Diagnostic accuracy of brush biopsy-based cytology for the early detection of oral cancer and precursors in Fanconi anemia.

BACKGROUND: Individuals with Fanconi anemia (FA) have a 500-fold to 700-fold elevated risk, much earlier onset, and limited therapeutic options for oral squamous cell carcinoma (SCC) compared with the general population. The early detection of SCC, or preferably its precursors, is mandatory to retain curative therapeutic options. Due to frequent synchronic and metachronic oral lesions, tissue biopsies, as usually recommended by guidelines, often are not feasible. In the current study, an alternative strategy for early detection using oral brush biopsy-based cytology was validated regarding its diagnostic accuracy. METHODS: Over a 12-year period, the oral cavities of a large cohort of 713 individuals with FA were inspected systematically and brush biopsy-based cytology of 1233 visible oral lesions was performed. In cases of inconclusive cytology, analysis of DNA ploidy was performed whenever possible. The results were correlated to a long-term clinicopathological follow-up reference standard. RESULTS: A total of 737 lesions were suitable for statistical analysis, including 86 lesions with at least high-grade oral epithelial dysplasia in 30 patients. For cytology, the sensitivity and specificity were 97.7% and 84.5%, respectively. Additional analysis of DNA ploidy increased the sensitivity and specificity to 100% and 92.2%, respectively. CONCLUSIONS: Careful inspection of the oral cavity of individuals with FA followed by brush biopsy-based cytology appears to identify visible oral, potentially malignant and malignant lesions that warrant treatment. Approximately 63% of SCC and precursor lesions are detected at a noninvasive or early stage. Negative cytology or a lack of DNA aneuploidy can exclude high-grade oral epithelial dysplasia or SCC with high accuracy and thus reduce the need for invasive diagnostic biopsies.

Loss of LLGL1 Expression Correlates with Diffuse Gastric Cancer and Distant Peritoneal Metastases.

Background: Loss of LLGL1 has been associated with loss of cellular adhesion and dissemination of cells from colorectal cancer and malignant melanoma. Regulation and relevance of LLGL1 were analyzed in gastric cancer patients with lymphatic and distant dissemination. Furthermore, LLGL1 expression was analyzed in relation to the cellular adhesion protein E-cadherin. Methods: LLGL1 and E-cadherin transcription levels were evaluated in 56 gastric cancer patients and five gastric cancer cell lines. IHC staining for LLGL1 was performed on 39 gastric cancer specimens. LLGL1 was stably transfected into LLGL1 negative gastric cancer cell line SNU16 (del(17) (p11.2)) for functional in vitro assays and a xenograft bioassay. Results: Gastric cancer specimens and cell lines displayed LLGL1 and E-cadherin expression levels with variable intensity. In gastric mucosa, LLGL1 exhibited weak cytoplasmic and strong cortical staining. Loss of LLGL1 expression occurred in 65% of gastric cancers and significantly correlated with loss of E-cadherin expression (P=0.00009). Loss of LLGL1 expression was associated with the diffuse type of gastric cancer (P=0.029) with peritoneal carcinomatosis (M1; P=0.006) and with female gender (P=0.017). Stable reexpression of LLGL1 in SNU16 cells significantly increased both plastic surface adhesion and extracellular matrix proteins laminin and fibronectin, but had no impact on in vitro proliferation, apoptosis, or invasion or on in vivo proliferation or differentiation in our xenograft bioassay. Conclusion: LLGL1 is coexpressed with E-cadherin. Loss of expression of either protein is associated with diffuse gastric cancer and peritoneal metastases. LLGL1 does not impact on proliferation or epithelial-mesenchymal transition (EMT) rather increasing cellular adhesion.

Automated detection of cancer cells in effusion specimens by DNA karyometry.

