Biology of Blood and Marrow Transplantation 13:1380-1392 (2007) 2007 American Society for Blood and Marrow Transplantation 1083-8791/07/1311-0001$32.00/0 doi:10.1016/j.bbmt.2007.08.007 Fifth Annual International Umbilical Cord Blood Transplantation Symposium, Los Angeles, California, May 11-12, 2007 John Wagner,1 Richard Champlin,2 Lawrence D. Petz3 1 University of Minnesota Minneapolis, Minnesota; 2MD Anderson Cancer Center, Houston, Texas; 3StemCyte International Cord Blood Center, Arcadia, California Symposium Summary The 39-member faculty for the Symposium included SESSION I. RISK FACTORS AFFECTING SURVIVAL IN leaders of the major transplantation centers from a round RECIPIENTS OF CORD BLOOD the globe: France, Italy, Japan, Spain, Australia, and the Dr. Vanderson Rocha reviewed the risk factors af- United States. Attendees were from Belgium, Czech fecting survival after umbilical cord blood transplan- Republic, France, Germany, Italy, Greece, The Nether- tation (UCBT) in children and adults with hemato- lands, Poland, Switzerland, United Kingdom, Israel, logical malignancy. He pointed out that factors Cyprus, Kuwait, Saudi Arabia, United Arab Emirates, associated with outcomes after allogeneic hematopoi- India, Australia, Hong Kong, Malaysia, Singapore, Japan etic stem cell transplantation (HSCT) are related to Korea, Taiwan, Canada, United States, Panama, Brazil, the donor, the graft, the patient, the transplant con- and Columbia. ditioning regimen, and prophylactic graft-versus-host The program was divided into 8 sessions: (1) disease (GVHD) therapy. All of these factors affect Risk factors affecting survival in recipients of cord hematopoietic and immune recovery, GVHD and blood, (2) Strategies to improve engraftment after graft-versus-leukemia (GVL), treatment related mor- UCBT, (3) Cord blood banking, manipulation and tality (TRM), relapse, and disease-free survival (DFS). unit selection, (4) Conditioning regimens, (5) Im- Favorable risk factors identiﬁed by the authors of a mune reconstitution and infections, (6) Non-hema- number of signiﬁcant publications include the diagno- topoietic stem cells, (7) The graft versus tumor sis, cell dose, cytomegalovirus (CMV) negative serol- effect, and (8) Governmental affairs. The following ogy, age, the HLA disparity, favorable remission sta- comments emphasize signiﬁcant aspects of selected tus, CD34 cell dose, and absence of acute GVHD presentations. Additional details are provided in the (aGVHD). For example, in a study of 191 children abstracts that follow. with acute myelogenous leukemia (AML) who re- The Fifth Annual International Cord Blood Trans- ceived unrelated cord blood transplants, DFS at 42 plantation Symposium was presented by the California months when transplanted in complete remission Blood Bank Society and the Cord Blood Forum, and was (CR) 1 was 67%, in CR2 it was 49% and for later partially supported by unrestricted educational grants stages of the disease was 24%. A study of 171 adults from StemCyte International Cord Blood Center and the who received an unrelated cord blood transplant iden- National Marrow Donor Program (NMDP). The Sym- tiﬁed favorable risk factors for survival and DFS as posium was also partially ﬁnancially supported by a remission status, ABO, and sex of patient (female contract between the Health Resources and Services Ad- recipients having more favorable outcomes). In an- ministration (HRSA), US Department of Health and other study of 262 adults with hematologic malignan- Human Services, and the California Blood Bank Society. cies who received a UCBT using a myeloablative Any opinions, ﬁndings, conclusions or recommendations conditioning regimen, favorable factors as determined expressed or presented at this conference are those of the in a multivariate analysis for DFS were CMV nega- presenters and do not necessarily reﬂect the views of the tivity, ABO compatibility, status of the disease at US Government. transplant, and the use of total body irradiation (TBI); 1380 Summary of IUCBT Symposium 1381 also, there was a trend for favorable outcome for transplant was the major determinant of DFS. Finally, patients receiving 2 107 nucleated cells/kg body the criteria for selecting the best CB unit for adults weight. In a study of children with acute lymphoblas- need urgent modiﬁcation. tic leukemia (ALL) in CR2 who received a UCBT, an Dr. Marcos de Lima reviewed the impact of host- important factor was whether the ﬁrst relapse was on versus-graft (HVG) HLA mismatch on outcomes of or off chemotherapy. The DFS for those whose re- CBT. The authors of this presentation developed the lapse occurred off therapy (n 65) was 45% and for hypothesis that HLA mismatches in the HVG direc- those whose relapse was while on therapy (n 73) the tion are likely to inﬂuence outcomes of unrelated DFS was 26%. In a recent analysis of 925 patients with CBT, in particular engraftment. They performed a malignant disorders who received a UCBT, multivar- retrospective study of 91 patients with malignant or iate analysis for TRM identiﬁed three favorable prog- non-malignant disorders who received CBT. The nostic factors: HLA 6 of 6 or 5 of 6, early and inter- conditioning regimen was myeloablative in 86 patients mediate phase of the disease, and the number of cells (95%). In pediatric patients the 1 year survival was infused being 2 107/kg. Multivariate analysis for 60% versus 70% (zero versus 1 mismatches, respec- overall survival (OS) also identiﬁed three favorable tively), P 0.3. In adult patients the corresponding prognostic factors: early and intermediate phase of the values were 45% vs. 39%, respectively. Thus, in this disease, the number of cells infused as 2 107/kg, small sample set, there was no statistically signiﬁcant and negative CMV serology. association between number of graft-versus-host Dr. Guillermo Sanz reviewed the outcomes in 92 (GVH) mismatches and survival. There was, however, adults with hematologic malignancies who underwent a higher cumulative incidence of grade II-IV aGVHD a single unit UCBT at a single institution after my- among adult recipients of larger grafts, with a similar eloablative conditioning using thiotepa, busulfan, cy- trend apparent in children. The authors concluded clophosphamide or ﬂudarabine (Flu), and anti-thymo- that evaluation of mismatches at a higher level of cyte globulin (ATG). The cumulative incidence of resolution and in other HLA loci (HLA-C, -DQ, myeloid engraftment, grade III-IV aGVHD, and non- -DP) is warranted to better evaluate these histocom- relapse mortality (NRM) at 100 days was 90%, 18%, patibility effects on engraftment, incidence of GVHD, and 25% respectively. The cumulative incidence of and immune reconstitution. A larger patient popula- relapse and the probability of DFS at 3 years were tion should be tested. 21% and 39% (median follow-up, 26 months). A mul- Dr. Mary Eapen discussed the risks and beneﬁts of tivariate analysis of prognostic factors of short term UCB vs. bone marrow (BM) unrelated donor HSCT outcome endpoints indicated that the absolute num- in children with acute leukemia. She pointed out that ber of CD34 cells infused had a marked effect on selection of CB over BM remains controversial. For neutrophil and platelet engraftment; the type of ATG bone marrow transplants (BMT), studies have shown (lympho vs. thymoglobulin) had a signiﬁcant effect on that there is signiﬁcantly higher mortality when there platelet engraftment and aGVHD; and the develop- is a mismatch at -A, -C or -DR loci. More speciﬁcally, ment of aGVHD had a signiﬁcant effect on platelet the data indicate that there is a 9-10% lower OS with engraftment. A multivariate analysis of prognostic fac- each additional mismatch. That is, with 8 of 8 HLA tors of long term outcome endpoints indicated that matched transplants, OS was 52%; with 7 of 8 HLA age, stage of disease, CD3 cells infused, condition- matched transplants OS was 43% and for 6 of 8 HLA ing regimen (Flu vs. no Flu), and development of matched transplants the OS was 33%. She reviewed a grade II-IV aGVHD all had a signiﬁcant effect on study of 116 BMT patients who were compared with NRM. Patients with advanced disease or who had a 503 CBT. All BM patients had an 8 of 8 allele matched diagnosis other than CML had a higher relapse rate donor whereas only 7% of donor-recipient CB pairs and the only variable that affected DFS was the stage were matched (low resolution at -A and -B loci and of disease at transplant. The authors concluded that high resolution at -DR), 40% had a one antigen mis- UCBT of adults with a single and adequate unit and match and 53% had a 2 antigen mismatch. The results using highly immunosuppressive conditioning can of the study indicated that, relative to matched BM, 1 provide the same engraftment rate and similar speed antigen mismatched CBTs had