Age Impacts Risk of Mixed Chimerism Following Reduced-Intensity Conditioning Hematopoietic Cell Transplantation for Non-Severe Combined Immune Deficiency Inborn Errors of Immunity.

Alemtuzumab, fludarabine, and melphalan containing-reduced intensity conditioning (RIC) is commonly used in patients undergoing allogeneic hematopoietic cell transplantation (HCT) for definitive treatment of high-risk inborn errors of immunity (IEI). Although survival is favorable, there is an increased risk of mixed chimerism leading to secondary graft failure. This study evaluated factors associated with the risk of developing mixed chimerism, particularly the influence of age in patients undergoing allogeneic HCT for non-severe combined immune deficiency (SCID) IEI who received a uniform RIC regimen that included intermediate schedule alemtuzumab, fludarabine, and melphalan. We hypothesized that age would impact the incidence of mixed chimerism. We retrospectively reviewed records of patients who underwent HCT for non-SCID IEI with a uniform RIC regimen that included intermediate schedule alemtuzumab (1 mg/kg divided over days -14 to -10), fludarabine (150 mg/m2 or 5 mg/kg if weight <10 kg divided over days -9 to -4), and melphalan (140 mg/m2 or 4.7 mg/kg if weight <10 kg on day -3) between 2010 and 2020 at our institution. Mixed chimerism was defined as <95% donor chimerism on 2 or more consecutive occasions in whole blood. Ninety-three patients who underwent RIC-HCT for non-SCID IEI using intermediate schedule alemtuzumab, fludarabine, and melphalan were categorized into 3 groups: age <1 year, age 1 to 5 years, and age >5 years. Forty-nine patients (52.7%) developed mixed chimerism, at a median of 34 days post-HCT (range, 10 to 1396 days). Mixed chimerism developed in 88.9% (n = 16/18) of the age <1 year group, in 57.1% (n = 20/35) of the age 1 to 5 years group, and in 35% (n =14/40) of the age >5 years group. Patients age <5 years were significantly more likely to develop mixed chimerism (χ2 (3, N = 93) = 14.8; P = .001). We observed a significantly increased cumulative incidence of developing mixed chimerism associated with age <1 year (P = .0002). Competing risk regression analysis showed a 3-fold higher risk of developing mixed chimerism for age <1 year (subdistribution hazard ratio (HR), 3.05; 95% confidence interval [CI], 1.11 to 8.38; P = .031,) compared to age >5 years and a significantly decreased risk of mixed chimerism in patients who developed acute GVHD prior to any intervention (OR, .24; 95% CI, .09 to .65; P = .005) There were no significant associations between mixed chimerism and graft source, graft type, CD34+ or CD3+ cell dose, HLA match, or underlying disease (hemophagocytic lymphohistiocytosis [HLH] versus non-HLH). Additionally, the need for secondary intervention was evaluated; 27 patients (29.0%) required 1 or more secondary interventions (donor lymphocyte infusion, CD34 boost, or second HCT). Patients age <1 year with mixed chimerism were significantly more likely than patients age >5 years to require secondary intervention for mixed chimerism (P = .004). Our study demonstrates that age <5 years, especially age <1 year, is associated with an increased risk of developing mixed chimerism in patients undergoing RIC-HCT for non-SCID IEI using intermediate-schedule alemtuzumab, fludarabine, and melphalan. Our data suggest tailoring regimen intensity based on age to reduce the incidence of mixed chimerism. Children age <5 years, particularly those age <1 year, require a higher-intensity regimen. Possible strategies include adding thiotepa or using a busulfan-based reduced toxicity regimen.

A Novel Type of Monocytic Leukemia Stem Cell Revealed by the Clinical Use of Venetoclax-Based Therapy.

