Stable Engraftment Research Articles

Transplantation of CD15-enriched murine neural stem cells increases total engraftment and shifts differentiation toward the oligodendrocyte lineage.

Neural stem cell (NSC) transplantation is a promising therapeutic approach for neurological diseases. However, only a limited number of cells can be transplanted into the brain, resulting in relatively low levels of engraftment. This study investigated the potential of using a cell surface marker to enrich a primary NSC population to increase stable engraftment in the recipient brain. NSCs were enriched from the neonatal mouse forebrain using anti-CD15 (Lewis X antigen, or SSEA-1) in a "gentle" fluorescence-activated cell sorting protocol, which yielded >98% CD15-positive cells. The CD15-positive cells differentiated into neurons, astrocytes, and oligodendrocytes in vitro, after withdrawal of growth factors, demonstrating multipotentiality. CD15-positive cells were expanded in vitro and injected bilaterally into the ventricles of neonatal mice. Cells from enriched and unenriched donor populations were found throughout the neuraxis, in both neurogenic and non-neurogenic regions. Total engraftment was similar at 7 days postinjection, but by 28 days postinjection, after brain organogenesis was complete, the survival of donor cells was significantly increased in CD15-enriched grafts over the unenriched cell grafts. The engrafted cells were heterogeneous in morphology and differentiated into all three neural lineages. Furthermore, in the CD15-enriched grafts, there was a significant shift toward differentiation into oligodendrocytes. This strategy may allow better delivery of therapeutic cells to the developing central nervous system and may be particularly useful for treating diseases involving white matter lesions.

Stable renal engraftment in a patient following successful tandem autologous/reduced-intensity conditioning allogeneic transplantation for treatment of multiple myeloma with del(17p) that developed as a post-transplantation lymphoproliferative disease following renal transplantation

Multiple myeloma (MM) developing after renal transplantation is rare. From January 1972 to December 2011, a total of 1,485 patients underwent renal transplantation in Nagoya Daini Red Cross Hospital; 14 (0.9%) of these recipients developed post-transplantation lymphoproliferative disorders (PTLDs) including two plasma cell neoplasms. Here, we report the clinical course of a 35-year-old male with immunoglobulin G k-type MM of recipient origin that developed 5 years after renal transplantation from a human leukocyte antigen (HLA)-haploidentical female sibling donor, which was performed to address dialysis-dependent chronic glomerulonephritis. Cytogenetic analysis revealed significant del(17p) abnormalities in myeloma cells. After non-response to bortezomib treatment, the patient achieved partial response with a thalidomide-containing salvage regimen and underwent successful tandem autologous/reduced-intensity conditioning allogeneic hematopoietic stem cell transplantation (HSCT) from an unrelated male donor matched for seven of eight HLAs. At the 8-month follow-up time point, the patient's performance status remained good, and the transplanted kidney remains functional without rejection. To the best of our knowledge, this is the first report of a successful use of allogeneic HSCT for a patient who developed MM as a PTLD after renal transplantation. This patient has a transplanted kidney and transplanted hematopoietic cells that currently coexist without rejection.

Indications and outcomes of reduced-toxicity hematopoietic stem cell transplantation in adult patients with hematological malignancies

Hematopoietic stem cell transplantation (HCT) utilizing non-myeloablative (NMA) and reduced-intensity conditioning (RIC) regimens (collectively referred to as reduced-toxicity HCT, RT-HCT) has become a viable therapeutic option for patients with hematological malignancies who are ineligible for standard myeloablative conditioning transplantation (MA-HCT). RT-HCT has been shown to induce stable engraftment with low toxicity, and to produce similar overall and progression-free survival (PFS) when compared to MA-HCT in acute myeloid leukemia and myelodysplastic syndrome. The best results for RT-HCT have been reported for patients with disease that is in remission, indolent and chemosensitive, and with a strong graft-versus-malignancy effect. Chronic graft-versus-host disease seems to correlate with a lower relapse rate and better PFS. RT-HCT is inferior when performed in poor risk or advanced disease, due to high relapse rates. A search for novel strategies that includes the most appropriate conditioning regimens and post-transplant immunomodulation protocols with more intensive anti-malignancy activity but limited toxicity is in progress. This review provides an update on the results of clinical studies of RT-HCT, and discusses possible indications and investigative strategies for improving the clinical outcomes of RT-HCT for the major hematological malignancies.

