Clinical array-based karyotyping of breast cancer with equivocal HER2 status resolves gene copy number and reveals chromosome 17 complexity

<p>Abstract</p> <p>Background</p> <p><it>HER2 </it>gene copy status, and concomitant administration of trastuzumab (Herceptin), remains one of the best examples of targeted cancer therapy based on understanding the genomic etiology of disease. However, newly diagnosed breast cancer cases with equivocal HER2 results present a challenge for the oncologist who must make treatment decisions despite the patient's unresolved HER2 status. In some cases both immunohistochemistry (IHC) and fluorescence <it>in situ </it>hybridization (FISH) are reported as equivocal, whereas in other cases IHC results and FISH are discordant for positive versus negative results. The recent validation of array-based, molecular karyotyping for clinical oncology testing provides an alternative method for determination of HER2 gene copy number status in cases remaining unresolved by traditional methods.</p> <p>Methods</p> <p>In the current study, DNA extracted from 20 formalin fixed paraffin embedded (FFPE) tissue samples from newly diagnosed cases of invasive ductal carcinoma referred to our laboratory with unresolved HER2 status, were analyzed using a clinically validated genomic array containing 127 probes covering the HER2 amplicon, the pericentromeric regions, and both chromosome 17 arms.</p> <p>Results</p> <p>Array-based comparative genomic hybridization (array CGH) analysis of chromosome 17 resolved HER2 gene status in [20/20] (100%) of cases and revealed additional chromosome 17 copy number changes in [18/20] (90%) of cases. Array CGH analysis also revealed two false positives and one false negative by FISH due to "ratio skewing" caused by chromosomal gains and losses in the centromeric region. All cases with complex rearrangements of chromosome 17 showed genome-wide chromosomal instability.</p> <p>Conclusions</p> <p>These results illustrate the analytical power of array-based genomic analysis as a clinical laboratory technique for resolution of HER2 status in breast cancer cases with equivocal results. The frequency of complex chromosome 17 abnormalities in these cases suggests that the two probe FISH interphase analysis is inadequate and results interpreted using the HER2/CEP17 ratio should be reported "with caution" when the presence of centromeric amplification or monosomy is suspected by FISH signal gains or losses. The presence of these pericentromeric copy number changes may result in artificial skewing of the HER2/CEP17 ratio towards false negative or false positive results in breast cancer with chromosome 17 complexity. Full genomic analysis should be considered in all cases with complex chromosome 17 aneusomy as these cases are likely to have genome-wide instability, amplifications, and a poor prognosis.</p

Genome-wide expression assay comparison across frozen and fixed postmortem brain tissue samples

<p>Abstract</p> <p>Background</p> <p>Gene expression assays have been shown to yield high quality genome-wide data from partially degraded RNA samples. However, these methods have not yet been applied to postmortem human brain tissue, despite their potential to overcome poor RNA quality and other technical limitations inherent in many assays. We compared cDNA-mediated annealing, selection, and ligation (DASL)- and <it>in vitro </it>transcription (IVT)-based genome-wide expression profiling assays on RNA samples from artificially degraded reference pools, frozen brain tissue, and formalin-fixed brain tissue.</p> <p>Results</p> <p>The DASL-based platform produced expression results of greater reliability than the IVT-based platform in artificially degraded reference brain RNA and RNA from frozen tissue-based samples. Although data associated with a small sample of formalin-fixed RNA samples were poor when obtained from both assays, the DASL-based platform exhibited greater reliability in a subset of probes and samples.</p> <p>Conclusions</p> <p>Our results suggest that the DASL-based gene expression-profiling platform may confer some advantages on mRNA assays of the brain over traditional IVT-based methods. We ultimately consider the implications of these results on investigations of neuropsychiatric disorders.</p

