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Background Caulobacter spp. are Gram-negative bacteria that have rarely been found to be pathogenic in humans. Case Description This report describes the first case, to our knowledge, of meningitis in an adult patient caused by Caulobacter spp. A 75-year-old man was operated for a glioblastoma with no evident signs of primary infection in the wound site. Eight days after surgery, the patient developed signs and symptoms of meningitis. Caulobacter was then isolated on 3 separate occasions in the patient's cerebrospinal fluid. Thereafter, specific antibiotic therapy began. After 2 weeks of therapy, the patient was discharged with complete resolution of any related symptoms. Conclusions Caulobacter spp. can cause adult meningitis even where there is no evidence of surgical site infection.

During June 9–September 30, 2015, five cases of louseborne relapsing fever were identified in Turin, Italy. All 5 cases were in young refugees from Somalia, 2 of whom had lived in Italy since 2011. Our report seems to confirm the possibility of local transmission of louse-borne relapsing fever.

A proper diagnostic strategy of invasive fungal infections (IFI) is a very important component in the management of infectious complications in hematological patients. A good diagnostic approach should be adapted to the patient in relation to the underlying disease, stage of disease, localization of infection and immune status. None of the diagnostic markers can be entirely adopted for medical decision making, and sometimes it’s useful to use the combination of several microbiological tests.The diagnosis of IFI must therefore have a multidisciplinary approach that includes clinical suspicion, microbiological results and radiological evidence.

Male gender was recently reported as an adverse prognostic factor in patients with diffuse large B-cell lymphoma (DLBCL) treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone). We conducted a retrospective study of adult patients with DLBCL initially treated with rituximab containing regimens between 2001 and 2007. Patients were identified from the clinical archives of 43 Italian and Brazilian institutions. The principal endpoint was overall survival (OS). One thousand seven hundred and ninety-three patients were fully eligible for the study. Thirty-eight percent, 27%, 22% and 12% of patients had an International Prognostic Index (IPI) score of 0-1, 2, 3 and 4-5, respectively; 53% were males. After a median follow-up of 36 months (1-106), the 5-year OS was 76% (95% confidence interval 74-78%). In univariate analysis, male gender was an adverse prognostic factor with a hazard ratio of 1.52. In multivariate analysis, when adjusted by IPI, again gender maintained its prognostic relevance, showing an independent additive effect. In conclusion, in patients with DLBCL treated with rituximab containing regimens, gender may increase the predictive power of the IPI. Based on these results, given possible differences in blood clearance of rituximab between males and females, the benefit of higher doses of rituximab in males should be explored.

In Italy, the incidence of invasive meningococcal disease (IMD) has remained stable since 2007 (around 0.3 cases/100,000 inhabitants). However, as reported for other European countries, an increase of serogroup Y Neisseria meningitidis has been observed. In this study we report IMD cases from 2007 to 2013 in Italy and investigate the clinical and epidemiological features of cases affected by serogroup Y. Molecular characteristics of serogroup Y strains are also described. During the study period, the proportion of IMD cases due to serogroup Y increased, ranging from 2% in 2007 to 17% in 2013 (odds ratio (OR): 8.8), whereby the five to 14 years age group was mostly affected (p lpxL1 gene; however, no associations among lpxL1 mutations, ST and age group were identified. Overall, these findings generate scientific evidence for the use of the quadrivalent meningococcal conjugate vaccine in the five to 14 years age group.

BACKGROUND AND OBJECTIVES: Cytokine-induced killer (CIK) cells have shown anti-leukemic activity and little graft-versus-host disease (GVHD) in several animal models. The safety of these cells in autologous settings has been shown. We performed a phase I study of allogeneic (donor's) CIK cells in patients relapsing after allogeneic haematopoietic stem cell transplantation (HSCT). DESIGN AND METHODS: Eleven patients with acute myelogenous leukemia (n=4), Hodgkin's disease (n=3), chronic myelomonocytic leukemia, (n=1), pre-B acute lymphoblastic leukemia (n=1) and myelodysplasia (n=2), all of whom had relapsed after sibling (n=6) or matched unrelated donor (n=5) HSCT, entered this study. RESULTS: Before CIK administration, six patients had received other salvage treatments including chemotherapy (n=5), radiotherapy (n=1) and unmanipulated donor lymphocytes (n=6) without any significant tumor response. The median number of CIK infusions was two (range 1-7) and the median number of total CIK cells was 12.4x106/kg (range 7.2-87.4). The infusions were well tolerated and no acute or late infusion-related reactions were recorded. Acute GVHD (grade I and II) was observed in four patients, 30 days after the last CIK infusion, and progressed into extensive chronic GVHD in two cases. Disease progression and death occurred in six patients. One patient had stable disease, one had hematologic improvement and three achieved complete responses. INTERPRETATION AND CONCLUSIONS: This study shows that the production of allogeneic CIK cells is feasible under clinical-grade conditions, well tolerated and may contribute to clinical responses.

Background Early diagnosis of invasive pulmonary aspergillosis (IPA) is important as prompt treatment with antifungal drugs may increase patient survival. Our study investigated the efficiency of routine testing of the Aspergillus galactomannan antigen (AGA) test in combination with chest CT scans for IPA diagnosis. Patients and Methods From February 2002 to June 2004, 74 hemato-oncologic patients undergoing allogeneic stem cell transplantation were prospectively studied with serum AGA twice weekly from admission until death or discharge and weekly afterward when possible. Chest CT scans were performed when fever of unknown origin had lasted beyond 3 days of antibacterial therapy. Results Seven patients were classified with possible IPA and two patients, proven IPA. Fourteen patients showed positive results for AGA (OD index ≥ 1.0 on two subsequent sera). The sensitivity and specificity of the test were 100% and 93%, respectively; the positive and negative predictive values were 64% and 100%, respectively. All patients with possible/proven IPA showed abnormal CT signs; in four cases, imaging signs followed AGA positivity (median 5 days), whereas in five cases they preceded serologic positivity (median, 8 days). In the nine patients with IPA, antifungal therapy was promptly instituted, including lipid formulations of amphotericin B (n = 5) or caspofungin (n = 4). In only two of the nine patients (22%) with IPA, the primary cause of death was fungal infection. Conclusions The combination of AGA detection and early chest CT scans might be considered useful tools to detect minimal changes of IPA. Based on these findings, aggressive antifungal therapy should be initiated.

Objective Identification of a clinical grade method for the ex vivo generation of donor-derived T cells cytotoxic against both myeloid and lymphoblastic cells still remains elusive. We investigated rapid generation and expansion of donor derived-allogeneic T-cell lines cytotoxic against patient leukemic cells. Materials and Methods Acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) blasts were cultured 5 days in Stem Span, granulocyte macrophage colony-stimulating factor, interleukin-4, and calcium ionophore. All B-precursor ALL (N22) and AML (N13), but not T-cell ALL (N3), differentiated into mature leukemia-derived antigen-presenting cells (LD-APC). All but one LD-APC generated cytotoxic T lymphocyte (CTL) from adult human leukocyte antigen (HLA)-identical (N8) or unrelated donors (N2). Results Upon in vitro culture, donor-derived CTL acquired a memory T phenotype, showing concomitant high CD45RA, CD45RO, CD62L expression. CD8 + cells, but not CD4 + cells, were granzyme, perforine, and interferon-γ−positive. Pooled CD4 + and CD8 + cells were cytotoxic against leukemic blasts (32%, 30:1 E:T ratio), but not against autologous or patient-derived phytohemagglutinin blasts. LD-APC from five ALL patients were used to generate CTL from cord blood. A mixed population of CD4 + and CD8 + cells was documented in 54% of wells. T cells acquired classical effector memory phenotype and showed a higher cytotoxicity against leukemia blasts (47%, 1:1 E:T ratio). Adult and cord blood CTL showed a skewing from a complete T-cell receptor repertoire to an oligo-clonal/clonal pattern. Conclusions Availability of these cells should allow clinical trials for salvage treatment of leukemia patients relapsing after allogeneic stem cell transplantation.

