Dose Of G-CSF Research Articles

Prediction of CD34+ hematopoietic stem cells in healthy allogeneic stem cell donors on the Optia cell separator based on CE2: Which formula is more correlated with actual CD34+?

Peripheral blood-derived hematopoietic stem cells (HSCs) are widely used for various adult stem cell transplants. To obtain sufficient HSCs from healthy volunteer donors during the apheresis process and ensure that the donors are exposed to fewer apheresis-related side effects, calculation methods have been developed for the prediction of processed blood volume or CD34+ count. However, there is no consensus on a formula to predict the volume of blood to be processed or the number of stem cells to be obtained. This study aimed to estimate the predicted blood volume and CD34+ cell counts using collection efficiency (CE)-based formulas and evaluate their accuracy compared to the actual CD34+ cell counts. It also seeks to identify the factors that affect CE. Data from 397 healthy, unrelated stem cell donors were retrospectively analyzed. An algorithm using four different CE2 metrics (1st quartile, mean, 3rd quartile, and median) was developed to predict the volume of blood to be processed using the Spectra Optia continuous mononuclear cell collection procedure. When employing the mean CE2 algorithm, the results revealed a strong correlation (r = .894, p < .001) between predicted and actual CD34+ values. The study also identified strong associations between pre-apheresis CD34+, pre-apheresis leukocyte count, the use of two doses of G-CSF, and low CE2. These findings suggest that the mean CE2 algorithm could be a potent, straightforward, and accurate tool for predicting CD34+ stem cell counts in healthy allogeneic stem cell donors and potentially optimizing stem cell collection procedures.

Significant Reduction of Radiation-Induced Death in Mice Treated with PrC-210 and G-CSF after Irradiation.

The search for single or combined radiation countermeasures that mitigate the development of Acute Radiation Syndrome (ARS) after radiation exposure remains a prominent goal of the U.S. government. This study was undertaken to determine whether PrC-210 and G-CSF, when administered 24-48 h postirradiation, would confer an additive or synergistic survival benefit and mitigate ARS in mice that had received an otherwise 96% lethal radiation dose. Our results show that optimum systemic doses of PrC-210 and G-CSF, when administered 24 h or later after a 96% lethal dose of whole-body irradiation, conferred: 1. strong individual survival benefits (PrC-210 44%, P = 0.003), (G-CSF 48%, P = 0.0002), 2. a profound combined 85% survival benefit (P < 0.0001) when administered together, and on day 14 postirradiation, 3. peripheral white blood cell/lymphocyte counts equal to unirradiated controls, 4. dense bone marrow cell density (>65% of unirradiated controls), 5. jejunal villi density that equaled 90% of unirradiated controls, and 6. spleen weights that equaled 93% of unirradiated controls. Our results show that PrC-210 and G-CSF given together 24 h after irradiation confer strong additive efficacy by protecting the immune system, and enabling recovery of the bone marrow, and they work synergistically to enable recovery of peripheral white blood cells in circulating blood.

Decade of insights: Bleomycin-induced pneumonitis and use of G-CSF in chemotherapy-treated germ cell testicular tumors and Hodgkin lymphoma—Real-world outcomes from an academic cancer center.

e17013 Background: Bleomycin induced pulmonary toxicity (BIP) occurs in 4-10% of treated patients. Preclinical reports suggest G-CSF use may increase BIP risk, due to neutrophil migration to lung tissues. Limited real-world clinical data is available and G-CSF use may reduce risk of treatment related toxicity with curative bleomycin combination treatment. Methods: We conducted a retrospective study of patients with GCT (Germ cell tumor) and HL (Hodgkin’s lymphoma) receiving bleomycin containing chemotherapy and at-least on dose of G-CSF at The James Cancer Center from 2010-2022 using the institutional database. BIP was diagnosed mainly using clinical symptoms complemented by radiological evidence when possible. We reviewed 361 patients, 145 in GCT group and 216 in HL group and clinically relevant information was recorded.Descriptive statistics for the study variables were presented using frequencies and percentages overall. The bivariate associations between G-CSF use and clinical outcomes were assessed using chi-square tests or fisher’s exact tests for the overall sample, and by chemotherapy type. We used SPSS 26.0 for all the statistical analyses, and an alpha level of 0.05 (two-sided) was used for all the tests. Results: Data on 361 patients, including 145 GCT and 216 HL patients treated with at least one dose of bleomycin was available. The median age was 36.3 years in the GCT group and 44.2 years in the HL group.144 patients in the GCT and 50 patients in the HL group received at least 1 dose of G-CSF. BIP occurred in 4.8% in the GCT and 7.4% of patients in the HL group. Only 2 out of 16 patients developing BIP in the HL group received G-CSF. All patients with BIP developed symptomatic disease. On bivariate analysis, no association was noted between BIP and G-CSF in either group (p-value =1.00 in GCT and p-value = 0.32 in HL). Febrile neutropenia (FN) was reported in 4 patients in the GCT group and 29 in the HL group. In the HL subgroup, 28 (28/29) patients developing FN did not receive G-CSF (p-value=0.0008). All patients who developed FN required hospital admission. Conclusions: Use of G-CSF in patients receiving bleomycin was not associated with an increased incidence of BIP. Incidence of FN was higher in patients not receiving G-CSF leading to hospital admissions. [Table: see text]

Complications and the impact of hematological parameters in donors involved in allogenic peripheral blood stem cell transplantation for hematological malignancies

Hematopoietic stem cell transplantation (HSCT) is a vital treatment option for many hematological malignancies, offering a more convenient alternative to bone marrow transplantation. In allogeneic HSCT, donors provide hematopoietic stem cells for transplantation into patients, necessitating careful consideration of the donor's health and hematological parameters to ensure successful engraftment and treatment outcomes. In a study involving 20 adult donors at Apeksha Hospital, Sri Lanka key hematological parameters, comorbidities, clinical histories, and medications were analyzed. Diabetes mellitus and hypertension were the most prevalent comorbidities among donors, while anemia, primarily linked to iron and vitamin B12 deficiencies, was common. Although baseline coagulation abnormalities were rare and no significant complications occurred during transplantation, early clinical issues such as pain and paresthesia correlated with abnormal peripheral blood smear findings. Notably, a 4-day single daily G-CSF dosing schedule was favored over a 5-day twice daily regimen due to fewer reported problems. While anemia and thrombocytopenia were infrequent, these findings underscore the critical importance of meticulous donor selection and vigilant monitoring throughout the transplantation process to optimize outcomes.

The Effect of Granulocyte Colony-Stimulating Factor on Endometrial Receptivity of Implantation Failure Mouse.

The purpose of this study was to investigate the effect of G-CSF on the endometrial receptivity of implantation failure mice.Sixty female mice were treated mifepristone to establish an implant failure model. The treatment groups received different doses of G-CSF. Endometrial tissue and serum were collected on day 5 after mating. The abundance of pinopodes on the endometrium was observed by scanning electron microscopy. The expressions of LPAR3, COX2, and HOXA10 were detected by RT-qPCR and Western blotting. Serum levels of E2, P, VEGF, LIF, TNF-α and IL-10 were measured by ELISA. The expressions of VEGF, CD34, CD57, TNF-α, and IL-10 were assessed by immunohistochemistry. Immunofluorescence analysis was performed to determine the number of CD57, Treg, and Th17 cells.G-CSF increased implantation and pregnancy rates of mifepristone-induced implantation failure mice, with the most significant effect seen at the intermediate dose. G-CSF increased the serum levels of E2 and P, the abundance of endometrial pinopodes, and the level of LIF in the endometrium. It also promoted the expression of VEGF, HOXA10, LPAR3, and COX2. Moreover, G-CSF reduced the level of CD57 cells and the ratio of Th17/Treg cells in endometrium. G-CSF reduced the inflammatory factor TNF-α, but IL-10 did not change significantly.G-CSF can enhance embryo implantation rate and pregnancy rate and improve endometrial receptivity by attenuating degeneration of pinopodes, upregulating estrogen and progesterone, facilitating angiogenesis, maintaining immune cell homeostasis, and reducing the production of inflammatory cytokines in implantation failure mouse.