Gene expression profile of circulating tumor cells in breast cancer by RT-qPCR

<p>Abstract</p> <p>Background</p> <p>Circulating tumor cells (CTCs) have been associated with prognosis especially in breast cancer and have been proposed as a liquid biopsy for repeated follow up examinations. Molecular characterization of CTCs is difficult to address since they are very rare and the amount of available sample is very limited.</p> <p>Methods</p> <p>We quantified by RT-qPCR <it>CK-19, MAGE-A3, HER-2, TWIST1, hTERT α+β+</it>, and <it>mammaglobin </it>gene transcripts in immunomagnetically positively selected CTCs from 92 breast cancer patients, and 28 healthy individuals. We also compared our results with the CellSearch system in 33 of these patients with early breast cancer.</p> <p>Results</p> <p>RT-qPCR is highly sensitive and specific and can detect the expression of each individual gene at the one cell level. None of the genes tested was detected in the group of healthy donors. In 66 operable breast cancer patients, <it>CK-19 </it>was detected in 42.4%, <it>HER-2 </it>in 13.6%, <it>MAGE-A3 </it>in 21.2%, <it>hMAM </it>in 13.6%, <it>TWIST-1 </it>in 42.4%, and <it>hTERT α+β+ </it>in 10.2%. In 26 patients with verified metastasis, <it>CK-19 </it>was detected in 53.8%, <it>HER-2 </it>in 19.2%, <it>MAGE-A3 </it>in 15.4%, <it>hMAM </it>in 30.8%, <it>TWIST-1 </it>in 38.5% and <it>hTERT </it>α⁺β⁺in 19.2%. Our preliminary data on the comparison between RT-qPCR and CellSearch in 33 early breast cancer patients showed that RT-qPCR gives more positive results in respect to CellSearch.</p> <p>Conclusions</p> <p>Molecular characterization of CTCs has revealed a remarkable heterogeneity of gene expression between breast cancer patients. In a small percentage of patients, CTCs were positive for all six genes tested, while in some patients only one of these genes was expressed. The clinical significance of these findings in early breast cancer remains to be elucidated when the clinical outcome for these patients is known.</p

Minimal residual disease and circulating tumor cells in breast cancer

Tumor cell dissemination in bone marrow or other organs is thought to represent an important step in the metastatic process. The detection of bone marrow disseminated tumor cells is associated with worse outcome in early breast cancer. Moreover, the detection of peripheral blood circulating tumor cells is an adverse prognostic factor in metastatic breast cancer, and emerging data suggest that this is also true for early disease. Beyond enumeration, the characterization of these cells has the potential to improve risk assessment, treatment selection and monitoring, and the development of novel therapeutic agents, and to advance our understanding of the biology of metastasis

Gene expression profiles of prostate cancer reveal involvement of multiple molecular pathways in the metastatic process

BACKGROUND: Prostate cancer is characterized by heterogeneity in the clinical course that often does not correlate with morphologic features of the tumor. Metastasis reflects the most adverse outcome of prostate cancer, and to date there are no reliable morphologic features or serum biomarkers that can reliably predict which patients are at higher risk of developing metastatic disease. Understanding the differences in the biology of metastatic and organ confined primary tumors is essential for developing new prognostic markers and therapeutic targets. METHODS: Using Affymetrix oligonucleotide arrays, we analyzed gene expression profiles of 24 androgen-ablation resistant metastatic samples obtained from 4 patients and a previously published dataset of 64 primary prostate tumor samples. Differential gene expression was analyzed after removing potentially uninformative stromal genes, addressing the differences in cellular content between primary and metastatic tumors. RESULTS: The metastatic samples are highly heterogenous in expression; however, differential expression analysis shows that 415 genes are upregulated and 364 genes are downregulated at least 2 fold in every patient with metastasis. The expression profile of metastatic samples reveals changes in expression of a unique set of genes representing both the androgen ablation related pathways and other metastasis related gene networks such as cell adhesion, bone remodelling and cell cycle. The differentially expressed genes include metabolic enzymes, transcription factors such as Forkhead Box M1 (FoxM1) and cell adhesion molecules such as Osteopontin (SPP1). CONCLUSION: We hypothesize that these genes have a role in the biology of metastatic disease and that they represent potential therapeutic targets for prostate cancer

Gene expression profiling of tumour epithelial and stromal compartments during breast cancer progression

