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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.

Supplementary Figure S1.

Cardiac lymphomas are extremely rare and have poor prognoses. Currently, there are no established guidelines for treatment and the main approaches include surgery, chemotherapy (CHT), possibly combined with radiotherapy (RT), and autologous stem cell transplantation (ASCT). RT’s role is controversial and is not considered a standard approach. We describe the case of a patient diagnosed with cardiac lymphoblastic B- cell lymphoma/ acute Lymphoblastic B-cell leukemia and successfully treated with a multimodality approach, including CHT, RT, and ASCT. In particular, the patient was referred to magnetic resonance-guided RT (MRgRT), currently the most advanced available technology in the RT field, which maximizes therapeutic efficacy by exploiting the best soft-tissue resolution of the magnetic resonance images and efficient gating protocols during treatment delivery.

PurposeOne of the main limiting factors of whole-brain radiation therapy (WBRT) for primary central nervous system lymphoma (PCNSL) is the impairment of neurocognitive functions (NCFs), which is mainly caused by radiation-induced injury to the hippocampus. With a view to preventing NCF impairment and personalizing treatment, we explored the feasibility of sparing the hippocampus during WBRT by correlating the sites of PCNSL lesions with the hippocampus. Methods and MaterialsPre-treatment MR images from patients who underwent WBRT between 2010 and January 2020 - and post-radiotherapy images in cases of relapse - were imported into the Varian Eclipse treatment-planning system and registered with the simulation CT. We constructed three 3-dimensional envelopes around the hippocampus at distances of 5, 10 and 15mm and also contoured primary lesions and recurrences.ResultsWe analyzed 43 patients with 66 primary lesions: 9/66 (13.6%) involved the hippocampus and 11/66 (16.7%) were located within 5mm of it. Thirtysix lesions (54.5%) were situated more than 15 mm from the hippocampus, while 10/66 (15.2%) were between 5 and 15 mm from it. The most common location was in deep brain structures (31%). Thirty-five of the 66 lesions relapsed: 14/35 (40%) in the same location and 21/35 (60%) in different sites. Globally, 16/35 recurrences (45.7%) were located in the hippocampus or within 5mm of it.ConclusionThese data show that routinely sparing the hippocampus is not feasible. This approach could be considered in selected patients, when the lesion is more than 15 mm from the hippocampus.

Background Overweight and obese patients with solid tumors receiving anti-programmed cell death-1 (PD-1)/PD-ligand-1(PD-L1) immune checkpoint inhibitors exhibit improved survival and higher risk of immune-related adverse events (irAEs) than those with a normal body mass index (BMI). In classic Hodgkin lymphoma (cHL), the impact of BMI on survival and immune-related toxicity is unknown. We evaluated for the first time associations of BMI with survival and irAEs in patients with relapsed/refractory (RR)-cHL undergoing PD-1 blockade. Methods Data from a multicenter study on 133 patients treated with the anti-PD1 antibody nivolumab (July 2015–December 2016) were retrieved from a prospective database. Progression-free (PFS), overall survival (OS), incidence and severity of irAEs according to BMI categories were estimated by Kaplan–Meier method, landmark-analyses and Cox regressions. Results Patients, mostly males (63%, n = 84) with a median age of 35 years (range, 15–82), advanced stage (75%), B symptoms (63%), bulky disease (24%), a median of 4 previous treatments (range, 1–9), received a median of 18 nivolumab doses (range, 1–57). No statistically significant differences across BMI subgroups emerged as to PFS, with 1-year rates of 67.1% for both normal weight (n = 66; 49.6%) and overweight (n = 31; 23.3%) patients. Underweight (n = 12; 9%) and obese (n = 24; 18%) patients had a 1-year PFS of 54.5% and 49%, respectively. In survival analyses, BMI either as a continuous (P = 0.5) or categorical (P for trend = 0.63) variable failed to associate with PFS. Response rates and time-to-response did not cluster in any BMI subset. No BMI-related differences in OS emerged across normal, overweight and obese patients but underweight patients had the worst survival. Occurrence of irAEs of whatever severity did not statistically associate with BMI. Conclusions In patients with RR-cHL receiving nivolumab, no statistically significant differences emerged in response rates, PFS and OS across BMI categories of normal weight, overweight and obese. Overweight/obese patients did not display an increased risk of irAEs. The exquisite sensitivity to anti-PD-1 antibodies, the unique cytokine milieu and effector pathways triggered by nivolumab in cHL, may represent biologic ‘equalizers’ counteracting the immunoregulatory effects of adiposity. Differently from solid tumors, BMI is not associated with treatment efficacy and immune-related toxicity and does not represent a predictive tool for PD-1-targeted immunotherapies in cHL.

