Unbiased quantitative assessment of Her-2 expression of circulating tumor cells in patients with metastatic and non-metastatic breast cancer.

Background Circulating tumor cells (CTCs) can provide the basis for a liquid biopsy and may guide the use of targeted therapies. We report on unbiased quantification of Her-2 protein expression of CTCs. Patients and methods Her-2 assessment of CTCs was carried out using the CellSearch(®) system in 103 metastatic (M1) and 88 non-metastatic (M0) breast-cancer patients. Expression of Her-2 on CTCs was determined by a manual review and an automated algorithm using Her-2- fluorescein isothiocyanate (FITC) fluorescence of leukocytes to determine the Her-2-expression threshold in each sample. Results Her-2 expression of CTCs varied greatly within and among patients compared with Her-2 expression of leukocytes. In M1 patients, a threshold of 75% of Her-2 positive CTCs in patients with ≥5 CTCs was set. Applying this threshold, 9% of M1 patients with Her-2-negative primary tumors had Her-2-positive CTC status and 29% of M1 patients with Her-2-positive primary tumors had Her-2-negative CTC status. No Her-2 discrepancy was observed between CTCs and primary tumors in M0 patients. Conclusions Our findings demonstrate that Her-2 expression is heterogeneous among CTCs within each patient. We show the feasibility of unbiased quantitative and reproducible assessment of treatment targets on CTCs, opening a path towards personalized treatment.

Challenges in the stratification of breast tumors for tailored therapies.

Studying the molecular stratification of breast carcinoma is a real challenge considering the extreme heterogeneity of these tumors. Many patients are now treated following recommendation established at several NIH and St Gallen consensus conferences. However a significant fraction of these breast cancer patients do not need adjuvant chemotherapies while other patients receive inefficacious therapies. High density gene expression arrays have been designed to attempt to establish expression profiles that could be used as prognostic indicators or as predictive markers for response to treatment. This review is intended to discuss the potential value of these new indicators, but also the current weaknesses of these new genomic and bioinformatic approaches. The combined analysis of transcriptomic and genomic alteration data from relatively large numbers of well annotated tumor specimens may offer an opportunity to overcome the current difficulties in validating recently published non overlapping gene lists as prognostic or therapeutic indicators. There is also hope for identifying and deciphering signal transduction pathways driving tumor progression with newly developed algorithms and semi quantitative parameters obtained in simplified in vitro or in vivo models for specific transduction pathways.

Zyxin is up-regulated during megakaryocytic differentiation of human UT-7/c-mpl cells.

To characterize genes involved in megakaryocytic commitment, we compared expression profiles of bipotent cells (UT-7/c-mpl) with those of the same cells induced to differentiate towards megakaryopoiesis in the presence of TPO. Using cDNA arrays, we showed that 12 out of 2260 genes changed their expression level after 6h of TPO stimulation. One of these genes encodes for zyxin, a cytoskeleton protein component. Zyxin is up-regulated at the mRNA and protein levels in UT-7/c-mpl cells in response to TPO confirming the reliability of the cDNA array technology. Similarly, when CD34 positive cells were induced to differentiate into megakaryocytes, zyxin mRNA was accumulated. Furthermore, when megakaryocytes were allowed to spread on fibrinogen, formation of stress fibers and lamellipodia was induced and zyxin was localized at the picks of actin stress fibers. These results suggest an important role for zyxin during megakaryocytic differentiation and more precisely in the regulation of the integrin mediated adhesion process in megakaryocytes.

Automatic quantitation of hybridization signals on cDNA arrays.

Large-scale hybridization of simple or complex cDNA probes to cDNA clones arrayed on high-density filters is a method frequently used to determine systematically the expression profiles of thousands of genes. Hybridization signal intensities, which reflect the level of transcription of the corresponding genes, are captured on phosphor screens with an imaging system. We describe a high-throughput system, Xdots-Reader, that performs automatic detection and quantitation of each signal on hundreds of images. Reproducibility of spot detection and quantitation within filters and between filters has been assessed in analysis of more than 850000 hybridization signals on 436 filters. The automatic analysis success was greater than 97%, with 424 of the 436 tested filters fully analyzed without any human intervention. XdotsReader is available from the Software Library at www.BioTechniques.com or at http://www. ami. univ-evry. fr/approximately tahi/XDotsReader. It runs on SUN workstations under UNIX (SunOS or Solaris) and on PC under LINUX. No particular hardware is required, and the software is compatible with any other software. It supports the main standard image formats.

Reverse transcription in the presence of dideoxynucleotides to increase the sensitivity of expression monitoring with cDNA arrays.

Complex cDNA targets conforming to the nomenclature proposed by Duggan et al. (Nat. Genet. 21 [1 Suppl.]: 10-14) (where the immobilized nucleic acid is called "probe" and free nucleic acid is called "target") were prepared by priming reverse transcription of mRNA with oligo-(dT)-primers or random oligonucleotide primers in the presence or absence of dideoxynucleotides. These targets were then hybridized to high-density filters spotted with 1339 cDNA clone inserts from a skeletal muscle library. The addition of dideoxynucleotides was found to significantly improve the efficiency and reproducibility of the reverse transcription reaction, and consequently, to improve detection of the least abundant transcripts in expression profiling experiments.

The Genexpress IMAGE knowledge base of the human brain transcriptome: a prototype integrated resource for functional and computational genomics.

Expression profiles of 5058 human gene transcripts represented by an array of 7451 clones from the first IMAGE Consortium cDNA library from infant brain have been collected by semiquantitative hybridization of the array with complex probes derived by reverse transcription of mRNA from brain and five other human tissues. Twenty-one percent of the clones corresponded to transcripts that could be classified in general categories of low, moderate, or high abundance. These expression profiles were integrated with cDNA clone and sequence clustering and gene mapping information from an upgraded version of the Genexpress Index. For seven gene transcripts found to be transcribed preferentially or specifically in brain, the expression profiles were confirmed by Northern blot analyses of mRNA from eight adult and four fetal tissues, and 15 distinct regions of brain. In four instances, further documentation of the sites of expression was obtained by in situ hybridization of rat-brain tissue sections. A systematic effort was undertaken to further integrate available cytogenetic, genetic, physical, and genic map informations through radiation-hybrid mapping to provide a unique validated map location for each of these genes in relation to the disease map. The resulting Genexpress IMAGE Knowledge Base is illustrated by five examples presented in the printed article with additional data available on a dedicated Web site at the address http://idefix.upr420.vjf.cnrs.fr/EXPR++ +/ welcome.html.