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Immunotherapy for Medullary Thyroid Carcinoma by Dendritic Cell Vaccination

Department of Endocrinology (M.S., J.F.) and German Diabetes Research Institute (J.S., M.L., V.F., W.A.S.), Heinrich-Heine-University Duesseldorf, D-40225 Duesseldorf, Germany

Address all correspondence and requests for reprints to: Matthias Schott, M.D., Department of Endocrinology, Heinrich-Heine-University Duesseldorf, Moorenstr. 5, D-40225 Duesseldorf, Germany. E-mail: schottmt{at}uni-duesseldorf.de

Abstract

Recent studies suggest that immunization with autologeous dendritic cells (DCs) pulsed with tumor antigen result in protective immunity and rejection of established tumors in various human malignancies. The objective of this study was to develop a DC vaccination therapy in patients with metastasized medullary thyroid carcinoma (MTC). Mature DCs were generated from peripheral blood monocytes in the presence of granulocyte macrophage colony-stimulating factor, IL-4, and TNF. After loading with calcitonin and carcinoembryonic antigen (CEA) peptide, 2–5 x 10⁶ DCs were repeatedly delivered by sc injections.

During follow-up (mean, 13.1 months) all patients developed a strong delayed-type hypersensitivity skin reaction caused by perivascular and epidermal infiltration with CD4+ memory T cells and CD8+ cytotoxic T cells. Clinical responses with a decrease of serum calcitonin and CEA were initially documented in three of seven patients. One of these patients had a complete regression of detectable liver metastases and a significant reduction of pulmonary lesions. T-cell response in this patient revealed a calcitonin- and CEA-specific immunreactivity.

Our data indicate that vaccination with calcitonin and/or CEA peptide-pulsed DC results in the induction of a cellular, antigen-specific immune response in patients with MTC, leading to clinical response in some patients. Our approach may represent the basis for the development of new therapeutic strategies not only in MTC but also in other endocrine malignancies.

MEDULLARY THYROID CARCINOMA (MTC), arising from the parafollicular, calcitonin-producing C cells, represents usually slowly growing tumor types occurring in both sporadic (56%) and familial (44%) forms including MEN type 2 (MEN 2) (1). When the tumor is confined to the thyroid gland itself, total thyroidectomy and central lymph node dissection may result in long-term cure (2, 3). In many instances, however, elevated plasma calcitonin and/or carcinoembryonic antigen (CEA) levels indicate persistent metastatic disease, although conventional diagnostic procedures such as computed tomography (CT) or magnetic resonance imaging scans fail to localize the respective lesions. Although the prognosis of patients with unresectable disease and/or distant metastases of MTC is generally better than with other neoplasms, it is still poor (1). The ineffectiveness of chemotherapy and external beam irradiation emphasize the need to establish novel therapeutic strategies. One such alternative could be an active immunotherapy using autologeous dendritic cells (DCs) pulsed with tumor antigen(s) to generate a cytotoxic immune response directed against the cancer cells.

Compelling evidence suggests that DCs are professional antigen-presenting cells (APCs) specialized to activate naive and memory CD4+ and CD8+ T cells (4, 5). When compared with other APCs, MHC-peptide complexes are expressed 10- to 100-fold higher on DCs (6). Additionally, DCs express a panel of accessory molecules that interact with receptors on T cells (7, 8). Consistent with their unique abilities, several studies have documented the generation of antitumor cytotoxic T cells after vaccination with tumor antigen-loaded DCs. Promising results were reported for metastasized melanoma (9, 10, 11), advanced prostate cancer (12, 13), B-cell lymphoma (14), and renal cell carcinoma (15, 16). Recently, our group demonstrated the induction of cellular immunity and a clinical response in two patients with parathyroid carcinoma and a neuroendocrine pancreas carcinoma (17, 18). Here, we report on a clinical trial of DC vaccination in metastasized MTC. For the first time, we demonstrate the induction of a specific immunoreactivity following DC vaccination, leading to a clinical response in three of seven treated cases.

Materials and Methods

Patients

Table 1 provides a summary of patient characteristics. Patients with histologically proven MTC with radiologically established disease or with postoperative elevated plasma calcitonin levels higher than 100 pg/ml as a marker of residual disease were included in the study. Genotyping revealed that six of seven patients were HLA-A2 positive. These patients were treated with double-pulsed (CEA and calcitonin) DCs. Because of the HLA-A2 restriction of the CEA peptide (YLSGANLNL), patient 2 (HLA-A1 positive) received only DCs pulsed with calcitonin. The study protocol was approved by the Ethical Review Board.

