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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.

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.

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.

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.

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

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.

Chemotherapy including high-dose methotrexate (HD-MTX), with or without radiotherapy, is standard treatment for primary cerebral nervous system lymphoma (PCNSL). It remains controversial whether addition of other drugs will add to therapeutic efficacy. We report here on 41 patients with PCNSL treated using a combined treatment modality, including HD-MTX (3.5 g/m2 for 2 cycles) prior to whole brain radiotherapy (WBRT). In 22 patients, the chemotherapy was intensified by adding high-dose cytosine arabinoside (HD-AraC) (2g/m2 for 4 doses for 2 cycles). Complete remission was obtained in 23 of 34 assessable patients (67%), and overall and disease-free survival rates were 24% and 46%, respectively, without differences between treatment groups. The addition of HD-AraC was complicated by severe infections in 17/22 (77%) patients, resulting in 3 toxic deaths. Our study indicates that addition of HD-AraC may not improve clinical outcome in PCNSL, while it increases toxicity. More targeted and less toxic therapies are warranted.

Epigenetic mechanisms affecting chromatin structure contribute to regulate gene expression and assure the inheritance of information, which are essential for the proper expression of key regulatory genes in healthy cells, tissues and organs. In the medical field, an increasing body of evidence indicates that altered gene expression or de-regulated gene function lead to disease. Cancer cells also suffer a profound change in the genomic methylation patterns and chromatin status. Aberrant DNA methylation patterns, changes in chromatin structure and in gene expression are common in all kind of tumor types. However, studies on leukemias have provided paradigmatic examples for the functional implications of the epigenetic alterations in cancer development and progression as well as their relevance for therapeutical targeting.

Background: Polymorphisms in genes involved in detoxification and DNA-repair pathways may modify the individual's risk for genomic damage, and, as a consequence, the risk of developing malignant diseases. Patients and methods: We performed a case-control study including 160 cases of acute myeloid leukaemia (AML) and 162 matched controls to test the impact of six genomic polymorphisms on the risk to develop AML and/or therapy-related AML. Results: We found a significantly higher prevalence of the polymorphic variants RAD51-G135C and CYP3A4-A-290G genes in AML cases, when compared with controls (P = 0.02 and P = 0.04), increasing the risk of AML 2.1-folds (95% CI: 1.1–4.0) and 3.2-fold (95% CI: 1.1–11.5), respectively. Carriers of both the RAD51-G135C and CYP3A4-A-290G variants were at highest AML risk (P = 0.003; OR:13,6; 95% CI: 2.0–585.5), suggesting a synergistic effect between these polymorphisms. Conclusions: These results suggest that polymorphic variants in DNA-repair and detoxification enzymes may co-operate in modulating the individual's risk of AML.

2-Cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) induces differentiation and apoptosis of tumor cells in vitro and in vivo. Here we assessed the effects of CDDO on CCAAT enhancer–binding protein alpha (CEBPA), a transcription factor critical for granulocytic differentiation. In HL60 acute myeloid leukemia (AML) cells, CDDO (0.01 to 2 μM) induces apoptosis in a dose-dependent manner. Conversely, subapoptotic doses of CDDO promote phagocytic activity and granulocytic-monocytic differentiation of HL60 cells through increased de novo synthesis of p42 CEBPA protein. CEBPA translational up-regulation is required for CDDO-induced granulocytic differentiation and depends on the integrity of the CEBPA upstream open reading frame (uORF). Moreover, CDDO increases the ratio of transcriptionally active p42 and the inactive p30 CEBPA isoform, which, in turn, leads to transcriptional activation of CEBPA-regulated genes (eg, GSCFR) and is associated with dephosphorylation of eIF2α and phosphorylation of eIF4E. In concordance with these results, CDDO induces a CEBPA ratio change and differentiation of primary blasts from patients with acute myeloid leukemia (AML). Because AML is characterized by arrested differentiation, our data suggest the inclusion of CDDO in the therapy of AML characterized by dysfunctional CEBPA expression.

