ALBERT

Description: Leukemia caused by retroviral insertional mutagenesis after stem cell gene transfer has been reported in several experimental animals and in patients treated for X-linked severe combined immunodeficiency. Here, we analyzed whether gene transfer into mature T cells bears the same genotoxic risk. To address this issue in an experimental “worst case scenario,” we transduced mature T cells and hematopoietic progenitor cells from C57BL/6 (Ly5.1) donor mice with high copy numbers of gamma retroviral vectors encoding the potent T-cell oncogenes LMO2, TCL1, or ΔTrkA, a constitutively active mutant of TrkA. After transplantation into RAG-1–deficient recipients (Ly5.2), animals that received stem cell transplants developed T-cell lymphoma/leukemia for all investigated oncogenes with a characteristic phenotype and after characteristic latency periods. Ligation-mediated polymerase chain reaction analysis revealed monoclonality or oligoclonality of the malignancies. In striking contrast, none of the mice that received T-cell transplants transduced with the same vectors developed leukemia/lymphoma despite persistence of gene-modified cells. Thus, our data provide direct evidence that mature T cells are less prone to transformation than hematopoietic progenitor cells.

Description: After the report of two cases of leukaemia caused by insertional mutagenesis of a retroviral vector in children with SCID, it became clear that safety issues of therapeutic gene transfer must be addressed more thoroughly. We analysed whether gene transfer into mature T cells and haematopoietic stem cells bear the same risk of generating T cell leukaemia through activation of specific T cell oncogenes, such as LMO2, TCL1 and ΔTrkA. To address this issue, we used the Rag-1 mouse model, which allows long term analysis of transplanted T cells and haematopoietic stem cells. We were able to transduce mature T cells and haematopoietic stem cells of C57BL/6 (Ly5.1) donor mice with oncoretroviral vectors expressing LMO2, TCL1 and ΔTrkA. Transduction efficacies of up to 70% were achieved for mature T cells and approximately 90% for haematopoietic stem cells. After transplantation into Rag-1-deficient recipients, stem cell transplanted animals developed T cell lymphomas/leukemia for all investigated oncogenes after characteristic incubation times, mostly of a CD8+CD4+ double positive phenotype. T cell lymphomas were characterised by gross thymic mass, splenomegaly and heavily enlarged lymph nodes, although none of the control- vector- transduced mice developed lymphoma/leukaemia. LM PCR analysis revealed mono- or oligoclonality of the tumours. T cell transplanted animals showed no signs of leukaemia development so far. However, after several attempts, one immortalized T cell progenitor clone could be generated after transduction with LMO2. Our results so far indicate that mature T cells are less susceptible to transformation by known T cell proto-oncogenes, but the studies are still ongoing.

Description: Hodgkin’s disease (HD) represents a malignant lymphoma in which the putative malignant Hodgkin and Reed-Sternberg (H-RS) cells are rare and surrounded by abundant reactive cells. Single-cell analyses showed that H-RS cells regularly bear clonal Ig gene rearrangements. However, there is little information on the clinical evolution of HD in a given patient. In this study, we used the single-cell polymerase chain reaction (PCR) to identify H-RS cells with clonal Ig gene rearrangements in biopsy specimens of patients with relapsed HD. The obtained clonal variable region heavy-chain (VH) gene rearrangements were used to construct tumor-clone-specific oligonucleotides spanning the complementarity determining region (CDR) III and somatically mutated areas in the rearranged VHgene. A number of biopsies were obtained during a period of 3 years from two HD patients. H-RS cells with identical VHrearrangements were detected in two separate infiltrated lymph nodes from one patient with nodular sclerosis HD. In a second patient with mixed cellularity HD subtype, clonal VH rearrangements with identical sequences were detected in infiltrated spleen and two lymph node biopsies. Despite the high sensitivity of the PCR method used (one clonal cell in 105 mononuclear cells), residual H-RS cells were not found in peripheral blood, leukapheresis material, purified CD34+ stem cells or bone marrow. The results show that different specimens from relapsed patients suffering from classical HD carry the same clonotypic IgH rearrangements with identical somatic mutations, demonstrating the persistence and the dissemination of a clonal tumor cell population. Thus, PCR assays with CDRIII-specific probes derived from clonal H-RS cells are of clinical importance in monitoring the dissemination of HD and tumor progression and could be useful for analysis of minimal residual disease after autologous stem cell transplantation. © 1998 by The American Society of Hematology.

