Small Tissue Specimens Research Articles

Three-dimensional microscopic analysis of clinical prostate specimens.

Microscopic evaluation of prostate specimens for both clinical and research purposes is generally performed on 5-μm-thick tissue sections. Because cross-sections give a two-dimensional (2D) representation, little is known about the actual underlying three-dimensional (3D) architectural features of benign prostate tissue and prostate cancer (PCa). The aim of this study was to show that a combination of tissue-clearing protocols and confocal microscopy can successfully be applied to investigate the 3D architecture of human prostate tissue. Optical clearing of intact fresh and formalin-fixed paraffin-embedded (FFPE) clinical prostate specimens allowed us to visualize tissue structures up to a depth of 800 μm, whereas, in uncleared tissue, detection of fluorescence was only possible up to 70 μm. Fluorescent labelling with a general nuclear dye and antibodies against cytokeratin (CK) 5 and CK8-18 resulted in comprehensive 3D imaging of benign peripheral and transition prostate zones, as well as individual PCa growth patterns. After staining, clearing, and imaging, samples could still be processed for 2D (immuno)histochemical staining and DNA analysis, enabling additional molecular and diagnostic characterization of small tissue specimens. In conclusion, the applicability of 3D imaging to archival FFPE and fresh clinical specimens offers unlimited opportunities to study clinical and biological topics of interest in their actual 3D context.

Claudin expression in follicle-associated epithelium of rat Peyer's patches defines a major restriction of the paracellular pathway.

Members of the tight junction protein family of claudins have been demonstrated to specifically determine paracellular permeability of the intestinal epithelium. In small intestinal mucosa, which is generally considered to be a leaky epithelium, Peyer's patches are a primary part of the immune system. The aim of this study was to analyse the tight junctional barrier of follicle-associated epithelium covering Peyer's patches (lymphoid follicles). Employing small intestinal tissue specimens of male Wistar rats, electrophysiological analyses including the Ussing chamber technique, marker flux measurements and one-path impedance spectroscopy were performed. Morphometry of HE-stained tissue sections was taken into account. Claudin expression and localization was analysed by immunoblotting and confocal laser scanning immunofluorescence microscopy. Almost twofold higher parameters of epithelial and transepithelial tissue resistance and a markedly lower permeability for the paracellular permeability markers 4 and 20 kDa FITC-dextran were detected in follicle-associated epithelium compared to neighbouring villous epithelium. Analysis of claudin expression and localization revealed a stronger expression of major sealing proteins in follicle-associated epithelium, including claudin-1, claudin-4, claudin-5 and claudin-8. Therefore, the specific expression and localization of claudins is in accordance with barrier properties of follicle-associated epithelium vs. neighbouring villous epithelium. We demonstrate that follicle-associated epithelium is specialized to ensure maximum restriction of the epithelial paracellular pathway in Peyer's patches by selective sealing of tight junctions. This results in an exclusive transcellular pathway of epithelial cells as the limiting and mandatory route for a controlled presentation of antigens to the underlying lymphocytes under physiological conditions.

