Yield Of CD34 Research Articles

Remarkable Clinical Efficacy, Stem Cell Mobilization Activity and Good Toxicity Profile of the Novel Begev Regimen (Bendamustine, Gemcitabine And Vinorelbine) Used As Salvage Therapy Prior to Autologous Stem Cell Transplant for Relapsed/Refractory Hodgkin Lymphoma

INTRODUCTION: High-dose salvage chemotherapy followed by autologous stem cell transplantation (ASCT) is the standard of care for refractory/relapsed (R/R) Hodgkin lymphoma (HL). Achieving complete remission (CR) prior to ASCT represents the strongest prognostic factor for R/R HL patients receiving salvage chemotherapy. Therefore, increasing the CR rate prior to ASCT represents a primary goal in these patients. Since Bendamustine monotherapy induces CR in a substantial proportion (25% to 35%) of R/R HL patients, the present phase II study aimed at investigating efficacy and toxicity of a novel salvage regimen combining Bendamustine, Gemcitabine and Vinorelbine (BeGEV) as second-line salvage chemotherapy in patients with relapsed/refractory HL.PATIENTS AND METHODS: HL patients who were refractory to, or have relapsed after one previous chemotherapy line were eligible. The primary endpoint was CR rate after four cycles of therapy. Secondary endpoints were: overall response rate (ORR), stem cell mobilization activity, and toxicity. Progression free survival (PFS) and overall survival (OS) were also evaluated. BeGEV regimen consisted of: Bendamustine (90 mg/sqm, days 2-3), Gemcitabine (800 mg/sqm, day 1 and 4) and Vinorelbine (25 mg/sqm, day 1) every 3 weeks for a total of 4 courses.RESULTS: Between August 2011 and March 2014, 59 consecutive patients with relapsed (46%) or refractory (54%) HL were enrolled. The median age was 33 years (range 18-68). Out of 59 enrolled patients, 43 (73%) achieved a CR and 6 (10%) a partial response (PR) for an ORR of 83%. One case (2%) showed stable disease (SD), while 8 patients (14%) progressed, and 1 (2%) was not evaluable for response. With a median follow-up of 16 months, the 2-year PFS and OS were 51% and 69%, respectively, without significant difference between relapsed and refractory patients. OS was higher for BeGEV-responding (CR+PR) patients compared with those who failed the induction regimen (2-year OS: 86% vs 0%, p<0.001). Fifty-seven out of 59 patients were evaluable for CD34+ cell mobilization. Mobilization failure was detected in 2 of 57 patients (3.5 %) while CD34+ cells were successfully harvested in 55 out of 57 evaluable patients (96.5 %). Forty-two patients (76%) required 1 leukapheresis to harvest the planned target CD34+ cell yield (3×106 CD34+ cells/Kg body weight) while 13 patients (24%) required 2 leukaphereses. The median total yield of CD34+ cells/Kg body weight was 8.8×106 CD34+ cells (range, 3-56). After ASCT, engraftment of neutrophils and platelets was recorded on day 11 (range 9-21) and day 12 (range 9 - 26), respectively. Among the 49 responding patients, 43 (88%) proceeded to ASCT (38/43 in CR, 5/6 in PR); the remaining 6 patients did not proceed to ASCT due to mobilization failure (n=2), physician's decision (n=2), early relapse (n=1), and patient's refusal (n=1). Hematological and non-hematological side effects were acceptable. Out of 204 administered cycles, 23 (11%) had to be delayed and 4 (2%) reduced, respectively; only 1 patient stopped therapy for toxicity. The most common grade 3-4 nonhematologic toxicities included febrile neutropenia (n=7), and infections (n=4). Among hematologic toxicities, grade 3-4 thrombocytopenia and neutropenia were experienced by 8 (13.5%) patients.CONCLUSIONS: This phase II study demonstrates that BeGEV is a highly effective salvage regimen able to induce a remarkable proportion of complete remission prior to ASCT in relapsed/refractory HL patients. These data provide a strong rationale for further development of the BeGEV regimen. DisclosuresLuminari:Roche: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees.

Biosimilar Filgrastim (Tevagrastim, XMO2) for AllogeneicHematopoietic Stem Cell Mobilization andTransplantation in Patients with Acute MyelogenousLeukemia/Myelodysplastic Syndromes.

Human recombinant granulocyte colony-stimulating factor (G-CSF), filgrastim (Neupogen; Amgen, Thousand Oaks, CA, USA), has been widely used for the mobilization of CD34(+) hematopoietic stem cells (HSC) from healthy donors. The experience with biosimilar G-CSF agents in this area is limited. We performed a prospective study assessing Tevagrastim (biosimilar filgrastim, XMO2; Teva, Israel) for mobilization of CD34(+) peripheral blood HSC in HLA-matched healthy sibling donors for transplantation in 24 patients with acute myelogenous leukemia (AML) and high-risk myelodysplastic syndromes (MDS) (NCT01542944). Results were compared to a historical control group of sibling donors who received filgrastim for stem cell mobilization for allogeneic stem cell transplantations in patients with AML and MDS. The healthy donors received Tevagrastim or filgrastim in a dose of 10 μg/kg body weight (BW) subcutaneously for 4 days. The target yields of CD34(+) cells was 5× 10(6) CD34(+) cells/kg BW of the recipient. A median 10.2× 10(6) (range, 2.52 to 35.4) and 9.35× 10(6) (range, 3.7 to 30.6) CD34(+) cells per kg BW were collected in the Tevagrastim and filgrastim groups, respectively. All patients promptly engrafted with a median day of absolute neutrophil count (ANC) of >.5× 10(9)/L and >1× 10(9)/L of 13 days (range, 10 to 21) and 13.5 days (range, 10 to 22), respectively in the Tevagrastim group and 12 days (range, 10 to 18) and 13 days (range, 10 to 18) in the filgrastim group, respectively. Platelets reached counts of >20× 10(9)/L and >50× 10(9)/L within a median of 14 days (range, 11 to 33) and 17 days (range, 12 to 33) in the Tevagrastim group and 13 (range, 10 to 29) and 15 (range, 10 to 32) days in the filgrastim group, respectively. The donors developed only mild and transient side effects, which were not different between the Tevagrastim study group and the filgrastim historical control group. Similarly, the transplantation-related toxicitiesand outcomes did not differ between the patients who underwent transplantation with Tevagrastim-mobilized grafts and the filgrastim historical controls. In summary, we observed a similar yield of CD34(+) stem cell mobilization in the Tevagrastim study group and the filgrastim historical control group with similar engraftment kinetic, hematopoietic reconstitution, and transplantation outcome. Tevagrastim administration was safe with minimal side effects and toxicity not different than historical controls. The lack of significant differences for all parameters of stem cell collection, engraftment, and safety with the biosimilar XMO2 Tevagrastim demonstrate the "similarity" of the biosimilar and recombinant human G-CSF inthis indication.

