Successful Stem Cell Transplantation Research Articles

Successful Stem Cell Transplantation from Varicella Infected Donor.

Successful Stem Cell Transplantation from Varicella Infected Donor.

CD38 promotes hematopoietic stem cell dormancy.

A subpopulation of deeply quiescent, so-called dormant hematopoietic stem cells (dHSCs) resides at the top of the hematopoietic hierarchy and serves as a reserve pool for HSCs. The state of dormancy protects the HSC pool from exhaustion throughout life; however, excessive dormancy may prevent an efficient response to hematological stresses. Despite the significance of dHSCs, the mechanisms maintaining their dormancy remain elusive. Here, we identify CD38 as a novel and broadly applicable surface marker for the enrichment of murine dHSCs. We demonstrate that cyclic adenosine diphosphate ribose (cADPR), the product of CD38 cyclase activity, regulates the expression of the transcription factor c-Fos by increasing the release of Ca2+ from the endoplasmic reticulum (ER). Subsequently, we uncover that c-Fos induces the expression of the cell cycle inhibitor p57Kip2 to drive HSC dormancy. Moreover, we found that CD38 ecto-enzymatic activity at the neighboring CD38-positive cells can promote human HSC quiescence. Together, CD38/cADPR/Ca2+/c-Fos/p57Kip2 axis maintains HSC dormancy. Pharmacological manipulations of this pathway can provide new strategies to improve the success of stem cell transplantation and blood regeneration after injury or disease.

Effect of the delivery route and dose of multipotent mesenchymal stem cells on the efficacy of cell therapy (review)

Multipotent mesenchymal stem cells (MMSCs) are known to be excellent therapeutic agents. Apart from their ability to differentiate into various cell types, and thus participate in the repair of injured tissues and organs, they can influence the regeneration process through secretion of paracrine factors. Thus, MMSC therapy represents a special type of medical intervention that has both a systemic range of therapeutic efficacy and local activity on individual sites of an organ. Over the past decades, MMSC therapy has continuously been in a cautious transition from research development to clinically approved therapies. Clinical trial data has shown that this therapy is rarely associated with severe adverse events, is well tolerated and quite safe in the short-term period. However, it has a number of limitations for use, mainly due to the risk of malignant transformation. The success of stem cell transplantation in the treatment of various diseases has been confirmed both in preclinical studies and in clinical practice. The main issues that arise when assessing the therapeutic efficacy of MMSC-associated therapy are the type of cells (adipogenic, bone marrow, etc.), delivery route, number of cells injected, and the optimal number of injections. There is a growing body of experimental and clinical evidence suggesting that both an adequate delivery route and an adequate dose can increase the likelihood of success of MMSC-associated. Each cell delivery route has costs and benefits. However, there is generally contradictory evidence on the comparative efficacy of different cell delivery routes. The optimal dose of transplanted cells is also debated, as high MMSC doses may increase the risks of complications and may not have the proper effect both when administered systemically and locally. These aspects require further systematization of available data to maximize the effect of cell therapy by selecting the safest and most appropriate approaches.

National Long-Term Follow-up of Children with Sickle Cell Disease Diagnosed By Newborn Screening in the Netherlands: Overview of the Morbidity and Mortality

Background: Sickle cell disease (SCD) is a genetic red blood cell disorder associated with increased morbidity and mortality. Newborn screening for SCD was introduced in January 2007 in the Netherlands to enable early diagnosis and initiation of preventive measures. The objective of this study was to assess the effect of newborn screening on the morbidity and mortality of children living with SCD in the Netherlands, 16 years after its implementation. Methods: In this national cohort study all children with SCD diagnosed by newborn screening were included. Patients were followed between 1 January 2007 and 31 March 2023 until end of follow-up, death or successful stem cell transplantation. Following informed consent, data were collected from medical files on SCD genotype, treatment, mortality and development of SCD-related complications. The incidence rate of SCD-complications was presented as lifetime-event per 100 person-years for the total duration of follow-up and for the age categories: 0-4, 5-11, and 12-16 years. Results: In total, 391 out of 540 eligible patients (72%) with SCD were included in all hospitals with patients with SCD under treatment, accounting for 3047 person-years. The mean age at the end of follow-up was 7.8 years (SD±4.5). The majority of patients had SCD genotype HbSS (58%), followed by HbSC (28%). As for the mortality, five patients died during follow-up, primarily at or below the age of 2 years (4/5, 80%). In three patients, the cause of death was related to SCD, including infection (n=2; aged 1.2 and 2.0 years) and acute splenic sequestration (n=1; 2.0 years). The SCD-related complications are presented in Table 1. The most common SCD complication in this cohort was vaso-occlusive crisis (including dactylitis) with hospitalization. SCD-related complications that mainly developed within the first 4 years of life, were vaso-occlusive crisis, acute hemolytic crisis, aplastic crisis, severe infections, splenic sequestration and ischemic cerebral infarction. Acute chest syndrome occurred equally in all age categories. Other SCD-related complications developed to a lesser extent in our cohort, and at a later age in adolescence or adulthood. Conclusion: In this cohort of neonatally screened children with SCD, a substantial number of SCD-complications occurred in the first 4 years of life. This study underlines the importance of parental education and 24/7 availably, high level acute specialized clinical care for these children from birth on. Further analysis to identify risk factors at young age for a severe phenotype will be conducted, to enable more targeted interventions in a timely manner. Hopefully, this will lead to reduction of disease burden, improving the quality of life in children living with SCD in the Netherlands.