lower GVHD and of engraftment to that obtained in children. Also, the similar rates of TRM, relapse, and LFS. Matched number of CD34 cells in the UCB unit is the most CBTs had less GVHD and TRM and higher survival relevant factor for engraftment. This factor must be rates. However, small patient numbers prevent deﬁ- used for the UCB unit of choice. The degree of HLA nite conclusions. Two antigen mismatched CBTs had mismatching does not seem as important in adults as lower GVHD, higher rates of early TRM, lower rates in children. Several outcomes after UCBT (speed of of relapse and similar rates of LFS. The authors of the engraftment and risk of aGVHD, TRM, and relapse) study concluded that the data support use of 0-2 an- were modulated by the conditioning regimen. Age had tigen mismatched CB grafts regardless of the avail- a major inﬂuence on NRM. The stage of the disease at ability of a matched BM donor in pediatric patients 1382 Summary of IUCBT Symposium with acute leukemia. Also, that larger repositories of in this analysis (although too few patients were in the CB are needed to meet the increasing demand for 6/6 and 3/6 categories) and G-CSF may be important. larger, partially HLA matched units. A more recent and larger multi-center study Dr. Eliane Gluckman presented the results of a (COBLT) included 316 patients between ages of 0.1 study of unrelated CBTs in non-malignant diseases (n and 17.9 years (median age 4.6 years) who were 268). The diagnosis in 48% of patients was a BM transplanted between 1999 and 2004. The results in- failure syndrome, 30% with severe combined immu- dicated that cell dose, HLA match (5-6/6 versus 4/6) nodeﬁciency disease (SCID), and 22% had a meta- and donor age are signiﬁcant factors. Eurocord regis- bolic disorder. HLA matching was done with low try data indicate that engraftment is affected by CMV resolution for -A and -B and high resolution for serostatus, stage of the disease, and cell dose infused -DRB1: 17% of patients were matched, 43% had a ( 2 107/kg). There is an interaction between cell one antigen mismatch, 36% had 2 antigen mismatches dose and HLA match, i.e., one needs more cells to and 4% had greater than 2 antigen mismatches. For achieve engraftment with increased HLA mismatches. neutrophil recovery, favorable prognostic factors were When donor and recipient are matched (6 of 6) there (1) 1 HLA disparity, (2) cell dose infused 3 is no clear impact of cell dose, but when there are 107/kg, and (3) diagnosis of SCID. For platelet recov- mismatches, the affect of cell dose becomes apparent. ery, favorable prognostic factors were (1) 1 HLA Early results from the NY Blood Center suggest that disparity, (2) diagnosis other than BM failure syn- Flu may have a favorable affect on engraftment. A drome, and (3) cell dose infused 3 107/kg. For multivariate analysis in adults indicated that nucleated grade II-IV aGVHD, only one favorable prognostic cell dose 2.5 107/kg, HLA mismatch, absence of factor was identiﬁed, HLA disparities 3. For sur- Flu, and use of methotrexate (MTX) adversely af- vival, favorable factors were (1) negative CMV serol- fected neutrophil recovery. Different strategies are ogy, (2) diagnosis other than BM failure syndrome, (3) being developed to enhance engraftment including cells dose infused 2 107/kg, and (4) no more than optimization of the peri-transplant therapy, improv- 1 HLA disparity. Further data indicated that the re- ing HSC homing (e.g., CD26 inhibition, PTH, intra- quirements for cell dose and HLA matching are dif- bone marrow injection), co-infusion of haplo TCD ferent in malignant and non-malignant diseases. In peripheral blood stem cells (PBSC) with UCB, use non-malignant disease the optimal cell dose is 5 reduced intensity conditioning (RIC), and increased 107 at collection and 4 107 at infusion. Also, HLA cell dose through improved collection volumes, ex- mismatches play a major role for engraftment, vivo expansion, or infusion of two partially HLA GVHD, and survival, and the effect of HLA mis- matched units. matches is partially abrogated by increasing cell dose. Dr. Mary Laughlin discussed the inﬂuence of HLA disparity and graft lymphocytes on allogeneic engraft- ment and survival after UCB transplants in adults. She reported the results of a single institution prospective SESSION II. STRATEGIES TO IMPROVE phase II study in adults receiving unrelated UCB fol- ENGRAFTMENT AFTER UCBT lowing a myeloablative conditioning regimen. The Dr. John Wagner reviewed factors having an im- study was designed to test the hypothesis that UCB pact on engraftment after UCBT. These include nu- graft lymphocytes may have an impact on rates and cleated cell dose, CD34 dose, HLA match, GVHD, kinetics of donor engraftment. A secondary objective history of prior chemotherapy, graft T cell dose, use was to prospectively determine the relationship be- of growth factors, host microenvironment, use of tween degree and nature of HLA mismatch in UCB post-transplant cytotoxic agents, and development of grafts and GVHD/patient survival. Results of this post-transplant infection. He reviewed transplant data early phase II study indicated that graft nucleated cell from 1994-2000 at the University of Minnesota (n and CD34 hematopoietic progenitor cell dose are 102), which indicated a superiority of CD34 and predictors of allogeneic engraftment and survival in CFU-GM dose over nucleated cell dose in predicting UCB adult recipients. Univariate analyses demon- engraftment. There was a linear relationship between strated that UCB graft infused cell doses of CD34 cell dose and the days to neutrophil recovery. CD34 , CD3 and CD34 HLADR CD38 Also, patients with the lowest cell doses (speciﬁcally progenitors correlated with donor neutrophil en- 1.7 105 CD34 /kg) had the highest risk of graft graftment. Also, allele matching for HLA class II failure. For platelet recovery, CD34 again had a resulted in improved event free survival (EFS) and signiﬁcant effect as did CMV serostatus. Lessons decreased risk for aGVHD. learned from this early study were that CD34 cell Dr. John Wagner reviewed results at the University dose is a major factor. These data led to a change in of Minnesota using double UCBT. As cell dose is a minimum cryopreserved cell dose from 1 to 2.5 107 limiting factor in the successful use of UCBT, two CB nucleated cells/kg. HLA match was not a clear factor units were used to increase cell dose. The units must Summary of IUCBT Symposium 1383 be partially HLA matched with each other and with Dr. Ronald Hoffman discussed the expansion of the patient. No more than 2 antigen mismatches are human UCB SCID-repopulating cells using chroma- allowed with the patient and between the two grafts. tin-modifying agents. The lack of net stem cell expan- The combined cell dose of the two units must be at sion following in vitro exposure to cytokines could least 2.5 107 cells/kg. The preparative therapy in- result from inability of cytokines to promote HSC cludes TBI, Flu and Cy, and the post-transplant im- divisions. The remaining bone marrow repopulating mune suppression is cyclosporine A (CSA) and myco- potential of such expanded cell populations might be phenolate mofetil (MMF); all patients received growth from cytokine unresponsive HSC. Previous ex-vivo factor. In the initial phase I study, 23 of 23 patients attempts to alter HSC fate divisions in the presence of receiving two units had neutrophil engraftment at a a variety of cytokine combinations or feeder layers median of 23 days. Transient “double” chimerism have resulted over time in a loss of cells expressing the occurs and in most, the unit that predominates early stem cell phenotype and progressive loss of the num- on “wins.” Predicting which unit will win is not yet ber of primitive progenitors cells quantiﬁed using a possible as there is no association with infusion order, variety of surrogate stem cell assays. Further, silencing mononuclear cell dose, CD34 dose, CD3 dose, gran- of genes has been shown to be accompanied by DNA ulocyte-macrophage colony forming units (GM- methylation of a gene’s promoter and by histone CFU) dose, HLA, or ABO match. In a multivariate deacetylation in regions controlling the genes of in- analysis, the only factor predictive of neutrophil en- terest. Studies were carried out to investigate whether graftment was the combined CD34 cell dose. Using the addition of two chromatin-modifying agents, double UCBT, engraftment is comparable to that in 5-aza-2=-deoxycytidine (5azaD) and trichostatin A children, 93% of adults are now eligible for UCBT as (TSA), to CB CD34( ) cells in culture results in compared to 30% with a single UCB unit, if the expansion of the numbers of SCID repopulating cells threshold is 2.5 107/kg). (SRC). Results indicated that the program of HSC in A retrospective analysis was done to