Perivascular, but not parenchymal, cerebral engraftment of donor cells after non-myeloablative bone marrow transplantation

Myeloablative (MyA) bone marrow transplantation (BMT) results in robust engraftment of BMT-derived cells in the central nervous system (CNS) and is neuroprotective in diverse experimental models of neurodegenerative diseases of the brain and retina. However, MyA irradiation is associated with significant morbidity and mortality and does not represent a viable therapeutic option for the elderly. Non-myeloablative (NMyA) BMT is less toxic, but it is not known if the therapeutic efficacy observed with MyA BMT is preserved. As a first step to address this important gap in knowledge, we evaluated and compared engraftment characteristics of BMT-derived monocytes/microglia using several clinically relevant NMyA pretransplant conditioning regimens in C57BL/6 mice. These included chemotherapy (fludarabine and cyclophosphamide) with or without 2Gy irradiation, and 5.5Gy irradiation alone. Each regimen was followed by transplantation of whole bone marrow from green fluorescent protein-expressing wild type (wt) mice. While stable hematopoietic engraftment occurred, to varying degrees, in all NMyA regimens, only 5.5Gy irradiation resulted in significant engraftment of BMT-derived cells in the brain, where these cells were exclusively localized to perivascular, leptomeningeal, and related anatomic regions. Engraftment in retina under 5.5Gy NMyA conditions was significantly reduced compared to MyA, but robust engraftment was identified in the optic nerve. Advancing the therapeutic applications of BMT to neurodegenerative diseases will require identification of the barrier mechanisms that MyA, but not NMyA, BMT is able to overcome.

Orai1 Function Is Essential for T Cell Homing to Lymph Nodes

In T lymphocytes, Ca(2+) release-activated Ca(2+) (CRAC) channels composed of Orai1 subunits trigger Ag-induced gene expression and cell proliferation through the NFAT pathway. We evaluated the requirement of CRAC channel function for lymphocyte homing using expression of a dominant-negative Orai1-E106A mutant to suppress Ca(2+) signaling. To investigate homing and motility of human lymphocytes in immunocompromised mouse hosts, we transferred human lymphocytes either acutely or after stable engraftment after a second transfer from the same blood donor. Human and mouse lymphocyte homing was assessed, and cells were tracked within lymph nodes (LNs) by two-photon microscopy. Our results demonstrate that human T and B lymphocytes home into and migrate within the LNs of immunocompromised NOD.SCID mice similar to murine lymphocytes. Human T and B cells colocalized in atrophied or reconstituted mouse LNs, where T cells migrated in a random walk at velocities of 9-13 μm/min and B cells at 6 μm/min. Expression of Orai1-E106A inhibited CRAC channel function in human and mouse T cells, and prevented homing from high endothelial venules into murine LNs. Ca(2+) signals induced by CCL21 were also inhibited in T cells expressing Orai1-E106A. With CRAC channels inhibited, the high-affinity form of LFA-1 failed to become active, and T cells failed to migrate across endothelial cells in a transwell model. These results establish a requirement for CRAC channel-mediated Ca(2+) influx for T cell homing to LNs mediated by high-affinity integrin activation and chemokine-induced transendothelial migration.

Role for IL2 adjunctive cotherapy for suppression of a solid tumor with designer T cells: Phase I trial data in prostate cancer.

