ALBERT

Description: Introduction: Filgrastim is widely used for mobilizing CD34+ cells into the peripheral blood that are easily collected by apheresis for allogeneic transplantation. With case reports documenting splenomegaly with life-threatening complications in normal donors, we prospectively evaluated spleen size using ultrasonography and clinical examination during PBPC mobilization and collection in a single-arm trial. Methods: Subjects ≥18 yrs eligible to be PBPC donors per institutional guidelines enrolled. Splenic assessments were done before, during, and after PBPC mobilization. Filgrastim dose and schedule and leukapheresis (LK) procedures were per institutional practice. The primary endpoint was fold change from baseline in splenic volume in post-baseline measurements during mobilization (measured by ultrasound [US]). Spleen size by US was measured in 3 dimensions similarly by all centers: longitudinal (craniocaudal), transverse, and diagonal (perpendicular to transverse in transverse image) diameters. Splenic volume was estimated by taking the cross-product of 3 dimensions and multiplying by 0.52, approximating the volume of an ellipse. Physical examination was performed on US days, assessing spleen palpability. US and palpation results were blinded from each other at assessment times. Timepoints included baseline (before first filgrastim dose), first LK (done before LK, typically day 4 or 5 of filgrastim), 2 and 4 days after first LK, and 7 days after last LK. Timepoints in the post-amendment cohort (n=219) were reduced to facilitate enrollment and were baseline and day of first LK (before LK). Results: 309 donors enrolled, median age 44yrs (range 18 to 74), 56% male. Mean daily filgrastim dose was 11.4mcg/kg (SD=3.0). Median number of LK was 1.5 (range 1 to 4). In all donors, the median increase in each measured dimension on first LK day was 1.4cm, 1.4cm, and 0.6cm (12.8%, 12.6%, and 15.0%), and the median fold volume increase from baseline to first LK was 1.47, resolving to near baseline 1 week after last LK. There was no apparent relationship between volume fold change and filgrastim dose, ANC, or CD34+ yield. Of 861 splenic palpation assessments reported in all donors, 98% were reported as nonpalpable (842 assessments), and 2% were palpable (19 assessments, 2 at baseline). Reporting of palpable spleens did not correlate with increased spleen size. Tenderness or guarding upon splenic palpation was reported in 2 donors with a spleen considered palpable and in 6 donors with nonpalpable spleens. No donor experienced a splenic rupture. Adverse events related to filgrastim were generally mild to moderate. Conclusion: During PBPC mobilization with filgrastim in normal donors, the spleen increased a median of approximately 50% from baseline to day of first LK and returned to near baseline 1 week after last LK. Size change was not associated with significant clinical sequelae. Timepoint Median fold change from baseline in splenic volume (Q1, Q3) *statistically significant (p

Description: Graft versus host disease (GVH) incidence and severity increases with older patient age. Host dendritic cells (DC) are responsible for initiating GVH, presumably as a result of activation at the time of transplant conditioning (as they are short lived after transplantation). Age-related alterations in host DC function have not been studied in a transplant setting. In this study, young (8–14 wk) and older (12–16 mo) mice were treated with the conditioning regimen of lethal irradiation (1100 cGy) and assessed at 6 h later for changes in DC costimulatory molecule expression and cytokine secretion. Older mice had slightly higher numbers of splenic classical DC (cDC; CD11chi Cl II+ B220−) and plasmacytoid DC (pDC; CD11clo Cl IIlo B220+) both before and after irradiation. No differences were observed in CD40, CD80, or CD86 expression between young and older mice, either before or after irradiation. IL-12, IFNγ, and TNFα producing cDC were more numerous in young untreated mice than in older mice. However, these cells decreased in young mice and increased in older mice after irradiation, resulting in generally higher numbers of cells producing these cytokines in older mice. IL-12, IFNγ, and TNFα producing pDC were more frequent in older mice than in young mice prior to irradiation, and both older and young mice showed increased frequency of cells producing these cytokines after irradiation. Overall, the highest numbers of cDC and pDC producing IL-12, IFNγ, and TNFα were present in older mice after irradiation. In both cDC and pDC populations, older mice had a higher frequency of IL-10+ cells prior to irradiation. After irradiation, young mice had a higher frequency of IL-10+ cells. These findings indicate that the balance of cytokine production in older mice after conditioning with irradiation is shifted to immunostimulatory cytokines rather than immunosuppressive cytokines. Murine GVH can be prevented by treatment with regulatory DC (rDC) in young animals (Sato et al., Immunity 2003). To determine whether this treatment is feasible in older animals, older BALB/c mice were transplanted with young C57Bl/6 BM. A subset of each group received age-matched host derived rDC. As expected, among mice that did not receive rDC, old recipients got more rapid and severe GVH than young recipients. This correlated with an increased frequency of residual host DC at the time of GVH in the older group. rDC were used to successfully treat GVH in older (as well as young) transplant recipients. These results provide the foundation for a cell based therapeutic approach to the treatment of GVH in older individuals.

