Manuela Giachelia

Anemia in Hodgkin's Lymphoma: The Role of Interleukin-6 and Hepcidin

PURPOSE Cytokines play a pivotal role in Hodgkins lymphoma (HL). Because interleukin-6 (IL-6) induces expression of hepcidin, one of the principal regulators of iron metabolism, we studied the contribution of hepcidin in anemia in HL at diagnosis. PATIENTS AND METHODS Plasma samples from 65 patients with HL were analyzed for hepcidin levels using a combination of weak cation exchange chromatography and time-of-flight mass spectrometry; cytokine levels were analyzed using enzyme-linked immunosorbent assays and parameters of iron metabolism and acute-phase reaction. RESULTS Hepcidin plasma levels were significantly higher in HL patients when compared with controls, independent of the presence of anemia (P = .001). In the subset of patients with anemia, hepcidin levels inversely correlated with hemoglobin levels (P = .01). Analyzing parameters of iron metabolism, hepcidin levels showed a positive correlation with ferritin (P < .001) and an inverse correlation to iron and iron-binding capacity. Hepcidin strongly correlated to IL-6 levels (P < .001) but not to IL-10 or thymus and activation-regulated cytokine (TARC)/chemokine (C-C motif) ligand 17 (CCL17) levels. In a multivariate regression analysis, IL-6 and fibrinogen levels were independently associated with hepcidin. Higher hepcidin levels were observed in patients with more aggressive disease characteristics: stage IV disease (P = .01), presence of B symptoms (P = .03), and International Prognostic Score > 2 (P = .005). CONCLUSION Our findings suggest that in HL, hepcidin is upregulated by IL-6. Elevated hepcidin levels result in iron restriction and signs of anemia of chronic inflammation, although hepcidin-independent mechanisms contribute to development of anemia in HL.

The viral load of Epstein-Barr virus (EBV)-DNA in Peripheral Blood Predicts for Biological and Clinical Characteristics in Hodgkin Lymphoma

Purpose: The Epstein–Barr virus (EBV) is present in the malignant Hodgkin/Reed–Sternberg (HRS) cells of 20% to 40% cases of Hodgkin lymphoma (HL) in Western countries. We were interested in the detection and quantification of cell-free plasma EBV-DNA as an indicator of biological and clinical characteristics in EBV-associated HL. Experimental Design: EBV was detected in peripheral blood compartments (whole blood, plasma, and mononuclear cells) at diagnosis by real-time PCR for the EBNA (EB nuclear antigen) region (n = 93) and in HRS cells by in situ hybridization for EBV-encoded small RNAs (EBER; n = 63). These data were correlated to histological and clinical characteristics, EBV serology, circulating cell-free DNA, and interleukin (IL)-6 levels. Results: Detection of EBV-DNA in plasma had a high specificity (90%), but a relatively low sensitivity (65%) to predict for EBV association. The viral load was higher in patients with advanced stage disease, older age in the presence of B-symptoms, and international prognostic score more than 2. The presence of EBV in HRS cells and higher plasma EBV-DNA copy numbers correlated to an increased frequency of tumor-infiltrating CD68+ macrophages in lymph node biopsies. Plasma EBV-DNA load correlated to circulating cell-free DNA and IL-6 levels, and inversely correlated to lymphocyte counts and EBNA1 antibody titers. Conclusion: Although the presence of EBV-DNA in peripheral blood cannot be regarded as a surrogate marker for EBER, the plasma EBV-DNA load at HL diagnosis is an indicator of disease activity and biological characteristics associated with negative prognosis. Moreover, the inverse correlation to EBNA1 antibody titers and lymphocyte counts may indicate a reduction in immunosurveillance, favoring the expansion of EBV-HRS cells in HL. Clin Cancer Res; 17(9); 2885–92. ©2011 AACR.

Cell-free circulating DNA in Hodgkin's and non-Hodgkin's lymphomas

BACKGROUND Levels of cell-free circulating DNA have been correlated to clinical characteristics and prognosis in patients with cancers of epithelial origin, while there are no data on patients with B-lymphoproliferative diseases. PATIENTS AND METHODS Cell-free DNA levels in the plasma samples of 142 patients with lymphomas [45 with Hodgkins lymphoma (HL), 63 with diffuse large B-cell non-Hodgkins lymphoma (DLBCL), 24 with follicular, and 10 with mantle cell non-Hodgkins lymphoma (NHL)] at diagnosis and of 41 healthy individuals were determined using a quantitative PCR for the beta-globin gene. RESULTS Levels of circulating DNA in patients with HL, DLBCL, and mantle cell NHL were significantly higher than in controls (P < 0.01 for all). Increased levels of plasma DNA were associated with advanced stage disease, presence of B-symptoms, elevated lactate dehydrogenase levels, and age >60 years (P = 0.009; <0.0001; <0.0001; 0.04, respectively). In HL, histological signs of necrosis and grade 2 type of nodular sclerosis were associated with increased plasma DNA. Elevated plasma DNA levels were associated with an inferior failure-free survival in patients with HL (P = 0.01) and DLBCL (P = 0.03). CONCLUSION Quantification of circulating DNA by real-time PCR at diagnosis can identify patients with elevated levels that are associated with disease characteristics indicating aggressive disease and poor prognosis.

