Allele-specific disparity in breast cancer

Background

Molecular alterations critical to development of cancer include mutations, copy number alterations (amplifications and deletions) as well as genomic rearrangements resulting in gene fusions. Massively parallel next generation sequencing, which enables the discovery of such changes, uses considerable quantities of genomic DNA (> 5 ug), a serious limitation in ever smaller clinical samples. However, a commonly available microarray platforms such as array comparative genomic hybridization (array CGH) allows the characterization of gene copy number at a single gene resolution using much smaller amounts of genomic DNA. In this study we evaluate the sensitivity of ultra-dense array CGH platforms developed by Agilent, especially that of the 1 million probe array (1 M array), and their application when whole genome amplification is required because of limited sample quantities.

Methods

We performed array CGH on whole genome amplified and not amplified genomic DNA from MCF-7 breast cancer cells, using 244 K and 1 M Agilent arrays. The ADM-2 algorithm was used to identify micro-copy number alterations that measured less than 1 Mb in genomic length.

Results

DNA from MCF-7 breast cancer cells was analyzed for micro-copy number alterations, defined as measuring less than 1 Mb in genomic length. The 4-fold extra resolution of the 1 M array platform relative to the less dense 244 K array platform, led to the improved detection of copy number variations (CNVs) and micro-CNAs. The identification of intra-genic breakpoints in areas of DNA copy number gain signaled the possible presence of gene fusion events. However, the ultra-dense platforms, especially the densest 1 M array, detect artifacts inherent to whole genome amplification and should be used only with non-amplified DNA samples.

Conclusions

This is a first report using 1 M array CGH for the discovery of cancer genes and biomarkers. We show the remarkable capacity of this technology to discover CNVs, micro-copy number alterations and even gene fusions. However, these platforms require excellent genomic DNA quality and do not tolerate relatively small imperfections related to the whole genome amplification.     

High-resolution array comparative genomic hybridization of single micrometastatic tumor cells

Only few selected cancer cells drive tumor progression and are responsible for therapy resistance. Their specific genomic characteristics, however, are largely unknown because high-resolution genome analysis is currently limited to DNA pooled from many cells. Here, we describe a protocol for array comparative genomic hybridization (array CGH), which enables the detection of DNA copy number changes in single cells. Combining a PCR-based whole genome amplification method with arrays of highly purified BAC clones we could accurately determine known chromosomal changes such as trisomy 21 in single leukocytes as well as complex genomic imbalances of single cell line cells. In single T47D cells aberrant regions as small as 1–2 Mb were identified in most cases when compared to non-amplified DNA from 10⁶ cells. Most importantly, in single micrometastatic cancer cells isolated from bone marrow of breast cancer patients, we retrieved and confirmed amplifications as small as 4.4 and 5 Mb. Thus, high-resolution genome analysis of single metastatic precursor cells is now possible and may be used for the identification of novel therapy target genes.     

A Robust Method to Analyze Copy Number Alterations of Less than 100 kb in Single Cells Using Oligonucleotide Array CGH

Comprehensive genome wide analyses of single cells became increasingly important in cancer research, but remain to be a technically challenging task. Here, we provide a protocol for array comparative genomic hybridization (aCGH) of single cells. The protocol is based on an established adapter-linker PCR (WGAM) and allowed us to detect copy number alterations as small as 56 kb in single cells. In addition we report on factors influencing the success of single cell aCGH downstream of the amplification method, including the characteristics of the reference DNA, the labeling technique, the amount of input DNA, reamplification, the aCGH resolution, and data analysis. In comparison with two other commercially available non-linear single cell amplification methods, WGAM showed a very good performance in aCGH experiments. Finally, we demonstrate that cancer cells that were processed and identified by the CellSearch® System and that were subsequently isolated from the CellSearch® cartridge as single cells by fluorescence activated cell sorting (FACS) could be successfully analyzed using our WGAM-aCGH protocol. We believe that even in the era of next-generation sequencing, our single cell aCGH protocol will be a useful and (cost-) effective approach to study copy number alterations in single cells at resolution comparable to those reported currently for single cell digital karyotyping based on next generation sequencing data.     

