Colon Cancer

A. J. M. van den Eertwegh

A.J.M. van den Eertwegh (u)

Department of Medical Oncology, Vrije Universiteit Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands

e-mail: vandeneertwegh@Vumc.nl

Abstract

Colorectal cancer is one of the leading causes of cancer-related mortality. After a series of clinical trials, the adjuvant 5-FU-based chemotherapy has established a definitive role in the management of stage III colon cancer. While the precise role for chemotherapy in stage II disease remains under investigation, less toxic treatment modalities such as active specific immunotherapy (ASI) have emerged as potentially attractive alternatives. In a randomized trial it was demonstrated that ASI, using autologous tumor cells and BCG, had a significant clinical benefit in the adjuvant treatment of stage II colon cancer. In addition, it was found that the delayed-type cutaneous hypersensitivity reactions (DTH) against autologous cells correlated significantly with survival, confirming its suitability for immunomon- itoring. Nowadays, 5-FU-based chemotherapy is a standard treatment for stage III colon cancer and because there is preclinical evidence for synergism between chemotherapy and ASI, we performed a phase II study to investigate whether the combined treatment with 5-FU and ASI is tolerable and feasible. We demonstrated that 5-FU treatment hardly affected the DTH response against autologous tumor cells and that the combination treatment was well tolerated. A randomized phase III trial is now warranted to show that the combination of ASI and chemotherapy is superior to chemotherapy alone.

Colon Cancer

Colorectal cancer is a leading cause of cancer death in Western societies [5]. Most patients present with surgically resectable disease, but unfortunately in a signifi- cant number of these patients metastases will occur in the years following surgery.

Only a low percentage of patients with recurrent disease may be cured by radical resection of metastases and the majority will ultimately die of their disease [5]. In general, surgical cure of colorectal cancer is only possible if the malignancy has not spread beyond the regional lymph nodes. Stage II colon cancer patients, whose Recent Results in Cancer Research, Vol. 165

Springer-Verlag Berlin Heidelberg 2005c

tumors invade through the muscularis propria or serosa, have a 5-year survival rate of approximately 75%, while the survival rate of stage III patients with lymph node metastases is between 25% and 60%, depending on the number of positive nodes [5]. Different types of adjuvant treatments have been developed as auxiliary weapons to surgery aiming at eradicating the so-called micro-metastases [19]. So far, chemotherapy has been the only modality that has evolved to a standard treat- ment for stage III colon cancer. Preclinical studies suggest that immunotherapy is most effective in patients with low residual disease [3], and although the clin- ical results are very promising, particularly for ASI, this new modality is not yet a standard adjuvant treatment for colon cancer [8].

Adjuvant Chemotherapy

The early adjuvant trials were retrospective and under-powered and failed to show any therapeutic benefit with respect to recurrence rate or survival [4]. In 1990 the intergroup trial showed that adjuvant treatment with 5-FU/levamisole resulted in 33% reduction in the odds of death and a 41% decrease in recurrences as compared with surgery alone or surgery plus levamisole [16]. These results were confirmed by three large randomized phase III trials investigating 5-FU and leucovorin as adjuvant treatment [17, 20, 25]. Nowadays, 5-FU/leucovorin is considered as standard adjuvant therapy for stage III colon cancer patients.

For the adjuvant treatment of stage II colon cancer, the precise role of chemother- apy is still not defined. The intergroup 0035 trial failed to demonstrate a survival benefit from 5-FU/leucovorin chemotherapy in stage II patients, although a reduc- tion in recurrences comparable with that documented for stage III patients was observed [18]. Even in an international multicenter pooled analysis of stage II colon cancer trials with a total accrual of 1,016 patients, no significant benefit in overall survival could be demonstrated [7]. Several possible reasons for this discrepancy in clinical benefit have been suggested. Because of the relatively low frequency of death in stage II patients, most adjuvant trials contain an insufficient num- ber of patients to be able to address the study’s primary questions with adequate statistical power. Patients participating in these trials are relatively old and have co-morbidity and consequently a relatively large percentage of non-cancer-related deaths. This dilutes the observed survival difference and makes the detection of a significant survival benefit very difficult.

