Specific Monoclonal Antibody Against Bcr/Abl Out-of-Frame Alternative Proteins as Diagnostic Tool in Chronic Myelogenous Leukemia Patients.

More recently, alternative splicing of specific genes are investigated for their therapeutic potential. In particular, we reported the existence of BCR-ABL alternative splicing isoforms, in about 80% of Philadelphia-positive patients, which lead to the expression of aberrant proteins. These fusion proteins are characterized by an orphan initial and correct Bcr portion attached to a 112 amino acid sequence, arising from the impairment in the reading frame (reading of ABL exon 4 and 5). We demonstrated that these Abl-out-of-frame (OOF) isoforms could have an immunological role with therapeutic implications. The aim of this study was to characterize a new monoclonal antibody (MAb) specific for Abl-OOF protein portion, for diagnostic use, to detect this biomarker in Philadelphia chromosome-positive chronic myelogenous leukemia (CML) patients and to generate novel approaches in the immunotherapy. 5F11G11 MAb recognizes the OOF protein portion of the native full-length Bcr/Abl-OOF protein expressed in cells transiently transfected, as demonstrated by immunoprecipitation and immunofluorescence. In addition, we demonstrate the MAb's ability to recognize the alternative hybrid Bcr/Abl fusion protein expressed in leukemic cells from CML patients, to support the possible use of 5F11G11 MAb as a diagnostic tool to select patients with Philadelphia chromosome-positive CML that could be eligible for an immunotherapeutic approach with this new antigen.

The Wilms' tumor (WT1) gene expression correlates with the International Prognostic Scoring System (IPSS) score in patients with myelofibrosis and it is a marker of response to therapy.

The Wilms tumor gene WT1 is a useful marker of clonal hematopoiesis and it has been shown to be a good marker of residual disease and it reflects the response to therapy. Although myelofibrosis is characterized by mutations of JAK2 and calreticulin (CALR), these mutations are not useful to monitor response to therapy. In this study we demonstrated that in patients affected by myelofibrosis WT1 correlates with the International Prognostic Scoring System (IPSS) score at diagnosis. Furthermore WT1 is a good marker of response to JAK2 inhibitors especially for patients without blasts and for patients who develop anemia or thrombocytopenia not for progression but as therapy related toxicity. Finally, WT1 transcript reduction can mirror a benefit of therapy on the disease burden. This study demonstrated that WT1 is a good marker for monitoring the response to therapy in patients affected by myelofibrosis.

New alternative splicing BCR/ABL-OOF shows an oncogenic role by lack of inhibition of BCR GTPase activity and an increased of persistence of Rac activation in chronic myeloid leukemia.

In Chronic Myeloid Leukemia 80% of patients present alternative splice variants involving BCR exons 1, 13 or 14 and ABL exon 4, with a consequent impairment in the reading frame of the ABL gene. Therefore BCR/ABL fusion proteins (BCR/ABL-OOF) are characterized by an in-frame BCR portion followed by an amino acids sequence arising from the out of frame (OOF) reading of the ABL gene. The product of this new transcript contains the characteristic BCR domains while lacking the COOH-terminal Rho GTPase GAP domain. The present work aims to characterize the protein functionality in terms of cytoskeleton (re-)modelling, adhesion and activation of canonical oncogenic signalling pathways. Here, we show that BCR/ABL-OOF has a peculiar endosomal localization which affects EGF receptor activation and turnover. Moreover, we demonstrate that BCR/ABL-OOF expression leads to aberrant cellular adhesion due to the activation of Rac GTPase, increase in cellular proliferation, migration and survival. When overexpressed in a BCR/ABL positive cell line, BCR/ABL-OOF induces hyperactivation of Rac signaling axis offering a therapeutic window for Rac-targeted therapy. Our data support a critical role of BCR/ABL-OOF in leukemogenesis and identify a subset of patients that may benefit from Rac-targeted therapies.

