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UNITED STATES OF AMERICA

FOOD AND DRUG ADMINISTRATION

CENTER FOR BIOLOGICS EVALUATION AND RESEARCH

BIOLOGICAL RESPONSE MODIFIERS ADVISORY COMMITTEE

34^th^ MEETING

THURSDAY,

FEBRUARY 27, 2003

The Advisory Committee met at 8:00 a.m. in the Kennedy Ballroom of the
Holiday Inn-Silver Springs, 8777 Georgia Avenue, Silver Spring,
Maryland, Dr. Daniel R. Salomon, Chairman, presiding.

[PRESENT]{.underline}:

DANIEL R. SALOMON, M.D. Chairman

JONATHAN S. ALLAN, D.V.M. Member

DAVID M. HARLAN, M.D. Member

KATHERINE A. HIGH, M.D. Member

MARY M. HOROWITZ, M.D. Temporary Voting Member

JOANNE KURTZBERG, M.D. Member

ALISON F. LAWTON Industry Representative

JEANNE V. LINDEN, M.D. Temporary Voting Member

RICHARD C. MULLIGAN, Ph.D. Member

MAHENDRA S. RAO, M.D., Ph.D. Member

DAVID F. STRONCEK, M.D. Temporary Voting Member

ANASTASIOS A. TSIATIS, Ph.D. Member

GAIL DAPOLITO Executive Secretary

[NIH REPRESENTATIVE]{.underline}

LIANE HARVATH, Ph.D.

[GUEST SPEAKERS]{.underline}

NELSON J. CHAO, M.D., M.B.A.

PABLO RUBINSTEIN, M.D.

JOHN WAGNER, M.D.

[PRESENT FROM FDA]{.underline}:

PHILIP NOGUCHI, M.D.

RUTH SOLOMON, M.D.

ELLEN LAZARUS, M.D.

STEPHEN LITWIN, M.D.

KAREN D. WEISS, M.D.

I-N-D-E-X

[Page]{.underline}

**Morning Session**

Welcome and Introductory Remarks 4

Daniel Salomon, M.D., Chair

Meeting Statement 4

Gail Dapolito, Executive Secretary

Presentation of Certificate of Appreciation 11

to Dr. Salomon

Karen Weiss, M.D. &

Philip Noguchi, M.D.

**Topic I: Unrelated allogeneic hematopoietic stem/**

**progenitor cells from placental/umbilical**

**cord blood for hematopoietic reconstitution**

FDA Introduction

Ellen Lazarus. M.D. 15

Stephen Litwin, M.D. 20

Guest Presentations

John Wagner, M.D. 45

Nelson J. Chao, M.D., M.B.A. 75

Question & Answer Period 87

Pablo Rubinstein, M.D. 120

Open Public Hearing

Mr. Steve Barsh 159

Mr. Glen Ackerman 165

Ms. Faraday Niakani 167

Mr. Joel Ruderman 167

Ms. Kathleen Kim 169

Mr. Herb Lee 171

Mr. Steven Sprague 174

Ms. Heidi Shaw-Tweten 179

Ms. Serls 180

**Afternoon Session**

Committee Discussion of Questions 209

I-N-D-E-X (con\'t)

**Topic II: Update CBER Research Programs** Page

Division of Monoclonal Antibodies

Overview

Keith Webber, Ph.D. 326

Laboratory of Cell Biology

Kathy Clouse, Ph.D. 329

Ingrid Markovic, Ph.D. 335

David Frucht, M.D. 340

Laboratory of Immunobiology

Ezio Bonvini, M.D. 346

Laboratory of Molecular and

Development Immunology

Steven Kozlowski, M.D. 351

P-R-O-C-E-E-D-I-N-G-S

8:10 a.m.

CHAIRMAN SALOMON: On the record. Good morning, everybody, on this post
snow morning or pre snow morning depending on what happens with the
weather in the few hours or day. For those of you who are here, I
certainly want to say for the Committee and for the FDA that we very
much appreciate your attendance, weather notwithstanding and welcome.

We started a little bit late just to give people a little extra time to
get here given the roads. I assume the roads are on the icy side. Given
that I haven\'t left the hotel, I can only surmise from past experience.
I\'m going to keep my introductory remarks short. We have a relatively
long day today. The title of this talk is \"Stem Cell Redux.\" That
doesn\'t officially go anywhere. This is my challenge to Gail which is
to at least have a real title for every meeting instead of Meeting No.
34 which just doesn\'t do anything for me. Welcome and with that I will
turn to Gail to give an introductory statement.

EXEC. SECRETARY DAPOLITO: Good morning, Dr. Salomon and the Committee.
This announcement is part of the public record for the Biological
Response Modifiers Advisory Committee Meeting on February 27, 2003.
Pursuant to the authority granted under the Committee Charter, the
Director of FDA\'s Center for Biologics Evaluation and Research has
appointed Drs. Mary Horowitz, Harvey Kline, Jeanne Linden and David
Stroncek as temporary voting members for today\'s discussion.

Based on the agenda, it was determined that there are no products being
approved at this meeting. The Committee participants were screened for
their financial interests to determine if any conflicts of interests
exist. The Agency reviewed the agenda and all relevant financial
interests reported by the meeting participants.

The Food and Drug Administration prepared general matters waivers for
special Government employees who required a waiver under 18 USC 208.
Because general topics impact on so many entities, it is not prudent to
recite all potential conflicts of interest as they apply to each member.
 FDA acknowledges that there may be potential conflicts of interest but
because of the general nature of the discussion before the Committee,
these potential conflicts are mitigated.

We also note for the record that Ms. Alison Lawton serves as the
non-voting industry representative member acting on behalf of regulated
industry. She is employed by Genzyme and thus has interest in her
employer and other similar firms.

With regards to FDA\'s invited guests, the Agency has determined that
the services of these guests are essential. The following interests are
being made public to allow meeting participants to objectively evaluate
any presentation and/or comments made by the guests.  Dr. Nelson Chao is
employed by Duke University. Dr. Pablo Rubinstein is employed by the
National Cord Blood Program of the New York Blood Center. Dr. John
Wagner is employed by the University of Minnesota.

Members and consultants are aware of the need to exclude themselves from
discussions involving specific products or firms for which they have not
been screened for conflicts of interest.  Their exclusion will be noted
for the public record. With respect to all other meeting participants,
we ask in the interest of fairness that you state your name, affiliation
and address any current or previous financial involvement with any firm
whose product you wish to comment upon. Waivers are available by written
request under the Freedom of Information Act. We also ask as a courtesy
to the Committee discussion and your neighbors in the audience that you
silence cell phones and pagers. Thank you.

CHAIRMAN SALOMON: So as usual given that the membership changes
particularly with ad hoc members, I always like everyone to introduce
themselves briefly and just explain in a sentence or two what your
general area of expertise is. Richard, do you want to start?

DR. MULLIGAN: I\'m Rich Mulligan from Harvard Medical School and
Children\'s Hospital. My lab works on gene transfer and stem cells.

DR. ALLAN: I\'m Jon Allan from Southwest Foundation for Biomedical
Research. I work with SIV pathogenesis. I\'m basically a virologist.

DR. KURTZBERG: I\'m Joanne Kurtzberg. I direct the Pediatric Bone Marrow
Transplant Program at Duke University and also the Carolinas Cord Blood
Bank at Duke.

DR. TSIATIS: I\'m Butch Tsiatis from North Carolina State University and
I\'m a biostatistician.

DR. STRONCEK: Dave Stroncek from the Department of Transfusion Medicine,
NIH Clinical Center. I\'ve been involved with unrelated stem cell donors
on and off for a number of years.

DR. HOROWITZ: Mary Horowitz from the Medical College of Wisconsin, Bone
Marrow Transplant Unit. I\'m the Director of the International Bone
Marrow Transplant Registry. I spend most of my life analyzing the
outcomes of hematopoietic stem cell transplants.

MS. LAWTON: I\'m Alison Lawton. I\'m the industry rep. I\'m also head of
registry at Genzyme Corporation.

DR. RAO: My name is Mahendra Rao from the National Institute on Aging. I
work on neural stem cells.

CHAIRMAN SALOMON: Dan Salomon, I\'m from Scripps Research Institute.
I\'m working on organ and cell transplantation, gene therapy and
xenotransplantation.

EXEC. SECRETARY DAPOLITO: Gail Dapolito, Executive Secretary of the
Committee. I would also like to introduce Rosanna Harvey, the Committee
Management Specialist.

MS. WOLFSON: Alice Wolfson. I\'m the consumer representative on the
committee.

DR. HARLAN: David Harlan. I work for National Institutes of Diabetes,
Digestive and Kidney Diseases where I study diabetes and immunological
therapies for transplant.

DR. HIGH: Kathy High. I am at the University of Pennsylvania and the
Children\'s Hospital of Philadelphia. My interests are in the molecular
basis of blood coagulation and gene transfer for hemophilia.

DR. LINDEN: Jeanne Linden from the New York State Department of Health.
I\'m a pathologist and transfusion medicine physician. In addition to
many other facilities, we license stem cell banks to do business in New
York.

DR. HARVATH: I\'m Liana Harvath. I\'m from the National Heart, Lung and
Blood Institute, Division of Blood Diseases and Resources and have had a
long standing interest in leucocyte biology and before going to NHLBI I
worked at FDA for 19 years and worked in the stem cell policy area.

DR. LITWIN: I\'m Stephen Litwin. I\'m a medical officer at the Food and
Drug Administration.

DR. LAZARUS: I\'m Ellen Lazarus. I\'m also a medical officer in the new
Office of Cellular, Tissue and Gene Therapies. I\'m a recent transplant
from the Office of Blood and I\'m at the FDA.

DR. SOLOMON: I\'m Ruth Solomon. I\'m from the Division of Human Tissues
in the Office of Cellular, Tissue and Gene Therapies. I\'m a
pathologist.

DR. NOGUCHI: Phil Noguchi, Acting Director of the Office of Cellular,
Tissue and Gene Therapies.

CHAIRMAN SALOMON: Thank you all. It just dawned on me as we went around
here that this is more like the original BRMAC that I joined where it
was a very strong group in stem cell transplantation and particularly at
that time in bone marrow transplantation. That was a lot of what we were
doing. It\'s interesting how the pendulum swings. It\'s fun to have
everybody back again. Phil, I think you are up next.

DR. NOGUCHI: I\'d like to ask Karen Weiss to join me at the podium. As
with any committee, we like to think of the committee not just as a set
of people to give us advice but as really a team of colleagues that
takes time to develop. We need to have time to plan, a time to practice,
a time to actually come together and then to make some decisions.
Unfortunately as with all teams, we have rotations through this.
Tomorrow is Dan Salomon\'s last meeting.  Both Karen and I have enjoyed
his leadership of this committee over a number of years and we would
just like to say a few words but it won\'t be too embarrassing. Karen.

DR. WEISS: Good morning, everybody. My job this morning is fairly
straight forward and quite pleasant for a change. I have the distinction
of acknowledging and thanking you, Dr. Salomon, on behalf of the Office
of Therapeutics Research and Review currently still in CBER for all that
you have done for the agency, first as a member of the BRMAC and more
recently over the many years as chair of this advisory committee.

During the many hours of deliberations at these meetings where the
Agency sought and received the Committee\'s advice on important cutting
edge areas of medicine and basic science, you have been a tireless
champion of the public health. I specifically recall one meeting, it
might have been the islet cell meeting or it might have been one of
those many meetings on gene therapy and long term follow-up that never
would end ? anyway, it was one of those meetings where another Committee
member was rotating off the Committee. As typical, the Agency officials
came up and acknowledged this member and presented him with a plaque.

Subsequently the meeting happened and as typical of all of the meetings
I think that have characterized your term, the topic was quite complex
and complicated and controversial. The FDA questions as typical were
quite convoluted and long. As most people know, my former boss who has
recently left the Agency, Dr. Jay Siegel, was a master at editing and
making questions long and convoluted but also like you, a tireless
champion of the public health. It was one of those types of meetings and
I think somewhat frustrated, you kept pushing and challenging the
Committee to be more responsive and more direct in their advice to the
Agency. I think finally you said look guys at the end of the day when my
term is up I want to get one of those plaques too so you have to do
better.

Dan, you certainly have earned your plaque. But more than just the
plaque, you have certainly earned mine and the Office and the Agencies\'
deep respect and appreciation for all that you have done over the years
in your championship for what was right and for providing the best
advice to the Agency and ultimately for enhancing the public health from
in utero transplantation to islet cell transplantation to
haplo-identical transplantation to stem cell factor for peripheral blood
progenitor cell transplantation as well as for the more recent advice on
gene therapy issues including and not last of course the long term
follow-up where we kept pushing. We brought the issue back not once, not
twice but three times. We finally got some really good solid advice. For
all of that, I wish to personally thank you. You have left behind a
legacy. It\'s been a great experience having you on the Committee and as
the Chair. Thank you very much.

DR. NOGUCHI: I\'m not going to spend too much more time on this. Karen
has actually gone through many if not all the highlights over a
relatively brief but an extraordinarily exciting time. I\'ll just give
him my one little dig here. A number of years ago actually Dan had been
on fellowship from his position at Florida to the NIH. Of course like
any good FDA person, I tried to recruit him to FDA. He finally was
gracious enough to tell me in person that well I have this fairly nice
job at Scripps. It\'s in San Diego. My wife is from there. It\'s really
hard to turn down the FDA but ?-

At that point, I said don\'t worry. You\'re always going to be working
with us.  Even though, Dan, this is your last meeting for the BRMAC as
chair, don\'t worry. We\'ll keep asking you back.

We have here two letters for you. One is from the Agency representing
our appreciation for just an outstanding job done. It\'s also a bill for
what you are going to do for us in the future. First I would like to
give you that. Then this is our plaque that you so richly deserve and
wanted after all these years. Dan, my very best and thank you so much
for helping us with all these difficult issues.

CHAIRMAN SALOMON: As my swan song here, all I can say is the privilege
of serving as chair of this Committee over the last several years is
something that I can\'t imagine having not had that experience. So for
me, it\'s been a privilege. The quality of the people that I\'ve met and
friendships that I\'ve made during that as well and just the
intellectual stimulation and the feeling of doing something right, it\'s
just a remarkable mix for me. I thank everybody here. The FDA has been
so supportive and so flexible and so interesting.

All I can say is that if you think about it as a theme in America, the
idea of voluntary service to the Government is something that began with
the very start of this country. It is something that\'s extremely
precious as part of being an American. So it\'s been a real privilege to
participate in something as fundamental as that. Thank you.

What we can do now is get started with the FDA introduction to the
topic. For that I introduce Ellen Lazarus.

DR. LAZARUS: Thank you, Dr. Salomon. Welcome everyone. As you know, the
focus of the first meeting this morning is on unrelated allogeneic
hematopoietic stem progenitor cells from placental umbilical cord blood
for hematopoietic reconstitution hereinafter referred to as cord blood
or in my slides UCB.  The CBER presenters and the invited speakers will
be presenting efficacy data for the use of minimally manipulated cord
blood for hematopoietic reconstitution for recipients of particular age
groups.

A brief overview of the field of cord blood research and
transplantation. This history begins in the waning years of the 20^th^
century. In 1988, Dr. Gluckman and colleagues performed the first
reported transplant of cord blood in a child with Fanconi\'s anemia
using an HLA matched sibling donor. Then in the early to mid-90\'s, more
reports were published demonstrating the feasibility and efficacy of
related and unrelated HLA matched and mismatched cord blood
transplantation.

By 1993, there were three public cord banks established located in New
York, Dusseldorf and Milan. In 1997, Drs. Broxmeyer and Cooper published
a report extending their earlier observations on the effects of frozen
storage of cord blood. In this study, they assessed recovery of
nucleated cells and proliferative capacity of the thawed hematopoietic
cells in vitro demonstrating potential functionality of cord blood
cryo-preserved for up to 10 years. They have actually recently published
a report extending their observations to cord blood cryo-preserved for
15 years.

Then in 1998, Drs. Rubinstein and colleagues published their landmark
study demonstrating that stored cord blood is a potentially useful
source of hematopoietic stem cells for patients who lack an HLA matched
related donor. Next slide.

Now more the 2000 unrelated donor cord blood transplants as estimated
have been performed since the beginning of this history and evidence is
accumulating in the published literature suggesting comparable efficacy
between HLA matched bone marrow and cord blood for pediatric patients
with acute leukemia. Some preliminary reports of successful unrelated
donor cord blood transplant in pediatric patients with certain immune
deficiency disorders and in-born errors of metabolism have been
published. In contrast the experience with the use of cord blood for
transplantation in adults with hematopoietic malignancies is more
limited.

Now one of the potential advantages of cord blood as a source of
hematopoietic stem cells for transplantation is the relatively rapid
availability of a particular unit once it\'s been identified in a
registry as being a good match for the patient. But it is difficult to
exactly determine the number of facilities storing unrelated donor cord
units and the exact number of units stored within. However we can use
some Internet resources to come up with some reasonable estimates.

We have the very informative National Marrow Donor Program (NMDP)
website that lists approximately 30 cord banks. Not all of these
facilities participate in the program. Some of these listed banks store
unrelated donor cord units exclusively while others store both unrelated
donor units as well as units for family or autologous use.

Numerous cord banks have been established outside the U.S. as well as
evidenced by the listing on the Bone Marrow Donors Worldwide website.
Using these sources of information, it can be estimated that there are
over 60,000 units of cord blood potentially available for unrelated
transplantation in the U.S. There are over 130,000 cord blood units
stored in facilities worldwide.

Now the quality of the stored cord blood products is essential to assure
successful hematopoietic reconstitution which depends on the number of
functional progenitor cells transplanted. The assurance of quality
begins in the moments after delivery of the infant when first after
assuring safety of the donor and mother. The collection staff must
optimize collection of the cord blood.

Now many of the establishments that collect, process and store cord
blood products follow voluntary standards published by AABB, FACT,
NetCord, and NMDP. Nevertheless, the quality and characteristics of
stored cord blood may vary due to both uncontrollable donor factors such
as placental size and weight and controllable factors such as collection
methods, product containers and different processing and storage
systems. Next slide.

We are actually rather limited in the assays that we can use for
determining the number and quality of the hematopoietic progenitor cells
in each of these units. Nucleated cell counts are performed and these
counts include all differentiated and undifferentiated nucleated cells.
Viability assays are invariably performed either using dye exclusion
methods or flow cytometry.

Colony forming unit (CFU) culture assays are performed on many of these
products and in these assays colonies from individual cells grow. These
cells are plated on the semisolid medium. The limitation of these assays
we\'re all familiar with. The reading of them is somewhat subjective.
They are difficult to standardize. They take 10 to 14 days approximately
to grow. In contrast, CD34 cell enumeration by flow cytometry is fast.

Our speakers today will give more information about the role of these
different assays in the manufacturing and selection of cord blood units.
So that was just a very brief summary of medical and manufacturing
issues that we need to keep in mind and will be used to inform our
discussion today.

The purpose of our meeting is to review first CBER analyses of clinical
outcome data that were submitted to the FDA regarding safety and
efficacy of cord blood for hematopoietic reconstitution. We will have
invited speakers present data from recent studies of cord blood
transplantation, Drs. Wagner, Rubinstein and Chao. We will seek the
Advisory Committee comments on factors the Agency should consider in
determining safety and efficacy of cord blood and on the role of CD34
cell count in selection of cord blood and other measures of quality that
should be considered. So I look forward to a very interesting discussion
today. I\'ll turn the podium over to my colleague, Dr. Steve Litwin.

DR. LITWIN: Good morning. Transplants using allogeneic stem cells are a
therapeutic adjunct in a large number of life-threatening diseases.
First slide please. They include hematologic and non-hematologic
malignancies and an increasing number of in-born errors of metabolism,
phagocytic and histiocytic disorder storage diseases.  The IBMTR, and
this is an estimate from the year 2000, estimates that there are
approximately 15,000 related and unrelated transplants during a year.

Despite the increasing activity which has leveled off somewhat, the
restrictions on transplantation, particularly allotransplantation,
remain on the supply side. What I mean by that is that the restrictions
are on the availability of suitable well matched units for donor
transplant which would in FDA terminology lower the risk. The emphasis
then is on the sources of stem cells. Next slide please.

These are the classic sources of stem cells. With the development of
growth factors, it was possible to mobilize stem cells from tissue
sources into the blood and collect them by phoresis. The most recent
addition to available sources of stem cells is umbilical cord blood.
Next slide please.

This now serves as a useful and valuable alternative to bone marrow and
peripheral blood. There is obviously much less patient experience to
date. About two years ago, CBER set up a working group whose mandate was
to look at the efficacy of stem cells from different sources with
emphasis on umbilical cord blood and particularly with interest in the
two problems which we are going to deal with today. That is the
enumeration of umbilical cord blood in grafting cells and finally the
age related risk of UCB transplants.

It is my task today to focus on the latter which is the age related
risk. I would like to do that by presenting data that had been submitted
to the docket from the New York Blood Center and which was analyzed by
FDA statisticians.  Before I do that, I would like to emphasize some
salient features of umbilical cord blood and talk somewhat about the
differences in engraftment kinetics. Next slide please.

There are three things I would like to emphasize. First of all, cord
blood units have a smaller number of engrafting cells available than do
the classic sources, bone marrow and peripheral blood. Secondly, cord
blood may represent a different mix of stem cell populations. Finally,
cord blood cells may have different biological properties mainly
suggested by laboratory studies. Next slide please.

This is data taken from a study of umbilical cord blood from a single
study by Wagner, et al. The total nucleated cells are listed on the top
because this still is the method generally used by many centers to
estimate the potential usefulness of a cord blood unit as a donor
source. You look at the number of CD34 positive cells which are listed
here, which may differ between studies, but are listed here as 280,000
per kilogram of body weight of the recipient. This can be compared to
the number of CD34 cells which are usually included in the Infusaid in
the peripheral blood transplant which is roughly a log larger. That is
about 10 times larger between two and five million CD34 cells per
kilogram.

Perhaps the good news is in the number of T-cells, CD3 positive cells,
which are listed here for the cord blood unit in this study as eight
million per kilogram of body weight of the recipient. That\'s five to
ten times lower than the amount given in an T-cell non-manipulated
peripheral blood transplant. Next slide please.

Because of this factor, that is the limitation on the number of
available engrafting cells, the majority of the umbilical cord blood as
a transplant source to date has been confined to younger recipients.
These are data once again from the IBMTR. They are divided into the
younger age group on the left, less than 20 years, and the older age
group on the right.

If we just look at the period of time from 1998 to 2000, you can see in
the younger group that there was a predominance of bone marrow
transplants as opposed to peripheral blood, 69 percent vs. 17 percent.
About 15 percent of the transplants and this is up to year 2000 were
from cord blood sources. In the older age group there\'s a closer to
equal number of sources from bone marrow and peripheral blood, 53
percent vs. 43 percent. A very small number were from sources from cord
blood. Next slide please.

The second point is that umbilical cord blood may represent the
different mixture of stem cells of different levels of maturity and
different lineages. The incidents of graft-versus-host disease is
suggestively lower after cord blood transplants. At this particular
point in time because of the lack of comparative studies, there remain
many questions about whether the anti-tumor that is allogeneic effect
noted in allo-transplants is present to the same diminished or increased
three in cord blood and particularly whether cord blood cells\' ability
to reconstitute the immune responses to infectious agents and to other
wide variety of antigens needed in a reconstituted recipient. Next slide
please.

The third point is based mainly on laboratory data which suggests that
cord blood cells have different biological properties. They seem to have
increased growth and engraftment potential. Colonies formed in culture
are larger. Cord blood cells can repopulate immunodeficient mice without
cytokine support in sharp contrast to peripheral blood as a source of
stem cells in these laboratory studies. A subset of cells, CD34
positive, 38 negative, have a higher comparative cloning efficiency from
cord blood in response to cytokines and there are several reports that
suggest that cord blood cells may be more responsive to ex vivo
expansion, a relatively critical area in experimental growth and
experimental efforts to try to increase the supply. Next slide please.

The engraftment kinetics of cord blood cells also differ. In the first
vertical column marked HLA mismatched greater than two, the percentages
in each of three studies are shown. These are three illustrative studies
of cord blood used as a source of stem cells. You can see that the
percentages of mismatched greater than two mismatches at the HLA loci
differ here. These studies are different.

Despite that, what is evident is the relatively high incidents of
engraftment failure, the 12 percent, 29 percent and 19 percent, can be
compared. These are only illustrative figures. I don\'t mean that they
represent the whole field. They can be compared to the anticipated one
to five percent one would expect in most homologous and well matched
allotransplants. The neutrophil engraftment time, that\'s the fourth
column, is about twice as long in cord blood as it would be anticipated
from the historical literature from peripheral blood or bone marrow. The
platelin engraftment time is two to three times as long as once again
one would anticipate from historical literature.

Interestingly enough, the incident of acute graft-versus-host disease
grades three and four are relatively similar than these three selected
studies. At 20 percent, these figures are lower than one would
ordinarily anticipate from studies of patients at this particular
clinical status but there is no comparative study to make any definitive
statements. Next slide please.

I would like to turn now to some of the statistical analyses that we
performed. To begin with, these were done by Dr. Jean Wang of our
Department of Bio Statistics with Dr. Peter Lachenbruch serving as a
consultant. The source were primary data on 562 cord blood transplants
which were collected at the New York Blood Center. The recipients for
the most part were myeloablated and for the most part were under 20
years of age. The data was collected from 1992 to 1998. Next slide
please.

There were four clinical outcome studied, neutrophil engraftment both
the presence of neutrophil engraftment, yes, no and the time to
neutrophil engraftment, platelin engraftment once again similarly,
disease-free survival and severe acute graft-versus-host disease, grades
three and four.  The co-variate studies were multiple age, weight, the
total number of nucleated cells per kilogram of body weight of the
recipient and the HLA disparities. I bolded and enlarge the age because
we spent most of our time focusing on this aspect of it. The remainder
are competing risks and that is acknowledged. Next slide.

The goals were threefold: first of all, to compare the risk of bad
clinical outcomes between the very youngest members of this 562 patient
group and the oldest members in the study; and if there was an increased
risk to see the trend or shape of that risk as to whether it was gradual
cline or a very sharp point of demarcation by age that was a cut point;
and finally to determine if different clinical outcomes, the four
outcomes we looked at, showed the same trend. Next slide please.

The methodology for the most part that I will show you, there was a
substantial number of analyses done and I have chosen three to try to
illustrate the points I\'m making, was simple logistic regression
analysis and time-to-event analysis on the same clinical outcomes. Some
of the other approaches were listed. Next slide please.

The strategy for the most part was to compare different age cohorts.
I\'ll go into some of the limitations on that data in a moment. The data
presented in three different types of analyses. The clinical outcomes
were compared between the different age cohorts and trends searched for.
The clinical outcomes in consecutive age cohorts were also looked at and
sharp differences were searched for. Finally the clinical outcomes in
dichotomous age cohorts, that is the population was divided into two
parts and comparative differences were looked for. Next slide please.

This is the first set of data. These essentially are the clinical
outcomes by the age cohort. I apologize for the slide. There was no
other way I could get that much information on a slide. I know most of
you in the audience cannot read that. That\'s why I bolded the top and
the bottom, the youngest and the oldest, in red. You will have to take
my word for it that there are differences but I will show you the graphs
on the same data in just a moment.

If you look then at the youngest members of the group, the first column
shows the age group in years. The first group is 0, 1 and 2 years of
age. The second group is 3, 4 and 5 years of age, etc. Look at the
clinical outcomes. Then compare them to the bottom which 18, 19 and 20
years of age and 21 on up. One can see that the poorer clinical outcomes
are found in each of these four. That is 82 plus percent ANC engraftment
as compared to the older group there were about 49 percent engraftment
and so on.

The next thing we did besides the inspection and comparison of the upper
and lower age groups is to look at the trend. Once again I know you
can\'t see this but it appears to be in at least three of these four
clinical outcomes a gradual change. I\'ll show the graphs in just a
moment. Next slide please.

This is the same data. This is neutrophil engraftments starting in the
youngest age group and going to the oldest age group bearing from 80
percent success rate to roughly a little over 40 percent success rate.
Next slide please.

The same is true and perhaps even more dramatically in platelin
engraftment where if you look hard enough you can imagine that there\'s
a sharp cut point somewhere around the middle of that range. I don\'t
want to make any claims for statistical significance to this except to
say that it would seem apparent that the increase in risk from the
youngest to the oldest is quite clear. Next slide please.

This is disease-free survival. Once again we see the increased risk from
one to the other age cohort. Next slide please.

Acute graft-versus-host disease (aGVHD). Here the negative clinical
outcome would be an increase in the number of patients with grade three
and four. This is also evident but the pattern is clearly less marked
here and very hard to interpret. Next slide please.

The second approach that we took was to look at consecutive age cohorts.
If you look at the bottom of the slide, on the lower left side of each
of the panels, we have the age cohorts which were compared. On the upper
left panel to the left, it\'s age cohorts age zero through two compared
to three through five. It\'s a three year age cohort. If we look along
all of the panels we get comparisons of consecutive age groups looking
for a change.

The most marked differences are in the first panel that is the ages zero
through two, three to five. In the middle ages nine through 11 compared
to 12 through 14. Then in the last one. The first two that I showed you
are statistically significant. The last is not mainly because the
numbers in this comparison are much smaller. Next slide please.

Finally, a third approach was to divide the population in half and to
compare the two paths. In this case, we looked at the ages zero to 12
and ages 12 onward.  The ages zero to 12 are here in red. The ages of 12
onward are in green. The difference between these dichotomous age
cohorts is significant. The figures are once again hard to read but they
are significantly different. The next slide please.

These are the same data from this approach. In addition to doing a
Kaplan-Meier proportion and percent of these two dichotomous cohorts,
and you see the figures here, we also did odds ratio. This is the
technique frequently used in oncology. The odds ratio above two are
generally considered significant.

As you can see the odds ratios are high with the exception of acute
graft-versus-host disease which because it has a reversal that is a
higher number here would be negative outcome is probably less than half.
We did not invert these. Next slide please.

To finish up, we were concerned with whether comparing age cohorts was a
fair tactic. That is whether the cohorts of age even from the same study
might have different characteristics, whether they were balanced. We
looked for a number of features. Here is a comparison between the
dichotomous cohorts for the number of mismatches. There are differences
just to illustrate one which was present in 44 percent of the younger
age dichotomous cohort and present in 27 percent of the older age
cohort. Next slide please.

The same comparison was done for diagnostic groups. The term here
\"acquired\" is acquired bone marrow failures. Once again in the younger
group, the major differences are the higher number of genetic disorders
for which the transplant was performed 29 percent versus eight percent
in the older age group and to the consequent lower number of leukemic
states for which it was performed. Next slide please.

These data were extremely informative. However they were not definitive.
There are a number of potential sources of bias. The retrospective
analysis of data was done for data that were not essentially primarily
structured for the questions that were posed when the study was
designed. There are fewer patients in the older age groups. There are
certainly some imbalances which we are still analyzing between the age
cohorts. There is anticipated variability because the underlying
transplantation protocols and the disease states are widely varied in
the transplant field and this would be anticipated. Finally, we know
that competing risks will exist for the weight of the subject or the
number of total nucleated cells transplanted. If I showed you data that
emphasized that, I\'m sure it would look very similar.

In conclusion then, next slide please. The risks of negative clinical
outcomes increase from young children to young adults with age. The
comparative data between ages zero and 20 plus indicate a climb of
increasing risk with age, most marked for engraftment, also present for
event-free survival, less so for acute graft-versus-host disease. There
is no sharp cut point demonstrable but there\'s a trend to change
somewhere midway in this region of the zero to 20 age group and the
presence of competing risks are observed. Thank you.

CHAIRMAN SALOMON: Thank you, Stephen and Ellen, for really nice
introductory presentations. We are exactly on time. That\'s vaguely
discomforting. I mean there is no triumph in finishing on time if you
are on time the whole day. I initially said that we would have no
questions or very limited questions after this.

I have to take the Chair\'s prerogative to ask one though. I think you
were good, Stephen, pointing out all the different possible biases at
the end of looking at outcome parameters like for example disease-free
survival. Given that, one, it\'s very reasonable to presume that the
disease states for very young children and 20 to 29 years old are
dramatically different and therefore the risks are different. So I think
there will be more discussion of that.

The other point that I would like to ask you about is the incidence of
acute graft-versus-host disease.  That\'s a little surprising in that
it\'s essentially the same cord blood that you are getting. Of course,
you are getting more total but the same amount per kilo. Really young
children shouldn\'t have any reduced immune response since in organ
transplantation we have the most trouble with five years and younger.
Even if you throw out some renal vascular issues in the really young
children, a lot of it is a very aggressive type of rejection. It\'s just
a little bit surprising to see that kind of a difference. Do you or
anyone else have any comments about that?

DR. LITWIN: We focused on the zero to 20 age group. I think there is a
lot of literature suggesting that acute graft-versus-host disease is
certainly at a much higher risk in an older age population.

But it would seem from this data, and this was only a touch of data
essentially speaking, that that increased risk with age is not down in
the zero to 20 range but maybe somewhat higher. The transplanters
notoriously have been hesitant to transplant patients under 50 and 55
and now it\'s 60.

I would suggest that, and there are probably people who know much more
about this than I do, that that risk continues to increase with older
age.  Whereas the risks in engraftment and in disease-free survival
perhaps may be more focused at the younger age group. I think Dr.
Rubinstein probably has some feelings about that. I know he is published
on that issue.

DR. KURTZBERG: Two comments. First in response to that, I think it\'s
well known even from marrow and stem cell transplantation that younger
patients have a lower risk of acute and chronic GVHD and the younger the
patient as well as the younger the donor the lower the risk. Second I
wanted to ask Dr. Litwin. Did you do an analysis of chronic GVHD,
because that\'s an important question to ask about cord blood?

DR. LITWIN: It\'s not finished. We did some analyses. They did not seem
to be increased. We are still looking at that.  There is data on that
within this population group. It does not show much.

DR. KURTZBERG: I think it shows a lower incident compared to bone marrow
transplants.

DR. LITWIN: We don\'t have many people over 20 so it\'s hard to do.

DR. KURTZBERG: No, I just mean in the entire cohort. Did you look at the
incidence of chronic GVHD?

DR. LITWIN: Not yet, no.

DR. HARLAN: You said that if you made similar graphs showing weight
versus success rate no matter what that you would imagine that they
would look the same. I wonder within any single age group if you tried
to break it down by weight as opposed to age to see if it\'s a weight
thing like I suspect, in a dose of stem cell per weight as opposed to
age.

DR. LITWIN: We tried to break that down but we found that the data were
similar for weight. We did not focus on it obviously but we were not
successful in dissociating the two.

CHAIRMAN SALOMON: I guess what I come away with is if it\'s true,
particularly based on Dr. Kurtzberg\'s comments, that the incidence of
acute GVHD follows the age of the recipient is not a direct factor of
the use of the cord blood, then one of the questions that we\'re going
to get to at the end of the day is what is the potential use of cord
blood in older individuals.

One way of interpreting this data would be you go from about a 17
percent incidence of acute GVHD to about 40 percent in the 20 to 29 age
cohort. So it would be bad to use cord blood in that group.  But if
it\'s really tracking with the recipient age, then this has nothing to
do with cord blood. One could then take that data and make a different
conclusion. Joanne.

DR. KURTZBERG: The comment that GVHD does track with age is true of any
stem cell source, but you have to consider that these are mismatched
transplants and to compare the incidence of GVHD as it would be to other
adult stem cell sources. In that case, it is lower in both the younger
children and the adults as compared to more traditional stem cell
sources. So there are two points. GVHD does relate to age, but there\'s
a difference here as you compare it to other stem cell sources.

CHAIRMAN SALOMON: And that\'s perfect. That\'s what I\'m trying to set
the stage for since that\'s where we want to go in the afternoon.

DR. HOROWITZ: I would say that the data in bone marrow transplants very
much cuts at puberty. Because somewhere in adolescence it is not a
continuous relationship between age and outcome. There is a threshold
effect. There are fewer differences between 20 year olds and 40 year
olds than there are between 10 year olds and 20 year olds. Many of these
data could be reproduced in the population of patients who receive bone
marrow transplants.

DR. LITWIN: I didn\'t mean to imply that there was a gradual change. I
really meant to say that we failed to show a sharp cut point. I think
that\'s somewhat different. What the truth may be may be lost in the
variability which is inherent in this study and other limitations.

DR. TSIATIS: I have some questions about the analysis. Was the amount of
follow-up the same for all the different age groups? That was one
question and then I have a follow-up.

DR. LITWIN: Yes.

DR. TSIATIS: They all followed the same amount of time. The second
question is it was hard sometimes looking at the graphs because they
were small. When I looked at the nine to 11 group in those pair-wise
comparisons, the nine to 11 group the survival curve in one comparison
looked different than the nine to 11 group in the other comparison. When
I actually just plotted the survival at 1,000 days, it looked to me like
the biggest cut point was less than three and greater than three rather
than 10 or 13 that you used.

DR. LITWIN: Dr. Wang was the statistician who did these studies.

CHAIRMAN SALOMON: Can you come to the microphone please.

DR. WANG: All the survival curves applied the same statistical methods,
Kaplan-Meier survival curve.  So the data just shows the group 10, 11,
12 is a significant difference from the consecutive age group 13, 14 and
15.

DR. TSIATIS: The question I had was that you had two comparisons with
nine, ten and 11 comparing to a lower group and then to a higher group.
The survival curve for the nine, ten and 11 in those two analyses looked
different to me.

DR. WANG: No, I think that the analysis is applied the same.

DR. TSIATIS: I understand. I\'m saying the pictures that were shown
looked like one had a survival rate of about 40 percent and the other
one looked like it had closer to 20 percent. I\'m just wondering about
the same cohort in two different curves looked different to me. The only
thing is I\'m a little concerned about the quality of the analysis.

DR. WANG: Can you show me the ones that you are talking about?

CHAIRMAN SALOMON: What I was going to suggest is ? that\'s a key point
to bring up, Butch. Perhaps to give staff a chance between now and
through the break to review the curves and maybe make a response to that
when we come back. Would that be appropriate?

DR. LACHENBRUCH: This is Tony Lachenbruch. Butch, perhaps we could meet
at the break and we\'ll settle it, I hope.

DR. WANG: Sorry about that.

CHAIRMAN SALOMON: No, it\'s okay. I think it\'s an important function of
the group to do a reality check on the way the data looks. I think you
need a little bit of time to go over it and give us feedback. Butch, if
you would take responsibility for it and just give the group a response
to that.

I had one last question. This is the dumb questions chairs get to ask.
But if one looks at the clinical outcomes by age cohort in the zero
through two group, you have a 58 percent platelet engraftment that then
falls in the successive age cohorts. Does it really mean 40 percent of
these children never get platelets and are life-long dependent on
platelet transfusion? Of course not, so I just don\'t understand that
number.

DR. LITWIN: A lot of them are censored because of events including
death. That is a limitation of the data that there is no question that
these are risky transplants.

CHAIRMAN SALOMON: I just wanted to correct an impression that someone
might get just looking at these numbers that it\'s only effective in
reconstituting the marrow in 60 percent of the kids. I don\'t think
it\'s that. I think that if the kids survive, I would think that these
are relatively effective in 100 percent of the cases. Is that fair? I
just want to make sure that impression is correct.

DR. HOROWITZ: I think that\'s fair that most recipients who survive
become platelet transfusion independent but it\'s a high competing risk
environment. So in part with cord blood transplants, because the
recovery is slower, patients succumb to the competing risks more
frequently before they get platelet recovery, not that they succumb to
the competing risk more frequently overall.

CHAIRMAN SALOMON: Steve, did you have a comment?

DR. LITWIN: No.

CHAIRMAN SALOMON: Joanne.

DR. KURTZBERG: Since I take care of several hundred surviving cord blood
patients, mostly children but a few adults, I mean I can tell you that
surviving patients fully engrafted are not transfusion independent at
all and have normal counts. It may take up to even 18 months
post-procedure to get to a normal platelet count of 150,000.

MS. LAWTON: I just have one question. Did you look at the two separate
cohorts specifically by disease for the patients to see whether there
were any differences in outcomes for the same disease in the two
different age cohorts?

DR. LITWIN: No, we did not. We thought the numbers representing the
different diseases were too small to get us the type of data that we
were after. We did not.

DR. HOROWITZ: Related to that, did you make an attempt to identify
patients by risk category? Even though there are multiple diseases, you
can sort patients into low, intermediate, high risk of failure based on
a wealth of data in other transplant setting.

DR. LITWIN: No, we did not.

CHAIRMAN SALOMON: I think that\'s a great start because what comes out
of this first discussion is just how extraordinarily difficult this very
important type of analysis is. You have to do this analysis, and yet we
point out how difficult it is and then how careful one has to be about
the interpretations one makes from it because of this complex and
competing risk factor model that\'s operant here. So I think that\'s
going to be a challenge for us to come to grips with later this
afternoon. I think that was a very good beginning.