We discuss the case of a 74-year old male patient with mantle cell lymphoma, who faced severe CMV infection after the fifth cycle of first-line chemo-immunotherapy with a dose-reduced bendamustine and rituximab regimen.

We analyzed the prognostic impact of levels of free light chains (FLC) in 106 patients with DLBCL, selecting 61 patients with a monoclonal (M) protein in serum, and 45 patients without a M protein as an IPI-matched control group. Patients with a M protein had higher levels of FLC, but these were not of prognostic significance in this group. The presence of a M protein nullified associations of κ-FLC with several laboratory parameters indicating immune system activation observed in patients without a M protein. Patients without M protein and κ-FLC50 mg/L had a significant inferior event-free survival (

Thrombocytopenia is a severe complication for patients with myelodysplastic syndrome (MDS). Eltrombopag increases platelet count in MDS patients but its combination with azacitidine elicited controversial results. We aimed to quantify the colony forming units of megakaryocytes (CFU-Mk) obtained from CD34+ bone marrow cells isolated from patients with MDS and from healthy donors that were cultured in vitro in the presence or absence of azacitidine and with or without the sequential addition of eltrombopag to the culture medium. CD34+ bone marrow cells from 6 MDS patients and 3 controls were expanded in vitro and cultured for 3 days with or without azacitidine. Subsequently, a CFU-Mk assay was performed in presence or absence of eltrombopag. The addition of eltrombopag in the CFU-Mk assay after mock treatment of CD34+ cells increased the number of CFU-Mk in both controls and patients. On the contrary, using azacitidine pretreated CD34+ cells, eltrombopag minimally increased CFU-Mk in controls and produced heterogeneous response in MDS patients with no change in two patients and CFU-Mk increase in four patients. In vitro CFU-Mk assay suggest that some MDS patients are likely to benefit from the sequential addition of eltrombopag after azacitidine treatment, in the context of a personalized medicine.

This study aimed to define the maximum tolerated dose (MTD) of temozolomide (TMZ) concurrent with radiotherapy (RT) after high-dose methotrexate (HD-MTX) for newly diagnosed primary central nervous system lymphoma (PCNSL). Adult patients with PCNSL were treated according to a response-adapted strategy. HD-MTX (3.5 g/m2) was followed by concomitant RT and escalating TMZ (50-60-75 mg/m2/day, 5 days/week). The total radiation dose was modulated according to the patient's response to HD-MTX. All patients received 30 Gy to the whole brain plus leptomeninges to C2, including the third posterior of the orbital cavity (clinical target volume 2; CTV2), plus 6, 10, or 16 Gy to the primary site, including the residual mass (CTV1), if a complete response (CR), partial response (PR)/stable disease (SD), or progressive disease (PD) was observed, respectively. Acute toxicities were graded according to the RTOG-EORTC criteria. Dose-limiting toxicity (DLT) was defined as grade 4 hematological toxicity or grade 3-4 hepatic toxicity, although 75 mg/m2/day was the maximum dose regardless of DLT. Neurocognitive function was evaluated using the Mini-Mental State Examination. Three patients were enrolled at each TMZ dose level (total = 9 patients). Twelve lesions were treated. Six patients received 2 cycles of HD-MTX, while 3 received only 1 cycle because of hepatic or renal toxicity. All patients completed chemoradiotherapy without interruptions. No DLT events were recorded. TMZ appears to be tolerable at a dose of 75 mg/m2/day when administered concomitantly with radiotherapy and after HD-MTX.

