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Calum MacAulay

Publications and source records attributed to Calum MacAulay.

At least 19 recordsLinked to original sources
PubMed2006

High resolution analysis of non-small cell lung cancer cell lines by whole genome tiling path array CGH.

Chromosomal regions harboring tumor suppressors and oncogenes are often deleted or amplified. Array comparative genomic hybridization detects segmental DNA copy number alterations in tumor DNA relative to a normal control. The recent development of a bacterial artificial chromosome array, which spans the human genome in a tiling path manner with >32,000 clones, has facilitated whole genome profiling at an unprecedented resolution. Using this technology, we comprehensively describe and compare the genomes of 28 commonly used non-small cell lung carcinoma (NSCLC) cell models, derived from 18 adenocarcinomas (AC), 9 squamous cell carcinomas and 1 large cell carcinoma. Analysis at such resolution not only provided a detailed genomic alteration template for each of these model cell lines, but revealed novel regions of frequent duplication and deletion. Significantly, a detailed analysis of chromosome 7 identified 6 distinct regions of alterations across this chromosome, implicating the presence of multiple novel oncogene loci on this chromosome. As well, a comparison between the squamous and AC cells revealed alterations common to both subtypes, such as the loss of 3p and gain of 5p, in addition to multiple hotspots more frequently associated with only 1 subtype. Interestingly, chromosome 3q, which is known to be amplified in both subtypes, showed 2 distinct regions of alteration, 1 frequently altered in squamous and 1 more frequently altered in AC. In summary, our data demonstrate the unique information generated by high resolution analysis of NSCLC genomes and uncover the presence of genetic alterations prevalent in the different NSCLC subtypes.

Carcinoma, Non-Small-Cell Lung↗
PubMed2006

Epigenomics: mapping the methylome.

DNA methylation is integral to normal development and disease processes. However, the genomic distribution of methylated sequences--the methylome--is poorly understood. We have recently developed a platform technology for rapid assessment of methylation status throughout the human genome in a high-resolution, high-throughput manner. This is achieved by coupling a methylated DNA immunoprecipitation (MeDIP) method for isolating methyl cytosine rich fragments with array-based comparative genomic hybridization (array CGH). Using a combination of whole genome tiling path BAC arrays and CpG island microarrays, DNA methylation profiles are obtained simultaneously at both genome-wide and locus-specific levels. A comparison between male and female DNA using MeDIP-array CGH revealed unexpected hypomethylation of the inactive x-chromosome in gene-poor regions. Furthermore, comparisons between cancer and noncancer cell types yielded differential methylation patterns that link genetic and epigenetic instability offering a new approach to decipher misregulation in cancer. Finally, we provide new data showing epigenomic instability in lung cancer cells with concurrent regions of genetic and epigenetic alterations harboring known oncogenes.

Chromatin Immunoprecipitation↗
PubMed2006

Gain of a region on 7p22.3, containing MAD1L1, is the most frequent event in small-cell lung cancer cell lines.

Small-cell lung cancer (SCLC) is a highly aggressive lung neoplasm, which accounts for 20% of yearly lung cancer cases. The lack of knowledge of the progenitor cell type for SCLC precludes the definition of a normal gene expression profile and has hampered the identification of gene expression changes, while the low resolution of conventional genomic screens such as comparative genomic hybridization (CGH) and loss of heterozygosity analysis limit our ability to fine-map genetic alterations. The recent advent of whole genome tiling path array CGH enables profiling of segmental DNA copy number gains and losses at a resolution 100 times that of conventional methods. Here we report the analysis of 14 SCLC cell lines and six matched normal B-lymphocyte lines. We detected 7p22.3 copy number gain in 13 of the 14 SCLC lines and 0 of the 6 matched normal lines. In 4 of the 14 cell lines, this gain is present as a 350 kbp gene specific copy number gain centered at MAD1L1 (the human homologue of the yeast gene MAD1). Fluorescence in situ hybridization validated the array CGH finding. Intriguingly, MAD1L1 has been implicated to have tumor-suppressing functions. Our data suggest a more complex role for this gene, as MAD1L1 is the most frequent copy number gain in SCLC cell lines.

B-Lymphocytes↗
PubMed2005

A stepwise framework for the normalization of array CGH data.

