Immunotherapy: New Strategies for the Treatment of Adrenocortical Carcinoma

Adrenocortical carcinoma (ACC) is a rare malignancy associated with a dismal prognosis. As stated at the 2002 National Institute of Science Consensus Conference, the likelihood of having a malignant adrenal tumor is clearly related to the size of an adrenal mass - 6 % of tumors greater than 4 cm are malignant, whereas as many as 25 % of masses greater than 6 cm present as ACC. At the time of diagnosis, approximately 40 - 70 % of tumors have already undergone metastatic spread [1]. ACC has a very poor prognosis with an overall 5-year survival of less than 10 - 25 % [1] [2]. The treatment options for metastatic ACC are limited. Although ACC is potentially curable in early stages with surgical resection, only 30 % of tumors have been reported as confined to the adrenal gland at the time of initial presentation [3]. Mitotane (o,p'DDD), an analogue of DDT insecticide, has been used in the treatment of ACC either alone or in combination with other agents. Although early studies in the era before modern imaging modalities reported high response rates, it now is clear that mitotane has a response rate in about a quarter of all patients at best. Moreover, these tumors also fail to respond to conventional therapy, including chemotherapy, with agents such as doxorubicin, vincristine or etoposide, or with radiation [4]. Other therapies such as image-guided percutaneous radiofrequency ablation may help to treat local metastases; however, this does not have any influence on clinical outcome in these patients [5].

The ineffectiveness of these modalities coupled with the potentially harmful effects of such treatments emphasize the need to search for novel tumor therapies. This includes alternative strategies such as gene therapy using adenoviral and retroviral vectors, immunotherapy with DNA vaccination, antigen-presenting dendritic cells (DCs), or a combination of these regimens. A gene therapy-based approach using transcomplementing adenoviral vectors resulted in a greater tumor volume reduction in an ACC mouse model [6]. A comparable in vitro study with various adenoviral deletion mutants induced an impairment of adrenocortical steroidogenesis [7]. Finally, active immunotherapy with autologous DCs pulsed with tumor antigens may serve as a promising adjuvant therapy in the treatment of ACC. DCs are antigen-presenting cells involved in the induction of primary immune responses. Several protocols have been generated for the purpose of obtaining large numbers of activated human DCs from either CD34+ bone-marrow cells or peripheral blood monocytes by culturing in media supplemented with GM-CSF and IL-4, followed by stimulation with TNF-α or monocyte-conditioned medium [8] [9] [10]. The unique ability of DCs to activate naive and memory CD4- and CD8-positive T-cells suggested that they could be used for the induction of specific anti-tumor immunity. Within the past few years, many in vitro and in vivo studies in rodents and humans have demonstrated that immunization with these cells pulsed with tumor antigens results in protective immunity and rejection of established tumors in various malignancies. Important data were reported in diverse carcinomas with as yet identified tumor-specific antigens or shared antigens such as malignant melanoma [11] [12], B-cell lymphoma [13], prostate cancer [14] and other malignancies. Because of the lack of specific tumor antigens in endocrine cancers such as in ACC, there are two alternatives for DC vaccination: first, preparations of autologous tumor cells could be used for antigen delivery within DC therapy. This approach was applied in our own pilot studies in neuroendocrine pancreas carcinoma and parathyroid carcinoma patients in advanced disease stages resulting in tumor lysate (TL)-specific anti-tumor immunity [15] [16]. Second, cell-specific antigens, such as specific proteins involved in the synthesis of hormones or hormones themselves, might be used for inducing a cytotoxic T-cell response against ACC. This approach has already been reported successful in inducing a Th1-dominated cellular immune and clinical responses in metastasized medullary thyroid carcinomas [17] [18].

Based on these results, a multitude of adrenocortical enzymes such as 11β-hydroxylase (P450c11), 21-hydroxylase (P450c21) and P450scc (side chain cleavage enzyme) as well as regulatory proteins such as steroidogenic acute regulatory protein (StAR) and DAX-1 may serve as specific targets for cytotoxic immune response in ACC. Recently, we have shown that 21-hydroxylase, which represents a key enzyme essential for normal zona glomerulosa and fasciculata function, is expressed in cortisol-producing (but not aldosterone-producing) carcinomas [19]. Similar data were demonstrated for the P450 side chain cleavage enzyme with some expression in non-functional adrenal carcinomas [20]. Steroidogenic acute regulatory protein (StAR) may prove to be an interesting and complementary target for anti-tumor immune response; this protein is responsible for the transport of cholesterol through the outer to the inner mitochondrial membrane during the process of steroid biosynthesis. StAR expression has not only been demonstrated in adrenocortical adenomas but also in carcinomas [21], and can therefore be used within an immunotherapy approach. DAX-1, which is essential for normal fetal development of the adrenal cortex, has been reported to act as a transcriptional repressor of steroidogenic StAR, suppressing steroidogenesis. Since DAX-1 is expressed in an intermediate fashion in non-aldosterone-producing adrenal carcinomas, it may also serve as a potent target within the adrenal gland [22]. As proof of principal, we investigated StAR as a potential target for an immune based therapy in a syngen mouse tumor model using DNA vaccination [23]. BALB/c mice were vaccinated three times within 3 weeks with cDNA expression vectors encoding for StAR or a control peptide followed by vaccine virus boosting. At four weeks, mice were subcutaneously inoculated with SP-2.0 myeloma tumor cells stably expressing StAR or control proteins. This vaccination strategy induced a specific cytotoxic T-cell response as measured by ELISPOT. Compared to control experiments vaccination against StAR protein resulted in a highly efficient protection against SP-2.0 tumor formation. No side effects on normal adrenocortical function were observed. These results demonstrate that tissue specific antigens such as StAR may serve as a potential target for immune therapy-based treatment strategies.

The key question besides the choice of target protein to be used is, however, how to present these antigens to the immune system in order to induce an efficient anti-tumor response. Transfection of autologous DCs with antigen-specific plasmid-DNA or RNA, respectively, promises to be a very elegant method since it has been successfully demonstrated in prostate cancer [14]. This approach would then use the advantage of efficient antigen transfection together with the highly effective antigen-presenting capability of DCs. Alternatively, these potent antigens could also be delivered by retroviral or adenoviral vectors coding for these targets by subcutaneous injections or gene-gun delivery. As mentioned, we have shown that adenoviral vectors themselves can also impair adrenocortical steroidogenesis [7]. Therefore, this approach would even help to ameliorate the hormonal effects in patients with ACC suffering from Cushing's syndrome or virilization in cases of ineffective anti-tumor response. Finally, both approaches could also be used in combination. In animals, DCs transduced with adenoviruses or retroviruses coding for antigens were successfully used to induce protective and therapeutic anti-tumor response against various tumor types [24] [25].

In summary, tissue-specific enzymes or regulatory proteins might be used in the future as targets to induce an immune response associated with the elimination of endocrine carcinoma cells in spite of the lack of defined tumor antigens in ACC. DCs, which might be pulsed with proteins or transfected with adenoviruses, should than represent an elegant vehicle to present these antigens to the immune system in order to induce a strong immune response.

References

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