Projects

Project 1. Characterization of the primary tumor, liver metastases and temporal T-cell immunity in the SECApatients

1.1 Recent studies have addressed the importance of regulatory T-cells (T regs) in suppressing T-cell immunity to tumor associated antigens. There is evidence that T regs may be the a main obstacle of successful tumor immunotherapy and that patients who exhibit allograft tolerance have increased levels of T regs. Temporal levels of T regs are measured at both preoperatively and at fixed intervals postoperatively. Lymphoid infiltration in tumors will be assesed, as this has shown to be an independent prognostic factor in CRC.

1.2 Cytokine profiles will be assessed at Ltx and at defined time points at follow up. The cytokine profiles will be evaluated in regard to immunosuppressive treatment, to the cancer disease and in recognition of the liver as an active immunological organ participating in local and systemic host defence. Relevant cytokines will be quantified by Bioplex assays.

1.3 T-cell immunity will be assessed by mRNA from the tumor cells at time of Ltx (RNA-later). The RNA will be used to transfect dendritic cells and monocytes in order to measure T-cells ability to recognize this material post transplant.

1.4 T-cell receptor clonotype mapping will be performed. CD4+ and CD8+ T cells will be isolated and used for blocking experiments. Results will be correlated to the clinical course of the patients.

1.5 Samples of tissue from the metastases as well as normal liver tissue will be collected and frozen (-70ºC) for later investigations. The properties of this tissue and the possible relation to the clinical course of the patients will be explored. The exact modalities of analysis are not yet fully established as de-freezing is a nonreversible event that needs meticulous planninng. The analyses will be performed after all patients have been included in the trial. There are several possible paths to follow:

1.6 Tumor gene expression profiles will be determined in metastases and in normal liver tissue (oligonucleotide micro array analysis)

1.7 Proteome profiles will be assessed. Clinical proteomics is a field which has been reported to have high sensitivity and specificities for early detection of several solid tumours, and is promising in the search for novel biomarkers in CRC. Analyses of the proteomic profiles will be performed by MALDITOF mass spectrometry and peptide sequencing using linear ion trap mass spectrometry (q-Trap).

1.7 DNA-Ploidy pattern and chromosomal instability will be assessed by flow cytomtery complemented by image analytic techniques. Abnormalities in DNA content (aneuploidy) have been independently associated with adverse prognosis in CRC. Analyses of microsatelite stability and chromosomal instability will be performed as these factors also have shown to be of prognostic value.

1.8 The role of disseminated tumour cells ((DTC, micro metastases). The prognostic value of disseminated tumour cells in bone marrow (BM) has been investigated in several studies comprising patients with epithelial cancers such as breast-, colorectal- and gastric cancer. The presence of DTC in BM seems to be of predictive value for the development of metastases not only in bones but also in other distant organs even in tumours that rarely develop skeletal metastases such CRC. Concerning breast cancer it has been shown that the detection of disseminated tumours cells in bone marrow by immunocytochemical methods is a strong and independent prognostic factor. Although not completely unequivocal, possibly because of a certain degree of methodological concern and lack of standardization, identification of DTC in BM also seems to predict disease free survival for patients with CRC. The incidence of DTC in BM in patients with isolated liver metastases from CRC is low when compared to patients with primary CRC or disseminated CRC disease, but their presence is associated with an poor prognosis. There is strong evidence, at least from breast cancer that DTC in the peripheral blood from patients with N0 disease is correlated with a high incidence of systemic relapse and mortality. In CRC the inconsistency of detection is larger and a more standardized methodology and sets of markers are called for. Nevertheless, it is probable that such a correlation will be found using a combination of markers. Little is known about the molecular characteristics of isolated DTC cells. Recent technological advances involving enrichment of the relevant cell population, amplification procedures for DNA and RNA as well as high-throughput molecular screening tools now render such characterization feasible. Samples containing from DTCs in BM, systemic- and portal blood give the possibility to compare molecular characteristics of cells from these compartments with each other and with the hepatic metastatic tissue. A novel aspect is that peripheral blood is collected for DTCs at Ltx, both at induction of anaesthesia and after manipulation of the liver in order to assess the potential effect of manipulation of the liver. Samples from BM, portal- and systemic blood will be processed at the Department of Tumour Biology immediately after sampling, using standard procedures for mononuclear cell isolation ie. immunobead rosetting and preparation of cytospins for immunocytochemical analysis. Tumour cells isolated by immunobead rosetting will be further enriched using the CellPick system followed by RNA/DNA isolation and molecular profiling.