Analysis of genome-wide methylation and gene expression induced by 5-aza-2'-deoxycytidine identifies BCL2L10 as a frequent methylation target in acute myeloid leukemia

Epigenetic changes play a role in the pathogenesis of myeloid malignancies, and hypomethylating agents have shown efficacy in these diseases. We studied the apoptotic effect, genome-wide methylation, and gene expression profiles in HL60 cells following 5-aza-2′-deoxycytidine (decitabine; DAC) treatment, using microarray technologies. Decitabine treatment resulted in a decrease in global DNA methylation, corresponding to 4876 probeset IDs with significantly reduced methylation levels, while the expression of 2583 IDs was modified. The integrated analysis identified 160 genes demethylated and up-regulated by decitabine, mainly including development and differentiation pathway genes. Gene targets of Polycomb group protein regulation were overrepresented in this group. Apoptosis was induced by decitabine, and apoptosis-specific PCR arrays more precisely indicated decitabine-induced up-regulation of 13 apoptosis-related genes, in particular DAP-kinase 1 and BCL2L10. Correspondingly, in primary patient samples, BCL2L10 was hypermethylated in 45% of AML, 43% of therapy-related myeloid neoplasms, 12% of MDS, and in none of the controls. In conclusion, decitabine induces global demethylation and gene expression, in particular of Polycomb target genes involved in development and differentiation pathways. The apoptotic gene BCL2L10 is a frequent target for aberrant promoter methylation in patients with acute leukemia, de novo and therapy-related.

Clinical significance of interleukin-10 gene polymorphisms and plasma levels in Hodgkin lymphoma

We studied plasma levels of IL-10 and five single nucleotide polymorphisms in the interleukin-10 (IL-10) gene promoter in patients with Hodgkin lymphoma (HL) to address potential genotype-phenotype correlations. Patients with elevated IL-10 levels were more likely to have advanced stage disease and inferior event-free survival. Homozygous carriers of the variant alleles at position -592 (AA) and -1082 (GG) of the IL-10 promoter had higher IL-10 plasma levels, independent of male gender and advanced stage of disease which also determined increased IL-10 production. This analysis indicates that the genetic background can modulate plasma levels of IL-10, and ultimately prognosis in HL.

Anemia in diffuse large B-cell non-Hodgkin lymphoma: the role of interleukin-6, hepcidin and erythropoietin

Abstract Anemia is a frequent sign in patients with diffuse large B-cell lymphoma (DLBCL) at diagnosis. We determined erythropoietin, hepcidin and interleukin-6 (IL-6) in plasma samples of 53 patients with DLBCL. The majority of patients (40/53, 75%) showed defective endogenous erythropoietin production, in particular when anemia was present (p = 0.01). Hepcidin plasma levels were significantly higher in patients compared to controls (p = 0.006), particularly in those with characteristics associated with a more active disease, including elevated lactate dehydrogenase (LDH) (p = 0.0004), B-symptoms (p = 0.07) and an age-adjusted international prognostic index (IPI) score > 1 (p = 0.01). Hepcidin levels correlated strongly to ferritin (r = 0.77, p < 0.0001) and weakly to IL-6 concentrations (r = 0.30, p = 0.03), but not to hemoglobin values. IL-6 inversely correlated to hemoglobin values in both univariate and multivariate analysis (p = 0.04), including hepcidin and erythropoietin as variables. Our findings suggest that elevated hepcidin levels and inadequate erythropoietin response are frequent in DLBCL, but elevated IL-6 plays the major role for the development of anemia.

Interleukin-6 plasma levels are modulated by a polymorphism in the NF-κB1 gene and are associated with outcome following rituximab-combined chemotherapy in diffuse large B-cell non-Hodgkin lymphoma.

Abstract Peripheral blood cytokines are known prognostic parameters in diffuse large B-cell lymphoma (DLBCL) treated with chemotherapy, but their role after the introduction of rituximab is unknown. Seven polymorphisms in the promoter regions of IL-6, IL-10 and NF-κB1 genes were assessed in 167 patients with DLBCL and 99 controls and correlated with interleukin-6 (IL-6) and IL-10 plasma levels. Outcome was analyzed in 137 patients treated with rituximab-based chemotherapy. The NF-κB1 − 94ATTG deletion was associated with increased IL-6 and IL-10 in DLBCL. High IL-6 concentration correlated with unfavorable prognostic factors included in the international prognostic index (IPI) and predicted for inferior progression-free (p = 0.007) and overall survival (p = 0.02). IL-6 levels remained a significant outcome predictor also including IPI as a covariate (p = 0.006 for progression-free survival). Our data suggest that the NF-κB1 genetic background influences IL-6 production in DLBCL, and that high IL-6 concentration is an independent prognostic factor also in the “rituximab era.”