High-dose therapy is an effective standard treatment for multiple myeloma patients. Evidence that intermediate-dose therapy improves survival is limited. At diagnosis, about 70% of patients are older than 65. Intermediate-dose regimen is very well tolerated in older patients. In a multicenter study, 194 patients were randomized to receive at diagnosis either conventional chemotherapy (6 courses of oral melphalan and prednisone [MP]) or intermediate-dose therapy (2 courses of melphalan at 100 mg/m(2) [MEL100]) with stem cell support. Response rate was higher after MEL100. Near-complete remission (nCR) was 6% after MP and 25% after MEL100 (P = .0002). At 3 years, MEL100 increased event-free survival (EFS) from 16% to 37% and overall survival (OS) from 62% to 77% (P < .001). Similar results were observed in patients aged 65 to 70: nCR was 8% after MP and 25% after MEL100 (P = .05); at 3 years, MEL100 improved EFS from 18% to 31% (P = .01) and OS from 58% to 73% (P = .01). Patients aged 65 to 70 had a median OS of 37.2 months (MP) versus 58 months (MEL100). Intermediate-dose melphalan improves response rate, EFS, and OS in myeloma patients, specifically in those aged 65 to 70. It constitutes a more effective first-line regimen than standard treatment for elderly patients.

Occult Hepatitis B virus (o-HBV) infection has been reported in HB surface antigen (HBsAg)-negative liver donors whose risk of transmitting HBV justifies a specific prophylaxis in liver recipients. The clinical significance of o-HBV infection in HBsAg-negative recipients and their need for prophylaxis is unknown. Liver samples collected during surgery from 23 HBsAg-negative patients (9 liver donors and 14 recipients) and 20 HBsAg-positive recipients (controls) were studied by polymerase chain reaction with an independent set of primers mapping the core and surface HBV genes. Intrahepatic HBV DNA was detected as core and surface genes in all the HBsAg-positive recipients, in none of the HBsAg-negative donors and in 9/14 (64%) of the HBsAg-negative recipients (2 HCV negative, 7 HCV positive). The intrahepatic amount of HBV was significantly lower in HBsAg-negative than in HBsAg-positive livers (median values 1.36 Log10/μg DNA vs. 3.66 Logs, p

Nonmyeloablative hematopoietic stem cell transplantation (NST) has been explored in hematological malignancies and solid tumors in an attempt to minimize treatment-related toxicity. Whether this approach is associated with reduced risk of infectious complications is unclear. The aim of the current study was to evaluate the infectious complications in a series of 32 consecutive adult patients who received NSTat our institution. Peripheral blood stem cell grafts (n = 30) or marrow grafts (n = 2) were infused from human leukocyte antibody (HLA)-matched sibling (n = 30), partially matched related (n = 1), or unrelated (n = 1) donors. Neutropenia developed in two-thirds of patients and lasted 16 days. Acute graft-versus-host disease (GVHD) grade II to IV was observed in 25% of patients, whereas 35% of patients had signs of extensive chronic GVHD. Twenty-two patients (69%) had at least one significant infectious episode. Bacteremia occurred in 19% of patients (n = 5 gram-positive, n = 1 gram-negative microorganisms). Cytomegalovirus (CMV) infection was observed in 10 out of 28 (36%) evaluable patients; 4 of these had recurrent or persistent CMVantigenemia requiring a second-line treatment, but eventually the viremia cleared. No patients experienced CMV disease. Fungal infections were documented in five (16%) patients, comprising invasive fungal infections in two cases and mucosal fungal infections in three. Four patients died of transplant-related causes, and three of these died before day +100. Infection was considered the primary cause of death in one patient (pulmonary aspergillosis) and contributed to death in another two. The actuarial probability of nonrelapse mortality at 100 days was 10% (95% confidence interval, 3-26%). Our preliminary results suggest that NST is associated to a low incidence of bacteremia or fungal and viral infections. Whether these findings would translate into an improved overall survival needs to be confirmed in larger prospective studies.

Gemtuzumab ozogamicin (GO) is a humanized anti-CD33 antibody conjugated to the anticancer agent calicheamicin, approved for the treatment of CD33+-relapsed acute myeloid leukemia. We have investigated the effects of GO on 4 human myeloid leukemia lines of different French-American-British (FAB) types (KG-1, THP-1, HL-60, and NB-4), observing 3 different types of response. Exposure to GO (10-1000 ng/mL) induced G2 arrest (up to 80% of the cells) followed by apoptosis (45% of the cells) in HL-60 and NB-4 cells. By contrast, in THP-1 cells we observed a strong G2 arrest (up to 75% of the cells) with little apoptosis. Finally, the KG-1 line was completely resistant to the same concentrations of GO. These different responses did not correlate with the levels of expression of either CD33 or multiple-drug resistance proteins, although the higher cyclosporin A (CsA)–inhibitable efflux activity of KG-1 cells may play a role in the resistance of this line to the drug. We could show that Chk1 and Chk2 phosphorylation, but not p53 or p21 expression, correlated with G2 arrest, implicating the ataxia-telangiectasia mutated/ataxia-telangiectasia related (ATM/ATR)–Chk1/Chk2 pathway in the cell cycle response to GO. However, apoptosis was associated with caspase 3 activation. Freshly isolated acute myeloid leukemia (AML) cells showed patterns of response to GO in vitro similar to those observed with the cell lines, including phosphorylation of Chk2 and caspase 3 activation. Our results suggest that the different molecular pathways induced by the drug in vitro may reflect, at least in part, the variable response to GO obtained in vivo.

We recently described a two-step negative selection procedure whereby peripheral blood stem cells (PBSCs) were efficiently purged of contaminating neoplastic cells by a combination of monoclonal antibodies. Here, we report 60 newly diagnosed multiple myeloma (MM) patients treated with a double transplant programme and randomized to receive either unmanipulated or in vitro purged PBSCs. We demonstrated that this technique is feasible and safe without significant loss of either CD34+ or CD3+ cells. Haematological engraftment and immunological reconstitution were rapid without treatment-related mortality. Using polymerase chain reaction (PCR), we compared the level of minimal residual disease (MRD) in PBSC before and after in vitro purging and in vivo after transplant. A median of one tumour cell per 10(2) normal cells (range 10(1)-10(5)) was seen in the unmanipulated aphereses with a 3-4 log reduction after manipulation in vitro. However, despite this tumour debulking, all patients remained PCR positive in vivo. At 3 years, the estimated event-free survival was 40% in the control arm and 72% in the experimental arm (P = 0.05), whereas the estimated overall survival was 83% in both arms. This suggests that autologous transplantation using efficiently purged PBSCs can be performed safely, but confirms the need for innovative protocols for MRD eradication in vivo.

BACKGROUND: Primary refractory disease is a main challenge in the management of non-Hodgkin's Lymphoma (NHL). This survey was performed to define the rate of refractory disease to first-line therapy in B and T-cell NHL subtypes and the long-term survival of primary refractory compared to primary responsive patients. METHODS: Medical records were reviewed of 3,106 patients who had undergone primary treatment for NHL between 1982 and 2012, at the Hematology Centers of Torino and Bergamo, Italy. Primary treatment included CHOP or CHOP-like regimens (63.2%), intensive therapy with autograft (16.9%), or other therapies (19.9%). Among B-cell NHL, 1,356 (47.8%) received first-line chemotherapy with rituximab. Refractory disease was defined as stable/progressive disease, or transient response with disease progression within six months. RESULTS: Overall, 690 (22.2%) patients showed primary refractory disease, with a higher incidence amongst T-cell compared to B-cell NHL (41.9% vs. 20.5%, respectively, p

BACKGROUND: The soluble pattern-recognition receptor known as long pentraxin 3 (PTX3) has a nonredundant role in antifungal immunity. The contribution of single-nucleotide polymorphisms (SNPs) in PTX3 to the development of invasive aspergillosis is unknown. METHODS: We screened an initial cohort of 268 patients undergoing hematopoietic stem-cell transplantation (HSCT) and their donors for PTX3 SNPs modifying the risk of invasive aspergillosis. The analysis was also performed in a multicenter study involving 107 patients with invasive aspergillosis and 223 matched controls. The functional consequences of PTX3 SNPs were investigated in vitro and in lung specimens from transplant recipients. RESULTS: Receipt of a transplant from a donor with a homozygous haplotype (h2/h2) in PTX3 was associated with an increased risk of infection, in both the discovery study (cumulative incidence, 37% vs. 15%; adjusted hazard ratio, 3.08; P=0.003) and the confirmation study (adjusted odds ratio, 2.78; P=0.03), as well as with defective expression of PTX3. Functionally, PTX3 deficiency in h2/h2 neutrophils, presumably due to messenger RNA instability, led to impaired phagocytosis and clearance of the fungus. CONCLUSIONS: Genetic deficiency of PTX3 affects the antifungal capacity of neutrophils and may contribute to the risk of invasive aspergillosis in patients treated (Funded by the European Society of Clinical Microbiology and Infectious Diseases and others) .with HSCT., Supported by grants from the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) (to Dr. Carvalho); the German Ministry for Education and Science (03Z2JN21, to Dr. Kurzai); the European Commission (FP7-HEALTH-2009-260338, to Dr. Romani; FP7-HEALTH-2011-280873, to Dr. Mantovani), the European Research Council (ERC-2008-AdG-233417, to Dr. Mantovani; ERC-2011-AdG-293714, to Dr. Romani), Associazione Italiana per la Ricerca sul Cancro (99629, to Dr. Mantovani); and Fundacao para a Ciencia e Tecnologia, Portugal (SFRH/BPD/46292/2008, to Dr. Carvalho; SFRH/BD/65962/2009, to Dr. Cunha; and SFRH/BPD/70783/2010, to Dr. Almeida).