Now I\'m comfortable. We\'re about almost 15 minutes running late. It\'s
this idea of being on time for my last meeting. It was not just going to
work. With that, I would like to introduce at this point John Wagner to
make the first of three guest presentations on umbilical cord blood.

DR. WAGNER: I think it\'s important for me to go back and actually
introduce to you some of the background information that really leads us
to then have some sources of comparison by which to evaluate cord blood.
Let me just start off because I have quite a few slides to show. Next
slide.

Obviously when we are going back and thinking about how cord blood is
successful or not successful, we have to do it in the context of what we
know about bone marrow transplantation in general. Obviously, you have
all seen this before that there are a number of obstacles that we are
trying to overcome in terms of facilitating transplants for our
patients.

One is that there is a restriction on the basis of HLA. There\'s a long
search interval. Many patients will enter at either a higher risk
category or will die while waiting for a transplant. For those that do
have a transplant donor, there is still a high risk of non-relapse
mortality as a result of graft-versus-host disease and opportunistic
infection. There is poor survival in adults in most diseases with the
exception of CML in chronic phase. When we are thinking about the
success or failure of cord blood, we have to keep these parts in mind.
Next slide.

The National Merit Donor Program is trying to overcome some of these
obstacles by improving the level of HLA typing, by initiating a new
study in terms of looking at the ultra-urgent searches and seeing if we
can find donors more quickly, and that\'s been an overwhelming success.
There\'s been a focus on research in non-relapse mortality. Obviously
the reason why we are here today is that one of the things that has
occurred as a result of these limitations with bone marrow transplants
is we are exploring alternative stem cell sources. Next slide.

The first thing we need to do is we need to evaluate what are the risk
factors that will predict outcome after bone marrow transplantation.
Before I go on to the next slide, I would like to show one transparency
because it relates to the conversations we just had. Although we
certainly could provide you with a lot more information, let me first
talk to you about survival.

We actually could do this for all factors whether it be
graft-versus-host disease, engraftment or survival. Actually to make a
point that was addressed a few minutes ago, is what do you expect in
terms of engraftment? Dr. Litwin, the one thing that you said that was a
bit misleading although not incorrect was that it depends upon how you
evaluate engraftment. Now most of the reports today would record it as a
cumulative incidence. There you would expect a graft failure rate with
unmanipulated bone marrow of 11 percent, not one to five percent. So you
have to have that in the context of what you expect with cord blood
transplant. That\'s for all ages put together.

Now in terms of survival, we do a Kaplan-Meier probabilities, and I\'m
going to show the multi-variate in a second. However, this is a critical
piece of information in which to evaluate the success of cord blood. As
you can see here, there is a profound effect of age on the overall
outcome. This is now mixing patients with all different types of
malignant disease.

As reported by the National Merit Donor Program this was just done as of
this past week, you can see here that there\'s a tremendous impact of
age on the outcome in this analysis where you see those under the ages
of 14 having the best outcome. I would suggest to you that there is a
break point between 14 and 15 in that younger patients doing better than
the older patients.

Then when you see between 15 and 45 there is not a tremendous difference
at least in the way we have divided the patients up in survival. Then
once again in those are over the age of 46, the outcome is inferior.
This is mixing patients with CML as well as acute leukemia. CML patients
will do better than those with acute leukemia.

This is also mixing patients with a variety of stages of disease at the
time of transplant. If you would like, I have all the data here to be
able to show you at the break perhaps which were the other variables
that impacted on outcome.

We can turn this off and now go back to the results of the multi-variate
analysis that was performed on the same dataset by the National Merit
Donor Program.  What you can do is see the relative risk in terms of age
and its impact on survival. With the relative risk assigned to the
younger age group of one, you can see with each increment we find that
there is a significantly higher risk of death as a result of age alone.

You also see that when you look at the effective diagnosis, and although
I didn\'t put it all out and I can show you the actual data, is if that
you assign a relative risk of one to patients who have CML in chronic
phase, every other disease and disease stage has a significantly worse
outcome compared to CML in chronic phase. When you look at the impact of
Karnofsky status and if you assign it a relative risk of one to those
with the worst Karnofsky performance status you can see that those with
the better Karnofsky performance will do better.

This is the result of a multi-variate analysis demonstrating that there
are factors that we have to keep in mind: age, diagnosis, Karnofsky
performance as well as HLA and CMV. We have to do this exact same
analysis when we are looking at cord blood because what we are trying to
do is not to compare really young patients to old patients, because old
patients will always do worst regardless of the stem cell source. What
we have to do is we have to compare the results of cord blood with bone
marrow transplantations because bone marrow transplants are the standard
of care, are the gold Standard by which we compare everything else. Next
slide.

So graphically in this older analysis where we compared ?this was now
the analysis of 5,000 and some bone marrow transplant patients performed
several years ago, what you can see here that there is a tremendous
impact of HLA mismatch and outcome. The more closely matched patients
are on the top two lines. The more mismatched patients are on the lower
two lines. This was done to compare T-cell depletion versus
unmanipulated marrow. You can see the overall comparisons are really
virtually identical between the two cohorts. Mismatch itself has a
negative impact regardless of the GVHD prophylaxis. Next slide.

Then perhaps more relevant to the current discussion is that once again
you see when you just use the break point of 18 above or below, you can
see that there is an impact of age. Also patients don\'t do any
differently comparing their GVHD prophylactic regimens if you segregate
them on the basis of being older or younger. Next slide.

This is a result that I\'m just going to show you, the multi-variate
analysis of our randomized trial that was performed by the NHLBI and the
analysis was just completed. I presented this at ASH.

Once again you can see that these are the risk factors that we have to
look at in terms of predicting the ultimate outcome of transplant. We
have to perform this exact same analysis when we are looking at cord
blood again, not comparing younger versus older but comparing what
results we have with the cord blood versus with marrow. So these are
risk factors that we know exist. Next slide.

As a result of these outcomes, we think that there\'s been a variety of
us, several of us in this room, that have looked at a variety of new
directions in trying to improve the outcome of transplantation. As I
mentioned, we are trying to shorten the search time. We\'re developing
new preparative therapies. We\'re trying to figure out new ways of
speeding immune recovery but we are looking at new stem cell sources.

The reason why we have chosen cord blood is because we have data now
that suggest that there is less graft-versus-host disease. Yes, there is
slower engraftment. There appears to be less HLA restriction and it\'s
immediately available. So when we are talking about the outcomes,
remember we are now comparing outcomes in our minds to what we expect
with bone marrow transplants. Next slide.

Let me now review with you our past experience at the University of
Minnesota. The reason for this analysis is specifically to address the
issue of CD34. It will be self-explanatory as we go through. Next slide.

For purpose of this analysis, HLA typing is A and B serologic level and
DRB-1 at high resolution. Next slide.

In this patient population there were 102 patients. This includes both
adult and pediatric patients but the median age was 7.4 years. Obviously
it\'s predominantly a pediatric population. Although there were
approximately 25 patients that were in the adult age range within this
group of patients. These are patients that only received a full prep.
Next slide.

The median cell dose is 3.1 times 10^7^ nucleated cells per kilogram.
 Again to get to the issue, is CD34 analysis important, we now have
looked at the infused CD34 cell dose at the time of thawing the cord
blood unit. We then did a CD34 analysis so that we know exactly what
went into the patient. This is not the data that was provided by the
cord blood banks.

We similarly looked at the CD3 cell dose that was infused. You can see
here that the incidence of engraftment is 88 percent. This is exactly
the same incidence of engraftment which you would predict with
unmanipulated bone marrow which is 89 percent. The median time to
engraftment is 23 days as compared to 21 days with methotrexate
cyclosporine we see in the unrelated marrow transplant setting. Next
slide.

When we looked at the impact of age on ultimate outcome, you find that
there may be a separation based on the older age patients compared to
younger age patients, but there is nothing significantly different
between these two populations. Next slide.

We also found no difference between those that received more matched
grafts as compared to those receiving more mismatched grafts. What I
should point out to you is that when we did this analysis, we looked at
HLA looking at the graft rejection vector. Though I didn\'t show you the
actual proportion of patients on this slide, which I probably should
have done, approximately 45 percent of the patients received a two
antigen mismatched graft. Approximately 40 percent received a one
antigen mismatched graft. There is only a small proportion that received
a matched graft in this analysis. Next slide.

However where we started seeing a difference is now looking at cell
dose. Once again, it\'s not age specifically. It\'s related to the cell
dose of the graft that goes in. Although this did not quite reach
statistical significance, you do see a beginning of a separation in
terms of the impact of nucleated cell dose on the incidence of
engraftment and the speed of recovery.

This being the incidence and then ultimately the speed as you are
comparing the graft what would happen at 50 percent incidence and what
is the time to recovery. You can see there is a difference. Those
receiving higher cell numbers have a more rapid recovery. Next slide.

Where we started seeing a dramatic difference in outcome and predicting
the time to ultimate engraftment, you can see that CD34 has a very
linear relationship with the time to recovery. I would suggest to you
that this shows you the principle that CD34 is extraordinarily important
in predicting outcome. However there is a problem with this analysis.
That is that CD34 analysis in my institution may not be the same result
as another institution. We\'ll get back to that in a second. Next slide.

Perhaps it\'s more easy to see the impact of CD34 on the outcome of
engraftment when you look at this slide where the patients were divided
into quartiles. Those receiving the lowest cell dose of 1.7 CD34
positive cells per kilogram, that is with a factor of 10^5^, you can see
that this group of patient does exceedingly badly. These patients have a
very delayed recovery with a median time of recovery of 32 days in
comparison to 18 days in the highest cell dose group. The ultimate
engraftment is only 70 percent. The incidence of graft failure then is
30 percent. I would suggest to you that this is an unacceptable outcome.
Next slide.

Then we look at the impact of growth factor. The reasons why I\'m
highlighting are these are factors that actually fell out in the
multi-variate analysis. You can see here that in this uni-variate
presentation those receiving growth factor did better than those not
receiving growth factor. Next slide.

As you can see in the multi-variate analysis, HLA matching,
predominately we are comparing one antigen mismatch versus two antigen
mismatch. It\'s misleading to believe that we are really comparing to
the six-of-sixes, and perhaps Pablo Rubinstein will address this issue.
So all I can tell you is that our data would suggest to us in our
limited numbers of patients that there is no difference in neutrophil
engraftment comparing one antigen versus two antigen mismatch.

There could be something very different in the six-of-six matches but I
can\'t address that because we had too few at our institution. However
when you do look at the impact of CD34 cell dose, you find that there is
an extraordinary impact. The higher the cell dose could better the
outcome. Next slide.

There appears to be a trend, although not quite significant in the
multiple regression analysis, that growth factor seems to be helpful in
predicting a better outcome in terms of engraftment. Next slide.

Now the second question that a number of you were asking beforehand is
what is the impact of cord blood transplant on graft-versus-host
disease. To put it in the context of what you expect, any pediatric
population receiving an unrelated bone marrow, we would anticipate at
our institution to have overall probability of grade three to four GVHD
of approximately 20 percent. That\'s four patients receiving one antigen
mismatch bone marrow transplant.

When you look at the impact of cord blood on GVHD, keep in mind that
looking at the GVHD vector, approximately half the patients received a
two or three antigen mismatch graft. Yet the ultimate incidence of grade
three to four GVHD is only 10 percent. So it would suggest that the risk
of GVHD is indeed lower. Next slide.

What is the impact of age? Now let me switch back. This is now grade two
to four, not three to four as I was showing you previously. When looking
at the risk of age, you find that patients that are older tend to have
more graft-versus-host disease but no difference in that age zero to
one. Now why is that the case? I can\'t really answer that. It may be a
statistical issue or patient number issue but nonetheless there the
p-value is still not significant. Next slide.

Then you look at the impact of HLA disparity. It\'s nothing that we can
detect but once again it may be related to our patient numbers. Remember
predominantly, we\'re comparing one versus two, but there\'s not even a
suggestion of a difference within this cohort. The advantage of looking
at a single institution is the fact that whether we grade GVHD the same
as they grade it at Seattle or Duke or wherever, it doesn\'t matter
because at least we are doing it consistently within the institution
although it may be different between institutions. Next slide.

When we look at the multiple regression analysis, we find that no
factor, and though I only highlight these because they are of interest,
we found no factor that influenced or predicted the risk of acute
graft-versus-host disease. Next slide.

As Joanne Kurtzberg asked, what is the impact upon chronic
graft-versus-host disease? To put it in the context of bone marrow
transplants and assuming I can show you data and I have that available
of what you would expect, you would anticipate for a pediatric age group
to be somewhere in the order of about 30 to 40 percent having chronic
graft-versus-host disease even in children. Yet we find an incidence of
10 percent. I would suggest to you that at least within our dataset this
is quite a significant difference. However soon we need to compare
between bone marrow transplant in a more formal way. Next slide.

When you look at the impact by age, we find that adults have a higher
risk of chronic graft-versus-host disease as compared to the younger age
groups. But practically speaking, it\'s still quite low compared to what
we would have expected with matched bone marrow and these are mostly
mismatched unrelated cord bloods. Next slide.

We find no impact of HLA once again. Although again numbers may be
prohibitive in doing this analysis. Next slide.

Importantly I think that the biggest obstacle to successful bone marrow
transplants is non- relapsed mortality. The question is will cord blood
reduce this risk. When we did the analysis of looking at
treatment-related mortality, the first question was whether or not HLA
mismatching had a negative impact. I would predict that yes there will
be although not quite significant in this analysis.

With time, I\'m sure we\'re going to show that a two antigen mismatch
has a higher treatment-related mortality as compared to the one antigen
mismatch group. Again remember there are too few in the HLA matched
group to really have anything of statistical significance in this
dataset. Next slide.

We find that age has a tremendous impact at least in this uni-variate
analysis. Age has an independent risk in terms of its impact upon
treatment related mortality. Next slide.

What I would suggest to you is that it certainly is not just being
older. It\'s also having a lower cell dose. Although not all the
patients receiving the lower cell dose were adults, and in fact half of
them were children, you can find here that it\'s really cell dose that
impacts upon the outcome and those receiving the higher cell dose have a
better outcome with actually quite respectable, perhaps even lower than
what we would expect in general, of an incidence of transplant-related
mortality. It\'s on the order of 20 to 30 percent. However, when you get
to this lower cell dose, there you have an unacceptable risk of
treatment-related mortality. Next slide.

Survival by age is shown here. It\'s actually not a surprise compared to
what the slide I just showed you of treatment related mortality. You
find that those under the age of zero to one have very different
survival, but at least within our dataset the reason why these results
are so positive is because they are predominantly non-malignant
diseases. So you can\'t look at these survival curves without having a
comparable population. This population here is predominantly
non-malignant diseases, whereas these two are predominantly malignancy.
That by itself has a tremendous impact in addition to the other factors
that we\'ve presented. Next slide.

We find now a separation in terms of matching. Those with two antigen
mismatch do have an inferior survival as compared to one antigen
mismatches. Actually that has a p-value that is significant. Next slide.

Then you find that the impact of CD34 is obvious here. Those receiving
the lowest cell doses have a markedly inferior survival. Next slide.

When we did the multiple regression analysis, several factors fell out
as being important. This time HLA match is important. CD34 cell dose is
important. Having had graft-versus-host disease is important in
predicting the ultimate probability of survival. Next slide.

So now as a clinician, we have this problem of now trying to balance HLA
matching and cell dose. How do you put the two together, because both
are critical factors in choosing which is the appropriate cord blood
unit for our patients needing transplantation? What I would suggest to
you is that really what we observed \-- there is a couple of points to
make. One is that the curves are pretty similar in appearance overall.
You find that those with the higher cell doses tend to do better than
those receiving the lowest cell dose here.

But what is different is those receiving a two antigen mismatched graft
the curves are just pushed down. So rather than having a one year
survival of approximately 80 percent in the higher doses, it\'s 60
percent in the two antigen mismatch grafts. Those receiving a low cell
dose in the one antigen mismatch group have an overall one year survival
of 40 percent as compared to 11 percent. It tells us that both HLA as
well as cell dose are two factors that we have to look at simultaneously
in deciding which graft is appropriate.

The other thing that this tells me or at least suggests to me is that if
you have a two antigen mismatch graft, part of that negative impact of
being more mismatched can be in part overcome by increasing cell dose.
Does that make sense? So even though HLA is important and cell dose is
important, if you are forced to use an HLA mismatch graft, the higher
the cell dose at least will help reduce the negative impact of HLA. Next
slide.

Of those that died, what were the causes of death? It comes out just
like in bone marrow transplant. Malignancy and infection are the two
most common reasons for death in those that died. This is not the
proportion of all patients but only for those that died and what were
the causes. You get the overall impression that these are the two
problems that are to be addressed. In part, the relapse issue is
obviously related to patients, this is including both malignancy and
non-malignancy patients, and so many of those analyses need to be done
in a different way. Next slide.

What we need now is not to compare younger cord blood patients to older
cord blood patients but to figure out ways of comparing bone marrow
transplants and cord blood transplants, being able to match for those
factors that we have already identified as being critical to the outcome
after bone marrow transplantation. What we could do, and what\'s going
to be presented this morning, is looking at these retrospective
analyses, because obviously there is no perspective randomized trial to
compare it to, all we can do is look at institutional matched pair data
and we can look at registry data. Some of that will be presented by
others, but let me just tell you what I can talk to you about which is
our institutional matched pair data. Next slide.

What we did is we did two analyses. One is that we compared all of our
cord blood patients to similar patients that had a matched bone marrow
donor. We looked at engraftment, acute graft-versus-host disease and
survival. Next slide.

What we did is we matched these patients on the basis of age, plus or
minus three years. So we took all the cord blood patients and then we
found a comparable bone marrow transplant recipient at our institution.
We matched for age, their diagnosis and disease stage, and for patients
with malignancy we matched them on the basis of their risk group. Again
what we are doing is comparing predominantly mismatched core bloods only
to patients receiving a matched bone marrow either with methotrexate
cyclosporine as their GVHD prophylaxis or T-cell depletion by
counterflow elutriation. Next slide.

To make a long story short, and I\'ll go through these slides pretty
quickly, is that we find that there is a negative impact on engraftment
by using cord blood, that patients receiving matched marrow will engraft
more rapidly but ultimate engraftment is not that different. Next slide.

When we look at graft-versus-host disease, I was actually quite
surprised to see that there was no difference in outcome. The advantage
of this analysis is the fact that however we grade graft-versus-host
disease, and it was actually Dr. Stella Davies that did this blindly,
she took all of our patients and then she performed a retrospective
grading of all the patients. What you find out is that there was no
difference in the ultimate risk of grade three to four graft-versus-host
disease. You can see for children predominantly we have a lower risk of
graft-versus-host disease. Next slide.

So it was actually in contrast to what I expected before we did this
analysis. I thought that cord blood would have a lower risk of
graft-versus-host disease. When you look at overall survival, certainly
cord blood was no different but it certainly was not inferior to what we
expected with bone marrow transplants. Next slide.

Then we repeated the analysis but this time now looking at those
receiving T-cell depleted grafts. Again what you find is that cord blood
is slow in terms of recovery; however, ultimate engraftment is no
different by day 45. Next slide.

You find that there is no difference in risk of graft-versus-host
disease as you might have expected in this category. Next slide.

You find that overall survival is virtually superimposable between the
two cohorts. Next slide.

I then repeated this analysis but this time another factor was matched
between the bone marrow dataset and the cord blood dataset. Next slide.

What\'s different this time is I also matched on the degree of HLA
disparity. So if the cord blood patient was mismatched, so was the bone
marrow comparative group. Does that make sense? Otherwise they are all
the same. Next slide.

Once again you find the same outcome in terms of incidence of neutrophil
recovery. Next slide.

Here you now see a difference in terms of the probability of grade three
to four GVHD with cord blood having a lower risk of GVHD as compared to
mismatched marrow predominantly. Next slide.

And you find that there is a trend towards worst survival in the marrow
recipients receiving predominantly mismatched grafts. So what you have
done now is you have taken really the worst patients with bone marrow
transplants and comparing those to cord blood. Next slide.

This is probably the more accurate comparative group. We did the same
analysis now using T-cell depletion as our comparative group. Next
slide.

This latter part data is not been published whereas the first part was
published in *Blood* several years ago. You find no difference in GVHD.
Next slide.

But you find a dramatic difference in terms of survival. Next slide.

At least my interpretation of this is that mismatched cord blood is
associated with a rate of GVHD that is not too different compared to
matched marrow but is superior to mismatched marrow. Umbilical cord
blood is at least as good as HLA matched marrow in terms of survival but
may be better than mismatched marrow. But remember that this is still
predominantly a pediatric population. Next slide.

There is other data that we can look at. I\'m going to briefly go over
this. That is there is another retrospective analysis performed by
Euro-Cord where they compared patients only with acute leukemia and they
are all pediatric. They compared them to methotrexate cyclosporine with
bone marrow transplants and T-cell depletion predominantly by Campak.
Next slide.

Now, what we should point out to you, you find here now looking at the
cord blood category there are some major differences. Patients receiving
cord blood are predominantly more mismatches compared to bone marrow
group. You can find where the mismatches are occurring are really not
class one antigens but predominantly class two. Next slide.

When you look at overall recovery now looking at the Kaplan-Meier
recovery here they compared 96 percent with methotrexate cyclosporine at
18 days median time to neutrophil of 500. When you compare that to cord
blood once again it appears pretty consistent that cord blood is slower.
In this analysis by Kaplan-Meier it was suggested indeed the risk of
graft failure is higher. Again we need to be looking at cumulative
incidence versus Kaplan-Meier results because their results are
different. Next slide.

Can you go back one? One thing they bring up is the incidence of
platelet recovery. You can see here to address your point. There is no
difference in terms of probability of platelet recovery as compared to
bone marrow transplants, but the time to get there is significantly
delayed. Next slide.

In terms of graft-versus-host disease, you can see here that there is a
difference between cord blood and methotrexate cyclosporine where the
incidence is statistically lower using cord blood, about as good as
T-cell depletion, but it\'s statistically lower in terms of grade three
to four graft-versus-host disease as well to two to four as you can see
here. Then the incidence of chronic GVHD is significant as with cord
blood as compared to methotrexate cyclosporine. Next slide.

However the one thing that did fall out that was quite intriguing is
that the early transplant-related mortality is significantly greater in
patients receiving a cord blood transplant. This is driven by the higher
number of HLA two antigen mismatch grafts being used with cord blood. So
early death is higher, at least in this analysis, in recipients of cord
blood. Next slide.

Even though you see the early deaths are different and then you look at
those patients after day 100 and looking thereafter in terms of the
impact of cord blood on survival, there is no difference. However what I
should point out to you is that cord blood never quite makes up for that
early death. So therefore that early death rate does have a prolonged
impact on ultimate outcome for long term. Next slide.

One other issue is that because of the fact that there is a reduced risk
of graft-versus-host disease there was great concern that there would be
a higher risk of relapse. However in the IBMTR data analysis and in the
Euro-Cord analysis, there is no difference in the risk of relapse
between cord blood versus bone marrow at least in acute leukemia. There
is not enough patients receiving transplants with cord blood in CML to
make any firm conclusion at this point. However you can see that there
is a higher risk of relapse in those receiving T-cell depletion. Next
slide.

This is the dataset from the IBMTR comparing bone marrow versus cord
blood, mixing the diseases together, but there is not even a suggestion
that there is an increased risk of relapse, although that was of
concern. Next slide.

This was just a summary which I have already gone through. Next slide.

I think that one thing we need to discuss perhaps is how do you
interpret the data. But I think before we begin doing that and comparing
what we observed at the University of Minnesota in a matched pair
analysis, which was limited in patient numbers compared to the registry
analysis, we should save that until after the subsequent presentations.

However we have to keep in mind the difficulties with a registry
analysis that you have multiple institutions involved, multiple grading
schemes for graft-versus-host disease; there is different cell dose
requirements and limits between different institutions; and different
degrees of HLA disparity between the different cohorts. It makes it
difficult to interpret in some instances. Next slide.

In conclusion, unrelated donor bone marrow transplant is an effective
treatment for young patients regardless of diagnosis. This is bone
marrow transplant. Several factors clearly predict survival after
unrelated donor bone marrow transplant, age, disease stage, HLA matched,
performance status and CMV sero status. Therefore we have to use these
same parameters in which to evaluate cord blood. Next slide.

Survival is significantly worse for each decade of age. However, there
seems to be two critical break points in bone marrow transplants at the
ages of 10 and 45. Next slide.

In terms of cord blood transplants, what I would say is my conclusion is
that cell dose predicts engraftment, rate of hematopoietic recovery and
survival regardless of whether you use nucleated cell dose or CD34.
However nucleated cell dose does not have the same impact as CD34, but
the problem with CD34 is that the way I do the analysis may be different
than another institution.

What I have been able to show to you is the proof of principle. CD34 is
a critical determinant, but I can\'t provide you with that absolute
number that is applicable to every institution. It is only applicable to
the University of Minnesota. So I would say to you that we need CD34
analyses being done but by the banks not by the institutions themselves.

HLA matched is the most significant predictor of survival and recipients
with an adequate cell dose. This is not what I would have told you a
year ago. I would have said to you it\'s cell dose. I think now what I
would interpret this as is that if you have a cell dose that is
adequate, for me greater than 1.7 times 10^5^ CD34 per kilo, then HLA
becomes the most critical determinant. The risk of acute and chronic
GVHD is low despite HLA disparity. Next slide.

The negative effect of HLA disparity on survival can be ameliorated by
increasing the cell dose. This is a practical point in terms of choosing
which graft to go after. Next slide.

Now how would I envision regulating unrelated donor cord blood? From my
point of view, cell dose needs to be part of that regulation. But cell
dose less than 1.5, I would think, is not acceptable for routine use
regardless of the age. Mismatch of more than two HLA antigens is not
acceptable for routine use regardless of age only because of the fact
that we don\'t have enough data presented thus far, but perhaps Dr.
Rubinstein will present more data that would tell us differently. But
until I have seen that data with three and greater mismatches, I don\'t
think that we can say that it should be used routinely. Until the method
of CD34 quantitation is standardized, a specific dose cannot be stated
as universal threshold. Next slide.

Age is a risk factor for survival regardless of stem cell dose. I think
that the use of double cord bloods or co-infusion of haploidentical stem
cells with cord blood or co-infusion of mesenchymal stem cells with cord
blood or other cell populations or enhancing graft and GVLR immune
recovery remain investigational or not for routine use, again regardless
of age. I don\'t think that age is the important factor here. It\'s cell
dose and HLA. Next slide.

In terms of activity in adults, don\'t be misled to think that cord
blood transplants are not being done for adults. I polled a variety of
cord blood banks and there are mothers that have not yet been presented
here.

What you can see here is that there have been more than 1,103
transplants done in adults, and this is a dataset that we need to
evaluate before we can make any great recommendations about the impact
of cord blood transplantation in adults. Nonetheless, the type of
analysis that needs to be done has yet to be done. Next slide. That\'s
it. Thank you.

CHAIRMAN SALOMON: I think we\'ll do the questions after just because I
think these are linked. I would like to invite Nelson Chao to do the
next one. The only reason for the deviation from the printed schedule is
that Pablo Rubinstein\'s slides have to be loaded I understand. We will
do that during the break. Dr. Chao.

DR. CHAO: Thank you for the invitation to be here. As an adult
transplanter looking at some of the kid\'s data, it makes us dream of
getting data like that. What I wanted to present is the results of a
prospective multi-institutional NHLBI sponsor trial that was presented
at ASH. Next slide please.

The COBLT study is a prospective pilot analysis of unrelated cord blood
transplantation in subjects requiring allogenated transplantation.
There?s one stratum that was dedicated to adult patients and there were
34 patients enrolled. The primary endpoint of the study was to look at
survival at 180 days post transplant. The secondary endpoints including
engraftment, GVHD, relapse and long term survival.

The eligibility criteria for these patients were for malignant and
non-malignant diseases suitable for allogenated transplantation. The
subjects with relapse or refractured disease were eligible for
enrollment. We needed a pre-cryo preserved cord blood unit containing
10^7^ total nucleated cells. The recipient had to match at least four or
greater HLA alleles. Subjects with active CNS disease, Kamofsky
performance status of less than 70, primary myelofibrosis were suitable,
but related donors were excluded. As we discuss this further later on,
please keep this in mind that these were patients who did not have an
HLA matched sibling and did not have an HLA matched unrelated donor
either. Next slide please.

These are the baseline characteristics. The median age was 34.5 so
clearly an adult population of the range of 18 to 55. Male/female was
balanced. Ethnicity, three-quarters were Caucasian and 12 percent
African-Americans.

Performance status again is important to note. Twenty-six percent had a
100 percent performance status. Twenty-one or 62 percent had performance
status of 80 to 90 and 12 percent a performance status of 70. Next
slide.

The primary diseases were primarily malignancies, hematological
malignancies, 56 percent AML, 26 percent ALL, nine percent CML, one with
non-Hodgkins lymphoma, one with myelodysplasia and one with PNH. Next
slide.

Because these patients in a sense were in a pilot study, these were very
poor risk patients, so of 94 percent of these patients, 32 of them had
poor risk disease, 33 subjects with malignancies, 15 had refractory
disease and 11 of them were second or greater remission. Again, keep
this in mind as you look at this data that these are patients who really
didn\'t have any other options for allogenated transplant and were in
fact a very high risk group of patients. Next slide.

The conditioning regimen for these patients consisted of total body
radiation, cyclophosphamide in the majority of the patients, 79 percent;
and busulfan/melphalan in the other 21 percent. HLA matched were A and B
with intermediate matching DRB1 at high resolution. There was only a
single patient that was six-out-of-six HLA matched. Ten patients were
five-out-of-six and 23 patients were four-out-of-six. Next slide.

These are the graft characteristics. At freezing, the median was 2.3
times 10^7^ per kilo. Infusion in terms of nucleated cells, 1.7 times
10^7^ per kilo of the recipient weight. Next slide.

This is a summary of the neutrophil engraftment to an ANC greater than
500 per cubic millimeter. Of the 34 patients enrolled, 29 were valuable.
They had survived to day 14 after cord blood. So four died actually even
before the transplant itself and one died on day six. This is a good
example of the severity of this patient population in terms of disease
status. Twenty-nine were valuable.

Deaths before engraftment were six. Deaths occurred between 15 and 40
days after cord blood engraftment and died before day 42 was a single
patient. So 22 engrafted, so survived 42 days or greater. The number of
primary graft failures were eight, so 28 percent. Secondary graft
failure was a single patient. The time to get to 500 neutrophil was a
median of 28.5 days with a range of 13 to 55. The cumulative incidence
was 72 percent. Next slide.

This just shows graphically looking at the probability of the 29
patients are valuable to the cumulative incidence of neutrophil
engraftment. Next slide.

In acute graft-versus-host disease, there were 13 patients with grade
zero, five with grade one, five with grade two, six with grade three and
none with grade four. Keep in mind that there was a single
six-out-of-six HLA match patient population. Certainly for the adult
transplanters, I think this is a remarkable and quite surprising low
instance of acute graft-versus-host disease for HLA mismatched unrelated
transplants. Next slide.

The primary cause of death is, as one would expect in these patients,
infection in 39 percent, relapse in 35 percent. So again, the
predominant cause of death was infection and the recurrence of the
underlying disease. Seventy percent organ failure and three percent
graft failure, three percent acute GVHD and hemorrhage in a patient.
Next slide.

Survival, there are four subjects alive between 373 days and 743 days
after transplant. The Kaplan-Meier survival estimates, excluding the
four patients that died before they actually got the transplant, was 47
percent 100 days and 30 percent at 180 days. Next slide.

This is a Kaplan-Meier curve again excluding the four patients who died.
Next slide.

Obviously there are only a very few patients in all this analysis, so
its stratification looking at p-values, there is not a whole lot of
power here. But if you look at age, degree of matching and cell dosage
there really is no significant difference. Next slide.

Again there is no difference by survival by age, although there\'s a
hint that perhaps some of the younger patients do better. There really
was not enough patients. Next slide.

So cord blood was associated with acceptable engraftment and risk of
graft-versus-host disease in adults. This was a multi-center study.
There\'s a high treatment-related mortality, and relapse rates observed
clearly reflect the poor risk subjects enrolled. These are all patients
with fairly advanced leukemias. The role of cord blood will require
additional studies and subjects with standard risk factors for
transplantation. Next slide.

I just want to spend a couple of minutes asking whether we could do
better. We clearly can do better I think if we start with a better
patient population. A lot of the morbidity and the early deaths were
related to the type of patients who enrolled.

There is a lot of research going on both in the pediatric group and the
adult groups looking at ways to make cord blood work better. We could
combine units for example. We could think of ex vivo expansion. A lot of
the problems with the infectious complications we think are related to
the low rate and the low pace of immune recovery. There are other things
that one could think of such as IL-7 or LIF or adoptive T-cell transfer
and so, as I mentioned earlier, better candidates. Next slide.

We actually have been looking at the idea whether one could use a
non-ablative regimen in umbilical cord blood. A lot of the morbidity
that we\'ve seen in our adult patients had been related to the toxicity
of the preparatory regimen and then the prolonged delay in terms of
myeloid recovery. We have been exploring a regimen with fludarabine
cyclophosphamide in ATG. Again these are non-myeloablative doses so the
fludarabine dose and cyclophosphamide dose are taken from patients with
CLL who can get this kind of a regimen monthly for over a year and the
GVHD prophylaxis with cyclosporine and methoprednisone. Next slide.

The rationale was that we could actually get a lot of these cells into
peripheral blood by mobilization; that anti-lymphocyte drugs can be used
without too much toxicity; and large doses of hematopoietic precursors
can lead to chimera. So that was the rationale in terms of using
peripheral blood in these non-ablative transplants. Next slide.

You have a patient here with A type with leukemia cells which has this
type of a pattern. You have donor cells and then you end up with an
intermediate stage with a mixed chimera. Then in the peripheral blood in
the bone marrow patients, we can actually given the donor leukocyte
infusions and actually convert them over to complete chimera. Cord blood
obviously you are really waiting for the cord blood itself to take over.
Next slide.

In terms of engraftment, there\'s clearly a balance between a lot of
these factors that we can control such as stem cell dose and T-cell
dose, as well as in the host there is a degree of immuno-suppression you
need to prepare them for and then the post transplant factors such as
GVHD prophylaxis. Next slide.

If you look at the non-myeloablative in terms of stem cell peripheral
blood, you can see that the conventional way of doing what the ablative
regimen gives you about a two week period of aplasia. Whereas in the
non-myeloablative, the host cells come down a little slower because the
type of chemo is not as intense and the donor cells come back a little
sooner so that the native period then is much shorter, and partly
because the host cells are recovering as well because we have a mixed
chimera state. As the donor cells come back later, you are actually
being protected by the host cells that recover from the preparatory
regimen. Next slide.

The potential benefits then are lower toxicity, lower anticipated late
effect, potentially treat older patients, treat patients with co-morbid
conditions. You can do this as an outpatient. The hope is that you have
fewer complications and less infections. Next slide.

I\'m going to show you some of the preliminary data in cord blood
patients. You can see in fact that this does happen. If you look at
these four cord blood patients, this patient really had really a
transient engraftment and then lost the graft.

You can see at two weeks, rather than waiting for the 26 or 28 days of
engraftment, you have a white count now that\'s already 3.5. You have a
platelet count of 29. This is still transfused. You have 6.5 percent
donor by chimerism studies in this patient. By three months you can see
that the patient is now 100 percent donor and the patient really didn\'t
suffer much from neutropenia.

There is another patient at two weeks who is still neutropenic but
actually by six weeks is 100 percent donor ready. The fourth patient
here again is 37 percent donor, and this patient by six months becomes
100 percent donor. It\'s a way of trying to potentially look at using
the recipient\'s own cells to recover partially as the endogenous cord
blood grows. Next slide.

Part of the reason for this really is to try to get these patients
immune recovery to come back sooner. We had done a previous study
comparing adult cord blood, which is shown in these diamonds, with the
pediatric recipients shown in these open squares. As you can see the
pediatric patients always recover faster.

If you look at absolute lymphocyte count, the adult patients are not
recovering to about a year out. Whereas in these orange dots here these
non-ablative recipients clearly by a year have recovered their absolute
lymphocyte count in the normal range. Next slide.

If you look at naive phenotype for T-cells or memory T-cells, again a
very rapid engraftment of these. These are donor cells because we know
at these time points that they are 100 percent donor. Next slide.

If you look at spectrotyping looking at all their T-cell receptors, what
you see normally is a nice Gaussian distribution with about eight peaks
in each of these T-cell receptors. Next slide.

You can see in the immunoscope recovery comparing a patient with
myeloablated cord blood versus a non-myeloablated cord blood. that
there\'s a very nice and rapid response of the T-cells. Again as I
mentioned, these are all donor T-cells because by chimerism studies at
this point they are all 100 percent donor. If you go back to the cord
blood and you look what you put in, you get the same type of repertoire.
Next slide.

This is just an example of two patients. This is pre-transplant at three
months, six months and a year. This is very different from what we see
from unrelated cord blood with ablative regimen and much more similar to
what we see with like an HLA match sibling. Next slide.

The other thing we can do actually is look at these T-cell receptor
incision circles which are a marker for new thymic immigrants. Again
these are the pediatric population looking months after transplant. You
can see that the kids recover very nicely these TREC-positive T-cells.
Whereas many months after transplant, the adult patient population,
it\'s really not until two years that we start to see a few patients
with positive TREC signal. It\'s not until three years when a lot of
these patients recover on the adult side. But within these non-ablative
transplants, actually we\'re starting to see some signal actually at the
one year mark. Next slide.

We think that the T-cell recovery following cord blood transplantation
occurs to the peripheral expansion of these adoptively transferred donor
cells. The recipients of these non-myeloablative regimen have a much
more rapid recovery in all the cell lines including T-cells and that
these non-myeloablative regimens may be one of the ways at least that we
can use cord blood in these patients who don\'t have donors. Thank you.

CHAIRMAN SALOMON: Thank you very much. Can I take some questions for
both Drs. Wagner and Chao at this point? Dr. Wagner, if you can step up
to the podium that might make things easier. I had one question just to
start things for Dr. Wagner, though Dr. Chao may comment as well, just
to finish this data gathering piece. You stopped short of telling us
exactly why the cord blood transplant patients died. What was it that
killed them in the group that didn\'t survive?

DR. WAGNER: For those patients that died it was predominantly relapse
and infection.

CHAIRMAN SALOMON: So just to make a point that will come back in the
discussion later, the delay in immune reconstitution, T-cell and
neutrophil, that occurs with the cord blood transplants as compared to
the bone marrow which was consistent, is it fair then to say that\'s one
of the major Achilles? heels of cord blood transplantation as that
specifically increases the risk?

DR. WAGNER: No, so far we have not presented any data this morning that
actually compares immune reconstitution between bone marrow versus cord
blood. You\'ve not seen any of that. However, what we do is if you look
at cord blood patients, at least within our own dataset at University of
Minnesota, and you look at those with an adequate cell dose, that is
excluding that cohort that has less than 1.7, the risk of
transplant-related mortality is very low which is the infection is very
low. But there has been no comparison between the two arms of marrow
versus cord blood. That\'s yet not been done.

CHAIRMAN SALOMON: But I thought you showed data that compared the time
to ANC reconstitution comparing bone marrow and cord blood to make sure
I\'m clear on this.

DR. WAGNER: That\'s true but that\'s not immune recovery.  That\'s
neutrophil recovery. So neutrophil recovery and immune recovery \-- at
least in my mind I think of it two different ways. One is looking at
lymphocytes and one is looking at neutrophils. I didn\'t present
anything on immune recovery. However it is indeed true that neutrophil
recovery is indeed delayed.

When you look at those patients, the fact is that some of those patients
will die of infection. You are right. But most patients don\'t die that
quickly. They die later on, as do bone marrow transplant patients,
because of their immune recovery. Their lymphocyte recovery is markedly
delayed. I can\'t tell you and I don\'t believe that it\'s any different
between marrow versus cord blood.

So yes, delayed neutrophil recovery occurs up front but most of the
patients don\'t die of infection during that period of time. They die
later after they have neutrophil recovery because that\'s not all that
you need. You need the lymphocytes as well.

CHAIRMAN SALOMON: Again just in terms of adjectives, immune recovery,
you made the definition that it\'s lymphocyte. I happen to think of the
innate and adaptive immune system and I\'m comfortable thinking about
neutrophils as being apart of the immune system.

DR. WAGNER: You are not incorrect except that \-- so the definition is
clear. When I talk about immune recovery, I\'m talking about lymphocyte
and lymphocyte subset recovery because that\'s where patients die. It\'s
because of that delayed lymphocyte recovery that causes death. It\'s not
really deaths up front, because with the antibiotics we have now even
though a neutrophil recovery is slow, that\'s not where they die.

CHAIRMAN SALOMON: Dr. Chao, do you want a chance to respond?

