[Digitalization and multiplex IHC as predictive biomarkers for novel immune-therapeutics : New challenges for immunohistochemistry]. / Digitalisierung und Multiplex-IHC als prädiktive Biomarker für neue Immuntherapeutika : Neue Herausforderungen an die Immunhistochemie.

Molecular biology assays, for example next-generation sequencing, whole-exosome sequencing, and RNAseq, are well-established tools in precision oncology. Novel therapeutic concepts like bi- or multivalent antibodies may require different diagnostic approaches. In this context, multiplexed immunohistochemistry (IHC) in combination with digital image analysis offers a wide range of application possibilities.Many therapeutics currently tested in early clinical phases or preclinical development aim at the modulation of the immune response to the tumor. The respective diagnostic procedures have to address questions, e.g. regarding the spatial relationship of target and effector cells or the presence and ratio of certain cell subpopulations. These questions are also increasingly raised by the more classical therapeutics, such as monoclonal antibodies or antibody-drug conjugates.While it is hard to identify the same or adjacent cells in serial sections, multiplexed IHC assays combine oligoparametric analysis and cellular context. Establishment and validation of such assays is more complex than for common single-plex procedures with regard to specificity, accuracy and precision. Digital image analysis algorithms as an emerging tool for standardized evaluation of (multiplexed) IHC assays need to be set up in parallel with the wet lab procedures.This article focusses on the potential new role for multiplexed in situ assays in a yet-to-be-established precision pathology as a discipline of precision oncology.

Metabolic impact of an NADH-producing glucose-6-phosphate dehydrogenase in Escherichia coli.

In Escherichia coli, the oxidative branch of the pentose phosphate pathway (oxPPP) is one of the major sources of NADPH when glucose is the sole carbon nutrient. However, unbalanced NADPH production causes growth impairment as observed in a strain lacking phosphoglucoisomerase (Δpgi). In this work, we studied the metabolic response of this bacterium to the replacement of its glucose-6-phosphate dehydrogenase (G6PDH) by an NADH-producing variant. The homologous enzyme from Leuconostoc mesenteroides was studied by molecular dynamics and site-directed mutagenesis to obtain the NAD-preferring LmG6PDH(R46E,Q47E). Through homologous recombination, the zwf loci (encoding G6PDH) in the chromosomes of WT and Δpgi E. coli strains were replaced by DNA encoding LmG6PDH(R46E,Q47E). Contrary to some predictions performed with flux balance analysis, the replacements caused a substantial effect on the growth rates, increasing 59 % in the Δpgi strain, while falling 44 % in the WT. Quantitative PCR (qPCR) analysis of the zwf locus showed that the expression level of the mutant enzyme was similar to the native enzyme and the expression of genes encoding key enzymes of the central pathways also showed moderate changes among the studied strains. The phenotypic and qPCR data were integrated into in silico modelling, showing an operative G6PDH flux contributing to the NADH pool. Our results indicated that, in vivo, the generation of NADH by G6PDH is beneficial or disadvantageous for growth depending on the operation of the upper Embden-Meyerhof pathway. Interestingly, a genomic database search suggested that in bacteria lacking phosphofructokinase, the G6PDHs tend to have similar preferences for NAD and NADP. The importance of the generation of NADPH in a pathway such as the oxPPP is discussed.

HER2 staining intensity in HER2-positive disease: relationship with FISH amplification and clinical outcome in the HERA trial of adjuvant trastuzumab.

BACKGROUND: Trastuzumab treatment improves survival of HER2-positive primary breast cancer. HER2 staining intensity varies widely in HER2-positive tumours. PATIENTS AND METHODS: We investigated whether differences in immunohistochemical (IHC) staining intensity for HER2 in HER2-positive tumors (IHC 3+ or FISH ratio ≥2.0) was associated with prognosis or benefit from trastuzumab treatment in patients randomized to 1 year or no trastuzumab in the HERceptin Adjuvant (HERA) trial. Median follow-up was 2 years. The nested case-control analysis, included 425 patients (cases) with a disease-free survival (DFS) event and two matched controls (no DFS event) per case. Tissue sections stained for HER2 were assessed for HER2 staining intensity by image analysis. RESULTS: HER2 staining intensity varied widely and correlated with HER2 gene copy number (Spearman, r = 0.498, P < 0.001) or less closely with HER2/CEP17 FISH ratio (r = 0.396, P < 0.001). We found no significant difference in DFS in the observation arm according to staining intensity (odds ratio [OR] change per 10 unit change in intensity: 1.015, 95% confidence interval [CI] 0.930-1.108) and no impact of staining intensity on benefit derived from 1-year trastuzumab (OR: 1.017, 95% CI 0.925-1.120). CONCLUSIONS: Variability in HER2 staining in HER2-positive tumours has no role in clinical management with adjuvant trastuzumab. HERA TRIAL NO: NCT00045032.