BACKGROUND: In two-channel competitive genomic hybridization microarray experiments, the ratio of the two fluorescent signal intensities at each spot on the microarray is commonly used to infer the relative amounts of the test and reference sample DNA levels. This ratio may be influenced by systematic measurement effects from non-biological sources that can introduce biases in the estimated ratios. These biases should be removed before drawing conclusions about the relative levels of DNA. The performance of existing gene expression microarray normalization strategies has not been evaluated for removing systematic biases encountered in array-based comparative genomic hybridization (CGH), which aims to detect single copy gains and losses typically in samples with heterogeneous cell populations resulting in only slight shifts in signal ratios. The purpose of this work is to establish a framework for correcting the systematic sources of variation in high density CGH array images, while maintaining the true biological variations. RESULTS: After an investigation of the systematic variations in the data from two array CGH platforms, SMRT (Sub Mega base Resolution Tiling) BAC arrays and cDNA arrays of Pollack et al., we have developed a stepwise normalization framework integrating novel and existing normalization methods in order to reduce intensity, spatial, plate and background biases. We used stringent measures to quantify the performance of this stepwise normalization using data derived from 5 sets of experiments representing self-self hybridizations, replicated experiments, detection of single copy changes, array CGH experiments which mimic cell population heterogeneity, and array CGH experiments simulating different levels of gene amplifications and deletions. Our results demonstrate that the three-step normalization procedure provides significant improvement in the sensitivity of detection of single copy changes compared to conventional single step normalization approaches in both SMRT BAC array and cDNA array platforms. CONCLUSION: The proposed stepwise normalization framework preserves the minute copy number changes while removing the observed systematic biases.

Calibration↗
PubMed2005

Chromosome 5p aberrations are early events in lung cancer: implication of glial cell line-derived neurotrophic factor in disease progression.

Lung cancer is the most widely diagnosed malignancy in the world. Understanding early-stage disease will give insight into its pathogenesis. Despite the fact that pre-invasive lesions are challenging to isolate, and often yield insufficient DNA for the analysis of multiple loci, genomic profiling of such lesions will lead to the discovery of causal genetic alterations, which may be otherwise masked by the gross instability associated with tumors. In this study, we report the identification of multiple early genetic events on chromosome 5p in lung cancer progression. Using a high-resolution 5p-specific genomic array, which contains a tiling path of DNA segments for comparative genomic hybridization, nine novel minimal regions of loss and gain were discovered in bronchial carcinoma in situ (CIS) specimens. Within these regions we identified two candidate genes novel to lung cancer. The 0.27 Mbp region at 5p15.2 contains a single gene, Triple Functional Domain, which we determined to be differentially expressed in tumors. The 0.34 Mbp region at 5p13.2 contains Glial Cell Line-Derived Neurotrophic Factor (GDNF), which is a ligand for the RET oncogene product and is normally expressed during lung development (but absent in adult lung tissue). Our data showed not only that GDNF is overexpressed at the transcript level in squamous non-small-cell lung carcinoma, but also that the GDNF protein is present in early-stage lesions. Reactivation of the fetal lung expressed GDNF in early lesions and its amplification in CIS suggests an early role in tumorigenesis. These results highlight the value of examining the genomes of pre-invasive stages of cancer at tiling resolution.

Chromosome Deletion↗
PubMed2005

High-resolution array CGH increases heterogeneity tolerance in the analysis of clinical samples.

Recent advances in array comparative genomic hybridization (array CGH) technology are revolutionizing our understanding of tumor genomes. Marker-based arrays enable rapid survey at megabase intervals, while tiling path arrays examine the entire genome in unprecedented detail. Tumor biopsies are typically small and contain infiltrating stromal cells, requiring tedious microdissection. Tissue heterogeneity is a major barrier to high-throughput profiling of tumor genomes and is also an important consideration for the introduction of array CGH to clinical settings. We propose that increasing array resolution will enhance detection sensitivity in mixed tissues and as a result significantly reduce microdissection requirements. In this study, we first simulated normal cell contamination to determine the heterogeneity tolerance of array CGH and then validated this detection sensitivity model on cancer specimens using the newly developed submegabase resolution tiling-set (SMRT) array, which spans the human genome with 32,433 overlapping BAC clones.