216 Background: We created chimeric antigen receptors (CAR) against prostate specific membrane antigen (PSMA). When expressed in patient T cells, these “designer T cells” (dTc) kill prostate cancer cells in vitro and in vivo in animal models (Ma et al. Prostate2004:61:12-25). A Phase I trial was performed. Methods: Patient T cells are retrovirally transduced and expanded ex vivoto span doses of 10^9 to 10^10 T cells. Patients undergo prior non-myeloablative (NMA) conditioning to create a “hematologic space” into which dTc are infused for stable engraftment and prolonged in vivo efficacy. Continuous infusion low dose IL2 (LDI: 75 kiu/kg/d) is administered. Results: Infused T cells were documented. Safety was shown and partial responses (PR) obtained in 2/5 subjects (40%), with PSA suppressions of 50-70% over 1-2+ mo and PSA progression delay up to 150d. Against expectation, response correlated inversely with engraftment (p=0.06) but directly with plasma IL2 (p=0.03) that was as much as 20-fold lower in non-responders than responders. Low plasma IL2 correlated with high engraftments (p<0.01), prompting an hypothesis that high engrafted numbers of activated T cells absorb out IL2 to a level too low to sustain dTc activation for tumor killing. Conclusions: Encouraging early results were obtained with anti-PSMA dTc in adoptive immune therapy of advanced prostate cancer. It is proposed that adequate higher plasma IL2 will reveal the expected greater potency of higher dTc doses, thereby potentially inducing PSA reductions of 100%, with durable remissions. A study redesign tests moderate dose IL2 (MDI) (300 kiu/kg/8h bolus) versus LDI in a randomized Pilot at the 10^10 cell dose, which is now open to enrolment. To refer patients, please contact the study referral manager by telephone: 401-456-2507 or email: RDavies@rwmc.org. This trial received partial funding from US Army/DOD. Preclinical work was supported by Prostate Cancer Foundation. Clinical trial information: NCT00664196.

Significantly Higher Rates of Donor Attrition in Minority Ethnic, Female and Older Donors: A Review of 7542 Confirmatory Typing Requests at Anthony Nolan

s / Biol Blood Marrow Transplant 19 (2013) S109eS128 S122 United Kingdom; 7 Royal Marsden NHS Foundation Trust, London, United Kingdom Graft failure is a highly unsatisfactory outcome of allogeneic transplant. Several factors have been identified which may predict for graft failure, however factors influencing the success of a second allogeneic transplant in this setting are not well studied. Methods: The BSBMT analysed 130 patients receiving a second allogeneic transplant for graft failure. Patients were identified from the BSBMT Data Registry and transplant centres were approached to provide follow up data. Results: The median age of patients was 9 (range 4m-69), with 53 (40%) being adults (>18). The disease was malignant (47%) or non-malignant (53%). The majority of patients had conditioning for the second transplant (68%), which included serotherapy (Alemtuzumab/ATG) in almost all cases. This was myeloablative (30%) or reduced intensity (39%) (compared to 56% and 43%, respectively, at first transplant). The donor was a sibling (24%), unrelated donor (58%) or other relative (18%). In 74% the same donor was used. The stem cell source was bone marrow (41%), PBSC (54%), both (2%) and UCB (3%) (compared to 53%, 38%, 1% and 7%, respectively, at first transplant). Stable engraftment occurred in 88% of patients following the second transplant. Factors associated with a significant increase in engraftment failure were older age (p1⁄40.003), male recipients (p1⁄40.025), reduced intensity conditioning (compared to myeloablative or none) (p1⁄40.031), the use of a different donor (p1⁄40.026) and transplant for a malignant diagnosis (p1⁄40.005). Age (HR1⁄411.52, p1⁄40.001) and the use of a different donor (HR1⁄48.17, p1⁄40.003) remained significant in the multivariate analysis. The donor type, stem cell source and the time post first transplant did not impact on engraftment. Overall survival was 60% at 5 years in the entire group. OS was significantly worse in adult patients (37% vs 74%, p<0.0001), those with malignant disease (40% vs 78%, p1⁄40.0001) and those transplanted beyond 3months after the first transplant (47% vs 65%, p1⁄40.02). No other factors tested were significant, and only malignancy remained significantly associated with OS in multivariate analysis (HR1⁄42.53, p1⁄40.018). Conclusion: These encouraging results suggest that successful engraftment and long term survival is possible following a second transplant for graft failure. Although outcomes are superior in children and those with nonmalignant disease, over a third of adult patients and those with malignant diseases can also achieve long term survival.

Haploidentical transplantation in patients with acquired aplastic anemia

Haploidentical SCT (haplo-SCT) has been considered a therapeutic option in patients with acquired severe aplastic anemia (SAA) failing at least one course of immune suppressive therapy with antithymocyte globulin and lacking an HLA-matched related or unrelated donor. The platforms of both ex vivo T-cell-depleted and unmanipulated grafts have been explored in children and adults. Overall, the primary objective of a stable haploidentical hematopoietic engraftment with a low rate of GVHD is unmet in a significant proportion of patients undergoing haplo-SCT for SAA. Haploidentical transplants for refractory SAA should be performed in a specialist center with major experience in hematopoietic SCT procedures and preferably performed within the framework of a local clinical protocol designed specifically to address the prevention of graft rejection and GVHD.