Evaluation of a Quality Improvement Process for Health-System Retention of Long Acting Growth Factors Prescriptions in the Pediatric Oncology Population

OBJECTIVE Granulocyte-colony stimulating factor (GCSF) products are often used in pediatric patients with malignant diagnoses to reduce the time that the patient is neutropenic. Long-acting GCSF products have been shown to be non-inferior to daily dosing of GCSF products, and are becoming more desired by patients and families. Insurance companies often require a prior authorization prior to approving the use of the long-acting GCSF products. This process has proven challenging leading to treatment delays and missed doses. The purpose of this study is to evaluate a quality improvement process for the prescribing and dispensing of long-acting GCSF to better serve pediatric patients within a single health care system. METHODS This is a single-center, retrospective chart review with the purpose of collecting data to compare prescription retention before and after the improvement intervention. Study timeline includes all doses of long-acting GCSF prescribed for pediatric oncology patients between June 2020–June 2021 compared with July 2021–March 2022. On June 30, 2021, educational information was provided to the appropriate stakeholders regarding the change in practice. RESULTS A total of 31 patients were included in the review, with 22 patients prior to the intervention (115 prescriptions), and 9 patients after the intervention (43 prescriptions). There was a 37.8% increase in health system prescription retention (15.7% vs 53.5%). CONCLUSIONS Pharmacist directed long-acting GCSF prescription destination and a dedicated prior-authorization team led to an increase in prescription retention for patients regardless of payer mandated outpatient pharmacy.

One Year Follow-up on the First Patient Treated with Nula-Cel: An Autologous CRISPR/Cas9 Gene Corrected CD34+ Cell Product to Treat Sickle Cell Disease

Introduction Sickle cell disease (SCD) is the most common serious genetic disease in the world with over 500,000 births per year globally. It is estimated that there are &amp;gt;100,000 patients in the US with SCD and at least 20% have severe disease. Patients with SCD have at least one HBB gene with an adenine to thymidine variant at position 6 of the coding region resulting in a glutamic acid to valine amino acid change ( HbS). The success of allogeneic hematopoietic stem cell transplantation demonstrates that reconstitution of the hematopoietic compartment with HSCs that contain at least one non- HbS allele can cure the disease. Homology directed repair gene editing (HDR-editing) has achieved high frequencies of conversion of the thymidine to the non-pathologic adenine in CD34+ hematopoietic stem and progenitor cells from patients with SCD. Nulabeglogene autogedtemcel (nula-cel) is an investigational drug product in which HDR-editing of plerixafor-mobilized CD34+ HSPCs corrects the underlying variant that causes SCD. This is the first drug to directly correct a disease-causing variant. We report on the 1 year follow up of the first and only patient to receive nula-cel. Results Patient 1 is now a 23 year old female with homozygous SCD. She was consented and enrolled on a Phase I//II trial to test the safety and efficacy of nula-cel. In the 2 years prior to enrollment, the patient averaged 6 VOC's and 4 hospitalizations per year. Two aphereses of plerixafor-mobilized CD34+ cells were performed followed by fresh CD34+ cell purification. The purified CD34+ cells were pooled and cryopreserved. A 5-day manufacturing process starting with 9.3 x 10 6 cells/kg of cryopreserved CD34+ cells resulted in a yield of 8.75 x 10 6 CD34/kg with an on-target allele correction frequency of 33%. In August, 2022 the thawed drug product was infused after the patient received AUC-adjusted busulfan myeloablative conditioning chemotherapy. The initial cell viability was 77% giving a viable infused cell dose of 6.74 x 10 6 CD34/kg. Follow-up exploratory apoptois studies, however, showed the viable CD34+cell dose may have been as low as 3.5 x 10 6 CD34+/kg. Neutrophil engraftment occurred at transplant Day +40. Because of the lack of platelet recovery and continued platelet and RBC transfusion requirements, the patient was started on eltrombopag on day +106 (marrow cellularity of 5%). Since eltrombopag usage was not part of the protocol, its use prompted the reporting of an SAE but the trial was never put on FDA hold. The last dose of GCSF was D+140, the last platelet transfusion was D+181 and the last RBC transfusion was D+263. With rising platelet counts and hemoglobin and a stable ANC &amp;gt;1500, eltrombopag was discontinued on D+322. The blood counts have continued to slowly increase after discontinuation of the eltrombopag. On D+307, with a Hgb of 8.5 g/dl, hemoglobin electrophoresis demonstrated HgbA=12.5%, HgbF &amp;gt;78% and HgbS=4.5%. On D+349 the blood tests show Hgb=9.1 g/dl, Platelet=77,000/ul, ANC=1788/ul with an absolute reticulocyte count of 119,130/ul. There are no signs or symptoms of hemolysis. Table 1 shows the time course of gene marking with stable allele gene correction and INDEL frequencies since D+49. No change in off-target INDEL frequency nor change in on-target INDEL spectrum has occurred and no evidence of oligoclonal or clonal hematopoiesis has been observed. Clinically, the patient has shown marked improvement in quality of life with zero VOE's or other manifestations of SCD. The patient still has not reached a steady state and updated results will be presented. Conclusion We describe the clinical results of the first patient treated with an autologous cell product (nula-cel) in which the pathologic variant was directly corrected. The mechanism of action is fundamentally different than other clinical gene editing and gene therapy programs as the level of the pathologic HgbS is directly decreased thereby removing the dominant negative effect that HgbS has on red blood cells. The high HgbF is an unexpected but potentially effective mechanism of action for the clinical benefit and is being further studied. Longer follow up to assess the durability of the curative clinical impact is underway. Improvements in cell manufacturing are being implemented to shorten the duration of transfusion needs and increase the level of gene correction of engrafted cells. Plans to treat the next patients with an improved nula-cel product are in place.