A-075 Development and Validation of Image-based Cell Counter Microscanner C3 for CD34-positive Hematopoietic Stem Cell Enumeration

Abstract Background Accurate measurement of CD34-positive hematopoietic stem cells (HSCs) is critical for successful peripheral blood stem cell transplantation (PBSCT). Flow cytometry is by far the gold standard for measuring CD34-positive stem cells. However, its complicated procedure requires time, cost and highly-trained operators, thus the necessity of simpler and cheaper alternative method is emerging. We have developed a new image-based cell counter, Microscanner C3 (MSC3, Biozentech, South Korea) as an alternative method for enumeration of CD34-positive HSCs and evaluated its performance. Methods 100 µl of apheresis product was stained with CD45-BB515 and CD34-PE antibodies (Becton Dickinson, USA) at room temperature in the dark. Red blood cell lysis with 500 µl of OptiLyze C solution (Beckman Coulter, USA) was followed by centrifugation. After the removal of supernatants, the sample was resuspended with 500 µL PBS, which then infused to filter paper imbedded BZ-1 microchip (Biozentech, South Korea). Thirty-fields of the loaded microchip was auto-scanned by MSC3 and its dedicated software analyzed the images and yielded CD34-positive cell fraction out of CD45-positive cells as percentage. Results of thirty-six samples were compared to that of FacsLyric (Beckton Dickinson, USA) flow cytometry and accuracy and imprecision (pentaplicate measurements for three samples), repeatability (two experimenters performed triplicate measurements for three samples each), linearity (triplicate measurements for five levels), limit of detection (hexaplicate measurements for five levels) and specificity (using whole blood specimen from three donors with monoclonal gammopathy, thrombocytosis or monocytosis) was evaluated. Results CV% values of inaccuracy, imprecision and repeatability did not exceed 15%. The method was found to be linear across the measuring range. LOD was evaluated to be 7 cells/µL and no background interference was detected. The difference between the measured fraction of MSC3 and FacsLyric was within allowable limit, with an R-value greater than 0.98. Conclusion We developed a reliable method for quantifying CD34-positive HSCs based on image analysis. The limitation involved in MSC3 applications in CD34-positive HSCs is that, like dual-platform flow cytometry, the results should be combined with the results of conventional hematology analyzer in order to define absolute HSC numbers. However, with its simpler and faster procedures, MSC3 exhibited a great potential to be used as an alternative to conventional flow cytometry in CD34-positive HSCs enumeration and other applications as well.

Stem cell mobilizating effect of heparin in patients undergoing autologous stem cell transplantation.

Adequate stem cell collection is essential for successful stem cell transplantation. Heparin enhances stem cell mobilization by competing with heparin sulfate proteoglycans. Heparin is also used as an anticoagulant before leukapheresis. Here, we evaluated the effects of heparin on stem cell mobilization in patients who underwent autologous stem cell transplantation (ASCT). We evaluated patients who underwent ASCT. Patients were divided into two groups: those who received heparin plus citrate (heparinized patients) and those who received citrate only (nonheparinized patients) for anticoagulation. Univariate and multivariate analyses were also performed. The collection efficiency 2 (CE2) for CD34+ cells was calculated and compared between heparinized and nonheparinized patients. This study included 1017 patients. There were 478 (47%) heparinized and 539 (53%) nonheparinized patients. The number of collected CD34+ cells was significantly higher in heparinized patients (P < .00001). The multivariate analyses showed that using heparin was an independent positive factor for collected CD34+ cells (adj-R2 = 0.744; F = 369.331, P < .00001). CE2 was significantly higher in heparinized patients than in nonheparinized patients (66.8% vs 52.1%; P < .00001). The rate of collecting at least 2 × 106 /kg CD34+ cells was 3.3 times higher for heparinized patients in poor mobilizers (P < .00001). Heparinized patients had significantly higher total nucleated and mononuclear cell counts (P < .00001 and <.00001, respectively). Heparin enhances stem cell collection and increases CE2. The use of heparin may reduce the need for other strategies to increase stem cell mobilization.