Mature DCs were generated from peripheral blood mononuclear cells (PBMCs) as described recently (19). PBMCs were allowed to adhere to plastic dishes, and adherent cells were cultured for 6 d with granulocyte macrophage colony-stimulating factor (800 U/ml; PromoCell) and IL-4 (500 U/ml). On d 6, DCs were washed and cultured with granulocyte macrophage colony-stimulating factor (800 U/ml), TNF (1000 U/ml; PromoCell), and antigen. DCs (5 x 10⁶) were pulsed with CEA and calcitonin (Novartis, Nuernberg, Germany) (10 µg and 100 µg/ml per 1 x 10⁶ DCs). During the first two cycles keyhole limpet hemocyanin (Calbiochem, La Jolla, CA) was administered as a CD4+ helper antigen as well (100 µg/ml) (19).

Treatment of patients and clinical response

Antigen-pulsed DCs (1–5 x 10⁶ cells in 100 µl 0.9% NaCl) were administered by intracutaneous injections in the upper arm. The first four treatments were administered weekly, whereas the following vaccinations were given at intervals of 4–8 wk (Table 2). Metastatic lesions were evaluated by CT scans and ultrasonic examinations. WHO definitions were used for complete response (no metastatic lesion detectable) and partial response (reduction of tumor sites >50%) for at least 3 months. Stable disease was defined as a change of less than 25% in tumor size and tumor markers without occurrence of new lesions for a minimum of 6 wk. Measurements of CEA (Roche, Mannheim, Germany) and calcitonin (Nichols, Bad Nauheim, Germany) were performed by commercial kits according to the manufacturer’s instructions. A reduction in tumor markers of more than 25% for at least 1 month was considered as an antitumor effect ("mixed response"), and an increase of more than 25% in these markers was considered as progression (20). A partial response as judged by tumor markers was defined as a reduction by at least 50% for a minimum of 12 wk.

Freshly prepared PBMCs were resuspended in RPMI 1640 supplemented with 10% autologeous serum, penicillin (100 U/ml), and streptomycin (100 µg/ml) and were incubated with 1% Lymphocult-T (Biotest, Dreieich, Germany) and 100 µg/ml calcitonin and CEA (separately) for 5 d. Then, 1 x 10⁵ cells were cultured in round-bottom 96-well tissue culture plates in triplicates in the presence of CEA (1–100 µg/ml), calcitonin (1–100 µg/ml), keyhole limpet hemocyanin (1–100 µg/ml), or ovalbumin (1–100 µg/ml) for an additional 5 d. Cells were pulsed with 1 µCi [³H] thymidine per well (Amersham Pharmacia Biotech, Braunschweig, Germany) 18 h before harvesting. Thymidine incorporation was assessed using a microscintillation counter (Canberra Packard, Dreieich, Germany). Cellular proliferation was expressed in stimulation indices (SIs) using ovalbumin as a control protein.

Delayed-type hypersensitivity (DTH) and immunhistochemistry

DTH skin tests were performed with CEA/calcitonin-pulsed DCs. DCs were injected intradermally into the upper arm. A positive skin test reaction was defined as erythema and induration more than 5 mm in diameter 24 h after injection. In some cases, a biopsy (diameter, 5 mm) of the DTH site was taken. Cryostat sections were incubated with anti-CD4 and anti-CD8 monoclonal antibodies (BD PharMingen, San Diego, CA) and stained with alkaline-phosphatase labeled secondary antibody and fast red (DAKO Corp., Hamburg, Germany).

Statistical analysis

The results were analyzed for statistical significance by paired t test using Prism computer software (GraphPad Software Inc., San Diego, CA).

Results

Clinical response to DC vaccination

DC vaccination was well tolerated by all patients. There were no serious adverse effects or any clinical signs of autoimmune reaction. During follow-up (mean, 13.1 months) three of seven patients (43%) developed a clinically measurable response after DC vaccination, of whom one (patient 3) had a partial response (PR) and two other patients (1, 7) developed a mixed response (Table 2). Patient 3 developed complete regression of all detectable liver metastases and a significant regression of pulmonary metastases (Fig. 1). In parallel, there was a substantial decrease of the tumor marker calcitonin from 8895 pg/ml to 2870 pg/ml (68%) after treatment and of the serum CEA from 208 µg/liter to 63 µg/liter (70%) (Fig. 2). Another patient (no.1) developed a mixed response with a steady decrease of serum calcitonin by 30% (with a nadir in June 1999) during the first 5 months of DC immunotherapy, but returned to pretreatment tumor marker levels during follow-up. A CT scan performed after 1 yr revealed liver metastases without signs of progression. Four of seven patients (patients 2, 4, 5, and 6) had a stable disease without biochemical or morphological (CT scan, ultrasound) signs of growing tumor masses. Patient 7 showed a decrease of the calcitonin levels (36%) within the first 2 months of therapy. However, a progression of liver metastases was observed after 10 months of immunotherapy. Therefore, vaccination therapy was finished and chemotherapy was started.

View larger version (121K): [in this window] [in a new window]   Figure 1. Clinical response to DC vaccination. CT scan of patient 3 before (September 1999, left) and 13 months after (October 2000, right) initiation of DC vaccination shows complete regression of detectable liver metastases (top) and a significant reduction of pulmonary lesions (bottom). Arrows, sites of lesions before vaccination.

