ALBERT

Description: T-cell and B-cell reconstitution was studied in nine patients who received fluorescence activated cell sorter (FACS)-sorted autologous CD34+ hematopoietic progenitor cells (HPC). The mean numbers of T cells (CD3+), B cells (CD19+) and CD34+ HPC administered to each patient were .004, .002, and 1.8 × 106 cells/kg, respectively. After high-dose myeloablative chemotherapy (busulfan, cyclophosphamide, etoposide) CD34+ HPC were infused and lymphoid reconstitution was monitored using flow cytometry and reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of VDJ T-cell receptor (TcR) sequences. Restoration of normal numbers of peripheral blood T cells and B cells among recipients of FACS-sorted CD34+ HPC was delayed compared to recipients of non-T-cell–depleted PBSC autografts. In both patient groups, the circulating T cells were primarily CD4−, CD8+, αβ TcR+, and CD45RO+, CD45RA− during the first 2 months after transplant. Subsequent increases in the frequency of CD45RA+ CD45RO− T cells occurred at 2 to 3 months after transplant, suggesting maturation of CD34+hematopoietic progenitors to “naive” T cells. Analysis of the TcR repertoire after hematopoietic reconstitution demonstrated decreased diversity of Vβ TcR expression associated with global decreases in the absolute number of total peripheral blood T cells and most Vβ TcR+ subsets. Three of nine recipients of FACS-sorted CD34+ HPC demonstrated significant increases in the percentage of γδ+ peripheral T cells and CD5+ B cells at 3 to 9 weeks after transplantation, and all patients had transient oligoclonal expansions of T cells expressing specific Vβ TcR. Transplantation with highly purified CD34+ HPC results in reduced diversity of the peripheral T-cell repertoire during the early post-transplant period compared with patients receiving unmanipulated or MoAb-depleted transplants.

Description: Nonmyeloablative (NM) regimens for allogeneic (allo) hematopoietic cell transplantation (HCT) may be effective and associated with a low incidence of acute graft-versus-host disease (GVHD) in high-risk patients (pts) with hematologic malignancies. We report the results of NM bone marrow transplantation (BMT) from HLA-matched volunteer unrelated donors (VUDs) in 14 pts (median age 45 yr; range, 19–58 yr) with relapsed and/or refractory hematologic malignancies (1 ALL, 3 AML, 3 MDS/AML, 3 Hodgkin’s disease [HD], 2 multiple myeloma, 2 NHL). Thirteen pts had previous HCT: 10 autologous (auto), 1 related allo, 1 VUD, 1 auto and VUD. The NM regimen included alemtuzumab (20 mg/day x 5, days −8 through −4), fludarabine (30 mg/m2/day x 5, days −7 through −3) and melphalan (140 mg/m2 on day −2), as described by Kottaridis PD et al, Blood2000; 96: 2419 and Chakraverty R et al, Blood2002; 99: 1071. All pts received single-agent cyclosporine for prophylaxis of acute graft-versus-host disease (GVHD). Median doses of CD34+ and CD3+ cells were 2.79 (range, 1.35–4.87) x 106/kg and 2.8 (range, 1.99–6.39) x 107/kg, respectively. In 12 evaluable pts, median times to absolute neutrophils 〉0.5 x 109/L and platelets 〉20 x 109/L were 15 (range, 9–41) and 21 days (range, 15–41), respectively, after NMBMT. Two pts had primary graft failure (actuarial probability 16.4%; 95% confidence interval [CI], 0–37.6%). Two pts developed acute GVHD (1 grade I, 1 grade II) at 19 and 76 days, respectively, after NMBMT. The actuarial probability of all grades of acute GVHD is 16.1% (95% CI, 0–36.7%) and of grade II or greater GVHD is 9.1 % (95% CI, 0–26.2%). Two pts (1 with grade II acute