Annarosa Cuccaro

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.

Circulating tumor DNA reveals genetics, clonal evolution and residual disease in classical Hodgkin lymphoma

The rarity of neoplastic cells in the biopsy imposes major technical hurdles that have so far limited genomic studies in classical Hodgkin lymphoma (cHL). By using a highly sensitive and robust deep next-generation sequencing approach for circulating tumor DNA (ctDNA), we aimed to identify the genetics of cHL in different clinical phases, as well as its modifications on treatment. The analysis was based on specimens collected from 80 newly diagnosed and 32 refractory patients with cHL, including longitudinal samples collected under ABVD (adriamycin, bleomycin, vinblastine, dacarbazine) chemotherapy and longitudinal samples from relapsing patients treated with chemotherapy and immunotherapy. ctDNA mirrored Hodgkin and Reed-Sternberg cell genetics, thus establishing ctDNA as an easily accessible source of tumor DNA for cHL genotyping. By identifying STAT6 as the most frequently mutated gene in ∼40% of cases, we refined the current knowledge of cHL genetics. Longitudinal ctDNA profiling identified treatment-dependent patterns of clonal evolution in patients relapsing after chemotherapy and patients maintained in partial remission under immunotherapy. By measuring ctDNA changes during therapy, we propose ctDNA as a radiation-free tool to track residual disease that may integrate positron emission tomography imaging for the early identification of chemorefractory patients with cHL. Collectively, our results provide the proof of concept that ctDNA may serve as a novel precision medicine biomarker in cHL.

CD68+ cell count, early evaluation with PET and plasma TARC levels predict response in Hodgkin lymphoma

Early response evaluation with [18F]fluordeoxyglucose (FDG) positron emission tomography after 2 cycles of chemotherapy (interim PET) has been indicated as the strongest predictor for outcome in classical Hodgkin lymphoma (HL). We studied the prognostic role of the number of tumor‐infiltrating CD68+ cells and of the plasma levels of TARC (thymus and activation‐regulated chemokine) in the context of interim PET in 102 patients with classical HL treated with Adriamycin, Bleomycin, Vinblastine, Dacarbazine (ABVD). After 2 ABVD cycles, interim PET according to Deauville criteria was negative (score 0–3) in 85 patients and positive (score 4–5) in 15 patients (2 patients technically not evaluable). TARC levels were elevated in 89% of patients at diagnosis, and decreased after 2 cycles in 82% of patients. Persistently elevated TARC levels in 18% of patients were significantly associated with a positive PET result (P = 0.007). Strong predictors for progression‐free survival (PFS) were a negative interim PET (85% vs. 28%, P < 0.0001) and CD68+ cell counts <5% (89% vs. 67%, P = 0.006), while TARC levels at diagnosis and at interim evaluation had no prognostic role. In multivariate analysis, interim PET, CD68+ cell counts and presence of B‐symptoms were independently associated with PFS. We conclude that although TARC levels are a biomarker for early response evaluation, they cannot substitute for interim PET as outcome predictor in HL. The evaluation of CD68 counts and B‐symptoms at diagnosis may help to identify low‐risk patients regardless positive interim PET.

PROGNOSTIC FACTORS IN HODGKIN LYMPHOMA

Hodgkin lymphoma (HL) is among the neoplastic diseases that has the best long-term outcome after cytotoxic treatment. Cure rates approach 80–90%; however, 15–20% of patients will be resistant to therapy (primary refractory) or relapse after treatment. Prognostic factors should help to stratify treatment according to the risk profile and identify patients at risk for failure. Significance of prognostic factors partly depends on the efficacy of the treatments administered, since new effective therapies can variably counterbalance the adverse effects of some unfavorable clinical determinants. As a consequence, some prognostic factors thought to be important in the past may become meaningless when modern successful therapies are used. Therefore, the value of prognostic factors has to be updated periodically, and then adapted to new emerging biomarkers. Besides the prognostic role of PET imaging, tissue and circulating biomarkers, as the number of tumor-infiltrating macrophages, cytokine and chemokine levels and profiling of circulating nucleic acids (DNA and microRNAs) have shown promise.

