Fifth Annual International Umbilical Cord Blood Liaison by liaoqinmei


									Biology of Blood and Marrow Transplantation 13:1380-1392 (2007)
   2007 American Society for Blood and Marrow Transplantation

Fifth Annual International Umbilical Cord Blood
Transplantation Symposium, Los Angeles, California,
May 11-12, 2007

             John Wagner,1 Richard Champlin,2 Lawrence D. Petz3

              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);

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-
                                                          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
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
                                                            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
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
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
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
                                                           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
                                                              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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