Olga Ritz

Recurrent mutations of the STAT6 DNA binding domain in primary mediastinal B-cell lymphoma

Primary mediastinal B-cell lymphoma (PMBL) is a separate entity of aggressive B-cell lymphoma, characterized by a constitutive activation of janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway, also observed in Hodgkin lymphoma. Although many cancers exhibit constitutive JAK-STAT pathway activation, mutations of STAT genes have not been reported in neoplasms. Here, we show that MedB-1 PMBL-derived and L1236 Hodgkin-derived cell lines and 20 of 55 (36%) PMBL cases harbor heterozygous missense mutations in STAT6 DNA binding domain, whereas no mutation was found in 25 diffuse large B-cell lymphoma samples. In 3 cases, somatic origin was indicated by the absence of the mutations in the nontumoral tissue. The pattern of STAT6 mutations was different from the classical features of somatic hypermutations. The mutant STAT6 proteins showed a decreased DNA binding ability in transfected HEK cells, but no decrease in expression of STAT6 canonical target genes was observed in PMBL cases with a mutated STAT6 gene. Although the oncogenic properties of STAT6 mutant proteins remain to be determined, their recurrent selection in PMBL strongly argues for their involvement in the pathogenesis of this aggressive B-cell lymphoma.

Biallelic deletion within 16p13.13 including SOCS-1 in Karpas1106P mediastinal B-cell lymphoma line is associated with delayed degradation of JAK2 protein.

Activity of Janus kinase 2 (JAK2) in the JAK2/STAT5 signaling pathway is critically controlled by suppressor of cytokine signaling‐1 (SOCS‐1). We have previously shown that SOCS‐1 is biallelically mutated in the primary mediastinal B‐cell lymphoma (PMBL) cell line MedB‐1, resulting in impaired JAK2 degradation and sustained phospho‐JAK2 action. SOCS‐1 is frequently mutated in PMBL tumor primaries. Here, we report that the PMBL cell line Karpas1106P has a biallelic deletion of the SOCS‐1 region on chromosome 16p13.13. By fluorescence in situ hybridization and microsatellite analysis, this deletion was narrowed down to a range of 650 kb to 1.48 Mb. Like MedB‐1, Karpas1106P harbors gains of the JAK2 gene on chromosomal region 9p24 and elevated levels of JAK2 mRNA. Nevertheless, JAK2 protein was not increased but constitutively phosphorylated in Karpas1106P cells. In analogy to MedB‐1 cells, Karpas1106P cells exhibited a retarded degradation of de novo synthesized JAK2 protein revealed by pulse/chase experiments. Therefore, we conclude that loss of SOCS‐1 function either by mutation or by the complete deletion of the gene plays an important role in the dysregulation of JAK/STAT signaling in Karpas1106P and PMBL.

STAT6 activity is regulated by SOCS-1 and modulates BCL-XL expression in primary mediastinal B-cell lymphoma.

STAT6 activity is regulated by SOCS-1 and modulates BCL-XL expression in primary mediastinal B-Cell lymphoma

ABF-1 is frequently silenced by promoter methylation in follicular lymphoma, diffuse large B-cell lymphoma and Burkitt's lymphoma.

Activated B-cell factor 1 (ABF-1) is a member of the bHLH transcription factor family. Its expression was detected in lymph nodes, appendix and other tissues, but not in thymus and peripheral blood lymphocytes (PBL). It contains a transrepression domain and was shown to inhibit transactivation of the related E2A transcription factors.1 E2A proteins are necessary for B-cell survival and proliferation.2 They are highly expressed in many types of B-cell non-Hodgkins lymphomas (B-NHL), that is, follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL) and Burkitts lymphoma (BL).3, 4 In contrast, expression of the E2A antagonist ABF-1 is low or absent in these types of tumor. Interestingly, an inverse situation is observed in classical Hodgkins lymphoma (cHL) where the levels of E2A are relatively low and ABF-1 expression is high.3 Indeed, ABF-1 expression was found to be limited to lymphoblastoid cell lines (LCL),5 cHL3 and primary effusion lymphoma.6 We therefore hypothesized that stable downregulation ABF-1 expression in FL, DLBCL and BL might endow a selective advantage to these tumors. Along with genomic mutations robust and stable gene inactivation has been shown to be achieved by epigenetic gene silencing.7 We therefore determined whether this mechanism contributes to the regulation of ABF-1 expression in distinct types of B-NHLs.

