Hematopoietic Stem Cell Collection Research Articles

Hematopoietic, Immune Recovery and Outcomes after Autologous Stem Cell Transplantation Following Plerixafor Plus G-CSF Versus Chemotherapy and G-CSF Based Mobilization in Pediatric Patients

Background: High-dose chemotherapy (HDC) and autologous stem-cell transplantation (ASCT) has shown survival benefits for chemo-sensitive high risk pediatric solid tumors. Collection of sufficient hematopoietic stem cells is required for ASCT. Plerixafor, a CXCR4 receptor antagonist, has been used when granulocyte colony-stimulating factor (G-CSF) and chemotherapy based (conventional) mobilization may not enable us to collect enough cells, especially in recurred or heavily treated patients. The experiences of plerixafor in pediatric patients are lacking as compared with adults, and immune reconstitution and outcomes in pediatric patients after ASCT with plerixafor based mobilization have not been reported in the literature as far as we have known. Therefore, we aimed to compare engraftment, hematopoietic and immune recovery and transplantation outcomes after ASCT between plerixafor plus G-CSF based and conventional mobilization group in pediatric patients.Methods: Propensity score matching (PSM) by optimal method with 1:1 ratio was performed to determine the control group. Age, sex, diagnosis, transplantation year, conditioning regimen, and number of transplantation were used as matching variables. We evaluated available parameters such as the day of neutrophil and platelet engraftment, absolute neutrophil counts (ANCs), absolute lymphocyte counts (ALCs), platelet and hemoglobin counts and lymphocyte subsets (CD3, CD4, CD8 [T cell] counts). Mann-Whitney U test was used to compare between two groups.Results: There were a total of 233 (21 plerixafor plus G-CSF based and 212 conventional mobilization group) patients who underwent HDC and ASCT in Seoul National University Children's Hospital between 2009 and 2016. By creating matched samples using PSM, 42 (21 plerixafor plus G-CSF based and 21 conventional mobilization group) patients were analyzed. The diagnoses were osteosarcoma in 14, brain tumor in 10, lymphoma in 6, neuroblastoma in 5, Ewing sarcoma in 4 and retinoblastoma in 3 patients. We used plerixafor plus G-CSF in patients who previously failed peripheral blood stem cell mobilization by chemotherapy and G-CSF. Patients received G-CSF (10 μg/kg) for 4 days, without prior chemotherapy. Then plerixafor (240 ug/kg) and G-CSF (10 ug/kg) were administered subcutaneously, at 10 and 2 hours before each apheresis. All 21 patients using plerixafor were mobilized successfully, and the median number of CD34+ cells were 10.65 (range 2.35-28.97) × 106/kg after 1 to 4 cycles of apheresis without serious complications. Fifty six ASCTs were performed, including 14 tandem transplantation. The median age at ASCT was 6 (range 3.7-22.4) years. All patients in conventional mobilization group achieved engraftment, but 2 patients in plerixafor plus G-CSF based mobilization group died due to acute respiratory distress syndrome before platelet engraftment and 1 patient died of sudden cardiac arrest before neutrophil and platelet engraftment. The engraftment tended to be faster in conventional mobilization group, but there was no statistically significant difference except for the day of ANC more than 0.5 × 109/L for 3 consecutive days (p=0.040). The ALC was lower in plerixafor plus G-CSF based mobilization group at one and six months after ASCT, respectively (p <0.001, p=0.041). The CD3, CD4 and CD8 count was lower in plerixafor plus G-CSF based mobilization group at one month after ASCT, respectively (p <0.001, p=0.007, p<0.001). The CD8 count was lower in plerixafor plus G-CSF based mobilization group (p=0.016), but the CD3 and CD4 count showed no statistical significant between-group differences at one year after ASCT, respectively (p=0.098, p=0.473). There was no significant between-group differences of overall survival (85.2% in plerixafor plus G-CSF based and 68.8% in conventional mobilization group, p=0.496) with a median follow-up duration of 22.7 (range 0.1-90.2) months.Conclusion: Hematopoietic and immune recovery tended to be slower in plerixafor plus G-CSF based mobilization group, but was done to a similar extent over time. In addition, transplant outcomes are similar between two groups. Our study suggests that plerixafor could be used safely and enabled more patients to procced to successful ASCT in pediatric patients. DisclosuresNo relevant conflicts of interest to declare.

Long-Acting, Oxidation-Resistant IL-33 Mobilizes High-Quality Hematopoietic Stem and Progenitor Cells More Efficiently Than Wild-Type IL-33, G-CSF or AMD3100