Cyclophosphamide plus granulocyte-colony stimulating factor for hematopoietic stem cell mobilization in patients with multiple myeloma.

We retrospectively reviewed the results of cyclophosphamide (3 g/m(2) ), doxorubicin and dexamethasone plus granulocyte-colony stimulating factor (G-CSF) (ID-CY/DOX group), low-dose cyclophosphamide (2 g/m(2) ) plus G-CSF (LD-CY group) and G-CSF alone (G-CSF group) for stem cell mobilization in patients with multiple myeloma. A total of 89 patients with 93 mobilizations were included. Apheresis was started when total white blood cell (WBC) count >10 × 10(9) /L for ID-CY/DOX and LD-CY groups and after eight doses of G-CSF (5 μg/kg twice daily) for G-CSF group. For five mobilizations in ID-CY/DOX group, the rate of successful mobilization (≥4.0 × 10(6) /kg CD34+ cells) was 80%. For 78 mobilizations in LD-CY group, the successful rate was 80.8%. For 10 mobilizations in the G-CSF group, the successful rate was 50%. The mean yield of CD34+ cells was higher in ID-CY/DOX and LD-CY groups as compared with that in G-CSF group (P = 0.026 and 0.020, respectively). There was no difference in the yield of CD34+ cells between ID-CY/DOX and LD-CY groups (P = 0.831). After autologous stem cell transplantation, the days to neutrophil and platelet engraftment were similar in these three groups (P = 0.713 and 0.821, respectively). In conclusion, we observed that ID-CY/DOX and LD-CY plus G-CSF for stem cell mobilization resulted in a higher successful rate and higher stem cell yields than G-CSF alone and their engraftment time were similar. Total WBC count >10 × 10(9) /L can be used as a guide to start apheresis in CY-based stem cell mobilization. J. Clin. Apheresis 31:423-428, 2016. © 2015 Wiley Periodicals, Inc.

Optimizing stem cell mobilization: lessons learned.

Granulocyte colony-stimulating factor is the pivotal component of mobilization regimens and the growth factor most often used for peripheral blood progenitor cell collections. When used alone or after chemotherapy, products with adequate yields of CD34(+) cells are obtained after leukapheresis, resulting in optimal blood count recovery after transplant. For patients who have had extensive prior treatment with chemotherapy and/or radiation, or treatment with specific agents, the yields may be limited. For these patients, plerixafor in combination with growth factor can be used to augment progenitor cell yield and ensure successful collection of target goals, with preservation of the integrity of the graft. Progenitor cells can similarly be collected by leukapheresis from patients for autologous use and from allogeneic donors, after a period of growth factor administration. Many chemotherapy regimens can be used before growth factor administration that serve a dual role of reducing tumor burden and enhancing the progenitor cell collection. Given modern methods, a high success rate exists for procurement of adequate stem cell products.

Peripheral Blood Stem Cell Mobilization and Engraftment after Autologous Stem Cell Transplantation with Biosimilar rhG-CSF

Biosimilar versions of filgrastim [recombinant human granulocyte colony-stimulating factor (rhG-CSF)] are now widely available. To date, biosimilar rhG-CSF has demonstrated a comparable quality, safety and efficacy profile to the originator product (filgrastim [Neupogen(®)], Amgen Inc., CA, USA) in the prevention and management of neutropenia. Biosimilar rhG-CSFs have also been used to induce peripheral blood stem cell (PBSC) mobilization in patients undergoing autologous stem cell transplantation (AHSCT). The authors have examined the effectiveness of a biosimilar rhG-CSF (Zarzio(®), Sandoz Biopharmaceuticals, Holzkirchen, Germany) in two retrospective studies across two medical centers in Hungary. In Study 1, 70 patients with hematological malignancies scheduled to undergo AHSCT received chemotherapy followed by biosimilar rhG-CSF (2 × 5 μg) for facilitating neutrophil, leukocyte, and platelet engraftment. In study 2, 40 additional patients with lymphoid malignancies and planned AHSCT received chemotherapy followed by biosimilar rhG-CSF for PBSC mobilization. The effectiveness of treatment was assessed by the average yield of cluster of differentiation (CD) 34+ cells and the number of leukaphereses required. In Study 1 (patients undergoing AHSCT), the median age was 56 years and most patients were male (60%). The conditioning regimens were mainly high-dose melphalan (n = 41) and carmustine (BiCNU(®), Bristol-Myers Squibb, NJ, USA), etoposide, cytarabine and melphalan BEAM (n = 21). Median times to absolute neutrophil and leukocyte engraftment were 9 (range 8-11 days) and 10 (8-12) days, respectively. Median time to platelet engraftment was 10.5 days (7-19 days). In Study 2, the patients' median age was 54 years and the majority (57.5%) were female. The median time interval between day 1 of mobilizing chemotherapy and first leukapheresis was 12 (9-27) days. In the autologous PBSC grafts, the median number of CD34+ cells harvested was 5.2 × 10(6)/kg (2.22-57.07 × 10(6)/kg). The median yield of CD34+ cells per leukapheresis product was 2.47 × 10(6)/kg. In total, 58 leukaphereses were performed in 40 successfully harvested patients. In line with previous studies with originator rhG-CSF, the findings of this study indicate that biosimilar rhG-CSF following AHSCT is effective and generally well tolerated in the engraftment setting. In addition, biosimilar rhG-CSF is comparable to the originator rhG-CSF in terms of kinetics of PBSC mobilization and yield of CD34+ cells. In conclusion, the authors have demonstrated that the use of biosimilar rhG-CSF is effective and safe in autologous PBSC mobilization and engraftment after AHSCT.