Description: BACKGROUND: Graft versus host disease (GVHD) remains a major cause of mortality after unrelated transplants. Reduced intensity conditioning regimens (RIC) followed by allogeneic stem cell transplantation (SCT) with in vivo T-cell depletion using Campath-1H have been shown to decrease the incidence of GVHD. We report our experience with such an approach in 19 patients (pts) with advanced hematologic malignacies who undergone unrelated SCT. PATIENTS AND METHODS: Between October 2001 and June 2003, 19 pts with a median age of 56 years (range 18–70) not eligible for ablative SCT received Campath-1H 20 mg/m2, fludarabine 25 mg/m2 × 5 days and either cyclophosphamide 1 g/m2 × 2 days (n=16pts), melphalan 140mg/m2 (1 pt) or busulfan 0.8 mg/m2 × 8 doses (n=2pts) followed by G-CSF stimulated peripheral blood (n=13) or unmanipulated bone marrow (n=6). GVHD prophylaxis consisted of cyclosporin at least until T+60 and mycophenolate mofetil through T+30. DLI was allowed for residual disease/progressive disease or mixed chimerism after T+ 60, in the absence of acute GVHD. Seven pts had a previous autologous transplant. Ten pts had myeloid diseases (AML=6, CML Ph negative=1, CML accelerated phase =1, therapy related MDS=1, myelofibrosis=1); 9 pts lymphoid (HD=3, NHD=5, PLL=1); Only 5 pts were in CR at transplant. Six pts received bone marrow witha median CD34+ cells infused of 3.7 × 106/Kg, and 13 pts received peripheral blood with a median of 4.5 × 106/Kg CD34+ cells infused. RESULTS:19 pts achieved ANC〉0.5 × 106/L within a median of 12 days (range 8–16); 2 pts rejected the graft and 1 had autologous reconstitutuion; 10/19 pts achieved sustained platelet engraftment at a median of 16 days (range7–27). 2/17 pts with sustained engraftment developed acute GVHD (grade IV T+30 and grade III T+53); 7/14 at risk pts had CMV reactivation; TRM at T+100 was 16% (disease progression in 2 and GVHD in 1). Nine pts received DLI (5 for mixed chimerism and 4 pts for persistent/progressive disease). After DLI, 5 pts had GVHD limited to skin. None of the pts receiving DLI for persistent/progressive disease responded. Response to transplant was as follows: CR in 3/6 AML pts, CR in 1/1 pt with myelofibrosis, CR in 3/3 HD pts, CR in 3/5 NHL pts. Five pts (26%) remain alive in CR at T+ 36, 42, 50, 56 and 66 months. Causes of death include disease progression/relapse in 7 pts, CMV disease in 1 pt, sepsis in 2 pts, GVHD in 3 pts (2 underwent a second transplant), other malignancy in 1 pt. CONCLUSION: These results confirm that unrelated SCT with in vivo T -cell depletion using Campath-1H is well tolerated. This approach is associated with low TRM even in pts with advanced disease, and some can achieve long-term disease control. Disease progression/relapse remains a major impediment to this approach.