High-throughput DNA sequence analysis reveals stable engraftment of gut microbiota following transplantation of previously frozen fecal bacteria

Fecal microbiota transplantation (FMT) is becoming a more widely used technology for treatment of recurrent Clostridum difficile infection (CDI). While previous treatments used fresh fecal slurries as a source of microbiota for FMT, we recently reported the successful use of standardized, partially purified and frozen fecal microbiota to treat CDI. Here we report that high-throughput 16S rRNA gene sequencing showed stable engraftment of gut microbiota following FMT using frozen fecal bacteria from a healthy donor. Similar bacterial taxa were found in post-transplantation samples obtained from the recipients and donor samples, but the relative abundance varied considerably between patients and time points. Post FMT samples from patients showed an increase in the abundance of Firmicutes and Bacteroidetes, representing 75–80% of the total sequence reads. Proteobacteria and Actinobacteria were less abundant (< 5%) than that found in patients prior to FMT. Post FMT samples from two patients were very similar to donor samples, with the Bacteroidetes phylum represented by a great abundance of members of the families Bacteroidaceae, Rikenellaceae and Porphyromonadaceae, and were largely comprised of Bacteroides, Alistipes and Parabacteroides genera. Members of the phylum Firmicutes were represented by Ruminococcaceae, Lachnospiraceae, Verrucomicrobiaceae and unclassified Clostridiales and members of the Firmicutes. One patient subsequently received antibiotics for an unrelated infection, resulting in an increase in the number of intestinal Proteobacteria, primarily Enterobacteriaceae. Our results demonstrate that frozen fecal microbiota from a healthy donor can be used to effectively treat recurrent CDI resulting in restoration of the structure of gut microbiota and clearing of Clostridum difficile.

Availability of Haematopoietic Niches for Transplanted Stem Cells

Following transplantation, donor haematopoietic stem cells (HSCs) must reach specific parts of haematopoietic stroma tissue known as stem cell niches to become engrafted and to start blood cell production. Regularly, they have to compete with the host’s HSCs for a limited number of niches. The exact mechanisms of HSC engraftment as well as of niche “opening” to incoming HSCs by conditioning treatments are not well-known yet. Significant and stable engraftment of syngeneic donor HSCs can be achieved in untreated mice only after transplantation of very large numbers of marrow cells. Engraftment can be largely facilitated by the stem cell mutations reducing numbers of the host HSCs. Pre-transplantation manipulations of the host haematopoietic tissue enhance engraftment depending on how much they damage HSCs. Ionizing radiation appears to be the most effective in this respect despite proliferative quiescence of a majority of HSCs. The review summarizes major achievements in deciphering biological principles of the HSCs and their engraftment after transplantation obtained in experimental research studying murine haematopoiesis.

Selective Development of Myogenic Mesenchymal Cells from Human Embryonic and Induced Pluripotent Stem Cells

Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are promising sources for the cell therapy of muscle diseases and can serve as powerful experimental tools for skeletal muscle research, provided an effective method to induce skeletal muscle cells is established. However, the current methods for myogenic differentiation from human ES cells are still inefficient for clinical use, while myogenic differentiation from human iPS cells remains to be accomplished. Here, we aimed to establish a practical differentiation method to induce skeletal myogenesis from both human ES and iPS cells. To accomplish this goal, we developed a novel stepwise culture method for the selective expansion of mesenchymal cells from cell aggregations called embryoid bodies. These mesenchymal cells, which were obtained by dissociation and re-cultivation of embryoid bodies, uniformly expressed CD56 and the mesenchymal markers CD73, CD105, CD166, and CD29, and finally differentiated into mature myotubes in vitro. Furthermore, these myogenic mesenchymal cells exhibited stable long-term engraftment in injured muscles of immunodeficient mice in vivo and were reactivated upon subsequent muscle damage, increasing in number to reconstruct damaged muscles. Our simple differentiation system facilitates further utilization of ES and iPS cells in both developmental and pathological muscle research and in serving as a practical donor source for cell therapy of muscle diseases.