Polymorphism of CD44 Influences the Efficacy of CD34+ Cells Mobilization in Patients with Hematological Malignancies

In the last decade, peripheral blood was the main source of hematopoietic stem cells (HSC) for autologous and allogeneic transplantation. The exact mechanisms of HSC mobilization are still not clear and the efficacy of the procedure is hardly predictable. Ligand-receptor interactions of adhesion molecules, such as SDF1/CXCR4, VLA4/VCAM-1, or CD44/osteopontin, play an important role in homing of HSC in the hematopoietic niche. There is some evidence that disruption of the ligand-receptor complex leads to the egress of HSCs to the peripheral blood. The aim of the present study was the evaluation of constitutive polymorphism of genes encoding cytokines and receptors present in the HSC niche and their impact on the efficacy of mobilization of HSCs in patients with hematological malignancies. We enrolled 110 patients (60 females and 50 males) in the study. The median age of the patients was 55 (range, 22 to 69) years. The group consisted of patients with multiple myeloma (n = 74), non-Hodgkin lymphoma (n = 19), Hodgkin lymphoma (n = 15), or acute myeloid leukemia (n = 2). The mobilization procedures comprised chemotherapy and subsequent granulocyte-colony stimulating factor (G-CSF) at a dose of 10 μg/kg daily. The poor mobilizers group was defined according to Italian National Bone Marrow Transplant Registry criteria: patients with peak CD34+ in the peripheral blood < 20/μL or total yield < 2 × 106 CD34+ cells/kg body weight in maximum 3 aphereses. Genotyping was performed using standard PCR-based assays. The group of patients (N = 108) who achieved minimal threshold for collections (CD34+ at least 10/μL) proceeded to apheresis. The median total yield of CD34+ in this group was 5.6 × 106 cells/kg body weight, whereas the median number of cells collected during the first apheresis was 3.3 × 106 cells/kg body weight. Median number of days of G-CSF treatment before first apheresis was 10. Fifteen patients fulfilled the criteria for poor mobilizer. The group of poor mobilizers had higher frequency of TT genotype in rs13347 (CD44) gene (CC+ CT versus TT P = .047). Patients homozygous for T allele had a lower total yield of CD34+ cells/kg body weight than the group with allele C (median, 3.7 × 106/kg versus 5.8 × 106/kg; P = .019) and a lower number of CD34+ cells gathered during first apheresis (.95 × 106/kg versus 3.3 × 106/kg, P = .04). Multivariate logistic regression analysis revealed that the CD44 TT genotype was the only factor associated with 5-fold higher risk of poor mobilization (P = .037). Polymorphic variants of CXCR4 and VCAM-1 did not significantly influence the efficacy of HSCs mobilization in our group of patients. In conclusion, our results indicate that among investigated single nucleotide polymorphisms (SNPs), only CD44 rs13347 has an impact on the efficacy of HSCs mobilization in patients with hematologic malignancies. CD44 SNPs analysis may be helpful for predicting the poor mobilizers population who may benefit from newer modalities using adhesion molecules inhibitors.

The impact of recent vincristine on human hematopoietic progenitor cell collection in pediatric patients with central nervous system tumors

Central nervous system (CNS) malignancies represent 20% of all childhood cancers. To improve outcomes in infants and children with high-risk disease, treatment can include adjuvant chemotherapy and early autologous peripheral blood human progenitor cell collection (AHPCC), followed by high-dose chemotherapy and stem cell rescue. In many protocols, postoperative chemotherapy includes the administration of weekly vincristine (VCR) between induction chemotherapy cycles, regardless of scheduled AHPCC. We observed anecdotal AHPCC failures in children receiving midcycle VCR (MC-VCR). The study was an 8-year retrospective chart review of all children with a CNS malignancy and who underwent AHPCC. Information included patient demographic and clinical data, mobilization regimen, VCR administration, product yields, infusion toxicity, and patient charges. Data were analyzed relative to MC-VCR administration. Graphics and statistical analysis (t-test, chi-square, linear regression) were performed with commercial software. Twenty-four patients and 47 AHPCCs were available for analysis. Nine patients (37%) received MC-VCR within 7 days of scheduled AHPCC. MC-VCR was associated with delayed marrow recovery (17.9 days vs. 14.9 days, p=0.0012), decreased median peripheral CD34 counts (75 × 10(6) CD34/L vs. 352 × 10(6) CD34/L, p=0.03), decreased median CD34 yields (2.4 × 10(6) CD34/L vs. 17.8 × 10(6) CD34/kg, p=0.08), more AHPCCs per mobilization (2.9 vs. 1.1, p=0.01), and an increased rate of remobilization (33% vs. 6%). Mean patient charges were 2.5× higher in patients receiving MC-VCR than controls (p=0.01). MC-VCR should be withheld before scheduled AHPCC to optimize CD34 collection.

Factors Influencing Peripheral Blood Progenitor Cell Yield After 2nd Mobilization With G-CSF In Allogeneic Donors

