Performance of Idylla KRAS assay on extracted DNA and de-stained cytology smears: Can we rescue small sample?

OBJECTIVE: KRAS is a frequently mutated gene in cancers, and with recent FDA-approved targeted therapy for the G12C mutation, testing for KRAS variants is essential. We evaluated the performance of the Idylla KRAS assay on extracted DNA and cytology smears in order to expand the utility of the assay. METHODS: In total, fifty-seven human samples were analyzed. Idylla results from sixteen DNA extracted from formalin-fixed, paraffin-embedded tissues (FFPE DNA) and thirty cytology smears were compared to the reference method. We evaluated the performance of the Idylla assay using corresponding cytology smears to rescue cellblocks or surgical blocks that were quantity not sufficient (QNS) for next generation sequencing (NGS). RESULT: In the FFPE DNA cohort, 10 ng DNA input yielded valid results in all 16 samples, with 15 of 16 (93 %) concordant with NGS findings. In the cytology smear cohort, the Idylla KRAS assay demonstrated 100 % concordance with previous NGS results in 30 cases. In the QNS cohort, the assay was valid in all cases and KRAS mutations were identified in 3 of 11 cytology smears, including one G12C mutation. CONCLUSION: The Idylla KRAS assay is a high-performing, feasible, and convenient option for testing extracted DNA and cytology smears. It rescues QNS samples allowing it to be integrated into the molecular workflow as an initial screening test with remarkably quick turnaround times.

Acute pulmonary effects of aerosolized nicotine.

Nicotine is a highly addictive principal component of both tobacco and electronic cigarette that is readily absorbed in blood. Nicotine-containing electronic cigarettes are promoted as a safe alternative to cigarette smoking. However, the isolated effects of inhaled nicotine are largely unknown. Here we report a novel rat model of aerosolized nicotine with a particle size (~1 µm) in the respirable diameter range. Acute nicotine inhalation caused increased pulmonary edema and lung injury as measured by enhanced bronchoalveolar lavage fluid protein, IgM, lung wet-to-dry weight ratio, and high-mobility group box 1 (HMGB1) protein and decreased lung E-cadherin protein. Immunohistochemical analysis revealed congested blood vessels and increased neutrophil infiltration. Lung myeloperoxidase mRNA and protein increased in the nicotine-exposed rats. Complete blood counts also showed an increase in neutrophils, white blood cells, eosinophils, and basophils. Arterial blood gas measurements showed an increase in lactate. Lungs of nicotine-inhaling animals revealed increased mRNA levels of IL-1A and CXCL1. There was also an increase in IL-1α protein. In in vitro air-liquid interface cultures of airway epithelial cells, there was a dose dependent increase in HMGB1 release with nicotine treatment. Air-liquid cultures exposed to nicotine also resulted in a dose-dependent loss of barrier as measured by transepithelial electrical resistance and a decrease in E-cadherin expression. Nicotine also caused a dose-dependent increase in epithelial cell death and an increase in caspase-3/7 activities. These results show that the nicotine content of electronic cigarettes may have adverse pulmonary and systemic effects.

Dynamic telecytology compares favorably to rapid onsite evaluation of endoscopic ultrasound fine needle aspirates.

BACKGROUND AND AIMS: Rapid onsite evaluation (ROSE) has been demonstrated to correlate with final cytologic interpretations and improves the diagnostic yield of endoscopic ultrasound (EUS)-fine needle aspiration (FNA); however, its availability is variable across centers. The aim of this prospective study was to evaluate whether remote telecytology can substitute for ROSE. METHODS: Consecutive patients who underwent EUS-FNA for diverse indications at a high volume referral center were enrolled and all samples were prospectively evaluated by three methods. ROSE was performed by a cytopathologist in the procedure room; simultaneously dynamic telecytology was done by a different cytopathologist in a remote location at our institution. The third method, final cytologic interpretation in the laboratory, was the gold standard. Telecytology was performed using an Olympus microscope system (BX) which broadcasts live images over the Internet. Accuracy of telecytology and agreement with other methods were the principle outcome measurements. RESULTS: Twenty-five consecutive samples were obtained from participants 40-87 years old (median age 63, 48 % male). There was 88 % agreement between telecytology and final cytology (p < 0.001) and 92 % agreement between ROSE and final cytology (p < 0.001). There was consistency between telecytology and ROSE (p value for McNemar's χ(2) = 1.0). Cohen's kappa for agreement for telecytology and ROSE was 0.80 (SE = 0.11), confirming favorable correlation. CONCLUSION: Dynamic telecytology compares favorably to ROSE in the assessment of EUS acquired fine needle aspirates. If confirmed by larger trials, this system might obviate the need for onsite interpretation of EUS-FNA specimens.

