["Digital Pathology in Diagnostics" guideline : Reporting on digital images]. / Leitfaden "Digitale Pathologie in der Diagnostik" : Befunderstellung an digitalen Bildern.

BACKGROUND: The digitization of medicine is gaining momentum in pathology. Long-known technologies have reached such a degree of maturity that their use in primary diagnostics in routine pathology will be possible. In spite of the complexity of technological solutions and the far-reaching consequences in terms of diagnostic reliability, as well as due to the high investments, the decision for a specific product may become highly sophisticated for a pathologist. AIM: An implementation guide for Digital Diagnostics in Pathology is presented to describe technical and legal conditions for making this new technology feasible for the single pathologist. RESULTS AND DISCUSSION: For more than two years, the Digital Pathology Commission of the Federal Association of German Pathologists developed and discussed this implementation guide for digital diagnostics, especially for the use of virtual microscopy in the daily pathology routine in Germany. The key is the principal comparability of diagnostic reliability between conventional stained microscopic slides and their digital images, which have to be shown by the potential user. In eight chapters, the validation procedure as well as technical minimum requirements in slide scanners, the visualization pipeline, archiving, and integration in the pathology workflow are described.

[Implementation of the "Digital Pathology in Diagnostics" guideline : Support systems and their functionality]. / Umsetzung des Leitfadens "Digitale Pathologie in der Diagnostik" : Unterstützende Systeme und ihre Funktionalität.

BACKGROUND: The "Digital Pathology in Diagnostics - Assessment of Digital Images" guideline describes the technical and legal framework under which the use of this digital technology is justifiable for the individual pathologist. The focus is on conducting a validation study, defining minimum requirements for the generation and management of whole slide images, and ensuring the functionality and quality of the virtual microscopy solution used. By establishing a special web-based service, supportive services can be provided to assist the pathologist in the introduction of virtual microscopy and quality assurance. AIM: Presentation of the Digitale-Pathologie.de server and description of its services in the context of the guideline. RESULTS AND DISCUSSION: In the context of several scanner contests at the Charité in Berlin, as well as with the introduction of virtual microscopy in practice, many experiences were collected, which will be presented systematically. Essentially, this will provide support for the application of the guideline in practice. The following fields are discussed: implementation of the guideline recommendations, planning and evaluation of the validation study, and ensuring the correct color calibration of the slide scanner being used. Via the Digitale-Pathologie.de server, the possibility for self-monitoring and anonymously benchmarking will be offered. For example, errors in setup can be detected quickly and optimal settings can be identified.

Cancer beyond organ and tissue specificity: next-generation-sequencing gene mutation data reveal complex genetic similarities across major cancers.

Cancer medicine relies on the paradigm that cancer is an organ- and tissue-specific disease, which is the basis for classifying tumors. With the extensive genomic information now available on tumors it is possible to conduct analyses to reveal common genetic features across cancer types and to explore whether the established anatomy-based tumor classification is actually reflected on the genetic level, which might provide important guides to new therapeutic directions. Here, we have conducted an extensive analysis of the genetic similarity of tumors from 14 major cancer entities using somatic mutation data from 4,796 cases available through The Cancer Genome Atlas (TCGA) based on all available genes as well as different cancer-related gene sets. Our analysis provides a systematic account of the genetic similarity network for major cancer types and shows that in about 43% of the cases on average, tumors of a particular anatomic site are genetically more similar to tumors from different organs and tissues (trans-similarity) than to tumors of the same origin (self-similarity). The observed similarities exist not only for carcinomas from different sites but are also present among neoplasms from different tissue origin, such as melanoma, acute myeloid leukemia, and glioblastoma. The current WHO cancer classification is therefore reflected on the genetic level by only about 57% of the tumors. These results provide a rationale to reconsider organ- and tissue-specificity in cancer and contribute to the discussion about whether personalized therapies targeting specific genetic alterations may be transferred to cancers from other anatomic sites with similar genetic properties.