Cytogenetically balanced translocations are associated with focal copy number alterations

Current cytogenetic methods (e.g., G-banding and multicolor chromosomal painting) allow detection of translocation events but lack the resolution to (a) locate the breakpoints precisely at the chromosome band level or (b) discriminate balanced translocations from translocations with copy number alterations not previously reported, or imperfectly balanced translocations. In this study, we demonstrate that cytogenetically balanced translocations are in fact frequently associated with segmental gain or loss of DNA. The recent development of a whole genome tiling path BAC array has enabled tiling resolution analysis of genomic segmental copy number status. Combining tiling resolution BAC array comparative genomic hybridization (array CGH) with G-Banding analysis and multicolor chromosomal painting approaches such as spectral karyotyping (SKY) facilitates high-resolution mapping of genomic alterations associated with imperfectly balanced translocations. Using a refined version of our CGH array we have deduced the copy number status throughout the genomes of three cytogenetically well-characterized prostate cancer cell lines (PC3, DU145, LNCaP) to determine whether translocations are associated with focal gains and losses of DNA. At 78 kb tiling resolution we identified the boundaries of 170, 80, and 34 known and novel copy number alterations (CNA) in these cell line genomes, respectively. Thirty-three of the 36 known translocations (92%, P < 0.001) in DU145 were associated with segmental CNA. Likewise, 80% (P < 0.001) of the known translocations showed association in LNCaP. Although many translocation breakpoints exhibit segmental alteration in PC3, the pattern of chromosomal rearrangements is too complex for use in comprehensive association with CNA boundaries. Our results reveal that imperfectly balanced translocations in tumor genomes are a phenomenon that occurs at frequencies much higher than previously demonstrated. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Detection of single base alterations in genomic DNA by solid phase polymerase chain reaction on oligonucleotide microarrays.

DNA microarray technology holds significant promise for human DNA diagnostics. A number of technical approaches directed at the parallel identification of mutations or single nucleotide polymorphisms make use of polymerase-based specificity, like minisequencing or allele-specific primer elongation. These techniques, however, require separate laborious sample amplification, preparation, and purification steps, making large-scale analyses time and cost consuming. Here, we address this challenge by applying an experimental setup using simultaneous solid and liquid phase PCR on polyethyleneimine-coated glass slides, a novel microarray support allowing on-chip amplification reactions with exquisite specificity. A gene-specific oligonucleotide tiling array contains covalently attached allele-specific primers which interrogate single nucleotide positions within a genomic region of interest. During a thermal cycling reaction amplification products remain covalently bound to the solid support and can be visualized and analyzed by the incorporation of fluorescent dyes. Using the described procedure we unequivocally defined the presence of point mutations in the human tumor suppressor gene p53 directly from a natural DNA source. This semi-multiplex solid phase amplification format allowed the rapid and correct identification of 20 nucleotide positions from minute amounts of human genomic DNA. Our results suggest that this approach might constitute a vital component of future integrated DNA chip devices used in gene analysis.     

High resolution array-CGH analysis of single cells

Heterogeneity in the genome copy number of tissues is of particular importance in solid tumor biology. Furthermore, many clinical applications such as pre-implantation and non-invasive prenatal diagnosis would benefit from the ability to characterize individual single cells. As the amount of DNA from single cells is so small, several PCR protocols have been developed in an attempt to achieve unbiased amplification. Many of these approaches are suitable for subsequent cytogenetic analyses using conventional methodologies such as comparative genomic hybridization (CGH) to metaphase spreads. However, attempts to harness array-CGH for single-cell analysis to provide improved resolution have been disappointing. Here we describe a strategy that combines single-cell amplification using GenomePlex library technology (GenomePlex^® Single Cell Whole Genome Amplification Kit, Sigma-Aldrich, UK) and detailed analysis of genomic copy number changes by high-resolution array-CGH. We show that single copy changes as small as 8.3 Mb in single cells are detected reliably with single cells derived from various tumor cell lines as well as patients presenting with trisomy 21 and Prader–Willi syndrome. Our results demonstrate the potential of this technology for studies of tumor biology and for clinical diagnostics.     