Invasive fungal infections in chronic lymphoproliferative disorders: a monocentric retrospective study

Invasive fungal infections (IFIs) are serious, life-threatening complications frequently observed in hematological malignancies, in particular in patients affected by acute myeloid leukemia (AML) or in allogeneic hematopoietic stem cell transplantation (HSCT) recipients.[1][1] Epidemiological data

Vitamin D deficiency and supplementation in patients with aggressive B-cell lymphomas treated with immunochemotherapy

Vitamin D deficiency has been reported to be a negative prognostic factor in elderly patients with aggressive B‐cell lymphomas. In vitro data suggest that vitamin D supplementation may enhance rituximab‐mediated cytotoxicity. We prospectively assessed 25‐hydroxyvitamin D [25(OH)D] levels at diagnosis in a cohort of 155 patients with aggressive B‐cell lymphomas of whom 128 had diffuse large B‐cell lymphoma (DLBCL) not otherwise specified. 25(OH)D levels were deficient (<20 ng/mL) in 105 (67%), insufficient (20–29 ng/mL) in 32 (21%), and normal (≥30 ng/mL) in 18 (12%) patients with a seasonal variation. Patient characteristics associated with lower 25(OH)D levels were poor performance status, overweight, B‐symptoms, elevated LDH, lower albumin and hemoglobin levels. As a result of a change in practice pattern, 116 patients received vitamin D3 (cholecalciferol) supplementation that included a loading phase with daily replacement and subsequent maintenance phase with a weekly dose of 25,000 IU until end of treatment. This resulted in a significant increase in 25(OH)D levels, with normalization in 56% of patients. We analyzed the impact of 25(OH)D levels on event‐free survival in patients treated with Rituximab‐CHOP. 25(OH)D levels below 20 ng/mL at diagnosis and IPI were independently associated with inferior EFS. Moreover, patients with normalized 25(OH)D levels following supplementation showed better EFS than patients with persistently deficient/insufficient 25(OH)D levels. Our study provides the first evidence that achievement of normal 25(OH)D levels after vitamin D3 supplementation is associated with improved outcome in patients with DLBCL and deficient/insufficient 25(OH)D levels when receiving rituximab‐based treatment.

FDG-PET/CT at the end of immuno-chemotherapy in follicular lymphoma: the prognostic role of the ratio between target lesion and liver SUVmax (rPET)

AimTo retrospectively investigate the prognostic role of the ratio between target lesion and liver SUVmax (rPET) in patients with follicular lymphoma (FL) submitted to FDG-PET/CT at the end of immuno-chemotherapy (PI-PET), and to compare rPET with International Harmonization Project criteria (IHP), Deauville Score (5p-DS) and FL International Prognostic Index at diagnosis (FLIPI).MethodsEighty-nine patients with FL undergoing PI-PET were evaluated. The receiver operating characteristic (ROC) approach was applied to identify the optimal cut-point of rPET with respect to 5-years progression free survival (PFS). The prognostic significance of rPET was compared with IHP, DS and FLIPI. Positive predictive value (PPV) and negative predictive value (NPV) were calculated using the presence of adverse events as gold standard.ResultsThe ROC analysis for rPET as predictor of progression showed an optimal rPET cut-point of 0.98. Patients with positive values of IHP, DS and rPET had a PFS of 50, 30 and 31%. PPV were of 56, 80 and 80%, NPV of 83, 86 and 88%, respectively. DS and rPET differed only in two patients. FLIPI was not predictive of progression and relapse.ConclusionsrPET is a prognostic factor in patients with FL submitted to PI-PET. Although it has a similar prognostic power as DS, it can have methodological advantages over visual analysis. PI-PET with different evaluation systems has a stronger prognostic power than FLIPI at diagnosis, so it could be useful to identify patients with FL at risk for early relapse after immuno-chemotherapy.

Interleukin-31 and thymic stromal lymphopoietin expression in plasma and lymph node from Hodgkin lymphoma patients

Hodgkin Lymphoma (HL) is a tumor of B-cell origin characterized by Hodgkin and Reed-Stenberg (H/RS) cells embedded in an inflammatory tissue where numerous cytokines/chemokines contribute to shape the microenvironment, leading to the typical clinical symptoms. We investigated: i) the expression of Interleukin-IL-31 (IL-31) and Thymic Stromal Lymphopoietin (TSLP), two Th2-related cytokines with tumor-promoting and pruritogenic functions, and of the respective receptors in HL invaded lymph nodes by flow cytometry, and ii) the potential association of IL-31/TSLP plasma concentrations with clinical characteristics by ELISA. H/RS cells and the major immune cell types infiltrating HL lymph nodes expressed intracytoplasmic and surface IL-31/TSLP, and their receptors. A subgroup of patients showing at diagnosis elevated IL-31 and TSLP plasma levels had an International Prognostic Score>2, indicative of high risk of relapse, and a subsequent positive interim PET-scan, indicative of insufficient response to chemotherapy. No correlation was found between IL-31/TSLP plasma levels and overall or event-free survival. In conclusion, IL-31/TSLP and their receptors are expressed in HL cells and in immune cells infiltrating affected lymph nodes, where both cytokines may contribute to local immune suppression. The clinical impact of IL-31 and TSLP plasma levels has to be further defined in larger patient cohorts.