Purpose2nd collection of Peripheral Blood Progenitor Cells (PBPC) from the same donor is increasingly required for the treatment of complications after allogeneic transplantation, but information regarding the efficacy of this procedure is still limited. MethodsWithin our program 212 allogeneic donors underwent a 2nd cycle of G-CSF-application and PBPC leukapheresis between 1996 and 2012. Demographical data and details of donation are shown in table 1. G-CSF (lenograstim) was administered at a median daily dose of 7.5µg/kg for 4.5d; leukapheresis was performed on day 5 and 6, if necessary. Peripheral CD 34+ counts and leukapheresis yields after both mobilization cycles were compared. The influence of age, sex, BMI, G-CSF schedule and the interval between the donations on the efficacy of the 2nd PBPC mobilization were analyzed, too.Table 1Characteristics of PBPC donors with 2 mobilization cyclesGender145 male (68.4 %);67 female (31.6%)Age, years (mean, min-max)37 (1-70)Relationship to the recipient171 unrelated (80.7%)41 related (19.3%)Body Mass Index (mean, min-max)24.8 (17.2-43.2)Schedule of G-CSF-application29 single dose174 devided doseInterval between 1st and 2nd mobilization, months (mean, min-max)7.3 (0.6-105.0) ResultsMedian CD 34+ counts in peripheral blood were 53.2/µl at day 5 of 1st mobilization compared to 49.5/µl at day 5 of 2nd mobilization. Between 1st and 2nd donation, the median yield of CD 34+ cells/kg recipient b.w. decreased from 7.3 x 106 to 6.6 x 106 (p = 0.026, see table 2). The number of aphereses necessary in both donation episodes did not differ in the majority of donors (n=175; 82.5%). A strong correlation was observed between the amount of CD 34+ cells per recipient b.w. after the 1st and 2nd PBPC donation (r=0.89). The majority (65.4%, n=17) of donors with poor mobilization (<3 x 106 CD 34+ cells/kg recipient b.w.) after the 1st leukapheresis reached similar low CD 34+ counts after the 2nd mobilization. The interval between both mobilization cycles did not affect the results of the 2nd donation. According to an adjusted model, the other variables (age, sex, BMI and G-CSF-schedule) had no additional influence on mobilization efficacy in the 2nd cycle of G-CSF application.Table 2Comparison of the results of the first and second PBPC mobilization (median values and range)Parameter1stMobilization2ndMobilizationpCD 34+ concentration in peripheral blood on day 5 of G-CSF application53.2/µl (9-243)49.5/µl (6-207)0.099Yield of CD 34+ cells/ kg recipient weight (in 1 or 2 leukaphereses)7.3 x106 (1.3-68.6)6.6 x106 (0.9-65.1)0.026Donors with 2 leukaphereses60430.068 ConclusionsIn the largest group of healthy donors reported so far, we could demonstrate a slight reduction (about 10%) of PBPC yield after the 2nd mobilization that might not be clinically significant. Demographic variables affected the results of both mobilization episodes in the same way and the interval between them was of no significance. Our data support the assumption, that stem cell mobilization is probably determined by genetic factors and the outcome of a 2nd donation can easily be predicted by the results of the 1st one. Disclosures:No relevant conflicts of interest to declare.

The Use Of Tevagrastim (Biosimilar Filgrastim XMO2) For Hematopoietic Stem Cell Mobilization In HLA Matched Sibling Donors For Allogeneic Stem Cell Transplantation To AML/MDS Patients

▪ IntroductionHuman recombinant G-CSF Filgrastim (Neupogen) has been widely used for the mobilization of CD34+ hematopoietic stem cells (HSC) from healthy donors. The experience with biosimilar G-CSF agents is limited and the only publication, to our knowledge, is the recent study by Schmitt M and co-authors (BMT 2013; 48, 922–925). This study involved 11donors and pts with various disease categories. The Biosimilar G-CSF was found to be comparable in efficacy and safety to Neupogen. Developing new cost effective mobilizing reagents if important.We, therefore, initiated a prospective study assessing Tevagrastim (biosimilar Filgrastim XMO2) for mobilization of CD34+ PB HSC in healthy sibling donors (MRD) for transplantation in pts with AML/MDS (NCT01542944). Materials and methods24 pts with AML or high-risk MDS undergoing allo-SCT from MRD were investigated. The study was approved by the National Regulatory Authorities and both patients and donors signed an informed consent. The donors, median age 46 years (range, 25–64), F- 14; M- 10 received Tevagrastim in a standard dose of 10 μg/kg BW s.c. BID for 4 days. On the morning of the 5th day they underwent conventional leukapheresis. The target yields of CD 34 cell was 5 × 106 CD34+ cells/kg BW of the recipient. If one leukapheresis was insufficient a second was performed and, the last dose of Tevagrastim was administered on the evening of the 5th day. The conditioning was myeloablative Bu/Cy (n=10), reduced toxicity Flu/Treo (n=7), Flu/Bu4 (n=3) or RIC Flu/Bu2 (n=4).The study parameters were: a CD34+ cell count,both absolute numbers and the CD34+ cells per kg BW of the recipient, the number of leukapheresis procedures, the number of CD3+ T lymphocytes in the graft, post transplantation engraftment including the WBC, the neutrophils and the platelets, as well as side effects. Follow up was 100 days post allo-SCT. ResultsEfficacy 77-1982 × 106 (median 749 × 106) CD34+ were collected. The number of CD34+ cell per kg BW of the pts was 0.93-35.4 × 106 (median 10.2 × 106). Collections contained 144-709 × 108 (median 299 × 108) CD3+ T-cells, 1.74-11.6 ×108 (median 4.4 ×108) per kg BW of the pts. The mean number of leukapheresis procedures was 1.3. Engraftment was: ANC >0.5× 109/L and >1× 109/L within a median of 13 days (range, 10–21) and 13.5 days (range, 10–22), respectively. PLT reached counts of >20× 109/L and >50× 109/L within a median of 16 days (range, 12–33) and 17 days (range, 12–33) from allo-SCT, respectively. The median days of isolation was 10 (range, 6-21). As for blood support, the median number of PC and PLT transfusions was 5 (range, 2-20) and 21 (range, 0-180), respectively. 18/22 (81.8%) pts achieved full donor chimerism at 1 month after transplantation (2 pts are too early to evaluate). SafetyOverall Tevagrastim was found to be safe with minimal transient side effects. Neither allergic reactions nor severe adverse events were observed in the donors. 12/24 donors reported transient arthralgias and 2 developed flu-like syndrome while receiving Tevagrastim. As for transplantation related toxicity in the 24 pts transplanted with Tevagrastim mobilized HSC grafts, side effects were not different than those we observed in historical controls. The main side effects were mucositis (n-15, grade II -9, grade III-IV- 6), infection complications (n-20) and fluid retention (n-8). One pt suffered from VOD (Grade-I) that resolved with conventional therapy. Five pts developed acute GVHD (grade II-III) that responded to conventional therapy. In total TRM was 1/24 at d 100. 2 pts died from leukemia progression. ConclusionsOur study with 24 AML/MDS pts, indicates that the G-CSF biosimilar XM02, Tevagrastim is safe and efficient for stem cells mobilization in HLA matched normal sibling donors. The CD34 yield and post transplantation engraftment are similar to those achieved with the human recombinant G-CSF Filgrastim (Neupogen). We have not seen significant differences in the graft CD34+ and CD3+ T lymphocytes cell count, the number of leukapheresis procedures and the regeneration of WBC, neutrophils and platelets in comparison with our historical controls. All patients promptly engrafted, and the donors developed only expected side effects like arthralgias and flu-like syndrome. Neither graft rejection nor side effects occurred more frequently than expected from the standard G-CSF. Disclosures:Nagler:Teva : Consultancy, Honoraria, Research Funding.