Impact of filaggrin mutations on Raman spectra and biophysical properties of the stratum corneum in mild to moderate atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is associated with null mutations in the filaggrin (FLG) gene. OBJECTIVE: To assess the impact of FLG null mutations on biophysical properties and the molecular composition of the stratum corneum (SC) in healthy individuals and AD patients. METHODS: A total of 196 French adults, including 97 with a history of mild to moderate AD, were genotyped for the three major European FLG mutations. Components of the natural moisturizing factor (NMF), lipids and water content in the SC were determined using Raman spectroscopy. In addition, trans-epidermal water loss, capacitance and pH of the SC were measured. RESULTS: Stratum corneum concentrations of total NMF, water, ornithine and urocanic acid (UCA) were significantly lower in AD patients than in healthy controls. Null mutations of FLG were detected in 4% of controls and 10% of AD patients. FLG mutations were associated with increased SC levels of lactate, reduced concentrations of most other NMF components and higher disease severity in AD patients. In AD patients without FLG mutations, the content of NMF constituents decreased with increasing disease severity. The concomittant presence of low concentrations of histidine, alanine and either glycine or pyrrolidone-5-carboxylic acid (PCA) in the SC was associated with FLG mutations with 92% specificity. CONCLUSIONS: Our findings suggest a low prevalence of FLG mutations in mild AD and support an important role for filaggrin in determining the physicochemical parameters of the SC. The combined measurement of several filaggrin breakdown products in the SC may be useful to specifically predict the presence of FLG mutations.

Advanced wastewater treatment with ozonation and granular activated carbon filtration: Inactivation of antibiotic resistance targets in a long-term pilot study.

The inactivation of antibiotic resistant bacteria (ARB) and genes (ARGs) in an advanced plant combining ozonation and granular activated carbon (GAC) filtration applied for effluent after conventional activated sludge treatment at a full-scale urban wastewater treatment plant was investigated for over 13 consecutive months. The nitrite compensated specific ozone dose ranged between 0.4 and 0.7 g O3/g DOC with short-time sampling campaigns (0.2-0.9 g O3/g DOC). Samples were analysed with culture-dependent methods for bacterial targets and with qPCR for genes. The log removal values were correlated with a decrease of the matrix UV absorption at 254 nm (ΔUV254) and indicated a range of ΔUV254 that corresponds to a sufficient membrane damage to affect DNA. For trimethoprim/sulfamethoxazole resistant E. coli, sul1, ermB and tetW, this phase was observed at ΔUV254 of ~30 % (~0.5 g O3/g DOC). For ampicillin resistant E. coli and blaTEM-1, it was observed around 35-40 % (~0.7 g O3/g DOC), which can be linked to mechanisms related to oxidative damages in bacteria resistant to bactericidal antibiotics. GAC treatment resulted in a further abatement for trimethoprim/sulfamethoxazole E. coli, sul1 and tetW, and in increase in absolute and relative abundance of ermB and blaTEM-1.

Airway inflammation induced after allergic poly-sensitization can be prevented by mucosal but not by systemic administration of poly-peptides.

BACKGROUND: Patients with multiple sensitizations require alternative forms of treatment, as the efficacy of conventional immunotherapy is unsatisfactory. OBJECTIVE: In the present study, we sought to compare the efficacy of a subcutaneously (s.c.) and a mucosally applied polyvalent vaccine to reduce allergic immune responses within airway and lung tissues. METHODS: Female BALB/c mice were intraperitoneally immunized with recombinant (r)Bet v 1, rPhl p 1 and rPhl p 5, followed by an aerosol challenge of birch and phleum pollen extract. For tolerance induction, either a mixture of the immunodominant peptides or a hybrid peptide of the respective antigens was s.c. injected or intranasally applied before poly-sensitization. RESULTS: Mucosal but not systemic pre-treatment with poly-peptides led to significant suppression of eosinophils and IL-5 production in bronchoalveolar lavages, as well as IL-5, IL-4, IL-13 and eotaxin levels in lung cell cultures. Lung histology showed a clear reduction of cellular infiltration and mucus production only in intranasally pre-treated mice. In accordance, also the systemic immune response, characterized by IgE-dependent basophil degranulation and IL-4 levels in vitro, was significantly reduced by mucosal antigen application, but only marginally influenced by subcutaneous pre-treatment. Both treatment routes led to up-regulated CTLA4 expression in splenocytes, whereas only after mucosal pre-treatment Foxp3 expression levels were enhanced in lung CD3(+) T cells. Furthermore, intranasal but not subcutaneous application of the peptides enhanced IL-10 levels in the lungs, indicating regulatory mechanisms operating in local tolerance induction. CONCLUSION: Mucosal application of peptides is superior to systemic application in preventing both local and systemic poly-allergic T helper2 immune responses, suggesting mucosal tolerance induction as an attractive strategy for the primary and secondary prevention of allergic multi-sensitization and lung pathology.

Evaluation of a novel internally controlled real-time PCR assay targeting the 16S rRNA gene for confirmation of Neisseria gonorrhoeae infections.