The BCL2 inhibitor venetoclax has recently emerged as an important component of acute myeloid leukemia (AML) therapy. Notably, use of this agent has revealed a previously unrecognized form of pathogenesis characterized by monocytic disease progression. We demonstrate that this form of disease arises from a fundamentally different type of leukemia stem cell (LSC), which we designate as monocytic LSC (m-LSC), that is developmentally and clinically distinct from the more well-described primitive LSC (p-LSC). The m-LSC is distinguished by a unique immunophenotype (CD34-, CD4+, CD11b-, CD14-, CD36-), unique transcriptional state, reliance on purine metabolism, and selective sensitivity to cladribine. Critically, in some instances, m-LSC and p-LSC subtypes can co-reside in the same patient with AML and simultaneously contribute to overall tumor biology. Thus, our findings demonstrate that LSC heterogeneity has direct clinical significance and highlight the need to distinguish and target m-LSCs as a means to improve clinical outcomes with venetoclax-based regimens. SIGNIFICANCE: These studies identify and characterize a new type of human acute myeloid LSC that is responsible for monocytic disease progression in patients with AML treated with venetoclax-based regimens. Our studies describe the phenotype, molecular properties, and drug sensitivities of this unique LSC subclass. This article is featured in Selected Articles from This Issue, p. 1949.

Efficacy and Safety of CD34+ Stem Cell Boost for Delayed Hematopoietic Recovery After BCMA Directed CAR T-cell Therapy.

Chimeric antigen receptor (CAR) T-cell therapy has revolutionized treatment outcomes for patients with relapsed refractory multiple myeloma (RRMM). Despite supportive care with growth factors and thrombopoietin (TPO) mimetics, nearly half of the patients experience severe and prolonged cytopenias after CAR T infusion, which have become a major therapeutic challenge for patients with RRMM. Given the successful use of CD34+ autologous hematopoietic stem cells for treatment of non- or delayed engraftment after allogeneic and autologous stem cell transplantations, there is a need to explore the role of previously stored autologous stem cells as a boost for post-CAR T cytopenias in RRMM. We performed a multicenter retrospective analysis of adult patients with RRMM who received previously collected and stored CD34+ stem cell boosts after CAR T-cell therapy between 2 July 2020 and 18 January 2023. Indications for boost were determined per physician discretion and primarily included cytopenias and related complications. Overall, a total of 19 patients received stem cell boost, at a median dose of 2.75 × 106 CD34+ cells/kg (range 1.76-7.38), given at a median of 53 days (range 24-126) after CAR T infusion. Eighteen (95%) patients successfully recovered hematopoiesis after stem cell boost with median time for neutrophil, platelet, and hemoglobin engraftment of 14 (range 9-39), 17 (range 12-39), and 23 (range 6-34) days after the boost, respectively. Stem cell boosts were well tolerated, with no patients experiencing infusion reactions. Although infections were common and severe before stem cell boost, only 1 patient experienced a new infection after boost. All patients had experienced independence from use of growth factors, TPO agonists, and transfusions at the last follow-up. Autologous stem cell boosts can be effectively and safely used to promote hematopoietic recovery for post-CAR T cytopenias in patients with RRMM. Stem cell boosts can be a very effective rescue measure for post-CAR T cytopenias and related complications, as well as supportive care needs.

ENPP4 and HOXA3 as potential leukaemia stem cell markers in acute myeloid leukaemia.

