Evaluation of Oxidative Stress Markers in Hematopoietic Stem Cell Transplantation Patients

[Introduction] Oxidative stress caused by the increased production of reactive oxygen species (ROS) or decreased efficacy of the antioxidant system is implicated in the pathogenesis of various disease entities, such as atherosclerosis, cardiovascular disease, renal failure, malignant tumors, and autoimmune diseases. Chemotherapy and radiation therapy are associated with increased formation of ROS. Conditioning regimens preceding hematopoietic stem cell transplantation (HSCT) usually consist of high-dose chemotherapy and/or total body irradiation (TBI). A limited number of studies demonstrated that the conditioning therapy given to HSCT patients creates a high oxidative stress and decreases the antioxidant defense system. The objective of this study was to look for further evidence of oxidative stress status in HSCT patients. [Methods] In this study, urine samples were collected from 32 HSCT patients before and after conditioning therapy and from 15 healthy controls. The patients included 19 male and 13 female with a median age of 58 years (27-68 years). Twenty patients received allogeneic HSCT (9 uBMT, 1 rPBSCT and 10 CBT) and 12 patients received autologous PBSCT. The conditioning regimens for allo-HSCT included TBI12Gy+Ara-C+G-CSF (n=3), TBI12Gy+Ara-C+CY (n=1), TBI12Gy+VP-16+CY (n=1), Flu+L-PAM+TBI3Gy (n=6), Flu+BU+TBI3Gy (n=1), Flu+Ara-C+G-CSF+BU+TBI4Gy (n=3), Flu+L-PAM (n=2), and BU+CY (n=3). The diagnosis included AML (n=9), MDS (n=5), DLBCL (n=1), FL (n=2), T-ALL (n=1) and MM (n=2). Fifteen patients (75%) had high-risk disease at transplantation. Twelve multiple myeloma (MM) patients received L-PAM 200mg/m2 + autologous PBSCT. We measured urinary 8-hydroxydeoxyguanosine (8-OHdG) by competitive immunochromatography using a novel automatic oxidative stress analyzer, ICR-001 (Techno-Medica). 8-OHdG, which originates from damaged DNA repaired by non-specific endonucleases and specific glycosylates and is eliminated into urine, is widely used as a sensitive biomarker of oxidative stress. [Results] Urinary 8-OHdG significantly increased immediately after conditioning therapy. In 20 allo-HSCT patients, urinary 8-OHdG levels on day 0 were significantly higher than pre-conditioning levels and healthy controls (mean: 458.2 vs. 90.9 vs. 15.7 ng/mgCr). In 12 auto-HSCT patients, urinary 8-OHdG levels on day 0 were also higher than pre-conditioning levels (mean: 273.6 vs. 107.2 ng/mgCr). No significant correlation was found between urinary 8-OHdG levels and serum ferritin levels during pre- and post-transplant period. Allo-HSCT patients with high urinary 8-OHdG levels on day 0 (over 300ng/mg/Cr; N=10) had significantly shorter survival than those with low urinary 8-OHdG levels on day 0 (under 300ng/mg/Cr; N=10) (1-year OS, 11.1% vs. 80.0%, respectively; P=0.01) (Figure. 1). On the other hand, post-conditioning urinary 8-OHdG levels were not associated with outcome in auto-HSCT patients. In univariate analysis, a high 8-OHdG level on day 0, CBT, and RIC regimen were associated with poor OS in allo-HSCT patients. [Conclusion] In the present study we demonstrated that conditioning therapy results in increased oxidative stress in HSCT patients. In particular, our data proved that high urinary 8-OHdG levels on day 0 were associated with poor prognosis in allo-HSCT patients. These results suggest that oxidative stress may have an important role in allo-HSCT and also may be a useful prognostic biomarker. Since our results are based on a small-sized analysis, further large prospective studies are warranted to verify this conclusion. Disclosures No relevant conflicts of interest to declare.

Recovery Of Natural Killer Cells and Monocyte Subsets Following Autologous Peripheral Blood Stem Cell Transplantation Predicts Longer Progression Free Survival Among Multiple Myeloma Patients

