size differences between the targeted nodule and the tumor in thyroidectomy specimen: a cause for a false-negative diagnosis of papillary thyroid carcinoma on fine-needle aspiration.

BACKGROUND: Missed papillary thyroid carcinoma (PTC) diagnoses on fine-needle aspiration (FNA) can result from many causes. To the authors' knowledge, the issue of whether the detection of PTC is correlated with nodule heterogeneity has not been studied to date. METHODS: The authors identified all thyroidectomy specimens with a diagnosis of PTC that had undergone at least 1 prior FNA in the study institution between 1998 and 2003. The tumor size at the time of the resection, the ultrasound (US)-determined nodule size, and other parameters were compared between the 2 groups in which PTC was or was not diagnosed on FNA. RESULTS: Of a total of 89 specimens, 47 were diagnosed on FNA with an average tumor size of 1.7 cm and an US-determined nodule size of 2.1 cm (a difference of 0.4 cm). Forty-two specimens with a smaller average tumor size of 0.9 cm (P < .0001) and a US-determined nodule size of 2.4 cm (a difference of 1.5 cm) were missed. The differences with regard to the US-determined nodule size and tumor size between the 2 groups were significant (0.4 cm vs 1.5 cm; P < .0001). In the missed group, 29 specimens were found to have PTC foci that measured < or = 1.0 cm and 26 had a reasonable size difference (RSD; defined as a PTC size outside the range of +/-50% of the US-determined nodule size) as the indicator of the mixed nature of nodules targeted for FNA, whereas in the diagnostic group, 9 foci measured < or = 1.0 cm and 6 had RSD. There was no cytologic evidence with which to render a diagnosis of PTC on further review in the missed group. CONCLUSIONS: The major reason for a missed diagnosis of PTC on FNA is because of inadequate tumor sampling due to the heterogeneity of the nodule targeted for FNA. This is illustrated by the RSD noted between the targeted nodule and the actual PTC tumor focus in the resection specimen.

Utility of WT-1, p63, MOC31, mesothelin, and cytokeratin (K903 and CK5/6) immunostains in differentiating adenocarcinoma, squamous cell carcinoma, and malignant mesothelioma in effusions.

To distinguish carcinoma, either adenocarcinoma (ADC) or squamous cell carcinoma (SCC), and malignant mesothelioma (MM) in effusion can be a diagnostic challenge based on morphology alone. This study evaluates the utility of WT-1, p63, MOC31, mesothelin, and cytokeratin (K903 and CK5/6) immunostains in effusions when ADC and SCC of the lung are in the differential diagnosis with MM. A cohort of 43 effusions consisting of lung ADC (N = 10), SCC (N = 15), and MM (N = 18, mostly (16) pleural based), was subjected to immunostains using the above mentioned antibodies. WT-1 was positive in 100% MM, 0% ADC, and 0% SCC cases while p63 was positive in 0% MM, 30% ADC, and 80% SCC cases. Stain for MOC31 was positive in 100% ADC, 67% SCC, and 35% MM cases. Similarly, mesothelin antibody stained 100% ADC, 60% SCC, and 47% MM cases. Antibodies for K903 and CK5/6 stained 100% SCC cases but fewer ADC cases (40 and 10%, respectively). In conclusion, in this cohort of mostly pleural malignant effusion, MM can be identified with positive staining for WT-1 and negative staining for p63. Conversely, negative staining with WT-1 and positive staining for p63 exclude MM. Used as part of an immunostain panel, cytokeratin markers (CK5/6 and K903) are useful in differentiating SCC from ADC when MM is already excluded, and MOC31 might have limited value in differentiating ADC from MM. A negative stain with MOC31 can exclude lung ADC. Mesothelin, on the other hand, is not useful in the differential diagnosis of ADC, SCC, and MM.

Cellular adequacy for thyroid aspirates prepared by ThinPrep: how many cells are needed?

