From Fluorescence In Situ Hybridization Research Articles

Identification and Reporting of Cell of Origin, Double-/Triple-Hit and Double Expressor Lymphoma in a Real-World Cohort of Diffuse Large B-Cell Lymphoma Patients

Background The distinction of diffuse large B-cell lymphoma (DLBCL) into cell-of-origin (COO) subgroups (germinal center B-cell-like [GCB] or activated B-cell-like [ABC]) based on gene expression profiling is associated with prognosis and has potential therapeutic implications to mitigate the worse outcome for patients with DLBCL. Other phenotypic and molecular/cytogenetic features such as concurrent translocations of oncogene MYC and BCL2 and/or BCL6 (so-called double-/triple-hit lymphoma, DHL/THL) and coexpression of MYC and BCL2 proteins (so-called double-expressor lymphoma, DEL) are also recognized to have great prognostic impact (Swerdlow SH et al. Blood 2016;127(20):2375-2390). This study investigated the prevalence of COO, DHL/THL and DEL in a real-world cohort of patients with DLBCL who had documented results of diagnostic testing. Methods This study used the Flatiron Health electronic health record-derived de-identified database to abstract information on patients diagnosed with DLBCL between 2011-2019. Information on diagnostic testing from immunohistochemistry (IHC) for expression of MYC, BCL2, BCL6, CD10 or MUM1, and from fluorescence in situ hybridization (FISH) or karyotype analysis for rearrangement of MYC, BCL2 or BCL6 was abstracted from pathology reports or clinical visit notes, where available. We calculated the proportions of COO subgroups (GCB vs. ABC) that were derived from IHC testing results according to Hans algorithm (Hans C et al., Blood 2004;103(1):275-282), DHL/THL, and DEL. We also examined concordance of COO classification derived from IHC testing results with that directly reported by the healthcare providers. Differences in patient characteristics between IHC testing results-derived COO subgroups (GCB vs. ABC) were assessed using chi-square tests. Results 4400 patients had documented results of IHC and 73% (n=3194) can be classified into either GCB or ABC DLBCL (GCB/ABC ratio of 1.38). 3205 patients had documented results of FISH or karyotype analysis and 8% (n=245) were DHL/THL; only 33 patients were DEL. Within the GCB DLBCL patients derived from IHC testing results (n=1854), 163 patients were DHL/THL and 11 were DEL, whereas 24 DHL/THL and 18 DEL were identified within the ABC type (n=1340). When comparing COO classification derived from IHC testing results (n=3194) with that directly reported by the healthcare providers (n=2765), additional 695 and 439 patients can be classified as GCB and ABC DLBCL by IHC, respectively (Table). Univariate analysis showed that patients who were non-White ethnic group, diagnosed in academic centers, with lower body mass index but elevated serum lactate dehydrogenase levels and worse ECOG performance status, and without transformation from a prior indolent lymphoid malignancy, were more likely to be associated with ABC DLBCL (for all variables, p<0.05). There were no clinically meaningful and/or statistically significant differences in IHC testing results-derived COO classification (GCB vs. ABC) by age, gender, year of DLBCL diagnosis, geographic location of residency, type of insurance plan, tumor group stage, documentation of extranodal site or any other primary cancer history at the time of diagnosis. Discussion In this large real-world DLBCL cohort, a lower-than-expected proportion of DEL patients were identified vs. the 20-35% reported in the literature (Karube K and Campo E. Hematology 2015;52(2):97-106). This is likely due to our cohort of patients requiring clear evidence of coexpression for MYC and BCL2 (≥40% and >50%, respectively) that are not related to underlying chromosomal rearrangements, and few pathologists reported levels of percent staining for IHC testing among those with documented positive results of MYC/BCL2 protein coexpression. In addition, results from this study showed that only half of cases had COO classification documented by healthcare providers, despite available IHC results. Although this study indicated lack of details in the reporting of diagnostic testing (e.g. COO identification, levels of percent staining, methods for DLBCL subgroup identification), findings should be interpreted with caution, as patients with DLBCL might have been tested but not documented in the electronic health record system or might have biomarker testing performed at sites outside of the Flatiron Health network. Disclosures Yang: F. Hoffmann-La Roche: Current Employment. Zhang:F. Hoffmann-La Roche: Current Employment. Xiao:F. Hoffmann-La Roche: Current Employment. Hammer:Roche: Consultancy, Honoraria, Research Funding; Caris Lifesciences: Honoraria; PER Med education: Honoraria; PathEdEx: Current equity holder in private company. Prime:F. Hoffmann-La Roche: Current Employment.

