ALBERT

Description: Severe anemia is one of the most lethal complications in children infected with Plasmodium falciparum. The pathogenesis of this anemia is not completely understood. Experimental data from malaria-infected humans and animal models suggest that uninfected red cells have a shortened life span. This study looked for changes in the red cell surfaces of children with severe malarial anemia that could explain this accelerated destruction. A prospective case-control study was conducted of children with severe P falciparum anemia (hemoglobin of 5 g/dL or lower) admitted to a large general hospital in western Kenya. Children with severe anemia were compared with children who had symptoms of uncomplicated malaria and with asymptomatic children. Cytofluorometry was used to quantify in vitro erythrophagocytosis and to measure red cell surface immunoglobulin G (IgG) and the complement regulatory proteins CR1, CD55, and CD59. Red cells from patients with severe anemia were more susceptible to phagocytosis and also showed increased surface IgG and deficiencies in CR1 and CD55 compared with controls. Red cell surface CD59 was elevated in cases of severe anemia compared with asymptomatic controls but not as compared with symptomatic controls. The surface of red cells of children with severe P falciparum anemia is modified by the deposition of IgG and alterations in the levels of complement regulatory proteins. These changes could contribute to the accelerated destruction of red cells in these patients by mechanisms such as phagocytosis or complement-mediated lysis.

Description: PURPOSE: Rheumatoid arthritis (RA) has been associated with an increased risk of non-Hodgkin’s lymphoma (NHL). We describe the characteristics of NHL patients with antecedent RA. METHODS: Records of the Nebraska Lymphoma Study Group (NLSG) registry between 1982–2004 and the Mayo Clinic lymphoma registry between 1988–1998 were systematically reviewed for RA diagnosis and classified by the quality of supporting evidence. We abstracted available follow-up data through July 1, 2004. Definite cases had either a documented RA diagnosis from a trained rheumatologist or met 4 of 7 American College of Rheumatology criteria. Probable RA cases had a non-rheumatologist diagnosis of RA in addition to one of the following: 1) radiographic joint erosions; 2) disease-modifying anti-rheumatic drug (DMARD) use or; 3) RA-associated deformities (i.e., ulnar drift, swan-neck deformities). Five-year overall survival (OAS), event-free survival (EFS), and rates of treatment response were determined. RESULTS: We identified 74 RA cases (54 definite and 20 probable) with concomitant NHL. These patients had a mean age of 67 + 8 years and were predominantly Caucasian (82%) and female (64%). Of the probable RA cases, a majority had documented DMARD use (n=17). Approximately half of the cases used methotrexate (55%) and were deceased (52%) by the common closing date. The most common NHL subtypes included B-DLCL (n=31), B-FL-1/B-FL-2 (n=12), B-FL-3 (n=6), and B-BL/B-BLL (n=4). Of the remaining 21 cases, most were of B-cell origin (n=13) and included B-EMZL (n=3), B-LPL (n=3), B-UCL (n=2), B-UCL-LG (n=2), B-DFL-2 (n=1), B-MCL (n=1), and B-SLL (n=1). The remaining NHL subtypes included NHL-NOS (n=7) or T-PTCL-LC (n=1). B-symptoms were present for 6 (19%) patients with B-DLCL and none of the patients with B-FL-1/B-FL-2. Complete response rates to NHL treatment were 48% in B-DLCL and 33% in B-FL-1/B-FL-2. For B-DLCL and B-FL-1/B-FL-2, respectively, the five-year OAS was 54% (95% CI 36%–69%) and 65% (95% CI 31%–85%) while the five-year EFS was 48% (95% CI 36%–69%) and 56% (95% CI 24%–79%). For all NHL cases the 5-year OAS was 58% (95% CI 47%–69%) and the 5-year EFS was 49% (95% CI 37%–59%). CONCLUSIONS: Lymphomas developing in the context of RA most often include diffuse large B-cell and follicular subtypes. Complete response rates to standard treatment for these major subtypes ranged from 33 to 48%. For RA patients developing NHL, five-year overall- and event-free survival rates are approximately 50%.