Biomarkers, Tumor↗
PubMed2005

Spectrally programmable light engine for in vitro or in vivo molecular imaging and spectroscopy.

A spectrally and temporally programmable light engine can create any spectral profile for hyperspectral, fluorescence, or principal-component imaging or with medical photonics devices employing spectroscopy, microscopy, and endoscopy. Multispectral imaging feasibility was demonstrated by capturing nine images at wavelengths from 450 to 650 mm (25-nm FWHM) with a CCD-camera-equipped bronchoscope coupled to the light engine. Selected wavelength regions were combined to produce a color endoscopy image.

Bronchoscopes↗
PubMed2005

Optical technologies for cervical neoplasia: update of an NCI program project grant.

Cervical cancer is the second most common cancer in women worldwide and the leading cause of cancer mortality in women in developing countries. In the United States, over $6 billion is spent annually in the evaluation and treatment of low-grade lesions, many of which do not develop into full-blown cancer. In developing countries, however, the chief concern is that cervical cancer goes undetected because of the cost of testing and the lack of resources and trained personnel to screen and diagnose the disease. The goal of the National Cancer Institute Program Project Grant CA82710 is to assess the emerging technologies of fluorescence and reflectance spectroscopy and quantitative cytology and histopathology for the diagnosis of cervical neoplasia. All of these technologies should decrease mortality, morbidity, and the cost of treating cervical cancer.

Computational Biology↗
PubMed2004

A tiling resolution DNA microarray with complete coverage of the human genome.

We constructed a tiling resolution array consisting of 32,433 overlapping BAC clones covering the entire human genome. This increases our ability to identify genetic alterations and their boundaries throughout the genome in a single comparative genomic hybridization (CGH) experiment. At this tiling resolution, we identified minute DNA alterations not previously reported. These alterations include microamplifications and deletions containing oncogenes, tumor-suppressor genes and new genes that may be associated with multiple tumor types. Our findings show the need to move beyond conventional marker-based genome comparison approaches, that rely on inference of continuity between interval markers. Our submegabase resolution tiling set for array CGH (SMRT array) allows comprehensive assessment of genomic integrity and thereby the identification of new genes associated with disease.

Chromosomes, Artificial, Bacterial↗
PubMed2004

SeeGH--a software tool for visualization of whole genome array comparative genomic hybridization data.

BACKGROUND: Array comparative genomic hybridization (CGH) is a technique which detects copy number differences in DNA segments. Complete sequencing of the human genome and the development of an array representing a tiling set of tens of thousands of DNA segments spanning the entire human genome has made high resolution copy number analysis throughout the genome possible. Since array CGH provides signal ratio for each DNA segment, visualization would require the reassembly of individual data points into chromosome profiles. RESULTS: We have developed a visualization tool for displaying whole genome array CGH data in the context of chromosomal location. SeeGH is an application that translates spot signal ratio data from array CGH experiments to displays of high resolution chromosome profiles. Data is imported from a simple tab delimited text file obtained from standard microarray image analysis software. SeeGH processes the signal ratio data and graphically displays it in a conventional CGH karyotype diagram with the added features of magnification and DNA segment annotation. In this process, SeeGH imports the data into a database, calculates the average ratio and standard deviation for each replicate spot, and links them to chromosome regions for graphical display. Once the data is displayed, users have the option of hiding or flagging DNA segments based on user defined criteria, and retrieve annotation information such as clone name, NCBI sequence accession number, ratio, base pair position on the chromosome, and standard deviation. CONCLUSIONS: SeeGH represents a novel software tool used to view and analyze array CGH data. The software gives users the ability to view the data in an overall genomic view as well as magnify specific chromosomal regions facilitating the precise localization of genetic alterations. SeeGH is easily installed and runs on Microsoft Windows 2000 or later environments.

Chromosome Mapping↗
PubMed2004

Exploratory analysis of quantitative histopathology of cervical intraepithelial neoplasia: objectivity, reproducibility, malignancy-associated changes, and human papillomavirus.