Testing NTRK testing: Wet-lab and in silico comparison of RNA-based targeted sequencing assays.

NTRK fusions involving three neurotrophic tyrosine receptor kinase genes NTRK1, NTRK2, and NTRK3 and a variety of fusion partners were identified as oncogenic drivers across many cancer types. Drugs that target the chimeric protein product require the identification of the underlying gene fusion. This advocates the diagnostic use of molecular assays ranging from fluorescence in situ hybridization (FISH) and reverse transcription polymerase chain reaction (RT-PCR)/Sanger approaches to targeted next-generation sequencing (NGS). Immunohistochemistry may be used as a screening tool and adjunct diagnostic assay in this context. Although FISH and RT-PCR/Sanger approaches are widely adopted in routine diagnostics, current experience with targeted RNA-based NGS is limited. Here, we report on the analysis of major assays (TruSight TST170 and TruSight RNA Fusion [Illumina]; Archer FusionPlex Solid Tumor, Archer FusionPlex Lung, and Archer FusionPlex Oncology [Archer]; Oncomine Comprehensive Assay v3 RNA and Oncomine Focus RNA [Thermo Fisher Scientific]) that are commercially available. The data set includes performance results of a multicentric comparative wet-lab study as well as an in silico analysis on the ability to detect the broad range of NTRK fusions reported until now. A test algorithm that reflects assay methodology is provided. This data will support implementation of targeted RNA sequencing in routine diagnostics and inform screening and testing strategies that have been brought forward.

Wastewater biofilm formation on self-assembled monolayer surfaces using elastomeric flow cells

In anaerobic wastewater treatment, microbial biofilm is beneficial for efficient substrate utilization and for preventing the wash-out of key microorganisms. By providing solid supports, biofilm formation can be accelerated due to the early initial adhesion of residing microbes. Alteration in surface properties is therefore one such approach that helps us understand microbial interfacial interaction. Here, self-assembled monolayers of alkanethiols with carboxyl (-COOH), hydroxyl (-OH), and amine (-NH2) terminal moieties on gold (Au) substrates were employed to study the initial adhesion of wastewater microbes. An elastomeric flow cell was also utilized to simulate the environment of wastewater bioreactor. Results from fluorescence in situ hybridization (FISH) portrayed more enhanced microbial adhesion after 2 h on -NH2 functional group with the calculated surface coverage of 12.8 ± 2.4% as compared to 7.7 ± 1.6% on -COOH, 11.0 ± 2.0% on -OH, and 1.2% on unmodified Au surfaces. This might be because of concomitant electrostatic attraction between negatively-charged bacteria and positively-charged (-NH3+) functional groups. Nevertheless, the average surface coverage by individual biofilm clusters was 28.0 ± 5.0 μm2 and 32.0 ± 9.0 μm2 on -NH2 and -OH surfaces, respectively, while -COOH surfaces resulted in higher value (60.0 ± 5.0 μm2) and no significant cluster formation was observed on Au surfaces. Accordingly, the average inter-cluster distance observed on -NH2 surfaces was relatively smaller (3.0 ± 0.6 μm) as compared to that on other surfaces. Overall, these data suggest favorable initial biofilm growth on more hydrophilic and positively-charged surfaces. Furthermore, the analysis of the mean fluorescence intensity revealed preferred initial adhesion of key microbes (archaea) on -OH and -NH2 surfaces. Indeed, results obtained from this study would be beneficial in designing selective biointerfaces for certain biofilm carriers in a typical wastewater bioreactor. Importantly, our elastomeric flow cell integrated with SAM-modified surfaces demonstrated an ideal platform for high-throughput investigation of wastewater biofilm under controlled environments.