DR. CHAO: So in the adult population, I think all of us would be much
happier if we got a neutrophil recovery in ten days. Although I would
like to say if you look at a different group of patients who we are
studying where we\'re looking at a non-myeloablative regimen using
Campak in a matched sib setting looking at a very similar patient
population with very advanced disease, that the neutrophil recovery
really doesn?t \-- we have many patients who recover their neutrophils
and go on to die from complications of either fungal infection or other
complications which are much more related I think to the disease that
they are entering with rather than necessarily just the graft effects.

DR. WAGNER: I just want to make one more comment to really finalize this
point. Though I have not shown you that and though I could and it was
introduced by Nelson, it is that looking at the non-myeloablative
approach at our institution we have done 47 patients using
non-myeloablative approach.  The median date of recovery after cord
blood transplantation is day 11.

However, you have a mixture of both host and cord blood neutrophils. So
they have neutrophils very early and actually never become fully
neutropenic. Yet the single most important cause of death is infection
because of the lymphocyte recovery.

CHAIRMAN SALOMON: Butch, Joanne and then Mahendra.

DR. TSIATIS: This is a question for Dr. Wagner. First of all, I just
wanted to say that I really enjoyed his analysis. I think with the kind
of observational data that you have, this mass analysis is exactly the
right thing to do to make these kind of comparisons.

This question I have is a minor issue. I was intrigued by your
multi-variate analysis where you looked at some of the different factors
that affect survival. But I was wondering. Did you include a
multi-variate analysis where you had age, cell dose and HLA matching
together?

DR. WAGNER: They all were. Age and HLA were always forced into the
analysis.

DR. TSIATIS: When you forced age with cell dose and HLA matching, did
the effect of age get diminished?

DR. WAGNER: As you notice, in terms of cord blood results, age fell out.

DR. TSIATIS: So it did fall out.

DR. WAGNER: Yes.

DR. TSIATIS: So it was no longer significant.

DR. WAGNER: I only showed you those that were significant.

DR. TSIATIS: And it became insignificant when you included cell dose and
HLA matter.

DR. WAGNER: It became insignificant in the survival.

DR. TSIATIS: In survival.

DR. WAGNER: However in the transplant-related mortality, age fell out as
an independent risk factor but it didn\'t fall out in survival. I would
bet that as we enlarge the number of patients in the dataset, age will
fall out as being an independent risk factor as it would with marrow.

DR. TSIATIS: Thank you.

DR. KURTZBERG: I wanted to go back and comment on immune reconstitution
because it really is different than neutrophil recovery and it relates
to endogenous host thymic function. It\'s influenced by age strongly. As
well, it\'s influenced by preparative regimen. So patients getting TBI
are going to have more injury to the thymus and more delayed immune
reconstitution. Patients who are younger have a more active thymus and
can contribute more quickly to immune reconstitution.

Much of the data, you?re seeing particularly in the very young kids
where they get high cell doses, they also don\'t get TBI because the
disease there transplants \-- Remember the younger group had more
genetic and non-malignant diseases and most groups do not radiate those
patients. There are just many factors contributing to this that are
host- or treatment-related rather than graft source related.

CHAIRMAN SALOMON: Following up on that, Dr. Chao\'s data on TREC
analysis would fit that really nicely where there was reconstitution
forming quite late.

DR. RAO: I was curious about the HLA mismatch data which fell out as an
absolute criteria in the survival data. What was the speculation of what
the mismatch didn\'t seem to affect the graft?

DR. WAGNER: I think that the answer is going back to the issue of
infection. I believe that the HLA mismatch has probably an impact upon
the speed of immune recovery, at least the way I define immune recovery.
That is I think what\'s happening is that \-- this is speculation
because we have not proven this \-- but I would speculate that with
higher levels of HLA mismatch because we did not see an impact on the
other outcomes like graft-versus-host disease and engraftment. Again
that\'s really comparing one versus two in our dataset. I bet it\'s
related to delayed immune recovery and that\'s what is impacting the
overall survival and non-relapse mortality.

DR. RAO: Do you think that\'s just a general consensus in the field?

DR. WAGNER: We can ask the people in here, but I believe that\'s true
because that\'s clearly true for bone marrow transplants. So why would
we believe that it would be different for cord blood?

DR. HOROWITZ: I think definitely it\'s true in general for
transplantations. Recipients of HLA mismatch transplants do worse than
recipients of HLA match transplants in general, and it\'s hard to
ascribe it to any one single transplant-related outcome like the acute
GVHD, chronic GVHD, engraftment, immune reconstitution. They do worse in
everything and it ends up to a significant effect on the bottom line,
which is survival. Certainly slower immune reconstitution contributes
importantly to it.

CHAIRMAN SALOMON: Dr. Harvath and then Dr. Harlan. And then I think we
have someone from the \--

DR. HARVATH: John, I wanted to get clarification from you about the data
that you presented with adult cord blood transplant experience at
Minnesota. Is there any overlap in the patients in your analysis with
the patients that were analyzed by FDA? I mean, were any of the same
patients analyzed in both?

DR. WAGNER: Yes, I\'m sure they were because the dataset that the FDA
has is from the New York Blood Center, I believe. That would have
incorporated at least a proportion of our patients, although the
majority of our patients were done after 1998. So remember the dataset
that you are reporting is from 1994 to 1998.

DR. HARVATH: Right.

DR. WAGNER: Which is very different.

DR. HARVATH: Did your conditioning regimen and use of growth factors
change, let?s say, from the experience presented and analyzed by FDA
with the experience after that time?

DR. WAGNER: That\'s indeed true and I hadn\'t thought about that until
now. In March 1998, we did not use growth factor. Which we then
subsequently after comparing our results with Duke, we realized that
growth factor may have an important impact upon outcome, and then we
modified our use after March 1998.

DR. HARVATH: I had one other question. This is really whether you are
planning an analysis or perhaps have already done it on the importance
of the antigen matching and, for example, class one versus class two.
Would you believe that\'s an important consideration that should be done
with cord blood transplants?

DR. WAGNER: Absolutely it\'s an important consideration to be done. I
have not yet done it. But really when you consider the numbers of
patients required for such an analysis, only the registries could do
that at this point. That\'s a critical piece of information, but
there\'s no point in me doing it.

DR. HARVATH: Mary, are you going to do that?

DR. HOROWITZ: In the study that we did in collaboration with the New
York Blood Center comparing bone marrow and cord blood transplants in
children, we did not see a difference in disparity of class one versus
class two. That study had about 300 cord blood transplants. That\'s
totally inadequate to answer that question.

Trying to answer that question in the National Marrow Donor Program with
bone marrow transplants is difficult even with thousands of patients,
trying to sort out the effect of one locus versus another. I would say
that we haven\'t found it, but that\'s not a definitive statement that
it doesn\'t exist.

CHAIRMAN SALOMON: Dr. Harlan and then I think Dr. Rubinstein.

DR. HARLAN: I\'m going to ask that hard question, maybe a stupid one
because I want you to speculate biologically what you think is happening
based on three different observations that you have shown us. One is the
CD34 stem cell dose. While it quite clearly from your empirical data is
important, it would only be two cell cycles to make up the difference
between the low dose and the high dose. 1.7 to 5.4 would be made up with
two cell divisions.

The second observation is the relevance of the mismatch. The third
observation is the importance of this non-myeloablative regimen. It
seems to allow for a quicker recovery. So I\'m wondering what you think
biologically if there\'s a grand unifying hypothesis for that. Basically
what you want is that stem cell that\'s marked within the CD34
population to start dividing and to repopulate the bone marrow. I\'m
asking for you to connect those dots for us if you can. I\'m sure you
have thought about it a lot.

DR. WAGNER: I think that in terms of the first question about why is it
that cord blood all we are asking for it to have one or two cell
divisions to go into cell cycle and then give you more rapid recovery.
At least I can tell you in culture, because obviously like others we are
interested in ex vivo expansion. Even if we can get a two-fold
expansion, then we have addressed the issue of cell dose. But number
one, remember that CD34 only provides us limited information. It\'s not
truly the stem cell.

Secondly there is something biologically different between cord blood
and marrow. It takes significantly longer to get the cord blood cells
into cell cycle. So yes, you\'re right. It only needs several cell
divisions but it takes a while to get there. That\'s an artificial
system but still it\'s different between marrow versus cord blood. It\'s
probably about three times longer for cord blood as compared to marrow.
That may in part relate to it.

I think that what we are seeing is that also when you look at the cells
on day 21 bone marrows, early days we have actually had people report
back that they had leukemia when in fact they were transplanted for
non-malignant diseases. If you look at the bone marrow specimens, there
is a high proportion of very primitive cells in cord blood recipients. I
can actually show you some slides of that a little bit later.

There is something very different. We reported on that to show that the
cord blood even when it recovers even morphologically it doesn\'t
differentiate as quickly as do bone marrow cells. It may be related to
what you are putting in rather than anything else. What was your second
question?

CHAIRMAN SALOMON: Actually he asked about myeloablative related to the
non-myeloablative. I wonder if Dr. Chao would want to take that one.

DR. CHAO: There is two bits of data I think that are helpful. We have
mouse model for cord blood as well as a model where we used KTLS cells.
These are KIT lineage negative Scouwin positive cells but that\'s
assorted cells where we can put ten to 50 cells in a mouse and
reconstitute them.

If you look at those animals, there really is a tremendous delay in the
time to get to lymphocyte counts which are normal. If you use a congenic
strain of 45.1 versus 45.2, you can very nicely see that the donor cells
ultimately take over and the majority of the cells you get early on are
the residual resecant cells which are radio resistant. That\'s one piece
of information. If you compare that instead of using a cord blood
source, what we use is fetal blood day 20 from the animals, if you use a
marrow source, those cells seem to come up faster.

I think the other piece of clinical data that\'s useful to think about
is the SCID transplants that Dr. Buckley does without any ablation. If
you look at the time it takes to first start to see lymphocytes, it\'s
about 100 days in these SCIDs. There is going to be a delay in that
time. I think what we would like to see with these non-ablation
transplants is this bridging with the residual host cells which may
allow you to overcome some of the lack of responder cells in the host.

DR. RUBINSTEIN: This is in respect to the use of CD34 as a marker. I
have a brief comment and a question for Dr. Wagner on this. We have seen
and looked at the correlation between CD34 and total nucleated cells and
also the correlation between colony-forming cells and total nucleated
cells. These correlations are very strong. It seems that total nucleated
cells are also perhaps not as close to the true stem cell as CD34 but it
is also an portent of engraftment ability.

The question is really whether you have looked at the correlation
between the total nucleated cells you recover after thawing the cord
blood and the more specific CD34 cell. This is a critical issue for us
from the point of view of banking because it relates to our ability to
give an estimate that is meaningful for the transplant physician.
Clearly the performance of CD34 counting varies quite a bit by technical
reasons and other conditions that Dr. Wagner alluded to. This would be
extremely useful from a practical point of view.

DR. WAGNER: To answer the question what is the correlation between total
nucleated cells and CD34 cells, there is a good correlation between the
two. But as you can guess, with any other biological correlations, there
is a great spread. For every CD34, it\'s not a one-to-one correlation.

However when you look at the uni-variate analyses that we have done,
total nucleated cell dose does not predict neutrophil recovery. It does
not come out as quite significant. It does predict survival but does not
predict nucleated cell recovery. It\'s at a p-value of 0.06. I showed
you that slide.

However, if you look at CFUGM and you look at CD34, though I\'ve not
shown you the GMs, they are both very profound predictors of the time to
neutrophil recovery. Both of them do correlate with total nucleated cell
dose, but the other two are much more predictive of what you can expect
in terms of days to recovery.

Now CFUGM is also a good predictor. I didn\'t show that to you because
you either do a CD34 or you do a CFUGM. The two are virtually
superimposable if I showed you the curves, except that CD34 is rapidly
available. CFUGM takes 14 days.

DR. RUBINSTEIN: Yes. Our data concurs exactly on that but there is a
slight difference, and that may be related to the numbers. We do show
significant effect of total nucleated cell dose on the rapidity of
engraftment. Again, we also can show with the multi-variate analyses,
colony-forming ability displaces total nucleated cell dose from
significance.

DR. WAGNER: Correct. Yes, and I agree with that. It\'s just that I think
that if you have to choose one, there\'s more variability with total
nucleated cell doses compared to CFUGM in terms of predicting outcome.
So I think it\'s a better predictor.

CHAIRMAN SALOMON: We\'ll get back to this point this afternoon where one
of the specific questions is using this.

DR. CREER: We actually have some data that will show exactly that
correlation. The difference is really post-processing versus post-thaw.
The correlation is significantly different between CD34 and TNC and data
obtained post-thaw. That\'s what most of the studies are coming out from
the transplant center perspective that have been correlated with
clinical outcomes and not post-processing data coming out of the bank.
 There is a big issue there. But my question really is -?

CHAIRMAN SALOMON: Before you ask your question, for anyone stepping up
to the mike just identify yourself so that the transcriptionist can tell
who you are.

DR. CREER: I\'m Michael Creer. I\'m the Director of the St. Louis Cord
Blood Bank. The question I really have is when we looked at outcomes, we
focused a lot on survival as the main thing. We haven\'t really asked
the question so much about the quality of that survival.

I\'m wondering if anybody has ever looked at the quality of life
following survival from a cord blood transplant as opposed to either
bone marrow or peripheral blood. It seems like the lower incidence of
graft-versus-host disease, the fact that once stable in graft in
supported care seems to be diminished, there is no transfusion
dependency, that these people are probably much better off in the long
term. Is there any evidence to suggest that that?s really indeed the
case?

DR. WAGNER: No one has actually formally done that analysis, at least as
it has not been reported yet. It\'s certainly a key question that we
need to address. However one point I can make to you is that we clearly
demonstrated in the randomized trial with T-cell depletion that we were
able to markedly reduce the risk of acute graft-versus-host disease. We
found at least nothing that we can tell so far at one year and perhaps
at three years that there is any difference in quality of life.

What you did was you changed what the problems were. Rather than just
chronic GVHD, you still have opportunistic infection as a barrier. The
fact that you get rid of graft-versus-host disease doesn\'t mean that
you\'ll have improved quality of life, but still the question needs to
be asked and it\'s not yet been done formally as far as I\'m aware of.

MR. COELHO: My name is Phil Coelho. I\'m the Chairman of ThermoGenesis
Corporation. I\'m aware of a study also on adults, the use of cord blood
adults, done at the University of Tokyo\'s Medical Center and Transplant
Center. It\'s 46 adults, a little higher than the COBLT study, 29 of
which were high risk, 19 low risk. I know the study has been provided to
the FDA and some of the other panel members here.

In that case, the survivals were much better. The high \[sic\] risk
patients, all 17 of them, survive at one year. Fifty-seven percent of
the high risk patients remain alive at the end of one year. I\'m not
sure what were the differences in the regimen utilized under the COBLT
study and this study, but the results were so different. Perhaps the
lower size of the patients helped the cell dose, but I\'d be curious for
those who are aware of this study if they would have some comments in
that regard.

CHAIRMAN SALOMON: Before they answer when you say \"much better,\"
you\'re not comparing to a control group in that study. You\'re just
comparing one study to the COBLT study.

MR. COELHO: They are similar in that respect.  There is no control
group. They are just simply the use of cord blood on adults, but the
percentage of high risk is not too dissimilar. The cell dose was a
little higher in Tokyo because the patients were a little smaller, but
they were all adults in both studies and neither of which are published.

CHAIRMAN SALOMON: Dr. Wagner, I want Dr. Chao to comment. You had a
slide that had Japan on it with the adult cord blood. Was that this
study or is it a different study?

DR. WAGNER: No, all I did a couple of weeks ago was just contacted them
asking what was the total number of adults that have been transplanted.
So all I have is just the total number. I have a much higher number than
what\'s been reported.

CHAIRMAN SALOMON: Dr. Chao, do you want to comment?

DR. CHAO: I\'m aware that the study exists although it\'s not published.
It\'s hard for me to answer because I can\'t compare what the
differences are. One can speculate that the HLA pool is different. Out
of these 36 patients, 35 were high risk, so they were all high risk.
It\'s hard to know HLA, GVH prophylaxis, cell dose, disease status.

MR. COELHO: These actually were not good matches and most of them were
four-out-of-six. Again there, like here in the United States, most of
these patients are waiting for a bone marrow match. So with a homogenous
population, you would of course expect the bone marrow.

So those who are unable to get a bone marrow match tended to go to the
cord blood. These were predominantly four-of-sixes. I think a few
five-of-sixes. No perfect matches. Again others here are aware of this
study and could comment more knowledgeably than myself.

CHAIRMAN SALOMON: Dr. Kurtzberg.

DR. KURTZBERG: I actually had the ability to review both studies as a
reviewer for one of the major journals. I know one of them is in press.
But there are two Japanese studies that look at adults, one that Phil is
referring to and a second with 13 adults with myelodysplasia. In both
studies, the median cell dose was between two and three times 10^7^ per
kilo versus in the COBLT study of a number below two.

Otherwise the matching was predominantly four-of-six. There weren\'t any
differences in the preparative regimens or anything that was obvious
from what was presented in the data. But the event-free survivals are
markedly different. In the MDS paper, it was 85 percent at one year for
13 patients. In the this paper, it was 57 percent with the high risk
patients and 100 percent in the low risk patients. This is a small
number but the trends are certainly superior to what we saw in COBLT. It
may be that cell dose threshold is really breaking somewhere between 1.5
and 2.5 and that\'s the crux of the matter.

CHAIRMAN SALOMON: Just for the record, you are talking about nucleated
cell dose not CD34.

DR. KURTZBERG: Yes, I\'m talking about nucleated cell dose of the graft
from the cryo-preserved dose that the bank provides.

CHAIRMAN SALOMON: Do you have a specific comment?

DR. LACHENBRUCH: I had one comment and a couple of questions. Comment
one refers to all of the papers where many tests were being done. We all
are aware that multiple testing is a potential hazard in the sense that
we get too many false leads. It might be appropriate to consider some
adjustments for the multiple tests.

For Dr. Wagner, in your matching you had UCB versus bone marrow. There
might be some reasons for getting one or the other that I\'m sure you
people here are far more aware of than I that might have an effect on
survival. Essentially, you are creating a comparison group in this way.

For Dr. Chao, it appeared that there\'s a very rapid failure rate in the
early period of time followed by something else. This might suggest a
mixture of survival distributions which becomes considerably more
complicated but certainly not outside the realm of possibility. I would
just offer that as a possible suggestion.

DR. WAGNER: You\'re right in that when you do a retrospective analysis
comparing patient populations, even though it\'s a matched paired
analysis there could be different reasons for going to cord blood. If
anything because cord blood is the newcomer, typically they would be
higher risk patients. If anything, it actually makes their argument in a
stronger way, but that\'s pure speculation. The data is the data and the
data shows that there is no significant disadvantage and, if anything,
an advantage to bone marrow at least within our dataset. In fact, it is
so compelling to our group that we have all but stopped doing bone
marrow transplants in the adults because the results are so positive.
That\'s for acute leukemia.

It would be a different story if we were still doing patients with CML
because there, bone marrow transplants still is the standard by which
not everything else needs to be compared. There needs to be more
patients to be done with cord blood to be able to say that it\'s as
effective.

I have to make one comment about the COBLT study which I was a
participant of and still am a participant of. There is high risk and
then there is really high risk. These patients were the worst risk
patients you could ever have chosen for a transplant procedure simply
because when we designed the study back in 1998, it was appropriate then
to put the high risk adults in the cord blood trial. We have learned a
lot since that study has come on based in part we know cell dose effect
and we know HLA effect. But those patients, as Nelson pointed out, four
died before the transplant even took place. That\'s extraordinary. That
doesn\'t happen that commonly if you take the typical high risk patient.

Nonetheless you can just say this is your speculation and that I\'m
biased and that\'s probably indeed true, but I can tell you \-- and in
fact at the break I\'ll make another transparency and show you those
same results with bone marrow transplants. You pick these really bad
patients. You get a really bad outcome. The outcome with bone marrow
transplants is 14 percent.

DR. CHAO: I just have one comment that I want to make. As a transplanter
taking care of these adult patients, in many ways you can think of these
patients who went on to the cord blood as patients who really didn\'t
have a chance to fail. In other words, if you look for a donor and you
have somebody who has his third relapse of leukemia and you know that
remission is going to last two or three weeks and you look for an
unrelated donor at that point, you know you\'re not going to find one
because it takes a while to find a donor.

Certainly three or four years ago when we started to do this, there was
a long delay in trying to get to an unrelated donor transplant. Whereas
in the cord blood there really was no delay. You end up actually pulling
a larger group of patients who in fact may be relapsing as you are
trying to get them on to the study, which I think reflects the poor
status of these patients. That early drop-off is in fact the reflection
of the type of patients that were picked.

DR. McNIECE: Ian McNiece from Johns Hopkins. Considering that we are
talking about how to evaluate products and which product to choose for a
transplant, John, on your comparison of the six-of-six marrow to a
six-of-six cord blood, if you only had a five-to-six bone marrow
available, wouldn\'t a better comparison be six-to-six cord blood to the
outcome of five-to-six marrow? I think the analysis probably would come
out in better favor of the cord obviously.

We have to evaluate from a patient perspective if we are going to be
choosing products what\'s actually available for that patient. I would
like your comment now.

But in the same light, if we are going to evaluate CD34 levels, what
percentage of products actually have that available? If you are going to
consider putting that as a criteria, if those data are unavailable, then
we need to be very careful in terms of listing that as a minimum
requirement.

DR. WAGNER: Let\'s go to the first one first because that\'s easier. The
first question, that analysis that you are asking for is really a
detailed analysis comparing bone marrow versus cord blood. Matched and
mismatched bone marrow versus matched and mismatched cord blood has been
done. At least it\'s been done and reported by Mary Horowitz at ASH a
year ago.

Pablo, will you presenting that data today? Yes, so I purposely didn\'t
address that detailed analysis because it will be presented shortly.
It\'s a very good question. It\'s a pivotal question, but just wait a
little bit. In terms of your second question, what was it again?

DR. McNIECE: If we are going to list CD34 levels as a criteria for
choosing a product, what percentage of products would actually have that
available, and is that a reasonable criteria to put down if it\'s not
available to the majority of products, which I presume it\'s probably
not?

DR. WAGNER: There are two parts to that question. One is how many would
be excluded from cord blood transplants based on that cell dose. The way
I did that analysis, basically it would come out that 75 percent of our
patients would have met that higher dose limit. Also, as you know at our
institution, that\'s the pivotal reason why we are now doing double cord
blood transplants and why we are doing non-myeloablative therapy.

It\'s because there is a significant number that would never have the
opportunity of cord blood transplant because of this limited cell dose
issue. The fact is that can\'t be a reason to change the criteria. The
fact is that if a dose of 1.7 is inadequate, it\'s inadequate. It
doesn\'t mean that you can then say it would exclude too many people.

DR. McNIECE: But I wouldn\'t want to put it in there if it\'s not
available for a lot of products because it would exclude too many
patients.

DR. WAGNER: But if it\'s inadequate then it shouldn\'t be allowed as a
routine thing. So yes, if I cut off a cell dose at a certain level and
it means that half the patients are excluded, that\'s appropriate if the
outcome is so poor with that low cell dose.

DR. KURTZBERG: John, I don\'t think you understand the question. A lot
of banks don\'t have that data available on their cryo-preserved units.
You are basing your analysis on a \--

DR. WAGNER: That\'s true.

DR. KURTZBERG: So what Ian is saying, is it realistic to make that a
criteria if the data isn\'t available for more than half of the units or
maybe greater than that.

DR. WAGNER: Is that what you are asking?

DR. McNIECE: Yes.

DR. WAGNER: That\'s different.

CHAIRMAN SALOMON: Then what I would like to do again is say that\'s a
key question that we\'ll get back to this afternoon.

DR. WAGNER: But I did answer that. That was in my last slide. The last
slide says that CD34 should not be used.

CHAIRMAN SALOMON: The last question or comment before the break goes to
Dr. Mulligan.

DR. MULLIGAN: I was going to harp on this issue one last time. If you
say that the most important thing is the cell dose and if you get up to
a certain point that\'s going to be very important, does the correlation
between the CD34 count versus absolute count change as you get up
higher? You would think it would as you have more and more cells. I
guess I\'m asking, would you need to have a CD34 test if indeed the
legislation was that you had to have a certain total number? That is
once you get to a certain number I would think that the CD34 issue would
be less.

DR. WAGNER: So what you are saying that if you exceed a certain cell
dose, that we can be pretty assured that the CD34 would have been above
the minimum. I would bet that\'s probably correct. I don\'t know that
answer. Maybe Pablo does. That probably is a good point and maybe that
would help us. It\'s only at the lower cell doses that you would need
the CD34 perhaps if you wanted to do it that way.

DR. MULLIGAN: This is important if we are weighing the various kinds of
advice that we give if we say the most important issue is to have this
ample cell dose. Once you are up at this cell dose, it\'s not really
much of an issue. Another pass which are the units that don\'t have very
much where you have to be more careful, I would think, in terms of
holding back what\'s there.

DR. WAGNER: Sure, that\'s a good point. I have never evaluated that, but
it could be done.

DR. McNIECE: Could I just make a comment? If you look at peripheral
blood progenitor cells, the data is exactly the opposite. The total cell
numbers don\'t relate to numbers of CD34 cells. You could have a very
high cell dose with a low percentage of CD34 and a low absolute number
of CD34. So the two are not connected in any way.

There is a driver because the calculation for absolute CD34s is based
upon your total white count. So there is some correlation. However you
could have a high white count and very low percentage CD34 level in the
cord blood as opposed to a similar cell count with a very high CD34
level. So they are not linked.

CHAIRMAN SALOMON: Okay. We\'re ending this. We are going to a break.
These are really very important questions. I\'m not trivializing it, but
part of the first part here is to set a database and then discuss it.
Everyone wants to discuss it. That\'s great. Save it for the afternoon.

DR. WANG: I would like to take this opportunity to clarify the
questions.

CHAIRMAN SALOMON: We will do that right after the break.

DR. WANG: No, there is no problem of the quality of analysis. The
problem is because of the scale of leveling.

CHAIRMAN SALOMON: I want everyone to be able to really pay attention to
what you are saying. We will do right after the break. Five minutes. I
know if I say five minutes no one is going to be back here in five
minutes. Off the record.

(Whereupon, the foregoing matter went off the record at 10:40 a.m. and
went back on the record at 11:07 a.m.)

CHAIRMAN SALOMON: Back on the record. So what I promised that we would
start immediately after the break is a short response to the questions
about data presentation that Butch came up with. Butch, do you want to
just make an introductory statement and we\'ll review it?

DR. TSIATIS: Yes, my understanding is \-- the concern I had was
apparently a mislabeling of two of the graphs. We have cleared that up.

CHAIRMAN SALOMON: Is that it?

DR. LACHENBRUCH: I think that\'s all that needs to be said. We have a
couple of other things that we\'ll be in direct communications with
Butch on.

CHAIRMAN SALOMON: Good. As long we are giving everyone a hard time about
mislabeling, I think John Wagner had a mislabeled slide as well because
he was talking about zero to one HLA match on one of his slides and I\'m
sure that wasn\'t what you intended. What I would like to do is get
started again with introducing Dr. Rubinstein.

DR. RUBINSTEIN: Thank you very much for the invitation and opportunity
to share with you the overall data of the National Cord Blood Program.
These graphs show the distribution of recipients around the world.
Basically it\'s useful to illustrate that the data that I will be
talking to you comes from a group of 1317 patients given cord blood from
our program. First the purpose of telling you that some of the reports
that have been published by other groups, including the Euro-Cord data,
include some of this data, so adding them all up may not be an accurate
representation of the number of patients. Next slide please.

These are the patient characteristics. They tell about the U.S. versus
non-U.S., the gender and these other percentages of each of the
different sub-categories in this analysis. These are risk factors and
for these we use IBMTR criteria. Next slide please.

First let me show you very briefly the data for engraftment. We are
going to use here the cumulative rates with competing risks. Competing
risks are presented in this table over the days after the transplant.
They are four types receiving a backup transplant having autologous
reconstitution, suffering relapse or death. These are the numbers. Next
slide.

These slides presented are cumulative data and here give the Cox
regression for the analysis. The effect of the cell dose in the timing
and probability of reaching ANC 500 by cell dose is given here and
dependent on the cell dose. So these are people who received 100 million
cells per kilo. These are better than 50 to 100, 25 to 50 and under 25.
The lowest has been seven. That\'s 10^6^ per kilo. These are all
significant, taking as a reference the lower group. All the others are
significantly different. Next slide.

Here we have the effect of age. You can see that there is a neat
stratification also but it\'s not nearly as good as the previous one.
And using as a reference those older than 16, the 12 to 15 group is
barely different and six to 11 group is also not significantly worse and
the only one that is not significantly better. The only one that is
significantly better is the young kids under five. Next slide.

This is understandable. There is a correlation between cell dose and
patient weight and therefore age. For the group between birth and maybe
12 years, there is correlation there. That becomes blurred afterwards.
The results show that as you grow older the probability of getting a
match with 100 million cells becomes essentially zero and vice versa.
You cannot see the red behind here but I assure you that only two
patients received less than 25 million cells per kilo and were younger
than two. Next slide.

This is a multi-variate analysis taking into account these two factors.
We left HLA out of this analysis although statistically it is
significant because the significance comes all from one group and
that\'s a six-out-of-six matches. There are only 56 of these. It is
significantly better though as a group, looks in a uni-variate and
that\'s a group in the multi-variate. The results using as a reference
the lowest of those or the younger group shows very clearly that the TNC
is the predominant factor. Next slide.

One of the aspects that I think is important to this Committee is how
useful is the pre-freeze TNC content to predict the results in the end.
In this slide, we analyze it with respect to what the transplant centers
can expect to find.

These are the data obtained at the transplant center plotted against
what we found for the same cord blood. This data indicates that the
correlation is quite respectable. This is despite the intervention of
freezing, storage, transportation, thawing and counting. Counting is
also important because the methods vary a lot and particularly how to
estimate the dead cells. If you look at these there is an apparent
concentration of points that are directly on the diagonal. That was a
little surprising. Next slide.

This slide will show why that happened. This is a distribution of the
percent recoveries. There is a group here at the 100 percent recovery.
Cladd said \"They are busted.\" I added the exclamation point. It is a
reasonable relationship between what we found and what the people in the
transplant center can expect to find. That was reassuring. Next slide.

Now we will talk about survival. I begin with all the patients in our
group. All of these patients were given cord blood from our bank. Next
slide please.

This is the overall curve of survival. This curve looks hairy. That\'s
because we marked where the patients are censored. There are a number of
patients still on the path right here. That can be seen here. The number
of patients that remain at different points in the curve are shown and
the decrease is quite rapid. That is not only because some patients died
but because of all the other courses of censoring, particularly that
they have not been observed for more than that time. Next slide.

Here again is a survival and the TNC, total nucleated cell, dose. We see
that for the group as a whole, 100 million cells has an overall survival
probability of 51 percent at three years. The important message here is,
one, that there is a very nice stratification of results with dose but
also, number two, that the lowest dose under 25 million total nucleated
cells has a probability of survival of less than half of the 100. Next
slide.

Patient age has a similar distribution, but here the age of 12 to 15 or
16 and over are overlapping. These are the ages. Overall these curves
are bunched together with respect to those we saw for the cell dose. All
of these groups are however significantly different in their Cox
regression by age compared to the young patients, six years and under.
Next slide.

Now HLA. Here we see the expected stratification and it works exactly as
it would be expected. Using six-out-of-six matches as a reference, here
note we are only talking about 56 patients in this group. The
four-out-of-six is different and the three-out-of-six is different, but
the five-out-of-six is nearly the same.

So we made another regression using five-out-of-six as the reference
point asking the question is four-out-of-six clearly different, and it
is. That is the place at which most of the trouble for a transplant
physician lies in the decision to use a product of six with more cells
or five-out-of-six cells with fewer cells.

In the analysis of HLA, there is one point that I would like to refer to
because it was surprising to us to see the results of it. It\'s the
effect of blanks. Blanks are important because when one of the two
participants, the donor or recipient, has a blank then the reaction of
donor against patient or patient against unit is different.

If you have a five-out-of-six match, one mismatch, and the mismatch in
which there is a blank in one of the two, and if the blank is in the
unit, then the patient who has an antigen that is not present in the
unit will not be able to see these as different. That\'s a three. It\'s
most likely a homozygote three. It will not react against this. There is
no rejection mismatch.

But there is a mismatch if you look from the point of view of
graft-versus-host. This is what some people have referred to as the
vectors. The graft-versus-host disease vector would have one mismatch.
Next slide will show us the effect of these.

These are all five-out-of-six matches. When the mismatch is
p-directional, so both donor and patient are heterozygote for the locus,
then you have this effect as we saw before, about 40 percent survival.
When the mismatch has a blank and the blank is in the direction against
graft-versus-host disease and not in the direction of rejection, the
survival is 63 percent which is the same as when you have a perfect
match. It\'s actually a little better. Next slide will show you when you
have two of these.

These are patients that are four-out-of-six but have two blanks in the
unit. There are only four such patients but all of them are alive. There
is only one censor here to use. These are the six-out-of-six matches.
These are the five-out-of-six as we just saw in the previous slide.
These are not due to a patient with lower risk factors. In fact, two of
the four are older than 12, and 37 percent of them have a mean count of
37 million cells per kilo for this group.  

Despite that all of them are alive. Of course, there is only four and
that doesn\'t mean very much but it\'s an interesting phenomena. We have
used it in multi-variate analysis. You will see these in a little while.
Next slide.

These are other factors that are important. One is where the transplant
takes place. Here we compare the U.S. patients. There are almost 1,000
of them, with foreign transplant centers about 200. One thing that
worries us is the follow-up of this group is not quite as good as the
U.S. patients. You can see this straight line here that immediately
sparks suspicion. Next slide.

The risk factors. As I explained, we used for this estimation patients
with ALL, AML or CML or patients who don\'t have these diseases. The
known ALL, AML or CML patients are used as a reference. That\'s the
black line here. You can see that the low risk patients are almost the
same as the non-malignant results.  Then the intermediate patients have
a slightly lower survival.  The advanced patients have a much lower
survival basically parallel to the results that we heard earlier this
morning. Next slide.

It has been known for many years that even after you correct for the
known factors of HLA, matching and so on, Caucasoids tend to do better
than other patients. We have the same here. You can see that this is
significant. Next slide.

The survival by the existence of a latent viral infection or at least
the presence of CMV antibody in the recipient prior to the transplant is
significant. The relative risk is not very large from 0.3 but it\'s
extremely clear that there is a significant difference. Next slide.

This is a very important one and this is the ABO match. These are
patients where the unit has not been modified. So they are not washed.
The patient is not plasmaphoresed to remove antibody and there is
essentially no difference between them. Next slide.

So the multi-variate analysis shows that TNC is important. As you can
see, the 50 to 100 million is not a significant difference to the 100
million and over. You can see it for the rest. Age is independently
important. The risk groups are significantly different. HLA, the
five-out-of-six, in the overall analysis is not significant but
four-out-of-six is and so on. Next slide.

It has the same data but here we incorporated in the six-out-of-six
those that don\'t have a rejection direction mismatch. If instead of
counting matches as six-out-of-six, we counted mismatches, in the
rejection direction this would be still zero. By doing that, we see that
the five-out-of-six now becomes significantly different for survival.
The overall significance is strengthened. Next slide.

We tried to look very briefly at the survival of the older patients in
our subset. We arbitrarily split it at 12 years. Next slide.

This shows the effect of TNC dose. You can see here that it is much less
evident than in the data as a whole. It\'s important to realize that we
don\'t have 100 million per kilo. There are only 14 in the group between
50 and 99 million per kilo. So there is not much and that\'s why we
cannot see the difference. But perhaps with more numbers the difference
would become significant. We are at the borderline of significance. The
Cox regression however shows that the seven to 24 million group is again
at the borderline but is significant. Next slide.

These is HLA.  Again six-out-of-six appear to be better but the
probability is not definitively there. In the Cox regression, we don\'t
see a significant difference even for the three-out-of -six group which
is not difficult to understand because of the numbers. There are only 10
six-out-of-six patients.

The next group shows that even this subset of patients, these
five-out-of-six matches with a mismatch in the direction of GVHD and not
in the direction of rejection, are also doing well. Again the numbers
preclude much speeches. Next slide.

Now the risk factor again is quite evident and the advanced patients are
doing poorly, again about 50 percent as well as the early patients. This
is significant. As you can see the significance comes from the advanced
group. Next slide.

The multi-variate analysis for this subset is again showing significant
difference for the lower dose group. There HLA is not significant in
this. The other factors are as expected. Next slide.

We changed the HLA, and now incorporating those other five patients we
have significance for the five-out-of-six and the four-out-of-six,
although you can see the relative risk is almost the same. Next slide.

This is a brief overview of the data of IBMTR analysis of comparison
between cord blood and bone marrow transplants in patients with ALL, AML
and CML and myelodysplasia in patients under 16 years of age. Next
slide.

This briefly describes the patients. The male/female ratio as has been
seen is exactly the same. There seems to be a little difference in age.
As you can see, there are more younger patients in cord blood. The
difference is barely significant. At ethnicity, there is more of a
difference. CMV antibody and risk. Although in the risk there is some
difference but it\'s not very important. Next slide.

Here at the end of that comparison the biggest difference, of course,
comes in the bone marrow and cord blood for HLA where we have many more
mismatched transplants. Only six percent in that comparison are full
matches in the cord blood and in the cell dose, which doesn\'t require
any comment. Next slide.

This slide looks at the evolution and survival of these patients. Given
bone marrow in the black lines, the six-out-of-six matches and the five-
out-of-six matches are very similar. The cord blood, the six-out-of-six
matches is only 20, probably to survival rate of 68 percent. These are
49 and 46 percent. The cord blood at four-out-of-six or
three-out-of-six, which is the largest group in cord blood, has an
overall survival of 30 percent.

So that analysis shows that using bone marrow six-out-of-six as a
reference, there is no significant difference for the five-out-of-six
and the cord blood has a lower risk but is not quite significant. The
five-out-of-six and the three-out-of-six have increasing difference,
with the three- and the four-out-of-six being significantly different
from the bone marrow. But the five-out-of-six is not either in bone
marrow or cord blood transplants. Next slide.

This looks at the same data but this time from point of view of cell
dose. Here are the patients who received cord blood under 25 million.
These are all in the bone marrow which are used as a reference. This is
cord blood over 25 million.

Now you see the numbers are different as the majority are here, and this
curve is very close to that of the bone marrow. It\'s lower. It\'s not
significant now but presumably with many more patients it would be
significant. The difference, relative risk, is not a major league
difference. Next slide.

At five years survival, the effect of the total nucleated cell dose in a
restricted population of 12 to 15 years old, which are the oldest
patients here, we again have bone marrow and the cord blood with more
than 25 million cells. There is no significant difference here. The cord
blood under 25 million is significant. Next slide.

Again this is the same data but this time for patients between six and
11 years old. There is absolutely overlapping in most parts of this
curve for bone marrow and cord blood with over 25 million. Next slide.

This is the youngest patients. Here the two curves are different. Bone
marrow seems to be better. So if we are going to disallow transplants
for some age group, I think this would have to be the one, the youngest
of all patients. Next slide.

This makes a somewhat artificial grouping here. These are patients who
are in the younger group and have the cell dose over 25 million. It
looks at HLA matching. So those that have zero or one HLA mismatches are
identical to the bone marrow in most of its extension. Whereas those
that have two mismatches are clearly different.  This is reflected by
the Cox regression. Next slide.

In conclusion of all this data, the effect of histo-compatibility and
age on patient survival are similar in cord blood and bone marrow
transplants. So the trends that are caused by histo-compatibility and
age are similar. The survival of cord blood recipients can be improved
by using higher cell doses and particularly by better HLA matches.

If you put this evidence from the point of view of regulating or
providing guidance for the selection of tissue for a transplant, the
consequence is that we could identify safe levels for the selection of
these transplants. Age does not appear to be the factor that needs to be
considered. Thank you very much.

CHAIRMAN SALOMON: Thank you very much. This is an expanded dataset that
obviously matches nicely with that presented this morning. Any
questions? The only thing I\'m taking off the table is nucleated cell
counts until this afternoon.

DR. RAO: You showed the data when you reanalyzed it with the mismatches
in cord blood. It looked like now there was a significance between one
and two mismatches. Is that because those mismatches that you included
in the expanded set were so much better? Remember in the data that you
showed with cord blood transplants that the survival was much better
when they had two mismatches but they were in the direction of the
graft-versus-host, the blanks.

DR. RUBINSTEIN: That\'s right.

DR. RAO: Then subsequently they included them in that analysis and then
it became significant that the mismatch was better. Do you think that\'s
fair given that it was actually even better than the bone marrow
complete match?

DR. RUBINSTEIN: No. When you have mismatches that are in that direction,
that makes them better. We like those. I understood your question as
asking whether in this comparison those four or five patients are
included in the group with two mismatches. The answer to that question
is yes.

DR. RAO: So when it\'s included then you conclude that the one mismatch
is now significant as a difference.