Description: We report 28 patients (pts) who experienced late onset severe neutropenia (NCI/CTC grade III/IV) after rituximab. Rituximab had been given for the treatment of DLCL(N=15), follicular lymphoma (N=9), mantle cell lymphoma (N=2), CLL (N=2). Rituximab was administered as a front line treatment (N=11), for recurrent disease (N=17), before ASCT (N=1) or after ASCT (N=5). Rituximab was given as a single agent (N=14), with CHOP (N=9) or with other regimen (N=5). Rituximab was given at a dose of 375mg/m2 at weekly intervals over a period of 4 weeks when used as a single agent, or as a single dose of 375mg/m2 with chemotherapy. Characteriscs of neutropenia are summarized in Table. At the time of the neutropenia, all the pts were in CR or VGPR. In 3 pts, the neutropenia had relapsed. All 3 pts were still in CR, and had not been retreated with rituximab. 3 patients were retreated with rituximab for a recurrence of their NHL after the outbreak of neutropenia and 1 pt experienced a further episode of neutropenia after the reintroduction of rituximab. Tests for anti PMN antibodies were performed in 6 pts. In 4 pts, antibodies bound to the surface of neutrophils were detected by the direct neutrophil immunofluorescence test. No antibody could be detected in the serum. A direct toxic effect of rituximab can be ruled out as granulocytes and uncommitted hematopoietic precursor stem cells do not express CD 20. We propose the following hypothesis. The rituximab-induced depletion of the normal B-lymphocyte population was followed by the acquisition of a new immune repertoire under non physiological condition which could promote the transient production of autoantibodies. Some of these antibodies might target either neutrophils or hematopoietic precursors. Some of these antibodies might also be directed against other cells since other delayed-onset cytopenia have been reported after the administration of rituximab, pure red aplasia in particular. An other possible mechanism can be proposed. Rituximab administration could lead to the production of antibodies directed against the complex formed when rituximab is bound to the FcγRIIIb receptor on the PMN. This hypothesis does not account for the delay between the administration of rituximab and the onset of the neutropenia. Characteristics of neutropenic episodes. Characteristics N *: interval between the last infusion of rituximab and the nadir of neutropenia. **: 1 pt was lost to follow-up for 4.5 months but had a normal blood count subsequently. Median time to neutropenia (range) (weeks)* 15 (4–33) Nadir PMN 10x9/L median (range) 0.135 (0–0.760) Bone marrow aspiration 14 Hypocellular marrow 7 Normocellular marrow with severe reduction in mature neutrophils 6 Normal 1 Fever/sepsis 6/1 Patients treated with G-CSF/duration (range) (days) 12/6 (3–21) Duration of neutropenia (range) (days) Patients treated with G-CSF N=12 4 (2-53) Patients not treated with G-CSF N=16 12 (4–105)** Follow-up from the nadir of neutropenia (range) (month) 6 (0.5–36) NHL/CLL status at follow-up CR or CRu 22 VGPR 1 Progression 5 Status of PMN count at follow-up Normal 26 Neutropenia 2

Description: Limited cell dose within a single UCB graft provides the rationale for multiple UCB unit transplantation. Our single institution phase I study testing the safety and efficacy of multiple UCB unit infusion targeted a nucleated cell dose of minimum ≥ 5x107cells/kg, and patients were transplanted with 3-5 unrelated UCB units. Seven adult patients (median 56 years; range 20–69) with advanced hematologic malignancies (4 AML, 1 ALL, 2 NHL) were enrolled and treated with non-myelablative conditioning including Fludarabine 150mg/m2, Cyclophosphamide 2gm/m2, and ATGAM 60mg/kg. UCB grafts were not T-depleted. All UCB units were 1-2 HLA antigen mismatched with the patient, and HLA matching between units was not required. The patients were transplanted sequentially and received a median infused total nucleated cell dose: 5.4x107/kg (range 4.2–8.9), CD3+: 1.4x107/kg (range 1.4–3.4), and CD34+: 2.2x105/kg (range 1.9–5.3). Three of the 7 patients demonstrated UCB donor engraftment while 3 patients had autologous recovery, and one patient died prior to engraftment (day=56). Mixed lymphocyte culture (MLC) was performed including proliferation and cytokine production in order to evaluate impact of graft-graft and patient-graft immune reactivity on donor engraftment. Cryogenically preserved pre-transplant patient and corresponding UCB graft samples were thawed for MLC with readouts including proliferation (CFSE staining) and cytokine production (cytometric bead assay-CBA)(Becton Dickinson, Franklin Lakes, NJ) including pro-inflammatory TH1 cytokines (IFN-γ, TNF-α, IL-2) and anti-inflammatory TH2 cytokines (IL-10, IL-5, IL-4). We hypothesize that increased proliferation and a strong TH1 response may be detrimental to engraftment which was confirmed by preliminary analysis (n=4). We observed higher rates of proliferation as well as higher TH1 cytokine production within the MLC of patients who did not attain donor engraftment. Table 1: TH1 Cytokine Output and Proliferation of Patient and Graft Mixed Lymphocyte Cultures Patient Number Number of UCB Units Donor Engraftment IFNγ(pg/mL) TNFα (pg/mL) IL-2(pg/mL) % Proliferation Pt #1 5 No 940 226 79 16 Pt #2 3 No 234 56 46 20 Pt #3 3 Yes 32

