ALBERT

Description: Some autoimmune disorders are increasingly recognized as risk factors for non-Hodgkin lymphoma (NHL) overall, but large-scale systematic assessments of risk of NHL subtypes are lacking. We performed a pooled analysis of self-reported autoimmune conditions and risk of NHL and subtypes, including 29 423 participants in 12 case-control studies. We computed pooled odds ratios (OR) and 95% confidence intervals (CI) in a joint fixed-effects model. Sjögren syndrome was associated with a 6.5-fold increased risk of NHL, a 1000-fold increased risk of parotid gland marginal zone lymphoma (OR = 996; 95% CI, 216-4596), and with diffuse large B-cell and follicular lymphomas. Systemic lupus erythematosus was associated with a 2.7-fold increased risk of NHL and with diffuse large B-cell and marginal zone lymphomas. Hemolytic anemia was associated with diffuse large B-cell NHL. T-cell NHL risk was increased for patients with celiac disease and psoriasis. Results for rheumatoid arthritis were heterogeneous between studies. Inflammatory bowel disorders, type 1 diabetes, sarcoidosis, pernicious anemia, and multiple sclerosis were not associated with risk of NHL or subtypes. Thus, specific autoimmune disorders are associated with NHL risk beyond the development of rare NHL subtypes in affected organs. The pattern of associations with NHL subtypes may harbor clues to lymphomagenesis.

Description: The activated B cell–like (ABC) subgroup of diffuse large B-cell lymphoma (DLBCL) is characterized by constitutive activation of the nuclear factor-κB (NF-κB) pathway. In this study, we showed that the NF-κB pathway induced the expression of the cytokines interleukin (IL)-6 and IL-10 in ABC DLBCL cell lines, which also have high levels of total and phosphorylated signal transducer and activator of transcription (STAT) 3 protein, suggesting autocrine signaling. Using RNA interference for STAT3, we defined a gene expression signature of IL-6 and IL-10 signaling through STAT3. Based on this signature, we constructed a molecular predictor of STAT3 signaling that defined a subset of ABC DLBCL tumors with high expression of STAT3, IL-6, and/or IL-10 and their downstream targets. Although the STAT3-high and STAT3-low subsets had equivalent expression of genes that distinguish ABC DLBCL from germinal center B cell–like DLBCL, STAT3-high ABC DLBCLs had higher expression of signatures that reflected NF-κB activity, proliferation, and glycolysis. A small-molecule inhibitor of Janus kinase signaling, which blocked STAT3 signature expression, was toxic only for ABC DLBCL lines and synergized with an inhibitor of NF-κB signaling. These findings suggest that the biological interplay between the STAT3 and NF-κB pathways may be exploited for the treatments of a subset of ABC DLBCLs.

Description: Long-term survival in Diamond Blackfan Anaemia (DBA) is compromised by transfusional iron overload and/or side effects of high-dose steroids as well as increasing recognition of the increased risks of aplasia and malignancy. Allogeneic haemopoietic stem cell transplantation (HSCT) can cure the haematological manifestations of DBA, but the optimal conditioning protocol is yet to be defined, particularly avoiding irradiation in view of the increased risk of malignancy. Four consecutive children with DBA were conditioned with oral busulfan 14 mg/kg (days-9 to -6), cyclophosphamide 200 mg/kg (days-5 to -2) and horse ALG 45 mg/kg (days-4 to -2) prior to HLA-matched sibling donor HSCT. Graft-versus-host disease (GvHD) prophylaxis was provided with ciclosporin from day-1 for six months to a target range of 100–150 ng/mL and methotrexate 10 mg/m2 on days +4 and +7. P1: 10 years old boy who was transfusion dependent for two years having lost the response to steroids. In addition, he had deafness, short stature, gastrooesophageal reflux and cleft palate. He received bone marrow (8 × 108 TNC/kg). P2: 10 years old boy who was steroid dependent and had developed diffuse liver steatosis and bone fractures. In addition, he had short stature and undescended testis. He received bone marrow (1.91 × 108 TNC/kg). P3: four year old girl who was transfusion dependent with no associated abnormalities. She received a combination of cord blood (0.83 × 107 TNC/kg) and bone marrow (0.738 × 108 TNC/kg) from a sibling born following pre-implantation genetic diagnosis and HLA typing. P4: six year old boy born at 34 weeks of gestation who was transfusion dependent. He received a combination of cord blood (1.4 × 107 TNC/kg) and bone marrow (2.73 × 108 TNC/kg). Neutrophil and platelet engraftment were prompt in all 4 patients (neutrophils: day 15–22; platelets day 10–33). Stable complete donor haemopoiesis was confirmed in all four patients using peripheral blood genomic DNA microsatellites markers by genescanning on day +28, 6 months and 1 year post-transplantation. The conditioning protocol was well tolerated by all patients with no life-threatening infections, though one patient had moderate veno-occlusive disease responding to defibrotide followed by gastrointestinal haemorrhage and another suffered reversible ciclosporin-associated posterior leucoencephalopathy and stage IV, steroid-responsive, acute gut GvHD. All four patients are alive with full donor haemopoiesis and normal haematological parameters at 11, 12, 15 and 24 months post-transplantation. Sibling allogeneic HSCT with this non-TBI protocol is a reasonable and feasible option for DBA patients suffering from unacceptable steroid side effects or chronic transfusion therapy.

