Alexandra Papoudou-Bai

Promoter methylation of argininosuccinate synthetase-1 sensitises lymphomas to arginine deiminase treatment, autophagy and caspase-dependent apoptosis

Tumours lacking argininosuccinate synthetase-1 (ASS1) are auxotrophic for arginine and sensitive to amino-acid deprivation. Here, we investigated the role of ASS1 as a biomarker of response to the arginine-lowering agent, pegylated arginine deiminase (ADI-PEG20), in lymphoid malignancies. Although ASS1 protein was largely undetectable in normal and malignant lymphoid tissues, frequent hypermethylation of the ASS1 promoter was observed specifically in the latter. A good correlation was observed between ASS1 methylation, low ASS1 mRNA, absence of ASS1 protein expression and sensitivity to ADI-PEG20 in malignant lymphoid cell lines. We confirmed that the demethylating agent 5-Aza-dC reactivated ASS1 expression and rescued lymphoma cell lines from ADI-PEG20 cytotoxicity. ASS1-methylated cell lines exhibited autophagy and caspase-dependent apoptosis following treatment with ADI-PEG20. In addition, the autophagy inhibitor chloroquine triggered an accumulation of light chain 3-II protein and potentiated the apoptotic effect of ADI-PEG20 in malignant lymphoid cells and patient-derived tumour cells. Finally, a patient with an ASS1-methylated cutaneous T-cell lymphoma responded to compassionate-use ADI-PEG20. In summary, ASS1 promoter methylation contributes to arginine auxotrophy and represents a novel biomarker for evaluating the efficacy of arginine deprivation in patients with lymphoma.

The prolyl-hydroxylase EGLN3 and not EGLN1 is inactivated by methylation in plasma cell neoplasia

EGLN1 and EGLN3 are members of the egg‐laying‐defective 9 (EglN) prolyl‐hydroxylases which during normoxia catalyse hydroxylation of the hypoxia‐inducible factor (HIF)‐1α, thereby promoting its ubiquitination by a complex containing the von Hippel–Lindau (VHL) tumour suppressor. EGLN3 also has pro‐apoptotic activity in some cell types. Analyses of a well‐characterised series of cases of plasma cell dyscrasias, including multiple myeloma (MM), Waldenström’s macroglobulinaemia (WM) and monoclonal gammopathy of undetermined significance (MGUS) surprisingly demonstrated that the CpG island of EGLN3, and not EGLN1, is frequently methylated in these disorders. Multiple myeloma patients with a methylated EGLN3 promoter showed trends towards an increased risk of death, bone lytic lesions, anaemia, advanced stage of disease and the presence of extramedullary disease. Those individuals with methylation in the EGLN3 CpG island also had significantly lower albumin levels. These data suggest that the prolyl‐hydroxylases may be a novel class of potential tumour suppressors in plasma cell neoplasia that warrant further investigation with regard to their potential utility as biomarkers. Moreover, we observed that EGLN3 is also methylated at high frequency in B‐cell lymphoma subtypes, implying that loss of EGLN3 is an important epigenetic event not only in plasma cell neoplasias but also in B‐cell neoplasias.

B-cell differentiation immunophenotypes in classical Hodgkin lymphomas.

The bcl6/CD10/MUM1/CD138 B-cell differentiation immunophenotypes were analysed in 101 cases of classical Hodgkin lymphomas (cHL) aiming to elucidate their histogenesis. Three major bcl6/CD10/MUM1/CD138 immunophenotypes were distinguished on the basis of the immunohistochemical positivity of Hodgkin and Reed-Sternberg (H/RS) cells: (a) the late germinal center (GC)/early post-GC B-cell-like immunophenotype (bcl6−/CD10−/MUM1+/CD138−); 59/101 cases (59%), (b) the post-GC B-cell-like immunophenotype (bcl6−/CD10−/MUM1+/CD138+); 24/101 cases (24%) and (c) the indeterminate immunophenotype (bcl6+/CD10−/MUM1+/CD138−: 14 cases and bcl6+/CD10−/MUM1+/CD138+: four cases); 18/101 cases (18%). The above findings indicate that H/RS cells in most cHL display bcl6/CD10/MUM1/CD138 immunophenotypes consistent with late GC/early post-GC or post-GC B-cell differentiation. In addition, H/RS cells in a small fraction of cHL display indeterminate bcl6/CD10/MUM1/CD138 immunophenotypic profiles which are characterized by simultaneous expression of GC, late GC/early post-GC and post-GC B-cell differentiation proteins. These immunophenotypic profiles do not correspond to the differentiation immunophenotypes of normal B-cells and their identification in a part of cHL suggests that the differentiation process of H/RS cells is not complete in a fraction of these cells and/or is still ongoing at the time of observation.