INTRODUCTION: Acute myeloid leukaemia (AML) is a heterogeneous malignant disease with a high degree of treatment failure using chemotherapy. Leukaemia stem cells (LSCs) are CD34+CD38- early progenitors associated with poor prognosis in AML. A unique LSC phenotype that excludes rare normal haematopoietic stem cells (HSC) is still elusive. This study aimed to determine expression of selected potential LSC markers in normal and leukaemic myeloid cells and correlate prognosis in AML patients. MATERIALS AND METHODS: Flow cytometry and RT-qPCR measured expressions of ALDH, IL3RA/CD123, CLEC12A/CLL-1/CD371, HOXA3 and ENPP4. Normal cord blood (n=3) and blood monocytes (n=5) represented HSC and mature cells, respectively. Myeloid leukaemia cell lines (THP-1, KG-1a, K562 and HL-60) represented progenitor cells at various stages of maturation. AML samples included chemo-resistant (n=8), early relapse (n=2) and late relapse (n=18). RESULTS: Combining protein/gene expressions, CD34+CD38- was a feature of immature cells seen in cord blood, KG-1a, and K562 but not more mature cells (blood monocytes and HL-60). Normal cells expressed CD371 while mature cells (blood monocytes and HL-60) lacked CD123. ENPP4 was not expressed on normal cells while HOXA3 was expressed only on cord blood and THP-1. In AML, CD123, HOXA3, ENPP4 (but not CD371) were significantly increased in the CD34+CD38- fraction of chemo-resistant patients while ALDH was associated with chemo-resistance. CONCLUSION: CD34+CD38- presented an immature phenotype and with ALDH were associated with poor prognosis. CD123, HOXA3 and ENPP4 further enriched the LSC population. ENPP4 has not been reported and has the advantage of not being expressed on HSC and normal monocytes.

Subclones of bone marrow CD34+ cells in acute myeloid leukemia at diagnosis confer responses of patients to induction chemotherapy.

BACKGROUND: Acute myeloid leukemia (AML) is a hematopoietic malignancy with a prognosis that varies with genetic heterogeneity of hematopoietic stem/progenitor cells (HSPCs). Induction chemotherapy with cytarabine and anthracycline has been the standard care for newly diagnosed AML, but about 30% of patients have no response to this regimen. The resistance mechanisms require deeper understanding. METHODS: In our study, using single-cell RNA sequencing, we analyzed the heterogeneity of bone marrow CD34+ cells from newly diagnosed patients with AML who were then divided into sensitive and resistant groups according to their responses to induction chemotherapy with cytarabine and anthracycline. We verified our findings by TCGA database, GEO datasets, and multiparameter flow cytometry. RESULTS: We established a landscape for AML CD34+ cells and identified HSPC types based on the lineage signature genes. Interestingly, we found a cell population with CRIP1high LGALS1high S100Ashigh showing features of granulocyte-monocyte progenitors was associated with poor prognosis of AML. And two cell populations marked by CD34+ CD52+ or CD34+ CD74+ DAP12+ were related to good response to induction therapy, showing characteristics of hematopoietic stem cells. CONCLUSION: Our study indicates the subclones of CD34+ cells confers for outcomes of AML and provides biomarkers to predict the response of patients with AML to induction chemotherapy.

Higher Cryopreserved CD34+ Cell Dose Is Associated with Decreased Hepatic Veno-Occlusive Disease/Sinusoidal Obstruction Syndrome after Single-Unit Cord Blood Transplantation in Adults Given Prophylactic Ursodeoxycholic Acid and Intravenous Heparin.

Hepatic veno-occlusive disease (VOD), also known as sinusoidal obstruction syndrome (SOS), is one of the most serious complications to occur early after allogeneic hematopoietic cell transplantation (HCT). However, detailed data on VOD/SOS after cord blood transplantation (CBT) are not available. The objective of this retrospective study was to evaluate the incidence, risk factors, and clinical impact of VOD/SOS after single-unit unrelated CBT for adult patients at our institution. We retrospectively evaluated the incidence, risk factors, and outcomes of VOD/SOS after 390 single-unit unrelated CBTs performed in 332 adults under a prophylactic strategy of ursodeoxycholic acid (UDCA) and i.v. heparin at our institution between 1998 and 2021. VOD/SOS was observed in 24 of the 390 CBTs. The cumulative incidence of VOD/SOS was 5.9% at 30 days and 6.2% at 100 days after CBT. Multivariate analysis showed that cryopreserved CD34+ cell dose ≥1.0 × 105/kg was significantly associated with a decreased risk of VOD/SOS after CBT (hazard ratio [HR], 0.33; 95% confidence interval [CI], 0.12 to 0.91; P = .032). In multivariate analysis, the development of VOD/SOS was significantly associated with higher overall mortality (HR, 6.19; 95% CI, 3.61 to 10.65; P < .001), treatment failure (HR, 4.79; 95% CI, 2.95 to 7.76; P < .001), and nonrelapse mortality (HR, 12.60; 95% CI, 6.90 to 23.00; P < .001). Our study shows that the incidence of hepatic VOD/SOS was relatively low after unrelated single-unit CBT under a prophylactic strategy of UDCA and i.v. heparin. A higher cryopreserved cord blood CD34+ cell dose was associated with a reduction in VOD/SOS, suggesting that selection of a higher cord blood unit CD34+ cell dose could be efficient in preventing hepatic VOD/SOS in adults undergoing single-unit CBT.