Introduction: Tenascins are a family of large glycoproteins present in extracellular matrix, overexpressed in tumors compared to healthy tissues. Tenatumomab (ST2146) is an anti tenascin-C monoclonal antibody that is currently under development for radio immunotherapy (RIT) in tenascin-C expressing cancer. T-cell non-Hodgkin lymphomas (T-NHL) are a heterogeneous and rare group of poor prognosis malignancies lacking of standard treatments. Aim of the study was to evaluate the expression of tenascin-C in a cohort of T-NHL. Material and Methods: Under an IRB-approved protocol, 100 patients with a diagnosis of T-NHL in charge at the Hematology Unit of "Papa Giovanni XXII" Hospital were included in the study. Paraffin-embedded tumor samples were investigated using standard immunohistochemistry (IHC) for tenascin-C expression using tenatumomab antibody. Slides were assessed by two independent investigators. Staining was scored using the four different levels "no staining", "weak", "moderate" and "strong" (0-3). A grading system was used to express the proportion of involved areas in each case, as follows: 0 (0% to 25%), 1 (26% to 50%), 2 (51% to 75%), 3 (76% to 100%). Pattern of expression (stromal, vascular and/or cytoplasmic) was as well recorded. Tenascin C gene expression data were extracted from publicly available datasets. Association among variables was assessed with Chi square or Fisher exact test. Results:Of the 100 patients evaluated, 75 cases were peripheral T-NHL (PTCL) and 25 cutaneous T-NHL (CTCL). Specific diagnosis was, anaplastic large cell lymphoma (ALCL) ALK negative (n=21) or positive (n=19), PTCL-not otherwise specified (NOS, n=20), mycosis fungoides (n=13), angioimmunoblastic T-cell lymphoma (AITL, n=9), CD30+ primary cutaneous T-NHL (n=6) and other subtypes (n=12). Tenatumomab revealed the expression of tenascin-C in all the patients, with a staining that was weak, moderate and strong in 21, 51 and 28 of the cases. There was no significant different distribution among histologies (P=0.334). A high (>50%, grade 2-3) proportion of involved areas in pathologic samples were shown in half of the patients and this proportion was higher in ALCL ALK- (81%), AITL (78%) and ALCL ALK+ (58%) while was lower for PTCL NOS (30%) and CTCL (24%) (P=0.0019). A stromal pattern of expression was present in all the cases, vasculature was stained in 47 patients while in 21 tenascin-C was as well cytoplasmic. Vascular pattern was revealed in 56% of PTCL and 20% of CTCL (P=0.0018). To further evaluate the presence of tenascin C in T-NHL, gene expression datasets from published reports were retrieved and expression values of tenascin-C gene were extracted. Significant overexpression was present in T-NHL compared to normal tissues in both of the two considered datasets (Piccaluga et al., 2007 and Iqbal et al., 2010). Conclusions: Tenatumomab revealedthat tenascin-C is uniformly present in T-NHL with a high proportion of cases showing a strong and diffuse expression. Thus, tenascin-C represent an attractive target for RIT in this category of poor prognosis rare diseases. Disclosures Petronzelli: Sigma Tau S.p.A: Employment.

Relationships between viruses and autoimmune diseases such as systemic lupus erythematosus (SLE) are still elusive. Recent reports demonstrated the association of some viral infections with peculiar clinical events in the general population, such as cytomegalovirus (CMV) with arterial damage and Parvovirus B19 (PV-B19) with hematologic abnormalities. We planned to look for this kind of viral imprinting in SLE, hypothesizing that traces of specific features of some viral infections might be found in some subsets of seropositive SLE patients. In 60 SLE patients recruited at our nephrologic center, serology for CMV, PV-B19, Epstein-Barr virus viral capsid antigen (EBV-VCA), Epstein-Barr nuclear antigen (EBNA) and Epstein-Barr virus early antigen (EBV-EA) was performed. χ2 and ANOVA were employed to compare the frequency and titers of antiviral antibodies in SLE patients with groups of transplant, hemodialysis and blood donor subjects. χ2, Fisher’s test, Bonferroni and Scheffe’s test were employed to compare the different biochemical/clinical features between seropositive and seronegative SLE patients. Univariate and multivariate analysis (logistic regression models) were employed to evaluate the odds ratio (OR) of different risk factors for vascular events (including Raynaud’s phenomenon, deep venous thrombosis) and hematologic abnormalities (including severe anemia, leukopenia and thrombocytopenia). Anti-CMV (82%), anti-PV-B19 (60%), anti-EBV-VCA (92%) and EBV-EA (45%) IgG antibodies were frequent in SLE, with higher prevalence in comparison with the blood donor group and higher titers in comparison with transplant and hemodialysis groups. CMV seropositivity was a highly significant risk factor for Raynaud’s phenomenon (OR +α in univariate and multivariate analysis = 13.51 using a correction of 0.5 in case of a zero event), but not for venous vascular events (OR = 1.31). An increased though not significant risk factor was found for antiphospholipid antibodies (OR = 2.71, p = 0.19), while the presence of nephrotic syndrome during the follow-up was a significant protective factor (OR = 0.15, p = 0.035). There was no significantly increased OR for PV-B19 seropositivity in cases with severe anemia (OR = 2.09, p = 0.29). No significant associations were found with the status of EBV reactivation. In conclusion, our results support the hypothesis that viral infection may imprint the course of SLE leading to specific clinical subsets (i.e. CMV and ‘vascular’ SLE, with more frequent Raynaud’s phenomenon and a less frequent typical histological renal picture responsible for nephrotic syndrome). Further prospective studies are justified to validate these correlations, mainly dealing with associations between acute viral infections and vascular events, thus eventually leading to a better understanding of mutual relationships between viruses and SLE.

OBJECTIVE: To retrospectively determine DNA levels in blood polymorphonuclear leukocytes (PMNLs) of 21 heart transplant patients who suffered from HCMV infection and who were monitored by the antigenemia assay (pp65 test) during follow-up, by use of a quantitative competitive polymerase chain reaction (PCR) assay for human cytomegalovirus (HCMV) DNA. METHODS: Quantitation of HCMV DNA by PCR was expressed as genome equivalents (GE) per 200 000 PMNLs. RESULTS: Ten patients experienced symptomatic HCMV infection (five primary infections and five reactivations) with mild symptoms and received ganciclovir treatment, whereas 11 asymptomatic HCMV infections were not treated. Therapy was discontinued when a 90% reduction of the pretreatment antigenic load was achieved in a symptomless patient. The mean HCMV DNA and antigenic loads were significantly higher in symptomatic than in asymptomatic patients: 4.6 x 105 plus minus 4.7 x 105 GE and 1.1 x 104 GE (p0.0001) and 390 plus minus 350 versus 25 plus minus 12 pp65-positive PMNLs (p0.0001), and in primary than in secondary infections (583 plus minus 403 pp65-positive PMNLs versus 85 plus minus 111, p=0.002 and 5.2 x 105 plus minus 5.2 x 105 GE instead of 1.5 x 105 plus minus 3.2 x 105 GE, p=0.02). A single course of 14--21 days of ganciclovir caused a marked decrease of HCMV DNA and antigenemia in eight of 10 patients in whom a 90% reduction of the antigenic load correlated with a 98% DNA reduction of the pretreatment levels. In two primary infections, a 90% antigenic reduction was achieved by 21 days of ganciclovir treatment, but those data only correlated with a DNA load reduction of 28% and 60% of the pretreatment levels. Fifteen and 12 days later, respectively, the two patients relapsed and underwent a second ganciclovir course, at the end of which a 90% reduction of the antigenic load correlated with a98% DNA drop. GCV was discontinued and the patients recovered completely. In those two patients we retrospectively found persistent high DNA levels before the second ganciclovir course, whereas the antigenic load slowly increased after an apparent reduction. CONCLUSIONS: Our data suggest that: (1) DNA levels have the same trend as the pp65 antigen test---they are significantly higher in symptomatic and in primary HCMV-infected patients than in asymptomatic patients and those with secondary infection; (2) a 90% antigenic load reduction from the pre-treatment level may be a less reliable predictor of the efficacy of anti-HCMV therapy than DNA load, at least in primary infection, in which a much higher viral load and much more severe disease are present; and (3) a DNA load reduction of98% of the pretreatment value is required for therapeutic success.