Heavy chain disease: our experience

Heavy chain disease (HCD) is a rare lymphoproliferative disorder of plasma cells, described for the first time in 1964 by Franklin [1], characterized by production of incomplete monoclonal immunoglobulin heavy chains without associated light chains [2]. HCDs involve the three main immunoglobulin classes, the most common α-HCD, γand μ-HCD. Originally, Franklin’s disease (γ-HCD) was considered to be a lymphoproliferative disease, however, HCDs have variable clinical presentations and different histopathologic features. γ-HCD has been reported to occur equally in men and women, but in a recently reported series there was a clear female predominance [3]. The diagnosis of γ-HCD requires the evidence of a deleted immunoglobulin heavy chain without a bound light chain in the serum or urine [3]. These alterations typically result in loss of a large portion of the constant-1 (CH1) domain of the immunoglobulin heavy chain molecule responsible for light chain binding. In the absence of an associated light chain, the CH1 domain of the normal heavy chain binds to heat-shock protein 78 (hsp78) and undergoes degradation in the proteasome compartment of cells; normal heavy chains unassociated with light chains are therefore never detected in serum or urine. In the HCDs, the altered structure of the CH1 domain prevents the heavy chain from binding both the light chain and hsp78, thereby allowing it to bypass degradation by the proteasome and be secreted into the serum or urine [4]. In addition, the altered heavy chain may function as part of the transmembrane B-cell receptor and facilitate antigen-independent aggregation and downstream signaling by the receptor, thereby conferring a growth advantage to the heavy-chain expressing cells [5]. The clinical course of γ-HCD is extremely variable and ranges from an asymptomatic benign, or stable process to a rapidly progressive neoplasm leading to mortality within a few weeks. Prognosis is variable and the mean survival time has been reported to be 7.4 years (range, 1 month to >21 years) [3, 6]. We describe three cases of γ-HCD associated with chronic lymphocytic leukemia, peripheral T-cell lymphoma, not otherwise specified and small cell lung carcinoma, respectively. All three patients were male. The Ethics Committee of the “Policlinico Universitario A. Gemelli, Roma” approved this study. The first case is a 75-year-old man, who was admitted to the hospital for a 2-months history of progressive worsening of neurological symptoms, i.e. vision changes, dysphagia and amnesia. He had surgery for adenocarcinoma of the rectum 6 years before, with local recurrence after 5 years. In the last hospital stay before admission, he was treated with radiotherapy and chemotherapy with 5-fluorouracil. Total-body computed tomography scanning was negative for local, nodal or other organ involvement. Franklin’s disease was diagnosed 2 years before with no underlying lymphoproliferative or autoimmune disorder. Laboratory tests, including blood leukocyte count and CD4+ to CD8+ cells ratio, were within normal range, but a severe deficiency of serum IgA (0.39 g/L, 0.70–4.00) and IgM (<0.19 g/L, 0.40–2.30) concentrations and a mild IgG deficiency (6.45 g/L, 7.00–16.00) were found. Serum electrophoresis was negative but serum immunofixation electrophoresis (IFE) showed two γ-heavy chain monoclonal components (MCs) migrating in β, not associated with corresponding light chain, and one MC in the γ-region (small MC IgMλ); urine IFE interestingly showed a typical pattern with a broad band of incomplete γ-heavy chain (Figure 1) [7]. *Corresponding author: Umberto Basile, School of Medicine, Department of Laboratory Medicine, Catholic University of the Sacred Heart, Largo A. Gemelli 8, 00168 Rome, Italy, E-mail: umberto.basile@policlinicogemelli.it. http://orcid.org/0000-0002-8328-2570 Francesca Gulli, Cecilia Napodano and Krizia Pocino: Department of Laboratory Medicine, Catholic University of the Sacred Heart, Rome, Italy Annarosa Cuccaro and Stefan Hohaus: Institute of Hematology, Catholic University of the Sacred Heart, Rome, Italy