GVHD) developed extensive chronic GVHD. Five pts developed cytomegalovirus (CMV) reactivation, for an actuarial probability of CMV reactivation of 58.5% (95% CI, 19.9–97.1%). One pt with CMV reactivation died with CMV infection at 122 days after NMBMT. Six pts died with other infections at a median of 74 days (range, 52–209) after NMBMT: 2 adenovirus (1 associated with graft failure), 2 toxoplasmosis, 1 Aspergillus (associated with graft failure), 1 Pseudomonas (associated with chronic GVHD). One pt died with pulmonary failure and chronic GVHD 267 days after NMBMT. Actuarial nonrelapse mortality (NRM) is 64.3% (95% CI, 35.5–93.1), and infection-associated mortality is 52.4% (95% CI 25.0–79.8%). Three pts (2 AML, 1 HD) relapsed at 181, 182 and 202 days, respectively, after NMBMT; actuarial relapse rate is 46.4% (95% CI, 26.4–66.4%). Three pts (1 ALL, 1 AML, 1 NHL) are alive without relapse at 164+, 184+ and 843+ days, respectively, after NMBMT; actuarial event-free survival (EFS) is 10.2% (95% CI, 0–28.8%). We conclude that opportunistic infection is the major cause of NRM after VUD NMBMT with a preparative regimen of alemtuzumab, fludarabine and melphalan and contributes significantly to the poor EFS in pts with relapsed/refractory hematologic malignancies thus treated. Efforts to improve immune reconstitution and infection prophylaxis after NMBMT with this regimen are warranted.

Description: High-dose melphalan (MEL) and autologous hematopoietic cell transplantation (HCT) is now a standard therapy in patients (pts) with multiple myeloma (MM). This HCT procedure is sufficiently tolerable to be performed on either an outpatient (OP) or inpatient (IP) basis. We compared the infection profiles in 16 consecutive pts who underwent OP HCT and 31 consecutive pts who underwent IP HCT for MM over a 24-month period. The median age of each group was comparable (OP 58 yr, range 33–72 yr; IP 59 yr, range 32–74 yr). All pts received MEL 200 mg/m2 before HCT and anti-infective prophylaxis with oral acyclovir, fluconazole and levofloxacin. Empiric treatment of first neutropenic fever in the OP group was imipenem and vancomycin (VAN) and in the IP group was cefepime with addition of VAN for persistent fever and/or positive cultures with VAN-sensitive bacteria. Median time to neutrophils 〉0.5 x 109/L after HCT was 10 days in both groups (range 8–12 days for OP and 9–12 days for IP). Fever during neutropenia occurred in 7/16 (43.8%) of OP and 16/31 (51.6%) of IP HCT pts. Positive blood cultures (BCs) were identified in 3/7 (42.9%) of the febrile OP and 5/16 (31.3%) of the febrile IP HCT pts. Positive BCs in febrile OP HCT pts were coagulase-negative Staphylococcus (CNS) (2 pts) and Pseudomonas (1 pt). Positive BCs in febrile IP HSCT pts were CNS (2 pts), Streptococcus viridans (1 pt), Enterococcus (1 pt) and Bacillus (1 pt). One febrile OP HCT pt had cytomegalovirus pneumonia, and one afebrile IP HCT pt had a positive BC for CNS. Four febrile IP HCT pts with negative BCs had other infections, including cellulitis (2), facial abscess (1), and Clostridium difficile-associated colitis (CDAC) (1). Two other IP HCT pts (1 with fever and positive BC for CNS, 1 without fever) also had CDAC, for an overall incidence of CDAC of 3/31 (9.7%) after IP HCT. In contrast, no OP HCT pts had CDAC. Overall, the incidence of infections was 4/16 (25%) after OP HCT and 11/31 (35.5%) after IP HCT. We conclude that the infection profile of OP HCT compares favorably with that of IP HCT in pts with MM and is associated with a lower incidence of CDAC.