Prolonged CXCR4 Receptor Occupancy By Motixafortide Following a Single Subcutaneous Injection Is Associated with Extended Mobilization of CD34+ Cells in Peripheral Blood for > 24 Hours

Background: Interaction between CXCL12 (SDF-1) and its receptor, CXCR4, plays a critical role in the retention of CD34+ hematopoietic stem and progenitor cells (HSPCs) in the bone marrow. Motixafortide (motix) is a novel selective CXCR4 antagonist with high affinity for CXCR4 (IC 50 0.42 - 4.5nM), slow dissociation (Kd = 3.38e-5 s-1), and high plasma protein binding (&amp;gt;99%). In the Phase 3 GENESIS trial (NCT03246529), motix added to G-CSF (G) resulted in significantly higher mobilization of HSPCs in patients with multiple myeloma (MM) prior to autologous stem cell transplantation, with 88.8% of patients collecting ≥6x10 6 CD34+ cells/kg (local lab) in 1 leukapheresis procedure (LP) 10-14 hours following a single dose of motix (1.25mg/kg). Of note, GENESIS trial patients requiring additional LPs to meet HSPC mobilization goals underwent a second LP at 34-38 hours post-motix, with a total of 92.5% of patients meeting their collection goal. Here, we report in-vitro receptor occupancy, as well as clinical pharmacokinetics (PK) and pharmacodynamics (PD) of peripheral blood (PB) mobilization of CD34+ cells/ μL in healthy volunteers (HV) and patients with MM following motix administration. Methods: CXCR4 receptor occupancy and dissociation rate were assessed by incubating increasing doses of motix (3-500nM) with Jurkat cells in vitro followed by washing and incubation 0-72 hours later with either an anti-CXCR4 12G5 fluorophore-conjugated monoclonal antibody (mAb) (competes with motix for binding to CXCR4) or an anti-CXCR4 1D9 fluorophore-conjugated mAb (binds to CXCR4 but does not compete with motix) followed by flow cytometry. In HV (NCT05293171), PB samples were collected following a single subcutaneous (SC) administration of motix (1.25mg/kg). PK timepoints were pre-dose and at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, 16, and 24 hours post-motix; and PD timepoints were pre-dose and at 0.75, 2, 4, 8, 12, 16, and 24 hours post-motix. In patients with MM, PB samples were collected following SC motix (1.25mg/kg) added to G. PK timepoints were pre-motix (post-G) and 0.25, 0.5, 1, 2, 4, 8, and 12 hours post- motix; and PD timepoints were pre-motix (post-G) and 12 hours post-motix. Results: In vitro studies demonstrated complete CXCR4 receptor occupancy at 3 nM (6.48 ng/mL) of motix, with increasing concentrations resulting in longer receptor occupancy of &amp;gt;72 hours. Due to high protein binding, a minimum total plasma concentration of 800 ng/mL is required to achieve a free fraction of 3nM. This level was achieved following administration with doses ≥ 1.0 mg/kg (Fig 1A). PK analysis in HVs and in patients with MM dosed with 1.25 mg/kg demonstrated that motix was rapidly absorbed (maximum concentration within 0.25-1 hours). Mean maximal plasma concentrations were 2245 ± 791 ng/mL (1040nM) in HVs and 1800 ± 695 ng/mL (834nM) in patients with MM. Notably, the half-life of motix was 1-3 hours. However, despite the short half-life, the high CXCR4 receptor affinity and slow dissociation rate of motix led to an extended PD effect. In HVs who received motix as monotherapy, median PB CD34+ counts increased from baseline of 1.6 CD34+ cells/µl to 17 CD34+ cells/ μL within 4 hours post-motix, reached maximal levels of 43 CD34+ cells/μL at 16 hours post-motix and remained elevated at 36 CD34+ cells/μL at the last observation time point 24 hours post-motix (Fig 1B). In MM patients who received motixafortide after 4 doses of G-CSF, there was a 6.6 fold increase in median PB CD34+ cell counts, from 15.8 cells/μL (post-G-CSF, pre-motix) to 116.0 CD34+ cells/μL at 12 hours post-motix (Fig 1B). Conclusions: In vitro studies demonstrated complete receptor occupancy by motix starting at a concentration of 3nM. Increasing concentrations resulted in longer duration of receptor occupancy. Mobilization of CD34+ cells to PB was observed within 2 hours after motix injection (1.25mg/kg), peaked at 12-16 hours, and remained elevated (&amp;gt;20 CD34+ cells/μL) for &amp;gt;24 hours post-motix. These PK/PD characteristics of motix result in rapid and robust PB CD34+ HSPC mobilization (median 116.0 CD34+ cells/μL); enabling 88.8% of patients to collect ≥6x10 6 CD34+ cells/kg in 1 LP with 1 dose of motix added to G. Furthermore, the extended PD effect of motix supports a flexible administration window that allows for LP to be performed &amp;gt; 24 hours post-motix administration.

Venetoclax Plus Azacitidine, Low Dose Cytarabine, Aclarubicin and G-CSF (VA-CAG Regimen) for Newly Diagnosed Young Patients with Acute Myeloid Leukemia: A Prospective, Multicenter, Single-Arm Phase 2 Clinical Trial

Background: Venetoclax, in combination with hypomethylating agents or low-dose cytarabine, is approved for newly diagnosed acute myeloid leukemia (AML) patients who are over 75 years of age or not eligible for intensive chemotherapy, while the efficacy of venetoclax-based regimens in young fit AML patients still needs to be further explored. Although the combination of anthracycline and cytarabine has achieved a remission rate of about 70% in these patients, the low complete response rate in adverse-risk patients, strong adverse reactions, and long duration of myelosuppression limited the clinical benefit. We presumed that venetoclax plus azacitidine in combination with low-intensity regimens CAG (G-CSF priming, low dose cytarabine, and aclarubicin) could have a better composite complete remission with an acceptable safety profile. Methods: Patients who were newly diagnosed with AML aged from 18 to 65 years without active infection, bleeding and severe cardiopulmonary insufficiency were enrolled. Participants received induction as azacitidine 75 mg/m 2 subcutaneously once daily on days 1-7, cytarabine 10mg/m 2 subcutaneously q12h on days 1-7, aclacinomycin 12 mg/m 2 on days 1,3,5,7, granulocyte colony-stimulating factor 5µg/kg on days 1-7, and venetoclax orally once daily (100 mg d1, 200 mg d2, 400 mg d3-21). Venetoclax dose was adjusted based on concomitant CYP3A inhibitors. The efficacy were evaluated by bone marrow morphology and flow cytometry on day 28 cycle 1. The primary endpoints were safety and the composite complete remission rate. This study was approved for ethical review and registered with ClinicalTrials.gov (NCT05662956). Results: Twenty-six patients from eight sites were enrolled between January 1, 2023, and June 20, 2023. The median age was 52 years (range, 18-63years). Thirteen patients (50.0%) had adverse risk, 7 patients (26.9%) had intermediate risk, and 6 patients (23.1%) had favorable risk. All 26 patients (100%) achieved complete remission after cycle 1, and 21 patients (80.8%) was MRD negative by flow cytometry. Two patients (7.7%) were treated with venetoclax for 14 days due to ineffective platelet transfusion and severe infection during treatment. Twenty-two patients (84%) used azole drugs to prevent fungal infection. The median myelosuppression time was 13 days (range, 5-22days), and the median red blood cell and platelet transfusions were 5.5u (range, 0-11.5u) and 30u (range, 0-170u), respectively. Eleven patients (42.3%) experienced infection (including pulmonary fungal infection, oral cold herpes, novel coronavirus infection, etc.), and two of them (7.7%) had a breakthrough invasive fungal infection, but with good management. The most common grade 3/4 adverse events included bone marrow suppression (92.3%), liver injury (19.2%), gastrointestinal reaction (11.5%) and coagulation dysfunction (3.8%).No treatment-related death occurred. Conclusion: VA-CAG regimen achieved high rates of complete remission and MRD negative in young adult fit patients with newly diagnosed AML. This combined regimen has an acceptable safety profile, with low incidence of infection and transfusion dependence.

