Gisella Volpe

Distribution of human herpesvirus-8 sequences throughout the spectrum of AIDS-related neoplasia

Objective:AIDS frequently associates with certain malignancies, including Kaposis sarcoma, non-Hodgkins lymphoma (NHL), and anogenital neoplasia. In this study we aimed to define the frequency of infection by human herpesvirus (HHV)-8 throughout the spectrum of AIDS-related neoplasia in Europe. Design:A tumour panel representative of the distinct types of AIDS-related neoplasms was tested for the presence of HHV-8 DNA sequences. Autologous uninvolved tissues were also tested in selected cases. Methods:The presence of HHV-8 DNA sequences was assayed by a combination of polymerase chain reaction followed by oligohybridization and Southern blot hybridization of genomic DNA with an HHV-8-specific probe. Results:HHV-8 sequences were detected in 100% of AIDS-related Kaposis sarcoma (all 35 cases). Among AIDS-related NHL, HHV-8 sequences selectively clustered with body-cavity-based lymphomas (BCBL; all three cases), although they were consistently negative in small non-cleaved cell lymphomas (none in 18 cases), diffuse large cell lymphomas (none in seven), or anaplastic large cell lymphomas (none in three). No HHV-8 sequences were found in cases of anogenital neoplasia (out of 14) or Hodgkins disease (out of three). HHV-8 DNA sequences were also positive in the uninvolved skin of all six AIDS-related Kaposis sarcoma patients, but not in the circulating lymphocytes of a BCBL patient. Positivity for HHV-8 sequences occurred in patients belonging to all major AIDS risk categories. Conclusions:These data confirm that HHV-8 sequences associate at high frequency with selected types of AIDS-related neoplasia, namely Kaposis sarcoma and BCBL, although they are consistently absent in other types of AIDS-NHL and AIDS-related anogenital neoplasia.

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.

Integrin-Mediated Signal Transduction in Human Endothelial Cells: Analysis of Tyrosine Phosphorylation Events

Adhesion of human umbilical endothelial cells to fibronectin resulted in increased tyrosine phosphorylation of a group of proteins with molecular mass ranging from 100 to 130 kDa and of a 70 kDa protein. This pattern of tyrosine phosphorylation was also observed when endothelial cells adhered to vitronectin, collagen IV, collagen I and laminin or to culture dishes coated with antibodies directed to either beta 1, alpha 3, alpha 5, alpha 6 or beta 3 integrin subunits. Increased phosphorylation of the 100-130 kDa proteins was detectable as early as 30 sec after adhesion, reached maximal level after 15 min, and remained high as long as the cells adhere to culture dishes. The 70 kDa protein was phosphorylated with a slower kinetics and its phosphorylation increased over a period of 3 h. Using specific monoclonal antibodies, the major component of the 100-130 kDa complex was identified as the focal adhesion tyrosine kinase p125FAK. The phosphorylation of the p125FAK was also observed by inducing beta 1 integrin clustering in non adherent HEC, indicating that this is a primary signalling event induced by integrins. Using tyrosine kinase inhibitors, we show a direct correlation between integrin-stimulated tyrosine kinases and assembly of focal adhesions and actin fibres.

Molecular heterogeneity of B-lineage diffuse large cell lymphoma

B‐lineage diffuse large cell lymphoma (B‐DLCL) arising de novo is characterized by a marked degree of clinical heterogeneity. To determine whether or not the clinical heterogeneity of de novo B‐DLCL is reflected by heterogeneity in the molecular features of these tumors, we investigated the pattern of distribution of several genetic lesions in 70 cases of de novo B‐DLCL at diagnosis. The panel of genetic lesions tested comprised the molecular alterations most frequently detected in B‐DLCL, including rearrangements of BCL2, BCL6, and MYC as well as deletions of 6q and mutations of TP53. One or more genetic lesions were detected in 39/70 cases of B‐DLCL. Isolated structural alterations of BCL2, BCL6, 6q or TP53 were detected in 8/70, 10/70, 11/70, and 3/70 cases, respectively. No isolated MYC lesions were detected. Six cases carried different combinations of two genetic lesions, including lesions of BCL2 + BCL6 (1 case), BCL2 + MYC (1 case), BCL2 + 6q (2 cases), or BCL6 + 6q (2 cases). One case had accumulated three genetic lesions, namely a rearrangement of BCL2 and BCL6 and a mutation of TP53. Overall, these data show that multiple distinct patterns of genetic lesions may associate with de novo B‐DLCL, indicating that the molecular pathogenesis of this group of lymphomas is characterized by a high degree of molecular heterogeneity. Genes Chromosom Cancer 16:21–30 (1996).