BACKGROUND: The average sensitivity of conventional cytology for the identification of cancer cells in effusion specimens is only approximately 58%. DNA image cytometry (DNA-ICM), which exploits the DNA content of morphologically suspicious nuclei measured on digital images, has a sensitivity of up to 91% for the detection of cancer cells. However, when performed manually, to our knowledge to date, an expert needs approximately 60 minutes for the analysis of a single slide. METHODS: In the current study, the authors present a novel method of supervised machine learning for the automated identification of morphologically suspicious mesothelial and epithelial nuclei in Feulgen-stained effusion specimens. The authors compared this with manual DNA-ICM and a gold standard cytological diagnosis for 121 cases. Furthermore, the authors retrospectively analyzed whether the amount of morphometrically abnormal mesothelial or epithelial nuclei detected by the digital classifier could be used as an additional diagnostic marker. RESULTS: The presented semiautomated DNA karyometric solution identified more diagnostically relevant abnormal nuclei compared with manual DNA-ICM, which led to a higher sensitivity (76.4% vs 68.5%) at a specificity of 100%. The ratio between digitally abnormal and all mesothelial nuclei was found to identify cancer cell-positive slides at 100% sensitivity and 70% specificity. The time effort for an expert therefore is reduced to the verification of a few nuclei with exceeding DNA content, which to our knowledge can be accomplished within 5 minutes. CONCLUSIONS: The authors have created and validated a computer-assisted bimodal karyometric approach for which both nuclear morphology and DNA are quantified from a Feulgen-stained slide. DNA karyometry thus increases the diagnostic accuracy and reduces the workload of an expert when compared with manual DNA-ICM.

Predictive scores in primary biliary cirrhosis: a retrospective single center analysis of 204 patients.

GOALS: The aim of this study was to assess the long-term outcome of primary biliary cirrhosis (PBC) patients and to test the clinical value of various outcome models, such as the Mayo Risk Score (MRS), in a large single-center cohort in Germany. BACKGROUND: PBC is a chronic autoimmune liver disease with a female gender predominance and a peak incidence in the fifth decade of life. PBC is characterized by portal inflammation and immune-mediated destruction of intrahepatic bile ducts in liver histology and the presence of antimitochondrial antibodies in the serum of nearly 95% of patients. In 5% to 20% of patients an overlap syndrome with autoimmune hepatitis (AIH) is diagnosed. Ursodeoxycholic acid is widely accepted as the standard medical treatment. STUDY: A total of 204 patients with PBC or PBC/AIH were retrospectively analyzed with regard to their clinical, biochemical, serological, and histologic features. PBC was diagnosed on the basis of the American Association for the Study of Liver Diseases criteria. Specific PBC scores, such as the MRS, the European and the Yale model, as well as nonspecific scores such as the Child-Pugh, the Model for End-stage Liver Disease, and Aspartate Aminotransferase to Platelet Ratio Index score were analyzed for their utility to predict the clinical outcome of patients. RESULTS: One hundred eighty-four patients with PBC alone and 20 with primary biliary cirrhosis/autoimmune hepatitis overlap were followed up for an average of 7.0 (range, 0.5 to 33.2) years. Importantly, baseline values of serum bilirubin, alkaline phosphatase, immunoglobulin M (IgM) and IgG, as well as antimitochondrial antibodies titers did not allow in properly predicting patient's outcome. The MRS proved clinical applicability. Patients with an R-value <6 did not develop liver-related complications. The Aspartate Aminotransferase to Platelet Ratio Index score had a significant correlation with the histologic degree of liver fibrosis, with limited value of scores between 1.0 and 1.5. Patients with a Model for End-stage Liver Disease score ≥8 (n=17) had a significantly higher risk to undergo liver transplantation or liver-related death. Outcome was less favorable than predicted by the European model. All scores showed low positive predictive values, limiting their applicability in clinical practice. CONCLUSIONS: Herein, we demonstrate that clinical risk scores in PBC should be interpreted with care. The MRS proved to be helpful to predict a favorable outcome. Novel approaches to predict outcome are needed to identify patients who may benefit from alternative, intensified treatment regimens.

Analysis of SHOX2 methylation as an aid to cytology in lung cancer diagnosis.

BACKGROUND/AIM: The Epi proLung® BL Reflex Assay [short stature homeobox gene two methylation assay (SHOX2 assay)] (Epigenomics AG, Berlin, Germany) utilizes quantitative methylation-sensitive real-time polymerase chain reaction (QMSP) for the quantification of methylated short stature homeobox gene two (SHOX2) DNA. In the present study, the diagnostic utility of the SHOX2 assay was tested with regard to cytology for different cytological diagnostic categories to assess whether it can complement the cytological examination and the DNA methylation marker panel targeting the gene promoters of adenomatous polyposis coli 1A (APC), cyclin-dependent kinase inhibitor-2A (p16(INK4A)) and Ras association domain family protein 1 (RASSF1A) regarding lung cancer detection in bronchial aspirates. MATERIALS AND METHODS: Prospectively collected DNA from 169 patients (cytological diagnosis: 47 tumor-positive, 56 equivocal and 66 tumor-negative) was analyzed for SHOX2 DNA methylation utilizing QMSP. Patients were followed-up for a period of 11 months maximum. RESULTS: When equivocal diagnoses were categorized as tumor-positive, cytology and SHOX2 DNA methylation achieved 72% and 64% sensitivity and 63% and 98% specificity, respectively. SHOX2 DNA methylation identified 66% of the patients with cancer subsequent to a cytological equivocal diagnosis. SHOX2 complements the cytological diagnosis and the methylation marker panel. CONCLUSION: The assay could be of use for the improvement of diagnostic accuracy if applied subsequent to equivocal or negative cytology (sensitivity=69%, specificity=98%). Furthermore, the SHOX2 assay can complement a methylation-based marker panel.

Rectal cancer: dynamic contrast-enhanced MRI correlates with lymph node status and epidermal growth factor receptor expression.

PURPOSE: To evaluate correlations between dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and clinicopathologic data as well as immunostaining of the markers of angiogenesis epidermal growth factor receptor (EGFR) and CXC-motif chemokine receptor 4 (CXCR4) in patients with rectal cancer. MATERIALS AND METHODS: Presurgical DCE-MRI was performed in 41 patients according to a standardized protocol. Two quantitative parameters (k21 , A) were derived from a pharmacokinetic two-compartment model, and one semiquantitative parameter (TTP) was assessed. Standardized surgery and histopathologic examinations were performed in all patients. Immunostaining for EGFR and CXCR4 was performed and evaluated with a standardized scoring system. RESULTS: DCE-MRI parameter A correlated significantly with the N category (P = 0.048) and k21 with the occurrence of synchronous and metachronous distant metastases (P = 0.029). A trend was shown toward a correlation between k21 and EGFR expression (P = 0.107). A significant correlation was found between DCE-MRI parameter TTP and the expression of EGFR (P = 0.044). DCE-MRI data did not correlate with CXCR4 expression. CONCLUSION: DCE-MRI is a noninvasive method which can characterize microcirculation in rectal cancer and correlates with EGFR expression. Given the relationship between the dynamic parameters and the clinicopathologic data, DCE-MRI data may constitute a prognostic indicator for lymph node and distant metastases in patients with rectal cancer.

SHOX2 DNA methylation is a tumour marker in pleural effusions.

BACKGROUND: The published sensitivity of cytological examination for malignant pleural effusions (MPE) ranges between 50% and 71%. The Epi proLung® BL Reflex Assay (Epigenomics AG, Berlin, Germany) has been reported as being highly sensitive and specific for lung cancer using bronchial aspirates. We hypothesize the assay to be of use in the detection of MPE. MATERIALS AND METHODS: To test our hypothesis, we performed a retrospective cohort study on pleural effusion specimens of 1,270 patients (472 cases and 798 controls). The assay is based on quantification of methylated Short Stature Homeobox gene two (SHOX2) DNA in the specimen measured via multiplex real-time Polymerase Chain Reaction (PCR) on bisulfite-converted DNA. RESULTS: Surprisingly, the assay detects metastases of lung cancer, as well as metastases of other malignant tumours. With a re-defined cut-off criterion, the test achieved a sensitivity of 39.5% with a specificity of 96.2%. CONCLUSION: This assay is able to detect MPE while not limited to the detection of lung cancer.

Deficiency of the promyelocytic leukemia protein fosters hepatitis C-associated hepatocarcinogenesis in mice.

Overwhelming lines of epidemiological evidence have indicated that persistent infection with hepatitis C virus (HCV) is a major risk for the development of hepatocellular carcinoma (HCC). We have recently shown that HCV core protein mediates functional inactivation of the promyelocytic leukemia (PML) tumor suppressor pathway. However, the role of PML in HCC development yet remains unclear. To clarify the function of PML in liver carcinogenesis and HCV-associated pathogenesis we crossed PML-deficient mice with HCV transgene (HCV-Tg) expressing mice and treated the resulting animals with DEN/Phenobarbital, an established protocol for liver carcinogenesis. Seven months after treatment, livers were examined macroscopically and histologically. Genetic depletion of the tumor suppressor PML coincided with an increase in hepatocyte proliferation, resulting in development of multiple dysplastic nodules in 100% of the PML-deficient livers and of HCCs in 53%, establishing a tumor suppressive function of PML in the liver. In animals expressing the HCV-transgene in PML-deficient background, HCC development occurred even in 73%, while only 7% of their wildtype littermates developed HCC. The neoplastic nature of the tumors was confirmed by histology and expression of the HCC marker glutamine synthetase. Several pro- and antiapoptotic factors were tested for differential expression and liver carcinogenesis was associated with impaired expression of the proapoptotic molecule TRAIL in PML-deficient mice. In conclusion, this study provides first in vivo evidence that the tumor suppressor PML acts as an important barrier in liver carcinogenesis and HCV-dependent liver pathology.

Frozen section diagnosis in donor liver biopsies: observer variation of semiquantitative and quantitative steatosis assessment.

Donor livers are not generally accepted for liver transplantation if intraoperative frozen section histology on wedge biopsies provides evidence for more severe steatosis. In this reliability study, assessment of steatosis in donor liver biopsies by different approaches (frozen sections vs. paraffin sections; macrovesicular steatosis vs. microvesicular steatosis), different observers, and different evaluation methods (conventional microscopy vs. point grid analysis on digital microphotographs) was compared. One hundred twenty consecutive donor liver biopsies were investigated. Intraoperative diagnosis was made on hematoxylin and eosin (H&E)-stained frozen sections. The residual portion of each biopsy was analyzed later on H&E-, diastase-resistant PAS-, and Elastica van Gieson-stained paraffin sections. Microvesicular steatosis and macrovesicular steatosis were classified semiquantitatively into 5 % steps. Additionally, point grid counting was applied on ten digital microphotographs per slide. The values for steatosis revealed a wide range of data between 0 and 70 or 85 % (mean values, 12.0-18.3 %), considering all types of specimens. The results of the two observers were highly correlated for macrovesicular steatosis (r ≥ 0.925) and for microvesicular steatosis (r ≥ 0.880). The values for macrovesicular and microvesicular steatosis, however, showed poor correlation (r ≤ 0.581). The rate of agreement between the two observers ranged between 84.2 and 95.8 % (κ, 0.763-0.937), depending on the threshold setting. For point grid analysis, significantly lower mean values and ranges for both types of steatosis compared to conventional histopathology were found (p < 0.001). Comparing the results of point grid analysis with those of conventional histopathology, a relatively loose correlation was found (r, 0.581-0.779). Intraoperative histology remains a reliable and highly relevant method for the assessment of steatosis in liver donor grafts. It represents one important component in the decision-finding whether or not a donor liver should be accepted and should possibly be combined with results of preoperative computed tomography imaging. Considering our data, macrovesicular and microvesicular steatosis should be analyzed separately due to the limited correlation between them.

Preservation of HUGL-1 expression as a favourable prognostic factor in pancreatic carcinoma.

AIM: The expression of the human homologue of Drosophila tumour suppressor gene lgl (HUGL-1) in pancreatic cancer was retrospectively assessed in 97 patients with surgically treated pancreatic cancer in order to correlate the HUGL-1 profile with patients' survival. MATERIALS AND METHODS: Immunohistochemistry was performed on 4-µm-thick paraffin sections from representative tumour blocks using a standard protocol. The expression of HUGL-1 was evaluated semiquantitatively as negative (0), weak (1), medium (2) or strong (3). The results were correlated with clinicopathological parameters and with patients' survival, considering an observation period of 17 (mean) ± 16 (SD) months. RESULTS: In normal and inflammatory tissue, a uniform and relatively strong staining was observed in ductal epithelium, ganglion cells and some acinar epithelia. The endocrine islets exhibited a weak positivity. Human pancreatic cancer revealed variable intensities of HUGL-1 expression. A total of 69 tumour specimens were classified as negative and 28 as positive. The HUGL-1 expression was not correlated with clinical variables (age, gender), staging or tumour grading. HUGL-1 positivity proved to be prognostically favourable (p=0.0241) conferring a higher survival rate, especially for patients who had survived more than 12 months. The presence of distant metastases (M1) at diagnosis had a weak significant influence on survival (p=0.0474). The other staging parameters (T, N, UICC stage), tumour grading and clinical variables (age, gender) gave no significant prognostic information. In a multivariate Cox model, only HUGL-1 expression passed the entry limits. CONCLUSION: Preservation of HUGL-1 expression in pancreatic adenocarcinoma is a good prognostic factor that contributes to a better overall survival.

The functional cancer map: a systems-level synopsis of genetic deregulation in cancer.

BACKGROUND: Cancer cells are characterized by massive dysegulation of physiological cell functions with considerable disruption of transcriptional regulation. Genome-wide transcriptome profiling can be utilized for early detection and molecular classification of cancers. Accurate discrimination of functionally different tumor types may help to guide selection of targeted therapy in translational research. Concise grouping of tumor types in cancer maps according to their molecular profile may further be helpful for the development of new therapeutic modalities or open new avenues for already established therapies. METHODS: Complete available human tumor data of the Stanford Microarray Database was downloaded and filtered for relevance, adequacy and reliability. A total of 649 tumor samples from more than 1400 experiments and 58 different tissues were analyzed. Next, a method to score deregulation of KEGG pathway maps in different tumor entities was established, which was then used to convert hundreds of gene expression profiles into corresponding tumor-specific pathway activity profiles. Based on the latter, we defined a measure for functional similarity between tumor entities, which yielded to phylogeny of tumors. RESULTS: We provide a comprehensive, easy-to-interpret functional cancer map that characterizes tumor types with respect to their biological and functional behavior. Consistently, multiple pathways commonly associated with tumor progression were revealed as common features in the majority of the tumors. However, several pathways previously not linked to carcinogenesis were identified in multiple cancers suggesting an essential role of these pathways in cancer biology. Among these pathways were 'ECM-receptor interaction', 'Complement and Coagulation cascades', and 'PPAR signaling pathway'. CONCLUSION: The functional cancer map provides a systematic view on molecular similarities across different cancers by comparing tumors on the level of pathway activity. This work resulted in identification of novel superimposed functional pathways potentially linked to cancer biology. Therefore, our work may serve as a starting point for rationalizing combination of tumor therapeutics as well as for expanding the application of well-established targeted tumor therapies.

The recipient CYP2D6 allele 4-associated poor metabolizer status correlates with an early fibrosis development after liver transplantation.

CYP2D6 is part of the cytochrome P450 system, which catalyzes biotransformation of endogenous substrates and xenobiotics. Approximately 10% of the Caucasian population has two null alleles, resulting in a poor metabolizer (PM) status. Mostly, allele four (CYP2D6*4) is responsible for the PM status, which is suspected to be associated with an accelerated fibrosis progression (FP). The aim of the present study was to analyze the role of the CYP2D6*4 genotype for FP after liver transplantation (LT). Genotypes were determined in liver biopsies (donor) and peripheral blood (recipient) by fluorescence resonance energy transfer. Data were correlated with clinical variables and risk factors for fibrosis. We analyzed 517 LTs performed between 1997 and 2009. Overall donor and recipient allele frequencies were comparable (18.0%, 20.5%; P = 0.43). The donor genotype did not correlate with FP. In contrast, recipients carrying CYP2D6*4, showed a significant higher risk for an accelerated FP (P = 0.011) in HCV positive (P = 0.038) and HCV negative patients (P = 0.033). Results were confirmed by multivariate analysis (Hazard ratio 1.65; P = 0.001). The CYP2D6*4-associated PM status of the donor liver seems to have no influence on FP after LT. Recipients, carrying the allele, have an elevated risk for an accelerated FP.

Alterations in lipid, carbohydrate and iron metabolism in patients with non-alcoholic steatohepatitis (NASH) and metabolic syndrome.

BACKGROUND: NASH (non-alcoholic steatohepatitis) is considered the hepatic manifestation of the metabolic syndrome (MS). We aimed to analyze lipid, carbohydrate, and iron metabolism in NASH. PATIENTS, METHODS: 37 patients with MS (17 M/20 F, 51+/-15 years), elevated transaminases; 25 patients had histologically proven NASH (NAS score≥5), 12 patients had toxic background (nonNASH). 37 age, sex, BMI-matched healthy controls. Lipid variables, LDL-subfractions, iron, ferritin, transferrin (T), transferrin saturation (TS), and hepcidin (H) were measured in patients/controls. Oral glucose tolerance tests were performed. RESULTS: NASH patients with steatosis gr. 2 and 3 (>33% hepatic fat) had higher sd-LDL (mg/dl) concentrations than patients with steatosis gr. 1 (<33%) (p=0.002), nonNASH patients (p=0.03) and controls (p=0.001). Sd absolute (mg/dl) correlated directly with the steatosis grade only in patients with NASH and steatosis >33% (p=0.04). NASH-patients showed higher insulin, C-peptide and IRI values than nonNASH patients (p=0.034; 0.032; 0.04). H was increased in patients versus controls (p<0.001). H correlated with ferritin in MS-patients (p=0.01), correlated directly with sd-LDL (mg/dl) (p=0.017) and IRI (p<0.001) and indirectly with HDL (p=0.05) in NASH. No associations between hepatic inflammation/iron content on liver biopsy and variables of lipid metabolism were found but hepcidin correlated with hepatic inflammation in all patients and with NAS scores in NASH. CONCLUSIONS: NASH-patients show insulin resistance and increased sd-LDL subfractions, suggesting an atherogenic profile. The correlation of H with sd-LDL and IRI, without relation to hepatic iron content suggests a putative link between inflammation, carbohydrate and lipid metabolism in NASH.

Equivocal cytology in lung cancer diagnosis: improvement of diagnostic accuracy using adjuvant multicolor FISH, DNA-image cytometry, and quantitative promoter hypermethylation analysis.

BACKGROUND: Sometimes, cytological lung cancer diagnosis is challenging because equivocal diagnoses are common. To enhance diagnostic accuracy, fluorescent in situ hybridization (FISH), DNA-image cytometry, and quantitative promoter hypermethylation analysis have been proposed as adjuncts. METHODS: Bronchial washings and/or brushings or transbronchial fine-needle aspiration biopsies were prospectively collected from patients who were clinically suspected of having lung carcinoma. After routine cytological diagnosis, 70 consecutive specimens, each cytologically diagnosed as negative, equivocal, or positive for cancer cells, were investigated with adjuvant methods. Suspicious areas on the smears were restained with the LAVysion multicolor FISH probe set (Abbott Molecular, Des Plaines, Illinois) or according to the Feulgen Staining Method for DNA-image cytometry analysis. DNA was extracted from residual liquid material, and frequencies of aberrant methylation of APC, p16(INK4A) , and RASSF1A gene promoters were determined with quantitative methylation-specific polymerase chain reaction (QMSP) after bisulfite conversion. Clinical and histological follow-up according to a reference standard, defined in advance, were available for 198 of 210 patients. RESULTS: In the whole cohort, cytology, FISH, DNA-image cytometry, and QMSP achieved sensitivities of 83.7%, 78%, 79%, and 49.6%, respectively (specificities of 69.8%, 98.2%, 98.2%, and 98.4%, respectively). Subsequent to cytologically equivocal diagnoses, FISH, DNA-image cytometry, and QMSP definitely identified malignancy in 79%, 83%, and 49%, respectively. With QMSP, 4 of 22 cancer patients with cytologically negative diagnoses were correctly identified. CONCLUSIONS: Thus, adjuvant FISH or DNA-image cytometry in cytologically equivocal diagnoses improves diagnostic accuracy at comparable rates. Adjuvant QMSP in cytologically negative cases with persistent suspicion of lung cancer would enhance sensitivity.

Feulgen staining remains the gold standard for precise DNA image cytometry.

AIM: The suitability of Papanicolaou staining and of hematoxylin staining for DNA single-cell cytometry was investigated in comparison to Feulgen staining. MATERIALS AND METHODS: Ten normal cervical smears and ten cervical smears containing cells of a squamous cell carcinoma in situ were analyzed. The integrated optical density (IOD) of 200 epithelial cells, chosen per random, was determined using a CM-1 TV-image analysis system (Hund, Wetzlar, Germany). Various DNA cytometric variables, accepted by the European Society for Analytical and Cellular Pathology (ESACP), and the mean nuclear area were calculated. Two measurements were performed after Papanicolaou staining (wavelengths: 530 nm and 590 nm), followed by measurements after hematoxylin re-staining (wavelength: 590 nm) and after Feulgen restaining (wavelength: 570 nm). RESULTS: All histograms of Feulgen-stained normal squamous epithelia revealed a regular DNA distribution. The corresponding histograms after Papanicolaou staining or hematoxylin staining showed a wide scatter of values instead of a clear-cut diploid peak and an increased number of values >4c. Similar findings were observed in the carcinomatous smears. In particular, the mean values of the dispersion parameters (2cDI, entropy, ploidy imbalance and 2,5cEE) were significantly increased as compared to Feulgen staining. CONCLUSION: Diagnostic or prognostic conclusions cannot be drawn from DNA measurements on Papanicolaou-stained or hematoxylin-stained specimens; Feulgen staining remains the gold standard for such purposes.

Diagnostic efficiency of differentiating small cancerous and precancerous lesions using mucosal brush smears of the oral cavity--a prospective and blinded study.

The aim of this study was to evaluate the diagnostic accuracy of oral brush biopsy to identify early malignancy. One hundred and eighty-six brush biopsies of suspicious mucosal lesions were obtained, haematoxilin and eosin (H&E)-stained and compared with the histology of conventional excision biopsies of the same site performed concomitantly. The sensitivity for identifying squamous cell carcinoma (SCC) was 88.5%. High-risk lesions including squamous intraepithelial neoplasia (SIN II, SIN III) and SCC were identified with a sensitivity of 86.4%, using a pap-analogous classification, which is considered to be carcinomatous, as well as moderate and severe dysplastic cells positive. Depending on the cytopathologic definition for malignancy and the tumour size, the test accuracy varied: Extending the cytopathologic criteria for malignancy by defining all dysplastic or malignant cytopathologic findings as positive, the sensitivity was increased to 95.2% at the expense of the specificity, which was reduced from 94.9% to 82.3%. Separately analysing SCCs of less than 20 mm, the sensitivity was reduced by 9.5% to 78%. Although small malignant lesions seem to be less reliable by the conventional oral brush biopsy, it is a useful screening instrument for early diagnosis of suspicious, epithelial lesions and could therefore contribute to improved cancer prognosis.

Confocal laser endomicroscopy for the diagnosis of urothelial bladder neoplasia: a technology of the future?

OBJECTIVE: To evaluate bladder urothelium by confocal laser endomicroscopy (CLE) and correlate microscopic findings with standard histopathology. PATIENTS AND METHODS: Fresh bladder urothelium tissue specimens were topically stained with acriflavine for instantaneous microscopic imaging. A single-line laser in a handheld CLE probe delivered an excitation wavelength of 488 nm providing a high resolution of 0.7 µm and an adjustable imaging depth of 0-250 µm. Resection specimens of 18 patients were investigated with 1000-fold magnification and ex vivo findings were compared with targeted histopathology (haematoxylin and eosin staining). RESULTS: Typical tumour growth patterns such as altered nuclear to cytoplasmic ratio, pseudopapillar tissue stratification and neoangiogenesis were readily visible. Nuclear and subnuclear architecture of healthy bladder tissue could be discriminated against neoplastic tissue. CONCLUSIONS: In addition to white light cystoscopy, CLE is a promising novel tool for the in vivo microscopic visualization of bladder cancer; first results of the present study show its potential to define microscopic characteristics of bladder cancer tissue. Further in vivo studies are necessary to determine sensitivity and specificity of the technique.

Effectiveness of autofluorescence to identify suspicious oral lesions--a prospective, blinded clinical trial.

Regular screening through white light inspection of the entire oral mucosa is the most important examination method to identify precancerous lesions and early oral carcinoma. Additionally, the physiologic autofluorescence of the oral mucosa has been described as a novel screening method for the detection of mucosal lesions that are not visible by white light. This study aimed to evaluate the sensitivity and specificity of the autofluorescence examination. Seventy-eight patients were examined in this study. All of them suffered from suspicious oral mucosal lesions. Two different investigation methods were applied: the standard examination by white light and an examination by a novel light source of 400 nm that evoked a green light emission (>500 nm) in normal mucosa. It was proposed that malignant oral mucosal lesions show different autofluorescence characteristics than the green autofluorescence of healthy mucosa. Red autofluorescence indicated SCC with a sensitivity of 20% and a specificity of 98%. The results showed that dysplasia and carcinoma could be identified with a sensitivity of 96% and a specificity of 18% by using the autofluorescence method. The sensitivity decreased according to the grade of mucosal keratosis and was influenced by the localisation of the lesion. In conclusion, benign as well as malignant oral lesions could not be distinguished by a diminished autofluorescence signal. A red autofluorescence signal, however, could indicate cancerous processes of the oral mucosa.

Liver-specific Ldb1 deletion results in enhanced liver cancer development.

BACKGROUND & AIMS: LIM-domain-binding (Ldb) proteins have been demonstrated to be essential not only to key embryonic developmental processes but also to carcinogenesis. We have previously demonstrated Ldb1 to be of high biological and developmental relevance, as a targeted deletion of the Ldb1 gene in mice results in an embryonic lethal and pleiotropic phenotype. METHODS: We have now established a liver-specific Ldb1 knock out to investigate the role of Ldb1 in carcinogenesis, in particular in hepatocellular carcinoma (HCC) development, in vivo. RESULTS: These mice demonstrated a significantly enhanced growth of liver cancer by means of tumor size and number, advocating for an essential role of Ldb1 in HCC development. In addition, proliferation and resistance against apoptosis were increased. In order to identify the functional disturbances due to a lack of Ldb1, we performed a 15k mouse gene microarray expression analysis. We found the Myc oncogene to be regulated in the microarray analysis and were able to further confirm this regulation by demonstrating an over-expression of its downstream target Cyclin D1. Furthermore, we were able to demonstrate a down-regulation of the tumor suppressor p21. Finally, the liver stem cell marker EpCAM was also identified to be over expressed in Ldb1(-/-) knock out mice. CONCLUSIONS: We have established a significant role of Ldb1 in cancer development. Furthermore, we provided evidence for a myc/cyclin D1, p21, and EpCAM-dependent signalling to be key downstream regulators of this novel concept in HCC development.

Liver transplanted patients with preoperative autoimmune hepatitis and immunological disorders are at increased risk for Post-Transplant Lymphoproliferative Disease (PTLD).

BACKGROUND: Long term immunosuppression and therapy of acute rejections result in a 20-120-fold increased risk to develop Non Hodgkin lymphoma (NHL). Since immunosuppressive therapy and immunological disorders are major risk factors for the development of NHL in the non-transplant population we aimed to analyze risk factors for PTLD in our cohort of liver transplanted (LT) patients. METHODS: We analyzed retrospectively 431 patients liver transplanted between 1998 and 2008. RESULTS: PTLD was diagnosed in eleven of 431 patients (2.6%). PTLD, especially late PTLD, was significantly more frequent in patients who received steroids before LT (Kaplan-Meier: p<0.001). Moreover PTLD in immunocompromised patients with preoperative steroid treatment occurred at a significantly younger age (49.5+/-4.7 years) compared to patients without steroids (60.6+/-5.1 years; p=0.006). Multivariate analysis revealed pretransplant steroid treatment and liver transplantation for autoimmune hepatitis as main risk factors for the development of PTLD after liver transplantation (p<0.001). CONCLUSION: Liver transplanted patients who received steroids before LT due to immunological disorders and patients with autoimmune hepatitis seem to be at particular high risk to develop PTLD. Prospective cohort studies including immunoepidemiologic investigations of abnormalities of cellular, humoral and innate immunity should be carried out to identify predictive factors and patients at risk.