Description: Activity of the nuclear factor-κB (NF-κB) family of transcription factors is tightly regulated by its inhibitor, IκBα, through cytoplasmic localization of latent NF-κB: IκBα complexes. This arrangement is essential for efficient signal-inducible activation and regulation of biologic functions. Maintenance of cytoplasmic localization of latent NF-κB: IκBα complex requires continuous nuclear export that is dependent on the N-terminal nuclear export sequence (N-NES) of IκBα. While these mechanisms have been elucidated through in vitro studies, the biological significance of this “nucleocytoplasmic shuttling” has yet to be evaluated in vivo. To address this, we derived mice harboring germ-line M45A, L48A, and I52A amino acid substitutions in the N-NES of IκBα. In splenic B-cells, the disrupted N-NES caused constitutive nuclear accumulation of IκBα and inactive c-Rel containing complexes but surprisingly not IκBα: p65 complexes. Since p65 contains a NES sequence and c-Rel does not, nuclear export of N-NES mutant IκBα:NF-κB complexes appear to be NF-κB family member dependent. Functionally, NF-κB activity in splenic B-cells after stimulation with IgM or LPS was clearly reduced in the mutants compared to wild-type by electrophoretic mobility shift assay. B-cell development in the bone marrow of mice harboring the mutation was impaired, showing a preponderance of pro/pre B-cells and few mature B-cells compared to their wild type littermates (p 〈 0.001). Concordantly, there were significantly fewer B-cells in the spleen (p 〈 0.05) and lymph nodes (p 〈 0.01) of the mutant mice. Additionally, populations of T2, follicular (FO), and marginal zone (MZ) B-cells, which represent mature B-cells in the spleen, were also reduced in the mutant mice (p 〈 0.001). To demonstrate that this B-cell maturation defect in IκBα mutant mice was B-cell intrinsic, sublethally irradiated Jak3-deficient mice were transplanted with BM from either wild-type or mutant mice. B-cell development in mice transplanted with mutant donors was impaired relative to those with wild-type donors in a fashion identical to that of the primary mutants described above. Finally, severe phenotypes in inguinal lymph nodes and Peyer’s patch development were present, with mutant mice frequently lacking these secondary organs/tissues, the underlying mechanisms of which are currently being investigated. In conclusion, our findings uncover an in vivo mechanism controlling NF-κB localization and its essential role in the generation of mature B-cells and certain secondary lymphoid organs.

