Characterization of Lysophospholipase D Activity in Mammalian Cell Membranes.

Lysophosphatidic acid (LPA) is a lipid mediator that binds to G-protein-coupled receptors, eliciting a wide variety of responses in mammalian cells. Lyso-phospholipids generated via phospholipase A2 (PLA2) can be converted to LPA by a lysophospholipase D (lyso-PLD). Secreted lyso-PLDs have been studied in more detail than membrane-localized lyso-PLDs. This study utilized in vitro enzyme assays with fluorescent substrates to examine LPA generation in membranes from multiple mammalian cell lines (PC12, rat pheochromocytoma; A7r5, rat vascular smooth muscle; Rat-1, rat fibroblast; PC-3, human prostate carcinoma; and SKOV-3 and OVCAR-3, human ovarian carcinoma). The results show that membranes contain a lyso-PLD activity that generates LPA from a fluorescent alkyl-lyso-phosphatidylcholine, as well as from naturally occurring acyl-linked lysophospholipids. Membrane lyso-PLD and PLD activities were distinguished by multiple criteria, including lack of effect of PLD2 over-expression on lyso-PLD activity and differential sensitivities to vanadate (PLD inhibitor) and iodate (lyso-PLD inhibitor). Based on several lines of evidence, including siRNA knockdown, membrane lyso-PLD is distinct from autotaxin, a secreted lyso-PLD. PC-3 cells express GDE4 and GDE7, recently described lyso-PLDs that localize to membranes. These findings demonstrate that membrane-associated lyso-D activity, expressed by multiple mammalian cell lines, can contribute to LPA production.

"Equivocal" high-risk HPV DNA tests performed on ThinPrep specimens after ASC-US diagnoses are associated with an increased incidence of CIN3: a cytologic/histologic review of 315 cases.

OBJECTIVE: The Hybrid Capture II high-risk HPV test (HC II hrHPVT) improves early detection of cervical neoplasia in Pap tests. However, weakly positive HC II results may be reported as indeterminate or "equivocal," for which there is little clinical guidance. This study is designed to evaluate the clinical outcome of equivocal HC II hrHPVTs and concurrent atypical squamous cells of undetermined significance (ASC-US) on ThinPrep Pap specimens through correlation with 2-year follow-up cervical biopsies. MATERIALS AND METHODS: Over a 5-year period, ThinPrep Pap tests diagnosed as ASC-US were grouped according to their hrHPVT results (i.e., positive, negative, or equivocal) and correlated with histologic follow-up. All equivocal and representative positive and negative hrHPVTs were included. Biopsies showing high-grade dysplasia were reviewed by two pathologists. RESULTS: Of 9,012 ASCUS Pap tests, 945 had corresponding hrHPVTs and follow-up cervical biopsies. High-grade squamous intraepithelial lesion (HSIL-cervical intraepithelial neoplasia grades 2/3, CIN2/3) was identified in 20.3% (14/69) of biopsies after equivocal hrHPVTs (CIN2-5.8%, CIN3-14.5% (p=.0261); 16.7% (25/150) after positive hrHPVT (CIN2-12%, CIN3-4.7%); and 5.4% (5/93) of biopsies after negative hrHPVT (CIN2-4.3%, CIN3-1.1%). CONCLUSION: ASC-US in association with equivocal and positive HC II results respectively shows similar incidences of CIN2/3 on 2-year follow-up cervical biopsy. Additionally, a significant proportion of CIN3 biopsies are in the equivocal HC II cohort. As clinical decision making would be impacted by this finding, laboratories should consider evaluating the clinical performance of their HC II assay via correlation with subsequent cervical biopsies.

The use of stained cytologic direct smears for ALK gene rearrangement analysis of lung adenocarcinoma.