Description: The spectrum of entities, the therapeutic strategy, and the outcome of mature aggressive B-cell non-Hodgkin lymphomas (maB-NHLs) differs between children and adolescents on the one hand and adult patients on the other. Whereas adult maB-NHLs have been studied in detail, data on molecular profiling of pediatric maB-NHLs are hitherto lacking. We analyzed 65 cases of maB-NHL from patients up to 18 years of age by gene expression profiling, matrix comparative genomic hybridization (CGH), fluorescent in situ hybridization (FISH), and immunohistochemistry. The majority of the analyzed pediatric patients were treated within prospective trials (n = 49). We compared this group to a series of 182 previously published cases of adult maB-NHL. Gene expression profiling reclassified 31% of morphologically defined diffuse large B-cell lymphomas as molecular Burkitt lymphoma (mBL). The subgroups obtained by molecular reclassification did not show any difference in outcome in children treated with the NHL-Berlin-Frankfurt-Muenster (BFM) protocols. No differences were detectable between pediatric and adult mBL with regard to gene expression or chromosomal imbalances. This is the first report on molecular profiling of pediatric B-NHL showing mBL to be much more prominent in children than suggested by morphologic assessment. Based on molecular profiling mBL is a molecularly homogeneous disease across children and adults.

Description: Aberrant activities of JAK/STAT signaling pathways have been observed in several hematologic malignancies. Here, we show high expression of JAK2 in the tumor cells of lymphocyte-predominant Hodgkin lymphoma in 85% of cases and activation of JAK2 in 39% of cases. STAT6, which is a target of JAK2, was activated in 50% of the cases. SOCS1 controls JAK2 activity and degradation. Mutations in SOCS1 of either somatic or germ-line origin were observed in micromanipulated tumor cells of 50% of cases. Most mutations truncated SOCS1 or caused replacement of amino acids in functional important regions. Activating mutations in exon 12 of JAK2, which are frequent in myeloproliferative diseases, were not observed. In lymphocyte-predominant Hodgkin lymphoma SOCS1 function may thus be frequently impaired by mutations, and this may contribute to high JAK2 expression and activation of the JAK2/STAT6 pathway.

Description: Mantle cell lymphoma (MCL) is a prime example of a well-defined entity based on morphology, phenotype, genetics and also clinical features. Although, most patients have an adverse clinical course, some have a better survival than others. The most consistently reported adverse histopathological prognostic parameter is a high mitotic rate. Recently, it has been shown that hypermutation in the immunoglobulin heavy chain gene occurs in a subset of mantle cell lymphomas. It is, however, unclear, how hypermutation needs to be defined and whether the mutational status is stable over time within a given case. Also it is not clearwhether hypermutation might be influenced by therapy and how it is related to other relevant biological features of MCL, like genetic imbalances, breakpoint of the cyclin D1 gene, and proliferative and apoptotic rate. In the present study we analyzed a series of typical MCL with respect to mutational status, and compared the results with clinicopathological and genetic data to determine whether the presence of mutation does indicate a sub entity with clinical or pathological relevance. For this study 28 specimens from 24 patients were selected and stained for cyclin D1 and CD5 and were reviewed by four experienced pathologists at a multiheaded microscope. Morphological features were assessed, mitotic figures were counted, clonal IGH-VJ gene rearrangements of the MCL were identified according to the Biomed-2-protocol. All, but one case, were clonal by PCR. The negative case was excluded from further molecular studies. Mutation in the VH gene segment was absent in 7 cases, another 6 cases carried 0.5% mutation frequencies; in 10 cases more than 1.5% mutation frequencies were present, in 6 of these more than 2%. We found a preferential usage of VH3-21 (26%) and VH4-34 (17%) in tumor cells of both, mutated and unmutated cases. Of two cases from our series in which sequential biopsies were available tumor cells harbored mutations in the VH genes (1.5% mutation frequency and 2.2% mutation frequency, respectively), but there was no change of mutation status over time, the duration being as long as 7 years in one case. In our study there was a statistically significant difference between MCL with a Ki-67-positivity =35% in regard to a longer survival time in the former whereas mutation status of both groups did not correlate with Ki-67-positivity. Further, no significant correlations were found between mutation status and the other morphologic and genetic features analyzed. In conclusion, our results provide additional evidence that mutation status does not permit definition of sub entities in MCL that are associated invariably with a certain prognosis. Mutation status in MCL is better interpreted as a feature within the spectrum of disease that seems to have little clinical or pathological relevance. Margit Schraders and Sabine Oeschker contributed equally to this work.

Description: The pathogenesis of Hodgkin lymphoma (HL) is still largely unknown. Based on a search for footprints of pathogenetic mechanisms in global RNA expression data of Hodgkin/Reed-Sternberg (HRS) cell lines, we analyzed the expression and activation of 6 receptor tyrosine kinases (RTKs) in classic HL. Immunohistochemistry revealed that the RTKs platelet-derived growth factor receptor A (PDGFRA), DDR2, EPHB1, RON, TRKB, and TRKA were each expressed in HRS cells in 30% to 75% of patients. These RTKs were not expressed in normal B cells, the origin of HRS cells, or in most B-cell non-Hodgkin lymphoma (NHL). In the majority of patients at least one RTK was expressed, and in most patients several RTKs were coexpressed, most prominently in Hodgkin lymphoma of the nodular sclerosis subtype. Phosphotyrosine-specific antibodies revealed exemplarily the activation of PDGFRA and TRKA/B and an elevation of cellular phosphotyrosine content. Immunohistochemistry for RTK ligands indicated that DDR2 and TRKA are likely activated in a paracrine fashion, whereas PDGFRA and EPHB1 seem to be activated by autocrine loops. Activating mutations were not detected in cDNA encoding the RTKs in HRS cell lines. These findings show the unprecedented coexpression of multiple RTKs in a tumor and indicate that aberrant RTK signaling is an important factor in HL pathogenesis and that it may be a novel therapeutic target.

