Amplification and protein expression of chromosome 12q13-15 genes in osteosarcomas of the jaws.

Overexpression and amplification of several genes (MDM2, CDK4 and SAS) located on chromosome 12q13-15 have been noted to occur in various human sarcomas. As a result, two major growth regulation pathways may be inhibited. MDM2 may down regulate the p53-mediated growth control and CDK4 may affect pRB-mediated events. To determine the frequency of alterations in these genes and their correlation with clinicopathologic features, we analyzed the MDM2 and CDK4 protein levels by immunohistochemistry and assessed MDM2, CDK4 and SAS amplification by real-time PCR in nine osteosarcomas of the jaws. Positive staining for CDK4 and MDM2 was observed in eight cases (88.8%) and five cases (55.5%), respectively. Intense CDK4 staining was noted in four cases (two high grade, one intermediate grade and one low grade). Intense MDM2 staining was observed in the same four previous cases, as well as, one additional high-grade tumor. Individual DNA amplification for CDK4, MDM2 and SAS was observed in six cases for each gene. Co-amplification was observed in five cases that showed CDK4 and MDM2 concomitant amplification and four cases that displayed amplification for all of the genes. In addition, among the five cases that presented CDK4 and MDM2 amplification, strong overexpression of CDK4 and MDM2 was observed in three and in four cases, respectively (three high grade and one intermediate grade). These results suggest that 12q13-15 genes are involved in neoplastic disease and concurrent amplification and overexpression of these genes might help to define high-grade tumors.

The frequent mutation Gly/Asp in CDR1H may determine a cross-reactive idiotope in anti-I cold agglutinins.

Variable domains (VH) of all known anti i/I cold agglutinin (CA) heavy chains are codified by the VH4-21 gene. While anti-i CAs are the expression of gene rearrangement without mutations represented by amino acid changes, anti-1 CAs present, among others, a frequent somatic mutation of Gly by Asp at position 31. The hydropathy profile calculated for the CDR1H (position 30 to position 35), as well as some adjacent positions of the heavy chain belonging to anti-i and anti-I antibodies, showed the conformational changes accompanying the replacement of Gly by Asp. A MoAb (LP91), which had been obtained in BALB/c mice immunized with a Fabmu fragment from a monoclonal IgMkappaIIIb anti-I CA (protein KAU), proved capable of inhibiting human adult erythrocyte cryoagglutination by anti-I CAs but not that of fetal erythrocytes by anti-i CAs. Western blot analysis disclosed that such MoAbs recognized a sequential epitope located in the Fd fragment of all anti-I CAs employed in this study. With the purpose of checking whether Asp(31) was involved in the epitope recognized by the MoAb, two peptides, D and G, were synthesized which mimicked the CDR1H structure of anti-I and anti-i, respectively; the MoAb only reacted with peptide D by ELISA. Subsequent experimental results indicate that the Gly/Asp mutation could be associated with the diverse specificity presented by these autoantibodies, a change determining a characteristic epitope/idiotope, recognized by LP91 in the CDR1H.