Transcriptional profiling of transurethral resection samples provides insight into molecular mechanisms of hormone refractory prostate cancer.

The molecular mechanisms for hormone-resistant prostate cancer progression still remain elusive, mainly due to the limited availability of corresponding tissue. As transurethral resection (TUR) is a common palliative therapy for patients with hormone refractory prostate cancer (HRPC) who have subvesical obstruction, we aimed to demonstrate that TUR samples can be used to identify significantly affected biological pathways during the switch to HRPC using oligonucleotide microarray analysis. Among the most significantly deregulated pathways in HRPC, we observed an induction of oxidative phosphorylation and a repression of cytoskeletal components.

Validation of a flow cytometric acridine orange micronuclei methodology in rats.

Our laboratory has previously reported a flow cytometric acridine orange method for detection of micronucleus (MN) in the rat using cyclophosphamide as a test compound. To replace the manual method of scoring and satisfy Good Laboratory Practice (GLP) requirements, an extensive validation of the flow method was required. Therefore, manual scoring and flow cytometric determination of MN were compared using vincristine, chlorambucil, methotrexate, and doxorubicin compounds known to induce MN formation with various mechanisms of action. 1,2-Dimethylhydrazine (1,2-DH), a compound with negative or equivocal MN findings also was evaluated. The flow method consistently demonstrated dose- and time-dependent responses for MN production at all concentrations of vincristine, methotrexate, clorambucil, and doxorubicin. In contrast, manual scoring of slides failed to detect an increase in MN at the lowest doses of doxorubicin (1mg/kg) at 24 or 48 h, and methotrexate at 48 h, or any dose of methotrexate (50, 100, or 250 mg/kg) at 24h. Additionally, a dose-response for methotrexate at 48 h, and chlorambucil at 24 h were missed using manual scoring. For 1,2-DH, the flow method showed a low level (< 1.4-fold) increase in MN at all doses and times. In contrast, the manual method showed five-seven-fold increases at 24 h, but a < two-fold increase at 48 h in the highest dose only. These data may suggest that the flow method has a greater sensitivity and possibly accuracy than manual scoring. Significant decreases in polychromatic erythrocytes (PCE) were seen using both methods at approximately the same dose for all compounds. However, absolute flow cytometric PCE values were consistently higher than manual. Additional cytotoxicity parameters obtained by the flow method allowed a more complete assessment of cytotoxicity than PCE alone. Furthermore, data reported here combined with improved throughput, shortened data turnaround and reporting times, and possibly better precision due to evaluation of much larger numbers of cells clearly demonstrate the usefulness of flow cytometry method in the routine micronucleus evaluation.

Child maltreatment and the early onset of problem behaviors: can a program of nurse home visitation break the link?

This study investigated the relationship between child maltreatment and the early onset of problem behaviors in the Elmira Nurse Home Visitation Program. Participants were predominantly low-income and unmarried mothers and their first-born children who were randomized either to receive over 2 years of home-visitation services by nurses or to be placed in a comparison group. Data were drawn from a follow-up study that took place when the children were 15 years of age. Results demonstrated that, in the comparison group. child maltreatment was associated with significant increases in the number of early onset problem behaviors reported by the youth. For the youth in the nurse-visited group there was no relationship between maltreatment and early onset problem behaviors. We suggest that this finding was due to the effects of the intervention in reducing the number as well as the developmental timing of the maltreatment incidents. Results suggest that prenatal and infancy home visiting by nurses can moderate the risk of child maltreatment as a predictor of conduct problems and antisocial behavior among children and youth born into at-risk families.

A simplified and reliable HPV testing of archival Papanicolaou-stained cervical smears: application to cervical smears from cancer patients starting with cytologically normal smears.

