Radiation exposure determination in a secure, cloud-based online environment.

Rapid sample processing and interpretation of estimated exposures will be critical for triaging exposed individuals after a major radiation incident. The dicentric chromosome (DC) assay assesses absorbed radiation using metaphase cells from blood. The Automated Dicentric Chromosome Identifier and Dose Estimator System (ADCI) identifies DCs and determines radiation doses.

Differentially accessible, single copy sequences form contiguous domains along metaphase chromosomes that are conserved among multiple tissues.

Background: During mitosis, chromatin engages in a dynamic cycle of condensation and decondensation. Condensation into distinct units to ensure high fidelity segregation is followed by rapid and reproducible decondensation to produce functional daughter cells. Factors contributing to the reproducibility of chromatin structure between cell generations are not well understood.

Estimating partial-body ionizing radiation exposure by automated cytogenetic biodosimetry.

Purpose: Inhomogeneous exposures to ionizing radiation can be detected and quantified with the dicentric chromosome assay (DCA) of metaphase cells. Complete automation of interpretation of the DCA for whole-body irradiation has significantly improved throughput without compromising accuracy, however, low levels of residual false positive dicentric chromosomes (DCs) have confounded its application for partial-body exposure determination.

Materials And Methods: We describe a method of estimating and correcting for false positive DCs in digitally processed images of metaphase cells.

Meeting radiation dosimetry capacity requirements of population-scale exposures by geostatistical sampling.

Background: Accurate radiation dose estimates are critical for determining eligibility for therapies by timely triaging of exposed individuals after large-scale radiation events. However, the universal assessment of a large population subjected to a nuclear spill incident or detonation is not feasible. Even with high-throughput dosimetry analysis, test volumes far exceed the capacities of first responders to measure radiation exposures directly, or to acquire and process samples for follow-on biodosimetry testing.

RADIATION DOSE ESTIMATION BY COMPLETELY AUTOMATED INTERPRETATION OF THE DICENTRIC CHROMOSOME ASSAY.

Accuracy of the automated dicentric chromosome (DC) assay relies on metaphase image selection. This study validates a software framework to find the best image selection models that mitigate inter-sample variability. Evaluation methods to determine model quality include the Poisson goodness-of-fit of DC distributions for each sample, residuals after calibration curve fitting and leave-one-out dose estimation errors.

Accurate cytogenetic biodosimetry through automated dicentric chromosome curation and metaphase cell selection.

Accurate digital image analysis of abnormal microscopic structures relies on high quality images and on minimizing the rates of false positive (FP) and negative objects in images. Cytogenetic biodosimetry detects dicentric chromosomes (DCs) that arise from exposure to ionizing radiation, and determines radiation dose received based on DC frequency. Improvements in automated DC recognition increase the accuracy of dose estimates by reclassifying FP DCs as monocentric chromosomes or chromosome fragments.

Expedited Radiation Biodosimetry by Automated Dicentric Chromosome Identification (ADCI) and Dose Estimation.

Biological radiation dose can be estimated from dicentric chromosome frequencies in metaphase cells. Performing these cytogenetic dicentric chromosome assays is traditionally a manual, labor-intensive process not well suited to handle the volume of samples which may require examination in the wake of a mass casualty event. Automated Dicentric Chromosome Identifier and Dose Estimator (ADCI) software automates this process by examining sets of metaphase images using machine learning-based image processing techniques.

Radiation Dose Estimation by Automated Cytogenetic Biodosimetry.

The dose from ionizing radiation exposure can be interpolated from a calibration curve fit to the frequency of dicentric chromosomes (DCs) at multiple doses. As DC counts are manually determined, there is an acute need for accurate, fully automated biodosimetry calibration curve generation and analysis of exposed samples. Software, the Automated Dicentric Chromosome Identifier (ADCI), is presented which detects and discriminates DCs from monocentric chromosomes, computes biodosimetry calibration curves and estimates radiation dose.

Clinical Next-Generation Sequencing Pipeline Outperforms a Combined Approach Using Sanger Sequencing and Multiplex Ligation-Dependent Probe Amplification in Targeted Gene Panel Analysis.