Prior research has shown that a timely immune recovery following peripheral blood stem cell transplantation (PBSCT) may improve progression free and overall survival for lymphoma and leukemia patients (pts). However, this has not been studied in multiple myeloma (MM) pts. We conducted an exploratory investigation of the extent to which the recovery of Natural Killer (NK) cell and monocyte (Mo) subsets predicted progression free survival (PFS) among MM pts following autologous PBSCT. Patients (N = 37) were treated with 200mg/m2 of melphalan, followed by infusion of >2 x106 CD34 cells/kg, and were followed prospectively for 3 to 36 months. Immunophenotyping and multicolor flow cytometry were used to identify NK and Mo subsets at Day 100 post-PBSCT. Disease progression or relapse was defined using IMWG criteria. Cox proportional hazard regression models were used to examine relationships between cell subpopulation counts and time from PBSCT to MM progression. All models were adjusted for ISS stage and risk (high, defined as plasma cell leukemia; loss of p53, myc rearrangement, t(14;16), t(14:20) by FISH; minus 13 by cytogenetics; versus standard) at diagnosis, and hazard ratios were standardized to correspond to a 2 standard deviation difference in the logarithm of NK or Mo cell counts. Compared to a healthy reference sample, MM patients showed good recovery of all Mo subsets with normal counts by Day 100, but CD56+CD3- NK cell numbers remained low, with the subset of NK cells expressing high CD16 particularly suppressed. During the follow-up period, 25 patients (67.6%) progressed or relapsed. Pts with higher Day 100 CD56+CD16hi NK cell counts had a longer PFS (hazard ratio = .351, p = .033). A similar effect was seen for CD56+CD16lo NK cells (hazard ratio = .321, p = .079). Higher CD14+ Mo counts were also associated with longer PFS (hazard ratio = .230, p = .007). An examination of Mo subpopulations showed that higher numbers of Mo expressing low or no CD16 predicted a longer time to disease progression (hazard ratio = .207, p = .011), but the inflammatory subsets with moderate or high CD16 expression were not significantly associated with PFS. Results suggest that MM patients who have lower NK cell and CD14+ Mo counts may be at risk for a poorer outcome following autologous PBSCT, with these subsets adding prognostic significance above and beyond cytogenetic risk and ISS stage. Subpopulations with greater regulatory and less inflammatory function had the most salient effects. These NK cell and Mo subsets may inhibit residual MM growth and progression and could serve as novel markers of the clinical outcome following PBSCT for MM. Disclosures: No relevant conflicts of interest to declare.

Higher Percentage of CD34+CD38− Cells Detected by Multiparameter Flow Cytometry From Leukapheresis Products Predict Unsustained Complete Remission in AML

Abstract 1485 Aim: Myeloablative chemotherapy followed by autologous PBSCT remains one treatment strategy in adult AML patients. Relapse has been shown higher for those who received the highest CD34+ PB doses. Although highly active against the leukemic bulk, intensive chemotherapy often spare the hardier leukemia stem cells (LSCs) responsible for relapse. Detection of MRD in harvest products may reflect inadequate in vivo purging at least in part responsible for relapses. Although recent data have challenged the CD34+CD38− phenotype of LSCs, this cell population remains generally considered enriched for LSCs. In this setting, MRD remaining during CR should be relatively enriched in CD34+CD38− leukemic cells and their persistence should correlate with disease recurrence. Methods: CD34+ cells were harvested after CR achievement in 123 AML patients [median age: 53 y (25–72)] treated by induction chemotherapy in our Institution between 10/1994 and 04/2003. Patients were included in different clinical trials planning autologous SC harvest in CR and autologous SCT in absence of donor or allogeneic SCT indication. Seventy-one of them received effectively autologous PBSCT. Harvests performed in 15 normal donors were used as controls. CD34/CD38 cell profile was analyzed in harvests in one single tube by multicolor flow cytometry using multiple MoAbs. The gating strategy was based on CD45low/SSC and CD34+CD45low cell populations from total FSC/SSC viable cells. Three populations of CD34+ were distinguished: CD34+CD38–; CD34+CD38low; and CD34+CD38+. Results: Patients from the entire cohort with higher percentage of CD34+ cells (cut-off level: 1%) in PBSC products were associated with shorter EFS [median: 5.6 months (3-y EFS: 13%) vs 13.6 (37%); p=0.0005] and OS [median: 10 months (3-y OS: 19%) vs 23.4 (47%); p=0.004]. This was also the case when analyzing only patients who received autologous SCT: [median EFS: 5 months (3-y EFS: 13%) vs 22.2 (48%); p=0.0006, and median OS: 9.1 months (3-y OS: 21%) vs 43.3 (57%); p=0.001]. Among CD34+ populations, only CD34+CD38– had a prognostic impact on EFS and OS. At a cut-off level of 0.9%, median EFS was 8.2 months (3-y EFS: 29%) for those with higher percentage vs 91.9 (62%) for those with lower percentage and median OS was 14.2 months (3-y OS: 36%) vs 95.4 (69%) respectively for the entire cohort. These results were confirmed in patients undergoing autologous SCT: median EFS was 7.3 months (3-y EFS: 31%) vs 91.1 (70%) (p= 0.05), and median OS was 14.4 months (3-y OS: 39%) vs 94.6 (80%). CD34+CD38low and CD34+CD38+ populations did not show any prognostic impact. Harvests from AML patients were divided into 3 groups: Group A: 51 patients with CR duration <1 y; Group B: 22 patients with CR duration >1 y; and Group C: 50 patients without relapse. Harvests from 15 normal donors (Group D) were used as controls. Significant differences were only observed when comparing Group A and Group D for total CD34+ cells (mean ± SEM 2.5 ± 0.5 vs 1.2 ± 0.3; p < 0.05) and CD34+CD38– (4.5 ± 0.7 vs 2.3 ± 0.5; p < 0.05). To confirm the prognostic value of CD34+CD38–, 19 patients (Group 1) with evidence of leukemic contamination in harvests (abnormal cytogenetics at presentation found in aphereses) were compared with 22 patients (Group 2) without evidence of contamination (abnormal cytogenetics at presentation not found in aphereses). Median EFS was 10.1 months (3-year EFS: 45%) in Group 2 vs 6.3 (13%) in Group 1 (p=0.01), and median OS was 36.6 months (3-year OS: 55%) vs 10.8 (23%), respectively (p=0.03). Harvests from 15 normal donors (Group 3) served as controls. Significant differences were noted in harvest products regarding CD34+CD38– between Group 1 and Group 3 (mean ± SEM 6.0 ± 1.5 vs 2.3 ± 0.5; p = 0.04) and Group 1 and Group 2 (6.0 ± 1.5 vs 2.4 ± 0.5; p = 0.03), while there were no differences between Group 2 and controls. There were no significant differences between groups regarding CD34+CD38low and CD34+CD38+. We also measured MFI of CD13, CD33, CD123, CD117 in CD34+ subpopulations. Phenotypes were compared among the different groups. Conclusions: Higher proportions of CD34+CD38− in apheresis products appear to reflect inadequate in vivo purging and distinguish samples as enriched in ‘leukemic cells’ from those with lower CD34+CD38− as largely constituted of ‘normal cells’. This could serve as detection of MRD and help to identify samples associated with high-risk of relapse after autologous SCT. Disclosures: No relevant conflicts of interest to declare.