determine vitro can be epigenetically modiﬁed to change their what factor – use of Flu or coinfusion of UCB most fate by agents affecting DNA hypomethylation and signiﬁcantly affected engraftment. The analysis in- acetylation. Epigenetics appears to play a role in de- cluded 147 patients with hematologic malignancy un- termining the transcriptional options of HSCs and dergoing ﬁrst transplants between 1994-2005. Results changing the behavior of these cells in vitro. Finally, of this study suggested that the combined cell dose is the use of chromatin modifying agents favors symmet- the principal factor responsible for both engraftment ric cell division of CB HSC. Thus, ex-vivo expansion and speed of neutrophil recovery. Although Flu is strategy using chromatin-modifying agents provides a associated with increased incidence of engraftment, it potential avenue by which to expand the number of is not statistically signiﬁcant. In the subgroup of pa- HSC in single CB unit for use as an alternative source tients receiving Flu/Cy/TBI, HLA-matching as well of HSC grafts for adult recipients. as cell dose is responsible for the speed of neutrophil Dr. Tracey O’Brien presented data indicating that recovery. GSK-3 regulates ex-vivo expansion and engraftment Dr. Hal Broxmeyer reported on the effect of CD26 of UCB HSC through activation of Wnt signaling. inhibition on CB engraftment. Inhibition of CD26/ Past experience has indicated in animal models that dipeptidylpeptidase IV (DPPIV) with small peptides, short term reconstituting HPC are expanded at the such as Diprotin A or Val-Pyr enhance the capacity of expense of longer term repopulating cells required for human CB CD34 cells and mouse Sca1 c-kit lin durable engraftment. In clinical trials engraftment has BM cells to respond to the chemotactic activity of been no faster despite infusion of expanded cell num- stromal cell derived factor in vitro, and they enhance bers suggesting loss of function and/or homing of donor mouse BM HSC homing and engraftment of HSC/HPC in the expansion process. There are data lethally irradiated recipient mice in both a competitive indicating that Wnt signaling positively regulates self- and a non-competitive assay. Recent studies indicate renewal of murine HSC. An alternate approach to that inhibition of CD26/DPPIV in human CB achieve Wnt activation is by inhibition of glycogen CD34 cells enhances their engraftment of nonobese synthase kinase-3 inhibition, which prevents degra- diabetic (NOD)/SCID mice. Also, Diprotin A pro- dation of -catenin. GSK-3 inhibition suppresses duces enhancement of CD34 cord blood and G-CSF ex-vivo proliferation of UCB cells induced by cyto- mobilized adult peripheral blood CD34 cells, respec- kines but improves the repopulating capacity of UCB. tively, by pretreating donor CD34 cells, or treating We have explored the role of GSK-3 inhibition and the recipient mice themselves. Pretreatment of le- Wnt activation as a means to expand ex-vivo human thally irradiated mice with Diprotin A enhances en- UCB cells. We have shown that GSK-3 inhibition graftment of untreated donor mouse cells. Future suppresses ex-vivo proliferation of UCB cells induced studies to enhance HSC engraftment will encompass by cytokines but improves the repopulating capacity studies in higher animals and human clinical trials. of UCB. Using a synthetic GSK-3 inhibitor (6-bro- 1384 Summary of IUCBT Symposium moindirubin 3’-oxime; BIO) co-cultured with UCB engraftment 20K occurred on days 53 and 32, CD34 cells, we demonstrate that GSK-3 inhibi- and 50K on days 68 and 41 in the ﬁrst and tion suppresses ex-vivo expansion of committed second patient, respectively. In patient #1 weekly chi- HPCs, whereas increasing the pool of the most prim- merism data indicated that all cells were from the itive progenitor/stem cells. UCB C34 cells cultured expanded unit on day 7 and again on day 14; by in BIO results in the accumulation of -catenin and its day 21 there was no longer engraftment by the relocation from the cytoplasm to the cell nucleus. In expanded cells. In patient #2 the non-manipulated addition up-regulation of c-myc and HoxB4 genes both unit did not begin to contribute to the engraftment known -catenin targets involved in the regulation of until after day 14 and, until that time, 100% of the stem cell renewal, was observed. Using the ‘human- engrafted cells were from the expanded unit. Using ized’ NOD/SCID mouse model we have demon- the same preparative regimen and post-transplant im- strated both expansion and preservation of function munosuppression, the median time to engraftment with increased numbers of engrafting BIO treated was 27 days for 9 patients receiving double CBTs but human CD34 UCB (120-fold expansion) compared without using expanded cells. to control cells (45-fold expansion). In addition, in- Dr. Elizabeth Shpall discussed CB expansion and creased output of myeloid, lymphoid and megakaryo- indicated that to date no infusional toxicity has been cytic progenitor cells was observed in NOD/SCID associated with transplantation of expanded CB; time mice that received BIO-treated UCB cells. Thus, us- to platelet engraftment may be reduced but neutrophil ing a humanized NOD/SCID mouse model we have engraftment is still three weeks; CD34 and total cell shown that activation of the Wnt pathway through losses with positive selection are substantial; and GSK-3 inhibition improves the repopulating ability therefore alternative cord expansion strategies are of human UCB hematopoietic progenitor/stem cells. needed. Mesenchymal stem cells (MSC) can differen- Dr. Colleen Delaney discussed Delta1: A notch up tiate into bone, adipocytes, cartilage, functional mus- on CBT? Notch signaling is generally capable of in- cle, and liver cells. MSC may provide cellular and ﬂuencing the ability of a cell to progress from a less to extracellular components of the stem cell “niche” ab- a more differentiated state in response to speciﬁc sig- sent in current liquid expansion protocols, thereby nals, e.g. growth factors. A role for Notch in hemato- improving CB expansion, engraftment, and poten- poiesis was initially suggested by detection of the hu- tially reducing the rate or severity of GVHD. Studies man Notch1 gene in CD34 or CD34 lin- human are underway in which CB MNC are co-cultured with hematopoietic precursors. Retrovirus-mediated ex- MSC. The co-culture system, compared with the liq- pression of a constitutively active form of Notch 1 uid culture system, has produced superior results in resulted in enhanced self-renewal of hematopoietic expansion of TNC, CFU, CD34 , CD133 , CAF- progenitors, and activation of endogenous Notch re- Cwk2 and CAFCwk6. A clinical trial is soon to begin ceptors in primary murine hematopoietic progenitors to determine if cells expanded in the co-culture system (LSK cells) by culture in the presence of immobilized can improve time to neutrophil and platelet engraft- Notch ligand (Delta1) resulted in a multi-log increase ment and engraftment failure rate; secondary end- in short-term lymphoid and myeloid repopulating points are GVHD, infections, immune reconstitution, cells. Similarly, culture of CB progenitors in the pres- day 100, and OS. ence of Notch ligand results in 100 fold increase in Dr. David Scadden discussed methods of enhancing CD34 cells with short-term and possibly long term the engraftment of stem cell grafts. The stem cell NOD/SCID repopulating ability. Optimization of the niche hypothesis suggests that stem cell persistence notch-mediated expansion system was carried out with depends upon engagement of a specialized anatomi- the goal of providing cells that rapidly engraft and cally deﬁned microenvironment. A niche is an archi- overcome the delay in hematopoietic recovery after tectural place where stem cell survival and replication HSCT. A clinical trial is underway using double CBT are fostered and where interaction between heterolo- with a fully myeloablative Cy/Flu/TBI conditioning gous elements is dynamic and may be transient. Mod- regimen. An unmanipulated unit is infused ﬁrst fol- ifying parathyroid hormone (PTH) receptor geneti- lowed by expanded cells given 4 hours later. Three cally can lead to an increase the number of stem cells patients have been enrolled in the study and data are in a mouse by about 2 fold. PTH given as a once daily available on the ﬁrst two. Both were adults with AML dose immediately following transplantation in a mu- in CR1. Cell expansion resulted in a 791 fold expan- rine model resulted in improved cellularity in the sion in patient #1 and a 743 fold expansion in patient marrow, an increase in the stem cell pool, and greater #2; CD34 expansion was 210 fold for patient #1 and ability to tolerate the transplant. Data indicate that 174 fold for patient #2. Patient #1 engrafted on day PTH in an animal