Prevalence of CD30 immunostaining in neoplastic mast cells: A retrospective immunohistochemical study.

Mastocytosis is a rare disease characterized by clonal neoplastic proliferation of mast cells (MCs). It ranges from skin lesions as cutaneous mastocytosis (CM) which may spontaneously regress to highly aggressive neoplasms with multiorgan involvement corresponding to some aggressive systemic mastocytosis (ASM), mast cell leukemia (MCL), and/or mast cell sarcoma (MCS).There is increasing evidence of CD30 expression in neoplastic MCs of the bone marrow. This expression has been described almost exclusively in aggressive forms of systemic mastocytosis (SM).The aim of the present study is to evaluate CD30 expression both in cutaneous and systemic forms of mastocytosis. Forty-two mastocytosis cases were reviewed, including cutaneous (n = 29) and systemic (n = 13) forms to assess the prevalence of CD30 expression. Thirty-nine out of 42 (92.8%) cases were CD30 positive. In cases of CM, 28/29 (96.5%) cases were CD30 positive, 11/13 cases of SM (84.6%) were positive for CD30. MCs in normal skin biopsies and in urticaria lesions were CD30-negative. This study found that CD30 is also frequently expressed in CM as well as in systemic forms. This finding is a major departure from the prevailing concept that CD30 expression is often related to aggressive systemic forms of mastocytosis.

Impact of Expert Pathologic Review of Lymphoma Diagnosis: Study of Patients From the French Lymphopath Network.

Purpose To prospectively assess the clinical impact of expert review of lymphoma diagnosis in France. Materials and Methods From January 2010 to December 2013, 42,145 samples from patients with newly diagnosed or suspected lymphomas were reviewed, according to the 2008 WHO classification, in real time by experts through the Lymphopath Network. Changes in diagnosis between referral and expert review were classified as major or minor according to their potential impact on patient care. Results The 42,145 reviewed samples comprised 36,920 newly diagnosed mature lymphomas, 321 precursor lymphoid neoplasms, 314 myeloid disorders, and 200 nonhematopoietic neoplasms, with 4,390 benign lesions. There were 4,352 cutaneous and 32,568 noncutaneous lymphomas. The most common mature noncutaneous lymphomas were diffuse large B-cell lymphomas (32.4%), follicular lymphomas (15.3%), classic Hodgkin lymphomas (13%), peripheral T-cell lymphomas (6.3%) of which angioimmunoblastic T-cell lymphomas (2.3%) were the most frequent, and mucosa-associated lymphoid tissue lymphomas (5.8%). A diagnostic change between referral and expert review occurred in 19.7% of patients, with an estimated impact on patient care for 17.4% of patients. This rate was significantly higher for patients sent with a provisional diagnosis seeking expert second opinion (37.8%) than for patients sent with a formal diagnosis (3.7%). The most frequent discrepancies were misclassifications in lymphoma subtype (41.3%), with 12.3% being misclassifications among small B-cell lymphoma entities. Fewer than 2% of changes were between benign and malignant lymphoid conditions. Minor changes (2.3%) mostly consisted of follicular lymphoma misgrading and diffuse large B-cell lymphoma subtype misclassification. Conclusion To our knowledge, this study provides the largest ever description of the distribution of lymphoma entities in a western country and highlights how expert review significantly contributes to a precise lymphoma diagnosis and optimal clinical management in a proportion of patients.

Clinical analysis and prognostic significance of haemophagocytic lymphohistiocytosis-associated anaplastic large cell lymphoma in children.