Safety and Efficacy of High-Dose Cyclophosphamide, Etoposide, and Ranimustine (CEM) Regimen Followed by Autologous Peripheral Blood Stem Cell Transplantation for Patients with High Risk De Novo Diffuse Large B-Cell Lymphoma or Diffuse Large B-Cell Lymphoma Associated with Follicular Lymphoma.

Abstract 3121 Background We evaluated the safety and efficacy of the CEM regimen containing ranimustine (MCNU) with autologous peripheral blood stem cell transplantation (PBSCT) in adult patients with relapsed or high-risk de novo DLBCL or DLBCL associated with follicular lymphoma (FL/DLBCL) in our institution. Patients and methods We retrospectively analyzed 55 consecutive patients who underwent autologous PBSCT following the CEM regimen between March 1999 and June 2011 for the treatment of relapsed or high-risk DLBCL and FL/DLBCL. High-risk DLBCL was defined as partial or no response to initial treatment or high-intermediate/high risk disease according to international prognostic index (IPI) at initial diagnosis. Age-adjusted IPI was used for patients under 60. The CEM regimen consisted of CY (60 mg/kg on days -7 and -6), etoposide (500 mg/m2 on days -6 to -4) and MCNU (250 mg/m2on day -3 and -2), followed by PBSCT. Results Median age at transplant was 51 years (range, 23–66), and median time from diagnosis to transplant was 5 months (range, 3–241). Of them, 7 patients received radiation therapy before transplant, and 11 had no history of rituximab use before transplant. The 36 patients in CR1 at transplant were with high or high-intermediate risk according to international prognostic index at diagnosis. Other 15 patients in CR2, and 4 patients who were not in CR1/2 at transplant were included. CY dose was reduced in five patients by physicians choice. The median number of infused CD34-positive cells was 3.98 × 106/kg (range, 1.36–26.87). The median post-transplant days of neutrophil recovery and platelet were 11 days (range, 9–20) and 12 days (range, 7–53), respectively. Common grade 3/4 non-hematological treatment-related toxicities within the first 100 days after transplant were nausea (24%), vomit (4%), stomatitis (15%), and diarrhea (9%). Infections included febrile neutropenia (85%), sepsis (15%) which contained catheter-related bacteremia (n=4) and pneumonia (n=2). Other less common severe toxicities were acute renal impairment (n=3), and pleural effusion (n=1). No sever cardiac, neurologic toxicity, or veno-occlusive disease of the liver was observed in any of the patients. Late-onset adverse effects during follow-up period includes chronic renal impairment (n= 5), therapy-related myelodysplastic syndrome (refractory anemia) (n=1, 120 months), and prostate cancer (n=1, 83 months). Serious late cardiac or pulmonary impairment was not diagnosed in this cohort. Median follow-up duration of 42 patients surviving at the time of the analysis was 52 months (range, 1–159). Relapse or disease progression after PBSCT was documented in 21 cases (range, 0–81 months after PBSCT). No therapy related mortality (TRM) associated with PBSCT was observed, and all the 13 deaths were due to disease relapse/progression. The 5-year estimated overall survival (OS) and progression-free survival (PFS) were 70.6% (95% CI, 54.0–82.1)% and 57.0% (95% CI, 39.5–71.2)% for all patients, respectively. In a univariate analysis for factors affecting OS and PFS, no significant factors associated with unfavorable OS and PFS was found including age at transplant, gender, history of rituximab use, histology, disease status at transplant, and previous BM involvement. Conclusion We conclude that the CEM regimen using MCNU would be a tolerable, effective conditioning regimen of autologous PBSCT for high-risk or relapsed de novo DLBCL or FL/DLBCL. Although no TRM was observed, relapse was the major cause of treatment failure. Late-onset complications in long-term survivors can occur, and should be carefully monitored. Disclosures: No relevant conflicts of interest to declare.