In order to develop a clinically feasible gene marking approach, we have used the recently described PINCO retroviral expression system, composed of the enhanced green fluorescence protein (EGFP) cDNA driven by Moloney MLV LTR and packaged in the Phoenix amphotropic cell line. Two T, five B, one erythromyeloid and three myeloid cell lines were successfully infected with % GFP+ cells ranging from 4% to 79%, showing a lineage-dependent difference in infection susceptibility, with the myeloid cells being the least efficiently infected. We also infected normal mononuclear peripheral cells cultured in PHA and rhIL-2 for 2 d, and obtained an average of 30% GFP+ cells, all present within the CD3+ population, with CD4+ and CD8+ cells being equally infected. Finally, the tonsillar purified B population showed lower levels of infectivity (6%) whereas high susceptibility was shown by normal human umbilical vein endothelial cells (57%). Highly purified CD34+ cells were also susceptible, varying from 6% to 10% GFP+ cells. Immature myeloid/erythroid progenitors have been infected which stably expressed the GFP protein during further differentiation in culture. The GFP+ T cells were FACS-sorted rapidly upon infection, subsequently cultured and the fluorescence intensity monitored. In all cases the difference in percentage of GFP+ cells did not correlate with the percentage of S/G2/M cycling cells as determined at the moment of infection or with the expression levels of Ram-1 amphotrophic receptor. The improved safety of this retroviral system, the rapidity of the technique, the high efficiency of infection with respect to normal T lymphocytes (in this last case higher than previously reported) and the lack of need for in vitro selection make this system favourable for clinical development.

Patients with acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS) during induction chemotherapy and those receiving allogeneic stem-cell transplantation (HSCT) are at high risk of inva...

The peripheral blood lymphocyte to monocyte ratio (LMR) at diagnosis can be clinically relevant in patients with diffuse large B-cell lymphoma (DLBCL). We reviewed the outcome of 1,057 DLBCL patients followed from 1984 to 2012 at four centers. LMR was analyzed as a clinical biomarker by receiver-operating characteristic (ROC) analysis and Harrell's C-statistics. Patients were characterized by a median age of 61 years, International Prognostic Index (IPI) score of >2 in 39%, and were treated with a rituximab-containing chemotherapy in 66%. LMR proved strongly predictive for survival in patients treated with rituximab-based programs, but not in those receiving chemotherapy alone. Additionally, an LMR value of ≤2.6 (as determined by ROC analysis) was associated with a worst performance status, a higher lactate dehydrogenase (LDH) level, an advanced clinical stage, and a higher IPI score (P = 0.000). In patients treated with rituximab-supplemented chemotherapy programs, an LMR value of

Purpose We compared the risk factors, the diagnostic tools and the outcome of filamentous fungal infections (FFIs) in hematological patients (HAEs) and non-hematological patients (non-HAEs). Methods Prospective surveillance (2009–2011) of proven and probable FFIs was implemented in 23 Italian hospitals. Results Out of 232 FFIs, 113 occurred in HAEs and 119 in non-HAEs. The most frequent infection was invasive aspergillosis (76.1 % for HAEs, 56.3 % for non-HAEs), and the localization was principally pulmonary (83.2 % for HAEs, 74.8 % for non-HAEs). Neutropenia was a risk factor for 89.4 % HAEs; the main underlying condition was corticosteroid treatment (52.9 %) for non-HAEs. The distribution of proven and probable FFIs was different in the two groups: proven FFIs occurred more frequently in non-HAEs, whereas probable FFIs were correlated with the HAEs. The sensitivity of the galactomannan assay was higher for HAEs than for non-HAEs (95.3 vs. 48.1 %). The overall mortality rate was 44.2 % among the HAEs and 35.3 % among the non-HAEs. The etiology influenced the patient outcomes: mucormycosis was associated with a high mortality rate (57.1 % for HAEs, 77.8 % for non-HAEs). Conclusions The epidemiological and clinical data for FFIs were not identical in the HAEs and non-HAEs. The differences should be considered to improve the management of FFIs according to the patients’ setting.

The presence of human cytomegalovirus DNA was investigated in 103 unfixed endomyocardial biopsies, performed during the first 4 months in 17 heart transplant recipients by polymerase chain reaction. Results were correlated with human cytomegalovirus systemic infection, as detected by the test for the viral lower matrix phosphoprotein pp65 (antigenemia) and by polymerase chain reaction for viral DNA in blood leukocytes (DNAemia). Three patients out of 17 did not develop cytomegalovirus infection and 14 did : 5 had symptomatic disease treated with ganciclovir and 9 developed asymptomatic infection and were not treated. Viral DNA was detected in 24 out of 103 biopsies (23%) from 13 patients : 5 with symptomatic infection during the acute phase of disease (mean levels of pp65 : 125±232 pp65 positive leukocytes/200,000 examined cells) and 8 patients with asymptomatic infection when the mean antigenemia was 5±15/200,000 (4 patients) or when only DNAemia was present in blood (4 patients). No histological evidence of myocarditis was shown in viral DNA-positive biopsies. No difference in acute rejection was found in viral DNA-positive and viral DNA-negative biopsy specimens in symptomatic and asymptomatic infected patients. Our experience suggests that during systemic symptomatic and asymptomatic cytomegalovirus infection, polymerase chain reaction can detect a relatively frequent myocardial involvement, but this involvement is not associated with myocarditis or with a higher incidence of acute rejection. The presence of viral DNA in myocardial biopsies can be a result of high viremia, but it can also be due to a low level of viral DNA in circulating infected leukocytes. Polymerase chain reaction is the most sensitive method for cytomegalovirus DNA detection in biopsies, but its results need to be evaluated together with morphology-preserving methods and systemic markers of infection in order to make a correct diagnosis.

Introduction: Despite the growing interest about the optimal treatment of older patients with cancer, only few data are available for indolent non-Hodgkin lymphomas (NHL). Aim of the study was to evaluate the impact of comorbidities on treatment choice and survival in a cohort of older indolent NHL patients. Material and Methods: In an IRB-approved protocol, we reviewed the records of 742 patients with age≥60 years diagnosed of indolent NHL between January 1990 to December 2012 at our Center. Patients not receiving a treatment (N=177) and those treated with local therapy for stage I disease (N=138) were excluded from the analysis. Clinical information was gathered from the electronic charts, comorbidity was assessed using the cumulative illness rating scale-geriatrics (CIRS-G). Association between categorical variables was assessed with chi-squared test or Fisher's exact test. Overall survival (OS) and progression free survival (PFS) were the end-point of the study, defined respectively as the time from diagnosis to death or last visit and time to death or relapse/progression, which one occurred first, or last visit. Differences among groups in terms of OS and PFS were verified with both log-rank test in univariate setting and Cox proportional hazard model in multivariable analysis. Results: A total of 427 patients with a median follow-up of 4.04 years (range 0.1-20) were evaluated in the study. Patients were diagnosed as follicular NHL (n=207), marginal zone NHL (MALT N=93, splenic N=32, nodal N=38) and lymphocytic NHL (N=57). Median age was 69.9 years (range 60-93.9) with 11% of the patients older than 80 years, M/F ratio was 0.81. At least one comorbidity were present in 363 patients (85%), CIRS total score 2 was recorded in 161 patients (38%). At least one score 3-4 comorbidity was present in 176 (41%) and the most frequent categories involved were vascular (N=191, 45%), metabolic/endocrine/breast (N=102, 24%) and heart (N=71, 17%). Patients were further categorized in fit (N=224, 52%) or unfit/frail (N=203, 48%) on the basis of age (>80 years) and CIRS parameters (>1 grade 3-4 or >4 grade 2 categories). Patients categorized according these comorbidity scores were balanced for main clinical factors, with the exception of age. First line treatment was CHOP/CHOP-like (N=179, 42%), CVP (N=112, 26%), chlorambucil (N=104, 24%) or other (N=32, 7%) with the addition of rituximab in 53% of the cases. Severity index>2 (P=0.0146), presence of >1 score 3-4 category (P=0.0167) and frail/unfit status (P2 (P=0.0310), presence of >1 score 3-4 category (P=0.0239) and frail/unfit status (P Conclusions: Age and comorbidity scores influenced treatment intensity as well as the decision to add rituximab, this was associated to a significant impact on overall outcome of indolent NHL. Figure 1. Figure 1. Disclosures Rambaldi: Celgene: Research Funding; Amgen: Honoraria; Novartis: Honoraria; Roche: Honoraria; Pierre Fabre: Honoraria.

