In vitro schedule-dependent interaction between melphalan and oxaliplatin in human colorectal cancer cell lines.

OBJECTIVE: In order to determine the applicability of oxaliplatin in isolated liver perfusion, we identified the interaction between combinations of oxaliplatin and melphalan in 13 human colorectal cancer cell lines. METHODS: Cytotoxic activity was determined by the MTT-assay. Three different administration schedules of the two drugs were compared and median effect isobologram analysis was applied to the results, to determine the presence of synergism, additive effects, or antagonism as described by Chou and Talalay. RESULTS: Resistance to melphalan did not correspond to resistance to oxaliplatin. All combinations of melphalan and oxaliplatin showed synergistic or additive interaction in the majority of the cell lines. One hour of oxaliplatin followed by 1h of melphalan showed the lowest percentage of cell viability, with synergy in 10 out of 13 cell lines at 50% cell viability. Simultaneous treatment showed the highest cell viability, with antagonism in six cell lines, additivity in two cell lines, synergism in five cell lines at 50% cell viability. One hour of melphalan followed by 1h of oxaliplatin showed synergy in six cell lines, antagonism in another six, and additivity in one cell line. CONCLUSIONS: Our findings suggest a schedule-dependent synergistic interaction between melphalan and oxaliplatin. Therefore, oxaliplatin should be considered as a new, potentially valuable additional agent to the currently commonly used melphalan in isolated hepatic perfusion in colorectal cancer patients.

Prediction of the immunogenic potential of frameshift-mutated antigens in microsatellite instable cancer.

Microsatellite instable (MSI) cancers express frameshift-mutated antigens, the C-terminal polypeptides of which are foreign to the immune system. Consequently, these antigens constitute a unique pool of tumor-specific antigens that can be exploited for patient diagnosis and selective, immune-mediated targeting of cancers. However, other than their sequence, very little is known about the characteristics of the majority of these proteins. We therefore developed a methodology for predicting their immunogenic behavior that is based on a gene-expression system, in which each of the proteins was fused to a short C-terminal polypeptide comprising two epitopes that can be readily detected by T-cells and antibodies, respectively. In this manner, accumulation of the antigens and processing of peptides derived thereof into MHC can be monitored systematically. The antigens, which accumulate in the cells in which they are synthesized, are of primary interest for cancer immunotherapy, because peptide epitopes derived thereof can be presented by dendritic cells in addition to the tumor cells themselves. As a result, these antigens constitute the best targets for a coordinated immune response by both CD8+ and CD4+ T-cells, which increases the likelihood that tumor-induced immunity would be detectable against these antigens in cancer patients, as well as the potential value of these antigens as components of anticancer vaccines. Our data indicate that, of 15 frameshift-mutated antigens examined in our study, 4 (TGFbetaR2-1, MARCKS-1, MARCKS-2 and CDX2-2) are of primary interest, and 4 additional antigens (TAF1B-1, PCNXL2-2, TCF7L2-2 and Baxalpha+1) are of moderate interest for further tumor immunological research.

Prognostic value of tumor-infiltrating dendritic cells in colorectal cancer: role of maturation status and intratumoral localization.

The clinical significance of tumor-infiltrating dendritic cells has been reported in a variety of human solid tumors as shown by the correlations found between the presence of tumor-infiltrating dendritic cells and clinical prognosis. In this study, we evaluated whether there is an association between the presence and maturation status of tumor-infiltrating dendritic cells, T lymphocytes, and clinical course in 104 primary tumor samples of patients with colorectal cancer. Dendritic cells were identified with four different markers (S-100, HLA class II, CD208, and CD1a) in double immunohistochemistry, with laminin as second marker to support the exact localization. Tumor-infiltrating dendritic cells showed a distinct infiltration pattern based on their maturation status. CD1a-positive dendritic cells resided in the advancing tumor margins in relatively high numbers, whereas mature CD208-positive dendritic cells were sparsely present in the tumor epithelium but mainly distributed in the tumor stroma and advancing tumor margin. Furthermore, high infiltration of CD1a-positive dendritic cells in the tumor epithelium was significantly correlated to the infiltration of CD4 lymphocytes (P = 0.006). Patients with relatively high numbers of mature CD208-positive infiltrating dendritic cells in the tumor epithelium had a shorter overall survival (P = 0.004). In addition, patients with relatively high numbers of CD1a-positive dendritic cells in the advancing margin of the tumor had a shorter disease-free survival (P = 0.03). We found that tumor-infiltrating dendritic cells had preferential infiltration sites within a tumor, affected local tumor cell-immune cell interactions, and correlated to the clinical prognosis of colorectal cancer patients.