DR. RUBINSTEIN: Yes.

DR. RAO: Maybe that\'s not fair. It may not be different because you?ve
changed the bar.

DR. RUBINSTEIN: You are correct but just to be accurate in your
presentation of the data, traditionally those are two antigen
mismatches. So that\'s why they are included. But because they are so
few, they still cannot compensate and make that subset equal to the one
mismatch.

CHAIRMAN SALOMON: How do you do HLA typing in these studies? In organ
transplantation now, we pretty much have gone over to molecular typing
so that the danger of finding these blanks and not being able to
distinguish homozygocity for the allele versus an unusual allele is not
as big an issue any longer.

DR. RUBINSTEIN: It is and it isn\'t. It is not a big issue in the sense
that we now know that they are homozygote for sure. When you use
serologic data, you cannot tell that. You cannot say that. Or when you
use low-resolution DNA data. That is correct.

Now this data is done at the serological level of resolution, which for
us is serology with all the known splits. All the accepted splits are
in. It is confirmed by medium resolution DNA typing for class I. It is
at the sequence level of resolution for class II. It is different for
the two classes. We have analyzed the data to see where the increased
resolution available from the splits in serology whether that makes the
data any better. It doesn\'t. We are currently analyzing the result of
testing all of our five-out-of-six matches by sequence analysis of class
I. That data is not available yet but it will be very soon.

CHAIRMAN SALOMON: So is it fair to say then that unlike organ
transplantation if you look at splits and you take those out as
mismatches, you actually call it split match, although traditionally by
serological data a split would be conservatively called a mismatch. This
is data from Terasaki.

DR. RUBINSTEIN: It is.

CHAIRMAN SALOMON: It is, yes. But if you take the splits and call them
matches, you actually do fine.

DR. RUBINSTEIN: It doesn\'t make the data any worse.

CHAIRMAN SALOMON: Right. Is the same thing true for cord blood
transplantation?

DR. RUBINSTEIN: Yes, that is correct.

CHAIRMAN SALOMON: So the splits act just like an organ. They act as a
match.

DR. RUBINSTEIN: Exactly. The increased resolution there does not seem to
help you to select a better subset of the match.

CHAIRMAN SALOMON: Dr. Wagner, if you want to step to the mike.

DR. WAGNER: While I can\'t answer that for cord blood transplants, a
split is a mismatch in bone marrow transplants.

DR. RUBINSTEIN: Absolutely.

CHAIRMAN SALOMON: That\'s what I was asking you. That\'s not been true
for organ.

DR. WAGNER: A split is a mismatch.

DR. KURTZBERG: I think the jury is out on cord blood. I think a split is
a mismatch in cord blood as well.

CHAIRMAN SALOMON: Just so we are clear about it when you say a split is
a mismatch you are saying you get worse results. Obviously we realize
scientifically a split is a split. It\'s always a mismatch. I didn\'t
mean to confuse anyone but you get worse results with a split.

DR. WAGNER: Yes.  Everyone has shown that. It\'s been shown by multiple
people.

DR. KURTZBERG: And it\'s a moving target in terms of what you called a
match in 1995 and what you call a match in 2000 and what you call a
match now. It\'s really difficult and I know Pablo has made that
analysis. The definitions change as the technology improves and as the
selection of donors change as the technology changes.

CHAIRMAN SALOMON: Just so nobody misunderstands why I brought that up,
because in organ transplantation the argument has been on the table that
one could improve the distribution of organs because of this data
showing that splits can be treated like matches. It is a relevant point.
If you don\'t get away with that, then that\'s too bad. Then you have to
deal that.

DR. RUBINSTEIN: The point I think is that for class II we have clear cut
and definitive evidence that in class II, increasing the resolution and
getting the subtypes helps. The subtypes contribute to the matching. You
should indeed not use low resolution class II data. Our data for class I
does not show improvement by going to a more refined typing. The
analysis is not complete. There may be some procedure. I would rather
suspect that it will not contribute very much because of the very high
probabilities that we get with the best matches. If it mattered, I
don\'t know how much better it could be.

DR. CREER: Just one other comment on that. This is Dr. Creer again from
St. Louis. The specific set of primers that are often used to PCR
amplify these HLA target gene regions that we use for typing sometimes
do not include primers that will amplify relatively rare types,
particularly those likely to occur in specific ethnic groups. So they
will appear as blanks but when studied with sequence based allele-level
resolution will actually be found to be not homozygous.

Without actual sequencing of every one of those blanks, you\'re not
sure. You can indirectly perhaps answer the question if you look at the
ethnic background of those \"blanks\" and compare that to the
distribution by ethnicity of the total group to see if you are selecting
out more rare ethnic groups. If that is the case, then perhaps that
might be something contributing to it.

I know your numbers are small and the donor base is such that it\'s
difficult to get these low ethnic representation, but the recipients,
when they\'re blanks, that\'s when the problem I think becomes even more
so and they have a rare ethnic group. They will appear in that
particular way, the way you are grading your GVH or rejection vector.
You might not have that totally correctly identified.

DR. RUBINSTEIN: Yes, I think that is absolutely correct a priori and
there is no argument. It is interesting however that in the data itself
the results were so good. If there was a residual heterozygocity, and
that procedure heterozygocity was important, it should have been worse,
at least a little worse, than the full matches.

DR. HOROWITZ: I think it\'s an interesting phenomenon that deserves
further study, but I think we have to be really careful about going off
on biological speculation when you have such small numbers of patients
in which the phenomenon is observed. I would say the jury is out on
whether those four patients with 4/6 matches having 100 percent survival
is a real phenomenon or a random event.

I think Dennis Confer is here from NMDP. I think the most recent data
looking at class I and looking at high resolution DNA typing and
disparities only detectable by that method, as opposed to serologic or
low resolution DNA typing, suggests that class I may be as important as
class II in terms of the necessity in matching for bone marrow
transplantation.

It takes a lot of numbers to be able to look at those things, but I
would say based on the fact that with more and more numbers of cord
blood we see that everything that\'s been true about bone marrow is true
about cord blood. That HLA matching is important. All the other
prognostic factors are important. I would be surprised if as we get more
numbers of patients we didn\'t see the same phenomenon there.

Then there\'s splits versus high resolution DNA typing. The typing for
these pairs are to the level of splits identifiable with current
reagents at the time the transplant was done. It is a moving target. The
level of resolution of the donor/ recipient designation as matched or
mismatched is different over time in all of these registries\' studies.
I would be very hesitant to say that class I isn\'t going to matter in
cord blood transplants.

DR. RUBINSTEIN: No, class I does matter, but whether there is additional
improvement by matching at the more refined splits and more refined DNA
subsets, that is where we are not sure yet. I would expect it as you
said. But regarding those unusual one directional mismatches, I must say
that there is data in bone marrow. There are 12 patients from Seattle
that have been reported only in the book, not in a formal publication,
in which the same phenomenon occurs.

DR. HOROWITZ: Yes, we\'ve looked at it in our database which is a lot
bigger and we don\'t see that same phenomenon. I don\'t know either.

DR. RUBINSTEIN: It probably then is not significant.

CHAIRMAN SALOMON: Dueling numbers, that\'s a theme here. John.

DR. ALLAN: To change the subject, in one of the latter slides you
presented, you said that the zero to five age group, the kids who got
the bone marrow did better than did the kids who got the cord blood. Do
you know why that might be true?

DR. RUBINSTEIN: No, I don\'t know the answer to that. I think overall
the variability of these numbers is quite substantial. I don\'t think
one can expect them to be always precise just purely on a statistical
basis.

DR. ALLAN: Could it possibly be cell dose, just that the kids got more
cells from the bone marrow?

DR. RUBINSTEIN: It could be that. It could be also that the patients who
came to cord blood were a little later. There are a number of other
possibilities but the numbers per se don\'t tell the whole story.

DR. HOROWITZ: They also tended to be the most mismatched. There is a lot
of selection biases in selecting a bone marrow transplant versus a cord
blood transplant in clinical practice. If you have an infant, since kids
seem to tolerate mismatch, tolerate transplant the younger they are,
clinicians are less reluctant to take a more mismatched cord blood in
that setting.

If you look at the under fives, there the disparity and mismatch was
greatest. There are a lot of two and three antigen mismatch in that very
young group of children that got cord blood transplants in that study.
So part of it is because that was a more mismatched group.

CHAIRMAN SALOMON: From the FDA, any additional comments, because we\'re
going to go to the public comment here. Given that the intention of this
morning was to set a base upon which we will discuss the questions in
the afternoon, is there anything that you feel hasn\'t come out here?

DR. LAZARUS: Right, no, I think that all of these presentations and
questions are leading very nicely into the discussion that we\'re going
to have in the afternoon FDA session and that is where we will ask them.

DR. HARLAN: Just a quick comment with regard to the apparent disparity
between what you are talking about with splits and solid organ and bone
marrow transplant. A major difference I think is the problem with cold
ischemia in an organ transplant and that there is such a major effect of
how long that organ is cold on its ultimate survival that you might be
unable to see a difference in the split in that setting, where you could
see it in the bone marrow. So it could explain this apparent disparity.
Immunologically speaking, the null hypothesis should be, I think, that
the closer the match, of course, the better it will do. It just may be
hard to see that in the organ transplant setting.

CHAIRMAN SALOMON: It\'s taken me several years to get used to the idea
of calling a six-out-of -six HLA matches an allo antigenic, but of
course it\'s true but it just drives me nuts. I finally can do it
without choking. Definitely what I\'m saying is that the rules for bone
marrow, the bar is much higher for these guys than for organ
transplantation. I just have accepted that.

I had one question. Here again the dumb questions chair\'s get to ask,
but just biologically I think about the known mechanism for
graft-versus-host disease. We have been talking about HLA as if it\'s
all the same. Certainly class II antigen mismatches and class I antigen
mismatches have a whole lot to do with the CD8 and CD4 cell activation
pathways. Can we make some comment? When you say that class II antigen
mismatches are really important, I\'m fine with that as the data shows
it. What\'s the biology? What is the difference between that and a
patient who has a problem with a class I mismatch?

DR. RUBINSTEIN: The first point is that we are talking now specifically
about the contribution of these very refined splits. We can see a DF4 in
32 different varieties, for example.

CHAIRMAN SALOMON: I\'m not talking about splits now. I\'m just talking
about class II is relevant, right? So if you had a group of children who
received cord blood transplants, or adults for that matter, who received
class II mismatched transplants and had an impact of that class II
mismatching. Then you compared that to a group that had class I
mismatching. I\'m done with the splits thing. The question is do you
know much about the biology? How different is the biology in a class II
mismatch problem and a class I mismatch problem in terms of GVH? Do they
have more chronic GVH for example? Do they have more infections? Do
their T-cells not come up as quickly? It\'s just an interesting
question.

DR. RUBINSTEIN: Yes, it is a very interesting question and you are
right. We know that the biological mechanism of activation of T-cells is
different in the two groups. I would like to just answer your question
with empirical data. When we compared mismatches at class I or class II
as we defined them, which is class I defined by serology and class II at
highest resolution, both of them are important. Whether they are equally
important or not, I suggested by our data it\'s still not possible to be
sure because the numbers are not sufficient to discriminate the slight
effect in which class II could be stronger or class I could be stronger.
I think to some extent it is difficult to generalize the answer to that
because it will depend on what is the balance of factors that would
influence the death or survival of these patients. The empirical data so
far indicate that if there is a difference between class I and class II
as defined, it\'s not a major concern.

DR. CREER: One comment. Eppy Petersdorf has actually done a detailed
structural characteristics of difference in class I antigen mismatches
that are serologic and molecular versus molecular only. The molecular
only mismatches tend to map predominantly to the antigen binding group,
whereas the combination of serological and molecular mismatches map to
both the combination of T-cell receptor contact and antigen binding
group and are associated with a higher incidence of rejection of grafts
than those that are molecular only. That was a very large number of
patients. I can\'t remember how many that was. That was NMPD data but it
was many thousands required to actually map those particular sites.

The general consensus that I get without having a lot of data to show it
is that class II antigen mismatches are more predictive of
graft-versus-host disease as opposed to class I. The data that I\'ve
seen suggests that there is no difference. That still seems to be a bias
that we see in transplant centers that search us in trying to match cord
blood transplants.  They believe that class II antigen mismatches are
more predictive for graft-versus-host.

CHAIRMAN SALOMON: Certainly experimental data, unless someone wants to
correct me, would not agree with that. Class I mismatching is a powerful
driving force in transgenic animals at least for GVH and the immunology
of it. That\'s why I asked if anyone knew anything about the biology.
Dr. Chao.

DR. CHAO: I think the data would suggest that there is really no
difference whether they are mismatched class I or class II. Both would
contribute to graft-versus-host disease. The other part that is
different in these patients is a lot of work done by Jim Ferrard
demonstrating the importance of cytokines and amplification of that in
numerous positive graft-versus-host reaction which always happens.
Whether that turns into disease or not depends a lot on the preparatory
regimen of the patient because each patient can get total body radiation
or very high doses of chemotherapy. A lot of this might be number\'s
game, but I think with larger numbers there is no doubt that class I and
class II difference probably contribute equally to GVH.

CHAIRMAN SALOMON: And again I think that\'s really interesting. I would
love to know more about the biology event. Is there any difference? It
may have the same negative impact of class II mismatch and class I
mismatch. It would really be interesting to know. They can\'t be the
same mechanisms though, right?

One would guess that a class II mismatch would lead to a mechanism
predominantly being driven through cytokine activation driving then
other HLA minors that aren\'t in the typical class I mismatch or
possibly molecular mismatches that aren\'t defined by the serology.
Whereas class I mismatches might not have that same cytokine drive. You
just wonder whether those patients might be more or less susceptible to
chronic GVH. Maybe this isn\'t as big a point.

DR. CHAO: Probably not but I think there are major mismatches. In these
patients who are mismatched either class I or class II, it\'s major HLA
mismatch plus minors. That major mismatch I think is a direct antigen
presentation versus an indirect antigen presentation. I would suspect
that is probably similar.

DR. CONFER: Dennis Confer from National Marrow Donor Program. I don\'t
want to trivialize it, but it\'s all important. Everything is important.
Right now, our data suggests that ABC and DRB1 are all important and
they are all related to graft-versus-host disease. They are all related
to adverse outcomes and we can\'t pick out any that are more important
than the others.

The thing that I remind you is we\'d like to simplify it and be able to
come up with simple models. Animal models that are simple don\'t readily
translate into the human situation. But remember HLA identical sibling
transplant is a great model here because there you have identical
regions of the chromosome no. 6 in the donor and the recipient because
they have inherited them from the parents. They are genotypically
matched all the way along this and they can still get all the way from
no graft-versus-host disease to fatal graft-versus-host disease.

The only situation currently that doesn\'t cause a risk for fatal
graft-versus-host disease is identical twin transplant where literally
everything is matched. It is all important. Sorting it out is going to
be a monumental task.

CHAIRMAN SALOMON: Your comments are well taken and it actually brings up
an interesting thing that typically is a theme in these meetings and
hasn\'t come up as much today. That is the potential use or lack of
utility of animal models to study this, because whenever you get into
these gray areas of considering biologicals, particularly cell
transplantation, to the extent that one has confidence in these
surrogate models one can advance significantly ahead of clinical trials
and ensure safety, etc. What I\'m hearing from you is a certain amount
of concern that these animal models are predictive. Any other comments
on this area?

DR. HARVATH: I wanted to just make a point about and ask Dr. Rubinstein
because he\'s had such vast experience in cord blood banking in the last
decade, is we heard your composite data just now and then we heard the
earlier data of your first 530 some odd cord blood unit transplants.
Could you help us understand how the product processing advancements may
have changed, perhaps becoming better, allowing you to get higher volume
product, perhaps better yields and so on from that first experience of
the first 530, 540 transplants to now your over 1,000 units?  

We know from your papers that you are constantly refining the
technology. If you could comment about the importance of the kinds of
measures that are needed to get at the quality of that product. So if
you could help us understand whether there is something that we should
think about when we are addressing these FDA questions regarding the
quality of the product and the advances you\'ve made from the beginning
of your bank to the current time.

DR. RUBINSTEIN: There have been a number of steps in arriving at the
current procedure. We don\'t consider the current procedure to be
optimal. There will be further improvements. Most of the improvements in
the banking have been in improving of the processing of the cord blood.
We now can routinely obtain 92 percent recovery of all the mononuclear
cells in our final product. The average is a little above 95 percent,
but we set the limit at 92 percent. That was not always the case.

Another factor that we have seen recently or really documented the
importance of this factor recently is the intervention of something we
call transient warming events in the management of the cord blood. These
events are when you expose liquid nitrogen temperature cord blood, so
one that has been frozen as minus 196, and now you take that cord blood
from one place and put it in the air and put it in another place. This
is unavoidable at the time of shipping because there you must transfer
the cord blood to another vessel.

But in our earlier experience when we used the standard doors for liquid
nitrogen conservation, every time we took the rack out to select a unit
the other two, three or four units that are in the same rack get
exposed. So each unit would be exposed as many as four times.  That does
not happen anymore. We have a system where each unit is stored in a
unique destination. So you can access that unit and that unit alone.  We
don\'t modify any other.

We have been ordinary about using all the units under the level of
nitrogen from the start. But we estimate that the average number of
transient warming events for our older units was about four. Now it\'s
only one. The significance of these warming events was demonstrated. We
reported these at an ISH meeting a couple of years ago.

CHAIRMAN SALOMON: Okay, last comment.

DR. WAGNER: So now that we are discussing banking issues and the
importance of specific banking manipulations on the outcome, one thing
as you know that I\'ve tried to address with the banks is really the
issue of identity of the unit. How do we verify identity of the unit? As
you know over the summer, we had a problem where we had a graft that
already went through the entire screening process and yet it was the
wrong unit from which they said it was, the unit that we thought.

So in fact what happened is that the patient had undergone a preparative
therapy and on the day of the infusion we found that the ABO typing was
mismatched from what the bank said the ABO typing was. That then led to
HLA typing of that unit. We found out that the entire unit, even though
the numbers match and the HLA in the computer matched the HLA of the
patient, it was not at all the same unit we believed it to be. How do we
verify or how do we improve the safety from a transplanter\'s
perspective of the unit identity?

DR. RUBINSTEIN: I think that\'s an excellent question and one where
everybody who has done banking of any type has had problems. This is a
real critical problem. We suggested from the beginning, and we have
followed these rules, every unit that is collected has a segment that
accompanies that unit throughout its life.

Before a transplant is made, the typing has to be repeated on that
segment. This process has been facilitated now immensely by the
availability of DNA techniques, because the segments of the cord blood
units will not always freeze well at the same rate as the content of the
unit itself. So the viability of the cells in the segment for serologic
procedures cannot be guaranteed. With the DNA techniques, you can now
have an absolutely convincing definitive evidence of identity.

DR. WAGNER: So that leaves us as a transplant center where we have units
that are said to be HLA matched or suitably matched with the patient.
Then we are faced with a dilemma in those cases where there is not an
attached segment: should they be used or should they not be used.

In fact, what we\'ve instituted as a result of this incident, which by
the way the patient died \-- what happened subsequently is that for
those units that do not have an attached segment we will have a back-up
unit available that will not be as good, but we\'ll have a back-up. Then
we do a thaw the day of transplant and we will do a rapid HLA typing for
class I prior to its infusion. The majority of cases it will be
correctly identified, but we have to have that strategy now in place now
recognizing that this is potential complication.

One other issue is related to things that have occurred over the past
couple of months, and that is as new infectious disease screens come
down the pike that are considered to more sensitive, how do we evaluate
older cord blood units that were stored with different methods of
infectious disease screening?

CHAIRMAN SALOMON: I don\'t want to say that you can\'t answer that but
not now. That\'s a very good question. These are all good questions and
I would rather focus them all together in one part this afternoon. Thank
you very much, Dr. Rubinstein. I would like to go on at this point to
the public hearing before lunch.

I have to say this meeting has the largest number of people speaking in
the public hearing part, which is fine. It is certainly part of the
whole purpose of these public meetings. It\'s to encourage people and
make sure that we demonstrate free access. The only thing I would say is
that there are a lot of people so if we could work together to keep
these on time and to the point we\'ll get through them. Otherwise things
could get really bogged down.

I would like to start with Mr. Steve Barsh. I know that you through your
hard work brought together quite a number of families with some very
well-behaved children I must say. Mr. Barsh, if you would make your
introductory comments. I\'m going to start with Mr. and Mrs. Ackerman
after you.

MR. BARSH: I get to say good afternoon. My name is Steve Barsh. I\'m a
parent of a child who went through an unrelated cord blood transplant
recently. I would like to thank the FDA and BRMAC for the opportunity to
present today. I would like to thank you for having the courage to allow
experimental medical technologies such as cord blood transplantation.

We first learned a little over two years ago that our son, Spencer, had
ALD or adrenoleukodystrophy. Fifty percent of ALD children have a deadly
cerebral onset of this rare neurodegenerative disease leading to a loss
of function and death by the age of ten. The remaining 50 percent have a
severe to deadly form as adults. A parents\' worse nightmare, this is
the disease behind the film \"Lorenzo\'s Oil.\"

Spencer was one year old when he was diagnosed. Spencer started repeated
MRI studies, and on February 14^th^ MRI studies, just 12 months ago,
changes were seen in the brain stem area indicating a deadly cerebral
onset had begun.  Cord blood and bone marrow transplants are the only
accepted therapies, if you call them accepted, not exactly from an
insurance point of view, but viable therapies when an cerebral onset of
ALD has begun and is caught in time. We had already been looking for a
perfect six-out-of-six bone marrow match and we couldn\'t find one. Plus
we were looking at a minimum of three to six months before we could even
get to transplant while Spencer would be deteriorating.

Even if you find someone on the donor list, and this has been brought up
this morning, it doesn\'t mean that you can get to them quickly. It\'s a
heartbreaking and gut wrenching process of calling in potential donors,
having them screened, confirming they will go through surgery, etc. You
are losing precious time waiting.

ALD, like many metabolic diseases, can move very quickly along its
destructive path and deficit changes can sometimes be seen weekly and
even daily. This is particularly true for rare metabolic diseases such
as Hurler\'s, metachromatic leukodystrophy, Tay Sach\'s and others. In
metabolic diseases, lessening the time to transplant equal less deficits
and usually a better transplant outcome.

Time is enemy number one. In cord blood, the unit has already been
typed, checked, prepped and is waiting in a freezer right now. It\'s
felt that 95 percent of the U.S. population needing a stem cell
transplant already has an acceptable match in a cord blood bank today.

If it\'s not already obvious, we decided on a cord blood transplant.
Spencer\'s pre-transplant work-up testing started just four weeks after
his bad MRI, only five days after my wife and I made the decision that
he should be transplanted. He was a little over two and a half years of
age at that time. He fully engrafted, by the way since it\'s a big issue
for you, on day plus 17^th^. His cord blood transplant was just 10
months ago.

Today is day plus 314, but who\'s counting? He\'s doing extremely well.
We are seeing improvements in deficit reversal on a weekly and even
daily basis. We feel so lucky when we think about those kids with rare
metabolic diseases and disorders whose deficits are instead worsening.
This is because they are being told that there is no treatment option,
it\'s too late, or they are deteriorating waiting to find a bone marrow
match as they mistakenly think it\'s the only option.

Four months ago at only six months post transplant, physicians began to
see improvements in Spencer\'s MRI as well as his clinical presentation.
It appears that Spencer is actually remyelinating as the earlier
progenitor stem cells from the cord blood are differentiating into other
cell types in his brain. The MRI and clinical observations have been
confirmed both by Duke and a diverse team at Children\'s Hospital of
Philadelphia (CHOP).

The CHOP team included Dr. Peter Phillips, Director of Pediatric
Neuro-oncology and Dr. Gihan Tennekoon, Acting Chief, Division of
Neurology. I believe that the non-technical word that they\'ve used was
\"amazing,\" going on to say that they\'ve never seen anything like
this. We usually don\'t see MRI improve.

Cord blood works. These treatment options must be available to children
who have these devastating diseases, including malignant diseases. It\'s
imperative that cord blood banks be licensed so they can reimbursed
reasonable, break-even fees, not having to live by a set fee that leaves
them begging for donations and funds to keep their doors and freezers
open.

Finally I would like to raise the point of informed consent, an issue
that seems to be important in the past to this Committee. Before Spencer
was transplanted, we received a document from our transplant team that
clearly delineated the risks we were taking and what was going to be
done was experimental. It was given to us days in advance so we could
have ample time to review it and digest what it said.

The transplant team scheduled several review meetings with us and was
not satisfied until every single question we had was answered and they
felt that we had a solid understanding of the process and the risks. The
issues we faced were frightening and we knew that this was a life and
death decision. The transplant team helped us understand everything,
including that we might very well lose our son.

We looked at like any other decision. What was the risk versus what was
the reward? We knew the risk of not intervening, certain death. Based on
our understanding, we were willing to take the risk. Interestingly,
there were families who we became very close with who lost their
children during transplantation. One of those families might actually be
speaking today. No one expressed the lack of understanding of the risk
they had taken. They felt they did the best thing possible for their
child given the few medical options they had.

In closing, it\'s important to remember that you can get to transplant
with cord blood extremely quickly which is very critical in many
metabolic diseases. In some of the discussion I heard this morning, it
seemed to be something that was overlooked. Comparing six-out-of-six and
bone marrow versus cord blood, on a metabolic disease these kids
deteriorate quickly and time is of the essence.

Also I would like to highlight that the early progenitor stem cells in
Spencer now appear to be differentiating into other cell types that are
repairing problems in his brain. Again just \"amazing\" is the term that
we hear again and again.

Public cord blood banks must be licensed so they can operate at
break-even not a continual deficit potentially having to close. They
shouldn\'t be wasting precious time and energy scrounging for money so
they can save lives. We now have a normal three year old son. His name
is Spencer Barsh and cord blood saved his life. Thank you for your time.

CHAIRMAN SALOMON: Thank you, Mr. Barsh. I certainly hope that you can
come back here in 10 years and tell us about your normal 13 year old
son. Mr. and Mrs. Ackerman. We have Connor. That\'s the young man. Then
after that would be Mr. and Mrs. Niakani. Please be ready to go.

MR. ACKERMAN: Good afternoon. My wife couldn\'t make it today. I\'m Glen
Ackerman. This is my son, Connor. Three years ago almost to the day,
Connor was diagnosed with AML leukemia, subtype M5, and he had the T9
and 11 abnormalities. The doctors gave him a zero chance of survival
without a transplant. We were unable to find a suitable bone marrow
match, and without going into the long story we ended up selecting a
cord blood. We didn\'t know much about it at the time, but with some
help from some friends we ended up at Duke University. We had a
four-of-six match. That was in June. He was diagnosed March 3, 2000. He
go the cord blood June and he engrafted 37 days after transplant.

Without going into the entire history, he has done well post-transplant.
We have had a few bouts in the hospital. For the most part, he has done
well. He\'s only on right now amoxicillin 250 mg twice a day, basically
as a prophylactic. He\'s in the 3^rd^ grade. He\'s an average student,
plays soccer and swims on a swim team.

I would just like to say I think you should license cord blood to help
out other young children. As a career Naval aviator, I just want to put
in a plug for the military here. We have numerous folks at remote
locations around the world and the speed at which you can get cord blood
transplant is very important to them. We also have a significant
minority population in the military and my understanding is it\'s
difficult to match bone marrow. That\'s another consideration for cord
blood.

Finally I\'ve worked a lot because we had numerous bone marrow drives
for Connor, all of which obviously were negative. However, I know
there\'s a DOD bone marrow organization with C.W. Bill Young I believe.
So I know the DOD is involved with bone marrow and with all the military
hospitals around the world and with all the military folks, I think you
have a ready source of cord blood out there. With the right funding and
licensing you could make it work for other folks. Thank you.

MS. NIAKANI: My name is Faraday Niakani and my husband and my son are
not with me today. My son, who is nine and a half years, old was
diagnosed with ALL at the age of four. He went through treatment for two
and a half years. He had relapse. We couldn\'t find a bone marrow match.
Our only option was to have a cord blood transplant. Time was running
out and we went ahead with it.

His first transplant did not work. He had to get a second cord blood
transplant which the second one took. It was a success. I believe that
we should go ahead and license cord blood because it\'s vital. I have
him. We have many complications, but this is a big challenge that I
face. I don\'t know what else. Thank you.

CHAIRMAN SALOMON: Mr. and Mrs. Ruderman.

MR. RUDERMAN: Hello, my name is Joel Ruderman. This is my wife, Lori.
 This is our little miracle, Hunter. He is almost four years of age.
This is our youngest son, Isaac. Hunter, At 10 months of age \-- I still
remember the date, March 24, 2000 \-- was diagnosed with leukemia. He
started a protocol here in Washington, D.C. in which he was just on
chemotherapy. He relapsed almost within a month. At about 11 months he
had already relapsed from chemotherapy. We were looking for a
transplant.

We had spoken to the doctors and the things that they had pointed out to
us was he needed to have a transplant immediately. So we searched around
and we investigated cord blood transplants, which clearly we knew very
little about at the time. We decided to go down to North Carolina to
have the transplant done on Hunter.

Everything since has been a success. He engrafted at seven days on July
18 and has just continued to improve ever since. His match was only a
four-out-of-six, which from my understanding would not have worked for a
normal bone marrow transplant. He has just moved forward. He is
obviously a success and the reason why we believe licensing should go
forward.

MS. RUDERMAN: It saves children. Thank you.

CHAIRMAN SALOMON: Thank you. I wonder if there\'s an impact on this on
behavior of children, because they are so good. Next would be Ms.
Kathleen Kim.

MS. KIM: Hi, my name is Kathleen Kim. My daughter, Emily Kim, is
currently eight years old. She was diagnosed with ALL when she was
three. She went into remission quickly after diagnosis and went through
the three and a half years of chemotherapy and was off treatment for
about 11 months before she relapsed again. This time it was CNS relapse.
We were told that the best option for her was a bone marrow transplant.
Being Asian, we also knew that the possibility of finding a perfect
match, much less a suitable match, was pretty slim.

As it turned out we found six potential donors in the U.S. or actually
in the world but four of them turned out to be four-of-six matches. The
fifth was unavailable. The sixth was a five-out-of-six match at the ERB
level or mismatch. It was taking a long time to find a donor despite the
17 bone marrow drives that we ran. We obviously did not find the right
match.

My husband and I decided rather than sitting back and hoping that
eventually someone would come along and be able to help Emily that we
did some research and found out about several of the transplant centers
across the country that specifically specialized in cord blood
transplants. We took Emily down to Duke last August 2001. She got her
transplant in October. While it was not a complication-free transplant,
I\'m happy to say that the complications were not life threatening.

She is a happy, fairly healthy eight year old who just the other day
said to me \"Mommy, can I do something?\" My response was \"no.\" Her
response was \"But I don\'t have cancer anymore.\" I was thrilled when
Dr. Kurtzberg contacted me. Actually she didn\'t contact me; I contacted
her with my five million questions and asked me to speak today because I
really had a hard time deciding between a bone marrow transplant and a
cord blood transplant.

A five-out-of-six mismatch is not a terrible thing with bone marrow as
I\'ve seen today from the statistics. However, as Steve Barsh mentioned,
time is of the essence. You don\'t want to wait for your child to
relapse and you don\'t know how long it\'s going to take for the
transplant to take place. There are so many factors that go into bone
marrow transplants. You need the donor to agree. It has to work in their
time schedule. They have to be healthy enough to be able to do the
procedure. We couldn\'t wait.

In retrospect, to be honest, I wish we had just jumped right into the
cord blood transplant because it would have saved us a lot of time and
heartache and frustration and anxiety and so many nights of not
sleeping. I have no doubt in my mind that the cord blood transplant
saved my daughter\'s life.

As Steve mentioned, I think it\'s imperative to license the cord blood
because there are many kids particularly Asians who don\'t have a lot of
options. A cord blood transplant may be their only option. Thank you for
giving us the opportunity to speak today. Hopefully these parents and
our words will touch you and you will take these stories into
consideration when you think about licensing the cord blood. Thank you.

CHAIRMAN SALOMON: Mr. Herb Lee.

MR. LEE: Hi. My name is Herb Lee. I had not intended to come here today
to say anything. I was just going to stop by and see what\'s going on
here. But as I sat back and I saw some of what was being presented, I
felt as though I needed to make some comment.

Our son, Andrew, was diagnosed with leukemia when he was three and a
half years old. After two years of chemotherapy, he relapsed. We of
course looked at the options and initiated a search for a possible bone
marrow transplant. As Mrs. Kim mentioned being Asian the chances are
very slim of finding a match. From my understanding just a standard
match is maybe one out of 30,000. There are only maybe 30,000 Asians
registered as far as I know.

We decided that since we could not find a match for Andrew for bone
marrow to continue chemotherapy and wait and see if there is something
else that could up. After almost another two years of chemotherapy,
Andrew relapsed again.

This time we told that it was leukemia but something called mixed
lineage leukemia which is a little bit different. The prognosis was as
the doctors put it very poor and when I said what does very poor mean in
this case they said he was not going to survive. I guess very poor is
not good.

Again we initiated a search for a transplant and the results for bone
marrow were zero. As I looked at the presentations as I was sitting
back, it looks like comparing bone marrow and cord blood and risks and
factors and that kind of thing, I am not a medical person, for us it was
very simple. The risk was that he was going to die if we didn\'t do
anything.

The difference between the risks of bone marrow and cord blood well
there was no choice there. We had zero for bone marrow. There was some
chance with cord blood. As a parent of a seriously ill child, the only
thing that really matters is having hope when the doctors tell you that
the prognosis is not good. Cord blood gave us hope.

Unfortunately Andrew passed away due to a virus infection but he
actually went through the transplant very well. He had a good time out
there in some ways I\'d say. But it gave us hope as a parent. I think
it\'s given life to a lot of children and a lot of families without cord
blood. There are some circumstances to me all the data means nothing
because there\'s only one way to give some families hope. Thank you.

CHAIRMAN SALOMON: I believe that the split now is that these were
families with children that had undergone cord blood. If I\'m wrong I\'m
sure someone will correct me. I believe the next three people are adults
that have had cord blood transplants. I think that\'s an interesting
distinction here. The first would be Mr. Steven Sprague.

MR. SPRAGUE: Good afternoon, ladies and gentlemen. I\'m certainly happy
to have this opportunity today. My name is Steve Sprague and after
listening to your presentation this morning, I\'ll introduce myself to
you as a lucky duck. Any long term cord blood transplant survivor is
happy to be anywhere to be able to do anything.

My real purpose here this afternoon is to try to put a smiley face on
some of the numbers and slides and statistics that you saw this morning.
I just want to take a few minutes to talk to you in patient-speak.

I know you probably can\'t tell but I\'m an aging baby boomer. But I was
already a medical veteran before I got my leukemia diagnosis when I was
47. I was a diabetic. I had survived a heart attack. I had had quadruple
by-pass surgery in 1993.

Despite of all that, I was totally unprepared for a battle with cancer.
That began in November 1995. For those of you who know leukemia, those
were the pre-Glivec days for CMLers like me. In those days, chemo only
really stalled what was inevitable for a cure and that was the allusive
bone marrow transplant. We refer to it as the healing hell those us who
have gone through it.

I was resigned when I got my diagnosis to a slowly moving leukemia but
for whatever reason 18 months after my diagnosis I went into blast
crisis, the end stage of this disease. That was in May 1997. I found
myself suddenly in a real pickle. Six weeks after hospitalization, my
oncologist got me into first remission while obviously began marrow
donor search. I was an only child so of course they had to find an
unrelated matching donor if I was even going to have a transplant to
consider as an option.

To make a long story short, as unbelievable as it was to me at the time,
I was not one of the lucky ones to find a marrow donor match. The doctor
looked me square in the eye and said Mr. Sprague, you enjoy your
remission for as long as you can. Get your affairs in order. We\'ll keep
looking and we\'ll try to figure something else out.

At that point in this story, it was a typically sad cancer tale for
adult leukemians. Then a series of events that\'s even hard for me to
understand or to describe even now, I\'m convinced that those of us in
that situation it\'s really all about fate. It\'s about the miracles
about modern medicine that you folks deal with on a daily basis. It\'s
about faith. It\'s certainly about being in the right place at the right
time.

Here\'s what I mean. I was beginning to lose my remission just at the
same time that my oncologist, Dr. Andy Peccora, up in New Jersey was
planning to start a new clinical trial, first one of its kind in the
area of end stage adult CMLers using stem cells from umbilical cord
blood. Part of that trial in my case would be to try to expand the stem
cells ex vivo using one of the experimental medical devices. This was
the Aastrom Replicell System.

Now look at me. I was a 239 pounder back then. That was a whole lot of
expanding if this thing was going to work. Equally astonishing to me the
New York Cord Blood Center found a perfect six-out-of-six cord blood
match for me in a matter of days. So I went into the hospital October
30, the day before Halloween. Magic, miracles happened and 40 days later
I was discharged certainly by the grace of God but a lot of other things
as well. That was in December 1997. A new working immune system, no
leukemia and no hair. Of course I didn\'t have much when I went in.

Fast forward a little bit and here I am now before you five years, two
months, 19 days and I count every one of them, still a 100 percent donor
cells. All female chromosomes because my donor was a female infant, not
a single filly chromosome and still no hair.

I certainly had a lot of time to think about things as a post transplant
patient advocate. I learned a lot of things not only about myself but
about life and about death and about perspectives, appreciations and
priorities and the most important one that I have come to learn about is
hope. Several others have spoken about that word. It\'s an important
issue for those in the transplant community.

My point is simply this. Part of that hope for desperate patients that
are seeking transplant, patients like I was once, involves options.
Heading down the transplant trail as I don\'t have to tell anybody here
sometimes against overwhelming odds is a risky endeavor even in the best
of circumstances. But that first critical step can\'t be taken without
some kind of a stem cell match.

I\'m not exactly sure what you as a committee have in mind here but I\'m
obviously convinced that a cord blood transplant is an option that has
to be available to any adult willing to undertake the risk and should
not be restricted just to children. For me I just walk around as living
proof that cord blood can work successfully for adults and even for very
large, very ill middle aged adults. Sometimes as I\'ve discovered the
gain ends up well worth the taking of the risk.

I would ask you not to deny this life restoring option to adults who may
have no other hope. Had that been the case for me five years ago, I
think you all know how that story would have ended.

I would remiss if I didn\'t take this opportunity to thank everybody in
this room for what you do and the way you do it so well and certainly to
the FDA for giving the public, the survivor community, an opportunity to
be heard and to try to convey to you from a patient perspective what
going through transplant is all about. Thank you.

CHAIRMAN SALOMON: Thank you and approximately half of us are sympathetic
about the hair. The next speaker would be Ms. Heidi Shaw-Tweten.

MS. SHAW-TWETEN: Hello, I\'m Heidi. I\'m from Rochester, Minnesota. I
just wanted to come here and tell you my story. When I was 10 months
old, I was diagnosed with Diamond-Blackfan anemia. With that you don\'t
make any red blood cells. So the only treatment for that would be either
to have bone marrow transplant or have blood transfusions. Since I had
no family members that were a good match for a transplant and the risk
of graft-versus-host with a bone marrow transplant was too great, I had
monthly transfusions.

By the time I was 20, I had made a lot of antibodies against the blood
so the blood was just breaking down. The quality of my life had really
diminished. I had to come in about every two weeks for transfusions and
I was still tired most of the time. I had to look for other options.

Six years ago I had an unrelated cord blood transplant that matched
four-out-of-six. At that time I only had grade one graft-versus-host
disease which is totally resolved. Also I\'m off all the antibody
anti-rejection meds for about five years and all my counts are normal.
Since then, my quality of life had greatly increased. A year after
transplant, I was already skiing in Colorado. I have graduated from
nursing school and I\'ve worked at a major medical center and been
married for the last two and a half years. In June, my husband and I are
also planning on adopting also a baby boy.

Without the opportunity of having a cord blood transplant, I probably
wouldn\'t be here talking to you today. So I\'m very grateful for the
opportunity of the cord blood transplant and I hope others have that
option. Thanks.

CHAIRMAN SALOMON: And then Ms. Gayle Serls.

MS. SERLS: I\'m last.

CHAIRMAN SALOMON: No, you are actually not last. There are two other
people after you.

MS. SERLS: A cord blood transplant has extended my life so now that I
have menopause and hot flashes.

CHAIRMAN SALOMON: Approximately half of us would sympathize with you.

MS. SERLS: So that\'s really a good thing now or otherwise I might not
have experienced this. In May 1995 I was at my grandmother\'s funeral
when she died at the age of 98 and had been a very active and alert
person. I thought that\'s going to be me. I\'m going to live to be at
least 98. At that time, I was 45 years old and I was fit and I was
strong. I was confident that my body would carry me on for the next 50
some years.

Then two months later I was diagnosed with acute lymphocytic leukemia.
Where did that come from? It was out of nowhere. I had been a bit tired
but all of a sudden all of my glands had swollen. So here I was. I went
into the hospital and three weeks later I found out I had the
Philadelphia chromosome which is a nasty little bugger and was set out
to kill me. I knew that I was in big, big trouble.

