Anke M. E. Claessen

Active specific immunotherapy for stage II and stage III human colon cancer: a randomised trial

BACKGROUND Colon cancer is curable by surgery, but cure rate depends on the extent of disease. We investigated whether adjuvant active specific immunotherapy (ASI) with an autologous tumour cell-BCG vaccine with surgical resection was more beneficial than resection alone in stage II and III colon cancer. METHODS In a prospective randomised trial, 254 patients with colon cancer were randomly assigned postoperative ASI or no adjuvant treatment. ASI was three weekly vaccinations starting 4 weeks after surgery, with a booster vaccination at 6 months with 10(7) irradiated autologous tumour cells. The first vaccinations contained 10(7) BCG organisms. We followed up patients for time to recurrence, and recurrence-free and overall survival. Analysis was by intention to treat. FINDINGS The 5.3 year median follow-up (range 8 months to 8 years 11 months) showed 44% (95% CI 7-66) risk reduction for recurrence in the recurrence-free period in all patients receiving ASI (p=0.023). Overall, there were 40 recurrences in the control group and 25 in the ASI group. Analysis by stage showed no significant benefit of ASI in stage III disease. The major impact of ASI was seen in patients with stage II disease, with a significantly longer recurrence-free period (p=0.011) and 61% (18-81) risk reduction for recurrences. Recurrence-free survival was significantly longer with ASI (42% risk reduction for recurrence or death [0-68], p=0.032) and there was a trend towards improved overall survival. INTERPRETATION ASI gave significant clinical benefit in surgically resected patients with stage II colon cancer. ASI has minimal adverse reactions and should be considered in the management of stage II colon cancer.

Reversal of mucosal tolerance by subcutaneous administration of interleukin‐12 at the site of attempted sensitization

Oral feeding of proteins causes peripheral T‐cell tolerance, as revealed by reduced delayed‐type hypersensitivity (DTH) reactivity after immunization. This type of tolerance can be due both to passive T‐cell anergy and active immunosuppression. Using ovalbumin‐fed mice we studied whether putatively immunostimulatory cytokines could break this state of mucosal tolerance. Cytokines were administered locally at the site of attempted sensitization. It was found that neither interleukin‐2 (IL‐2), interferon‐γ (IFN‐γ) nor granulocyte–macrophage colony‐stimulating factor (GM‐CSF) could restore the response to immunization. In contrast, local administration of IL‐12 at the site of attempted immunization resulted in full recovery of DTH reactivity. The dichotomy between the two Th1 stimulatory cytokines IFN‐γ and IL‐12 was also reflected by different effects on ovalbumin‐specific antibody isotypes. Although both IFN‐γ and IL‐12 downregulated serum IgG1‐levels in tolerant mice, suggesting decreased ovalbumin‐specific Th2 function, only local administration of IL‐12 led to increased serum IgG2a levels. These results support the view that potentiation of Th1 effector function is critical for reversal of mucosal tolerance.

Oral tolerance is determined at the level of draining lymph nodes

In the skin and in the epithelium of the oral mucosa a comparable network of Langerhans cells can be found. Antigen application on either epithelium leads to rapid emigration of Langerhans cells to the draining lymph nodes. Application on the oral mucosa leads to tolerance induction while application on the skin results in sensitization of the animal. Here we show that there are no differences in the antigen presentation capacity of oral mucosa- and skin-derived dendritic cells. However, measurement of IFN-gamma and IL-5 production, as representatives of Th1 and Th2 cytokines respectively, in total lymph node suspensions after sensitization via the skin or oral mucosa demonstrated a skewing of the response towards Th2 after antigen application on the oral mucosa. Together with our previous studies, in which it was shown that oral tolerance induction is not inherent to oral mucosa-derived dendritic cells, we postulate that oral tolerance is determined at the level of draining lymph nodes influenced by local cytokine profiles.

Cell-mediated immunity is enhanced by cytostatic drugs continuously released at the site of antigenic stimulation

SummaryImmunopotentiation by cytostatic drugs continuously released from osmotic minipumps, was investigated in a guinea-pig contact-sensitivity model. These pumps are designed to release their content within a period of 7 days. Vepesid (VP-16) and 5-fluorouracil were released into oxazolone-stimulated lymph nodes by subcutaneous implantation of pumps containing either of these drugs. The pumps were implanted at the intended sensitization site, 2 days before sensitization. Strong potentiation of T-cell-mediated immunity, evaluated by delayed-type hypersensitivity measurements, was observed with both drugs tested. Daily injections with VP-16 also caused an enhancement of the immune response. However, daily injections with 5-fluorouracil, a drug assumed to be cell-cycle-specific in its action, failed to potentiate delayed hypersensitivity to oxazolone. Intralesional administration of cytostatic drugs has been put forward as an effective treatment modality in various types of cancer. Therapeutic effects may depend on both local tumorcytotoxic and immunopotentiating activities. Our present results suggest that osmotic minipumps can be applied to broaden the applicability and effectiveness of local chemotherapy.

Tumor infiltrating leukocytes (tils) during progressive tumor growth and BCG-mediated tumor regression

SummaryTumor regression was induced by intralesional injection with BCG, 7 days after inoculation of line 10 hepatocellular carcinoma cells into strain 2 guinea pigs. Tumor-infiltrating leukocytes (TILS) were characterized immunohistochemically with 11 monoclonal antibodies (Mo Abs) during the induction phase of line 10-immunity, and during immune-mediated regression of the tumor, at days 12 and 28 after tumor cell inoculation, respectively.At day 5 after BCG-injection (day 12 after tumor cell inoculation), there were no major differences between the TIL subpopulations of the BCG-treated and untreated tumors. The TILS were mainly T-cells, as identified by MoAbs against Pan T-cells (CT5), T-cyto-toxic/suppressor cells (CT6) and T-helper/inducer cells (HI 55). A limited number of macrophages was also present. However, at day 21 after BCG-treatment (28 days after tumor cell inoculation), the fibrous stroma was increased dramatically in most of the BCG-treated tumors, and as a result, the tumor cell islets were smaller than in control tumors. In the BCG treated tumors, the numbers of T-cells and macrophages were increased. In growing and regressing tumors, MHC class I and II antigens were strongly expressed in TILS and in the tumor stroma. Line 10 tumor cells prior to inoculation expressed no MHC class I or II antigens. In the centers of the tumor islets at days 12 and 28, expression of these antigens was not found. However, MHC class I and II antigens were expressed on tumor cells at sites where they lay close to the fibrous stroma or TILS. This observation was made in progressively growing tumors and was most apparent in BCG-treated tumors.

Reversal of orally induced T-cell tolerance by subcutaneous administration of interleukin-12 at the site of attempted sensitization.

Feeding of proteins causes peripheral T-cell tolerance, as revealed by reduced delayed-type hypersensitivity (DTH) reactivity after immunization. Using ovalbumin-fed mice, we studied whether putatively immunostimulatory cytokines could reverse this state of mucosal tolerance. It was found that local administration of neither IL-2, IFN-gamma, nor GM-CSF resulted in reversal of tolerance. In contrast, subcutaneous administration of IL-12 at the site of attempted immunization resulted in complete recovery of DTH reactivity. The dichotomy between the two Th1-stimulatory cytokines IFN-gamma and IL-12 was also reflected by different effects on ovalbumin-specific antibody isotypes. Although both IFN-gamma and IL-12 downregulated serum IgG1-levels in tolerant mice, suggesting decreased ovalbumin-specific Th2 function, only local administration of IL-12 led to increased serum Th1-related IgG2a levels. These results support the view that potentiation of Th1 effector function is critical for reversal of mucosal tolerance.