Evaluation of Angiogenesis by Using CD105 and CD34 in Sudanese Breast Cancer Patients.

<b>Background and Objective:</b> Angiogenesis is a mechanism by which new blood vessels are developed in healing and tumour tissues, where it is necessary for regeneration growth, tumour cells survival and metastasis. This study aimed to assess the angiogenesis mechanism among Sudanese females with breast cancer using anti-CD34 and anti-CD105 markers. <b>Materials and Methods:</b> Three hundred female representative Formalin-Fixed Paraffin-Embedded (FFPE) breast tissue blocks were included in this study. Of the 300 representative tissue blocks, 200 were breast cancer patient's tissues (confirmed cases) and 100 were normal breast tissues (controls). Their ages mean±SD, 47.3±12.9 years. <b>Results:</b> The results showed the MVD of CD34 significantly increased in malignant lesions as compared to normal breast tissues. The mean of MVD CD34 and MVD CD105 showed statistical differences among different histologic types of breast cancer. Also, a strong positive correlation was detected between the manual and automated MVD counting methods. Also, the current study revealed no significant differences were observed in mean MVD counting for both markers and menopausal status or the age groups of the study population. <b>Conclusion:</b> The MVD is a good tool for assessing prognostic markers. The CD105 marker has a high specificity to the new evolving tumour vessels and is a useful predictor for angiogenesis and breast cancer metastasis.

Post-Transplant CD34+ Selected Stem Cell "Boost" for Mixed Chimerism after Reduced-Intensity Conditioning Hematopoietic Stem Cell Transplantation in Children and Young Adults with Primary Immune Deficiencies.

Mixed chimerism and eventual graft loss occurs in a proportion of children with primary immune deficiencies receiving alemtuzumab, fludarabine, and melphalan reduced-intensity conditioning (RIC) regimens before allogeneic hematopoietic stem cell transplantation (HSCT). We investigated the usefulness of a CD34+ selected stem cell "boost" without conditioning to treat mixed chimerism in children and young adults who received predominantly an alemtuzumab, fludarabine, and melphalan RIC regimen for primary immune deficiencies and reported the outcomes. Patients with a primary immune deficiency disorder who were either enrolled on a prospective CD34+ boost study for treatment of mixed chimerism from 2011 to 2014 (n = 9) or treated with a CD34+ boost on a clinical basis from 2014 to 2016 (n = 3) were included in this analysis. Response to a CD34+ boost was defined as a rise in donor chimerism by ≥15% with donor chimerism of at least 20%, stabilization was defined as a rise in chimerism by <15% with donor chimerism ≥ 20%, and no response was defined as any decline in donor chimerism or need for a second HSCT after a CD34+ boost. Twelve patients received alemtuzumab, fludarabine, and melphalan. Median age was 4.5 years (range, .9 to 20.6), and median whole blood donor chimerism before the boost was 25% (range, 3% to 61%). Three patients (25%) met criteria for response, 1 patient (8%) was considered to have stabilization, and 8 patients (67%) had no response 12 months after the boost. None of the patients developed any complications from a CD34+ boost, including no acute graft-versus-host disease (GVHD). All patients are alive with a median follow-up of 32 months (range, 8 to 79). We conclude that a CD34+ selected stem cell boost can be considered for treatment of mixed chimerism after alemtuzumab, fludarabine, and melphalan RIC HSCT in children and young adults with primary immune deficiencies. Approximately one-third of patients can be expected to benefit from a CD34+ selected stem cell boost and may avoid the need for a second HSCT. Lack of any GVHD or toxicity makes a stem cell boost an attractive option compared with donor lymphocyte infusions for treatment of mixed chimerism.