Management of pulmonary arterial hypertension associated to thalassemia: when pulmonary endarterectomy is the best therapeutical option? A case report.

Pulmonary arterial hypertension (PAH) has been reported with nearly all forms of the inherited as well as the acquired hemolytic anemias. Although screening studies suggested that PAH has emerged as major complication of thalassemia patients, its impact on survival is unknown; the pathophysiology of the PAH in these patients is multifactorial, and a thorough diagnostic evaluation is essential. Understanding the PAH pathogenesis, diagnostic options, prevention is critical for clinicians who care for the thalassemic patients; there are virtually no high-quality data on the safety/efficacy of PAH treatment strategy in this patient population. We are reporting the case of a thalassemic patient suffering from progressive severe PAH, not responding to medical treatment and related to chronic thromboembolic disease. After carefully considering all the options, we decided to proceed with vascular disobliteration by pulmonary endarterectomy (PEA), the first line choice in these cases. This intervention led to a significant improvement in the clinical status and in the functional parameters. Therefore, even if haemolytic anemia-associated-PAH is included in the group I of the Dana-point classification, an individualized approach is recommended as well as a particular management with disease-specific measures and a comprehensive evaluation of other causes of PAH; this current report supports the feasibility and effectiveness of PEA also in the thalassemic patients with surgically accessible chronic thromboembolic pulmonary hypertension.

Immunologic evaluation of peptides derived from BCR/ABL-out-of-frame fusion protein in HLA A2.1 transgenic mice.

Philadelphia chromosome-positive chronic myelogenous leukemia and acute lymphocytic leukemia express, besides the main BCR/ABL transcripts, novel BCR/ABL transcripts derived from alternative splicing between BCR exons 1, 13, or 14 with ABL exons 4 and 5. Their translational products present at C-terminus an amino acid portion derived from out-of-frame (OOF) reading of the ABL gene. The presence of OOF-peptide-specific T cells in chronic myelogenous leukemia patients was demonstrated and a first study in in vivo model demonstrated that OOF ABL portion was immunogenic in human leukcocyte antigen (HLA)-A2.1 transgenic mice. Here we immunized HLA A2.1 mice with novel peptides designed on the ABL OOF sequence, containing epitopes with high affinity for HLA A2.1 molecule. The specific immune response, cellular and humoral, obtained ex vivo against HLA A2.1-positive human chronic myelogenous leukemia cells using peptide 22-53 and the cytotoxic activity induced by peptide 32mer confirm the possibility to use the ABL OOF portion as target to evoke a specific and multiple immune response in Philadelphia positive leukemic patients in cytogenetic remission.

Characterization of a monoclonal antibody specific for novel Bcr/Abl out-of-frame fusion proteins.

The new tumor-specific antigens Bcr/Abl-OOF, identified in Philadelphia chromosome (Ph)-positive leukemia cells, are derived from an alternative splicing event involving BCR exons 1, 13, or 14 and ABL exons 4 and 5. The COOH-terminus of these transcription products contain an amino acid portion derived from an out-of-frame (OOF) reading of the ABL gene; these variants are expressed in Ph-positive chronic myelogenous leukemia (CML) and acute lymphocytic leukemia patients. Previously, we confirmed the presence of out-of-frame peptide-specific T cells in the peripheral blood of CML patients with the ability to lyse primary autologous CML cells. We also demonstrated that the out-of-frame Abl portion was immunogenic in HLA-A2.1 transgenic mice. Here we describe the production and characterization of monoclonal antibody 1D8G8, a new tool for localization and functional studies of the tumor antigen Bcr/Abl-OOF. This antibody recognizes the out-of-frame protein portion of the native full-length Bcr/Abl-OOF protein expressed in cells transiently transfected, as demonstrated by immunoprecipitation and immunofluorescence, and binds to a specific epitope of this antigen presented in association with HLA-A2.1 molecules at the surface of these cells, as demonstrated by flow cytometry. Thus this MAb could be useful to better understand how this new protein presents in Ph-positive cells beside the canonical Bcr/Abl fusion proteins.