BACKGROUND: Rearrangements involving the anaplastic lymphoma kinase (ALK) gene are present in approximately 5% of lung adenocarcinomas. Crizotinib is approved for the treatment of lung adenocarcinomas harboring ALK rearrangements. Patients with advanced stage lung cancer are not candidates for surgical resection of their primary tumors. For these patients, cytologic specimens often represent the only diagnostic tissue available. Cell blocks (CBs) are routinely used for molecular studies; however, insufficient CB cellularity can impede the performance of these assays. METHODS: Thirty-two cytology cases of lung adenocarcinomas were analyzed by fluorescence in situ hybridization (FISH) for ALK rearrangements. Diff-Quik-stained smears were examined to identify tumor cell-enriched areas that were marked using a diamond-tipped scribe. Paired ALK rearrangement FISH was performed using smears and CBs in each case. RESULTS: An ALK rearrangement was detected on direct smears and CB sections in 5 (16%) and 4 (13%), respectively, of the 32 cases studied. Concordant FISH results for smears and CBs were observed in 31 (97%) of 32 cases. In the 1 discordant case, an ALK rearrangement was detected on the direct smear but not in the CB. Reverse transcriptase-polymerase chain reaction analysis of this CB revealed the presence of an EML4-ALK rearrangement, thereby confirming a false-negative FISH result in the CB. CONCLUSIONS: Stained cytologic direct smears can be effectively used for ALK rearrangement analysis by FISH. This approach represents a useful safeguard when insufficient CB cellularity is encountered and could prevent delays in treatment in this era of precision medicine.

The application of immunocytochemistry to cytologic direct smears of metastatic merkel cell carcinoma.

Merkel cell carcinoma represents a highly aggressive cutaneous malignancy characterized by regional recurrences, lymph node metastases, distant metastases, and high mortality. As the cytomorphology of Merkel cell carcinoma can be mimicked by other malignancies, especially lymphoma and pulmonary small cell carcinoma, immunocytochemistry is often useful in confirming the diagnosis. Cell blocks, which are traditionally utilized for immunocytochemistry, occasionally exhibit insufficient cellularity. Hence, we prospectively investigated the application of CK20 immunocytochemistry to air-dried, unstained direct smears in the diagnosis of Merkel cell carcinoma fine needle aspirates (FNAs). Eight consecutive FNAs of Merkel cell carcinoma were prospectively examined in this series; seven (88%) cases exhibited immunoreactivity for CK20 in the tumor cells. The one CK20-negative Merkel cell carcinoma was immunoreactive for synaptophysin and CD56. This immunophenotype was identical to that of the original primary tumor. For comparison, air-dried direct smears prepared from three pulmonary small cell carcinoma FNAs were examined by CK20 immunocytochemistry. In all cases, no CK20 immunoreactivity was seen in any of the tumor cells. In conclusion, direct smears represent a feasible and robust source of cellular material for immunocytochemical studies to diagnose Merkel cell carcinoma. This methodology allows the cytologist to confirm on site that material for diagnostic immunocytochemistry is present thereby serving as a safeguard in instances where insufficient cell block cellularity is anticipated or encountered.

Ancillary techniques on direct-smear aspirate slides: a significant evolution for cytopathology techniques.

Numerous cytologic techniques aimed at effectively acquiring patient material for molecular testing have been proposed. Such techniques are becoming ever more important in an age of personalized medicine. In this commentary, the authors explored some more commonly proposed techniques to aid in the molecular testing of cytologic specimens. These techniques include the use of cell blocks, direct cytologic smears, filter paper storage, frozen samples, and enriched cellular techniques such as ThinPrep and cytospin preparations. Direct-smeared slides demonstrate excellent preservation of DNA, are easy to prepare, and are amenable to immediate adequacy at the time of the fine-needle aspiration (FNA) procedure as well as effective subsequent tumor purity estimation. Cell block methods cannot be assessed at the time of FNA and often demonstrate insufficiency, whereas filter paper and frozen techniques do not allow for the direct assessment of the presence and purity of tumor cells in the sample. Direct-smeared slides are emerging as the most effective preparation and storage medium of cytologic material to be used for molecular testing. Their cost-effectiveness, ease of use, and reliability have cemented them as the optimal solution for cytopathologists to fulfill the role of providing advanced molecular testing on patient samples.