▪Mobilization and collection of peripheral blood hematopoietic stem and progenitor cells (HSPC) has become increasingly important for hematopoietic cell transplantation. Granulocyte-colony stimulating factor (G-CSF) alone or in combination with the CXCR4-inhibitor AMD3100 are the most common mobilization approaches. These are however limited since they (a) fail to mobilize a sufficient number of cells in a significant fraction of patients, and (b) are biased towards myeloid immune reconstitution. A novel, single drug mobilization agent that results in a more balanced - myeloid and lymphoid - reconstitution would therefore be highly favorable to improve transplantation outcome.Interleukin (IL)-33 has recently been described as a potent mobilizer of HSPCs with long-term reconstitution potential. IL-33 is an intracellular protein that is believed to be enzymatically activated, released upon cell death, and inactivated upon oxidation. Whereas mobilization of HSPCs by G-CSF has been linked, at least in-part, to modulating the CXCR4-CXCL12 axis, the mobilizing function of IL-33 has been attributed to a different chemokine, CCL7.Here we asked whether IL-33 mobilizes HSPCs better than current clinical mobilizing agents, G-CSF and/or AMD3100, and how the quality of the mobilized cells compares. By flow cytometry and colony-forming assays, we observed for the first time that IL-33 treatment in mice mobilized Lin- Sca-1+ c-kit+ (LSK) cells and common myeloid progenitors (CMP/CFU-GEMMs) more efficiently than G-CSF or AMD3100 alone, and that IL-33 acts synergistically with both drugs. Next we engineered IL-33 variants lacking native cysteine residues, thereby rendering these variants resistant to oxidative inactivation. These variants had equal or better in vitro activity (a) in cell-based reporter assays and (b) in inducing CCL7 protein production by primary human umbilical vein endothelial cells (HUVECs). In vivo, these variants mobilized LSK cells and, interestingly also common lymphoid progenitor (CLP) and endothelial progenitor cells, more efficiently than wild-type IL-33 or G-CSF. We then engineered a one-armed IL-33-Fc fusion molecule comprising a single oxidation-resistant IL-33 fused to an Fc domain. Interestingly, a single dose of this oxidation-resistant IL-33-Fc variant was sufficient to significantly increase the mobilization of LSK, CLP, and endothelial progenitor cells, whereas oxidation-resistant IL-33 or G-CSF were not.In conclusion, our findings suggest that a single dose of long-acting, oxidation-resistant IL-33 may allow efficient mobilization of high-quality HSPCs including CLP cells, and may thus represent a novel mechanism to improve bone marrow transplantation.All authors were employed by Bayer at the time the studies were conducted. DisclosuresAlt:Bayer: Equity Ownership; Global Blood Therapeutics: Employment, Equity Ownership. Schopies:Bayer: Employment. Liu:Global Blood Therapeutics: Employment. Kauser:Global Blood Therapeutics: Employment, Equity Ownership. Bringmann:Cerus Corporation: Employment. Thompson:Bayer: Employment. Yuan:Bayer: Employment.

Second Autologous Stem Cell Transplantation in Poor Stem Cell Mobilizers Multiple Myeloma Patients

Second Autologous Stem Cell Transplantation in Poor Stem Cell Mobilizers Multiple Myeloma Patients

Prediction of Severe Acute Graft-Versus-Host Disease (aGVHD) Based on Whole Transcriptome Sequencing of Donor CD4 T Cells

Background: After decades of intensive research, GVHD remains the unrelenting nemesis of patients and physicians involved in allogeneic hematopoietic cell transplantation (AHCT). While some plasma biomarkers can predict response to GVHD therapy at day 14-post AHCT and at the onset of clinical GVHD, an ideal tool would enable prediction of GVHD prior to transplantation. Recent systems immunology analyses of human cohorts have shown that immune parameters are stable over time within a given person, but display substantial inter-individual variations. We therefore hypothesized that inter-individual variations in T-cell function could explain a fundamental question in GVHD pathogenesis: while all allogeneic donor-recipient pairs are histo-incompatible, only a fraction of recipients present severe aGVHD post-AHCT. In order to address this question, we searched for potential correlation between the whole transcriptome of donor CD4 T cells and the occurrence of aGVHD in their recipient.Methods and Patients: We performed RNA-sequencing on purified CD4 T cells from 330 AHCT donors from three centers (Boston, Calgary, Montreal). Donor and recipient were 8/8 HLA matched siblings. CD4 T lymphocytes were isolated from blood samples collected from donors prior to hematopoietic stem cell mobilisation and collection. Recipients with acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), chronic myeloid leukemia (CML), or myelodysplastic syndromes were included. Both myeloablative and reduced intensity conditioning were included. Peripheral blood and marrow graft were used as source of stem cells. Recipients received standard calcineurin based GVHD prophylaxis. Acute(a) and chronic(c) GVHD were graded according to Glucksberg and IBMTR classification. RNA was poly-A enriched and sequenced on an Ion Proton to an average depth of 40M reads. Expression profiles were computed using TopHat and cuffquant. A nested cross-validation protocol was used to train a logistic regression with either L1 or L2 regularization to predict the onset of acute GVHD of IBMTR grade C and above. An outer 10-fold cross-validation was used for the selection of hyper-parameters (number of genes considered, type and weight of the regularization) and an inner leave-one-out cross-validation was used for the predictor training. Area under the ROC curve (AUC) and relative risks were computed based on out-of-sample predicted risk of GVHD.Results: aGVHD developed in 43%. According to IBMTR classification, severe and potentially lethal aGVHD of grade ≥ C was diagnosed in 13.6% of recipients.Figure 1A presents the cross-validated performance of logistic regression classifier using either all annotated transcripts (solid line, AUC = 0.62) or expression profiles in which miRNA and snoRNA were removed (dashed line, AUC = 0.49). Discrimination between high-risk vs. low-risk donors was absolutely contingent upon the inclusion miRNA and snoRNA expression profile in our algorithm. Depending on the selected threshold applied to our predictive score, we can identify donors whose relative risk to trigger severe aGVHD is increased by 1.8- to 4-fold (Figure 1B depicting bootstrap-based confidence intervals (95%) for relative risks derived for all possible thresholds).Conclusion: Transcriptome sequencing of donor CD4 T cells can segregate donors that carry a high- vs. low-risk of causing severe aGVHD in their recipient. The key variable here was the miRNA and snoRNA expression profile. Considering that i) miRNA and snoRNA detected with our method (poly-A enriched RNA) are mostly located in introns, and ii) that intron retention regulates CD4 T-cell activity (PMID: 27369383), the most parsimonious mechanistic explanation is that the extent of intron retention in donor CD4 T cells determines the risk of severe aGVHD in recipients. Transcriptome sequencing of donor CD4 T cells could become a useful tool to optimize donor selection, personalized GVHD prophylaxis and improve AHCT outcome. [Display omitted] DisclosuresBusque:Novartis Canada Inc.: Honoraria; Pfizer: Honoraria; Bristol Myer Squibb: Honoraria; Paladin: Honoraria.