Lymphoma is listed among the neoplasias with a high risk of venous thromboembolism (VTE). Risk factors for VTE appear to differ from risk factors in solid tumors. We review the literature of the last 20 years for reports identifying these risk factors in cohorts consisting exclusively of lymphoma patients. We selected 25 publications. The most frequent studies were analyses of retrospective single-center cohorts. We also included two reports of pooled analyses of clinical trials, two meta-analyses, two analyses of patient registries, and three analyses of population-based databases. The VTE risk is the highest upfront during the first two months after lymphoma diagnosis and decreases over time. This upfront risk may be related to tumor burden and the start of chemotherapy as contributing factors. Factors consistently reported as VTE risk factors are aggressive histology, a performance status ECOG ≥ 2 leading to increased immobility, more extensive disease, and localization to particular sites, such as central nervous system (CNS) and mediastinal mass. Association between laboratory values that are part of risk assessment models in solid tumors and VTE risk in lymphomas are very inconsistent. Recently, VTE risk scores for lymphoma were developed that need further validation, before they can be used for risk stratification and primary prophylaxis. Knowledge of VTE risk factors in lymphomas may help in the evaluation of the individual risk-benefit ratio of prophylaxis and help to design prospective studies on primary prophylaxis in lymphoma.

In solid tumors and lymphomas, the neutrophil/lymphocyte (N/L) ratio at diagnosis has been shown to be a prognostic factor. The aim of our study was to validate the originally reported N/L ratio cut-point of 3.5 in patients with diffuse large B-cell lymphoma (DLBCL) registered in an Italian real-life database. The prognostic role of the N/L ratio at diagnosis on event-free survival (EFS) and overall survival (OS) was assessed in 505 patients with DLBCL. Patients with an N/L ratio3.5 (

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

Although sometimes presenting as an indolent lymphoma, mantle cell lymphoma (MCL) is an aggressive disease, hardly curable with standard chemo-immunotherapy. Current approaches have greatly improved patients' outcomes, nevertheless the disease is still characterized by high relapse rates. Before approval by EMA, Italian patients with relapsed/refractory MCL were granted ibrutinib early access through a Named Patient Program (NPP). An observational, retrospective, multicenter study was conducted. Seventy-seven heavily pretreated patients were enrolled. At the end of therapy there were 14 complete responses and 14 partial responses, leading to an overall response rate of 36.4%. At 40 months overall survival was 37.8% and progression free survival was 30%; disease free survival was 78.6% at 4 years: 11/14 patients are in continuous complete response with a median of 36 months of follow up. Hematological toxicities were manageable, and main extra-hematological toxicities were diarrhea (9.4%) and lung infections (9.0%). Overall, 4 (5.2%) atrial fibrillations and 3 (3.9%) hemorrhagic syndromes occurred. In conclusions, thrombocytopenia, diarrhea and lung infections are the relevant adverse events to be clinically focused on; regarding effectiveness, ibrutinib is confirmed to be a valid option for refractory/relapsed MCL also in a clinical setting mimicking the real world.

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 (

The transcription factor C/EBPα (CCAAT/enhancer binding protein α) is critical for granulopoiesis. Gene disruption in mice blocks early granulocyte differentiation and disruption of C/EBPα function has been implicated in human acute myeloid leukemia (AML), but no systematic structure-function analysis has been undertaken to identify the mechanisms involved in C/EBPα-mediated granulocyte differentiation. Here we demonstrate that loss of either of 2 key regions results in disruption of C/EBPα granulocytic development: the amino terminus and specific residues residing on the non-DNA binding face of the basic region. Mutation of either results in loss of C/EBPα inhibition of E2F and down-regulation of c-Myc, but only mutation of the basic region results in loss of physical interaction with E2F. In contrast, while the amino terminal mutant retains the ability to interact with E2F, this mutant fails to bind a C/EBPα site efficiently, fails to activate C/EBPα target genes, and is also defective in inhibition of E2F activity. These results further emphasize the importance of inhibition of proliferative pathways in granulopoiesis and demonstrate that several regions of the C/EBPα protein are involved in this mechanism.

Mutations of epigenetic regulators and of the spliceosome machinery in therapy-related myeloid neoplasms and in acute leukemias evolved from chronic myeloproliferative diseases

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.