A large single-center experience of EUS-guided FNA of the left and right adrenal glands: diagnostic utility and impact on patient management.

BACKGROUND: EUS-guided FNA of the left and right adrenals has been described, but data are very limited. OBJECTIVES: Our primary objective was to determine the impact of the diagnostic utility of EUS-guided FNA of adrenal glands on patient management. Our secondary objective was to determine predictors of malignant adrenal involvement. STUDY DESIGN: Observational study. SETTING: Tertiary referral center. PATIENTS: Patients with enlarged adrenal(s) on abdominal imaging underwent EUS-guided FNA. The left adrenal (n = 54) was sampled via the transgastric approach and the right adrenal (n = 5) via a transduodenal approach. RESULTS: Fifty-nine patients (63% men, median age 65 years) were evaluated. The median adrenal gland size was 25 x 17 mm. Adrenal tissue adequate for interpretation was obtained in all of the patients. EUS-guided FNA confirmed malignancy in 22 (37%) patients. Based on size (> or =30 mm) alone, EUS had an accuracy of 68%. Patients with malignant cytology had higher standard uptake value scores on positron-emission tomography compared with patients with benign adrenal masses (P < .001). Malignant masses were more likely to have an altered adrenal gland shape compared with benign masses (crude odds ratio [OR] 12.0; P < .001). On multivariable analysis, altered adrenal gland shape was a significant predictor of malignancy (adjusted OR 7.94; P = .015), whereas a size of 30 mm or larger (adjusted OR 1.30; P = .774) and hypoechoic nature (adjusted OR 12.05; P = .148) were not. All patients except 2 with malignant cytology were treated with systemic therapy without the need for additional invasive biopsies or surgery. No immediate complications were encountered. LIMITATIONS: Lack of surgical criterion standard; 1 experienced endosonographer. CONCLUSIONS: EUS-guided FNA of the adrenal glands is a minimally invasive and safe approach that documents or excludes malignant involvement. EUS-guided FNA should be the first next test to evaluate enlarged adrenal glands because it directs therapy and affects patient management.

Anal-rectal cytology: correlation with human papillomavirus status and biopsy diagnoses in a population of HIV-positive patients.

OBJECTIVES: We describe the cytological distribution of disease, correlate cytological diagnoses with human papillomavirus (HPV) DNA status and surgical biopsy diagnoses, determine if CD4 counts correlate with lesion severity, and compare anal-rectal data of HIV-infected patients (primarily men) with cervical data. MATERIALS AND METHODS: A retrospective search of the computerized database identified 118 HIV-positive patients who had anal-rectal cytology. Cytology results were compared with available follow-up data including repeat anal-rectal cytology tests, surgical biopsy, CD4 counts, and HPV DNA polymerase chain reaction-based genotyping. RESULTS: Cytological diagnoses included 3% unsatisfactory for diagnosis, 41% negative for intraepithelial lesion or malignancy (NILM), 23% atypical squamous cells of undermined significance (ASC-US), 31% low-grade squamous intraepithelial lesion (LSIL), and 2% high-grade squamous intraepithelial lesion (HSIL) (ASC-US/squamous intraepithelial lesion, 0.7:1). Two anal intraepithelial neoplasia (AIN) II, 10 AIN III, and 1 invasive squamous cell carcinoma were histologically detected (11%). The majority of AIN II was preceded by LSIL, 54%; ASC-US, 15%; and HSIL, 8%. The false-negative fraction was 23%. Sensitivity, specificity, negative predictive value, and positive predictive value were 92%, 8%, 33%, and 67%, respectively. Of those HPV tested concurrent with the first cytology specimen, 48% NILM, 78% ASC-US, and 100% LSIL were HPV positive. Mean CD4 counts (per microliter) were lower in patients with HSIL (243 [SD, 65]) compared with LSIL (400 [SD, 261]) and NILM (428 [SD, 232]). CONCLUSIONS: Anal-rectal cytology is a useful screening test. A high percentage of AIN II lesions were detected in this at-risk population, and the majority was detected following cytological abnormality.