model improves production of stem 9 and patient #2 engrafted on day 16. Both pa- cells, improves preservation of stem cells after chemo- tients had a consistent absolute neutrophil count therapy, and improves efﬁciency of engraftment of (ANC) above 100 by day 7 post-transplant. Platelet stem cells. Experiments in Cynomolgous monkeys (n Summary of IUCBT Symposium 1385 6) in which conditioning plus allogeneic transplants patients are alive at a median of 1,095 days (range were performed with and without daily PTH (d 7 to 758-1,328 days). Chronic GVHD was diagnosed in 6 49) indicated increased cellularity, and marked im- patients and was extensive in 5; the performance status provement in immunologic recovery. Patients who is 30-90% (median 70% - Lansky scale). Two deaths had failed mobilization protocols for autologous resulted from respiratory failure, one was from EBV- transplant were treated with PTH; 7 of 15 patients LPD, and one was due to Pseudomonas sepsis. Conclu- who had failed one prior mobilization had an adequate sions reached are that primary graft failure is a serious mobilization after PTH, and 2 of 5 patients who had risk of UCBT. A back-up donor source should be failed 2 mobilizations had a successful subsequent mo- identiﬁed before start of procedure; the lack of donor bilization procedure. The study of the effects of PTH chimerism early on is predictive of GF, an early 2nd is being extended to patients who are to receive a dual UCBT was a feasible therapeutic option, and using an allogeneic CB transplant to see if PTH can improve immunosuppressive preparative regimen resulted in the outcome. high engraftment rate. Survivors experienced a greater Dr. Ka Wah Chan discussed primary graft failure risk of extensive chronic GVHD (cGVHD) and mul- after unrelated donor CB transplants: risk factors and tiple infections and viral reactivation post-transplant. management. The deﬁnition of engraftment is recov- ery of ANC 500/ l with documented donor chimer- ism. Primary engraftment failure is deﬁned as no ev- idence of recovery of ANC 500/ l, (except those SESSION III. CORD BLOOD BANKING, MANIPULATION AND UNIT SELECTION with autologous recovery), or 5% donor cells on 2 chimerism studies (PCR/STR method), performed Dr. Eliane Gluckman discussed how to choose the weekly, beginning 3rd - 4th week. Patients who die best CB unit for unrelated CBT. It is important to before day 28 are not evaluable. Among 100 UCBT consider whether the transplant is for an adult or child transplants 6 died early, 83 engrafted and there were and one must also consider the diagnosis and stage of 11 primary graft failures. An analysis was performed of the patient’s disease. It is critical to be objective when possible risk factors leading to primary graft failure. comparing with other sources of stem cells. The cri- None of the following features were statistically dif- teria of choice are nucleated cells, CD34 cells, HLA ferent among patients who engrafted, and those who matching at high or low resolution, and other factors. did not: patient age or weight; TNC, CD34, or CFU Eurocord recommendations (2007) are to ﬁrst look at in the graft; a diagnosis of a malignant or non-malig- the number of cells that should be 3 107 NC/kg 5 nant disorder; inclusion or not of TBI in the prepar- and/or 2 10 CD34 /kg. Second, one should look ative regimen; or HLA match of 5 of 6 or 4 of 6. at HLA matches: 0-1 mismatch is better than 2 (avoid The approach to primary graft failure at the Texas 3-4 mismatches). Class I mismatches are preferable to Transplant Institute is as follows: A backup autolo- class II mismatches. Increase the number of cells to gous marrow is not collected. Instead, a second partially overcome HLA mismatches. Then one UCBT is used as rescue (sooner rather than later) should adopt to the graft indication: For malignant using the most readily available unit with ﬁrst priority diseases the cell dose is the most important factor for being cell dose but also important are HLA match and outcome. A minimum of 3 107 NC/kg at collection immediate shipment. The patient’s marrow is usually or 2 107/kg at infusion must be obtained. HLA aplastic, so emphasis of the preparative regimen is on mismatches increase the risk of engraftment delays, immunosuppression. The clinical outcomes of 11 pa- TRM and cGVHD and decrease the risk of relapse tients with primary graft failure were as follows: two resulting of an absence of the role of HLA mismatches were not transplant candidates because of active fun- for survival. The type of HLA mismatches did not gal pneumonia, or recurrent leukemia and these pa- inﬂuence outcomes and increasing cell dose abrogates tients died 35 and 62 days post transplant. Nine chil- the effect of HLA mismatches. For non-malignant dren went on to second unrelated donor CBT. The diseases the requirements for cell dose and HLA median time to the second UCBT was 55 days, range matching are different than in malignant disorders. In 33-95 days. Six patients were treated with a prepara- non-malignant disease cell dose is higher at 4.9 107 tive regime that included CY/Flu/TBI (2Gy), at collection and 3.5 107at infusion; HLA mis- ATG. Two patients with aplastic anemia were matches play a major role for engraftment, GVHD, treated with TBI (6Gy), CY and ECP or Campath. and survival, and are partially abrogated by increasing One patient received Campath and Flu. There was cell dose. The CB selection procedures from Dr. J.E. one early death (day 12) and the other 8 patients had Wagner are quite similar and are as follows: Sort by myeloid engraftment (median day 15), platelet en- HLA match showing only those CB units that are graftment (median day 92), and complete donor chi- potentially 4 of 6 matches, then sort by cell dose merism. However, late infectious complications af- within each HLA matched category, and then list only fected 7 of the 8 survivors. The current status is that 5 those units that have a cell dose 1.5 107/kg. Use a 1386 Summary of IUCBT Symposium single if 6 of 6 unit is identiﬁed with a cell dose 3.0 allow GVL to eradicate the malignancy. Some regi- 107/kg, use a single if 5 of 6 unit is identiﬁed with mens are truly nonmyeloablative and the patients are a cell dose 4.0 107/kg, and use a double cord if not capable of repopulating their marrows without a enough cells in a single cord, using units as closely transplant. Other regimens are RIC but are myeloa- matched as possible with a total cell dose 3.0 blative and the patient requires a transplant. Patients 107/kg. receiving truly nonmyeloablative regimens have a Dr. Juliet Barker’s presentation was entitled: A “no lower rate of aGVHD and there is a trend toward a wash” albumin-dextran dilution strategy for UCB lower rate of cGVHD. The results of nonmyeloabla- thaw for adolescent and adult UCB transplant recip- tive BMTs have indicated reduced toxicity and re- ients: Superior to wash? Some studies suggest that duced GVHD; similar infections occur, but these are washing the CB unit is not necessary in adults and that generally responsive to therapy. There is a lower rate albumin dilution is a good alternative. However, some of TRM and the use of such transplants can extend the UCBT centers are resistant to change. The potential use of HSCT to patients up to 75 years of age. Greater advantages of an albumin-dilution thaw strategy over immune suppression is needed to reliably achieve en- washing are reduced UCB manipulation; it is faster; it graftment in CBT. Regimens effective for matched is simpler; there is reduced cell loss (washing is re- sibling and matched unrelated transplants may not be sponsible for half of post-thaw cell loss); and there is sufﬁciently immunosuppressive for UCBT. In a Bu/Flu no risk of bag break in centrifuge. Advantages over regimen, there was a 3% rejection rate for sibs/ bedside thaw are that it is performed in the controlled matched unrelated donor (MUD), but 40% with environment of a cytotherapy lab, it avoids prolonged UCBT. RIC effective in UCBT include Cy/Flu/TBI exposure to high concentrations of DMSO at room and Melphalan-thiotepa-Flu. There is a potential ben- temperature, it allows for immediately taking and dis- eﬁt of HLA mismatches in UCBT in that the frequent tributing samples for CD34 analysis and research, use of mismatched units provides a target for GVL and it results in the ability to obtain cell count and and results in a lower relapse rate. Elderly patients perform trypan blue testing prior to infusion. A pro- with AML have a dismal prognosis, but a trial of spective study was performed with the hypothesis that reduced intensity BMT indicated no early TRM and a albumin dilution was comparable to washing in terms 60% survival at 60 months (patients must be in remis- of post-thaw TNC/CD34 cell dose and engraftment. sion prior to