To circumvent this problem, future studies should focus on stage II colon cancer patients with high-risk prognostic factors (e.g., microsatellite instability and p53 mutation, tumor-positive sentinel node), to make it easier to identify a subset of patients that may benefit from adjuvant chemotherapy.

Adjuvant Immunotherapy

Immunotherapeutical approaches for colorectal cancer have substantially evolved over the past years from treatments with nonspecific immune stimulants to more tumor-specific therapies. Nonspecific immune stimulating therapies such as Bacil- lus Calmette-Guérin (BCG) [21] and levamisole [16, 17] were tested in randomized trials and were found to have no clinical benefit in the treatment of high-risk colon cancer patients. Therefore, research is now moving towards more tumor-specific immunotherapies such as colon cancer-specific antibodies [9] or active vaccina- tion strategies. In this article we will review the promising results of ASI, using autologous tumor cells and BCG, in the adjuvant treatment of colon cancer.

Adjuvant Active Specific Immunotherapy

ASI differs from nonspecific immune-based therapies in that the goal is not gen- eral but rather specific activation of the immune system to eliminate tumor cells without affecting surrounding normal tissue [22]. It is generally assumed that specific vaccination should result in activation of the two main arms of the im- mune system, namely the humoral (antibody-producing B cells) and the cellular immune response (T cells). B cells recognize the tumor antigens in their native protein state at the cell surface, whereas T lymphocytes recognize proteins as peptide fragments, presented in the context of major histocompatibility complex (MHC) antigens on the surface of the tumor cells. There are two types of T cells, CD4 and CD8, which recognize antigens through a specific T-cell receptor. These antigens are presented by a group of specialized cells called antigen-presenting cells (APCs). A variety of cells are capable of processing and presenting antigens, including B cells, monocytes, macrophages, and dendritic cells (DCs). DCs are the most efficient APCs, expressing costimulatory molecules and high levels of MHC class I and class II molecules required for the activation of CD8- and CD4-positive T cells, respectively. CD4-positive T cells, also called helper T cells, secrete cy- tokines that regulate B cells, cytotoxic cells and other immune cells, but can also have a cytotoxic activity. CD8-positive cytotoxic T cells (CTL) are at this moment considered to be the most potent cells to eradicate specifically tumor cells. The purpose of most vaccination strategies is to activate this specific subset of T cells.

DCs are essential for the specific activation of T cells and these cells are found in the lymphoid organs, blood and skin. The most convenient way to load these APCs with tumor antigens is via an intradermal injection. An adjuvant such as BCG is co-administered to activate the skin DCs and these APCs will subsequently migrate to the draining lymph nodes, where they will encounter and activate the tumor-specific T cells.

A logical strategy of immunization is the use of whole tumor-cell preparations.

One of the main advantages of this approach is that all potential tumor antigens of the patients’ carcinoma are presented to the immune system. Hanna et al.

established a guinea pig hepatocarcinoma model for the study of ASI as adjuvant treatment [10]. They demonstrated the value of a vaccine prepared from viable

metabolically active tumor cells mixed with BCG organisms. A correct ratio of BCG organisms to tumor cells and an optimal vaccination schedule enabled them to control hematogenous and lymphatic metastases from surgically excised primary tumors.

On the basis of these preclinical studies, Hoover et al. conducted a trial using irradiated autologous tumor cells and BCG in patients with stage II and stage III colorectal cancer [14]. After surgical resection of their primary tumors, patients were randomized to vaccination or observation and stratified by both disease type and stage. Three to 4 weeks after surgery, patients were vaccinated with two weekly vaccinations with tumor cells and BCG. One week later, a third vaccine was admin- istered, not containing BCG. An intention-to-treat analysis showed no significant clinical benefit, but a subgroup analysis of overall and disease-free survival in colon cancer patients showed a significant trend for ASI being superior to surgery alone. Side effects were minimal and the most prominent were ulcerations at the site of the first two vaccinations caused by BCG. Immunized patients showed DTH reactions to autologous tumor cells that were stronger than background responses to autologous mucosal cells, suggesting the presence of tumor-specific immunity.