p53 and HLA class-I expression are not down-regulated in colorectal cancer liver metastases.

p53 overexpression occurs in more than 50% of colorectal carcinomas, which makes it an interesting target for immunotherapy. HLA class I expression on tumor cells is required for the presentation of p53 peptides and an effective T-cell mediated-immune response to ensue. To analyze to which extent p53 and HLA-I expression in a primary tumor reflects expression in liver metastases, we investigated p53, HLA-A and HLA-B/C expression in 82 colorectal carcinomas and 143 associated liver metastases of 82 patients. We used the monoclonal antibodies DO-7 (p53), HCA2 (HLA-A) and HC-10 (HLA-B/C) on formalin-fixed, paraffin embedded tissue. The percentage of expressing cells was estimated. P53 was overexpressed in 73% of the colorectal carcinomas and 66% of liver metastases. HLA-A was expressed in 98% and 96% and HLA-B/C in 100% and 94% of colorectal cancers and liver metastases respectively. There were no significant differences between the primary tumors and the liver metastases for each marker. The concordance was also very high in those cases in which more than one metastasis was available. Discordant cases consisted of tumors in which expression of p53 or HLA-A was lost in the liver metastases, whereas it was present in only a few tumor cells in the primary tumor. The combined analysis of p53 and HLA-I expression in liver metastases demonstrated that both molecules were expressed in 63% of the cases. P53 and HLA-I were expressed in the majority of primary tumors and their associated liver metastases. This allows to select patients for p53-immunotherapy on the basis of p53 and HLA-I expression in the primary tumor.

Immune system and prognosis in colorectal cancer: a detailed immunohistochemical analysis.

The efficacy of tumor cell-immune cell interactions depends on a number of factors, for example, the expression of HLA-I on tumor cells, the type of immune cell, the accessibility of tumor cells for immune cells and the expression of immunogenic epitopes. We assessed infiltration of CD4+, CD8+, CD56+ and CD57+ cells in the tumor epithelium, tumor stroma and advancing tumor margin of 93 colorectal carcinomas and correlated this to clinicopathological parameters, the expression of HLA-A and HLA-B/C on tumor cells, the presence of a basal membrane (BM)-like structure surrounding tumor nodules and the presence of microsatellite instability/mutator phenotype (absent MLH-1 expression). The median intraepithelial CD4+, CD8+, CD56+ and CD57+ cell infiltrations were 3, 23, 0 and 0 cells/mm(2) tumor, respectively. HLA-A/BC expression by tumor cells was normal in 28/43%, heterogeneous in 59/48% and absent in 13/9% of the cases. A BM-like structure surrounding the tumor nodules was absent, present and thick in 47, 38 and 15% of the cases. Six cases lost MLH1 expression. There was a positive correlation between leukocyte infiltration in the three compartments for CD4+, CD8+, CD56+ (partly) and CD57+ (all P<0.05) cell infiltration. Intraepithelial CD8+ cell infiltration inversely correlated with HLA-A (P=0.04) and HLA-B/C expression (P=0.04). Intraepithelial CD57+ cell infiltration inversely correlated with HLA-B/C expression (P=0.04). Moreover, intraepithelial infiltration of CD8+ and CD57+ cells was inversely correlated to the presence of a BM-like structure (P=0.003 and 0.04, respectively). Uni- and multivariate analyses showed that a lower tumor stage (P=0.004) and marked infiltration of CD8+ (P=0.04) and CD57+ cells (P=0.05) at the advancing tumor margin were independent prognostic factors for a longer disease-free survival. Loss of MLH1 expression was correlated with a significantly higher intraepithelial CD8+ and CD57+ cell infiltration. We conclude that infiltration of CD8+ and CD57+ cells are important prognostic factors in colorectal cancer. However, their interaction with tumor cells is inversely correlated to the presence of HLA-I on tumor cells and a thick BM-like structure around tumor islets. Our data indicate that NK cells might play an important role in the immune surveillance in colorectal cancer patients.