Hemophagocytic Lymphohistiocytosis: an Under-recognized and Life-threatening Condition.

Diagnosis of hemophagocytic lymphohistiocytosis is a challenge in Nepal because of limited resources and the high prevalence of tropical febrile illness mimicking hemophagocytic lymphohistiocytosis. We retrospectively reviewed medical records of 21 patients who were diagnosed with hemophagocytic lymphohistiocytosis from 2010 to 2015 at a single center in Nepal. Two patients had a mutation in their perforin gene and underwent successful haploidentical stem cell transplantation. Marrow hemophagocytosis was found only in 57% of the patients. Five patients had hematological malignancy and were treated with disease-specific chemotherapy. Seven patients developed hemophagocytic lymphohistiocytosis secondary to an infection, including visceral leishmaniasis, scrub typhus, and Epstein Barr virus. EBV-associated hemophagocytic lymphohistiocytosis was refractory to hemophagocytic lymphohistiocytosis 94 protocol, including the addition of rituximab. Malignancy and infection-associated hemophagocytic lymphohistiocytosis was more common. The most common clinical presentations included fever, splenomegaly, hyponatremia, liver function derangement, hyperfibrinogenemia, hyperferritinemia, and cytopenia. With a mortality of 29% in our study cohort, hemophagocytic lymphohistiocytosis should be considered a lethal disease, and clinicians should maintain a high index of suspicion to diagnose this disease. Keywords: Hemophagocytic lymphohistiocytosis; infection; malignancy.

Why and How Should Ethiopia Establish a Stem Cell Transplant Service? A Review Article.

Ethiopia is attempting to reduce cancer-related morbidity and mortality through a strategic national cancer control plan but according to Globocan 2020, hematologic malignancies particularly leukemia and non-Hodgkin's lymphoma rank among the top five leading causes of new cancer incidence and cause of death among all age groups in both sexes. Hematopoietic stem-cell transplantation (HSCT) is an advanced treatment modality that makes the only effective treatment for cancer and non-cancer-related hematologic diseases unresponsive to conventional therapy. Patients who need stem cell transplants must travel to abroad countries to get the treatment. Meanwhile, the Ethiopian National Specialty and Subspecialty Roadmap sets the goal of establishing HSCT centers in 2020-2029 GC, yet leaders and planners must start taking steps to put the setup in place. Setting up an HSCT facility is challenging for developing countries due to the high costs, limited infrastructure, and need for intensive medical staff training; however, several nations have been able to start successful stem cell transplant programs. This review summarizes the basic steps and requirements of the program in light of guidelines recommendations and lessons learned from other developing countries. It also highlights possible cost-effective opportunities, bottlenecks, and areas that will require work and investment to make the objective reality in Ethiopia. Provides key information to assist administrators and policymakers to set priorities in planning and making informed decisions to establish and maintain the service.

Allogeneic Hematopoietic Stem Cell Transplantation in Older Patients with Acute Myeloid Leukemia: Analysis of Outcomes and Contemporary Trends Spanning 10 Years