A novel HybProbe real-time LightCycler PCR assay was developed for confirmation of Neisseria gonorrhoeae in samples positive according to the COBAS AMPLICOR Chlamydia trachomatis/Neisseria gonorrhoeae PCR assay. The new assay amplifies 375 bp of the N. gonorrhoeae 16S rRNA gene and includes an internal amplification control introduced during DNA purification. The assay had 100% specificity because of the high specificity of the HybProbes and primers. Other Neisseria spp. failed to generate positive crossing-point values and melting peaks. The analytical sensitivity for N. gonorrhoeae DNA was 0.5 fg/PCR, corresponding to 0.3 CFU/PCR. Sensitivity was not impaired in the presence of higher DNA concentrations (>or=1000-fold) from Neisseria spp. other than N. gonorrhoeae. The sensitivity was similar to that reported for the COBAS AMPLICOR assay with cervical swab samples. To assess its clinical applicability as a confirmatory test, 38 (2.9%) of 1313 swabs that were positive according to the COBAS AMPLICOR assay were tested using the new in-house assay and the commercially available GenFlow Neisseria test. Twenty-one samples negative according to COBAS AMPLICOR also underwent confirmatory testing. Both confirmatory tests yielded identical results; the 21 negative samples remained negative, and only 11 (28.9%) of the samples positive according to COBAS AMPLICOR were positive after retesting, suggesting a low prevalence (0.84%) of N. gonorrhoeae infection in the study population. These data suggest that the novel real-time PCR assay is an excellent and easy to interpret confirmatory test for the existing COBAS AMPLICOR assay for N. gonorrhoeae.

[Virtual microscopy and routine diagnostics. A discussion paper]. / Virtuelle Mikroskopie und Routinediagnostik. Ein Diskussionspapier.

There are several areas of application for virtual microscopy in pathology. After broad use in education and research, we are now seeing its initial application in health care. We can predict that, on the basis of experience gained in digital radiology and early experience in pathology, VM will be established as routine. However, its introduction will follow a different course to that taken in digital radiology, which required that the computer be accepted as a "necessary evil" to record and display computer tomograms. Virtual microscopy needs to ensure that it supports the pathologist significantly in terms of access to archives, quantification of markers, display of biopsy stacks, cooperation with colleagues, etc. Consequently, the question of "When will virtual microscopy enter daily pathology practice?" may not only be answered on the basis of technical features. Of course, it is necessary that scanning speed goes below 1 min/cm(2) and that it remains financially viable, but more important is an optimal integration of virtual microscopy in daily pathology routine. This process will extend over several decades, as past developments in digital radiology have shown.

Molecular cloning and sequence analysis of the mouse protein C inhibitor gene.

The gene encoding mouse protein C inhibitor (mPCI) was isolated and its nucleotide sequence determined. Alignment of the genomic sequence with that of a cDNA obtained from mouse testis revealed that the mPCI gene (like the human counterpart) is composed of five exons and four introns with highly conserved exon/intron boundaries. It encodes a pre-polypeptide of 405 amino acids, which shows 63% identity with human PCI (hPCI). The putative reactive site is identical to that of hPCI from P5 to P3', suggesting a similar protease specificity. Also the putative heparin binding sites and 'hinge' regions are highly homologous in mouse and hPCI.

Thrombin stimulates expression of tissue-type plasminogen activator and plasminogen activator inhibitor type 1 in cultured human vascular smooth muscle cells.

The effect of thrombin on the fibrinolytic potential of human vascular smooth muscle cells (SMC) in culture was studied. SMC of different origin responded to thrombin treatment with a dose and time dependent increase in tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type-1 (PAI-1) levels in both cell lysates and conditioned media with maximum effects achieved at 10-20 IU/ml thrombin. PAI-1 antigen levels also increased in the extracellular matrix of thrombin treated SMC. PAI-2 levels in cell lysates of such SMC were not affected by thrombin. The effect was restricted to active thrombin, since DFP-thrombin and thrombin treated with hirudin showed no increasing effect on t-PA and PAI-1 levels in SMC. Enzymatically active thrombin also caused a four-fold increase in specific PAI-1 mRNA and a three-fold increase in t-PA mRNA. Furthermore we demonstrated the presence of high and low affinity binding sites for thrombin on the surface of SMC with a KD = 4.3 x 10(-10)M and 9.0 x 10(4) sites per cell and a KD = 0.6 x 10(-8) M and 5.8 x 10(5) sites per cell respectively. Thrombin could come in contact with SMC in case of vascular injury or following gap formation between endothelial cells. Our data support the idea that besides its known proliferative effect for SMC, thrombin could also modulate their fibrinolytic system.

Azelaic acid decreases the fibrinolytic potential of cultured human melanoma cells in vitro.