The application of immunocytochemistry to direct smears in the diagnosis of effusions.

Metastatic malignancy represents a common cause of effusions. Immunocytochemistry (ICC) is useful in confirming malignancy and gaining insight into the site of origin. Cell blocks are commonly utilized for this purpose; nonetheless, when the malignant cells are sparse, they may not be represented in cell blocks thereby precluding immunophenotypic characterization. Thus, we sought to investigate the utility of direct smear preparations as a platform for ICC in the diagnosis of effusions. Air-dried, unstained direct smears were prepared from 49 malignant effusions and 17 reactive effusions for comparison. ICC for EMA and MOC-31 highlighted the tumor cells in 91 and 98% of the malignant effusions tested, respectively. EMA immunoreactivity was focally observed within the calretinin-positive mesothelial cell population in 1 (6%) of the 17 reactive effusions. ICC for MOC-31 was negative in all reactive effusions. Site-specific immunomarkers were also evaluated. Immunoreactivity for Napsin-A and TTF-1 were observed in 78 and 67% of metastatic lung adenocarcinomas, respectively. ICC for PAX8 highlighted metastatic Müllerian and thyroid carcinomas in 100% of cases tested. CDX-2 immunoreactivity was observed in 25, 60, and 100% of metastatic gastric, pancreatic, and colorectal adenocarcinomas, respectively. Positivity for p63 was observed in 75% of metastatic urothelial cell carcinomas and the one case of pulmonary squamous cell carcinoma examined. Calretinin ICC highlighted the tumor cells in both malignant mesothelioma cases tested as well as the benign mesothelial cells in the reactive effusions. In conclusion, direct smears represent an effective platform for the performance of ICC in the diagnosis of malignant effusions.

Molecular diagnostics of melanoma fine-needle aspirates: a cytology-histology correlation study.

Patients with advanced-stage melanoma harboring a BRAF mutation are candidates for BRAF inhibition as a therapeutic strategy. The use of fine-needle aspiration (FNA) to diagnose metastatic melanoma is increasing. Studies examining the predictive value of BRAF mutation analysis on melanoma FNAs via correlation with follow-up excision findings are lacking. We examined 37 consecutive FNA cases of metastatic melanoma in which the aspirated lesion was subsequently excised. DNA was purified from Diff-Quik-stained FNA smears and tissue blocks from corresponding excisions in parallel. BRAF mutation status was successfully obtained from both specimen types in 34 (92%) of 37 cases. BRAF mutations were detected in 12 (35%) of 34 cases-11 V600E and 1 V600K. Results of BRAF mutational analysis were concordant in all 34 FNA smear/tissue excision pairs. Thus, melanoma FNA for molecular diagnostics represents a rapid, minimally invasive, and effective management strategy in this era of precision medicine.

Utility of PAX8 and PAX2 immunohistochemistry in the identification of renal cell carcinoma in diagnostic cytology.

The diagnosis of metastatic renal cell carcinoma (RCC) in cytology specimens may be difficult to confirm on the basis of cytomorphology alone. Often, immunohistochemistry serves as an important adjunct in confirming this diagnosis. Recently, PAX2 was shown to be useful in this regard. In this study, we sought to compare the utility of PAX8 to that of PAX2 immunohistochemistry in the diagnosis of RCC in cytology specimens. First, we verified the performance of PAX8 immunohistochemistry on a tissue microarray (TMA) composed of 54 cases of RCC; PAX8 immunoreactivity was seen in at least 10% of the tumor cells in all cases. Next, we applied PAX8 immunohistochemistry to cell block sections prepared from 24 cases of RCC, obtained from fine-needle aspirates and effusion specimens. PAX2 immunohistochemistry was performed for comparison. Immunopositivity was defined as the presence of nuclear staining in at least 10% of tumor cell nuclei. Immunoreactivity for PAX8 and PAX2 was seen in 21 (88%) and 20 (83%) of the 24 cases, respectively. The presence of either PAX8 or PAX2 immunostaining was present in 22 of 24 cases, thus showing a total sensitivity of 92%. Overall, the results indicate that PAX8 and PAX2 are diagnostically useful adjuncts in confirming the diagnosis of RCC in cytology specimens.