transplant). This is in contrast to 5% The study was initially restricted to RBC depleted survival at two years for standard chemotherapy. Fur- units but was subsequently modiﬁed to include RBC ther studies of reduced intensity transplants in this containing units. Nineteen patients were studied, each group of patients are in progress. Another category of of whom received a double CBT. Results indicated patients who have been helped by non-myeloablative that the albumin-dilution thaw of UCB was compara- conditioning regimes is indolent lymphoma. Using a ble to wash with centrifugation for TNC yield, infu- conditioning regimen of Flu, CSa and rituximab for sion reaction proﬁle, and engraftment. It was faster, patients with follicular lymphoma (FL), 90% are alive more efﬁcient, reduces technologist time, speeds time at 90 days, which is much improved over the results to patient infusion, and reduces potential for cell loss. using a myelablative regimen. Studies are also in Therefore, is it superior to wash for adult UCBT? It progress for MCL and chronic lymphocytic leukemia should facilitate center-center standardization and is (CLL). Certain diseases, such as chronic myelogenous to be investigated in CIBMTR sponsored multi-cen- leukemia (CML), (CLL) and LGL, are more sensitive ter prospective study of adult double unit UCBT. to graft-versus-malignancy than others and these dis- Finally, reduced cell loss from albumin-dilution strat- orders are the prime candidates for reduced intensity egy makes taking of a small sample for research ethi- transplants. cally acceptable. Unanswered questions are whether it Dr. Claudio Brunstein discussed nonmyeloablative should be utilized in smaller children (patients 40 kg UCBT. The hypothesis underlying nonmyeloablative were not tested). Also, the incidence of aGVHD UCB transplantation is that despite the differences seems to be a bit lower so that there is a possibility of in UCB alloreactivity, UCB has enough immune cells qualitative effects on GVHD. to mediate engraftment after nonmyeloablative con- ditioning. There is a growing number of reports of nonmyeloablative UCB transplants, especially for adult patients. At the University of Minnesota, a study SESSION IV. CONDITIONING REGIMENS was performed using Cy/Flu/TBI for the conditioning Dr. Richard Champlin reviewed nonmyeloablative regimen and CSa and MMF for post-transplant im- SCT for hematologic malignancies – considerations munosuppression. ATG was added for patients who for CBTs. The fundamental hypothesis for nonmy- had not received chemotherapy within 3 months of eloablative SCT is to intentionally decrease the inten- transplantation. Patients receive a double UCB trans- sity of the preparative regimen to reduce toxicity, and plant if no single graft is big enough. HLA matching Summary of IUCBT Symposium 1387 is done at the antigen level for -A and -B loci and at HLA disparity, 21% with 2 HLA disparities, and 12% the allele level for -DRB1. Using this study design with 3 HLA disparities. DFS was 42% when trans- about 80-85% of patients received two UCB units. planted in remission and 23% if not in remission. The sustained donor engraftment rate for 161 patients With TBI Flu ENDX the DFS was 44% and with was 87%. The incidence of grade II-IV aGVHD was other conditioning regimens it was 23%. Conclusions 54% and Grade III-IV was 18%. The 6 month TRM reached in this retrospective based registry study and was 18% and at 3 years it was 25%. The progression- high risk group of patients were that the results of free survival (PFS) for patients who received two units single RIC-UCBT are encouraging. Cell dose and was 39% and for those receiving one unit was 24% (n HLA remain important factors in this setting. For this 110, P .05). OS was 45% for those receiving reason probably results can be improved with double either 1 or two units (n 161). A further study was cords and better HLA matching. Importantly, the performed comparing the outcomes after reduced in- type of conditioning (Flu ENDX TBI) is associated tensity transplantation using UCB versus sibling with decreased TRM and better DFS. Use of busulfan PBSC for de novo and secondary AML. Results indi- was associated with increased TRM. Immunosuppres- cated that the OS was not signiﬁcantly different when sion after RIC-UCBT should be carefully evaluated in comparing the two HSC sources. In summary, UCB is high risk patients of relapsing without GVHD. an effective alternative source of HSC for NMA trans- Dr. Karen Ballen presented the results of a study of plantation. Patients who need nonmyeloablative outcomes of adult patients after double CBT using an transplant and who do not have a suitable sibling or RIC regimen which consisted of Flu, melphalan and unrelated donor should be considered for UCB trans- rabbit ATG. The minimum combined cell dose was plantation. Larger numbers of patients will help fur- 3.7 107NC/kg pre-freeze. GVHD prophylaxis in ther determine the role of UCB transplantation in protocol #1 consisted of CSa and MMF (21 patients), older patients and those with pre-existing high risk and in protocol #2 it consisted of sirolimus and ta- clinical features. crolimus (22 patients with 100 days follow up). The Dr. Vanderson Rocha discussed a RIC regimen after median age for protocol #1 was 49 years (range 24-63) single unrelated CBT for adults with hematological and, for protocol #2 was 53 years (19-64). All patients malignancies. Unrelated CBT using RIC regimen has had hematologic malignancies. For the two protocols been proposed in order to shorten the aplasia period the median days to engraftment for ANC 500 were and decrease TRM in adult patients with high risk of 20 or 21, respectively. Days to platelets 20K were 41 dying of toxicity. Since cell dose is a major limiting and 47 days; to platelets 100K were 65 and 125, factor in UCBT, concerns have been raised in the use respectively. There were two graft failures in protocol of CB cells in RIC. Also, another concern is the GVL #1 in patients who had aplastic anemia (AA) and MDS effect from lower incidence of GVHD after UCBT. and who had not received prior cytotoxic therapy. The There are no data available in single RIC UCBT aGVHD rate for grades II-IV were 40% and 14%, analyzing risk factors of outcomes. A retrospective respectively for the two protocols and, for grades study of 142 patients with hematologic malignancies III-IV, the rate was identical at 5%. The cGVHD rate was performed. The median age of patients was 44 was 34% and 20%, respectively and the extensive years (range, 16-76). Twenty-three conditioning reg- cGVHD rate was 20% and 6%, respectively. For imens and a number of regimens for GVHD prophy- protocols #1 and #2, the one year OS was 67% and laxis were used in this registry based study. The me- 73%, one year DFS was 67% and 51%, and relapse dian days to neutrophil recovery was 20 days (5-56) rate was 19% and 34%, respectively. The TRM was and for platelet recovery was 38 days (15-171). A 14% for both protocols. Chimerism analysis indicated multivariate analysis for neutrophil recovery indicated that a single CB donor predominated in 76% of pa- that conditioning with Flu ENDX TBI was a sig- tients by day 30. By day 90 one unit was detectable niﬁcant factor as was HLA 0-1 vs. 2-3-4. Other factors in 72%, both CB units in 18% and a single CB unit included in the model that were not signiﬁcant were and host cells in 10%. Predictors of the predominant cell dose, status of the disease and female sex. aGVHD unit were (1) ﬁrst unit infused, (2) higher CD34 occurred in 29% and cGVHD in 40%. TRM at day count and (3) higher NC/kg. A number of factors led 100 was 28% overall and was 14% with a cell dose of to some patients who had an indication for a trans- 2.8 107/kg and 39% with a cell dose of 2.8 plant but who did not receive a transplant: Six patients 7 10 /kg. Multivariate analysis indicated that the condi- had no allele level match, two patients had no antigen tioning regimen and cell dose were signiﬁcant factors. level match, grafts for 3 patients had an inadequate Relapse at one year was 53% for patients with ad- cell dose, there were insurance issues for 2 patients, vanced disease and 36% for those with early or inter- progressive disease in 4 patients, and another donor mediate disease. OS was 43% and DFS was 32%. The source in 9 patients. In conclusion, double CBT with DFS varied depending on the HLA match: 67% when this RIC regimen are well tolerated with TRM of patient and donor were HLA matched, 46% with 1 14%. Low relapse rate with low risk of GVHD sug- 1388 Summary of IUCBT Symposium gests preservation of GVL effect, and the risk of related to adenovirus infection and twelve (12) from GVHD may be lower with the use of sirolimus/ta- CMV infection, rendering these two viruses the cause crolimus. in 50% of all OI related deaths. A logistic regression model was used to investigate the impact of ten de- mographic and clinical characteristics on the risk of death from OI by 6 months post unrelatd CBT. These SESSION V. IMMUNE RECONSTITUTION AND INFECTIONS potential predictors were sex, race, age at unrelated CBT, CMV serology, HLA mismatches (2-3 vs 1), Dr. Bruce Blazar reviewed the topic of infection malignancy, TBI, total graft cell dose/kg, CD34 and immune reconstitution after HSCT: Challenges graft cell dose/kg and CD3 graft cell dose/kg. In and opportunities. Despite hematopoietic engraft- univariate analyses, sex, race and TBI did not predict ment and recovery of marrow function, recipients of 6-month death from OI. Malignancy (P .07) was hematopoietic cell transplants have prolonged defects marginally associated with a greater probability of in generation of functional T and B lymphocytes. 