Description: Median ages of pts with most hematological malignancies are beyond 60 years. The advent of nonmyeloablative conditioning regimens has extended the use of allogeneic HCT to these older pts. Here, we retrospectively investigated outcomes among 280 pts aged ≥60 years, given HCT between 1998 and 2006. Results were broken down in 3 age groups 60–64 (n=166), 65–69 (n=90), and ≥70 (n=24) years old, respectively. Pts aged ≥70 years had less preceding chemotherapy, less often failed autologous HCT, more often had related grafts, and had higher comorbidity scores compared to pts in the other two age groups (table 1). Acute leukemias were more frequent and lymphoma/multiple myeloma were less frequent among pts aged ≥70 years old, resulting in higher risk for relapse (Table 1). After HCT, non-relapse mortality, relapse, and progression free survivals at 5-years for all pts were 26%, 41%, and 32% respectively. Five-year Kaplan Meier rate of overall survival was 35%, of those 12% vs. 23% were with vs. without chronic GVHD requiring immunosuppressive therapy, respectively. There were no statistically significant differences in outcomes among the three age groups except for more infections and days of hospitalization among pts aged 65–69 years compared to the other two age groups (Table 2). In multivariate analyses of risk factors, high HCT-comorbidity index (1–2 and ≥3) independently predicted higher NRM (HR: 2.84 and 3.0, respectively, p=0.01) and, not surprisingly, previously defined high relapse risk predicted relapse (HR: 2.17, p=0.06); both predicted worse OS (p=0.005 and p=0.0009, respectively) and PFS (p=0.01 and p=0.02, respectively). Pts given unrelated grafts did as well as recipients of related grafts. In conclusion, nonmyeloablative allogeneic HCT can be curative among pts older than 60 years with resolution of chronic GVHD among a majority of pts. Comorbidities and relapse risk rather than age should be used for benefit-risk assessment. Continued enrollment of elderly pts in prospective studies including unrelated donors is warranted. Table 1: Pt characteristics Age groups, years (60–64) (n=166) (65–69) (n=90) (70–74) (n=24) Median Interval from Dx to HCT, months 19 15 8 Median (range) # of prior regimens 3 (0–14) 3 (0–13) 2 (0–10) Median CD34+ cell number x 106/kg 6.7 6.7 6.5 Median CD3+ cell number x 108/kg 2.9 3.1 3.4 % Prior radiotherapy 18 11 13 Prior HCT Failed 19 12 4 Planned 8 6 4 Positive patient CMV sero-status 58 65 79 HLA-matched related 54 53 83 Donor HLA-matched unrelated 41 44 17 HLA-mismatched unrelated 5 3 0 Conditioning 2 Gy TBI 12 13 21 2 Gy TBI + FLU 88 87 79 HCT-CI scores 0 27 27 15 1–2 30 39 25 3–4 30 17 40 ≥5 13 17 20 Diagnoses Acute leukemia 35 47 62 Chronic leukemia 15 13 13 Lymphoma/myeloma 21 23 8 Myelodysplastic/myeloproliferative 16 16 17 Others 3 1 0 Disease status at HCT CR 46 41 38 PR 15 30 21 Refractory 28 21 33 Relapse 11 8 8 Relapse risk Low 19 17 8 Standard 48 51 38 High 33 32 54 Table 2: Outcomes per age groups HCT Outcomes Age groups, years P* (60–64) (n=166) (65–69) (n=90) (70–74) (n=24) FUO indicates fever of unknown origin *Test for trend μBased on hazard ratio analysis Bacterial 1.4 2.3 1.7 .0004 Rates of Infection episodes per person Viral 0.7 1.1 0.7 NS within 100 days FUO 0.3 0.4 0.1 NS Fungal 0.3 0.2 0.3 NS Median (range) days of hospitalization 1 (0–60) 4.5 (0–179) 0.5 (0–47) NS % Acute GVHD Grades II–IV 52 49 54 NSμ Grades III–IV 16 12 13 NSμ Chronic extensive GVHD 39 45 54 0.05μ NCI-CTC criteria grades III–IV non-hematological toxicities 42 57 50 NSμ Pts with Full donor chimerism at 1-year CD3/CD33/BM 68/ 84/ 80 66/ 83/ 77 67/ 79/ 71 NSμ CR at 2-years 40 47 63 NSμ 5-years Relapse/progression 38 46 46 NSμ 5-years NRM 28 22 29 NSμ 5-years OS 37 36 23 NSμ 5-years PFS 34 31 25 NSμ Patients alive and off all immunosuppression 24 25 5 NSμ