Effects of lysophosphatidic acid on calpain-mediated proteolysis of focal adhesion kinase in human prostate cancer cells.

BACKGROUND: Calcium-mediated proteolysis plays an important role in cell migration. Lysophosphatidic acid (LPA), a lipid mediator present in serum, enhances migration of carcinoma cells. The effects of LPA on calpain-mediated proteolysis were, therefore, examined in PC-3, a human prostate cancer cell line. METHODS: Cultured PC-3 cells were used in studies utilizing pharmacologic interventions, immunoblotting, and confocal immunolocalization. RESULTS: Focal adhesion kinase (FAK), a tyrosine kinase involved in cell adhesion, is rapidly proteolyzed in serum-starved PC-3 cells exposed to the calcium ionophore, ionomycin; Nck, p130CAS, PKCα, and Ras-GAP are also degraded. Thapsigargin, which causes more moderate increases in intracellular calcium, induces partial proteolysis of these proteins. Calpain inhibitors block the proteolytic responses to ionomycin and thapsigargin. Ionomycin does not induce proteolysis in cells maintained in serum, suggesting a protective role for growth factors contained in serum. LPA causes minor FAK proteolysis when added alone, but protects against ionomycin-induced proteolysis in a time-dependent manner. LPA also protects against the cell detachment that eventually follows ionomycin treatment. The response to LPA is blocked by an LPA receptor antagonist. A similar effect of LPA is observed in ionomycin-treated Rat-1 fibroblasts. In PC-3 cells, the protective effects of LPA and serum are correlated with phosphorylation and redistribution of paxillin, suggesting roles for phosphorylation-mediated protein-protein interactions. CONCLUSIONS: The complex effects of LPA on calpain-mediated proteolysis of FAK and other adhesion proteins are likely to play a role in the ability of LPA to promote attachment, migration, and survival of prostate cancer cells.

The application and diagnostic utility of immunocytochemistry on direct smears in the diagnosis of pulmonary adenocarcinoma and squamous cell carcinoma.

The importance of subclassifying pulmonary nonsmall cell carcinoma (NSCLC) in cytologic material is becoming increasingly paramount. Occasionally, cell blocks traditionally used for ancillary studies are sparsely cellular or acellular. Hence, we investigated the diagnostic utility of immunocytochemistry for Napsin-A, TTF-1, and p63 on direct smears of NSCLC. Immunohistochemistry for Napsin-A was initially tested on a tissue microarray (TMA) composed of pulmonary adenocarcinoma. Subsequently, in 25 cases, immunocytochemistry for Napsin-A, TTF-1, and p63 was performed on cytologic direct smears. Smears were prepared from tumor cells scraped from lung resection specimens (n = 10), endobronchial ultrasound-guided transbronchial fine-needle aspirates (n = 13), and pelleted cell material from pleural effusions (n = 2). Immunohistochemistry utilizing the TMA revealed Napsin-A positivity in 73% of pulmonary ADCs. Next, immunocytochemistry on direct cytologic smears demonstrated a Napsin-A(+)/TTF-1(+) immunophenotype in 15 of 18 adenocarcinomas; p63 was completely negative (n = 12) or only focally positive (n = 3) in these 15 adenocarcinomas. The remaining three adenocarcinomas were negative for all three markers. All six squamous cell carcinomas were Napsin-A(-)/TTF-1(-) and diffusely p63(+). In conclusion, direct smears represent a feasible and robust source of cellular material for immunocytochemical studies to diagnose pulmonary ADC and SQC. Our method allows the cytologist to confirm on site that material for diagnostic immunocytochemistry is present thereby serving as a safeguard in instances where the cell block is of insufficient cellularity.

