To identify new risk variants for chronic lymphocytic leukemia (CLL), we conducted a genome-wide association study of 299,983 tagging SNPs, with validation in four additional series totaling 2,503 cases and 5,789 controls. We identified four new risk loci for CLL at 2q37.3 (rs757978, FARP2; odds ratio (OR) = 1.39; P = 2.11 x 10(-9)), 8q24.21 (rs2456449; OR = 1.26; P = 7.84 x 10(-10)), 15q21.3 (rs7169431; OR = 1.36; P = 4.74 x 10(-7)) and 16q24.1 (rs305061; OR = 1.22; P = 3.60 x 10(-7)). We also found evidence for risk loci at 15q25.2 (rs783540, CPEB1; OR = 1.18; P = 3.67 x 10(-6)) and 18q21.1 (rs1036935; OR = 1.22; P = 2.28 x 10(-6)). These data provide further evidence for genetic susceptibility to this B-cell hematological malignancy.

/pdf/common-variants-at-2q37-3-8q24-21-15q21-3-and-16q24-1-3umbbzmyw1.pdf

Common variants at 2q37.3, 8q24.21, 15q21.3 and 16q24.1 influence chronic lymphocytic leukemia risk

Approximately 8% of breast cancers show increased copy numbers of chromosome 17 centromere (CEP17) by fluorescence in situ hybridization (FISH) (ie average CEP17 >3.0 per nucleus). Currently, this pattern is believed to represent polysomy of chromosome 17. HER2-amplified cancers have been shown to harbour complex patterns of genetic aberrations of chromosome 17, in particular involving its long arm. We hypothesized that aberrant copy numbers of CEP17 in FISH assays may not necessarily represent true chromosome 17 polysomy. Eighteen randomly selected CEP17 polysomic cases and a control group of ten CEP17 disomic cases, as defined by dual-colour FISH, were studied by microarray-based comparative genomic hybridization (aCGH), which was performed on microdissected samples using a 32K tiling-path bacterial artificial chromosome microarray platform. Additional FISH probes were employed for SMS (17p11.2) and RARA (17q21.2) genes, as references for chromosome 17 copy number. Microarray-based comparative genomic hybridization revealed that 11 out of the 18 polysomic cases harboured gains of 17q with involvement of the centromere, one displayed 17q gain sparing the centromeric region, and only one could be defined as polysomic. The remaining five cases displayed amplification of the centromeric region. Among these, one case, showing score 2+ by immunohistochemistry and 8.5 HER2 mean copy number, was classified as not amplified by HER2/CEP17 ratio and as amplified by HER2/SMS ratio. Our results suggest that true chromosome 17 polysomy is likely to be a rare event in breast cancer and that CEP17 copy number greater than 3.0 in FISH analysis is frequently related to gain or amplification of the centromeric region. Larger studies investigating the genetic profiles of CEP17 polysomic cases are warranted.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/path.2574

Does chromosome 17 centromere copy number predict polysomy in breast cancer? A fluorescence in situ hybridization and microarray-based CGH analysis†

Cancers may be composed of multiple populations of submodal clones sharing the same initiating genetic lesions, followed by the acquisition of divergent genetic hits. Intra-tumour genetic heterogeneity has profound implications for cancer clinical management. To determine the extent of intra-tumour genetic heterogeneity in breast cancers, and whether the morphological diversity of breast cancers is underpinned by divergent genetic aberrations, we analysed the genomic profiles of microdissected, morphologically distinct components of six metaplastic breast carcinomas, tumours characterized by the presence of morphological areas with divergent differentiation. Each morphologically distinct component was separately microdissected and subjected to high-resolution microarray-based comparative genomic hybridization. Each component was also analysed by immunohistochemistry and in situ hybridization. Clonal relationship between the distinct components was tested by TP53 sequencing and human androgen receptor (HUMARA) X-chromosome inactivation assay. In the majority of cases, all morphologically distinct components from each case were clonal and displayed remarkably similar genetic profiles. In two cases, however, morphologically distinct components harboured specific genetic aberrations. In an adenosquamous carcinoma, the differences were such that only 20% of the genome harboured similar copy number changes. The squamous component displayed EGFR gene amplification, EGFR over-expression and lack of expression of hormone receptors, whereas the lobular component displayed the reverse pattern. The components of a biphasic spindle cell carcinoma harboured similar gains, losses, amplifications of 9p23 and 17q12 (HER2) and identical TP53 mutations, suggesting that these were relatively early events in the development of this tumour; however, each component displayed divergent focal amplifications. Importantly, the metastatic deposit of this case, despite harbouring a TP53 mutation identical to that found in the primary tumour, harboured additional specific focal amplifications. This proof-of-principle study provides direct evidence of intra-tumour genetic heterogeneity in breast cancers, and shows that in some cases morphological diversity may be underpinned by distinct genetic aberrations.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1002/path.2675

Molecular analysis reveals a genetic basis for the phenotypic diversity of metaplastic breast carcinomas.