Description: Adoptive transfer (AT) of autologous T cells genetically-redirected against tumor antigens has considerable potential as cancer immunotherapy [Kershaw, Nat Rev Immunol. 2005]. However, the in vivo persistence of AT T cells is critical for tumor control and requires the development (in vitro or in vivo) of a memory T cell subset. We investigated the generation of memory T cell subsets in a novel chimeric T cell receptor-expressing T cell product prior to, and after exposure to cognate antigen. Gene-modified T cells (LeY-T) express a chimeric receptor comprising a single chain variable fragment (scFv) specific for Lewis Y (LeY) antigen coupled to the intracellular signaling domains of CD3 zeta and CD28, capable of inducing T cell effector granule release and target killing [Westwood PNAS 2005]. To produce LeY-T cells, PBMC from healthy donors (n=20) or multiple myeloma patients (n=2) were cultured with anti-OKT3 (30ng/ml) and IL-2 (600IU/ml) for three days, followed by two rounds of transduction with retroviral supernatant. Subsequently, T cells were expanded in high dose IL-2 (600IU/ml) from day 5 onwards. T cells were harvested for this study on culture days 10–12, CD8+ and CD4+ T cells expressed the chimeric protein (50–60)%. LeY CD8+ T cell subsets were assessed as naïve (N), central memory (CM), effector memory (EM) or effector (E) based on three features:- phenotype (CD45RA, CCR7, CD28, CD27 and perforin); homeostatic cytokine (IL-15/IL-7) proliferation; response to Lewis antigen contact including cell proliferation and cytokine secretion. We repeatedly observed that CD8+ LeY-T cells analyzed directly from the initial expansion culture demonstrate an effector memory (EM) phenotype (CD45RA−/CCR7−/CD28+/perforinhi and variable CD27 expression) (Figure 1A). Furthermore in vitro expanded LeY CD8 T cells express IL- 15R beta (CD122) and the common gamma chain (CD132), they proliferate in response to IL-15 (86% cell division, division index 1.82), but less with IL-7 (30% cell division, division index 0.56). Baseline expanded CD8+ LeY-T cells respond to the presence of LeY antigen by proliferating and secreting IFN-gamma (4–8% of CD8 T cells) but not IL-2. Importantly, no IFN-gamma secretion was seen in control T cells transduced with empty vector (Figure 1B, OVCAR cells). Furthermore, no IFN-gamma was secreted by the control or the CD8+ LeY-T cells in response to the Lewis antigen negative cell line (Figure 1C, HCT116 cells). To explore the memory component further, we examined the functional status of the CD8+ LeY-T cells seven and 30 days following a 48-hour exposure to LeY antigen (OVCAR cells), and compared this to CD8+ LeY-T cell functional status at baseline. Thus, direct from transduction, expansion culture LeY CD8+ T cells were largely EM phenotype (95%) a small population of cells (1–5)% had a CM phenotype (CD45RA−/CCR7+/CD28+/perforinlo). In contrast, seven days after Lewis antigen contact the EM cells had decreased to (76–88)% and CM increased to (10–21)%; this distribution was retained up to day 30 post-antigen exposure. In addition, seven days after Lewis antigen exposure, CD8+ LeY-T cells retain the capacity to proliferate in response to Lewis antigen and to secrete IFN-gamma, at no stage do these cells secrete IL-2. In conclusion, the CD8+ LeY-T cells produced by in vitro transduction and expansion culture have an EM functional status direct from in vitro culture indicating that they are an appropriate starting population for in vivo adoptive transfer. After exposure to LeY expressed on tumor cell lines in vitro, CD8+ LeY T cells show further polarization to either EM or CM cells. These results suggest that the LeY-chimeric T cells have the potential to form long-term memory populations in vivo after adoptive transfer. Figure 1. LeY T cells have an effector memory phenotype and respond to Lewis antigen expressing cell lines by secreting IFN-gamma. Following the transduction culture, the CD8+ LeY-T cells (A) expressed high levels of perforin and an EM phenotype. In (B), LeY T or empty vector control T cells were co-cultured with tumour cells overnight and intracellular cytokine secretion assay performed. The LeY CD8+ T cells responded to Lewis antigen expressing OVCAR cells by secreting IFN-gamma, whereas no response was observed with the negative cell line HCT-116. Figure 1. LeY T cells have an effector memory phenotype and respond to Lewis antigen expressing cell lines by secreting IFN-gamma. Following the transduction culture, the CD8+ LeY-T cells (A) expressed high levels of perforin and an EM phenotype. In (B), LeY T or empty vector control T cells were co-cultured with tumour cells overnight and intracellular cytokine secretion assay performed. The LeY CD8+ T cells responded to Lewis antigen expressing OVCAR cells by secreting IFN-gamma, whereas no response was observed with the negative cell line HCT-116.