6-month death from OI. Malignancy without TBI was Chemoradiotherapy- and GVHD- induced epithelial also associated with a marginally higher probability of cell injury introduces a portal for bacterial, fungal, and 6-month death due to OI (P .04). A signiﬁcantly viral infections. Bacterial products result in macro- greater probability of 6-month OI-related death was phage activation and the release of pro-inﬂammatory associated with CMV positive serology (P .0001), cytokines which may contribute to or resemble greater HLA mismatch (P .006), and older age (P GVHD. An innate and adaptive immune system re- .0009). Higher total graft cell dose (P .001), CD34 sponse is needed to eliminate infectious pathogens. A cell dose (P .014) and CD3 cell dose (.014) were major cause of post-BMT immune deﬁciency is the associated with lower probability of death from OI at loss of thymopoietic capacity, and impaired T cell 6 months. Multivariate analyses were performed: recovery as a result of factors such as age, radiation, or Model 1 included; CMV (P .0004), HLA mismatch GVHD. Damage to thymic epithelial cells by pre- (P .042) and age (P .03). Model 2 included; CMV BMT conditioning impairs the ability of the thymus (P .0001), HLA mismatch (P .005) and malig- to generate mature T lymphocytes after BMT. Ap- nancy without TBI (P .04). Since total graft cell proaches to lessen the injury or hasten the repair of or dose, CD34 cell dose and CD3 cell dose were replace thymic epithelial cell or stromal elements or highly correlated, each of these variables was intro- their products represent a promising strategy to speed duced into models 1) and 2) separately. Total graft cell peripheral T cell reconstitution and function. dose was the strongest predictor when cell dose vari- Dr. Paul Szabolcs presented data regarding a mul- ables were added to Models 1 (P .0097) and 2 (P tivariate analysis of patient and graft speciﬁc factors among 330 recipients of unrelated cord blood trans- .004). CD34 cell dose contributed less signiﬁcantly plant (CBT) to predict risk of death from opportunis- to both models (P .02 both models). CD3 cell tic infections in the ﬁrst 6 months after CBT. Cord dose did not signiﬁcantly contribute to Model 1 and blood is unique because of a high frequency of highly was marginally signiﬁcant in Model 2 (P .05). The proliferative primitive stem cells paired with “naïve” percent concordance among these models ranged lymphocytes. To determine the impact of patient and from 71%-75%. Thus, 6-month death from OI after graft-speciﬁc factors on 6-month post-unrelated CBT single cord unrelated CBT in children can be pre- OI-related mortality we reviewed all consecutive pe- dicted by the following risk factors: older age, positive diatric patients transplanted at Duke University Med- CMV serology, 1 HLA mismatch, malignancy with- ical Center between June 1999 and Oct. 2005. Three out TBI, lower graft cell dose (total, CD34 and hundred thirty (330) consecutive pediatric recipients CD3 ). Sex, race, and TBI alone do not predict of single unrelated CB grafts were identiﬁed. Those 6-month death from OI. In summary, opportunistic receiving a second transplant for primary graft failure infections, (most being viral) are the most prevalent were not analyzed. Two hundred twenty (220) of the cause of death in the ﬁrst 6 months after UCBT. This 330 patients (67%) were alive at 6 months. Of the 110 analysis of 330 consecutive patients demonstrates that children who died by 6 months, 64 patients (58%) patient and graft-speciﬁc factors have a signiﬁcant were identiﬁed with OI (viral, fungal infections) im- impact on 6-month mortality from OI. Potential strat- plicated as a cause of death. The 46 patients who died egies to reduce OI related mortality are to increase prior to 6 months and for whom OI was not impli- engraftment with improved homing, an increased cell cated as a cause of death were omitted from the study dose and/or ex-vivo expansion of stem/progenitor data set, resulting in 284 patients. Of these 284 pa- cells; enhance immune recovery by improving surviv- tients, 200 patients (77%) were alive at 6 months and al/homeostatic expansion of infused lymphoid cells, 64 (23%) died at or before 6 months with cause of boosting thymic output, and reducing GVHD; and death related to OI. Twenty two (22) patients died increase T cell dose/adoptive therapy by ex-vivo ex- Summary of IUCBT Symposium 1389 pansion of broad unprimed T cell repertoire and/or itoring would be beneﬁcial for detection of virus in- antiviral, antifungal CTL generation in vitro. fection at early stage; that infection with multiple Dr. Krishna Komanduri presented data indicating pathogens was less in CBT-recipients than that in that delayed immune recovery after UCBT is charac- BMT/PBSCT recipients; and all the CBT-recipients terized by thymic regeneration failure. A prospective with multiple virus infections showed profound im- study of T cell immune reconstitution was performed. munodeﬁciency. Samples were obtained from 32 patients (of 34 en- rolled), median age: 33.5 years (7-57); 28 patients adults. The median follow-up was 221 days. Diag- SESSION VI. NON-HEMATOPOETIC STEM CELLS noses were AML/MDS (16), CLL (2), CML (2), HD (8), and non-hodgkin lymphoma (NHL) (4). Patients Dr. Mark Weiss presented a review lecture enti- were heavily pretreated. All but one patient received a tled, “Stem cells in the umbilical cord matrix: isolation myeloablative conditioning regimen. Primary im- and characterization.” Non-hematopoietic cells in the mune recovery endpoints were immunophenotypic umbilical cord or CB include mesenchymal stem cells analysis of T cells, B cells, NK cells; assessment of (MSCs), USSCs, BioE’s cells, and VSLE. From the functional T cell responses using cytokine ﬂow cytom- umbilical vein one can obtain endothelial cells and etry (stimulation with superantigen and CMV anti- MSCs. Wharton’s Jelly contains umbilical cord matrix gens); assessment of thymic function ( TREC as- cells and perivascular cells. The amniotic ﬂuid and the say); and naïve and memory maturation phenotyping. placenta and decidua also contain stem cells. The ISCT Results indicated decreased to absent thymic function working group deﬁnition of MSCs is that they are plas- at baseline, no detectable thymic recovery in 24 of 26 tic-adherent; their surface phenotype is CD34, CD45, subjects, only one subject with signiﬁcant thymopoi- HLA-DR negative, and CD29 (SH3) – CD105 (SH2), etic recovery, thymopoiesis reduced relative to other CD73 (SH4), HLA-class I positive; and they are mul- SCT recipients (despite lower age of CBT recipients, tipotent, able to differentiate to bone, cartilage, adi- 33 vs. 50). Thus, thymic regeneration failure charac- pose, and muscle. MSCs play a role in immune mod- terizes this group of cord blood SCT recipients. Im- ulation, hematopoietic support (ex-vivo expansion of proved CBT immune recovery can result from pro- HSCs, enable engraftment of HSCs when co-grafted), spective studies characterizing the effects of age, stem and homing to pathology. Animal studies suggest cell dose, and HLA matching on T cell recovery; that umbilical cord matrix cells may enable engraft- moving beyond “monofunctional” assays to examine ment of HSCs and may have therapeutic effects in functional T cell recovery; optimization of ex vivo disorders including breast cancer, Parkinson’s dis- expansion strategies to maximize the recovery of T ease, global ischemia brain damage (stroke), retinal cell progenitors (e.g., using NotchL); use of thymo- damage, and myocardial ischemia. The mechanism is protective (KGF) and thymopoietic (GH, leuprolide, not well-understood and may include immune modu- IL-7) agents; and infusing antigen-speciﬁc T cells in lation and growth factor/cytokine effects. donor grafts. Dr. Mariusz Z. Ratajczak discussed the morpho- Dr. Tomohiro Morio discussed a strategy to combat logical and molecular characterization of a