Haemophagocytic lymphohistiocytosis (HLH) has been rarely described in children treated for an anaplastic large-cell lymphoma (ALCL). We evaluated the incidence, the clinical and histological characteristics and the prognosis of HLH associated-ALCL. The medical, biological, cytological and histological data of patients treated for ALK-positive ALCL in the paediatric department of a single institution between 1975 and 2008 were analysed and assessed for HLH according to diagnosis criteria of the Histiocyte Society. Data concerning a series of 50 consecutive children with ALCL were reviewed. HLH-associated ALCL was observed in 12% of the patients. Lung involvement was significantly more frequent in HLH-associated ALCL patients than in the group without HLH (P = 0·004), as well as central nervous system (CNS) and bone marrow involvement (P = 0·001 and P = 0·007 respectively). The histological subtype in children with HLH-associated ALCL did not differ from that of the group without HLH. There was no significant difference between the two groups in 5-year EFS and OS (P = 0·91 and P > 0·99 respectively). In conclusion, HLH is not rare in paediatric ALCL. Despite a high incidence of visceral, CNS and bone marrow involvement, HLH does not seem to exert a significant impact on outcome in children treated for ALCL.

CD30-positive peripheral T-cell lymphomas share molecular and phenotypic features.

Peripheral T-cell lymphoma, not otherwise specified is a heterogeneous group of aggressive neoplasms with indistinct borders. By gene expression profiling we previously reported unsupervised clusters of peripheral T-cell lymphomas, not otherwise specified correlating with CD30 expression. In this work we extended the analysis of peripheral T-cell lymphoma molecular profiles to prototypical CD30(+) peripheral T-cell lymphomas (anaplastic large cell lymphomas), and validated mRNA expression profiles at the protein level. Existing transcriptomic datasets from peripheral T-cell lymphomas, not otherwise specified and anaplastic large cell lymphomas were reanalyzed. Twenty-one markers were selected for immunohistochemical validation on 80 peripheral T-cell lymphoma samples (not otherwise specified, CD30(+) and CD30(-); anaplastic large cell lymphomas, ALK(+) and ALK(-)), and differences between subgroups were assessed. Clinical follow-up was recorded. Compared to CD30(-) tumors, CD30(+) peripheral T-cell lymphomas, not otherwise specified were significantly enriched in ALK(-) anaplastic large cell lymphoma-related genes. By immunohistochemistry, CD30(+) peripheral T-cell lymphomas, not otherwise specified differed significantly from CD30(-) samples [down-regulated expression of T-cell receptor-associated proximal tyrosine kinases (Lck, Fyn, Itk) and of proteins involved in T-cell differentiation/activation (CD69, ICOS, CD52, NFATc2); upregulation of JunB and MUM1], while overlapping with anaplastic large cell lymphomas. CD30(-) peripheral T-cell lymphomas, not otherwise specified tended to have an inferior clinical outcome compared to the CD30(+) subgroups. In conclusion, we show molecular and phenotypic features common to CD30(+) peripheral T-cell lymphomas, and significant differences between CD30(-) and CD30(+) peripheral T-cell lymphomas, not otherwise specified, suggesting that CD30 expression might delineate two biologically distinct subgroups.

Technical note: Hapten synthesis, antibody production and development of an enzyme-linked immunosorbent assay for detection of the natural steroidal alkaloid Dendrogenin A.

We have recently discovered the existence of 5α-Hydroxy-6ß-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3ß-ol, called Dendrogenin A (DDA), as the first endogenous steroidal alkaloid ever described in mammals. We found that the DDA content of tumors and cancer cell lines was low or absent compared with normal cells showing that a deregulation in DDA biosynthesis was associated with cancer and therefore suggesting that DDA could represent a metabolomic cancer biomarker. This prompted us to produce antibodies that selectively recognize DDA. For this purpose, the hapten 5α-hydroxy-6ß-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3ß-o-hemisuccinate with a carboxylic spacer arm attached to the 3ß-hydroxyl group of DDA was synthesized. The hapten was coupled to bovine serum albumin and keyhole limpet hemocyanin for antibody production to develop an enzyme-linked immunosorbent assay (ELISA). The protein conjugates were injected into BALB/c mice to raise antibodies. The monoclonal antibodies that were secreted from the hybridoma cell lines established were assessed with indirect ELISA by competitive assays using dilutions of a DDA standard. The antibodies from the selected hybridomas had an IC(50) value ranging from 0.8 to 425 ng/ml. Three antibodies showed no cross-reactivity with structurally related compounds including histamine, cholesterol, ring B oxysterols and a regio-isomer of DDA. In this study, high-affinity and selective antibodies against DDA were produced for the first time, and a competitive indirect ELISA was developed.