Clinical Validation of Treatment Response Predictions Using a Genomics Driven Computational Biology Modelling Multiple Myeloma Algorithm

Background: Multiple myeloma (MM) is an incurable and heterogeneous haematological malignancy in which immune suppression and complex biology affect the disease and its response to treatment. Several new treatments have been approved for MM in recent years providing numerous options for patients with relapsed/refractory disease. However, there is no validated method for selecting the best treatment combination for each patient, making patient management difficult. The ability to predict treatment response based on disease characteristics could improve clinically outcomes.Aim: This was a validation of a genomics-informed response prediction using computational biology modelling (CBM) in patients with relapsed/refractory MM.Methods: Input data from fluorescence in-situ hybridization (FISH), karyotype, and a MM specific next generation sequencing capture array were analysed using CBM. This was a retrospective review of patients which were treated with different combinations based on patient/physician choice. The CBM uses PubMed and other online resources to generate patient-specific protein network maps of activated and inactivated pathways. The specific drug combination for each patient was simulated and the quantitative drug effect was measured on a composite MM disease inhibition score (i.e., cell proliferation, viability, apoptosis and paraproteins). The predicted outcomes were then compared to the clinical response (≥PR or < PR per IMWG) to assess the accuracy of this CBM predictive approach.Results: 27 patients were selected for the study; 3 failed CBM due to missing inputs and in 3 clinical response was not able to be assessed, leaving 21 eligible for the analysis. The median age at presentation was 57 years (range 37-76) and 52% were male. The median prior lines of MM therapy was 5 (range 1-15). 38% were refractory to bortezomib, 62% to lenalidomide, 52% to carfilzomib, 57% to pomalidomide, and 43% to daratumumab. 81% had a prior autologous stem cell transplant.The treatments modelled included IMiD-based regimens (n = 9), PI-based regimens (n = 6), chemo-based regimens (n = 3), selinexor (n = 2), PI/IMiD combination regimens (n = 1). Sixteen were clinical responders and 5 were non-responders. CBM predictions matched for 17 of 21 treatments overall, 15 of 16 clinical responders and 2 of 5 non-responders. The statistics of prediction accuracy against clinical outcome are presented in Table 1.Interestingly, the CBM identified drugs within the combination regimens which may not have impacted efficacy. For example, the CBM predicted that one patient treated with bortezomib, venetoclax, and dexamethasone would have had similar response if venetoclax had been omitted from the regimen.Conclusion: We have demonstrated that a CBM approach, which incorporates genomics, can help predict response in patients with relapsed or refractory MM. Prospective studies using the CBM as part of treatment decision-making will help determine its application into clinical settings. [Display omitted] DisclosuresVij:Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jansson: Honoraria, Membership on an entity's Board of Directors or advisory committees. Singh:Cellworks Research India Private Limited: Employment. Sauban:Cellworks Research India Private Limited: Employment. Husain:Cellworks Research India Private Limited: Employment. Lakshminarayana:Cellworks Research India Private Limited: Employment. Talawdekar:Cellworks Research India Private Limited: Employment. Mitra:Cellworks Research India Private Limited: Employment. Abbasi:Cell Works Group Inc.: Employment. Vali:Cell Works Group Inc.: Employment.

Improved pregnancy rates following transfer of euploid embryos screened by next-generation sequencing compared to array comparative genomic hybridization

Preimplantation genetic screening (PGS) improves implantation and decreases miscarriage rates per embryo transferred1. PGS has evolved from fluorescence in-situ hybridization (FISH) to array comparative genomic hybridization (aCGH), and recently to next-generation sequencing (NGS). Compared to aCGH, various NGS protocols using amplified DNA have been shown to have a sensitivity and specificity as high as 100% for detecting aneuploidy in blastocysts2,3. Some NGS protocols may be able to detect partial chromosomal gains and losses and identify more cases of mosaicism within trophectoderm cells than other techniques4-6.