BACKGROUND: As part of a project to evaluate emerging optical technologies for cervical neoplasia, our group is performing quantitative histopathological analyses of biopsy specimens from 1,190 patients. Objectives in the interim analysis are (a) quantitatively assessing progression of the neoplastic process of cervical intraepithelial neoplasia (CIN)/squamous intraepithelial lesions (SIL), (b) detecting malignancy-associated changes (MACs), and (c) phenotypically measuring human papillomavirus (HPV) detected by DNA testing. METHODS: The diagnostic region of interest (ROI) from immediately adjacent sections were imaged, and the basal lamina and surface of the superficial layer were delimited. Nonoverlapping quantitatively stained nuclei were selected from 1,190 samples with histopathological characteristics of normal (929), koilocytosis (130), CIN 1 (40), CIN 2 (23), and CIN 3/carcinoma in situ (CIS) (68). A fully automatic procedure located and recorded the center of every nucleus in the region of interest (ROI). We used linear discriminant analysis to assess the changes between normal and CIN 3/CIS. RESULTS: Scores computed from the cell-by cell features and the clinical grade of CIN/SIL were highly correlated, as were those of the architectural features and the clinical grade of CIN/SIL. We found even higher correlations between a combination of cell-by-cell and architectural scores, and clinical grade. Using these scores, we found MACs in the normal biopsy specimens from patients with high-grade CIN/SIL. Furthermore, the same scores correlated with the molecular detection of HPV. CONCLUSIONS: Quantitative histopathology can be used in large clinical trials as an objective and reproducible measure of CIN/SIL. Detectable phenotypic changes correlate well with CIN/SIL neoplastic progression. It can also be used to infer the presence of CIN/SIL (MACs) and molecular changes associated with increased risk of cancer development (high-risk HPV).

DNA, Viral↗
PubMed2004

In vivo pathology: microendoscopy as a new endoscopic imaging modality.

Confocal microendoscopy permits direct observation of pathologic change at the microscopic level rather than traditional inference based on indirect changes at the macroscopic (cell) level. The main benefit includes earlier detection of precancerous and cancer conditions through improved biopsy selection and examination and more cost-effective solutions to screening and surveillance. Numerous outstanding research and commercial groups with varying approaches to confocal microendoscopy are allocating significant efforts to making the technology commercially available. The initial instruments will likely be geared toward screening for and surveillance of esophageal and colon-related conditions. Future developments related to greater functionality, improved ease of use, and automated analysis are likely to facilitate adoption and use of the technology. Clinical gastroenterologists should look forward to the potential of confocal microendoscopy as a logical and needed modality to advance the field of gastroenterology.

Animals↗
PubMed2004

Quantitative histopathology and chromosome 9 polysomy in a clinical trial of 4-HPR.

OBJECTIVE: This trial examined the use of 4-hydroxyphenyl-retinamide (4-HPR), demonstrated to be a potent inhibitor of carcinogenesis in vitro and in animal models, in patients with cervical intraepithelial neoplasia (CIN) grades 2 to 3. Quantitative pathology and chromosome 9 polysomy were used to understand the biology and quantify the clinical histopathologic changes observed. METHODS: Patients were randomized to 4-HPR or placebo for 6 months and followed for six more months. Cervical biopsies were obtained at baseline, 6 months, and 12 months; the biopsies were read blinded three times by the study pathologist. Feulgen-stained sections were also obtained and analyzed using computer-assisted image cytometry. Chromosome 9 polysomy was performed on tissue slices using in situ hybridization and measured quantitatively. Statistical analyses were carried out in S-Plus (Insightful Corporation, Seattle, WA) and R. RESULTS: The interim analysis, planned for 40 patients, was carried out on 39. The 6- and 12-month analyses showed a statistically significant difference between the two study arms. When code was broken, the 4-HPR-treatment arm was found to have fared less well than placebo. Analyses of Feulgen-stained sections provided a quantitative measure of the increase of DNA content and texture features. Chromosome 9 polysomy was also measured using image analysis. The changes observed were consistent with those of cells displaying cancerous changes, indicating a lack of response. CONCLUSION: 4-HPR is not active at 200 mg/day. The interim analysis was helpful in directing the study; and, in this case, ending it. The intermediate endpoint biomarkers of quantitative histomorphometry and chromosome 9 polysomy yielded quantitative and repeatable results consistent with the findings of the clinical pathologist.

Anticarcinogenic Agents↗
PubMed2004

Optical computed-tomographic microscope for three-dimensional quantitative histology.