The Effects of Premedication with Desipramine on Bone Marrow Mobilization for Autologous Bone Marrow Transplantation in Multiple Myeloma

Background: Multiple myeloma (MM) management includes primary chemotherapy, followed by autologous bone marrow transplantation (ABMT) if the bone marrow gets cleared of cancerous cells. In some cases disease relapse may occur disease relapse may occur if complete clearance is not achieved. Transplantation was primarily performed through the bone marrow; however, peripheral blood has become more favored due to its safety and convenient collection in case of appropriate bone marrow mobilization which is a challenging issue. Objectives: The current study was aimed to investigate the effectiveness of desipramine application for bone marrow mobilization in multiple myeloma (MM) who were candidate for autologous bone marrow transplantation (ABMT). Methods: The current randomized clinical trial involved 122 MM patients who were candidate for ABMT. The participants were divided into two intervention groups: the first group (n = 63) received G-CSF only treatment, while the second group (n = 59) received a similar G-CSF treatment plus desipramine. The first group received 30 microgram intravenous G-CSF for five days and the second one was treated with G-CSF with similar pattern in combination with daily 100 mg desipramine initiated within three days before G-CSF treatment and continued until the last dose of G-CSF. CD34+ cells and complete blood cells and differentiation were assessed by the end of the interventions. Results: The findings of the study show that the number of CD34+ cells, white blood cells (WBC) and platelet (PLT) count were remarkably higher among the patients receiving the combination therapy compared to the G-CSF only treated group (P-value &lt; 0.001) within 5 days after the interventions; however, no significant differences were observed between the two groups when considering the stage of the disease and the frequency of chemotherapy sessions (P-value &gt; 0.05). Conclusions: Desipramine application led to significantly increased levels of CD34+ cells as the representatives of bone marrow mobilization. Besides, the patients treated with this regimen had higher serum levels of WBC and PLT; however, the disease stage and the number of chemotherapy sessions did not affect the response to the treatment.

Phase 1/2 Study of the Timing and Efficacy of 3 mg Peg-GCSF in Neo/Adjuvant Dose Dense Breast Cancer Treatment Protocols.

Amol PatelBackground Peg-GCSF has similar efficacy at a dose of 60 µg/kg and 100 µg/kg. The conventional 6 mg SC dose was based on the maximum tolerable dose. In Japan, 3.6 mg dose was approved on the basis of dose finding studies. Peg-GCSF is an integral part of dose-dense chemotherapy protocols. Dose finding and scheduling study of peg-GCSF have not been conducted in Indian patients. Materials and Methods We conducted two-center phase 1/2 clinical study addressing the timing and efficacy of peg-GCSF in Indian breast cancer patients (CTRI no: 2021/07/034751). Three groups of timing administration were studied, namely 1, 6, and 24 hours post chemotherapy. The phase 2 part was the expansion of the best timing group. The primary objective was dose density, which was defined as receiving chemotherapy on < 3 days of scheduled date. Adriamycin/epirubicin cyclophosphamide (AC/EC) was administered q2 weeks. The total leucocyte (TLC) and absolute neutrophil (ANC) kinetics were studied. Other outcomes were incidence of grade 4 neutropenia, febrile neutropenia (FN), and requirement of additional doses of G-CSF. Bone pain, fever, and myalgia were studied for adverse effects. Results From November 20 to December 21, 36 patients were enrolled. Patient characteristics are depicted in Table 1. Initially, three patients received the peg-GCSF in each timing group. One patient in each 1-hour and 6 hours needed G-CSF support for maintaining the dose density. The 24-hour group was carried to phase 2 part. Dose density was maintained in 97% of patients. None of the patient in 24-hour group had FN. Also, 4/30 patients had grade 4 neutropenia and required an additional dose of GCSF. Grade 3 or 4 bone pain was not noticed by any of the patients. During the first cycle, the mean ANC (cells/μL) was 5284, 20704, 3010, 6954 on D0, D + 3, D + 7, and D + 13, respectively (Fig. 1A-TLC and 1B-ANC). The mean ANC (cells/μL) rise on D + 3 in cycles 1, 2, 3, 4 was 23810, 29209, 32428,22455, respectively. Conclusion Dose density of AC/EC breast cancer protocol is maintained with peg-GCSF 3 mg. Post chemotherapy 24-hour timing of peg-GCSF administration remains as the standard. A phase 3 trial of 6 mg versus 3 mg is warranted.

P-371 Subendometrial administration of selectively enriched angiogenic precursor cells and growth factors derived from peripheral blood and bone marrow optimizes endometrialthickness and pregnancyoutcomes in thin-endometrium patients

Abstract Study question Can hysteroscopic instillation of progenitor cells combined with platelet derived growth factor concentrate into subendometrial region help patient with thin endometrium conceive? Summary answer Hysteroscopic subendometrial administration of stem cells and growth factors significantly improved endometrial thickness (EMT) and pregnancy outcomes in women with refractory thin endometrium What is known already Chronically thin endometrium is a challenge in ART, results in repeated expensive IVF cycles, cycle cancellations, unplanned cryopreservation of embryos and, surrogacy. Many studies have successfully used stem cells or platelet derivatives to rejuvenate reproductive tissues. When these growth-promoting cells and growth factors from platelets are delivered into basal layer which is origin of endometrial cells quality of the endometrium can be improved. While bone marrow is excellent source for obtaining stem cells main challenge is invasiveness of collection. This forms the basis for collecting mobilized endothelial progenitor cells from bone marrow into circulation for easy collection. Study design, size, duration This study is to evaluate effect angiogenic precursor cells and growth factors derived from peripheral blood and bone marrow in improving endometrial quality and pregnancy outcomes. In the present pilot study, 50 patients with persistent refractory thin endometrium were included and underwent frozen embryo transfer from September 2019-January 2023 at A4 fertility center, Chennai. Out of 50 patients, 43 were treated with peripheral blood and 7 were treated with bonemarrow derived cells and growth factors. Participants/materials, setting, methods 50 patients with EMT&amp;lt;6mm and &amp;gt;2 canceled cycles included. Two doses of subcutaneous G-CSF was given on Menstrual Cycle Days 3&amp;4, followed by 60ml venous blood or bone marrow aspirated on MCD-5. Seragen’s selective enrichment protocol was used to prepare circulating endothelial progenitor cells and growth factors concentrate. With a 2.9mm hysteroscope and single lumen ovum pickup needle harvested cells and growth factors were injected on all four walls of cavity. Main results and the role of chance EMT increased significantly following bone marrow and peripheral blood cells 5 days after sub-endometrial injection (5.8 ± 0.71 vs. 7.12 ± 0.8; P = 0.0001), with average increase of 1.30mm and 1.80mm respectively and considered optimal improvement when EMT≥ 7mm. 33 (66%) had an optimal response and there was a significant improvement in the endometrial thickness(mm).Cycle cancellations were significantly lesser in both peripheral blood and bone marrow group and LBR was significantly improved (p &amp;lt; 0.05). There was no significant difference in CPR, IR and LBR between peripheral blood and bone marrow groups (p &amp;gt; 0.05). Overall, clinical pregnancy and LBR reached up to 40% and 30%, respectively. No adverse reactions were reported. There was statistically significant probability of achieving pregnancy (p &amp;lt; 0.01) when treated with both peripheral blood and bone marrow derived cells. Likewise, there was also a statistically significant probability of getting pregnant (p &amp;lt; 0.05) when embryo transfer was planned in the subsequent cycle. While bone marrow group showed 100% HCG positive in all 5 out of 7 patients underwent embryo transfer and 57% (4/7) of clinical pregnancy, the sample size was small compared to peripheral blood group. Limitations, reasons for caution Further research is needed to provide opportunity for women with refractory thin endometrium to conceive without surrogacy. Clinician expertise in patient recruitment, dosage personalization support, and administration is paramount. Our study provides promising information for future randomized, controlled trials with large sample size in this field. Wider implications of the findings We have validated feasibility and efficacy of mobilized peripheral blood cells for improving endometrial pathologies when bone marrow aspiration is a challenge. Our findings show targeted delivery of personalizeddosage from minimally invasive autologous source could be new rayof hope in females failto improve despite all possible treatment options recommended surrogacy. Trial registration number Not applicable

Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome and tetralogy of fallot; Case report and literature review

WHIM (Warts, Hypogammaglobulinemia, Infections, and Myelokathexis) syndrome is a rare primary immunodeficiency disorder characterized by susceptibility to human papillomavirus (HPV) infections, neutropenia, and hypogammaglobulinemia. WHIM syndrome is caused by a gain-of-function mutation CXCR4, leading to increased responsiveness of neutrophils and lymphocytes to CXCL12. This results in an accumulation of atypical hypersegmented mature neutrophils in the bone marrow and peripheral blood neutropenia. This case report discusses a 10-year-old girl who was diagnosed with WHIM syndrome at the age of four years following Haemophilus influenzae meningitis. She was found to have Tetralogy of Fallot at birth, which was surgically repaired when she was one year old. She was also found to have significant neutropenia during surgery and received multiple doses of G-CSF with a good response. She presented to the hospital at the age of four years with fever, neck stiffness, lethargy, and headache and was diagnosed with H. influenzae meningitis. Her immunology workup revealed significant neutropenia and hypogammaglobulinemia, as well as low antibody levels to H. influenza type B, tetanus toxoid, and Streptococcus pneumonia, despite up-to-date vaccination. Furthermore, decreased levels of T cells and B cells were detected. The patient was started on IgG replacement therapy and Bactrim prophylaxis. Bone marrow biopsy revealed granulocytic hyperplasia and occasional hypersegmented neutrophils, suggesting myelokathexis. Genetic testing revealed a heterozygous CXCR4 mutation with a premature stop codon (p.Ser338Ter), confirming the diagnosis of WHIM syndrome. This case report highlights the association of WHIM syndrome with congenital heart defects, such as Tetralogy of Fallot. A literature review revealed three other cases of congenital heart defects in patients with WHIM syndrome, indicating a potential role for CXCR4 and CXCL12 in septum formation. The rarity of the disorder and the lack of a universal screening tool makes the diagnosis of WHIM syndrome difficult. Therefore, physicians should consider WHIM syndrome in patients with congenital heart defects, particularly if they have recurrent HPV infections, neutropenia, and hypogammaglobulinemia.

Peripheral Blood Allogeneic Stem Cell Mobilization: Can We Predict a Suboptimal Mobilization?

Allogeneic peripheral blood stem cells mobilization is now the basis of most stem cell transplants. In a very limited number of cases, mobilization is suboptimal leading to further collection procedures, to suboptimal cell doses infusion with delayed engraftment time, increased risks of transplant procedure and of related costs. To date we have no recognized and shared criteria for early estimating the probability of poor mobilization in healthy donors. We then analyzed allogeneic peripheral blood stem cell donations performed at the Fondazione Policlinico Universitario A.Gemelli IRCCS Hospital from January 2013 to December 2021 in order to identify premobilization factors associated with successful mobilization. The following data were collected: age, gender, weight, complete blood cell count at baseline, G-CSF dose, number of collection procedures, CD34+ cell count in peripheral blood on the first day of collection, CD34+ cell dose per kg body weight of recipient. Mobilization efficacy was defined according to the number of CD34+ cells in peripheral blood on day +5 of G-CSF administration. We classified donors as sub-optimal mobilizers or good mobilizers according to the achievement of the 50 CD34+ cell/μL threshold. We observed 30 suboptimal mobilizations in 158 allogeneic peripheral blood stem cell donations. Age and baseline white blood cell count were factors significantly associated with negative or positive impact on mobilization, respectively. We did not find significant differences in mobilization based on gender or G-CSF dose. Using cut-off values of 43 years and 5.5×109/L WBC count, we built a suboptimal mobilization score: donors who reach 2, 1 or 0 points have a 46%, 16% or 4% probability of suboptimal mobilization, respectively. Our model explains 26% of the variability of mobilization confirming that most of the mobilization magnitude depends on genetically determined factors; however, suboptimal mobilization score is a simple tool providing an early assessment of mobilization efficacy before G-CSF administration begins in order to support allogeneic stem cells selection, mobilization and collection. Through a systematic review, we looked for confirmation of our findings. According to the published articles, all the variables we included in our model are confirmed to be strongly related to the success of mobilization. We believe that score system approach could be applied in clinical practice to assess the risk of mobilization failure at baseline allowing for a priori intervention.

Cost-efficiency analysis of conversion to biosimilar filgrastim for supportive cancer care and resultant expanded access analysis to supportive care and early-stage HER2+ breast cancer treatment in Saudi Arabia: simulation study

AimsThis study estimated, for Saudi Arabia, the cost-efficiency of converting patients from reference Neupogen and Neulastim to one of two filgrastim biosimilars (Nivestim, Zarzio); the budget-neutral expanded access to supportive care with biosimilar filgrastim and therapeutic care to ado-trastuzumab emtansine thus afforded; and the number-needed-to-convert (NNC) to provide supportive or therapeutic treatment to one patient.Materials and methodsReplicating prior studies, we modeled the cost-efficiencies gained from converting varying proportions of a hypothetical panel of 4,000 patients undergoing six cycles of cancer treatment from Neupogen or Neulastim to one of the two biosimilar G-CSF formulations, using national cost inputs. Cost-savings in USD were used to estimate the additional doses of biosimilar G-CSF and expanded access to ado-trastuzumab emtansine on a budget-neutral basis, and NNC to purchase one additional dose of supportive or therapeutic treatment.ResultsSavings from conversion from reference to a biosimilar filgrastim were $3,086,400 (Nivestim) and $3,460,800 (Zarzio). With reference pegfilgrastim, savings from conversion were $11,712,240 (Nivestim) and $12,086,640 (Zarzio). Biosimilar conversion from reference to biosimilar filgrastim enabled expanded access to ado-trastuzumab emtansine ranging from 61 patients (5 days, Nivestim) to 191 patients (14 days, Zarzio). For supportive care, biosimilar conversion enabled expanded access ranging from 8,244 patients (5 days, Nivestim) to 25,882 patients (14 days, Zarzio). For biosimilar conversion from daily filgrastim, the NNC for treatment with ado-trastuzumab emtansine decreased as days of injections increased [5 days: 395 (Nivestim), 352 (Zarzio); 14 days: 141(Nivestim), 126 (Zarzio)]. Alternately, for biosimilar conversion from single-injection pegfilgrastim to daily biosimilar filgrastim, the NNC for treatment with ado-trastuzumab emtansine rose as days of injections increased, being highest under the 14-day scenario (146, Nivestim; 130, Zarzio).ConclusionThis simulation study demonstrated significant potential cost-savings from biosimilar conversion. These savings provide budget-neutral increased access to supportive and therapeutic cancer care.

