**5. How to learn more? A difficult interplay between the technique, the tumor and the patient**

Three main areas of elucidation arise at the moment. First of all, intrinsic improvements of the vaccine details itself are to be monitored by advanced immune monitoring techniques(28). Apart from the classical monitoring assays aimed at detecting a specific anti-tumoral immune response, we should move towards a more global appreciation of the immune system and changes under therapy, both conventional and vaccination therapy. Indeed, preliminary evidence is emerging about the importance of the global immune status of the patient at time of diagnosis and treatment steps. Radiochemotherapy leads to a 're-setting' of the patient's immune system, possibly already then priming it towards a favorable or unfavorable starting position for subsequent immunotherapy. The vaccine itself might cause quantitative and qualitative shifts in immune cell subtypes, not only regulatory or effector T cells, but also natural killer cells or natural killer T cells. We should start to include monitoring of local immune cells in the target environment, i.e. the brain and brain tumor micro-environment, rather than only in the blood, which is often not (at all) correlating with the local immune conditions in the target organs. Probably coinciding with shifts in cellular compartments of the immune system, cytokine environments might mimic the underlying changes and are good candidates to represent relevant switches in micro-environment facilitating or suppressing an effective tumor rejection by the immune system. Usually, a Th-1 proinflammatory environment is believed to be beneficial for tumor rejection, although other

Immunotherapy for Malignant Gliomas: A Roadmap for the Future 421

A too quick global 'dissemination' of today's technologies will probably kill the credits for further development as too many aspects of the tumor vaccine approach itself are rapidly

The combination of both objectives can be accomplished by performing further preclinical experiments and small-scale early-clinical trials to optimize the vaccine technology as such and a gradual implementation of solid techniques of DC-based vaccine production and administration in large, randomized trials with the appropriate control arms to stepwise introduce the best available DC vaccine at that moment. The latter element is imperfect in se, but nevertheless highly required, even already at the moment, given the unmet medical

It is hard to predict the outline of the final, optimal DC-based vaccine for the future. Nevertheless it is clear it will have to integrate all the aspects of the difficult interplay between the technique, the tumor and the patient. Indeed, all of these three elements are highly relevant for a successful immunotherapy approach and probably for any approach at all. As for the anti-glioma DC-based vaccine production itself, preliminary evidence is being reported that whole tumor cell –based preparations are superior to defined epitopes in terms of overall survival data. Which types of other therapies that might synergize the most with cancer vaccines is subject of further investigations. Many candidates from radio-and chemotherapy over anti-angiogenesis, blood-brain barrier disruption techniques or oncolytic virus therapy do exist or are emerging today. Immune modulators and strategies able to modify the tumor micro-environment will play a crucial role in the future cancer vaccines. For anti-glioma vaccines, again many candidates immune modulators are at the edge of a clinical application to improve the overall vaccine efficiency. Substances like galectin-1, transforming growth factor β, interleukin 10, interleukin 6 and vascular endothelial growth factor are known to hamper vaccine efficiency and might even be interconnected, so interference with either of these locally secreted factors could result in a dramaticly increased vaccine efficacy. Finally, the patient itself creates the background that might alter the impact of all the interventions according to pre-existing parameters. In that regard, the notion of inherent immune cycles is an intriguing finding that needs however further clarification. Nevertheless, it might explain the indirect evidence we have for the tremendous importance of the timing of immunotherapy interventions on the final outcome

All this implies that we should consequently move into the direction of 'individualized' or 'customized' cancer vaccines rather than mass-produced 'off the shelf' constructs. Therefore , we should not mix up the ideal medical tracts with the desired 'manufacturing profile' of a cancer vaccine. It is highly likely that clinical results in this area of research will proceed the elucidation of all the underlying mechanisms rather than vice versa. Also for regulating agencies of cellular therapies, all the above mentioned aspects hold an enormous challenge

[1] Weller RO, Engelhardt B, Phillips MJ. Lymphocyte targeting of the central nervous

system: a review of afferent and efferent CNS-immune pathways. Brain Pathol

need and the promising results for important subgroups of patients thus far.

evolving towards a theoretical optimum.

of the patient.

**7. References** 

but also a 'life-important' responsibility.

1996; 6(3):275-288.

types of cytokine profiles, like e.g. a Th-17 mediated immune response are gaining importance in the global picture. Moreover, a TLR-agonist matured, fully Th-1 polarized DC vaccine has not yet been applied in larger clinical trials. A pro-inflammatory environment however is only facilitating a tumor rejection, if there's no evolution to a chronic inflammatory state, like in chronic inflammatory diseases : in this particular situation indeed, the immune system exhibits important signs of immune exhaustion. Even this state can be detected and monitored to date in an increasingly accurate way e.g. by analyzing zeta-chain down regulation in the T cell compartments(29). Many other relevant cell types from tumor infiltrating macrophages (especially those with pro-tumor 'M2' phenotype – called 'alternatively activated macrophages'), or tumor infiltrating myeloid-derived suppressor cells (MDSC), both abundantly present in several glioma models and human glioma specimens are only beginning to be unraveled. Analogue to the regulatory T cell compartment, which has been recognized for years as a relevant player in the balance between tumor rejection and immune tolerance, the first preliminary reports arise about 'regulatory dendritic cells'. It goes without saying that the fast acquisition of growing knowledge on the complex interplay between all these immune cells will influence our future understanding and concept of the current vaccine approaches.

A second important source of contributing insights will come from the molecular analysis of the tumor specimens. The molecular profile of a malignant glioma with the characterization of MGMT promotor methylation status, 1p19q co-deletion, PTEN loss, IDH1 mutation etc is rapidly gaining access to the routine clinical assessment of a common high grade glioma. To the same extent, predictive (or prognostic) markers for immunotherapy will become available as there are already now the reports on tumoral HOX genes relevant for 'immune reactions' (30)or the suggestion of an 'immunotherapy prone' mesenchymal phenotype of the glioblastoma tumor cells(31).

A third emerging field is the 'predictive' radiology field. In era's of pseudo-progression and therapy-induced radiological changes on gadolinium(Gd)-enhanced MRI of the brain tumor patients, distinctive radiological techniques predicting a tumor response or a tumor progression are of utmost importance. Especially its non-invasive character turns MRI into a preferred monitoring tool for malignant glioma follow-up especially for the new therapy classes. Preliminary steps are being made to try to distinguish vaccine-induced radiological changes from tumor progression in Gd-enhancing lesions on the MRI, using perfusionweighted (regional cerebral blood volume-rCBV) and diffusion-weighted (apparent diffusion coefficient-ADC) images in combination with spectroscopic findings(18). In terms of correct patient counseling, these new monitoring paradigms are considered crucial for the near future.