BACKGROUND Follicular lymphoma (FL) is the most common indolent form of non-Hodgkin's lymphoma. However, FL is a heterogeneous disorder and in a proportion of patients, the disease is very resistant to standard frontline therapies. In the current analysis clinical features and outcome to primary treatment were evaluated in a large series of FL patients who were consecutively treated at the Hematology Centers of Bergamo and Torino, Italy between 1976 and 2012. The aim of the study was to define the rate of refractory disease and the long term survival of patients according to response to their primary treatment. METHODS Medical records of 597 FL patients were reviewed. In front line therapy, rituximab was employed in 330 patients (55%), front-line high dose therapy with autograft (HDS) was administered in 58 patients (9.7%). Primary refractory disease was defined as full refractoriness (stable or progressive disease) or progressive disease within six months after initial response. Univariate analysis was done for prognostic factors including gender, age at diagnosis (age≤60 and >60 years), histological grade, IPI score (low=0-2 versus high=3-5), bone marrow (BM) involvement, rituximab administration in 1st line treatment, lymphocyte to monocyte ratio at diagnosis (>2.6 vs ≤2.6), presence of primary refractory disease, and the administration of front-line HDS. Cox model was also used for multivariate analysis. RESULTS: A total of 375 patients (63%) were older than 60 years (range: 18-88) and 49% were males. There were 476 patients (79.7%) with stage III-IV, 286 patients (48%) with BM involvement, 185 (31%) had a high IPI score and 28 patients (5%) presented with high histological grade. Eighty-seven patients (13%) displayed primary refractory disease. At a median follow-up of 8 years, median overall survival (OS) was 25 years for all patients, 32.6 years for responsive patients compared to 5 years for primary refractory patients (p= CONCLUSION: FL patients who display responsive disease to their primary treatment have a very long life expectancy with median survival of 32.6 yrs. Similarly to the aggressive lymphoma subtypes, primary refractory disease is of major concern also for FL. Research studies should be focused on the early identification of primary refractory patients to promptly institute adapted therapy for this unfavorable subgroup, and possibly optimize treatment strategies for patients with high-risk FL. Table 1. Multivariate analysis for overall survival Parameter Hazard Ratio (95% Confidence interval) p-value Age (yrs): >60 vs. ≤ 60 1.54 (1.5-2.3) .03 Histologic grade: 1-2 vs 3 2.25 (0.5-9.1) .3 IPI *Score: low (0-2) vs high(3-5) 0.59 (0.4-0.9) .009 Primary Refractory: yes vs no 4.40 (3.0-6.5) < .0001 Rituximab 1st line: yes vs no 0.56 (0.4-0.8) .005 BM# involvement: yes vs no 1.44 (1.0-2.1) .06 *International prognostic index was used to have a uniform prognostic factors scoring system for patients treated over the three decades of the survey. # Bone marrow Figure 1. Overall Survival in 597 follicular lymphoma patients according to response to primary treatment Figure 1. Overall Survival in 597 follicular lymphoma patients according to response to primary treatment Disclosures No relevant conflicts of interest to declare.

Purpose High-dose chemotherapy with peripheral blood progenitor cell (PBPC) autograft is effective in high-risk lymphoma, particularly with the addition of rituximab; however, it is associated with risk of secondary malignancy. These issues have been addressed in a series of 1,347 patients with lymphoma treated with a high-dose sequential (HDS) program. Patients and Methods A total of 1,024 patients with B-cell lymphoma, 234 patients with Hodgkin's lymphoma, and 89 patients with T-cell lymphoma were treated with HDS between 1985 and 2005 at 11 Gruppo Italiano Terapie Innovative Linfomi centers. HDS was given as salvage treatment to 707 patients (52%); 655 patients (49%) received a modified HDS, with high-dose cytarabine and two consecutive PBPC harvests. Rituximab-supplemented HDS was given to 523 patients (39%). Results At a median follow-up of 7 years, the median overall survival (OS) was 16.2 years; in B-cell lymphoma the OS was significantly superior with rituximab HDS compared to HDS alone. The cumulative incidence at 5 and 10 years of secondary myelodysplasia/acute leukemia (sMDS/AL) were 3.09% and 4.52%, respectively, that of solid tumors were 2.54% and 6.79%, respectively. Factors associated with sMDS/AL were male sex and use of the second harvest PBPC for the graft; factors found to be associated with solid tumor were advanced age, post-HDS radiotherapy, and rituximab addition to HDS. Despite the increased risk of solid tumors, rituximab addition to HDS was still associated with survival advantages. Conclusion This analysis has relevant implications for the design and use of intensive chemoimmunotherapy with autograft. In addition, it offers useful insights toward the understanding and prevention of tumor development.

Invasive aspergillosis (IA) is increasingly recognized in the intensive care unit (ICU), and new risk factors associated with respiratory colonization or infection by Aspergillus spp. include steroid treatment and chronic lung obstructive disease [1, 2]. In a review of 289 autopsies in the ICU, IA was the leading cause of Goldman class I discrepancy (a missed major diagnosis with major impact on patient management and survival) [3]. The epidemiology of IA indicates an increasing number of infections in immunosuppressed patients/individuals undergoing transplantation of bone marrow, hematopoietic stem cells, or organ transplantations, and those receiving intensive chemotherapy or other immunosuppressive treatments. A broad group of patients who are admitted to ICU also have some form of immunosupppression and may be susceptible to invasive mould infections. For various reasons, figures about the true incidence of IA in ICU are difficult to generate. The most important reason is the difficulty encountered in making a definite diagnosis of IA (lack of sensitivity and specificity with regard to culture and radiology) [4]. Recently, galactomannan (GM) in bronchoalveolar lavage (BAL) fluid appears to be a promising tool for early diagnosis in non-neutropenic critically ill patients and has been associated in proven cases with sensitivity and specificity of 88 and 87%, respectively [5]. Pseudomembranous and obstructive Aspergillus tracheobronchitis are still considered to have a fatal outcome and have been reported in a wide variety of patients [6]. There has been only one report in a patient with diabetes which was treated by deoxycholate amphotericin B (AmB) and subsequent addition of oral itraconazole [7]. In this paper, we report a pseudomembranous and obstructive tracheobronchitis in a diabetic patient successfully treated with caspofungin and AmB.