Background: Allogeneic hematopoietic stem cell transplantation (SCT) is a highly effective consolidation strategy in acute myeloid leukemia (AML). Non-myeloablative and reduced-intensity (NMA/RIC) conditioning regimens have expanded the use of SCT to older patients (pts) (age ≥ 60 years). Age alone no longer appears to be a barrier for successful SCT, with reported 3-year overall survival (OS) rates in the 45-50% range (Maakaron 2022). Methods: We conducted a retrospective study spanning 10 years to evaluate SCT-related outcomes in older AML. All pts ≥ 60 years presenting to our institution with newly-diagnosed (ND) AML were included. Acute promyelocytic leukemia (APL) was excluded. Median OS and relapse-free survival (RFS) were calculated using the Kaplan Meier method. A landmark analysis was performed to evaluate time-to-event endpoints in pts who underwent SCT in first response compared to those who did not. Results: Between 7/19/2012 and 11/17/2021, we identified 1073 pts ≥ 60 years with ND-AML. The median age at diagnosis was 71 years (range 60-94), 622 (58%) were male, 189 (18%) had prior myelodysplastic/myeloproliferative neoplasm, and 220 (21%) had therapy-related AML. ELN 2017 risk was favorable in 215 (20%), intermediate in 234 (22%), and adverse in 538 (50%) pts (86 with data missing). Most pts (84%) were treated with low-intensity (LOW) regimens. Only 16% were treated with intensive (INT) chemotherapy (intermediate or higher dose cytarabine). Venetoclax (VEN) was included in 34% of the regimens. The CR/CRi rate was 60% in the overall cohort. The CR/CRi rate was 74%, 48%, and 72% in pts treated with INT, LOW, and LOW + VEN therapies, respectively. The median OS and RFS were 11.2 and 10.6 months for the full cohort, respectively. The rate of referral to the SCT service was 38% (413/1073) and increased over time (31% in 2012-2013, 52% by 2020-2021). This occurred in parallel to increasing rates of CR/CRi (53% in 2012-2013, 66% by 2020-2021), likely reflecting increased efficacy of VEN-containing regimens in the later years. We identified 198/1073 pts (18%) who underwent SCT at some point during therapy, 152/198 (77%) in first response (CR/CRi/MLFS) after frontline therapy, and 46/198 (23%) following one or more salvage regimens. Of all ND-AML pts, the SCT rate was 37%, 10%, and 22% in pts treated with INT, LOW, and LOW + VEN, respectively. The rate of undergoing SCT increased over time (15% in 2012-2013, 27% by 2020-2021). Clinical/SCT characteristics for the pts who underwent SCT are shown in table 1. The rate of grade 3/4 acute graft-vs-host disease (GVHD) was 11% and 19% of pts experienced chronic GVHD of any grade. Following SCT, 24% of pts experienced relapse of AML and 21% died without disease relapse. The 100-day mortality following SCT was 13%. We then performed a landmark analysis (using median time to SCT [4.3 months] as the landmark) comparing pts undergoing SCT in first response following frontline therapy at our institution (n=139) to responding pts who did not receive SCT in first response and who were alive and without relapse at the landmark (n=353). Both the median OS (31.0 vs 18.9 months, p=0.0208, figure 1) and median RFS (23.2 vs 10.0 months, p<0.0001) were superior in the pts who underwent SCT compared to those who did not. Pts undergoing SCT in first response were younger (median age 66 vs 72 yrs) and had better performance status (PS 2-4 in 10% vs 22%) compared to no-SCT pts. Distribution of ELN risk categories and best response (CR/CRi/MLFS) were similar in the SCT and no-SCT groups. In pts who responded to initial therapy and were referred for SCT, but ultimately did not undergo SCT in first response (n=161), the most common documented reasons for forgoing SCT were social reasons (pt preference, financial/insurance/caregiver issues) in 29%, disease relapse in 29%, being deemed unfit due to comorbidities in 22%, active uncontrolled infections in 11%, and other reasons in 11%. An adequate donor was documented in 66% of these pts. Conclusions: Our results support the use of SCT as a consolidative strategy in older AML pts. Pts who undergo SCT have superior OS and RFS compared to those who do not. However, fewer than 20% of pts undergo the procedure mostly due to inadequate disease control, comorbidities, and a variety of social reasons. Recent trends indicate higher rates of SCT referral and completion as more effective anti-leukemic therapies are allowing a greater proportion of older AML pts to undergo SCT. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

PIK3CD, IMMUNODEFICIENCY, MUTATIONS, GAIN-OF-FUNCTION

<h3>Introduction</h3> PIK3CD-GOF is an inborn error of immunity (IEI) with variable phenotypic characteristics even with the same variant. Lymphoproliferation, autoimmunity, and dysgammaglobulinemia may lead to suspicion of the diagnosis. Here we present four unrelated Mexican patients from nonconsanguineous families with a diagnosis of PIK3CD-GOF: <h3>Case Description</h3> (1) A 2-year-old female with a history of very early-onset inflammatory bowel disease (VEO-IBD) at 8 months, late omphalorrhexis, hyper-IgA, hyperleukocytosis, and low B and NK cells. (2) A 12-year-old male with a family history of lymphoma. Sepsis due to Listeria, periodontal abscess, systemic lupus erythematosus, Sjögren's syndrome, bronchiectasis, hepatosplenomegaly, lymphadenopathy, autoimmune hemolytic anemia, chronic EBV/CMV infection; leukocytosis and hyper-IgM. (3) A 2-year-old male with chronic rhinosinusitis, chronic suppurative otitis media, diarrhea; enlarged lymph nodes, splenomegaly, thrombocytopenia, low IgG and IgA and normal IgM with poor antibody response to pneumococcal vaccine. Hyper-IgM syndrome was suspected so he underwent successful stem cell transplantation. (4) An 11-year-old male child whose both parents were HIV+. Presented at 4-months with episodic fever and recurrent respiratory infections, bronchiectasis, mild mental retardation elevated IgM, low IgG, lymphopenia, and decreased lymphocyte proliferation. HIV were suspected in this patient. <h3>Discussion</h3> We identified heterozygous pathogenic variants related to PIK3CD: 3 of them in exon 24 (p.Glu1021Lys); and a novel variant in exon 7 (p.Ile262Val). PIK3CD-GOF is a combined immunodeficiency disorder (CID) with variable phenotype characteristics even with the same variant. Lymphoproliferation, autoimmunity, and dysgammaglobulinemia may raise suspicion. By behaving like a great simulator, genetic diagnosis is necessary to identify the specific pathogenic variant.