DNA methylation is one of the major epigenetic changes in human cancers, leading to silencing of tumor suppressor genes, with a pathogenetic role in tumor development and progression in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Methylation of key promoter regions, induced by cytotoxic therapy together with complex genetic changes, is important in the biology of therapy-related myeloid neoplasms (t-MN). We were interested in the characterization of the methylation pattern of AML and MDS de novo and therapy-related. We studied 385 patients (179 females, 206 males), of a median age of 66 years (range 16-98 years). There were 105 MDS, 208 de novo AML and 72 t-MN (45 MDS and 27 AML). Using a methylation-specific PCR, we studied the promoter methylation status of E-cadherin (CDH1), TSP1 and DAP-Kinase 1. These genes have been shown to be involved in the malignant transformation, interfering with angiogenesis, interaction with micro-environment, apoptosis and xenobiotic detoxification. We found no associations between promoter hypermethylation and gender or age at the time of initial diagnosis. In patients with MDS, there were no associations between hypermethylation and clinical characteristics, including IPSS score, WHO classification and cytogenetics. DAPK1 was more frequently methylated in t-MDS/AML when compared to de novo MDS and AML (39% vs 15.3% and 24.4%, p=0.0001), while methylation of CDH1 was similar in t-MDS/AML and AML (51% and 53.4%), but less frequent in de novo MDS (29%) (p=0.003). In the t-MDS/AML group, we found that the methylation pattern appeared to be related to the primary tumor, with DAPK1 more frequently methylated in patients with a previous lymphoproliferative disease (75% vs 32%, p=0.006). On the other hand, methylation of CDH1 was associated to radiotherapy for the primary malignancy (84.5% vs 38%, p=0.003). TSP1 hypermethylation was rare and not characteristic of t-MDS/AML. In 177 patients studied for concurrent methylation of several promoters, t-MN and AML de novo were significantly more frequently hypermethylated in 2 or more promoter regions than de novo MDS (20% vs 12.4%, p

Purpose Cytokines play a pivotal role in Hodgkin's 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.

Intravascular large B-cell lymphoma (IVLBCL) is a rare disease recognized by the WHO classification of lymphoid neoplasms as a subtype of diffuse large B-cell lymphoma (DLBCL), which is characteriz...

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.Cell-free DNA levels in the plasma samples of 142 patients with lymphomas [45 with Hodgkin's lymphoma (HL), 63 with diffuse large B-cell non-Hodgkin's lymphoma (DLBCL), 24 with follicular, and 10 with mantle cell non-Hodgkin's lymphoma (NHL)] at diagnosis and of 41 healthy individuals were determined using a quantitative PCR for the beta-globin gene.Levels of circulating DNA in patients with HL, DLBCL, and mantle cell NHL were significantly higher than in controls (P0.01 for all). Increased levels of plasma DNA were associated with advanced stage disease, presence of B-symptoms, elevated lactate dehydrogenase levels, and age60 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).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.

Glutathione S-transferases (GSTs) are phase II detoxification enzymes involved in the metabolism of carcinogens and anticancer drugs, known also to interact with kinase complexes during oxidative or chemical stress-induced apoptosis. We were interested whether their polymorphic variants may account for differences in outcome of patients with acute myeloid leukemia (AML) following chemotherapy. We studied the prognostic role of polymorphisms in three GST genes (GSTP1/M1/T1) in a large patient cohort of the German Austrian Acute Myeloid Leukemia Study Group, treated according to prospective multicenter clinical trials (AML HD98A: 254 patients; AML HD98-B: 100 patients), with a median follow-up of 46 months. Looking at short-term adverse drug reactions, homozygous carriers of the GSTP1*105 Val allele had a faster neutrophil and platelet recovery (P=0.002 and 0.02, respectively) and a reduced need of red cell and platelet transfusions (P=0.01 and 0.03, respectively). Response to induction chemotherapy did not vary according to GST polymorphisms. Multivariable Cox regression models revealed a significant better relapse-free (RFS) and overall survival for the GSTP1(*)105 Val (P=0.003 and 0.03, respectively), whereas GSTT1 and GSTM1 genotypes had no significant impact. The favorable impact of GSTP1(*)105 Val on RFS seems to be restricted to the subgroup of patients exhibiting a normal karyotype.