Histological Assessment of Cutaneous Acute Graft-Versus-Host Disease in a Preclinical Swine Model of Hematopoietic Cell Transplantation and Vascularized Skin Flap Tolerance

Abstract 1894Graft-versus-host disease (GVHD) remains a frequent complication of hematopoietic cell transplantation with skin being a principal target organ. Murine models have provided some insight into the mechanisms of this complex disease process. However, mouse skin differs from human skin, and results of studies in rodents may not translate well to the clinic. Miniature swine is a well-recognized animal model for preclinical studies of skin including dermal toxicology, transdermal drug delivery and wound healing. Unlike skin of rodents, dogs or non-human primates, porcine skin is similar to human skin in terms of structure of epidermal rete ridges (Figure 1A), hair follicle structure and density, and presence of sweat glands and subcutaneous fat.We have developed a miniature swine model of haploidentical hematopoietic cell transplantation that results in stable stem cell engraftment, hematopoietic chimerism and donor skin flap tolerance without GVHD. Swine leukocyte antigen (SLA)ad animals receive 100cGy total body irradiation and T cell depletion using porcine CD3 immunotoxin prior to cytokine mobilized peripheral blood hematopoietic cell transplantation from a haploidentical SLAac donor. Daily cyclosporine is administered at doses adjusted to maintain serum levels between 400–800 ng/ml starting one day prior to transplantation and continued for 30 days with taper until day 45.Mixed hematopoietic cell chimerism persists in these animals and donor vascularized skin flaps are accepted without the need for maintenance immunosuppression regardless of whether they are placed at the time of hematopoietic cell transplantation or greater than 100 days later. Despite the initial resistance to GVHD in this model, acute cutaneous GVHD could be induced through infusion of a high dose of donor leukocytes (DLI) including 15 million donor T cells/kg recipient body weight administered 463 days post hematopoietic cell transplantation and 314 days post vascularized donor skin flap transplantation. Conversion to full donor hematopoiesis was observed between 3–4 weeks post DLI. At this time a mild skin rash was evident in both donor and host skin which appeared to progress specifically within host skin. The presence of both donor and host skin on a tolerant animal whose peripheral blood consists of a mixture of both donor and host derived hematopoietic cells provides a novel model to evaluate specific targets of acute GVHD.In this study we assess histologically the manifestations of acute GVHD in both donor and host skin and compare it to typical histological manifestations of acute GVHD observed in patients. Specifically, recipient skin developed superficial lymphoid infiltrates that progressed to confluence along the dermal-epidermal junction by day 21. These infiltrates were associated with intraepidermal migration, prominent basal cell layer keratinocyte apoptosis, and satellitosis virtually indistinguishable from grade 3–4 human cutaneous acute GVHD (Figure 1B). Superficial portions of hair follicles were involved, as also seen in human disease. Of interest, mild disease was detected in the donor skin grafts by day 62 post-DLI, where selective lymphoid infiltration in the tips of the epidermal rete ridges and in association with target cell apoptosis/satellitosis was documented, a finding remarkably similar to early targeting events in humans.These findings emphasize the value of the porcine model for translational studies of cutaneous GVHD. Preliminary data suggest differences in cellular targeting between recipient and donor skin whereby the latter may be triggered by the presence of chimeric recipient-derived hematopoietic elements that have populated donor grafts. Further studies are now possible to explore the effector-target cell interactions in this clinically-relevant model of human cutaneous GVHD. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Restoration of Function with Human Islets in Humanized Diabetic Mice with Subsequent Rejection of Islet Allografts by Allogeneic Engrafted Human T Cells