Successful pseudo-autologous stem cell transplantation for donor-derived Burkitt lymphoma occurring 9 years after allogeneic transplantation.

Donor-derived hematological malignancies have been recognized as rare but serious late complications in allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients. Most cases in the literature were diagnosed as myelodysplastic syndrome or acute leukemia, with very few malignant lymphoma reported. We herein present another case of donor-derived Burkitt lymphoma that occurred 9 years after allo-HSCT under continued administration of immunosuppressants for chronic graft-versus-host disease (GVHD). The patient achieved a partial response after rituximab-combined intensive chemotherapy. To reduce the risk of relapse and to avoid organ toxicities due to repeated chemotherapies, we performed upfront high-dose chemotherapy followed by stem cell rescue using donor-derived CD34+ cells, called pseudo-autologous HSCT (pASCT), and adjusted immunosuppressants appropriately. The patient remained disease-free for 23months after pASCT without exacerbation of cGVHD. Although the observation period has been relatively short and longer follow-up is needed, pASCT may be a feasible option for donor-derived lymphoma even in patients with active cGVHD.

DAMP-modulating nanoparticle for successful pancreatic islet and stem cell transplantation

Cell therapy is targeted at many organs, but locally or systemically delivered cells are shortly able to survive resulting from the immune/inflammation reactions and irregular cell targeting. Here we explore the multimodal nanoparticle having anti-inflammation and magnetic guidance for successful cell transplantation. We design magnetic resonance (MR)-active glycyrrhizin-chitosan coated superparamagnetic iron oxide nanoparticle (SPIO@Chitosan-GL) to inhibit release of inflammatory damage-associated molecular pattern (DAMP) protein and to offer noninvasive monitoring after intrahepatic transplantation of pancreatic islets and mesenchymal stem cell (MSC) spheroids. Intracellular delivered SPIO@Chitosan-GL is not cytotoxic to pancreatic islets and MSC spheroids and attenuate DAMP release from them. Also, therapeutic cells labeled with SPIO@Chitosan-GL are magnetically localized to the intended lobe of liver during transplantation procedure. If necessary, partial hepatectomy can be performed to remove the localized therapeutic cells for protection of the remaining liver lobes from systemic inflammation. Therapeutically, the cells selectively localized in the liver can treat blood glucose in diabetic mice to normal levels with DAMP modulation, and are visualized using in vivo MR imaging for over 4 weeks. Collectively, DAMP-modulating SPIO@Chitosan-GL can be used in multimodal nanomedince for attenuating the inflammation reaction by transplanted cells and for noninvasively long-term monitoring of transplanted cells.

Biomaterials to enhance stem cell transplantation.

The successful transplantation of stem cells has the potential to transform regenerative medicine approaches and open promising avenues to repair, replace, and regenerate diseased, damaged, or aged tissues. However, pre-/post-transplantation issues of poor cell survival, retention, cell fate regulation, and insufficient integration with host tissues constitute significant challenges. The success of stem cell transplantation depends upon the coordinated sequence of stem cell renewal, specific lineage differentiation, assembly, and maintenance of long-term function. Advances in biomaterials can improve pre-/post-transplantation outcomes by integrating biophysiochemical cues and emulating tissue microenvironments. This review highlights leading biomaterials-based approaches for enhancing stem cell transplantation.

Successful Second Autologous Stem Cell Transplantation for Late Disease Progression after the First Procedure in POEMS Syndrome

Autologous stem cell transplantation (ASCT) is a good option in fit young patients affected by POEMS syndrome. Few data are published on how to treat these patients in case of relapse. In this paper we described our two patients who underwent a second ASCT. From our experience, we can confirm that a second transplant is a real treatment option with good results and limited side effects. It provided excellent response and long-term disease-free survival in POEMS patients who relapsed after a first transplant, including late relapses.

578 - Are We Crazy? Building a Successful Acute Leukemia and Stem Cell Transplant Program during the COVID-19 Pandemic

578 - Are We Crazy? Building a Successful Acute Leukemia and Stem Cell Transplant Program during the COVID-19 Pandemic

A single centre evaluation of sysmex XN-HPC for the enumeration of CD34+ cell in PBSC

Introduction: CD34+ cell is used to define cell harvest goals. Successful peripheral blood stem cell (PBSC) transplantation depends on the infusion of an appropriate number of HPCs to achieve rapid and durable haematologic recovery. In this study, we evaluated the use of the Hematopoietic Progenitor Cell count programme on the Sysmex XN haematology analyser (XN-HPC) as a new parameter for enumeration of CD34+ cell.

Quantification of Tryptophan, Indole, and Indoxyl Sulfate in Urine Using Liquid Chromatography-Tandem Mass Spectrometry.