Description: Myeloid cell factor-1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 protein family that associates with tumor cell survival and drug resistance in chronic lymphocytic leukemia (CLL). Increased Mcl-1 expression is associated with failure to achieve remission after chemotherapy with fludarabine and chlorambucil in CLL patients. However, the influence of Mcl-1 expression has not been examined in CLL trials using treatment regimens that contain rituximab, which has shown substantially improved outcomes for a large subset of patients and have become a mainstay in the therapy of newly diagnosed CLL. We previously reported that combination chemoimmunotherapy with pentostatin, cyclophosphamide, and rituximab (PCR) has significant clinical activity with low accompanying toxicity in previously untreated CLL patients, and is especially well tolerated in older patients in whom the use of fludarabine may be associated with prohibitive toxicities. As part of this study, we prospectively investigated association of Mcl-1 protein expression with response and progression-free survival. Eligible patients received a regimen consisting of pentostatin (2 mg/m2), cyclophosphamide (600 mg/ m2), and rituximab (375 mg/m2) given intravenously on day 1 of a 21-day cycle for a maximum of 6 cycles. Responses were assessed according to NCI 1996 criteria and included bone marrow evaluation and two-color flow cytometry two months after completion of therapy. Flow cytometry-negative status was defined as less than or equal to 1% CD5+/CD19+ cells. Of the 64 patients evaluated in this trial, clinical responses were seen in 58 (91%), with 26 (41%) complete responses (CR), 14 (22%) nodular partial responses (nPR), and 18 (28%) partial responses (PR). Lysates from peripheral blood cells obtained pre-treatment were analyzed for Mcl-1 expression by immunoblot. Mcl-1 as a continuous variable was not found to be associated with other commonly described risk factors. However, Recursive Partitioning Analysis (RPA) was devised employing Mcl-1 expression as a continuous variable, which revealed an optimal cut point of 0.85. At this cutoff, flow cytometry-negative status was significantly different between the two groups (p=0.01) and median PFS was significantly higher (p=0.02) in patients with Mcl-1 levels of

Description: To evaluate the hypothesis that host germ line variation in immune genes is associated with overall survival in diffuse large B-cell lymphoma (DLBCL), we genotyped 73 single nucleotide polymorphisms (SNPs) from 44 candidate genes in 365 DLBCL patients diagnosed from 1998 to 2000. We estimated hazard ratios (HRs) and 95% confidence intervals (CIs) for the association of SNPs with survival after adjusting for clinical factors. During follow-up, 96 (26%) patients died, and the median follow-up was 57 months for surviving patients. The observed survival of this cohort was consistent with population-based estimates conditioned on surviving 12 months. An IL10 haplotype (global P = .03) and SNPs in IL8RB (rs1126580; HRAG/GG = 2.11; CI, 1.28-3.50), IL1A (rs1800587; HRCT/TT = 1.90; CI, 1.26-2.87), TNF (rs1800629; HRAG/GG = 1.44; CI, 0.95-2.18), and IL4R (rs2107356; HRCC/CT = 1.97; CI, 1.01-3.83) were the strongest predictors of overall survival. A risk score that combined the latter 4 SNPs with clinical factors was strongly associated with survival in a Cox model (P = 6.0 × 10−11). Kaplan-Meier 5-year survival estimates for low, intermediate-low, intermediate-high, and high-risk patients were 94%, 79%, 60%, and 48%, respectively. These data support a role for germ line variation in immune genes, particularly genes associated with a proinflammatory state, as predictors of late survival in DLBCL.

