The nucleotide sequence of 48 recombinant DNA clones of the inverse PCR products was determined per tumor in FY'08. We found that 69-82% of the tumor DNAs contained at least one retroviral integration site (RIS) per tumor. This corresponds to an average of 5.2 RIS per tumor. A similar copy number of 2-3 acquired MMTV genomes were detected by a Southern blot analysis of these same tumors DNAs. In our tumor panels, of those tumors that had at least 1 RIS on average 58% had one or more candidate common integration sites (CIS). I define a CIS as a locus in which a MMTV genome has integrated in the gene or near the target gene in two or more independent mammary tumors, the integration event has been shown to alter the target genes expression relative to other tumors having no viral integrations at that site or in normal mammary gland tissue, and the integration either confers on normal mammary epithelial cells a transformed phenotype or in vivo contributes to mammary tumorigenesis. The most frequent CIS target genes were members of the Wnt, Fgf, and RSpo gene families. The overall similarity in the frequency with which the Wnt and Fgf, gene families are activated in the three tumor panels suggests to me that they represent the """"""""Core"""""""" MMTV CIS. In addition to the Core MMTV CIS, we have identified 70 low frequency (n=2-3 tumors) candidate CIS. Their distribution seems to be specific to particular strains of MMTV or mouse strains. In FY'09 we expanded the study by comparing the RIS from our four tumor panels with those published by John Hilkens (The Netherlands Cancer Institute, Amsterdam, The Netherlands) and in collaboration with Susan Ross (University of Pennsylvania Medical School, Philadelphia, PA) their respective tumor models. The Hilkens tumor panel was from Balb/cfC3H mice. The Ross panels were comprised of mammary tumors from Balb/c mice infected with either the Shackleford Hybrid Provirus (Mtv1 5'LTR-gag-pol/MMTV(C3H) env-3'LTR) or the Hybrid Virus with the mutated ITAM (17 in each group) and C3H/HeN mammary tumors infected with MMTV(C3H). In addition we compared these panels of MMTV RIS with MuLV RIS in the RTCGD database (NIH) of viral induced leukemia's and lymphomas. The rationale for undertaking this analysis was to amplify the possibility to identify new low frequency CIS by expanding the number of tumors and secondly, determine whether there is a specificity of CIS with respect to particular strains of virus and tissue. Using this approach we analyzed 1223 MMTV RIS that were near a gene and found 431 CIS. Of these CIS, 226 were also contained a MuLV RIS. This suggests that 50% of the MMTV CIS are not specific to breast cancer and may be affected by mutation in neoplasias of other tissue. Currently we are comparing these CIS to genes which have been found to be mutated in primary human tumors.
