Clonal analysis and tracking oncogenic events

Workpackage 3

Objectives

To exploit molecular imprints in the tumor genome for understanding origins and clonal history of the MSC, and generate tumor-specific antibody probes. To investigate aberrant gene expression in the tumor clone to identify pathways of importance in MSC evolution.

Description of work

Tumor-derived V genes encode the idiotype, and provide a powerful tool to assay the MSC in MM with absolute specificity. As part of coordinated research activity, idiotypic V genes will be identified in a selected cohort of patients where sufficient tumor material has been banked. V gene based probes will be designed to track putative MSC in phenotypic subpopulations. V genes will be assembled as scFv-fragment C DNA fusion vaccines to generate id-specific antibodies by vaccinating mice. Hybridoma clones will be screened with tumor M protein. Purified anti-id antibodies will be tagged with fluorescent probes to evaluate the existence, frequency and selection of putative B-MSCs for further analysis. Selected MM cell lines will be used to validate the approach, where a sIg+ve CD138-ve component has been described. Known oncogenic lesions (N-ras, p53) will be tracked at the single cell level. Expression of activation induced cytidine deaminase (AID) will be followed by RT-PCR in putative MSCs: this enzyme catalyses ds DNA breaks, and may be critical to tumor progression via the MSC. We have shown recently that some MM cell lines express AID constitutively (unpublished data). We will utilise commercially available antibodies for AID to track its expression. However, if these are unsatisfactory, we will generate new antibodies for use in flow cytometry to assay clonal cells. We will again use DNA fusion vaccines to generate anti-AID antibodies. We will assess a role for CD40:CD40L stimulation in selected tumor subpopulations with MSC by using a transfected line expressing CD40L (stocked). Any aberrant mutational activity of AID at selected loci will be assessed by amplification and cloning for DNA sequence analysis, selecting genes identified by GEP as important in tumor progression. We will utilise a full range of chromosomal analyses to characterize the MSC and track its evolution (in collaboration with Dr. Niklas Zojer, Vienna). Clearly, signature chromosomal translocations will be retained by the MSC, as will mutations in critical genes (e.g. N-ras) or patterns of aberrantly expressed genes. The latter category is noteworthy, and the cancer testis genes (e.g. SSX-2) will be tracked in the MSC.

Deliverables

• Will serve as a core facility to generate id-specific molecular probes and id-based antibodies.
• We will provide probes and carry out all necessary analysis of important subclonal fractions in MM of interest as MSC. We are able to identify V genes at the single cell level.
• We will establish Q-PCR assays for single cell and small population studies.
• We will generate specific antibodies to track aberrantly expressed genes if required by the DNA fusion vaccine protocol.
• We will develop a range of techniques for chromosomal analysis of the MSC.

Milestones

• Assembly and functional verification of anti-id scFv.FrC DNA fusion vaccines.
• Generation of anti-id antibodies following vaccination with DNA encoding Id.
• Characterization of Id sIg+ve B-MSC by flow cytometry (frequency and phenotype).
• Characterization of genetic features associated with the MSC.
• Characterization of patterns of critical gene expression associated with MSC evolution ( CTAs).
• Analysis of a role for AID in MSC evolution.
• Characterization of abberant chromosomal events in MSC evolution.