Having a diverse gut microbiota has been correlated with the short- and long-term success of allogeneic stem cell transplantation. Intestinal bacteria metabolize the amino acid tryptophan to indole. Indole is further oxidized and sulfonated in the liver to 3-indoxyl sulfate (3-IS), which is then excreted in urine. Urinary 3-IS is a potential biomarker for intestinal health and an early predictor of successful stem cell transplantation. We describe a rapid method for quantifying tryptophan, indole, and 3-indoxyl sulfate in urine specimens, in which urine samples are diluted with a formic acid solution and deuterated internal standards, and then injected on LC-MS/MS for analysis.

Detection of Systemic AL Amyloidosis By 124I-p5+14 PET/CT Imaging - Providing the Complete Picture for Diagnosis

Background: Immunoglobulin light chain-associated (AL) amyloidosis is associated with the deposition of fibrils in abdominothoracic organs, notably the heart, liver, spleen and kidneys, resulting in organ dysfunction and significant morbidity. The heterogeneous organ presentation of amyloid and the variable amounts of amyloid burden make accurate and rapid diagnosis challenging. At present, organ biopsy and inferences based on anatomic imaging or changes in serum and urine biomarkers are commonly used to assess organ involvement. Currently, there are no approved radiotracers in the US for the non-invasive detection of AL amyloid load in major organs.To address this, we have developed a synthetic peptide radiotracer, designated 124I-p5+14 (AT-01), suitable for PET/CT imaging. This peptide binds many forms of amyloid through multivalent electrostatic interactions with the amyloid-associated glycosaminoglycans and fibrils. Preclinical studies demonstrated the specific reactivity of the peptide with diverse forms of amyloid (Wall, J.S. et al. (2015) Molecules, 20, 7657). Based on these observations, peptide p5+14 was labeled with iodine-124 and evaluated in a Phase 1/2 PET/CT imaging trial of patients with systemic amyloidosis (NCT 03678259).Herein we describe 23 patients with systemic AL amyloidosis and 5 healthy subjects that have completed the study. Efficacy endpoints include patient- and organ-based sensitivity of 124I-p5+14 uptake in the heart, liver, spleen and kidney.Methods: Subjects, >18 years of age, with a confirmed diagnosis of AL amyloidosis based on organ or abdominal fat pad biopsy findings, with organ-related biomarker changes. Patients taking heparin therapy were excluded. Subjects received an IV infusion of <2 mg of 124I-p5+14 (<2 mCi) and images were acquired at 5-6 h post injection using a Biograph PET/CT with a low dose CT. Prior to imaging, the organ-based distribution of amyloid was determined based on review of the medical record. PET/CT images were evaluated for visual uptake of radiotracer by a reader blinded to patient-related clinical data. Patient and organ-specific sensitivity were determined based on comparison of visual interpretation of the images by a reader blinded to organ involvement and the organ involvement based on the clinical record.Results: Twenty-three patients with systemic amyloidosis and five healthy subjects completed the study. No subjects discontinued or were lost to follow-up. In healthy subjects, radioactivity was observed in the parotid, salivary and thyroid glands, saliva, stomach lumen and urine in the ureters and bladder, consistent with the biodistribution of free radioiodide. No uptake in abdominothoracic organs was observed. In contrast, patients with AL amyloidosis exhibited uptake in the heart (71% of patients), kidneys (61%), spleen (43%), liver (30%), pancreas, lung, bone marrow and other sites.The patient-based sensitivity (patients with visual uptake in at least one anatomic site) was 96% (22/23). The organ-based sensitivity (i.e., clinically positive heart, liver, spleen or kidney that exhibited visual uptake of 124I-p5+14) was 86% (13/14 heart; 3/3 liver; 1/1 spleen; 7/10 kidney). Of note, the one patient without cardiac uptake was 8 years post diagnosis and 7 years post successful stem cell transplant therapy. The percent of abdominothoracic organs detected by PET/CT imaging was 68% higher (47 vs 28) than was noted clinically (16/14 heart; 7/3 liver; 10/1 spleen; 14/10 kidney)Conclusion: PET/CT imaging of amyloidosis using 124I-p5+14 provides accurate detection of AL amyloid deposits in multiple organ systems. Clinically undetected amyloid was observed in numerous anatomic sites with amyloid visualized by 124I-p5+14 in 68% more abdominothoracic organs as compared to that documented in the clinical record. Non-invasive PET/CT imaging with 124I-p5+14 can improve detection of amyloid throughout the body providing a more comprehensive picture of the disease and may provide a method to monitor changes in disease progression.Acknowledgments: This study was supported in part by the National Heart Lung and Blood Institute, National Institutes of Health, through the Science Moving Towards Research Translation and Therapy (SMARTT) program and by contributions to the ACTP Gift Fund at the UTGSM. DisclosuresWall: Attralus Inc: Current holder of stock options in a privately-held company, Patents & Royalties: Inventor and patent holder, Research Funding. Martin: Attrlaus Inc: Current holder of stock options in a privately-held company. Stuckey: Attralus Inc: Current holder of stock options in a privately-held company. Guthrie: Attralus Inc: Current Employment, Current holder of stock options in a privately-held company. Kennel: Attralus Inc: Current holder of stock options in a privately-held company, Patents & Royalties: Inventor and patent holder.