novel pop- opportunistic infections after UCBT. Current data ulation of CXCR4 SSEA-4 Oct-4 very small em- indicate that the risk of serious infection after pediat- bryonic-like (VSEL) cells puriﬁed from human CB. ric UCBT is comparable to that with unmanipulated Recently we identiﬁed in murine BM a homogenous BM; there is a marked increased risk of EBV-related population of rare ( 0.01% of BMMNC) Sca-1 complications with the addition of ATG to a nonmy- CXCR4 lin CD45 cells that express, by RQ-PCR eloablative conditioning prior to URCBT; 24 of 40 and immuno-histochemistry, markers of pluripotent adult patients who underwent CBT developed VZV stem cells. They display several features typical for reactivation at a median of 5 months after CBT; there primary embryonic stem cells such as i) a large nuclei is a high incidence of HHV6 infection (7 of 10) with surrounded by a narrow rim of cytoplasm, and ii) a high viral load in CBSCT recipients; and positive open-type chromatin (euchromatin) that is typical for CMV antigenemia was seen in 19 of 24 sero-positive embryonic stem cells. These cells ( 2-4 m in diam- adults patients at a median of 42 d after CBT. A eter) were named very small embryonic-like (VSEL) PCR-based system for rapid and sensitive detection of stem cells. A new two step isolation procedure is used multiple viruses detects more than 20 viruses in one to purify a similar population of cells from human CB, run; it is rapid with less than 2 hours from extraction which is based on isolation of CB mononuclear cells to data analysis, sensitive with a detection level of at (CB MNC) by hypotonic lysis and multiparameter least 10 copies/sample; and costs less than $25. Expe- FACS sorting. In vitro cultures of CB-VSEL are able rience with the use of this system indicates that it to grow neurospheres that gave rise to neuronal lin- provides a economical, rapid, and sensitive method for eages (beta-III tubulin , nestin , O4 , MBP , monitoring multiple pathogens; that scheduled mon- GFAP ) and cardiomyocytes (beta-myosin heavy 1390 Summary of IUCBT Symposium chain , alpha-sarcomeric actin). Based on this we con- that augmentation of the cell dose by the infusion of clude that CB contains VSEL and that the majority of two partially HLA matched UCB units will speed these CB VSEL are lost during routine procedures hematopoietic recovery, reduce TRM, and improve employed currently for banking of CB MNC. We survival. Early studies showed that the time to engraft- conclude that CB tissue/organ regenerating potential ment was shifted from 32 days to 23 days with the use may be much higher than initially postulated if the of double CBTs vs. historical controls using a single proper fraction of CB MNC cells enriched in VSEL is UCB. The mechanism by which two units enhance employed. engraftment could be the augmentation of stem cell Dr. Mervin C Yoder discussed high proliferative dose although immunologic reactions. A study was potential endothelial progenitor cells found in UCB. performed of 126 patients with AML or ALL who UCB has been known to contain a population of received either a single or double UCBT using my- circulating cells that serve as progenitors for the en- eloablative conditioning. Seventy two patients re- dothelial lineage (EPCs). We have determined that a ceived a single unit and 54 received a double UCBT. rare population of cells in the umbilical cord blood Patients received a double CBT if there was not a emerges upon in vitro culture that displays clonal single unit available with an adequate cell dose. The colony forming ability. These endothelial colony total infused TNC was 3.2 107/kg for the single 7 forming cells (ECFCs) are present in adult peripheral cord transplants and 3.5 10 /kg for double CBTs blood as well, though vastly enriched in term and (NS). Total CD34 was also similar but CD3 cell preterm cord blood. ECFCs spontaneously form hu- dose was larger for the double CBTs (0.9 vs 1.3 man blood vessels upon implantation into immunode- 107/kg). Results indicated that the rates of sustained ﬁcient mice and participate as blood conduits. Recent neutrophil engraftment and TRM were similar be- studies indicate that ECFC can be derived from um- tween single and double CBTs. However, the inci- bilical arterial and venous endothelial cells as well as dence of grade II-IV aGVHD was higher with double endothelium from several other vascular sites. Thus, CBTs, although the incidence of grades III-IV human CB ECFCs may be useful as a source of cells to aGVHD was not different. The incidence of cGVHD regenerate human vasculature in vivo. was low and there was no difference between the two Tsuneo A. Takahashi discussed progress in regen- groups. The incidence of relapse for patients in CR1 erative medicine using CB: MSC isolation, and induc- and CR2 was higher for single UCB recipients than tion to bone and cartilage. MSC can be isolated from for double and this was not inﬂuenced by the diagnosis cord blood (CB-MSCs) and from chorionic villi of the (AML vs ALL). OS at 3 years was 72% for double placenta (PL-MSCs) using the explant culture CBTs and 47% for single (P .16). Considering method. They may differentiate into osteogenic, patients who survived for at least 9 months, all of the chondrogenic, and adipogenic cells. PL-MSCs were double UCBT patients were alive whereas there were induced to chondrocytes in vitro and produced a large 15 deaths among the single cord transplant patients (P amount of extracellular matrix of cartilage in vivo. .01) and 13 of 15 events were from relapse. Con- PL-MSCs could repair the cartilage defect in the knee clusions are that double UCBT promotes engraftment of a nude rat. MSCs could also be isolated from fresh achieving rates comparable to single UCBT with ad- CB and these cells expanded well with differentiation equate cell doses; the risk of grades II-IV aGVHD is ability to chondrocytes and osteocytes. Thus, both higher after double UCBT (although there is no dif- PL-MSCs and CB-MSCs are potential allogeneic ference in risk of grades III-IV aGVHD); and the risk stem cell sources for tissue engineering for bone and of cGVHD is similar. There is a reduced risk of cartilage. relapse associated with double UCBT and this was not driven by aGVHD. Patients in early disease status (CR1 and CR2) were the only ones to show beneﬁt. Dr. Jeffrey Miller discussed a novel triple UCBT SESSION VII. THE GRAFT VERSUS TUMOR EFFECT strategy to promote natural killer (NK) cell immuno- Dr. Michael Verneris discussed the impact of multi- therapy using a fully ablative preparative regimen fol- unit UCB transplantation on leukemia relapse. One of lowed by a double UCBT in patients with refractory the most common complications of transplantation is AML. The strategy was that appropriately chosen leukemia relapse. Variables that impact relapse in- donors will enhance NK cell alloreactivity if they clude patient characteristics, the conditioning regi- expand in vivo. The best strategy may be to combine men, HLA disparity, the type of graft, and graft ma- adoptive transfer and in vivo expansion followed by nipulation. Cell dose has a signiﬁcant impact on speed HCT. A trial was conducted with 19 poor prognosis of neutrophil recovery, probability of engraftment, AML patients. These patients had primary refractory the risk of TRM, survival, the inﬂuence of HLA dis- disease, relapsed disease not in CR after 1 or more parity, and access to transplantation. The rationale for cycles of standard re-induction therapy, secondary the use of multi-unit CB transplants is the hypothesis AML from MDS, relapsed AML 3 months after Summary of IUCBT Symposium 1391 HCT, and no active infections. Results indicated that Young Cell Transplantation Program (the Program) killer cell immunoglobulin-like receptor (KIR) ligand and the National Cord Blood Inventory. The aims of mismatched donor correlated with achieving CR (5 of the Stem Cell Therapeutic and Research Act of 2005 19). CR patients had higher numbers of functional are to increase the number of unrelated donor trans- NK cells after haplo NK cell infusions. Triple UCB plants, increase the public inventory of high quality strategy was developed using UCB derived NK cell CBUs from diverse populations and increase the num- double UCB transplant for patients with refractory ber of CBUs available for research. The Act autho- AML. Results indicated 9 of 10 patients engrafted; all rizes appropriations of $15 million/year for FY 2007- 9 achieved CR at day 14; 4 of 9 engrafted from the NK 2010; funding is temporary until 150,000 CBUs are cell unit #1; 2 are alive and in CR at days 120 and obtained. HRSA’s implementation approach is guided 262; 3 had TRM without evidence of relapse and by three principles: a single point of access for patients died on days 2, 62, and 67; and 5 relapses oc- and