Description: BACKGROUND: The success of allogeneic stem cell transplantation (SCT) is limited by transplant-related mortality (TRM). Death from pulmonary causes is a major contributor to TRM. Several factors influence TRM from pulmonary causes. We have analyzed the effect of cigarette smoking on pulmonary related TRM. METHODS: From January 2000 through April 2006 240 patients (pts) underwent allogeneic related or unrelated SCT. Diagnosis included ALL in 22 pts, AML in 90 pts, CLL in 11 pts, CML in 11 pts, Lymphoma in 49 pts, MDS in 29 pts, MM in 9 pts, SSA in 4 pts, solid tumors in 5 pts, other in 4 pts; median age of the pts was 50 years (range 18–70); 148 were males and 92 females; 48 pts received a reduced intensity regimen and 192 an ablative regimen; 117 pts received TBI; 123 pts were non-smokers (Group I) and 117 pts smokers. Amongst the smokers 86 pts provided pack/year information. In this group 46 pts (Group II) had a pack/year history 〈 30 and 40 pts (Group III) of ≥ 30. RESULTS: 70/123 pts in Group I are deceased with 44% surviving. In this group causes of death included disease progression/relapse in 17 pts, infection in 17 pts, organ failure in 13 pts, GVHD in 11 pts, other in 3 pts and pulmonary related TRM in 8 pts (6.5 ± 4%). 29/46 pts in Group II are deceased with 36% surviving. In this group causes of death included diseaseprogression/relapse in 10 pts, infection in 8 pts, organ failure in 1pt, GVHD in 3 pts, other in 2 pts and pulmonary related TRM in 5 pts (10.9 ± 9%). 31/40 pts in Group III are deceased with 22% surviving. In this group causes of death included disease progression/relapse in 11 pts, infection in 6 pts, organ failure in 5 pts, GVHD in 2 pts, other in 2 pts and pulmonary related TRM in 5 pts (12.5 ± 10 %). CONCLUSION: Smoking contributes to transplant-related mortality from pulmonary causes after allogeneic stem cell transplantation in a dose-related manner. In future analysis the interaction between smoking and other variables will be explored. Smoking and Outcomes Pt Group Surviving Pts (%) Deceased Pts (%) Pulmonary TRM Group I ( n=123) 44% 56% 6.5% Group Ii (n=46) 36% 64% 10.9% Group III ( n=40) 22% 78% 12.5%

Description: High dose therapy followed by autologous stem cell rescue for the treatment of multiple myeloma has been shown to be associated with improved outcome including increased complete response rates and survival. It is unclear what role, if any, autologous dendritic cell (DC) reconstitution patterns play in determining outcome. DC in peripheral blood (PB) can be divided into two subsets: DC1, which favor Th1 responses and DC2, which favor Th2 responses and in some cases appear to be immunosuppressive. In this companion study to a tandem stem cell transplant research protocol, PB DC subsets were studied just prior to the administration of mobilizing chemotherapy (baseline), at the time of stem cell collection, and on transplant d. 0, 15, 30, 60, and 90 following the first and second transplants. DC were identified as lineage (CD3/14/16/19) negative, HLA-DR+. Of these cells, DC1 = CD11c+CD123− or CD33hiCD4lo, and DC2 = CD11c−CD123+ or CD33lo/−CD4+. Precursor DC numbers (Pre-DC1 = CD45loCD34+HLA-DR+CD86+; Pre-DC2 = CD45loCD34+HLA-DR+CD10+) were also assessed in the stem cell products. GM-CSF (sargramostim) was used as part of the mobilization regimen as well as to enhance neutrophil recovery post-transplant. Twenty patients were enrolled and all are 〉90 d. post transplant #1. Six patients received a 2nd transplant. Median followup time is 25 months (range 3–56 months). Seven patients are in complete remission (CR), 7 have had a partial response, 4 have progressive disease, and 2 died of recurrent disease. In most cases, DC1 counts were higher than DC2 counts at all time points examined. At baseline, this was true for 70% of patients, indicating that immediately prior to transplant most patients maintain the DC1〉DC2 ratio found in healthy humans. DC subset numbers generally increased over baseline in mobilized PB, and correlated well with the patients’ baseline DC counts. Similarly, baseline DC subset numbers were highly correlated (R〉0.7) with the number of pre-DC1 or pre-DC2 found in the stem cell products. No patients with pre-DC110/ul) PB DC1 numbers at d. 90 consistently remain in CR. This study is the first to systematically examine DC subset reconstitution following autologous transplant for multiple myeloma. Our results provide preliminary evidence that the presence of high numbers of PB DC1 at d. 90 may predict a good long term prognosis, perhaps by promoting an effective endogenous immune response against residual disease. Furthermore, pre-DC1

