Replicative and non-replicative mechanisms in the formation of clustered CNVs are indicated by whole genome characterization.

Clustered copy number variants (CNVs) as detected by chromosomal microarray analysis (CMA) are often reported as germline chromothripsis. However, such cases might need further investigations by massive parallel whole genome sequencing (WGS) in order to accurately define the underlying complex rearrangement, predict the occurrence mechanisms and identify additional complexities. Here, we utilized WGS to delineate the rearrangement structure of 21 clustered CNV carriers first investigated by CMA and identified a total of 83 breakpoint junctions (BPJs). The rearrangements were further sub-classified depending on the patterns observed: I) Cases with only deletions (n = 8) often had additional structural rearrangements, such as insertions and inversions typical to chromothripsis; II) cases with only duplications (n = 7) or III) combinations of deletions and duplications (n = 6) demonstrated mostly interspersed duplications and BPJs enriched with microhomology. In two cases the rearrangement mutational signatures indicated both a breakage-fusion-bridge cycle process and haltered formation of a ring chromosome. Finally, we observed two cases with Alu- and LINE-mediated rearrangements as well as two unrelated individuals with seemingly identical clustered CNVs on 2p25.3, possibly a rare European founder rearrangement. In conclusion, through detailed characterization of the derivative chromosomes we show that multiple mechanisms are likely involved in the formation of clustered CNVs and add further evidence for chromoanagenesis mechanisms in both "simple" and highly complex chromosomal rearrangements. Finally, WGS characterization adds positional information, important for a correct clinical interpretation and deciphering mechanisms involved in the formation of these rearrangements.

Cytogenetically visible inversions are formed by multiple molecular mechanisms.

Cytogenetically detected inversions are generally assumed to be copy number and phenotypically neutral events. While nonallelic homologous recombination is thought to play a major role, recent data suggest the involvement of other molecular mechanisms in inversion formation. Using a combination of short-read whole-genome sequencing (WGS), 10X Genomics Chromium WGS, droplet digital polymerase chain reaction and array comparative genomic hybridization we investigated the genomic structure of 18 large unique cytogenetically detected chromosomal inversions and achieved nucleotide resolution of at least one chromosomal inversion junction for 13/18 (72%). Surprisingly, we observed that seemingly copy number neutral inversions can be accompanied by a copy-number gain of up to 350 kb and local genomic complexities (3/18, 17%). In the resolved inversions, the mutational signatures are consistent with nonhomologous end-joining (8/13, 62%) or microhomology-mediated break-induced replication (5/13, 38%). Our study indicates that short-read 30x coverage WGS can detect a substantial fraction of chromosomal inversions. Moreover, replication-based mechanisms are responsible for approximately 38% of those events leading to a significant proportion of inversions that are actually accompanied by additional copy-number variation potentially contributing to the overall phenotypic presentation of those patients.

Androgen receptor expression predicts beneficial tamoxifen response in oestrogen receptor-α-negative breast cancer.

BACKGROUND: Although the androgen receptor (AR) is frequently expressed in breast cancer, its relevance in the disease is not fully understood. In addition, the relevance of AR in determining tamoxifen treatment efficiency requires evaluation. PURPOSE: To investigate the tamoxifen predictive relevance of the AR protein expression in breast cancer. METHODS: Patients were randomised to tamoxifen 40 mg daily for 2 or 5 years or to no endocrine treatment. Mean follow-up was 15 years. Hazard ratios were calculated with recurrence-free survival as end point. RESULTS: In patients with oestrogen receptor (ER)-negative tumours, expression of AR predicted decreased recurrence rate with tamoxifen (hazard ratio (HR)=0.34; 95% confidence interval (CI)=0.14-0.81; P=0.015), whereas the opposite was seen in the AR- group (HR=2.92; 95% CI=1.16-7.31; P=0.022). Interaction test was significant P<0.001. Patients with triple-negative and AR+ tumours benefitted from tamoxifen treatment (HR=0.12; 95% CI=0.014-0.95 P=0.044), whereas patients with AR- tumours had worse outcome when treated with tamoxifen (HR=3.98; 95% CI=1.32-12.03; P=0.014). Interaction test was significant P=0.003. Patients with ER+ tumours showed benefit from tamoxifen treatment regardless of AR expression. CONCLUSIONS: AR can predict tamoxifen treatment benefit in patients with ER- tumours and triple-negative breast cancer.

Imatinib reduces cholesterol uptake and matrix metalloproteinase activity in human THP-1 macrophages.

BACKGROUND: Imatinib mesylate (Glivec®, formerly STI-571) is a selective tyrosine kinase inhibitor used for the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors. However, there are reports suggesting that imatinib could be atheroprotective by lowering plasma low-density lipoprotein (LDL). AIM: To investigate the potential inhibitory effect of imatinib on cholesterol uptake in human macrophages as well as its effect on matrix metalloproteinase (MMP) activity. METHODS AND RESULTS: Uptake of fluorescence-labeled LDL was analyzed using flow cytometry. Macrophages treated with imatinib showed a 23.5%, 27%, and 15% decrease in uptake of native LDL (p<0.05), acetylated LDL (p<0.01), and copper-modified oxidized LDL (p<0.01), respectively. Gel-based zymography showed that secretion and activity of MMP-2 and MMP-9 were inhibited by imatinib. Using GeneChip Whole Transcript Expression array analysis, no obvious gene candidates involved in the mechanisms of cholesterol metabolism or MMP regulation were found to be affected by imatinib. Instead, we found that imatinib up-regulated microRNA 155 (miR155) by 43.8% and down-regulated ADAM metallopeptidase domain 28 (ADAM28) by 41.4%. Both genes could potentially play an atheroprotective role and would be interesting targets in future studies. CONCLUSION: Our results indicate that imatinib causes post-translational inhibition with respect to cholesterol uptake and regulation of MMP-2 and MMP-9. More research is needed to further evaluate the role of imatinib in the regulation of other genes and processes.