Description: Abstract 3450 Objective: Allogeneic hematopoietic cell transplantation (HCT) following a variety of reduced-intensity conditioning regimens has been reported to produce encouraging results in patients with AML. In these studies disease relapse was the main cause of treatment failure, with 2–4 year relapse rates ranging between 32–61% resulting in overall survivals of 28–45% at 2–4 years. Our goal here was to examine whether pre-HCT variables could identify patients at high risk for relapse following nonmyeloablative allogeneic HCT, who thus would become candidates for additional interventions to reduce the risk of AML relapse. Methods: The data were derived from 274 consecutive Seattle Consortium patients (median age: 60 years) with de novo or secondary AML who underwent allogeneic HCT from related or unrelated donors after conditioning with 2 Gy total body irradiation (TBI) with or without fludarabine (90 mg/m2) as recently reported (Gyurkocza et al., JCO 2010 Jun 10;28(17):2859–2867). Cox regression was used to perform multivariate analysis of risk factors for relapse in a subset of 231 patients in morphologic leukemia-free state (defined as less than 5% marrow blasts) with (n=134) or without (n=97) peripheral blood cell count recovery (defined as platelets 〉 100,000/ml and neutrophils 〉 1,000/ml) at the time of HCT. In this multivariate model, AML beyond 1st complete remission (CR1), unfavorable cytogenetics (according to SWOG criteria), incomplete peripheral blood cell count recovery before HCT, and shorter time between diagnosis and HCT were associated with statistically significantly higher risk of relapse (Table 1). From this multivariate model we developed a relapse risk score that summarizes the contribution of multiple risk factors by assigning weights based on the relative magnitude of the log hazard ratios associated with the principal risk factors. From a starting score of 0, points were added or subtracted based on the following factors: 2nd CR: +1 point; 3rd or later CR: +2 points; unfavorable cytogenetics: +1 point; absence of pre-HCT peripheral blood cell count recovery: +0.5 points; time from diagnosis to HCT 〉 18 months, -2 points (Table 2). Patients were then stratified into 2 relapse risk groups according to whether the total risk score was ≤0 (low-risk) or 〉0 (high-risk). Results: Stratification of patients according to the proposed Relapse Risk Score resulted in a clear separation of the two risk groups, with 5-year relapse rates of 50% and 17% in the high- and low-risk groups, respectively (Figure 1A). Five-year overall survival rates were 26% and 50% in the high- and low-risk groups, respectively (Figure 1B). Conclusion: Our Relapse Risk Score may be a useful tool to identify patients with AML at high risk for relapse, who could potentially benefit from additional interventions to reduce the risk of relapse following allogeneic HCT. We are currently attempting validation in an independent cohort of patients with AML to make this Risk Score more generalizable. Relapse/Progression (A) and Overall Survival (B) rates stratified by Relapse Risk Score. Disclosures: Off Label Use: Off label usage of fludarabine, cyclosporine, tacrolimus and mycophenolate mofetil is discussed.