Polymorphism Of CD44 Influences Efficacy Of CD34+Cells Mobilization In Patients With Hematological Malignancies

▪ BackgroundIn the last decade peripheral blood was the main source of hematopoietic stem cells (HSC) for autologous (auto-SCT) and allogeneic (allo-SCT) transplantation. The exact mechanisms of HSC mobilization are still not clear and efficacy of the procedure is hardly predictable. Numerous clinical factors including age, number of previous intensive regimens, radiotherapy and type of disease can influence efficacy of CD34+ cell mobilization for auto-SCT in patients with hematologic malignancies. Recently it has been stated that ligand- receptor interactions of adhesion molecules such as SDF1/CXCR4, VLA4/VCAM-1 or CD44/osteopontin play important role in homing of HSC in hematopoietic niche. There is evidence that disruption of the ligand-receptor complex leads to egress of HSC to peripheral blood. Influence of single nucleotide polymorphisms in CD44, VCAM-1 and CXCR4 on efficacy of HSC mobilization was evaluated in healthy donors, but not in patients with hematological disorders.The aim of the present study was evaluation of constitutive polymorphism of genes encoding cytokines and receptors present in HSC niche and their impact on efficacy of mobilization of HSC in patients with hematological malignancies. Patients and Methods110 (60 females and 50 males) were enrolled to the study. The median age of the patients was 55 (range 22-69) years. All of the patients evaluated were eligible for autologous HSC mobilization and transplantation. The group consisted of patients with multiple myeloma (74), non-Hodgkin lymphoma (19), Hodgkin lymphoma (15) or acute myeloid leukemia (2). The mobilization procedures comprised chemotherapy and then G-CSF at a dose of 10µg/kg daily. ‘Poor mobilizers' group was defined according to GITMO criteria: patients with peak CD34+ in peripheral blood < 20/μL or total yield <2x 106 CD34+/kg in maximum 3 aphereses. Genotyping was performed using standard PCR-based assays. Three subgroups were established for the genotype in each polymorphism (homozygous more frequent, heterozygous and homozygous less frequent). ResultsThe group of patients (N=108) who achieved minimal threshold for collections (CD34+ at least 10/µl) proceeded to aphereses. Median total yield of CD34+ in this group of patients was 5,6 x 106//kg, while median number of cells collected during first apheresis was 3,3x 106//kg. Median number of days of G-CSF treatment before first apheresis was 10. Fifteen patients fulfilled the criteria for ‘poor mobilizer'.The group of ‘poor mobilizers' had higher frequency of TT allele in rs13347 (CD44) gene (CC+ CT vs TT p=0,047), the difference was even more pronounced in patients with multiple myeloma (N=72, p=0,027). TT homozygous genotype resulted in reduced CD44 mRNA expression at the time of apheresis in comparison with carries of C allele (median=40 in TT group, median=77 in CT and median=85 in CC group, p<0,001). Patients with TT allele had lower total yield CD34+/kg than the group with allele C (Median=3,7x 106//kg vs. 5,8x 106//kg, p=0.019) and lower number of CD34+cells gathered during first apheresis (0,95x 106//kg vs. 3,3x 106//kg, p=0.04). Multivariate logistic regression analysis including age, sex, diagnosis (multliple myeloma vs. others), number of previous treatment lines and CD44 polymorhism (TT vs CT+CC) revealed TT allele was the only factor associated with 5 fold higher risk of poor mobilization (p=0,037).Polymorphic variants of CXCR4 and VCAM-1 did not influence significantly efficacy of HSC mobilization in our group of patients.In conclusion, our results indicate that among investigated SNPs, only CD44 rs13347 has impact on efficacy HSC mobilization in patients with hematologic malignancies. CD44 SNPs analysis may be helpful for predicting of ‘poor mobilizers' population that may benefit from newer modalities using adhesion molecules inhibitors. Disclosures:No relevant conflicts of interest to declare.

Peripheral blood progenitor cell collection by two programs for autologous and allogeneic transplantation

In the Spectra apheresis instrument (Terumo BCT), both manual (Spectra-MNC) and automated (Spectra-Auto) programs have been widely used to collect peripheral blood progenitor cells (PBPCs). However, direct comparison of these programs remains extremely limited. We investigated 188 collections and products from autologous (patient) and allogeneic (donor) subjects and analyzed a subset of 89 allogeneic collections and products. Twenty-nine subjects who received apheresis for 2 consecutive days using both programs were also evaluated with a paired crossover comparison. The two programs processed similar volumes, but run time was longer with Spectra-Auto. Yield and efficiency of CD34+ cell collection were similar between these programs in the whole cohort, although white blood cell (WBC) and mononuclear cell (MNC) yields were higher with Spectra-MNC. In the allogeneic cohort, yield and efficiency of WBC collection were greater in Spectra-MNC. However, collected WBCs, MNCs, and CD34+ cells were similar between these programs in paired comparison. Regardless of program, preapheresis peripheral WBC, MNC, and CD34+ cell counts correlated with the number of cells collected. In contrast, preapheresis WBC counts in the whole cohort were negatively correlated with collection efficiencies of CD34+ cells in Spectra-MNC but not Spectra-Auto. The products collected using Spectra-MNC contained more contaminating platelets (PLTs) than Spectra-Auto, with a corresponding reduction in postdonation circulating PLTs. Spectra-MNC and Spectra-Auto showed distinct features that should be considered on a case-by-case basis. Similar investigations should be undertaken as new collection platforms are introduced.

Sex and Body Mass Index but Not CXCL12 801 G/A Polymorphism Determine the Efficacy of Hematopoietic Cell Mobilization: A Study in Healthy Volunteer Donors

Analyses of healthy donors of granulocyte colony-stimulating factor (G-CSF) mobilized hematopoietic stem and progenitor cells (HSPCs) and of patients undergoing autologous stem cell transplantation have suggested that individuals harboring the CXCL12-A allele mobilize a higher number of CD34 + HSPCs after G-CSF administration. We typed 463 healthy unrelated donors (376 men and 87 women) who had received daily subcutaneous injections at a mean dose of 7.36 ± 1.71 μg/kg G-CSF for 5 days for CXCL12 801 G/A using a real-time PCR assay. Interestingly, the median concentration of mobilized CD34 + cells on day 5 was almost identical in donors with the A-allele (79/μL; range, 11 to 249/μL) and the G/G-group (82/μL; range, 15 to 268/μL). In addition, the allelic distribution was not different in donors (n = 11) who mobilized less than 20/μL CD34 + cells. No difference in the overall yield of CD34 + cells in the apheresis product and in the number of CD34 + cells/kg recipient could be detected between both groups. In a multivariate regression model for the endpoint CD34 + cells/μL at day 5, only male sex (regression coefficient, 11.5; 95% confidence interval, 1.7 to 21.2, P = .021) and body mass index as continuous variables (regression coefficient, 3.5; 95% confidence interval, 2.5 to 4.5, P = .0001) but not age, smoking status, or CXCL12 allelic status represented independent variables. Our data derived from a large well-controlled cohort contradict previous analyses suggesting an association between CXCL12 allelic status and the yield of CD34 + HSPC after G-CSF mobilization. Concentration of CD34 + cells in the peripheral blood, the most objective parameter, could not be predicted by CXCL12 genotype.