The progression of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) marks a critical step in the evolution of breast cancer. There is some evidence to suggest that dynamic interactions between the neoplastic cells and the tumour microenvironment play an important role. Using the whole-genome cDNA-mediated annealing, selection, extension and ligation assay (WG-DASL, Illumina), we performed gene expression profiling on 87 formalin-fixed paraffin-embedded (FFPE) samples from 17 patients consisting of matched IDC, DCIS and three types of stroma: IDC-S ( 10 mm from IDC or DCIS). Differential gene expression analysis was validated by quantitative real time-PCR, immunohistochemistry and immunofluorescence. The expression of several genes was down-regulated in stroma from cancer patients relative to normal stroma from reduction mammoplasties. In contrast, neoplastic epithelium underwent more gene expression changes during progression, including down regulation of SFRP1. In particular, we observed that molecules related to extracellular matrix (ECM) remodelling (e.g. COL11A1, COL5A2 and MMP13) were differentially expressed between DCIS and IDC. COL11A1 was overexpressed in IDC relative to DCIS and was expressed by both the epithelial and stromal compartments but was enriched in invading neoplastic epithelial cells. The contributions of both the epithelial and stromal compartments to the clinically important scenario of progression from DCIS to IDC. Gene expression profiles, we identified differential expression of genes related to ECM remodelling, and specifically the elevated expression of genes such as COL11A1, COL5A2 and MMP13 in epithelial cells of IDC. We propose that these expression changes could be involved in facilitating the transition from in situ disease to invasive cancer and may thus mark a critical point in disease development

Optimised and Rapid Pre-clinical Screening in the SOD1G93A Transgenic Mouse Model of Amyotrophic Lateral Sclerosis (ALS)

The human SOD1G93A transgenic mouse has been used extensively since its development in 1994 as a model for amyotrophic lateral sclerosis (ALS). In that time, a great many insights into the toxicity of mutant SOD1 have been gained using this and other mutant SOD transgenic mouse models. They all demonstrate a selective toxicity towards motor neurons and in some cases features of the pathology seen in the human disease. These models have two major drawbacks. Firstly the generation of robust preclinical data in these models has been highlighted as an area for concern. Secondly, the amount of time required for a single preclinical experiment in these models (3–4 months) is a hurdle to the development of new therapies. We have developed an inbred C57BL/6 mouse line from the original mixed background (SJLxC57BL/6) SOD1G93A transgenic line and show here that the disease course is remarkably consistent and much less prone to background noise, enabling reduced numbers of mice for testing of therapeutics. Secondly we have identified very early readouts showing a large decline in motor function compared to normal mice. This loss of motor function has allowed us to develop an early, sensitive and rapid screening protocol for the initial phases of denervation of muscle fibers, observed in this model. We describe multiple, quantitative readouts of motor function that can be used to interrogate this early mechanism. Such an approach will increase throughput for reduced costs, whilst reducing the severity of the experimental procedures involved

TIEG1/KLF10 Modulates Runx2 Expression and Activity in Osteoblasts

Deletion of TIEG1/KLF10 in mice results in a gender specific osteopenic skeletal phenotype with significant defects in both cortical and trabecular bone, which are observed only in female animals. Calvarial osteoblasts isolated from TIEG1 knockout (KO) mice display reduced expression levels of multiple bone related genes, including Runx2, and exhibit significant delays in their mineralization rates relative to wildtype controls. These data suggest that TIEG1 plays an important role in regulating Runx2 expression in bone and that decreased Runx2 expression in TIEG1 KO mice is in part responsible for the observed osteopenic phenotype. In this manuscript, data is presented demonstrating that over-expression of TIEG1 results in increased expression of Runx2 while repression of TIEG1 results in suppression of Runx2. Transient transfection and chromatin immunoprecipitation assays reveal that TIEG1 directly binds to and activates the Runx2 promoter. The zinc finger containing domain of TIEG1 is necessary for this regulation supporting that activation occurs through direct DNA binding. A role for the ubiquitin/proteasome pathway in fine tuning the regulation of Runx2 expression by TIEG1 is also implicated in this study. Additionally, the regulation of Runx2 expression by cytokines such as TGFβ1 and BMP2 is shown to be inhibited in the absence of TIEG1. Co-immunoprecipitation and co-localization assays indicate that TIEG1 protein associates with Runx2 protein resulting in co-activation of Runx2 transcriptional activity. Lastly, Runx2 adenoviral infection of TIEG1 KO calvarial osteoblasts leads to increased expression of Runx2 and enhancement of their ability to differentiate and mineralize in culture. Taken together, these data implicate an important role for TIEG1 in regulating the expression and activity of Runx2 in osteoblasts and suggest that decreased expression of Runx2 in TIEG1 KO mice contributes to the observed osteopenic bone phenotype