We showed previously that CCAAT/enhancer binding protein α (C/EBPα), a tissue-specific transcription factor, is a candidate tumor suppressor in lung cancer. In the present study, we have performed a transcriptional profiling study of C/EBPα target genes using an inducible cell line system. This study led to the identification of hepatocyte nuclear factor 3β (HNF3β), a transcription factor known to play a role in airway differentiation, as a downstream target of C/EBPα. We found down-regulation of HNF3β expression in a large proportion of lung cancer cell lines examined and identified two novel mutants of HNF3β, as well as hypermethylation of the HNF3β promoter. We also developed a tetracycline-inducible cell line model to study the cellular consequences of HNF3β expression. Conditional expression of HNF3β led to significant growth reduction, proliferation arrest, apoptosis, and loss of clonogenic ability, suggesting additionally that HNF3β is a novel tumor suppressor in lung cancer. This is the first study to show genetic abnormalities of lung-specific differentiation pathways in the development of lung cancer.

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.

DNA methylation was first described almost a century ago; however, the rules governing its establishment and maintenance remain elusive. Here we present data demonstrating that active transcription regulates levels of genomic methylation. We identify a novel RNA arising from the CEBPA gene locus that is critical in regulating the local DNA methylation profile. This RNA binds to DNMT1 and prevents CEBPA gene locus methylation. Deep sequencing of transcripts associated with DNMT1 combined with genome-scale methylation and expression profiling extend the generality of this finding to numerous gene loci. Collectively, these results delineate the nature of DNMT1-RNA interactions and suggest strategies for gene-selective demethylation of therapeutic targets in human diseases.

Vitamin D deficiency has been reported to be a risk factor in elderly patients (pts) with diffuse large B cell lymphoma (DLBCL) treated with Rituximab-containing chemotherapy (R-CHOP) (Bittenbring et al, J Clin Oncol 32:3242, 2014). In vitro data suggest that vitamin D supplementation could enhance rituximab-mediated cytotoxicity. In a single-center study, we prospectively measured 25-OH Vitamin D levels at diagnosis in a cohort of 156 pts with aggressive B cell non-Hodgkin lymphoma (DLBCL NOS, 129 pts; primary mediastinal large B cell lymphoma, 9 pts; B cell lymphoma, unclassifiable with intermediate characteristics between DLBCL and Burkitt lymphoma, 8 pts; T-cell/histiocyte-rich large B cell lymphoma, 4 pts; other forms, 6 pts) who were candidates for Rituximab-containing chemotherapy (R-CHOP or equivalent). Pts with deficient/insufficient vitamin D levels were offered supplementation. We used the formula of Singh (JABFM 27:495, 2014) to calculate the need of Vitamin D supplementation. Vitamin D levels were controlled during supplementation. Event Free Survival (EFS) was defined as time from diagnosis to relapse, disease progression or change of therapy for any reason or death. Vitamin D levels were considered deficient (30 to 100 ng/ml) in 18 pts (12%). Looking at pts characteristics, there was no difference in vitamin D levels according to sex (p=0.5), age (p=0.8) or stage (p=0.5), while poor performance status (ECOG > 2) and high LDH levels were significantly associated with lower vitamin D levels (p=0.002 and p=0.0007). We observed a weak, but significant negative correlation between Vitamin D levels and Hb and albumin levels (p=0.003 and p=0.0001, respectively). In addition, there was a significant seasonal variation with lowest vitamin D levels in the second trimester (p=0.001). We implemented an oral substitution regimen with Vitamin D3 (cholecalciferole) to increase vitamin D levels early during treatment. Vitamin D (cholecalciferole 25000 U) was given once a week following a loading phase of daily doses of 25000 U for 1 week in patients with insufficient Vitamin D levels and for 2 weeks with deficient Vitamin D levels. Vitamin D substitution was stopped at end of treatment. This supplementation resulted in substitution of Vitamin D over the treatment period of 4951 U/d in patients with insufficient Vitamin D levels (median of calculated need in 86 pts: 4374 U/d) and of 5612 U/d for patients with deficient Vitamin D levels (median of calculated need 6379 U/d in 52 pts). A total of 116 patients received Vitamin D supplementation. A second determination of Vitamin D levels after a median of 1.7 month in 84 pts showed a significant increase of Vitamin D levels from a median of 17 ng/ml to 33 ng/ml (p=0.001). Supplementation resulted in normalization of Vitamin D levels in 46/84 pts (55%). No episodes of hypervitaminosis or hypercalcemia were observed. We analyzed the prognostic impact of vitamin D levels at diagnosis and after supplementation. Pts with vitamin D levels in the normal range either at diagnosis or due to supplementation (n=61) had a significant better EFS at 18 months when compared to pts with persistently deficient/insufficient vitamin D levels (n=44) (88% versus 77%, p=0.03). Vitamin D levels at diagnosis before supplementation only showed a trend for impact on EFS (p=0.09). We conclude that Vitamin D deficiency is frequent in pts with aggressive B-cell lymphomas also in central Italy. Vitamin D supplementation results in improved vitamin D levels. Our data suggest that outcome in pts with DLBCL treated with rituximab-containing chemotherapy may be improved by vitamin D supplementation. Disclosures No relevant conflicts of interest to declare.