physicians to all sources of blood stem cells, col- curred (all were in CR at engraftment). In conclusion, lection of high-quality diverse CBU expeditiously, and activated NK cells may facilitate engraftment, strate- collection of complete data on clinical outcomes of gies to better promote NK cells in vivo may improve transplants. The National Cord Blood Inventory clinical results, and the DCBT distant from the NK (NCBI) will be funded through one-time, 10-year cell unit may not be needed. Better strategies to make contracts awarded competitively with HRSA funds for primary AML targets susceptible to NK cell killing 3 years; funded cord blood banks must participate in are being studied. the Program for 10 years; and the NCBI CBUs Dr. Hans G. Klingemann discussed ex-vivo expan- must be available through the Program in perpetuity sion and mRNA transfection of CB derived natural (or until they are no longer viable). Contracts for the killer cells. NK cells are a third subset of lymphocytes ﬁrst cohort of banks were awarded November 2, 2006, (in addition to T-and B cells). They are independent to six banks (New York Blood Center, Duke Univer- of V(D)J recombination and receptor, kill virus in- sity, MD Anderson, Puget Sound Blood Center, Uni- fected and transformed cells without priming, have an versity of Colorado, and StemCyte). About 10,500 important regulatory function (INF- g and other cy- CBU are to be collected with ﬁrst year of funding for tokines), kill MHC class I negative cells, express in- the ﬁrst cohort and these are to be comprised of hibitory receptors speciﬁc for “self” MHC class I, and 63% racial and ethnic minorities. Request for Pro- express many different activating receptors. CB is used posals for a second cohort of banks was published because it is a universal source of NK-cells, it is rich in April 12 with responses due May 15, 2007. HRSA will NK cell progenitors, and NK cells are believed to be hold annual competitions for new cohorts of banks, as more active than peripheral blood NK cells. A prob- funding permits. Infrastructure contracts include a lem is that there are only a limited numbers in a given contract for the stem cell therapeutic outcomes data- CB unit. A study was designed to develop an opti- base, which was awarded September 15, 2006, to the mized expansion system for CB NK cells suitable for Center for International Blood and Marrow Trans- clinical application, to make expanded cord NK cells plant Research; contracts for an Ofﬁce of Patient target speciﬁc by transfection of mRNA for a chimeric Advocacy/Single Point of Access, Bone Marrow Co- antigen receptor recognizing CD19. mRNA is advan- ordinating Center, and a Cord Blood Coordinating tageous compared to cDNA transfection in that there Center, which were awarded September 25, 2006, to is no genomic integration (mRNA stays in cytosol), the National Marrow Donor Program. An Advisory there is more efﬁcient transfection, expression is Council on Blood Stem Cell Transplantation is being highly uniform, expression can be controlled by established whose purpose is to consider and make changing amount of input mRNA, and cells can be recommendations to the Secretary of HHS on matters efﬁciently and simultaneously loaded with several dif- related to the Program. Ongoing work includes: com- ferent transcripts. Results indicate augmented cyto- plete the transition from the former National Bone toxicity of NK cells transfected with mRNA express- Marrow Donor Registry structure to C.W. Bill Young ing scFV-CD19. This is an important step to make Cell Transplantation Program, launch a web site for NK cells targeted to tumor cells. the Program (mid-Summer 2007), begin collection of blood stem cell transplant outcomes data (mid-Sum- mer 2007), complete establishment of the Advisory Council and convene its ﬁrst meeting, and future SESSION VIII. GOVERNMENTAL AFFAIRS cycles of funding for NCBI collections. Further, im- Robert Baitty, representing the U.S. Department of plement the Related Cord Blood Donor Demonstra- Health and Human Services (HHS), Health Re- tion Project, revisit interim program requirements sources and Services Administration (HRSA), Divi- with Advisory Council and public input, and deﬁne sion of Transplantation, Blood Stem Cell Transplan- targets for composition of NCBI and the size and tation Program, presented an update on the C.W. Bill composition of adult donor registry in consultation 1392 Summary of IUCBT Symposium with the Advisory Council. Finally, to encourage re- cellular therapies, disseminate data within the pro- search in collaboration with other Federal agencies gram, make transplant data publicly available, perform and the Advisory Council to improve transplant out- analyses of optimal size for the adult donor registry comes, reﬁne approaches to CB transplants for adult and cord blood unit inventory, and conduct and sup- patients, better deﬁne CBU characteristics required port research. In summary, the C.W. Bill Young for good outcomes, improve reliability and compara- Transplantation Program provides a blueprint for or- bility of measures used in CBU selection, and to col- ganizing and managing of unrelated donor hemato- lect and report data regarding new uses of stem cells poietic cell transplantation, and establishes a clear from CBU and adult donors. emphasis on Cord Blood as an important source of Dr. Dennis Confer discussed the development of hematopoietic and non-hematopoietic cells for treat- the National Cord Blood Center. The contracts for ment of disease. the Single Point of Access/Ofﬁce of Patient Advocacy Dr. Ellen Lazarus, representing the FDA, Ofﬁce of (OPA/SPA) and the Cord Blood Coordinating Center Cellular, Tissue and Gene Therapies, Division of Hu- were awarded to the National Marrow Donor Program man Tissues, presented an update on FDA Regulatory (NMDP). The contract for the Stem Cell Therapeutic Activities for Cord Blood. A draft guidance was pub- Outcome Database was awarded to the CIBMTR. lished January 16, 2007, and represents FDA’s current The OPA/SPA provides a uniform, consolidated thinking but does not establish legally enforceable search report (all searches are submitted here), pro- responsibilities. Its purpose is to recommend ways for vides advocacy services for patients, implements a plan cord blood banks to apply for licensure for speciﬁed to increase transplants, and facilitates access to trans- indications, to explain applicable regulations in Title plantation services for patients. The Cord Blood Co- 21 of the Code of Federal Regulations, and to provide ordinating Center facilitates transplants of cord blood other information about the manufacture of human from NCBI banks and from other participating banks, cord blood progenitor cells (HPC-C) and how to collects standardized CBU information from the comply with the applicable regulatory requirements. banks and provides this to the OPA/SPA, and coordi- The Guidance may be used when applying for a bio- nates search and distribution of CBUs. The Cord logics license. The FDA will review the application, Blood Coordinating Center will also develop an inter- schedule a prelicense inspection as soon as possible bank technical proﬁciency program, support public after receiving the complete application, and if the and professional education and recruitment activities, application is not complete, will identify and advise collaborate with CBBs to make CBUs available for the establishment of additional information that they research, improve operating efﬁciencies in collabora- will need to submit. The Draft Guidance Sections tion with the network, support contingency planning include factors of signiﬁcance include HPC-C de- for emergencies, and require transplant centers to scription and characterization, manufacturer informa- submit outcomes data to the SCTOD. The Center for tion, methods of manufacturing, and other informa- International Blood and Marrow Transplant Research tion. Applicable regulations and post-marketing (CIBMTR) represents an afﬁliation between the re- activities include applicable CGTPs and CGMPs and search programs of former IBMTR/ABMTR at the clinical outcome data collection. An Advisory Com- Medical College of Wisconsin and NMDP to support mittee meeting was held on 4/30/07, at which CB clinical research in hematopoietic cell transplantation issues under consideration were addressed. The next and related ﬁelds. The CIBMTR as the SCTOD steps area will be reviewing comments to the docket contractor must establish electronic data capture sys- and recommendations of the Advisory Committee, tems, collect data on all allogeneic transplants with a and ﬁnalization of the Guidance. License applications recipient or donor in the U.S, collect data on other will be accepted at any time.
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