Abstract 1220 Poster Board I-242 Background The outcome of B-cell diffuse large B-cell lymphoma (DLB-CL) patients has definitely improved since the introduction of therapeutic programs combining chemotherapy and the anti CD20 rituximab. However, the outcome of patients with adverse prognostic factors still needs a substantial improvement and intensive therapy with autologous stem cell transplantation (ASCT) may represent a feasible option. In a previous Phase II study the combination of rituximab and high-dose (HD) sequential chemotherapy, delivered with multiple autologous peripheral blood progenitor cell (PBPC) support (R-HDS regimen), proved effective and well tolerated in previously untreated patients with DLB-CL and age-adjusted International Prognostic Score (aaIPI) score 2-3 (Tarella et al.: Leukemia 2007). Based on this study, a multicenter Phase III study was planned to compare the R-HDS regimen to a standard dose dense, rituximab supplemented R-CHOP-14 chemotherapy program. Patients and Methods The multicenter phase III trial R-HDS 0305 (Clinical Trials.gov.number NCT00355199) was planned to include 240 patients with DLBCL without CNS with stage '2 II B, bulk, age 18-60 years, with ECOG-PS=0-3 and aaIPI 2-3 or age 61-65 years with ECOG-PS=0-2 and IPI 3-5. The control group received 8 courses of R-CHOP-14, supported by GCSF ± involved field radiotherapy (IFRT), if they achieved at least a PR after 4 cycles. Cases refractory to R-CHOP-14 were rescued with R-HDS. Experimental arm (RHDS) included: 3 courses of doxorubicin-containing chemotherapy (APO), followed by sequential administration of cyclophosphamide (CTX) 7 g/sqm, Cytarabine (Ara-C) 2g/sqm every 12 hours for 6 days, etoposide 2 g/sqm plus cisplatin 100 mg/sqm; the program was completed by mitoxantrone plus melphalan (60 and 180 mg/sqm) or BEAM conditioning regimen with ASCT± IFRT. Rituximab (375 mg/sqm) was given for a total of 6 doses, twice after CTX and Ara-C, as in vivo purging before CD 34+ cells harvest, and twice after ASCT. The primary outcomes of the study were CR, DFS, OS, EFS and toxicity. From June 2005 to February 2009, 165 patients were enrolled in the study (R-CHOP-14=83; RHDS=82). The planned interim analysis has been carried out on 119 patients who have been randomized up to March 2008. Results The median age was 49 years (range, 18-65), 11 patients (9.2%) were> 60 years and the M/F was 1.38. Patients in two arms presented with comparable adverse features such as advanced stage (87%), BM infiltration (24%), bulky disease (67%), elevated LDH (81%) and beta 2-microglobulin (mean 3.9 mg/dl, SD = 4.5), poor ECOG-PS (60%), B-symptoms (62%), and ≥1 extranodal site (63%). Until now 10 patients (8%) did not complete the planned program because of toxicity or PD, 6 and 4 cases, respectively. One patient resistant to R-CHOP-14 was shifted to R-HDS. Overall, 24 patients (20%) deceased during the course of the study, 20 (17%) died from lymphoma, 2 (2%) due to therapy complications and 2 (2%) from hepatitis B reactivation. Moreover, 6 patients (5%) recovered from severe adverse events: 1 interstitial pneumonia, 1 graft failure, 1 sepsis, 1 colon diverticulitis, 1 shock, 1 prolonged aplasia. The main G>2 hematological toxicity were: anemia, granulocytopenia and thrombocytopenia, in 42%, 60 % and 45 % of patients, respectively. Grade >2 gastrointestinal, hepatic and infectious toxicity were recorded in 32 %, 11 % and 20% of patients, respectively. One-hundred and eight of 119 patients (91%) were alive and assessable for the response rate and clinical outcome at final restaging. Eighty-nine patients (82%) achieved CR, 8 PR and 7 had SD or PD. With a median follow up of 18.1 months (SD= 9.3, range 0.3–40.6 months) the 2year OS, DFS and EFS of the 119 evaluable patients are 80%, 84% and 74%, respectively. Conclusion This interim analysis shows that in high-risk DLBCL patients aged =/ Disclosures No relevant conflicts of interest to declare.

We describe the case of a 19-year-old boy with acute leukaemia who developed primary hepatic zygomycosis. The patient presented with febrile neutropenia and severe abdominal tenderness. Despite the administration of antibiotics and liposomal Amphotericin-B (L-AmB), the CT scan demonstrated an increase in the size of liver lesions. A wide surgical resection was carried out and liver specimens demonstrated a branching, filamentous fungus that was identified as Rhizomucor pusillus by both phenotypic and molecular methods. The patient was treated with L-AmB combined with posaconazole, and deferasirox was subsequently added given the potential synergistic effect of this iron chelator in combination with L-AmB. Three months after surgical intervention, an allogeneic stem-cell transplantation was successfully carried out. The present case confirms that an early surgical management combined with antifungal agents is crucial to optimise the outcome of patients with zygomycosis and the use of deferasirox is a promising alternative.

We have investigated the activity of ITF2357, a novel hydroxamate histone deacetylase inhibitor, on multiple myeloma (MM) and acute myelogenous leukemia (AML) cells in vitro and in vivo. ITF2357 induced apoptosis in 8/9 MM and 6/7 AML cell lines, as well as 4/4 MM and 18/20 AML freshly isolated cases, with a mean IC(50) of 0.2 microM. ITF2357 activated the intrinsic apoptotic pathway, upregulated p21 and downmodulated Bcl-2 and Mcl-1. The drug induced hyperacetylation of histone H3, H4 and tubulin. When studied in more physiological conditions, ITF2357 was still strongly cytotoxic for the interleukin-6 (IL-6)-dependent MM cell line CMA-03, or for AML samples maximally stimulated by co-culture on mesenchymal stromal cells (MSCs), but not for the MSCs themselves. Interestingly, ITF2357 inhibited the production of IL-6, vascular endothelial growth factor (VEGF) and interferon-gamma by MSCs by 80-95%. Finally, the drug significantly prolonged survival of severe combined immunodeficient mice inoculated with the AML-PS in vivo passaged cell line already at the 10 mg/kg oral dose. These data demonstrate that ITF2357 has potent anti-neoplastic activity in vitro and in vivo through direct induction of leukemic cell apoptosis. Furthermore, the drug inhibits production of growth and angiogenic factors by bone marrow stromal cells, in particular IL-6 and VEGF.

Clinical trials have shown that high dose chemotherapy (HDT) with peripheral stem cell autotransplantation is presently the best treatment for patients with symptomatic multiple myeloma (MM). In the context of an outcomes research project, we analyzed the feasibility of this strategy in clinical practice in a large cohort of consecutive, unselected patients with newly diagnosed MM and looked at the major determinants of response of patients enrolled in a HDT with tandem autotransplantation (Total Therapy I, TTI) program.Two hundred and fourteen patients were treated outside of a clinical trial and regularly followed-up at our Center for symptomatic MM. Ninety-seven patients (45%) received conventional chemo-radiotherapy regimens, 110 (51%) entered the TTI program and the remaining 7 patients (3.3%) were enrolled in other programs involving HDT with autotransplantation.Patients enrolled in HDT with tandem autotransplantation programs were 14 years younger and less likely to have co-morbidities than patients treated with conventional therapy. Median overall survivals of the two groups were 60 and 33 months, respectively. Thirteen percent of the patients enrolled in the TTI program did not receive the first HDT with autotransplantation, mostly because of disease progression, and another 16% did not proceed to the second HDT with autotransplantation mainly because of infections or drug-related complications. Most patients achieved complete remission after the second autotransplantation, with acceptable toxicity. However, only patients with a major reduction of the myeloma burden at the end of induction therapy enjoyed significantly prolonged event-free and overall survivals.Approximately one third of patients with newly diagnosed symptomatic MM completed the TTI program. These data suggest the need to improve the induction therapy in order to increase both the number of patients able to proceed to autotransplantation programs and to enhance the rate of early response.

Summary Gemtuzumab ozogamicin (GO) is a humanized anti-CD33 antibody conjugated with the cytotoxic drug calicheamicin and approved for the treatment of relapsed acute myeloid leukaemia. As approximately 18% of acute lymphoblastic leukaemias (ALL) are also CD33 positive, we have investigated the cytotoxic activity of GO on CD33+ ALL cells in vitro and in vivo. 10 ng/ml GO induced 30–95% inhibition of thymidine uptake and 30–70% cell death in four freshly isolated and one in vivo passaged CD33+ ALL-cell cultures. Furthermore, an in vivo model of a CD33+ ALL carrying the Philadelphia chromosome [t(9;22)] was established. 5 × 106 ALL-2 cells inoculated in the tail vein of severe combined immunodeficient mice engrafted into haematopoietic organs, reaching a mean of 70%, 61% and 69% human CD45+ cells in bone marrow, spleen and liver, respectively, at 35 d. To test the therapeutic activity of GO, 50 or 100 μg immunotoxin was inoculated i.p. on days 7, 11 and 15 following tumour-cell inoculation. GO treatment dramatically inhibited expansion of ALL-2 cells in all tested organs and increased survival of tumour-injected animals by 28–41 d, relative to controls. These data demonstrated that GO is active both in vitro and in vivo against CD33+ ALL cells.