Background: Islet transplantation is a curative therapy for type 1 diabetes. However, its success is limited by graft loss which, in part, appears to be cellular mediated rejection. In this study, we used a humanized mouse model for in vivo studies of human islet transplantation. Our aim was to assess the capacity of engrafted human cells to reject allogeneic human islets. Methods: NOD/LtSz-scidL2Rgnull mice with streptozotocin-induced diabetes were transplanted with 2000 human islets. BSL< 10mmol/L demonstrated successful islet function. Transplanted and control mice were reconstituted with 5-10 × 106 human peripheral blood mononuclear cells (hPBMC)/human spleen mononuclear cells (hSPMC). In vivo reconstitution was assessed using flow cytometry weekly. Results: Both hPBMC and hSPMC engrafted in NOD/LtSz-scidL2Rgnull mice (n=6 by hPBMC and n=10 by hSPMC) by week 3 as assessed by presence of hCD45+ cells. Although both dose and time after reconstitution were important. In mice reconstituted with hPBMC, almost all cells were hCD3+/hCD45+ T cells (at week 5-6) (99.2%±0.3 double positive in peripheral blood (PB) and 99.1%±0.8 double positive in spleen). In mice reconstituted with hSPMC CD19+ B cells (8.9%±5.1 in mouse spleen, n=3) and CD56+ NK cells (6.8%±7.2 in PB, and 3.7%±2.5 in spleen) were detected by week 5-6 although the majority of engrafted cells were CD3+ T cells (93.7%±9.2 in PB,90.6%±4.5 in spleen). CD3+ T cells included CD4+ and CD8+ subsets as well as NKT and gamma delta T cells. However, the proportion of Tregs (hCD4+Foxp3+) present in reconstituted mice was reduced in both the PB (2.3%±2) and spleen (4.5%±1.5) when compared to the proportion present in hPBMC (8.7%). Moreover, the proportion of CD4+Foxp3+ T cells decreased further by week 9 after reconstitution when GVHD had occurred (0.4%±0.4)(p< 0.05). Functional islet allografts were rejected with a median survival time of 25 days in both the hPBMC mice and hSPMC mice in the absence of GVHD at the time of rejection. Both CD4+ and CD8+ T cell were found in rejecting islet grafts. Conclusions: Stable and high-level hematopoetic cell engraftment (CD3) was achieved with hPBMC and hSPMC in NOD/LtSz-scidIL2Rγnull. CD3 T cells are capable of rejecting human islet allografts in this humanised mouse model.

Thymidine Kinase Suicide Gene-mediated Ganciclovir Ablation of Autologous Gene-modified Rhesus Hematopoiesis

Despite the genotoxic complications encountered in clinical gene therapy trials for primary immunodeficiency diseases targeting hematopoietic cells with integrating vectors; this strategy holds promise for the cure of several monogenic blood, metabolic and neurodegenerative diseases. In this study, we asked whether the inclusion of a suicide gene in a standard retrovirus vector would allow elimination of vector-containing stem and progenitor cells and their progeny in vivo following transplantation, using our rhesus macaque transplantation model. Following stable engraftment with autologous CD34(+) cells transduced with a retrovirus vector encoding a highly sensitive modified Herpes simplex virus thymidine kinase SR39, the administration of the antiviral prodrug ganciclovir (GCV) was effective in completely eliminating vector-containing cells in all hematopoietic lineages in vivo. The sustained absence of vector-containing cells over time, without additional GCV administration, suggests that the ablation of TkSR39 GCV-sensitive cells occurred in the most primitive hematopoietic long-term repopulating stem or progenitor cell compartment. These results are a proof-of-concept that the inclusion of a suicide gene in integrating vectors, in addition to a therapeutic gene, can provide a mechanism for later elimination of vector-containing cells, thereby increasing the safety of gene transfer.

Controlled Angiogenesis in the Heart by Cell-Based Expression of Specific Vascular Endothelial Growth Factor Levels

Vascular endothelial growth factor (VEGF) can induce normal angiogenesis or the growth of angioma-like vascular tumors depending on the amount secreted by each producing cell because it remains localized in the microenvironment. In order to control the distribution of VEGF expression levels in vivo, we recently developed a high-throughput fluorescence-activated cell sorting (FACS)-based technique to rapidly purify transduced progenitors that homogeneously express a specific VEGF dose from a heterogeneous primary population. Here we tested the hypothesis that cell-based delivery of a controlled VEGF level could induce normal angiogenesis in the heart, while preventing the development of angiomas. Freshly isolated human adipose tissue-derived stem cells (ASC) were transduced with retroviral vectors expressing either rat VEGF linked to a FACS-quantifiable cell-surface marker (a truncated form of CD8) or CD8 alone as control (CTR). VEGF-expressing cells were FACS-purified to generate populations producing either a specific VEGF level (SPEC) or uncontrolled heterogeneous levels (ALL). Fifteen nude rats underwent intramyocardial injection of 10(7) cells. Histology was performed after 4 weeks. Both the SPEC and ALL cells produced a similar total amount of VEGF, and both cell types induced a 50%-60% increase in both total and perfused vessel density compared to CTR cells, despite very limited stable engraftment. However, homogeneous VEGF expression by SPEC cells induced only normal and stable angiogenesis. Conversely, heterogeneous expression of a similar total amount by the ALL cells caused the growth of numerous angioma-like structures. These results suggest that controlled VEGF delivery by FACS-purified ASC may be a promising strategy to achieve safe therapeutic angiogenesis in the heart.