Genistein and resveratrol, alone and in combination, suppress prostate cancer in SV-40 tag rats.

BACKGROUND: Chemoprevention utilizing dietary agents is an effective means to slow the development of prostate cancer. We evaluated the potential additive and synergistic effects of genistein and resveratrol for suppressing prostate cancer in the Simian Virus-40 T-antigen (SV-40 Tag) targeted probasin promoter rat model, a transgenic model of spontaneously developing prostate cancer. METHODS: Rats were fed genistein or resveratrol (250 mg/kg AIN-76A diet) alone and in combination, and a low-dose combination (83 mg genistein + 83 mg resveratrol/kg diet). Histopathology and mechanisms of action studies were conducted at 30 and 12 weeks of age, respectively. RESULTS: Genistein, resveratrol, and the high-dose combination treatments suppressed prostate cancer. The low-dose combination did not elicit protection against prostate cancer and was most likely below the effective dose for causing significant histopathological changes. Total genistein and resveratrol concentrations in the blood reached 2,160 and 211 nM, respectively in rats exposed to the single treatments. Polyphenol treatments decreased cell proliferation and insulin-like growth factor-1 (IGF-1) protein expression in the prostate. In addition, genistein as a single agent induced apoptosis and decreased steroid receptor coactivator-3 (SRC-3) in the ventral prostate (VP). CONCLUSIONS: Genistein and resveratrol, alone and in combination, suppress prostate cancer development in the SV-40 Tag model. Regulation of SRC-3 and growth factor signaling proteins are consistent with these nutritional polyphenols reducing cell proliferation and increasing apoptosis in the prostate.

Characterization of SV-40 Tag rats as a model to study prostate cancer.

BACKGROUND: Prostate cancer is the second most frequently diagnosed cancer in men. Animal models that closely mimic clinical disease in humans are invaluable tools in the fight against prostate cancer. Recently, a Simian Virus-40 T-antigen (SV-40 Tag) targeted probasin promoter rat model was developed. This model, however, has not been extensively characterized; hence we have investigated the ontogeny of prostate cancer and determined the role of sex steroid receptor and insulin-like growth factor-1 (IGF-1) signaling proteins in the novel SV-40 Tag rat. METHODS: The SV-40 Tag rat was histopathologically characterized for time to tumor development, incidence and multiplicity and in the ventral, dorsal, lateral and anterior lobes of the prostate. Immunoassay techniques were employed to measure cell proliferation, apoptosis, and sex steroid receptor and growth factor signaling-related proteins. Steroid hormone concentrations were measured via coated well enzyme linked immunosorbent assay (ELISA) kits. RESULTS: Prostatic intraepithelial neoplasia (PIN) and well-differentiated prostate cancer developed as early as 2 and 10 weeks of age, respectively in the ventral prostate (VP) followed by in the dorsolateral (DLP). At 8 weeks of age, testosterone and dihydrotestosterone (DHT) concentrations in SV-40 Tag rats were increased when compared to non-transgenic rats. High cell proliferation and apoptotic indices were found in VP and DLP of transgenic rats. Furthermore, we observed increased protein expression of androgen receptor, IGF-1, IGF-1 receptor, and extracellular signal-regulated kinases in the prostates of SV-40 Tag rats. CONCLUSION: The rapid development of PIN and prostate cancer in conjunction with the large prostate size makes the SV-40 Tag rat a useful model for studying prostate cancer. This study provides evidence of the role of sex steroid and growth factor proteins in prostate cancer development and defines appropriate windows of opportunity for preclinical trials and aids in the rational design of chemoprevention, intervention, regression, and therapeutic studies using prostate cancer rodent models.

p16 Improves interobserver agreement in diagnosis of anal intraepithelial neoplasia.