High levels of topoisomerase IIα protein expression in diffuse large B-cell lymphoma are associated with high proliferation, germinal center immunophenotype, and response to treatment

Gene copy number and protein expression of topoisomerase IIα were correlated to benefit from anthracyclines in various tumors. A retrospective series of 69 patients with DLBCL managed with CHOP chemotherapy were studied for immunohistochemical TopoIIα expression and numerical gene abnormalities by fluorescent in situ hybridization (FISH). The results were analyzed in relation to the expression of cell cycle proteins (Ki67, p53, HDM2, p21, p14, pRb, p16, and cyclins A, B1, D1, D2, D3, and E) and BCL6/CD10/MUM1/CD138 B-cell differentiation immunophenotype and outcome. High levels of TopoIIα protein were found in 91% of DLBCL cases. No evidence of TopoIIα gene amplification or deletion was found. The TopoIIα expression showed significant positive correlations with the proliferation index Ki67 (p = 0.002), cell cycle proteins pRb and cyclin D2 (p = 0.018 and p = 0.028, respectively), and the germinal center proteins bcl6 and CD10 (p = 0.010 and p < 0.0001, respectively). TopoIIα expression was significantly higher in germinal center B-cell like (GCB) DLBCL than in non-germinal center B-cell like (non-GCB) DLBCL (p = 0.048). TopoIIα protein was significantly associated with response to chemotherapy (χ2, p = 0.024), but not with relapse-free or overall survival (p = 0.5). On multivariate analysis, only stage of disease retained independent prognostic significance (HR 0.33 for early stage, p = 0.008). Although TopoIIa gene copy number abnormalities were not found in DLBCL, high levels of protein expression are associated with GCB-cell differentiation immunophenotype, high proliferation, and response to treatment.

Immunohistological analysis of cell cycle and apoptosis regulators in thymus

The combined expression patterns of cell cycle and apoptosis regulators have not been analyzed in details in human thymus to the best of our knowledge. Our objective was to provide multiparametric and combined immunohistological information regarding the expression levels and the topographical distribution of major cell cycle and apoptosis regulators in postnatal human thymus. Ki67 and cyclins A, B1, D3 and E were frequently expressed by thymocytes with higher expression in cortical than medullary thymocytes. The expression of cyclin D2 was low in thymocytes. Thymic epithelial cells (TEC) exhibited low expression of Ki67 and cyclins. Bid was frequently expressed by thymocytes, Bcl-xL by cortical thymocytes and Bcl-2 by medullary thymocytes. The expression levels of Bim and survivin in thymocytes were low. The expression levels of Bax and Mcl-1 were higher in medullary than cortical thymocytes and TEC. Bak and Bad were mainly expressed in medullary TEC and Hassall Bodies (HB). c-FLIP and Fas were frequently expressed in TEC and FasL was mainly expressed by medullary TEC and HB. Cleaved caspase-3 was expressed by scattered thymocytes at the cortex and the corticomedullary junction and very rarely at the medulla. The different expression profiles and immunotopographical distribution of cell cycle and apoptosis regulators in thymocytes and TEC indicate that their expression is tightly regulated during thymic cell differentiation and that they are differentially involved in the cell survival/death regulation of thymocytes and TEC. Furthermore, this study indicates decrease of the proliferation and caspase-dependent apoptosis of thymocytes from the cortex to the medulla.

Rare variants in the spectrum of human herpesvirus 8/Epstein-Barr virus–copositive lymphoproliferations

We report 2 rare variants in the spectrum of human herpesvirus 8 (HHV8)/Epstein-Barr virus (EBV)-copositive lymphoproliferations arising in HIV-seronegative patients, including a large B-cell lymphoma arising in the setting of multicentric Castleman disease and a germinotropic lymphoproliferative disorder. In the first case, histology revealed features of multicentric Castleman disease and a proliferation of large lymphoid cells forming clusters or arcs or rings replacing the periphery of follicles or sheets of frank lymphoma outside the follicles. In the second case, a proliferation of large lymphoid cells totally or partially invaded follicle germinal centers. In both cases, the large cells were positive for EBV-encoded small RNA, HHV8 (LANA-1), MUM1/IRF4, and CD38 and negative for CD45, CD79a, CD10, BCL6, and CD138. In the large B-cell lymphoma, the large cells did not express detectable cytoplasmic immunoglobulin light- and heavy-chains, whereas in the germinotropic lymphoproliferative disorder, the large cells expressed μ heavy chain. The present cases broaden the spectrum of HHV-EBV--copositive lymphoproliferations.

Expression patterns of the activator protein-1 (AP-1) family members in lymphoid neoplasms.

Abstract The activator protein-1 (AP-1) is a dimeric transcription factor composed of proteins belonging to the Jun (c-Jun, JunB and JunD), Fos (c-Fos, FosB, Fra1 and Fra2) and activating transcription factor protein families. AP-1 is involved in various cellular events including differentiation, proliferation, survival and apoptosis. Deregulated expression of AP-1 transcription factors is implicated in the pathogenesis of various lymphomas such as classical Hodgkin lymphomas, anaplastic large cell lymphomas, diffuse large B cell lymphomas and adult T cell leukemia/lymphoma. The main purpose of this review is the analysis of the expression patterns of AP-1 transcription factors in order to gain insight into the histophysiology of lymphoid tissues and the pathology of lymphoid malignancies.