Modeling Relapsed, Refractory Acute Lymphoblastic Leukemia from a Child with Neurofibromatosis

Neurofibromatosis type 1 (NF-1) is an autosomal dominant disorder affecting approximately 1:3000 individuals globally. While approximately 50% are familial, with over 3000 causative germline variants in the neurofibromatosis (NF1) gene identified, the remainder occur sporadically. These mutations lead to haploinsufficiency of NF1 and neurofibromin, a tumor suppressor and important negative regulator of RAS signaling. Children with NF-1 have a higher risk of developing juvenile myelomonocytic leukemia and acute myeloid leukemia, but rarely develop acute lymphoblastic leukemia (ALL).A 9-year-old male presented in 2015 with persistent migratory subcutaneous swellings and multiple bony aches with lytic lesions on bone imaging. He had a high white cell count with eosinophilia (WCC 43.4 x 10 9/L, eosinophils 23.87 x 10 9/L) with no circulating blasts, 10% marrow blasts (CD10+/CD19+/CD34+) and was CNS negative. Although previously undiagnosed, NF-1 was clinically suspected due to typical skin changes.He was diagnosed with iAMP21 ALL and NF-1 was confirmed with the identification of a germline NF1 donor splice site mutation (c.1845G>A:p.L615=). Bone marrow cells were sorted by flow cytometry on CD19 positivity and underwent transcriptomic sequencing. This revealed a P2RY8-CRLF2 gene fusion, with no other clinically relevant variants, while a custom Taqman low density array indicated high-risk B-ALL subtype Ph-like ALL. Multiplex ligation-dependent probe amplification (MLPA) confirmed iAMP21 and also identified IKZF1 exon 2-3 and BTG1 deletions.Treatment followed the high-risk B-ALL arm of the AEIOP-BFM ALL2009 protocol due to persistent end-consolidation MRD in addition to iAMP21 and the Ph-like phenotype. He relapsed three years later off treatment and was refractory to both salvage chemotherapy and blinatumomab. The iAMP21, P2RY8-CRLF2 gene fusion, IKZF1 exon 2-3 and BTG1 deletions remained detectable. Whole exome sequencing of CD19 positive samples from diagnosis, relapse and mesenchymal stem cells (germline control) was performed, identifying a NF1 c.7400dupT:p.L2467 frameshift (fs) mutation only at relapse.To understand the implications of NF1 p.L2467fs, the P2RY8-CRLF2 gene fusion was first transduced into the interleukin 3 (IL3) dependent murine pro-B cell line Ba/F3. P2RY8-CRLF2 alone is not transforming and is thought to be a secondary event in iAMP21 ALL, providing an ideal model to study the cumulative effect of the NF1fs. The NF1fs was then introduced to the P2RY8-CRLF2 cells by CRISPR/Cas9. A proliferation assay was performed without IL3 and demonstrated the P2RY8-CRLF2+NF1fs cell line was IL3 independent, indicative of leukemic transformation, whereas all other lines were not (vs Ba/F3, p = 0.001).Neurofibromin can be constitutively phosphorylated at the c-terminus, negatively regulating NF1-GAP activity, suppressing RAS signaling and inducing cell cycle arrest. Therefore, to demonstrate loss of function due to the c-terminus NF1 p.L2467fs and increased RAS signaling, western blotting for pERK was performed. Significant upregulation of pERK was confirmed in P2RY8-CRLF2+NF1fs in comparison to Ba/F3 control cells (p=0.007) (Figure 1).The MEK inhibitors trametinib and mirdametinib are in clinical trials for NF-1 patients and have shown efficacy in ALL models with RAS mutations. In a 3-day cell death assay, only P2RY8-CRLF2+NF1fs demonstrated sensitivity to trametinib (LD 50 P2RY8-CRLF2 = >6.4 µM, NF1fs = >6.4 µM, P2RY8-CRLF2+NF1fs =1.7µM; p < 0.001) and mirdametinib (LD 50 P2RY8-CRLF2 = >16 µM, NF1fs = >16 µM, P2RY8-CRLF2+NF1fs = 8.3 µM; p < 0.0001) (Figure 2).Here, we have demonstrated a LOF NF1fs mutation using an in-vitro model of ALL. Germline NF1 haploinsufficiency and a second hit NF1 mutation in ALL is limited to one report of monozygotic twins with neurofibromatosis. We propose that NF1 p.L2467fs caused bi-allelic LOF and therefore contributed to relapse in this patient. An understanding of the genomic complexities that lead to relapse may also inform personalized treatment strategies. While this patient subsequently achieved remission with inotuzomab and underwent successful stem cell transplantation, the sensitivity to MEK inhibitors is an exciting development for neurofibromatosis patients with ALL. [Display omitted] DisclosuresYeung: Amgen: Honoraria; BMS: Honoraria, Research Funding; Pfizer: Honoraria; Novartis: Honoraria, Research Funding. White: BMS: Honoraria, Research Funding; Novartis: Research Funding.