OBJECTIVES: Evaluation of anal intraepithelial neoplasia (AIN) is subjective. Previous studies have shown p16 and Ki-67 expressions to correlate with AIN grade. Biomarkers like p16 and Ki-67 may improve interobserver agreement. The objectives were (1) to determine the extent of interobserver agreement in evaluating AIN on routine hematoxylin and eosin (H&E) sections and (2) to test whether p16 and/or Ki-67 staining improve interobserver diagnostic agreement. MATERIALS AND METHODS: Seventy-seven anal specimens were retrieved. Sections were stained with monoclonal antibodies against p16 and Ki-67. Blind to the original diagnoses, 4 pathologists assessed H&E alone, p16 alone, Ki-67 alone, and all 3 simultaneously. Diagnoses were normal/reactive, AIN I/HPV, AIN II, and AIN III. Agreement was calculated using kappa and S statistics. RESULTS: Pathologists were board certified and had 2 to 25 years (mean = 13.6 years) of experience. Fair agreement was observed using H&E diagnosis alone (kappa = 0.38, S = 0.56). The p16 diagnostic evaluation demonstrated the highest agreement (kappa = 0.57, S = 0.73). Interobserver agreement for Ki-67 alone and for H&E/p16/Ki-67 combined were comparable to that of H&E alone (kappa = 0.4, S = 0.54 and kappa = 0.44, S = 0.62, respectively). When the pathologists' diagnoses for all diagnostic evaluations were compared with consensus diagnoses, the lowest average magnitude of disagreement was seen with Ki-67 alone, followed by p16 alone, H&E/p16/Ki-67 combined, and H&E alone. CONCLUSIONS: Interobserver agreement for diagnosis of AIN was fair when based solely on H&E preparation. p16 alone improved interobserver agreement and demonstrated superior agreement when compared with H&E, Ki-67, and H&E/p16/Ki-67 combined.

SIRT1 is significantly elevated in mouse and human prostate cancer.

Evidence suggests that the histone deacetylase, SIRT1, is a mediator of life span extension by calorie restriction; however, SIRT1 may paradoxically increase the risk of cancer. To better understand the relationship among SIRT1, energy balance, and cancer, two experiments were done. First, a transgenic mouse model of prostate cancer (transgenic adenocarcinoma of mouse prostate; TRAMP) was used to determine the role of energy balance on SIRT1 expression and the effect of cancer stage on SIRT1 and hypermethylated in cancer-1 (HIC-1). Second, immunohistochemistry was done on human prostate tumors to determine if SIRT1 was differentially expressed in tumor cells versus uninvolved cells. Results show that SIRT1 is not increased in the dorsolateral prostate (DLP) of calorie-restricted mice during carcinogenesis. In contrast, when examined in the DLP as a function of pathologic score, SIRT1 was significantly elevated in mice with poorly differentiated adenocarcinomas compared with those with less-advanced disease. HIC-1, which has been shown to regulate SIRT1 levels, was markedly reduced in the same tumors, suggesting that a reduction in HIC-1 may be in part responsible for the increased expression of SIRT1 in prostatic adenocarcinomas. Furthermore, immunostaining of human prostate tumors showed that cancer cells had greater SIRT1 expression than uninvolved cells. In conclusion, DLP SIRT1 expression from calorie-restricted mice was not altered during carcinogenesis. However, SIRT1 expression was increased in mice with poorly differentiated adenocarcinomas and in human prostate cancer cells. Because SIRT1 may function as a tumor promoter, these results suggest that SIRT1 should be considered as a potential therapeutic target for prostate cancer.

