ALBERT

Description: BK virus is a highly ubiquitous polyoma virus with a peak seroprevalence in childhood ranging from 60–100%. After primary infection, which is often asymptomatic or occasionally associated with fever and upper respiratory symptoms, the virus remains latent in the kidneys. During heavy immunosuppression, as occurs following agents such as Alem or anti-thymocyte globulin (ATG), the virus reactivates and can cause significant uroepithelial cell lysis with resultant hemorrhagic cystitis (HC). Between April 2003 and June 2004, 25 patients (15 males, 10 females) undergoing allogeneic (13 MSD, 12 VUD) hematopoietic stem cell transplantation for a variety of indications (18 MDS/AML, 3 NHL, 1 CLL, 2 MM, 1 CML) were found to have BK viruria. Patients ranged in age from 20–67 years. 16 patients underwent ablative conditioning (12 busulfex [Bu]-fludarabine [Flu]-Alem, 1 Bu-Flu-ATG, 2 total body irradiation (TBI)-cyclophosphamide (Cy), 1 TBI-etoposide) whereas 9 patients underwent reduced-intensity (RI) conditioning (5 Blu-Flu-Alem, 2 Bu-Flu-ATG, 1 Flu-Cy, 1 Cy-ATG). Patients treated on ablative regimens containing Bu received a total dose of 12.8 mg/kg whereas those treated with RI conditioning received 6.4 mg/kg. Those who received Flu got 30 mg/m2/d for 5 doses. Alem-based regimens contained 30 mg/d for 3 days or 20 mg/d for 5 days. Graft-versus-host disease (GVHD) prophylaxis consisted of: tacrolimus (FK) alone (n=16), FK + methotrexate (MTX) (n=7), FK + steroids (n=1), cyclosporine-MTX (n=1). 23 patients had BK viral loads in urine measured by PCR with peak levels ranging from 1500 copies/ml to 〉 200,000,000 copies/ml. BK virus was first detected in the urine anywhere from day −7 to day +85 post-transplant. The time to peak level of BK viruria ranged from day −2 to day + 85 (median 17 days). 40 % (10/25) of these BK viruric patients developed symptomatic HC and 16 % (4/25) developed uretostenosis and/or hydronephrosis (2 required ureteral stent placement). Only 1 of 14 patients treated with multiple doses of Cidofovir (range 3–26 doses) either cleared their viruria by qualitative assay or had greater than a 3 log reduction by quantitative PCR within 4 weeks of starting antiviral therapy; 2 of the 13 patients did have a 2 log reduction in viral load within 4 weeks of initiation of antiviral therapy. It is interesting that 27% (3/11) of patients not treated with Cidofovir cleared their BK viruria between 21–54 days post-transplant. Of note, an additional 6 patients were identified who received Alem; overall 21/23 patients who received Alem developed BK viruria. In conclusion, we found an extremely high incidence of BK viruria (91%) in patients receiving Alem as conditioning therapy for allogeneic SCT. This was associated with ureteral stenosis or hydronephrosis in 4 patients. Therapy with cidofovir given at two different doses had no impact on the excretion of BK virus.

Description: Chakravarti, (Blood, 2002, p1071) and others have described the use of alemtuzamab, a monoclonal antibody that binds to CD52 on T cells, B cells, monocytes and dendritic cells in vivo for control of graft vs host disease in reduced intensity or fully-ablative matched related and unrelated transplant regimens for hematologic malignancies. Control of GVHD has been good, but high relapse rates and infectious complications have been seen. We report our experience with 42 pts who underwent matched related (27) or unrelated (15) bone marrow (8), blood stem cell (33), or both (1) transplants for hematologic malignancies. There were 16 women and 26 men, median age 48, and median f/u is 9 months. All AML pts had high-risk disease defined as beyond CR1 (11), CR1 with high risk cytogenetics (5), AML with tri-lineage dysplasia (6), or failed prior autologous transplant (1) Three had 〉10% marrow blasts at time of transplant. All other pts had advanced and multiply recurrent disease. All pts received alemtuzamab either at 20 mg/m2/d x 5 (17), or 30 mg/m2/d x 3 (25) between days -8 through -4. The conditioning regimens also included 12.8 mg/kg IV busulfan, (27), 6.4 mg/kg IV busulfan (8), melphalan 140 mg/m2 (6), or Cy/TBI (1). All non-TBI patients received 30 mg/m2/d x 5 of fludaribine. Median recovery of ANC to 〉500/ul and platelets 〉 20,000/ul w/o transfusion were 13 and 14 days, respectively. TRM included 1 pt each with intracranial hemorrhage at day 8, multi-organ failure/sepsis at day 28, and IPS/ARDS at day 119. Two of the 3 deaths were in MRD transplants; no serious episodes of VOD were observed. Grade 2–4 GVHD was seen in 4/14 evaluable MUD pts and 1 (sex mismatched) of 26 evaluable MRD pts. CMV reactivation has been seen in nearly all CMV sero+ pts, and bk reactivation was seen in 25 pts with hemorrhagic cystitis in 10. One case of PTLD, 3 graft failures, and one severe case of disseminated adenovirus infection resulting in nephritis, cystitis and transient cardiomyopathy were also seen. Relapse/# evaluable for different diseases are as follows: AML 12/22, Gleevec-resistant CML 3/3, MDS 0/4, MM 3/3, NHL 0/3, ALL 0/3, CLL 0/2. Relapse rates in evaluable MRD pts, 15/26 (58%), were statistically higher compared to MUD pts, 3/14 (21%) (p = 0.05). RR in marrow, 2/8, vs BSC, 16/32, were comparable. Alemtuzamab use has been effective at controlling GVHD and infectious complications can be reduced with early and pre-emptive use of ganciclovir. Nevertheless, high relapse rates remain a significant problem and preclude routine use of this approach in MRD patients with advanced myeloid diseases. Current use of this agent has been individualized for donor (MRD vs MUD) and disease (myeloid vs other) status.〉