Description: Abstract 4631 Background Peripheral T cell lymphomas (PTCLs) are a heterogeneous group of tumors representing approximately 12% of lymphoid neoplasms, basically subdivided into specified and not specified (NOS) forms. PTCL/NOS, corresponding to about 60%–70% of PTCLs, cannot be further classified on the basis of morphology, phenotype, or conventional molecular studies. Clinically, PTCLs/NOS are highly aggressive lymphomas, with a poor response to therapy, and dismal overall survival (20-30%). Their pathobiology is poorly known, though recent gene expression profiling (GEP) studies have provided some hints for better understanding their pathogenesis. In particular, GEP and immunohistochemical studies on tissue-microarrays (TMAs) demonstrated PDGFRA to be systematically activated in almost all PTCLs/NOS, by nominating it as potential therapeutic target. Aims In this study, we aimed to identify the determinants of PDGFRA activation in PTCL/NOS. Specifically, we studied PDGFRA locus in order to identify possible mutations, translocations, or copy number variations and we explored the possible existence of an autocrine/paracrine loop sustaining an otherwise integer kinase. Methods The PDGFRA locus (4q1.1-4q1.3) was studied by FISH and wide-genome SNPs analysis (Affymetrix 500K Array). Direct sequencing of all PDGFRA exons and introns as well as of the promoter region was also performed in 90 cases. IHC and ELISA were adopted in order to study the expression of PDGF-A, PDGF-B and PDGF-C on tissue sections and in supernatants from PTCL/NOS cell cultures, respectively. Finally, the expression of PDGFRA and its activated (phosphorilated) form, p-PDGFRA, was assessed by IHC on TMAs, and by flow-citometry in PTCL/NOS cultured cells as well as in a FIP1L1-PDGFRApos chronic eosinophilic leukemia cell line (EOL-1) before and after the exposure to an anti-PDGF ligand neutralizing antibody (R&D System), given at various concentrations (20-40-60-80 ug/mL). Vitality assessments, proliferation/cell cycle assay (by In Situ Cell Proliferation kit, FLUOS – Roche) and evaluation of PDGFRA and p-PDGFRA were performed at 24, 48, 96 hours. A human PDGF peptide (R&D Sytems) was added to cultured cells for 6 hours to evaluate whether PDGFRA de-phosphorilation was really due to PDGF ligand remotion. Results First, FISH, SNPs analysis and direct sequencing showed preserved integrity of PDGRA locus. Thus we tested the hypothesis of an autocrine/paracrine stimulation. PDGF-A, PDGF-B and PDGF-C were found to be expressed by neoplastic cells at IHC in 93-95% of cases. In addition, PDGF-AA was found to be secreted by cultured neoplastic cells by ELISA. Notheworthy, PTCL cells secreted much more ligand than any other cell taken as control. We then tested whether PDGFRA phosphorylation was actually due to the presence of a PDGF ligand. Indeed, PTCL cells treated with anti-PDGF ligand neutralizing antibody at various concentrations showed PDGFRA dephosphorilation ranging from 30% up to 90% in a time dependent manner. Notably, the effect was specific as in EOL-1 PDGFRA phosphorylation was not modified at all. In addition, PTCL cells treated with a minimum of 20ug/mL of anti-PDGF ligand neutralizing antibody for 48h showed a 70% blockade of proliferation in comparison to untreated cells (BrdU assay). A further addition of 20 ug/ml of inhibitory antibody at 48 hours, increased the proliferation arrest up to 80% at 96 hours. Finally, the addition of a natural human PDGF peptide to cells previously treated with the anti-PDGF antibody, could restore PDGFRA phosphorylation confirming that PDGFRA de-phosphorilation was due to ligand remotion. Conclusions Taken together, our data demonstrate that PDGFRA activity is sustained by an autocrine loop in PTCL/NOS. In fact, though, in vivo, a possible additive paracrine effects mediated by reactive components cannot be excluded, we provide evidence that the phenomenon is largely due to neoplastic cells. Importantly, as PDGFRA signaling abrogation was associated to proliferation arrest, PDGFRA was confirmed as potential therapeutic target. Acknowledgments: this work was supported by Centro Interdipartimentale per la Ricerca sul Cancro “G. Prodi”, BolognAIL, AIRC, FIRB, RFO, Fondazione Cassa di Risparmio in Bologna, Fondazione della Banca del Monte e Ravenna, Progetto Strategico di Ateneo 2006, and Vanini-Cavagnino grant. Disclosures: No relevant conflicts of interest to declare.