p53 dysregulation in B-cell malignancies: More than a single gene in the pathway to hell.

TP53 deletion or mutation is frequent in B-cell malignancies and is associated with a low response rate. We describe here the p53 landscape in B-cell malignancies, from B-Acute Lymphoblastic Leukemia to Plasma Cell Leukemia, by analyzing incidence of gain or loss of function of actors both upstream and within the p53 pathway, namely MYC, RAS, ARF, MDM2, ATM and TP53. Abnormalities are not equally distributed and their incidence is highly variable among malignancies. Deletion and mutation, usually associated, of ATM or TP53 are frequent in Diffuse Large B-Cell Lymphoma and Mantle Cell Lymphoma. MYC gain, absent in post-GC malignancies, is frequent in B-Prolymphocytic-Leukemia, Multiple Myeloma and Plasma Cell Leukemias. RAS mutations are rare except in MM and PCL. Multiple Factorial Analysis notes that MYC deregulation is closely related to TP53 status. Moreover, MYC gain, TP53 deletion and RAS mutations are inversely correlated with survival. Based on this landscape, we further propose targeted therapeutic approaches for the different B-cell malignancies.

A simple competitive assay to determine peptide affinity for HLA class II molecules: a useful tool for epitope prediction.

We have designed a cytometry-based competition assay to evaluate peptide binding to empty recombinant HLA class II molecules. The efficiency of this assay was evaluated using recombinant HLA-DP0401 molecules (HLA-DP) produced in insect cells and 13 peptides from human telomerase reverse transcriptase (hTERT). We demonstrate that our method allowed accurate measurements of peptide Ki values and can thus discriminate strong, moderate and poor HLA-DP binders. In parallel, we showed that among hTERT peptides, the most immunodominant in healthy individuals were those with moderate affinity for HLA-DP while no T cell response could be evidenced against peptides with very strong or very low affinities for HLA-DP. This strongly suggests that the precise determination of peptide affinity with our method can improve HLA class II epitope prediction.

A processed pseudogene codes for a new antigen recognized by a CD8(+) T cell clone on melanoma.

The M88.7 T cell clone recognizes an antigen presented by HLA B*1302 on the melanoma cell line M88. A cDNA encoding this antigen (NA88-A) was isolated using a library transfection approach. Analysis of the genomic gene's sequence identified it is a processed pseudogene, derived from a retrotranscript of mRNA coding for homeoprotein HPX42B. The NA88-A gene exhibits several premature stop codons, deletions, and insertions relative to the HPX42B gene. In NA88-A RNA, a short open reading frame codes for the peptide MTQGQHFLQKV from which antigenic peptides are derived; a stop codon follows the peptide's COOH-terminal Val codon. Part of the HPX42B mRNA's 3' untranslated region codes for a peptide of similar sequence (MTQGQHFSQKV). If produced, this peptide can be recognized by M88.7 T cells. However, in HPX42B mRNA, the peptide's COOH-terminal Val codon is followed by a Trp codon. As a result, expression of HPX42B mRNA does not lead to antigen production. A model is proposed for events that participated in creation of a gene coding for a melanoma antigen from a pseudogene.

The cancer germ-line genes MAGE-1, MAGE-3 and PRAME are commonly expressed by human myeloma cells.

In this study, we have investigated the mRNA expression of the cancer germ-line genes MAGE, BAGE, GAGE, RAGE and the tumor-overexpressed gene PRAME by human myeloma cell lines and malignant plasma cells from patients with multiple myeloma (MM). By reverse transcription-PCR, we show that all myeloma cell lines (n = 16) express at least one of these genes, except RAGE-1 that was never expressed. We show that malignant plasma cells from the majority of MM patients (n = 21) expressed MAGE-1, MAGE-3 and PRAME. On the contrary, polyclonal reactive plasma cells did not express any of these genes. By flow cytometry, we show that mage-1 protein is expressed within myeloma cells and cell lines and that anti-mage-1.HLA-A1 cytotoxic T lymphocytes efficiently killed MAGE-1+HLA-A1+ MDN myeloma cells. Taken together, our data show that mage-1 and mage-3 could constitute specific targets for tumor immunotherapy of MM patients.

Identification of a gp100 epitope recognized by HLA-A3 restricted melanoma infiltrating lymphocytes.

The large majority of known melanoma-associated antigenic peptides presented by MHC class I molecules are presented by the most frequent allele, HLA-A*0201. Thus although a significant percentage of Caucasians express HLA-A3, no melanoma-associated antigenic peptide presented by this allele has yet been identified. We show here that the T cell clone M45-10 isolated from tumor infiltrating lymphocytes recovered from a melanoma biopsy recognizes the gp100-derived peptide ALLAVGATK presented by HLA-A*0301. Since gp100 is expressed on most melanoma cells, our results imply that the gp100-based anti-melanoma strategies developed for individuals expressing HLA-A2 will also be applicable to those expressing HLA-AS (about one Caucasian in four). gp100 is therefore a particularly promising melanoma antigen, as different peptides derived from it can be presented by at least two different frequently encountered HLA class I molecules.

T cell response to Epstein-Barr virus transactivators in chronic rheumatoid arthritis.

Rheumatoid arthritis is a multistep disorder associated with autoimmune features of yet unknown etiology. Implication of viruses such as Epstein-Barr virus (EBV) in rheumatoid arthritis pathogenesis has been suspected on the basis of several indirect observations, but thus far, a direct link between EBV and rheumatoid arthritis has not been provided. Here we show that a large fraction of T cells infiltrating affected joints from a patient with chronic rheumatoid arthritis recognizes two EBV transactivators (BZLF1 and BMLF1) in a major histocompatibility complex-restricted fashion. Responses to these EBV antigens by synovial lymphocytes from several other chronic rheumatoid arthritis patients were readily detectable. Thus these results suggest a direct contribution of EBV to chronic rheumatoid arthritis pathogenesis. They also demonstrate for the first time the occurrence of T cell responses against EBV transactivating factors, which might be central in the control of virus reactivation.

Predominant occurrence of somatic mutations of the NF2 gene in meningiomas and schwannomas.

The NF2 gene is a putative tumor-suppressor gene that, when it is altered in the germline, causes neurofibromatosis type 2, a tumor-susceptibility disease that mainly predisposes to schwannomas and meningiomas. The recent isolation of the NF2 gene on chromosome 22 allows the identification of somatic mutations in human tumors. We have searched for mutations of the NF2 gene in 331 primary human tumors using a screening method based on denaturing gradient gel electrophoresis, which allows the detection of mutations in 95% of the coding sequence. Mutations were observed in 17 of 57 meningiomas and in 30 of 89 schwannomas. No mutations were observed for 17 ependymomas, 70 gliomas, 23 primary melanomas, 24 pheochromocytomas, 15 neuroblastomas, 6 medulloblastomas, 15 colon cancers, and 15 breast cancers. All meningiomas and one-half of the schwannomas with identified NF2 mutations demonstrated chromosome 22 allelic losses. We conclude that the involvement of the NF2 gene in human tumorigenesis may be restricted to schwannomas and meningiomas, where it is frequently inactivated by a two-hit process.

Characterization of several DNA polymorphic markers in the LIF gene region.

We describe seven restriction fragment length polymorphisms (RFLPs) in the Leukemia Inhibitory Factor (LIF) gene region. These new markers, found using a cosmid contig, have been used to map precisely the chromosome 22 long arm.