Maurilio Ponzoni

Mutations of multiple genes cause deregulation of NF-kB in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL), the most common form of lymphoma in adulthood, comprises multiple biologically and clinically distinct subtypes including germinal centre B-cell-like (GCB) and activated B-cell-like (ABC) DLBCL. Gene expression profile studies have shown that its most aggressive subtype, ABC-DLBCL, is associated with constitutive activation of the NF-κB transcription complex. However, except for a small fraction of cases, it remains unclear whether NF-κB activation in these tumours represents an intrinsic program of the tumour cell of origin or a pathogenetic event. Here we show that >50% of ABC-DLBCL and a smaller fraction of GCB-DLBCL carry somatic mutations in multiple genes, including negative (TNFAIP3, also called A20) and positive (CARD11, TRAF2, TRAF5, MAP3K7 (TAK1) and TNFRSF11A (RANK)) regulators of NF-κB. Of these, the A20 gene, which encodes a ubiquitin-modifying enzyme involved in termination of NF-κB responses, is most commonly affected, with ∼30% of patients displaying biallelic inactivation by mutations and/or deletions. When reintroduced in cell lines carrying biallelic inactivation of the gene, A20 induced apoptosis and cell growth arrest, indicating a tumour suppressor role. Less frequently, missense mutations of TRAF2 and CARD11 produce molecules with significantly enhanced ability to activate NF-κB. Thus, our results demonstrate that NF-κB activation in DLBCL is caused by genetic lesions affecting multiple genes, the loss or activation of which may promote lymphomagenesis by leading to abnormally prolonged NF-κB responses.

Hematopoietic stem cell gene transfer in a tumor-prone mouse model uncovers low genotoxicity of lentiviral vector integration

Insertional mutagenesis represents a major hurdle to gene therapy and necessitates sensitive preclinical genotoxicity assays. Cdkn2a−/− mice are susceptible to a broad range of cancer-triggering genetic lesions. We exploited hematopoietic stem cells from these tumor-prone mice to assess the oncogenicity of prototypical retroviral and lentiviral vectors. We transduced hematopoietic stem cells in matched clinically relevant conditions, and compared integration site selection and tumor development in transplanted mice. Retroviral vectors triggered dose-dependent acceleration of tumor onset contingent on long terminal repeat activity. Insertions at oncogenes and cell-cycle genes were enriched in early-onset tumors, indicating cooperation in tumorigenesis. In contrast, tumorigenesis was unaffected by lentiviral vectors and did not enrich for specific integrants, despite the higher integration load and robust expression of lentiviral vectors in all hematopoietic lineages. Our results validate a much-needed platform to assess vector safety and provide direct evidence that prototypical lentiviral vectors have low oncogenic potential, highlighting a major rationale for application to gene therapy.

Prognostic Scoring System for Primary CNS Lymphomas: The International Extranodal Lymphoma Study Group Experience

PURPOSE To identify survival predictors and to design a prognostic score useful for distinguishing risk groups in immunocompetent patients with primary CNS lymphomas (PCNSL). PATIENTS AND METHODS The prognostic role of patient-, lymphoma-, and treatment-related variables was analyzed in a multicenter series of 378 PCNSL patients treated at 23 cancer centers from five different countries. RESULTS Age more than 60 years, performance status (PS) more than 1, elevated lactate dehydrogenase (LDH) serum level, high CSF protein concentration, and involvement of deep regions of the brain (periventricular regions, basal ganglia, brainstem, and/or cerebellum) were significantly and independently associated with a worse survival. These five variables were used to design a prognostic score. Each variable was assigned a value of either 0, if favorable, or 1, if unfavorable. The values were then added together to arrive at a final score, which was tested in 105 assessable patients for which complete data of all five variables were available. The 2-year overall survival (OS) +/- SD was 80% +/- 8%, 48% +/- 7%, and 15% +/- 7% (P =.00001) for patients with zero to one, two to three, and four to five unfavorable features, respectively. The prognostic role of this score was confirmed by limiting analysis to assessable patients treated with high-dose methotrexate-based chemotherapy (2-year OS +/- SD: 85% +/- 8%, 57% +/- 8%, and 24% +/- 11%; P =.0004). CONCLUSION Age, PS, LDH serum level, CSF protein concentration, and involvement of deep structures of the brain were independent predictors of survival. A prognostic score including these five parameters seems advisable in distinguishing different risk groups in PCNSL patients. The proposed score and its relevance in therapeutic decision deserve to be validated in further studies.

