Circulating tumour cells as a predictive factor for response to systemic chemotherapy in patients with advanced colorectal cancer.

Circulating tumour cells (CTC) can be traced in patients with different types of cancer. The aim of this study was to detect CTC in patients with advanced colorectal cancer and whether CTC are still detectable after systemic chemotherapy. Blood from 34 patients with advanced colorectal cancer was analysed for the presence of CTC before chemotherapy was given and after 3 months. Eleven patients demonstrated a tumour remission after chemotherapy. In 6 cases CTC were detectable before but not after initiation of chemotherapy. Ten patients demonstrated a progression. In 5 cases CTC were detected before and after chemotherapy. Our data suggest that the detection of CTC will help to identify patients responding to chemotherapy or with a risk of a therapy failure.

Tumor-associated gene expression in disseminated tumor cells correlates with disease progression and tumor stage in colorectal cancer.

BACKGROUND: A possible correlation of disease progression and tumor stage in colorectal cancer patients with tumor-associated gene expression in disseminated tumor cells (DTC) was evaluated. Detection of DTC and expression of tumor-associated genes might be of clinical value with respect to individual patient prognosis, monitoring of therapy and as a surrogate tumor staging parameter. PATIENTS AND METHODS: In a multicenter study, a total of 196 peripheral blood samples were collected from 76 patients with tumor stage Dukes' A to D and analyzed using a DTC detection assay consisting of immunomagnetic selection and expression analysis of the tumor-associated genes CEA, EGFR and GA733-2. DTC detection rates were assessed prior to surgery and post surgery in patients with tumor stage Dukes' A, B and C, and compared with results in metastatic patients. CEA serum protein levels were determined and compared with DTC and CEA expression, respectively. RESULTS: In a comparison analysis, EGFR and CEA expression was detected in 88% (p = 0.001) and 0% (p = 0.002) prior to surgery, in 66% (p = 0.001) and 20% (p = 0.002) post surgery, as well as in 15% (p < 0.0001) and 66% (p < 0.0001) of blood samples collected from metastatic patients, respectively. Expression of tumor-associated genes in DTC prior to surgery and in follow-up samples indicated an ongoing metastatic process. DTC detection rates in patients with Dukes' A (14%), Dukes' B (13%) and Dukes' C (40%) prior to surgery correlated statistically with the expected recurrence rate. There was no correlation between DTC expressing CEA and elevation of CEA serum protein levels. CONCLUSION: EGFR and CEA gene expression correlated with disease progression and tumor stage. Detection of CEA expression in DTC might have a predictive value in colorectal cancer and may help to identify patients at a greater risk of relapse. DTC in peripheral blood collected prior to surgery as well as in follow-up samples have a prognostic clinical value.

Gene expression analysis identifies novel genes participating in early murine liver development and adult liver regeneration.

Adult liver tissue regeneration may recapitulate molecular events of liver organogenesis. As gaps in our understanding of the fundamental processes that govern development and regeneration of the liver still exist, we studied gene expression in the developing liver at embryonic day 9.5 post coitum (E d9.5 p.c.). Microarray data from E d9.5 p.c. as well as previously published data from embryonic day 11.5 post coitum (E d11.5 p.c.) and embryonic day 13.5 post coitum (E d13.5 p.c.) were subjected to cluster analysis. This led to the identification of 130 genes which were characterized by continuous expression at all stages of liver development with peak expression of 44 genes at E d9.5 p.c. Five of these genes, previously not known to be associated with early liver development or with adult liver regeneration were selected for further analysis. The expression of the genes was studied by real-time polymerase chain reaction at 0, 2, 4, 6, 12, 24 and 48 hr after partial hepatectomy in the adult liver. Two of the genes, growth arrest protein 43 (GAP43) and paired-like homeodomain transcription factor 2 (Pitx2) were exclusively detected at 24 hr, whereas the genes Twist1, Midkine, and zinc finger protein of cerebellum 1 (Zic1) each showed a specific expression profile in the regenerating liver with peak expressions at 4, 24, and 6 hr, respectively. In summary, we were able to identify novel genes, that may act as regulators during liver formation as well as in the regeneration phase of adult liver. This information may contribute to the development of new targets for the treatment of liver diseases in the future.

Increased populations of regulatory T cells in peripheral blood of patients with hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide with a poor prognosis and one for which immunotherapy remains a viable option. Experimental tumor models have shown that regulatory T cells, a functionally unique subset of T cells, can suppress effective antitumor immune responses. This suppression might explain the poor outcome of some of the immunotherapy protocols currently being used. A better understanding of the role of regulatory T cells in HCC is important for design of future immunotherapy-based clinical protocols. We have studied regulatory T cells from 84 patients with HCC and 74 controls, including healthy donors, patients with chronic hepatitis B virus and hepatitis C virus infection and nonviral liver cirrhosis. Regulatory T cells were identified by fluorescence-activated cell sorting using a panel of antibodies and by real-time PCR analysis for Foxp3 expression. Functional studies were done to analyze their inhibitory role. Finally, regulatory T cells were analyzed in tumors and ascites from patients with HCC. Patients with HCC have increased numbers of CD4+CD25+ regulatory T cells in their peripheral blood, which express high levels of HLA-DR, GITR, and low or no CD45RA. These cells were anergic toward T-cell receptor stimulation and, when cocultured with activated CD4+CD25- cells, potently suppressed their proliferation and cytokine secretion. There were also high numbers of regulatory T cells in tumor-infiltrating lymphocytes of HCC patients comparable with the increase in their peripheral blood. Our data suggest that the increase in frequency of regulatory T cells might play a role in modulation of the immune response against HCC and could be important in design of immunotherapeutic approaches.