FTY720 Markedly Increases Alloengraftment but Does Not Eliminate Host Anti-Donor T Cells that Cause Graft Rejection on Its Withdrawal

The immunomodulator FTY720 (FTY) is beneficial in models of graft-versus-host disease, solid organ transplantation, and autoimmunity and has been approved for use in patients with multiple sclerosis. FTY modifies the homing and migration of many cell types. We report that FTY has profound positive and negative effects on allogeneic bone marrow (BM) engraftment in sublethally irradiated recipients. FTY increased donor hematopoietic progenitors in the BM, resulting in high donor engraftment in the B cell, myeloid cell, and natural killer cell, but not T cell, lineages. Donor T cell progenitors within the thymus of FTY-treated recipients were dramatically reduced, resulting in a lack of donor T cell reconstitution. In addition to preventing the ingress of donor (and host) T cell progenitors, FTY prevented the egress of fully functional host CD4+CD8- and CD4-CD8+ thymocytes that on cessation of FTY administration were able to exit from the thymus and contribute to a rapid and complete rejection of a well-established donor BM graft. When used in combination with anti-CD40L mAbs to block the CD40L:CD40 costimulatory pathway, FTY markedly enhanced anti-CD40L mAb-mediated alloengraftment promotion. In contrast to FTY alone, the combination of anti-CD40L mAb and FTY resulted in a surprisingly stable, multilineage, long-term donor chimerism. These data illustrate FTY's profound migration modulating effects and suggest a use in combinatorial therapy in achieving stable alloengraftment under nonmyeloablative conditions.

A novel protocol for haploidentical hematopoietic SCT without in vitro T-cell depletion in the treatment of severe acquired aplastic anemia

Mismatched related donors of hematopoietic SCT (HSCT) for severe aplastic anemia (SAA) present challenges mainly associated with graft failure and GVHD. The greater the HLA disparity, the poorer the OS. About 19 consecutive SAA/very SAA (VSAA) patients who received HSCT from haploidentical family donors in our center are reported in this study, 18/19 pairs had 2-3 loci mismatched. All 19 cases failed to respond to previous therapy and were heavily transfused before transplantation. The conditioning regimen before HSCT included BU, CY and thymoglobulin. The recipients received CsA, mycophenolate mofetil (MMF) and short-term MTX for GVHD prophylaxis. The source of stem cell grafts was a combination of G-CSF-primed BM and G-CSF-mobilized peripheral blood stem cells. All patients achieved 100% donor myeloid engraftment; the median time for myeloid engraftment was 12 days (ranging from 10-29 days) and for platelets was 18 days (ranging from 8-180 days) with a cumulative platelet engraftment incidence of 84.21 ± 10.53%. The cumulative incidence was 42.1 ± 11.3% for grade II-IV acute GVHD and 56.2 ± 12.4% for chronic GVHD. The OS was 64.6 ± 12.4% with a median 746-day (90-1970) follow-up for surviving patients. These limited retrospective analysis data suggest that HLA-haploidentical HSCT for SAA patients without an HLA-identical sibling donor might be feasible. Further research to increase OS by decreasing GVHD while maintaining stable engraftment will be needed in the future.

In vivo selection of autologous MGMT gene-modified cells following reduced-intensity conditioning with BCNU and temozolomide in the dog model