Long-term outcome of adults with systemic anaplastic large-cell lymphoma treated within the Groupe d'Etude des Lymphomes de l'Adulte trials.

PURPOSE: Systemic anaplastic large-cell lymphoma (ALCL) is a T-cell lymphoma, whose anaplastic lymphoma kinase (ALK) expression varies according to age. Long-term outcomes of chemotherapy-treated adults are not definitively established and should be evaluated. PATIENTS AND METHODS: Patients treated in three Groupe d'Étude des Lymphomes de l'Adulte prospective clinical trials with confirmed systemic ALCL after immunohistopathologic review and defined ALK expression status were analyzed. RESULTS: Among the 138 adult patients with ALCL, 64 (46%) were ALK positive, and 74 (54%) were ALK negative. Median follow-up was 8 years. At diagnosis, significantly more patients younger than 40 years old were ALK positive than ALK negative (66% v 23%, respectively; P < .001). Comparing patients with ALK-positive and ALK-negative ALCL, ß(2)-microglobulin was ≥ 3 mg/L in 12% and 33% (P = .017); International Prognostic Index was high (score, 3 to 5) in 23% and 48% (P = .03); complete response rates to first-line treatment were 86% and 68% (P = .01); and 8-year overall survival (OS) rates were 82% (95% CI, 69% to 89%) and 49% (95% CI, 37% to 61%), respectively (P < .001). The survival difference mostly affected patients age ≥ 40 years. Multivariate analysis identified ß(2)-microglobulin ≥ 3 mg/L (P < .001) and age ≥ 40 years (P = .029), but not ALK status, as prognostic for OS. These two variables distinguished four survival risk groups, with 8-year OS ranging from 84% to 22%. CONCLUSION Results of this long-term study enabled refinement of the prognosis of adult systemic ALCL, with ALK prognostic value dependent on age, and could provide guidance for eventual treatment adjustment.

Anaplastic lymphoma kinase as a therapeutic target.

INTRODUCTION: Anaplastic lymphoma kinase (ALK), a tyrosine kinase receptor, has been initially identified through its involvement in chromosomal translocations associated with anaplastic large cell lymphoma. However, recent evidence that aberrant ALK activity is also involved in an expanding number of tumor types, such as other lymphomas, inflammatory myofibroblastic tumor, neuroblastomas and some carcinomas, including non-small cell lung carcinomas, is boosting research progress in ALK-targeted therapies. AREAS COVERED: The first aim of this review is to describe current understandings about the ALK tyrosine kinase and its implication in the oncogenesis of human cancers as a fusion protein or through mutations. The second goal is to discuss its interest as a therapeutic target and to provide a review of the literature regarding ALK inhibitors. Mechanisms of acquired resistance are also reviewed. EXPERT OPINION: Several ALK inhibitors have recently been developed, offering new treatment options in tumors driven by abnormal ALK signaling. However, as observed with other tyrosine kinase inhibitors, resistance has emerged in patients treated with these agents. The complexity of mechanisms of acquired resistance recently described suggests that other therapeutic options, including combination of ALK and other kinases targeted drugs, will be required in the future.

ALK+ALCLs induce cutaneous, HMGB-1-dependent IL-8/CXCL8 production by keratinocytes through NF-κB activation.