Imatinib resistance in CML.

Imatinib is, at present, the first-choice treatment for patients with chronic myeloid leukaemia in chronic phase. Despite the impressive rate of complete haematological response and complete cytogenetical remissions, some cases show primary resistance or relapse after an initial response (secondary or acquired resistance). The most common mechanisms responsible for this resistance are BCR/ABL kinase domain mutations, BCR/ABL amplification and over-expression and clonal evolution with activation of additional oncogenic pathways. Here, we describe the molecular basis of imatinib resistance, the significance of molecular monitoring and the current efforts to overcome imatinib resistance, ranging from the development of new drugs to the stimulation of an immune response against the disease.

Alternative BCR/ABL splice variants in Philadelphia chromosome-positive leukemias result in novel tumor-specific fusion proteins that may represent potential targets for immunotherapy approaches.

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 approximately 80% 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.

Rational approaches to the design of therapeutics targeting molecular markers: the case of chronic myelogenous leukemia.

Progress in understanding the molecular basis of signal transmission and transduction has contributed substantially to clarifying the mechanisms of leukemogenesis and of leukemia progression and has led to the identification of a number of specific molecular targets for treatment. Chronic myeloid leukemia (CML) has provided one of the best models, as the identification of a leukemia-specific hybrid tyrosine kinase (BCR-ABL, p210, p190) has led to the identification and the successful therapeutic application of a powerful tyrosine kinase inhibitor, imatinib. The BCR-ABL fusion gene is the result of a reciprocal translocation between the long arms of chromosomes 9 and 22, t(9;22)(q34;q11), which characterizes more than 95% of the cases of CML. The resulting chimeric proteins (P210 and P190), which retain a constitutively activated tyrosine kinase activity, have a causative role in the genesis of the leukemia process. In agreement with this observation, BCR-ABL tyrosine kinase inhibitors have recently emerged as powerful new therapeutic tools, obtaining extraordinary results in early chronic-phase CML as well as in more advanced phases of the disease. Although these results represent a remarkable breakthrough, there are still numerous issues, such as the emergence of resistance, that remain unsolved and that will need further investigation. In spite of its low incidence, CML remains a paradigmatic model for understanding the pathogenesis and therapeutic options of human leukemias.

From genes to therapy: the case of Philadelphia chromosome-positive leukemias.

The Philadelphia chromosome (Ph-chromosome) has long represented the only cytogenetic abnormality known to be associated with a specific malignant disease in humans, being present in more than 95% of patients with chronic myelogenous leukemia. This abnormality is the result of a reciprocal translocation between the long arms of chromosome 9 and 22, t(9;22)(q34;q11), and its presence is not restricted to chronic myelogenous leukemia, but can also be found in 30% of cases of acute lymphoblastic leukemia in adults. In the 1980s, the molecular counterpart of the chromosomal rearrangement was identified to consist of the juxtaposition of parts of the BCR and ABL genes to form a BCR-ABL hybrid gene. The resulting chimeric proteins (P210 and P190), which retain constitutively activated tyrosine kinase activity, have demonstrated a causative role in the genesis of the leukemic process. Although many aspects of the BCR-ABL driven transformation remain unsolved, great advances in understanding the molecular pathology of Ph-positive leukemias resulted in meaningful improvement in the clinical setting. Molecular tools to diagnose disease (PCR, FISH, and southern blot) and to monitor minimal residual disease after potential curative treatment are now in current practice, and new powerful therapeutic tools have emerged that target the molecular oncogenic pathways activated in Ph-positive cells. Among them, specific ABL tyrosine kinase inhibitors recently obtained extraordinary results in many clinical protocols. This review summarizes the most recent advances in this field with special focus on the putative mechanisms of the transformation and progression of chronic myelogenous leukemia and on the major impact that understanding the molecular biology of these diseases is having in clinical practice.