Advances in next-generation sequencing (NGS) have facilitated parallel analysis of multiple genes enabling the implementation of cost-effective, rapid, and high-throughput methods for the molecular diagnosis of multiple genetic conditions, including the identification of BRCA1 and BRCA2 mutations in high-risk patients for hereditary breast and ovarian cancer. We clinically validated a NGS pipeline designed to replace Sanger sequencing and multiplex ligation-dependent probe amplification analysis and to facilitate detection of sequence and copy number alterations in a single test focusing on a BRCA1/BRCA2 gene analysis panel. Our custom capture library covers 46 exons, including BRCA1 exons 2, 3, and 5 to 24 and BRCA2 exons 2 to 27, with 20 nucleotides of intronic regions both 5' and 3' of each exon.

A unified analytic framework for prioritization of non-coding variants of uncertain significance in heritable breast and ovarian cancer.

Background: Sequencing of both healthy and disease singletons yields many novel and low frequency variants of uncertain significance (VUS). Complete gene and genome sequencing by next generation sequencing (NGS) significantly increases the number of VUS detected. While prior studies have emphasized protein coding variants, non-coding sequence variants have also been proven to significantly contribute to high penetrance disorders, such as hereditary breast and ovarian cancer (HBOC).

Prioritizing Variants in Complete Hereditary Breast and Ovarian Cancer Genes in Patients Lacking Known BRCA Mutations.

BRCA1 and BRCA2 testing for hereditary breast and ovarian cancer (HBOC) does not identify all pathogenic variants. Sequencing of 20 complete genes in HBOC patients with uninformative test results (N = 287), including noncoding and flanking sequences of ATM, BARD1, BRCA1, BRCA2, CDH1, CHEK2, EPCAM, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, PALB2, PMS2, PTEN, RAD51B, STK11, TP53, and XRCC2, identified 38,372 unique variants. We apply information theory (IT) to predict and prioritize noncoding variants of uncertain significance in regulatory, coding, and intronic regions based on changes in binding sites in these genes.

Genomic signatures for paclitaxel and gemcitabine resistance in breast cancer derived by machine learning.

Increasingly, the effectiveness of adjuvant chemotherapy agents for breast cancer has been related to changes in the genomic profile of tumors. We investigated correspondence between growth inhibitory concentrations of paclitaxel and gemcitabine (GI50) and gene copy number, mutation, and expression first in breast cancer cell lines and then in patients. Genes encoding direct targets of these drugs, metabolizing enzymes, transporters, and those previously associated with chemoresistance to paclitaxel (n = 31 genes) or gemcitabine (n = 18) were analyzed.

Reversing chromatin accessibility differences that distinguish homologous mitotic metaphase chromosomes.

Background: Chromatin-modifying reagents that alter histone associating proteins, DNA conformation or its sequence are well established strategies for studying chromatin structure in interphase (G1, S, G2). Little is known about how these compounds act during metaphase. We assessed the effects of these reagents at genomic loci that show reproducible, non-random differences in accessibility to chromatin that distinguish homologous targets by single copy DNA probe fluorescence in situ hybridization (scFISH).

Canadian anaplastic lymphoma kinase study: a model for multicenter standardization and optimization of ALK testing in lung cancer.

Introduction: Fluorescence in situ hybridization (FISH) is currently the standard for diagnosing anaplastic lymphoma kinase (ALK)-rearranged (ALK+) lung cancers for ALK inhibitor therapies. ALK immunohistochemistry (IHC) may serve as a screening and alternative diagnostic method. The Canadian ALK (CALK) study was initiated to implement a multicenter optimization and standardization of laboratory developed ALK IHC and FISH tests across 14 hospitals.

Intensity integrated Laplacian-based thickness measurement for detecting human metaphase chromosome centromere location.

Accurate detection of the human metaphase chromosome centromere is an important step in many chromosome analysis and medical diagnosis algorithms. The centromere location can be utilized to derive information such as the chromosome type, polarity assignment, etc. Methods available in the literature yield unreliable results mainly due to high variability of morphology in metaphase chromosomes and boundary noise present in the image.