The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of HSC gene therapy

gamma-Retroviral vectors (gammaRVs), which are commonly used in gene therapy, can trigger oncogenesis by insertional mutagenesis. Here, we have dissected the contribution of vector design and viral integration site selection (ISS) to oncogenesis using an in vivo genotoxicity assay based on transplantation of vector-transduced tumor-prone mouse hematopoietic stem/progenitor cells. By swapping genetic elements between gammaRV and lentiviral vectors (LVs), we have demonstrated that transcriptionally active long terminal repeats (LTRs) are major determinants of genotoxicity even when reconstituted in LVs and that self-inactivating (SIN) LTRs enhance the safety of gammaRVs. By comparing the genotoxicity of vectors with matched active LTRs, we were able to determine that substantially greater LV integration loads are required to approach the same oncogenic risk as gammaRVs. This difference in facilitating oncogenesis is likely to be explained by the observed preferential targeting of cancer genes by gammaRVs. This integration-site bias was intrinsic to gammaRVs, as it was also observed for SIN gammaRVs that lacked genotoxicity in our model. Our findings strongly support the use of SIN viral vector platforms and show that ISS can substantially modulate genotoxicity.

Intravascular lymphoma: clinical presentation, natural history, management and prognostic factors in a series of 38 cases, with special emphasis on the 'cutaneous variant'.

Despite its recognition as a distinct, extremely rare entity, no large studies of intravascular lymphoma (IVL) have been reported. The clinico‐pathological characteristics of 38 human immunodeficiency virus‐negative patients with IVL diagnosed in Western countries were reviewed to better delineate clinical presentation, clinical variants, natural history and optimal therapy. The IVL is an aggressive and usually disseminated disease (Ann Arbor stage IV in 68% of cases) that predominantly affects elderly patients (median age 70 years, range: 34–90; male:female ratio 0·9), resulting in poor Eastern Cooperative Oncology Group Performance Status (ECOG‐PS >1 in 61%), B symptoms (55%), anaemia (63%) and high serum lactate dehydrogenase level (86%). The brain and skin are the most common sites of disease. In contrast to previous reports, hepatosplenic involvement (26%) and bone marrow infiltration (32%) were found to be common features in IVL, while nodal disease was confirmed as rare (11% of cases). Patients with disease limited to the skin (‘cutaneous variant’; 26% of cases) were invariably females with a normal platelet count, and exhibited a significantly better outcome than the remaining patients, which deserves further investigation. Overall survival was usually poor; however, the early use of intensive therapies could improve outcome in young patients with unfavourable features. ECOG‐PS >1, ‘cutaneous variant’, stage I and chemotherapy use were independently associated with improved survival.

Infusion of suicide-gene-engineered donor lymphocytes after family haploidentical haemopoietic stem-cell transplantation for leukaemia (the TK007 trial): a non-randomised phase I–II study