Although it is well established that ThinPrep introduces artifacts to thyroid aspirates, no criteria have been established for adequacy of such specimens. This study evaluates the adequate number of cells needed to establish the correct diagnosis based on ThinPrep alone. A total of 218 thyroid aspirates prepared by TP with surgical pathology follow-up were reviewed. The cellularity was calculated as follows: Count the total number of clusters, randomly select 10 clusters and count each, calculate the average number per cluster and multiply by the total number of clusters. A minimum number of 6 clusters with 10 cells each was arbitrary established to assume adequacy for a definitive diagnosis. Cytologic diagnoses were classified as: Nondiagnostic (ND), cystic contents, thyroiditis, nodular hyperplasia (NH), follicular/Hurthle (F/H) cell lesion, F/H cell neoplasm, and carcinoma: qualify. Histologic diagnoses were classified as: Cyst (colloid or otherwise), thyroiditis, NH, F/H adenoma, F/H carcinoma, carcinoma: qualify. Appropriate treatment triage was considered to be clinical for the former 4 cytologic categories and surgical for the latter 3 with ND warranting repeat aspiration. The results were subjected to logistic regressions analysis and contingency tables correlating the number of cells with the cytologic and histologic diagnosis as well as with treatment triage. Cellularity of sample was ranked in 10 deciles according to the number of cells and in 4 quartiles according to the number of clusters. The agreement percentage, for both diagnostic and treatment, was computed for each decile and quartile. 146 (67%) cases had cells and received a diagnosis while 72 (33%) were acellular. Of the 146 cases, 21 contained histiocytes or colloid only. 91/146 (62.3%) were correctly diagnosed and 123/146 (84.3%) would have been correctly triaged for treatment based upon the cytologic diagnosis. Samples with 180 cells or fewer had an agreement rate below 50%. Agreement rate increases to 80% when cellularity is 180-320. Above 320 agreement rate remains high but not uniformly. Total number of clusters did not play an independent role and only the number of cells per cluster had a significant correlation with diagnostic agreement. A 25-cell increase in average cells per cluster increases the odds of agreement between diagnoses by 65%.

Methylation profiling of mesothelioma using real-time methylation-specific PCR: a pilot study.

We tested whether methylation profiles generated by real-time methylation-specific PCR (MSP) can be useful in differentiating benign, reactive mesothelial cell proliferation (RM) from malignant mesothelioma (MM). Forty-two of the 63 cases (67%) yielded informative results for RARbeta2, GPC3, CDKN2A (p16), TERT, and CCND2 (cyclinD2) gene methylation. DNA methylation of any gene was observed in much higher frequency in MM cases than RM cases (63% vs. 33%, P < 0.05). Individual gene methylation was higher in the MM than the RM cases for most of the genes; however, this was not statistically significant (RARbeta2: 58% vs. 33%, P > 0.05; GPC3: 36% vs. 27%, P > 0.05; CDKN2A: 4% vs. 0%; TERT: 4% vs. 0%), while CCND2 methylation was not detected in any case. Although preliminary, we demonstrate that real-time MSP can be applied to archival specimens and gene methylation profiling may have potential to be a useful ancillary tool to help distinguish MM from RM.

Diagnosis of foregut and tailgut cysts by endosonographically guided fine-needle aspiration.

Foregut, hindgut, and tailgut cysts are uncommon developmental anomalies. Clinical and radiological diagnosis can present many challenges, especially in adult patients or when the lesions are in unique locations. Thus, diagnosis has traditionally been provided upon surgical resection. We describe the diagnoses of a gastric foregut cyst and a retrorectal tailgut cyst by endosonographically guided fine-needle aspiration in two adults. The common cytologic features of the specimens are ciliated epithelial cells, proteinaceous material with degenerated debris, histiocytes, and benign appearing epithelium of squamous and/or gastrointestinal type that lack cytologic atypia. The identification of ciliated columnar cells is the key finding. Cytologic diagnosis via endosonographically guided fine-needle aspiration of foregut/hindgut cyst is accurate and less traumatic than surgical biopsies.

Methylation profiling of urothelial carcinoma in bladder biopsy and urine.

OBJECTIVE: To test DNA methylation profiling in detection of urothelial carcinoma in urine. STUDY DESIGN: Thirty-three bladder specimens were analyzed for the DNA p16INK4a, RASSF1, APC, GSTP, E-Cad and CyclinD2 genes to determine if there is a difference in gene methylation between benign and malignant cases. Urine samples were analyzed in a feasibility study. Finally, methylation profiles of urine samples were obtained and compared with follow-up biopsy diagnoses. RESULTS: We found methylated genes in 18% benign, 37% urothelial carcinoma in situ and 93% infiltrating urothelial carcinoma cases (p = 0.001). Methylation profiles from the 18 urine samples revealed a significantly higher prevalence of methylated genes in carcinoma cases than benign cases (100% vs. 50%, p = 0.025). We analyzed methylation profiles in 37 cytologically atypical urine samples with malignant or benign diagnosis on surgical follow-up andfound that only APC (55% in malignant vs. 0% in benign, p=0.025) and CyclinD2 were differentially methylated (35% in malignant vs. 0% in benign, p=0.2) while p14ARF, p16INK4a, RASSF1, GSTP and E-Cad had similar methylation profiles. CONCLUSION: These results suggest that methylation of p14ARF, p16INK4a, RASSF1, GSTP and E-Cad genes may not accurately identify carcinoma, but methylated APC and CyclinD2 might be useful biomarkers for urothelial carcinoma in urine.