Background: Advanced cancer and chemotherapy are both associated with immune system suppression. We initiated a clinical trial in patients receiving chemotherapy for metastatic colorectal cancer to determine if administration of GM-CSF in this setting was immunostimulatory.Methods: Between June, 2003 and January, 2007, 20 patients were enrolled in a clinical trial (NCT00257322) in which they received 500 ug GM-CSF daily for 4 days starting 24 hours after each chemotherapy cycle. There were no toxicities or adverse events reported. Blood was obtained before chemotherapy/GM-CSF administration and 24 hours following the final dose of GM-CSF and evaluated for circulating dendritic cells and adaptive immune cellular subsets by flow cytometry. Peripheral blood mononuclear cell (PBMC) expression of γ-interferon and T-bet transcription factor (Tbx21) by quantitative real-time PCR was performed as a measure of Th1 adaptive cellular immunity. Pre- and post-treatment (i.e., chemotherapy and GM-CSF) samples were evaluable for 16 patients, ranging from 1 to 5 cycles (median 3 cycles, 6 biologic sample time points). Dendritic cells were defined as lineage (-) and MHC class II high (+).Results: 73% of patients had significant increases in circulating dendritic cells of ~3x for the overall group (5.8% to 13.6%, p = 0.02) and ~5x excluding non-responders (3.2% to 14.5%, p < 0.001). This effect was sustained over multiple cycles for approximately half of the responders, but tachyphylaxis over subsequent chemotherapy cycles was noted for the remainder. Treatment also led to a significant reduction in the proportion of circulating regulatory T-cells (Treg; p = 0.0042). PBMC Tbx21 levels declined by 75% following each chemotherapy cycle despite administration of GM-CSF (p = 0.02). PBMC γ-interferon expression, however was unchanged.Conclusions: This clinical trial confirms the suppressive effects of chemotherapy on Th1 cellular immunity in patients with metastatic colorectal cancer but demonstrates that mid-cycle administration of GM-CSF can significantly increase the proportion of circulating dendritic cells. As the role of dendritic cells in anti-tumor immunity becomes better defined, GM-CSF administration may provide a non-toxic intervention to augment this arm of the immune system for cancer patients receiving cytotoxic therapy.Trial Registration: ClinicalTrials.gov: NCT00257322. © 2012 Martinez et al; licensee BioMed Central Ltd.

Background

Wnt signalling dysregulation has been implicated in cancer, including colon and gastric cancer. Initiation of Wnt signalling is modulated by soluble Wnt antagonists (sWAs), including soluble frizzled related proteins, dickkopf (Dkk) proteins, and Wnt inhibitory factor-1 (Wif1).

Aims

To evaluate the role of sWAs in upper (gastric) and lower (colon) gastrointestinal tract tumorigenesis.

Methods

Dkk1-3, Wif1, and FrzB expression was evaluated by in situ RNA hybridisation on normal and malignant human gastric and colon tissues. Expression was graded semiquantitatively.

Results

Wif1, Dkk1, and Dkk2 were not expressed in normal gastric tissue. Dkk3 was expressed in some samples, with stronger expression in deep gastric glands. FrzB was expressed in several normal gastric samples, but not in matched tumour specimens. In contrast, Dkk1 and FrzB were not expressed in normal colon. Wif1 was expressed in most colon samples, with stronger expression at crypt bases. Dkk3 and Dkk2 expression was also concentrated at crypt bases. There were no differences between sWA expression in malignant colon and matched normal tissue.

Conclusions

sWA expression differed between upper and lower gastrointestinal tract. The loss of FrzB in gastric cancer suggests that it acts as a tumour suppressor. The graded expression of Dkk3 in gastric tissue, and Dkk2, Dkk3, and Wif1 in colon tissue, with increased expression in the deep gastric glands/colonic crypt bases, where gastrointestinal stem cells reside, suggests that sWAs may be crucial Wnt signalling regulators in these tissues, and may contribute to stem cell pool maintenance. sWAs are important components of the gastrointestinal proliferative regulatory network.

Aims

Signalling through the Wnt pathway is integrally associated with colon carcinogenesis. Although activating mutations in the genes for adenomatous polyposis coli (APC) and beta-catenin are clearly associated with colon cancer, less is understood about the role of the upstream secreted ligands (Wnts) and their receptors (frizzled, Fz) in this process. In other systems, increased Wnt signalling has been shown to alter the expression of components of this pathway. This study was designed to test the hypothesis that colon cancer is characterised by aberrant expression of specific Wnt genes and Fz receptors.

Methods

The expression of Wnt genes was assessed by in situ, antisense RNA hybridisation in paraffin wax embedded samples of normal and malignant human colon tissues with probes specific for the individual Wnt genes. The expression of Fz1 and Fz2 was determined by immunoperoxidase based antibody staining on human tissues.

