Identification of biomarkers for metastatic MCC
- with prognostic impact independent of specific treatments (endpoint: disease-specific overall survival)
- with predictive impact on treatment outcome (endpoint: tumor response and progression-free survival)
- with predictive impact on the in WP4 ex vivo measured immune response (correlation with induction/enhancement of specific immune responses)
The present clinical trial provides the unique possibility to prospectively collect rare patient material (genomicDNA, tumor tissue, serum) from metastatic MCC patients within a multicenter setting. These samples from two different treatment arms will allow determining predictive biomarkers for the immune modulating therapy, and additionally serve as a high quality validation set. The respective discovery set to identify a panel of candidate biomarkers of interest has already been collected within the consortium and consists of annotated biological materials from advanced MCC patients. This set will also allow establishing appropriate assays. The resulting IMMOMEC biobank takes advantage of and will thereby expand the internet based AchieverTM medical systemwhich has been previously developed in the FP7 EU project OISTER to manage a multicenter melanoma biobank.
Prospective collection of patient material. We will coordinate the standardized collection, processing, quality control and storage of tumor tissue biopsies (formalin-fixed paraffin-embedded (FFPE), as well as cryopreserved samples) and sera taken from study patients at distinct time points before and after treatment onset. Whole blood will serve as a source for genomic DNA. Additionally, we will gather FFPE samples of the primary tumors of the respective patients from different pathology archives in order to analyze the impact of the primary’s characteristics compared to those of the metastases. Respective SOPs have been previously established for banking of biomaterial from melanoma patients. PBMCs will be collected within WP4.
Identification of candidate prognostic biomarkers from retrospectively collected patient materials. Biobanked patient materials already summoned in the consortium will be compiled and tested for its respective quality for further molecular analysis. Thus, we anticipate to have biological materials from an additional 50 MCC patients with known clinical course available for the following analyses:
- extent and composition of the inflammatory infiltrate (CD4+ / CD8+ T-cells, regulatory T cells, NK cells, macrophages, myeloderived suppressor cells)
- expression of molecules known to be associated with immune recognition (MHC class-I, MHC class-II, TAP-1and -2)
- detection of MCV and corresponding viral antigens (e.g. large T antigen)
- expression of tenascin C as one crucial target of this study’s therapeutic within the tumor and stromal tissue
- vessel density as well as activation status of vascular endothelial cells
- previously described tissue-based prognostic factors of MCC which have until now not been validated in independent sample series (tumor cell size, mitotic index, expression of bcl-2, Ki-67, p63, and somatostatin receptors)
All analyses will be performed by immunohistochemistry mostly using tissue micro arrays to minimize the necessary biological material. The obtained data will be correlated with the survival data of the corresponding patients using biostatistics (ZMF core facility of the MUG).
- presumed functional single nucleotide polymorphisms (SNP) of genes associated with immune response such as cytokines and cytokine receptors will be analyzed by Taqman-based real time assays (46).
- cytokine profiling by multiplexed assays on a fluorescent bead platform (Luminex)
- quantification of previously described serum-based prognostic factors which have until now not been validated in independent sample series (e.g. neuron-specific enolase or serum amyloid A (SAA) as a new prognostic serum marker in skin cancer)
- antibodies to MCV-specific antigens such as the small or large T antigens
These markers will be tested by multivariate analysis together with known clinical prognostic factors of MCC like primary tumor diameter, gender, age, and immune suppression for their value as prognostic markers in MCC.
Testing and validation of biomarkers in prospectively collected patient materials. In this project step, the candidate markers of interest identified in the above analyses will be tested and validated in the highly valuable samples collected within the clinical trial. Bioinformatic testing will be done in the whole patient cohort as well as in the two different treatment arms. This approach will allow the identification of prognostic as well as predictive markers for the respective therapy. For a broader analysis of predictive biomarkers we will perform additional analyses of 5 responders and otherwise matched-pairs from the non-responder group:
- next-generation sequencing of metastases for detection of possible intrinsic factors
- SNP analyses using a chip containing 9000 SNPs spanning 1000 genes involved in immune responses and inflammation (Affymetrix)
- Determination of autoantibodies to more than 9,000 full length proteins (Protoarray® protein microarray) in sera before and after therapy. This analysis should unveil additional MCC associated antigens. Subsequently, the strongest potential biomarkers identified by these analyses will be tested in the whole patient cohort.
Identification of biomarkers predictive of the ex vivo immune response. All of the above collected data will be correlated with the immunomonitoring results of WP 4 to determine whether any of the biomarkers predicts the induction of immune responses.