MP30-05 DETECTION OF TRANSLOCATION TUMOURS ON RENAL CELL CARCINOMA TISSUE MICRO ARRAYS BY FISH

You have accessJournal of UrologyKidney Cancer: Evaluation/Staging II1 Apr 2014MP30-05 DETECTION OF TRANSLOCATION TUMOURS ON RENAL CELL CARCINOMA TISSUE MICRO ARRAYS BY FISH Christine Stoehr, Ramona Erber, Judith Frohnauer, Guido Martignoni, Frank Becker, Jens Bedke, Paolo Fornara, Susanne Füssel, Mieczyslaw Gajda, Bastian Gunawan, Volker Jung, Wolf Wieland, Mathias Meinhardt, Axel Merseburger, Arne Strauss, Heiko Wunderlich, Britta Meyer, Sven Hauke, Kerstin Junker, and Arndt Hartmann Christine StoehrChristine Stoehr More articles by this author , Ramona ErberRamona Erber More articles by this author , Judith FrohnauerJudith Frohnauer More articles by this author , Guido MartignoniGuido Martignoni More articles by this author , Frank BeckerFrank Becker More articles by this author , Jens BedkeJens Bedke More articles by this author , Paolo FornaraPaolo Fornara More articles by this author , Susanne FüsselSusanne Füssel More articles by this author , Mieczyslaw GajdaMieczyslaw Gajda More articles by this author , Bastian GunawanBastian Gunawan More articles by this author , Volker JungVolker Jung More articles by this author , Wolf WielandWolf Wieland More articles by this author , Mathias MeinhardtMathias Meinhardt More articles by this author , Axel MerseburgerAxel Merseburger More articles by this author , Arne StraussArne Strauss More articles by this author , Heiko WunderlichHeiko Wunderlich More articles by this author , Britta MeyerBritta Meyer More articles by this author , Sven HaukeSven Hauke More articles by this author , Kerstin JunkerKerstin Junker More articles by this author , and Arndt HartmannArndt Hartmann More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.813AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Translocation tumours were acknowledged as a new renal cell carcinoma (RCC) subtype in the 2004 WHO classification. Diagnosis is usually performed by IHC analysis for transcription factor E3 (TFE3) and/or Cathepsin K (CTSK) overexpression. As not every translocation variant results in overexpression, several cases might have remained undiscovered. To overcome this problem, a dual color break-apart FISH assay was recently developed for the TFE3 locus. Another translocation tumour variant was discovered in RCC which involves rearrangements of ALK and EML4, a genetic alteration predominantly described in NSCLC, and can be detected by a three color FISH assay. Here, we tested the applicability of both FISH methods on small tissue specimens of a cohort of RCC that had been characterized for TFE3 and CTSK-overexpression. METHODS Two sets of tissue micro arrays ((TMA), FFPE material), that were already described earlier and comprised 252 renal cell carcinoma punches from patients ≤35 yrs and ≥80 yrs, or 169 punches ≤45 yrs and ≥75 yrs, respectively, were analysed for breaks or rearrangements of TFE3 and ALK/EML4. TFE3 status was analysed by ZytoLight® SPEC TFE3 Dual Color Break Apart Probe and ALK/EML4 status by ZytoLight® SPEC ALK/EML4 TriCheck™ Probe (ZytoVision, Bremerhaven, Germany). The resulting fluorescent signals were evaluated and compared to the IHC results we had obtained earlier. RESULTS TMA evaluation was compromised by tissue limitations. Twelve translocation tumours could be found by FISH analysis. Three of the five RCC punches with strong positivity for TFE3 in IHC could be confirmed by TFE3-FISH. One of these cases had also demonstrated weak CTSK positivity. Five of the ten cases which demonstrated strong CTSK positivity were TFE3 translocation negative. One papillary tumour, pT1a N0 M0 G2 R0, demonstrated rearrangement of ALK. CONCLUSIONS Results of IHC were not substantially confirmed using FISH on TMA. On the one hand, the TFE3 or ALK/EML4 status of many cases was not available due to tissue limitations. On the other hand, relying on IHC alone might create false positive TFE3 translocation tumors. Therefore, application of FISH in this context is recommended to detect genomic rearrangements directly. Moreover, by detecting one case with ALK rearrangement among the evaluable cases, we confirm that this type of translocation is very rarely found in RCC patients. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e315 Peer Review Report Advertisement Copyright & Permissions© 2014MetricsAuthor Information Christine Stoehr More articles by this author Ramona Erber More articles by this author Judith Frohnauer More articles by this author Guido Martignoni More articles by this author Frank Becker More articles by this author Jens Bedke More articles by this author Paolo Fornara More articles by this author Susanne Füssel More articles by this author Mieczyslaw Gajda More articles by this author Bastian Gunawan More articles by this author Volker Jung More articles by this author Wolf Wieland More articles by this author Mathias Meinhardt More articles by this author Axel Merseburger More articles by this author Arne Strauss More articles by this author Heiko Wunderlich More articles by this author Britta Meyer More articles by this author Sven Hauke More articles by this author Kerstin Junker More articles by this author Arndt Hartmann More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Quantitative analysis of microscopic X-ray computed tomography imaging: Japanese quail embryonic soft tissues with iodine staining.

