Genomic profiling using array comparative genomic hybridization define distinct subtypes of diffuse large b-cell lymphoma: a review of the literature

ABSTRACT: Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin Lymphoma comprising of greater than 30 % of adult non-Hodgkin Lymphomas. DLBCL represents a diverse set of lymphomas, defined as diffuse proliferation of large B lymphoid cells. Numerous cytogenetic studies including karyotypes and fluorescent in situ hybridization (FISH), as well as morphological, biological, clinical, microarray and sequencing technologies have attempted to categorize DLBCL into morphological variants, molecular and immunophenotypic subgroups, as well as distinct disease entities. Despite such efforts, most lymphoma remains undistinguishable and falls into DLBCL, not otherwise specified (DLBCL-NOS). The advent of microarray-based studies (chromosome, RNA, gene expression, etc) has provided a plethora of high-resolution data that could potentially facilitate the finer classification of DLBCL. This review covers the microarray data currently published for DLBCL. We will focus on these types of data; 1) array based CGH; 2) classical CGH; and 3) gene expression profiling studies. The aims of this review were three-fold: (1) to catalog chromosome loci that are present in at least 20 % or more of distinct DLBCL subtypes; a detailed list of gains and losses for different subtypes was generated in a table form to illustrate specific chromosome loci affected in selected subtypes; (2) to determine common and distinct copy number alterations among the different subtypes and based on this information, characteristic and similar chromosome loci for the different subtypes were depicted in two separate chromosome ideograms; and, (3) to list re-classified subtypes and those that remained indistinguishable after review of the microarray data. To the best of our knowledge, this is the first effort to compile and review available literatures on microarray analysis data and their practical utility in classifying DLBCL subtypes. Although conventional cytogenetic methods such as Karyotypes and FISH have played a major role in classification schemes of lymphomas, better classification models are clearly needed to further understanding the biology, disease outcome and therapeutic management of DLBCL. In summary, microarray data reviewed here can provide better subtype specific classifications models for DLBCL.     

Genomic profiling of submucosal-invasive gastric cancer by array-based comparative genomic hybridization

Genomic copy number aberrations (CNAs) in gastric cancer have already been extensively characterized by array comparative genomic hybridization (array CGH) analysis. However, involvement of genomic CNAs in the process of submucosal invasion and lymph node metastasis in early gastric cancer is still poorly understood. In this study, to address this issue, we collected a total of 59 tumor samples from 27 patients with submucosal-invasive gastric cancers (SMGC), analyzed their genomic profiles by array CGH, and compared them between paired samples of mucosal (MU) and submucosal (SM) invasion (23 pairs), and SM invasion and lymph node (LN) metastasis (9 pairs). Initially, we hypothesized that acquisition of specific CNA(s) is important for these processes. However, we observed no significant difference in the number of genomic CNAs between paired MU and SM, and between paired SM and LN. Furthermore, we were unable to find any CNAs specifically associated with SM invasion or LN metastasis. Among the 23 cases analyzed, 15 had some similar pattern of genomic profiling between SM and MU. Interestingly, 13 of the 15 cases also showed some differences in genomic profiles. These results suggest that the majority of SMGCs are composed of heterogeneous subpopulations derived from the same clonal origin. Comparison of genomic CNAs between SMGCs with and without LN metastasis revealed that gain of 11q13, 11q14, 11q22, 14q32 and amplification of 17q21 were more frequent in metastatic SMGCs, suggesting that these CNAs are related to LN metastasis of early gastric cancer. In conclusion, our data suggest that generation of genetically distinct subclones, rather than acquisition of specific CNA at MU, is integral to the process of submucosal invasion, and that subclones that acquire gain of 11q13, 11q14, 11q22, 14q32 or amplification of 17q21 are likely to become metastatic.     