Group consensus review minimizes the diagnosis of "follicular lesion of undetermined significance" and improves cytohistologic concordance.

We conducted a group consensus review of thyroid aspirates that were previously interpreted as "atypia of undetermined significance/follicular lesion of undetermined significance" (AUS/FLUS) and followed by surgical interventions. The study aimed to investigate if consensus review would minimize the diagnosis of AUS/FLUS with an optimal interobserver agreement and also promote a better cytohistologic concordance. A group of reviewers who were blinded to the corresponding histologic findings simultaneously evaluated a total of 50 aspirates at a multiheaded light microscope. Using the Bethesda System for Reporting Thyroid Cytopathology as a guideline, a consensus interpretation was reached upon review of each aspirate. Interobserver agreement was calculated and recorded. The cytohistologic correlation was then performed between the consensus interpretation and the corresponding histologic diagnosis. The consensus review reclassified 26 (52%) aspirates as non-neoplasia/benign, 10 (20%) as follicular neoplasm/suspicious for a follicular neoplasm, 1 (2%) as papillary thyroid carcinoma, and 2 (4%) as nondiagnostic. Eleven (22%) aspirates remained AUS/FLUS. The interobserver agreement across the five diagnostic categories ranged from 71.6% to 100% with an average level of 88.8%. Cytohistologic concordance was achieved in 24 of 26 (92.3%) and 9 of 11 (81.8%) aspirates that were reclassified as non-neoplasia/benign and neoplasia/malignancy, respectively. A diagnostic accuracy of 89.2% (33/37) was obtained in reclassified cases. In conclusion, the group consensus review minimized AUS/FLUS, offered an optimal level of interobserver agreement, and most importantly, promoted excellent cytohistologic concordance in reclassified cases and, therefore, could play a substantial role in the future in reducing reaspiration and/or unnecessary surgeries.

Application of immunocytochemistry and BRAF mutational analysis to direct smears of metastatic melanoma.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Ovarian steroid cell tumor with biallelic adenomatous polyposis coli inactivation in a patient with familial adenomatous polyposis.

Familial adenomatous polyposis (FAP) is an autosomal dominant cancer predisposition syndrome that accounts for approximately 0.5-1% of all colorectal cancer cases. It is caused by germline mutations in the gene encoding the adenomatous polyposis coli (APC) tumor suppressor. Somatic APC inactivation due to mutation or loss of heterozygosity (LOH) promotes the development of adenomatous polyps by stabilizing the transcriptional coactivator ß-catenin. Although colorectal cancer is by far the most common malignancy seen in FAP patients, the widespread use of prophylactic colectomy in these patients has increased the clinical importance of extracolonic tumors that are part of the neoplastic spectrum in FAP. Many of these tumors exhibit LOH or somatic APC mutation, strongly supporting a causative role of APC inactivation in their pathogenesis. Here we describe a 47-year-old female FAP patient with clinical manifestations of virilization who was found to have an ovarian steroid cell tumor, a rare neoplasm not known to be associated with FAP. Immunohistochemical analysis of the ovarian tumor demonstrated strong nuclear ß-catenin staining consistent with somatic APC inactivation, and molecular analysis confirmed biallelic APC inactivation in the tumor. Our findings provide the first evidence that ovarian steroid cell tumors may be an extracolonic manifestation of FAP and implicate ß-catenin activation as an oncogenic mechanism in ovarian steroid cell tumorigenesis.

The application of molecular diagnostic studies interrogating EGFR and KRAS mutations to stained cytologic smears of lung carcinoma.