The Impact of Foundation of the National Turkish Donor Registry, Turkok on Donor Matching Kinetics

Purpose: Turkish Stem Cell Coordination Center (TURKOK) was established by the Turkish Ministry of Health in April 2015 in order to facilitate unrelated hematopoietic stem cell (HSC) transplantation in Turkey. The donor recruitment task has been linked to the Turkish Crescent's (National Blood Product Provider) nationwide distributed branches. As of July 2017, more than 300.000 volunteer donors are registered into the database. As of July 2017 they have facilitated more than 300 HSCTs. Transplant Registry Istanbul (TRIS) and Transplant Registry Ankara (TRAN) are two University affiliated donor search - co-ordination centers, which are officially responsible for the search, matching and organization of the delivery for unrelated donor HSC in Turkey. The foundation of TURKOK should have theoretically decreased our time spent to find a suitable matched donor. The aim of this study is to compare the time frames between admission, confirmation, work-up, and infusion of HSC between TURKOK and other WMDA or NMDP facilitated International Volunteer Donor Registries.Subjects & Methods: In 2015 1000 allogeneic HSCTs were peformed in Turkey and only 18% were from MUDs. This is a single center analysis of a dedicated active HSCT center, which is performing >70 allogeneic HSCTs per year and utilizing alternative donor sources >50%. Patient charts were reviewed retrospectively and 51 patients who admitted to our center within 2015-2017 for various high risk hematological malignancies (mostşy acute leukemia, in which allogeneic HSCT was indicated, were identified for this study. Patients were divided into two groups: (1) Patients who had received HSC from matched unrelated donors (MUD-T) (10/10-9/10) facilitated by TURKOK (N=36) ; (2) 15 patients who had received HSC from International Registries facilitated by TRIS or TRAN (MUD-I) . The duration between application and finding a suitable match (tAM), duration between matching and confirmation (tMC) and time from application to HSC transplantation (tATX) were calculated for each of these two groups. The time duration between admission and donor identification and admission to HSCT were not distributed normally. The mean values were compared between two groups by Mann-Whitney U test. p < 0.05 was considered statistically significant.Results: In 51 HSC recipients the overall median durations for tAM, tMC and tATX were 71 (range, 1-172), 33 (range, 1-181) and 97 (range, 13-172), respectively. In MUD-I overall median durations for tAM, tMC and tATX were 90 (range, 1-170), 40.5 (range, 1-181) and 98 (range, 13-172), whereas in MUD-T the timelines for tAM, tMC and tATX were 37 (range, 4-172), 17 (range, 6-151) and 96 (range, 73-128), respectively. The p values for comparative analysis betwen MUD-I and MUD-T for tAM, tMC and tATX were p=0.005, p=0.007 and p>0.05, respectively.Conclusion: In this relative limited size of HSC recipients from MUDs we observed accelerated kinetics for tAM and tMC, which means having a National Registry have a positive impact on finding an available match and confirmation process. On the other hand this improvement did not translate into rapid HSCT. The tATX between MUD-I and MUD-T are not significant. TURKOK has to establish and improve the organization of confirmed donor for collection of HSCs. The decreased timeline from application to confirmation is encouraging, but the time spent from confirmation to work/up and collection are still high. As TURKOK is a recently established donor bank, there is room for improvement. DisclosuresNo relevant conflicts of interest to declare.

Improved collection of hematopoietic stem cells and progenitors from Fanconi anemia patients for gene therapy purposes

Difficulties in the collection of hematopoietic stem and progenitor cells (HSPCs) from Fanconi anemia (FA) patients have limited the gene therapy in this disease. We have investigated (ClinicalTrials.gov, NCT02931071) the safety and efficacy of filgrastim and plerixafor for mobilization of HSPCs and collection by leukapheresis in FA patients. Nine of eleven enrolled patients mobilized beyond the threshold level of 5 CD34+ cells/μL required to initiate apheresis. A median of 21.8 CD34+ cells/μL was reached at the peak of mobilization. Significantly, the oldest patients (15 and 16 years old) were the only ones who did not reach that threshold. A median of 4.27 million CD34+ cells/kg was collected in 2 or 3 aphereses. These numbers were markedly decreased to 1.1 million CD34+ cells/kg after immunoselection, probably because of weak expression of the CD34 antigen. However, these numbers were sufficient to facilitate the engraftment of corrected HSPCs in non-conditioned patients. No procedure-associated serious adverse events were observed. Mobilization of CD34+ cells correlated with younger age, higher leukocyte counts and hemoglobin values, lower mean corpuscular volume, and higher proportion of CD34+ cells in bone marrow (BM). All these values offer crucial information for the enrollment of FA patients for gene therapy protocols.