Description: High dose therapy followed by autologous stem cell rescue for the treatment of multiple myeloma has been shown to be associated with improved outcome including increased complete response rates and survival. It is unclear what role, if any, autologous dendritic cell (DC) reconstitution patterns play in determining outcome. DC in peripheral blood (PB) can be divided into two subsets: DC1, which favor Th1 responses and DC2, which favor Th2 responses and in some cases appear to be immunosuppressive. In this companion study to a tandem stem cell transplant research protocol, PB DC subsets were studied prior to the administration of mobilizing chemotherapy (baseline), at the time of stem cell collection, on the day of transplant and at post transplant d. 15, 30, 60, and 90 following the first transplant. GM-CSF was used as part of the mobilization regimen as well as to enhance neutrophil recovery post-transplant. DC were identified as lineage (CD3/14/16/19) negative, HLA-DR+. Of these cells, DC1 were identified as CD11c+CD123− or CD33hiCD4lo, and DC2 were identified as CD11c−CD123+ or CD33lo/−CD4+. Sixteen patients have been enrolled and 11 are at least 90 d. post transplant #1. Median followup time is 12 months (range 7–25 months). 5 patients currently are in complete remission, 3 have had partial responses, 2 have stable disease, and 1 died of recurrent disease. In most cases, DC1 counts were higher than DC2 counts at all time points examined. At baseline, this was true for 77% of patients, indicating at baseline most patients maintain the higher DC1 numbers found in healthy humans. DC numbers increased in mobilized PB, and correlated well with the patients’ baseline counts. GM-CSF was more efficient at increasing DC2 than DC1, as has been reported for G-CSF. Post-transplant, DC recovery to at or near baseline levels occurred in most patients by d. 60. There was a trend for patients who are currently disease-free and those with partial responses to have a lower DC1:DC2 ratio (i.,e., DC2 reconstitution occurred more vigorously than DC1 reconstitution). The patient who died of progressive disease had the highest DC1 counts at several time points, and among the lowest DC2 counts leading to a consistently high DC1:DC2 ratio at 30, 60, and 90 days post-transplant. This finding suggests that in autologous transplant for multiple myeloma, proportionately increased numbers of DC2 are not associated with an increased risk of relapse, as was previously reported for allogeneic bone marrow transplant, but instead are associated with an improved likelihood of complete remission. Mobilization with a growth factor that favors the DC2 subset, such as GM-CSF, may promote the post-transplant production of a DC population that is able to assist in maintaining a complete response to high dose chemotherapy. If these trends are maintained upon final analysis of the patient population, additional means of selectively increasing DC2 numbers in this transplant setting would warrant further investigation.

Description: Donor-derived T cells have been proposed to play a role in pathogenesis of chronic graft-versus-host disease (cGVHD). The impact of ex vivo T-cell depletion (TCD) on cGVHD was analyzed in a randomized multicenter trial involving unrelated donor marrow transplants. A total of 404 patients diagnosed with hematologic malignancies received a total body irradiation-based myeloablative conditioning regimen. GVHD prophylaxis included TCD plus cyclosporine (CSA) or unmodified grafts with CSA plus methotrexate (M/C). Median recipient age was 31.2 years (range, 0.5-55.6 years); median follow-up time since randomization was 4.2 years. The mean number of T cells infused was 1 log lower on the TCD arm. The incidence of cGVHD at 2 years was similar between the TCD and M/C arms, 29% versus 34% (P = .27), respectively. Survival at 3 years from diagnosis of cGVHD was also similar, (TCD 51% versus M/C 58%; P = .29). The proportion of patients with cGVHD who discontinued immunosuppression at 5 years was not different (TCD 72% versus M/C 63%; P = .27), and incidence of serious infections and leukemia relapse were similar on both treatment arms. In spite of a significant reduction of acute GVHD, TCD did not reduce the incidence of cGVHD or improve survival in patients who developed cGVHD.