Rapid three-dimensional imaging of embryos to better understand the complex process of morphogenesis has been challenging. Recently introduced iodine staining protocols (I2 KI and alcoholic iodine stains) combined with microscopic X-ray computed tomography allows visualization of soft tissues in diverse small organisms and tissue specimens. I2 KI protocols have been developed specifically for small animals, with a limited number of quantitative studies of soft tissue contrasts. To take full advantage of the low X-ray attenuation of ethanol and retain bound iodine while dehydrating the specimen in ethanol, we developed an ethanol I2 KI protocol. We present comparative microscopic X-ray computed tomography analyses of ethanol I2 KI and I2 KI staining protocols to assess the performance of this new protocol to visualize soft tissue anatomy in late stage Japanese quail embryos using quantitative measurements of soft tissue contrasts and sample shrinkage. Both protocols had only 5% shrinkage compared with the original harvested specimen, supporting the use of whole mounts to minimize tissue shrinkage effects. Discrimination within and among the selected organs with each staining protocol and microscopic X-ray computed tomography imaging were comparable to those of a gray scale histological section. Tissue discrimination was assessed using calibrated computed tomography values and a new discrimination index to quantify the degree of computed tomography value overlaps between selected soft tissue regions. Tissue contrasts were dependent on the depth of the tissue within the embryos before the embryos were saturated with each stain solution, and optimal stain saturations for the entire embryo were achieved at 14 and 28days staining for I2 KI and ethanol I2 KI, respectively. Ethanol I2 KI provided superior soft tissue contrasts by reducing overstaining of fluid-filled spaces and differentially modulating staining of some tissues, such as bronchial and esophageal walls and spinal cord. Delineating the selected soft tissues using optimal threshold ranges derived from the quantitative analyses of the contrast enhancement in optimally stained embryos is possible. The protocols presented here are expected to be applicable to other organisms with modifications to staining time and contribute toward rapid and more efficient segmentation of soft tissues for three-dimensional visualization.

Epithelial to mesenchymal markers and clinical outcomes on erlotinib in stage IV non-small cell lung cancer patients.

e19117 Background: An epithelial phenotype in NSCLC is associated with improved sensitivity to EGFR tyrosine kinase inhibitors (TKI). The best method to identify this subset is unknown (Richardson Anticancer Research 2012, Byers Clin Cancer Res 2012). This retrospective study correlates E-cadherin (Ecad) and vimentin (vim) immunohistochemistry (IHC) expression with outcomes in advanced NSCLC patients (pts) treated with erlotinib (E). Methods: Advanced NSCLC pts that received E were included if sufficient tumor was available from diagnosis. IHC scores for E-cad and vim were generated by multiplying frequency (0-4) by intensity (0-4). Log Rank was used to correlate IHC expression with progression free and overall survival (PFS, OS). Results were compared to a subset of pts with tissue from primary surgical NSCLC resection who later received E for recurrent disease. Results: 159 advanced NSCLC pts treated with E had tissue from diagnosis and IHC analysis. There was no correlation with PFS or OS on E and high/low vim or Ecad expression. Subtracting the IHC scores (vim minus ecad) created a difference score. A low difference score (n = 62) correlated with prolonged PFS (2.6 vs 1.9 months, p = .014 HR 1.52) compared with a high score, n = 97. Low difference score trended toward prolonged OS (p=.46) 33 of the patients had tissue available from primary surgical resection. The invasive front was examined for membranous E-cad and cytoplasmic vim (Allred score 0-8). Patients with low vim (< 4) and Ecad (>5), n= 19, trended toward prolonged PFS and OS on E compared with patients with high vim (>5) and low Ecad (<6), n=10 (4.2 vs 1.6 months and 15.5 vs 6.5 months, respectively, p=NS). Conclusions: In this retrospective analysis, using unselected, frequently small tissue specimens, the expression of ecad or vim alone by IHC did not correlate with outcomes for E treated patients. A complicated difference score (vimentin score minus ecadherin score) did correlate with PFS on E. Examining EMT markers at the invasive edge of resected NSCLC tumors might more accurately assess EMT activity and its relationship to outcomes when these pts are recommended EGFR-TKIs.

The use of FTA cards for preserving unfixed cytological material for high‐throughput molecular analysis

Novel high-throughput molecular technologies have made the collection and storage of cells and small tissue specimens a critical issue. The FTA card provides an alternative to cryopreservation for biobanking fresh unfixed cells. The current study compared the quality and integrity of the DNA obtained from 2 types of FTA cards (Classic and Elute) using 2 different extraction protocols ("Classic" and "Elute") and assessed the feasibility of performing multiplex mutational screening using fine-needle aspiration (FNA) biopsy samples. Residual material from 42 FNA biopsies was collected in the cards (21 Classic and 21 Elute cards). DNA was extracted using the Classic protocol for Classic cards and both protocols for Elute cards. Polymerase chain reaction for p53 (1.5 kilobase) and CARD11 (500 base pair) was performed to assess DNA integrity. Successful p53 amplification was achieved in 95.2% of the samples from the Classic cards and in 80.9% of the samples from the Elute cards using the Classic protocol and 28.5% using the Elute protocol (P = .001). All samples (both cards) could be amplified for CARD11. There was no significant difference in the DNA concentration or 260/280 purity ratio when the 2 types of cards were compared. Five samples were also successfully analyzed by multiplex MassARRAY spectrometry, with a mutation in KRAS found in 1 case. High molecular weight DNA was extracted from the cards in sufficient amounts and quality to perform high-throughput multiplex mutation assays. The results of the current study also suggest that FTA Classic cards preserve better DNA integrity for molecular applications compared with the FTA Elute cards.