Genome-wide mapping of copy number variation in humans: comparative analysis of high resolution array platforms

Accurate and efficient genome-wide detection of copy number variants (CNVs) is essential for understanding human genomic variation, genome-wide CNV association type studies, cytogenetics research and diagnostics, and independent validation of CNVs identified from sequencing based technologies. Numerous, array-based platforms for CNV detection exist utilizing array Comparative Genome Hybridization (aCGH), Single Nucleotide Polymorphism (SNP) genotyping or both. We have quantitatively assessed the abilities of twelve leading genome-wide CNV detection platforms to accurately detect Gold Standard sets of CNVs in the genome of HapMap CEU sample NA12878, and found significant differences in performance. The technologies analyzed were the NimbleGen 4.2 M, 2.1 M and 3×720 K Whole Genome and CNV focused arrays, the Agilent 1×1 M CGH and High Resolution and 2×400 K CNV and SNP+CGH arrays, the Illumina Human Omni1Quad array and the Affymetrix SNP 6.0 array. The Gold Standards used were a 1000 Genomes Project sequencing-based set of 3997 validated CNVs and an ultra high-resolution aCGH-based set of 756 validated CNVs. We found that sensitivity, total number, size range and breakpoint resolution of CNV calls were highest for CNV focused arrays. Our results are important for cost effective CNV detection and validation for both basic and clinical applications.     

Single-nucleotide polymorphism array coupled with multiple displacement amplification: accuracy and spatial resolution for analysis of chromosome copy numbers in few cells

When coupled with multiple displacement amplification (MDA), microarray-based comparative genomic intensity allows detection of chromosome copy number aberrations even in single or few cells, but the actual performance of the system and their influencing factors have not been well defined. Here, using single-nucleotide polymorphism (SNP) array, we analyzed copy number profiles from DNA amplified by MDA in 1-10 cells and estimated the accuracy and spatial resolution of the analysis. Based on the concordance of SNP copy numbers for DNA with and without MDA, the accuracy of the system can be significantly enhanced by using MDA-amplified DNA as reference and also by increasing the cell numbers. Analyses under different smoothing treatments revealed a practical resolution of 2?Mb for 10 cells and 10?Mb for a single cell. When both cells with known chromosomal duplication and deletion were analyzed, this platform detected a copy number "loss" more accurately than a "gain" (P < 0.01), particularly in single-cell MDA products. Together, we demonstrated that SNP array coupled with MDA is reliable and efficient for detection of copy number aberrations in a small number of cells, and its accuracy and resolution can both be significantly enhanced with increasing the number of cells as MDA template.     

Impact of whole genome amplification on analysis of copy number variants

Large-scale copy number variants (CNVs) have recently been recognized to play a role in human genome variation and disease. Approaches for analysis of CNVs in small samples such as microdissected tissues can be confounded by limited amounts of material. To facilitate analyses of such samples, whole genome amplification (WGA) techniques were developed. In this study, we explored the impact of Phi29 multiple-strand displacement amplification on detection of CNVs using oligonucleotide arrays. We extracted DNA from fresh frozen lymph node samples and used this for amplification and analysis on the Affymetrix Mapping 500k SNP array platform. We demonstrated that the WGA procedure introduces hundreds of potentially confounding CNV artifacts that can obscure detection of bona fide variants. Our analysis indicates that many artifacts are reproducible, and may correlate with proximity to chromosome ends and GC content. Pair-wise comparison of amplified products considerably reduced the number of apparent artifacts and partially restored the ability to detect real CNVs. Our results suggest WGA material may be appropriate for copy number analysis when amplified samples are compared to similarly amplified samples and that only the CNVs with the greatest significance values detected by such comparisons are likely to be representative of the unamplified samples.     

Allelic imbalance analysis of oral tongue squamous cell carcinoma by high-density single nucleotide polymorphism arrays using whole-genome amplified DNA

Multiple displacement-based whole-genome DNA amplification is a promising tool to obtain sufficient DNA from small tissue specimens for various genetic analyses, such as SNP array-based analysis. Using Affymetrix 10 K and 100 K SNP mapping array, we evaluated the performance of the Phi29 DNA polymerase-based genome amplification. Greater than 99% concordance in genotyping calls were achieved between amplified and non-amplified DNAs for both arrays. By utilizing the Affymetrix GeneChip Chromosome Copy Number Tool, the allelic imbalance profiles for the advanced stage oral tongue squamous cell carcinoma (OTSCC) were generated based on 10 K and 100 K SNP mapping array results. The results from these two array platforms agree closely, but more precise allelic imbalance patterns can be revealed from the 100 K SNP mapping array data. Furthermore, our data suggested a frequent loss at 3p11–p12 for advanced stage OTSCC.     

wuHMM: a robust algorithm to detect DNA copy number variation using long oligonucleotide microarray data