Description: In systemic vasculitis, interactions between antineutrophil cytoplasm autoantibodies (ANCAs) and neutrophils initiate endothelial and vascular injury. ANCAs directed against either myeloperoxidase (MPO) or proteinase 3 (PR3) can activate cytokine-primed neutrophils by binding cell surface–expressed MPO or PR3, with the concurrent engagement of Fcγ receptors (FcγR). Because roles for phospholipase D (PLD) and phosphatidylinositol 3 kinase (PI3K) have been demonstrated in FcγR activation of neutrophils, this study investigated the hypothesis that ANCA stimulation of neutrophils involved a similar engagement of FcγR and activation of PLD and PI3K. Pretreatment of tumor necrosis factor (TNF) α-primed neutrophils with antibodies against FcγRII and FcγRIII inhibited MPO-ANCA and PR3-ANCA induced superoxide generation, confirming that FcγR ligation is involved in ANCA-mediated neutrophil activation. However, although stimulation of TNF-α–primed neutrophils by conventional FcγR ligation, either using antibody-mediated cross-linking of FcγR or aggregated IgG, induced PLD activation, ANCA stimulation did not. Moreover, although ANCA-induced neutrophil activation results in significant PI3K activation—as assessed by phosphatidylinositol 3,4,5-triphosphate generation—conventional FcγR ligation, but not ANCA, activates the p85/p110 PI3K subtype. Inhibition of ANCA-induced superoxide generation with pertussis toxin suggests that ANCAs activate the p101/p110γ PI3K isoform. In addition, the kinetics of activation of protein kinase B differs between conventional FcγR ligation and ANCA stimulation of neutrophils. These results demonstrate that though ligation of FcγRIIa and FcγRIIIb may be necessary, it is likely that ANCAs require other membrane cofactors for neutrophil activation.

Description: Numerous studies have reported significant associations between ABO blood group and risk for cardiovascular disease. These studies have consistently demonstrated that thrombotic risk is significantly reduced in blood group O individuals. Nevertheless, the biological mechanisms through which ABO influences hemostasis have remained poorly understood. Exciting recent data have provided novel insights into how these ABO effects are modulated, and highlighted that ABO group significantly influences platelet plug formation at sites of vascular injury (primary hemostasis). In particular, ABO affects multiple aspects of von Willebrand factor (VWF) biology. In keeping with their reduced thrombotic risk, plasma VWF levels are approximately 25% lower in normal group O compared to non-O individuals. In addition, blood group O VWF demonstrates enhanced susceptibility to ADAMTS13 proteolysis. Finally, preliminary findings suggest that the ability of group O VWF to interact with platelets may also be reduced. Although the molecular mechanisms underlying these ABO effects on VWF have not been fully elucidated, it seems likely that they are mediated in large part by ABO(H) carbohydrate structures carried on both the N- and O-linked glycans of VWF. Interestingly, ABO(H) determinants are also expressed on a number of different platelet surface glycoprotein receptors. Recent studies support the hypothesis that ABO group not only exerts major quantitative and qualitative effects on VWF, but also may affect specific aspects of platelet function. Given the huge morbidity and mortality associated with thrombotic disorders, defining the mechanisms underlying these ABO effects is not only of scientific interest, but also of direct clinical importance.