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."

We sought to determine the mechanisms of downregulation of the airway transcription factor Foxa2 in lung cancer and the expression status of Foxa2 in non-small-cell lung cancer (NSCLC).A series of 25 lung cancer cell lines were evaluated for Foxa2 protein expression, FOXA2 mRNA levels, FOXA2 mutations, FOXA2 copy number changes and for evidence of FOXA2 promoter hypermethylation. In addition, 32 NSCLCs were sequenced for FOXA2 mutations and 173 primary NSCLC tumors evaluated for Foxa2 expression using an immunohistochemical assay.Out of the 25 cell lines, 13 (52%) had undetectable FOXA2 mRNA. The expression of FOXA2 mRNA and Foxa2 protein were congruent in 19/22 cells (p = 0.001). FOXA2 mutations were not identified in primary NSCLCs and were infrequent in cell lines. Focal or broad chromosomal deletions involving FOXA2 were not present. The promoter region of FOXA2 had evidence of hypermethylation, with an inverse correlation between FOXA2 mRNA expression and presence of CpG dinucleotide methylation (p0.0001). In primary NSCLC tumor specimens, there was a high frequency of either absence (42/173, 24.2%) or no/low expression (96/173, 55.4%) of Foxa2. In 130 patients with stage I NSCLC there was a trend towards decreased survival in tumors with no/low expression of Foxa2 (HR of 1.6, 95%CI 0.9-3.1; p = 0.122).Loss of expression of Foxa2 is frequent in lung cancer cell lines and NSCLCs. The main mechanism of downregulation of Foxa2 is epigenetic silencing through promoter hypermethylation. Further elucidation of the involvement of Foxa2 and other airway transcription factors in the pathogenesis of lung cancer may identify novel therapeutic targets.

Background Therapy-related myeloid neoplasms (t-MN), including myelodysplastic syndromes and acute myeloid leukemia (t-MDS and t-AML) are associated to clinical and biologic unfavorable prognostic features, including high levels of DNA methylation. Methods We retrospectively evaluated 50 t-MN patients (34 MDS and 16 AML) selected among all patients receiving azacitidine (AZA) at 10 Italian Hematology Centers. Patients had developed a t-MN at a median of 6.5 years (range 1.7- 29) after treatment of the primary tumor (hematological neoplasm, 27 patients; solid tumor, 23 patients). Results The overall response rate was 42% (complete remission: 10 patients, partial remission: 2 and hematological improvement: 8 patients) and was obtained after a median of 3 cycles (range 1–6). Median overall survival (OS) was 21 months (range 1–53.6+) from AZA start. OS was significantly better in patients with less than 20% blasts, in normal karyotype t-AML and when AZA was used as front-line treatment. This was confirmed by the multivariate analysis. Conclusions This study reports efficacy of AZA in the largest series of therapy-related MN patients treated with 5-AZA. Our data show that blasts and karyotype maintain their important prognostic role in t-MN also in the azacitidine era.

Primary pancreatic lymphoma (PPL) is an extremely rare disease which occurs in pancreas, accounts for less than 1% of extra-nodal malignant lymphomas and 0,5% of cases of pancreatic masses. We report the case of PPL in a 15 year-old boy suffering from Maturity onset Diabetes of the young type 3 (MODY3) diagnosed at the age of 1 year