Description: Abstract 2254 Background: Lenalidomide (LEN) is extensively used in combination with dexamethasone (DEX), with or without bortezomib (BOR), for initial treatment of multiple myeloma (MM). However, several studies have shown that previous exposure to LEN is associated with impaired collection of autologous peripheral blood stem cells (PBSCs) after mobilization with either granulocyte-colony stimulating factor (G-CSF) or chemotherapy plus G-CSF. The effects of previous exposure to LEN on the collection of autologous PBSCs after mobilization with G-CSF plus plerixafor (PLX; Mozobil®) are not well described. Methods: We evaluated the outcomes of autologous PBSC mobilization and collection in 15 consecutive patients (pts) with MM who received previous treatment with at least 2 21-day cycles of LEN in combination with either DEX (10 pts) or DEX and BOR (5 pts) and who underwent initial autologous PBSC mobilization with G-CSF (10 micrograms/kg/dose subcutaneously daily × 4 days) and PLX (0.24 mg/kg/dose subcutaneously, after fourth dose of G-CSF), with additional doses of G-CSF and PLX for subsequent apheresis sessions. Results: Median age of pts was 58 yr (range, 35–67). Patients received a median of 5 cycles (range, 2–12) of LEN. The median number of apheresis sessions was 1 (range, 1–2). The median level of CD34+ cells in the peripheral blood at day 1 of apheresis was 84.36 per microliter (range, 12.46–167.37), and the median yield of CD34+ cells was 6.52 × 106/kg (range, 1.43–17.11) after day 1 of apheresis and 7.46 × 106/kg (range, 2.32–17.11) after day 2 of apheresis. No LEN-treated pts failed mobilization (defined as collection of less than 2 × 106 CD34+ cells/kg). Eleven pts (73.3%) collected at least 6 × 106/kg CD34+ cells (i.e., sufficient for 2 transplants) after 1 or 2 aphereses, with 8 pts (53.3%) reaching that target after 1 apheresis. Of the 4 pts (26.7%) in whom fewer than 6 × 106/kg CD34+ cells were collected after 2 aphereses, 3 pts collected at least 4 × 106 CD34+ cells/kg, and only 1 pt (who received only 2 cycles of LEN) collected 2.32 × 106/kg CD34+ cells. The number of cycles of LEN did not correlate with concentration of CD34+ cells in peripheral blood on day 1 of apheresis (r2=0.099), yield of CD34+ cells on day 1 of apheresis (r2=0.108), or total yield of CD34+ cells after 2 days of apheresis (r2=0.067). Pts who received 4 or fewer cycles of LEN had higher median circulating CD34+ cells on apheresis day 1, higher median CD34+ cell yield with day 1 apheresis and higher total CD34+ cell yield after 2 aphereses than pts who received more than 4 cycles of LEN (Table). Five of 6 pts who received 4 or fewer cycles of LEN collected at least 6 × 106 CD34+ cells/kg after 1 apheresis, compared with 3 of 9 pts who received more than 4 cycles. However, none of these differences were statistically significant (Table). Conclusions: Previous treatment with LEN does not impair collection of autologous PBSCs after initial mobilization with G-CSF and PLX. However, there was a trend to more robust autologous PBSC collections and decreased need for second aphereses in pts who received fewer cycles of LEN. These findings are consistent with the recommendation of the International Myeloma Working Group to collect autologous PBSCs from MM pts after 4 or fewer cycles of LEN-containing regimens. As mobilization with G-CSF with or without chemotherapy leads to inconsistent autologous PBSC collection in LEN-treated MM pts, our experience supports the use of G-CSF plus PLX as an upfront mobilization strategy for these pts. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2145 Poster Board II-122 Introduction: Plerixafor (AMD3100; PLX), a bicyclam molecule that reversibly inhibits binding of the CXCR4 chemokine receptor to stromal cell derived factor-1, is used clinically in combination with G-CSF for mobilization of hematopoietic stem cells. Although the effects of PLX plus G-CSF on mobilization of CD34+ cells have been well studied, the impact of PLX plus G-CSF on mobilization of lymphocytes and lymphocyte subsets remains unclear. Additionally, comparative effects of PLX on the content and subpopulations of lymphocytes in apheresis products from patients (pts) with non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM) have not been previously reported. Patients and Methods: Thirteen pts (7 NHL, 6 MM) underwent autologous PBSC mobilization with subcutaneous (sq) G-CSF (10 micrograms/kg/day x 4 days) and sq PLX (0.24 mg/kg on the fourth day of G-CSF administration), followed by large-volume apheresis approximately 15 hr after PLX dose. Pts who did not attain target CD34+ collection on first day of apheresis (day 1) received up to 3 additional daily doses of PLX plus G-CSF and up to 3 additional days of apheresis. Aliquots of apheresis products were obtained after each collection for differential WBC count, from which the absolute lymphocyte count (ALC) was calculated, and for flow cytometric quantitation of lymphocyte subsets. Cell doses were expressed per kg of pt weight. A nonparametric (Mann-Whitney) tests was used for all statistical analyses. Results: Five pts (1 NHL, 4 MM) collected target CD34+ cell doses (median 7.46 × 106 cells/kg; range, 7.22-22.7 × 106 cells/kg) with 1 apheresis. The remaining 8 pts collected target CD34+ cell doses after a total of 2 (2 NHL, 2MM), 3 (3 NHL) or 4 (1 NHL) aphereses. Doses of CD34+ cells, ALC, CD4+ cells, CD19+ cells and CD56+ cells were significantly higher in day 1 PBSC apheresis products from MM pts than from NHL pts, but neither CD3+ nor CD8+ cell dose was significantly different in the two groups (Table 1). For the pts undergoing 2 or more aphereses, comparison of day 1 and day 2 apheresis products showed significantly decreased doses of ALC, CD3+, CD4+ and CD8+ cells, but no significant differences in doses of CD34+, CD19+ or CD56+ cells (Table 2). Conclusions: PLX plus G-CSF effectively mobilizes lymphocytes and lymphocyte subsets as well as CD34+ cells. Pts with MM had significantly higher doses of specific lymphocyte subsets, notably CD4+ and CD56+, than did pts with NHL, potentially reflecting differences in previous treatments (e.g., lenalidomide plus dexamethasone in MM pts compared with ICE or ESHAP in NHL pts). These findings are relevant to the understanding of the effects of infused lymphocyte subsets on outcomes of and immune reconstitution after autologous PBSC transplantation. Disclosures: No relevant conflicts of interest to declare.

Description: Substantial barriers exist to the engraftment of hematopoietic cells in mice after in utero transplantation. Although high levels of donor-derived hematopoiesis have been reported in SCID mice, the majority of chimeric recipients exhibit decreasing levels of donor cells over time. To directly test whether the natural killer cell and macrophage activity of the recipients represents a barrier to sustained engraftment, fetal NOD/SCID mice were injected on day 13.5 of gestation with an enriched congenic hematopoietic progenitor cell population. Forty-four percent of pups showed the presence of Ly5.1+ donor cells 4 weeks after transplantation. The mean number of donor-derived nucleated cells in the peripheral blood (PB) was 30%. Although the majority of circulating donor cells were lymphocytes, up to 15% expressed myelomonocytic markers. Serial PB samples from individual mice indicated that the percentage of circulating donor cells increased from 17% to 55% between 4 and 24 weeks. At 6 months posttransplantation, an increased frequency of multilineage, donor-derived cells was also observed in the bone marrow (BM) and the spleen of chimeric recipients. The engraftment of pluripotent hematopoietic stem cells was evaluated by transplanting BM from chimeric mice into irradiated congenic recipients. Irradiated secondary recipients also exhibited multilineage donor-derived hematopoiesis in the PB, BM, and spleen for up to 6 months. These results show that the in utero transplantation of lineage-depleted BM cells into NOD/SCID recipients produces a high frequency of sustained engraftment of allogeneic hematopoietic stem cells.