The efficacy of four methods to recover DNA from Papanicolaou (Pap)-stained archival cervical smears for optimal detection of human papillomavirus (HPV) DNA by GP5+/bioGP6+ polymerase chain reaction (PCR) was investigated. Two of the methods were based on proteinase K treatment and two based on treatment with guanidinium thiocyanate (GTC). The quality of the DNA as measured by PCR assays amplifying different sizes of the beta-globin gene appeared to be superior for the GTC-based assays. Using competitive beta-globin PCR assays, one of the GTC-based, assays, provisionally named High Pure PCR Template Preparation (HPPTP) assay, yielded by far the highest quantity of amplifiable DNA. It allowed the recovery of 2.2 x 10(5) to 3 x 10(5) genome equivalents in smears containing 5 x 10(5) to 20 x 10(5) nucleated cells, indicating a mean efficiency of 26% (range of 15-44%). In contrast, the other methods revealed markedly lower efficiencies varying from 1% to 10%. The use of the HPPTP assay as a reliable processing procedure was validated by demonstrating a complete agreement in HPV detection and 93% agreement in HPV typing between 39 archival Pap-stained and paired fresh-frozen cervical smears. This method was applied to 40 archival smears from ten cervical cancer patients (selected from a group of 200 patients) which had a history of 3-6 smears with the first smear being Pap 1 or 2 taken at least 5 years before cancer was diagnosed. The average time period between the first Pap 1/2 smear that contained the same HPV type as in the corresponding carcinoma and diagnosis of cervical cancer was 12.0 +/- 2.9 years. All subsequent smears were invariably positive for the same HPV type which was also found in the cervical cancer biopsy. In conclusion, the HPPTP assay provides a reliable and efficient means to extract DNA from Pap-stained archival cervical smears for the detection of HPV DNA by PCR and would be the method of choice for future HPV analysis of archival Pap-stained cervical smears.

Use of acridine orange in: flow cytometric evaluation of erythropoietic cytotoxicity.

Cytotoxic insult to bone marrow frequently impairs the proliferating and maturational abilities of erythroid cells. Typically, a ratio of enucleated, immature polychromatic erythrocytes (PCE) to mature normochromatic erythrocytes (NCE) is used to assess cytotoxicity in the micronucleus (MN) assay. The effects of cyclophosphamide (CP) on PCE/NCE ratio in rat bone marrow and spleen were assessed by a newly developed flow cytometric procedure using glutaraldehyde-fixed, acridine orange (AO)-stained cells, and compared to manual scoring of PCE/NCE in Wright stained slides. Comparison of methods showed that manual and flow cytometric determination of PCE were not statistically different. Several other parameters of cytotoxicity could be simultaneously assessed because the method allowed use of unfractionated whole bone marrow/spleen cell samples. Absolute numbers of total nucleated cells (TNC), a ratio of TNC to total erythrocytes (TE), and determination of RNA content within the PCE population demonstrated dose- and time-dependent effects with CP treatment. Shifts in RNA content were particularly sensitive, correctly identifying all CP-treated from control specimens, even in those samples where PCE/NCE ratio was similar. The AO methodology provided a more rapid, statistically-superior, and thorough approach in the assessment of bone marrow and spleen cytotoxicity than the conventional manual method of scoring PCE/NCE ratio alone.

Use of acridine orange in: flow cytometric assessment of micronuclei induction.

The micronucleus assay is a widely accepted method for evaluation of clastogens and aneugens. In the current study, acridine orange (AO) supravital staining was adapted for flow cytometric usage to assess micronucleated cells in rat bone marrow and spleen. Cyclophosphamide was used as a positive control test compound and results were compared to manual scoring in Wright-stained slides. In bone marrow, both manual and flow cytometric methods demonstrated positive dose response-trends for micronucleated polychromatic erythrocytes (MNPCE). Significant elevations in MNPCE were observed at all doses of cyclophosphamide, and comparisons between methods in bone marrow were not statistically different. The flow cytometric method was more sensitive in spleen samples, showing dose- and time-related increases in micronuclei compared with manual scoring. AO proved to be a sensitive discriminator of RNA and DNA, allowing distinct separation of polychromatic erythrocytes (PCE), normochromic erythrocytes (NCE), total nucleated cells (TNC), and micronucleated populations within both PCE and NCE regions. These results support the use of AO-based flow cytometry to provide a rapid and sensitive indicator of micronuclei inducers.

Comparison of flow cytometric and manual bone marrow differentials in Wistar rats.