Identification of protein marker in vaginal wall tissues of women with stress urinary incontinence by protein chip array.

We sought to investigate protein biomarkers for stress urinary incontinence (SUI) in vaginal tissues using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) and examine if this is a reliable methodology to examine proteins in small tissue specimens. We compared protein expression profile of vaginal tissue from women with SUI and continent controls. A 22.6kDa peak was identified by subsequent weak cation-exchange, reverse-phase fractionation, gel electrophoresis, and trypsin digestion, then analyzed by matrix assisted laser desorption/ionization mass spectrometry (MALDI MS) and MALDI MS-MS. Biomarker identity and expression level were confirmed by Western-blotting and immunohistochemistry. Expression of the 22.6kDa protein, identified as SM-22α, was significantly higher in women with SUI versus controls. A 3×3-mm tissue sample was sufficient for identification. Western-blot/immunohistochemistry confirmed the SELDI-TOS MS findings. SM-22α, a marker for myofibroblasts, was identified as a biomarker of SUI. Differential protein profiling by SELDI-TOF MS is a powerful and reliable tool for urogynecological research as it allows us to study an array of proteins simultaneously using small tissue samples.

Comparing normal and torn rotator cuff tendons using dynamic shear analysis

This study reports the application of a novel method for quantitatively determining differences in the mechanical properties of healthy and torn rotator cuff tissues. In order to overcome problems of stress risers at the grip-tendon interface that can obscure mechanical measurements of small tendons, we conducted our investigation using dynamic shear analysis. Rotator cuff tendon specimens were obtained from 100 patients during shoulder surgery. They included 82 differently sized tears and 18 matched controls. We subjected biopsy samples of 3 mm in diameter to oscillatory deformation under compression using dynamic shear analysis. The storage modulus (G') was calculated as an indicator of mechanical integrity. Normal tendons had a significantly higher storage modulus than torn tendons, indicating that torn tendons are mechanically weaker than normal tendons (p = 0.003). Normal tendons had a significantly higher mean shear modulus than tendons with massive tears (p < 0.01). Dynamic shear analysis allows the determination of shear mechanical properties of small tissue specimens obtained intra-operatively that could not be studied by conventional methods of tensile testing. These methods could be employed to investigate other musculoskeletal tissues. This pilot study provides some insight into mechanisms that might contribute to the failure of repair surgery, and with future application could help direct the most appropriate treatment for specific rotator cuff tears.

Characterization of Ocular Tissues Using Microindentation and Hertzian Viscoelastic Models

The authors applied a novel microindentation technique to characterize biomechanical properties of small ocular and orbital tissue specimens using the hertzian viscoelastic formulation, which defines material viscoelasticity in terms of the contact pressure required to maintain deformation by a harder body. They used a hard spherical indenter having 100 nm displacement and 100 μg force precision to impose small deformations on fresh bovine sclera, iris, crystalline lens, kidney fat, orbital pulley tissue, and orbital fatty tissue; normal human orbital fat, eyelid fat, and dermal fat; and orbital fat associated with thyroid eye disease. For each tissue, stress relaxation testing was performed using a range of ramp displacements. Results for single displacements were used to build quantitative hertzian models that were, in turn, compared with behavior for other displacements. Findings in orbital tissues were correlated with quantitative histology. Viscoelastic properties of small specimens of orbital and ocular tissues were reliably characterized over a wide range of rates and displacements by microindentation using the hertzian formulation. Bovine and human orbital fatty tissues exhibited highly similar elastic and viscous behaviors, but all other orbital tissues exhibited a wide range of biomechanical properties. Stiffness of fatty tissues tissue depended strongly on the connective tissue content. Relaxation testing by microindentation is a powerful method for characterization of time-dependent behaviors of a wide range of ocular and orbital tissues using small specimens, and provides data suitable to define finite element models of a wide range of tissue interactions.