Genomic profiling of rectal adenoma and carcinoma by array-based comparative genomic hybridization

Background

Aortopathies are a group of disorders characterized by aneurysms, dilation, and tortuosity of the aorta. Because of the phenotypic overlap and genetic heterogeneity of diseases featuring aortopathy, molecular testing is often required for timely and correct diagnosis of affected individuals. In this setting next generation sequencing (NGS) offers several advantages over traditional molecular techniques.

Methods

The purpose of our study was to compare NGS enrichment methods for a clinical assay targeting the nine genes known to be associated with aortopathy. RainDance emulsion PCR and SureSelect RNA-bait hybridization capture enrichment methods were directly compared by enriching DNA from eight samples. Enriched samples were barcoded, pooled, and sequenced on the Illumina HiSeq2000 platform. Depth of coverage, consistency of coverage across samples, and the overlap of variants identified were assessed. This data was also compared to whole-exome sequencing data from ten individuals.

Results

Read depth was greater and less variable among samples that had been enriched using the RNA-bait hybridization capture enrichment method. In addition, samples enriched by hybridization capture had fewer exons with mean coverage less than 10, reducing the need for followup Sanger sequencing. Variants sets produced were 77% concordant, with both techniques yielding similar numbers of discordant variants.

Conclusions

When comparing the design flexibility, performance, and cost of the targeted enrichment methods to whole-exome sequencing, the RNA-bait hybridization capture enrichment gene panel offers the better solution for interrogating the aortopathy genes in a clinical laboratory setting.     

BAC to the future! or oligonucleotides: a perspective for micro array comparative genomic hybridization (array CGH)

The array CGH technique (Array Comparative Genome Hybridization) has been developed to detect chromosomal copy number changes on a genome-wide and/or high-resolution scale. It is used in human genetics and oncology, with great promise for clinical application. Until recently primarily PCR amplified bacterial artificial chromosomes (BACs) or cDNAs have been spotted as elements on the array. The large-scale DNA isolations or PCR amplifications of the large-insert clones necessary for manufacturing the arrays are elaborate and time-consuming. Lack of a high-resolution highly sensitive (commercial) alternative has undoubtedly hindered the implementation of array CGH in research and diagnostics. Recently, synthetic oligonucleotides as arrayed elements have been introduced as an alternative substrate for array CGH, both by academic institutions as well as by commercial providers. Oligonucleotide libraries or ready-made arrays can be bought off-the-shelf saving considerable time and efforts. For RNA expression profiling, we have seen a gradual transition from in-house printed cDNA-based expression arrays to oligonucleotide arrays and we expect a similar transition for array CGH. This review compares the different platforms and will attempt to shine a light on the ‘BAC to the future’ of the array CGH technique.     

High-resolution mapping of genotype-phenotype relationships in cri du chat syndrome using array comparative genomic hybridization

We have used array comparative genomic hybridization to map DNA copy-number changes in 94 patients with cri du chat syndrome who had been carefully evaluated for the presence of the characteristic cry, speech delay, facial dysmorphology, and level of mental retardation (MR). Most subjects had simple deletions involving 5p (67 terminal and 12 interstitial). Genotype-phenotype correlations localized the region associated with the cry to 1.5 Mb in distal 5p15.31, between bacterial artificial chromosomes (BACs) containing markers D5S2054 and D5S676; speech delay to 3.2 Mb in 5p15.32-15.33, between BACs containing D5S417 and D5S635; and the region associated with facial dysmorphology to 2.4 Mb in 5p15.2-15.31, between BACs containing D5S208 and D5S2887. These results overlap and refine those reported in previous publications. MR depended approximately on the 5p deletion size and location, but there were many cases in which the retardation was disproportionately severe, given the 5p deletion. All 15 of these cases, approximately two-thirds of the severely retarded patients, were found to have copy-number aberrations in addition to the 5p deletion. Restriction of consideration to patients with only 5p deletions clarified the effect of such deletions and suggested the presence of three regions, MRI-III, with differing effect on retardation. Deletions including MRI, a 1.2-Mb region overlapping the previously defined cri du chat critical region but not including MRII and MRIII, produced a moderate level of retardation. Deletions restricted to MRII, located just proximal to MRI, produced a milder level of retardation, whereas deletions restricted to the still-more proximal MRIII produced no discernible phenotype. However, MR increased as deletions that included MRI extended progressively into MRII and MRIII, and MR became profound when all three regions were deleted.     

Epstein-Barr virus-induced gene 3 (EBI3): a novel diagnosis marker in Burkitt lymphoma and diffuse large B-cell lymphoma

The distinction between Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL), two types of mature aggressive B-cell lymphomas that require distinct treatments, can be difficult because of forms showing features intermediate between DLBCL and BL (here called BL/DLBCL). They can be discriminated by the presence of c-myc translocations characteristic of BL. However, these are not exclusive of BL and when present in DLBCL are associated with lower survival. In this study, we show that Epstein-Barr virus-induced gene 3 (EBI3) is differentially expressed among BL and DLBCL. Analysis of gene expression data from 502 cases of aggressive mature B-cell lymphomas available on Gene Expression Omnibus and immunohistochemical analysis of 184 cases of BL, BL/DLBCL or DLBCL, showed that EBI3 was not expressed in EBV-positive or -negative BL cases, whereas it was expressed by over 30% of tumoral cells in nearly 80% of DLBCL cases, independently of their subtypes. In addition, we show that c-myc overexpression represses EBI3 expression, and that DLBCL or BL/DLBCL cases with c-myc translocations have lower expression of EBI3. Thus, EBI3 immunohistochemistry could be useful to discriminate BL from DLBCL, and to identify cases of BL/DLBCL or DLBCL with potential c-myc translocations.     

DNA profiling by array comparative genomic hybridization (CGH) of peripheral blood mononuclear cells (PBMC) and tumor tissue cell in non-small cell lung cancer (NSCLC)

Lung tumor cell DNA copy number alteration (CNA) was expected to display specific patterns such as a large-scale amplification or deletion of chromosomal arms, as previously published data have reported. Peripheral blood mononuclear cell (PBMC) CNA however, was expected to show normal variations in cancer patients as well as healthy individuals, and has thus been used as normal control DNA samples in various published studies. We performed array CGH to measure and compare genetic changes in terms of the CNA of PBMC samples as well as DNA isolated from tumor tissue samples, obtained from 24 non-small cell lung cancer patients. Contradictory to expectations, our studies showed that the PBMC CNA also showed chromosomal variant regions. The list included well-known tumor-associated NTRK1, FGF8, TP53, and TGFβ1 genes and potentially novel oncogenes such as THPO (3q27.1), JMJD1B, and EGR1 (5q31.2), which was investigated in this study. The results of this study highlighted the connection between PBMC and tumor cell genomic DNA in lung cancer patients. However, the application of these studies to cancer prognosis may pose a challenge due to the large amount of information contained in genetic predisposition and family history that has to be processed for useful downstream clinical applications.     

From array to array: Confirmation of genomic gains and losses discovered by array-based comparative genomic hybridization utilizing fluorescence in situ hybridization on tissue microarrays

The combination of array-based comparative genomic hybridization (CGH) with fluorescence in situ hybridization utilizing custom-designed bacterial artificial chromosome (BAC) probes applied to tissue microarrays represents a powerful compendium of techniques–greatly enhancing the throughput of genomic analysis and subsequent target validation. Such approach can be automated at various levels and allows managing large volume of targets and samples in a few experiments. As such, this approach facilitates discovery, validation and implementation of findings in the process of identification of new diagnostic, prognostic and potentially therapeutic molecular markers.     