The host genetic background of DNA repair mechanisms is an independent predictor of survival in diffuse large B-cell lymphoma

Several drugs used for diffuse large B-cell lymphoma (DLBCL) treatment rely on DNA damage for tumor cell killing. We verified the prognostic impact of the host DNA repair genotype in 2 independent cohorts of DLBCL treated with R-CHOP21 (training cohort, 163 cases; validation cohort, 145 cases). Among 35 single nucleotide polymorphisms analyzed in the training series, MLH1 rs1799977 was the sole predicting overall survival. DLBCL carrying the MLH1 AG/GG genotype displayed an increased death risk (hazard ratio [HR] = 3.23; P < .001; q =0 .009) compared with patients carrying the AA genotype. Multivariate analysis adjusted for International Prognostic Index identified MLH1 AG/GG as an independent OS predictor (P < .001). The poor prognosis of MLH1 AG/GG was the result of an increased risk of failing both R-CHOP21 (HR = 2.02; P = .007) and platinum-based second-line (HR = 2.26; P = .044) treatment. Survival analysis in the validation series confirmed all outcomes predicted by MLH1 rs1799977. The effect on OS of MLH1, a component of the DNA mismatch repair system, is consistent with its role in regulating the genotoxic effects of doxorubicin and platinum compounds, which are a mainstay of DLBCL first- and second-line treatment.

Association of the OCTN1/1672T variant with increased risk for colorectal cancer in young individuals and ulcerative colitis patients.

Background: Ulcerative colitis (UC) is associated with colorectal cancer. Chronic inflammation may also play a role in the pathogenesis of sporadic colorectal cancer (SCC), particularly in younger patients (<55 years). We evaluated whether single nucleotide polymorphisms of the OCTN1 and OCTN2 genes are associated with UC, SCC, and with UC cases with cancer progression (UCCP). Methods: We evaluated the OCTN1 and OCTN2 polymorphisms in 200 patients with UC, 59 patients with UCCP, 200 patients with SCC, and 200 controls (HC). IL‐8 expression was also assessed by real‐time polymerase chain reaction (PCR). Additionally, we transfected human colon carcinoma Caco2 cells, homozygous for OCTN1/1672T variant, with the OCTN1/1672C allele and NF‐&kgr;B activity was evaluated by luciferase based reporter assay and IL‐8 mRNA expression by real‐time PCR. Results: OCTN2 polymorphisms did not present a significant association with any group of patients compared to normal controls. Conversely, homozygosity for the OCTN1/1672T variant was significantly associated with UC (P = 0.047 vs. HC), with UCCP (UCCP vs. HC, P < 0.001), and with SCC developing in early age (<55 years) (P = 0.021 vs. HC). Importantly, IL‐8 mRNA expression was higher in UC and UCCP patients homozygous for the OCTN1 1672T variant compared to the other genotypes. Moreover, in Caco2 cells transfection of the OCTN1/1672C variant reduced the activity of the proinflammatory factor NF‐&kgr;B. Conclusion: Our data demonstrate that OCTN1 could have a role in modulating the severity of chronic inflammation associated with SCC in early age and in UC patients, and that its polymorphisms may help to predict malignant progression of IBD. (Inflamm Bowel Dis 2012;)

Glutathione-S-transferase genotypes influence prognosis in follicular non-Hodgkin's Lymphoma

Polymorphisms in detoxification enzymes of the glutathione S-transferase (GST) family have been associated with risk and prognosis of several cancer types. We studied deletions of GSTM1 and GSTT1, and the GSTP1 Ile105Val polymorphism in 89 patients with follicular lymphoma (FL). Patients with a GSTM1 or GSTT1 deletion had a significantly worse event-free survival, when compared with patients with undeleted genotype (p = 0.03 and p = 0.03, respectively). Outcome was even worse in patients with a double negative genotype, in comparison with patients with only one GST deletion or normal genotype (p = 0.01). In the multivariate analysis, the GSTM1/GSTT1 genotype tended to have a prognostic significance independent from the Follicular Lymphoma International Prognostic Index (FLIPI) score. In particular, GSTM1/T1 deletions identified patients with negative prognosis in the low (<3) FLIPI score group (p = 0.01). Larger prospective studies including homogeneously treated patients will be needed to confirm these results.