The absence of a survival benefit in the rectal cancer group was thought to be caused by the radiotherapy that was given close to the vaccination-draining lymph nodes. However, it is important to realize that because of the low number of vac- cinated rectal cancer patients, this low-powered study does not allow a reliable analysis of the efficacy of ASI in rectal cancer. Nowadays, rectal cancer patients are being irradiated before removal of the carcinoma, which precludes ASI in its current form.

These promising results were reason to conduct a large phase III study with stage II and stage III colon cancer patients under the auspices of the Eastern Cooperative Oncology Group (ECOG) [13]. This study differed from the Hoover study in that each site performed its own vaccine manufacturing because of large numbers of subjects and wide geographic distribution of the sites involved. From a practical point of view, this meant that some centers were making only a few vaccines in a year. The fact that these centers were not making vaccines on a daily basis could have had a negative effect on the quality of vaccines. In an intent-to-treat analysis of all randomized patients, there were no significant differences between the two treatment arms in time to recurrence or overall survival. In the ECOG study, 12%

of all vaccines failed to meet quality control specifications (cell number/viability), and 15% of the vaccinated patients failed to have adequate DTH reactions. It was hypothesized that the poor quality of a part of the vaccinations could have caused the disappointing results of this study. Therefore, a survival analysis was performed on patients who were treated with vaccines that met standardized criteria and developed antitumor immunity (DTH response to third vaccine >5 mm) and compared to control patients. In this subgroup analysis, a significant improvement in overall survival was demonstrated in patients treated with ASI, suggesting that optimal immunization strategies are essential for a successful adjuvant treatment of colon cancer patients. This was supported by the observation that the size of DTH response to autologous tumor cells correlated with survival, which was also observed by our group in metastatic melanoma [1]. The development of a DTH

response could also reflect the immune status of patients. However, in a separate study it was demonstrated that stage II + III colon cancer patients had normal skin reactions against recall antigens, indicating that these patients with no or a low tumor burden had a normal immune status, which makes the latter possibility not so likely [13].

A third phase III study was conducted by the Vrijt University Medical Cen- ter Amsterdam in the Netherlands involving 254 patients with stage II and stage III colon cancer [24]. This pivotal study differed from the previous clinical trials in that treated patients received a booster with irradiated tumor cells alone, ad- ministered 6 months after surgical resection. In contrast to the previous study, a centralized manufacturing laboratory supported the 12 participating hospitals, which prepared 98% quality-approved vaccines, and 97% of the vaccinated pa- tients had DTH responses greater than 5 mm. In an intent-to-treat analysis, ASI significantly reduced the rate of disease recurrence by 44% in patients with stage II and stage III colon cancer, but the overall survival was not significantly better. The major impact was seen in stage II disease in which there was 61% risk reduction for recurrences and a trend toward improved overall survival. These data may indicate that the centralized method of vaccine manufacturing, or a facility that makes vaccines on a daily basis, is very important for vaccine quality and success- ful immunization. It is also possible that the extra vaccination after 6 months may have contributed to the positive results in the latter trial. The relatively low number of stage II patients in this trial did not make it possible to detect a small survival difference. Furthermore, the absence of a significant survival benefit could also be explained by the same arguments as were discussed for the adjuvant chemotherapy trials. The relatively high non-colon-cancer-related mortality in this aged patient group together with the relatively good overall survival rate of stage II colon cancer patients requires a very large randomized study to detect a survival benefit for any adjuvant treatment.

Therefore, a meta-analysis was performed that included the above-mentioned three randomized trials [12]. In the intent-to-treat meta-analysis of all 723 pa- tients who received either a three- or a four-vaccine regimen, recurrence-free survival was significantly improved by ASI. In the meta-analysis of patients who met quality control specifications and protocol eligibility, recurrence-free survival was significantly improved and disease-specific survival approached significance when compared with controls. In general, patients with a distant recurrence will eventually die from colon cancer. However, even though recurrences were signif- icantly reduced by ASI, no significant survival benefit could be demonstrated in the intent-to-treat meta-analysis, which might be explained by the same statistical difficulties for chemotherapy trials in stage II colon cancer, as discussed above. In conclusion, these studies showed that ASI has minimal side effects and that the most pronounced clinical benefit can be seen in stage II colon cancer.