Myelodysplastic syndromes (MDS) represent a group of diseases associated with bone marrow failure arising from the interaction between a clonal and deranged hematopoietic stem cell (HSC) and a deregulated bone marrow microenvironment. Several abnormalities have been described in the development of myelodysplastic stem cells which give origin to dysplastic hematopoiesis, including increased apoptosis, decreased survival, depletion of early hematopoietic cells and abnormal differentiation. Gene expression profiling (GEP) studies have allowed a clear discrimination between normal and myelodysplastic HSCs. Moreover, distinct gene expression signatures have been associated with disease stage (early versus advanced), prognosis, morphology and presence of recurrent chromosomal abnormalities, such as monosomy 7/deletion 7q, trisomy 8 and deletion 5q. Most interestingly, GEP has allowed the identification of crucial genes and biologic pathways deranged in MDS including among all interferon signaling, ribosomal protein biogenesis, immune response, Wnt/ β-catenin signalling, cell cycle control and DNA damage response. These pathways beside shedding light on the comprehension of MDS pathobiology, may represent the basis for further investigations and realization of a more targeted approach in the therapeutic management of these diseases. Strictly correlated to GEP, miRNA profiling identified distinct miRNAs deregulated in MDS, which can significantly influence expression of coding transcripts in myelodysplastic stem cells.

Abnormal gene promoter methylation contributes to deregulate gene expression of hematopoietic progenitors in myelodysplastic syndromes (MDS). We analyzed the gene expression profile of myelodysplastic and normal CD34+ hematopoietic stem cells (HSCs) treated in vitro with decitabine. We identified a list of candidate tumor suppressor genes, expressed at low levels in MDS HSCs and induced by hypomethylating treatment only in MDS, but not in normal HSCs. Real-time RT-PCR confirmed reduced CD9 expression in MDS CD34+ and bone marrow mononuclear cells, compared to normal controls. CD9 was specifically up-regulated by decitabine treatment in myelodysplastic CD34+ cells.

Therapy-related Myelodysplastic Syndromes/Acute Myeloid Leukemias (t-MDS/AML) are one of the most compelling long term adverse events occurring in cancer survivors treated with chemo-radiotherapy regimes. Beside several well-described genetic lesions, a growing amount of data suggests that abnormalities in DNA methylation profile contribute to multistep secondary leukemogenesis. Two distinct alterations of normal DNA methylation patterns may occur in cancer: a global hypomethylation resulting in chromosomal instability and loss of genetic integrity, and promoter specific DNA hypermethylation which leads to silencing of tumor suppressor genes. Cytotoxic drugs and radiation have been shown to affect tissue DNA methylation profile. Radiation is able to induce a stable DNA hypomethylation in both target and bystander tissues. Gene promoter methylation is a common finding in t-MDS/AML and has been associated to a shorter latency period from the treatment of the primary tumor. Among the studied genes, p15 methylation correlated to monosomy/deletion of chromosome 7q, suggesting that it could be a relevant event in alkylating agent-induced leukemogenesis. We found frequent methylation of DAPK in the t-MDS/AML group, especially in patients with a previous lymphoproliferative disease. In patients studied for concurrent methylation of several promoters, t-MDS/AML were significantly more frequently hypermethylated in 2 or more promoter regions than de novo MDS or AML suggesting that promoter hypermethylation of genes involved in cell cycle control, apoptosis and DNA repair pathways is a frequent finding in t-MDS/AML and may contribute to secondary leukemogenesis. However, how the epigenetic machinery is disrupted after chemo/radiotherapy and during secondary carcinogenesis is still unknown, warranting further studies.

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.

The authors describe a case of dissecting aneurysm of the abdominal aorta observed by magnetic resonance imaging (MRI) in a patient with diffuse aneu rysmal disease. A dissected atherosclerotic plaque confined to the infrarenal aorta occasioned an antegrade and an unusual retrograde dissection. MRI properly indicated this condition, showing the tear inside the aortic wall, ex cluding the thoracic entry of dissection, and assessing aortic and iliac artery aneurysmal involvement.

The genetic background may modify the individual's risk of developing cancer. We performed a case-control study to test the impact of genomic polymorphisms of 9 genes involved in xenobiotics detoxification and DNA repair in myelodysplastic syndromes (MDS). Frequencies of polymorphic variants of RAD51, XRCC3, NQO1, GSTA1, GSTM1, GSTT1, CYP3A4 and XPD enzymes were similar in patients and controls. On the other hand, the GSTP1-105Val allele was associated to an increased risk of MDS (O.R. 1.66; p = 0.04) and to higher probability of overall survival in the low/intermediate-1 IPSS risk group (p = 0.008). The GSTP1-Ile105Val polymorphism is likely to influence MDS risk and prognosis.