View larger version (19K): [in this window] [in a new window]   Figure 2. Tumor marker levels during therapy. Plasma calcitonin (lower panel) and CEA (upper panel) levels obtained at various time points during follow-up. Patient 3 developed a PR (decrease of tumor markers >50%), patients 1 and 7 had a mixed response, whereas patients 2, 4, 5, and 6 developed no response after DC vaccination.

To monitor T lymphocyte reactivities in vivo, we used DTH skin reaction as a standard technique for detecting antigen-specific immunity. Following the third to fifth immunization with CEA- and calcitonin-pulsed DCs, a significant reaction with an erythema and induration was observed in all patients, suggesting the presence of CEA- and/or calcitonin-specific memory T lymphocytes. In patient 3, who developed a strong clinical response, the last immunization with CEA- and calcitonin-pulsed DCs was given separately. At both injection sites a strong DTH skin reaction was observed. Immunohistochemistry of the skin biopsy revealed a strong perivascular and epidermal infiltration with CD4+ helper T lymphocytes as well as CD8+ cytotoxic T cells. In addition, we performed T-cell proliferation assays using PBMCs. Before therapy there was no PBMC reactivity, neither against CEA (SI_{1 µg/ml} 0.9 - SI_{10 µg/ml} 1.21 vs. ovalbumin) nor against calcitonin (SI, 0.6–0.95). Of special interest was the characterization of the immune reaction of patient 3, who developed a partial tumor remission. After 12 vaccinations there was a significant PBMC proliferation toward CEA peptide (SI, 1.78–2.72; P < 0.001 vs. pretreatment) and calcitonin (SI, 1.51 to 2.74; P < 0.001 vs. pretreatment), indicating the induction of a specific T-cell response against both antigens.

Discussion

Here, we report on a prospective study of DC vaccination therapy in patients with metastasized MTC. For the first time, we describe the induction of a T cell-dependent immunity following DC vaccination using cell-specific proteins. A clinical response was observed in three of seven patients, of whom one patient showed a significant regression of liver and pulmonary lesions.

A major problem of all endocrine malignancies, including MTC, is the lack of defined tumor antigens. Feasible targets for the induction of cytotoxic immunity may, however, represent the CEA and the C cell-specific hormone calcitonin. CEA is a tumor-associated antigen that occurs among a wide range of cancers, including MTC. The generation of an immune response against the CEA antigen has been described to be difficult in previous studies using CEA as a vaccine (21, 22). This might be explained by the expression of CEA in fetal tissue and the colon, causing a state of immune tolerance in adults. Breaking of peripheral tolerance might, however, be achieved by using highly activated APCs such as DCs for antigen delivery. Recent in vitro studies have demonstrated that autologeous DCs loaded with CEA peptide or CEA RNA are able to induce cytotoxic T lymphocytes in patients with CEA-expressing malignancies in a HLA-dependent manner (23, 24). Another approach to induce C cell-specific immunity in MTC may represent the immunization with naked plasmid DNA coding for the antigen. By using an animal model Haupt et al. (25) have demonstrated that vaccination with plasmid DNA encoding preprocalcitonin was followed by an antibody response and a significant T-cell proliferation in some animals. In light of these promising results, here we used both antigens for our vaccination strategy to widen the spectrum of immunreactivity and to possibly minimize the escape of tumor cells from immune surveillance.

In line with previous studies in other tumors (9, 26), here we demonstrate that the reduction of tumor mass and/or tumor marker emerged within the first weeks or months of vaccination. In patient 3, eradication of liver metastases was already detectable 5 months after the start of vaccination (CT scan not shown). Patients 1 and 7 also had a decrease of the calcitonin and CEA levels within the first couple of months. Careful clinical examination of our patients did not reveal any marker that could explain why some patients respond to the vaccination whereas others did not show any benefit. It is obvious that additional studies are required to answer this question.

In conclusion, this is the first trial on a DC vaccination in patients with MTC demonstrating a beneficial effect in some patients. Our observations strongly suggest that DC immunotherapy using CEA and/or calcitonin represents a promising therapeutic approach in yet untreatable forms of MTC. We have now started a randomized trial to evaluate the optimal DC vaccination technique in patients with MTC.

Acknowledgments

Footnotes

M.S. and J.S. contributed equally to this work.

Abbreviations: APC, Antigen-presenting cells; CEA, carcinoembryonic antigen; CT, computed tomography; DC, dendritic cell; DTH, delayed-type hypersensitivity; MEN 2, MEN type 2; MTC, medullary thyroid carcinoma; PBMC, peripheral blood mononuclear cell; PR, partial response; SI, stimulation index.

Received April 9, 2001.

Accepted July 16, 2001.

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Schott, M. Articles by Feldkamp, J. Search for Related Content PubMed PubMed Citation Articles by Schott, M. Articles by Feldkamp, J.