Anaplastic large-cell lymphomas (ALCLs) bearing the t(2;5) translocation (ALK(+)ALCLs) are frequently characterized by skin colonization and associated with a poor prognosis. Using conditional transgenic models of anaplastic lymphoma kinase-positive (ALK(+)) lymphomas and human ALK(+)ALCL cell lines, in the present study, we show that high-mobility-group box-1 (HMGB-1), a proinflammatory cytokine, is released by ALK(+) cells, and demonstrate extracellular HMGB-1-stimulated secretion of the IL-8 chemokine by HaCaT keratinocytes through the involvement of MMP-9, PAR-2, and the NF-κB pathway. Furthermore, we demonstrate that, in vitro, IL-8 is able to induce the invasiveness of ALK(+) cells, which express the IL-8 receptors CXCR1 and CXCR2. In vitro and in vivo, HMGB-1 inhibition achieved by glycyrrhizin treatment led to a drastic reduction in ALK(+) cell invasiveness. The pathophysiological relevance of our observations was confirmed by demonstrating that the HMGB-1 and IL-8 receptors are expressed in ALK(+)ALCL biopsies. We have also shown that IL-8 secretion is correlated with leukemic dissemination of ALK(+) cells in a significant number of patients. The results of the present study demonstrate for the first time a relationship among the pro-inflammatory mediators HMGB-1, MMP-9, PAR-2, and IL-8. We propose that these mediators create a premetastatic niche within the skin, thereby participating in ALK(+) lymphoma epidermotropism.

Prognostic impact of morphologic and phenotypic features of childhood ALK-positive anaplastic large-cell lymphoma: results of the ALCL99 study.

PURPOSE: The prognostic value of pathologic characteristics of childhood ALK-positive anaplastic large-cell lymphomas (ALCL), such as histologic subtypes, immunophenotype, and presence of the t(2;5) translocation or its variants, was assessed. PATIENTS AND METHODS: All 375 patients with systemic ALK-positive ALCL included in an international trial launched by the European Intergroup for Childhood Non-Hodgkin's Lymphoma were reviewed by an international panel of pathologists based on conventional hematoxylin and eosin-stained and immunostained sections and classified according to the 2001 WHO classification. RESULTS: A small-cell (SC) or lymphohistiocytic (LH) component was observed in 114 (32%) of 361 patients, whereas ALCL of common type was diagnosed in 235 (65%) of 361 patients. Regarding the histologic subtyping of patients within the two categories of ALCL (with v without SC/LH component), the concordance between the national and international reviews was quite good, with a κ index equal to 0.67 (95% CI, 0.57 to 0.75). The presence of an SC/LH component was significantly associated with a high risk of failure (hazard ratio [HR], 2.0; 95% CI, 1.3 to 3.0; P = .002) in the multivariate analysis controlling for clinical characteristics, as well as the perivascular pattern (HR, 1.7; 95% CI, 1.1 to 2.7; P = .01), whereas CD3 positivity was significantly associated with a high risk of failure only in univariate analysis. CONCLUSION: Our study, which to our knowledge includes the largest series of childhood systemic ALK-positive ALCL so far, demonstrates the adverse prognostic value of SC and/or LH morphologic features. Combining these histologic characteristics with other biologic or clinical factors might have a high potential for future risk stratification and treatment.

[Impact of the pathology in modern medicine]. / Représentation en sciences du vivant (9) émergence d'une spécialité médicale nouvelle : la pathologie.

In this review, the authors emphasize the pivotal role of the pathology in the setting of a revolution which progressively transforms medical sciences into basic sciences. Several key aspects of this specialty will be discussed together with the main perspectives in the fields of oncologic disorders.

MiR-29a down-regulation in ALK-positive anaplastic large cell lymphomas contributes to apoptosis blockade through MCL-1 overexpression.

Although deregulated expression of specific microRNAs (miRNAs) has been described in solid cancers and leukemias, little evidence of miRNA deregulation has been reported in ALK-positive (ALK(+)) anaplastic large cell lymphomas (ALCL). These tumors overexpress the major antiapoptotic protein myeloid cell leukemia 1 (MCL-1), a situation that could compensate for the lack of BCL-2. We report that ALK(+) ALCL cell lines and biopsy specimens (n = 20) express a low level of miR-29a and that this down-modulation requires an active NPM-ALK kinase. Murine models (transgenic mice and mouse embryonic fibroblast [MEF] cells), which allow conditional NPM-ALK fusion protein expression, showed an increase of miR-29a expression in the absence of NPM-ALK. Concordant results were observed after the abolition of NPM-ALK kinase activity (siALK or PF-2341066) in NPM-ALK(+) ALCL cell lines. In addition, we showed that low expression of miR-29a, probably through methylation repression, plays an important regulatory role in MCL-1 overexpression that could promote tumor cell survival by inhibiting apoptosis. Enforced miR-29a expression was found to modulate apoptosis through inhibition of MCL-1 expression in ALCL cell lines and in a xenografted model, with a concomitant tumor growth reduction. Thus, synthetic miR-29a represents a potential new tool to affect tumorigenesis in these lymphomas.