Intensified organics and nitrogen removal in the intermittent-aerated constructed wetland using a novel sludge-ceramsite as substrate

In this study, a novel sludge-ceramsite was applied as main substrate in intermittent-aerated subsurface flow constructed wetlands (SSF CWs) for treating decentralized domestic wastewater, and intensified organics and nitrogen removal in different SSF CWs (with and without intermittent aeration, with and without sludge-ceramsite substrate) were evaluated. High removal of 97.2% COD, 98.9% NH4+-N and 85.8% TN were obtained simultaneously in the intermittent-aerated CW system using sludge-ceramsite substrate compared with non-aerated CWs. Moreover, results from fluorescence in situ hybridization (FISH) analysis revealed that the growth of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in the intermittent-aerated CW system with sludge-ceramsite substrate was enhanced, thus indicating that the application of intermittent aeration and sludge-ceramsite plays an important role in nitrogen transformations. These results suggest that a combination of intermittent aeration and sludge-ceramsite substrate is reliable to enhance the treatment performance in SSF CWs.

Inferential modeling of 3D chromatin structure.

For eukaryotic cells, the biological processes involving regulatory DNA elements play an important role in cell cycle. Understanding 3D spatial arrangements of chromosomes and revealing long-range chromatin interactions are critical to decipher these biological processes. In recent years, chromosome conformation capture (3C) related techniques have been developed to measure the interaction frequencies between long-range genome loci, which have provided a great opportunity to decode the 3D organization of the genome. In this paper, we develop a new Bayesian framework to derive the 3D architecture of a chromosome from 3C-based data. By modeling each chromosome as a polymer chain, we define the conformational energy based on our current knowledge on polymer physics and use it as prior information in the Bayesian framework. We also propose an expectation-maximization (EM) based algorithm to estimate the unknown parameters of the Bayesian model and infer an ensemble of chromatin structures based on interaction frequency data. We have validated our Bayesian inference approach through cross-validation and verified the computed chromatin conformations using the geometric constraints derived from fluorescence in situ hybridization (FISH) experiments. We have further confirmed the inferred chromatin structures using the known genetic interactions derived from other studies in the literature. Our test results have indicated that our Bayesian framework can compute an accurate ensemble of 3D chromatin conformations that best interpret the distance constraints derived from 3C-based data and also agree with other sources of geometric constraints derived from experimental evidence in the previous studies. The source code of our approach can be found in https://github.com/wangsy11/InfMod3DGen.

Chromosomal diversification and karyotype evolution of diploids in the cytologically diverse genus Prospero(Hyacinthaceae)

BackgroundProspero (Hyacinthaceae) provides a unique system to assess the impact of genome rearrangements on plant diversification and evolution. The genus exhibits remarkable chromosomal variation but very little morphological differentiation. Basic numbers of x = 4, 5, 6 and 7, extensive polyploidy, and numerous polymorphic chromosome variants were described, but only three species are commonly recognized: P. obtusifolium, P. hanburyi, and P. autumnale s.l., the latter comprising four diploid cytotypes. The relationship between evolutionary patterns and chromosomal variation in diploids, the basic modules of the extensive cytological diversity, is presented.ResultsEvolutionary inferences were derived from fluorescence in situ hybridization (FISH) with 5S and 35S rDNA, genome size estimations, and phylogenetic analyses of internal transcribed spacer (ITS) of 35S rDNA of 49 diploids in the three species and all cytotypes of P. autumnale s.l. All species and cytotypes possess a single 35S rDNA locus, interstitial except in P. hanburyi where it is sub-terminal, and one or two 5S rDNA loci (occasionally a third in P. obtusifolium) at fixed locations. The localization of the two rDNA types is unique for each species and cytotype. Phylogenetic data in the P. autumnale complex enable tracing of the evolution of rDNA loci, genome size, and direction of chromosomal fusions: mixed descending dysploidy of x = 7 to x = 6 and independently to x = 5, rather than successive descending dysploidy, is proposed.ConclusionsAll diploid cytotypes are recovered as well-defined evolutionary lineages. The cytogenetic and phylogenetic approaches have provided excellent phylogenetic markers to infer the direction of chromosomal change in Prospero. Evolution in Prospero, especially in the P. autumnale complex, has been driven by differentiation of an ancestral karyotype largely unaccompanied by morphological change. These new results provide a framework for detailed analyses of various types of chromosomal rearrangements and karyotypic variation in polyploids.