The use of growth factors, CD34(+) cells and fibrin for the management of chronic venous ulcers.

Chronic venous ulceration represents a very common event. Current standard treatment includes local wound care with the application of compression. We report the effects of platelet-rich plasma in patients with chronic venous ulcers, which is able to stimulate fibroblasts, macrophages and mesenchymal cells and growth factors in order to achieve re-epithelialisation and neovascularisation within the microenviroment of the wound. We also documented the efficacy of this method as the sole treatment without surgical procedures.

Sonic hedgehog-modified human CD34+ cells preserve cardiac function after acute myocardial infarction.

RATIONALE: Ischemic cardiovascular disease represents one of the largest epidemics currently facing the aging population. Current literature has illustrated the efficacy of autologous, stem cell therapies as novel strategies for treating these disorders. The CD34+ hematopoetic stem cell has shown significant promise in addressing myocardial ischemia by promoting angiogenesis that helps preserve the functionality of ischemic myocardium. Unfortunately, both viability and angiogenic quality of autologous CD34+ cells decline with advanced age and diminished cardiovascular health. OBJECTIVE: To offset age- and health-related angiogenic declines in CD34+ cells, we explored whether the therapeutic efficacy of human CD34+ cells could be enhanced by augmenting their secretion of the known angiogenic factor, sonic hedgehog (Shh). METHODS AND RESULTS: When injected into the border zone of mice after acute myocardial infarction, Shh-modified CD34+ cells (CD34(Shh)) protected against ventricular dilation and cardiac functional declines associated with acute myocardial infarction. Treatment with CD34(Shh) also reduced infarct size and increased border zone capillary density compared with unmodified CD34 cells or cells transfected with the empty vector. CD34(Shh) primarily store and secrete Shh protein in exosomes and this storage process appears to be cell-type specific. In vitro analysis of exosomes derived from CD34(Shh) revealed that (1) exosomes transfer Shh protein to other cell types, and (2) exosomal transfer of functional Shh elicits induction of the canonical Shh signaling pathway in recipient cells. CONCLUSIONS: Exosome-mediated delivery of Shh to ischemic myocardium represents a major mechanism explaining the observed preservation of cardiac function in mice treated with CD34(Shh) cells.

Mesenchymal stem cells provide better results than hematopoietic precursors for the treatment of myocardial infarction.

OBJECTIVES: The purpose of this study was to compare the ability of human CD34(+) hematopoietic stem cells and bone marrow mesenchymal stem cells (MSC) to treat myocardial infarction (MI) in a model of permanent left descendent coronary artery (LDA) ligation in nude rats. BACKGROUND: Transplantation of human CD34(+) cells and MSC has been proved to be effective in treating MI, but no comparative studies have been performed to elucidate which treatment prevents left ventricular (LV) remodelling more efficiently. METHODS: Human bone marrow MSC or freshly isolated CD34(+) cells from umbilical cord blood were injected intramyocardially in infarcted nude rats. Cardiac function was analyzed by echocardiography. Ventricular remodelling was evaluated by tissue histology and electron microscopy, and neo-formed vessels were quantified by immunohistochemistry. Chronic local inflammatory infiltrates were evaluated in LV wall by hematoxylin-eosin staining. Apoptosis of infarcted tissue was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. RESULTS: Both cell types induced an improvement in LV cardiac function and increased tissue cell proliferation in myocardial tissue and neoangiogenesis. However, MSC were more effective for the reduction of infarct size and prevention of ventricular remodelling. Scar tissue was 17.48 +/- 1.29% in the CD34 group and 10.36 +/- 1.07% in the MSC group (p < 0.001 in MSC vs. CD34). Moreover, unlike MSC, CD34(+)-treated animals showed local inflammatory infiltrates in LV wall that persisted 4 weeks after transplantation. CONCLUSIONS: Mesenchymal stem cells might be more effective than CD34(+) cells for the healing of the infarct. This study contributes to elucidate the mechanisms by which these cell types operate in the course of MI treatment.