HuR-mediated control of C/EBPbeta mRNA stability and translation in ALK-positive anaplastic large cell lymphomas.

The CCAAT/enhancer-binding protein ß (C/EBPß) plays a major role in the pathogenesis of anaplastic large cell lymphomas (ALCL) that express the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) tyrosine kinase (ALK(+)). Although ALK-mediated C/EBPß transcriptional activation has been reported, C/EBPß mRNA possesses U- and AU-rich domains in its 3'-untranslated region (3'-UTR) that might be privileged targets for posttranscriptional control in ALK(+) ALCLs. The purpose of this study was to explore this possibility. By using human ALCL-derived cells and a murine model of ALK-transformed cells, we show that the AU-binding protein HuR binds to the 3'-UTR of C/EBPß mRNA, as previously reported in adipocytes, and that NPM-ALK enhances this interaction. Interaction between HuR and C/EBPß mRNA impacts on C/EBPß gene expression at both the mRNA and protein levels. Indeed, C/EBPß mRNA stability following HuR silencing is reduced and reaches the value observed in ALK-inactivated cells. Remarkably, HuR expression is not modified by NPM-ALK, but its association with actively translating polysomes is dramatically increased in ALK(+) cells. HuR/polysomes association diminishes when NPM-ALK activity is inhibited and is accompanied by a concomitant decrease of C/EBPß mRNA translation. Finally, we show that HuR and NPM-ALK colocalized in cytoplasmic granules and HuR is phosphroylated on tyrosine residues in ALK(+) ALCL cells. Our study thus demonstrates that C/EBPß is indeed regulated at the posttranscriptional level by HuR in ALK(+) cells, leading us to propose that part of NPM-ALK oncogenic properties relies on its ability to modify HuR properties in the cytoplasm and hence to alter expression of key actors of transformation.

Matrix metalloproteinase-9 is upregulated in nucleophosmin-anaplastic lymphoma kinase-positive anaplastic lymphomas and activated at the cell surface by the chaperone heat shock protein 90 to promote cell invasion.

Many anaplastic large cell lymphomas (ALCL) express the chimeric oncogene NPM-ALK, which drives malignant transformation and invasion. In this study, we show that NPM-ALK expression increases matrix metalloproteinase-9 (MMP-9) expression. Accordingly, we found that 100% of a large panel of ALK(+) ALCL biopsies examined were also MMP-9(+), in contrast to only 36.3% of ALK(-) tumors. Mechanistic studies revealed that Rac1 drove MMP-9 secretion. The MMP inhibitor GM6001 and MMP-9 blocking antibodies abolished the invasiveness of NPM-ALK(+) cells. Interestingly, the hyaluronan receptor CD44 acted as a docking surface for MMP-9 and the chaperone heat shock protein 90 on the cell surface, where MMP-9 was cleaved and activated. Membrane-associated MMP-9 was localized to invadopodia, which display a strong gelatinase activity. Taken together, our observations strengthen the concept that chaperones have a major extracellular role in the regulation of protein activation status, and reveal new factors that are crucial for spreading and invasion of ALK(+) ALCL. They also point out new factors crucial for ALK(+) ALCL.

Conditional TPM3-ALK and NPM-ALK transgenic mice develop reversible ALK-positive early B-cell lymphoma/leukemia.

