M. Heberer

Abstract 3384: High ALDH activity is associated with high tumorigenicity in prostate cancer

OBJECTIVE: Tumor initiation and progression might be driven by rare populations of cells endowed with stem-properties, and therefore defined as cancer stem cells (CSC). High Aldehyde dehydrogenase (ALDH) activity has been suggested to selectively identify CSC in several tumor types including breast cancer, and, possibly, prostate cancer (PCA). In this study, we investigated presence and potential CSC characteristics of cells with high ALDH activity (ALDH bright) in PCA cell lines, fresh surgical specimens, and primary cultures. MATERIALS AND METHODS: PC3, Du145, VCaP, and LNCaP PCA cell lines were evaluated. Surgical specimens, including Benign Prostate Hyperplasia (BPH) and PCA, were used directly for gene expression studies. Surgical samples were also enzymatically digested for functional analysis and the establishment of primary cultures. ALDH activity was tested using ALDEFLUOR® technology. Cells were sorted from individual cell lines by flow cytometry and evaluated for CSC properties, including spheroid formation ability, clonogenicity, stemness-related gene expression, ALDH specific isoforms expression, and tumorigenicity upon injection in NOD/SCID mice. RESULTS: PCA cell lines and primary cultures displayed heterogeneous ALDH activity and expression of specific ALDH isoforms. Despite a higher expression of Oct4A and Klf4 stemness associated genes, ALDH bright cells isolated from Du145 and PC3 did not show improved spheroid formation or clonogenic capacity, as compared to their dim counterparts. Interestingly, however, ALDH bright cells were associated with a significantly higher tumorigenic capacity in vivo as compared to ALDH low cells. Nevertheless, ALDH bright cells lost their higher tumorigenic capacity following serial “in vivo” passages. Most importantly, a well defined ALDH bright population could also be detected in cells isolated from PCA specimens (n>20). ALDH activity was consistent with a strong expression of several ALDH specific isoforms in PCA tissues. Notably, a significant increase of defined ALDH isoforms such as ALDH1A3 (p=0.001) was detectable in tissues obtained from patients with PCA as compared to BPH or normal specimens. CONCLUSIONS: ALDH bright subsets can be detected in cells isolated from fresh PCA tissue samples, PCA cell lines and primary cultures. These populations appear to be associated with increased “in vivo” tumorigenicity rather than enhanced “in vitro” stem properties in the cell lines investigated. Importantly, expression of ALDH specific isoforms appears to be increased in clinical PCA as compared to BPH and “normal” samples, thereby suggesting a putative role of ALDH in PCA tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3384. doi:1538-7445.AM2012-3384

Immunkompetenz bei Melanompatienten des Stadiums III–IV: Konsequenzen für eine aktiv spezifische Immuntherapie

Introduction: Patients with advanced cancer display some immunodepression. We assess here humoral and cytotoxic T lymphocyte responsiveness in melanoma patients undergoing active specific immunotherapy trials and correlate immunocompetency with the response rate to immunotherapy. Methods: 30 melanoma patients (TNM stages III and IV; n = 20 intradermal vaccination [1, 2]; n = 10 ongoing intranodal vaccination trial) were admitted to immunization trials with a recombinant vaccinia (rVV) virus encoding 3 tumor associated epitopes (TAA: gp100280–288, Mart-127–35 and tyrosinase1–9) and CD80/CD86 costimulatory molecules. Booster immunizations were performed with the same TAA peptides. Frequencies of CTL precursor (CTLp) specific for TAA or influenza matrix 58–66 (IM) control epitope were evaluated by limiting dilution analysis prior and after immunization. Humoral response against rVV vector was measured by ELISA prior and after vaccination. Depending on specific responses to immunization (CTLp > 2fold pre-treatment) patients were ranked as good responders (responsive to 3 or 2 antigens) or poor responders (unresponsive or responsive to 1 antigen only). Results: All stage III patients in both trials showed CTL responses against all 3 antigens. In contrast (p < 0.05), 6/13 stage IV patients were good responders. Specific humoral response for the rVV was increased in all stage III patients, but only in 6/13 stage IV patients (p < 0.05). A significant correlation (p < 0.05) was noted between a high humoral response against the rVV and the good responder status. The CTLp for influenza in melanoma stage IV patients was 4 times lower than in healthy volunteers, but for a patient who showed a long lasting complete response with a CTLp for influenza in normal range. The currently ongoing intranodal vaccination trial shows more good responders than the intradermal protocol (6/7 Vs 6/13 good responders, p < 0.05). Conclusion: Stage III melanoma patients and immunocompetent stage IV patients are more likely to respond to active specific immunotherapy. Immunocompromission in stage IV melanoma patients hampers the induction of tumor specific CTL responses. Conversely, induction of rVV specific humoral responses does not prevent but correlates with the generation of CTL specific for TAA. The intranodal immunization shows a higher response rate than the intradermal protocol. In summary, cellular and humoral immunocompetency are a marker for the response to immunotherapy and might therefore be an inclusion criterium in immunotherapy trial.

