Giuseppina Massini

Glutathione S-transferase P1 Genotype and Prognosis in Hodgkin's Lymphoma

Purpose: Glutathione S-transferase P1 (GSTP1) is a member of the GST enzyme superfamily that is important for the detoxification of several cytotoxic drugs and their by-products. A single nucleotide polymorphism results in the substitution of isoleucine (Ile) to valine (Val) at codon 105, causing a metabolically less active variant of the enzyme. We assessed the impact of the GSTP1 codon 105 genotype on treatment outcome in patients with Hodgkins lymphoma. Experimental Design: The Ile105Val polymorphism in the GSTP1 gene was analyzed using a PCR-RFLP technique. Ninety-seven patients with Hodgkins lymphoma were included and associations with patient characteristics and treatment outcome were analyzed. Results: The GSTP1 Ile105Val polymorphism was associated in a dose-dependent fashion with an improved failure-free survival in patients with Hodgkins lymphoma (P = 0.02). The probability of 5-year survival for patients homozygous for the 105Val/105Val GSTP1 genotype was 100%, for heterozygous patients 74% (95% confidence interval, 56-85), and for patients homozygous for the 105Ile/105Ile genotype 43% (95% confidence interval, 23-61). The Cox multivariate analysis showed that GSTP1 codon 105 genotype was an independent prognostic factor. Conclusions: The GSTP1 genotype predicts clinical outcome in patients with Hodgkins lymphoma.

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.

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.

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.”

Phosphorylated STAT5 Represents a New Possible Prognostic Marker in Hodgkin Lymphoma

An important pathogenetic mechanism in Hodgkin lymphoma (HL) is the interaction between the neoplastic and reactive cells mediated by a complex network of cytokines with activation of cytokine signal transduction (STAT) pathways. We studied the prognostic impact of the phosphorylation status of STAT5 in HL. By using immunohistochemical analysis, we found phosphorylated STAT5 (pSTAT5) in 35 (38%) of 93 lymph node biopsy specimens of patients with HL. The detection of pSTAT5 in Hodgkin and Reed-Sternberg (HRS) cells in classical HL (cHL) was not associated with any clinical and biologic features evaluated, including Epstein-Barr virus status. The primary end point for analysis of clinical outcome was freedom from treatment failure (FFTF). At a median follow-up of 5 years, pSTAT5+ patients with cHL had a better FFTF than pSTAT5-patients (77% vs 56%; P = .03), which translated into a reduced risk for failure for pSTAT5+ patients with a hazard ratio of 0.2 (95% confidence interval, 0.06-0.73; P = .015). Our data suggest that the phosphorylation status of STAT5 of HRS cells in cHL could be a prognostic marker in HL.

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.

Quantification of DAPK1 promoter methylation in bone marrow and peripheral blood as a follicular lymphoma biomarker

Hypermethylation of DAPK1 promoter gene was found to be a frequent epigenetic alteration in follicular lymphoma (FL). We evaluated whether the quantification of DAPK1 methylation in the bone marrow (BM) and peripheral blood of FL patients at diagnosis and during follow-up provides important prognostic information. DAPK1 methylation was quantitated by real-time MethyLight PCR in 107 patients at diagnosis, at end of therapy, and during follow-up. Information on BCL2-IGH rearrangement and clinical characteristics were available for all patients. Aberrant DAPK1 methylation was found in 22 of 26 (85%) lymph node biopsy samples, 62 of 107 (58%) BM specimens, and 25 of 63 (40%) peripheral blood samples at diagnosis. DAPK1 methylation was greater in patients with BM infiltration and a higher Follicular Lymphoma International Prognostic Index score. The presence of aberrant DAPK1 methylation in BM significantly reduced progression-free survival following immunochemotherapy, independent of Follicular Lymphoma International Prognostic Index score. Residual or increased methylation after treatment was associated with an increased risk for relapse. With watchful waiting, greater DAPK1 methylation at diagnosis was associated with a shorter time to antilymphoma treatment. Our study indicates that quantification of DAPK1 methylation represents a prognostically relevant FL biomarker, with promising implications for risk assessment.

Hypoxia-inducible factor-1α(Pro-582-Ser) polymorphism prevents iron deprivation in healthy blood donors.

BACKGROUND Frequent blood loss induces progressive depletion of iron stores, leading to iron deficiency and, ultimately, to overt iron-deficient anaemia. The erythropoietin-mediated bone marrow response to anaemia is under the control of hypoxia-inducible factors (HIF), the master regulators of oxygen and iron homeostasis. Since the HIF-1α(Pro-582-Ser) variant is associated with elevated trans-activation capacity of hypoxia responsive elements of target genes, we investigated whether the HIF-1α(Pro-582-Ser) polymorphism might influence the response to repeated blood withdrawals. MATERIALS AND METHODS Using polymerase chain reaction analysis and DNA sequencing, we retrospectively investigated the presence of HIF-1α(Pro-582-Ser) in a series of 163 blood donors. Haematological findings, serum ferritin levels and frequency of donations were compared according to the mutational status of the HIF-1α gene. RESULTS We found that male carriers of the HIF-1α(Pro-582-Ser) polymorphism had higher haemoglobin and ferritin levels than individuals homozygous for the wild-type allele. Moreover, the HIF-1α(Pro-582-Ser) polymorphism protected regular blood donors from developing iron deficiency and anaemia and predicted uninterrupted donation activity. DISCUSSION These findings show for the first time that the HIF-1α(Pro-582-Ser) polymorphism significantly affects red blood cell and iron homeostasis after blood loss, conferring to male carriers a resistance to anaemia. Regarding the female gender, large series of individuals should be investigated to establish whether there is an effect of the HIF-1α(Pro-582-Ser) polymorphism in this population. Although these data need to be confirmed in prospective studies, they could have important implications in blood donor selection and donation procedures.