Preclinical drug trials frequently require the evaluation of animal bone marrow, a time-consuming process requiring the skills of a highly trained hematologist. In the present study, a flow cytometric technique was developed that could effectively replace the need for manual bone marrow differentials in rats. Peroxidase activity, measured indirectly with 2'7'-dichlorofluorescein, was coupled with the use of species-specific T- and B-lymphocyte antibodies and cell size to produce a flow cytometric analysis of rat bone marrow. Accurate identification of lymphocyte, proliferating and maturing erythroid and myeloid, and megakaryocyte populations was confirmed by cell sorting. Flow cytometry yielded differentials that were indistinguishable from manual differentials and published reference ranges. Enumeration of lymphocyte numbers with monoclonal markers is a key advantage of flow cytometric differentials because misidentification of lymphocytes in poorly prepared or stained bone marrow smears is a common problem. The most apparent advantage is increased throughput and reproducibility. Operator training for analysis using flow cytometry can be readily accomplished within a few days as opposed to the extensive training required for individuals performing manual bone marrow differentials. This methodology provides a high-volume, rapid, and relatively low-cost tool for the reliable evaluation of rat bone marrow differentials that has been heretofore unavailable.

Flow cytometric evaluation of bone marrow differentials in rats with pharmacologically induced hematologic abnormalities.

Previously, flow cytometric determination of peroxidase activity, cell size, and reactivity to lymphocyte antibodies were used to produce bone marrow differentials in untreated rats. In the present study, abnormal hematologic profiles were induced with erythropoietin (EPO), recombinant murine stem cell factor (rm-SCF), granulocyte-macrophage stimulating factor (GM-CSF), and cyclophosphamide (CP). Manual and flow cytometric data showed comparable levels of erythroid and myeloid hyperplasia in EPO- and rm-SCF/GM-CSF-treated animals, respectively. In CP-treated animals, flow cytometric data revealed significant decreases in cellularity at concentrations of CP > or = 5 mg/kg. In contrast, 20 mg/kg CP were necessary to induce microscopically apparent hypoplasia in histologic bone sections, showing that the automated methodology was a more sensitive indicator of bone marrow hypocellularity than was the more conventional manual method. Megakaryocyte counts were consistently higher by flow cytometer than by manual counts performed on cytocentrifuge preparations made from the same cell suspensions but were similar to megakaryocyte counts performed on histologic sections of femur, indicating that the automated methodology produced a more accurate reflection of true megakaryocyte numbers. Induction of hematologic abnormalities in the present study showed that manual bone marrow differentials can be replaced with the more efficient and reliable flow cytometric method in most preclinical toxicology studies.

[Risk factors for squamous epithelial carcinoma of the mouth, the oropharynx, the hypopharynx and the larynx]. / Risikofaktoren bei Plattenepithelkarzinomen der Mundhöhle, des Oropharynx, des Hypopharynx und des Larynx.

This case-control-study, carried out in two University clinics, comprised 200 men (mean age 57.5 +/- 10.8 [range 33-89] years) with squamous epithelioma of the larynx (44.5%), oral cavity (23.5%), oropharynx (24%) or hypopharynx (8%) and 800 controls. Enquiries were directed at social status, life style and occupational exposure to substances such as asbestos, solvents, wood dust and cement. The peak incidence of these cancers was from 50 to 60 years of age. The proportion of unmarried or divorced men among the cancer patients was more than twice as high as in the controls (25.8% vs 11.8%; P less than 0.001). The proportion of cancer patients who had completed technical college or university education was significantly lower than in the controls (9.6% vs 24.4%; P less than 0.001). Tobacco and alcohol consumption by the cancer patients was roughly twice as great as in the controls: the cancer patients admitted to an average cigarette consumption of 43.2 +/- 27.9 pack years as compared with 20.1 +/- 26.7 pack years for the controls (P less than 0.001), and an alcohol intake of 69.2 +/- 58.1 g/d as compared with 29.8 +/- 27.5 g/d for the controls (P less than 0.001). Both these factors--drinking alcohol and smoking cigarettes--acted independently of one another to raise the relative risk of squamous epithelioma of the upper respiratory or digestive tract. The effects of alcohol and tobacco on cancer risk were multiplicative rather than merely additive. Enquiries into diet failed to reveal any clear differences tending to incriminate any particular food. Long-term exposure to cement dust was linked with an increased risk of cancer.

[Psychosocial conditions of psychotropic drug poisoning of patients treated in the Gdansk Toxicology Center]. / Psychospoleczne uwarunkowania zatruc lekami psychotropowymi u chorych leczonych w Gdanskim Osrodku Toksykologicznym.

The purpose of the study was in analysis of both causes and social conditioning leading to attempts of self-intoxications by the use of psychotropic drugs. The study included 234 patients treated in the Gdansk Toxicological Center during years 1982-1987. In a majority, the intoxications referred to young subjects up to 30 year of age, more frequently women than men. Only 10% of subjects studied have had university education. The alcohol or drug dependence and various psychiatric disturbances has been noted in a majority of cases studied. The lack of real suicidal determination was characteristic pattern of group observed.