Abstract 2895: Phylogenetic models of tumor progression from fluorescence in situ hybridization (FISH) data on many single cells of a solid tumor.

Abstract Purpose: Characterizing the common pathways through which tumors progress is critical to understanding the molecular basis of cancer and developing effective treatments. Algorithms for phylogenetics, i.e., evolutionary tree building, can be used to infer progression pathways of single tumors when there is widespread intra-tumor heterogeneity from cell to cell. We describe computational methods to compute likely evolutionary histories from single-cell copy number data and apply the methods to single-cell gene copy number data derived from paired primary and metastatic samples of cervical cancers. Materials and methods: The inputs to our methods are lists of cell types for each patient sample, where a cell type is an array of copy numbers of gene probes measured by FISH. In the cervical cancer data set, copy numbers of four genes (LAMP3, PROX1, PRKAA1 and CCND1) were measured on up to 250 cells each of paired primary and metastatic samples from 16 patients. An example of a cell type is (4, 1, 3, 2). Our algorithms derive maximum parsimony phylogenetic trees explaining how all observed cell types might have evolved from a common diploid ancestor (2,2,2,2), seeking to explain the observed cells with as few copy number changes as possible across the tree. Due to the computational difficulty, we apply a heuristic algorithm to infer an approximately optimal tree by identifying likely unobserved ancestral cell states through a variant of the median joining method and then find the most parsimonious tree connecting these states. Our software also includes a method of computing consensus trees that facilitate the comparison of the tree models for each primary/metastasis sample pair. Results and Conclusion: Our methods allow us to build models of evolution of single tumors at the cellular level that provide insights into possible progression pathways and varying selective pressures in primary and metastatic sites. Analysis of tree geometry shows significant variability in evolutionary patterns between primary and metastatic sites consistent with more specific selective pressures in the metastases. Analysis of specific patterns of gene gain or loss in each environment shows a complicated portrait of tumor evolution with substantial variability from patient to patient. Citation Format: Salim A. Chowdhury, Alejandro A. Schäffer, Stanley E. Shackney, Darawalee Wangsa, Kerstin Heselmeyer-Haddad, Thomas Ried, Russell Schwartz. Phylogenetic models of tumor progression from fluorescence in situ hybridization (FISH) data on many single cells of a solid tumor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2895. doi:10.1158/1538-7445.AM2013-2895

The progression of PGS: technological advancements and application improves implantation and clinical pregnancy rates by enhancing diagnostic accuracy in identifying the euploid embryo

For more than two decades, embryologists and clinicians have attempted to use pre-implantation genetic screening (PGS) to detect embryonic chromosomal abnormalities in hopes of transferring only reproductively competent embryos. Coinciding with the evolution of the technology, our center has progressed from cell stage biopsy (D3) to blastocyst biopsy (D5) and from fluorescence in situ hybridization (FISH) with 5-12 Probes (Genzyme, Reprogenetics and RMAGenetics), to 24 chromosomal array single nucleotide polymorphism (SNP) (Gene Security Network) and finally to the more recent real-time quantitative polymerase chain reaction (qPCR) of 24 chromosomes (RMAGenetics) for embryonic screening.

Comprehensive analysis of HER2 expression and gene amplification in gastric cancers using immunohistochemistry and in situ hybridization: which scoring system should we use?