Moral Distress: Its Manifestations in Healthy Donors during Peripheral Blood Hematopoietic Stem Cell Harvesting

Hematopoietic stem cell donors (HSCDs) may have ambivalent feelings about donation. These feelings are related to moral obligation to help a sick relative and/or fear about the donation procedure. This ambivalence can produce moral distress (MD) and anxiety, which are usually unnoticed by the treating physician. The aim of this study was to evaluate the incidence of MD and anxiety in a group of related HSCDs for allogeneic transplantation. In this prospective observational study, to assess MD and anxiety, we applied 3 self-answered questionnaires-a questionnaire developed to assess MD (MDQ), State Trait Anxiety Index (STAI), and Edmonton Symptom Assessment System (ESAS)-before, during, and after hematopoietic stem cell donation. A total of 60 consecutive related HSCDs with a mean age of 38.2 years were included. Thirty-six were male. Hematopoietic stem cell collections were done by apheresis, performed as an outpatient process in all cases. The incidence of MD during the donation process was 56%. The proportion of HSCDs with moderate to high state anxiety decreased significantly from before donation (63%) to after donation (30%). Higher scores for MD correlated with higher scores on the STAI questionnaire (r=0.448; P < .005). Thirty-seven donors (62%) had at least 1 physical symptom even before the stem cell mobilization process started, mainly anxiety (33%), difficulty sleeping (33%), and fatigue (30%). The number of symptomatic donors increased during donation (100%) and decreased after the procedure (80%). We conclude that MD and anxiety symptoms experienced by HSCDs are very common and can be explained by mixed feelings about the donation process. Providing comprehensive psychological support before starting the donation process and guaranteeing respect for the donor's autonomy are needed to decrease the negative impact of the donation experience.

CAMRELIZUMAB COMBINED WITH GEMOX IN PATIENTS WITH RELAPSED OR REFRACTORY HODGKIN LYMPHOMA

Objective: PD-1antibody has shown remarkable efficacy in relapsed or refractory Hodgkin lymphoma (R/R HL). Here we designed a clinical trial to evaluate the efficacy and safety of PD-1 antibody camrelizumab combined with GEMOX chemotherapy in R/R HL patients. The preliminary results had been obtained. Method: This is an open-label, single-center, non-randomized, phase 2 study (NCT04239170). The main inclusion criteria include age≥18, ECOG < 2, histopathology confirmed classical HL, number of previous chemotherapy regimen ≥1 but <3, having measurable lesion(s) assessed by Lugano 2014 criteria and preparing to receive autologous stem cell transplantation (ASCT). The treatment schedule is shown in Figure 1. The primary endpoint is the proportion of patients who achieved PET-CT-confirmed complete response (CR) according to Lugano 2014 criteria. Results: From March 2020 to February 2021, 14 patients were enrolled. One patient was excluded because of the diagnosis of diffused large B-cell lymphoma in the second biopsy. The remaining 13 patients ranged in age from 22 to 48, with a male-to-female ratio of 8:5. Among the 13 patients, nine patients failed to response in the last treatment. Among the 4 relapsed cases, 2 of them recurred within one year and 3 of them had extranodal invasion. At the first tumor response evaluation, 61.5% (8/13) patients achieved CR and 30.8% (4/13) patients achieved PR, resulting an ORR of 92.3%. Only one patient had stable disease (SD). The 5 patients who had PR or SD received an additional cycle of camrelizumab plus GEMOX treatment, and 3 of them had a second tumor response results, in which one patient who had PR in the first evaluation was found to achieve CR. The remaining 2 patients were still waiting for evaluation. Up to now, collection of the autologous hematopoietic stem cells was completed in seven patients and the average number of CD34+ cells was 2.24 × 106/kg. Most adverse events (AEs) were of grade 1 to 2. No grade 4 or above AEs occurred. AEs of grade 3 included neutropenia (2.3%) and transfusion reaction (2.3%). Conclusion: The preliminary data obtained so far suggests that camrelizumab combined with GEMOX chemotherapy has encouraging clinical efficacy and tolerable toxicity in R/R HL patients and merits continuous study. Abbreviations: ASCT, autologous stem-cell transplantation *Patients who wait longer than 4 weeks to receive ASCT may be considered to receive 1 or 2 camrelizumab monotherapy. Experiment design Keywords: Combination Therapies No conflicts of interest pertinent to the abstract.