Discordant CD34+ cell results in peripheral blood and hematopoietic progenitor cell–apheresis product: implications for clinical decisions and impact on patient treatment

A 50-year-old male with T-cell lymphoma presented for autologous peripheral blood stem cell transplantation. After granulocyte-colony-stimulating factor (G-CSF) mobilization, his peripheral blood CD34+ cell count was 166 × 10(6) /L on the day before collection, which predicted a high yield of CD34+ cells in the apheresis product. The first two collections had yields much lower than expected, triggering an investigation and changes to the apheresis collection methods since mobilization appeared adequate from the peripheral CD34+ values. Changes to the apheresis collection variables and instrumentation did not improve the yields in the next three collections. The laboratory investigation demonstrated that there was an interfering substance in the patient's plasma that was causing falsely high peripheral blood CD34+ cell values and that the low CD34+ cell yields in the collections were consistent with the actual peripheral blood CD34+ cell value. Based on this finding and after discussion with the clinical service, the patient then received plerixafor to increase the number of circulating CD34+ cells before Collections 6 and 7. The patient went on to achieve the target CD34+ cell dose and subsequently underwent a successful autologous transplant with full hematopoietic engraftment. This case demonstrates the importance of timely and critical review of laboratory results in the context of the specific patient. This case exemplifies how diligent review of laboratory results and open communication among the various teams can positively affect patient outcomes.

Remobilization of hematopoietic stem cells with high‐dose methotrexate and cytarabine in patients with non‐Hodgkin's lymphoma and multiple myeloma after failure to mobilize with chemotherapy and cytokines

High-dose chemotherapy supported by autologous stem cell transplantation is an effective treatment for patients with relapsed or refractory non-Hodgkin's lymphomas (NHLs) and fit patients with multiple myeloma (MM). However, failure rates of hematopoietic stem cell mobilization are estimated to be between 5 and 30%, respectively. Thus, we investigated the efficacy of the combination chemotherapy of high-dose methotrexate (MTX) and cytarabine with granulocyte-colony-stimulating factor (G-CSF) as a remobilization method in those who failed a prior mobilization and collection with chemotherapy and G-CSF. Mobilization failure was defined as a collection of fewer than 5 × 10(6) CD34+ cells after three to five apheresis procedures. MTX (3500 mg/m(2) in a 120-min infusion) on Day 1 and cytarabine (3000 mg/m(2) infusion for 120 min) on Day 4 and Day 5 were followed by G-CSF (10 μg/kg daily). A total of eight patients (six NHL and two MM; median age, 55 years) who had failed in prior mobilization with conventional chemotherapy and G-CSF underwent the second mobilization as described in the method. Successful collection of CD34+ cells (> 5 × 10(6) /kg) was achieved in six patients (75%) with three to five apheresis procedures. The total yield of CD34+ cells/kg body weight was 6.28 × 10(6) /kg (median; range, 1.53 × 10(6) -10.09 × 10(6) /kg). This preliminary result warrants further investigation of high-dose MTX and cytarabine plus G-CSF as a means to remobilize stem cells in those with prior failure to mobilize stem cells with chemotherapy and G-CSF.

Current and historical perspectives on methodological flaws in processing umbilical cord blood

Umbilical cord blood (UCB) hematopoietic stem cells (HSC-CD34+) are valuable for treating malignant or nonmalignant disease. Processing UCB by HESPAN-6% and anti-CD34-Miltenyi particles provides insufficient cells for treating adults. Physicochemical-electrokinetic studies on UCB-mononuclear cells (MNCs) under conditions of delayed processing, ice or very low temperatures, and some cell separation media identified artifacts introduced by procedures. Adsorption of biomaterials from cell damage by temperature, degradation products after using enzymes, harsh reagents, dithiothreitol, and HESPAN affect cell properties and distribution. Miltenyi particles internalized by cells could release iron that accumulating in liver or spleen would then risk toxicity. Summary topics included the effects of temperature, HESPAN (fast sedimenting agent), glycoproteases, DNase, and dithiothreitol risk affecting cell receptors in recognition, "homing," leading to possible unintended iatrogenic bioeffects should such cells be transfused into humans. The loss of undetectable and uncaptured low CD34 antigen-bearing cells by Miltenyi particles seems to occur when the current methods of isolation of CD34+ cells and other cells are critically assessed. The purpose here is to highlight and suggest avoiding the procedural flaws involved. Preventing ice temperatures avoids ice-damaged platelets releasing biomaterials that are adsorbed on cells altering UBC-MNCs/HSC properties and cell loss. Omitting the positive selection with antibody-linked Miltenyi particles obviates the use of harsh reagents to release the cells. Internalized Miltenyi particles are a toxicity hazard that needs investigations. Achieving approximately 5% yields of CD34+ cells (153 × 10(5) /110 mL cord-placenta blood) is a major advance holding great promise, for the first time increasing the prospect of stem cell therapy of 70-kg adults, using a single UCB donation (with dose of 1.5 × 10(5) cells/kg) and considerably cheaper cultured red blood cells manufacture (multiple packs/2 × 10(12) ).