Description: Haemopoietic stem cell transplantation (HSCT) is the only proven curative treatment available for haemoglobinopathies. To reduce transplant-related mortality and morbidity associated with graft-versus-host disease (GvHD) and facilitate donor engraftment we have used low-dose alemtuzumab in 43 consecutive patients (36 with thalassaemia and 7 with sickle cell disease; median age 6 years; range 2 to 16 years) transplanted since 2000. Donors were HLA-matched family donors in all cases. All patients were conditioned with oral busulfan 14 mg/kg (days -9 to -6), cyclophosphamide 200 mg/kg (days -5 to -2) and alemtuzumab 0.3 mg/kg (days -8 to -6) with post-HSCT methotrexate (10 mg/m2 days +4 and +7) and ciclosporin for six months. Hypertransfusion was initiated six weeks before conditioning and maintained until day +28 to suppress endogenous haemopoiesis and minimise graft rejection. Patients with thalassaemia were Pesaro class I or II. Patients with sickle cell disease were transplanted for stroke or recurrent vasooclusive crisis and/or acute chest syndrome not responding to hydroxycarbamide. The source of stem cells was bone marrow in all cases except one patient who received PBSC with a median cell dose of 3.08 x 108 TNC/kg (range 1.69 – 10.90 x 108 TNC/kg). Median follow up was 25.8 months (range 6.5 – 94.3 months). Chimerism studies were performed on peripheral blood genomic DNA using 8 microsatellites markers by genescanning. All patients are alive (43/43), 42 with long-term transfusion-independence and donor haematological values (5 years DFS 97.7%). One patient failed to engraft and was re-infused with autologous cells. Donor haemopoiesis was ≥ 95% on day +28 in all 42 patients who engrafted and was maintained at ≥ 95% in the majority of patients at 12 months post-HSCT. Eleven patients (11/42; 31%) have stable mixed chimerism at ≥ 12 months post-HSCT: donor haemopoiesis 90–94% (n=4);

Description: BACKGROUND: The aggresome/autophagy pathway is the primary mechanism for disposal of ubiquitinated proteins for cells exposed to proteasome inhibition. Preclinical evidence shows that combining inhibition of the proteasome with bortezomib (Bz) and inhibition of autophagy with the anti-malarial drug hydroxychloroquine (HCQ) leads to enhanced cytotoxicity in myeloma cells. METHODS: Patients with relapsed or refractory myeloma are enrolling on a standard 3+3 dose escalation design. Patients receive a 2-week run-in of single-agent oral HCQ (at escalating doses), followed by addition of standard dose bortezomib (on days 1, 4, 8, and 11 of 21-day cycles). Dose-limiting toxicity (DLT) is defined as grade ≥3 toxicity probably related to study therapy and occurring during the first 5 weeks, with the exception of any anemia or lymphopenia, neutropenia responsive to growth factor, platelets 〉10,000/mm3 not associated with bleeding, or gastrointestinal complaints relieved by symptomatic therapy. Response is assessed on day 1 of each cycle according to International Working Group criteria. We use electron microscopy (EM) to characterize changes in autophagic vesicles (AVs) in serial samples of peripheral blood mononuclear cells (PBMCs) and CD138-selected bone marrow plasma cells (BMPCs) RESULTS: Ten patients have enrolled in the first 3 dose levels (see table). The median age is 63.5 (range 43–68), and 5 (50%) are male. The median number of prior lines of therapy is 4 (range 1–8). Seven patients had received prior Bz. No DLTs have been observed. Adverse events have generally been those expected with Bz: thrombocytopenia (30%, grade 3: 20%), anemia (70%, grade 3: 30%), and fatigue (70%, all grade 1/2), as well as neutropenia (20%, grade 3:10%). Grade 2 treatment-emergent peripheral neuropathy occurred in 1 patient (grade 1 in 3 patients). No patients developed retinal toxicity. Four patients had pulmonary infections: 1 viral influenza, 1 H. influenzae, and 2 lobar pneumonias; all resolved with appropriate antibiotics and did not preclude continued therapy. One patient died from complications of C. difficile colitis that occurred at the end of the 4th cycle, after antibiotic therapy for an upper respiratory infection. One patient