Background In Hodgkin's lymphoma (HL), the production of cytokines by Reed–Sternberg cells and the surrounding tissue is thought to contribute to the biology of the disease. Cytokine expression can be altered by common single nucleotide polymorphisms (SNPs) in the 5′-promoter regions. Patients and methods We studied polymorphic allele variants of the cytokine genes interleukin (IL)-10 (T-3575A, G-2849A, C-2763A, A-1082G and C-592A), IL-6 (G-174C) and tumor necrosis factor-α (C-863A and G-308A) in 184 patients with HL, and analyzed for associations with treatment outcome. Results Carriers of the IL-10-592AA and the IL-6-174GG genotypes had a significantly lower probability of freedom from treatment failure (FFTF) with adjusted hazard ratios (HRs) for failure of 2.92 [95% CI (confidence interval) 1.58–5.41, P = 0.001] and of 1.75 (95% CI 1.04–2.92, P = 0.03), respectively. Reconstructing haplotypes from the five SNPs in the IL-10 promoter revealed that homozygous carriers of the IL-10.4 haplotype (T-G-C-A-A) had a worse FFTF (HR, 2.35; 95% CI 1.2–4.6, P = 0.01). In the Cox multivariate analysis, the IL-10-592AA, the IL-6-174GG genotypes and stage were independent prognostic factors. Conclusions Our study indicates that cytokine genotypes predict clinical outcome in patients with HL and points to the importance of the genetic background of the host for treatment response.

BRCA1 plays a pivotal role in the repair of DNA damage, especially following chemotherapy and ionising radiation. We were interested in the regulation of BRCA1 expression in acute myeloid leukaemia (AML), in particular in therapy-related forms (t-AML). Using real-time PCR and Western blot, we found that BRCA1 mRNA was expressed at barely detectable levels by normal peripheral blood granulocytes, monocytes and lymphocytes, whereas control BM-mononuclear cells and selected CD34+ progenitor cells displayed significantly higher BRCA1 expression (P=0.0003). Acute myeloid leukaemia samples showed heterogeneous BRCA1 mRNA levels, which were lower than those of normal bone marrows (P=0.0001). We found a high frequency of hypermethylation of the BRCA1 promoter region in AML (51/133 samples, 38%), in particular in patients with karyotypic aberrations (P=0.026), and in t-AML, as compared to de novo AML (76 vs 31%, P=0.0002). Examining eight primary tumour samples from hypermethylated t-AML patients, BRCA1 was hypermethylated in three of four breast cancer samples, whereas it was unmethylated in the other four tumours. BRCA1 hypermethylation correlated to reduced BRCA1 mRNA (P=0.0004), and to increased DNA methyltransferase DNMT3A (P=0.003) expression. Our data show that reduced BRCA1 expression owing to promoter hypermethylation is frequent in t-AML and that this could contribute to secondary leukaemogenesis.

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 Hodgkin's lymphoma. Experimental Design: The Ile105Val polymorphism in the GSTP1 gene was analyzed using a PCR-RFLP technique. Ninety-seven patients with Hodgkin's 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 Hodgkin's 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 Hodgkin's lymphoma.