Expanding probe repertoire and improving reproducibility in human genomic hybridization.

Diagnostic DNA hybridization relies on probes composed of single copy (sc) genomic sequences. Sc sequences in probe design ensure high specificity and avoid cross-hybridization to other regions of the genome, which could lead to ambiguous results that are difficult to interpret. We examine how the distribution and composition of repetitive sequences in the genome affects sc probe performance.

Structural and genic characterization of stable genomic regions in breast cancer: relevance to chemotherapy.

Background: Cancer genomes accumulate frequent and diverse chromosomal abnormalities as well as gene mutations but must maintain the ability to survive in vivo. We hypothesize that genetic selection acts to maintain tumour survival by preserving copy number of specific genes and genomic regions. Genomic regions and genes that remain unaltered in copy number and expression, respectively, may be essential for maintaining tumour survival.

In situ hybridization to metaphase chromosomes and interphase nuclei.

In situ hybridization is used to determine the chromosomal map location and the relative order of genes and DNA sequences within a chromosomal band. It can also be used to detect aneuploidy, gene amplification, and subtle chromosomal rearrangements. Fluorescence in situ hybridization (FISH), probably the most widely used method, is described in the first basic protocol.

In situ hybridization and detection using nonisotopic probes.

Nonisotopic in situ hybridization can be used to determine the cellular location and relative levels of expression for specific transcripts within cells and tissues. RNA in specimen preparations is hybridized with a biotin- or digoxigenin-labeled probe, which is generally detected by fluorescence or enzymatic methods. Fluorescence in situ hybridization (FISH), probably the most widely used method, is described here, along with amplification of weak FISH signals.

Human metaphase chromosome FISH using quantum dot conjugates.

Fluorescence in situ hybridization (FISH) is a powerful, molecular technique with a wide range of applications in medicine and biology. In medicine, FISH uses genomic and cDNA probes to determine the chromosomal position of genes and DNA sequences, which enables detection of ploidy levels and identification of subtle chromosomal rearrangements. Because of its exquisite sensitivity, FISH often enhances conventional cytogenetic analysis and it can provide either diagnostic or prognostic results for particular chromosomal disorders.

Determination of genomic copy number with quantitative microsphere hybridization.

We developed a novel quantitative microsphere suspension hybridization (QMH) assay for determination of genomic copy number by flow cytometry. Single copy (sc) products ranging in length from 62 to 2,304 nucleotides [Rogan et al., 2001; Knoll and Rogan, 2004] from ABL1 (chromosome 9q34), TEKT3 (17p12), PMP22 (17p12), and HOXB1 (17q21) were conjugated to spectrally distinct polystyrene microspheres.

A 9-year-old male with a duplication of chromosome 3p25.3p26.2: clinical report and gene expression analysis.

We describe a 9-year-old male referred for genetic evaluation for Prader-Willi syndrome (PWS). PWS is the most common genetically defined cause of life-threatening obesity and results from a functional loss of paternally expressed genes from the chromosome 15q11-q13 region. The patient presented with pervasive developmental disorder, delayed speech, and rapid onset of obesity at age 4 years, all features similar to PWS.

Dendrimer FISH detection of single-copy intervals in acute promyelocytic leukemia.

Acute promyelocytic leukemia (AML-M3) is characterized by a translocation between chromosomes 15 and 17 [t(15;17)]. The detection of t(15;17) at the single cell level, is commonly done by fluorescence in situ hybridization (FISH) using recombinant locus specific genomic probes greater than 14 kilobases kb in length. To allow a more thorough study of t(15;17), we designed small (0.

Distortion of quantitative genomic and expression hybridization by Cot-1 DNA: mitigation of this effect.

Cross-hybridization of repetitive sequences in genomic and expression arrays is reported to be suppressed with repeat-blocking nucleic acids (C(o)t-1 DNA). Contrary to expectation, we demonstrated that C(o)t-1 also enhanced non-specific hybridization between probes and genomic targets. When added to target DNA, C(o)t-1 enhanced hybridization (2.