Successful autologous peripheral blood stem cell collection using large volume leukapheresis in patients with very low or undetectable peripheral blood CD34+ progenitor cells

Autologous stem cell transplantation provides some patients with hematolymphoid and solid organ malignancies an opportunity for cure. Management of peripheral hematopoietic stem cell (HSC) collections differs among institutions, especially if a very low pre-procedure peripheral blood CD34+ cell count (PBCD34) is demonstrated. This study retrospectively analyzed results of large-volume peripheral HSC collections in 91 patients over approximately two years. Fifteen patients with PBCD34 < 10 × 10e6/l (eleven with undetectable PBCD34) were compared to 76 patients with higher counts on the first collection day (adequate mobilizers). The poor mobilizer group had significantly lower pre-collection WBC and platelet counts as well as collection yields. However, most patients with PBCD34 < 10 × 10e6/l (80 %) collected the minimum target for HSC transplant (2.0 × 10e6 CD34+ cells/kg) in <5 consecutive days of collection, and those who did collect the minimum successfully underwent autologous transplantation, with hematopoietic engraftment and long-term survival comparable to the adequate mobilizers. Successful HSC collection may often be achieved regardless of d 1 PBCD34 counts.

Disease severity impacts plerixafor-mobilized stem cell collection in patients with sickle cell disease

Recent studies suggest that plerixafor mobilization and apheresis in patients with sickle cell disease (SCD) is safe and can allow collection of sufficient CD34+ hematopoietic stem cell (HSC) collection for clinical gene therapy applications. However, the quantities of plerixafor-mobilized CD34+ cells vary between different SCD patients for unknown reasons. Twenty-three participants with SCD underwent plerixafor mobilization followed by apheresis, processing, and HSC enrichment under a phase 1 safety and efficacy study conducted at 2 institutions. Linear regression or Spearman's correlation test was used to assess the relationships between various hematologic and clinical parameters with total CD34+ cells/kg collected. Median CD34+ cells/kg after 2 or fewer mobilization and apheresis cycles was 4.0 × 106 (range, 1.5-12.0). Similar to what is observed generally, CD34+ yield correlated negatively with age (P < .001) and positively with baseline (P = .003) and preapheresis blood CD34+ cells/µL (P < .001), and baseline white blood cell (P = .01) and platelet counts (P = .03). Uniquely for SCD, CD34+ cell yields correlated positively with the number of days hydroxyurea was held (for up to 5 weeks, P = .01) and negatively with markers of disease severity, including hospitalization frequency within the preceding year (P = .01) and the number of medications taken for chronic pain (P = .002). Unique SCD-specific technical challenges in apheresis were also associated with reduced CD34+ cell collection efficiency and purification. Here, we describe factors that impact plerixafor mobilization success in patients with SCD, confirming known factors as described in other populations in addition to reporting previously unknown disease specific factors in patients with SCD. This trial was registered at www.clinicaltrials.gov as #NCT03226691.

YF-H-2015005, a CXCR4 Antagonist, for the Mobilization of Hematopoietic Stem Cells in Non-Hodgkin Lymphoma Patients: A Randomized, Controlled, Phase 3 Clinical Trial

Background: YF-H-2015005, a novel CXCR4 antagonist, has been proven to increase the quantities of circulating hematopoietic stem cells (HSCs), which results in an adequate collection of HSCs in non-Hodgkin lymphoma (NHL) patients.Methods: This was a multicenter, double-blind, randomized (1:1), placebo-controlled phase III clinical trial. All patients received granulocyte colony-stimulating factor (G-CSF) for up to 8 consecutive days. YF-H-2015005 or placebo was administrated on the evening of day 4 and continued daily for up to 4 days. Apheresis was conducted 9–10 h after each dose of YF-H-2015005 or placebo. The primary endpoint was the proportion of NHL patients procuring ≥5 × 106/kg CD34+ HSCs within ≤4 apheresis sessions.Results: In total, 101 patients with NHL were enrolled. The proportions of patients achieving primary endpoint were 57 and 12% in YF-H-2015005 and placebo groups, respectively (P < 0.001). Moreover, a higher proportion of YF-H-2015005-treated patients reached a minimum target collection of ≥2 × 106/kg CD34+ HSCs in ≤4 apheresis days compared to placebo-treated patients (86 vs. 38%, P < 0.001). Furthermore, the median time to collect ≥2 or 5 × 106/kg CD34+ HSCs were 1 and 3 days in YF-H-2015005-treated patients, but 4 days and not reached in placebo-treated patients, respectively. No severe treatment emergent adverse events were observed in both YF-H-2015005 treatment and placebo groups.Conclusions: YF-H-2015005 plus G-CSF regimen was a tolerable combination with high efficacy, which might be used to rapidly mobilize and collect HSCs in NHL patients.