Cancer progression in the transgenic adenocarcinoma of mouse prostate mouse is related to energy balance, body mass, and body composition, but not food intake.

Calorie restriction can inhibit or delay carcinogenesis, reportedly due to a reduction in calorie intake rather than by concurrent changes in body mass and/or composition. Our objective was to test the hypothesis that body mass and/or composition have an important effect, independent of energy intake, on the benefits or hazards associated with calorie restriction or overeating, respectively. In the first experiment, transgenic mice that spontaneously develop prostate cancer [transgenic adenocarcinoma of mouse prostate (TRAMP)] were housed at 27 degrees C or 22 degrees C and pair fed the same diet for 21 weeks (95% of ad libitum intake at 27 degrees C). In the second experiment, TRAMP mice were housed at 27 degrees C or 22 degrees C and fed the same diet ad libitum for 21 weeks. Despite a similar calorie intake, pair-fed mice at 27 degrees C (PF27) were heavier (28.3 +/- 3.3 versus 17.6 +/- 1.6 g at 21 weeks; P < 0.001; mean +/- SD) and had greater fat (6.4 +/- 2.1 versus 1.9 +/- 0.3 g; P < 0.001) and lean mass (P < 0.001) than pair-fed mice at 22 degrees C. Furthermore, PF27 mice had greater levels of serum leptin (P < 0.001), lower levels of adiponectin (P < 0.05), and a greater frequency of prostatic adenocarcinoma (P < 0.05). In contrast, ad libitum-fed mice housed at 22 degrees C consumed approximately 30% more calories than ad libitum-fed mice at 27 degrees C, but there was no difference between groups in body composition or cancer progression. These results imply that the ability of calorie restriction to inhibit or delay cancer incidence and progression is mediated in part by changes in energy balance, body mass, and/or body composition rather than calorie intake per se, suggesting that excess calorie retention, rather than consumption, confers cancer risk.

Endoscopic ultrasound-guided fine needle aspiration is useful for nodal staging in patients with pleural mesothelioma.

Patients with malignant pleural mesothelioma and negative N2 stage lymph nodes may benefit from extrapleural pneumonectomy with adjuvant therapy. The objective of this study is to describe the use of EUS-FNA to determine N2 stage status in patients with mesothelioma and its impact in the management of such patients. Six patients (mean age, 62 yr; median age, 63 yr; range, 52-70 yr; 5 men; 1 woman) underwent EUS-FNA for staging of N2 lymph nodes from July 2000 to July 2006. Follow-up included operative notes, treatment summaries, and surgical pathology. Eight sites were aspirated: four subcarinal lymph nodes, three aorto-pulmonary window lymph nodes, and one paraesophageal mass. Two of 8 (25%) aspirates were positive for metastatic disease in two different patients. Two false negative EUS-FNAs were observed and were attributed to sampling error not diagnostic error. No complications were observed. EUS-FNA is a safe N2 node staging technique in patients with mesothelioma. A positive N2 lymph node by EUS-FNA may be a contraindication to definitive surgery in patients with malignant mesothelioma.

Endoscopic ultrasound-guided fine-needle aspiration: sampling, pitfalls, and quality management.

Endoscopic ultrasound-guided fine-needle aspiration (EUS FNA) is an accurate and versatile technique in the diagnosis of gastrointestinal tract lesions as well as other organ sites. EUS FNA is performed ideally with cytopathologic adequacy evaluation, and diagnosis at the time of tissue procurement. In this article, we review the most relevant issues in the process of EUS FNA-based diagnostics. Specifically, we describe the technical aspects of specimen collection, processing, and appropriate selection of ancillary studies. We also illustrate the most commonly encountered diagnostic pitfalls, and methods for their avoidance. Lastly, we discuss quality management, which emphasizes the communication between the endoscopist and the pathologist.

Impact of expected changes in national papanicolaou test volume on the cytotechnology labor market: an impending crisis.