A novel optical computed-tomographic microscope has been developed allowing quantitative three-dimensional (3D) imaging and analysis of fixed pathological material. Rather than a conventional two-dimensional (2D) image, the instrument produces a 3D representation of fixed absorption-stained material, from which quantitative histopathological features can be measured more accurately. The accurate quantification of these features is critically important in disease diagnosis and the clinical classification of cancer. The system consists of two high NA objective lenses, a light source, a digital spatial light modulator (DMD, by Texas Instrument), an x-y stage, and a CCD detector. The DMD, positioned at the back pupil-plane of the illumination objective, is employed to illuminate the specimen with parallel rays at any desired angle. The system uses a modification of the convolution backprojection algorithm for reconstruction. In contrast to fluorescent images acquired by a confocal microscope, this instrument produces 3D images of absorption stained material. Microscopic 3D volume reconstructions of absorption-stained cells have been demonstrated. Reconstructed 3D images of individual cells and tissue can be cut virtually with the distance between the axial slices less than 0.5 microm.

Algorithms↗
PubMed2004

Quantitative histopathological analysis of cervical intra-epithelial neoplasia sections: methodological issues.

OBJECTIVES: As part a Program Project to evaluate emerging optical technologies for cervical neoplasia, our group is performing quantitative histopathological analysis of biopsies from 1,800 patients. Several methodological issues have arisen with respect to this analysis: (1) Finding the most efficient way to compensate for staining intensity variation with out losing diagnostic information; (2) Assessing the inter- and intra-observer variability of the semi-interactive data collection; and (3) the use of non-overlapping cells from the intermediate layer only. METHODS: Non-overlapping quantitatively stained nuclei were selected from 280 samples with histopathological characteristics of normal (199), koilocytosis (37), CIN 1 (18), CIN 2 (10) and CIN 3 (16). Linear discriminant analysis was used to assess the diagnostic information in three different feature sets to evaluate and compare staining intensity normalization methods. Selected feature values and summary scores were used to evaluate intra- and inter-observer variability. RESULTS: The features normalized by the internal subset of the imaged cells had the same discriminatory power as those normalized by the control cells and by both normalization methods seem to have additional discriminatory power over the set of features which do not require normalization. The use of the internal subset decreased the image acquisition time by approximately 50% at each center, respectively. The intra- and inter-observer variability was of a similar size. Good performance was obtained by measuring the intermediate layer only. CONCLUSION: The use of intensity normalization from a subset of the imaged non-overlapping intermediate layer cells works as well as or better than any of the other methods tested and provides a significant timesaving. Our intra- and inter-observer variability do not seem to affect the diagnostic power of the data. Although this must be tested in a larger data set, the use of intermediate layer cells only may be acceptable when using quantitative histopathology.

Cell Nucleus↗
PubMed2004

Quality assurance system using statistical process control: an implementation for image cytometry.

AIMS: Optical technologies have shown some promise for improving the care of cervical neoplasia. We are currently evaluating fluorescence and reflectance spectroscopy and quantitative cyto-histopathology for cervical neoplasia screening and diagnosis. Here we describe the establishment and application of a quality assurance (QA) system for detecting system malfunctions and assessing the comparability of four image cytometers used in a multicenter clinical trial. METHODS: Our QA system involves three levels of evaluation based on the periodicity and complexity of the measurements. We implemented our QA system at three image cytometers at the British Columbia Cancer Agency and one at M.D. Anderson Cancer Center. The measurements or tasks were performed daily, monthly, and semi-annually. The current and voltage of the lamp, the calibration image characteristics, and the room temperature were checked daily. Long-term stability over time, short-term variability over time, and spatial response field uniformity were evaluated monthly. Camera linearity was measured semi-annually. Control charts based on statistical process control techniques were used to detect when the system did not perform optimally. RESULTS: Daily measurements have shown good consistency in room temperature, lamp and calibration behaviour. Monthly measurements have shown small coefficients of variation between and within the four devices. There have been greater differences between sessions than within sessions. Comparability among the four systems is reasonably good. Semi-annual measurements have shown stable camera linearity. QA events were detected using the QA system. Multiple examples of event detection leading to correction of system malfunction are described in this report. CONCLUSIONS: QA programs are critical for ensuring data integrity and therefore for the conduct of multicenter clinical trials.

Female↗