Addition of Sorafenib to Cladribine, High-Dose Cytarabine, G-CSF, and Mitoxantrone (CLAG-M) in Adults with Newly-Diagnosed Acute Myeloid Leukemia (AML) and High-Grade Myeloid Neoplasms Independent of FLT3-Mutation Status: Final Results of a Phase 1/2 Study

BACKGROUND: We previously showed that cladribine, cytarabine, G-CSF and dose-escalated mitoxantrone (CLAG-M) led to a higher rate of complete remissions without measurable residual disease (MRDneg CR) than 7+3. Recently, a randomized trial demonstrated addition of the multikinase inhibitor sorafenib to 7+3 improved event free survival (EFS) irrespective of FLT3 mutation status. Here, we report the results of a phase 1/2 study (NCT02728050) evaluating addition of sorafenib to CLAG-M (CLAGM+S) in adults with newly-diagnosed AML or other high-grade myeloid neoplasms (≥10% blasts). Phase 1 results were reported previously. METHODS: Patients aged 18-60 years were eligible if they had a treatment-related mortality score (TRM) of ≤13.1 (corresponding to a predicted <13.1% risk of death by day 28 following intensive induction chemotherapy) and adequate organ function. Patients with uncontrolled infection or concomitant illness with expected survival <1 year were excluded. Cohorts of 6-12 patients were assigned to 1 of 6 dose levels of mitoxantrone (10, 12, 15, or 18 mg/m2/day, days(d) 1-3) and sorafenib (200 to 400 mg twice daily [BID] on d10-20 with induction and 8-27 with consolidation). G-CSF dose was 300 or 480 μg/day (weight-based, d0-5), cladribine 5 mg/m2/day (d1-5), and cytarabine 2 g/m2/day (d1-5). Up to 4 cycles of post-remission therapy with CLAG+S (mitoxantrone omitted) were allowed, followed by 12 months of maintenance sorafenib. Patients were taken off trial if they underwent allogeneic transplant. As primary endpoint in Phase 2, the rate of MRDneg CR following CLAGM+S was compared to a matched historical control population who received CLAG-M alone. Secondary endpoints included rate of CR, overall response rate (ORR defined as CR+CR with incomplete hematologic recovery [CRi]), overall survival (OS), EFS and the toxicity profile. EFS and OS were estimated using the Kaplan-Meier method and compared using Cox regression models. RESULTS: 84 patients were enrolled from 12/8/2016-10/7/2021. In Phase 1, CLAG-M with mitoxantrone at 18 mg/m2/day and sorafenib 400 mg BID was established as the recommended phase 2 dose (RP2D). Sixty-one patients with a median age of 50 [range: 21-60] years were treated at the RP2D. ELN2017 risk category was favorable in 28%, intermediate in 36%, and adverse in 34%. FLT3-ITD and TKD mutations were present in 28% and 7% respectively. Mortality within 28 days was 2%. At the RP2D, a response of MRDneg CR occurred in 49/61 patients (80%, 95% CI: 68-89%). Additional responses of MRDpos CR and CRi (3 patients each) led to an ORR of 90%. The MRDneg CR rate in patients with and without FLT3-mutations was similar (90% vs. 83%, p=0.30). Median times to an absolute neutrophil count ≥500/μL and a platelet count of ≥50,000/μL were 29 and 25 days. Besides infections/neutropenic fever, rash was the most common grade ≥3 adverse event. With a median follow up of 35 months, median OS was not reached; 12-month OS was 82%. There was no difference in OS and EFS in the FLT3-mutated and FLT3-wild type patients. We next compared CLAGM-S to a historical cohort of 71 newly diagnosed patients treated with CLAG-M alone, matched for mitoxantrone dose, age and TRM score (Table 1). In univariate analysis, rates of MRDneg CR, OS and EFS were similar (p=0.83, 0.16, and 0.37 respectively). Likewise, there was no difference in OS (p=0.54) or EFS (p=0.63) in FLT3-mutated patients who received CLAGM+S vs CLAG-M. In subgroup analysis, we observed a benefit favoring CLAGM+S for both OS (p<0.01) and EFS (p=0.03) among ELN2017 intermediate risk patients (Figure 1). No benefit of CLAGM+S vs CLAG-M alone in either OS or EFS was observed among adverse risk patients. Analysis of the favorable-risk cohort was limited by small sample size. Multivariate models comparing CLAGM-S to CLAG-M alone (reference) and controlling for age, secondary disease, FLT3 status and cytogenetic risk showed improved OS (HR=0.38, 95%CI 0.17-0.84, p= 0.02) and EFS (HR= 0.43, 95% CI 0.22-0.85 p=0.02) following CLAGM-S; MRDneg CR was also more likely with CLAGM-S though not statistically significant (OR= 2.56, 95% CI 0.68-9.61, p= 0.16). CONCLUSION: Addition of sorafenib to CLAG-M produces high rates of MRDneg CR in newly diagnosed AML patients ≤60 and prolongs OS and EFS in multivariate analysis compared to historical, matched control cohort receiving GLAG-M alone, a benefit that was particularly evident in patients with intermediate-risk disease. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Accumulation of Specific Somatic Leukemia-Associated Mutations in Congenital Neutropenia Precedes Malignant Transformation - New Preconditions for Treatment Decisions

Severe congenital neutropenia (CN) and Shwachman-Diamond Syndrome (SBDS) are pre-leukemia bone marrow failure syndromes with a high cumulative incidence of developing MDS or AML. Clonal hematopoiesis (CH) due to nonsense mutations in the intracellular part of CSF3R or missense TP53 mutations has been reported previously in CN or SBDS patients, respectively. So far, studies of CH in CN were conducted by sequencing CSF3R only (Germeshausen et al., 2007, Klimiankou et al., 2019) or in ELANE-CN patients (Jun Xia et al., 2018). Precise characterization of the timeline and occurrence of somatic mutations found at the neutropenia, pre-leukemia or leukemia stage in CN is essential to make a timely and correct decision on the requirement of bone marrow transplantation prior to transformation to overt leukemia. We therefore aimed to assess the acquisition of somatic leukemia-associated mutations in a large number of genes in a worldwide largest and most representative cohort of CN patients. We performed error-corrected sequencing with fixed single-stranded unique molecular indexes (UMIs) on panel of 31 genes and "hotspot" regions in 340 genes reported to be mutated in de novo MDS/AML. We analyzed 146 CN patients representing most of genetic subtypes of CN, 21 SBDS patients, 29 CN patients who progressed to MDS or AML, and 18 healthy individuals. The median age of CN and SBDS patients was 13,73 and 10,5 years, respectively. For 53 CN patients, we collected sequential samples with a median observation time of 1,1 years. We found that the prevalence of CH with at least 1 somatic DNA lesions in CN group was 49,5 % which is much higher than in healthy individuals of the same age. The prevalence of CH with CSF3R mutations varied between 20 and 38 % in patients with genetic defects in ELANE (n = 75), HAX1 (n = 26), SRP54 (n = 8), JAGN1 (n = 6), and G6PC3 (n = 5). The percentage of CH positive cases without CSF3R mutations ranged between 12 % and 50 %. CN patients acquired somatic coding mutations most frequently in CSF3R (49 %) following by TP53 (4,1 %), ASXL1 (4,1 %), DNMT3A (2,1 %), TET2 (2,1 %), JAK2 (1,37 %), SETBP1 (1,37 %), ARID1B (1,37 %), GNAS (1,37 %), and ZRSR2 (1,37 %). Interestingly, in the SRP54-CN group, we found 1 patient with CSF3R mutation, 1 patient with TP53 mutations, and 1 patient with both mutations. Out of 3 CLPB-CN patients, 1 patient has CSF3R and 1 patient acquired TP53 mutations. High prevalence of CSF3R (67 %), RUNX1 (44 %), SETBP1 (22 %), ASXL1 (17 %), ZRSR2 (11 %), NPM1 (11 %) and PTPN11 (11 %) mutations was detected in CN patients with overt MDS/AML, arguing for a specific pathway which initiates and drives leukemic transformation in CN. Comparison of CN patients who later developed MDS/AML with MDS/AML-free CN cohort revealed enrichment of CSF3R (Fisher's exact test; OR 3,42; 95% CI, 1,27, - 9,36, p = 0,01), and ASXL1 (Fisher's exact test; OR 7,4; 95% CI, 0,93 - 59,16, p = 0,03) mutations. In contrast, RUNX1 and SETBP1 mutations were associated with overt MDS/AML in CN. These data argue that the acquisition of CSF3R and ASXL1 mutations are early events in CN leukemogenesis, whereas RUNX1 and SETBP1 mutations may have stronger leukemogenic potential and therefore are rarely found at the neutropenia stage. Moreover, mutation burden at the neutropenia stage in CN patients who later progressed to MDS/AML was significantly higher (p = 0.016, by Fisher's exact test), as compared to CN group who haven't developed leukemia so far. The number of mutations in samples prior AML/MDS was dependent on duration to overt AML/MDS with gradual accumulation of genetic lesions over time. CH was detected in 66,7 % of SBDS patients with a high prevalence of TP53 mutations (9 out of 14 patients with CH) followed by missense mutations in IDH1 (n = 1), FOXP1 (n = 1), and PTPN11 (n = 1). Our results are in line with previous publications about dominant role of TP53 mutation in SBDS patients. Prevalence of CH didn't differ between CN patients depending on G-CSF dose (median = 4,06 μg/kg/d) in two biggest CN cohorts harboring ELANE or HAX1 mutations. Taken together, CN per se contributes to high level of CH already in the young age, as compared to healthy population. Acquisition of multiple genetic lesions, especially RUNX1, SETBP1, ASXL1, TP53, PTPN11 in association with CSF3R mutation might be a strong indicator of the advanced pre-leukemia stage in CN patients at neutropenia stage and requires closer patient follow up.