Azelaic acid (AZA) has been used successfully in the treatment of lentigo maligna melanoma. Since it is generally accepted that the fibrinolytic potential of tumour cells is related to their malignant phenotype, it was the aim of this study to investigate the effect of AZA on the fibrinolytic potential of three different human melanoma cell lines (Bowes, GUBSB and MJZJ). Melanoma cells were incubated with AZA in doses ranging from 10(-2) M to 4 x 10(-2) M for 5, 8 and 24 h. The expression of tissue-type plasminogen activator (t-PA), urokinase-type PA (u-PA) and PA inhibitor-1 (PAI-1) in such treated cells was investigated by specific ELISAs on the protein level and by Northern blotting on the mRNA level. AZA caused a time and dose dependent decrease in the fibrinolytic potential of all three cell lines investigated by decreasing t-PA antigen in Bowes, by decreasing u-PA antigen in GUBSB and by increasing PAI-1 antigen in MJZJ cells, respectively. There was no significant difference between the viability of cells in control cultures and those treated with AZA. The effect of AZA on specific mRNA for t-PA in Bowes cells, u-PA in GUBSB and PAI-1 in MJZJ was consistent with its effect on the secretion of these fibrinolytic proteins by the respective cells. The results show that AZA decreases the fibrinolytic potential of the three human melanoma cell lines in vitro. This decrease may be operative in the mechanism by which AZA has been shown to affect malignant melanoma in vivo.

Improvement of breast cancer prognostication using cell kinetic-based silver-stainable nucleolar organizer region quantification of the MIB-1 positive tumor cell compartment.

Recently, it was stated that the proliferative activity (P) of a cell population could be indirectly calculated by multiplying the MIB-1 immunopositivity and silver-stainable nucleolar organizer region (AgNOR) features extracted exclusively in MIB-1 positive (pos.) nuclei: P=MIB-1 x AgNOR(MIB-1pos.). To study the prognostic significance of this hypothesis, MIB- immunohistochemistry and AgNOR staining were applied on a series of 89 cases of breast cancer with an 8-year follow-up period. The mean MIB-1 immunopositivity (MIB-1mean) was evaluated immunohistometrically on paraffin sections using a TV image analysis system CM-2 (Hund, Wetzlar, Germany). Later, a combined MIB-1/AgNOR staining was applied and evaluated using a TV image analysis system AMBA (IBSB, Berlin, Germany). The AgNOR features of 150 randomly chosen tumor nuclei were investigated, irrespective of their MIB-1 status (AgNOR count, AgNOR area). Later, a second measurement was performed on 100 MIB-1 positive tumor nuclei exclusively (AgNOR countMIB-1pos., AgNOR areaMIB-1pos.). AgNOR count and AgNOR countMIB-1pos. showed a different data distribution [2.7+/-0.7 (mean+/-SD) vs 3.9+/-1.1; r=0.315, P=0.014]. Similar results were obtained for AgNOR area and AgNOR areaMIB-1pos. (5.1+/-2.1 microm2 vs 7.5+/-2.4 microm2; r=0.501, P<0.001). Kaplan-Meier survival curves revealed significant differences for MIB-1mean (P=0.0018) and AgNOR areaMIB-1pos. (P=0.0340). In Cox models, both parameters provided independent prognostic information. Using their combination, the P, three groups of patients with statistically different survival could be separated (P=0.0014). Thus, the combination of MIB-1-immunopositivity and AgNOR measurements in MIB-1 positive nuclei appears to be more useful in breast cancer prognosis than the exclusive application of one of the two methods. By this combined application, probably effects of tumor biology are represented more precisely.

Location of nucleolar organizer regions (AgNORs) in the nuclei of astrocytic tumors.

Seventy-nine astrocytic tumors classified according to the WHO classification and stained with a modified AgNOR staining technique were analyzed with the help of an automatic image analyzing system. With increasing malignancy there is an increase of AgNORs per cell. Furthermore, AgNORs move from a more central to a more peripheral position within the nucleus, thus leading to a significant increase of the AgNOR count at the periphery of the nucleus. An inverse relation was found at the center. With regard to AgNORs, there are little differences between anaplastic astrocytomas and small cell glioblastomas. The most striking differences were seen between differentiated (grade 2) and anaplastic (grade 3) astrocytomas.

Fetal autopsy: a review of recent developments.

