ALBERT

Description: Purpose: Despite therapeutic advancements, biological markers that predict the natural history of primary central nervous system lymphoma (PCNSL) are lacking and age and performance status are the only consistently identified independent prognostic variables. BCL6 rearrangements and deletion of the tumor suppressor gene R-PTP-κ (PTPRK) at 6q22 are thought to be common genetic abnormalities in PCNSL but their prognostic significance is unknown. The aim of this study is to determine the prevalence and survival impact of del(6)(q22), BCL6, immunoglobulin heavy chain (IGH), and MYC gene rearrangements and prevalence of Epstein-Barr virus (EBV) infection in PCNSL affecting immunocompetent patients. Patients and methods: Seventy-six specimens from 76 HIV-negative, immunocompetent patients with PCNSL newly diagnosed and treated at Mayo Clinic between 1985 and 2006 were studied. Interphase fluorescence in-situ hybridization (FISH) was performed using two-color break apart probes (BAP) for BCL6 and MYC, a two-color dual-fusion probe for IGH-BCL6, and a two-color probe for del(6)(q22) on thin sections of paraffin-embedded tumor samples. Two-color IGH BAP FISH probes were also used to confirm IGH rearrangements in cases showing extra IGH signals without fusion using the IGH-BCL6 probe. In situ hybridization was performed using probes that recognize EBV-encoded RNA (EBER) on paraffin-embedded tumor samples. Survival data were analyzed for patients diagnosed after 1997 (n=53), corresponding to the change to high dose methotrexate as the standard of care. Survival was calculated from the date of tissue diagnosis to date of death or last contact. Survival curves were estimated using the Kaplan-Meier method. The log-rank test was used to compare survival across groups. Two-tailed p-values

Description: Background: In deferasirox 1-yr core trials, doses were initially assigned according to baseline liver iron concentration. However, these trials demonstrated that transfusional iron intake has a profound impact on the outcome of chelation therapy and should therefore be considered when assigning deferasirox dose. A large number of patients (pts) were initially assigned 5 and 10 mg/kg/d doses, which were insufficient to balance iron intake from ongoing transfusions. Generally, deferasirox 20/30 mg/kg/d effectively maintained/reduced body iron. This analysis from the 4-yr extension trials evaluates the impact on serum ferritin (SF) of subsequent dose increases in pts who initially received 5/10 mg/kg/d, and the long-term effects of 20 and 30 mg/kg/d doses. Methods: Data for this analysis were pooled from 4 extension trials (106–109E). In the extensions, deferasirox doses were modified based on efficacy and safety markers, including iron burden and transfusional iron intake. SF was measured monthly. Results: In total, 227 pts initially received deferasirox 5 or 10 mg/kg/d, while 182 and 243 received 20 and 30 mg/kg/d, respectively. Underlying diseases included β-thalassemia (n=421), sickle cell disease (n=132), MDS (n=47) and other anemias (n=52). To date, pts have been receiving treatment for a median 3.4 (range: 0–4.5) yrs. Overall, median SF was maintained in the 20 mg/kg/d cohort (Table). In the 30 mg/kg/d cohort, SF levels decreased overall from baseline to month 42 (3734 ng/mL to 2025 ng/mL). However, levels plateaued at around 24 mos in this cohort, reflecting a decrease in mean dose to around 25 mg/kg/d. Median baseline SF in the 5/10 mg/kg/d dose group was 2051 ng/mL, which steadily increased during the first 18 mos of treatment. Subsequent dose increases during the extension phase generally resulted in decreased SF levels, which returned to baseline and below during the remainder of the study. Conclusions: In regularly transfused pts who initially received deferasirox 5/10 mg/kg/d in the core 1-yr clinical trials, SF steadily decreased below baseline once doses were increased to an appropriate level in the extensions. This highlights the importance of ensuring that pts receive the correct deferasirox dose to achieve the goal of therapy, based on iron burden and transfusional iron intake. If a pt is not achieving their therapeutic goal based on SF trends, deferasirox dose should be increased in steps of 5 or 10 mg/kg/d. This analysis confirms that deferasirox 30 mg/kg/d effectively reduces body iron, whereas doses of 20–25 mg/kg/d are generally effective in maintaining iron levels. Median change from baseline in SF (ng/mL) during deferasirox treatment of up to 3.4 years Initial dose, mg/kg/d 5/10 (n=227*) 20 (n=182*) 30 (n=243*) Month Mean dose ± SD† Change in SF (ng/mL) Mean dose ± SD† Change in SF (ng/mL) Mean dose ± SD† Change in SF (ng/mL) *Baseline; †At time point Baseline 2051 2375 3734 1 9.4 ± 1.7 90 19.5 ± 2.6 30 29.2 ± 4.3 −212 6 10.3 ± 3.9 399 18.9 ± 3.5 −15 28.2 ± 5.7 −532 12 13.0 ± 5.4 613 19.0 ± 4.0 −118 26.8 ± 6.6 −716 18 18.5 ± 6.7 831 18.2 ± 7.7 174 23.1 ± 8.6 −676 24 21.7 ± 6.6 635 21.5 ± 6.5 −125 24.5 ± 7.6 −901 30 22.6 ± 7.0 317 22.0 ± 8.5 −205 24.4 ± 8.0 −959 36 21.1 ± 8.7 −211 22.8 ± 7.7 −159 24.3 ± 8.5 −1002 42 21.8 ± 9.3 −65 23.2 ± 8.2 −204 25.8 ± 9.8 −955 EOS 1345 1667 2025

Description: Inefficient transduction, poor long term expression, and engraftment failure of ex vivo manipulated cells have slowed the practical advancement of gene therapy trials. Thus, the ability to select for or amplify a population of cells that has been modified to express a gene of interest might enhance the effectiveness of gene therapy. Strategies for in vivo expansion of genetically modified cells that have been studied to date have relatively high toxicity or low efficacy in selection of hematopoietic stem cells. We hypothesized that resistance to the purine analog 6-thioguanine (6TG) could be programmed via lentiviruses, and that treatment with 6TG would allow for selection of genetically modified cells in vitro and in vivo. Using short hairpin RNAs, we achieved efficient knockdown of hypoxanthine phosphoribosyl transferase (HPRTkd), the enzyme required for 6TG cytotoxicity, in the murine hematopoietic progenitor cell line FL5.12. In so doing we were able to provide Fl5.12 cells with resistance to 6TG. In the presence of 6TG, HPRTkd cells continued to proliferate for at least 30 days, whereas control transduced cells ceased proliferating after 7-10 days. 6TG treatment of mixed cultures of GFP+-HPRTkd cells and untransduced cells resulted in selective outgrowth of HPRTkd cells. Knockdown of HPRT in FL5.12 cells was found to attenuate the checkpoint activation, cell cycle arrest and apoptosis seen in control transduced cells when treated with 6TG. Knockdown of HPRT in murine primary hematopoietic cells also allowed for selection of transduced cells with 6TG ex vivo. Furthermore, and most importantly, after transduction of whole bone marrow and transplantation into sub-lethally irradiated recipient mice, a single, short course of treatment with 6TG resulted in up to 12 fold greater percentages of circulating transduced granulocytes as compared to untreated controls. These results suggest that genetically modified hematopoietic stem cells can be selected in vivo using 6TG. This strategy may be useful for therapy of a variety of hematopoietic diseases, particularly those that affect hematopoietic progenitors. The benefits of this strategy include the following: 1) the use of a lentivirus with a self inactivating long terminal repeat, 2) a very short cassette encoding drug resistance, making the vector easier to manipulate, and 3) a very well tolerated and relatively non-toxic medication for selection.

Description: Enucleation is the hallmark of erythropoiesis in mammals. Previously, we determined that yolk sac-derived primitive erythroblasts mature in the bloodstream and enucleate between E14.5–16.5 of mouse gestation (Kingsley, et al. Blood104:19, 2004), however, it is unclear by what mechanism or even where primitive erythroblasts enucleate. Definitive erythroblasts in the fetal liver and adult marrow enucleate by nuclear extrusion, generating reticulocytes and small, nucleated cells with a thin rim of cytoplasm referred to as “extruded nuclei”, that rapidly lose cell membrane phosphatidylserine asymmetry and are engulfed by macrophages. Since the cell membrane plays an important role in the biology of these cells, “extruded nucleus” is a misleading term. We propose that these cells be termed “pyrenocytes”, derived from the Greek “pyren” (the pit of a stone fruit) and “cyte” (cell), reflecting their highly condensed nucleus and high nuclear to cytoplasmic ratio. Careful examination of murine fetal blood by immunohistochemistry and multispectral imaging flow cytometry (Amnis ImageStream) revealed a transient population of εy-globin-positive pyrenocytes temporally coincident with the enucleation of primitive erythroblasts (E14.5–15.5). A high percentage (40%) of these circulating primitive pyrenocytes were annexin V-positive, suggesting that they, like their definitive counterparts, generate a signal that can facilitate engulfment by macrophage cells. At E14.5–15.5, the highest frequency of macrophage cells within the conceptus is found in the liver. Immunohistochemical studies with anti-εy-globin and F4/80 antibodies revealed that many primitive erythroblasts in the liver, but not the spleen, are in close proximity to macrophages. Furthermore, the frequency of nucleated primitive erythroblasts was higher in the liver than in the bloodstream at E15.5. These results, taken together, suggest that late-stage primitive erythroblasts do not passively flow through the liver but may, rather, interact with macrophage cells there. Surprisingly, primitive erythroblasts, but not co-circulating fetal definitive erythrocytes, can reconstitute erythroblast islands by attaching to fetal liver-derived macrophages in vitro. α4 integrin blocking antibodies, but not isotype control antibodies, reduce erythroblast island reconstitution, indicating that these primitive erythroid-macrophage interactions are mediated in part by α4 integrin. Finally, we found that unlike definitive erythroblasts, late-stage primitive erythroblasts fail to autonomously enucleate in vitro unless co-cultured with macrophage cells. We conclude that primitive erythroblasts in the mammalian fetus enucleate by nuclear extrusion as a semi-synchronous cohort and generate a transient population of pyrenocytes. Furthermore, our studies suggest that this process occurs in the fetal liver in association with macrophage cells. Continued investigation of the differences and similarities between primitive and definitive erythropoiesis will lead to an improved understanding of the terminal steps of erythroid maturation.

Description: Background: The efficacy and safety of deferasirox was established during five 1-year core trials. As many patients will require lifelong chelation therapy, assessing long-term efficacy and safety is important. In the core trials, initial doses were assigned by baseline liver iron concentration (LIC). A clear dose response was observed: 5/10 mg/kg/d doses were generally insufficient to balance iron intake from ongoing transfusions, while 20 and 30 mg/kg/d maintained or reduced iron balance, respectively. This analysis evaluates serum ferritin (SF) levels and cumulative safety data during 4-year extension trials. Methods: In the extensions, dose modifications were based on safety and efficacy parameters. Safety and SF were assessed monthly. Due to methodological differences in study 105, the SF data from this trial are not included in this analysis. Results: 1034 patients with β-thalassemia (n=750), sickle cell disease (n=185), MDS (n=47) and other anemias (n=52) have received deferasirox. 703 patients received deferasirox in the core trials and have been on treatment for a median of 3.4 years; 331 crossed over to deferasirox in the extensions. 210 (20%) patients have discontinued treatment due to: AEs (74), consent withdrawal (64), unsatisfactory therapeutic effect (29) and other (43). Only 17 (1.6%) patients have discontinued in the past 12 months (9 AEs; 1 death; 4 lost to follow-up; 2 withdrew consent; 1 other). 16 patients have died, 6 in the core and 10 in the extensions. 5 patients (4 β-thalassemia, 1 sideroblastic anemia) aged 18-24 years with inadequate chelation and severe iron overload before study entry died with cardiac failure in the extensions. Other deaths were due to other complications/progression of the underlying disease. At month 42, mean (SD) dose in the 5/10, 20 and 30 mg/kg/d dose groups was 21.8 (9.3), 23.2 (8.2) and 25.4 (9.8) mg/kg/d, respectively. Median SF levels (ng/mL) are shown in the Table (n=652, deferasirox cohort only). Drug-related AEs during deferasirox treatment were generally transient, of mild/moderate severity, and showed a reduction in frequency from the core trials. No patient has developed progressive increases in serum creatinine or values 〉2 x ULN. 2 patients discontinued due to stable creatinine increases of 1.5 x ULN and confounding circumstances (concomitant cyclosporine and multiple infections, respectively). 1 patient discontinued due to recurrent episodes of proteinuria. 12 patients discontinued due to increases in transaminases (4 core study, 8 extension [3 crossover patients]). Drug-induced liver toxicity was likely in 2 patients with early onset and positive rechallenge. Increasing LIC due to under-chelation was the likely explanation in at least some patients. Conclusions: Over 3.5 years’ treatment, deferasirox 20/30 mg/kg/d maintained/reduced SF levels in patients with various transfusion-dependent anemias. There was no increase in frequency of drug-related AEs or changes in markers of liver or renal function that differed significantly from the 1-year core trials. Initial dose, mg/kg/d *Dose adjustments Month 5/10 (n=227) 20 (n=182) 30 (n=243) Baseline 2051 2375 3734 12* 2650 2161 2649 24 2481 2508 2271 36 1439 1844 2071 42 1345 1667 2025

Description: EBV is known to be detectable within a number of different tumors, though the exact relationship between the malignant cells and the EBV virus is unknown in most cases. In post-transplant lymphoproliferative disorders, which are clearly EBV-driven, EBV PCR of peripheral blood has proven to be a clinically useful tumor marker. The goal of our study is to determine the utility of novel EBV PCR panel in EBV associated malignancies. Subjects with suspected active or untreated EBV-associated malignancies were evaluated with a 6 assay EBV PCR panel targeting LMP-1, EBER-1 and EBNA-1 within both whole blood cells and plasma. EBV positive subjects and EBV negative control tumor samples are tested for LMP and EBER using traditional histochemical staining. LDH, SPEP, and traditional EBV serology are also measured. Of the 40 patients who were evaluated to date, 18 subjects showed evidence of elevated blood EBV viral load: 3/5 nasopharyngeal carcinoma, 3/4 angioimmunoblastic lymphomas, 3/6 Hodgkin’s lymphomas, 4/10 peripheral T-cell lymphomas, 3/3 allogeneic bone marrow transplant lymphoproliferative disorders, 1/4 diffuse large B-cell lymphomas, 1/1 non-langerhans histiocytosis, and 0/7 low-grade B-cell NHL. Median DNA PCR copies/ml and ranges in EBV positive patients: EBNA plasma 500 (0–134,000), EBNA whole blood 700 (0–6,200), EBER plasma 200 (0–19,700), EBER whole blood 200 (0–1,000), LMP plasma 0 (0–600), LMP whole blood 0 (0–100). Patients were monitored with EBV PCR as they proceeded through treatment and 12 subjects are evaluable for response. Changes in viral load appear to be highly correlated with clinical tumor response (p=0.0013). 7/7 patients achieving CR had concurrent resolution of their plasma and whole blood EBV viral load. 5/5 patients with persistent disease had persistent elevated plasma and whole blood viral load. EBV histologic studies on the tumor samples are pending. Our data suggests that EBV PCR may be useful in the management of patients with EBV positive malignancies. Further patient enrollment and evaluation is ongoing.