Chemotherapy with BCNU and temozolomide (TMZ) is commonly used for the treatment of glioblastoma multiforme (GBM) and other cancers. In preparation for a clinical gene therapy study in patients with glioblastoma, we wished to study whether these reagents could be used as a reduced-intensity conditioning regimen for autologous transplantation of gene-modified cells. We used an MGMT(P140K)-expressing lentivirus vector to modify dog CD34+ cells and tested in 4 dogs whether these autologous cells engraft and provide chemoprotection after transplantation. Treatment with O6-benzylguanine (O6BG)/TMZ after transplantation resulted in gene marking levels up to 75%, without significant hematopoietic cytopenia, which is consistent with hematopoietic chemoprotection. Retrovirus integration analysis showed that multiple clones contribute to hematopoiesis. These studies demonstrate the ability to achieve stable engraftment of MGMT(P140K)-modified autologous HSCs after a novel reduced-intensity conditioning protocol using a combination of BCNU and TMZ. Furthermore, we show that MGMT(P140K)-HSC engraftment provides chemoprotection during TMZ dose escalation. Clinically, chemoconditioning with BCNU and TMZ should facilitate engraftment of MGMT(P140K)-modified cells while providing anti-tumor activity for patients with poor prognosis glioblastoma or alkylating agent sensitive tumors, thereby supporting dose-intensified chemotherapy regimens.

Late graft failure in FA – case report and review of the literature

Hematological disorders in patients with FA can only be cured by allogeneic HSCT. Severe infections in primary and early secondary graft failures pose a particular risk. Whereas most graft failures occur within 100 days, those observed after day +100 are infrequent. Here, we present our analysis of a secondary graft failure more than five yr after a first allogeneic HSCT. In this patient, isolated thrombocytopenia over a period of 12 months resulted in a chimerism subset analysis revealing a considerable decrease in the CD34-positive donor cell fraction. After a second fludarabine-based preparative regimen, the patient received PBSC from the same donor. Chimerism returned to full donor in all subsets. This clinical course demonstrates that isolated thrombocytopenia can precede complete graft failure for several months. Our review of the literature on late graft failures in patients with FA after day +100 reveals the absence of fludarabine in the preparative regimen as a potential risk factor. Further clinical research is necessary to identify more suitable approaches for ensuring safe and stable engraftment.

Abstract LB-352: Reversal of resistance to endocrine therapy by mTOR pathway targeting in a panel of patient derived breast cancer xenografts

Abstract Introduction: Resistance to endocrine therapy (ET) is a major complication of luminal breast cancers. The aims of this study were: 1) to evaluate the response to endocrine and targeted therapies in a large series of luminal breast cancer xenografts, 2) to generate hormone-resistant xenograft models and 3) to use these models as a support to study ET resistance reversal by mTOR targeting Methods: More than 400 tumor fragments obtained directly from patients with ER+ve breast cancer have been grafted in Swiss nude mice. After stable engraftment, xenografted tumors have been validated by pathology and immunohistochemistry (IHC) examination (ER, PR, HER2 and Ki67). In vivo response to different endocrine treatments (tamoxifen, fulvestrant, ovariectomy and letrozole) was evaluated. ET-resistant variants were generated from tumors growing under continuous therapeutic pressure. Models with intrinsic or acquired resistance to ET were tested with the mTOR inhibitor RAD001, alone and in combination with ET. Molecular analyses will be performed. Results: 12 luminal breast cancer xenografts have been characterized and consistently showed maintenance of the luminal phenotype The genetic profile measured by aCGH for both the patient and xenograft tumors showed a similar profile, stable through the in vivo mice generations. Different patterns of response to ET were observed, thus exhibiting heterogeneity similar to what is observed in the clinic. Five xenograft models with acquired resistance to ET were generated after several months of continuous endocrine therapy and were maintained as independent hormono-resistant variants. Two of them were derived from tumors resistant to tamoxifen and 3 from tumors resistant to ovariectomy, used as a surrogate to estrogen deprivation. IHC analysis showed that expression of ER and PR receptors was conserved in the variant models, while proliferation status assessed by Ki67 expression was generally higher. For all 5 models, resistance to ET was confirmed in further engraftment and ET experiments. RAD001 treatment resulted in a strong anti-tumor effect in the tamoxifen resistant xenograft models and an additive effect was observed when RAD001 was given in combination with tamoxifen or fulvestrant.The PI3K/mTOR pathway status and gene expression profiles will be analyzed in all experiments. Detailed results will be presented at the meeting. Conclusion: We have established a large panel of primary luminal breast cancer xenografts, recapitulating the biological and clinical features of patient tumors. ET resistant variants have been generated, which serve as a preclinical platform to test reversal of resistance to ET, e.g; by mTOR pathway targeting. Molecular studies at the gene and protein expression levels will give an insight of the mechanisms associated with acquired resistance to endocrine treatments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-352. doi:1538-7445.AM2012-LB-352