Effect of different cryopreservation time on quality of umbilical cord blood cells

This study was aimed to explore the effect of different cryopreservation time on recovery rate of cord blood stem cells, and analyze the influence of cord blood cells after thawing on the engraftment speed of cord blood cells in patients. 20 cord blood units were stored at -196°C for 1 - 10 years. The cell viability, content of total nucleated cell (TNC), CD34(+) cells and the colony forming units of granulocyte/macrophage (CFU-GM) were assessed after thawing, the impact of cell recovery on engraftment speed in patients was analyzed. The results showed that as compared with data provided by Umbilical Cord Blood Bark, the different cryopreservation time had no effect on yield of cord blood stem cells after thawing. The cell viability was (92.75 ± 2.55)% after thawing, the yields of TNC, CD34(+) cells and CFU-GM were 89.9%, 84.8% and 84.3%, compared with that of pre-freezing, their differences were statistically significant (P = 0.000), however, loss of cells had no effect on the time of neutrophils and platelets engraftment. The TNC and CD34(+)cell count after thawing correlated closely with that of pre-freezing (r = 0.954 and r = 0.931, P = 0.000), but CFU-GM content poorly correlated with that (r = 0.285, P = 0.223). It is concluded that cryopreservation and thawing process can damage the cord blood stem cells, leading to cell loss, but not affect transplant results.

Mobilization of PBSC for allogeneic transplantation by the use of the G-CSF biosimilar XM02 in healthy donors

The human recombinant G-CSF filgrastim has been widely used for the mobilization of CD34+ stem cells of healthy donors (HD). In 2008, the G-CSF biosimilar XM02 (Ratiograstim, Tevagrastim and Biograstim) was approved by the European Medicines Agency (EMA) for the mobilization of PBSC. However, there is limited experience in the application of biosimilar G-CSF for the mobilization of PBSC especially in HD. Therefore, we investigated in two cohorts (n=22), the efficacy and safety of PBSC mobilization by either biosimilar G-CSF or reference G-CSF. We observed a similar yield of CD34+ stem cells as well as CD3+ T-cells and nucleated cells in both groups and a safe engraftment in all patients with similar reconstitution of hematopoiesis in all hosts. In summary, we found a comparable efficacy and safety of biosimilar G-CSF when compared with reference G-CSF.

Successful Mobilization, Intra-Apheresis Recruitment, and Harvest of Hematopoietic Progenitor Cells by Addition of Plerixafor and Subsequent Large-Volume Leukapheresis

Background: In patients failing successful conventional mobilization of hematopoietic progenitor cells (HPC) plerixafor (Mozobil®) seems to be an alternative. We report a series of 14 patients with multiple myeloma or NHL successfully mobilized and harvested by plerixafor together with large-volume leukaphereses (LVL). Methods: In a first series (GI), 5 patients were mobilized with G-CSF and plerixafor. In the second series (GII), 9 patients were mobilized by chemotherapy, G-CSF, and plerixafor. Results: In GI and GII, addition of plerixafor led to a significant (p < 0.01) increase of leukocytes and CD34+ cells in peripheral blood (PB). In GII, the median number of CD34+ cells in PB before and after addition of plerixafor was significantly (p = 0.019) higher compared to GI (9 vs. 5 and 50 vs. 24 cells/µl, respectively). In GI and GII, a median number of three or one aphereses was performed. In GII, the median yield (6.7 × 10⁶ CD34+ cells/kg) of the first apheresis and the median intra-apheresis recruitment of CD34+ cells were significantly (p < 0.05) higher compared to GI (2.94 × 10⁶ CD34+ cells/kg). All patients transplanted, 5 in GI and 8 in GII, exhibited successful engraftment. Conclusions: Plerixafor and G-CSF mobilization or the addition of plerixafor during non-optimal chemotherapy and G-CSF mobilization together with LVL enabled, independent of leukocyte count and even without detectable CD34+ cells before addition of plerixafor, sufficient harvest of HPC numbers for transplantation. Addition of plerixafor during chemotherapy and G-CSF mobilization led to an increased intra-apheresis recruitment and a significantly higher yield of CD34+ cells compared to plerixafor and G-CSF steady-state mobilized patients.

Plerixafor use for peripheral blood stem cell mobilization in Korea

Peripheral blood stem cell (PBSC) mobilization is a prerequisite for the success of autologous stem cell transplantation (ASCT), and the infusion of adequate number of CD34+ hematopoietic progenitor cells is correlated with successful engraftment [1]. Traditionally, chemotherapeutic agents such as cyclophosphamide in combination with granulocyte-colony stimulating factor (G-CSF) have been used for mobilization of hematopoietic stem cells from bone marrow into peripheral blood. However, approximately 10-30% of patients who are at risk of mobilization failure are unable to mobilize minimum required number of PBSCs (≥2.0×106 CD34+ cells/kg) for the success of ASCT [2], and consequently fail to undergo ASCT. Plerixafor (AMD3100), a small molecule inhibitor of stromal cell-derived factor-1α binding to the CXCR4 receptor, was introduced as a solution for patients who show inadequate mobilization of CD34+ PBSCs. Results of Phase I/II trials demonstrated the efficacy of plerixafor, and a subsequent Phase III study showed that the use of plerixafor in combination with G-CSF significantly increased the number of CD34+ PBSCs, compared to G-CSF alone [3]. Plerixafor has been approved for patients with lymphoma and multiple myeloma who show poor PBSC mobilization, and its use is expected to increase the likelihood of successful ASCT. However, it is still difficult to define 'poor' mobilization because the total number of CD34+ cells can be influenced by the number of apheresis. There have been variable parameters to evaluate the extent of mobilization, such as cumulative apheresis yield as well as the number of CD34+ cells per apheresis. In addition, the total yield of CD34+ cells depends on the number of courses of apheresis, which is, in turn contingent on physicians' discretion. Recently, the Italian Group for Stem Cell Transplantation (GITMO) proposed consensus criteria for 'proven poor mobilizers' as follows: (a) circulating CD34+ cell peak of less than 20/µL up to 6 days after mobilization with G-CSF or up to 20 days after adequate mobilization such as using G-CSF 10 µg/kg if used alone or 5 µg/kg after chemotherapy, (b) less than 2.0×106 CD34+ cells per kg after fewer than three apheresis [4]. In Korea, use of plerixafor in combination with G-CSF is approved in patients with non-Hodgkin lymphoma and multiple myeloma for PBSC mobilization, and reimbursement is provided to those who have failed previous mobilization attempts with chemotherapy and G-CSF. However, the appropriate time for the initiation of plerixafor is still subject to ambiguity due to the lack of consensus on usable criteria for determining mobilization failure. Despite CD34+ PBSC count being considered the most powerful predictive factor of mobilization failure, the measurement of circulating CD34+ cells is not feasible in a majority of institutes in Korea. Therefore, at the recent meeting of the Consortium for Improving Survival of Lymphoma (CISL), the board members adopted a consensus definition of 'mobilization failure' as a count of less than 2.0×106 CD34+ cells/kg after three courses of apheresis. Thus, if the total number of CD34+ cells does not reach the minimum requirement for ASCT, the use of plerixafor should be considered, rather than continuation of apheresis with G-CSF. Furthermore, in specific patient populations which are expected to have low possibility of adequate cell yield such as elderly and heavily-pretreated patients, the board members of CISL discussed early administration of plerixafor. While the risk factors of mobilization failure have not yet been clearly identified, the recent GITMO working group proposed consensus criteria to define 'predicted poor mobilizers' as follows: (1) they failed a previous collection attempt (not otherwise specified); (2) they previously received extensive radiotherapy or full courses of therapy affecting PBSC mobilization; and (3) they met two of the following criteria: advanced disease (≥two lines of chemotherapy), refractory disease, extensive bone marrow involvement or cellularity <30% at the time of mobilization; age ≥65 years (4). A recent study on identifying prognostic factors for prescribing plerixafor-based PBSC mobilization showed that an age of 65 years, a diagnosis of non-Hodgkin's lymphoma, and treatment with four chemotherapy regimens were significantly associated with mobilization failure. Given the congruence between these prognostic factors and GITMO criteria of 'predicted poor mobilizers', it is evident that PBSC mobilization is impacted by the existence of risk factors and hence, the early use of plerixafor for high-risk groups should be carefully considered [5]. Further study is warranted to prevent mobilization failure by maximizing the benefit of plerixafor in Korean clinical setting. Plerixafor is a new class agent with a novel mechanism of action and has the potential to lead to a paradigm shift in PBSC mobilization and success of ASCT. In the era of plerixafor, considerable changes in clinical practices associated with PBSC mobilization can be expected in Korea.