EGFR and KRAS mutation analyses are of increasing importance for guiding the treatment of non-small cell lung carcinomas. Insufficient cellularity of cell blocks can represent an impediment to the performance of these tests. We investigated the usefulness of cytologic direct smears as an alternative specimen source for mutation testing. Tumor cell-enriched areas from freshly prepared and archived rapid Romanowsky-stained direct smears in 33 cases of lung carcinoma were microdissected for DNA isolation and evaluated for EGFR and KRAS mutations. EGFR mutations were detected in 3 adenocarcinomas; 2 tumors had the L858R substitution and 1 an exon 19 deletion. KRAS mutations affecting codon 12, 13, or 61 were detected in 11 cases (8 adenocarcinomas and 3 non-small cell carcinomas). EGFR and KRAS mutations were mutually exclusive. Hence, archived and freshly prepared direct smears represent a robust and valuable specimen source for molecular studies, especially when cell blocks exhibit insufficient cellularity.

Differential expression of FAK and Pyk2 in metastatic and non-metastatic EL4 lymphoma cell lines.

The murine EL4 lymphoma cell line exists in variants that are either sensitive or resistant to phorbol 12-myristate 13-acetate (PMA). In sensitive cells, PMA causes Erk MAPK activation and Erk-mediated growth arrest. In resistant cells, PMA induces a low level of Erk activation, without growth arrest. A relatively unexplored aspect of the phenotypes is that resistant cells are more adherent to culture substrate than are sensitive cells. In this study, the roles of the protein tyrosine kinases FAK and Pyk2 in EL4 phenotype were examined, with a particular emphasis on the role of these proteins in metastasis. FAK is expressed only in PMA-resistant (or intermediate phenotype) EL4 cells, correlating with enhanced cell-substrate adherence, while Pyk2 is more highly expressed in non-adherent PMA-sensitive cells. PMA treatment causes modulation of mRNA for FAK (up-regulation) and Pyk2 (down-regulation) in PMA-sensitive but not PMA-resistant EL4 cells. The increase in Pyk2 mRNA is correlated with an increase in Pyk2 protein expression. The roles of FAK in cell phenotype were further explored using transfection and knockdown experiments. The results showed that FAK does not play a major role in modulating PMA-induced Erk activation in EL4 cells. However, the knockdown studies demonstrated that FAK expression is required for proliferation and migration of PMA-resistant cells. In an experimental metastasis model using syngeneic mice, only FAK-expressing (PMA-resistant) EL4 cells form liver tumors. Taken together, these studies suggest that FAK expression promotes metastasis of EL4 lymphoma cells.

Minimizing the diagnosis of "follicular lesion of undetermined significance" and identifying predictive features for neoplasia.

We used proposed standard morphologic criteria as a guideline to conduct a 10-year retrospective review of thyroid fine-needle aspiration specimens that were originally interpreted as "follicular lesion of undetermined significance" and followed by surgical intervention. We sought to investigate whether the indeterminate diagnosis could be minimized by assessing various cytomorphologic features and identifying the features predictive of neoplasia. Using the standard morphologic criteria, we semi-quantitatively assessed a total of 24 cytomorphologic features in 123 aspirates and recorded an overall interpretation on completion of the review. Cyto-histologic correlation was evaluated and logistic regression model was performed to identify cytomorphologic features predictive of neoplasia. Although 32 of 123 aspirates remained in the indeterminate category, the retrospective review reclassified 64 aspirates as non-neoplasia and 27 aspirates as neoplasia. Histologic confirmation was achieved in 47 (73.4%) non-neoplastic and 15 (55.6%) neoplastic aspirates with a diagnostic accuracy of 68.1%. Furthermore, our analysis demonstrated that neoplasia is positively associated with the presence of syncytial tissue fragments, isolated microfollicles, follicles with scalloped borders, scant cytoplasm, irregular nuclear membranes, nuclear overlapping, coarse chromatin, and increased cellularity. On the contrary, the presence of honeycombing tissue fragments, background colloid, and histiocytes inversely correlated with neoplasia. Overall, using proposed standard morphological criteria can minimize the diagnosis of "follicular lesion of undetermined significance," and allow for more accurate cyto-histologic correlation, and thereby playing a substantial role in reducing unnecessary surgical intervention.