I have two children and they needed a mother. Let me tell you. My
insurance company stepped in and they said you could go to Johns Hopkins
and talk with them. I thought that\'s wonderful. That\'s where the bone
marrow transplant program was born I believe and I felt confident that I
would get very good treatment.

They told me I was too old. You are too old to have a bone marrow
transplant. I said okay what are we going to do. They offered an
autologous transplant and then that would be supplemented with something
that would boost my immune system and I forget the name of what they
were going to do. While I was encouraged, I knew I would accept my own
cells and I thought I could live through this and maybe extend my life
and if it doesn\'t work totally something else will come along that will
extend my life further.

At any rate, they wanted me to be in remission. The day that I got on
the plane to go to Johns Hopkins to start this procedure, I had a lump
right here. The next day I reached Baltimore and was in my room.  The
next morning I woke up and I looked like I had the mumps. I knew it was
bad.

I went to see the doctor on my regular appointment and the first thing I
said was the leukemia is back. She just started shaking her head. I\'ll
never forget that. I said what else can we do. Is there peripheral stem
cell? I was just trying to think of words. She said there\'s just not
enough time. That was the darkest day. I had hope up until that point
and then all of a sudden I had no hope.

I got on the plan and went back to Durham and back to Duke. Peter
Jennings and my mother helped save my life. I was in Duke. I was on
really heavy or big doses of chemotherapy and for the very first time I
was sick. I was really sick. I had colitis and something had happened
and my eyeballs felt like they were inflamed and I was on morphine.

My mother called me one night when I was in the hospital and she said I
just heard on Peter Jennings that cord blood is being used to help
patients with leukemia. I immediately called a friend because I was
afraid I would forget it because I was on morphine and it was like
minute to minute. She called the American Red Cross and the American Red
Cross referred her right back to Duke, the hospital I was in.

Before I left the hospital, Dr. Mary Laughlin came to my room. We talked
and I thought we were talking if I could be a candidate but she was
talking like we\'re going to do this. I got back into remission. I got
out of the hospital. Two weeks later, I was back in the hospital and I
had my cord blood transplant on May 1^st^.

Now I\'m 46 years old. I don\'t remember what day the cells came in. I
had very little graft-versus-host disease. I actually had a good time in
the hospital. I was waited on hand and foot. I had very little
complications that I remember. I was out of the hospital in about eight
to nine weeks and by the middle of August I was off all medications.
Since that point, I have had no side effects, no other medications.
Nobody told that there might be complications after you get out of the
hospital.

If it wasn\'t for this cord blood transplant which was a four-out-of-six
match, as everyone has said before it\'s time when time is of the
essence this is the thing that you can turn to. This is the thing that
will give you hope. When I heard in the hospital that I had another
option, I had gone from zero to maybe 10 percent of saving my life. That
was the biggest 10 percent that I will ever realize. It didn\'t matter
if it was 90 or 100 percent. At that point, my spirit rallied. My body
rallied. I was set to go.

It\'s a wonderful thing. I\'m on the opposite end of the scale. I\'m six
feet tall with 145 pounds. It worked. I hope that it will stay available
to help other people like me regain their life. I\'m very thankful to be
here. Thank you for letting me speak.

CHAIRMAN SALOMON: Again I want to thank all of you. We\'ll go on with
two more presentations but these will gear back to more traditional
format of data presentation. I just want to review five points that I
wrote down that I thought came out of the aggregate of this public
session. I just wanted to put them out there because perhaps these would
be things that we would turn to perhaps this afternoon in terms of
appropriately contributing their input into our public discussion.

The first was and in no particular order a mention of
licensing/reimbursement issues. There were several very pertinent
references to racial distributions specifically for Asians but I think
that it would be very appropriate to expand those considerations to
African-Americans, Hispanics and Native Americans. I\'m sure up here
I\'m missing a major racial distribution and I apologize to them.

A third issue was speed to the procedure, time to the procedure. That
was made specifically at first in the case of metabolic diseases but
then echoed in several others with respect to these precious gaps
between remissions particularly in the adult diseases. Relative to that,
my fourth point was also mentioned which was the impact on these
families and also on the patients that I just can?t imagine. As a
physician, I can get in touch with exactly the stress of running bone
marrow drives and not knowing what\'s going to happen tomorrow as
opposed to the whole concept of an efficient banking procedure.

It was interesting that the mention of the military and just take that a
little broader and say what sorts of things could enhance the overall
process and the diversity of these pools.  That might be something to
consider. Lastly something came up at the end about high age limitations
for transplantation. That was kind of interesting about the idea that
you might be too old. Usually the range is somewhere around 55 or 60 for
certain transplants even though that\'s gray. The possibility that with
cord blood transplantation older patients might be candidates that
wouldn\'t normally be for bone marrow. That\'s something I would like to
hear a little bit of comment on later because I wouldn\'t necessarily
have guessed that one. It certainly didn\'t come up in this morning\'s
discussion. Would anyone add anything to that list?

DR. WEISS: I think the other issue was informed consent. That was
brought up.

CHAIRMAN SALOMON: Informed consent is such an big issue that I totally
agree to the extent that we probably should never have a discussion
without taking a moment to do a reality check on informed consent.

DR. KURTZBERG: I think the last issue is that for people who don\'t have
a bone marrow donor this is the only available donor source they may
have. A high proportion will find a donor that gives them an option for
transplantation.

DR. TSIATIS: Maybe you know more as a chair but I\'ve sat in a number of
these committees and I never know from the questions what the FDA is
getting at. Maybe when they read the questions this afternoon, they can
tell what the background is a little bit. It sounds like there are some
issues with licensure and things that I don\'t know about and I guess
we\'re asked to discuss it. It would be nice to have more background.

DR. HARLAN: The only other point that I heard from the public comment
was the concept not proved but the concept that these cells might allow
or differentiate into tissues other than bone marrow or support the
differentiation of other tissues into good phenotype.

CHAIRMAN SALOMON: Now there are two last speakers. If we can go to Dr.
Michael Creer from the St. Louis Cord Blood Bank and trying to keep
these presentations around five minutes if we can.

DR. CREER: Thus far the issue of safety of umbilical cord blood for
transplantation has been presented primarily from perspective of the
transplant center. I would like to address it from the perspective of a
cord blood banker.

There are three major issues that we address in the cord blood bank with
regard to safety. They relate to the donor, the biological properties of
the product and the way we handle it in the lab. With regard to donor
suitability and selection, we have adopted the general guidelines that
we use for screening whole blood donors with slightly more restrictions
applied to that including serological and nucleic acid based testing for
pathogenic viruses, viral exposure in the donors and have now achieved
unprecedented level of safety in those products. I don\'t believe that
it\'s a significant problem in any way, shape or form at this particular
point in time.

We are now faced with impractical objective of achieving zero risk which
we cannot do. I think the main limitation that we have here is the
request by many centers to actually screen patients or screen products
for inherited metabolic disorders which we cannot possibly achieve
practically. There is one issue there that is a whole other subject to
address.

With regard to the biologic properties of the product, we know
ultimately that what will determine outcome will be the content of a
hematopoietic stem cells/progenitor cells (HSC/HPC). I think at this
particular point in time we have to acknowledge that we have no
practical way of measuring that. The only things we have available are
surrogate markers to currently use surrogate markers or total nucleated
cell count and CD34 cell count.

We have seen that the one thing that does distinguish umbilical cord
blood from all other transplant products at this time is the fact that
there is a dose limited effect. The characterizing dose is therefore the
single most important task of the cord blood banker. We currently do
that based on measurements of total nucleated cell count and CD34. We
know biologically that among these two measurements we expect CD34 to be
a much stronger correlate of the actual HPC/HSC content. Our inability
to demonstrate that may relate not only to the fact that CD34 is maybe
not the perfect marker but perhaps the methods we use to measure it are
inadequate and that\'s the main problem.

I would address how we go about characterizing products and what this
might potentially impact us in terms of what we think we know about
transplants outcomes and their relationship to products. I\'ve shown
here the sequence of events that occur between the time of collection
and the time of transplantation. Products are characterized in the cord
blood bank at two separate stages, immediately after collection and
immediately before controlled rate freezing and putting them to sleep in
liquid nitrogen.

These measurements preprocessing are primarily used solely for purpose
of quality assurance within the laboratory to optimize processing
procedures. The post processing information that we obtain on the
product is all that\'s available to the transplant center for search of
cord blood products.

We then put the cord blood product to sleep by a process of controlled
rate freezing, leaving it in liquid nitrogen which is the most stressful
event on the product that could ever possibly occur during the sequence.
Product is then removed from that state and thawed. At that point the
product is recharacterized by the transplant center and infused into the
patient.

It\'s appropriate to correlate clinical outcomes with what actually gets
into the patient so it\'s very clear that we need to correlate the
clinical outcomes with the information that we obtain after thawing.
However the information that we obtain after thawing may not be reliably
extrapolated and utilized by the transplant center to select products
because they could be very different.

To give you some idea of the potential problem this might create the
question is could they potentially be different because the technology
does not exist to allow us to characterize them or is it because we have
not adopted uniform standards for processing. I think that what we can
say right now is that adequate technology exists to provide very precise
measurements of total nucleated cell count with very good cord blood of
variation, a measure of variability in the measurement.

However CD34 cell counts cannot be accurately determined at this point
in time with values that range over a fivefold range from 0.4 as you can
see to 2.0 at about a 1.4 percent overall cell level which is comparable
to what we generally find in stem cell products. When you go down to
about a half of a percent, the range is over fourfold different. That\'s
just instrument variability. These are analyses conducted on the same
products and the exact same material by different labs.

In order to get a better idea of what the variability might be within
stem cell processing labs, I want to clearly acknowledge the people from
the American Red Cross particularly Dr. Gary Moroff and Dr. Rebecca
Haley for allowing us to participate in the study designed to assess the
variability in laboratory cord blood bank laboratory processing and
total nucleated cell count and CD34 characterization.

Utilizing a mean plus or minus 25 percent target for characterizing
total nucleated cell count in CD34 and in products that have not been
cryopreserved but remain in the liquid phase, you can see that with TNC
we can get very good precision and nine out of nine labs agree. However,
agreement among labs for CD34 is considerably lower.

If you look at the values obtained after thawing the product, TNC values
become quite a bit more variable and this is due again to differences in
recovery between labs. CD34 looked pretty good although there were two
outliers that had to be rejected just to get that kind of agreement. So
there seems to be even on the same product thawed the exact same time by
each different individual laboratory. You can see after we do the
characterization post thaw, there is more variability in results. Next
slide please.

In order to determine what the product characteristics might be that we
report to the physicians from the cord blood bank side and what\'s
actually going into the patient, I\'ve taken this data from Dr.
Wagner\'s publication regarding the correlation of TNC with CD34. I\'ve
compared it with the TNC versus CD34 correlation that we have obtained
in the products processed in our lab between January 2001 and January
2003.  

The slope of the lines are different. They both show a very strong
correlation between CD34 cell count and TNC. What you can see with the
individual products that there is a significant difference. What you get
by CD34 measurement in a product tells you something totally different
than TNC. Even though individual products show an overall correlation
what the actual values in any individual product can be quite different.

When you take the products post thaw what you see is that the variation
is even greater. The CD34 cell count in some products can be extremely
high. Whereas the nucleated cell dose that you would predict from that
would be very low. Based on all the information that we\'ve seen so far,
you would have two different very disparate predications about outcome
in products based on that kind of information. Next slide please.

In order to determine what kind of a problem this might be overall, I
have summarized the post thaw recovery results from 60 different
transplant centers that we exported products to and compared them with
CD34 recovery. In the blue line and I apologize for the contrast here
you can see that the TNC recovery varies between a low of zero which one
particular institution reported zero recovery while the CD34 recovery is
about 55 percent.

Overall the average is around 70 percent with variations extending from
a low of 55 to a high of about 98. However if you look at CD34 recovery,
it\'s very variable from a low of 20 to a high of about 90 percent.

You can see that I\'ve ordered the transplant centers on the bottom to
show increasing recoveries. Then you will see a break in the line and a
solid at the bottom indicating that almost two-thirds of the
laboratories do not even measure or report to us CD34 counts post thaw.
Next slide please.

To look at some other potential problems we have with these values I
took this information, downloaded it from a website, I won\'t tell what
particular cord bank claims this but you can see what we have is
misleading information being presented to physicians in a variety of
different formats with regard to what they might expect in a cord blood
product.

Some people claim 100 percent nucleated cell recovery. At one
institution with 25 to 50 percent average over other institutions and
the data I have from our 60 sample would indicate that those are clearly
unrealistic claims on the front end but the 25 to 50 on the back end. I
have never seen one that bad on average. Also viabilities of 99 to 100
percent I think are also clearly not what you would expect. So the
information out there for many of the physicians particularly the
inexperienced transplant physicians who might be trying to initiate a
cord blood search is something that we have to address and maybe
establish some standards in terms of recording and advertising. Next
slide please.

In terms of what we might have for information with regard to
correlation of transplant outcome, we have now exported approximately
520 products from the St. Louis cord blood bank that shows the trend in
transplant exports over time. As you can see, virtually over 45 percent
nearly 48 percent of all of our products have been in people for one and
a half years. So transplant outcomes from our particular bank are going
to very limited in time. Next slide please.

To give you some idea of the problems to the particular cord blood bank
centers with regard to utilizing transplant outcomes to optimize
processing, this just shows some of the responses that we get from our
transplant centers with regard to reporting. Only 58 percent of all the
transplant centers actually report to us complete post thaw data. Only
50 percent of them actually report to us any kind of outcome information
between zero and three months. Only 34 percent give us outcome
information extending between three and six months and it drops to 21
and then down to 10 percent for transplant outcome information extending
up to one year. As I mentioned earlier, part of that is because a lot of
the products we have exported have not been a year out in terms of their
transplant but you can see that there is a great deal of loss of
information that comes back to the cord blood banks from the transplant
centers. So we don\'t know if we are doing the right thing or not. We
have no ability without adequate reporting to help us improve our
processing. Next slide please.

In summary I would say that currently there is no standardization of
methods used to characterize cord blood, hematopoietic progenitors, cell
composition, resulting considerable variability and results were posed
by different cord blood banks and transplant centers. There are
significant differences in post thaw data, correlating outcomes with
cord blood characteristics and post processing data provided to
transplant centers to guide product selection. Inexperience transplant
centers may be mislead by unsubstantiated claims of cord blood
processing efficiency and post thaw recovery. There is a significant
number of cord blood transplants for which outcomes have not been
reported and we have no idea what\'s going on with those right now.
Finally failure to report clinical outcomes to cord blood bank impedes
our progress in optimizing quality of safety in cord blood processing
and is a major problem that I would like this committee to try to
address. Thank you.

CHAIRMAN SALOMON: Thank you very much. The last speaker of this morning.
You really want to ask a question, Alison. Okay.

MS. LAWTON: Just one really quick question because you talked about
standardization and obviously the methods used for testing. But one
question I have just for my clarification.  What consistency is there in
the collection and the processing and the storage and shipment for all
these different banks?

DR. CREER: The collection model varies. There is a point of contention
about whether or not the best way to collect this is to have individual
obstetricians collect it or whether it should be done by an ex vivo
procedure. We have not seen significant differences in terms of overall
yield comparing the two methods between our center and other centers
that use two different ways of collecting. The infection rates seems to
be the same. Overall sale yield seems to be the same. I don\'t think the
collection component is that critical.

With regard to transplant to the laboratory, we have not seen a
significant loss in viability with products received up to 12 to 18
hours. The components of collection with regard to how you do and how
fast you get it to the lab seem to be problems that we can overcome and
that don\'t seem to have a real significant impact on the product
quality.

The issue with regard to the processing we all utilize pretty much the
same standard approach to red cell depletion and plasma depletion of our
products. The approaches that we use have been published in the
literature. I think efficiencies for banks with experience are quite
comparable. So what we actually get back from with the products that we
begin with is very comparable across labs.

Contamination rates are very similar. I think the real problem that
results in the variability is in the laboratory assessment of what the
progenitor cell content is in the product which is the ultimate
determinant of outcome. We have no practical way to assess that.

The surrogate markers that we are currently using are imprecise and
being utilized at different points in time and are giving us misleading
information about how to correlate that with transplant outcomes. Until
we can get around that, our ability to show statistical significance
with regard to transplant outcomes for these product characteristics is
minimal. That\'s what I think we need to address. We have got to get
more tight data or variability will never allow us to show what the real
dose effect is and give us the ability to give guidelines to other
transplant centers so that they know what they should get and to be able
to predict that from what we\'ve already published. That\'s critical. If
we try to extend this or license this, I don\'t think we can do it until
we can give firm guidelines.

CHAIRMAN SALOMON: I think that there are a lot of complicated issues
here. I\'m not sure they are all just based on typing things. We\'ll get
into the details of that this afternoon.

DR. KURTZBERG: One sentence. There is an NIH sponsored on-going current
study called COBLT which is collecting data prospectively and which has
standardized approaches that will answer some of this question.

CHAIRMAN SALOMON: The last speaker of this morning is Mary Laughlin who
is at Case Western Reserve and is going to talk about a retrospective
analysis based on data from the International Bone Marrow registry
comparing bone marrow to cord blood.

DR. LAUGHLIN: Good morning. Thanks for the opportunity to present this
data. This is a study that is coordinated by the IBMTR to analyze
alternative donor transplants for adults with hematologic disorders.
This is a comparison of unrelated donor bone marrow or peripheral blood
and unrelated donor cord blood. The study chairs including clinicians
Dick Champlin and John Wagner and myself are collaborating with Mary
Horowitz and her group at IBMTR as well as Pablo Rubinstein and Cladd
Stevens at the New York Blood Center. Next slide please.

The primary objective of this study is to determine one and two year
survival after allogeneic transplant in adults with hematologic
disorders lacking in HLA identical sibling donor comparing these
transplants using bone marrow or peripheral blood from HLA matched or
mismatched adult donors with that from umbilical cord blood. Secondary
objectives of this trial are to monitor the incidence and kinetics of
hematopoietic recovery, the incidence of severity of graft versus host
disease, the transplant related mortality, disease of recurrence and
disease of free survival. This study has been on-going since March of
last year. Next slide please.

This slide outlines the patient demographics of the current study
cohort. I\'d like to begin with the caveat that this is a preliminary
data analysis and that as such has not yet been subjected to peer
review. The current study cohort includes 186 adults over age 16 who
have undergone myeloablative conditioning and infusion of minimally
manipulated single unit cord blood. These patients are compared with 451
adults who have also received full myeloablative conditioning and
infusion of neither six to six or one antigen mismatched adult derived
bone marrow or peripheral blood graft.

There are no differences as far as demographics of sex. There are
differences in demographics of age with a higher proportion of the cord
blood patients being a younger age group compared to the bone marrow
group.

There are also differences in weight. The cord blood patients are
slightly lower weight than the bone marrow cohort. Further differences
in race with a higher proportion of minority groups represented in the
core blood study group. Disease entities, there\'s a higher
representation of CML in the bone marrow group. Next slide.

Further demographics to share with you before we go into the analysis
includes disease status. There were differences in that.  In the cord
blood patients, there was a higher proportion of patients with advanced
disease as defined by relapse, primary induction failure, blast crisis
or advanced myelodysplasia. A higher proportion overall of the cord
blood patients received TBI based conditioning compared to the bone
marrow group. GVHD prophylaxis also differed.

The majority of the cord blood patients received a combination of
cyclosporine and steroids due to concerns of administration of
methotrexate and its potential impact on engraftment. The nucleated cell
dose in these patients was fully a log less than that of the adult
derived grafts. HLA compatibility was one antigen mismatch in 21 percent
of the patients. The majority of the patients, 69 percent, received two
antigen mismatch grafts and ten percent of the patients received three
antigen mismatched grafts. There were four patients who had
six-out-of-six matched grafts.  They were not included in the analysis
due to the very small numbers. Next slide please.

This is a uni-variate analysis of outcomes after transplant. Treatment
related mortality was lower in adults transplanted with fully matched
adult derived grafts. Importantly in comparing bone marrow from adult
donors that is mismatched at one antigen which is considered a current
standard of care in multiple transplant centers, treatment related
mortality was similar in comparing adults transplanted with HLA
mismatched cord blood.

Other parameters, including leukemia free and overall survival, again
was better in the patients receiving fully matched bone marrow grafts.
However importantly equivalent in comparing these two patient
populations, those receiving bone marrow with one antigen mismatch from
adult donors and HLA mismatch. Next slide please.

Cox proportional analysis was performed and the model was to test the
main effect of the graft source. Potential confounding variables that
were built into the model included patient age, sex, disease status, the
graft cell dose and HLA compatibility and TBI versus non-TBI
conditioning. Next slide please.

This Cox proportional analysis in the analysis of relative risk of
treatment related mortality using matched bone marrow from adult donors
as a reference point demonstrates that if you received a graft from an
adult donor that was mismatched at one antigen or various levels of
mismatch cord blood grafts you demonstrated an equivalent likelihood of
treatment related mortality which was all higher than that of the
reference group.

Age was also a factor in predicting treatment related mortality as a
continuous variable and disease stage in the aspect patients with more
advanced disease demonstrated a higher treatment related mortality not
unexpectedly. Next slide.

The relative risk of relapse again using patients infused with matched
bone marrow as a reference point was lower in all patients receiving HLA
mismatched grafts whether they were from an adult donor or derived from
cord blood. There seemed to be a lowering risk of relapse as increasing
HLA disparity was observed.

Further disease stage not unexpectedly predicted a higher relative risk
of relapse. Here patients with more advanced disease had a 6.4 fold
higher risk of relapse. Next slide.

The relative risk of overall mortality again using matched bone marrow
as reference point was notably higher in patients receiving HLA mismatch
grafts either from adult donors or from cord blood. Again age broke out
in the Cox proportional analysis as a predictor of overall mortality, as
a continuous variable and patients with more advanced disease were more
likely to die of their procedure. Next slide.

In an analysis where now the reference group, the patients receiving
fully matched bone marrow from adult donors, is excluded. We see no
differences in comparing cord blood of various HLA disparity with single
antigen mismatched grafts infused from adult donors. Next slide.

So in summary we find in our retrospective analysis and again I want to
emphasize that this is a preliminary data analysis that cord blood is
equivalent to one antigen HLA mismatched grafts from adult unrelated
donors in all transplant outcomes including treatment related mortality,
relapse, leukemia free survival and overall survival. We also found that
HLA matched grafts from adult donors is associated with improved
transplant outcomes compared with one antigen mismatched adult derived
grafts and cord blood.

The Cox regression analysis thus far does not identify HLA mismatched
cord blood to be associated with poorer transplant outcomes as observed
in recipients of adult derived grafts. In addition, cord blood cell dose
in this dataset was not associated with improved transplant outcomes in
these adult recipients but I wish to emphasize the narrow dose range
that may be contributing to this observation.  Thanks for your
attention.

CHAIRMAN SALOMON: Thank you, Mary. I don\'t know about you but I\'m
hungry. How about we go to lunch and try and get back here in an half
hour and I\'ll start the meeting as soon as we have a quorum. See you in
a little bit. Off the record.

(Whereupon, at 1:21 p.m., the above-entitled matter recessed to
reconvene at 2:13 p.m. the same day.)

A-F-T-E-R-N-O-O-N S-E-S-S-I-O-N

2:13 p.m.

CHAIRMAN SALOMON: Welcome back from a fast lunch to the afternoon
meeting where we are going to initiate a discussion of the four
questions from the FDA. Before we get started particularly in responding
to David\'s questions about just wanting a little more context on the
questions which is fine and that should be an on-going process as we go
along, I would like to start off with Phil Noguchi. Phil, do you want to
make some comments?

DR. NOGUCHI: Yes. Thank you, Dan, and thanks everyone for getting a
little heartburn over the quick lunch. What we\'ve seen today is we came
to this meeting with a certain set of data that has been evaluated. We
certainly have seen a lot more data being presented. We have had an
extraordinary public meeting where we are seeing it\'s not just the
numbers. It\'s not just the curves but at the end of those curves, there
are people. There are people who have benefitted from the emergence of
this technology.

You have also heard from Dr. Rubinstein and Dr. Creer that it isn\'t
just the outcomes and it isn\'t just the patient and it isn\'t just the
source of the blood and the blood banks that produce them and the
centers but it\'s all of them together. What we have seen and are seeing
that the datasets are very interesting. They are very promising in some
aspects but they are incomplete and we are asking for advice on how we
can go further and begin to identify those things that are critical
versus those things that are yet to be developed in the future.

As we step through the questions, those concepts of what do we now need
will become very clear. Thank you, Dan, for allowing some exploration of
many of the concepts within the questions during the morning session.
With that, let\'s get stared.

CHAIRMAN SALOMON: The first question I have taken the liberty of
modifying it slightly with permission of the FDA. The question 1(a) is
gone. It will be discussed under two with a slight modification to the
way we will phrase two. For right now, it goes as follows. Does everyone
have the questions?

What factors should the Agency consider in determining the safety and
effectiveness for the use of placental umbilical cord blood for
hematopoietic reconstitution? That\'s obviously front and center today.
With revision, the first thing is specific disease indications including
specific types of cancers and severity of diseases for which there are
data on the use of cord blood, time to engraftment, HLA matching issues
and the incidence of graft versus host disease.

I must say in the context of this morning\'s discussions I feel that we
certainly aired enough data. I\'m not sure I\'ve had time to digest all
that data. It was definitely put there. We will need some help perhaps
in synthesizing it down which is part of it. What kind of things do we
think in discussion points?

Let me underline one thing though. In a way, you can all relax. None of
this is going to lead to any kind of vote. What we want to provide the
FDA is a high quality discussion that\'s balanced and shows what things
we feel comfortable as a group saying and others that the field feels is
just not known yet and in so perhaps establish priorities for future
questions in consideration. Really everyone is encouraged to participate
and feel that in the end there has to be some big pressure to come to
final conclusion. Does anyone want to start with 1(b) specific disease
indications? Mary.

DR. HOROWITZ: The thing that struck me in listening to the presentations
this morning and preparing for coming to this meeting is that the
results of cord blood transplantation are very similar to the results of
bone marrow peripheral blood transplantation. All the things that effect
bone marrow transplantation, and I\'ll use bone marrow to refer to both
bone marrow and peripheral blood, will effect cord blood
transplantation.

Some patients who get bone marrow transplants have a lower probability
of a good outcome than others but still it\'s an acceptable approach in
a variety of diseases. I haven\'t heard anything or seen anything that
would make me think that the spectrum diseases for which cord blood
transplantation should and can be used as any different from the
spectrum of diseases for which bone marrow transplantation should be
used.

What I\'m struggling with is where are the differences in cord blood
transplantation versus bone marrow transplantation that require some
special consideration. We are not talking about the general safety,
effectiveness of hematopoietic stem cell transplantation in general.  We
are talking about cord blood transplantation and trying to find out
what\'s a little different here and there\'s not much I think actually
that\'s very different in cord blood transplantation than the general
field of allogeneic hematopoietic stem cell transplantation.

The differences have to do with we are operating at the limits of cell
dose. There are special consideration for stem cell product where we are
operating at the shoulder of the curve where we have special area
actually includes inadequate for cell dose where we don\'t have to worry
about that too much in bone marrow transplantation. We are in the
adequate range by in large.

The other thing is that we have an experience with HLA mismatching in
the unrelated donor setting that we actually don\'t have in bone marrow
transplantation.  We have a fairly extensive experience with multiply
mismatched cord blood transplants that gives us actually a more precise
indication of what the outcome is with this stem cell product than we do
with bone marrow transplantation. The data that was presented today says
it makes a difference but actually the data with two antigen mismatched
cord blood transplants looks pretty good when you compare it one antigen
bone marrow transplants.

I would say the specs on the disease issue should be no different than
our usual specs on disease for bone marrow transplantation but we
probably need to have some special considerations about cell dose.

CHAIRMAN SALOMON: That\'s an excellent place to start. There are a lot
of implications that flow from the first statement. Bone marrow
transplantation, mobilized peripheral blood stem cell transplantation
and placental umbilical cord blood transplantation are essentially all
the same.

When we started there certainly is a scientific premise that would
suggest that they shouldn\'t be the same. In other words, if you had no
data, the hypothesis would be that these would be distinct. Certainly
the umbilical cord from the others because you have these immature
T-cells so you should have less graft versus host disease. I would like
some comment on that.

There are some biological reasons to suggest that there may be
differences. There\'s the issue in the so-called SCID repopulating
populations that suggest that these proliferate better, that they are
more primitive cells. Dr. Wagner picked up on that in a little bit of a
different way in human patients.

Given that as where we started, I agree with you that the data we saw
today when you get down to clinical outcome parameters which is what the
data was that overall the things that are going to drive therapy with
these tools are not distinguishing. How about some discussion along that
spectrum of things? Richard and then David.

DR. MULLIGAN: I\'m very impressed by those last comments. That
succinctly really summed up much of what we did here. That\'s the crux
of the entire issue we have here. Two things. Is there equivalence or
not and data? If we can come to some consensus that there is, that\'s
very important. Very important to separate this dosage. It sounds like
all the safety issues, the uncertainty of the questions really have to
do with this issue of the limitation that you have per patient. I would
propose that we leave out that, the limitation, to try wean out all
those issues and deal with the other part first and directly deal with
the issue of the dosage.

CHAIRMAN SALOMON: Precisely, yes. David.

DR. HARLAN: I\'m just going to agree but I propose a way that would
codify all of this pretty quickly is just to say that the indications
for umbilical cord transplant are identical to anything that exists for
HSC or peripheral blood transplantation except\... The only except I
heard was if you have an HLA identical bone marrow donor immediately
available because there the dosage is better and I thought the results
were better in that one exception. Correct me if I\'m wrong.

CHAIRMAN SALOMON: That\'s fair. Then what we are really saying though is
if you had an HLA identical cord blood donor it would be the same too.

DR. HARLAN: There\'s a difference. There you have the dose problem. The
one advantage that you have in bone marrow and peripheral blood is you
don\'t have the limitation of dose.

DR. HOROWITZ: Although I should point out that in the pediatric study
where dose is not as limiting there was some data suggesting that
perhaps if you had HLA identical cord blood transplant your outcome may
be better than with a HLA identical bone marrow transplant. Dose has to
figure into that. There are not sufficient data with HLA identical cord
blood transplants in adults to be able to make a statement one way or
the other in my opinion.

CHAIRMAN SALOMON: Don\'t forget to remind me that I have this one point
I have to make.

DR. WAGNER: I\'ll try to remember that. In terms of addressing this
point, are you saying that if there is an HLA matched unrelated marrow
donor then that should be chosen in preference to a cord blood donor
except maybe with the caveat of six-out-of-six cord blood donor?

DR. HARLAN: Based upon the data I heard ?-

DR. WAGNER: But that\'s not exactly what I said though. Basically what
the data suggests is that umbilical cord blood at least if it\'s matched
in one antigen mismatched that appears to be equivalent to a
six-out-of-six marrow.

It wasn\'t saying one was better than the other. In fact, the cord blood
six-out-of-six looked preferable to all other stem cell sources. However
the one antigen mismatched cord blood appeared to be equivalent the
six-of-six marrow. Whether you look at the IBMTR New York Blood Center
dataset or whether you look at the Minnesota dataset, that seemed to be
the case. You can\'t say you should select one over the other because
they were quite comparable in outcome.

Now you could argue that if you have your choices of six-of-six marrow
donor versus a four-out-of-six cord blood donor particularly if the cell
dose is low then you can argue perhaps in that setting that you should
choose the marrow donor in preference. What makes this all difficult is
the fact that it\'s this issue of timing of transplant. Therefore to
come up with a strategy that says if you have a potential six-out-six
you must wait for that. Practically speaking that can\'t happen. That\'s
where the dilemma comes up. Sometimes you just have to go ahead and use
the more mismatched graft because you simply can\'t wait. That\'s
something to keep in mind.

DR. KURTZBERG: I agree with John said. I can\'t stress that enough. I do
a lot of work with the children with inborn errors in metabolism. We can
go forward in two weeks with the cord blood and we can\'t do that with a
bone marrow donor unless it\'s in the family.

I don\'t think you can tell a family with two matched siblings there is
no regulation that says which one you are going to choose. If you have
equivalent donors then you as the transplanter have the authority given
the context of the entire situation to pick the best donor. So then I
wouldn?t, when you have equivalency, put a restriction on which goes
first. I think that\'s really important.

DR. HARLAN: As the subject matter expert, the one that has done a lot of
these, you should be the one that suggests. Are there any limitations
that should be placed on umbilical cord blood in your expertise?

DR. KURTZBERG: I think cell dose is the thing that should be defined as
safe or unsafe. It should be targeted to a certain minimal level. I
think we have the data to make those recommendations.

DR. HOROWITZ: Go back to the statement. The indications which I think of
as the underlying disease for which the transplant is going to be done
are the same for bone marrow, peripheral blood and cord blood
transplantation. In the absence of any compelling data which I didn\'t
hear today that one thing is really better than another because of all
the difficulties in these kinds of analyses that have to do with why one
went to a cord blood rather than a bone marrow transplant which have to
do with the issue that Joanne brings up which is you can\'t wait all the
time, CMV positivity of the donor, it would be hazardous to try and
regulate that decision making process.

CHAIRMAN SALOMON: I didn\'t take David\'s comments as he was suggesting
that an outcome of this was that we said you can do this or this but you
couldn\'t not that. Number one we all know that no one is going to allow
that sort of advice to be given to the FDA. You don\'t want that in any
medicine. We all realize that there are so many different things that
impact on the decision processes that we want to just come up with some
general views.

What I thought David was challenging was just is there any situation in
which one could argue that a bone marrow or peripheral blood was
different. That was the premise. So far, no one has come up with that.
In the absence of that, we can make a very clear statement.

I have one. That was the point I wanted to be reminded of. It would be
perhaps in some of these diseases where we are playing out this new
hypothesis that these stem cells might hone to disease tissues such as
occurs in the ALD children that maybe in that instance there might
actually be a rationale for using an umbilical cord blood. Again I know
there was no data shown today that will allow a decision on that. I\'m
just throwing that out as the one thing I can even think of now right
now.

DR. KURTZBERG: It\'s a very exciting field. There is a lot of early
information that is suggestive that cord blood might be a good stem cell
source but there are again so many complex decisions that go on in
making a decision to treat those children that it might get in the way
of their care if that kind of marrow restriction was placed on their
care. I believe they should have cord blood but I believe more
importantly they should have a transplant. I don\'t think we are at the
point where we have enough information to say it has to be this kind or
that kind.

CHAIRMAN SALOMON: And I wasn\'t going anywhere that. I was just saying
that it was the only thing I could think of that might eventually be a
distinguishing feature in terms of the diseases that might be treated
because that\'s what we are trying to swear. Mary.

DR. HOROWITZ: It should be noted that differentiation into
non-hematologic tissues has been demonstrated for bone marrow and for
blood stem cell transplants not just cord blood transplants without a
lot more data of one being better than the other.

CHAIRMAN SALOMON: Of course, do you believe that data too?

DR. HOROWITZ: It depends. We can talk about that later.

DR. KURTZBERG: Frankly, I don\'t think there will ever be a study done
where you analyze bone marrow versus cord blood. The other logistics
that go into that are just too daunting.

DR. RAO: Everybody said that bone marrow and cord blood are effectively
equivalent. Is the data quite clear for this one mismatch in bone marrow
with one mismatch in cord blood or is that still just a matter of timing
and availability that one would say there is no choice? In other words,
preference of one or the other?

DR. KURTZBERG: I\'ll start. There are so many biases. I\'ve done 500
transplants myself but I\'m biased. I accept more mismatch. I use a
certain type of typing. I have a certain timeframe. I only use a certain
bank. So whatever comes out of my center is biased by those decisions
that I\'ve made for better or worse. In my data, it is not clear that
the one-antigen or two-antigen mismatch make a difference.

Again you have to realize that the majority of patients who come to this
are people without donors. They are not people who are deciding between
a bone marrow donor and cord blood donor. They are people either who
don\'t have a bone marrow donor or because of time, don\'t have time to
wait for a bone marrow donor.

So you are talking about availability of a therapy versus no therapy.
What we have to do is decide if this is safe, if it\'s equivalent enough
to the other stem cell sources that might be available to allow these
people access to a similar therapy that they otherwise could not have.

DR. RAO: Are you saying something like it\'s a choice after when
compatible bone marrow is not available? That\'s why I asked this
question about the one mismatch because then the possibility of bone
marrow becoming available would be that much less.

DR. KURTZBERG: In the majority of patients that come to cord blood, they
have looked for a bone marrow donor and have not found them at least in
a timely fashion. But yes there will be some who have a five-out-of-six
match and make a choice between one or the other.

DR. HARLAN: Joanne, you said something that tickled a thought in my mind
too and that is all of the cases that we heard in the public comment
were people who really had no other choice. In the 500 that you
transplanted, are these otherwise fatal diseases for which there is
really no other choice and should that enter in our recommendations at
all?

DR. KURTZBERG: I think in general any people who go to any sort of stem
cell transplantation don\'t have a casual disease.

DR. HARLAN: I mean not a casual disease but these are rapidly terminal
diseases that we heard about today. The area where I could see this
becoming a slippery slope is if whatever recommendations that we make
mean to people okay we start using this for something that\'s maybe not
quite so terminal. Then the risk benefit equation changes considerably.

DR. KURTZBERG: You can go first.

DR. HOROWITZ: I think that\'s unlikely. Whether you are talking about a
bone marrow transplant or a cord blood transplant, as long as you have
informed consent I know the patients and their families won\'t take it
likely. The relative efficacy of stem cell transplant versus
non-transplant therapy is beyond the scope of this conversation and
probably unresolvable in a lot of instances.

Most of the patients who go to unrelated donor transplantation
regardless of graft source have diseases for which you can get
reasonable agreement that there aren\'t a lot of other options. These
are not transposes that they can be done easily. They tend to be done in
larger tertiary care centers. I don\'t think we have to worry about a
slippery slope here.

DR. HARLAN: I can tell you from my experience that we do. I deal with
diabetes all the time and patients say I don\'t care what I have to do I
want to get rid of this disease. They will do anything even if the risk
benefit ratio isn\'t supported.

DR. HOROWITZ: I doubt that there would a transplanter that would do it
for diabetes.

DR. KURTZBERG: This is not different whether you use cord blood or you
use bone marrow or you use peripheral blood. This issue is generic to
stem cell transplantation not different for cord blood.

CHAIRMAN SALOMON: That was the point that I wanted to bring us back to
and that is notwithstanding that I totally agree with David in that this
is a classic where in your field that it\'s probably not a very slippery
slope. But outside of you and stuff you haven\'t thought about yet,
it\'s real slippery.  The key thing here is if there is no critical
difference between hematopoietic stem cell of any of these sources at
least as far as the data shows today if that\'s going to be the
conclusion that we are rapidly getting to and then we\'re going to talk
about the matching and then we\'re going to talk about the dose then
risk benefit issues for those on the slippery slope, and I agree it\'s
there, it won\'t matter what they are arguing, the safety and efficacy
issues are still going to be the same for as long as we are using a stem
cell. David.

DR. STRONCEK: I do worry about the slippery slope too. You have to
remember with unrelated bone marrow donors there are criteria that
centers have to meet. It\'s fairly liberal but there still is a criteria
and the centers have to be qualified. Right not, there is no such
criteria for cord blood transplant centers. I agree that right now the
centers doing it are very good, very academic centers. It doesn\'t mean
that as this becomes more routine that other centers that aren\'t
qualified won\'t do it and they won\'t get as good outcomes.

DR. HOROWITZ: Related to that, I find it very concerning, the data of
the St. Louis Cord Blood Bank that they are not getting outcome data on
half of the transplants that they are facilitating. There?s a lot of
ways to get outcome data. You can\'t get an unrelated donor marrow from
NMDP unless you agree to provide outcome data. I don\'t think that the
criteria for obtaining a cord blood should be any less stringent than
that or else how can you evaluate the field and how can you evaluate the
outcome from that cord blood bank if half the data aren\'t there and you
don\'t know whether that\'s a representative omission or not?

CHAIRMAN SALOMON: I think that\'s a very important point. Let\'s hold
that for question three which is outcome parameters for clinical data
which I think is a nice segue to your question of how you can get the
outcome data. I think that\'s really important.

The slippery slope thing is worth discussing just a little bit. Two
things in the field right now that we heard about today relate to this
as well. One is a non-myeloablative preparative regimen and two, less
HLA matching. One thing that could happen here is that centers that can
do non-myeloablative regimen which are much less taxing than fully
myeloablative regimen, number one then would feel emboldened to go in
directions that would be defined and then if you open up and say one
good thing about cord blood stem cells is that you can break down some
of these HLA match criteria then again the pressure is going to be I can
go to this cord blood registry and I can find a two to three antigen
mismatch cord blood for me and cure my fill in the blank, diabetes,
lupus, rheumatoid arthritis, allergy. I do think we need to consider
that a little bit.