Purpose: To characterize the molecular genetic profiles of grade 3 invasive ductal carcinomas of no special type using high-resolution microarray-based comparative genomic hybridization (aCGH) and to identify recurrent amplicons harboring putative therapeutic targets associated with luminal, HER-2, and basal-like tumor phenotypes. Experimental Design: Ninety-five grade 3 invasive ductal carcinomas of no special type were classified into luminal, HER-2, and basal-like subgroups using a previously validated immunohistochemical panel. Tumor samples were microdissected and subjected to aCGH using a tiling path 32K BAC array platform. Selected regions of recurrent amplification were validated by means of in situ hybridization. Expression of genes pertaining to selected amplicons was investigated using quantitative real-time PCR and gene silencing was done using previously validated short hairpin RNA constructs. Results: We show that basal-like and HER-2 tumors are characterized by "sawtooth" and "firestorm" genetic patterns, respectively, whereas luminal cancers were more heterogeneous. Apart from confirming known amplifications associated with basal-like (1q21, 10p, and 12p), luminal (8p12, 11q13, and 11q14), and HER-2 (17q12) cancers, we identified previously unreported recurrent amplifications associated with each molecular subgroup: 19q12 in basal-like, 1q32.1 in luminal, and 14q12 in HER-2 cancers. PPM1D gene amplification (17q23.2) was found in 20% and 8% of HER-2 and luminal cancers, respectively. Silencing of PPM1D by short hairpin RNA resulted in selective loss of viability in tumor cell lines harboring the 17q23.2 amplification. Conclusions: Our results show the power of aCGH analysis in unraveling the genetic profiles of specific subgroups of cancer and for the identification of novel therapeutic targets.

https://clincancerres.aacrjournals.org/content/clincanres/15/8/2711.full.pdf

Tiling path genomic profiling of grade 3 invasive ductal breast cancers.

Structural variations are among the most frequent interindividual genetic differences in the human genome. The frequency and distribution of de novo somatic structural variants in normal cells is, however, poorly explored. Using age-stratified cohorts of 318 monozygotic (MZ) twins and 296 single-born subjects, we describe age-related accumulation of copy-number variation in the nuclear genomes in vivo and frequency changes for both megabase- and kilobase-range variants. Megabase-range aberrations were found in 3.4% (9 of 264) of subjects ≥60 years old; these subjects included 78 MZ twin pairs and 108 single-born individuals. No such findings were observed in 81 MZ pairs or 180 single-born subjects who were ≤55 years old. Recurrent region- and gene-specific mutations, mostly deletions, were observed. Longitudinal analyses of 43 subjects whose data were collected 7–19 years apart suggest considerable variation in the rate of accumulation of clones carrying structural changes. Furthermore, the longitudinal analysis of individuals with structural aberrations suggests that there is a natural self-removal of aberrant cell clones from peripheral blood. In three healthy subjects, we detected somatic aberrations characteristic of patients with myelodysplastic syndrome. The recurrent rearrangements uncovered here are candidates for common age-related defects in human blood cells. We anticipate that extension of these results will allow determination of the genetic age of different somatic-cell lineages and estimation of possible individual differences between genetic and chronological age. Our work might also help to explain the cause of an age-related reduction in the number of cell clones in the blood; such a reduction is one of the hallmarks of immunosenescence.

/pdf/age-related-somatic-structural-changes-in-the-nuclear-genome-5bkv8agmac.pdf

Age-Related Somatic Structural Changes in the Nuclear Genome of Human Blood Cells

Screening for gene copy‐number alterations (CNAs) has improved by applying genome‐wide microarrays, where SNP arrays also allow analysis of loss of heterozygozity (LOH). We here analyzed 10 chronic lymphocytic leukemia (CLL) samples using four different high‐resolution platforms: BAC arrays (32K), oligonucleotide arrays (185K, Agilent), and two SNP arrays (250K, Affymetrix and 317K, Illumina). Cross‐platform comparison revealed 29 concordantly detected CNAs, including known recurrent alterations, which confirmed that all platforms are powerful tools when screening for large aberrations. However, detection of 32 additional regions present in 2–3 platforms illustrated a discrepancy in detection of small CNAs, which often involved reported copy‐number variations. LOH analysis using dChip revealed concordance of mainly large regions, but showed numerous, small nonoverlapping regions and LOH escaping detection. Evaluation of baseline variation and copy‐number ratio response showed the best performance for the Agilent platform and confirmed the robustness of BAC arrays. Accordingly, these platforms demonstrated a higher degree of platform‐specific CNAs. The SNP arrays displayed higher technical variation, although this was compensated by high density of elements. Affymetrix detected a higher degree of CNAs compared to Illumina, while the latter showed a lower noise level and higher detection rate in the LOH analysis. Large‐scale studies of genomic aberrations are now feasible, but new tools for LOH analysis are requested. © 2008 Wiley‐Liss, Inc.

Screening For Copy-Number Alterations And Loss-Of-Heterozygosity In Chronic Lymphocytic Leukemia - A Comparative Study Of Four Differently Designed, High Resolution Microarray Platforms

A. C. Syvaenen

Papers

Screening For Copy-Number Alterations And Loss-Of-Heterozygosity In Chronic Lymphocytic Leukemia - A Comparative Study Of Four Differently Designed, High Resolution Microarray Platforms