Autologous stem cell transplantation in autoimmune and rheumatic diseases: from the molecular background to clinical applications.

Autoimmune diseases have a multifactorial origin. Because of disturbances of the immune system, autoreactive T and B cells target self-antigens, leading to permanent organ damage. Despite novel therapeutic protocols, the disease course is chronic and in many instances the outcome is lethal. The efficacy of stem cell therapy has been observed in autoimmune animal models and in autoimmune diseases related to haematological abnormalities. Although the therapy is more than 30 years old, its broad spread has been delayed by the serious side-effects due to the conditioning treatments based on oncological protocols. Evaluation of the data of patients who have undergone autologous stem cell therapy reinforced the view that protocols used for conditioning treatments, mostly causing lymphoablation, and procedures carried out in specialist centres significantly reduced mortality, with an almost optimal therapeutical efficacy. New, multicentre investigations have been launched to compare the efficacy of various protocols. In this review, we summarize certain aspects of the molecular background of autologous stem cell transplantation and also depict the response to therapy in various autoimmune and rheumatic diseases.

Autologous stem cell transplantation with selected CD34+ cells and unmanipulated peripheral blood stem cells in patients with relapsed and refractory Hodgkin's lymphoma: a single centre experience.

With the aim to evaluate the long term outcome after high-dose chemotherapy and autologous stem cell transplantation (HDCT+ASCT) in patients with relapsed or refractory Hodgkin's lymphoma (HL) we performed a retrospective analysis of patients transplanted at our centre. Between January 1993 and December 2005, 126 consecutive patients with relapsed or refractory HL in the age of 16 to 65 years underwent HDCT+ASCT at our centre and were enrolled in this retrospective analysis. Patients were autografted with either CD34+ positively selected or unmanipulated periferal blood stem cells (PBSC). With a median follow up of 69 months (3-162 months), the actuarial 5-y PFS and OS for all patients after HDCT+ASCT were 59% and 72%, respectively. In patients transplanted from 1996 the actuarial 5-y PFS and OS for CD34+ selected group were 64% and 79% and for unmanipulated PBSC group 63% and 66%, respectively. A total of 42/126 (33%) patients died. Treatment related mortality (TRM) was 3% (4 patients). In univariate analysis, chemosensitive disease and increased LDH were the strongest prognostic factors for PFS and OS. Our results confirm the efficacy of HDCT+ASCT in relapsed or refractory HL with acceptable toxicity. The use of CD34+ positively selected stem cells for autografting is feasible, safe and effective procedure.

Human umbilical cord blood cells or estrogen may be beneficial in treating heatstroke.

