Background. Expression of the pancarcinoma Thomsen‐Friedenreich (TF) carbohydrate antigen or, more correctly, hapten, in colorectal carcinomas is not generally agreed on. Furthermore, its suggested role in liver metastasis so far has not been substantiated by direct immunohistochemical evidence. Methods. Cryostat sections from 52 primary tumors (20 with adjacent transitional mucosa), 22 liver metastases of colorectal carcinomas, and 17 samples of normal mucosae were examined immunohistologically with a panel of at least two monoclonal antibodies (mAbs) each to TF, to its precursor, Tn, and to sialosyl‐Tn, among them two newly developed anti‐TF mAbs. Results. Of the primary colorectal carcinomas, 60% expressed TF. Staining was more intense with TF‐alpha/beta‐reactive than with exclusively TF‐alpha‐ or TF‐beta‐reactive mAbs. Normal and transitional mucosae were negative. Liver metastases were positive for TF in a significantly higher percentage of cases (91%) than primary carcinomas. Patients with TF‐positive primary tumors had a significantly higher risk to develop liver metastases compared with patients with TF‐negative tumors (57% vs. 14%, respectively). Tn and sialosyl‐Tn were expressed concomitantly in most primary (85%) and metastatic (95%) colorectal carcinomas. These antigens also were detected in transitional mucosae (Tn in 25%, sialosyl‐Tn in 55% of cases). Normal mucosae were negative. Conclusions. These results prove unequivocally the presence of exposed TF epitopes in a majority of colorectal carcinomas in which both anomers of TF are expressed. These data further suggest that TF favors liver metastasis and that its expression in primary colorectal carcinomas is a significant risk factor for the development of liver metastasis. Cancer 1995; 76:1700–8.

A group of 23 colorectal cancer patients were treated by a new type of active specific immunotherapy (ASI) following complete surgical resection of liver metastases (RO resection). For ASI treatment we used a vaccine consisting of 1 x 10(7) autologous, irradiated (200 Gy) metastases-derived tumor cells incubated with 32 hemagglutination units (HU) of Newcastle disease virus (NDV). The adjuvant vaccine therapy was started 2 weeks after surgery and was repeated five times at 14-days intervals followed by one boost 3 months later. The delayed-type hypersensitivity (DTH) skin reactions to the vaccine were measured as well as the DTH reactions to a challenge test of 1 x 10(7) non-virus-modified autologous tumor cells from liver metastases or 1 x 10(7) autologous normal liver cells. In addition 32 HU NDV alone and a standard antigen test (Merieux test) were applied pre- and post-vaccination. The vaccination was well tolerated. In 13 of 23 patients an increasing reactivity against the vaccine was observed during the vaccination procedure. Nine patients (40%) experienced an increased DTH reactivity against autologous tumor cells following vaccination, while 17% or fewer showed an increased reactivity to Merieux test antigens, NDV, or normal liver cells. The increased antitumor response was not correlated to responsiveness to NDV alone, autologous liver cells, enzymes and culture medium used for vaccine preparation or standard antigens (Merieux test). After a follow-up of at least 18 months 61% of the vaccinated patients developed tumor recurrence in comparison to 87% of a matched control groups from the same institution that had been only surgically treated. The results of this phase II trial are encouraging and should stimulate further prospective randomized studies.

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Active specific immunotherapy was performed in a phase I study in 20 colorectal cancer patients after surgical resection of the tumor. An autologous tumor cell vaccine surface modified by Newcastle disease virus (NDV) was used, which showed the following characteristics. After mechanical and enzymatic dissociation of the tumor tissue an average of 5 x 10(7) cells/g tissue was obtained. According to trypan blue dye exclusion assay the average viability was 72%. Following irradiation (200 Gy) the inactivation of proliferative activity of the cells could be demonstrated by the absence of incorporation of 3H-labelled thymidine. The cells were, however, still metabolically active as shown by the incorporation of [3H]-uridine and a mixture of 3H-labelled amino acids. Epithelium-specific antigens (detected by mAb HEA125) were expressed on more than 75% cells of the cell suspension indicating a high amount of (epithelium-derived) tumor cells. In order to increase the immunogenicity of the tumor cells the suspended cells were infected by the nonlytic, apathogenic Ulster strain of NDV. The successful modification of tumor cells with NDV could be shown by electron microscopy. Three weeks postoperatively cells were thawed, virus-modified, and inoculated intradermally in the upper thigh. Several cell and virus concentrations were tested in each patient. As control, tumor cells without NDV, NDV alone and normal colon mucosa were used. The number of tumor cells ranged from 2 x 10(6) up to 2 x 10(7) cells and NDV concentrations from 4 to 64 hemagglutination units (HU) were tested. Sixteen patients responded with a delayed-type hypersensitivity (DTH) skin reaction to the vaccine. The best DTH reaction, measured 24 h following vaccination, was obtained using a vaccine consisting of 1 x 10(7) tumor cells and 32 HU NDV (median induration of 8 mm). Response to NDV alone was seen in 2 patients only (median induration of 3 mm); 12 patients responded to tumor cells (1 x 10(7) alone (median induration of 4 mm). Of 10 patients tested with normal colorectal mucosa, 4 responded with a median induration of 3.5 mm. DTH responses to the vaccine of 1 x 10(7) tumor cells and 32 HU NDV increased throughout the repeated vaccinations to a median induration of 9.5 mm at the end of the therapy. No severe side-effects in the course of the immunotherapy, except for mild fever in 4/20 patients, were observed. The results of our phase I study show that this type of autologous colorectal tumor cell vaccine is ready for a large clinical trial to prove its efficacy.

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