淋巴瘤自体造血干细胞动员和采集中国专家共识（2020年版）

淋巴瘤自体造血干细胞动员和采集中国专家共识（2020年版）

Peripheral Venous Access for Collection of Immune Effector Cells and Hematopoietic Stem Cells Is Feasible and Safe in Older Children and Young Adult Patients

Introduction: Collection of hematopoietic stem cells (HSC) for HSC transplantation (HSCT) and immune effector cells (IEC) for cellular manufacturing (e.g. CAR T-cells) in children and young adults has unique challenges. In order to maintain adequate flow rates, central venous catheters (CVCs) are often utilized at pediatric institutions. However, CVCs are associated with additional procedural risks (anesthetic complications, thrombotic events) and resource utilization (anesthesia, interventional radiology). In adults collection through peripheral venous catheter (PVC) access has become increasingly standard practice, but has not yet been routinely used in pediatrics. The aim of this study was to determine if it is feasible and safe to utilize PVC access in a select population of older children and young adults to reduce the need for CVC insertions. Methods: All individuals undergoing HSC and IEC collections at our institution (patients and sibling donors) between February 2019 and July 2020 were considered for PVC collection if in the treating physicians assessment the patient would be able to tolerate the procedure (arms outstretched for up to 6 hours) and venous vessels were deemed suitable for an 18 gauge PVC. For blood return patients received either a second PVC (minimum 20 gauge) or if present their port/central line was used. For patients screened after April 2020, assessment by ultrasound was performed prior to the procedure to determine venous vessel size. All patients had topical analgesia applied prior to PVC insertion. All collections were carried out using the Continuous mononuclear cell collection (CMNC) protocol on an Optia apheresis device. Collection-related parameters such as demographics, collection efficiency (CE2), collection time, flow rate and product quality were collected. Results: A total of 27 patients were screened for PVC collection from February 2019 until July 2020. The median age was 16.1 years (range 9.7-27.3) and median weight 67.7 kg (range 29.4-115.4). Ten out of 27 patients had ultrasound screening prior to collection on which one patient was deemed not suitable for PVC collection due to a venous anomaly. Cells were collected successfully by PVC in 24/26 (92%) of patients, 2/26 (8%) patients required CVC insertion after an unsuccessful PVC attempt due to inability to maintain an adequate flow rate. Average PVC collection flow rate was 50.3 ml/min (range 34-91) with median collection time of 244.5 min (range 87-397 min). Median collection efficiency (CE2) for CD3 cells was 59% (range 21-88), for CD34 cells 55% (range 28-69), product cell viability was 100%. Collection and product parameters were comparable to institutional collections performed utilizing CVCs. PVC collections were well tolerated, no patient had to stop the procedure due to discomfort or patient safety concerns. Conclusion: PVC collection of IEC and HSC is feasible and safe in older children and young adults. Ultrasound screening prior to PVC insertion and collection attempt can reduce failure rate and streamline the collection process. Disclosures Krueger: Novartis: Consultancy, Other: Travel support; no honoraria, Speakers Bureau; Kite, a Gilead Company: Other: Travel support; Atara: Other: Travel support.

A Report of Fourcases of Severethrombocytopenia with Rh G-CSF Mobilized Allogeneic Peripheral Blood Stem Cell Transplant Donors

Background and Objective: Granulocyte colony-stimulating factor (G-CSF) is used to mobilize progenitor cells into the peripheral bloodin healthy donors. Thrombocytopenia is one of common side effects of G-CSFmobilization, but seriousthrombocytopenia is rare. Herein,we report serious thrombocytopeniain 4 allogeneic peripheral blood stem cell transplantation (Allo-PBSCT) healthy donors induced by recombinant human G-CSF (rhG-CSF) mobilization. Methods: The donor received 10ug/kg/d G-CSF via subcutaneous injection.The blood parameterswere examined every day, Abdominal ultrasound detected the spleen size before, during, and after rhG-CSF mobilization. Results: From April 2015 to May 2020, 1127 donors received hematopoietic stem cell mobilization, the percentage of serious thrombocytopenia in healthy donors was 0.35%.The count of white blood cell increased to the 4-7 timesbasic level of (35.4x109/L)after rhG-CSF mobilization 4 days, the lowestcount ofplatelet was 22x109/Lafter single postharvest. After mobilization, length of spleen increased 0.6-1.3 cm (median 10.1 cm), thickness ofspleenincreased 0-1.3 cm (median 3.4 cm). There were no obvious bleeding symptomsafter mobilization in four cases of donor.The quality of stem cell collection was relatively poor in 3 of the 4 donors, the number of mononuclear cells (MNC) was 7.47-18.58×108/kg, and the value of CD34+ was 0.82-2.42×106/kg (CD34+ proportion: 0.11-0.16%). The quality of stem cell collection in the other 1 donor was satisfactory, the number of MNC was 9.29-11.75×108/kg, and the proportion of CD34+ was 0.26~0.38%. Platelet count of 4 donors recovered to the basal level 3-10 days after drug withdrawal. Conclusion:The severe thrombocytopenia caused by rhG-CSF mobilization healthy donor is very rare.while, the mechanismis unknown. Some studies suggested that it may be related to the insufficient expression of megakaryocytes proliferation-related genes, such as PF4 and PTFN4. Other studies indicated that rhG-CSF may aggravate the immune-mediated thrombocytopenia, which leads to hypersplenism and extensive activation of reticuloendothelial cells, thus increasing the consumption of platelets.In addition, the probability of severe thrombocytopenia was increased in allergic donors after rhG-CSF mobilization.In our report, although there was thrombocytopenia, mobilization was not affected.After the mobilization of rhG-CSF, platelets changes should be closely monitored to keep alert of donor bleeding tendency. While ensuring safe collection of hematopoietic stem cells by donors, patients should not be placed in extreme danger of not being able to receive allogeneic hematopoietic stem cells on time. Key words:Severe thrombocytopenia;Healthy donor; rhG-CSF; Mobilization; Allogeneic peripheral blood stem cell transplantation. Disclosures No relevant conflicts of interest to declare.