With the new screening and treatment guidelines and the prospect of human papillomavirus vaccination for adolescents, the current total volume of Papanicolaou (Pap) tests will be significantly reduced. We used available data to assess the current supply and demand in the cytotechnology labor market and how an expected change in Pap test volume impacts this market. Cytotechnologists' data were obtained from the American Society for Clinical Pathology (ASCP) Board of Registry and the Center for Medicare and Medicaid Services. Data for wages and vacancies were obtained from American Society for Cytotechnologists and ASCP Surveys. Cytotechnology training program data were obtained from annual reports of the Cytotechnology Programs Review Committee of American Society for Cytopathology. In the current market, the demand for cytotechnologists increases by 3.6% and the supply by 4.0% each year. At any given time, there is a vacancy rate of 3%. In the coming years, the demand will decrease remarkably with a projected total demand for cytotechnologists of 5,623 instead of 8,033 by the year 2010 and of 8,538 instead of 14,146 by the year 2026. The cytotechnology market faces an impending crisis. There is a high need for prospectively collected accurate data on demand for and supply of cytotechnologists.

Hedgehog pathway expression in heterogeneous pancreatic adenocarcinoma: implications for the molecular analysis of clinically available biopsies.

Recent studies suggest that hedgehog (HH)-pathway signaling is required for the initiation and continued growth of pancreatic adenocarcinoma (PAC). Definitive gene expression analysis of PAC remains difficult, owing to the host desmoplastic stromal interaction and subsequent tumor heterogeneity. The primary goal of this study was to evaluate the effect of heterogeneity within a series (n=5) of matched clinical PAC biopsies [snap-frozen, formalin-fixed paraffin-embedded (FPE), endoscopic ultrasound-guided fine-needle aspirate (EUS-FNA)]. Differential expressions, specific to tumor cells, were evaluated by comparisons of uninvolved pancreas (n=9), EUS-FNA (n=14), and macrodissected (tumor-cell-enriched) biopsies (n=16). To determine whether treatment modulates gene expression, a unique (independent) set of synchronous EUS-FNA samples (n=4) was obtained before, and 2 weeks after, chemoradiation. mRNA levels were evaluated using real-time quantitative polymerase chain reaction formatted in a TaqMan low-density array, which was capable of simultaneously quantifying 46 independent genes in the HH pathway. Protein levels for Patched, Smoothened, and glioma-associated oncogene 1 (Gli-1) in FPE tissues were determined, using immunohistochemistry. A significant concordance (P<0.0001) was observed in the HH-pathway mRNA levels between matched surgically resected (both snap-frozen and FPE) and EUS-FNA biopsies. HH-pathway mRNA levels changed (increased) only after macrodissection, suggesting localization to tumor cells. Immunohistochemical staining for Patched, Smoothened, and Gli-1 confirmed the increased (P<0.001) levels of protein in the PAC cells, compared with cells from uninvolved pancreas. EUS-FNA biopsies that were obtained before and during chemoradiation demonstrated no significant changes in HH-pathway gene expression. Collectively, these studies demonstrate presence of HH-pathway expression in all the clinical PAC biopsies examined, suggesting that this is a significant tumor-associated target and offering the possibility that specific molecular profiling might be attempted from these heterogeneous tissues.

Cytologic criteria to reduce error in EUS-FNA of solid pseudopapillary neoplasms of the pancreas.

INTRODUCTION: Standardization of error classification in pathology remains an important issue. This study assesses the extent of error in cytopathologic diagnosis of solid pseudopapillary neoplasms (SPN) of the pancreas. Because of morphologic overlap of SPN and pancreatic neuroendocrine neoplasms (NET), we compared cytologic characteristics to determine which best distinguishes these entities. MATERIALS AND METHODS: We collected cases diagnosed as SPN either by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) by cytology or surgical pathology from January 2000 to March 2013. An equal number of NET cases were randomly selected. Cytology and surgical pathology cases were evaluated for diagnostic errors and patient impact. Cytologic features in SPN and NET were scored based on presence of previously described characteristics. RESULTS: A total of 17 patients with EUS-FNA were diagnosed with SPN by cytology or surgical pathology. Of those, 14 had surgical follow-up and 13 had adequate cell blocks and immunohistochemistry. There were 5 discrepancies between cytology and surgical pathology (5 of 14, 36%). There were no false positives or false negatives, but 5 misclassifications: 4 diagnosed as NET on cytology, and 1 as NET versus SPN. All misclassification errors were associated with no harm. When compared with NET, fine chromatin, nuclear grooves, pseud papillae, pink stroma, and hyaline globules are statistically significantly associated with SPN. CONCLUSIONS: EUS-FNA of pancreatic SPN has excellent positive and negative predictive value, with no false positives or false negatives in this 12-year study. Only misclassification errors as pancreatic NET were made with minimal impact. We suggest that the presence of 3 of 5 major cytologic criteria offer accuracy in diagnosing SPN to prevent misclassification.