Non-Myeloablative Transplant in Severe Sickle Cell Disease Is Safe & Effective. Single Center Experience from Saudi Arabia

Background:Sickle cell disease (SCD) is a severe monogenic disorder in the world with >300,000 newly born afflicted children per year. Prevalence of this disorder is high in sub-Saharan Africa, Mediterranean basin, Middle East and India. Patients with severe disease phenotype manifested with stroke or other complications may benefit from allogeneic hematopoietic stem cell transplant (SCT). Recent reports in pediatric patients using myeloablative regimen showed excellent outcomes but similar progress in adults has been hampered by higher rates of transplant related mortality (TRM) and infertility.Methods:We conducted a study of adult patients at our institution with SCD whom underwent a matched related SCT during the period of 2014-2017. Medical records of eligible patients were retrospectively reviewed and extracted. All patients received hydroxyurea (HU) at maximally tolerated doses with hyper-transfusion 2-3 months prior to SCT. Iron chelation was given for patients with ferritin > 1000 ug/L. The preperative regimen was non-myeloablative (NMA) consisting of alemtuzumab 1 mg/kg divided over 5 days (day -7 to -3) followed by Total Body Irradiation (TBI) 300 cGy on day -1. Stem cells were collected from peripheral blood via apheresis after 5 days of GCSF with target CD34 cells of 10×106/kg. Graft vs. host disease (GVHD) prophylaxis consisted of sirolimus starting from day -2 and continued for one year post SCT with dose modification based on chimerism. Event free survival (EFS) was defined from time to SCT to graft failure, development of GVHD or death from any cause. EFS and overall survival (OS) via time to end point analysis was computed using the Kaplan-Meir test.Results:A total of 18 adults patients were eligible and further analysed. Median age was 23.5 (14 - 39) years with 14 males and 4 females. All patients were on HU prior to SCT. Median ECOG performance status was 1 (0-1). Indications for transplant were recurrent vaso-occlusive crisis (VOC) refractory to standard therapy in 61%, stroke or other central nervous system (CNS) complication in 38%, recurrent avascular necrosis of joints in 38%, recurrent acute chest syndrome in 22% and severe disease requiring recurrent intensive care unit admission (ICU) in 17%. All donors were related siblings fully matched at 10/10 human leucocyte antigen (HLA) loci with median age of 23.6 years. A total of 11 (61%) of donors were sickle cell carriers with average Hgb S 33%. Stem cell mobilization was with 5 ug/kg GCSF in all donors and the average stem cell collected 11.6 x 106/kg. No significant complications resulted from GCSF use in donors with sickle cell trait.17 patients were conditioned with alemtuzumab / TBI regimen and GVHD prophylaxis with sirolmus. One patient who failed prior transplant received fludarabine, busulfan and thymoglobulin with GVHD prophylaxis as cyclosporine and methotrexate. Median duration of neutrophil engraftment was 20 days and platelet engraftment 2.6 days with half of patient's platelet nadir > 20. Average hemoglobin (Hb) pre- and post-SCT was 83 g/L and 142 g/L respectively. Average hemoglobin (Hb) S pre- and post-SCT was 77% and 13% respectively. Peri-SCT adverse events were 9 (50%) patients experienced neutropenic fever, 12 (67%) mucositis, 6 (33%) diarrhea. None of the patients developed acute or chronic GVHD. No SCD crises developed post SCT. One patient developed aplastic anemia while retaining 71% donor cell chimerism and underwent a second successful SCT from the same donor with fludarabine, cyclophosphamide and thymoglobulin. All patients have stable mixed chimerism resulting in full donor myeloid engraftment and normalization of blood count. With a median follow up of 9.1 months (2.3-32), all patients are alive and free of sickle cell crises. At last follow up, OS and EFS were 100% and 88.9%, respectively as shown in Figure 1.Conclusion:Herein, we report that SCT in adults with severe SCD using NMA regimen is safe, feasible and results in amelioration of disease severity. These excellent results are encouraging given the high prevalence and debilitating nature of SCD in our region. Follow up is on-going to assess the feasibility of sirolimus tapering and sustainability of donor chimerism status over time. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.