DR. KURTZBERG: Why do people have an M.D. degree if people are going to
be that cavalier about performing a transplant? We\'re in a lot of
trouble if that\'s true. I don\'t know how anybody can regulate that. I
think transplanters are generally very responsible and take what they do
very seriously and understand that the risks are quite high regardless
of which one of themes you take to an ending. This is not done lightly.
(1) I just can\'t imagine that you could control it. (2) I can\'t
believe that the practice of medicine would go in that direction.

DR. HOROWITZ: Could we ask John Wagner to give his opinion on that?
Except for a small number of transplants that were presented this
morning, almost all the data that is in the literature or was presented
this morning has to do with myeloablative preparative regimen. So I
don\'t know how you can say much about the others right now.

DR. WAGNER: Right. There are two points. First, I think there is a risk
of slippery slope but we need to go back and say would you do a
non-myeloablative prep using bone marrow cells. If you are willing to do
that without any oversight, then yes you are willing to do unrelated
cord blood without any oversight as well. It may or may not stop if
someone really wanted to do it.

On the other hand in terms of specific disease indications and I know
this is going to be controversial but the only disease that makes me
nervous only because I don\'t know the data is CML. CML is a disease
which in chronic phase where the results of unrelated marrow transplants
if you have a six-of-six , the results are very good as has been
reported by a number of institutions.

Now it\'s not to say that cord blood is any different. The question is
whether or not we have the data to be able to say that it is the same.
Maybe you do at the large repositories of data could address that issue.
But because of the Seattle data first reported by them and then by other
institutions, if you have a six-of-six unrelated marrow donor and you
have CML in chronic phase and you have the right placement of the moon
in the sky, you can actually have the outcomes in excess of 70 percent
success. That\'s the only indication that is not known to me. I don\'t
know that it would be any different if we have cord blood. It\'s just
that I don\'t know the answer.

CHAIRMAN SALOMON: So that\'s the conservative/gold standard argument.
Phil.

DR. NOGUCHI: Before we leave this topic, what I do want to say is Dr.
Kurtzberg is absolutely right. Responsible transplanters have ethic and
a standard that is will established. However cord blood in its own right
is a limited study. That is you don\'t get more than one cord blood
sample per person normally per donor. It is being banked under various
conditions. If you use one and it happens to be a match for somebody
that\'s one less person that may be able to get a use of it for a well
established procedure for which bone marrow transplantation is useable.

I think it is not for an in-depth discussion today but unfortunately FDA
does have to consider all these other things. We think the data is
certainly very encouraging for the outcome for the kinds of
transplantations that are typically done by transplanters. But as this
data becomes more widely appreciated, there is going to be the push for
what other diseases can we do that for.

That\'s where we really do need to be very clear about what data exists
for what is available as well as recognizing that the difference between
cord blood and other bone marrow peripheral blood stem cell
transplantation is. It limited in terms of the numbers. It is limited in
terms of the dosing and everything else. The others are at least
theoretically you might be able to do it more than once from the same
donor.

CHAIRMAN SALOMON: Mary.

DR. LAUGHLIN: I had a couple of comments. One was I wanted to add a
caveat to Dr. Wagner\'s comments of the aspect of CML.

CHAIRMAN SALOMON: This is Mary Laughlin from Case Western.

DR. LAUGHLIN: The data is antedating the initiation of oral Glivec and
certainly the demographics of CML patients that are stepping forward
into unrelated transplant are not in chronic phase generally. The role
of cord blood may be even more preeminent now in the treatment of CML in
the Glivec phase because we are now more often seeing patients with
blast crisis or accelerated phase having grown through oral Glivec. That
was one comment I wanted to make.

The other from the standpoint of a leukemia physician in the trench is
this aspect of somewhat doing the math of 10 leukemics who needed
transplant to having a donor sister that matches them. The remaining
eight turning to unrelated sources of grafts whether they are from
adults donors or cord blood and then the kinetics of their disease
determining which of those grafts is optimal for their disease.

It would seem to me from the standpoint of a licensure mindset that
those diseases where stem cell transplant is a standard indication
doesn\'t require stringent mechanisms such as IMD but that perhaps by
these newer non-myeloablative approaches to extend into renal cell
cancers, autoimmune processes that the restrictions might be to incur
greater stringency in those less well defined indications for stem cell
transplant in general.

DR. KURTZBERG: Just relating to CML, this was our data for CML in kids.
These are kids in accelerated phase or blast crisis and two in chronic
phase. There were only 11 patients but this is mismatched cord blood in
terms of what we have with the median follow-up of 4.7 months but
actually I think that\'s probably about 19 months. This is not data that
would suggest to me that cord blood wouldn\'t be a reasonable source for
kids with CML who come to us.

CHAIRMAN SALOMON: Can you just educate me? This would be very good for
adults with blast phase CML. Is that also true for children?

DR. KURTZBERG: Yes, this is a mixture of accelerated phase blast crisis
and there are a couple in there with first or second chronic phase.

CHAIRMAN SALOMON: Are children less prone to problems?

DR. KURTZBERG: I don\'t think so.

CHAIRMAN SALOMON: So it\'s just as bad for them as it is for adults.

DR. KURTZBERG: Yes.

DR. WAGNER: The only point to make however is that in contrast to what
Mary Laughlin indicated, it\'s a very small subset in the narrow
transplant setting that fits all those perfect criteria. When you look
at that perfect criterion that\'s the only group that makes me somewhat
nervous. It\'s not to say that I know what\'s right or wrong. It\'s just
that I don\'t know what\'s right or wrong. That\'s my only point.

CHAIRMAN SALOMON: I think again to reassure you that\'s not what the FDA
is worried about. The FDA is not going to try and tell you that you
shouldn\'t do this or that in that specific group. I think you agree,
right, Phil? That\'s not ever going to come out in an FDA reg.

DR. NOGUCHI: No, FDA always recognizes the autonomy of the physician to
within his or her best judgment to make the best decision for the
patient absolutely.

CHAIRMAN SALOMON: Unless somebody has something to change, then I think
there?s a consensus here that based on clinical data so far regardless
of whatever we postulate from theoretical points of the biology of these
types of preparation, there doesn\'t seem to be any reason for the FDA
to consider in at least the spectrum of diseases a difference in any
sort of hematopoietic stem cell source. We are going to get now to a
couple of the key issues. Does everyone agree with that as a first
answer?

DR. NOGUCHI: That\'s with what the underlining assumption in terms of
standard bone marrow and peripheral blood stem cell experience with the
diseases for which it\'s being done.

CHAIRMAN SALOMON: That\'s right. I still think it\'s important that you
realize that there are these other indications. There are various levels
of concern. My bone marrow transplant guys don\'t realize that if I get
it in my mind to do this particularly the non-myeloablative I\'m going
to put them in the hospital and just do it. I don\'t need you.

I\'m just teasing, but if someone feels strongly about it, that\'s what
they are going to do. We need to consider a very separate group of
things. Again I don\'t know that it needs to be considered from any
hematopoietic stem cell which is the fundamental reason we\'re here
today.

As Rich directed us after Mary\'s comments the two key issues seem to be
the HLA typing and the dose. Let\'s talk about the HLA typing first. All
right. Let\'s talk about the dose.

DR. ALLAN: Actually I have a question that I was a little bit confused
about this morning. I don\'t remember who presented the data but it was
that there was less relapse in those patients that had greater mismatch.
Is that what it was?

CHAIRMAN SALOMON: That\'s classic in the GVL literature. As you increase
dose, you get more GVL but you also get graft versus leukemia and graft
versus host disease. That\'s the dark and light side of this.

DR. ALLAN: But in terms of survival, would you want to have more
mismatches because that way you get less relapse and might still have
survival?

DR. HOROWITZ: No. That analysis was the analysis of the adult bone
marrow versus cord blood transplant where there was a suggestion of less
relapse with increasing mismatch. I have to go back to the multiple
comparisons issue that was pointed out earlier quite rightfully. The
overall p-value for that whole category of variable was not significant.
The most significant p-value was 0.05. The number of comparisons was in
excess of 20 so I wouldn\'t make much out of that in this analysis. GVL
is nice. Survival is better. It\'s quite clear that more mismatch gives
you more transplant related mortality.

CHAIRMAN SALOMON: The only thing one could go based on today\'s
discussions would be if the premise could be put out for discussion that
if the cord blood allows you to get away with a more mismatch with
equivalent or less GVHD which is what the data showed us today, would
you potentially get a GVL benefit in the process? That might at some
point argue that cord blood would be a better source. I\'m just trying
to get these points into the discussion. I know we are back to HLA and
we said we were going to talk about dose.

DR. KURTZBERG: If you look at the composite groups that have presented,
there are a higher proportion of high risk patients but there is no
comparison group. You would have expected a higher relapse rate with a
standard matched sibling transplant in those groups than you see in the
cord blood patients. Both of them have alternative donor sources that
are not related matched have a lower relapse rate than a matched sibling
transplant. I don\'t know that you have the data to say more than that
but your job was in the data where the relapse rates were in the 20
percent range.

In our group and Pablo\'s group where the portion of the patients that
are high risk are already 80 percent you would have expected a 60 to 80
percent relapse rate. There is no comparison or randomized trial that
actually answers the question directly.

CHAIRMAN SALOMON: That could be one of the things that we can\'t really
comment on because the data is not out there. That might be worth
looking into. Of course, the null hypothesis would be that the same
biological mechanism gives you less GVHD, would give less GVL. It\'s
hard to know.

DR. WAGNER: In terms of this question about HLA and the importance of
HLA, I agree it\'s probably the easier of the two issues to talk about.
Maybe the way to get to it is actually to find what is unacceptable. Go
the other way around. Most of us here would agree that some degree of
matching is important. The question is how much matching is important.
From my point of view, what I would at least propose to you is that the
data that we\'ve seen for all intents and purposes has really been
focused on one and two antigen mismatches with some zero mismatches and
some three and beyond.

Just based on that alone, I think that we have to focus on saying
probably we don\'t have enough data yet unless Pablo would say
differently to say that a three antigen mismatch transplant probably
should not yet be done routinely until we have more data to say that
it\'s okay. Then everything beyond that in terms of more mismatches
probably there is really no data to comment on. At least three and
beyond should not be routine use.

CHAIRMAN SALOMON: Does everyone agree with that statement that three and
beyond is in a different class than two and less?

DR. HARLAN: Couldn\'t we even make it simpler? Isn\'t it the same
criteria that\'s currently used for bone marrow and peripheral blood? If
there is no data to suggest what criteria should be used, why not just
leave it alone?

CHAIRMAN SALOMON: I\'m glad you brought that up. Do the bone marrow
transplanters want to respond to that?

DR. KURTZBERG: No one would use the four-of-six antigen without some
additional method to prevent graft versus host disease. So it\'s not the
same.

DR. HOROWITZ: There\'s actually not very much experience in that.
There\'s enough experience to know that two antigen mismatching the bone
marrow setting is probably not a good thing. John points out that the
real difference in cord blood transplants between the two and the three
is not that we know that the three\'s are worse but there is ample
experience with the two antigen mismatch to say that has an acceptable
outcome to feel comfortable with it. There\'s just not enough experience
with the three and more antigen mismatch to feel comfortable with it.

DR. STRONCEK: John, what do you mean by antigen? Yes, as defined here.
For bone marrow and unrelated donors, people will do basically
sequencing so they get allele specific matches and then they start
talking about eight antigen and 10 antigen matches. Now with cord blood,
it would be theoretically nice to have everyone sequenced but that\'s so
costly. I don\'t think that happens when you match up your cord bloods,
does it?

DR. WAGNER: It\'s being done now. We treat cord bloods the same as we
treat unrelated marrow donors except that that\'s relatively new and
only in those cases where we can get DNA to be able to do the high
resolution 10 antigen testing. What we are doing is we are comparing
class I lower intermediate resolution, A and B and DRB1. That\'s really
the only dataset that\'s comparable to bone marrow dataset.

When we talk about what level of matching is the minimum, we\'re talking
about A, B and DRB1. The others are interesting but we don\'t even know
what the impact is for sure yet in bone marrow transplants much less in
cord blood. So what I would suggest to you that clearly with cord blood,
zero and one antigen mismatches at that level of typing is certainly
adequate. We have substantial data in the two antigen mismatched cord
blood transplant to at least be able to adequately inform our patients
as to what the outcomes should be. I would suggest that zero, one or two
at that level of typing should be considered adequate.

DR. STRONCEK: This is too technical. I\'m confused. When you do your
actual transplants, you get DNA from that cord and then you do really
high resolution typing but then when you do this group data you are
looking at lower resolution typing for A and B antigens and high
resolution for DRB-1. Is that what you said?

DR. WAGNER: It depends which analysis you are asking to be done. All
this data that\'s been presented was dataset that was several years old
and it\'s at the level of typing that was available then. We are just
now beginning to do a higher resolution typing prospectively for future
patients but there is no data analysis that we have at our institution
to be able to ask that question. As we pointed out in our discussions,
it would require a huge dataset before we can make any statement whether
it be bone marrow or cord blood as to what degree of mismatch should be
allowed.

DR. KURTZBERG: The standard of practice now for cord blood is not to do
high resolution DNA typing at A, B, C, D, RD, PD, Q.  The standard of
practice is A and B at medium resolution at the serologic level. DRbeta1
at high resolution DNA.

CHAIRMAN SALOMON: That is what I was asking Dr. Rubinstein during his
presentation exactly what he was talking about in the typing. What David
is getting at is really critical and that is if we are going toward just
a recommendation to the FDA that two or less mismatches seems to be
equivalent that\'s a very important one.  Then we have to be clear that
we are defining to them what does two mismatch mean. I think that\'s
where David was coming from. Dr. Rubinstein.

DR. RUBINSTEIN: With respect to the high resolution typing, the data
that I presented this morning has been typed for DR at the highest
resolution possible. Even the old transplants we have the DNA of the
donor and recipient in essentially all cases and we have retrospectively
typed them so we know where they are for class II at the sequence level.

For class I we are at the moment finishing the sequencing of A, B and C
for the patients who have one mismatch. For those patients we will have
an answer about whether we can increase discrimination possible with
better matches in parallel to an improvement of the each situation. It
will be retrospective but that\'s one point.

The second point is that all our cord bloods are now tested from the
start with sequence for DR. We do that because it turns out to be
cheaper than to do the sequence of an intermediate DNA level and then
prior to the transplant to come back and do the sequence.

CHAIRMAN SALOMON: I think that what I\'m hearing here is still what we
are talking about in this first recommendation which is relatively low
level typing not sequence based typing. But if the recommendation was
going to be that we should go on and get data on that, we don\'t really
have to make that recommendation because you are already doing that.
That means it\'s underway and we can look forward to that sort of
information.

DR. WAGNER: That\'s my point though. So you can still make that
recommendation as appropriate because one bank doesn\'t mean that
everyone\'s doing the same. Also with being very specific, it\'s low
resolution or low to intermediate at A and B. DRB is high.

CHAIRMAN SALOMON: Is everyone comfortable with our recommendation on HLA
matching? My understanding is that we are saying that for cord blood two
or less mismatches as defined currently by the field which is relatively
low level resolution is acceptable. And that, we acknowledge, is
distinct from current practice today for bone marrow and peripheral
blood allogeneic transplants. However as was pointed out, I don\'t know
if everybody got it, there are people doing that now coupling in
strategies to do aggressive T-cell depletion or to use antibodies and
other strategies. It\'s not that there isn\'t research going on to even
make that equivalent to hematopoietic cells.

DR. WAGNER: I may have missed it. Can you repeat what you said for DR?

CHAIRMAN SALOMON: I tried to get at this DR, A, B, C difference and I
thought that what I heard overwhelmingly is not make a distinction. It
was two mismatches.

DR. HOROWITZ: How we define them is different. We define DR with high
resolution. We define A and B with low to intermediate resolution.

DR. WAGNER: Right. That\'s the distinction I wanted to make sure was
made.

CHAIRMAN SALOMON: I have to say that was where I was going earlier. I am
surprised that you can\'t go further and distinguish the difference
between DR and A and B matching, not resolution but just in the effects
but you can\'t. I don\'t know I don\'t believe that but I\'m sure
that\'s true from the data.

DR. ALLAN: I just wanted a clarification. When you say two mismatches or
less and then John said that there is very little data on three
mismatches or more. If there is more data in the future, you could
change. But you are not going to have more data if you say two or less.
You are not going to have any three mismatches so you will never know
whether or not three would actually be useful.

CHAIRMAN SALOMON: I think the answer there, Jon, is that no one is
trying to recommend regulations on anybody. What we are saying as a
committee is that if someone wanted to do three or more, that would be
in a group that would have to be taken differently than if they wanted
to do two or less.

DR. ALLAN: You know how things operate. It gets around pretty quick.
Everybody goes you shouldn\'t do three so it\'s not going to get done.

DR. WAGNER: That certainly is possible. I\'m thinking from a different
point of view. What we are saying is not that you can\'t do these
things. It\'s just that in terms of what is acceptable based on what we
know today the category has data that seems to support it being
acceptable. This may be but whatever mechanism they can still study it.
It\'s just a matter of how you define what\'s routine versus what\'s not
routine.

DR. NOGUCHI: Perhaps just a better way of putting it from the way we
typically look at things is you can make statements about two or fewer
mismatches. You really can\'t make a statement three or more. We try to
avoid such things as an acceptable versus not acceptable because it
depends a lot on the clinical situation as Joanne and other rightfully
point out.

MS. LAWTON: I just wanted to add to that discussion if we are
recommending two or less and clearly you don\'t want to stop the
research to understand whether three could be acceptable. Presumably the
FDA will look on a case by case basis and proposal, regimen, etc. as to
whether that\'s appropriate research to continue to try and find out
because we want to make sure that we don\'t stifle that research.
That\'s all.

DR. NOGUCHI: Again, to say that it\'s acceptable versus not acceptable I
think we would be more in the category of saying we hope that you will
be able to make a statement. For two or less there is a set of outcomes
for which we can make some predictions that will be useable in the
clinic. For three or more you can\'t make any statement. Acceptable
versus not acceptable is really not quite what the FDA is looking for
here.

CHAIRMAN SALOMON: I think that\'s what we\'ve told you.

DR. LAUGHLIN: I guess I would also reemphasize John\'s data that as you
increase HLA disparity you can somewhat overcome or you can rescue the
less optimal transplant outcomes by increasing cell dose. It\'s
certainly been in our experience in the adult recipients within the
constraints of what you can attain cell dose that our outcomes in three
to six mismatches has been below the threshold that we consider
acceptable. There are some differences in the pediatric data versus the
adult data in that in the pediatric data you have a greater wobble cell
dose-wise that you don\'t have that luxury in the adult recipients.

CHAIRMAN SALOMON: Before we go on to focus on dose a little bit more,
incidence of graft versus host disease. I\'m not sure what to say about
the data I saw today. Part of it showed that there would be less graft
versus host disease with CD34 enriched populations from cord blood. Also
the statements that we made that you can get away with two mismatches or
less which would certainly argue strongly for that premise.

DR. WAGNER: The data that I\'ve seen and published has only demonstrated
that if you put all patients together that cord blood is associated with
less graft versus host disease. Data presented today which is the
cleanest dataset that Mary and I and others worked on was looking at HLA
identical sibling transplants with cord blood versus marrow. There you
were able to more easily tease out what was the contribution to graft
versus host disease. There clearly acute graft versus host disease was
significantly less in the setting with cord blood. So there is something
biologically different.

With unrelated transplants, there it\'s a little bit more complicated
because there is also other level of disparity which we can\'t account
for because we don\'t test for them. Nonetheless the data goes flows in
the same direction that would appear that there is a less risk of severe
graft versus host disease with unrelated cord blood as compared to
marrow.

The one exception of that was my dataset at the University of Minnesota
which is a matched pair of analysis. The surprise was that in children
when you compare mismatched cord blood with matched marrow in that very
specific setting there the results were equivalent. So it\'s at least as
equivalent if not better.

DR. KURTZBERG: Again that\'s a mismatched cord blood with matched
marrow. If you are thinking about availability, the people getting the
mismatched cord blood are not the people who have matched donor. The
other thing which John presented in his talk was the chronic GVH which
is really a marked difference between bone marrow as far as I am
concerned. It\'s true in children as well as in adults. Chronic GVH
rates and the severity of chronic GVH is lower with cord blood than with
bone marrow.

CHAIRMAN SALOMON: I have to say that so far the committee is saying in
concert that GVHD is less with cord blood. Acute GVHD. We?ve segued into
chronic. I would like to come back to chronic for a second. I think we
can agree that the weight of the data is pretty good that acute GVHD is
less with cord blood. That\'s a good thing. Dr. Rubinstein.

DR. RUBINSTEIN: When we look at what the transplant center tells us
about GVH, the picture is not so transparent. The centers that have the
most transplants who all agree with what we have heard that the
frequency of acute, severe GVHD is lower in cord blood. But the other
centers report higher frequencies. I\'m not certain what this means. I
didn\'t show this data in the morning. I don\'t think we are comfortable
with just giving frequency data. I think there is a problem in the
evaluation of these parameters.

CHAIRMAN SALOMON: Certainly if I was running a multi-center clinical
trial specifically with acute graft versus host disease, I would be much
more inclined to trust the GVHD evaluations in an experienced center
than a smaller center that didn\'t have that kind of experiences. There
is a lot of subjective, experiential based judgments on judging acute
GVHD. That may be very powerful. Mary.

DR. HOROWITZ: Even in big centers I might add. What you can be confident
of is that it\'s not worse than bone marrow transplants. I think that\'s
the only issue that this committee has to deal with. I feel much more
comfortable when we look at things like survival because there is no
inter-center variability with that one. Everything that we\'ve said up
to now can be held up by looking at the survival data which is the data
that we can be really sure of. When we start looking at the intermediate
outcomes of acute GVHD, chronic GVHD engraftment, we are bound to leave
out some that are important.

I go back to the conversation earlier about the T-cell depletion trail
where everyone assumed even though there was no survival difference
since there was less GVHD and the T-cell depletion arm, the quality of
life was going to better. It doesn\'t look like that\'s the case because
we can\'t think of every surrogate, of every cumulative outcome that
adds up to a person who is alive and feeling good. So we pick out some
of them. GVHD is of course a traditional one in transplantation. We can
feel really comfortable that it\'s not worse and I don\'t think we
should talk about it anymore. Then the recommendations because they are
held up with the survival data are stronger on HLA and then we\'ll get
to cell dose eventually and indications.

CHAIRMAN SALOMON: Yes, we\'ll get to cell dose eventually. I don\'t
totally agree with what you said in that survival data of course is very
comforting and you\'re right, it\'s clear. You\'re alive or dead. It\'s
kind of binary. But the problem is when you start comparing using
survival data as your only parameter then you do get into trouble
because there are differences in diseases.

There are differences in some of the therapies. A certain survival rate
would be very appropriate for one disease and totally unacceptable for
another. So I don\'t think we should get too much comfort from a single
variable.

DR. HOROWITZ: What you just said about differences in therapies and
differences in diseases is true about all the intermediate outcomes as
well. One of the things in evaluating GVHD is that GVHD prophylaxis
regimens may very much influence the maximum grade of GVHD when used by
different centers. It may decrease GVHD as T-cell depletion did with
bone marrow transplants and increase an adverse outcome that you don\'t
happen to be measuring.

CHAIRMAN SALOMON: All I\'m saying is that we just should realize that
there are multiple parameters and not just survival. That\'s the only
point I was trying to make.

DR. WAGNER: My only comment about graft versus host disease is the fact
that we can\'t throw away the sibling data which was the best way of
evaluating graft versus host disease and in the sibling data, works the
cleanest for both acute and chronic. It was less for cord blood. However
for the unrelated transplant patients as Pablo pointed out, there is a
marked discrepancy between institutions and by reports both for acute
and chronic graft versus host disease. As you have pointed out, Mary,
never have we seen a report where it says it\'s higher with cord blood.
I think that it\'s safe to say it\'s at least equivalent despite the
level of mismatching with cord blood in contrast to marrow.

CHAIRMAN SALOMON: I think that\'s clear. What I would like to put out is
that I\'m not comfortable at the moment saying anything about chronic
GVHD and this is for discussion. I really don\'t think there was any
data presented today that made me comfortable that there is enough known
about chronic GVHD in any of these. I think we should tell the FDA that
there isn\'t enough data to make any comment on that right now.

DR. WAGNER: There is plenty of data. We just didn\'t present it. The
only data that was presented today was my data which showed at risk of
10 percent. I would tell you that you can look in any transplant
literature and you will never see another report that says 10 percent
for bone marrow transplants for any disease because bone marrow
transplants is higher. It\'s always higher than 10 percent.

DR. KURTZBERG: The data for unrelated marrow ranges from 40 to 70
percent depending on the population. The data that John and I put
together and I can pull those slides up from an analysis of about 300
patients between our combined institutions held up the 10 percent
figure.

DR. WAGNER: So even if we graded differently, at least within our own
institution where we graded it and despite the fact that you might say
that the way we graded is different, we have two investigators, Stella
Davies and Dan Weisdorf, who blindly graded all the graft versus host
disease between the unrelated marrows and the cord blood. That\'s how
we\'ve presented it. At least it applied to the same criteria to both
arms.

DR. HOROWITZ: Did you see a difference in chronic GVHD in the same
subset, where you did see a difference in acute GVHD?

DR. WAGNER: Yes, we did except unfortunately I didn\'t bring the slide
to present to you. We did see a difference.

DR. HOROWITZ: In the pediatric study with the New York Blood Center
versus the IBMTR data, we did not see a difference. In those children in
that analysis, there was not a significant difference in chronic GVHD
between graft sources.

DR. WAGNER: And that\'s what the Europeans have presented.

DR. HOROWITZ: So I think it\'s inconclusive.

DR. WAGNER: But where we do agree is that it\'s at least not higher.

DR. HOROWITZ: It is not higher, right. I can agree with that.

DR. RUBINSTEIN: I could give you a few. In the aggregate data, the
chronic GVHD has gotten up to 30 percent. But if you make subsets in the
data, it\'s very clear that the results are very different. We can make
some extrapolations that people with more experience will grade these
patients lower.

DR. HARLAN: Dr. Horowitz moved about five minutes ago to table this
discussion. I second that motion because I think everybody is saying
that there is no?evidence that it\'s worse. Incomplete data. I just
think end of discussion.

CHAIRMAN SALOMON: We don\'t need to be that formal, guys. Fine, we\'ll
go on. I\'m just trying to make sure we answer everything here. Time to
engraftment. I tried to go there when I started talking about immune
reconstitution but just like allogeneic, you have your own term for
immune reconstitution too. Immune reconstitution now is T-cell function.
According to that hypothesis, what I\'m hearing from you is the
difference of reconstitution of the granulocytes is not really that
critical a determinate of outcomes. Now the question is is there a
difference in time to engraftment for immune reconstitution that
distinguishes cord blood from other hematopoietic stem cell sources.

DR. WAGNER: Now I have to sit down.

DR. KURTZBERG: I don\'t think that\'s ever been studied. I think there
is more data about immune reconstitution after alternative donor either
T-cell depleted marrow or cord blood than there is after standard marrow
transplantation with matched donors. There\'s never been any
head-to-head comparison. From what I know there is not a distinct
difference between the cord blood and the marrow.

CHAIRMAN SALOMON: One way we would look at it is that there doesn\'t
seem to be a significant difference at least from what I saw today in
infection related deaths when you compare cord blood to bone marrow
which would support a contention that there\'s no difference in
functional immune reconstitution as an impact on the patient. So that
would be supporting what you are saying.

On the other hand, Dr. Chao showed data where the TRECs appeared in the
subset of patients at one year and not until three years in a lot of
them and those were non-myeloablative. That may be changing the rule a
little bit.

DR. KURTZBERG: No, actually what he showed was cord blood children and
adults after ablated transplant where the children came in much sooner
than the adults. Then he had those dots on top of it which was the
non-myeloablative showing that in the non-myeloablative cord blood the
adults came in sooner.

DR. HOROWITZ: That\'s similar in bone marrow.

DR. KURTZBERG: That relates to the injury to the host as much as it does
to stem cells.

CHAIRMAN SALOMON: Again in terms of engraftment, what I\'m trying to get
at this morning and now is that I don\'t think that\'s an issue here. I
don\'t that engraftment is a big problem in thinking about hematopoietic
stem cells of cord blood, bone marrow or peripheral blood derivation.

DR. WAGNER: I don\'t know if I\'m comfortable with that statement.

DR. HOROWITZ: Hematopoietic recovery is slower. I don\'t think that
there\'s anyone in this room that would say that.

DR. WAGNER: It\'s unequivocal.

DR. HOROWITZ: Unequivocally slower. Hematopoietic recovery is slower but
long term immune reconstitution probably isn\'t different but that\'s
outside my area of expertise.

DR. WAGNER: Based on the data that I showed, if you look at those
patients with an adequate cell dose, the transplant related mortality or
the non-relapse mortality was low. It was only 20 or 25 percent as long
as you had an adequate cell dose.

DR. HIGH: That\'s the point I wanted clarification on. If you correct
for the dose, is there a difference in time to hematopoietic
reconstitution? There is.

DR. KURTZBERG: Yes, it\'s longer to neutrophils and it\'s longer to
platelets. Once it gets there, it\'s durable. The differences are in
weeks not in months and years.

DR. HIGH: We\'re edging in on cell dose.

DR. KURTZBERG: There is a relationship to cell dose. But regardless of
that, it\'s still delayed.

DR. TSIATIS: Was there an analysis shown that showed the relationship of
survival to time from engraftment?

CHAIRMAN SALOMON: There was actually the reverse. It didn\'t come out in
the multi-variate analysis.

DR. KURTZBERG: I don\'t think anybody has ever done it quite that way.
Maybe Pablo has. There\'s definitely a relationship. I think Gluckman\'s
looked at 60 days to an ANC of 500.  Other groups or programs have
looked at 42 days to an ANC of 500. Although engraftment occurs past
that point for some patients, the mortality is higher in that group.

DR. WAGNER: I\'ve never really analyzed it that way so I can\'t tell
you. But we could.

CHAIRMAN SALOMON: Dr. Rubinstein, do you have any comment on that?

DR. RUBINSTEIN: I think Joanne\'s statement is perfect. I will sit down.

DR. NOGUCHI: I think our experience with peripheral blood stem cells and
mobilization would suggest that the data really are inadequate to try to
get at any meaningful differences in overall survival but that with
peripheral mobilization and selection you do get delayed engraftment. It
may or may not have any major consequence on the long term outcomes. I
think that this data does appear to be along the same line. So I guess
the question is, at least from what we are hearing, is that there is
nothing major that jumps out at you that would suggest that delay of
engraftment is some reason to steer away from cord blood versus bone
marrow at any point.

CHAIRMAN SALOMON: Right, but I would say, again could be argued with,
from my impression here and also just based on my experience as a
physician that you have to face the fact that to anyone undergoing a
cord blood transplant is going to engraft slower, not talking about
anything specific but the ANC, by platelet count, etc. That\'s going to
probably contribute some morbidity and possibly some mortality.

It doesn\'t come out in the big statistics and that\'s important but
it\'s going to mean that they are going to get some more platelet
transfusions maybe. It may mean that they are going to longer antibiotic
prophylaxis maybe. It may be more than one readmission or a longer
primary admission. There will be price to pay, not a monetary price,
possibly, but \--.

DR. TSIATIS: I just feel that\'s a real jump without actually looking at
and doing a formal analysis of that relationship.

CHAIRMAN SALOMON: I don\'t think it\'s a big jump based on the fact that
that is what happens in hospital care. I do this all the time with organ
transplant patients. I\'m going on 20 years of experience here.

DR. TSIATIS: I don\'t know the data and I don\'t know the area. I can
see that time to engraftment is going to be important but when the
relative relationship of time to engraftment for cord blood is the same
as the relative relationship with bone marrow, again that\'s not clear.
To make a jump that one is slower than the other means that it\'s going
to have some bad effects again is a jump.

DR. KURTZBERG: It does have a monetary effect. The first in-patient stay
is longer. It costs more. In our setting, our average length of stay for
an autologous transplant in the pediatric population is 34 days, and
that includes the chemotherapy. For a matched sibling it\'s usually 39
or 40 days and for cord blood it\'s 48 days. You used to be as high as
60 to 70 days. We\'ve brought it down with an active outpatient clinic.

CHAIRMAN SALOMON: Remember, Butch, being the hospital is bad.

DR. KURTZBERG: It would cost more money too.

CHAIRMAN SALOMON: Anything that keeps you in the hospital longer is bad.

DR. WAGNER: Just two points. Remember this is still cell dose issue.
Those with the lowest cell doses have the very prolonged recovery
periods in terms of neutrophil recovery and they\'re the ones that do
badly. I\'m sure that somehow this very slow rate of recovery in that
cohort that\'s at the lowest cell doses, cell recovery is playing an
impact upon survival. So I bet you\'re right. There\'s an impact that\'s
predominantly affected by the lowest cell doses. The other thing is if
you look at the impact of cell dose in those that engrafted, the cell
dose effect is still there.

CHAIRMAN SALOMON: So cell dose.

DR. HARLAN: Just one comment. I\'m still not exactly clear despite what
you\'ve said and what Phil Noguchi said as to what the product of this
is going to be but I\'m envisioning now some document that could say
something like starting from where we started today that umbilical cord
blood is equivalent in all ways to traditional bone marrow transplant
with these exceptions: where we can have a bullet that says for instance
it looks like you can relax stringency slightly on HLA match; it looks
like graft versus host disease incidence is no greater and maybe less,
you have to have that proviso; and the third one is this last issue of
that time to engraftment appears to be consistently longer, whether it
has any relevance or not we can\'t say but these are observations.

CHAIRMAN SALOMON: That\'s about where we are right now. Yes.

DR. RUBINSTEIN: This is just about the last point that Dr. Wagner made.
In the overall data, when we do a multi-variate analysis after
engraftment, cell dose is no longer a significant predictor of survival.
If you take the patients not after the transplant but after they engraft
and then you ask the question is cell dose important, in the overall
data it isn\'t.

CHAIRMAN SALOMON: We are now talking about cell dose. This is what
everyone has been waiting for. The curves that show these family of
curves with improved survival, Dr. Rubinstein, you are saying is all
engraftment driven. Comments?

DR. KURTZBERG: I think there is no question that cell dose influences
engraftment but cell dose also influences survival.

CHAIRMAN SALOMON: So why do you say that and he says that?

DR. KURTZBERG: I think he would say that too but I\'ll let him talk for
himself.

DR. RUBINSTEIN: If you do the multi-variate analysis starting from the
transplant and then of course cord blood the survival is very dependent
on the cell dose because you must engraft if you are going to survive.
Immediately there is some difference in pieces of the data in both
directions. But once you engraft, the cell dose is no longer a
predictor.

CHAIRMAN SALOMON: That is not what you said, Joanne, if I heard you
right.

DR. KURTZBERG: To me doing that kind of analysis is the same as saying
you are only going to now analyze the leukemic survival after
achievement of remission. And I don\'t think that\'s a fair analysis. I
think you have to analyze from the day you start the therapy which is
before you start the transplant.

From that point, cell dose influences both engraftment and survival and
even after engraftment. In our data if engraftment is delayed, survival
is poorer even if that patient engrafted. I don\'t think Pablo\'s data
shows that. But I don\'t think it\'s a valid analysis either way. I
think the analysis has to be from the start of the therapy and take in
all competing risks. If you go from start of therapy then cell dose
impacts both engraftment and survival. That\'s the more important
analysis to do.

CHAIRMAN SALOMON: Mary, did you want to comment on that?

DR. HOROWITZ: We haven\'t looked at the data from time to engraftment.
John, I thought you said that even among patients who engrafted, cell
dose made the difference.

DR. WAGNER: When we looked at our dataset as Joanne just said, we both
looked at Duke and Minnesota but also more recently just looking at the
Minnesota dataset and the impact of cell dose occurs both from the very
beginning as well as from those that had already engrafted. If you start
the time clock after engraftment, there is still an impact of cell dose.
My guess is those with the very delayed recoveries even though they
recover they still are negatively impacted by cell dose. My
interpretation is still pretty much the same.

DR. KURTZBERG: We have analyzed immune reconstitution as it relates to
cell dose using one year as the endpoint and cell dose impacted immune
reconstitution as well.

DR. CREER: If you look at the survival curves too, you never really see
that they separate out based on cell dose until at least maybe 60 to 90
days which is the time beyond engraftment. They really don\'t begin to
disperse until after. I would say that even though there is delay in
engraftment, that delay is not the factor that is contributing to the
death. The problem determining survival is occurring after that 60 to 90
day period.

CHAIRMAN SALOMON: Let\'s take a different view of the cell dose because
I don\'t think this is the most critical thing. The part of the cell
dose is are there recommendations we want to make in this discussion to
the FDA that gives them some sort of a sense of what is an appropriate
cell dose and then think about the pros and cons of that sort of a thing
because it has to do with the size of the patient that one could do and
the availability of the numbers of units. If you set a really high cell
dose, you decrease the number of units that are going to acceptable.
This is an awfully critical piece of the conversation. I would like to
focus on that rather on this rather tenuous engraftment issue.

DR. LITWIN: Could we perhaps as we are entering this discussion talk
about numbers in terms of cell dose? That would be useful to see where
people are.

CHAIRMAN SALOMON: That\'s where I\'m going. I hope that\'s what I meant.
That didn\'t come out right. That is what I meant. Let\'s have some
discussion about numbers here with the realization that you are going to
pay a price when you make the numbers. The good news is you will get
better survival to a certain point but the bad news you will decrease
the number of useable units. Mahendra is saying let\'s start with Dr.
Wagner\'s number. You guys messed around with me a little bit because
some of you were talking about per kilo and then Dr. Rubinstein was
showing total. Your best data was 100 million total.

DR. WAGNER: That\'s per kilo.

CHAIRMAN SALOMON: One hundred million per kilo?

DR. KURTZBERG: In 10 kilo kids you can get 100 million.

DR. WAGNER: Yes. Basically again what I would say is we should step them
back and say what\'s unacceptable and work our way that way. Therefore I
would say that certainly anything less than a dose of 1.5 times 10^7^
per kilo is unacceptable. Now remember I\'m talking about nucleated
cells now, not CD34, because as I said in my talk CD34 in principle is a
better measure. On the other hand, it\'s not practical yet.

CHAIRMAN SALOMON: We\'ll get back to the CD34 in question four. So 15
million or more is what John is putting on the table per kilo.

DR. WAGNER: Per kilo, yes.

DR. KURTZBERG: I would actually say for adults that\'s reasonable but
for children I would make the bar higher.

DR. HOROWITZ: I would make it higher for both.

DR. KURTZBERG: For children, I think it should be 25 or even 30 and for
adults I would say 15.

DR. WAGNER: That doesn\'t make any sense. If a cell dose is acceptable,
it\'s not going to be different only because otherwise you could limit
the use of cord blood for adults but that may be appropriate. I mean the
fact is why would it biologically have to be higher for children than
adults. If anything it would be the other way around.

CHAIRMAN SALOMON: I can answer that but do you want to make a comment?

DR. McNIECE: Ian McNiece, Johns Hopkins. I would really warn against
going to some of these minimum doses. We have learned a lot through bone
marrow and peripheral blood stem cell transplants. With PPCs we have
optimal doses. We don\'t have minimal doses because the number of the
grafts even with lower doses still engraft. If you look at the
Kaplan-Meier probabilities, a number of those grafts still reach
neutrophil engraftment within 20 to 30 days. We ran a study with
expansion at the University of Colorado. Our median dose was 0.85 times
10^7^ per kilogram. We had engraftment in all patients. We gave as low
as 0.28 times 10^7^ per kilogram. We still got neutrophil engraftment.
These were expanded products. The quality was somewhat different. We are
talking about different things.

I would like to see a recommendation for an optimal dose but I would
certainly warn against giving a minimum dose. As we have heard, a lot of
times you don\'t have an option in terms of the graft. So you are going
to prevent people going forward with getting a transplant because of
your cell dose. What is their option?

CHAIRMAN SALOMON: Again, this comes back to what Phil said. We\'re not
trying to say acceptable and unacceptable. We\'re just talking about
optimal.

DR. McNIECE: Minimum is different than optimal.

CHAIRMAN SALOMON: That\'s right. You\'re right. We should talk about
optimal, not necessarily use the word minimal. That implies an
unacceptable dose. Rich.

DR. MULLIGAN: How about if we just first build a non-controversial dose
without using CD34 that everyone agrees on? What would that be?

DR. KURTZBERG: That\'s the discussion we are trying to have.

CHAIRMAN SALOMON: That\'s what the people are saying. We have 15 million
per kilo on the table as an optimal dose possibly. Then Joanne and Mary
raised the bar on that thinking it ought to be higher and John saying he
didn\'t think that you ought to play with the biology like that. If
it\'s optimal, it\'s optimal.