Bcl2-interacting killer CpG methylation in multiple myeloma: a potential predictor of relapsed/refractory disease with therapeutic implications

Abstract BIK (bcl2-interacting killer) is the founding member of the BH3-only bcl-2 family of pro-apoptotic proteins, which is suppressed in various cancers. In multiple myeloma (MM), BIK has been shown to be epigenetically silenced in vitro, but there is a lack of clinical data. We investigated the CpG methylation status of the BIK promoter in a well-characterized clinical series of patients with MM and investigated its clinical relevance. Forty patients with MM (21 male, 19 female; mean age 66) were studied. According to the International Staging System (ISS) they were classified as 16 patients with stage I, 12 patients with stage II and 12 patients with stage III disease. Methylation in the BIK CpG island was assessed by methylation-specific polymerase chain reaction (MSP) assay. Logistic regression analysis was used to investigate associations between gene methylation and age, ISS stage, performance status, extramedullary disease, bone disease, anemia (hemoglobin ≤10 mg/dL), serum albumin, β2-microglobulin level and relapsed/refractory disease. Methylation in the BIK CpG island was detected in 16 patients (40%), with a trend favoring male gender (odds ratio [OR] = 3.08, p = 0.09) and development of bone disease and extramedullary disease (OR = 1.6, p = 0.35 and OR = 3, p = 0.14, respectively). Patients with MM with methylated BIK CpG island had a statistically significant risk for disease evolution to relapsed/refractory disease (OR = 5.4, p = 0.03). This study provides clinical evidence that methylation-induced transcriptional silencing of the BIK pro-apoptotic gene may occur in MM, which might serve as a predictor of the development of relapsed/refractory MM. These findings warrant validation in larger cohorts of patients and suggest therapeutic utility for agents that enhance BIK expression.

The expression levels of JunB, JunD and p-c-Jun are positively correlated with tumor cell proliferation in diffuse large B-cell lymphomas.

We analyzed the expression of Jun family in relation to CD30 expression, cell proliferation and B-cell differentiation immunophenotypes [Germinal Center and non-Germinal Center] in diffuse large B-cell lymphomas (DLBCL). Expression and high expression of phosphorylated-c-Jun (p-c-Jun), JunB, JunD and CD30 (cut-off scores 20% and 50%, respectively) was found in 18/103, 49/103, 72/101 and 26/102 cases, respectively, and in 6/103, 27/103, 60/101 and 21/102 cases, respectively. The following significant positive correlations were observed: (a) JunB with cyclin A (p = 0.046), cyclin B1 (p = 0.033), cyclin E (p = 0.003), MUM-1 (p = 0.002) and CD30 (p < 0.001), (b) JunD with Ki67 (p = 0.002) and cyclin E (p = 0.014), (c) p-c-Jun with CD30 (p = 0.015), and (d) high p-c-Jun with cyclin A (p = 0.034). The positive correlation between expression of JunB, JunD and p-c-Jun and tumor cell proliferation in DLBCL, suggests that increased JunB, JunD and p-c-Jun expression may be involved in the pathogenesis of DLBCL by increasing tumor cell proliferation.

The collagen prolyl hydroxylases are novel transcriptionally silenced genes in lymphoma.

Background:Prolyl hydroxylation is a post-translational modification that affects the structure, stability and function of proteins including collagen by catalysing hydroxylation of proline to hydroxyproline through action of collagen prolyl hydroxylases3 (C-P3H) and 4 (C-P4H). Three C-P3Hs (nomenclature was amended according to approval by the HGNC symbols and names at http://www.genenames.org/ and Entrez database at http://www.ncbi.nlm.nih.gov/gene) leucineproline-enriched proteoglycan (leprecan) 1 (Lepre1), leprecan-like 1 (Leprel1), leprecan-like 2 (Leprel2) and two paralogs Cartilage-Related Protein (CRTAP) and leprecan-like 4 (Leprel4) are found in humans. The C-P4Hs are tetrameric proteins comprising a variable α subunit, encoded by the P4HA1, P4HA2 and P4HA3 genes and a constant β subunit encoded by P4HB.Methods:We used RT–PCR, qPCR, pyrosequencing, methylation-specific PCR, western blotting and immunohistochemistry to investigate expression and regulation of the C-P3H and C-P4H genes in B lymphomas and normal bone marrow.Results:C-P3H and C-P4H are downregulated in lymphoma. Down-regulation is associated with methylation in the CpG islands and is detected in almost all common types of B-cell lymphoma, but the CpG islands are unmethylated or methylated at lower levels in DNA isolated from normal bone marrow and lymphoblastoid cell lines. Methylation of multiple C-P3H and C-P4H genes is present in some lymphomas, particularly Burkitt’s lymphoma.Conclusions:Methylation of C-P3H and C-P4H is common in B lymphomas and may have utility in differentiating disease subtypes.