Oral SGLT2 Inhibitors in Glycogen Storage Disease Type Ib and G6PC3-Deficiency. Preliminary Results from an Off-Label Study of 21 Patients

Introduction Glycogen storage disease type Ib (GSDIb) and G6PC3-deficiency (G6PC3d) are two distinct genetic disorders associating neutropenia, bacterial infections susceptibility and inflamatory bowel disease (IBD), requiring GCSF therapy, and various immunotherapies. These neutropenias are caused either by a defect in the glucose-6-phosphate transporter of the ER or in the phosphatase G6PC3. These two defective proteins were recently shown to destroy a toxic metabolite, 1,5-anhydroglucitol-6-phosphate (1,5-AG6P), formed from the phosphorylation of a food derived polyol present in blood, 1,5-anhydroglucitol (1,5-AG), which otherwise accumulates in neutrophils, inhibits glycolysis and explains the neutropenia (Veiga-da-Cunha M, PMID: 30626647). As a direct result from this discovery, oral inhibitors of SGLT2 (iSGLT2s), the renal Na2+/glucose cotransporter 2 (e.g., empagliflozin or dapagliflozin) routinely used to treat type-2 diabetes were proposed to treat neutropenia in GSDIb and G6PC3d. Examples of such therapies have been successfully used in small cohorts of patients (Wortmann SB, PMID: 32294159; Boulanger, PMID: 35506446). We show preliminary results of the off-labelled study using iSGTL2s proposed to patients enrolled in the French severe chronic neutropenia registry (FSCNR). Methods: Patients (or their parents) with a genetic diagnosis of GSDIb or of G6PC3d enrolled in the FSCNR, signed an informed consent to participate to this observational study. Empaglifozin or mostly dapaglifozin were started at a dose of 0.4 mg/kg/day or 0.2 mg/kg/day respectively. The study was divided in 3 phases. During the pre-therapeutic period, information was collected on the medical history and various biological parameters (complete blood counts, 1,5-AG, concentration, bone marrow smears). The initiation period aims to adapt the iSGLT2 dose while GCSF and immunotherapy were tapered down. The maintenance therapy was an observational therapy period, during which iSGLT2 dose remained largely unchanged. Results Twenty-one patients (GSDIb n = 14; G6PC3d n =7) were enrolled and the median follow-up was 0.8 years. The median age at the start of therapy was 18.8 years [range 0.4 - 37.7 years]. Two are still in the initiation period, while 19 are in the maintenance therapy and their results are summarized in the table 1. During the initiation period, 6 patients continued taking the initial iSGTL2 dose, while a dosage increased appeared necessary in 13 others, both to lower GCSF dose and to optimally reduce 1,5-AG in plasma which was particularly high at onset. The only severe side-effect was related to neutrophilia observed in a patient with G6PC3d with transient digestive manifestations (recurrent vomits and stomach pains). Patients with GSDIb required a longer initiation period to achieve a clinical benefit. Three patients were not taking GCSF during the pre-therapeutic period. For the 16 patients treated with GCSF prior to iSGLT2 therapy (iSGTL2t), the mean dose of GCSF was 2.5 µg/kg/day; 9 of them stopped GCSF, 6 decreased their daily dose and 1 was unchanged. Overall, the mean dose of GCSF under iSGTL2t was reduced by 8-fold to 0.31 µg/kg/day. During the pre-therapeutic period 3 patients were treated for severe IBD which required anti-TNF therapy (and surgery for 2). Most remarkably all 3 patients stop IBD related treatments, without recurrence of symptoms, while on iSGTL2t. Comparing periods, we observed limited and non-statistically significative change among hematological parameters (table 1). Of note, stable values for neutrophils were found while the GCSF was either tapered down or withdrawn. Bone marrow inspection indicated a qualitatively change related to the aspect of the endoplasmic reticulum in neutrophils which appeared less damaged both seen on their morphology and on the electronic microscopy images. Maturation arrest was never observed and myeloid compartiment appears less overstimulated under iSGTL2t. Conclusion: iSGTL2s therapy in GSDIb and G6PC3d patients appeared to be a useful way to limit both GCSF therapy, infections and IBD manifestations. Initiation of iSGLT2s did not result in an instant effect. We often needed to adapt the dose of iSGTL2s both to spare GCSF therapy and to optimally lower 1,5-AG plasmatic level, indicating that its monitoring is useful to adapt the dosage. Further studies are required to define the best schedule of iSGTL2s in such subset of patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Extended Detection in Peripheral Blood Following Infusion of Chronic Granulomatous Disease Patient Autologous Granulocytes Corrected By mRNA Transfection in Patients with Chronic Granulomatous Disease