Results

Changes in the expression of some ligands and receptors were seen in colon cancer. For example, Wnt2 mRNA was detected in colon cancer but was undetectable in normal colonic mucosa. Differential expression of Wnt5a in normal mucosa was also noted, with increased expression at the base of the crypts compared with the luminal villi and slightly increased expression in colon cancer. Wnt7a exhibited minimal expression in both normal and malignant colon tissues, whereas other Wnt ligands including Wnts 1, 4, 5b, 6, 7b, and 10b were expressed equally and strongly in both normal and malignant colon tissues. In defining cellular responses and phenotype, the type and distribution of Fz receptors may be as important as the pattern of Wnt ligand expression. No expression of Fz receptor 1 and 2 was seen in normal colonic mucosa and in well differentiated tumours. However, poorly differentiated tumours exhibited a high degree of Fz receptor expression, especially at the margin of cellular invasion.

Conclusions

These data indicate that the expression of members of the Wnt signal transduction pathway, distinct from APC and beta-catenin, is integrally associated with the process of colon carcinogenesis. Wnt2, and possibly Wnt5a, may be involved in the progression from normal mucosa to cancer and the expression of Fz1/2 receptors may be involved in processes associated with tumour invasion. Altered expression of these Wnts and Fz receptors may prove useful as prognostic or diagnostic markers for patients with colon cancer.

A method was described for using steady-state and frequency-domain reflectance measurements to obtain broad wavelength quantitative data with deep tissue penetration. Fundamental information on tissue biochemistry and structure was provided using the wavelength-dependence of absorption and scattering parameters. Results showed that total hemoglobin content, water and lipid percentage can be measured using this technique.

Near-infrared (NIR) diffuse optical spectroscopy and imaging may enhance existing technologies for breast cancer screening, diagnosis, and treatment. NIR techniques are based on quantitative measurements of functional contrast between healthy and diseased tissue. In this study we measured the spectral dependence of tissue absorption (mu(a)) and reduced scattering (mu'(s)) in the breasts of 30 healthy women and one woman with a fibroadenoma using a seven-wavelength frequency-domain photon migration probe. Subjects included pre- and postmenopausal women between the ages of 18 and 64. Multi-spectral measurements were used along with a four-component fit to determine the concentrations of de-oxy and oxy-hemoglobin, water and lipids in breast. The scattering spectral shape was also quantified. Our measurements demonstrate that the measured concentrations of NIR analytes correlate well with known breast physiology. Although the tissue scattering at a single wavelength was found to have little value as a functional parameter, the dependence of the scattering on wavelength provided key insights into breast composition and physiology. Lipids and scattering spectra in the breast were found to increase and decrease, respectively, with increasing body mass index. Simple calculations are also provided to demonstrate potential penalties from ignoring the contributions of water and lipids in breast measurements. Finally, water is shown to be a possible indicator for detecting a fibroadenoma, whereas the hemoglobin saturation was found to be a poor indicator. Multi-spectral measurements, compared to measurements restricted to one or two wavelengths, provide additional information that may be useful in managing breast disease.

Constitutive activation of the Wnt/beta-catenin pathway has been implicated as the primary cause of colon cancer. However, the major transducers of Wnt signaling in the intestine, T-cell factor 1 (TCF-1) and TCF-4, have opposing functions. Knockout of TCF-4 suppresses growth and maintenance of crypt stem cells, whereas knockout of TCF-1 leads to adenomas. These phenotypes suggest that TCF-4 is Wnt-promoting, whereas TCF-1 acts like a tumor suppressor. Our study of TCF expression in human colon crypts reveals a mechanistic basis for this paradox. In normal colon cells, a dominant-negative isoform of TCF-1 (dnTCF-1) is expressed that is equally distributed between nuclear and cytoplasmic compartments. In colon cancer cells, TCF-1 is predominantly cytoplasmic. Localization is because of active nuclear export and is directed by an autocrine-acting Wnt ligand that requires Ca2+/calmodulin-dependent kinase II (CaMKII) activity for secretion and a downstream step in the export pathway. TCF-4 remains nuclear; its unopposed activity is accompanied by downregulation of dnTCF-1 and increased expression of full-length isoforms. Thus, the dnTCF-1 and TCF-4 balance is corrupted in cancer by two mechanisms, a Wnt/CaMKII kinase signal for nuclear export and decreased dnTCF-1 expression. We propose that dnTCF-1 provides homeostatic regulation of Wnt signaling and growth in normal colon, and the alterations in nuclear export and promoter usage contribute to aberrant Wnt activity in colon cancer.