had a minor response after previously progressing on single-agent Bz. EM analysis of PBMCs and BMPCs show no significant changes in AVs thus far with the lowest dose of HCQ. Enrollment is ongoing at higher doses of HCQ (escalating from 400 to 1200 mg daily). CONCLUSION: At the lowest dose levels tested, combined Bz and HCQ is tolerable, without evidence of synergistic toxicity. EM is a feasible pharmacodynamic assay for patient tumor and normal cells. Further dose escalation will determine whether combined autophagy and proteasome inhibition is achievable and safe with these agents. Updated clinical and correlative results will be presented. Dose Cohort Prior response to Velcade Best response on study Weeks on study Reason for discontinuation HCQ 200 mg qod PD Progression 11 Progression + Bz 1.0 mg/m2 SD Stable disease 32+ - - Stable disease 18 Lack of response ___________ _________ ___________ ________ __________________ HCQ 200 mg qod SD→PD Stable disease 14 Lack of response + Bz 1.3 mg/m2 PR→PD Minor response 14 Adverse event (unrelated) - Stable disease 21+ - ___________ _________ ___________ ________ __________________ HCQ 200 mg qd - Stable disease 9+ - + Bz 1.3 mg/m2 SD→PD Stable disease 8+ - PR→PD Progression 3 Progression PR→PD Not evaluable 4+ -

Description: Background: Retrospective studies have reported a correlation of higher ELISA optical density (OD) values for H-PF4 antibodies with occurrence of thrombotic events in patients (pts) with HIT; often with variable prophylactic direct thrombin inhibitor (DTI) use. Objectives: To compare positive OD (≥0.4) values with thrombotic risk and clinical outcome in pts with clinically-suspected or confirmed HIT. Design: Retrospective cohort study. Setting: IRB-approved, CAMC HIT Registry. Patients: 182 HIT pts, age ≥18 yrs with OD values ≥0.40 (EIA, GTI) enrolled from 3/11/05 to 12/27/07. Age 64.5±13.4 yrs; M/F (107/75); caucasian (96.2%); CV surgery (n=120), medical (n=45), gen/vasc surgery (n=17); heparin use: therapeutic (n=160), prophylaxis (n=17), catheter flushes (n=5). Measurements: mean OD±SD; objectively confirmed thrombosis (venous, arterial); all-cause mortality; length of stay (LOS); DTI use; composite outcome (new thrombosis, death, amputation); thrombotic composite outcome (new thrombosis, death from new thrombosis, amputation). Results: At HIT diagnosis, prevalent thrombosis occurred in 82 pts and isolated HIT occurred in 100 pts. Thrombotic presentations (n) included: venous alone (DVT-27, PE-9, DVT + PE-5), arterial alone (single event-28, multiple events-5), and venous plus arterial (8). Prevalent thromboses were symptomatic in 65 pts (79.3%). Fourteen pts developed a new thrombosis; 5 of them had isolated HIT. A total of 87 pts (47.8%) had “ever” thrombosis. The OD of the 182 pts was 1.12±0.76 with a cumulative thrombotic rate of 52.4%, 75.6% and 90.3% in OD ranges of 0.40–0.89, 0.4–1.49 and 0.4–2.29, respectively. No significant difference in rates of prevalent thrombosis was observed in any of 3 categorizations of OD values (% thrombosis): 0.40–0.99 vs. ≥1.00 (42.3% vs. 41.7%; P=0.457), 0.40–1.09 vs. ≥1.10 (47.9% vs. 40.0%; P=0.307) and 0.40–1.19 vs. ≥ 1.20 (47.5% vs. 40.0%; P=0.337). Among pts with “ever” vs. “never” thrombosis, there was no significant difference in OD values (1.14±0.84 vs. 1.10±0.69; P=0.692). Among pts with a new thrombosis, the OD was 1.45±1.36 (min 0.43, max 5.53). The OD was numerically higher in pts with isolated HIT who developed a new thrombosis vs. pts with “never” thrombosis (2.23±2.05 vs. 1.10±0.69; P=0.285). Males developed a higher rate of thrombosis than females (56.1% vs. 36.0%; P=0.008). Surgical pts comprised a higher proportion of “ever” thrombosis than medical pts (54.7% vs. 26.7%); P=0.001). All-cause mortality was significantly higher in the “ever” vs. “never” thrombosis groups (16.1% vs. 5.3%; P=0.017); highest among pts with isolated HIT (80.0%) and prevalent thrombosis who developed a new thrombosis (55.6%). HIT as cause or possible cause of mortality in pts with “never” thrombosis (1.1%) was significantly lower than those with “ever” thrombosis (10.3%, P=0.007) and those with new thrombosis (57.1%, P