DR. KURTZBERG: But 15 million is not optimal. I have trouble with that.
Fifteen million may be the lower threshold but it is not optimal.

DR. WAGNER: You know that\'s not what I\'m saying.

DR. KURTZBERG: I know but we just have to be careful how we choose our
words.

DR. WAGNER: What I said was specifically that working our way from the
bottom up I think that everyone would agree that 1.5 or below is
unacceptable. I\'m not saying that above 1.5 that there might not be a
better level. I\'m just working my way up to figuring out what that
threshold should be. The data that I presented would suggest to you that
a graft failure rate of 30 percent and a time to recovery of 32 to 35
days is pretty poor. Therefore routine use of cord blood \-- that cell
dose should not be considered for routine use.

DR. KURTZBERG: I agree with that. I also think you have to realize that
people have options sometimes where you might have a four-of-six unit
that gives 50 million and a six-of-six unit that gives 10 million. I\'m
not saying we have all these answers but a traditional transplanter who
has no knowledge of the impact of cell dose is going to pick the
six-of-six unit. That to me is substandard because of that cell dose.

CHAIRMAN SALOMON: That\'s excellent. We do have to realize that now
bringing in the idea that there is more than parameter to deal with is
critical. Alison.

MS. LAWTON: I have a couple of comments. I thought that I wrote down
here and I wanted to check if this is correct that, Dr. Rubinstein, you
presented data that showed at below 25 million it was statistically
significant that you did get a drop-off below that dose. Then the second
comment I would just make which is slightly different is this is
obviously a very difficult discussion on the dose. Maybe one way to go
is whatever dose or whatever discussion we come up with is that again it
becomes part of informed consent for the patient because if that patient
still doesn\'t have a choice they may still have a better chance with a
low dose than having no dose whatsoever. As long as they are informed of
that, maybe that\'s the way to go.

DR. RUBINSTEIN: I think those points are excellent but I wanted to say
that in discussing the dose, it\'s very important to make a precision
whether we are talking of the dose as seen in the bank before it is
frozen or in the clinic when it is thawed. You should anticipate some
loss there. In our experience and I showed the curve that represents all
of the replies to us from the transplant centers, the data shows that
apex of that distribution is about 70 to 75 percent, somewhere in that
area. The estimate of 15 million that John is making maybe more or less
equivalent to the threshold of 25 million that I was talking about.

CHAIRMAN SALOMON: So for clarification, I\'m glad you brought that up. I
was thinking that when you talk about 25 million, are you standardized
to that\'s the time of infusion after thawing? That\'s what I was
thinking.

DR. WAGNER: But remember you don\'t that up front.

DR. KURTZBERG: Let him answer that. Your numbers are at banking not at
thawing.

DR. WAGNER: No, it\'s infused.

DR. KURTZBERG: I\'m asking Pablo.

DR. RUBINSTEIN: When we represent to you the numbers in a unit, we tell
you the numbers that are pre-freeze.

DR. KURTZBERG: Right. As far as I\'m concerned, that\'s the only numbers
we can work with. I know what you are saying and you\'re right. But when
you select a unit for your patient, you don\'t know what it\'s fly out
at. You have to select it by what\'s there.

CHAIRMAN SALOMON: I\'m making a point here kind of dumbly. I want to get
this clear. When John presented us data about 15 million per kilo, he
was talking about thawed. When Dr. Rubinstein presented us data that
showed 100 million had the best results, he was talking about banked,
pre-thaw.

DR. WAGNER: No, this is actually a critical point that I was misleading
you to think that this is the cryo-preserved cell dose. It is in fact
the infused cell dose. So in fact it may then turn out to be closer to
the 2.0 to 2.5.

DR. KURTZBERG: This is a very slippery slope because transplant centers
get different recoveries. People with expertise in thawing may get a
better recovery. Different banks have different ways of counting and the
correlation. I don\'t think we should go there.

CHAIRMAN SALOMON: I think we have to. I don\'t think we can not go
there. But what I\'m hearing you say is that it should be based on the
bank number.

DR. KURTZBERG: Right, that\'s what I\'m saying.

CHAIRMAN SALOMON: That\'s what we should stay with is the bank numbers.
That\'s what I hear you saying.

DR. KURTZBERG: That\'s what I\'m saying.

DR. WAGNER: And I am saying the same thing. I agree with that. I wasn\'t
thinking about that when I told you the 1.5. I was giving you data that
was on infused but I agree with you entirely. It should be based on the
bank number and not the infused number because you don\'t have that up
front.

DR. CREER: This gives you an idea of the difference between the two. If
you look up there between all the units banks between 0.6 billion and
over 3.0 billion in our bank, the inventory distribution and the
selection of units based on their total cell count in each product and
if you look at the actual mean TNC dose, it\'s pretty much the same.

The products are being selected by the transplant centers to achieve a
specific TNC dose. That median dose in the range between 6.8 and a low
actually of 4.8 to a high of about 7.3. Those are much higher numbers
than you saw in any studies presented today because this is post
processing, not post thaw. That\'s where the big difference has been. We
have a median CD34 dose that we send out that based on post-processing
data that varies from a low of 3.7 to 6.8 which is well above all the
thresholds that you saw.

What I\'m saying is that the data you\'ve been seeing for outcomes
correlation is based on post-thaw information. That difference is very
significant with what we show them and what they are selecting
post-processing. And that\'s the problem.

DR. KURTZBERG: Now Pablo\'s data is all post-processing, what went in
the freezer.

DR. CREER: Right.

DR. KURTZBERG: John is saying that his data was post-thaw which was only
100 patients. All the other data you saw today was post-processing.

DR. CREER: And you get consistent recoveries on TNC but not on CD34.

CHAIRMAN SALOMON: We\'ll get back to the CD34 thing.

DR. KURTZBERG: Actually on TNC in the 500 units we thawed, we\'ve
recovered TNC from a range of 65 percent to 98 percent. It varies
somewhat on banks, somewhat on transportation and somewhat on I don\'t
know what but it\'s the same staff doing the same procedure.

DR. CREER: Single institution experience. When you look at every
institution, it varies from a low of 20 to a high of 90. What I\'m
saying is in a single institution you have very precise recoveries, very
precise calculations of CD34 and TNC. When you tease out the data and
look at correlation of outcomes by single institutional experience, you
get a different view than you do when you look at multi-institutional
studies where the variability and recovery changes things. That\'s the
difference.

Dr. Wagner sees a very clear correlation with CD34 because he gets
consistent recoveries at his institution. When you put multiple centers
in there who calculate CD34 using different techniques, get variable
recovery post-thaw, you can?t find a correlation because the variability
in the data is so great.  That\'s the problem.

CHAIRMAN SALOMON: Now we\'re talking about CD34 again which we will get
to in a minute.

DR. CREER: Well, it\'s the same for TNC but it\'s a little more
consistent.

CHAIRMAN SALOMON: Alison.

MS. LAWTON: I agree with you. I think we need to have a separate
discussion on CD34 but just on the TNC and just given the discussion, it
seems an obvious thing, and I know it\'s one of the things that we\'ll
get on to discuss, some standard data that we want to collect across all
centers and across everything that\'s done so that we start to see that
pre-freeze and post-thaw information and start to try to understand so
that people going to those centers and getting the units will understand
what they are getting in the future.

DR. McNIECE: I just come back to trying to put any minimum level. I
think some of the comments made earlier were we are going to try to
inhibit research. Coming back to the study that we did at the University
of Colorado with expansion, one of the problems we had with the FDA \--
we had to go through extension to get discussions with them was that
those very concerns about the minimum doses going back. Our studies
clearly demonstrate that expanded cells do behave differently than
unexpanded at least at cell doses. If you start to put down minimum
doses again, you are going to inhibit research. I think you need to be
careful.

CHAIRMAN SALOMON: Nobody is talking about minimum doses and no one is
talking about expanded cells which is not on the table today. That\'s
going to be a separate discussion.

DR. McNIECE: I understand that but I\'ve heard 15 million. I\'ve 25
million being raised as minimum levels for safety. I\'m just saying if
you start to put those doses down that\'s going to inhibit what research
projects can be done to look at improving engraftment and other
parameters because it\'s going to be impossible to do other things. We
don\'t know what cells in those grafts truly give you engraftment.
That\'s the whole problem now. We\'re dealing with gray matter that we
can\'t define.

CHAIRMAN SALOMON: If what you are saying is I don\'t want to talk about
cell doses because it might limit future research, then that point is
made. That\'s not what we are doing right now. We\'re discussing cell
doses. But you can go on record as saying that you don\'t want to talk
about cell doses because it makes you nervous that it might affect
research that you want to do and that\'s fine. I heard it.

DR. McNIECE: Okay.

DR. LITWIN: The FDA in the past in connection with approving cell
separation, cell selection devices has used the term and it\'s used the
term \"target.\" The target dose is not a minimal dose so it does not
preclude using lower doses. For those in the field who know those doses
of 1.4 million bone marrow per kilo, 2.0 million for peripheral blood, I
think it\'s well appreciated that these are certainly not maximal doses
and that you probably could do better. We, faced with the issue of
having to license a device, use the approach which may be somewhat
similar to what John was recommending that is if we looked at the data
which was limited but enough, we saw that below 2.0 million per kilo for
peripheral blood there were many bad results. There was a clear
difference.

Since the FDA is safety oriented in almost all circumstances we felt
that we made a reasonable choice as it\'s held up reasonably well in the
field. Although most peripheral blood transplants now are probably three
to five million, that two million per kilogram figure I think did
protect or at least give good advice. I would recommend that we use the
term \"target\" dose which would not constrain anybody without trying to
imply that we\'re giving the best possible dose under the best possible
circumstances.

CHAIRMAN SALOMON: Rich.

DR. MULLIGAN: Another way of looking at it is that maybe we just give
the information that we heard here which is essentially here\'s the
results. You give this amount and you get this survival percentage. You
use this amount and you have less. I don\'t think anyone would be trying
to say that you wouldn\'t do those transplants that were already done
for which we have statistics. I\'m not sure we have to go past that. We
now have a body of information that says you get this level of survival,
this dose and you get this level here. Based on that, you make the
decision.

CHAIRMAN SALOMON: Let me try and synthesize what we\'ve talked about so
far and then we can tear that apart. I think what we all agree on is
that dose is a critical thing. Whenever something is going to develop in
this field, dose is going to get put front and center and it\'s
appropriate. That\'s one thing I think we all agree on.

Second, clearly no one in this field is ready to say that there is a
given dose below which you shouldn\'t do a transplant. Everyone has
given a different version of why you can\'t do that but the bottom line
is that.

Your version was that it might inhibit research into expanded cells.
That\'s a very good point because expanded cells could have totally
different dose rule.

Another reason might be that adults would be dying and their only option
would be a transplant. It might very well violate an optimal dose range
or a target range to save a life and that would be very appropriate.
That\'s a good reason too.

Certainly size, the gentleman who was 239 pounds, he is standing there
as a slimmed down version of him but congratulations on that too.
Certainly none of us want to restrict a transplant on that basis. I
think there are a lot of reasons not to put any fixed number here. I
think we can tell the FDA that number is really important and that to
the extent that one can do it in the situations choosing a higher number
would be really of value except where the choice becomes more
complicated like a higher number but a worse HLA match and that it\'s
not going to be a single parameter decision but one that might need to
be integrated with more than one parameter. Is there anything else to
add to that?

DR. RUBINSTEIN: Really to support that since dose and HLA are completely
independent estimators and they involve different biological reasons or
mechanisms on how they determine, their effect is independent. We have
however tried to look at some sort of a equivalence to understand when
should we choose a higher dose and when we should choose a better HLA
match. If you compare the group that had the five-out-of-six, the group
that had the four-out-of-six match which are the two numerous groups in
this analysis and then you sort each one out by dose, it is remarkable
that at least in the data that we now have which doesn\'t mean that it
will be the same in another setting but for this data 25 million cells
make the difference and four-out-of-six becomes the same or gives the
same survival curve to five-out-of-six. If you have 25 million then you
are equivalent in terms of the overall survival.

CHAIRMAN SALOMON: From what I\'ve heard, I\'m comfortable with adding to
what I said that 25 million integrator per kilo would be a good target
dose.

DR. HOROWITZ: And that\'s only thing that I wanted to add. I don\'t
think we should be afraid to say that 25 million is a target dose. We
have seen a lot of data to suggest that there is some kind of threshold
effect somewhere in that range and we shouldn\'t be afraid to put it on
paper as a target.

CHAIRMAN SALOMON: So there\'s two things. One is 25 million as a target
dose. Twenty-five million as a target dose translates to 15 million
after you thaw it out. Then maybe I would be happier with 50 million as
a target dose and everything else would be covered by informed consent.

DR. NOGUCHI: I think you are hitting on upon some of the nuances of what
we really needed to set in the future. If what you are saying is that\'s
based on the post-processing but pre-thaw, then I think that should be
explicitly stated. It appears that the dataset in terms of post-thawing
is incomplete.

CHAIRMAN SALOMON: Remember I said there were two things that were really
bothering me. We\'ll get to the post-thaw in a second.

DR. NOGUCHI: Make sure of that.

CHAIRMAN SALOMON: What John is saying was that his was 15 million per
kilo post-thaw. We made a rough calculation that 25 million
post-processing was probably reasonable to say in a big center trial was
15 million post-thaw. If that\'s true, I didn\'t love his data in the 15
million post-thaw group. It was okay but it was much better in what I
would consider the 50 million post-processing group or the 25 to 30
million post-thaw. I\'m just trying to get this out. I\'m talking about
target dose. I\'m not trying to set any lower limit.

DR. KURTZBERG: But in this world that you live in, you are not going to
know your post-thaw recovery. There is some variability for a variety of
reasons that we don\'t have to go into. So you have to deal with what
went in the bank and use that to base your decision.

CHAIRMAN SALOMON: I\'m just saying I would rather see us set a target
dose a little higher than 25 million.

DR. KURTZBERG: But I think the worse case average number for post-thaw
recovery is 70 percent of the cryo-preserved count and that\'s being
very conservative. I think the math that you are doing is a little too
conservative.

DR. HARLAN: Could I try to elevate what Richard Mulligan said again?
Would anybody have a problem with saying below this number the survival
is this poor and just leave it at that? That way you say below it, it\'s
really bad. You should as well as you can above that. I like that.

CHAIRMAN SALOMON: I\'m okay with stopping here and just saying it\'s all
on the table to the FDA. It\'s really not important at this point for us
whether it\'s 30 million. I agree with that.

DR. MULLIGAN: Maybe just to preempt the CD34 discussion if we agree on
this, what do we have to talk about CD34 about?

CHAIRMAN SALOMON: We\'ll get back to CD34. So we are sticking with the
25 million post-processing. Now I said there were two things that bother
me and Phil picked up on it too. I don\'t think that we can dismiss what
the gentleman was saying from the St. Louis Blood Bank that there really
are in his experience very large variations in cells count from
post-processing to post-thaw.

What I would add now to this almost complete piece is that going forward
we ought to really be paying attention to the reproducibility of the
post-thaw. Joanne, what you are saying is that you have experience so
you are comfortable with this and that\'s great. I think as an overall
thing to the field there has to be some assurance that this is really
true. If it\'s really true that their center is getting 20 percent yield
I would be very uncomfortable with them selecting and then I\'m not sure
what to tell the FDA as to why are they getting 20 percent yield. Is it
something to do with the processing, the shipment, partial thawing as
Dr. Rubinstein mentioned or is it problems in these institutions?

DR. KURTZBERG: I know that Pablo from the start went to each institution
for their first thaw and probably still does that because you can read
about that but when you do it, it\'s different. COBLT actually adopted a
protocol for training for thawing and it\'s a certification protocol
where three units have to be done that are not going to be transplanted
to patients. As a person who lives on both sides of the fence, I
strongly recommend a thawing training certification requirement for any
center that\'s going to transplant.

CHAIRMAN SALOMON: I totally agree and that\'s where I was going. That\'s
probably one of the most important things we are telling the FDA right
now. If they are going to go forward with this, they are going to have
to in some sense certify not only the processing end of this but the
post-thaw end of it in the institutions. That\'s reality for cell
transplants. That\'s fine. Rich.

DR. MULLIGAN: May I add one thing and that is I would go with where the
data is. I mean the data that we are basing everything on comes from a
certain place. Then the data depends presumably upon the method that was
used in that case so that would be a benchmark as opposed to just some
other methods that has not been attached to some any statistics.

CHAIRMAN SALOMON: I agree with that too in the sense that we want to
consider. Yes, I remember that data. I think we have this one. Does
anyone want to add anything to it? Have we missed something? Mary, are
you comfortable now? Do you think that we de-emphasized at the end the
number?

DR. HOROWITZ: That\'s okay. That\'s fine as long as we make it clear
that there is an optimal dose.

CHAIRMAN SALOMON: There\'s a target dose.

DR. HOROWITZ: There\'s a target dose. There may be an optimal dose but
we don\'t know what it is. Most of the data that are available in terms
of numbers of patients studied suggest that 25 million is a reasonable
target dose. The only thing I want to make sure that we address is that
if we are going to be able to refine optimal dose, we are going to need
to have outcome data. So I hope that we will make that recommendation in
addition just certifying that transplant centers know how to file the
product, we need to be able to collect and analyze the outcome data for
their patients served.

That\'s another reason why we can\'t find correlations. If you only have
outcome data on half of the transplants you\'ve facilitated and you
don\'t really know whether that\'s a representative sample or not, then
you may find correlations that are there.

DR. KURTZBERG: I totally support that but I think the FDA could do
something helpful in this arena which is that there is no mechanism
within the health care system to support the collection of that data. If
it is required as part of standard patient care which I think it should
be for transplants, then the FDA can say this is part of the standard of
medicine in transplantation. Then the insurers may in the long run or
the hospitals may in the long run realize that they have an obligation
to support the collection of this data. Right now that doesn\'t happen.
Everybody scrambles in one way or another to make it happen. That\'s one
of the reasons that feedback is not as good as it should be.

CHAIRMAN SALOMON: The comment I have and then maybe Phil or somebody
else from the FDA wants to comment. Often times as you know Joanne, we
come to these situations in which we say we need a registry. We need
database. We have to be really careful that our recommendations unless
you really want them to be are that the FDA should have a database
because I don\'t think that\'s what you were saying. They don\'t have
the funding to do that. These are what we call unfunded mandates and
they are scary.

DR. KURTZBERG: The IBMTR has a database.

CHAIRMAN SALOMON: Right. The FDA can insist however \-- Well the FDA
should comment for themselves.

DR. KURTZBERG: But for me as a transplanter to provide that data back to
whoever there is no programmatic support to do that unless it\'s a
research project supported by a grant. But as part of routine day-to-day
hospital care, the collection of data is not part of what they fund.
They should.

CHAIRMAN SALOMON: We agree with that. The question now is \-- that\'s
the right thing to say. What that has to do with the FDA though is what
I\'m trying to put into context. The FDA is not going to maintain the
database and they are not going to fund the database and they are not
going to fund the collection of the data. That much I can say after five
years of doing this. The question is could the FDA somehow say that if
you want to participate in, down the road of course, a regulated or
licensed process involving cord blood, could you stipulate that it would
have to be shared with fill in the blank database which might be the
IMBTR?

DR. NOGUCHI: I think all things are possible. Part of the gestalt that
we\'re trying to develop here is what the impediments to getting the
data for which it appears that there is pretty good support within the
community to provide but the mechanisms are somewhat vague.

Sometimes we can\'t even find the wherewithal to pay our own employees
so yes, it\'s difficult for us to go beyond even our own basal
activities. However, the point is well taken. Our actions do have
effects, they can change attitudes and what we want to do is to do this
in judicious fashion.

If we can just go back to number because every time you talk about 25
million, Dan, you neglect to add that that\'s post-thaw that comes from
the bank.

DR. WAGNER: Post-processing.

DR. NOGUCHI: Excuse me. Post-processing coming from the bank. Dr.
Kurtzberg is correct. Her institution is very good but the goal is to
provide this at many different institutions, not all of which are going
to be at the same level of confidence. I just have to say from our point
of view we would be thinking about making sure and in terms of the
certification that\'s certainly a part of it but data is also a part of
it too. How can you begin as a physician to analyze where the deficits
in a cord blood comes from unless you actually know how good your
thawing procedure is? I do want to make that statement up front that we
think a part of this issue is the responsibility of a transplanting
center as well.

CHAIRMAN SALOMON: In fact, let me go back and say it\'s my fault then if
it didn\'t come across clearly. We were saying specifically to you that
this is important that you have to have a way of certifying that the
transplant center is getting at least 70 percent yield on thawing. If
not then there are systematic problems on the transplant center side. We
are specifically recommending to you that you see the problems both in
the processing and the thawing of the transplant side. Yes, we totally
agree with that.

DR. MCMANUS: John McManus, from M.D. Anderson. Related to that, now we
get a number of different cord blood from many of the different banks.
Many of them tell us how they want us to thaw the product. So will we
have to get certified in each one of those procedures or do we just go
with one standard one and say we no longer will do St. Louis\'s
procedure on how to thaw bank products?

CHAIRMAN SALOMON: That\'s a great point. There probably ought to be said
at the beginning of something like this at the time when the FDA is
really going to step up to regulate something like this, you are going
to have to set a gold standard and then you can use whatever thawing
procedure you want as long as you adequately reassure the authorities
that it fits the gold standard.

That\'s what we are doing in islet transplantation right now where you
have to show that you can get the right number of islets and they are
functional and cure a couple of patients. Then after that you can argue
to deviate from that in your IND.

DR. NOGUCHI: The idea is to identify those specifications that are
meaningful in terms of outcomes. We have heard some numbers thrown
around and we\'re not asking for what those numbers should be. I think
that the goal of post-thaw, you should get as high a viability as you
possibly can just as a general principle. Those are the principles we
would suggest you start from. If it\'s 20 percent I would say post-thaw,
likely everybody would be really concerned about what processes are
being done.

CHAIRMAN SALOMON: Unless the committee wants to disagree, we\'re telling
you that it should be around 70 percent or better if you want to talk
about a 25 million post-processing number as a target.

DR. NOGUCHI: I think if you are willing to do that, that\'s great.

CHAIRMAN SALOMON: I think that\'s what we\'re telling you. I\'m a little
bit cognizant now that I\'m under the pressure to get this done here
which is good. I love this. That\'s what I live for.

Question number two deals with age. We\'ve had discussion but we
haven\'t yet talked about age. There are two ways to go with this to set
it up. One way is to say that there\'s no evidence here that age is the
determining factor as long as you get adequate dose. If that\'s true,
we\'re done.

DR. KURTZBERG: Second.

CHAIRMAN SALOMON: I love it. That\'s good. Does anyone want to take that
alternatively? Is there a minority opinion on this?

DR. HARLAN: I agree actually. The only caveat is there was this question
about the increased incidence of GVHD and should we mention that. That
the older the patient, the higher the incidence of GVHD.

DR. HOROWITZ: You can say age does not appear to differentially impact
on cord blood transplantation and bone marrow transplantation as long as
cell dose is adequate.

CHAIRMAN SALOMON: All right. Question number three, this is really
important. What do you want to see for outcome parameters for trials?
This gets back to the argument Mary and I had about she likes binary
outcome parameters, live and dead. I understand. If I was running
something like the IBMTR, I would probably be happiest with that. So I
understand. What kind of outcome parameters besides survival? What have
we talked about? Disease-free survival/survival I guess is the same
thing. Time of engraftment.

DR. HOROWITZ: Time to hematopoietic recovery.

CHAIRMAN SALOMON: Time to hematopoietic recovery as opposed to ANC,
platelet and lymphocyte counts.

DR. HOROWITZ: Graft versus host disease, acute and chronic. Immune
reconstitution.

CHAIRMAN SALOMON: Well immune reconstitution you don\'t want to use that
term because you don\'t have a number for that. You don\'t do MLRs or
antigen stimulation tests. You have lymphocyte counts, leukocyte counts
and platelet counts.

DR. HOROWITZ: If we\'re talking about clinical trials. If we\'re talking
about clinical research that\'s different from all the other things that
we talked about. I think that it is hard to just say a point estimate
for immune reconstitution. There are a variety of things that came be
looked at in immune reconstitution. But I think if someone is really
going to do clinical research in this area, that group has to consider
some parameters of immune reconstitution. What\'s hard to say is what
they should be in every single trial.

TREC are one but they are not going to be done in every center. I don\'t
think you can ignore the immune reconstitution issue. I would say that
all of these outcomes are outcomes that have to be considered in all
trials of allogeneic hematopoietic stem cell transplant not just cord
blood transplantation.

CHAIRMAN SALOMON: That brings actually another view of this and that is
there is nothing in an outcome parameter that we currently have missed
in the field. There isn\'t any unique outcome parameter for cord blood
transplant. I\'m just putting that out for argument.

DR. HOROWITZ: Except for those processing things.

CHAIRMAN SALOMON: That\'s good. Let\'s say we\'re going to come back and
talk about the processing things because that\'s really important. How
about outcome parameters for the clinical trials not related to
processing? Mahendra.

DR. RAO: One thing that seemed to be important given what has been
reported as this whole issue of transdifferentiation if it\'s going to
be studied by a prospectively collecting data, should you be considering
looking at this liver contributions or looking at some other tissue?

CHAIRMAN SALOMON: That\'s a liver biopsy. Is that what you are
suggesting?

DR. KURTZBERG: I don\'t know any IRB that\'s going to approve screening
liver biopsies to look for hematopoietic cell differentiation in that
kid who\'s doing well. You could say that in livers that are biopsied
for medical indications you could also study it. But you are not going
to get that as a standard follow-up procedure.

CHAIRMAN SALOMON: I actually want a piece of that guy\'s coronary artery
if he\'s still here but I don\'t think he\'s going to let me do that
either.

DR. HOROWITZ: Those studies are hard to do trying to figure out what\'s
going in a recipient in a methodologically sound way. I don\'t think
that\'s something you can say across the board.

CHAIRMAN SALOMON: But his donor cells were actually X-chromosome. His
are Y.

DR. KURTZBERG: I have pictures of autopsy brains showing donor cells in
the brain with X, Y if you want to see them.

DR. HOROWITZ: But do you know whether they are from fusion or from real
transdifferentiation?

DR. KURTZBERG: They are confocal microscopy and they look like they are
not fusion. I don\'t think we should go into that here.

CHAIRMAN SALOMON: So what I\'m hearing is that even though we can all
think of some of these cutting edge sorts of things, those are going to
be very difficult and we\'re not suggesting that the FDA start sticking
those in. That would be going too far. Alison.

MS. LAWTON: One question I have and I\'m certainly not an expert in this
area but I know it\'s been discussed in detail is would you want to make
sure that you collect the information on the high resolution typing to
try and understand that further in your outcome data?

DR. RUBINSTEIN: I think it will be difficult to do but I would like to
have you consider the possibility of asking for data on donor type of
the reconstituted hematopoietic system. In many places, they just see
the reconstitution and consider it a success. It is not necessarily so.

CHAIRMAN SALOMON: So you are talking about percent. I know this is
another word that you like. Percent chimerism when it\'s 100 percent
one. That\'s what you want. I agree with that.

DR. MULLIGAN: That\'s a very important thing. In a mouse system, there
is definitely a well known thing where if you leave the irradiated
recipient and you don\'t give back any bone marrow, you die. When you
give back a low dose of bone marrow, you will get radio-protection but
you\'ll get largely host bone marrow reconstitution and that was exactly
the question I was next going to ask. Have people looked in detail to
see in fact whether long term reconstitution dose effects the amount of
actually donor reconstitution?

DR. KURTZBERG: Most of the centers doing this get chimerism on a regular
basis. I can only speak to my own data but we get chimerism every three
months the first year and yearly thereafter. Patients who have 100
percent donor chimerism maintain 100 percent donor chimerism.

DR. MULLIGAN: And do the survival statistics that we saw depend upon the
percent of donor reconstitution?

DR. KURTZBERG: In my data, people either engraft or they don\'t engraft.
We\'re not seeing mixed recovery except if you look two weeks
post-transplant you may see a mixture of patient and host. But everybody
is achieving 100 percent. So I can\'t really say that it impacts a lot
of them.

CHAIRMAN SALOMON: Everybody is achieving 100 percent. That\'s what Rich
is asking.

DR. KURTZBERG: Yes.

CHAIRMAN SALOMON: Therefore in a sense 100 percent engraftment is an
outcome variable.

DR. KURTZBERG: It is in the mini transplants and occasionally it\'s
possible that I have a host recovery. That\'s 100 percent too. It\'s not
usually a mix when you are using ablative regimen.

DR. MULLIGAN: So are you saying even at low doses when they seem to get
their neutrophil back it\'s always 100 percent or not?

DR. KURTZBERG: We are talking about ablative.

DR. MULLIGAN: Yes, but low dose.

DR. KURTZBERG: The lowest cell dose ablative patients who are grafted
and graft with donor cells. They are 100 percent donor cell.

CHAIRMAN SALOMON: Interestingly enough even in the experience now with
non-myeloablative stem cell transplants and I don\'t have data on the
cord blood but on peripheral blood and bone marrow, it\'s almost always
100 percent chimerism. Not always. I\'m talking about at a year or more.

DR. HOROWITZ: But non-myeloablative peripheral blood transplants
contrary to what people expected we don\'t see many patients with
durable mix chimerism. They revert to host. You may have transient mix
chimerism. Most of them revert to host or become 100 percent donor in
the long term.

CHAIRMAN SALOMON: This isn\'t published yet. We did 30 \-- a unique
genotyping method for doing it and it was 100 percent at nine months in
30 non-myeloablative allos. If you did 1,000, I\'m sure you\'d get some
but it\'s really unusual.

DR. KURTZBERG: One of the things Pablo is raising has to do with more
standards of practice in transplantation medicine again disregarding
stem cell source. It should be to me a no-brainer to check your cells
once your patient engrafts to make sure that they\'re donor but he\'s
right. Not everyone does that.

These are some minimal standards that might be address in another
meeting which don\'t just relate to cord.

DR. RUBINSTEIN: We could propose to the centers to do those studies for
them if they have difficulty in doing it. I wanted to mention that the
mouse model with cord blood transplants that is allogeneic mouse donor
of cord blood, they can achieve permanent chimerism in the sense that if
you do it from two donors, you will get a chimera that will be permanent
for the life of the animal. In humans, that doesn\'t seem to happen.
There may be chimerism but it eventually resolves for one or the other.

CHAIRMAN SALOMON: The other thing that I didn\'t see in enough data
would be some standardization of marking acute and chronic graft versus
host disease as outcome parameters. Again I don\'t mean to insult the
field and say they obviously know that they need to that. It was
interesting that you presented a lot of acute GVHD data but didn\'t
really step up with your chronic GVHD data. That has to be a part of it.

DR. KURTZBERG: Mary can probably speak to this better than I can but
there are committees as part of some of the national trials now trying
to define the universal criteria for grading and scoring chronic GVHD.
That\'s in progress but again it\'s related to all stem cell
transplantation. It\'s very hard.

CHAIRMAN SALOMON: In general, what we\'ve said now is we\'re going to
deal with this processing issue now. For clinical trials that
essentially I get the feeling that pretty much we are all comfortable
with the general outcome parameters for other hematopoietic stem cell
transplant trials and for that matter, donor chimerism is a relatively
important measurement for any hematopoietic stem cell trial.

DR. HOROWITZ: No, I\'m going to have to say something because the
clinical trials network and the transplant community have said that
chimerism studies for a standard myeloablative allogeneic transplant are
not necessary because they are a waste of money.

CHAIRMAN SALOMON: We\'re talking about clinical trials.

DR. HOROWITZ: In clinical trials, they are not always required. It
depends on the end point being looked at. If you talk about bone marrow
peripheral blood stem cell transplants, in myeloablative regimen, the
incidence of finding recipient cells is so low as to make it not worth
looking for. We could say that chimerism studies in cord blood
transplants might be something that should be considered as routine that
might not always be considered routine in bone marrow transplants.

CHAIRMAN SALOMON: Perfect. Processing.

DR. MULLIGAN: Just on that point, even though we\'re not going to talk
about the manipulation cell in in vitro amplification, I think that\'s
the area where this is going to be very important. When you begin to
change the stem cells or you begin purifying different parts of the stem
cell population then all bets are off.

CHAIRMAN SALOMON: I totally agree. I\'m thinking that this committee
will get those kind of protocols in the not too distant future. Kathy.

DR. HIGH: Are we on processing now?

CHAIRMAN SALOMON: We\'re on processing.

DR. HIGH: I just wanted to get one clarification. In your data about in
vitro functionality of ten year cryo-preserved umbilical cord blood and
15 year, is that a settled issue so now if it\'s 20 years or 25 years
it\'s okay? Do we know?

CHAIRMAN SALOMON: Remember that\'s based on SCID repopulating
efficiencies. It\'s not in vitro. It\'s in vivo. But I don\'t know.
That\'s really a good question. For the stem cell transplanting
clinicians, what do you guys think? If you had a routine patient and you
had offer of a three year old versus a 25 year old unit or a 15 year old
unit. What would you pick?

DR. KURTZBERG: I\'d pick the one with the highest cell dose.

DR. STRONCEK: From a laboratory medicine and transfusion medicine point
of view, if you haven\'t tested it, you don\'t know. When people have
looked at it, things seem to cryo-preserve pretty well. So it\'s likely
they\'ll be viable but you really don\'t know. You don\'t know what the
fall off is.

DR. RUBINSTEIN: We have some empirical data. Because we started
cryo-preserving and freezing this annulated cord blood in 1993 every
year we do give a few transplants from units that were collected in the
first year. I can tell you that the last three units that we
transplanted at the end of last year and beginning of this year
engrafted with the same kinetics that a newer unit would provide.

CHAIRMAN SALOMON: How old were those?

DR. RUBINSTEIN: Eight and a half years. One of them was nine years.

DR. CREER: We confirm that over five years, the same thing. No change in
rate of engraftment when matched for cell dose and no effect on outcome
over five years when matched for cell dose.

DR. RUBINSTEIN: So if this has not changed after nine years then the
likelihood that it would suffer an abrupt change some time in the near
future is not very much.

CHAIRMAN SALOMON: Liane.

DR. HARVATH: I would like to say that in general I would agree with the
caveat that we know that not all cryo-preservation facilities are
created equal. Both Pablo and Phil Coelho commented about transient
warming events. I think they have some pretty compelling data that if
you freeze your product in such a way that it has gone through a number
of cycles of transient warming events, you take a hit in the cell
viability.

We don\'t really have the in vivo data to show what that impact would be
on the outcome of engraftment. If a facility is incredibly rigorous and
they have well documented records of how they cryo-preserve and monitor
the status of their products than these data will probably show promise
in terms of cryo-preserving for a decade or perhaps more. Many of us
know of places where there have been freezer meltdowns and very good
facilities. Those accidents happen. We have to be really cautious about
the kind of message we send out as a committee making a recommendation
that we are very confident that this is always the case.

DR. HOROWITZ: I agree and I also agree with the fact that you don\'t
know until you know. There has to be some kind of program of
periodically testing a hypothesis.

CHAIRMAN SALOMON: Personally as a recommendation, I think David said it
well. In laboratory science, you don\'t know until you test it. That
probably covers your concerns as well.

DR. KURTZBERG: I agree but testing here is biologic. It\'s dictated by
the practice so it means collecting information.

CHAIRMAN SALOMON: Exactly. In terms of processing, we really would all
agree that if you are going to get into this in terms of regulating
licensing, INDing you are going to want to know this whole pathway.
That\'s going to be the processing step, the freezing step, the quality
of the long term freezing, some of the points of view you made. I see my
post-docs come in to our liquid nitrogen farm and pull the whole rack
out and set it up there and talking on the phone, 200,000 cells go into
that. There are a lot of issues here.

Another issue would be shipping. I\'ve done a lot of cell shipping and
we\'ve all had disasters doing that. That\'s a really big issue that in
the clinical trials there should be some standardization for how you
ship. Shipping dry ice, shipping liquid nitrogen, reassurance that you
are not losing temperature in the shipping process, time shipping, etc.
Then the thaw and what kind of yield you\'re getting. Does that cover
it? We\'ve really had a pretty good discussion of that today.

All right.  We get to finish on the CD34 question. Pablo\'s number one
is if you use the total nucleated count and you give enough as defined
empirically by the data and we\'ve had that discussion already then even
though we may admit that there could be differences in CD34, and there
might be some who think that\'s really important and some who think
that\'s less important, that it will all come out in the wash because
you gave enough. That might be the recommendation.

Just give enough by total nucleated count and make sure that the rest of
the processes are done well and you will be okay. Then you avoid all
these downstream issues with CD34 that have confounded this field for 10
years. I remember ISH obsessing about how to count CD34 cells. Here we
are ten years later and still these guys aren\'t sure how to count CD34
cells.

DR. HARVATH: Dr. Salomon, may I ask a question? I would like to ask a
question of my former colleagues at FDA. If you were to recommend
quantification or even make it a requirement of CD34 enumeration, are
there enumeration assays that have been licensed by FDA for the
quantification of CD34 in cord blood? If so, would that be something
that would need to done using licensed kits or ones that are alternative
methods?

DR. NOGUCHI: To my knowledge, we do not have such a kit licensed for
cord blood. I hope I\'m stating that correctly. It is of interest that
much of the discussion has been about the variability or non-variability
of CD34 which suggests to us that more work needs to be emphasized in
this area. As far as I know, we don\'t have something we could mandate
as a requirement which is why I think the extensive discussion here is
very helpful to us to enable us to really think about what are the
future needs.

DR. CREER: I think the main distinction that people have in the CD34
field is whether or not you actually gate out non-viable cells. The ISH
protocol requires that the immuno-phenotype labs that usually do most of
the CD34 counts. Transplant centers don\'t so they are not using a
gating strategy that eliminates non-viable cells. That\'s very key.
That\'s the most important part of it. You can just ask the transplant
center who is doing your CD34 and how they are doing it. If it\'s not
done by a processing lab but a path lab, the focus is entirely
different.

DR. HARLAN: I have a specific question and then a general statement. The
specific question is for the experts in the field. How well established
is it that the CD34 population is the best? I have heard about these
side population cells and CD34 positive and CD38 negative. Then the
general statement is maybe we should just suggest in our language that
assays to better characterize what the true stem cell population is and
just leave it very vague like that.

DR. KURTZBERG: I don\'t think it\'s practical at all to start requiring
banks or transplant centers in the clinical practice to look at side
populations, CD34 and all of that negative populations because those are
research questions. But even looking at 34 isn\'t standardized as you
are hearing. It\'s harder to do on the thawed cells than it is on the
fresh cells.

There are not established agreed upon protocols amongst different
transplant centers or banks. That\'s not to say that\'s not possible but
it\'s also a moving target. The technology is changing. The assays are
changing. The companies bring out new methods that sometimes can be
adapted for cord blood.

I think the real practical issue is that many of the units that are in
banks right now that may be good units for patients do not have
pre-cryo-preservation 34 numbers. So it\'s not practical at this point
to require that. A statement like this is of interest and it\'s been
shown in other stem cell transplants to be a reliable predictor of
engraftment and should be looked at or something like that but I don\'t
think we can go further than that.

DR. CREER: One important point is the CD34 count measured in peripheral
blood, bone marrow and umbilical cord blood are totally different
measures of stem cell activity. You can\'t take CD34 counts from one and
extrapolate it to the other. Peripheral blood has too many CD34 cells
that are already committed. It\'s much different than cord and bone
marrow.

CHAIRMAN SALOMON: Basically what I\'m hearing is that it\'s an
interesting scientific question but not one that steps up to the point
of displacing as a fundamental measurement than nucleated counts. Again
it comes to numbers. The numbers have been fine with total nucleated
counts. In a clinical trial, Mahendra whispered to me I think you would
want some data on this. It should be one of the questions you want to
solve for this field down the road but it\'s scientific one not a
clinical one right now.

DR. RUBINSTEIN: One issue that has been raised is the effect of
nucleated red cells in the total count and whether that is relevant to
the relationship between the cell count and the speed of engraftment or
other issues.

CHAIRMAN SALOMON: Thank you for bringing that up because that came up in
our conference call to ask that question. So what do you think? What are
these numbers when people are counting them? Are they counting nucleated
red cells? I think they are, right?

DR. RUBINSTEIN: Yes, most of the automated instruments at the moment
will count nucleated red cells into the TNC. But we studied the
relevance specifically of nucleated red cells on engraftment. We found
to our surprise that there is a very good correlation. It\'s almost as
good as the TNC as a whole. There is no problem in having people just
count TNC.

CHAIRMAN SALOMON: Just to be clear, are you saying that if you counted
the total nuclear red cells, the more nucleated red cells in a cord
blood unit the better the reconstitution?

DR. RUBINSTEIN: Yes.

DR. KURTZBERG: Pablo, I\'m aware of that data but every so often, we
have a unit that we bank that has more than 50 percent nucleated red
cells. I\'ve always wondered if those really are giving the same cell
dose so that the functional leukocyte dose is actually 50 percent of
what you are measuring. I wonder how much experience you might have with
those units.