Description: Cytokine-based expansion of umbilical cord blood (UCB) in vitro prior to infusion has been pursued in an attempt to overcome the limited cellular content of a single UCB unit. Thus far, these attempts have not shown improvement in kinetics of donor-derived hematopoietic recovery. Our studies have incorporated UCB expanded over a feeder-layer of human mesenchymal stem cells (huMSC), known to inhibit the differentiation of hematopoietic stem cells (HSC) observed in expansion with cytokines alone. Expansion conditions included: UCB expanded over a huMSC monolayer with the addition of cytokines (IL-3, IL-6, G-CSF, SCF, FLT-3L, EPO) and UCB expanded in the same cytokines alone. Day 12 culture readouts included: viable cell counts, 4-color flow analysis, and rates of human engraftment in NOD/SCID mice. In the current study the fold expansion was 6.4 fold in the huMSC + cytokines condition and 7 fold in the cytokines alone condition. Flow cytometry surface marker analysis proportions (absolute numbers) were notable for higher proportions and numbers of early HSC expressing CD133 in cultures incorporating huMSC stromal layer: Unexpanded MSC+ cytokines Cytokines CD34 0.68 (.068M) 0.74 (3.63M) 1.94 (5.39M) CD133 5.69 (.569M) 2.56 (12.54M) 0.74 (2.06M) CD3 49.6 (4.96M) 2.2 (10.78M) 0.42 (1.17M) CD56 17.4 (1.74M) 2.71 (13.28M) 1.06 (2.95M) CD69 0.80 (7.28M) 7.28 (35.67M) 24.4 (67.8M) UCB graft T and NK populations were maintained in huMSC culture conditions and the observed difference in CD69 expression supports the hypothesis that huMSC may have an inhibitory effect on T cell activation during UCB ex vivo expansion. To assess the human engraftment potential of the cultures, cells from each culture condition were injected by tail vein into NOD/SCID mice (no CD34 selection was performed). Mice receiving unexpanded UCB received 10M mononuclear cells each. Mice receiving culture expanded cells received cell doses in proportion to the fold expansion over the number of cells at the initiation of the cultures. Engraftment was assessed by the percentage of human CD45+ (≥0.4%) cells found within the bone marrow of mice at seven weeks post infusion. Mice were injected as follows: 7 mice with unexpanded UCB (2 of which died within a month of transplant), 7 mice with UCB expanded in huMSC + cytokines, and 3 mice with UCB expanded in cytokines alone. Flow analysis of mouse bone marrow cells revealed average CD45+ percentages of 1.79% for mice injected with unexpanded UCB, 2.66% for mice injected with cytokine alone cells, and 5.94% for mice injected with huMSC + cytokine cells. Human cell subset analysis was performed for CD3, CD19, and CD56 content. The percentages of gated CD45+ co-expressing CD3+ were 10.3% in the unexpanded UCB, 16.6% in the cytokine alone condition and 10.4% in the huMSC + cytokine condition. Cells co-expressing CD19+ were 7.86% in the unexpanded UCB, 8.31% in the huMSC + cytokine condition and dropped to 1.43% in the cytokine alone condition. Gated CD45+ cells co-expressing CD56+ were 16.4% in the unexpanded UCB, 8.8% in the huMSC + cytokines condition, and dropped to 2.6% in the cytokines alone condition. In conclusion, UCB expanded short-term in cytokines demonstrates maintenance of earlier HSC phenotype and improved human engraftment in NOD/SCID in cultures incorporating a huMSC monolayer platform.