NPM-ALK (nucleophosmin-anaplastic lymphoma kinase) and TPM3-ALK (nonmuscular tropomyosin 3-anaplastic lymphoma kinase) are oncogenic tyrosine kinases implicated in the pathogenesis of human ALK-positive lymphoma. We report here the development of novel conditional mouse models for ALK-induced lymphomagenesis, with the use of the tetracycline regulatory system under the control of the EmuSRalpha enhancer/promoter. The expression of either oncogene resulted in the arrest of the differentiation of early B cells and lymphomagenesis. We also observed the development of skin keratoacanthoma lesions, probably because of aberrant ALK expression in keratinocytes. The inactivation of the ALK oncogene on doxycycline treatment was sufficient to induce sustained regression of both hematopoietic tumors and skin disease. Importantly, treatment with the specific ALK inhibitor (PF-2341066) also reversed the pathologic states, showing the value of these mouse models for the validation of ALK tyrosine kinase inhibitors. Thus, our results show (1) that NPM-ALK and TPM3-ALK oncogenes are sufficient for lymphoma/leukemia development and required for tumor maintenance, hence validating ALK as potentially effective therapeutic target; and (2) for the first time, in vivo, the equal tumorigenic potential of the NPM-ALK and TPM3-ALK oncogenic tyrosine kinases. Our models offer a new tool to investigate in vivo the molecular mechanisms associated with ALK-induced lymphoproliferative disorders.

Cutaneous presentation of ALK-positive anaplastic large cell lymphoma following insect bites: evidence for an association in five cases.

BACKGROUND: Skin involvement is frequent in ALK-positive anaplastic large cell lymphomas. The role of an insect bite as a triggering event has been postulated but not well documented. DESIGN AND METHODS: We retrospectively investigated five cases of ALK-positive anaplastic large cell lymphoma who presented with skin lesions occurring after an insect bite. Biopsies were immunostained with antibodies against CD30, ALK, T- and B-cell antigens. RESULTS: Persistent skin lesions developed after solitary insect bites in three patients and after multiple bites in two. Regional lymphadenopathy developed within weeks after the bite in three cases. In four cases the correct diagnosis was delayed due to misinterpretation of the findings as a reactive infiltrate in the skin (n=2) or lymph nodes (n=2); all cases subsequently showed small numbers of cells with nuclear and cytoplasmic staining for ALK. The final diagnoses were lymphohistiocytic variant (n=3) and composite common/small cell type (n=2) anaplastic large cell lymphoma. The patients were treated and three were alive at the last follow-up. Two patients died, one of pneumonia and the other of disseminated disease. CONCLUSIONS: In these cases the sequence of events between the insect bites and the occurrence of both skin lesions and satellite lymphadenopathy suggest a direct relationship between the bite and the presentation with anaplastic large cell lymphoma. We postulate that insect bite-associated antigens could result in an influx of T lymphocytes, some bearing the t(2;5). The subsequent release of cytokines at the site of the bite could act as a 'second hit', eliciting activation of the latter cells, which would then express the oncogenic NPM-ALK protein and undergo uncontrolled proliferation.

Immunoexpression of Survivin in non-neoplastic lymphoid tissues and malignant lymphomas using a new monoclonal antibody reactive on paraffin sections.

Survivin is a member of the inhibitor of apoptosis gene family, which is also implicated in mitosis regulation. Most reports in the literature impute poor prognosis to neoplasms with overexpression of this protein. The purpose of the present study is to validate and compare the immunohistochemical reactivity of malignant lymphomas and reactive lymphoid tissue using a new mouse monoclonal antibody to Survivin produced in our laboratory, 6-78. Survivin was detected by immunohistochemistry on tissue microarrays. It was shown that the antibody anti-Survivin 6-78 reliably stains formalin-fixed, paraffin-embedded reactive and neoplastic lymphoid tissues, mostly in a nuclear pattern. We confirmed using this novel antibody that Survivin immunostaining has a tendency to be lower in reactive lymphoid tissues and low-grade B cell lymphomas than in aggressive lymphomas. This antibody may represent a useful tool for standardizing the study of the immunoexpression of Survivin in neoplasms.

Anaplastic lymphoma kinase-positive diffuse large B-cell lymphoma: a rare clinicopathologic entity with poor prognosis.