Asteroid bodies in lymph node cytology: infrequently seen and still mysterious.

Granulomatous inflammation is a relatively common finding in routine aspiration cytology of lymph nodes. However, asteroid bodies are very rarely encountered in cytologic preparations, and most morphologic descriptions result from observations made in histologic tissue sections. This brief report describes the cytologic findings in paratracheal aspirate smears from a 74-year-old Caucasian woman with the history of squamous-cell carcinoma of the right ankle metastatic to a right groin lymph node. At the time of removal of the metastatic tumor, the patient was noted to have multiple small, mildly FDG-avid lymph nodes in the supraclavicular, paratracheal, precarinal, pulmonic hilar, and axillary regions. A transbronchial fine-needle aspiration biopsy (FNAB) of a paratracheal lymph node showed granulomatous inflammation and numerous multinucleated giant cells containing asteroid bodies. No evidence of malignancy was present in any of the smears. Additional patient history elicited at the time of FNAB revealed a diagnosis 6 years previously of disseminated histoplasmosis infection. A concomitant workup for sarcoidosis was negative.

Retroperitoneal germ cell tumor diagnosed by endoscopic ultrasound-guided fine needle aspiration.

Isolated extragonadal germ cell tumors can be primary in nature or metastatic from a burned out testicular cancer. Accurate diagnosis is critical as appropriate therapy can be highly curative. We present the case of an isolated extragonadal germ cell tumor in the retroperitoneum diagnosed by endoscopic ultrasound-guided fine needle aspiration. This case underscores the importance of considering germ cell tumors in the differential diagnosis of an unexplained retroperitoneal mass, particularly since immunophenotypic staining may be necessary to establish the diagnosis.

Correlation between hybrid capture II high-risk human papillomavirus DNA test chemiluminescence intensity from cervical samples with follow-up histologic results: a cytologic/histologic review of 367 cases.

BACKGROUND: The Hybrid Capture II high-risk human papillomavirus (hrHPV) DNA test is a US Food and Drug Administration-approved nucleic acid hybridization assay using chemiluminescence for the semiquantitative detection of hrHPV in cervical samples. Patient samples and controls are used to calculate results as negative for hrHPV if <1.0, positive for hrHPV if >2.5, and "equivocal" if between 1.0 and 2.5. METHODS: The authors reported on the cervical histologic results of 209 patients demonstrating "equivocal" results for hrHPV from SurePath (204 patients) or ThinPrep (5 patients) vials, and compared patients in this cohort with atypical squamous cells of undetermined significance (ASC-US) cytology on the index cervical Papanicolaou (Pap) test (Group 1; n = 148 patients) with a patient cohort demonstrating unequivocal positive hrHPV test results (Group 2; n = 148 patients). The chemiluminescence intensity of hrHPV tests from patients in Group 2 were correlated with the presence and severity of dysplasia on subsequent histologic results, and patients were thereby stratified for their subsequent risk of cervical intraepithelial neoplasia (CIN) types II/III. RESULTS: Approximately 97% of hrHPV tests demonstrating "equivocal" results were found to be positive at the time of retesting, and 15% of biopsied cases demonstrated CIN II or III. Results of follow-up histology after an ASC-US diagnosis, expressed as a percentage of the biopsied cohort, were: CIN II/III: 16.5% in Group 1 and 22.4% in Group 2; CIN I: 27% in Group 1 and 23.5% in Group 2; and negative: 56.5% in Group 1 and 54.1% in Group 2. Chemiluminescence intensity did not appear to be correlated with the severity of dysplasia. CONCLUSIONS: The percentage of high-grade CIN in the "equivocal" hrHPV cohort is highly significant and therefore the management of these patients should be similar to the unequivocally positive population. After an unequivocal positive hrHPV test, the hrHPV chemiluminescence intensity does not appear to further predict the rate of high-grade CIN.