This current review summarized animal models of heatstroke experimentation that promote our current knowledge of therapeutic effects on cerebrovascular dysfunction, coagulopathy, and/or systemic inflammation with human umbilical cord blood cells (HUCBCs) or estrogen in the setting of heatstroke. Accumulating evidences have demonstrated that HUCBCs provide a promising new therapeutic method against neurodegenerative diseases, such as stroke, traumatic brain injury, and spinal cord injury as well as blood disease. More recently, we have also demonstrated that post- or pretreatment by HUCBCs may resuscitate heatstroke rats with by reducing circulatory shock, and cerebral nitric oxide overload and ischemic injury. Moreover, CD34+ cells sorted from HUCBCs may improve survival by attenuating inflammatory, coagulopathy, and multiorgan dysfunction during experimental heatstroke. Many researchers indicated pro- (e.g. tumor necrosis factor-alpha [TNF-alpha]) and anti-inflammatory (e.g. interleukin-10 [IL-10]) cytokines in the peripheral blood stream correlate with severity of circulatory shock, cerebral ischemia and hypoxia, and neuronal damage occurring in heatstroke. It has been shown that intravenous administration of CD34+ cells can secrete therapeutic molecules, such as neurotrophic factors, and attenuate systemic inflammatory reactions by decreasing serum TNF-alpha but increasing IL-10 during heatstroke. Another line of evidence has suggested that estrogen influences the severity of injury associated with cerebrovascular shock. Recently, we also successfully demonstrated estrogen resuscitated heatstroke rats by ameliorating systemic inflammation. Conclusively, HUCBCs or estrogen may be employed as a beneficial therapeutic strategy in prevention and repair of cerebrovascular dysfunction, coagulopathy, and/or systemic inflammation during heatstroke.

Clinical applications of CD34+ cell-selected peripheral blood stem cells.

Peripheral blood stem cells (PBSC) are increasingly used for stem cell transplantation after high dose chemotherapy. CD34+ cell selection has also been done for use in autologous transplantation studies Bone marrow (BM) may contain tumor cells at the time of harvesting, and on re-infusion, these cells could contribute to a subsequent relapse. Similarly, tumor cell contamination of PBSC collections has been found in a number of studies. Therefore, purging contaminating tumor cells may prevent cases of relapse. As most tumor cell types do not express CD34 antigen, one of the most widespread applications of CD34+ cell selection is likely to be in tumor cell purging. Similarly, CD34+ cell selection has aided allogeneic transplantation studies. Acute graft-versus-host disease (aGVHD) is a major cause of morbidity and mortality in cases of allogeneic transplantation. As aGVHD is mediated by donor T cells, removal of T cells from the graft by CD34+ cell selection may ensure prophylaxis against aGVHD. Further, high-dose immunosuppression followed by CD34+ cell-selected stem cell rescue is theoretically reasonable as a therapeutic tool for patients with autoimmune disease resistant to conventional therapy. However, patients given T cell-depleted transplantation have an increased risk of opportunistic infection as well as malignancies related to immunosuppression; therefore, close monitoring is warranted. We describe here clinical applications of CD34+ cell-selected PBSC for a variety of diseases, with special emphasis on the efficacy as well as drawbacks of this novel technique.

Xenotransplantation of immunodeficient mice with mobilized human blood CD34+ cells provides an in vivo model for human megakaryocytopoiesis and platelet production.

The study of megakaryocytopoiesis has been based largely on in vitro assays. We characterize an in vivo model of megakaryocyte and platelet development in which human peripheral blood stem cells (PBSCs) differentiate along megakaryocytic as well as myeloid/lymphoid lineages in sublethally irradiated nonobese diabetic/severe combined immunodeficient (NOD-SCID) mice. Human hematopoiesis preferentially occurs in the bone marrow of the murine recipients, and engraftment is independent of exogenous cytokines. Human colony-forming units-megakaryocyte (CFU-MK) develop predominantly in the bone marrow, and their presence correlates with the overall degree of human cell engraftment. Using a sensitive and specific flow cytometric assay, human platelets are detected in the peripheral blood from weeks 1 to 8 after transplantation. The number of circulating human platelets peaks at week 3 with a mean of 20 x 10(9)/L. These human platelets are functional as assessed by CD62P expression in response to thrombin stimulation in vitro. Exogenous cytokines have a detrimental effect on CFU-MK production after 2 weeks, and animals treated with these cytokines have no circulating platelets 8 weeks after transplantation. Although cytokine stimulation of human PBSCs ex vivo led to a significant increase in CFU-MK, CD34+/41+, and CD41+ cells, these ex vivo expanded cells provided only delayed and transient platelet production in vivo, and no CFU-MK developed in vivo after transplantation. In conclusion, xenogeneic transplantation of human PBSCs into NOD/SCID mice provides an excellent in vivo model to study human megakaryocytopoiesis and platelet production.