Optimal Immunohistochemical Markers For Distinguishing Lung Adenocarcinomas From Squamous Cell Carcinomas in Small Tumor Samples

The histologic subtype of non-small cell lung carcinoma is important in selecting appropriate chemotherapy for patients with advanced disease. As many of these patients are not operative candidates, they are treated medically after biopsy for diagnosis. Inherent limitations of small biopsy samples can make distinguishing poorly differentiated lung adenocarcinoma (ADC) from squamous cell carcinoma (SCC) difficult. The value of histochemical and immunohistochemical markers to help separate poorly differentiated ADC from SCC in resection specimens is well established; however, the optimal use of markers in small tissue samples has only recently been examined and the correlation of marker expression in small tissue samples with histologic subtype determined on resection specimens has not been well documented. We address this issue by examining the expression of 9 markers (p63, TTF1, CK5/6, CK7, 34βE12, Napsin A, mucicarmine, NTRK1, and NTRK2) on 200 cases of ADC and 225 cases of SCC in tissue microarray format to mimic small tissue specimens. The single best marker to separate ADC from SCC is p63 (for SCC: sensitivity 84%, specificity 85%). Logistic regression analysis identifies p63, TTF1, CK5/6, CK7, Napsin A, and mucicarmine as the optimal panel to separate ADC from SCC. Reduction of the panel to p63, TTF1, CK5/6, and CK7 is marginally less effective but may be the best compromise when tissue is limited. We present an algorithm for the stepwise application of p63, TTF1, CK5/6, CK7, Napsin A, and mucicarmine in situations in which separation of ADC from SCC in small specimens cannot be accomplished by morphology alone.

Abstract 794: Targeted-therapy related molecular biomarker characterization in NSCLC smokers and never-smokers by a novel qRT-PCR for microdissected tissues methodology

Abstract Several studies have focused on the identification of biologic markers that predict early recurrence and death in patients with non-small cell lung cancer (NSCLC); however, few studies have focused on indicators of outcome in ever-smoker and never-smoker lung cancer patients. We hypothesize that NSCLC primary tumors from smokers and never-smokers differ significantly in the expression of molecular abnormalities and their corresponding biomarkers. We also believe that the different pattern of molecular pathway abnormalities in NSCLCs based on smoking status may explain different response to targeted therapy in both patients’ populations. To test our hypotheses, and better understand the results of biomarker analysis in molecularly targeted clinical trials, we developed a novel qRT-PCR pre-amplification methodology for gene expression analysis of microdissected (MD) tissues to determine and compare the gene expression patterns of targeted therapy related molecular biomarkers and pathways from surgically resected tissue specimens obtained from NSCLC patients. Previously only 9 genes of interest could be analyzed from a typical MD sample (Erickson et al. 2009 Nature Protocols). Our new methodology allows for the use of 8 ng of total RNA for the analysis of up to 24 genes of interest or 24 ng of total RNA for the analysis of up to 72 genes of interest. We then applied this methodology to 40 MD stage I/II NSCLC adenocarcinoma tissues from tumor epithelium and matched normal bronchial epithelium from 20 smokers and 20 never-smokers for EGFR, KRAS, HER2N, BRAF, PIK-3, IGFR-1, LKB-1, mTOR, and AKT analysis. An average of 77.3 ng of total RNA (average RIN = 5.0) was recovered from ∼5000 MD epithelial cells per tissue sample. Gene expression was analyzed by relative quantitation using the 2−ΔΔCt method. Results show that analysis of the expression of multiple genes using a pre-amplification step is a feasible approach for small tissue specimens obtained from clinical trials with advanced lung cancer patients. Preliminary analysis revealed distinct gene expression patterns in smokers compared to never-smokers. Future plans include correlating gene expression with gene mutation, copy number variation, and immunohistochemistry analysis of these tissue specimens. This novel qRT-PCR methodology for gene expression analysis of MD tissues will be used to analyze fine needle aspiration and core needle biopsy tissue specimens from biomarker-driven clinical trial patients (e.g. BATTLE) for validation of profiles and analysis of multiple molecular pathway targets that can predict response to chemotherapy and targeted therapy. This work was funded by Department of Defense BATTLE Lung Cancer Program Initiative W81XWH-06-1-0303, Cohen-Reinauch BATTLE-2 Fund, and National Cancer Institute Intramural Program. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 794.

