Potential Targets for Immunotherapy Approaches Tumor-Specific Fusion Proteins that May Represent Positive Leukemias Result in Novel − Chromosome Alternative BCR / ABL Splice Variants in Philadelphia

Imatinib currently represents the standard treatment in the early chronic phase of chronic myelogenous leukemia (CML), thanks to the high percentage of cytogenetic complete remission achieved, but it is yet unclear to what extent it can eradicate leukemia. Therefore, different vaccination strategies have been suggested, mainly based on the exploitment of the junctional peptides spanning the fusion region of the Bcr/Abl proteins. To identify new potential immunologic targets, 63 Philadelphia chromosome–positive patients and 6 BCR/ABL–positive cell lines were tested in nested reverse transcriptase PCR to detect the presence of BCR/ABL transcripts arising from the alternative splicing of the main BCR/ ABL transcripts. We could detect BCR/ABL transcripts with junctions between BCR exon 1, 13, or 14 and ABL exon 4 in f80% of patients and 84% of cell lines, beside the main fusion transcripts. Translation products of these transcripts were characterized at their COOH terminus by a large amino acid portion derived from the out of frame (OOF) reading of ABL gene. These proteins were detected in BCR/ABL–positive cell lines by immunoprecipitation and immunohistochemistry. Finally, we determined whether OOF-specific CD8 T cells could be found in the peripheral blood of CML patients and whether they could acquire effector function following in vitro sensitization with OOF-derived peptides predicted to bind to human leucocyte antigen (HLA)-A2 and HLA-A3 molecules. We detected the presence of OOF-specific CD8 T cells in four of four patients studied, and in one case, these T cells exhibited specific cytotoxic activity against both peptide-pulsed targets and autologous primary CML cells. [Cancer Res 2007;67(11):5300–7] Introduction Imatinib is more effective than any other previous pharmacologic approach for chronic myelogenous leukemia (CML) and has become the golden standard for its front-line treatment (1, 2). In spite of the very high percentage of patients obtaining a complete cytogenetic response and of those obtaining a major molecular response (3), which corresponds to a decrease of >3 logs in the BCR/ABL amount compared with the mean amount normally observed at diagnosis (3, 4), patients achieving a complete molecular remission [undetectable BCR/ABL transcript at the nested reverse transcriptase PCR (RT-PCR) analysis] are ranging between 5% and 25% of the reported series (3, 5–7). Studies on bone marrow samples have shown that residual cells are part of the leukemic stem cell compartment (8) and consequently most of those who discontinue imatinib treatment relapse in a very short period (9). The mechanisms underlying the resistance of these cells are unknown, but their final eradication is believed to be needed to obtain a definitive cure for the disease. CML is considered one of the potential responsive malignancies to immunotherapy (10, 11). The evidence that leukemic cells present human leucocyte antigen (HLA)–associated immunogenic peptides derived from Bcr/Abl fusion protein has provided the basis for tumor vaccine development (12–14). Furthermore, the reinduction of complete molecular remission by means of donor lymphocyte infusions into patients relapsed after allogeneic bone marrow transplantation further confirms the role of T cells in the control of the disease (15, 16). Up to now, vaccination approaches have been based essentially on the use of Bcr/Abl junctional peptides, derived from amino acidic sequences spanning the e14a2 fusion junction between Bcr and Abl (17, 18). Other approaches tried to exploit the presence of proteins overexpressed by CML cells, such as proteinase-3 and prame (19, 20). In the present article, new potential tumor-specific antigens in BCR/ABL–positive leukemias are described. We report the presence of BCR/ABL transcripts arising from alternative splicing and of their corresponding proteins. In particular, those with junctions involving BCR exon 1, 13, or 14 on one side and ABL exon 4 or 5 on the other side result in the production of fusion proteins characterized by an initial and correct Bcr portion attached to a Note: G. Volpe, V.M. Fazio, and G. Saglio hold a patent related to the work that is described in the present study. Requests for reprints: Gisella Volpe, Department of Clinical and Biological Sciences, University of Turin, San Luigi Hospital, Regione Gonzole 10, 10043 Orbassano, Turin, Italy. Phone: 39-011-9026609; Fax: 39-011-9038636; E-mail: gisella.volpe@unito.it. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-3737 Cancer Res 2007; 67: (11). June 1, 2007 5300 www.aacrjournals. org Research Article American Association for Cancer Research Copyright © 2007 on February 20, 2013 cancerres.aacrjournals. org Downloaded from DOI:10.1158/0008-5472.CAN-06-3737