Project 2 Mechanisms of regulatory T cell immune suppression and characterization of the regulatory T cell – effector T cell interaction in vitro, with special emphasis on the potential of cytostatic drugs as immunosuppressants in SECA-patients and in transplanted patients with de novo malignancy.

The concept of transplantation of cancer patients raises several immunologic considerations. 1) Immunosuppressive treatment to prevent rejection may promote cancer recurrence. 2) High tumorload in the liver may lead to a relative immune suppression that possibly involves regulatory T cells (TR cells), thus the patients may need less intense immunosuppressive treatment than liver transplant recipients treated on other indications. 3) Cytostatic drugs with immunosuppressive effects may be an alternative to standard immunosuppressive treatment for the SECA patients.

1.1 The role of regulatory T cells in cancer immunity in SECA patients. Regulatory T cells have been shown to inhibit antitumor immune responses in various studies in patients with malignant diseases such as colorectal cancer, ovarial cancer, breast cancer and melanoma. In this project we aim to investigate the suppressive role of regulatory T cells in anti-tumor immune function in the SECA patients. This cohort of patients will be compared to patients with lower tumor burden and who are eligible for local liver resection (the Ullevål patient cohort). Anti-tumor immune function will be evaluated i) prior to transplantation; ii) four months after; iii) one year after transplantion and; iv) at the time of recurrence.

1.2 Identification of down-stream cell-signalling pathways involved in TR cell function. As a part of a broader strategy to test known anti-cancer drugs with immunosuppressive effects to prevent rejection and reduce risk of recurrence in the SECA-patients, we wish to assess the effects of known anti-cancer drugs, immunosuppressive drugs and small molecular compounds (Src-, PI3-, MEK and p38-kinase inhibitors) on TR cell function and effector T cell function. Compounds with a preferential, different or selective effect on either T cell subset, may point to important physiological differences in the intracellular signal transduction machinery with potential clinical value. Cell proliferation and intracellular cytokine production will be assessed by flow cytometry in CD4+ and CD8+ T cells.

1.3 The physiological role of regulatory T cells and the mechanisms of action in interaction with effector T cells. In this in vitro project, we will aim to elucidate the physiologic role of regulatory T cells and the mechanisms of action in interaction with effector T cells. Regulatory T cells have been extensively studied for the last ten years. However, several fundamental questions remain unanswered. The molecular interactions underlying the immunosuppessive activity of regulatory T cells are not known. Furthermore, the temporal and cellular requirements of the immunosuppressive activity of the regulatory T cells during the cell-cell interaction with the effector T cells during an ongoing immue response, have not been thoroughly dissected.

1.3.1 Kinetics of the immunosuppressive activity of regulatory T cells. In order to elucidate whether TR cells inhibit effector T cells in the early phases of TCR-engagement and T cell activiation, or later in the “production-phase” we will perform add-back CFSE proliferation experiments running for 5 days. TR cells will be added to anti-CD3/CD28-stimulated T cells at time zero, 30’, 1h, 2h, 4h, 6h, 12h and 24h.
1.3.2 Identification of the mode of action of regulatory T cells. Regulatory T cells have to be engaged through the TCR-complex to obtain immunosuppressive activity. Several lines of evidence indicate that thymic-derived naturally occurring TR cells require cell-contact to suppress effector T cells, whereas an extensive body of literature indicate potentially important roles of cytokines such as IL-10 and TGF-b. We aim to address these questions by PFA-fixation and BFA-treatment of TR cells prior to add-back experiments. Both these measures will prevent secretion of soluble factors. PFA-fixation at different time points after anti-CD3/CD28 activation of TR cells (5’, 30’, 1h, 2h, 6h and 12h) – and prior to add-back to effector T-cells – will reveal whether secretion of soluble factors are required of not. Furthermore, if cellular-contact dependent mechanisms are sufficient, the required time of activation prior to add-back of TR cells, will be important for identification of relevant plasma membrane proteins involved in the suppressive activity. This study will also allow for assessment of the persistency of the immunosuppressive properties, ie. whether the immunosuppressive activity occurs at a confined time window after TCR-engagement. In a similar manner, BFA-treatment to block the Golgi-apparatus, will be performed at different time points after anti-CD3/CD28 activation of TR cells – and prior to add-back to effector T cells. The rationale for this study is similar to the use of PFA, but with the important difference that BFA does not kill the cells.