In Vivo HSC Gene Therapy for Hemoglobinopathies: A Proof of Concept Evaluation in Rhesus Macaques

Current gene therapy or genome editing studies for hemoglobinopathies require highly sophisticated medical facilities to perform hematopoietic stem cell collections / selections and genetic modifications. In addition, patients receive high-dose chemotherapy to facilitate engraftment of gene-modified cells. Thus, current gene therapy protocols will not be accessible to most patients suffering from hemoglobinopathies. Here we describe a highly portable and scalable approach using in vivo hematopoietic stem cell (HSC) gene therapy to potentially overcome these limitations. The central idea of our in vivo HSC gene therapy approach is to mobilize HSCs from the bone marrow, and while they circulate at high numbers in the periphery, transduce them with an intravenously injected HSC-tropic, helper-dependent adenovirus HDAd5/35++ gene transfer vector system. Transduced cells return to the bone marrow where they persist long-term. Transgene integration is either achieved by a Sleeping Beauty transposase (SB100x) in a random pattern or by homology-directed-repair into a safe genomic harbor site. Currently, an in vivo selection system (involving the mgmtP140K gene/low-dose O6BG/BCNU) is employed to achieve 80-100% marking levels in peripheral blood cells. We demonstrated safety and efficacy of our approach in mouse models for thalassemia intermedia, Sickle Cell Disease, and hemophilia A, where we achieved a phenotypic correction. We now present data in 3 rhesus macaques. We show that treatment with G-CSF/AMD3100 resulted in efficient HSC mobilization into the blood circulation and subsequent intravenous injection of the HDAd5/35++ vector system (total 1-3 x1012 vp/kg, in two doses) was well tolerated. The longest follow up thus far is 24 weeks after in vivo HSC transduction with a human-gamma-globin expressing HDAd5/35++ vector. After in vivo selection with O6BG plus low dose (10 to 20 mg/m2) of BCNU, a dose that is up to 100-fold lower than what is used for autologous transplantation protocols, gamma-globin marking in peripheral red blood cells rose to ~90% and was stable for the duration of the study (see Figure). gamma-globin levels in red blood cells were ~18% of adult alpha1-globin (by HPLC). No abnormalities in genome and transcriptome analyses of animal #1 were found at the time of scheduled necropsy. We show that a new prophylaxis regimen (dexamethasone, IL-6R, IL-1bR antagonists, saline bolus IV) was able to mitigate all side effects associated with intravenous HDAd5/35++ vector administration. Analysis of day 3 bone marrow showed 30% transduced HSCs. Vector DNA biodistribution studies demonstrated very low or absent transduction of most tissues (including testes and CNS). Analysis of bone marrow showed efficient, preferential HSC transduction and re-homing of transduced CD34+/CD90+ cells to the bone marrow. At week 4, about 5% of progenitor colony-forming cells demonstrated stable transduction with integrated vector, and this frequency increased after starting the in vivo selection. The level of human mgmtP140K mRNA expression in PBMCs also increased after in vivo selection. In summary: Using a new and optimized prophylaxis regimen intravenous delivery of HDAd5/35++ was very well tolerated without any cytokine activation. We saw efficient transduction of HSCs and efficient in vivo selection of transduced progenitors with low dose O6BG/BCNU. This is the first proof-of-concept study that in vivo HSC gene therapy could be feasible in humans without the need of high-dose chemotherapy conditioning and without the need for highly specialized medical facilities. This approach would provide a major advance for the gene therapy and genome editing field and allow the necessary portability and accessibility to reach patients in places with limited medical resources. Figure 1 Disclosures Radtke: Forty Seven INC: Consultancy. Kiem:Umoja: Membership on an entity's Board of Directors or advisory committees; Rocket Pharma: Membership on an entity's Board of Directors or advisory committees; Vor Biopharma: Membership on an entity's Board of Directors or advisory committees; Enochian: Membership on an entity's Board of Directors or advisory committees; CSL: Consultancy; Magenta Therapeutics: Consultancy; Homology Medicines: Membership on an entity's Board of Directors or advisory committees. Lieber:Ensoma, Inc: Consultancy, Research Funding.

Experience with the use of bendamustine (Rozustin®): safety and efficacy

Background. Bendamustine is one of the main drugs included in the arsenal of cytostatic agents that are used in oncohematology. In Russia, the drug was registered at the end of 2010 under the commercial name Ribomustin (company Astellas). At the end of 2018, two national generics of bendamustine appeared on the Russian market – Rozustin® (Rafarma) and Kovada (Biocad).Materials and methods. In 2019 the department of hemoblastosis chemotherapy of the N. N. Blokhin National Medical Research Center of Oncology received the drug bendamustin (Rozustin®) (Rafarma, Russia). This article presents the first experience of using Rozustin® in patients with various lymphoproliferative diseases. Most often, the drug was used for relapsed or refractory classical Hodgkin’s lymphoma.Results and conclusion. The high effectiveness, acceptable toxicity profile compared to alternative “salvage” regimens, ease of implementation, and the possibility of a full collection of hematopoietic stem cells followed by high-dose chemotherapy and autologous stem cell transplant, confirm the good potential of the combined program with the inclusion of the drug bendamustine (Rozustin®).