Mesenchymal expression of Tbx4 gene is not altered in Adriamycin mouse model

The Adriamycin mouse model (AMM) is a reproducible teratogenic model of esophageal atresia/tracheo-esophageal fistula (EA/TEF). Tbx4 is a member of the T-box family of transcription factor genes, which is reported to play a key role in separation of the respiratory tract and the esophagus. Up-regulation of Tbx4 is reported to cause TEF in the chick. Optical projection tomography (OPT) is a technique that allows three-dimensional (3D) imaging of gene expression in small tissue specimens in an anatomical context. The aim of this study was to investigate the temporo-spatial expression of Tbx4 during the critical period of separation of the trachea and esophagus in normal and Adriamycin treated embryos using OPT. Time-mated CBA/Ca mice received intraperitoneal injections of Adriamycin (6 mg/kg) or saline on days 7 and 8 of gestation. Embryos were harvested on days 9-12, stained following whole mount in situ hybridization with labeled RNA probes to detect Tbx4 transcripts (n = 5 for each treatment/day of gestation). Immunolocalization with the endoderm marker Hnf3beta was used to visualize morphology. Embryos were scanned by OPT to obtain 3D representations of gene expression domains. Animal licence no. B100/4106. OPT elegantly revealed Tbx4 gene expression in both controls and in the disorganized pulmonary mesenchyme in the treated embryos. Although characteristic morphological abnormalities were observed in Adriamycin treated embryos, there was no significant difference in Tbx4 transcript distribution around lung primordia in comparison with control embryos. Although previously reported morphological abnormalities of notochord and esophagus were observed in AMM, Tbx4 gene expression was unaltered, suggesting that esophageal anomalies can occur in the presence of normal Tbx4 gene expression in this model.

Fgf10 gene expression is delayed in the embryonic lung mesenchyme in the adriamycin mouse model

The adriamycin mouse model is a well-established teratogenic model of esophageal atresia/tracheoesophageal fistula. Fibroblast growth factor 10 (Fgf10) plays a key role in branching of the lung buds during lung morphogenesis. Fgf10 knockout mice exhibit the absence of the lungs. Optical projection tomography (OPT) is a technique that allows three-dimensional (3D) imaging of gene expression in small tissue specimens in an anatomical context. The aim of this study was to investigate the temporo-spatial expression of Fgf10 during the critical period of separation of the trachea and esophagus in normal and adriamycin-treated embryos using OPT. Time-mated CBA/Ca mice received intraperitoneal injections of adriamycin (6 mg/kg) or saline on days 7 and 8 of gestation. Embryos were harvested on days 10-13, stained after whole mount in situ hybridization with labeled RNA probes to detect Fgf10 transcripts (n = 5 for each treatment/day of gestation). Immunolocalization with endoderm marker Hnf3 beta was used to visualize morphology. Embryos were scanned by OPT to obtain 3D representations of gene expression domains. Computer reconstructed specimens allowed precise staging of developing embryos according to Theiler Staging (TS) criteria. OPT elegantly displayed Fgf10 gene expression in the pulmonary mesenchyme around the tip of the lung buds in both controls and treated embryos in the same spatial territory. Fgf10 gene expression was first detected in the control embryos at TS17. However, Fgf10 gene expression in adriamycin-treated embryos was first only observed at TS18 in 67% of the specimens. Delayed Fgf10 gene expression during the critical period of separation of the trachea and esophagus may affect lung bud formation in the adriamycin model leading to tracheoesophageal malformations.

The Utilization of a Biopsy Needle to Obtain Small Muscle Tissue Specimens to Analyze the Gene and Protein Expression

Recent analytical methods such as real-time polymerase chain reaction (PCR) and Western blotting have now enabled us to analyze the gene and protein expression from small amounts of tissue. A fine needle muscle biopsy is thus expected to obtain a minimally sufficient amount of skeletal muscle to make a successful analysis. As a result, we used this fine needle muscle biopsy technique to obtain muscle tissue specimens from the vastus lateral muscle in 40 participants. The amount of tissue obtained by the fine needle was 5.2 +/- 3.2 mg (mean +/- standard deviation). The total RNA extracted was 3.0 +/- 1.4 microg and the total protein extracted was 2203 +/- 1541 microg. Furthermore, the skeletal muscle tissue specimens obtained by the regular needle technique and blood sample were used as the control. Those specimens were used to measure the gene expression of beta-myosin heavy chain slow (beta-MHC slow) by real-time PCR and the protein expression of monocalboxylate transporter 1 (MCT-1) by Western blotting. Beta-MHC slow gene expression was detected in both samples obtained by a fine and a regular needle biopsy, but not in a blood sample. Furthermore, the MCT-1 protein was detected in samples obtained by a fine needle muscle biopsy. These results indicated that the fine needle muscle biopsy is therefore a useful technique to obtain skeletal muscle specimens at least to analyze the gene and protein expression.