Multiple gene expression analyses in paraffin-embedded tissues by TaqMan low-density array: Application to hedgehog and Wnt pathway analysis in ovarian endometrioid adenocarcinoma.

Recent studies have shown the hedgehog and Wnt families of signaling proteins to be associated with tumor initiation, growth, and survival. However, these pathways remain unexplored in ovarian endometrioid adenocarcinoma (OEA). Here, we describe a novel TaqMan low-density array to examine the expression of 26 and 20 genes in the hedgehog and Wnt pathways, respectively, in six matched snap-frozen and formalin-fixed, paraffin-embedded (FPE) OEA specimens. Expression values were normalized to uninvolved ovarian epithelium. Gene expression in matched frozen and FPE tissues demonstrated significant concordance (r = 0.92, P < 0.0001). However, comparison of amplified and unamplified RNA from frozen OEA tissues revealed an altered molecular profile in amplified RNA. Amplification of RNA from FPE tissues was not successful. The expression of Desert hedgehog (DHH), Indian hedgehog (IHH), Hedge-hog interacting protein (HHIP), Wnt10B, Wnt9B, and Wnt inhibitory factor (WIF1) were tumor-specific with no detectable expression in normal ovarian epithelium. In addition, several genes were significantly (P < 0.025) down-regulated in OEA, including cyclin E2, Porcupine, c-Myc, and Axin 2 (4.8-, 3.6-, 2.9-, and 1.9-fold, respectively). TaqMan low-density array provides an effective multivariate technique for examining gene expression in RNA isolated from either snap-frozen or archival FPE tissues and can identify tumor-specific genes, possibly leading to novel treatments.

Diagnosis of deep-seated lymphoma and leukemia by endoscopic ultrasound-guided fine-needle aspiration biopsy.

We retrospectively studied the use of endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNAB) as a tool for the diagnosis of deep-seated lymphoma. An on-site assessment at the time of EUS-FNAB was performed by a cytopathologist using Diff-Quik (American Scientific Products, McGraw Park, IL) stain. In addition, Papanicolaou stains were performed on EUS-FNAB smears, immunohistochemical stains were performed on cell blocks, and additional samples were sent for flow cytometric analysis. Final cytologic diagnosis was correlated with surgical pathology and/or clinical follow-up. We evaluated EUS-FNAB specimens of deep-seated lymph nodes, spleen, stomach, and pancreas, and 1 EUS-guided needle core biopsy specimen of a lymph node. Thirteen cases of deep-seated lymphoma were diagnosed, including non-Hodgkin lymphomas and Hodgkin lymphoma. One case of hairy cell leukemia was diagnosed. EUS-FNAB is a minimally invasive, cost-effective, and useful tool for the primary diagnosis or staging of deep-seated lymphomas.

Suspicious cytologic diagnostic category in endoscopic ultrasound-guided FNA of the pancreas: Follow-up and outcomes.