Target Sequence Accessibility Limits Activation-Induced Cytidine Deaminase Activity in Primary Mediastinal B-Cell Lymphoma

Activation-induced cytidine deaminase (AID) initiates somatic hypermutation (SHM) and class switch recombination (CSR) in activated B lymphocytes and is potentially implicated in genomic instability of B-cell malignancies. For unknown reasons, B-cell neoplasms often lack SHM and CSR in spite of high AID expression. Here, we show that primary mediastinal B-cell lymphoma (PMBL), an immunoglobulin (Ig)-negative lymphoma that possesses hypermutated, class-switched Ig genes, expresses high levels of AID with an intact primary structure but does not do CSR in 14 of 16 cases analyzed. Absence of CSR coincided with low Ig germ-line transcription, whereas high level germ-line transcription was observed only in those two cases with active CSR. Interleukin-4/CD40L costimulation induced CSR and a marked up-regulation of germ-line transcription in the PMBL-derived cell line MedB-1. In the PMBL cell line Karpas 1106P, CSR was not inducible and germ-line transcription remained low on stimulation. However, Karpas 1106P, but not MedB-1, had ongoing SHM of the Ig gene and BCL6. These genes were transcribed in Karpas 1106P, whereas transcription was undetectable or low in MedB-1 cells. Thus, accessibility of the target sequences seems to be a major limiting factor for AID-dependent somatic gene diversification in PMBL.

Downregulation of internal enhancer activity contributes to abnormally low immunoglobulin expression in the MedB‐1 mediastinal B‐cell lymphoma cell line

Primary mediastinal B‐cell lymphoma (PMBL) is a highly aggressive tumour with a unique pattern of clinical, morphological, immunological and genetic features distinct from other diffuse large B‐cell lymphomas. PMBLs are characterized by a mature B‐cell phenotype, but they typically lack immunoglobulin (Ig) gene expression. The PMBL cell line MedB‐1 shares many characteristic properties of the primary tumour, including low‐level Ig production despite a functionally rearranged IgVH gene and absence of ‘crippling’ mutations. In this study, a search was undertaken for reasons for downregulated Ig expression. Similar levels of the B‐cell‐specific transcription factors BOB.1/OBF.1 and PU.1 were found in MedB‐1 cells to those in the Ig‐producing UM‐1 lymphoblastoid cell line. However, MedB‐1 lacked the Oct2 transcription factor. Reporter assays showed that Ig‐type promoters were active in MedB‐1 cells. In contrast, activity of the intronic heavy chain enhancer was dramatically reduced. Ectopic expression of Oct2 was able partially to restore enhancer activity but transcription from the endogenous IgVH gene could not be rescued. Therefore, the role of epigenetic factors in the downregulation of Ig was investigated. Methylated histone 3 lysine 9, a reliable marker of chromatin silencing, was not detected in MedB‐1 promoter and enhancer regions. Inhibition of DNA methyltransferase and of histone deacetylases also did not reactivate Ig production. These data suggest the existence of alternative mechanisms of Ig inhibition in MedB‐1 cells, different from chromatin silencing and the lack of Oct2. Copyright

Validation of a manual protocol for BRAF V600E mutation-specific immunohistochemistry

Detection of BRAF V600E has diagnostic, prognostic, and therapeutic relevance. The recently developed BRAF V600E mutation-specific antibody has evolved into a feasible alternative to DNA analysis. The plethora of immunohistochemical protocols makes implementation tedious and, here we tested a set of manual and automated protocols and compared test performance with sequencing results. For assays, we employed formalin-fixed, in part decalcified, and paraffin-embedded tissue samples. Empiric testing of manual protocols included 10 variables in 17 protocols. Automated immunohistochemical staining and BRAF pyrosequencing served as independent test methods. Test performance measures were compared without considering 1 method as a standard. Four well-fixed samples (2WT/2Mut) were used for testing of all protocols and indicated 2 correctly classifying procedures. Practical performance assessment employed 33 independent tissue samples, composed of 27 leukemias (by pyrosequencing: 8 wild-type; 18 mutated; 1 noninformative) and 6 melanomas (V600E; V600K; wild-type, 2 each). Manual V600E staining was positive in 20 cases (19 of 20 V600E-containing samples plus the 1 sample that was noninformative), whereas all wild-type and V600K cases were immunonegative. Manual or automated staining as well as pyrosequencing would have missed an equal number of V600E-mutated cases and the correlation coefficient for these methods was 0.75 to 0.93 (substantial to almost perfect); the Youden index was 0.95. Detection of V600E-mutated BRAF at the protein level in routine and decalcified tissue samples is possible, and the presented manual protocols should expedite implementation in routine diagnostic practice. Our results indicate that both molecular techniques should be considered complementary.