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: Congenital dyserythropoietic anemia type I (CDA I) is an inherited disorder characterized by macrocytic anemia and occasionally also by distal bone malformations. The disease has recently been shown to be caused by mutations in the CDAN1 gene, encoding codanin-1. The aim of the study was to characterize the CDAN1 mutations in 12 French patients with CDA I. The clinical data of the 12 French patients (10 kindreds) with CDA I were reviewed. Each of the 28 CDAN1 exons was amplified and sequenced. Half of the patients had a severe disease with prominent neonatal manifestations, complex bone disease, or both. Nine disease-causing mutations were identified: 6 described previously (P1130L, P671L, F869I, R681X, R713W, S1034F) and 3 novel mutations (R687W, F52L and IVS 8 G to A). Seven were missense mutations located in exons 14 to 28. Twenty seven per cent were identified in exon 14. No patient was homozygous for null type mutations. Only in two monozygotic twin patients did we fail to uncover a mutation. Mutations P671L, F52L and R713W were found on four, two and two alleles, respectively. One of the P671L alleles carried a second mutation (R687W). In conclusion the CDAN1 gene is apparently involved in most cases of CDA I in the French patients explored. Our study supports the previous findings that homozygosity for the null type mutations may be lethal, and that CDAN1 mutations are localized mainly in exons 12–28. In the present small group of patients, no apparent phenotype-genotype correlations were observed, in particular concerning abnormalities of the bones. Analysis of a larger number of patients from different ethnic groups is required to further characterize phenotype-genotype correlation in CDA I.

Description: Graft failure (GF), either primary or after transient engraftment, following matched, related allogeneic hematopoetic stem cell transplantation (HSCT) without T-cell depletion is usually less than 3% for hematological diseases. Graft failure using non-T-cell depleted unrelated donors in chronic myelogenous leukemia (CML) as reported by the national marrow registry is approximately 9.7%. For aplastic anemia, the incidence of graft failure is estimated to be 10%. In this report, we present 3 patients (Pts) with GF following unrelated ablative allogeneic HSCT (two with peripheral blood stem cells and one with bone marrow). Pt. 1 had transient engraftment and Pts 2 & 3 had primary GF. Their relevant demographics are as follows: TABLE 1 Disease Unrelated Donor Age/Sex GVHD: BMT1 GVHD: BMT2 PTLD Pt1 CML, 1st Chronic Phase Matched 29/F None Grade II Yes Pt2 MDS Matched 57/M None None No Pt3 MDS/AML 1ag Mismatched 55/M None None No All patients were initially conditioned with alemtuzumab (A) 30mg/day on days −8 to −6, fludarabine (F) 30mg/m2/day on days −7 to −3, and busulfan .8mg/kg x 16 doses (two patients) or x 8 doses (one patient) on days −5 to −2. Conditioning for HSCT #2 included cyclophosphamide 50mg/kg/day, Mesna 50mg/kg/day, and thymoglobulin 2.5mg/kg/day on days −5 to −2. Graft versus host disease (GVHD) prophylaxis included tacrolimus for both HSCT transplants, and low dose methotrexate (3 doses) combined with prednisone (2mg/kg) on days 0–18 followed by a two week taper for HSCT #2. The stem cell product and engraftment kinetics are shown below. Significant toxicities after HSCT#2 have included successful resolution of PTLD in patient #1 (who also experienced infection with pulmonary respiratory syncitial virus, BK viruria, and fatty infiltration of the liver). BK viruria was noted in patient #2, as well as hypertension and a modest pericardial effusion with global hypokinesis (but with a normal ejection fraction). Hepatomegaly was documented, but no vaso-occlusive disease was established. Patient #3 developed a cardiac arrhythmia, hypertension, and BK viruria and remains Plt transfusion dependent. Patient #1 had grade II GVHD of the skin and gut, but patients 2 and 3 had none. GVHD has been

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.