Our aim in this observational, prospective, noncontrolled study was to detect, in 29 patients who underwent abdominal aortic aneurysm (AAA) surgery, correlations between the incidence of postoperative organ failure and intraoperative changes in arterial and portal blood lactate; changes in intramucosal sigmoid pH (pHi); differences between sigmoid Pco(2) and arterial Pco(2) (DeltaCO(2)); and hemoglobin (Hb). Hb, arterial blood lactate concentrations, pHi, and DeltaCO(2) (air tonometry) were recorded at the start of anesthesia (T0), before aorta clamping (T1), 30 minutes after clamping (T2), and at the end of surgery (T3). Portal venous lactate concentrations were recorded at T1 and T2. Patients were stratified into two groups: group A patients had no postoperative organ failure, and group B patients had one or more organ failures. As compared with group A (n = 16), group B patients (n = 13) had a lower pHi value at T2 and T3 and a higher DeltaCO(2) at T3. A pHi value of7.15 was a predictor of organ failure, with a sensitivity of 92.3%, a specificity of 68.8%, and positive and negative predictive values of 70.6% and 91.7%, respectively, whereas a DeltaCO(2) value of28 mm Hg predicted later organ failure with a sensitivity of 92.3%, a specificity of 62.5%, and positive and negative predictive values of 66.6% and 90.9%, respectively. Portal venous lactate concentrations were larger in group B at T2 (P0.001), and an increaseor=5 g/dL predicted later postoperative organ failure with a sensitivity of 92.3%, a specificity of 100%, and positive and negative predictive values of 100% and 94.1%, respectively. The comparison of the receiving operator characteristic curves to test the discrimination of each variable and the logistic regression analysis revealed that the increase in portal lactate was the best predictor for the development of postoperative organ failure. Hb concentration was significantly smaller in group B at T0 (13.8 +/- 1.0 g/dL versus 12.2 +/- 2.2 g/dL) and T2 (10.9 +/- 1.2 g/dL versus 9.1 +/- 1.9 g/dL). In conclusion, both pHi and DeltaCO(2) are reasonably sensitive prognostic indices of organ failures after AAA surgery, but they are less specific and accurate than portal venous lactate.

Glutathione S-transferases (GSTs) are enzymes involved in the detoxification of several environmental mutagens, carcinogens, and anticancer drugs. GST polymorphisms resulting in decreased enzymatic activity have been associated with several types of solid tumors. We determined the prognostic significance of the deletion of 2 GST subfamilies genes, M1 and T1, in patients with acute myeloid leukemia (AML). Using polymerase chain reactions, we analyzed theGSTM1 and GSTT1 genotype in 106 patients with AML (median age, 60.5 years; range, 19-76 years). The relevance ofGSTM1 and GSTT1 homozygous deletions was studied with respect to patient characteristics, response to therapy, and survival. Homozygous deletions resulting in null genotypes at theGSTM1 and GSTT1 loci were detected in 45 (42%) and 30 (28%) patients, respectively. The double-null genotype was present in 19 patients (18%). GST deletions predicted poor response to chemotherapy (P = .04) and shorter survival (P = .04). The presence of at least one GST deletion proved to be an independent prognostic risk factor for response to induction treatment and overall survival in a multivariate analysis including age and karyotype (P = .02). GST genotyping was of particular prognostic value in the cytogenetically defined intermediate-risk group (P = .003). In conclusion, individuals with GSTM1 or GSTT1 deletions (or deletions of both) may have an enhanced resistance to chemotherapy and a shorter survival.