Oral potentially malignant lesions: first‐level micro‐histological diagnosis from tissue fragments sampled in liquid‐based diagnostic cytology

Scalpel biopsy may under-diagnose oral dysplasia/carcinoma in potentially malignant lesions (PMLs) because samples represent only one or a few sites. It is possible that small tissue specimens obtained from over the whole area of PMLs, by scraping with a dermatological curette, could be treated histologically and used as 'micro' biopsies. This study values the accuracy of micro-biopsies in the detection of dysplasia/carcinoma in oral PMLs. A prospective study was carried out on 164 patients with PMLs, with both scalpel and micro-biopsies, for the presence of dysplasia/carcinoma. The most severe diagnosis (obtained by either method) was used as the reference standard. The presence/absence of the basement membrane zone (BMZ) in the micro-biopsy specimens correlated with the site, the clinical features of the PMLs and the operator. Micro-biopsy gave six of 164 (3.66%) inadequate specimens. Of 158 of 164 adequate samples, dysplasia/carcinoma was diagnosed in 85 of 158 cases; micro-biopsy diagnosis was in agreement with scalpel biopsy in 144 of 158 (91.14%) cases and showed a better sensitivity than did scalpel biopsy (97.65% vs. 85.88%), corresponding to two of 158 false-negative cases by micro-biopsy vs. 12 of 158 by scalpel biopsy. The BMZ was observed in 110 of 158 (69.62%) of all micro-biopsies and had no relationship with the sampling site, the clinical features of the PMLs or the operator. The negative predictive value (97.33%) suggests that micro-biopsy may well be an effective first-level diagnostic procedure for PMLs (especially in follow-ups and multiple lesions); moreover, in carcinoma (17% of cases) a definitive diagnosis could be made without further investigation.

Loss and Reduction of Fus1 Protein Expression is a Frequent Phenomenon in the Pathogenesis of Lung Cancer

FUS1, a novel tumor-suppressor gene located in the chromosome 3p21.3 region, may play an important role in lung cancer development. Currently, FUS1-expressing nanoparticles have been developed for treating patients with lung cancer. However, the expression of Fus1 protein has not been examined in a large series of lung cancers and their sequential preneoplastic lesions. Using tissue microarrays, we examined Fus1 immunohistochemical expression in 281 non-small cell lung carcinoma (NSCLC) and 22 small cell lung carcinoma tissue specimens and correlated the findings with patients' clinicopathologic features. To investigate the expression of Fus1 in the early sequential pathogenesis of NSCLC, we studied Fus1 expression in 211 histologically normal and mildly abnormal bronchial epithelia, and 118 bronchial and alveolar preneoplastic lesions obtained from patients with lung cancer. Loss and reduction of expression was detected in 82% of NSCLCs and 100% of small cell lung carcinomas. In NSCLCs, loss of Fus1 immunohistochemical expression was associated with significantly worse overall survival. Bronchial squamous metaplastic and dysplastic lesions expressed significantly lower levels of Fus1 compared with normal (P = 0.014 and 0.047, respectively) and hyperplastic (P = 0.013 and 0.028, respectively) epithelia. Our findings show a high frequency of Fus1 protein loss and reduction of expression in lung cancer, and suggests that this reduction may play an important role in the early pathogenesis of lung squamous cell carcinoma. These findings support the concept that FUS1 gene and Fus1 protein abnormalities could be used to develop new strategies for molecular cancer therapy for a significant subset of lung tumors.

Visualizing expression patterns of Shh and Foxf1 genes in the foregut and lung buds by optical projection tomography

Congenital malformations of the foregut are common in humans. The respiratory and digestive tubes are both derived by division of the foregut primordium. Sonic hedgehog (Shh) and Fork head box F1 (Foxf1) genes encode regulatory molecules that play a pivotal role in gut and lung morphogenesis and are therefore important candidate genes to be examined in models of foregut developmental disruption. Optical projection tomography (OPT) is a new, rapid and non-invasive technique for three-dimensional (3D) imaging of small biological tissue specimens that allows visualization of the tissue distribution of RNA in developing organs while also recording morphology. To explore the application of OPT in this context, we visualized Shh and Foxf1 gene expression patterns in the mouse foregut and lung buds at several stages of development. Time-mated CBA/Ca mice were harvested on embryonic days 9-12. The embryos were stained following whole mount in situ hybridization with labelled RNA probes to detect Shh and Foxf1 transcripts at each stage. The embryos were scanned by OPT to obtain 3D representations of gene expression domains in the context of the changing morphology of the embryo. OPT analysis of Shh and Foxf1 expression in the foregut and lung buds revealed extra details of the patterns not previously reported, particularly in the case of Foxf1 where gene expression was revealed in a changing pattern in the mesenchyme around the developing lung. Shh expression was also revealed in the epithelium of the lung bud itself. Both genes were detected in complementary patterns in the developing bronchi as late as E12, showing successful penetration of molecular probes and imaging at later stages. OPT is a valuable tool for revealing gene expression in an anatomical context even in internal tissues like the foregut and lung buds across stages of development, at least until E12. This provides the possibility of visualizing altered gene expression in an in vivo context in genetic or teratogenic models of congenital malformations.