Analysis of microsatellite instability in chronic lymphoproliferative disorders.

Microsatellite instability (MSI) represents one specific pattern of genomic instability and is one of the genetic lesions most frequently detected in human neoplasia. Although MSI has been found to be associated with a wide variety of solid cancers, its involvement in lymphoid malignancies is virtually unexplored. In this study, we have investigated the presence of MSI in chronic lymphoproliferative disorders by comparing the pattern of nine microsatellite repeats (two tetranucleotides, two trinucleotides, and five dinucleotides) on autologous germline and tumor DNA of 23 patients, including 17 with B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma (B-CLL/SLL), four with hairy cell leukemia, one with lymphoplasmacytoid lymphoma, and one with T-cell chronic lymphocytic leukemia. All samples at diagnosis displayed a germline pattern of the microsatellites examined, thus suggesting that MSI is not involved in the pathogenesis of these lymphoproliferations. Also, no microsatellite alterations were observed in consecutive samples of B-CLL/SLL obtained from the same patient at various stages of the disease both before and after chemotherapy. Conversely, alterations in 3/9 microsatellite repeats were detected in one case of Richters syndrome which had evolved from a pre-existent B-CLL/SLL phase. Overall, the low frequency of MSI among chronic lymphoproliferative disorders adds further weight to the common view that the mechanisms and patterns of genomic instability in lymphoid neoplasia differ markedly from those commonly observed in solid cancers.

Rational Approaches to the Design of Therapeutics Targeting Molecular Markers: The Case of Chronic Myelogenous Leukemia

Abstract: 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.

AIDS-related non-Hodgkin's lymphomas : Molecular genetics, viral infection and cytokine deregulation

AIDS-related non-Hodgkins lymphomas (AIDS-NHLs) are almost invariably derived from B cells and are grouped into three distinct histologic categories, including small-non-cleaved-cell lymphoma (SNCCL), diffuse large-cell lymphoma (DLCL), and anaplastic large-cell lymphoma (ALCL). In addition, AIDS-NHLs presenting solely as a body cavity effusion are thought to be a peculiar clinicopathologic entity and are defined as body-cavity-based lymphoma (BCBL). At the biologic level, AIDS-related lymphomagenesis is characterized by activation of proto-oncogenes, inactivation of tumor suppressor genes, viral infection of the tumor clone, and deregulated cytokine production. Distinct AIDS-NHL types associate with specific molecular pathways. The first pathogenetic pathway clusters with AIDS-SNCCL, and is characterized by a relatively mild degree of host immunodeficiency. AIDS-SNCCL consistently associates with c-myc rearrangements and p53 inactivation in 100 and 60% of cases, respectively, whereas infection by Epstein-Barr virus (EBV) is restricted to 30% of the cases. Production of high levels of interleukin-10 is an additional peculiar feature of EBV-positive AIDS-SNCCL. The second pathogenetic pathway associates with AIDS-DLCL, which is usually accompanied by marked host immunodeficiency. AIDS-DLCL is characterized by EBV infection in the large majority of cases and by the mutually exclusive presence of bcl-6 rearrangements and c-myc translocations in 40% of the cases. A third pathway characterizes AIDS-BCBL, which associates in virtually all cases with infection by EBV and with the presence of DNA sequences of the recently identified Kaposi sarcoma herpesvirus in the apparent absence of other known genetic lesions. Finally, the pathogenetic features of AIDS-ALCL are still under investigation.

From Genes to Therapy: The Case of Philadelphia Chromosome-Positive Leukemias

Abstract: 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.

Out of frame peptides from BCR/ABL alternative splicing are immunogenic in HLA A2.1 transgenic mice

New, potentially tumor-specific antigens have been described in Bcr/Abl positive leukemias. Besides the main BCR/ABL hybrid fusion transcripts, a small number of transcripts derived from alternative splicing between BCR exons 1, 13, and 14 with ABL exons 4 and 5 have been identified. These variants are expressed in chronic myelogenous leukemia and acute lymphocytic leukemia patients. The transcriptional products were characterized at their C-terminus by a large amino acid portion derived from out of frame (OOF) reading of the ABL gene. This OOF peptide is expressed only in leukemic cells and has no homology with known human proteins. In order to study an in vivo model, three 39-amino acid peptides, each corresponding to a third of the whole human OOF peptide sequence, were tested for their capacity to elicit specific immune responses in HLA A2.1 transgenic mice. Peptides A and B, but not C, induced the production of specific antisera, while A and C induced the generation of specific cytotoxic T lymphocytes.

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.