BACKGROUND Procedures to prevent severe graft-versus-host disease (GVHD) delay immune reconstitution secondary to transplants of haploidentical haemopoietic stem cells for the treatment of leukaemia, leading to high rates of late infectious mortality. We aimed to systematically add back genetically engineered donor lymphocytes to facilitate immune reconstitution and prevent late mortality. METHODS In a phase I-II, multicentre, non-randomised trial of haploidentical stem-cell transplantation, we infused donor lymphocytes expressing herpes-simplex thymidine kinase suicide gene (TK-cells) after transplantation. The primary study endpoint was immune reconstitution defined as circulating CD3+ count of 100 cells per muL or more for two consecutive observations. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00423124. FINDINGS From Aug 13, 2002, to March 26, 2008, 50 patients (median age 51 years, range 17-66) received haploidentical stem-cell transplants for high-risk leukaemia. Immune reconstitution was not recorded before infusion of TK-cells. 28 patients received TK-cells starting 28 days after transplantation; 22 patients obtained immune reconstitution at median 75 days (range 34-127) from transplantation and 23 days (13-42) from infusion. Ten patients developed acute GVHD (grade I-IV) and one developed chronic GVHD, which were controlled by induction of the suicide gene. Overall survival at 3 years was 49% (95% CI 25-73) for 19 patients who were in remission from primary leukaemia at the time of stem-cell transplantation. After TK-cell infusion, the last death due to infection was at 166 days, this was the only infectious death at more than 100 days. No acute or chronic adverse events were related to the gene-transfer procedure. INTERPRETATION Infusion of TK-cells might be effective in accelerating immune reconstitution, while controlling GVHD and protecting patients from late mortality in those who are candidates for haploidentical stem-cell transplantation. FUNDING MolMed SpA, Italian Association for Cancer Research.

Myeloid sarcoma: clinico-pathologic, phenotypic and cytogenetic analysis of 92 adult patients.

Myeloid sarcoma (MS) is a rare neoplasm whose knowledge is largely based on case reports and/or technically dated contributions. Ninety-two MSs in adulthood with clinical data available were evaluated both morphologically and immunohistochemically. Seventy-four cases were also studied by fluorescent in situ hybridization on tissue sections and/or conventional karyotyping on bone marrow or peripheral blood. Histologically, 50% of the tumors were of the blastic type, 43.5% either monoblastic or myelomonocytic and 6.5% corresponded to different histotypes. CD68/KP1 was the most commonly expressed marker (100%), followed by myeloperoxidase (83.6%), CD117 (80.4%), CD99 (54.3%), CD68/PG-M1 (51%), CD34 (43.4%), terminal-deoxy-nucleotidyl-transferase (31.5%), CD56 (13%), CD61/linker for activation of T cells (2.2%), CD30 (2.2%) and CD4 (1.1%). Foci of plasmacytoid monocyte differentiation were observed in intestinal cases carrying inv16. Chromosomal aberrations were detected in about 54% of cases: monosomy 7(10.8%), trisomy 8(10.4%) and mixed lineage leukemia-splitting (8.5%) were the commonest abnormalities, whereas t(8;21) was rare (2.2%). The behavior was dramatic irrespective of presentation, age, sex, phenotype and cytogenetics. Most if not all, long survivors received bone-marrow transplantation. The present report expands the spectrum of our knowledge showing that MS has frequent monoblastic/myelomonocytic differentiation, displays distinctive phenotypic profile, carries chromosomal aberrations other than t(8;21), and requires supra-maximal therapy.

High-dose cytarabine plus high-dose methotrexate versus high-dose methotrexate alone in patients with primary CNS lymphoma: a randomised phase 2 trial

BACKGROUND Chemotherapy with high-dose methotrexate is the conventional approach to treat primary CNS lymphomas, but superiority of polychemotherapy compared with high-dose methotrexate alone is unproven. We assessed the effect of adding high-dose cytarabine to methotrexate in patients with newly diagnosed primary CNS lymphoma. METHODS This open, randomised, phase 2 trial was undertaken in 24 centres in six countries. 79 patients with non-Hodgkin lymphoma exclusively localised into the CNS, cranial nerves, or eyes, aged 18-75 years, and with Eastern Cooperative Oncology Group performance status of 3 or lower and measurable disease were centrally randomly assigned by computer to receive four courses of either methotrexate 3.5 g/m(2) on day 1 (n=40) or methotrexate 3.5 g/m(2) on day 1 plus cytarabine 2 g/m(2) twice a day on days 2-3 (n=39). Both regimens were administered every 3 weeks and were followed by whole-brain irradiation. The primary endpoint was complete remission rate after chemotherapy. Analysis was by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00210314. FINDINGS All randomly assigned participants were analysed. After chemotherapy, seven patients given methotrexate and 18 given methotrexate plus cytarabine achieved a complete remission, with a complete remission rate of 18% (95% CI 6-30) and 46% (31-61), respectively, (p=0.006). Nine patients receiving methotrexate and nine receiving methotrexate plus cytarabine achieved a partial response, with an overall response rate of 40% (25-55) and 69% (55-83), respectively, (p=0.009). Grade 3-4 haematological toxicity was more common in the methotrexate plus cytarabine group than in the methotrexate group (36 [92%] vs six [15%]). Four patients died of toxic effects (three vs one). INTERPRETATION In patients aged 75 years and younger with primary CNS lymphoma, the addition of high-dose cytarabine to high-dose methotrexate provides improved outcome with acceptable toxicity compared with high-dose methotrexate alone. FUNDING Swiss Cancer League.