One of the few measurable clinical results obtained by the use of somatic cells in onco hematology is the clear-cut effect donor leukocyte infusions (DLI) in patients with chronic myeloid leukemia (CML) who relapse after an allogeneic bone marrow transplantation (BMT). From then on much research has focused on the use of cells to treat different aspects of oncologic diseases from leukemia relapse to development in BMT recipients.In this review we critically and schematically summarize the cell-based therapies which have led to a clinical application and recapitulate the results.Although the overall numbers of successfully treated patients is small, therapy has been shown to be safe and effective in a variety of clinical contexts in oncohematology.Preliminary data will have to be validated in well designed clinical trials with cells generated by reproducible methods and in accreditated structures working according to Good Manufacturing Practices (GMP).

Cytomegalovirus (CMV) infection is an important cause of morbidity in solid organ recipients. Early markers to identify the progress of the infection and patients at high risk are required in order to apply a strategy of pre-emptive therapy. The efficacy of pre-emptive therapy relies on accurate laboratory tests to monitor CMV infection. The evaluation of CMV DNA kinetics by the polymerase chain reaction (PCR) is widely used for the management of CMV infection but markers predicting the progression of the infection and standardization of the technique are essential for the clinical interpretation of PCR results. A commercially available PCR system, the COBAS AMPLICOR Monitor (Roche Diagnostics, Brachburg, NJ), was used for the quantitation of CMV DNA in weekly blood samples (n = 504) from 47 solid organ recipients in the first 6 months after transplantation. PCR results were evaluated according to the development of clinical disease in order to find a DNA threshold and time points predicting the progression of CMV infection. Week 4 from transplantation was the earliest time point to note a significant difference between those patients who eventually developed CMV disease (n = 30) and those who remained asymptomatically infected (n = 17). At week 4, viral loads were significantly higher in patients who developed CMV disease than in asymptomatic infections (median value: 4 log(10)/10(6) leukocytes vs. 2.8, P0.0001). At week 4, a DNA level/=4 log(10)/10(6) leukocytes was associated with a 45.37 odds ratio for CMV disease. Any increase/=1 log from the first DNA detection to week 4 correlated with the clinical progression of CMV infection (odds ratio 1.74). In those patients who were treated with anti-CMV therapy, a97% reduction of the baseline viral load was associated with a complete therapeutic success. In conclusion, CMV infection is a highly dynamic process and the quantitation of CMV DNA by PCR is a powerful marker to control successfully the infection, but a strict follow-up of the recipient and standardized PCR tests are mandatory for the best management of the infection.

Several trials have shown the superior impact of high-dose melphalan (usually 200 mg/m(2), MEL200) vs standard therapy in myeloma patients. Intermediate-dose melphalan (100 mg/m(2), MEL100) is also superior to the standard dose, but has not been clinically compared with MEL200. A total of 90 patients at diagnosis were treated with two MEL100 courses. Their clinical outcome was compared with that of a control group of 90 pair mates matched for serum beta2-microglobulin levels and Durie and Salmon clinical stage. These patients were treated at diagnosis with two MEL200 courses. Patient characteristics were similar in both groups except that the median age of the MEL100 group was significantly higher (P0.0001). Complete remission was 35% after MEL100 and 48% after MEL200 (P=0.08). Median event-free survival (EFS) was 32 months in the MEL100 group and 42 months in the MEL200 group (P0.005), but overall survival (OS) was not different. Transplant-related mortality was not significantly different. Haematological and extra-haematological toxicity was significantly reduced after MEL100. Despite the significant age difference, tandem MEL100 was less toxic than tandem MEL200, and MEL100 was inferior to MEL200 in terms of EFS but not in terms of OS. The intensified nonmyeloablative MEL100 regimen is an effective first-line treatment.

Non-myeloablative stem cell transplantation (NMT) has been increasingly used in compromised patients who would otherwise have been unable to undergo allotransplant. There is little understanding of the kinetics of immune reconstitution and its influence on infective complications following NMT. The aim of present study was to evaluate lymphocyte subset reconstitution over the first 12 months post-transplant in 15 adult patients receiving NMT with comparison to that of 30 patients grafted with a conventional hemopoietic stem cell transplantation (HSCT). NMT recipients were conditioned with fludarabine-based conditioning regimens. Peripheral blood stem cell (PBSC) was the source of stem cells in 13 NMT recipients and in 24 conventional HSCT recipients. Absolute numbers of helper (CD4+) T cells, naive (CD4+ CD45RA+) and memory (CD4+ CD45RO+) T cells as well as suppressor (CD8+) T cells, CD19+ B cells and NK cells were comparable in the two groups at all time points after transplantation. A median value of 200 CD4+ T cells/microl was achieved at 2 months post-transplant by the NMT and HSCT recipients. The CD4:CD8 ratio remained severely depressed throughout the study period. Almost all CD4+ lymphocytes expressed CD45RO antigen in the both groups of patients B lymphocytes showed low counts throughout the entire study period in both groups. Bacteremia and CMV antigenemia occurred respectively in 13 and 36% of the patients in the NMT group and in 15 and 39% of the patients in the HSCT group. Our preliminary data indicate that patients receiving a NMT have a lymphocyte reconstitution similar to that observed in patients who received a conventional HSCT. The incidence of bacteremia and CMV infection were not significantly different between the groups. Nevertheless, due to the small sample size, these results should be considered suggestive rather than definitive.

We recently described a two-step negative selection procedure whereby peripheral blood stem cells (PBSCs) were efficiently purged of contaminating neoplastic cells by a combination of monoclonal antibodies. Here, we report 60 newly diagnosed multiple myeloma (MM) patients treated with a double transplant programme and randomized to receive either unmanipulated or in vitro purged PBSCs. We demonstrated that this technique is feasible and safe without significant loss of either CD34+ or CD3+ cells. Haematological engraftment and immunological reconstitution were rapid without treatment-related mortality. Using polymerase chain reaction (PCR), we compared the level of minimal residual disease (MRD) in PBSC before and after in vitro purging and in vivo after transplant. A median of one tumour cell per 10(2) normal cells (range 10(1)-10(5)) was seen in the unmanipulated aphereses with a 3-4 log reduction after manipulation in vitro. However, despite this tumour debulking, all patients remained PCR positive in vivo. At 3 years, the estimated event-free survival was 40% in the control arm and 72% in the experimental arm (P = 0.05), whereas the estimated overall survival was 83% in both arms. This suggests that autologous transplantation using efficiently purged PBSCs can be performed safely, but confirms the need for innovative protocols for MRD eradication in vivo.

Background/Aims : Interferon (IFN) with ribavirin combination therapy (CT) was proposed for the treatment of hepatitis C recurring in liver transplants. We assessed the efficacy of two protocols of CT in transplanted patients with recurrent severe hepatitis C virus (HCV) hepatitis. Methods : Fifty-seven patients (68% genotype 1b) were treated with IFN alfa-2b 3 million units three times weekly and oral ribavirin 800mg/die for 6 or 12 months. Study end-points were the end of treatment (ETVR) and the 12-month post-therapy sustained virologic response (SVR; negative HCV-RNA). Results : ETVR was induced in 9/27 (33%) and in 7/30 patients (23%) treated, respectively, for 6 and 12 months ( P =0.4); a SVR was induced in six (22%) of the former and five (17%) of the latter ( P =0.4). HCV genotype non-1 patients responded better than genotype 1 (SVR: 43% in genotype non-1 versus 12% in genotype 1, P : 0.02). In ETV responders the hepatitis activity index improved by >2 points in biopsies taken after therapy compared to pre-therapy biopsies. Anemia and leukopenia required reduction of therapy in 51% of the patients. Conclusions : CT is efficacious in controlling HCV disease in about 20% of transplants with recurrent hepatitis C. Six months of therapy are as efficacious as 12 months.

8506 Background: CoR is an anti-CD19 antibody maytansinoid conjugate. CD19 is expressed in the majority of B cell lymphomas. Phase I program showed clinical activity in pts with both indolent and a...

8531 Background: Early results of a randomized phase III GITMO/FIL study (www.clinicaltrials.gov NCT00435955) comparing CHOP-R vs. R-HDS + ASCT as primary treatment in134 FL pts, aged ≤60 yrs with aaIPI>1/IIL score>2, showed superior disease control with R-HDS but no OS advantage (Ladetto et al, Blood 2008). We here present an updated analysis from July 2013 at a median follow-up (MFU) of 9.5 yr including 125 pts of 134 originally randomized pts (61 CHOP-R/64 of R-HDS). Methods: Clinical features (CF) and treatment schedules have been already reported. Briefly, median age was 51 yrs. (22-60), M/F ratio 74/51, aaIPI 2-3 90%, retrospective FLIPI >2 60%, high LDH 49%, bulky disease 62%, B-symptoms 45%, BM+ 86%. CF were balanced among the two arms. Analysis was intention to treat and EFS the primary endpoint. Minimal residual disease was done by nested and RQ-PCR. Results: CR rate was 70.4% (57% with CHOP-R and 83% with R-HDS, p < .001); 64% patients achieved a Molecular Remission (MR). At MFU, 88 patients (7...