Description: BACKGROUND: Mantle cell lymphoma (MCL) typically has a poor outcome with overall survival of only 3–4 years. Higher treatment response and event-free survival has been demonstrated with aggressive high dose chemotherapy followed by autologous hematopoietic stem cell support, though long term cure rates remain unclear(Dreger P. Hematol J. 2000;vol.2). Modest response rates have also been reported with the monoclonal antibody (MoAb) rituximab and ALEMTUZUMAB (Foran, JM. JCO 2000; vol. 2. Faderl S. Blood 2003; vol. 9). We therefore combined dose-dense therapy with MoAbs to explore response rate and event free survival (EFS) in mantle cell lymphoma. The strength of this trial design is ability to follow all patients from induction chemotherapy through high dose therapy and transplant in order to gauge clinical outcome on all enrolled patients, not just the subpopulation who is able to proceed to high dose therapy. PATIENTS AND METHODS: Induction therapy consisted of 1 cycle of high dose cytarabine (3gm/m2 IV over 1 hour Q12H for 8 doses), mitoxantrone (10mg/m2 daily for 3 days), and ALEMTUZUMAB 30mg IV 3 times a week for 6 weeks with growth factor support. All responding patients were mobilized with cyclophosphamide 4gm/m2 and G-CSF 10 mcg/kg/day and/or bone marrow harvest. The transplant preparative regimen was carmustine 15mg/kg over 2 hours day -6, etoposide 60mg/kg over 4 hours day -4, and cyclophosphamide 100mg/kg over 2 hours day -2 followed by autologous reinfusion on day zero. Consolidation was given with rituximab 375mg/m2 weekly for 4 doses at 6 weeks and 6 months post transplant. RESULT: 9 patients with advanced disease (7 stage IV, 1 stage III, 1 stage IIA) and median age of 60 (48 – 65 years) have been accrued and treated since February 2003. Four were newly diagnosed and 5 had relapsed/refractory disease. Seventy eight percent (7/9) had complete response and 22% (2/9) had partial response (PR) following induction therapy. One patient had severe infection after induction and was unable to proceed to transplant. Another had constitutional decline preventing further therapy and each died within 4 months of withdrawal from the protocol. Both had relapse/refractory disease at accrual. The remaining 7 patients proceeded to the transplant phase. With a median follow-up of 7 months (range 3–16 months), all 7 patients remain in CR for 1 –16 months. Significant induction therapy toxicity included neutropenia in all 9 patients with average duration of 10.7 days, non-disseminated CMV reactivation in 44% of patients, one overwhelming fungal infection, and one patient with delay in engraftment. Figure Figure CONCLUSION: Our preliminary data show a high induction and transplant phase completion rate, manageable toxicity, and excellent overall response rate in this group of elderly patients with advanced disease. Larger numbers of patients and longer follow-up is needed to confirm these promising results.

Description: Cytochrome P450 1B1 (CYP1B1), a drug-metabolizing extrahepatic enzyme, was recently shown to be overexpressed in multiple types of cancer. Such tumor-associated genes may be useful targets for anticancer therapy, particularly cancer immunotherapeutics. We identified HLA-A*0201–binding peptides and a naturally processed and presented T-cell epitope capable of inducing CYP1B1-specific cytotoxic T lymphocytes (CTLs) in HLA-A2 transgenic mice. Furthermore, the induction of CYP1B1-specific T cells was demonstrated in healthy donors and cancer patients. These T cells efficiently lysed target cells pulsed with the cognate peptide. More important, HLA-A2–matched tumor cell lines and primary malignant cells were also recognized by CYP1B1-specific CTLs. These findings form the basis of a phase 1 clinical trial exploring a DNA-based vector encoding CYP1B1 for widely applicable cancer immunotherapy conducted at the Dana-Farber Cancer Institute.

Description: CD38 is a transmembrane glycoprotein expressed on the surface of leukemic cells in a significant percentage of patients with B-cell chronic lymphocytic leukemia (B-CLL). A recent study suggested that CD38 expression has prognostic value in CLL. Peripheral blood samples from 218 patients with B-CLL were analyzed by flow cytometry for CD38 expression on CD5/19+ leukemic cells. Various patient characteristics were studied including age, sex, Rai and Binet stages, splenomegaly, hepatomegaly, hemoglobin (Hgb) level, β-2 microglobulin (β2M) level in the serum, number of nodal sites involved with disease, and length of survival. The Kaplan-Meier method was used to construct survival curves, and the log-rank statistic was used to compare these curves. CD38 was expressed in 20% or more of leukemic cells in 43% of the patients. Patients with high CD38 expression (20% or more) had significantly shorter survival times (P =.00005). Multivariate analyses showed that CD38 expression is an important prognostic factor associated with high incidence of lymph node involvement (P = .004), lower hemoglobin level (P = .001), hepatomegaly (P = .05), and high β2M level (P = .00005). CD38 expression identified a group of patients with aggressive disease that was considered by Rai staging to be early-stage disease (Rai stages 0-II). Patients with CD38+ samples have significantly aggressive disease regardless of their clinical stage. Measurement of CD38 expression by flow cytometry should become a routine test in the evaluation of patients with CLL.