PURPOSE: Anaplastic lymphoma kinase (ALK) -positive diffuse large B-cell lymphoma (DLBCL) is a rare variant of DLBCL that has been described only in small case reports. To shed more light on the clinical and pathologic features and outcome of these tumors, we reviewed data from 38 patients. PATIENTS AND METHODS: We retrospectively analyzed 38 patients with ALK-positive DLBCL treated with cyclophosphamide, doxorubicin, vincristine, prednisone (CHOP) or CHOP-like regimens from different institutions to better define the presenting features, clinical course, and response to treatment. RESULTS: The histologic findings in all patients were similar. All patients expressed ALK fusion proteins, but virtually all were CD30 and CD20 negative. The median age was 43 years with a 5:1 ratio of males to females. Most patients (60%) followed an aggressive clinical course with advanced stage at diagnosis, frequent marrow infiltration, and poor outcome. Overall survival was 20.3 months (95% CI, 12.2 to 42.6 months). Of note, the median survival was only 12.2 months (95% CI, 9.1 to 32.5 months) in patients with advanced-stage disease. CONCLUSION: ALK-positive DLBCLs display clinicopathologic features that distinguish them from common DLBCL. Conventional therapy, as used for typical DLBCL, is of limited efficacy. Recognition of this new entity and the characteristic lack of CD20 expression are paramount. Novel front-line intensive chemotherapy regimens should be evaluated in this group of patients.

Regulation of DNA polymerase beta by the LMP1 oncoprotein of EBV through the nuclear factor-kappaB pathway.

The repair DNA polymerase beta (Polbeta), when overexpressed, plays a critical role in generating genetic instability via its interference with the genomic replication program. Up-regulation of Polbeta has been reported in many tumor types that exhibit genetic aberrations, including EBV-related B-cell lymphomas. However, the mechanisms responsible for its overexpression have never been examined. Here, we report that both expression and activity of Polbeta, in EBV-immortalized B cells, are induced by several natural genetic variants of LMP1, an oncoprotein associated with the vast majority of EBV-related tumors. Conversely, we found that the expression of Polbeta decreased when LMP1 signaling was down-regulated by a dominant negative of LMP1 or an inhibitor of the nuclear factor-kappaB (NF-kappaB) pathway, the main transduction pathway activated by LMP1, strongly supporting a role of NF-kappaB in the LMP1-mediated Polbeta regulation. Using electrophoretic mobility shift assay experiments from several EBV-immortalized B-cell nuclear extracts, we identified an LMP1-dependent p50/c-Rel heterodimer on a proximal kappaB binding site (-211 to -199nt) of the Polbeta promoter. This result was correlated with a specific Polbeta kappaB transcriptional activity. Taken together, our data enlighten a new mechanism responsible for Polbeta overexpression in EBV-infected cells, mediated by LMP1 and dependent on NF-kappaB activation.

Antitumor activity of pyridoisoquinoline derivatives F91873 and F91874, novel multikinase inhibitors with activity against the anaplastic lymphoma kinase.

The anaplastic lymphoma kinase (ALK) is a validated target for the therapy of different malignancies. Aberrant expression of constitutively active ALK chimeric proteins has been implicated in the pathogenesis of anaplastic large-cell lymphoma (ALCL) and has been detected in other cancers such as inflammatory myofibroblastic tumors, diffuse large B-cell lymphomas, certain non-small-cell lung cancers, rhabdomyosarcomas, neuroblastomas and glioblastomas. In the course of a screening program aimed at identifying kinase inhibitors with novel scaffolds, the two pyridoisoquinoline derivatives F91873 and F91874, were identified as multikinase inhibitors with activity against ALK in a biochemical screen. F91873 and F91874 also inhibited nucleophosmin-ALK and signal transducer and activator of transcription 3 phosphorylation in the ALCL cell line COST with the same potency. Both F91873 and F91874 behaved as ATP noncompetitive inhibitors and inhibited cell proliferation of the ALK(+) ALCL cell lines COST, PIO, and Karpas299 ALCL. This growth inhibition effect was associated with a G1-phase cell cycle arrest. Furthermore, administration of F91874 to severe combined immunodeficient mice bearing COST tumor xenografts resulted in a significant antitumor efficacy at 15 mg/kg/day, illustrating the potential utility of such compounds in the treatment of ALK-related pathologies.