Verbesserung der immunstimulatorischen Wirkung von Tumorantigenen durch liposomale Vektoren

Introduction: Immunodominant epitopes from tumor associated antigens (TAA) are currently used as soluble peptides (SP) in clinical trials in spite of an acknowledged low immunogenicity. We asked whether encapsulation into liposomes (LP) could improve their immunogenic capacity. Materials and Methods: The HLA-A2 restricted melanoma associated epitope Mart26–35 was synthesized and included into sterically stabilized liposomes, characterized by prolonged bio-availability in biological fluids in comparison with conventional reagents. Proliferation, cytokine production and cytotoxicity induced by either LP or SP in Mart26-35 specific CTL clones were evaluated by 3H-thymidine incorporation, ELISA and 51Cr release assays. To assess the induction of specific CTL, peripheral blood mononuclear cells (PBMC) from healthy donors were stimulated with autologous antigen presenting cells and LP or SP. Cytotoxic activity was evaluated by 51Cr release. Results: Proliferation of CTL clones could be comparably stimulated by saturating doses (> 100 ng/ml) of either SP or LP. At limiting concentrations (< 50 ng/ml), however, only LP were effective. IFN-γ production by specific CTL was also enhanced by LP as compared to SP stimulation. Furthermore, LP displayed a significantly higher capacity of sensitizing target cells to the cytotoxic effects of specific CTL clones as compared to SP. Most importantly, LP were able to induce specific cytotoxic activity in healthy donors PBMC, following a limited number of restimulation courses (n = 2). In the same conditions SP was totally ineffective. Conclusion: These data indicate that the specific immunostimulatory capacity of Mart26-35 melanoma associated epitope can be significantly improved by encapsulation into sterically stabilized liposomes.

Rekombinante Vakzinia Viren als effiziente Vektoren biologisch aktiver, humaner B7-Kostimulationsmoleküle

Antigen-prasentierende Zellen (APC) konnen eine klonale Expansion spezifischer T-Lymphozyten induzieren. Dazu sind mindestens zwei verschiedene Signale erforderlich. Als erstes Signal wird der Antigen-HLA-Komplex prasentiert, der den T-Zellrezeptor aktiviert. Das zweite Signal wird meistens durch die Kostimulationsmolekule B7-1 und B7-2 vermittelt (Linsley et al. 1994). Diese Molekule, die vorwiegend an der Oberflache von APC zu finden sind, werden der IgG Superfamilie zugerechnet und aktivieren die T-Zell Rezeptoren CD28 und CTLA-4. Im Gegensatz zur Stimulation des T-Zell Rezeptors unterliegt das zweite Signal weder der HLA-Restriktion noch ist es antigenspezifisch. Die Induktion von CD4+ T-Helfer Zellen durch diese beiden Signale (Aktivierung von T-Zell Rezeptor und Kostimulation) fuhrt zu einer spezifischen, klonalen Proliferation. Wir haben in vitro gepruft, ob die Kostimulation von APC durch Infektion mit B7-kodierenden VV verbessert werden kann.

Induktion Tumor spezifischer zytotoxischer T-Lymphozyten durch nicht replizierende, Melan-A27–35 exprimierende rekombinante Vaccinia Viren in vitro

Heute sind Immunogene Epitope von humanen Tumor-assoziierten Antigenen (TAA) bekannt, die fur eine adjuvante Tumorvakzination genutzt werden konnen. Die Anwendung antigener Molekule und die Entwicklung von immuntherapeutischen Strategien, mit denen spezifische zytotoxische T-Lymphozyten gegen TAA induziert werden, konnte eine erfolgreiche spezifische Immuntherapie von Tumorleiden darstellen. Wir haben deshalb gepruft, ob mit einem rekombinanten Vaccinia Virus (recVV), welches das HLA-A2.1 restringierte immunodominante TAA Melan-A kodiert, Antigen-spezifische zytotoxische T-Lymphozyten (CTL) in vitro induziert werden konnen.