A basal membrane-like structure surrounding tumour nodules may prevent intraepithelial leucocyte infiltration in colorectal cancer.

Epithelial tumours consist of an epithelial compartment and a stromal compartment, which are sometimes separated by a basal membrane-like structure. We sought to determine whether these factors have prognostic value in 84 curatively resected stage II and III colorectal cancer by immunohistochemically staining tumours for leucocytes (CD45) and extracellular matrix, and to assess the presence of a basal membrane-like structure. Leucocyte infiltration was also assessed in hematoxylin-eosin (HE) stained sections. Most leucocytes were located in the tumour stroma. A relatively high intraepithelial leucocyte infiltration was significantly correlated with a lower level of tumour recurrence (P=0.03) and a longer disease-free survival (P=0.05), whereas leucocytes located in the tumour stroma (P=0.92) or at the advancing margin (p=0.06) were not. Intraepithelial leucocyte infiltration was also significantly correlated with leucocyte infiltration in the tumour stroma (P=0.02) and at the advancing tumour margin (P=0.005), and as assessed in HE-stained tumour sections (P=0.05), but each of these parameters on its own did not have a prognostic value in predicting disease-free survival. Moreover, the presence of a basal membrane-like structure surrounding the tumour epithelium was inversely correlated with the number of intraepithelial leucocytes (P=0.05), suggesting that this membrane-like structure functions as a barrier to intraepithelial leucocyte infiltration. We conclude that leucocytes must be in the direct vicinity of tumour cells to affect tumour growth. The presence of an extracellular matrix barrier seems to prevent this interaction.

Down-regulation of HLA-A expression correlates with a better prognosis in colorectal cancer patients.

To evaluate the prognostic impact of human leukocyte antigen class I (HLA-I) expression on immune surveillance in colorectal cancer, we studied 88 curatively resected tumors for HLA-A and HLA-B/C expression and correlated these data to clinical and histopathological parameters. HLA-A was normal (all tumor cells had HLA expression) in 32%, reduced (HLA-negative and -positive tumor cells coexisted) in 56%, or absent (no tumor cells expressed HLA) in 12% of evaluable cases. HLA-B/C was normal in 47%, reduced in 47%, and absent in 7% of the cases. Considering both markers, total HLA-I expression was normal in 27%, reduced in 63%, absent in 7%, and could not be evaluated in 3% of the cases due to absent HLA-A expression in tumor and normal cells. Down-regulation of HLA-A expression significantly correlated with a lower tumor stage (p = 0.005), mucinous tumors (p = 0.05), a lower incidence of recurrences (p = 0.03), and a longer disease-free survival (p = 0.02). Down-regulation of HLA-B/C expression correlated with a lower tumor stage (p < 0.001) and a longer disease-free survival (p = 0.04). In multivariate analysis, HLA-A down-regulation was the only prognostic factor correlated with a longer disease-free survival (p = 0.02). Six tumors were negative for HLA-A and -B/C and did not recur during follow-up. Therefore, we analyzed microsatellite instability (MSI) in these cases. Three of these six tumors indeed showed down-regulation of MLH-1, MSH-2, or MSH-6, indicating a MSI-high phenotype. Beta-2-microglobulin protein expression was lost in five of six of the HLA-I-negative cases, but frame shift mutations in three repetitive sequences in beta2-microglobulin were absent. In contrast, loss of MLH-1, MSH-2, and MSH-6-protein expression was only observed in two of nine matched controls with reduced or normal HLA-A and -B/C expression. Our data showed that HLA-I was down-regulated in 72% of colorectal cancers and provided independent prognostic information for a longer disease-free survival. The better prognosis may be caused by elimination of HLA-negative cells by natural killer cells or by an attenuated tumor aggressiveness, as is seen in tumors with a MSI-high phenotype.