DR. RUBINSTEIN: They have typically extremely high colony counts, much
higher than would be predicted by the CD34 counts. In our experience,
they engraft as if they had a lot more white cells than we find in those
units.

DR. CREER: One thing we\'ve noticed is red cell depletion removes
nucleated red cells. So pre-processing and post-processing differ a lot.
You almost never recover after thaw nucleated red cells. You have to be
very careful when you make that correlation to state specifically at
what point in time your correlated nucleated red cell count with
engraftment potential because I think it should be at a pre-processing
stage and not later after you have selectively removed them or lost
them. That\'s very important. I agree with you. I think nucleated red
cells are a marker for erythroid potential. There are erythroid colonies
that you get when you have a higher nucleated red cell count.

DR. RUBINSTEIN: I am always talking about these numbers pre-freezing.
It\'s an interesting observation. The reason we looked into that is
there\'s a German group that raised the alarm about these nucleated red
cells. In their opinion, when you thaw these units, those nucleated red
cells lyse together with the rest of the red cells. We have done those
experiments and we find that these cells don\'t lyse like the others.
That\'s one of the problems with nucleated red cells. They still
maintain some metabolic ability to resist stochatic lysis so those cells
can be counted after thawing.

DR. HALEY: Rebecca Haley from the American Red Cross. In our cord blood
banks by agreement among the investigators, what we do with nucleated
red cells is we make a notation on the cord blood information record
when we have more than 20 per hundred white cells.

Whether they survive freezing or not, we are worried that the
transplanter may get the wrong idea about how many white cells there are
in the unit. Whether they survive freezing or not, they probably are not
going to contribute to overall engraftment. They may be an indicator of
engraftment as Dr. Rubinstein has said but we think in the interest of
full disclosure the transplanter needs to know what\'s there. We have
made it a practice to say what\'s there.

DR. RUBINSTEIN: I think that should be required. In our information, I
guess this is what we would call a label. In the label there is complete
description of the cell count including the nucleated red cell counts.

CHAIRMAN SALOMON: Then I think we\'ve answered the CD34 question.

DR. HARLAN: One other thought occurred to me. Dr. Wagner raised it about
the typing immediately pre-transplant because of the mistake that they
had uncovered. Should we include that?

CHAIRMAN SALOMON: That\'s a good point. Obviously that\'s been in the
news recently, the possibility of errors in typing and John had
experience that quite pre-dated that. It\'s easy for all of us to agree
we don\'t want that to happen. Joanne.

DR. KURTZBERG: Pablo has stated that they have a contiguous segment on
their bag that they type before they release the unit from the bank
which is one practice. There are some transplant centers that would also
like to retype themselves. That\'s practical if there are enough
segments on the bag but not if there aren\'t enough segments on the bag
and that varies from product to product and from bank to bank.

The actual typing though is not fast turn around typing. It\'s not
available at every transplant center. You don\'t want to keep the
product in the thawed state while you are waiting to check. John did ABO
which just by chance happened to be different in that patient.  That\'s
not going to be something that you can depend on to be 100 percent
accurate. There are issues that relate to that. The requirement of the
unit that it has at least a single segment that is still attached and
can be used for final confirmation of identity is really important.

CHAIRMAN SALOMON: When it gets to the host institution.

DR. KURTZBERG: I don\'t know because that\'s not always going to be
possible. Let\'s say that the bank has done some other testing on that
unit. Let\'s say I asked them for a segment to test for Hurler enzyme.
So they used one up already and maybe they have their internal QC that
uses the second and then they only have one left. Then the bank and/or
the transplant institution have to compete for that last segment. I\'m
not sure I know the right answer but if the programs are reputable that
one should trust the other. Some confirmation has to be done either at
the point it\'s shipped or at that point it\'s received.

CHAIRMAN SALOMON: Let me explore that for a second. If you call me up
and I\'m a bank. I say I have confirmed it. You say when. I say I
confirmed it on a separate segment within a week of freezing it which
was three years ago and I confirmed it before offering it to you.

DR. KURTZBERG: I think the confirmation has to be done at the time it\'s
being either offered or received not at the time it\'s being put in the
freezer.

CHAIRMAN SALOMON: That\'s what I was asking.

DR. KURTZBERG: Most places have something that they do at the time it is
put in the freezer but that isn\'t confirmation. You need confirmation
at the time received. If you say it has to be done by both, the bank and
the transplant center, there aren\'t always going to be enough segments
to do that.

CHAIRMAN SALOMON: I was trying to follow you through. One of the ways to
deal with the time issue is that you initiate your confirmation during
the shipping which gives you a little time.  Then by the time it arrives
at the host institution I can confirm from the bank that it was what I
sent you.

DR. NOGUCHI: Dan, this topic itself is a very generic one for a whole
lot of things for all cellular therapies. It\'s important to discuss it
but I suggest this is not the time to do it in detail. A statement or
consensus that even simple things about is it what you think it is and
do you know what you think you did it\'s an important one to say but to
put a bookmark and say in the general area that errors will be made and
we need to mitigate them as much as possible is really where we want to
go. For the purposes of today, that\'s not one that we need immediate
feedback on other than to say yes, be careful.

CHAIRMAN SALOMON: Dr. Rubinstein.

DR. RUBINSTEIN: Maybe this is beating a live horse but I have a question
and a suggestion on this issue. Sometimes we have done the confirmation
from the segment and for a number of reasons the transplant center
chooses not to use that unit. We don\'t ship it to them. So we store it
and we note in the computer what was found and we have all the data
available. I would suggest that those units should be available to other
patients.

CHAIRMAN SALOMON: What Phil is telling us is you\'re right. We\'re not
going to solve these. These are really important details but if the FDA
is comfortable that we\'ve gotten to the level of detail they are
comfortable with then we\'ve done our job today.

DR. RUBINSTEIN: The suggestion is that perhaps when we do the
confirmatory typing on the segment we might provide the transplant
center with a little bit of that DNA for their confirmation if they want
it.

CHAIRMAN SALOMON: Anyone feel like we missed something important today?
Joanne? You have your light on. You\'re okay?

DR. KURTZBERG: No.

CHAIRMAN SALOMON: Ellen, Stephen, Ruth, are we okay? Phil.

DR. NOGUCHI: Just a very quick and succinct thank you for the Committee
for dealing with these extraordinarily complex and really perplexing
issues. Given the advice, we\'re going to study this very carefully and
review the transcripts and move on from here. As we always say, we
can\'t do this without all your input. I would also say the public
hearing today was immensely helpful in making sure that all voices have
been heard including those who have benefit from it and including the
presentation on new outcome data.

CHAIRMAN SALOMON: Joanne. Then from my point of view, I would like to
thank all of you for all the hard work today. I always try to end a
session by thanking Gail Dapolito and Rosanna Harvey and Bill Fress and
the rest of the FDA staff that worked so hard and then sit quietly
making sure I don\'t screw up. These things don\'t happen without them.
I really appreciate that.

(Applause.)

CHAIRMAN SALOMON: We are now going to take a break for ten minutes. When
we come back, we\'re going to have in open session presentations from
investigators involved in the most recent site visit. I encourage all of
you to stay since these are always very succinct and informative about
the research going on intramurally in the FDA. Then we will go to a
closed session. Anyone on the committee who is going to be here tomorrow
for the gene therapy discussions we will start on time. Some people have
arrived at 3:00 p.m. today. Otherwise I\'ll give the talks and we\'ll
get this done. Thanks. Ten minute break. Off the record.

(Whereupon, the foregoing matter went off the record at 4:45 p.m. and
went back on the record at 4:57 p.m.)

CHAIRMAN SALOMON: Back on the record. What we are going to do in this
first part of the open session is review the SIF of the Division of
Monoclonal Antibodies. This was chaired by Dr. Mahendra Rao. We can do
this one of two ways. Mahendra, do you want to introduce these people
and take over this piece of it?

DR. RAO: You do it.

CHAIRMAN SALOMON: Just do it? Okay. Then if you will allow me to
continue in chair\'s role then I would like to introduce Keith Webber
who will provide an overview of the Division. Then we\'ll go on to
presentations from the Laboratory of Cell Biology, the Laboratory of
Immunobiology and the Laboratory of Molecular and Developmental
Immunology. Keith.

DR. WEBBER: As introduction, I\'m Keith Webber. I\'m the Director of the
Division of Monoclonal Antibodies. The Division is to review at the site
visit. First I\'d like to thank everybody who is here and let you know
that we certainly appreciate your participation in the site visit
review. Without a doubt, it\'s one of the most important aspects of our
program to keep the quality and integrity of our research programs as
high as I believe they are. We definitely all appreciate your help in
that manner. I\'m going to quickly give you a thumbnail sketch of the
Division and what we do.  Then I\'ll introduce the folks who are under
review within our Division.

Essentially Division has about 17 research reviewers who are as the name
implies do both research and review. They spend about half their time on
each of those aspects. They split 50-50 on doing research and regulatory
review work on product files. We also have six full time reviewers in
the Division who as their name implies are 100 percent regulatory review
personnel. We also have about 20 additional staff that includes
post-docs in laboratories, laboratory technicians and secretaries who
are very important to the function of our Division.

With regard to our regulatory load, the Division currently has about 20
licensed products. Those are naked antibodies, radio-labeled antibodies.
We have an antibody drug conjugate that we consult with Center for Drugs
on. Also some of the fusion proteins like Enbrel are within our Division
and then the latest one, Alefacept. We also have about 460 prelicensing
files. Those are the ones that are under clinical trial or in INDs or
they are master files that are supporting either license applications or
other INDs. Next slide please.

This is a very abbreviated diagram of the layout of the Division. Within
the Director\'s office, we have Patrick Swann who is the supervisor of
the full time reviewers in the Regulatory Science and Policy branch. Dr.
Ezio Bonvini is the acting deputy director of the Division. His
responsibilities are primarily for oversight of the research activities
of the Division.

The research activities in our Division are distributed into three
laboratories. The Laboratory of Cell Biology which is led by Dr.
Kathleen Clouse.  This laboratory focuses primarily on studying the
modulation of cytokine networks within monocytes, macrophages and other
immune cells. The Laboratory of Immunobiology is headed by Dr. Ezio
Bonvini. They study primarily the signaling activities within immune
cells that control their growth and differentiation as well as
neoplastic progression tumor cells.

We also have a Laboratory of Molecular and Developmental Immunology
which is alternatively headed by Steve Kozlowski and/or Margie Shapiro
as acting lab chiefs of that laboratory. This laboratory focuses
primarily on studying the humoral and cellular immune response and
control of those processes. Next slide.

The folks who are being reviewed currently are Dr. Kathleen Clouse here
which I\'ve already introduced. Her research is primarily focused on
studying the interactions between HIV and the cytokine network of
macrophages. She will briefly tell you about that today. Next slide.

Ingrid Markovic here who is studying the mechanism of entry of HIV into
immune cells particularly T-cells and macrophages. Next slide.

Dr. David Frucht who is a tenure track investigator in the Laboratory of
Cell Biology. He is studying the role of T-bet and Stat4 in
antigen-presenting cells. He is also one of the folks in our division
who is involved in doing counter bioterrorism work. Next slide.

Dr. Jerry Feldman is another tenure track investigator who is in the
Laboratory of Immunobiology. He\'s studying the effect of immune
complexes on interferon-gamma signaling pathways mostly in dendritic
cells. He is also the Office of Therapeutic point person for issues
regarding transmissible spongiform encephalopathy agents and he is doing
some research as well with regard to prion protein effects on
interferon-gamma expression.

Finally last but not least I should say that Dr. Kozlowski who is in the
Laboratory of Molecular and Developmental Immunology and is studying the
effects of T-cell activation. Since the hour is late, I\'m not going to
go further unless there are any questions for me specifically. We will
move on directly to Dr. Clouse\'s presentation.

DR. CLOUSE: I\'d like to thank you for the opportunity to give you a
summary of our research program concerning cytokines network and HIV
pathogenesis. The focus of our research program is actually to
investigate the modulation of cytokine networks in HIV disease and to
determine the impact of these changes on the disease process and to use
this information to evaluate immune-based therapies for HIV as well as
other infectious diseases.

The research can be covered under three specific aims. The first is to
determine the effect of HIV on cytokine production. The second is to
determine the effect of cytokines on HIV replication. The last is to
identify novel agents that block HIV fusion and entry and therefore
prevent a variate cytokine production.

I\'ll focus initially on the first specific aim to determine the effect
of HIV on cytokine focusing on human macrophages. The macrophages play a
pivotal role in HIV pathogenesis. They are the first cell type infected
with HIV in vivo. They serve as a vector for the spread of infection not
just to other macrophages but also to T-cells. They function as
regulatory cells that control the pace and intensity of disease
progression. They do this by the production of cytokines. Furthermore,
they serve as a reservoir for HIV which is quite resistant to the
effects of current highly acted antiretroviral therapy.

Our initial work to determine what cytokine genes were turned on by
HIV-I infected macrophages showed that they did not produce
pro-inflammatory cytokines or GM-CSF or other cytokines and chemokines
induced by GP-120 alone. They do however produce macrophage colony
stimulating factor which is important because M-CSF functions as a
survival factor for the macrophage and infected macrophages are known to
survive longer than their uninfected counterparts. M-CSF also
facilitates infection of macrophages in part via the up regulation of
the HIV receptors CD4 and the chemokine co-receptor CCR5.

We then went on to show M-CSF antagonists can actually inhibit HIV-I
replication in macrophages. Shown here as a typical infection of
macrophages with a CCR-5 M-tropic strain of HIV-I, you can see
substantial virus replication over the course of 21 days. We monitor
replication by measuring reverse transcriptase activity. However in the
presence of either polyclonal or monoclonal antibodies to M-CSF or
soluble M-CDF receptors, there is a dramatic inhibition of HIV-I
replication.

We then went on to do studies to look at the specificity of the M-CSF
induction by viruses. We used a panel of single-stranded RNA viruses. We
found viruses such as measles virus and respiratory syncytial virus all
though they replicated macrophages and they are functional in that they
produced pro-inflammatory cytokines and beta chemokines, they do induce
M-CSF production.

In contrast, M-tropic strands of HIV-I, HIV-II and also SIV replicate
and induce M-CSF production in infected human macrophages. Furthermore,
exogenous M-CSF enhances replication of these lentiviruses. The
replication of both HIV-I and HIV-II in macrophages can be inhibited
using M-CSF antagonists. This has led us to propose the following
cytokine network for sustaining a macrophage HIV reservoir where you
have an HIV infected macrophage which produces M-CSF but can function in
an autocrine and paracrine manner.

Beta chemokines are produced as well as infectious HIV particles. Beta
chemokines such as mip-1 alpha and beta attract uninfected cells to the
area. They are rendered more susceptible to infection by M-CSF. They are
infected by the released virus particles and the network continues.
Pivotal for maintaining this network is the production of M-CSF.

The second area of research dealt with the effect of cytokines on HIV
replication. For this we focused on the effect of interferon-alpha on
HIV replication and human macrophages and T-lymphocytes. We specifically
addressed the question of whether interferon-alpha therapy is beneficial
or detrimental for HIV disease.

It\'s pretty well known that interferon-alpha therapy is effective early
in the course of HIV disease but as the disease progresses it becomes
less effective and may actually hinder HIV patients. There are many
reasons that are thought to be the cause of this. We looked at a number
of isolated interferon-alpha components as well as the number of hybrid
interferon-alpha molecules and looked at their effect on virus
replication in both macrophages and T-cells. We have come up with the
following few effected macrophages with an M-tropic strain of HIV-I that
uses the CCR-5 chemokine co-receptor. You see again substantial virus
replication. But in the presence of interferon-alpha, there\'s a dose
dependent inhibition of virus replication. This does not represent a
block in virus release but an actual block in virus replication.

In contrast if you infect T-cells with a T-tropic strain of HIV-I that
uses the CXCR-4 receptor again you see substantial virus replication.
This is accompanied by death of the CD4 cells. But when you add an
interferon-alpha here, you actually see an enhancement of HIV
replication. This enhancement does not correlate with an increase in the
proliferation of the T-cells or an up-regulation of the receptor or the
co-receptor for HIV. We feel we now have a model that we can actually
use to determine one of the mechanism that may contribute to the
exacerbation of HIV-I disease mediated by interferon-alpha.

I would like to acknowledge the people involved in these first studies.
The people in my laboratory. Dr. Judy Beeler\'s lab collaborated on the
measles and RSV studies. People in Indira Hewlett\'s lab on the HIV-II.
Vanessa Hirsch\'s lab contributed SIV islets. All of the
interferon-alpha are being done in collaboration with the laboratory of
Dr. Kathy Zoon. The final area of research concerning the identification
of novel agents that block HIV fusion and entry will be presented by Dr.
Ingrid Markovic in my lab.

DR. MARKOVIC: As Dr. Clouse mentioned part of our laboratory\'s efforts
are dedicated to try to understand the mechanism of HIV entry and
eliciting the way how this process can be blocked. Shown here is the
schematic representation of HIV entry, this here being the viral
particle with the envelope surviving the virus.

Embedded in the envelope is gp120 and gp41 which is the protein which is
responsible for fusion between viral envelope and the target membrane.
When the virus approaches the target membrane it binds to CD4 which is
its primary receptor. Following that binding, there is a confirmation on
change in gp120 which induces the exposure of the co-receptor binding
site allowing gp120 to now bind to the co-receptor which is CXCR4 or
CCR5 depending on the type of the virus. That would finally lead into
the final confirmation on changes, this being the six helix bundle stage
which brings the viral and the target membrane in the close up position
and allows the fusion to take place.

Now previously there were a number of ways which people had thought
about in order to block the viral entry. Primarily those were trying to
block gp120 and binding site or blocking using a receptor ligands trying
to block the CD4 binding site and CXCR4 binding site. Another way was
trying to interfere with the confirmation on change so the six helix
bundle stage does not form.

There were problems associated with these different approaches whether
there are the formation of escaped mutants like here or the fact that
chemokine ligands can actually induce replication of the HIV-I, the
viruses inside the cell. Our approach was trying to actually use
inhibitors of cell by cell like interchange of gp120, gp41 and try to
understand the mechanism first whether this is possible and second what
would the mechanism of this action be.

The hypothetical role that we had in mind is how far this thiodisulfide
interchange could be applied in HIV entry, this here being the gp120,
gp41 in its initial confirmation with disulfide bonds in the original
form. In the presence of CD4 and CXCR4 in the target membrane in a
critical thiodisulfide oxidoreductase, gp120 would undergo unfolding and
reduction of critical disulfides opening now the free-style groups. We
actually have the data to show the distance remedied indeed does
actually. As a logical consequence of this intermediates we think that
protein now undergo final refolding to achieve the fusion competent
confirmation. The disulfides will be restructured in a different
orientation where the previously unpaired systems can now be bonded.

We decided to focus our interest on protein disulfide isomerase as a
punitive agent which would be this thiodisulfide isomerase. The reason
being that it\'s the most widely published enzyme because it is present
in many different cell types. The function of this enzyme is to
characterize reduction, formation, and isomerization of disulfide bonds.
It does that by a two active catalytic site which is shown here in green
which are basically two free cell groups. It localized in the ER as well
as the cell surface. It\'s been shown by our lab as well confirmed by
other labs that it can be inhibited by DTNB, Bacitracin and anti-PDI
monoclonal as well as polyclonal antibodies.

The first question that we had we wanted to confirm whether PDI is
indeed present at the cell surface and if so does it actually
co-localize with the CD4 and the CXCR4. Our approach was to use co-focal
microscopy. Here shown is the triple labeling where the green indicates
PDI, the red region indicates CXCR4 and the blue is CD4.

We did this in two different conditions. In the absence of soluble
gp120, we can pretty much see that PDI is isolated in distinct regions
on the membrane and it does not co-localize with either the receptor or
the co-receptor. The magenta color up here indicates co-localization of
CD4 and CXCR4 which is occurs obviously in the absence of gp120.

However when we added gp120 to those cells we now see that there is a
majority of the PDI is now co-localized in this white region which
indicate triple co-localization of PDI with CD4 and CXCR4. This
indicated to us that probably gp120 acts as some sort of an assembly
mechanism bringing PDI into conflict with CD4 and CXCR4.

Next we wanted to see using cell-cell fusion assay which was our assay
to estimate where the viral entry takes place. I should say to
substitute for viral entry assay. We wanted to see whether the
inhibitors of PDI such as antibody DTNB and Bacitracin can actually
block this process. Shown here are just the most effective doses of each
inhibitor namely antibody DTNB and Bacitracin which have been very
effective in blocking fusion in comparison to the fusion of cells which
were in control or that were treated with isotype antibody control. I
should say that we have a dose response for each of these inhibitors.
This is just the most effective dose of each of those inhibitors.

We next proceeded to look at the infection assays again trying to
estimate the potential of these inhibitors to inhibit infection. On the
X axis it stays Post-Infection and the Y axis is the Reverse
Transciptase Activity which are indicative of the level of the
infection. The three bell-shaped curves are control to antibody control
namely polyclonal and monoclonal antibody controls. This is a typical
bell-shaped curve that you get in control with HIV infection. However in
presence antibodies, both poly- and monoclonal we see a significant
delay in the onset of infection whereas DTNP and Bacitracin completed
abolished infection.

Next we decided to form a working model. Actually this is a working
model that we have now in our minds when we are thinking about this
problem. Namely this is gp120 and gp41 bound to CD4 and the target
membrane. This induces conformational change in the protein and exposes
a certain high affinity site which allows PDI to bind to gp120 which in
turn induces subsequent confirmation on changes allow it to bind to the
co-receptor and finally the fusion can take place.

I would just like to acknowledge that all this work was done in
laboratory headed by Dr. Kathleen Clouse. These are our collaborators
from CBER, USUHS, and NIAID. Thank you.

DR. FRUCHT: I\'m David Frucht, one of the newest investigators in the
DMA. The specific aims of my program are to divide expertise in
evaluating the safety and efficacy of new biologics that effect cytokine
networks. Specifically I\'ve been concentrating on doing reviews on
hematological and infectious disease related biologics.

As part of this Aim 1, Aims 2 and 3 also fit under Aim 1 which is to
establish research programs to establish the role of key transcription
factors and the function of antigen presenting cells (APCs) and to
determine the role of cytokine signaling and host responses to
anthrax-lethal toxins. That\'s a new project I just started in the last
couple of months.

Perhaps the most important process that occurs during immune responses
is the differentiation of T-helper cells into either TH1 or TH2 effector
cells. TH1 effector cells make interferon-gamma and are important for
cellular immune responses, control of intracellular infections like
Tuberculosis and Anthrax, Toxoplasma, Salmonella and also are important
for tumor surveillance. TH2 effector cells on the other hand make IL-4
and are important for humoral immunity, control of helminthic infections
and regulation of allergic responses. We now know that this process is
regulated by cytokines. IL-12 signals via Stat4 to induce TH1
differentiation whereas IL-4 signals via Stat4 signal transduction
molecule to induce TH2 differentiation.

Two other transcription factors are also very important. T-bet in TH1
cells which is important for programing TH1 cells to make interferon
gamma and GATA-3 which programs TH2 cells to make IL-4. My program
mainly deals with TH1 differentiation specifically with Stat4 and T-bet.

Instead of concentrating on T-cells, I\'ve concentrated on
antigen-presenting cells. Antigen-presenting cells are absolutely
required for TH differentiation. In fact if you take a T-cell and you
stimulate it through the T-cell receptor in absence of antigen
presenting cells you don\'t get cytokines, production or proliferation,
in physiological conditions. So antigen presenting cells are very
important.

We also know evolutionarily that they are very important because the
bugs actually target antigen-presenting cells like Tuberculosis and
Salmonella. Also the toxins made by bacteria also specifically target
antigen-presenting cells such as Anthrax-lethal toxins.

Our first major finding was that Stat4 is produced by antigen-presenting
cells and is required for IL-12 dependent signaling and interferon gamma
production. In this experiment using antigen presenting cells for
wild-type mice and Stat4 knockout mice \-- and Stat4 knockout mice have
the genes deleted to they don\'t express Stat4 messenger protein \-- you
can see clearly that in the wild-type mice following stimulation with
IL-12 or Il-12 in combination with Il-18 you get high levels of
interferon gamma in the wild-type samples. However you see no induction
of interferon gamma in the Stat4 knockout samples.

We also went on to show that Il-12 dependent nitric oxide production
depends on Stat4 as well. This is important because it allows control of
intracellular infections such as Toxoplasma.

Our second major finding was that T-bet message RNA is induced by
interferon gamma in antigen-presenting cells. As you can see in this
Northern blot looking at messenger RNA, high levels of T-bet message RNA
are induced following interferon gamma stimulation, higher levels than
we see even in our NK cell line which was our positive control.

Now you might recall from one of my first slides that T-bet regulates
interferon gamma production. What we\'ve shown is that T-bet message is
present in antigen-presenting cells. It\'s regulated by interferon
gamma. We have very good evidence that this positive feedback sustains
interferon gamma production by antigen-presenting cells. What wasn\'t
previously known was that interferon gamma regulates T-bet message in
T-cells as well. This positive feedback loop is absolutely critical for
TH1 differentiation in the absence of IL-12. These are experiments that
we did in collaboration with Bill Paul.

I would also like to mention that we\'ve been limited by the fact that
we could only look at message levels of T-bet because a good antibody
didn\'t exist. But now there are some commercial antibodies that we are
testing and looking at right that might let us make more definitive
statements about these positive feedback groups. Also we\'re going to be
getting the T-bet knockout mice by June. We are very happy about that.

In the very basic model of TH or cellular immunity, antigen-presenting
cells make IL-12 which signals to Stat4 to promote TH1 differentiation.
TH1 cells also express high levels of T-bet and T-bet allows the
programing of TH1 cells to make interferon gamma. What we showed is that
APCs express T-bet. It\'s regulated by interferon gamma and this
positive feedback loop likely sustains a long term interferon gamma
production by APCs. It\'s more important probably in T-cells, where
T-bet is also regulated by interferon gamma. Again this wasn\'t
recognized before and is absolutely essential for TH1 differentiation in
circumstances where IL-12 is not present.

We also showed that Stat4 is expressed by antigen presenting cells and
allows signal transduction response to IL-12. One of the genes that\'s
turned on is interferon gamma. Interferon gamma present at that time of
antigen presentation can be very important in promoting cellular
immunity at a very early stage. We call this the \"jump start\" model.
This model appeared in \"Trends in Immunology\" in a paper we wrote
recently. I\'m happy to say that it seems to reaching some level of
acceptance because in Laurie Glincher\'s recent review and annual
reviews of immunology, she highlighted some of our data in this regard.

I would like to conclude by talking about our last project which just
started a few months ago looking at the role of cytokine signaling and
host responses to anthrax-lethal toxin. Anthrax-lethal toxin
specifically targets macrophages. It kills them through apoptosis by
leading MAP kinase kinase. This is pro-survival pathway.  We have
developed toxin potency assays for rapid screening of potential
therapeutics. We are very interested in looking at particular cytokine
pathways; pro-inflammatory cytokine pathways like IL-1, TNF and IL-12;
suppressive pathways like IL-4 and Il-10 and pro- survival pathways like
GN-TSF and M-CSF and IL-7.

Finally I would like to acknowledge that this represents the work of a
lot of people especially in my lab. I would like to make a special point
that we just found out today by letter that Carla Lankford won an award
from the American Federation for Medical Research, Henry Christian
Award, for her work on the positive feedback model for T-bet during the
short time she was in my lab. Thank you very much.

DR. BONVINI: Mr. Chairman, Mr. Rao, distinguished members of the
Advisory Committee. My name is Ezio Bonvini. I\'m here to represent Dr.
Gerald Feldman who could not be here because he\'s attending a
regulatory conference in San Diego.

My task before you today is to briefly summarize the work of Gerry in
the lab. I\'ll start by mentioning the two projects he\'s involved with.
The major focus is represented by the PRIF program. That deals with the
signaling interference or crosstalk we will call it between
immunocomplex interferon gamma in monocytes. Monocytes are an important
first line of defense against pathogens and the critical antigen
processing and presenting cells such as we heard in the regulation of
the immune response.

I will only briefly touch the second aspect which is something that Dr.
Feldman started recently in our laboratory and that is the effect of
prion proteins on monocyte reliability cells. This is dictated by the
fact that studying immunocomplexes and their role in regulating immune
responses is quite an important area of interest within the FDA. That\'s
because not only are autoimmune diseases directly linked to
immunocomplexes but also because the use of antibody therapeutics has
forced us scientists of the FDA and the field in general to be more and
more aware of the IC receptor or the C portion of the antibody. The ?-
IC receptor pertain to both the effector function of antibody and the
generation of adverse effects.

As I stated earlier, monocytes are critical regulatory cells in a
disease and they are activated by a number of cytokines including
interferon gamma. The path of activation by interferon gamma has been
fairly well illustrated by work of numerous investigators, including Dr.
Feldman when he worked with Dr. Finland in the laboratory at CBER. What
this pattern shows is that interferon gamma hits a specific receptor on
the surface of monocytes and triggers the activation of a Janus Tyrosine
kinase family, JAK-1, JAK-2 which in turn activates a transcription
factor, Stat1, which is translocated to the nucleus where induces a
number of activation of a number of genes.

Now in the work by other scientists and in particular Emil Unanue and
Bob Schreiber showed that immunocomplexes interfere with the response to
interferon gamma in a negative fashion. With the knowledge of how the
interferon gamma signal perceives, Dr. Feldman set up to understand
where the effect was and by using a retrograde approach he went on to
show that immunocomplex pretreatment will down-regulate the entire
JAK-STAT activation pathway but he did so without affecting interferon
gamma binding, receptor expression or the forceful relation of the alpha
chain of the interferon gamma receptor. However the inhibition
correlated well with the inhibition or the reduction in activation of
the JAK-STAT pathway.

There are several IC receptors on the surface of the monocyte. We now
can put some names on these receptors. By the process of exclusion, Dr.
Feldman showed that this receptor, the CD64, FC gamma receptor 1 is the
one important for inducing inhibitory response. Surprisingly the CD32
which is the FC gamma receptor 2(b) which is the prototypical inhibitory
receptor was not involved. The FC gamma receptor 1 hits the Stat kinase
which also went on to activate a tyrosene phosphatase, SHIP-1, which
presumably serves as an inhibitory effect early on upstream with the
activation of JAK kinase.

We think that this is the first mechanistic evidence of how the CD64
exerts an inhibitory crosstalk on the interferon gamma receptor. I think
that this is an important first step to appreciate the potential impact
of immunocomplices and therapeutic antibodies on FCS.

I mentioned that Dr. Feldman got involved in a second project and that
stemmed from his leading role within the Office of Therapeutics in the
issue pertaining to the disease CJD. Because of his interest in
monocytes, he went on to study monocyte-derived dendritic cells. The
characteristics of dendritic cells are actually quite important because
they are found in the plaque associated with CJD disease, were they
producing inflammatory cytokines and they probably have a role in the
transport of prion protein from gut to the lesions.

He went on to show that dendritic cells are activated by a prion-derived
peptide in the treatment of dendritic cells for this prion derived
peptide-induced deactivation which leads to the induction of
pro-inflammatory cytokines which leads to the further activation and
maturation of these cells. This is fairly similar to what happened in
monocyte induction by him and by Murphy\'s group recently about these
cells responding to chemotaxis.

However there are quite important differences. For instance, the
dendritic cells show no constant response whatever in response to this
peptide, whereas monocytes do. The two cell types show differential
sensitivity to cartoxis toxin treatment which is a tool that we use to
determine the involvement of G-protein in this signaling. In the case of
monocytes, this prion peptide was involved in the activation of a
chemotaxis receptor which was not involved in the dendritic cells. That
led us to conclude that dendritic cells have a functionally different
response to prion peptide as do monocytes. Perhaps more importantly, the
prion peptide can use a differential signaling mechanism possibly which
is as specific.

I would like to acknowledge the people in this project, the laboratory
of Michelle Jessen. Dr. Feldman would like to encourage a couple of
collaborators, Petra Lenz from DNCI and John O\'Shea from NINLS. Thank
you very much for your attention.

DR. KOZLOWSKI: Hi. I\'m Steve Kozlowski in the Division of Monoclonal
Antibodies. I\'m also involved in the regulation of these products and I
would like to talk about the activation of T-cells, a critically
important issue both in immuno-protection and immunopathology.

Clearly to activate a T-cell, you need to have an antigen reach it.
That\'s generally done through an antigen-presenting cell. This doesn\'t
happen though in isolation. It happens in the context of many other
cells in the tissue in which it occurs. It occurs also after an
activation and signaling of both these cells through their passageway to
get to each other and through soluble mediators that are present when
they interact; and finally, last but not least, through the direct
interaction between the antigen-presenting cell and the T-cell.

We approached studying this through two opposite ends. We looked at
isolated T-cell ligands in a very minimalist in vitro activation system
and we looked at the opposite side, in vivo outcome of T-cell
activation. The way we linked these two different ends was to use a very
similar system. We used a transgenic T-cell mouse which had CD8 cells
primarily all sharing the same receptor specificity which is
allo-reactivity against the H2LODMHC molecule in the context of
endogenous peptide.

In moving to this first project that isolated T-cell ligands, T-cells
are activated through presentation of antigen to the specific T-cell
receptor. This has been called signal 1. It\'s been called signal 1
because it is the critical attribute of the immune cells that allows
them to have both specificity and memory for specific antigens, the
antigen-specific derived signal.

However important that signal is though, it\'s been found that it\'s
often not enough and there are other additional signals necessary for
full T-cell activation. In a model that\'s many decades old now, we
would say that the antigen specific signal plus another signal gives you
activation, whereas the antigen specific signal alone can be even
negative much less not necessarily positive.

The providing of the signal 2 has been termed co-stimulation. A classic
molecule for this is CD8 on the T-cells and its ligand, B7 molecule that
allow full activation of the T-cells. However although this is a prime
example of this signal, there are a myriad of other molecules and I\'m
showing you a small subset of them which also can function as a signal
too in some way or form. There are also negative second signals which
can actually down regulate T-cell activation.

All of these molecules are potential sites for therapeutic intervention.
In fact I would say the vast majority of these molecules have antibodies
to block them under IND or being licensed. So it\'s critical to
understand the role of these.

One of the questions we had from this is although there\'s a lot of data
to say signal 1 alone is not enough is that all was true. In other
words, is it absolutely true that you always need a second signal to
activate a T-cell?

Using a cell-free system, T-cell contained, but antigen-presenting,
cell-free system where you activate it with purified MHC peptide in the
absence of any presenting cells, we asked the question could we activate
T-cells and was the second signal absolutely necessary? We found for
naive CD8 T-cells, and they were sorted to be a naive phenotype, they
could be activated in the absence of antigen-presenting cells with
purified ligand.

Now activated may not be fully activated but it occurs in a large
fraction of cells when you look by flow cytometry for markers of
activation. It\'s associated with high levels of IL-2 production. These
cells mature to become lytic effectors. We would define this as full
activation.

Furthermore if we add blocking antibodies to LFA-1 or B7 molecules,
which both are forms of co-stimulation, we don\'t alter the activation.
Using the system though required using a high dose of extremely potent
peptide.

Under physiological conditions, second signals may be necessary but in
some aberrant situations which may lead to autoimmunity or to failure of
immunosuppression in some circumstances, this isolated signal, when
alone, can operate. By using weaker signals we can add back other
molecules. We have already added back I-chem 1 and we are looking to add
back other molecules to test in this isolated system the interaction
between these second signals.

I want to make a point though here. With all the things that we added
back including IL-4, we have never seen a TH2 response. We have never
seen these cells make IL4 in vitro. They will make interferon gamma but
not IL4.

Although data with isolated ligands has its advantages, nonetheless the
question is can you go back and say these interactions are relevant. So
despite the fact that antigen MHC, co-stimulators, soluble mediators,
even changes in cell surface typology are good readouts and markers of
T-cells, nonetheless, what happens in vivo? To look in vivo, we used an
adoptive transfer model. We look though at our outcome as cytokine
production and C2.

I want to talk a little bit about what David Frucht mentioned before but
type I versus type II cytokines. There is type I cytokines, T-cytokines
that family would be interferon gamma and type II IL4. They have very
different responses on the overall immune phenotype and they tend to
polarize. In fact, David Frucht gave you an example of an interferon
group. Cytokines tend to feedback positively and that allows a
polarization effect to one type or another. This has tremendous impact
because it alters disease outcomes both in disease models and in some
examples of human disease.

There are a number of factors that can affect these things including
cytokine, antigen dose, co-stimulatory molecules, chemokines and antigen
presenting cells can all affect this polarization which affects disease
outcome. As a broader range of what\'s regulated all of these things are
potential therapies under development in some way or another. All of
them may have impact on diseases. Again in vivo it\'s not any of these
in isolation. It\'s all of them together in concert that have an effect.

To look at this we adoptively transferred the same T-cells, purified the
same way that we used for our in vitro experiments. We generated a
graft-versus-host disease model. We took these cells. We isolated the
spleens. We purified the T-cells. We added 2x10^6^ purified T-cells. I
can tell you these were counted after processing. We never froze them.

These were then transferred into an F1 mouse which was selected so that
it would not reject the cells that were transplanted and so that it
would allow the allo response to be generated against them. Then we
immunized these mice with antigenic peptide that it sees endogenously
but at much higher levels and much stronger response.

Then we looked at tissues, generally at five days later, but sometimes
at different time points. The first thing we looked at we looked at was
whether these cells were migrating to these tissues so we stained with
an antibody specific for clonotypic T-cell receptor and looked in these
tissues. We found that primarily we saw two CT-cells in spleen, some in
mesenteric lymph nodes and in testes. We didn\'t see them in any of the
other tissues.

Then to look at the immune phenotype as I mentioned before we looked at
cytokine families. We first looked at IL4. We stained spleen and testes
for IL4-producing cells. Strikingly, and very unexpectedly, because in
vitro we could not make these cells make IL4, we found large numbers of
IL4-positive cells in the spleen. However, in the testes we found none.
We looked at interferon gamma, the type 1 T-cytokine, and we found
staining of interferon gamma positive cells in the testes but none in
the spleen. We saw a complete and total opposite polarization depending
on the tissue that you looked at.

We further went on to do double staining and proved that although not
all these cytokine producing T-cells are the adoptively transferred
T-cells, many of them are. The exact same clonotypic T-cell is making
the opposite cytokine in the different tissues.

This has a number of implications. Tissue specific immune responses to
identical epitopes in which we have clonotypic T-cells exactly the same
receptor making opposite cytokines, depending on the organ they were in.
It\'s dependent on peptide. If we do this without adding the antigen, it
doesn\'t happen. It occurs with peptides of a 30-fold difference in
avidity so it\'s not some narrow range of dose and not only have we
shown it by immunohistochemistry but also by ELISA.

I think the take-home from this is modulating the immune response
phenotype, even though it may ameliorate pathology in one organ, it
might exacerbate it in another. You really need to know what\'s going on
in all the tissues. In fact for toxicology issues for the FDA it may be
very important to look in models that not only evaluate infiltrates in
tissues but look at specific immunophenotypes of the responses in these
tissues.

Ultimately the goal of understanding both individual ligand-receptor
interactions and in vivo responses is trying to integrate all this
together. The immune system is a myriad of interactions. Many cytokines
were defined by the term pleiotropies and redundances.

Many of them do the same things but many of them do slightly overlapping
differences in terms of exactly what they do. If you start thinking
about in vitro figuring this out, I did a little back of the envelope
calculation here. There are about 80 cytokines and say 15 of them are
involved in immune responses. There are 40 chemokines that are known and
say 10 of them are involved in immune response. And say out of a dozen
or so co-stimulatory molecules, five of them. That\'s 10^20^
possibilities and that\'s not even looking at doses. So you clearly
can\'t mimic this entirely in vitro.

The question is you need to be able to make correlations. I think our
strategy is to use an identical system in vivo and in vitro in terms of
the T-cell and the T-cell receptor to look at it at difference scales of
activation, both from individual ligands to whole tissue responses and
to try and assess the contribution of different ligands and compare them
to what we see in the microenvironment.

In my lab, Julia Goldstein, Trina Chen, Elena Govina have done most of
the work with the in vitro system. Lee Zhang has done all these adoptive
transfer experiments. Thank you.

CHAIRMAN SALOMON: Thank you, Steve. Just to show you how young I am, we
were post-docs together at the NLI ten years ago. That\'s true. I think
now we go to the closed session, right? I want to thank everyone who has
presented this afternoon. I\'ve said it again and again but it can\'t be
said enough. The tremendous respect all of us have for the high quality
intramural work that\'s being done at the FDA. I know you don\'t get
enough appreciation. I know it\'s a battle.

Appreciation equals money. I get it. The fact that you get so much good
quality work done while there is so much competing for your time and you
get really relative to what we have in Research Institute like at
Scripps or Harvard is just fantastic. You do have our respect. It\'s
really an important part of the FDA mission. Off the record.

(Whereupon, at 5:49 p.m., the above-entitled matter concluded.)