It has been reported that HER2 expression is different in gastric and breast cancers, and a gastric cancer scoring system (GCSS) has recently been suggested. We investigated HER2 protein expression using GCSS and a breast cancer scoring system (BCSS) and correlated it with HER2 gene amplification. HER2 status was evaluated in 1091 cases by analyzing tissue microarrays constructed using 2 different cores from each case. Polyclonal (HercepTest) and monoclonal (Pathway) antibodies were used for immunohistochemistry (IHC), and results were scored by BCSS and GCSS. Gene amplification was evaluated by automated dual-color silver-enhanced in situ hybridization (SISH) in all cases and correlated with the results from fluorescence in situ hybridization (FISH) in 590 cases. The concordance between the IHC results using polyclonal and monoclonal antibodies was high (κ = 0.785). The results of dual-color SISH and FISH showed very high concordance as well (κ = 0.918). GCSS was significantly more sensitive for detecting SISH positivity than was BCSS in both antibodies (polyclonal, P = .003; monoclonal, P < .001), but specificity was higher in BCSS than GCSS (polyclonal, P = .004; monoclonal, P < .001). It has been recently shown that HER2-overexpressing patients with unresectable gastric cancer benefited from trastuzumab therapy. Because IHC is recommended before gene amplification studies in HER2 testing, GCSS should be used for evaluating HER2 expression in gastric cancers.

Rapid and high-throughput multiplex ligation-dependent probe amplification for diagnosis of chromosome aneuploidy

To assess the diagnostic value of multiplex ligation-dependent probe amplification (MLPA) for detection of common chromosome aneuploidy in amniotic fluid (AF) cells in order to obtain an accurate, rapid, cost-effective and high-throughput method in routine prenatal clinical practice. The MLPA test was performed on 500 AF samples by using kit P095 and the results were obtained by using analysis software RH-MLPA-v511. The results were compared with that from fluorescence in situ hybridization (FISH) and traditional karyotyping (TK). The technical critical issues were analyzed in routine diagnostic application. The absolute specificity and sensitivity of the MLPA test to detect the aneuploidy were 100%. For the 500 AF samples, the success rate of the MLPA tests was 97%. Among them 92% were finished within three working days and 5% required more days for repeating. The test failure rate was 3%. The results confirmed that for the 38 detectable aneuploid samples, the probe reliability weighted mean ratio values were more than 4SD compared to normal diploids and the 2 suspected trisomy samples were more than 2SD. In this study, authors analyzed hybridization efficiencies of 8 probes for chromosome 21, and the presence of a trisomy was considered if at least 4 of the 8 probes gave probe ratio of >1.3. Thedata suggested that MLPA is a rapid, simple and reliable method for large scale testing for aneuploidy of chromosomes 13, 18, 21, X, or Y in AF. The MLPA technology is complementary to AF culture and valuable for prenatal diagnosis.

Microscopical techniques reveal the in situ microbial association inside Aplysina aerophoba, Nardo 1886 (Porifera, Demospongiae, Verongida) almost exclusively consists of cyanobacteria

Sponges (Porifera) are aquatic, sessile filter feeders. As such they are permanently exposed to bacteria in the seawater. Molecular data recovered from sponges by PCR shows a high diversity in bacterial DNA. Hence, sponges are considered to live in close association with a diverse and abundant bacterial community. To recover the spatial distribution of bacteria in sponges we retrieved histological sections of Aplysina aerophoba fixed in situ. By combining signals from fluorescence in situ hybridization (FISH), light microscopy and scanning electron microscopy we revealed a detailed histological picture of the spatial organization of the sponge microbial association within the sponges. Our histological results confirm a high abundance of cyanobacteria inside A. aerophoba while other living bacteria are almost absent. This detailed insight into sponge microbiology could only be achieved by the combination of careful sample preparation and different microscopical and histological methods. It also shows the need to confirm molecular datasets in situ and with a high spatial resolution.

Current use and future needs of biodosimetry in studies of long-term health risk following radiation exposure.