MYC/BCL2 protein coexpression contributes to the inferior survival of activated B-cell subtype of diffuse large B-cell lymphoma and demonstrates high-risk gene expression signatures: a report from The International DLBCL Rituximab-CHOP Consortium Program

Diffuse large B-cell lymphoma (DLBCL) is stratified into prognostically favorable germinal center B-cell (GCB)-like and unfavorable activated B-cell (ABC)-like subtypes based on gene expression signatures. In this study, we analyzed 893 de novo DLBCL patients treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). We show that MYC/BCL2 protein coexpression occurred significantly more commonly in the ABC subtype. Patients with the ABC or GCB subtype of DLBCL had similar prognoses with MYC/BCL2 coexpression and without MYC/BCL2 coexpression. Consistent with the notion that the prognostic difference between the 2 subtypes is attributable to MYC/BCL2 coexpression, there is no difference in gene expression signatures between the 2 subtypes in the absence of MYC/BCL2 coexpression. DLBCL with MYC/BCL2 coexpression demonstrated a signature of marked downregulation of genes encoding extracellular matrix proteins, those involving matrix deposition/remodeling and cell adhesion, and upregulation of proliferation-associated genes. We conclude that MYC/BCL2 coexpression in DLBCL is associated with an aggressive clinical course, is more common in the ABC subtype, and contributes to the overall inferior prognosis of patients with ABC-DLBCL. In conclusion, the data suggest that MYC/BCL2 coexpression, rather than cell-of-origin classification, is a better predictor of prognosis in patients with DLBCL treated with R-CHOP.

Definition, Diagnosis, and Management of Intravascular Large B-Cell Lymphoma: Proposals and Perspectives From an International Consensus Meeting

Intravascular large B-cell lymphoma (IVLBCL) is a rare form of diffuse LBCL characterized by preferential intravascular growth of malignant lymphocytes, aggressive behavior, and an often fatal course. IVLBCL usually affects elderly patients with poor performance status, elevated lactic dehydrogenase serum levels, anemia, and B symptoms. It displays some differences in clinical presentation among diverse geographical areas, mostly between patients diagnosed in Western countries and Japan. In addition, data from the literature suggest that pathologic diagnostic criteria as well as clinical features of this disease may be broader than described in current classification scheme(s). Under the sponsorship of the International Extranodal Lymphoma Study Group, clinicians and pathologists with interest in IVLBCL, coming from Western and Eastern countries, joined to reach a consensus on defining features as well as to focus on the most urgent unresolved issues in IVLBCL. To this end, a representative group of IVLBCL patients coming from both the aforementioned geographical areas were collectively analyzed. Additional features of IVLBCL were proposed both under clinical and pathologic stand points. At the meeting, it emerged that IVLBCL may have additional histopathologic/cytologic definition criteria with respect to those currently recommended, some clinical features are not randomly distributed worldwide, recent therapeutic approaches, such as anti-CD20-containing regimens, may improve outcome, and kidney, spleen, and liver involvement may show peculiar histopathologic features. Finally, a provisional practical diagnostic approach to hemophagocytosis-associated patients and a proposal for the most useful criteria in the settings of differential diagnosis are included.