Characterization of chromosomal aberrations in diffuse large B-cell lymphoma (DLBL) by G-banding and spectral karyotyping (SKY)

Cytogenetic chromosome analysis by classical G-banding was supplemented by spectral karyotyping (SKY) in 12 cases of diffuse large B-cell lymphoma (DLBL). SKY is a fluorescence in-situ-based, genome-wide screening technique allowing identification of genetic material even in highly condensed metaphase chromosomes of poor morphology. By simultaneous hybridization of whole chromosome painting probes onto tumor chromosome spreads genetic rearrangements are visualized permitting the clarification of even complex karyotype alterations and the identification of genetic material of previously unknown origin, so-called marker chromosomes. Taking the SKY results into account, we reevaluated the G-banding karyotypes initially carried out, thus generating a more precise karyotype in ten of twelve (83%) cases investigated. In particular, thirteen chromosomal rearrangements not correctly recognized by classical cytogenetics were identified, the genetic origin of seven marker chromosomes was elucidated and three structural genetic rearrangements were redefined. We found SKY to be a valuable technique to establish a definite karyotype in addition to classical cytogenetics.     

CGcgh: a tool for molecular karyotyping using DNA microarray-based comparative genomic hybridization (array-CGH)

Microarray-based comparative genomic hybridization (array-CGH) is a technique by which variations in copy numbers between two genomes can be analyzed using DNA microarrays. Array CGH has been used to survey chromosomal amplifications and deletions in fetal aneuploidies or cancer tissues. Herein we report a user-friendly, MATLAB-based, array CGH analyzing program, Chang Gung comparative genomic hybridization (CGcgh), as a standalone PC version. The analyzed chromosomal data are displayed in a graphic interface, and CGcgh allows users to launch a corresponding G-banding ideogram. The abnormal DNA copy numbers (gains and losses) can be identified automatically using a user defined window size (default value is 50 probes) and sequential student t-tests with sliding windows along with chromosomes. CGcgh has been tested in multiple karyotype-confirmed human samples, including five published cases and trisomies 13, 18, 21 and X from our laboratories, and 18 cases of which microarray data are available publicly. CGcgh can be used to detect the copy number changes in small genomic regions, which are commonly encountered by clinical geneticists. CGcgh works well for the data from cDNA microarray, spotted oligonucleotide microarrays, and Affymetrix Human Mapping Arrays (10K, 100K, 500K Array Sets). The program can be freely downloaded from . Y. S. Lee and A. Chao contributed equally to this work.     

T-helper (Th)1/Th2 imbalance in patients with previously untreated B-cell diffuse large cell lymphoma

T-helper (Th)1/Th2 imbalance has been observed in a variety of pathological conditions, including malignant diseases. We evaluated the Th1/Th2 balance in peripheral blood Th cells by means of intracellular cytokine analysis in 19 patients with previously untreated B-cell diffuse large cell lymphoma (DLCL) and in 18 patients with B-cell DLCL who had achieved complete remission (CR) after chemotherapy. The mean percentage of Th2 in CD4 cells in patients with DLCL (5.00 +/- 2.20) and that of Th1 in CD4+ cells in patients in CR (32.42 +/- 11.30) were significantly increased in comparison with those in healthy volunteers, respectively (Th1; 23.02 +/- 9.45, Th2; 3.25 +/- 0.90; P<0.01). The mean ratio of Th1/Th2 was significantly lower in patients with DLCL (4.74 +/- 0.52) than in patients in CR (9.31 +/- 1.06; P<0.01) and in healthy volunteers (7.25 +/- 0.65; P<0.01). We conclude that the Th1/Th2 balance was polarized to Th2 in untreated DLCL patients and to Th1 in patients in CR, which suggests that a Th1/Th2 imbalance could play a role in lymphomagenesis and durable remission.     