OBJECTIVE: To analyze recent changes in fetal autopsy in response to developments in prenatal medicine. METHOD: During the period 1988 through 1997, 783 fetuses (75% induced abortions, 18% spontaneous abortions, and 7% stillbirths, all between the 12th and 40th week of gestation) with prenatally diagnosed congenital malformations and chromosomal aberrations were analyzed. We divided the autopsies into two periods: period A (1988-92, n=370) and period B (1993-97, n=413). All fetuses were analyzed before completion of 20 weeks of gestation. The malformations of the organ systems were presented according to their frequency for fetuses independent of the weeks of gestation. RESULTS: An autopsy was performed prior to the completion of 20 weeks of gestation for 24% of the fetuses in period A and 45% in period B (P<0.0001). The number of diagnosed congenital heart malformations increased from 16% in period A to 23% in period B. The number of congenital heart malformations before completion of 20 weeks of gestation was only 21% in period A as compared to 42% in period B. CONCLUSIONS: In period B, clinical questions were raised in a more concrete form and ultrasound images gave more detailed information than in period A. Access to prenatal findings prior to postmortem examination has helped investigators to develop a specific, clinically oriented, autopsy strategy based on the prenatal findings which makes it possible to diagnose very small malformations and to select sectional planes of special interest. The use of an interdisciplinary database is required for communication.

Computer-assisted image analysis of nucleolar organizer regions (NORs): a pilot study of astrocytomas and glioblastomas.

Computer-assisted image analysis was used to measure nucleolar organizer regions (NORs) of 22 astrocytomas and glioblastomas. Image analysis provides reproducible information about number and size of NORs together with further karyometric data, which can be compared and processed with other patient-related data. Our study exhibits a statistical relationship between number and size of NORs and malignancy of the measured gliomas.

[What is the contribution of yeast genetics to tumor biology and tumor diagnosis?]. / Welchen Beitrag leistet die Hefegenetik zur Tumorbiologie und Tumordiagnostik?

This short review article discusses methods and results of oncogene research in yeast. Current knowledge of the sequence, expression and biological function of ras-homologous genes of the yeast Saccharomyces cerevisiae is presented, as well as the implications of these findings for oncogene research in mammals. We review recent examples of highly conserved eukaryotic genes involved in growth control and mitosis control, including recent work from our own laboratories.

[Electronic communication in medicine]. / Elektronische Kommunikation in der Medizin.

Rapid developments in information and communication technology allow a fast transfer of extensive data. Geographical distances do not longer hinder an intense cooperation in diagnostics. The review shows the variety of possibilities for medical applications. Technique, routine application, experiences and the legal conditions are presented for telepathology as an example.

[Study of efficiancy of teleconsultation: the Telepathology Consultation Service of the Professional Assoziation of German Pathologists for the screening program of breast carcinoma]. / Studie zur Effektivität der Telekonsultation: Der Telepathologie-konsultationsservice des Berufsverbandes Deutscher Pathologen im Rahmen des Mamma-Screening-Programms.

AIMS: In the autumn a German screening program was started for detecting breast cancer in the population of women fifty and above. For the first time in this program, quality assurance rules were established: All statements of the radiologists and pathologists have to be confirmed by a second opinion. This improvement in quality is combined with a delay in time and additional expence. A new Telepathology Consultation Service was developed based on the experiences of the Telepathology Consultation Center of the UICC to speed up the second opinion process. METHODS AND MATERIAL: The complete web-based service is operated under MS Windows 2003 Server, as web server the Internet Information Server, and the SQL-Server (both Microsoft) as the database. The websites, forms and control mechanism have been coded in by ASP scripts and JavaScript. A study to evaluate the effectiveness of telepathological consultation in comparison to conventional consultation has been carried out. Pathologists of the Professional Association of German Pathologists took part as well as requesting pathologists and as consultants for other participants. RESULTS AND CONCLUSION: The quality of telepathological diagnosis was comparable to the conventional diagnosis. Telepathology allows a faster respond of 1 to 2 day (conventional postal delay). The time to prepare a telepathology request is about twice as conventional. This ratio may be inverted by an interface between the Pathology Information System and the Telepathology Server and the use of virtual microscopy. The Telepathology Consultation Service of the Professional Association of German Pathologists is a fast and effective German-language, internet-based service for obtaining a second opinion.

Technique and feasibility of a dual staining method for estrogen receptors and AgNORs.

A new staining method for dual demonstration of Estrogen receptors (ER) and argyrophilc Nucleolus-Organizer Regions (AgNORs) was developed. To rule out possible reciprocal effects, serial slides of 10 invasive ductale breast cancers were stained with either the single staining method or the simultaneous ER/AgNOR-staining method and investigated comparatively. By measuring the slides with the image analysis system AMBA, reciprocal effects could be excluded. It was proven that dual staining of both markers results in a reproducible and specific staining result. We concluded that it is justified to measure AgNORs in immunohistochemically stained cells.