BACKGROUND: The objective of the current study was to assess how the suspicious category is followed up in a large endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) service, its outcomes, and the predictors that are likely to be associated with the subsequent diagnosis of a neoplastic process. METHODS: For pancreatic EUS-FNA samples with the suspicious category diagnosis, the authors reviewed the electronic medical record for the method of follow-up and the risks associated with pancreatic malignancy. Logistic regression analysis was used to determine the risk factors that were likely to be associated with the diagnosis of a neoplastic lesion after a cytologic diagnosis of "suspicious." RESULTS: Of a total of 3832 EUS-FNA cases, 116 were diagnosed with suspicious cytology. A total of 90 of 98 neoplasms (92%) were identified, including 72 carcinomas (73%). Similar rates of neoplasia were detected after repeat FNA (34 of 37 neoplasms [92%]) and subsequent biopsy/surgical resection (44 of 46 neoplasms [96%]), but significantly fewer neoplasms were detected among patients with clinical follow-up (18 of 23 neoplasms [78%]). On multivariate analysis of the potential predictive variables listed above, the presence of a mass was found to be significantly associated with a higher rate of diagnosis of a neoplasm, whereas weight loss was significantly associated with a diagnosis of carcinoma. CONCLUSIONS: The diagnostic category of "suspicious" is associated with a high risk of benign and malignant neoplasms, regardless of the method of follow-up. The presence of a mass and weight loss are significant predictors of a subsequent diagnosis of a neoplasm after suspicious cytology. In patients with suspicious cytology and these findings, surgery is recommended for resectable masses and repeat FNA for unresectable masses.

Cytologic grade independently predicts survival of patients with pancreatic adenocarcinoma.

Our objectives were to devise a cytologic grading system and determine whether it would predict survival of patients with solid-type pancreatic adenocarcinoma. We evaluated 116 consecutive patients from July 2000 to November 2002; they were followed up until September 2003. We scored the following features on rapid Romanowsky-stained endoscopic ultrasound-guided fine-needle aspiration smears: cell group architecture, single cells, nuclear grade, mucus, bizarre cells, and necrosis. A cytologic grade (low vs high) was assigned. The Kaplan-Meier estimate of 6-month survival was 76% (SE, 7%) for patients with low-grade tumors vs 50% (SE, 6%) for patients with high-grade carcinoma. The median survival for patients with low-grade vs high-grade tumors was 1 year vs 6 months, respectively (chi2 = 4.45; P = .035). Cox proportional hazards regression showed tumor stage, cancer-specific treatment, and cytologic grade to be independent predictors of survival (P = .001). No other factors (age, mass location, placement of stent, presence of concomitant chronic pancreatitis, race, sex) predicted survival. We devised a grading system that independently predicted survival in patients with pancreatic adenocarcinoma.

Optimal molecular profiling of tissue and tissue components: defining the best processing and microdissection methods for biomedical applications.

Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of any tissue-based biological phenomenon. This article reviews current methods for obtaining anatomically specific signals from molecules isolated from tissues, a basic requirement for productive linking of phenotype and genotype. The quality of samples isolated from tissue and used for molecular analysis is often glossed over or omitted from publications, making interpretation and replication of data difficult or impossible. Fortunately, recently developed techniques allow life scientists to better document and control the quality of samples used for a given assay, creating a foundation for improvement in this area. Tissue processing for molecular studies usually involves some or all of the following steps: tissue collection, gross dissection/identification, fixation, processing/embedding, storage/archiving, sectioning, staining, microdissection/annotation, and pure analyte labeling/identification and quantification. We provide a detailed comparison of some current tissue microdissection technologies, and provide detailed example protocols for tissue component handling upstream and downstream from microdissection. We also discuss some of the physical and chemical issues related to optimal tissue processing, and include methods specific to cytology specimens. We encourage each laboratory to use these as a starting point for optimization of their overall process of moving from collected tissue to high quality, appropriately anatomically tagged scientific results. In optimized protocols is a source of inefficiency in current life science research. Improvement in this area will significantly increase life science quality and productivity. The article is divided into introduction, materials, protocols, and notes sections. Because many protocols are covered in each of these sections, information relating to a single protocol is not contiguous. To get the greatest benefit from this article, readers are advised to read through the entire article first, identify protocols appropriate to their laboratory for each step in their workflow, and then reread entries in each section pertaining to each of these single protocols.