Patients with Chronic Granulomatous Disease (CGD) have an inherited defect in production of microbicidal oxidants by neutrophils and monocytes that can result in life-threatening infections. Some of these infections, particularly those with mold fungi that invade across tissue boundaries, may persist and progress despite intensive medical therapy. In such a setting there is a long history of published single case and small series reports suggesting that a course of unmatched allogeneic donor granulocyte (gran) transfusions may result in improvement or even resolution of such refractory life-threatening infections in CGD patients. Donors for grans transfusion products are often challenging to find and not HLA-matched. Because of this most CGD patients can be provided only a limited course of grans transfusions before they develop limiting anti-HLA antibodies or lack of available donors. To address this problem, we developed an ex vivo correction of NADPH oxidase defective grans from CGD by mRNA transfection1. Apheresis grans transfected with mRNA restored NADPH oxidase function in vitro and following transplant into immunodeficient mice. Messenger RNA-transfected autologous grans from non-human primates (NHP) were well tolerated and were detected for up to 5 days in NHP peripheral blood following infusion. This pre-clinical work was transitioned to a Phase I, dose-escalating, feasibility and safety study (NIH Protocol 22-I-0001; ClinicalTrials.gov NCT05189925). We report here data from the first X-linked CGD subject infused with autologous gp91 mRNA-transfected grans at the first dose level. The patient received two daily doses of G-CSF before apheresis. From 26 x10e9 total nucleated cells in the apheresis product, ~7x10e9 grans were collected, though only 1.5x10e9 grans were subjected to electroporation transfection with gp91phox mRNA (CELLSCRIPT LLC). A single infusion of 70e6 (10e6 per kg) mRNA-transfected autologous grans where about 57% were DHR+ (40x10e6 DHR positive grans) was well tolerated without any adverse effects. NADPH oxidase functional (DHR+) grans were detected in peripheral blood (PB) at last assessment (72 hrs) after this single infusion of mRNA corrected autologous grans. Serial PB evaluation post infusion from study Subject detected oxidase (DHR)+ grans. Of note, DHR+ grans in Subject PB at ~ 43 hrs after infusion was 0.0037% compared to his pre-infusion DHR assays (0%) (Figure, top row). As a comparator, DHR assays performed pre- and also ~ 40 hrs post infusion of a clinical product of 40x10e9 allogeneic donor grans into another CGD patient being treated clinically for a refractory infection is shown (bottom row). There is 1000 fold more allogeneic DHR+ grans infused for the clinical treatment than the experimental DHR+ mRNA corrected autologous grans infused. Compared to their corresponding absolute neutrophil count, the study Subject (received 40x10e6 DHR+ mRNA transfected autologous grans) had 512 DHR+ neutrophils/ml while the CGD patient (received clinical 40x10e9 allogeneic grans) had 79,560 DHR+ neutrophils/ml. This indicates there are > 6 fold more mRNA-corrected autologous grans in circulation at 43 hrs per cell infused than following the clinical treatment with allogeneic cells infused. Our data demonstrates that mRNA-corrected autologous grans are not only well tolerated but are very well sustained viable and functional in the circulation relative to clinical unmanipulated allogeneic donor grans. Additional patients need to be studied at this dose level and the escalating dose levels to assure that this could be a safe alternative to non-HLA matched allogeneic grans transfusions. Figure shows FACS DHR analysis of peripheral blood from CGD patients after infusion of autologous mRNA-transfected granulocytes 40x106 (top) and donor granulocytes 40x109 (bottom). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Phenotype and Natural History of ELANE and NON ELANE Severe Congenital Neutropenia in Italy: Data from the National Registry

INTRODUCTION Severe Congenital Neutropenia includes several disorders characterized by absolute neutrophil count depletion appearing early in life, high propensity to potentially lethal infections and risk of myelodysplasia/acute leukemia. Mutations of ELANE and HAX 1 genes are often reported as the most frequent causes of SCN but many other genes are now recognized as causative of the disease. AIM of The STUDY. To describe the clinical history and the outcome of SCN patients included in the Italian Neutropenia Registry (INR) and to investigate differences between ELANE and non- ELANE mutated groups PATIENTS AND METHODS. Demographics, biochemical, clinical and genetic information were retrieved from the INR. OS probability was estimated by the Kaplan-Meier method. RESULTS from June 2000 to June 2022 844 patients were reported to the INR and 106 (52% females) were diagnosed with SCN. Median age at diagnosis was 1 year (IQR 0.4-4.1) and at last follow up was 11years (IQR 6.0-19.4). Subjects were Caucasian in 90% of cases and of other ethnicities in 10% . Consanguinity occurred in 4/49 (8%) of the patients in which data were available. Genetic testing performed in all patients, enabled a definite genetic diagnosis in 94% of the subjects whereas in the remaining 6% a genetic lesion was not found. Genetic lesions were distributed as follows: ELANE 54% of which 28% were cyclic , TAZ 10%, CXCR4 6%, SLC37A 4.6%, COH1 3%, JAGN1 3%, SDS 3%, G6PC3 3%, SRP54 2%, USB1 2%, HAX1 2%, CSFR3 1%, , GATA2 1%). Of 106 patients, 4 were also reported to specific Registries (SDS and GATA2) while 11% were lost to follow-up. The 15 and 20-year overall survival of the 94 patients in active follow-up were respectively 93 % (83.6%-97.0%) and 87.4% (69.2%-95.2%) without differences between the ELANE and non ELANE groups. In the whole cohort 7/106 (7%) patients died and of them 3/7 for infections due to non compliance to G-CSF therapy, 2/7 for organ damage associated to the disease and 2/7 for complication after bone marrow transplantation. The cumulative incidence of MDS/AL after 20 yrs after diagnosis was 10,9% (3,2%-34%) that favorably compares with that from other Registries. Total number of Infection episodes prior to SCN diagnosis, was 592 in 73 pts (42 in ELANE, 31 non- ELANE) and were: pharingytis and bronchitis (32%), skin and deep abscesses (22%), otitis/mastoidistis (17%), miscellaneous (29%) (enteritis, other upper respiratory , urinary tract infections and fever of unknown origin (14%), pneumonia (10%) and sepsis (5%)). In pre-diagnosis period otitis occurred more frequently in ELANE (64/316 episodes) vsnon ELANE (36/276) (p= 0,02). Sepsis were also more frequent, though non significantly in ELANE subset. On the contrary, urinary tract infections were more frequent in non-ELANE (27/35) vs ELANE (8/40) group ( p<0,001). The post-diagnosis frequency of infections by age (0-5yrs, 6-10yrs and 10-15 yrs) in the whole group is shown in fig 1. Apart from skin infections that increase in peri/pubertal period (11-15 years) we observed a progressive decrease of the number of all other infections with full disappearance of sepsis after the age of 5 years. No main differences was observed between ELANE and non- ELANE group. Eighty-six % of patients were on G-CSF; 27% required doses>7,5 mcg/kg/day (12 ELANE and 1 HAX1), 22% needed 5 mcg/kg/day (8 ELANE, 1 USB1, 1 HAX1) and 51% (11 ELANE, 4 SCL37A, 7 TAZ, 1 JAGN1, 2 G6PC3, 1 CSFR3) required lower than 5 mcg/kg/day. Doses higher than 5 mcg/kg/day were more frequently used in ELANE vs non-ELANE group ( p=0.01). Fourteen patients underwent bone marrow transplantation (12 ELANE, 1 SCN gene orphan, 1 HAX1 and 1 JAGN1) for leukemic transformation (3 patients), poor response to G-CSF (7) and center decision (4). The 10-year OS of transplanted subjects was 87.5% and was comparable with that of non-transplanted ones (93%) (p=0.21) CONCLUSIONS. A genetic diagnosis was achieved in almost all SCN patents of the INR. ELANE mutations are the major cause of SCN and are associated to a more severe phenotype requiring higher doses of G-CSF and more frequent need for transplant. The relatively low incidence of MDS/AL in the whole cohort is consistent with a high proportion (73%) of patients receiving G-CSF dose ≤ 5 mcg/kg/day. Infection pattern improves overtime after diagnosis with disappearance of sepsis in both ELANE and non-ELANE subsets possibly reflecting the tight monitoring received by patients reported to registries. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal