ALBERT

Description: Congenital dyserythropoietic anemia (CDA) type I is an inherited autosomal recessive macrocytic anemia associated with ineffective erythropoiesis and the development of secondary hemochromatosis. Distinct erythroid precursors with inter-nuclear chromatin bridges and spongy heterochromatin are pathognomonic for the disease. The mutated gene (CDAN1) encodes a ubiquitously expressed protein of unknown function, codanin-1. Based on the morphological features of CDA type I erythroblasts and the preliminary data on the Drosophila homolog, dlt, which was found to be required for cell survival and cell cycle progression, we investigated the location and the behavior of codanin-1 during the cell cycle. Using immunofluorescence and immune electron microscopy, we localized codanin-1 to the heterochromatin in interphase cells. During the cell cycle, high levels of codanin-1 were observed in S phase. At mitosis, codanin-1 underwent phosphorylation, which coincided with exclusion from condensed chromosomes. The proximal CDAN1 gene promoter region, never before characterized, was found to contain 5 putative E2F1 binding sites. E2F transcription factors are the main regulators of G1/S transition. Cotransfection of an E2F1 expression plasmid increased luciferase activity, confirming that E2F1 activates the transcription of CDAN1, and chromatin immunoprecipitation identified the codanin-1 promoter as a direct target of E2F1. Taken together, these data suggest that codanin-1 is a cell cycle-regulated protein active in S-phase. Based on the localization of codanin-1 to the heterochromatin and the spongy appearance of heterochromatin in CDA I, we suggest that codanin-1 may be involved in heterochromatin organization during DNA replication. This represents the first work towards understanding the function of the proteins involved in CDAs.

Description: Over the past decade, the use of granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood stem cells (PBSCs) during allogenic transplant has become common. Compared to the transplant of unmanipulated bone marrow, PBSCs showed earlier engraftment, but also showed more frequent and extensive chronic graft versus host disease (cGVHD). In an effort to monitor whether G-CSF stimulated bone marrow led to comparable engraftment and overall survival (OS), without increased cGVHD, we followed pts with matched related donors in two nonrandomized, sequential studies. One cohort received G-CSF mobilized PBSCs and the second received G-CSF stimulated bone marrow (GSBM). Eligible pts with ALL (2), AML (9), CML (19), MM (9) or recurrent/refractory NHL/HD (7) were enrolled from February 1994 through December 1998. All pts received busulfan (16mg/kg except MM 14mg/kg) and cyclophosphamide (120 mg/kg) followed by CSA and MTX for GVHD prophylaxis. Details of these studies have been previously published by Serody et al., Biol Blood Marrow Transplant.2000;6:434–40. At the time of the current analysis, the median follow-up for the surviving pts in the group that received PBSCs was 9 years, and 8 years for the group that received GSBM. At last count, 7/20 (35%) of the PBSC pts were still alive and 6 were disease-free. Median OS time and disease-free survival (DFS) time was approximately 3 years. In the GSBM group, 9/26 (35%) were alive and disease-free and one was alive with recurrence. Median OS time was 2 years with a median DFS time of 1.4 yrs. The DFS and OS difference between the PBSC and GSBM cohorts was not statistically significant (p = 0.6 and p = 0.9, respectively). We also divided pts into high risk (HR) (20 patients) and low risk (LR) (26 patients) groups. Analysis of the combined PBSC and GSBM cohorts showed that the LR pts did better than the HR pts. Median survival time for the combined HR pts was about 1 year while the median survival time for LR pts has not been reached (p=0.003). There was no significant difference in outcomes between the HR or LR pts treated with PBSC vs GSBM. At last count, the incidence of cGVHD was 12/20 (60%) in the PBSC cohort, and 11/26 (42%) in the GSBM group (not a statistically significant difference, p=0.37). Earlier, at one year post transplant (Serody et al, 2000), the percentages were 68% versus 37%, respectively, and statistically significant (p=0.049). While the current 18% incidence difference is not statistically significant, this percentage may represent a difference that is clinically relevant and that would be statistically significant with more pts. Unlike the cGVHD results, this latest analysis continued to demonstrate a lack of significant difference in OS and DFS results between the PBSC and GSBM cohorts.

Description: Busulfan is an alkylating agent that is frequently used as the ablative component of allogeneic stem cell transplants in conjunction with immunosuppressive drugs such as cyclophosphamide or fludarabine. Busulfan has recently become available for IV use and has been shown to be effective and less toxic due to more reliable pharmacology than the oral form of this drug. While prolonged administration of this drug has not been feasible in the past, the parenteral formulation now makes this possible and permits re-evaluation of the efficacy, toxicity, and optimal dosing of this compound. Such an approach may reduce the dose-limiting hepatic, pulmonary and CNS toxicities that are seen with this compound. Here we report on 15 patients treated on a study using a continuous 90 hour infusion of this drug at 12.8 mg/kg adjusted body wt along with 30 mg/m2 fludarabine qd x 5 and alemtuzamab at doses of 0, 30, or 90 mg depending on the disease (myeloid or non-myeloid) and donor (MRD or MUD) followed by SCT. GHVD prophylaxis also included tacrolimus for all pts and short-course methotrexate for MRD patients who did not receive alemtuzamab. Subjects included 11 males and 4 females, median age 38, 6 MUD, 9 MRD, and 9 AML, 2 APL, 2 CML, 1 MDS and 1 ALL pt. All were high-risk based on being in 2nd remission or beyond (4), or having MDS or trilineage dysplasia(4), poor cytogenetics (3), refractory disease (2), prior transplant (1), or advanced CML (1). There have been no grade 3 or 4 toxicities due to drug infusion or during the initial period of aplasia. Neutrophil and platelet engraftment occurred on day + 14 and day + 16 respectively. There was one graft failure in a 54 yo man with MDS/AML who received a sex mismatched MUD transplant and subsequently underwent successful re-transplant on day 50. There have been two cases of grade II, one grade III and one grade IV aGVHD in 2 MRD and 2 MUD pts. Relapses have occurred in 5 pts after a median f/u of 10 months. Pharmacokinetics were obtained on 11 pts and compared the AUC obtained with a test dose given the day prior to initiation of the 90 hour infusion and the AUC of the continuous infusion. Test dose AUC predicted the full dose AUC with 〉 90% accuracy (range 3–19%, precision and bias of 9.5 and 1.4%). Mean daily AUC was 3539 umol min (range 1936 to 5591) with minimal variation between day 1 and 4 for the full-dose infusion. This is similar to the mean AUC observed in a control group of 8 pts treated with q 6 hr dosing and reflects the 3-fold inter-patient variability that has been reported when the parenteral drug is administered by intermittent infusion. 3 of the 4 pts with AUC data who have relapsed have had AUC concentrations below the mean. No AUC targeting was undertaken in this study but the predictive capacity of the test-dose strategy and the low AUC suggest that targeted dosing would be feasible and desirable with this approach. Studies with targeted dosing and prolonged infusion are currently underway to identify better the risks, toxicities and MTD of this approach.

Description: The first joint common volume measurements by the Digisonde Portable Sounder (DPS-4) and a new Doppler type system has been run at the Pruhonice ionospheric observatory (49.99° N, 14.54° E) since January 2004. The measurement of the Doppler shift is carried out continuously on a frequency of 3.6 MHz, thus the radio wave is reflected predominantly from the ionospheric F layer. To compare digisonde measurements with the Doppler data, a phase path was calculated from both Doppler and digisonde records. Under stormy conditions and in the case where a sporadic E layer was present, a significant disagreement between both measurements has been found. The discrepancies could be related to the uncertainties of the observational inputs and to the interpretation of the digisonde data. The comparison of the phase paths shows that during geomagnetically quiet days, in the absence of the sporadic E layer, and when high quality ionograms are available and correctly scaled, the electron density N(h) profiles, calculated by the Automatic Real Time Ionogram Scaler with True height algorithm (ARTIST), can be considered reliable.