Copy number variants (CNVs) are currently defined as genomic sequences that are polymorphic in copy number and range in length from 1000 to several million base pairs. Among current array-based CNV detection platforms, long-oligonucleotide arrays promise the highest resolution. However, the performance of currently available analytical tools suffers when applied to these data because of the lower signal:noise ratio inherent in oligonucleotide-based hybridization assays. We have developed wuHMM, an algorithm for mapping CNVs from array comparative genomic hybridization (aCGH) platforms comprised of 385 000 to more than 3 million probes. wuHMM is unique in that it can utilize sequence divergence information to reduce the false positive rate (FPR). We apply wuHMM to 385K-aCGH, 2.1M-aCGH and 3.1M-aCGH experiments comparing the 129X1/SvJ and C57BL/6J inbred mouse genomes. We assess wuHMM's performance on the 385K platform by comparison to the higher resolution platforms and we independently validate 10 CNVs. The method requires no training data and is robust with respect to changes in algorithm parameters. At a FPR of <10%, the algorithm can detect CNVs with five probes on the 385K platform and three on the 2.1M and 3.1M platforms, resulting in effective resolutions of 24 kb, 2–5 kb and 1 kb, respectively.     

Comparison of sample preparation methods for ChIP-chip assays

A single chromatin immunoprecipitation (ChIP) sample does not provide enough DNA for hybridization to a genomic tiling array. A commonly used technique for amplifying the DNA obtained from ChIP assays is ligation-mediated PCR (LM-PCR). However; using this amplification method, we could not identify Oct4 binding sites on genomic tiling arrays representing 1% of the human genome (ENCODE arrays). In contrast, hybridization of a pool of 10 ChIP samples to the arrays produced reproducible binding patterns and low background signals. However the pooling method would greatly increase the number of ChIP reactions needed to analyze the entire human genome. Therefore, we have adapted the GenomePlex whole genome amplification (WGA) method for use in ChIP-chip assays; detailed ChIP and amplification protocols used for these analyses are provided as supplementary material. When applied to ENCODE arrays, the products prepared using this new method resulted in an Oct4 binding pattern similar to that from the pooled Oct4 ChIP samples. Importantly, the signal-to-noise ratio using the GenomePlex WGA method is superior to the LM-PCR amplification method.     

Quantitative high-resolution genomic analysis of single cancer cells

During cancer progression, specific genomic aberrations arise that can determine the scope of the disease and can be used as predictive or prognostic markers. The detection of specific gene amplifications or deletions in single blood-borne or disseminated tumour cells that may give rise to the development of metastases is of great clinical interest but technically challenging. In this study, we present a method for quantitative high-resolution genomic analysis of single cells. Cells were isolated under permanent microscopic control followed by high-fidelity whole genome amplification and subsequent analyses by fine tiling array-CGH and qPCR. The assay was applied to single breast cancer cells to analyze the chromosomal region centred by the therapeutical relevant EGFR gene. This method allows precise quantitative analysis of copy number variations in single cell diagnostics.     

Duplication of CXC chemokine genes on chromosome 4q13 in a melanoma-prone family

Copy number variations (CNVs) have been shown to contribute substantially to disease susceptibility in several inherited diseases including cancer. We conducted a genome-wide search for CNVs in blood-derived DNA from 79 individuals (62 melanoma patients and 17 spouse controls) of 30 high-risk melanoma-prone families without known segregating mutations using genome-wide comparative genomic hybridization (CGH) tiling arrays. We identified a duplicated region on chromosome 4q13 in germline DNA of all melanoma patients in a melanoma-prone family with three affected siblings. We confirmed the duplication using quantitative PCR and a custom-made CGH array design spanning the 4q13 region. The duplicated region contains 10 genes, most of which encode CXC chemokines. Among them, CXCL1 (melanoma growth-stimulating activity α) and IL8 (interleukin 8) have been shown to stimulate melanoma growth in vitro and in vivo. Our data suggest that the alteration of CXC chemokine genes may confer susceptibility to melanoma.     