High-resolution molecular characterization of 15q11-q13 rearrangements by array comparative genomic hybridization (array CGH) with detection of gene dosage

Maternally derived duplication of the imprinted region of chromosome 15q11-q14 leads to a complex neurobehavioral phenotype that often includes autism, cognitive deficits, and seizures. Multiple repeat elements within the region mediate a variety of rearrangements, including interstitial duplications, interstitial triplications, and supernumerary isodicentric marker chromosomes, as well as the deletions that cause Prader-Willi and Angelman syndromes. To elucidate the molecular structure of these duplication chromosomes, we designed a high-resolution array comparative genomic hybridization (array CGH) platform. The array contains 79 clones that form a gapped contig across the critical region on chromosome 15q11-q14 and 21 control clones from other autosomes and the sex chromosomes. We used this array to examine a set of 48 samples from patients with segmental aneuploidy of chromosome 15q. Using the array, we were able to determine accurately the dosage, which ranged from 1 to 6 copies, and also to detect atypical and asymmetric rearrangements. In addition, the increased resolution of the array allowed us to position two previously reported breakpoints within the contig. These results indicate that array CGH is a powerful technique to study rearrangements of proximal chromosome 15q.     

Copy number variants (CNVs) in primate species using array-based comparative genomic hybridization

A substantial amount of genomic variation is now known to exist in humans and other primate species. Single nucleotide polymorphisms (SNPs) are thought to represent the vast majority of genomic differences among individuals in a given primate species and comprise about 0.1% of the genomes of two humans. However, recent studies have now shown that structural variation msay account for as much as 0.7% of the genomic differences in humans, of which copy number variants (CNVs) are the largest component. CNVs are segments of DNA that can range in size from hundreds of bases to millions of base pairs in length and have different number of copies between individuals. Recent technological advancements in array technologies led to genome-wide identification of CNVs and consequently revealed thousands of variable loci in humans, comprising as much as 12% of the human genome [A.J. Iafrate, L. Feuk, M.N. Rivera, M.L. Listewnik, P.K. Donahoe, Y. Qi, S.W. Scherer, C. Lee, Nat. Genet. 36 (2004) 949–951, [3]]. CNVs in humans have already been associated with susceptibility to certain complex diseases, dietary adaptation, and several neurological conditions. In addition, recent studies have shown that CNVs can be successfully implemented in population genetics research, providing important insights into human genetic variation. Nevertheless, the important role of CNVs in primate evolution and genetic diversity is still largely unknown. This article aims to outline the strengths and weaknesses of current comparative genomic hybridization array technologies that have been employed to detect CNV variation and the applications of these techniques to primate genetic research.     

Application of array comparative genomic hybridization in 256 patients with developmental delay or intellectual disability

We used whole-genome exon-targeted oligonucleotide array comparative genomic hybridization (array CGH) in a cohort of 256 patients with developmental delay (DD)/intellectual disability (ID) with or without dysmorphic features, additional neurodevelopmental abnormalities, and/or congenital malformations. In 69 patients, we identified 84 non-polymorphic copy-number variants, among which 41 are known to be clinically relevant, including two recently described deletions, 4q21.21q21.22 and 17q24.2. Chromosomal microarray analysis revealed also 15 potentially pathogenic changes, including three rare deletions, 5q35.3, 10q21.3, and 13q12.11. Additionally, we found 28 copy-number variants of unknown clinical significance. Our results further support the notion that copy-number variants significantly contribute to the genetic etiology of DD/ID and emphasize the efficacy of the detection of novel candidate genes for neurodevelopmental disorders by whole-genome array CGH.     

Genome-wide screening of severe male factor infertile patients using BAC-array comparative genomic hybridization (CGH)

Male factor infertility is present in up to 50% of infertile couples, making it increasingly important in their treatment. Although most research into the genetics of male infertility has focused on the Y chromosome, male factor infertility may result from other genetic factors. We utilized the whole genome array comparative genomic hybridization (CGH) to identify novel genetic candidate associated with severely impaired spermatogenesis. We enrolled 37 patients with severe male factor infertility, defined as severe nonobstructive type oligozoospermia (≤5×10(6)/ml) or azoospermia, and 10 controls. Routine cytogenetic analyses, Yq microdeletion PCR test and whole genome bacterial artificial chromosome (BAC)-array CGH were performed. Array CGH results showed no specific gains or losses related to impaired spermatogenesis other than Yq microdeletions, and there were no novel candidate genetic abnormalities in the patients with severe male infertility. However, Yq microdeletions were detected in 10 patients. Three showed a deletion in the AZFb-c region and the other 7 had deletions in the AZFc region. Although we could not identify novel genetic regions specifically associated with male infertility, whole genome array CGH analysis with higher resolution including larger numbers of patients may be able to give an opportunity for identifying new genetic markers for male infertility.     