Prognostication in classical Hodgkin lymphoma (cHL) has mainly explored three areas of interest beyond the use of clinical risk factors in recent years: I. Early response evaluation with positron emission tomography scan (“interim PET”) after 2 cycles of treatment with doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) has become the most interesting functional imaging technique to accurately predict treatment response in cHL. II. An increased number of tumor-infiltrating macrophages stained with the CD68 antigen in the microenvironment of cHL has been shown to be a simple tissue biomarker with an adverse impact on outcome. III. Among circulating biomarkers, early changes in plasma levels of the chemokine TARC during therapy have been proposed as alternative biomarker for response evaluation. The aim of our study was to evaluate plasma levels of TARC at diagnosis and at interim PET in combination with the tissue biomarker CD68 and interim PET as predictors of outcome in cHL. We included 84 patients diagnosed with cHL (45 limited stage, 39 advanced stage) between 2007 and 2013 who were treated with ABVD in our department. Median age was 34 years (range 15-73), 43 patients were males and 41 females. PET-CT scan was performed at baseline and after two courses of chemotherapy. Interim PET results were assessed using the Deauville five-point scale. Treatment changed according to the PET result in 2 patients only. Plasma TARC levels were measured using ELISA (Human CCL-17/TARC DuoSet, R&D Systems Inc), and CD68+ cells were stained using the PGM1 antibody. When compared to normal individuals? (n=63), plasma TARC levels were elevated above the normal range (upper limit 162 U/ml) at diagnosis in 89% (73/82) patients with cHL. Significantly higher TARC levels were observed in patients with advanced stage disease (p=0.05) and patients with bulky disease (p5% vs 162 U/ml) in 18% of patients and were significantly associated with a positive PET result (p=0.007). Progression-free survival (PFS) at a median observation of 28 months was 76% (95% CI, 65-84%). Strong predictors for PFS were a negative interim PET (86% vs 25% , p5% remained independent predictive parameters for PFS (Hazard ratio 11.3, 95% C.I, 3.3-38.4, p In conclusion, baseline TARC levels are elevated in the vast majority of patients with classical HL in relation to tumor burden, and turn to normal in most patients after 2 courses of ABVD. Persistent elevated levels of TARC associate with a positive interim PET, but cannot substitute for it as outcome predictor. Interim PET and CD68+ cell counts in the microenvironment maintain their prognostic significance both in univariate and multivariate analysis. Disclosures No relevant conflicts of interest to declare.

Infection of EBV has been linked to the etiology of a number of lymphoproliferative diseases, and EBV+ diffuse large B cell lymphoma (DLBCL) of the elderly has been more recently described as a distinct entity. The viral load of EBV-DNA in plasma can be an indicator for EBV-association in some lymphoma types, as NK/T-cell lymphoma and Hodgkin lymphoma, and a marker for disease activity associated with negative prognosis. Here, we studied whether EBV-DNA in whole blood (WB), mononuclear cell fraction (MNC) and plasma of patients with DLBCL at diagnosis could be an indicator for EBV-association and a prognostic marker in patients treated with immunochemotherapy (R-CHOP). We analysed 247 patients with DLBCL (median age 64 years, range 15-92 years; 118 females, 129 males), diagnosed between 2006 and 2013. All patients were HIV-negative. EBV was detected using a commercial real-time PCR kit (BioQuant EBV, Biodiversity, Brescia, Italy) in peripheral blood (PB) compartments (WB n=240, plasma n=55, and MNC n=52). Lymph node samples from 61 DLBCL patients were analyzed for EBV infection through in situ hybridization for EBV-encoded small RNAs (EBER). EBV was detected in 58 of 240 WB samples (24%). The copy number varied between 2 x 102 and 4.9 x 106 copies/ml. The presence and copy number of EBV in WB and MNC were correlated (p60 years of age) had more often EBV in peripheral blood (30% vs 17%), and the viral load was higher in patients aged > 60 years (p 60 years (p=0.02), we performed a logistic regression analysis including age and HCV serology as parameters determining the EBV viral load in PB. In this analysis, HCV serology, but not age remained a significant factor for EBV viral load (p=0.004). Presence and viral load of EBV in PB was not related to other characteristics as gender, stage, performance status, LDH level, and IPI. In an univariate analysis including 201 patients treated with R-CHOP, the presence of EBV-DNA in WB was associated with a significantly shorter event-free survival (EFS): 67% (50%-79%, 95% C.I.) versus 80% at 2 years (p Our findings suggest that EBV can be frequently detected in peripheral blood at DLBCL diagnosis, that is not a surrogate marker for EBV-status in DLBCL, but associates with a worse outcome. Further studies are needed to explore the mechanisms of expansion of EBV-positive cells in patients with DLBCL. Disclosures: No relevant conflicts of interest to declare.

PURPOSE: 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). METHODS: 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. RESULTS: 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 (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4036. doi:1538-7445.AM2012-4036