Biodosimetry measurements can potentially be an important and integral part of the dosimetric methods used in long-term studies of health risk following radiation exposure. Such studies rely on accurate estimation of doses to the whole body or to specific organs of individuals in order to derive reliable estimates of cancer risk. However, dose estimates based on analytical dose reconstruction (i.e., models) or personnel monitoring measurements (i.e., film badges) can have substantial uncertainty. Biodosimetry can potentially reduce uncertainty in health risk studies by corroboration of model-based dose estimates or by using them to assess bias in dose models. While biodosimetry has begun to play a more significant role in long-term health risk studies, its use is still generally limited in that context due to one or more factors including inadequate limits of detection, large inter-individual variability of the signal measured, high per-sample cost, and invasiveness. Presently, the most suitable biodosimetry methods for epidemiologic studies are chromosome aberration frequencies from fluorescence in situ hybridization (FISH) of peripheral blood lymphocytes and electron paramagnetic resonance (EPR) measurements made on tooth enamel. Both types of measurements, however, are usually invasive and require biological samples that can be difficult to obtain. Moreover, doses derived from these methods are not always directly relevant to the tissues of interest. To increase the value of biodosimetry to epidemiologic studies, a number of issues need to be considered, including limits of detection, effects of inhomogenous exposure of the body, how to extrapolate from the tissue sampled to the tissues of interest, and how to adjust dosimetry models applied to large populations based on sparse biodosimetry measurements. The requirements of health risk studies suggest a set of characteristics that, if satisfied by new biodosimetry methods, would increase the overall usefulness of biodosimetry in determining radiation health risks.

Standing stocks and activity of Archaea and Bacteria in the western Arctic Ocean

This study examined the abundance, cell size, and activity of Bacteria and Archaea in the Chukchi Sea and the Canada Basin of the western Arctic Ocean in the spring (May—June) and summer (July—August) of 2002 and 2004. Data from fluorescence in situ hybridization (FISH) analyses indicate that bacterial abundance as a percent of total prokaryotes decreased with depth, whereas in contrast, Crenarchaeota increased from about 10%of prokaryotes in surface waters to as much as 40% in samples from 100 to 200 m. Euryarchaeota were detectable in only a few samples. Relative abundance of Crenarchaeota, expressed as a percent of total prokaryotes, correlated with ammonium concentrations, but relative bacterial abundance did not. Crenarchaeota cells were significantly larger than Bacteria by 1.5‐ to 2‐fold in the upper 200 m. Data collected from a combination of FISH and microautoradiography indicate that often the fraction of both Bacteria and Crenarchaeota assimilating organic compounds was high (up to 55%), and both microbial groups were more active in assimilating amino acids than other compounds. However, Crenarchaeota were usually less active than Bacteria in assimilating amino acids and glucose, but were nearly as active as Bacteria in assimilating protein and diatom extracellular polymers. The fraction of Bacteria and Crenarchaeota assimilating CO2 in surface waters was higher than expected by anaplerotic fixation alone, suggesting that many of these microbes are chemoautotrophic. These data add to a growing body of evidence indicating how the roles of Archaea and Bacteria differ in biogeochemical cycles of the oceans.

Acute lymphoblastic leukaemia: diagnosis and classification

Acute lymphoblastic leukaemia (ALL) is a heterogeneous disease with distinct biological and prognostic groupings. Diagnosis relies on traditional cytomorphological and immunohistochemical evaluation of the leukaemic blasts. Subsequently, cytogenetic analysis identifies clonal numeric and/or structural chromosomal abnormalities that may be present, thus confirming the subtype classification and providing important prognostic information for treatment planning. The major chromosomal abnormalities in ALL are t(9;22)(q34;q11), t(12;21)(p13;q22), t(4;11)(q21;q23), t(1;19)(q23;p13), 8q24 translocations and hyperdiploidy. Generally, hyperdiploidy, occurring most frequently in paediatric cases, is associated with a good prognosis, while hypodiploidy confers a poor prognosis. Among structural chromosomal abnormalities, the t(9;22)(q34;q11) resulting in the BCR/ABL fusion protein, and rearrangements of the MLL gene, confer a poor prognosis in both children and adults, while t(12;21)(p13;q22), resulting in the TEL/AML1 fusion protein, and del (12p) confer a good prognosis. More recently, additional diagnostic and prognostic information has been gained from fluorescence in situ hybridization (FISH) and DNA microarray techniques.

Identification of programmed cell death in situ in individual plant cells in vivo using a chromosome preparation technique.