Unrelated peripheral blood stem cell transplantation with 'megadoses' of purified CD34+ cells in three children with refractory severe aplastic anemia.

Three children with refractory severe aplastic anemia were transfused with high numbers of unrelated matched (n = 2) or C-locus haploidentical mismatched (n = 1) CD34-selected peripheral blood stem cells in the absence of an HLA-identical family donor. Two leukaphereses of the donors yielded a median number of 10.1 x 10(10) nucleated cells (range 9.7-15.4) with a median number of 9.89 x 10(8) CD34+ cells (range 7.46-26.1) and a median percentage of CD34+cells of 0.98% (range 0.77-1.7). After positive selection by magnetic cell sorting the patients received a median of 14.3 x 10(6) CD34+ cells/kg (range 11.7-24.3) and of 1.3 x 10(4) CD3+ cells/kg (range 0.57-5.8). Median time to ANC >/=0.5 x 10(9)/l was 7 days (range 7-12) and to platelets >/=20 x 10(9)/l 13 days (range 13-27). Chimerism analysis of peripheral blood after transplantation revealed permanent 100% donor hematopoiesis in all patients. The patient with the C-locus haploidentical mismatch presented with acute GVHD (grade III-IV) of the skin, liver and lower gastrointestinal tract (onset day +40) and died despite intensive immunosuppressive treatment on day +238. The two survivors developed lymphopoietic recovery of B and T lymphocytes within 3 months after transplantation. To our knowledge this experience represents the first report of transplantation with unrelated CD34+ enriched peripheral blood stem cell in children with refractory severe aplastic anemia. Bone Marrow Transplantation (2000) 25, 513-517.

Autologous peripheral blood stem cell transplantation as first line treatment of multiple myeloma: an Italian Multicenter Study.

BACKGROUND AND OBJECTIVE: The outcome of patients with multiple myeloma (MM) has not changed markedly since the introduction of melphalan and prednisone. In recent years several studies have investigated the role of intensive therapy followed by infusion of autologous peripheral blood stem cells (PBSC) together with the administration of hematopoietic growth factors. In this study we evaluated the feasibility and efficacy of a PBSC transplantation program for patients with de novo MM in a multicenter setting. DESIGN AND METHODS: In a non-randomized controlled trial 52 patients with de novo MM from 6 Italian centers underwent a three phase treatment strategy including 3 cycles of VAD-like chemotherapy for initial debulking, followed by high-dose cyclophosphamide (HD-CY) and collection of PBSC, that were transplanted after a conditioning regimen with melphalan plus busulfan. Maintenance treatment was a conventional dose of interferon, given until relapse. Actuarial survival and response duration curves were plotted according to Kaplan and Meier's method; the groups were compared using the log rank test. Response rates were compared by the c(2) test; multivariate analysis was performed according to the stepwise regression model. RESULTS: Overall 39/52 (75%) of patients responded, with a complete remission (CR) rate of 31%. After a median follow-up of 55 months, median duration of event-free survival (EFS) and overall survival (OS) are 21 and 57 months, with 24% and 48% probabilities of being event-free and alive after 6 years, respectively. Among the group of 39 responders, CR was significantly associated with prolonged response and survival (2 deaths and 6 relapses/16 patients) as compared with PR (11 deaths and 15 relapses/23 patients), and remained the only significant variable also in a multivariate analysis. Myelosuppression did not protract beyond one week in transplanted patients; extra-hematologic toxicity was very low. INTERPRETATION AND CONCLUSIONS: This multicenter study confirms the feasibility of an aggressive approach to de novo MM patients. Additional confirmation is given of the increased rate of CR, and the significant prolonged survival observed in complete responders. In this experience the association melphalan plus busulfan was shown to be effective, at least as part of conditioning regimens, in the transplant strategy.