Evaluation of Leukemic Stem Cell Persistence in Chronic Myeloid Leukemia (CML) Patients in Complete Molecular Remission Induced by First Line TKI Therapies

AbstractAbstract 3726Recent data showed that BCR-ABL expressing leukemic stem cells (LSCs) persist in vivo in patients in complete molecular remission (CMR) according to European Leukemia Net criteria (Chomel et al, Blood 2011, Chu et al, Blood 2011). It has also been established that, in this category of patients (CMR 4.5– 5), Imatinib Mesylate (IM) discontinuation leads to relapse in the majority of cases (60%), especially during the first 6 months. Although very likely, a formal link has not been established between these two phenomena and, in particular, the prevalence of LSC persistence in a cohort of patients treated with TKI as a first line therapy has not been established. To determine the prevalence of LSC persistence in CMR induced by first line TKI therapies, we have evaluated by clonogenic and long-term culture initiating cell (LTC-IC) assays, the presence of BCR-ABL expressing LSCs in marrow samples of 26 patients in CMR for > 2 years. CMR was induced by IM (n=22), Dasatinib (n=3) or Nilotinib (n=1) as a first line treatment. CD34+ cells were isolated from bone marrow aspirates (2–4 ml) using immunomagnetic columns and a clonogenic assay was performed. At week+2, clonogenic progenitors were counted and 20 to 40 individual hematopoietic colonies were plucked from methylcellulose for RNA extraction. In some cases, pooled CFU-Cs were also analyzed (20 pools of 10 hematopoietic colonies). The same CD34+ marrow sample was used to start LTC-IC assays, which were performed in murine MS5 cell feeders. At week+6 the cultures were sacrificed and the number of LTC-IC-derived clonogenic cells was determined. Hematopoietic colonies were plucked as decribed above for CFU-Cs for RNA extraction and BCR-ABL expression was quantified by q-RT-PCR. For 4 patients, the yield of CD34+ cells was not sufficient for stem cell assays (3 pts treated with IM; 1 with Dasatinib). qRT-PCR analyses of CFU-Cs revealed the presence of BCR-ABL expressing cells in 3 patients out of 16 tested so far. In all three patients in CMR induced by IM, this involvement can be estimated to approximately 100–360 leukemic CFU-GM/ml of bone marrow. Concerning LTC-IC assays, 2 patients out of 6 tested so far had evidence of BCR-ABL expressing LTC-IC-derived progenitors, one of these patients having also involvement of the clonogenic compartment. In both patients, there was a significant involvement of LTC-IC-derived progeny estimated to 1000–4000 LTC-IC derived progenitor/ml of bone marrow. In the 4 patients in whom LTC-IC compartment was found to be negative for LSCs, CMR was induced by IM in 3 Pts (IM 400 mg n= 2, IM 800 mg for accelerated phase n=1) and by Dasatinib (100 mg) in 1 Pt. Interestingly, in all q-RT-PCR assays, the amount of BCR-ABL mRNA (evaluated by the BCR-ABL/ABL ratio) in individual CFU-Cs appeared to be low, confirming our previous results (Chomel et al Blood 2012) and suggesting that these cells are not addict to BCR-ABL-TK activity for their survival. In 12 patients, the evaluation of LTC-IC compartment is underway. Overall, These data reveal that in patients in CMR induced by IM as a first line therapy, our strategy could identify two types of stem cell response, with some patients presenting a complete eradication of the leukemic stem cell compartment (within the limits of the sensitivity of the test) and others showing high numbers of BCR-ABL expressing stem cells despite the continuous CMR status on TKI therapy. It remains to be determined if the involvement of LTC-IC-compartment (in 2/6 analyzed so far) by LSC expressing low levels of BCR-ABL can be overcome by long-term TKI therapy in some patients. In addition, it will be of interest to determine if a correlation exists between LSC-positive patients the potential occurence of a molecular relapse upon discontinuation of TKI therapy. From the mechanistic point of view, the persistence of residual marrow LSCs in some CML patients in CMR strongly suggests the presence of active mechanisms of either LSC retention and/or LSC quiescence-promoting interactions within in the hematopoietic niche. Finally, the comparison of the LSC persistence phenomenon in CMR induced by TKI2 versus IM as first line treatment will be of major interest to determine the LSC eradication potential of these drugs. Disclosures:Rea:Bristol Myers-Squibb, Novartis, and Teva: Honoraria. Turhan:Novartis, Bristol Myers Squibb: Honoraria, Research Funding.