Predictive factors for the development of anemia after hematopoietic stem cell donation.

Although Hematopoietic Stem Cells (HSC) donation through bone marrow (BM) and peripheral blood (PB) are usually safe procedures, adverse events are expected. One of the most common events especially among BM donors (BMD) is the development of anemia. To protect the BMD and preserve the hemoglobin levels, many centers collect autologous pre-procedure blood, but the actual benefits of this procedure is controversial. This study analyzed retrospectively data to observe what factors may influence the occurrence of post-donation anemia and also evaluate the relevance of autologous red blood cell pre procedure donation (PAD). The development of immediately post donation anemia (IP) was higher in BMD than in PB donors (64.2% BMD and 10.7% PBD, P < .001) and also in late post donation (LP) (28.4% BMD and 3.6% PBD, P = .007). The study demonstrated an association between PAD and anemia in IP (72.7% with anemia and 27.3% without anemia, P = .006) and an association between the volume of red blood cells in the donated hematopoietic product and the development of anemia in LP (356.3 mL and 297.8 mL, P = .037). In conclusion, collection of HSC through BM is a risk factor for anemia and PAD is a risk factor for IP anemia.

Optimization of methods for collecting peripheral hematopoietic stem cells in children with cancer: literature review

Autologous hematopoietic stem cell transplantation (auto-HSCT) is a standard for the treatment of oncological, hematologic, and also some immune diseases, ensuring the restoration of blood counts after high-dose chemotherapy. In children, the success of mobilization and collection of hematopoietic stem cells (HSCs) is especially important. Mobilization schemes for children are decided on an individual basis, which requires the development and implementation of recommendations for improving the efficiency of mobilization and collection of HSCs. Mobilization schemes include the use of granulocyte colony-stimulating factor in the form of monotherapy or in combination with CXCR4 antagonists. These schemes are ineffective in some children, which requires re-mobilization or rejection of transplantation, which negatively affects the prognosis. When preparing a patient for HSCs collection, it is necessary to take into account all previous therapy, the patient’s age, weight and height indicators, and general somatic state. Harvesting the required amount of HSCs will allow for high-dose therapy followed by auto-HSCT, and thereby increase the effectiveness of treatment. It is necessary to optimize the protocol for mobilization of HSCs with a large bias for pediatric patients, which will clearly define the criteria for mobilization, give indications for this procedure and determine the criteria for technical collection, which will allow to obtain the optimal number of CD34+ cells, which will ensure the success of the treatment.

Apheresis of peripheral blood stem cells in children with very low body weight: a single centre experience

Polychemotherapy, accompanied by autologous hematopoietic stem cell transplantation, can improve the results of long-term survival of patients with cancer and some non-cancer diseases. Mobilizing and collecting hematopoietic stem cells in children with very low body weight can be a difficult task. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology, and Immunology. 19 children with extremely low body weight was included in the current study. The median age was 8 (5–14) months, the median of body weight 7.5 (5.8–8.8) kg. Apheresis was performed in an ICU, using sedative therapy and in compliance with the conditions for the prevention of anemia, hypovolemia, hypothermia. 19 hematopoietic stem cell apheresis were performed using the Spectra Optia MNC separator program. Mobilization of CD34+ cells was performed with filgrastim; three children were additionally given plerixaphor. All 19 hematopoietic stem cell apheresis were successful: the median of collected CD34+ cells was 18.7 × 106/kg (8.6– 60.6 × 106/kg), the median apheresis duration was 204 (161–351) min. Serious side effects during apheresis were not recorded, however, in 6 children (31%) we encountered difficulties in the process of installing central venous access. The collection of hematopoietic stem cells for the future high-dose chemotherapy with autologous hematopoietic stem cells is a feasible task even for very young children with extremely low body weight. Correct preparation for manipulation, taking into account all possible risk factors and technical features, can avoid serious complications.

Clinical Analysis of Autologous Peripheral Blood Hematopoietic Stem Cell Mobilization Regimen in Patients with Malignant Hematological Diseases

To analyze the factors influencing the mobilization and collection of peripheral blood hematopoietic stem cells (HSC) in the patients with malignant hematological diseases. The peripheral blood stem cells in 50 patients with hematological malignancies had been mobilized in the Hematology Department Zhongda Hospital. The factors, such as age, sex, mobilization regimen, disease status, cell separator were analyzed, and the effect of above-mentioned factors on stem cell mobilization was evaluated. And the correlation between WBC, Hb, Plt counts and CD34+ cell collection was anzlyzed. There was significant effect at mobilization regimen on the success rate and collection counts of CD34+ cells, while sex, age, interval between diagnosis and mobilization, previous chemotherapy regimen and bone marrow involvement showed no significant effect on stem cell collection. There was positively correlation of the WBC count, hemoglobin level of peripheral blood before apheresis with the collection of CD34+ cells . The WBC and MNC counts in peripheral blood before apheresis closely related with the success rate of mobilization. Chemotherapy plus combined with G-CSF mobilization regimens is superior to mobilization regimen of cytokine alone for collection of HSC. The WBC and MNC count in peripheral blood before apheresis can predict the optimal time for the PBSC collection and increase the success rate of mobilization and collection.