GeneCount: genome-wide calculation of absolute tumor DNA copy numbers from array comparative genomic hybridization data

Absolute tumor DNA copy numbers can currently be achieved only on a single gene basis by using fluorescence in situ hybridization (FISH). We present GeneCount, a method for genome-wide calculation of absolute copy numbers from clinical array comparative genomic hybridization data. The tumor cell fraction is reliably estimated in the model. Data consistent with FISH results are achieved. We demonstrate significant improvements over existing methods for exploring gene dosages and intratumor copy number heterogeneity in cancers.     

Cluster analysis of comparative genomic hybridization (CGH) data using self-organizing maps: application to prostate carcinomas.

Comparative genomic hybridization (CGH) is a modern genetic method which enables a genome-wide survey of chromosomal imbalances. For each chromosome region, one obtains the information whether there is a loss or gain of genetic material, or whether there is no change at that region. Usually it is not possible to evaluate all 46 chromosomes of a metaphase, therefore several (up to 20 or more) metaphases are analyzed per individual, and expressed as average. Mostly one does not study one individual alone but groups of 20-30 individuals. Therefore, large amounts of data quickly accumulate which must be put into a logical order. In this paper we present the application of a self-organizing map (Genecluster) as a tool for cluster analysis of data from pT2N0 prostate cancer cases studied by CGH. Self-organizing maps are artificial neural networks with the capability to form clusters on the basis of an unsupervised learning rule, i.e., in our examples it gets the CGH data as only information (no clinical data). We studied a group of 40 recent cases without follow-up, an older group of 20 cases with follow-up, and the data set obtained by pooling both groups. In all groups good clusterings were found in the sense that clinically similar cases were placed into the same clusters on the basis of the genetic information only. The data indicate that losses on chromosome arms 6q, 8p and 13q are all frequent in pT2N0 prostatic cancer, but the loss on 8p has probably the largest prognostic importance.     

SW-ARRAY: a dynamic programming solution for the identification of copy-number changes in genomic DNA using array comparative genome hybridization data

Comparative genome hybridization (CGH) to DNA microarrays (array CGH) is a technique capable of detecting deletions and duplications in genomes at high resolution. However, array CGH studies of the human genome noting false negative and false positive results using large insert clones as probes have raised important concerns regarding the suitability of this approach for clinical diagnostic applications. Here, we adapt the Smith–Waterman dynamic-programming algorithm to provide a sensitive and robust analytic approach (SW-ARRAY) for detecting copy-number changes in array CGH data. In a blind series of hybridizations to arrays consisting of the entire tiling path for the terminal 2 Mb of human chromosome 16p, the method identified all monosomies between 267 and 1567 kb with a high degree of statistical significance and accurately located the boundaries of deletions in the range 267–1052 kb. The approach is unique in offering both a nonparametric segmentation procedure and a nonparametric test of significance. It is scalable and well-suited to high resolution whole genome array CGH studies that use array probes derived from large insert clones as well as PCR products and oligonucleotides.     

Chromosome 22q11.2 deletion syndrome: prenatal diagnosis,array comparative genomic hybridization characterization using uncultured amniocytes and literature review

We present prenatal diagnosis of de novo 22q11.2 microdeletion syndrome using uncultured amniocytes in a pregnancy with conotruncal heart malformations in the fetus. We discuss the genotype–phenotype correlation and the consequence of haploinsufficiency of TBX1, COMT, UFD1L, GNB1L and MED15 in the deleted region. We review the literature of chromosomal loci and genes responsible for conotruncal heart malformations and tetralogy of Fallot.