Effects of sevoflurane and propofol on ischaemia–reperfusion injury after thoracic-aortic occlusion in pigs

Thoraco-abdominal-aneurysm surgery predicts high mortality. Propofol and sevoflurane are commonly used anaesthetics for this procedure. Halogenated anaesthetics induce organ protection similar to ischaemic preconditioning. We investigated which anaesthetic regimen would lead to a better protection against ischaemia-reperfusion injury induced by temporary thoracic-aortic occlusion. Following initial fentanyl-midazolam anaesthesia for surgical preparation, 18 pigs were randomly assigned to two groups: group one received propofol (n=9) and group two sevoflurane (n=9) before, during, and after lower body ischaemia in an investigator blinded fashion. Ten animals without aortic occlusion served as time controls (propofol, n=5; sevoflurane, n=5). For induction of ischaemia, the thoracic aorta was occluded by a balloon-catheter for 90 min. After 120 min of reperfusion, the study anaesthetics were discontinued and fentanyl-midazolam re-established for an additional 180 min. Goal-directed therapy was performed during reperfusion. Fluid and catecholamine requirements were assessed. Serum samples and intestinal tissue specimens were obtained. Severe declamping shock occurred in both study groups. While norepinephrine requirements in the sevoflurane group were significantly reduced during reperfusion (P<0.05), allowing cessation of catecholamine support in 4/9 animals, all 9/9 animals were still catecholamine dependent at the end of the experiment in the propofol group. Serum activities of lactate dehydrogenase, aspartate transaminase, and alanine aminotransferase were lower with sevoflurane (P<0.05). Small intestine tissue specimens did not differ histologically. Use of sevoflurane compared with propofol attenuated the haemodynamic sequelae of reperfusion injury in our model. Release of serum markers of cellular injury was also attenuated.

Gastrointestinal β2microglobulin amyloidosis in hemodialysis patients: biochemical analysis of amyloid proteins in small formalin-fixed paraffin-embedded tissue specimens

We present here a first report on the biochemical analysis of intestinal amyloid deposits found in two cases of hemodialysis-related amyloidosis. A new microtechnique was applied for extraction and immunochemical/chemical characterization of amyloid proteins in small amounts of fixed tissue, thus allowing precise identification of β2microglobulin amyloid (Aβ2M) in both cases studied. The molecular mass of the identified amyloid β2M was close to that of intact β2M (12 kDa), with no evidence of the products of proteolytic fragmentation of these molecules. The isoelectrofocusing of the purified Aβ2M demonstrated a shift to more acidic pI as compared to the normal β2M analyzed under the same experimental conditions. The obtained data suggest that the intestinal amyloid deposits in dialysis-related amyloidosis contain disease-specific β2M isoforms, which could play a role in the pathogenesis of amyloid disease. The new methodology used might be useful in obtaining precise diagnosis of amyloidosis that is necessary for appropriate therapy, and also provide new important information on the chemical structure of amyloid proteins.

Allelic imbalance analysis of oral tongue squamous cell carcinoma by high-density single nucleotide polymorphism arrays using whole-genome amplified DNA

Multiple displacement-based whole-genome DNA amplification is a promising tool to obtain sufficient DNA from small tissue specimens for various genetic analyses, such as SNP array-based analysis. Using Affymetrix 10 K and 100 K SNP mapping array, we evaluated the performance of the Phi29 DNA polymerase-based genome amplification. Greater than 99% concordance in genotyping calls were achieved between amplified and non-amplified DNAs for both arrays. By utilizing the Affymetrix GeneChip Chromosome Copy Number Tool, the allelic imbalance profiles for the advanced stage oral tongue squamous cell carcinoma (OTSCC) were generated based on 10 K and 100 K SNP mapping array results. The results from these two array platforms agree closely, but more precise allelic imbalance patterns can be revealed from the 100 K SNP mapping array data. Furthermore, our data suggested a frequent loss at 3p11-p12 for advanced stage OTSCC.