In stage III colon cancer patients, ASI did not result in a significant clinical benefit, which could be explained by the lack of statistical power of these studies.

Furthermore, the residual tumor load in stage III patients is probably larger than in stage II patients, which could be relevant since it is known that ASI is more effective in a minimal residual disease setting [10].

In preclinical models, ASI and chemotherapy were shown to have a synergistic antitumor effect [12]. Apart from the capacity to directly destroy micro-metastases, ASI has been demonstrated to disrupt the characteristically compact structure of metastatic foci, enabling chemotherapy to reach deeper into the cancer tissue.

Furthermore, chemotherapy reduces the tumor burden, thereby increasing the chances of ASI to eliminate the residual malignant cells. In preparation for a large phase III trial, we performed a feasibility study on the combination of ASI and chemotherapy in stage III colon cancer. We showed that the combination ASI and 5-FU/leucovorin did not result in more toxicity and that the ASI-induced antitumor immunity (DTH response) was hardly impaired by consecutive chemotherapy [2].

A randomized trial should prove that these two modalities have a synergistic antitumor effect.

Future Developments

One of the consequences of using colon tumor tissue for the production of au- tologous tumor cell vaccines is the inherent presence of small amounts of the normal colonic bacterial flora in the vaccines. The assumption that this does not present a clinically relevant threat to the health of the patients receiving the vac- cines is supported by the observation that no serious infections have occurred after more than 2,000 colon tumor vaccinations in the VU University Medical Center in the past 14 years. However, recent guidelines of the American Food and Drug Administration prescribe that anything that is injected into a patient needs to be sterile. To obtain sterility we slightly changed the production process of the vaccines. An important side effect of the adjuvant BCG is ulceration that can last more than 3 months. After healing, a scar at the site of vaccination remains visible. The last decade new potent adjuvants were developed which were in pre- clinical studies at least as good as BCG. Nowadays most vaccination studies use granulocyte-macrophage colony stimulating factor (GM-CSF) as an immunostim- ulating agent. This cytokine is able to recruit Langerhans cells and promote the maturation of these cells into potent APCs through up-regulation of MHC class II, co-stimulatory molecules and cytokine production [6]. It increases antibody re- sponses and cellular immunity after immunization. Another interesting adjuvant is Cytosine-phosphate Guanine (CpG) 7909, a single-stranded phosphorothioate oligodeoxynucleotide (ODN), which is a potent stimulator of both the innate and adaptive immune response [15]. CpG 7909 is a very potent adjuvant that promotes Th1 humoral and cellular immune responses with very little toxicity, particularly no ulcerations. Besides its enhancing effect on antibody production, CpG 7909 promotes the effector arm of the humoral response as well. The combination of GM-CSF and CPG7909 was found to give superior antitumor-immunity as com- pared to GM-CSF alone [23]. In the near future, we will investigate in a phase II trial whether the combination of CPG 7909 and GM-CSF at least equals the results of our previous studies using BCG alone. For this purpose the DTH response against autologous tumor cells will be used as measure for antitumor immunity. If results of the phase II study show that modified ASI is at least as effective as found in

previous studies, we will proceed with a randomized trial in stage II colon cancer comparing modified ASI with no treatment.

Conclusions

It took several decades before the toxic adjuvant treatment with 5-FU/leucovorin was established as a standard treatment for stage III colon cancer. Specific im- munotherapies have a low toxicity profile and randomized clinical trials suggest a promising role for ASI in the adjuvant treatment of colon cancer. ASI with autologous tumor cells have been investigated in three randomized trials and a meta-analysis confirmed that ASI has the greatest clinical in stage II colon can- cer. Because of new guidelines of the American Food and Drug Administration requiring sterile vaccines and the emerging of new potent adjuvants, it is expected that a new formulation of vaccines will be developed and studied in a large ran- domized trial to demonstrate its efficacy in colon cancer. In stage III colon cancer, it was demonstrated that a combined modality treatment with ASI and chemother- apy is well tolerated and that chemotherapy hardly affects the antitumor immunity induced by the tumor cell vaccination. A randomized phase III trial is warranted to show that the combination of ASI and chemotherapy is superior to chemotherapy alone.

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