Background/Aims : Treatment with hepatitis B virus immune globulins (HBIG) or lamivudine has reduced the rate of hepatitis B recurrence after liver transplantation to approximately 50%. Methods : To further decrease hepatitis B recurrence, 33 hepatitis B virus (HBV)-related cirrhotic patients were treated with lamivudine before liver transplantation and with lamivudine together with low-dose HBIG (46 500 IU the first month followed by 5000 IU/monthly) after surgery. Results : While on lamivudine, serum HBV DNA level decreased significantly in all patients and in 11 (33%) the Child–Pugh score improved. Twenty-six patients were transplanted. Among the 25 who survived for longer than 12 months, only one (4%) experienced a hepatitis B recurrence over an average follow-up of 31 months, a rate significantly lower ( P =0.0002) than the 50% recurrence rate among a historical control group of 12 patients. However, low-level HBV replication was detected sporadically throughout the follow-up in 64% of patients. Conclusions : Over the medium-term, combined prophylaxis with lamivudine and HBIG significantly decreases the risk of hepatitis B recurrence after liver transplantation. Though low-level HBV infection recurred in two thirds of patients, the pathogenic expression of HBV was prevented.

Introduction A randomized multicenter study of 134 Follicular Lymphoma (FL) patients, selected for age less than 60 yrs. and poor prognostic features according to age-adjusted IPI (2-3) and IIL-score (3 or greater) was conducted between March 2000 and May 2005, among 30 Italian Centers. The study compared efficacy and tolerability of CHOP-R vs. R-HDS with autograft as primary treatment in poor-risk FL. Initial results have been already reported (Ladetto M et al, Blood 2008), showing superior disease control with R-HDS without any survival advantage. We have recently updated the long-term outcome and the results at long-term are here presented at a median follow-up of 9.5 yrs. Patients and Methods Of the original 134 randomized patients, the long-term outcome has been updated for 125 patients, 61 of CHOP-R and 64 of R-HDS arms. Clinical characteristics at study entry and treatment schedules have been already reported. Briefly, the main features of the updated patients included: median age 51 yrs. (22-60), M/F ratio 74/51, aaIPI 2-3 90%, FLIPI >2 (retrospectively assigned) 60%, high LDH 49%, bulky disease 62%, B-symptoms 45%, BM involvement 86%. Clinical characteristics were balanced among the two arms. Treatment schedule consisted of: i. standard arm: 6 courses of cyclo-phosphamide/doxorubicin/vincristine/prednisone followed by 4-weekly rituximab courses (CHOP-R); ii. experimental arm: rituximab-supplemented high-dose sequential chemotherapy with autografting (R-HDS). The analysis was intention to treat with event-free survival as the primary endpoint. Minimal residual disease was evaluated post treatment in 58 patients with a bcl-2/IgH MBR or mcr translocation confirmed at diagnosis by nested PCR. The trial was registered at www.clinicaltrials.gov as no. NCT00435955. The long-term outcome has been updated in July 2013 by 28 out of 30 participating Centers accounting for 125 patients (93% of the whole series). Results Complete remission (CR) was achieved by 88 (70.4%) patients, including 35 (57%) with CHOP-R and 53 (83%) with R-HDS (p < .001); in addition, 37 out of 58 (64%) patients achieved a Molecular Remission (MR). At a median follow-up (MFU) of 9.5 yrs., 88 patients (70.4%) are alive. Overall, 19 patients died for lymphoma progression (11 in the CHOP-R, 8 in the R-HDS arms), there were nine deaths for secondary malignancy (3 in the CHOP-R, 6 in the R-HDS arms), nine more patients died for other causes, including four early toxic deaths. The overall survival projection for the whole series is 78% and 70% at 5 and 10 yrs., respectively. As shown in Figure 1, there were no main differences in the long-term OS between the two arms, with 5 and 10 yrs projections respectively of 75% and 70% for CHOP-R and 81% and 70% for R-HDS (p=0.96). Response to primary treatment had a major impact on the OS, with 5 and 10 yr survival projections respectively of 90% and 80% for patients achieving CR, and of 49 and 43 for those with less than CR (p < .001) (Figure 2A). Similarly, MR achievement was associated with prolonged overall survival, with 5 and 10 yr survival projections respectively of 89% and 83% for patients with PCR-ve on BM cells, and of 76 and 57 for those with persistent PCR-positivity (p = .03) (Figure 2B). Conclusion The long-term follow-up of the randomized CHOP-R vs. R-HDS trial indicate that: i. poor risk FL may now experience a prolonged survival, with approximately 70% of patients alive at 10 yrs., due to the combined efficacy of both primary chemo-immunotherapy and salvage treatments; ii. the superior disease control of R-HDS compared to CHOP-R does not translate in any survival advantage, with analogous OS regardless of which treatment is used; iii. also in FL like in other lymphoproliferative malignancies, achieving CR and MR is crucial not only for the disease control but also for long-term overall survival; iv. lymphoma progression remains the major cause of death, while secondary neoplasms, in particular secondary leukemias represent the second cause of treatment failure. Thus, efforts are still needed in order to increase the anti-tumor efficacy while reducing any potential late effect in treatment options for FL. Disclosures: Tarella: Roche Co.: support and honoraria for Conference participation Other. Ladetto:Roche: Honoraria, Research Funding, Speakers Bureau.

A retroviral vector has been constructed that contains the human CD20 cDNA under the control of the Moloney murine leukemia virus (Mo-MuLV) LTR. Freshly isolated mononuclear cells are infected for three consecutive days in the presence of PHA and hrlL-2, and a mean 15.9% of the cells (range, 6.5 to 31.7%) acquire a CD3+CD20+ phenotype. Transduced T lymphocytes grow and expand in vitro for up to 3 weeks like mock-infected cells and, as observed for the T lymphoblastoid CEM cell line, CD20 expression is maintained for several months with no change in the growth curve of the cells. CD20-expressing CEM and fresh T lymphocytes can be positively immunoselected on columns using different anti-CD20 antibodies. Exposure to monoclonal chimeric anti-CD20 IgG1(kappa) Rituximab antibody (Roche), in the presence of complement, results in effective and rapid killing of the transduced CD3+CD20+ human T cells in vitro. This approach represents a new and alternative method to gene manipulation with "suicide" genes for the production of drug-responsive T cell populations, a crucial step for the future management of graft-versus-host disease in bone marrow transplant patients.

OBJECTIVE: To establish whether polymerase chain reaction (PCR) for cytomegalovirus deoxyribonucleic acid (DNA) can provide clinical information for the management of the infection. METHODS: Leukocytes in 30 heart transplant recipients were monitored by pp65 antigen testing and PCR for 82 to 365 days after transplantation. RESULTS: Of the 30 patients, 26 developed cytomegalovirus infection, nine of whom were symptomatic. Altogether, 300 leukocyte samples were examined. The concordance between PCR and pp65 antigen test was 82.6%. In symptomatic patients after surgery, PCR detected cytomegalovirus infection after 38 plus minus 16 days and the pp65 antigen test, after 48 plus minus 15 days. Symptomatic infection correlated with a higher number of pp65-positive leukocytes than did asymptomatic infection: 310 plus minus 356 vs 24 plus minus 35 (p0.005)/200,000 examined, respectively. Clearance of virus was observed by PCR after 125 plus minus 73 days (range 29 to 225) in symptomatic, and after 82 plus minus 70 days (range 16 to 301) in asymptomatic, cases of infection. CONCLUSIONS: The positive predictive value of PCR for symptomatic infection was 34.6%. Our findings correlate with previous reports and show that the qualitative detection of cytomegalovirus DNA is not associated with overt disease whereas quantitation of pp65-positive leukocytes closely correlate with symptom onset. Insofar as the results are not quantitative, PCR is not a marker of clinically apparent infection. Careful monitoring of cytomegalovirus infection based on quantitative pp65 antigen assay can fulfill all clinical needs for early diagnosis and proper management of the infection