A Single Cell Level Based Method for Copy Number Variation Analysis by Low Coverage Massively Parallel Sequencing

Copy number variations (CNVs), a common genomic mutation associated with various diseases, are important in research and clinical applications. Whole genome amplification (WGA) and massively parallel sequencing have been applied to single cell CNVs analysis, which provides new insight for the fields of biology and medicine. However, the WGA-induced bias significantly limits sensitivity and specificity for CNVs detection. Addressing these limitations, we developed a practical bioinformatic methodology for CNVs detection at the single cell level using low coverage massively parallel sequencing. This method consists of GC correction for WGA-induced bias removal, binary segmentation algorithm for locating CNVs breakpoints, and dynamic threshold determination for final signals filtering. Afterwards, we evaluated our method with seven test samples using low coverage sequencing (4∼9.5%). Four single-cell samples from peripheral blood, whose karyotypes were confirmed by whole genome sequencing analysis, were acquired. Three other test samples derived from blastocysts whose karyotypes were confirmed by SNP-array analysis were also recruited. The detection results for CNVs of larger than 1 Mb were highly consistent with confirmed results reaching 99.63% sensitivity and 97.71% specificity at base-pair level. Our study demonstrates the potential to overcome WGA-bias and to detect CNVs (>1 Mb) at the single cell level through low coverage massively parallel sequencing. It highlights the potential for CNVs research on single cells or limited DNA samples and may prove as a promising tool for research and clinical applications, such as pre-implantation genetic diagnosis/screening, fetal nucleated red blood cells research and cancer heterogeneity analysis.     

Phenotypically concordant and discordant monozygotic twins display different DNA copy-number-variation profiles

The exploration of copy-number variation (CNV), notably of somatic cells, is an understudied aspect of genome biology. Any differences in the genetic makeup between twins derived from the same zygote represent an irrefutable example of somatic mosaicism. We studied 19 pairs of monozygotic twins with either concordant or discordant phenotype by using two platforms for genome-wide CNV analyses and showed that CNVs exist within pairs in both groups. These findings have an impact on our views of genotypic and phenotypic diversity in monozygotic twins and suggest that CNV analysis in phenotypically discordant monozygotic twins may provide a powerful tool for identifying disease-predisposition loci. Our results also imply that caution should be exercised when interpreting disease causality of de novo CNVs found in patients based on analysis of a single tissue in routine disease-related DNA diagnostics.     

Genomic profiling by DNA amplification of laser capture microdissected tissues and array CGH

Comparative genomic hybridization by means of BAC microarrays (array CGH) allows high-resolution profiling of copy-number aberrations in tumor DNA. However, specific genetic lesions associated with small but clinically relevant tumor areas may pass undetected due to intra-tumor heterogeneity and/or the presence of contaminating normal cells. Here, we show that the combination of laser capture microdissection, 29 DNA polymerase-mediated isothermal genomic DNA amplification, and array CGH allows genomic profiling of very limited numbers of cells. Moreover, by means of simple statistical models, we were able to bypass the exclusion of amplification distortions and variability prone areas, and to detect tumor-specific chromosomal gains and losses. We applied this new combined experimental and analytical approach to the genomic profiling of colorectal adenomatous polyps and demonstrated our ability to accurately detect single copy gains and losses affecting either whole chromosomes or small genomic regions from as little as 2 ng of DNA or 1000 microdissected cells.     

High resolution array in the clinical approach to chromosomal phenotypes

Array genomic hybridization (AGH) has recently been implemented as a diagnostic tool for the detection of submicroscopic copy number variants (CNVs) in patients with developmental disorders. However, there is no consensus regarding the choice of the platform, the minimal resolution needed and systematic interpretation of CNVs. We report our experience in the clinical diagnostic use of high resolution AGH up to 100 kb on 131 patients with chromosomal phenotypes but previously normal karyotype. We evaluated the usefulness in our clinics and laboratories by the detection rate of causal CNVs and CNVs of unknown clinical significance and to what extent their interpretation would challenge the systematic use of high-resolution arrays in clinical application. Prioritizing phenotype-genotype correlation in our interpretation strategy to criteria previously described, we identified 33 (25.2%) potentially pathogenic aberrations. 16 aberrations were confirmed pathogenic (16.4% syndromic, 8.5% non-syndromic patients); 9 were new and individual aberrations, 3 of them were pathogenic although inherited and one is as small as approx 200 kb. 13 of 16 further CNVs of unknown significance were classified likely benign, for 3 the significance remained unclear. High resolution array allows the detection of up to 12.2% of pathogenic aberrations in a diagnostic clinical setting. Although the majority of aberrations are larger, the detection of small causal aberrations may be relevant for family counseling. The number of remaining unclear CNVs is limited. Careful phenotype-genotype correlations of the individual CNVs and clinical features are challenging but remain a hallmark for CNV interpretation.