Quantitative gene expression profiling reveals a fetal hepatic phenotype of murine ES-derived hepatocytes.

To use embryonic stem (ES) cells in future therapeutical applications, differentiated hepatic phenotypes with specific liver functions would be necessary. We analyzed albumin (ALB), alpha-fetoprotein (AFP) and hepatic transcription factor (TF) gene expression in tissues derived from embryonic, fetal and adult liver, and compared the gene expression profiles with those from mouse ES cells after hepatic differentiation and from cultured adult hepatocytes. The mRNA expression of hepatocyte nuclear factor (HNF)-1alpha,beta, -3alpha,beta, -4alpha, -6, CCAAT/enhancer binding protein (C/EBP) alpha,beta, ALB and AFP relative to glyceralaldehyde-3-phosphate dehydrogenase (GAPDH) were studied by "real time" RT-PCR. ALBand AFP-expression was also determined by in situ hybridization (tissue) and immunofluorescence (ES-derived cells after hepatic differentiation, ES-HPC). Peak levels for HNF-1alpha, -3alpha, -4alpha and -6 were detected in early liver development at d9.5 and d11.5. C/EBPalpha and beta were most abundantly expressed in adult liver. ALB mRNA increased steadily from d10.5 on and was maximally present in adult liver. AFP was present at d9.5, peaked at d15.5 and dramatically declined in mature liver tissue. Based on immunofluorescence, ALB and AFP were expressed in approximately 20% of ES-HPC. While expression of HNF-3, 4 and 6 reached levels similar to adult hepatocytes, ALB and AFP expression was several orders of magnitude lowerthan in adult tissue or cells. Stages of liver organogenesis are characterized by specific expression patterns of developmentally regulated genes. With sophisticated differentiation protocols, hepatic gene expression can be induced in a proportion of ES cells with gene expression patterns similar to early fetal liver.

PCR-based quantification of amplified RNA from laser microdissected mouse liver samples.

New molecular methods such as quantitative reverse transcription-polymerase chain reaction (RT-PCR) and microarray gene expression analysis have been established recently to quantify gene expression in tissue samples. Such methods, although highly sensitive, require RNA quantities of at least several micrograms. These amounts are not available in many experiments concerning microdissected embryonic or regenerating structures. We combined laser-assisted tissue preparation, RNA amplification, and quantitative RT-PCR to estimate both accuracy and linearity of gene expression in small tissue samples. Our results show that mRNA isolated from laser-microdissected fetal liver tissue or regenerative nodules, which originated from EGFP-marked transplanted fetal cells, can be significantly increased with the amplification protocol. The quantitative expression ratio of the genes albumin and GAPDH was conserved after one and two rounds of amplification compared to nonamplified material. Furthermore, genes expressed at low levels such as the transcription factor C/EBPbeta become detectable after two rounds of amplification in small microdissected tissue samples.

Multi-stage analysis of differential gene expression in BALB/C mouse liver development by high-density microarrays.

The development of a complex organ such as the liver relies on precise temporal and spatial gene expression patterns during ontogenesis. The unique adult phenotype is a result of a cascade of transcriptional events that finally trigger gene expression in a liver-specific fashion. Development in mice starts at embryonic stage E8.5-9.5 with the expression of several genes typically associated with liver tissue. While the role of some genes and their expression is well studied, little is known about the complex expression pattern changes during embryonic and fetal liver development. High-density oligonucleotide microarrays, which allow simultaneous expression analysis of 12,488 mouse mRNA transcripts and EST sequences, were used to study the gene expression profiles in day 7.5 embryonic tissue, in micro-dissected fetal liver tissue from day 11.5 and day 13.5 embryos, and in adult liver. In pairwise comparisons of all stages, a total of 4242 distinct genes or ESTs were found to be differentially regulated. Cross-comparisons of data from all stages detected the highest number of differentially regulated genes in E11.5 fetal liver tissue versus adult liver (3063 genes) and the lowest number in E11.5 versus E13.5 fetal liver tissue (517 genes). Using adult liver as reference tissue, 212 genes were regulated exclusively in E7.5 embryonic tissue, 303 genes in E11.5 and 198 in E13.5 fetal liver tissue. Expression profiles of the 31 genes with significant regulation at all stages as well as of a number of known developmentally regulated genes were compared with published results and interpreted. The gene expression profiles detected by microarray hybridization were independently confirmed for selected genes by quantitative RT-PCR. Our data presented here suggest that a relatively small number of stage-specific genes exist, which may be of particular importance for liver development, growth and differentiation. Furthermore, the microarray approach led to the identification of a number of genes, which have not yet been associated with liver organogenesis and maturation.

Molecular and functional analysis of Popeye genes: A novel family of transmembrane proteins preferentially expressed in heart and skeletal muscle.

Popeye (Pop) genes encode novel transmembrane proteins, of which three family members are present in vertebrates, while in Drosophila a single gene is found. By northern blot analysis a restricted expression pattern is observed; Pop genes are predominantly expressed in the heart, skeletal and smooth muscle. Using homologous recombination, a null mutation was generated in the case of Pop1. The homozygous mutants are viable and do not display any obvious phenotype. They display an impaired ability to regenerate skeletal muscle while the hypertropic response of the heart after isoproterenol infusion revealed no difference between genotypes. Recently a function for Pop1 as a prototype of a novel class of cell adhesion molecules was proposed. Further work is required to substantiate these findings and to extend it to other members of the family.