A simple procedure, which combines a chromosome preparation technique with an in situ labelling technique modified from fluorescence in situ hybridization (FISH), has been developed for in situ detection of plant programmed cell death (PCD) at the single-cell level. After exposure of chromosomes and nuclei on slides by enzymolysis, Klenow or TdT was used to incorporate Bio-dUTP or fluorescein-dUTP at sites of DNA breaks. After Klenow-mediated labelling, the signals were amplified by a cascade of antigen-antibody reaction according to the detection system of FISH. This method enables in situ detection of plant PCD in vivo morphologically and biochemically at the chromosome, nuclear and DNA levels without cell culture and histological sectioning. This technique permits labelling of DNA breaks with high sensitivity due to increased chromosome and nucleus exposure to the labelling solutions, as well as due to the immunological amplification of the signals. Moreover, the changes in the cells were easier to be observed because the spatial obstacle of the cell wall and its autofluorescence were eliminated. It is potentially useful for in situ detection of PCD in plant root meristematic cells triggered by various environmental abiotic factors. It is proposed that the root tip is a versatile in vivo system for studying PCD induced by environmental abiotic factors.

Corrigendum: Genomic discrimination by in situ hybridization in Icelandic species of Elymus and Elytrigia (Poaceae: Triticeae)

(1) It was discovered that H9083 has been identified as a tetraploid species of Elymus (StStHH), not a diploid species of Psedoroegneria spicata as stated in the paper referenced above. Genomic DNA from this accession was used as probe DNA, combined with differently labelled genomic DNA from Hordeum (HH) and unlabeled blocking DNA from Elymus (NsNsXmXm), on species of Elymus and Elytrigia. Because of this, only St-genome specific sequences in the StH probe were free to hybridize to chromosomes, the same way as the St probe would have performed. However, the results of the combined probe are still to be interpreted as reported in the aforementioned paper, and results from fluorescence in situ hybridization (FISH) analyses using localized probes have confirmed and redefined the original genomic in situ hybridization (GISH) results.

Cyclic core dendrimer as a new kind of vector for gene transfer into mammalian cells.

Cyclic core dendritic polymer is a new type of synthetic polymers. The ability of generation 4 of the dendrimer with a core of 1,4,7,10-tetraazacyclododecane to function as an effective gene delivery vector was investigated. Results from fluorescence in situ hybridization (FISH) show that the pCH 110 plasmid DNA was transferred into human small intestine cancer metastatic ascites (HICMA) cells induced by this kind of dendrimer as a vector. The transferred LacZ, GFP and luciferase genes were highly expressed in the transfected HICMA, COS-7 and 293 cells. These studies demonstrate that the dendrimer can transfect mammalian cells in vitro which offers an alternatively efficient method for mammalian gene transfer.

Multiple Polysomies in Breast Carcinomas: Preferential Gain of Chromosomes 1, 5, 6, 7, 12, 16, 17, 18, and 19

Chromosome G-banding analysis of metaphase cells from 16 primary breast carcinomas revealed the presence of multiple polysomies in near-diploid as well as in polyploid cells. Chromosome 17 was preferentially gained in 7 tumors, followed in frequency by chromosomes 1, 12, and 19 (5 tumors each), and chromosomes 5, 6, 7, 16, and 18 (4 tumors each). Eleven of the 16 carcinomas had, apart from the clones exhibiting the numerical gains, other unrelated clones. Nine of these 11 cases had clones with structural chromosome aberrations, 5 of which had structural aberrations involving the short arm of chromosome 3. The biologic significance, if any, of this seemingly nonrandom coexistence of multiple polysomies with structural aberrations of 3p is at present not known. The pattern of numerical chromosome aberrations observed in the present study is comparable to previous results from fluorescence in situ hybridization (FISH) studies, with the use of centromeric probes on interphase cells. However, unlike FISH studies, which have been focused on chromosomes 1, 3, 7, 8, 11, 16, and 17, the cytogenetic results reveal that other chromosomes also may be nonrandomly gained as part of multiple polysomies in breast carcinomas. In addition, the tumors with multiple polysomies were generally of high histologic grade and with metastasis to axillary lymph nodes, suggesting that multiple whole-chromosome gains may be a pathway of genetic evolution or progression or both in some breast carcinomas.